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1090 IN VIVO BIOEQUIVALENCE GUIDANCES1
This chapter is divided into two sections that provide guidances related to the general conduct of bioequivalence tests and to bioavailability protocols for specific drugs. These statements were prepared by the Food and Drug Administration, Office of Generic Drugs (FDA, OGD), Division of Bioequivalence, and presented herein. For general background information on related issues, refer to In Vitro and In Vivo Evaluation of Dosage Forms 1088.

GENERAL GUIDANCES
Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design2
INTRODUCTION
The FDA Division of Bioequivalence in the Office of Generic Drugs usually evaluates bioequivalence by comparing the in vivo rate and extent of drug absorption of a test and reference formulation in healthy subjects. In a standard in vivo bioequivalence study design, study participants receive test and reference products on separate occasions, in either single or multiple doses, with random assignment to the two possible sequences of product administration. Samples of an accessible biologic fluid such as blood or urine are analyzed for drug and metabolite concentrations, and pharmacokinetic parameters (AUC, Cmax and Tmax) are obtained from the resulting concentration-time curves. These pharmacokinetic parameters are then analyzed statistically to determine if the test and reference products yield comparable values.
Standard statistical methodology based on the null hypothesis is not appropriate to assess bioequivalence. The Division of Bioequivalence has therefore employed a testing procedure termed the two one-sided tests procedure to determine whether average values for pharmacokinetic parameters measured after administration of the test and reference products are comparable. This procedure involves the calculation of a confidence interval for the ratio (or difference) between the test and reference product pharmacokinetic variable averages. The limits of the observed confidence interval must fall within a predetermined range for the ratio (or difference) of the product averages. The determination of the confidence interval range and the statistical level of significance are judgments made by the Division of Bioequivalence.
This guidance provides information about general pharmacokinetic and statistical analyses of bioequivalence data to be conducted by sponsors of abbreviated new drug and antibiotic applications and addresses three specific aspects of the statistical analysis as follows:
  1. Logarithmic transformation of pharmacokinetic data
  2. Sequence effect
  3. Outlier consideration.
This guidance became effective July 1, 1992. Any investigations initiated after this date should generally conform to the recommendations of the guidance. Sponsors following a different approach are encouraged to discuss the matter in advance with the FDA to prevent the expenditure of money and effort on preparing a submission that may later be determined to be unacceptable.
GENERAL METHODOLOGY
Pharmacokinetic Analysis—
Single-Dose Studies— At a minimum, the following pharmacokinetic parameters for the substances of interest should be measured in a single-dose bioequivalence study:
a. Area under the plasma/blood concentration-time curve from time zero to time t (AUC0–t), calculated by the trapezoidal rule, where t is the last measurable time point.
b. Area under the plasma/blood concentration-time curve from time zero to time infinity (AUC0–), where AUC0– = AUCt + Ct /Z, Ct is the last measurable drug concentration, and Z is the terminal elimination rate constant calculated according to an appropriate method. The terminal or elimination half-life of the drug (t½) should also be reported.
c. Peak drug concentration (Cmax) and the time to peak drug concentration (Tmax), obtained directly from the data without interpolation.
Multiple-Dose Studies— At a minimum, the following pharmacokinetic parameters for the substances of interest should be measured in a multiple-dose bioequivalence study:
a. Area under the plasma/blood concentration-time curve from time zero to time over a dosing interval at steady state (AUC0–), where is the dosing interval.
b. Peak drug concentration (Cmax) and the time to peak drug concentration (Tmax), obtained directly from the data without interpolation, after the last dose is administered.
c. Drug concentrations at the end of each dosing interval during steady state (Cmin).
d. Average drug concentration at steady state (Cav), where Cav = AUC0–/.
e. Degree of fluctuation (DF) at steady state, where DF = 100% × (Cmax – Cmin)/Cav.
Evidence of attainment of steady state for the test and reference products should be submitted in the bioequivalence study report.
Statistical Analysis— Parametric (normal-theory) general linear model procedures are recommended for the analysis of pharmacokinetic data derived from in vivo bioequivalence studies. An analysis of variance (ANOVA) should be performed on the pharmacokinetic parameters AUC and Cmax using appropriate statistical programs and models. For example, for a conventional two-treatment, two-period, two-sequence (2 × 2) randomized crossover study design, the statistical model often includes factors accounting for the following sources of variation:
  1. Sequence (sometimes called Group or Order)
  2. Subjects, nested in sequences
  3. Period (or Phase)
  4. Treatment (sometimes called Drug or Formulation).
The sequence effect should be tested using the [subject (sequence)] mean square from the ANOVA as an error term. All other main effects should be tested against the residual error (error mean square) from the ANOVA. The least square means (LSMEANS) statement should be used to calculate least square means for treatments. Estimates should be obtained for the adjusted differences between treatment means and the standard error associated with these differences.
The two one-sided hypotheses at the = 0.05 level of significance should be tested for AUC and Cmax by constructing the 90% confidence interval for the ratio between the test and reference averages.
LOGARITHMIC TRANSFORMATION OF PHARMACOKINETIC DATA
Statistical Assumptions— The assumptions underlying the ANOVA are:
  1. Randomization of samples
  2. Homogeneity of variances
  3. Additivity (linearity) of the statistical model
  4. Independency and normality of residuals.
In bioequivalence studies, these assumptions can be interpreted as follows:
  1. The subjects chosen for the study should be randomly assigned to the sequences of the study.
  2. The variances associated with the two treatments, as well as between the sequence groups, should be equal or at least comparable.
  3. The main effects of the statistical model, such as subject, sequence, period, and treatment effect for a standard 2 × 2 crossover study, should be additive. There should be no interactions between these effects.
  4. The residuals of the model should be independently and normally distributed. In other words, data from bioequivalence studies should have a normal distribution.
If these assumptions are not met, additional steps should be taken prior to the ANOVA including data transformation to improve the fit of the assumptions or use of a nonparametric statistical test in place of ANOVA. However, the normality and constant variance assumptions in the ANOVA model are known to be relatively robust [i.e., a small or moderate departure from each (or both) of these assumptions will not have a significant effect on the final result].
Rationale for Log Transformation— It is acceptable to use logarithms to the base 10 or natural logarithms (ln). The report must state unambiguously which logarithms were used, and the use must be consistent throughout.
Clinical Rationale— The primary comparison of interest in a bioequivalence study is the ratio of average parameter data from the test and reference formulations rather than the difference between them. Using log transformation, the general linear statistical model employed in the analysis of bioequivalence data allows inferences about the difference between the two means on the log scale, which can then be retransformed into inferences about the ratio of the two averages (means or medians) on the original scale. Log transformation thus achieves the general comparison based on the ratio rather than the difference.
Pharmacokinetic Rationale— Westlake observed that a multiplicative model is postulated for pharmacokinetic parameters in bioavailability/bioequivalence studies, i.e., AUC and Cmax (but not Tmax). Assuming that elimination of the drug is first order and only occurs from the central compartment, the following equation holds after an extravascular route of administration:
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where F is the fraction absorbed, D is the administered dose, and FD is the amount of drug absorbed. CL is the clearance of a given subject, which is the product of the apparent volume of distribution (V) and the elimination rate constant (Ke).3
The use of AUC as a measure of the amount of drug absorbed thus involves a multiplicative term (CL), which might be regarded as a function of the subject. For this reason, Westlake contends that the subject effect is not additive if the data is analyzed on the original scale of measurement.
Logarithmic transformation of the AUC data will bring the CL(VKe) term into the equation in an additive fashion:
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Similar arguments were given for Cmax. The following equation applies for a drug exhibiting one compartmental characteristic:
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where again F, D, and V are introduced into the model in a multiplicative manner. However, after logarithmic transformation the equation becomes
Click to View Image
Log transformation of the Cmax data also results in the additive treatment of the V term.
Statistical Rationale— Logarithmic transformation of the data from bioequivalence studies can be used to circumvent the use of estimates of the reference product average for computation of the confidence interval for the ratio of product averages. This is an advantage for the cases where a least square estimate for the reference product mean is not well defined. Standard parametric methods are ill-suited to making inferences about the ratio of two averages, though some valid methods do exist. Log transformation changes the problem to one of making inferences about the difference (on the log scale) of two averages, for which the standard methods are well suited.
Many biological data correspond more closely to a log-normal distribution than to a normal distribution. The plasma concentration data, including the derived parameters AUC and Cmax, tend to be skewed, and their variances tend to increase with the means. Log transformation is likely to remedy this situation and make the variances independent of the mean. In addition, frequency distributions skewed to the left (with a long tail to the right) are often made more symmetrical by log transformation.
This argument is actually less persuasive than the argument based on the additivity of the statistical model because it is based largely on the between-subject distribution of AUC and Cmax values. For crossover studies, it is largely the within-subject distribution of values that determines the validity and efficiency of the standard parametric methods of analysis. Despite the arguments regarding the effect of log transformation on normality of bioequivalence data, it is recognized that the limited sample size (20–30 subjects) in a bioequivalence study precludes a reliable determination of the underlying normal distribution of the data set either with or without log transformation.
General Procedures— Pharmacokinetic parameters AUC and Cmax should be log transformed. Firms are not encouraged to test for normality of data distribution after log transformation, nor should they employ normality of data distribution as a justification for carrying out the statistical analysis on the original scale. Robustness of a balanced study to non-normality of the data plus use of log transformation will be adequate in most cases.
If a firm believes that the data of a particular bioequivalence study should be statistically analyzed on the untransformed basis rather than the log scale, justification based upon a sound scientific rationale, as well as the statistical methods to be used, ought to be submitted to and reviewed by the FDA Division of Bioequivalence or a comparable regulatory authority.
Presentation of Data— The drug concentration in biological fluid at each sampling time point should be furnished untransformed for all the subjects who participated in the study. The derived pharmacokinetic parameters should also be furnished untransformed. The mean, the standard deviation, and the coefficient of variation for each variable should be computed and tabulated in the final report.
To facilitate bioequivalence comparisons, pharmacokinetic parameters for each individual should be displayed in parallel for the formulations tested. In particular, for AUC and Cmax, the difference (T R), ratio (T/R), and log of ratio (log T/R or ln T/R) between the test and reference values should be tabulated side by side for all the subjects. For each subject, the summary tables should indicate in which sequence (test, reference or reference, test) the subject received the product. Histograms showing the frequency distribution of the difference and ln ratio (or log ratio) for the major pharmacokinetic parameters (AUC and Cmax) are useful in the submission.
In addition to the arithmetic mean for the test and reference products, the geometric means (antilog of the means of the logs), means of the logs, and standard deviations of the logs should be calculated for AUC and Cmax. All means, including arithmetic mean, geometric mean, and means of the logs, as well as standard deviations and coefficients of variation, are to be included in the report.
Equivalence Criteria— For a broad range of drugs, the FDA Division of Bioequivalence used a range of 80% to 120% for the ratio of the product averages as the standard equivalence criterion when the study data are analyzed on the untransformed basis. This corresponds to a range of ±20% for the relative difference between the product averages.
When log-transformed data are used in the analysis of AUC and Cmax, using a range of 80% to 125% for the ratio of averages has an advantage over the 80% to 120% criterion in that for the analysis of log-transformed data, the probability of concluding equivalence is at a maximum if the ratio of averages is in fact 1.0 (i.e., exact equality). For the analysis of log-transformed data with a criterion of 80% to 120%, the maximum probability of concluding equivalence occurs when the ratio of product averages equals approximately 0.98. Thus, an equivalence criterion of 80% to 125% is used for the ratio of the product averages.
The 90% confidence interval for the difference in the means of the log-transformed data should be calculated using methods appropriate to the experimental design. The antilogs of the confidence limits constitute the 90% confidence interval for the ratio of the test and reference product averages.
SEQUENCE EFFECT
A major limitation of a conventional two-treatment, two-period, two-sequence crossover design is the confounding between (i) a true sequence or group effect, (ii) unequal residual or carryover effects, and (iii) treatment-by-period interactions. A true sequence effect (i.e., a difference between the average response for sequence group one and sequence group two) would not bias the determination of bioequivalence. Unequal residual effects, however, would bias this estimate. A treatment-by-period interaction based on an underlying physical basis (i.e., if there were actually something about the periods that caused the difference between the product averages to differ from one period to another), would lead to difficulties in interpreting the estimate of the ratio (difference) in the pharmacokinetic parameters between the test and reference formulations.
Even if there were no true sequence effect, no unequal residual effects, and no treatment-by-period statistical interaction, approximately 10 out of every 100 two-treatment crossover studies would be likely to show an apparent sequence effect, if the testing is carried out at the 10% level of significance.
If the ANOVA test for the presence of a sequence effect results in statistical significance, the actual cause cannot be determined from the data alone. In some cases, plausible causes might be evaluated by examining demographic or physiological subject data, but this examination is seldom conclusive.
On the basis of these considerations, an in vivo two-treatment, two-period, two-sequence crossover bioequivalence study showing a statistically significant sequence effect may be acceptable provided:
  1. It is a single-dose study;
  2. It includes only healthy, normal subjects;
  3. The drug is not an endogenous entity;
  4. More than adequate washout period has been allowed between the two phases of the study, and in the second phase, the predose biological matrix samples do not exhibit any detectable drug level in all subjects; and
  5. The study meets all scientific and statistical criteria such as:
    1. It is based upon an acceptable study protocol;
    2. It contains an acceptable validated assay methodology;
    3. The study data are acceptable;
    4. Appropriate statistical analyses of the data are performed; and
    5. Acceptable confidence intervals for the pharmacokinetic parameters are achieved.
Under all other circumstances, the sponsor may be asked to conduct another study. After regulatory review, multiple-dose studies or studies in patients demonstrating a statistically significant sequence effect may be acceptable provided they meet all other criteria listed above.
OUTLIER CONSIDERATION
Outliers are defined in bioequivalence studies as subjects having discordant values of one or more pharmacokinetic parameters when compared with other values in a study. Because bioequivalence studies are usually carried out as crossover studies, the most important type of outlier is where one or a few subjects differ notably from the rest of the subjects for the test product response versus the reference product response (e.g., test minus reference difference, test/reference ratio, or the log of the test/reference ratio).
The existence of an outlier could be indicative of the following problems with interchangeability of two formulations:
Product Failure— A subject obtained an unusually high or low response to one or the other of the products because of a problem with the specific dosage unit(s) administered. Examples include a modified- or extended-release dosage form exhibiting dose dumping or a dosage unit whose coating inhibited dissolution.
Subpopulation— A subject may be representative of a type of subject, present in the general population in low numbers, for whom the relative bioavailability of the two products is markedly different than it is for the majority of the population, and for whom the two products are not bioequivalent, even though they might be bioequivalent in the majority of the population.
In the case of product failure, it may make a difference whether the unusual response is observed on the test product or the reference product. In the case of a subpopulation, however, even if the unusual response is observed on the reference product, there may still be concern for lack of interchangeability of the two products.
Statistical tests exist for outlier identification. For detection of a single outlier, one important test is based on the absolute value of the “Studentized Residual.” Out of all the data in the study, the test focuses on the most extreme. Approximate critical values for this test have been published by Lund.9 In principle, however, outliers cannot be dropped from the analysis of the data solely on the basis of a statistical test. When one or more outliers are identified, one should provide scientific evidence or explanations to justify the exclusion of the subject data from statistical analysis.
Oral Extended-Release Dosage Forms4
This guidance describes in vivo bioequivalence studies and in vitro drug release testing recommended to applicants intending to submit Abbreviated New Drug Applications (ANDAs) for extended-release products administered orally.
NOMENCLATURE
Modified-Release Dosage Forms— A modified-release dosage form is one for which the drug release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed-release and extended-release dosage forms are two types of modified-release dosage forms. This guidance does not consider bioequivalence studies for delayed-release formulations.
Delayed-Release Dosage Forms— A delayed-release dosage form is one that releases a drug(s) at a time other than promptly after administration.
Extended-Release Dosage Forms— An extended-release dosage form is one that allows at least a twofold reduction in dosing frequency or significant increase in patient compliance or therapeutic performance as compared to that presented as a conventional dosage form (e.g., as a solution or a prompt drug-releasing, conventional solid dosage form).
The terms controlled release, prolonged action, and sustained release are used synonymously with extended release. This document uses the term extended release to describe a formulation that does not release active drug substance immediately after oral dosing and that also allows a reduction in dosage frequency. This nomenclature accords generally with the USP definition of extended release but does not specify an impact on dosing frequency. The terms controlled release and extended release are considered interchangeable in this guidance.
REGULATORY BACKGROUND and GENERAL REQUIREMENTS
The Drug Price Competition and Patent Term Restoration Act amendments of 1984 to the Food, Drug, and Cosmetic Act gave the Food and Drug Administration statutory authority to accept and approve for marketing ANDAs for generic substitutes of innovator products, including those approved after 1962. To gain approval, ANDAs for a generic extended-release formulation must demonstrate, among other things, that the formulation is both pharmaceutically equivalent and bioequivalent to the innovator extended-release product, which is also termed the reference listed product as identified in FDA's Approved Drug Products with Therapeutic Equivalence Rating, “The Orange Book” (USP DI, Volume III).
Pharmaceutical Equivalence— To be pharmaceutically equivalent, the generic and innovator formulations must (1) contain the same active ingredient; (2) contain the same strength of the active ingredient in the same dosage form; (3) be intended for the same route of administration, and (4) generally be labeled for the same conditions of use. The FDA does not require that the generic and reference listed extended-release products contain the same excipients or that the mechanism by which the active drug substance is released from the formulation be the same.
Bioequivalence Studies— Current regulations require that bioequivalence be demonstrated between a generic extended-release formulation and the reference listed product. The reference listed product is generally an extended-release product subject to an approved full New Drug Application (NDA). For approval, documentation of bioequivalence must be established through performance of a series of in vivo bioequivalence studies that are defined under the section In Vivo Bioequivalence Studies for Approval. Approval of an ANDA will rely on data derived from evaluation of a biobatch, which is to be manufactured in accordance with the Office of Generic Drugs Procedure and Policy Guide 22-90.
Quality control of the manufacture of an extended-release formulation after approval may be assessed, in part, through performance of in vitro dissolution tests. Preapproval submission of these data is required. Recommendations for the conditions under which this test may be performed are described under the section In Vitro Dissolution for Quality Control Preapproval. This section also describes how specifications for this test are developed by the applicant and approved by the Division of Bioequivalence. These data are required in the application for approval.
IN VIVO BIOEQUIVALENCE STUDIES FOR APPROVAL
In vivo bioequivalence studies recommended for approval for extended-release generic formulations are designed to document the following:
  • The drug product meets the extended-release claim made for it.
  • The drug product does not release the active drug substance at too rapid a rate (dose dumping).
  • Performance is equivalent between the generic and the reference listed product following single doses and dosing to steady state.
  • The influence of food on the vivo performance is comparable for the generic formulation relative to the innovator formulation.
The above objectives are generally met by three in vivo studies: (1) a single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions, comparing equal doses of the test and reference products; (2) a single-dose, randomized, three-treatment, three-period, six-sequence, crossover, limited food effects study, comparing equal doses of the test product administered under fasting conditions with those of the test and reference products administered immediately after a standard high-fat breakfast6 ; and (3) a multiple-dose, steady state, randomized, two-treatment, two-period, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference formulations. [NOTE—For safety reasons, this study may be performed in the nonfasting state. Applicants are encouraged to submit a study protocol describing the safety considerations requiring deviation from the fasting state to the Division of Bioequivalence for review prior to execution of the study.]
These studies are described in detail below. Under certain circumstances, the Division of Bioequivalence in the Office of Generic Drugs may require additional single-dose or multiple-dose steady state studies. The following general information relative to the three in vivo studies is provided:
  • FDA-designated reference product is identified by the symbol “+” in The Orange Book.
  • The assayed potency of the test product should not differ from that of the reference product by more than 5%.
  • The clinical laboratory conducting any in vivo study should retain an appropriately identified reserve sample of the test and reference products for a period of 5 years. Each reserve sample should consist of at least 200 dosage units. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
  • A single-dose two-way crossover study under fasting conditions is required for each strength of a generic extended-release tablet formulation with multiple strengths. The multiple-dose steady state study and the fasting/nonfasting single-dose three-way crossover study are to be conducted with the highest strength only.
For an extended-release bead type capsule formulation to be marketed in multiple strengths, a single-dose bioequivalence study under fasting conditions is required only on the highest strength, provided that the compositions of the lower strengths are proportional to that of the highest strength, and the capsules contain identical beads or pellets. Single-dose in vivo bioequivalence studies may be waived for the lower strengths on the basis of acceptable drug release profiles. Multiple-dose steady state and single-dose fasting/nonfasting studies are to be conducted on the highest strength of the capsule formulation.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
SINGLE-DOSE FASTING TWO-WAY CROSSOVER BIOEQUIVALENCE STUDY
Objective— The objective is to compare the rate and extent of absorption of a generic extended-release product with that of the reference-listed product when administered in equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with an adequate washout period (usually equal to at least 10 elimination half-lives of the drug) between the two phases of the study. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. More subjects may be required for a drug that exhibits high intra-subject variability in metrics of rate and extent of absorption. Subjects should be healthy, preferably nonsmoking, volunteers 18 to 50 years of age, and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be accepted on the basis of acceptable medical history, physical examination, and clinical laboratory tests. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all subjects before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water. They should continue fasting for 4 hours after administration of the test or reference treatment.
Restrictions— Study volunteers should observe the following restrictions:
  1. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  2. Subjects should take no Rx medications, including oral contraceptives, beginning two weeks prior to and no OTC medications beginning one week prior to initiation of the study and until after the study is completed.
  3. Water may be taken except for 1 hour before and after administration, when no liquid is allowed other than that needed for drug dosing.
  4. All meals during the study should be standardized, and the same meals should be served during both phases of the study.
Blood Sampling— In addition to the pre-dose (0 hour) sample, venous blood samples should be collected post-dose so that there are at least four sampling time points on the ascending part and six or more on the descending part of the concentration-time curve. The biological matrix (plasma, serum, or whole blood) should be immediately frozen after collection and, as appropriate, centrifugation, and kept frozen until assayed.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analysis of Blood Samples— The active ingredient should be assayed using a suitable analytical method validated with regard to specificity, accuracy, precision (both within and between days), limit of quantitation, linearity, and recovery. Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. If the analytical method is a chromatographic method, chromatograms of unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities.
Pharmacokinetic Analysis of Data— Calculation of area under the plasma concentration-time curve to the last quantifiable concentration (AUC0–t) and to infinity (AUC0–), Cmax, and Tmax should be performed according to standard techniques.
Statistical Analysis of Pharmacokinetic Data (see Statistical Procedures for Bioequivalence Studies Using a Standard Two-treatment Crossover Design)— The log transformed AUC and Cmax data should be analyzed statistically using ANOVA. These two parameters for the test product should be shown to be within 80% to 125% of the reference product using the 90% confidence interval.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examination and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
MULTIPLE-DOSE STEADY STATE, TWO-WAY CROSSOVER BIOEQUIVALENCE STUDY UNDER FASTING CONDITIONS
Objective— The objective is to compare the steady-state rate and extent of absorption of a generic extended-release formulation with that of the reference formulation when given as equal labeled doses.
Design— The study design is a multiple-dose, steady-state two-treatment, two-period, two-sequence crossover with an adequate washout period between the two phases of the study. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before beginning the study, the study protocol should be approved by an institutional review board.
Facilities and Selection of Subjects— See the appropriate section under Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
Procedure— Extended-release products that are administered once a day should be dosed following an overnight fast of at least 10 hours; subjects should continue fasting for 4 hours post-dose. For extended-release products that are dosed every 12 hours (b.i.d.), the morning dose should be given following an overnight fast of about 10 hours, and subjects should continue fasting for 4 hours post-dose; the evening dose should be administered 12 hours after the morning dose and after a fast of at least 2 hours and subjects should continue fasting for 2 hours post-dose. Each dose should be administered with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  2. Subjects should take no Rx medications, including oral contraceptives, beginning two weeks prior to and no OTC medications beginning one week prior to initiation of the study and until after the study is completed.
  3. Water may be taken except for 1 hour before and after administration, when no liquid is allowed other than that needed for drug dosing.
Blood Sampling— At least three trough concentrations (Cmin) on three consecutive days should be determined to ascertain that the subjects are at steady state prior to measurement of rate and extent of absorption after a single-dose administration in a dosing interval at steady state. The three consecutive trough samples should be collected at the same time of the day and should be comparable. For extended-release drug products administered more often than every 24 hours, assessment of trough levels just prior to 2 consecutive doses is not recommended because a difference in the consecutive trough values may occur due to circadian rhythm irrespective of whether or not steady state has been attained. Adequate blood samples should be collected at appropriate times during a dosing interval at steady state to permit estimation of the total area under the concentration-time curve, peak concentration (Cmax), and time to peak concentration (Tmax).
Subject Monitoring and Analysis of Blood Samples— See under Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
Pharmacokinetic Analysis of Data— The following pharmacokinetic data are to be reported for the evaluation of bioequivalence of the generic extended-release product with the reference listed product:
  1. Individual and mean blood drug concentration levels
  2. Individual and mean trough levels (Cmin)
  3. Individual and mean peak levels (Cmax)
  4. Calculation of individual and mean steady state AUCinterdose are recommended (AUCinterdose is AUC during a dosing interval at steady state)
  5. Individual and mean percent fluctuation [= 100 × (Cmax – Cmin)/Cmin]
  6. Individual and mean time to peak concentration (Tmax)
Statistical Analysis of Pharmacokinetic Data— The log transformed AUC and Cmax data should be analyzed statistically using ANOVA. These two parameters for the test product should be shown to be within 80% to 125% of the reference product using the 90% confidence interval. Fluctuation for the test product should be evaluated for comparability with that for the reference product. For further information on statistical analysis, see Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design.
Clinical Report, Side Effects, and Adverse Reactions— See under Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
SINGLE-DOSE, THREE-WAY CROSSOVER FASTING/NONFASTING BIOEQUIVALENCE STUDY
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of the reference listed formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with adequate washout period between the three phases of the study. An equal number of subjects should be randomly assigned to each dosing sequence. Before beginning the study, the study protocol should be approved by an institutional review board.
Facilities— See this section under Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
Selection of Subjects— A minimum of 18 subjects should be enrolled in this study. For other information on selection of subjects, see Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
Procedure— Each subject should receive the following three treatments:

TREATMENT 1: Generic extended-release product administered after a high fat content breakfast

TREATMENT 2: Innovator extended-release product (reference listed drug) administered after a high fat content breakfast.

TREATMENT 3: Generic extended-release product administered after fasting.
Following an overnight fast of at least 10 hours, subjects receiving treatments under nonfasting conditions should be served a high-fat breakfast then immediately dosed with Treatment 1 or Treatment 2 with 240 mL of water. Subjects receiving Treatment 3 should be dosed at the same time as Treatments 1 and 2 with 240 mL of water only. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Restrictions, Blood Sampling, Subject Monitoring, and Analysis of Blood Samples— See the appropriate section under Single-Dose Fasting Two-Way Crossover Bioequivalence Study.
Statistical Analysis of Pharmacokinetic Data— In general a comparable food effect will be assumed if the mean values of AUC0–t, AUC0–, and Cmax for the generic product administered with food differ by no more than 20% from the respective mean values for the reference listed product administered with food in the study.
Clinical Report, Side Effects, and Adverse Reactions— See under Single-dose Fasting Two-way Crossover Bioequivalence Study.
IN VITRO DRUG RELEASE FOR QUALITY CONTROL PREAPPROVAL
Drug Release Testing 724— Drug release testing should be conducted on 12 individual dosage units of the batches of the test and reference products used in the bioequivalence studies. The potential for pH dependence of drug release from an extended-release product is well recognized. Drug release profiles should therefore be generated in aqueous media at the following pH ranges: 1-1.5, 4-4.5, and 6.0-6.8. Early sampling times of 1, 2, and 4 hours should be included in the sampling schedule to provide assurance against premature release of the drug (dose dumping) from the formulation. Typical drug release conditions are shown below:
Apparatus 1 (for capsules): 100 rpm.
Apparatus 2 (for tablets): 50 and 75 rpm.
Temperature: 37 ± 0.5.
Units Tested: 12.
Medium: 900 mL of aqueous media at various pH values.
Times: 1, 2, and 4 hours, and every 2 hours thereafter, until 80% of the drug is released.
Tolerances: to be established based on data generated.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.
Specifications— Specifications for the drug release procedure to ensure quality control will be determined on a case-by-case basis. In general, further validation will be required to expand dissolution specifications beyond those established for the biobatch.

DRUG PROTOCOLS

Alprazolam Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Alprazolam is widely used in the management of anxiety disorders, panic disorder, social phobia, and depression. It is a triazolo analog of the 1,4-benzodiazepine class of compounds acting on the central nervous system (CNS). The exact mechanism of action of alprazolam is unknown; however, published literature indicates that this class of drugs exerts effects by binding to stereospecific receptors at several sites within the central nervous system. The drug effect is dose-related. The daily dose of alprazolam depends on the individual patient, and averages 1.5 to 4 mg. Although a major metabolite, -hydroxyalprazolam, shows some pharmacological activity, no alprazolam metabolites possess any clinically important role, and they do not accumulate to any significant degree.
Pharmacokinetics— Alprazolam is readily absorbed (more than 90%) after oral administration and is distributed widely with a volume of distribution of approximately 1 L per kg in healthy men and women. Peak concentrations in the plasma occur 1 to 2 hours after administration. With doses ranging from 0.5 to 3.0 mg given on an empty stomach, plasma peak levels of 8.0 to 37 ng per mL were observed. The mean plasma elimination half-life of alprazolam has been reported to be about 11.2 hours (range: 6.3 to 26.9 hours) in healthy adults. The drug absorption is slower when alprazolam is taken after a meal than on an empty stomach, but total absorption is unchanged.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Xanax® (Upjohn) 1-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, fasting, two-period, two-treatment, two-sequence crossover study comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsors should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, or OTC medications beginning 2 weeks before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1.0, 1.50, 2.0, 2.50, 3.0, 4.0, 6.0, 8.0, 12, 24, 36, 48, and 72 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following at least a minimum 1-week washout period, subjects should begin the second phase of the study.
Analytical Methods— Alprazolam should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data (Plasma)— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a period of not less than 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: phosphate buffer solution (pH 6.0); 500 mL.
Apparatus 1: 100 rpm.
Times: 10, 20, 30, and 45 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 80% in 30 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 0.25-, 0.5-, and 2-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 0.25-, 0.5-, and 2-mg strengths are proportionally similar in both active and inactive ingredients to another strength (1 mg), which has been demonstrated to be bioequivalent to a reference (1 mg) product in vivo.
  2. The 0.25-, 0.5-, and 2-mg strengths of the generic product meet dissolution requirements.
  3. The drug product follows linear kinetics over its range of available strengths.

Bumetanide Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Bumetanide is a potent diuretic indicated for the treatment of edema associated with congestive heart failure and hepatic and renal disease, including the nephrotic syndrome. Bumetanide is contraindicated in anuria, hepatic coma, states of severe electrolyte depletion, and in patients hypersensitive to the drug.
The major site of action of bumetanide is the ascending limb of the loop of Henle, where it inhibits the sodium-potassium-2 chloride absorptive pump. The usual daily dosing range for bumetanide is 0.5 to 2.0 mg.
Pharmacokinetics— Bumetanide is reported to be readily absorbed from the gastrointestinal (GI) tract with a Tmax of 0.5 to 2 hours, and a bioavailability of about 80% to 90%. Several pharmacokinetic studies have shown that bumetanide, administered orally, is eliminated rapidly in humans, with a half-life of between 1 and 2 hours. Plasma protein-binding is approximately 95%. Oral administration of bumetanide resulted in 36% to 60% recovery of the unchanged drug from urine. The volume of distribution (Vd) is approximately 0.2 L per kg. Following oral administration of bumetanide, the onset of diuresis occurs in 30 to 60 minutes. Peak activity is reached between 0.5 and 3 hours.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Bumex® (Roche Laboratories) 2.0-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with at least a three-day washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose (2.0 mg) of the test or reference product with 240 mL of water. The subjects should drink 240 mL of water at the following times: 2, 1, 0, 1, 2, 4, 6, 8, and 10 hours relative to dosing.
Restrictions— Study volunteers should observe the following restrictions:
a. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
b. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
c. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples in a volume sufficient for sample analysis and anticoagulated as appropriate should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 1.75, 2.0, 2.50, 3, 4, 5, 6, 8, and 12 hours post-dose. Plasma should be separated promptly, immediately frozen, and stored at –20 until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Urine Sampling— Urine may be collected over the following time intervals: 2 to 0, 0 to 1, 1 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10, and 10 to 12 hours post-dosing. The urine volume and pH should be noted. One 50-mL aliquot should be stored refrigerated for possible analysis of sodium and potassium. A 15-mL sample should be stored frozen at –20 for possible bumetanide assay.
Analytical Methods— High-performance liquid chromatographic methods have been reported for assaying bumetanide in human plasma and urine.
Bumetanide should be assayed using a suitable method fully validated with respect to stability, sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data (Plasma)— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Diuretic Effects and Bumetanide Urinary Excretion Data— Urine samples may be analyzed to determine the following parameters:
  1. The mean rate of excretion of sodium, potassium, water, and bumetanide for each collection period.
  2. The maximum excretion rate (excretion rate calculated for that time interval during which the rate is highest) for sodium, potassium, water, and bumetanide.
  3. Time to maximum excretion rate for sodium, potassium, water, and bumetanide.
  4. Cumulative excretion of sodium, potassium, water, and bumetanide over all sampling time intervals.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test product and the reference standard used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a period of not less than 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Times: 10, 20, 30, and 45 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 85% in 30 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 0.5- and 1.0-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 0.5- and 1-mg tablets are proportionally similar in both active and inactive ingredients to the 2-mg tablet, which has been demonstrated to be bioequivalent to the reference 2-mg product in vivo.
  2. The 0.5- and 1-mg tablets of the generic product meet dissolution test requirements.

Buspirone Hydrochloride Tablets—

In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Buspirone hydrochloride is an anti-anxiety agent. Clinically it is effective in the management of anxiety disorders or short-term relief of symptoms of anxiety. Buspirone has no anticonvulsant or muscle relaxant activity and has little sedative effect. It does not substantially affect psychomotor function. There is no evidence that the drug causes either physical or psychological dependence. The mechanism of action of buspirone is not known. Some in vitro preclinical studies indicate that buspirone has a high affinity for serotonin (5-HT1A) receptors and a moderate affinity for brain D2 receptors.
For the management of anxiety disorders, the usual initial adult dosage of buspirone is 10 to 15 mg daily, usually in 2 or 3 divided doses. Dosage is increased, as necessary, in increments of 5 mg daily to achieve an optimal therapeutic response. The maximum daily dose should not exceed 60 mg per day.
Pharmacokinetics— Buspirone is rapidly and almost completely absorbed from the GI tract. The drug undergoes extensive first-pass metabolism, with about 4% of a dose reaching the systemic circulation unchanged following oral administration. Following oral administration of a single dose of 20 mg in healthy volunteers, peak plasma buspirone concentrations of 1 to 6 ng per mL have been observed to occur within 40 to 90 minutes. Plasma concentrations of unchanged buspirone are low and exhibit substantial interindividual variation with oral administration of the drug. Approximately 95% of buspirone is bound to plasma proteins.
Buspirone is rapidly metabolized by oxidation to produce several hydroxylated derivatives and a pharmacologically active metabolite, 1-pyrimidinylpiperazine(1-PP). Because of rapid metabolism, less than 1% of the parent drug is excreted unchanged in the urine. The pharmacologically active metabolite has about 20% to 25% of the anxiolytic activity of buspirone. In humans, blood concentrations of 1-PP remain low even after chronic administration of buspirone. The contribution of 1-PP to the pharmacologic and toxic effect thus remains to be elucidated fully.
The average elimination half-life of unchanged buspirone after single doses of 10 to 40 mg is reported to be 2 to 3 hours. Buspirone exhibits linear kinetics following administration of doses (10 to 40 mg) in the therapeutic range. Although food increases the bioavailability of buspirone by decreasing first-pass metabolism, the total amount of drug (changed and unchanged) in plasma is not affected.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Buspar® (Bristol-Meyers Squibb) 10-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of buspirone and 1-PP from a generic formulation with that from a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing or a single-dose, two-treatment, four-period, four-sequence replicate design study with a 1-week washout period between each phase of dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered four 10-mg tablets of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 7, 8, 12, and 24 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Buspirone and 1-PP should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of buspirone and 1-PP from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and Phase II, and Phase II and Phase III.
The limited food effects study (with a minimum of 12 subjects) should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, four 10-mg tablets administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, four 10-mg Buspar® tablets administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, four 10-mg tablets administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–T, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing, whichever is greater, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile.
Times: 10, 20, 30, and 45 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest, lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 5-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 5-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 10-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 5-mg tablet of the generic product meets dissolution test requirements.

Captopril Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Captopril, an antihypertensive, inhibits the enzyme that converts angiotensin I, a relatively inactive decapeptide, to angiotensin II, a potent endogenous vasoconstrictor substance. This enzyme, angiotensin converting enzyme, or ACE, is a peptidyldipeptide carboxy hydrolase.
The recommended initial dosage for treatment of hypertension is 25 mg twice a day (b.i.d.) or three times a day (t.i.d.). This can be increased to 50 mg b.i.d. or t.i.d. after 1 or 2 weeks if the lower dose is ineffective. Doses of captopril higher than 50 mg b.i.d. are recommended only with concomitant administration of a thiazide diuretic.
Pharmacokinetics— Following oral administration, approximately 60% to 75% of the dose of captopril is rapidly absorbed from the gastrointestinal tract in fasting healthy adults or hypertensive patients. Peak blood levels of unchanged captopril occur about 1 hour after oral administration. Areas under the concentration-time curve and maximum blood concentrations after single oral doses of captopril appear to be dose-related over a range of 10 to 100 mg. Approximately 25% to 30% of the drug in the systemic circulation is bound to plasma proteins.
Because the presence of food in the GI tract is reported to reduce absorption of the drug by 30% to 40%, captopril is labeled to be dosed 1 hour before meals. Blood pressure reduction is usually maximal 60 to 90 minutes post-dose. The elimination half-life of captopril is reported to be about 2 hours.
About half the absorbed dose of captopril is rapidly metabolized, mainly to captopril-cysteine disulfide and to the disulfide dimer of captopril. Captopril and its metabolites are excreted in the urine. Renal excretion of unchanged captopril occurs via tubular secretion. In patients with normal renal function, more than 95% of the absorbed dose is excreted in the urine in 24 hours. About 40% to 50% of the drug excreted in the urine is unchanged captopril.
IN VIVO BIOEQUIVALENCE STUDY5
Product Information—
  1. FDA Designated Reference Product: Capoten® (Squibb) 100-mg tablets.
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing 100-mg doses of the test and reference products is required.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study Under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Written, informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10 hours, subjects should be given a single 100-mg dose of the test or reference product with 240 mL of water. Subject blood pressure and pulse rate should be measured immediately before drug administration (pre-dose) and at 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours post-dose.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1, 1.25, 1.50, 1.75, 2, 2.50, 3, 3.50 4, 4.50, 5, 6, 7, 8, and 10 hours post-dose. Whole blood or blood plasma may be used for the assay. In either case, the blood samples should be chemically stabilized immediately after collection (see Analytical Methods below). If plasma is to be assayed, it should be separated promptly from the stabilized whole blood. The whole blood or blood plasma samples should then be immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Analytical Methods— Captopril should be measured in an appropriate biological matrix such as whole blood or blood plasma. Several assay methods are available. The method chosen should be fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Because the thiol group of captopril is readily oxidized, a chemical stabilizer must be added to the blood samples immediately after collection. Stability of the samples under frozen storage conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a period of not less than 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product versus 12 units of the reference product. The biostudy lots should be used for those product strengths tested in vivo. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: 0.1 N hydrochloric acid; 900 mL.
Apparatus 1: 50 rpm.
Times: 10, 20, and 30 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 80% in 20 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 12.5-, 25-, and 50-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
1. The lower strengths are proportionally similar in both active and inactive ingredients to the 100-mg strength, which has been demonstrated to be bioequivalent to the reference product in vivo.
2. The 12.5-, 25-, and 50-mg strengths of the generic product meet the dissolution test requirements.

Cefaclor Capsules and Suspensions—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Cefaclor is a cephalosporin antibiotic that inhibits bacterial cell-wall synthesis in a manner similar to that of penicillin. Cefaclor is used in the treatment of otitis media caused by susceptible Streptococcus pneumoniae, Haemophilus influenzae (including ampicillin-resistant strains), staphylococci, and group A -hemolytic streptococci; of lower respiratory tract infections (including pneumonia) caused by susceptible Streptococcus pneumoniae, Haemophilus influenzae, or group A -hemolytic streptococci; of upper respiratory tract infections (including pharyngitis and tonsillitis) caused by susceptible group A -hemolytic streptococci; of urinary tract infections (including pyelonephritis and cystitis) caused by susceptible Escherichia coli, Proteus mirabilis, Klebsiella, or staphylococci; or of skin and skin structure infections caused by susceptible Staphylococcus aureus or group A -hemolytic streptococci.
The usual adult dosage is 250 mg every 8 hours. For more severe infections (such as pneumonia), doses may be doubled.
Pharmacokinetics— Cefaclor is well absorbed after oral administration to fasting subjects. Total absorption is similar in the fasted and the fed states. When it is taken with food, the peak concentration achieved is 50% to 75% of that observed in fasting subjects and generally appears about 1 hour later.
Following administration of 250-mg, 500-mg, and 1-g doses to fasting subjects, average peak serum levels of approximately 7, 13, and 23 µg per mL, respectively, were obtained within 30 to 60 minutes. Approximately 60% to 85% of the drug is excreted unchanged in urine within 8 hours, the major portion being excreted within the first 2 hours. The serum elimination half-life in subjects with normal renal function is 0.6 to 0.9 hour. In patients with severely reduced renal function, the plasma elimination half-life of the drug is 2.3 to 2.8 hours. Hemodialysis shortens the half-life by 25% to 30%.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
  1. FDA Designated Reference Product: Ceclor® (Eli Lilly) 500-mg capsules; or powder for oral suspension containing 375 mg per 5 mL (Eli Lilly).
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 capsules, whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing 500-mg doses of the test and reference capsules and 375-mg-per-5-mL doses of the test and reference suspensions is required.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence, crossover, limited food effects study comparing equal doses of the test product under fasting conditions as well as the test and reference products when administered immediately following a standard high-fat breakfast6 is required.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition, including sensitivity to cephalosporin or penicillin analogues, that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, or OTC medications beginning 2 weeks before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 2, 3, 4, 5, 6, and 8 hours post-dose. Due to the chemical instability of cefaclor, the blood samples should be analyzed as soon as they are drawn, or plasma samples should be promptly frozen at –70 until analysis. Following a minimum 1-week washout period, subjects should begin Phase II. For each subject, the sponsor should state the time elapsed between sample collection and its assay. An explanation should be given for any missing samples.
Analytical Methods— For the measurement of cefaclor in plasma samples, a microbiological assay, HPLC method, or other suitable method should be selected. The method used should be described in detail, and references, if any, should be cited. The method should include detailed calculation procedures for the assay results. The method chosen should be fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the analyte in plasma samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. It should be noted that cefaclor is most stable in the acid pH range at a temperature of 4. For analytical work, cefaclor reference standard stock solutions should be prepared fresh daily in pH 4.5 buffer.
Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study— The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design (total 18 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 500-mg cefaclor capsule or 375-mg-per-5-mL suspension administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 500-mg Ceclor® capsule or 375-mg-per-5-mL suspension (Eli Lilly) administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 500-mg cefaclor capsule or 375-mg-per-5-mL suspension administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should be dosed with Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during both phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–T, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product versus 12 units of the reference product. The biostudy lots should be used for those product strengths tested in vivo. The current official USP dissolution method (see Dissolution 711 should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Times: 10, 20, 30, and 45 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerance (Q) not less than 80% in 30 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 250-mg strength of the generic capsule may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 250-mg capsule is proportionally similar in both active and inactive ingredients to the firm's 500-mg capsule, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 250-mg capsule of the generic product meets dissolution test requirements.
Waiver of in vivo bioequivalence study requirements for the powder for reconstitution as a suspension for oral administration, 125-, 187-, and 250-mg-per-5-mL strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 125-, 187-, and 250-mg-per-5-mL suspensions are proportionally similar in their active and inactive ingredients to the firm's 375-mg-per-5-mL suspension.
  2. An acceptable in vivo bioequivalence study has been conducted for the 375-mg-per-5-mL suspension.

Cimetidine Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Cimetidine is a histamine H2-receptor antagonist, used for the treatment of endoscopically or radiographically confirmed duodenal ulcer, pathologic GI hypersecretory conditions (e.g., Zollinger-Ellison syndrome, systemic mastocytosis, and multiple endocrine adenomas), and active, benign, gastric ulcer.
Cimetidine inhibits competitively and selectively the interaction of histamine with H2 receptors. It inhibits basal (fasting) and nocturnal acid secretion, acid secretion stimulated by food, sham feeding, fundic distention, and various pharmacological agents.
Effective cimetidine concentrations between 0.5 and 1.0 µg per mL are required to suppress gastric acid secretion under basal or stimulated conditions. However, no correlation to the therapeutic response from pharmacokinetic data has been established between plasma concentrations of cimetidine or any of the pharmacodynamic parameters and duodenal ulcer healing rate.
For treatment of active duodenal ulcer, the usual adult oral dosage of cimetidine is 800 mg daily at bedtime. For maintenance therapy following healing of acute duodenal ulcer, the usual oral dosage of cimetidine is 400 mg daily at bedtime. For the treatment of pathologic hypersecretory conditions, the usual adult oral dosage is 300 mg, 4 times daily with meals and at bedtime. For the treatment of active benign gastric ulcer, the usual adult oral dosage is 800 mg at bedtime or 300 mg, 4 times daily with meals and at bedtime.
Pharmacokinetics— Following intravenous administration, the plasma concentration profile follows multicompartmental characteristics. The total systemic clearance is high (500 to 600 mL per minute) and is mainly determined by renal clearance. The volume of distribution is of the order of 1 L per kg. Elimination half-life is approximately 2 hours. Following oral administration of cimetidine, 2 plasma concentration peaks are frequently observed at about 1 hour and after about 3 hours, probably due to discontinuous absorption in the intestine or individual variation in gastric emptying (but not enterohepatic recycling since the biliary excretion rate in humans is less than 2%). The absolute bioavailability is about 60% in healthy subjects and around 70% in peptic ulcer patients. Absorption and clearance of cimetidine are linear after 200- and 800-mg doses. When given with food, the extent of absorption of the drug remains unchanged, but the time to reach the maximum peak concentration is delayed with only 1 peak in the plasma concentration curve observed at about 2 hours following dosing. Plasma protein binding of cimetidine is 20% and does not significantly affect the pharmacokinetics of the drug. Cimetidine distributes extensively into the kidney, lung, and muscle tissue but less than 1% into the cerebrospinal fluid.
Following IV administration, about 50% to 80% of the dose is recovered in urine as unchanged cimetidine. This fraction is less after oral doses. Approximately 2% of cimetidine is excreted in the bile. About 25% to 40% of the cimetidine dose is eliminated as metabolites, formed mainly in the liver. The metabolites are sulfoxide and 5-hydroxymethyl derivatives, and possibly guanylurea, although this latter compound may result from in vitro degradation. Elimination of cimetidine is accelerated in the presence of phenobarbital due to induction of its metabolism. Clearance of the drug is higher in children, who have greater renal elimination rates. With increasing age, the volume of distribution of the drug decreases, total plasma clearance decreases as a function of decreasing renal clearance, and plasma half-life increases.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
  1. FDA Designated Reference Product: Tagamet® (SmithKline Beecham) 800-mg tablets.
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, fasting, two-period, two-treatment, two-sequence crossover study comparing the 800-mg strength of the test and reference products. This protocol may also be used for lower strength tablets if an 800-mg tablet is not being manufactured. In this case, equal doses of the test and reference products should be used.
  2. A single-dose, two-way crossover, full food study comparing the 800-mg strength of the test and reference products. Due to the double-peak phenomenon, which is only observed under fasting conditions, the food study may be more reliable for determining the bioequivalency of the test product and therefore should be done with a full complement of subjects. This protocol may also be used for lower strength tablets if an 800-mg tablet is not being manufactured. In this case, equal doses of the test and reference products should be used.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 800-mg dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, or OTC medications beginning 2 weeks before drug administration until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1.0, 1.50, 2.0, 2.50, 3.0, 3.50, 4.0, 5.0, 6.0, 8.0, 10, 12, 16, 20, and 24 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following at least a minimum 1-week washout period, subjects should begin the second phase of the study.
Analytical Methods— Cimetidine should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data: See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a period of not less than 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study Under Postprandial Conditions— There should be at least 24 healthy volunteers. The postprandial study should be conducted in the same manner as described for the fasting study except under fed conditions. The subjects should be given a standard high-fat breakfast6 after an overnight fast of approximately 10 hours. The dose should be given with 240 mL of water approximately 30 minutes after beginning breakfast. The plasma cimetidine data should be obtained and analyzed in the same manner as described for the fasting study. Due to the double-peak phenomenon, which is only observed under fasting conditions, the postprandial study may be more reliable for determining the bioequivalency of a test product.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product versus 12 units of the reference product. The biostudy lots should be used for those product strengths tested in vivo. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: deaerated water; 900 mL.
Apparatus 1: 100 rpm.
Times: 10, 15, and 30 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 75% in 15 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 200-, 300-, and 400-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 200-, 300-, and 400-mg strengths are proportionally similar in both active and inactive ingredients to another strength (800 mg), which has been demonstrated to be bioequivalent to a reference (800 mg) product in vivo.
  2. The 200-, 300-, and 400-mg strengths of the generic product meet all applicable compendial requirements, including the specified dissolution and content uniformity testing requirements.
  3. The drug product follows linear kinetics over its range of available strengths.

Diclofenac Sodium Delayed-Release Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Diclofenac sodium is an orally administered nonsteroidal anti-inflammatory drug (NSAID), which also has analgesic and antipyretic properties. Currently approved indications for diclofenac sodium are for the acute or chronic treatment of the signs and symptoms of rheumatoid arthritis (RA), osteoarthritis, and ankylosing spondylitis. Doses above 200 mg a day in 3 or 4 divided doses have not been studied in RA patients. Diclofenac sodium inhibits prostaglandin synthesis, which may be involved in its mechanism of action. Due to possible cross-reactivity, diclofenac sodium is contraindicated in patients in whom aspirin or other NSAID has produced asthma, urticaria, or allergic reactions.
Pharmacokinetics— Diclofenac sodium is rapidly absorbed following oral administration with reported time of maximum concentration (Tmax) mean values of 1 to 3 hours and ranges of 1 to 5 hours under fasting conditions in normal volunteers. After single oral doses of 25-, 50-, and 75-mg delayed-release tablets, reported maximum concentrations (Cmax) in normal fasting subjects were 0.5 to 1, 0.9 to 1.5, and 1.9 to 2 µg per mL, respectively. Area under the plasma concentration-time curve (AUC) increases linearly over the dose range 25 to 150 mg; however, Cmax is less than dose-proportional with values of 1, 1.5, and 2 µg per mL after doses of 25, 50, and 75 mg, respectively.
Diclofenac sodium undergoes first-pass metabolism to give a systemic availability of 50% to 60%. Food may markedly delay the rate of absorption from delayed-release tablets but does not appear to change AUC significantly. In two single-dose (50 mg) studies, the nonfasting mean Tmax values were 5.4 hours (N = 12, range 2.5 to 12 hours) and 9.7 hours (N = 6, range 8 to 10 hours). The volume of distribution (Vd) of diclofenac sodium is about 0.12 to 0.17 L per kg and it is greater than 99% bound to plasma proteins. After an IV dose, elimination of diclofenac sodium from plasma appears triphasic; after oral dosing, reported terminal -phase half-life (t½) values are 1 to 2 hours with a range of 0.5 to 4.3 hours. The apparent elimination t½ of total radiolabeled compounds in patients with normal renal function is 25 to 33 hours.
The major route of elimination is hepatic clearance with 90% of a dose eliminated in 96 hours with about 65% of the dose in urine and 35% of the dose in bile. The dose is excreted as glucuronide and sulfate conjugates of unchanged drug and four metabolites. The principal metabolite (4¢-hydroxy) has about 1/40 the activity of the parent drug in animal models of arthritis. It is possible that this metabolite may contribute to the overall activity because it accounts for 30% to 40% of the dose.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Voltaren® (Geigy) 75-, 50-, or 25-mg delayed-release oral tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
  3. As stated in 21 CFR 320.22(d)(2)(iv), no waiver may be granted for the 25- or 50-mg strengths based on the 75-mg strength. Separate fasting studies must be performed for all of the strengths.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between phases. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 36 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medication, including oral contraceptives, or OTC medication beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 0.75, 1, 1.25, 1.50, 1.75, 2, 2.25, 2.50, 2.75, 3, 3.33, 3.67, 4, 5, 6, 8, 10, and 12 hours post-dose. Plasma serum should be separated promptly and immediately frozen until assayed. Following a 2-week washout period, subjects should begin the second phase of the study.
Analytical Methods— The active ingredient should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Any subject with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances. In addition, the following parameters should be tabulated: lag time (Tlag), the time of the last zero concentration before the first non-zero concentration; and adjusted Tmax (Tmaxadj), which is the observed Tmax minus Tlag.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of diclofenac sodium from a generic formulation with that from a reference formulation under nonfasting conditions, and to compare the rate and extent of absorption of the drug form from a generic product under fasting and nonfasting conditions when given in equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 2-week washout period between Phase I and Phase II, and Phase II and Phase III of dosing.
In view of the wide range of reported values for Tmax in the presence of food, it is recommended that the sponsor perform a pilot study to determine appropriate sampling times for the limited food effects study.
The limited food effects study (12 to 18 subjects) should be performed in the same manner as the single-dose fasting study, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 75-mg tablet, administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 75-mg Voltaren® tablet, administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 75-mg tablet, administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, and then should immediately receive Treatment 1 or Treatment 2, with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, with 240 mL of water. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0t, AUC0, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
There is currently no official USP dissolution method for a diclofenac sodium dosage form. A tentative method recommended by FDA is described below.
Drug Release Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The following method and tolerances are currently recommended for this product:
Method B 724—
Medium: 2 hours in 0.1 N hydrochloric acid followed by 45 minutes in pH 6.8, 0.05 M phosphate buffer prepared as follows. Dissolve 76 g of tribasic sodium phosphate in water to obtain 1000 mL of solution. Mix 250 mL of this solution with 750 mL of 0.1 N hydrochloric acid and, if necessary, adjust with 2 N hydrochloric acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05; 900 mL.
Apparatus 2: 50 rpm.
Times: ACID STAGE: 30, 60, and 120 minutes; BUFFER STAGE: 5, 10, 20, 30, 45, and 60 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): ACID STAGE: not more than 10% in 120 minutes; BUFFER STAGE: not less than 75% in 45 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
As stated in 21 CFR 320.22(d)(2)(iv), no waiver may be granted for the 25- or 50-mg strengths based on the 75-mg strength. Separate studies must be performed for all of the strengths.
The limited food effects study requirement for the 50- and 25-mg strengths of the generic diclofenac sodium product may be waived if the following conditions are met:
  1. The limited food study for the 75-mg strength demonstrates bioequivalence to the reference product.
  2. The formulations for the 50- and 25-mg strengths are proportionally similar to the 75-mg strength.

Diltiazem Hydrochloride Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Diltiazem hydrochloride is classified as a cardiovascular preparation and an anti-anginal calcium channel blocker. It is currently approved for angina pectoris due to coronary artery spasm and for chronic stable angina (classic effort-associated angina). Its therapeutic effects are brought about by its ability to block calcium entry into cardiac and vascular smooth muscle cells. It is known to dilate coronary arteries as well as to increase the tolerance of angina sufferers to physical exertion by reducing the demand for myocardial oxygen.
The dosage is adjusted according to the needs of adult patients. The starting regimen of 30 mg four times daily is increased gradually until the optimum response is achieved with safety. Safety and efficacy of diltiazem is not established for pediatric use. Diltiazem is prescribed to a pregnant woman only if the potential benefit justifies any potential risk to the fetus. A nursing mother taking diltiazem should not breast-feed her infant. The most commonly observed adverse reactions are edema, headache, nausea, dizziness, rash, and asthenia (weakness, lethargy).
Pharmacokinetics— After oral administration of diltiazem, 80% to 90% of the dose is absorbed. Diltiazem undergoes extensive first-pass metabolism and has 40% to 44% oral bioavailability. Diltiazem is 70% to 80% bound to plasma proteins. The peak plasma diltiazem level is reached within 2 to 3 hours after a single oral dose. Elimination half-lives of 3 to 5 hours and 5 to 9 hours have been reported for diltiazem. Diltiazem is metabolized by three major pathways into various metabolites. These pathways are O-deacetylation, N-demethylation, and O-demethylation. Desacetyldiltiazem (DAD) and N-monodemethyldiltiazem (NMD) are active metabolites.
Until 1987, the measurement of DAD and NMD was found to be difficult in single-dose (IV and oral) studies. In 1989, however, Boyd et al. showed the possibility of measuring DAD and NMD following a single oral dose.
At one time, DAD was thought to be the major metabolite of diltiazem. It is 40% to 50% active compared to the parent compound and shows, on average, only 20% of the plasma levels produced by diltiazem. DAD is 68% bound to plasma proteins. Following a single oral dose, average half-lives of 7.5 hours and 19.5 hours have been reported.
Recent studies have demonstrated that N-monodemethylation, leading to the formation of NMD, which is less active than DAD, is the major metabolic pathway. Average plasma levels of NMD are about 40% of those of diltiazem. NMD is 77% bound to plasma proteins and has an average half-life of 8 to 10 hours.
Diltiazem hydrochloride has a therapeutic concentration range of 50 to 200 ng per mL and is toxic above 1200 ng per mL.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Cardizem® (Marion) 120-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of diltiazem hydrochloride from a generic formulation with that from a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 120-mg dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 30, 36, and 48 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Diltiazem and its major metabolites, DAD and NMD, should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing, whichever is greater, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 30, 60, 90, and 180 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest, lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the immediate-release 30-, 60-, and 90-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 30-, 60-, and 90-mg tablets are proportionally similar in both active and inactive ingredients to the firm's 120-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 30-, 60-, and 90-mg tablets of the generic product meet dissolution test requirements.

Flurbiprofen Tablets In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Flurbiprofen is an orally administered nonsteroidal anti-inflammatory drug (NSAID) that also has analgesic and antipyretic properties. Labeled indications for flurbiprofen are for the acute or long-term treatment of the signs and symptoms of rheumatoid arthritis and osteoarthritis. Doses above 300 mg per day are not recommended. Flurbiprofen inhibits prostaglandin synthesis, which may be associated with its mechanism of action. Due to possible cross-reactivity, flurbiprofen is contraindicated in patients in whom aspirin or other NSAIDs have produced asthma, urticaria, or other allergic reactions.
Pharmacokinetics— Flurbiprofen is rapidly absorbed following oral administration, with reported time of maximum concentration values (Tmax) of 1 to 2 hours and ranges of 0.5 to 4 hours. After single oral doses of 50 and 100 mg, reported maximum concentrations (Cmax) in normal subjects were 5.7 to 7.4 µg per mL and 12.6 to 17.2 µg per mL, respectively. Food may decrease Cmax by 27% to 32% by delaying absorption but does not significantly change area under the plasma concentration-time curve (AUC). In another study, flurbiprofen absorption was found to be bimodal (with double peaks in some cases) after administration of the tablet form in the fasted (water) or fed (apple juice) state. In this study, the lag time before the onset of the second absorption phase was dependent on the gastric emptying time. The volume of distribution (Vd) of flurbiprofen is about 0.1 L per kg with values of Vd/F (F = oral availability) of 7.0 to 9.1 L after a 100-mg dose. Flurbiprofen is 99% bound to plasma proteins (albumin). The binding has been reported to be minimally nonlinear over the concentration ranges encountered clinically. In one study, however, AUC0– increased linearly over the dose range of 100 to 300 mg. Flurbiprofen elimination from plasma appears biphasic, with terminal -phase half-life (t½) values reported from 3 to 7 hours (mean = 5.7 hr; range 3 to 12 hours). The major route of elimination is hepatic clearance, with 95% of a daily dose excreted in urine in 24 hours, either as unchanged drug (20% to 25% of dose) or as the three metabolites—4¢-hydroxy (40% to 47%), 3¢-hydroxy-4¢-methoxy (20% to 30%), and 3¢,4¢-dihydroxy (5%). Other studies have reported 73% to 77% dose recovery in the urine up to 48 hours. Between 60% to 90% of flurbiprofen and its metabolites are present in urine as glucuronide and sulfate conjugates. The 4¢-hydroxy metabolite demonstrates low pharmacological activity in animal models.
Virtually all of the anti-inflammatory activity of flurbiprofen is present in the S(+)-enantiomer. Following oral doses of either the pure R(–)- or S(+)-enantiomer, only the administered enantiomer was detectable in plasma or urine. The available evidence from human studies indicates that R-flurbiprofen does not undergo the in vivo irreversible inversion to the S-enantiomer that has been observed with other NSAIDs of the 2-arylpropionic acid class. In three studies, significant differences were noted between the enantiomers for AUC with lower values for the R-enantiomer and with increased CL/F (CL = clearance) or decreased t½ for the R-enantiomer.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Ansaid® (Upjohn) 100-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of flurbiprofen from a generic formulation with that from a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocols should be approved by an institutional review board.
Facilities— The clinical and analytical laboratories used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects should be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test product or the reference product with 240 mL of water.
Restrictions— Study volunteers should be subject to the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected at pre-dose (0 hr), and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 18, 24, and 36 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Flurbiprofen should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data (Blood Plasma/Serum)— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of flurbiprofen from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and II, and Phases II and III. An equal number of subjects should be assigned to each of the 6 dosing sequences.
The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedures— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design (minimum of 12 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 100-mg tablet, administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 100-mg Ansaid® tablet (Upjohn), administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 100-mg tablet, administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used as in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed provided the AUC0–t, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examination reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing, whichever is greater, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method in the monograph (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 10, 20, 30, and 45 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest, lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 50-mg strength of the generic tablet may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 50-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 100-mg tablet, which has been demonstrated to be bioequivalent to the reference product.
  2. The 50-mg tablet of the generic product meets dissolution test requirements.

Gemfibrozil Capsules and Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Gemfibrozil is used clinically as a lipid-regulatory agent, which lowers serum triglycerides and produces a variable reduction in total serum cholesterol. The decrease occurs primarily in the very low-density lipoprotein (VLDL) and less frequently in the low-density lipoprotein (LDL). In addition, there is elevation of the high-density lipoprotein (HDL) concentration. Gemfibrozil is used in adult patients with all types of dyslipidemia (except type 1). The recommended dosage of gemfibrozil is 600 mg twice daily given 30 minutes before morning and evening meals.
The mechanism whereby gemfibrozil lowers plasma triglycerides and increases HDL cholesterol concentration is not well established. One mechanism by which the drug reduces circulating triglyceride concentration may be through suppression of free fatty acid mobilization from adipose tissue. Gemfibrozil also stimulates lipoprotein lipase activity.
Gemfibrozil is not recommended for use in patients with hepatic or severe renal dysfunction, including primary biliary cirrhosis and pre-existing gallbladder disease. The main adverse reactions are dyspepsia and abdominal pain. Gemfibrozil is metabolized to a number of compounds in humans. All the metabolites and the unchanged drug form glucuronide conjugates, which are excreted in urine. The metabolites have no lipid lowering activity.
Pharmacokinetics— Following oral administration of gemfibrozil, absorption is rapid and complete. After the administration of gemfibrozil (600 mg twice daily), mean peak plasma concentrations are about 10 to 15 µg per mL. Peak plasma concentrations are attained 1 to 2 hours after administration of single doses up to 2000 mg or after repeated doses up to 800 mg twice daily. Plasma drug concentration is directly proportional to dose and tends to rise during repeated administration, although steady state is achieved within 7 to 14 days with twice daily doses. The mean elimination half-life of gemfibrozil is 1.5 to 3.0 hours.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Lopid® (Parke-Davis Co.) 300-mg capsules and 600-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 600-mg dose (two 300-mg capsules or one 600-mg tablet) of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, and 14 hours post-dose. Plasma should be separated promptly, stored in light resistant containers, and immediately frozen until assayed. Following a minimum 2-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Gemfibrozil should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method in the monograph (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 15, 30, 45, and 60 minutes for capsules; and 10, 20, 30, and 45 minutes for tablets.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest, lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.

Glipizide Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Glipizide is an oral antidiabetic agent, which lowers blood glucose levels. It is approved for controlling blood glucose in noninsulin-dependent diabetes mellitus (NIDDM) patients whose blood glucose cannot be controlled by diet alone. Although the precise mechanism of hypoglycemic action of glipizide has not been clearly established, it appears to lower the blood glucose concentration mainly by stimulating endogenous insulin release from beta cells of the pancreas.
To achieve maximum reduction in the postprandial blood glucose concentration, glipizide should be administered 30 minutes before a meal. The recommended initial adult dose of glipizide is 5 mg daily, and the maximum recommended single daily dose is 15 mg. The optimum dosing regimen for each patient is obtained by titration. Glucose (urinary and blood) and blood glycosylated hemoglobin (Hb A1c) are used as the indicators of effective therapy. The most commonly observed adverse effects of glipizide are dizziness, sweating, tremors, and lightheadedness.
Pharmacokinetics— Glipizide is rapidly and completely absorbed after oral administration with a bioavailability of approximately 95%. After oral administration, the drug is 98.4% bound to plasma proteins, reaches Cmax in 1 to 3 hours, has an elimination half-life of 2 to 5 hours, and has a volume of distribution (Vd) of 11 L. Based on the mean plasma profiles, glipizide appears to exhibit either single compartment disposition or a very short distribution phase. The primary metabolites of glipizide are hydroxylation products and polar conjugates that are inactive and are excreted in both urine (70%) and feces. Biliary excretion is estimated to be approximately 30%. Less than 5% to 10% of the administered drug is excreted intact in urine. The extent of absorption of an oral dose of glipizide is unaffected by food in normal volunteers, but absorption is delayed by about 40 minutes. There are conflicting reports about the influence of food on Tmax. Plasma levels of glipizide in the range of 20 to 90 ng per mL have been reported as therapeutically effective.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Glucotrol® (Roerig) 10-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence, crossover, limited food effects study comparing equal doses of the test product under fasting conditions as well as the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose, Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given in equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of an acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure7 — Following an overnight fast of at least 10 hours, subjects should be administered a single dose (10 mg) of the test or reference product with 240 mL of a 20% glucose solution in water. After dosing, subjects should receive 60 mL of a 20% glucose solution in water every 15 minutes for 4 hours.
Restrictions— Study volunteers should observe the following restrictions:
  1. No additional water or fluids, except for the glucose solution, are allowed from 1 hour pre-dose to 1 hour post-dose.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medication, including oral contraceptives, or OTC medication beginning 2 weeks before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 1, 1.50, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, and 36 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a 1-week washout period, subjects should begin the second phase of the study. For each subject the sponsor should state the time elapsed between sample collection and its assay. An explanation should be given for any missing samples.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— The active ingredient should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study— The limited food effects study should be performed in the same manner as the single-dose fasting study, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the six dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 10-mg glipizide tablet administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 10-mg Glucotrol® tablet administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 10-mg glipizide tablet administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under nonfasting conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, and then should immediately receive Treatment 1 or Treatment 2, with 240 mL of a 20% glucose solution in water. Subjects receiving the treatment under fasting conditions should be dosed with Treatment 3, taken with 240 mL of a 20% glucose solution in water. The same lots of the test and reference products should be used in the fasting and nonfasting studies. After dosing, each subject in all 3 treatments should receive 60 mL of a 20% glucose solution in water every 15 minutes for 4 hours. No food should be allowed for at least 4 hours post-dose, and no additional water or fluids should be allowed from 1 hour pre-dose to 1 hour post-dose. Subjects should be served scheduled, standardized meals throughout the study, and the same meals should be served during both phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0 – t, AUC0–, and Cmax mean values for the test product administered under nonfasting conditions are within 20% of the respective mean values for the reference product administered under nonfasting conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a minimum of 5 years after approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method in the monograph should be followed and referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 15, 30, 45, and 60 minutes.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 5-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 5-mg tablet is proportionally similar in both active and inactive ingredients to the 10-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 5-mg tablet of the generic product meets the dissolution testing requirements.

Glyburide Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Glyburide is an oral antidiabetic agent, which lowers the blood glucose level. It is currently approved for controlling blood glucose in non-insulin-dependent diabetes mellitus (NIDDM) patients whose blood glucose cannot be controlled by diet alone. Glyburide acts mainly by stimulating endogenous insulin release from beta cells of the pancreas.
Glyburide is usually administered as a single daily dose each morning with breakfast or with the first main meal. The recommended initial adult dose is 2.5 to 5 mg daily for nonmicronized glyburide and 1.5 to 3 mg daily for micronized glyburide. The maximum recommended single daily dose is 10 mg for nonmicronized glyburide (up to 20 mg a day may be given in two divided doses) and 12 mg for micronized glyburide. The optimum dosing regimen of glyburide for each patient is obtained by titration. Glucose (urinary and blood) and blood glycosylated hemoglobin (Hb A1c) are used as the indicators of effective therapy. The most commonly observed adverse effects after administration of glyburide are dizziness or lightheadedness, which are due to hypoglycemia.
Pharmacokinetics— Glyburide is rapidly and completely absorbed after oral administration. As there is no significant first pass metabolism, 100% of the oral dose is bioavailable. After oral administration, nonmicronized glyburide reaches peak plasma or serum concentrations within 4 hours and exhibits an elimination half-life of 10 hours (more recently, half-lives of 1.4 to 5 hours have been reported). Micronized glyburide reaches peak concentrations within 2 to 3 hours and has a reported elimination half-life of about 4 hours. Glyburide concentration-time curves in plasma exhibit biphasic elimination. The primary metabolites of glyburide are hydroxylation products (4-trans-hydroxy and 3-cis-hydroxy derivatives) that are inactive and are excreted in urine (50%) and in feces (50%) via bile. Plasma levels of glyburide in the range of 20 to 90 ng per mL have been reported as therapeutically effective.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Micronase® (Pharmacia & Upjohn) 5-mg tablets, or Glynase® (Pharmacia & Upjohn) 6-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal 5-mg or 6-mg doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence, crossover, limited food effects study comparing equal doses of the test product under fasting conditions as well as the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of an acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose (5 mg or 6 mg) of the test or reference product with 240 mL of a 20% glucose solution in water. After dosing, the subjects should be given 60 mL of a 20% glucose solution in water, every 15 minutes for 4 hours.
Restrictions— Study volunteers should observe the following restrictions:
  1. No additional water or fluids, except the glucose solution, are allowed from 1 hour pre-dose to 1 hour post-dose.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks, and no OTC medications beginning 1 week, before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected predose (0 hours) and at 0.50, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 30, and 36 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a 1-week washout period, subjects should begin the second phase of the study. For each subject, the sponsor should state the time elapsed between sample collection and its assay. An explanation should be given for any missing samples.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— The active ingredient should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study— The limited food effects study should be performed in the same manner as the single-dose fasting study, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the six dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 5-mg glyburide tablet or a 6-mg micronized glyburide tablet, administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 5-mg Micronase® tablet or a 6-mg Glynase® tablet, administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 5-mg glyburide tablet or a 6-mg micronized glyburide tablet, administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under nonfasting conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, and then should immediately receive Treatment 1 or Treatment 2, with 240 mL of a 20% glucose solution in water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, with 240 mL of a 20% glucose solution in water. The same lots of the test and reference products should be used in the fasting and nonfasting studies. After dosing, each subject in all 3 treatments should receive 60 mL of a 20% glucose solution in water every 15 minutes for 4 hours. No food should be allowed for at least 4 hours post-dose, and no additional water or fluids should be allowed from 1 hour pre-dose to 1 hour post-dose. Subjects should be served scheduled, standardized meals throughout the study, and the same meals should be served during both phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0 – t, AUC0–, and Cmax mean values for the test product administered under nonfasting conditions are within 20% of the respective mean values for the reference product administered under nonfasting conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units, and should be retained for a minimum of 5 years after approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
There is currently no official monograph for a glyburide dosage form in USP.
Content Uniformity Test 905— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 1.25- and 2.5-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 1.25- and 2.5-mg tablets are proportionally similar in both active and inactive ingredients to the 5-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 1.25- and 2.5-mg tablets of the generic product meet the dissolution testing requirements.
Waiver of in vivo bioequivalence requirements for the 1.5- and 3-mg strengths of the generic micronized product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 1.5- and 3-mg tablets are proportionally similar in both active and inactive ingredients to the 6-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 1.5- and 3-mg tablets of the generic product meet the dissolution testing requirements.

Guanabenz Acetate—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Guanabenz acetate is a centrally acting antihypertensive agent. It appears to stimulate 2-adrenergic receptors in the CNS and cause inhibition of sympathetic outflow from the brain.
Guanabenz acetate is indicated in the treatment of hypertension and may be employed alone or in combination with a thiazide diuretic. It is contraindicated in patients with known hypersensitivity to the drug and should be used with caution in patients with hepatic or renal impairment, with cardiovascular diseases, during pregnancy, and in geriatric patients.
The side effects of guanabenz acetate are generally mild and may include a dry mouth, drowsiness or sedation, dizziness, weakness, and a headache. Abrupt withdrawal of guanabenz acetate (especially at doses 32 mg per day) may cause a rapid increase in serum catecholamine concentrations and systolic and diastolic blood pressures.
The initial dosage is 4 mg twice daily and may be gradually increased to a maximum of 32 mg twice daily.
Pharmacokinetics— Following an oral dose of guanabenz acetate, about 75% of the drug is absorbed. Because of extensive first-pass metabolism, the bioavailability is low—20% to 30% in monkeys—but the extent has not been established in man. A food effect on the drug absorption has not been determined.
Guanabenz acetate is rapidly and extensively distributed into the CNS and various organs. Following a 16-mg dose to fasting healthy individuals, peak plasma levels of unchanged drug are only 2.4 to 2.7 ng per mL at 2 to 5 hours with about 90% bound to proteins. For hepatically impaired patients, the Cmax is higher, 7.8 ng per mL. From the limited studies in humans, the concentration-time curves for the drug are best fit to a one- or two-compartment open model with a first-order absorption process and a lag time. The apparent steady-state volume of distribution is about 93 and 147 L per kg after 16- and 32-mg oral doses, respectively.
Therapeutic effects begin within 1 hour and last over 10 hours with a peak around 2 to 4 hours. At usual clinical doses, effects appear to be linearly related to dose.
The elimination half-life of guanabenz acetate averages 4 to 9 hours in healthy males. For liver impaired or hypertensive patients, the half-life averages 12 to 14 hours.
Guanabenz acetate metabolites—mainly from the liver—are excreted in urine (70% to 80% within 72 hours) and feces (10% to 30% in 6 days). One and four-tenths percent of the drug is excreted unchanged in the urine. Urinary metabolites include (E)-p-hydroxyguanabenz (11%), its glucuronide conjugate (25%), and the Z-guanabenz acetate (1.1%—the only active metabolite with about 25% parent drug activity).
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Wytensin® (Wyeth-Ayerst) 8-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of guanabenz acetate from a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered one 8-mg tablet of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 1, 1.50, 2, 2.50, 3, 3.50, 4, 6, 8, 12, 15, 24, 30, and 36 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Guanabenz acetate should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. [The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor.] The following method and tolerances are currently recommended for this product:
Medium: water; 1000 mL.
Apparatus 2: 50 rpm.
Times: 15, 30, 45, and 60 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerance (Q): not less than 75% in 60 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 4-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 4-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 8-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 4-mg tablet of the generic product meets dissolution test requirements.

Hydroxychloroquine Sulfate Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Hydroxychloroquine sulfate is indicated for the suppressive treatment and treatment of acute attacks of malaria due to Plasmodium vivax, P. malariae, P. ovale, and susceptible strains of P. falciparum. It is also indicated for the treatment of discoid and systematic lupus erythematosus and rheumatoid arthritis. Use of this drug is contraindicated in the presence of retinal or visual field changes attributable to any 4-aminoquinoline compound.
The exact mechanism of antimalarial activity of hydroxychloroquine and the mechanism of action of the drug in the treatment of rheumatoid arthritis and lupus erythematosus have not been determined. However, the actions of the drug are presumably the same as those of chloroquine. Dosage and administration are dependent on the indications.
Pharmacokinetics— Information in literature on the pharmacokinetics of hydroxychloroquine is scarce. The mean bioavailability of hydroxychloroquine after oral administration is 74%. A 200-mg dose given orally resulted in a Cmax in whole blood of 120 to 170 ng per mL and a Tmax of 3 to 4 hours. Terminal half-life ranged from 19 to 28 days. After intravenous administration, hydroxychloroquine was found to have a large volume of distribution (5,500 L from blood, 44,000 L from plasma) and a terminal elimination half-life of approximately 40 days. It may require 6 months to achieve a steady-state blood level at a 200 mg per day dosing regimen.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Plaquenil® (Sanofi Winthrop) 200-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 dosage units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence, crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-period, parallel study comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of the reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence, crossover study with a washout period of at least 4 months. An equal number of subjects should be randomly assigned to the two possible dosing sequences. As an alternative, the design may be a single-dose, randomized, two-treatment, one-period, parallel study with a blood sampling time of approximately 3 months. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 48 subjects be used for crossover studies or 96 subjects for parallel studies. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. The subjects should fast for 5 hours post-dose. Water may be taken except for 2 hours after drug administration.
  2. No xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until 24 hours post-dose.
  3. No alcoholic beverages should be consumed for 48 hours prior to and during the study period.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected into tubes containing an anti-coagulant pre-dose (0 hours), and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120 hours and at 8, 12, 15, 19, 22, 26, 29, 33, 36, 43, 50, 57, 64, 70, 80, and 90 days. Blood should be immediately frozen until assayed.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration. A physical examination and clinical laboratory tests should be repeated after completing the blood level study.
Analytical Methods— Hydroxychloroquine in whole blood should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). The method to be used should be specific enough to measure the parent drug without interferences from metabolites and endogenous or exogenous components in the blood. Stability of samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curves and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Pharmacokinetic Analysis— From the blood drug concentration-time data, the following pharmacokinetic parameters should be determined for hydroxychloroquine:
  1. Area under the concentration-time curve from time zero to time t (AUC0–t), calculated by the trapezoidal rule, where t is the last time point with measurable non-zero concentration.
  2. Area under the concentration-time curve from time zero to time infinity (AUC0–), where AUC0– = AUC0–t + Ct /(z), Ct is the last measurable non-zero concentration, and z is the terminal elimination rate constant calculated using an appropriate method.
  3. The terminal phase elimination rate constant, z, and the elimination half-life are calculated using an appropriate method.
  4. Peak drug concentration, Cmax, and the time to peak drug concentration, Tmax, are obtained directly from the data without interpolation.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment, Crossover Design under General Guidances; and an appropriate model should be chosen for the parallel study.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 15, 30, 45, and 60 minutes.
The percentage of label claim dissolved at each specified sampling interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest and lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Tests 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.

Indapamide Tablets— In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Indapamide is indicated for the treatment of hypertension, alone or in combination with other antihypertensive drugs. It is also indicated for the treatment of salt and fluid retention associated with congestive heart failure. The recommended daily dose of indapamide is 2.5 mg, to be taken in the morning, which may be increased to 5.0 mg taken once daily.
Like other thiazide diuretics, indapamide works in the cortical diluting segment of the distal renal tubule. It acts on the luminal surface of the tubule, probably by inhibiting the Na+-Cl co-transporter. Diuretic effects of indapamide are linearly related to dose in the range of 0.5 to 20 mg per day. The mechanism of antihypertensive action of indapamide is not established, and the magnitude of change in blood pressure does not correlate directly with change in extracellular fluid or plasma volume. A variety of actions of indapamide have been reported that may contribute to its antihypertensive activity. These effects include a decrease in the inward calcium current in the vascular smooth muscle, antipressor activity, and release of vasodilator prostaglandins such as prostaglandin E2 and prostacyclin. The net effect of indapamide may depend upon which of the various effects is dominant and may exhibit inter-patient variation.
Pharmacokinetics— The absorption of indapamide in healthy volunteers following oral administration is rapid and complete. The bioavailability of indapamide from oral solution and tablets is similar and is unaffected when the drug is given with food or antacids. The peak blood concentration, which exhibits linearity following administration of indapamide in doses of 2.5- to 10-mg, occurs 0.5 to 2 hours after drug administration. Steady-state blood concentration of indapamide is attained after 4 daily doses of 2.5 or 5.0 mg. Indapamide is readily taken up by erythrocytes. In human volunteers, a blood to plasma indapamide ratio of 5.7:1 at peak concentration has been reported, and its volumes of distribution estimated from blood and plasma concentrations are approximately 25 L per kg (V1) and 110 L per kg (V2), respectively. Indapamide is approximately 76% to 79% protein bound. Blood concentrations of indapamide exhibit biexponential decay with a rapid -phase and a prolonged -phase. The terminal-phase elimination half-life of indapamide is in the range of 13 to 18 hours.
In humans, the major route of indapamide elimination is the urine, where 60% to 70% of an orally administered dose may be excreted. The estimated total systemic clearance of indapamide is in the range of 20 to 23.4 mL per minute. Because renal clearance of indapamide is only 1.71 mL per minute, hepatic clearance (23.8 mL per minute) may be responsible for a major portion of systemic clearance. Fecal elimination accounts for 16% to 23% of an orally administered dose. Indapamide is extensively metabolized in the liver. Up to 7% of the administered dose is excreted unchanged; the remainder as metabolites. In human volunteers, anywhere from 5 to 19 metabolic products of indapamide have been reported.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Lozol® (Rhône-Poulenc Rorer) 2.5-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of an acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered two 2.5-mg tablets of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Because indapamide binds preferentially to erythrocytes, its concentration as determined using plasma may not reflect true amounts of drug in the body if the hematocrit is altered within or between treatments. Therefore, whole blood concentrations of indapamide should be assayed. Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 1, 1.50, 2, 2.50, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72 and 96 hours post-dose. Blood should be immediately frozen until assayed. Following a minimum 2-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Indapamide should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product versus 12 units of the reference product. The biostudy lots should be used for those product strengths tested in vivo. The current official USP dissolution method should be followed and should be referenced by the sponsor.
Times: 15, 30, 45, and 60 minutes.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 1.25-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 1.25-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 2.5-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 1.25-mg tablet of the generic product meets dissolution test requirements.

Ketoprofen Capsules—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Ketoprofen is a nonsteroidal anti-inflammatory drug with analgesic and antipyretic properties. It is indicated for the acute or long-term treatment of the signs and symptoms of rheumatoid arthritis and osteoarthritis. Ketoprofen is contraindicated in patients who are hypersensitive to aspirin and other nonsteroidal anti-inflammatory drugs. Monitoring of renal function is indicated in patients taking concomitant diuretics.
Ketoprofen curtails the inflammatory mechanism in rheumatoid arthritis and other disease states by reversibly inhibiting cyclooxygenase and lipoxygenase, enzymes involved in the synthesis of prostaglandins and leukotrienes. It is also a powerful inhibitor of bradykinin, a pain and inflammation mediator, and prevents the release of lysosomal enzymes, which can cause tissue destruction in inflammatory reactions.
For rheumatoid arthritis and osteoarthritis in adults, an initial dose of 75 mg 3 times or 50 mg 4 times a day is recommended. In patients who tolerate ketoprofen well, a maximum total daily dose of 300 mg can be used. Ketoprofen should be taken with food or milk.
Pharmacokinetics— Ketoprofen is rapidly and completely absorbed from the gastrointestinal tract with peak plasma concentration occurring in 0.5 to 2 hours. The mean plasma elimination half-life ranges from 2 to 4 hours. The absolute bioavailability of the 100-mg capsule of ketoprofen as compared to an intravenous formulation is 92%. Concurrent ingestion of food appears to affect the rate but not the extent of absorption of ketoprofen.
Ketoprofen is more than 95% bound to plasma albumin with a relatively small apparent volume of distribution (Vd = 0.1–0.2 L per kg). Substantial concentrations of ketoprofen have been found in synovial fluid, which is the proposed primary site of action for NSAIDS in rheumatoid arthritis. Ketoprofen exhibits a longer Tmax, a lower Cmax, and a 2.5-fold greater elimination half-life in synovial fluid than in plasma.
Two studies in humans have shown that 90% of an oral dose of tritiated ketoprofen given to healthy subjects was recovered in the urine over several days; an additional 1% to 8% of the labeled dose appeared in the feces. Virtually all of the material eliminated into urine after an oral dose is in the form of ketoprofen conjugates; less than 1% is the unchanged drug. Enterohepatic recirculation appeared to be negligible in humans. Ketoprofen metabolites are not active.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Orudis® (Wyeth Ayerst) 75-mg capsules.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers 18 to 50 years of age and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of an acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 75-mg tablet of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 10, 20, 30, and 45 minutes and at 1, 1.25, 1.50, 2, 2.50, 3, 4, 6, 8, 10, 12, 16, and 24 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Analytical Methods— The active ingredient should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of ketoprofen from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and Phase II, and Phase II and Phase III. An equal number of subjects should be assigned to each of the six dosing sequences.
The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects (12 to 18) should be assigned to each of the six dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 75-mg capsule administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 75-mg Orudis® capsule administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 75-mg capsule administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasting and nonfasting studies. No food should be allowed for at least 4 hours post-dose with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC 0–t, AUC0–, and Cmax mean values for the test product administered under nonfasting conditions are within 20% of the respective mean values obtained for the reference product administered under nonfasting conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing 711— Conduct dissolution testing on 12 dosage units of the test product and reference products, employing the biostudy lots. The following method and tolerances are currently recommended for this product:
Medium: 0.05 M pH 7.4 phosphate buffer; 1000 mL.
Apparatus 2: 50 rpm.
Times: 10, 20, and 30 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 80% in 30 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 25- and 50-mg strengths of the generic capsule may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 25- and 50-mg capsules are proportionally similar in both active and inactive ingredients to the firm's 75-mg capsule, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 25- and 50-mg capsules meet the dissolution test requirements.

Nadolol Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Nadolol is a long-acting, synthetic, nonselective beta-adrenergic receptor blocker. Currently approved indications include treatment of essential hypertension and treatment of angina pectoris.
For the treatment of hypertension, the usual initial dose is 40 mg of nadolol once daily. The dosage may be gradually increased in 40- to 80-mg increments until optimum blood pressure reduction is achieved. The usual maintenance dose is 40 or 80 mg once daily. Doses of up to 240 or 320 mg administered once daily may be needed.
For the treatment of angina pectoris, the usual initial dose is 40 mg of nadolol once daily. The dosage may be gradually increased in 40- to 80-mg increments at three- to five-day intervals until optimum clinical response is obtained or there is pronounced slowing of the heart rate. The usual maintenance dose is 40 or 80 mg once daily. Doses of up to 160 or 240 mg administered once daily may be needed.
Nadolol specifically competes with beta-adrenergic receptor agonists for available beta receptor sites; it inhibits both the beta-1 receptors located chiefly in the cardiac muscle and the beta-2 receptors located chiefly in the bronchial and vascular musculature, proportionately inhibiting the chronotropic, inotropic, and vasodilator responses to beta-adrenergic stimulation. Nadolol is contraindicated in cases of bronchial asthma, sinus bradycardia and greater than first degree conduction block, cardiogenic shock, and overt cardiac failure.
Pharmacokinetics— The absorption of nadolol from the GI tract is variable following oral dosing, averaging about 30%. About 20% to 30% of the drug is reversibly bound to plasma proteins. Peak blood serum concentrations are reached within 1 to 4 hours following oral administration. The elimination half-life is in the range of 12 to 24 hours, permitting once-daily administration. Steady-state serum levels are reached in 6 to 9 days with once-daily dosing. Because nadolol is excreted unchanged predominately in the urine, its half-life increases in patients with renal impairment.
Pharmacokinetically, the drug is described by an open, two-compartment model. The approved labeling for the drug states that the presence of food in the GI tract does not affect the rate or extent of absorption and that nadolol may be dosed without regard to meals.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Products: Corgard® (Bristol Laboratories) 40- and 160-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. Two single-dose, randomized, two-period, two-treatment, two-sequence crossover studies under fasting conditions comparing equal doses of the 40- and 160-mg test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the 40-mg test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocols for Conducting Single-Dose, Fasted Bioequivalence Studies—
Objective— The objective of the two studies is to compare the rate and extent of absorption of nadolol from generic formulations with that from reference formulations when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Subjects should have a resting pulse rate of at least 65 bpm. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose (40 or 160 mg) of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 18, 24, 36, 48, and 72 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 2-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Nadolol should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of nadolol from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 2-week washout period between Phase I and Phase II and Phase II and Phase III.
The limited food effects study (minimum 12 subjects) should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 40-mg tablet administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 40-mg Corgard® tablet administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 40-mg tablet administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing, whichever is greater, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 10, 20, 30, 40, 50, and 60 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range of dissolution (highest, lowest), and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 20-, 80- and 120-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 20-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 40-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo. The 80- and 120-mg tablets are proportionally similar in both active and inactive ingredients to the firm's 160-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 20-mg tablet of the generic product meets dissolution test requirements. The 80- and 120-mg tablets of the generic product meet dissolution test requirements.

Naproxen Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Naproxen is an orally administered nonsteroidal anti-inflammatory drug (NSAID), which also has analgesic and antipyretic properties. Currently approved indications for naproxen are (1) treatment of rheumatoid arthritis, osteoarthritis, juvenile arthritis, ankylosing spondylitis, tendinitis, bursitis, and acute gout; (2) relief of mild to moderate pain; and (3) treatment of primary dysmenorrhea. For adult arthritis and ankylosing spondylitis, the recommended dosage is 250-500 mg bid (morning and evening), up to 1500 mg per day for limited periods when a higher degree of anti-inflammatory activity is required and the dose is well tolerated. For juvenile arthritis, the recommended total daily dose of naproxen is 10 mg per kg in two divided doses. For acute gout, the recommended starting dose is 750 mg followed by 250 mg every 8 hours until the attack subsides. For the other indications, the recommended starting dose is 500 mg followed by 250 mg every 6 to 8 hours as required, not to exceed 1250 mg per day.
At plasma pH, naproxen exists predominantly as the naproxen ion that reversibly binds the enzyme cyclooxygenase and inhibits prostaglandin synthesis, affecting conditions where overproduction of prostaglandins occurs. Other mechanisms of action may be possible. Due to possible cross-reactivity, naproxen is contraindicated in patients in whom aspirin or other NSAID agents induce the syndrome of asthma, rhinitis, and nasal polyps, and in patients who have had any symptoms of allergic reactions to NSAIDs. The major adverse reaction is gastrointestinal irritation, upset, and dyspepsia, but serious gastrointestinal toxicity (ulceration, bleeding, and perforation) can occur with chronic use.
Pharmacokinetics— Naproxen is rapidly and completely absorbed following oral administration with a reported time of maximum concentration (Tmax) values of 2 hours and 2 to 4 hours. Because naproxen is insoluble in water at low pH but freely soluble at physiologic pH, the rate of gastric emptying and pH of the gastric and intestinal contents are more likely to be rate-limiting for naproxen absorption than dissolution in gastrointestinal fluids. After single oral doses of 100, 200, and 300 mg, reported maximum concentrations (Cmax) values were 12, 25, and 42 µg per mL, respectively. A 500-mg dose produces a Cmax of about 55 µg per mL. Food may delay absorption by decreasing the rate of gastric emptying, but does not significantly change Cmax or area under the plasma concentration-time curve (AUC). In general, the advantages of reduced gastrointestinal irritation are thought to outweigh any clinical effects of slowed absorption rate.
The volume of distribution of naproxen is about 0.09 to 0.16 L per kg. The drug distributes into synovial fluid to reach about 50% of plasma levels 3 to 4 hours after dosing. Naproxen is 99% bound to plasma proteins (albumin), and this binding is known to decrease with increasing plasma drug concentrations (99.6% bound at total plasma concentrations of 23 to 40 µg per mL; 97.4% bound at 473 µg per mL). Disproportional increments in naproxen AUC at doses >500 mg per day are attributed to nonlinear plasma protein binding. For this poorly extracted drug, the increased free fraction of naproxen at higher doses results in more unbound naproxen available for biotransformation and urinary excretion. Despite nonlinear disposition, the half-life of naproxen is independent of dosage or plasma concentration after both single or multiple doses; reported values are 12 to 16 hours. In humans, naproxen is metabolized to naproxen glucuronide (40%), an unknown conjugate (20%), and 6-desmethylnaproxen (28%). The latter moiety is itself conjugated with glucuronide (12%). Less than 10% of naproxen is excreted unchanged in the urine. About 95% of a dose appears in the urine after 5 days, with less than 5% fecal excretion.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Naprosyn® (Syntex) 500-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that from a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the 2 possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should preferably be healthy, preferably nonsmoking, volunteers aged 18 to 55 within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). If smokers are included, they should be identified as such. Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 500-mg tablet of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.50, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 18, 24, 36, 48, and 72 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Naproxen should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of naproxen from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and Phase II, and Phase II and Phase III. An equal number of subjects should be assigned to each of the 6 dosing sequences.
The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design (minimum 12 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 500-mg tablet administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 500-mg Naprosyn® tablet (Syntex) administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 500-mg tablet administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile.
Times: 10, 20 30, 45, and 60 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 375- and 250-mg strengths of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 375- and 250-mg tablets are proportionally similar in both active and inactive ingredients to the firm's 500-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 375- and 250-mg tablets of the generic product meet dissolution test requirements.

Pentoxifylline Extended-Release Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Pentoxifylline is a hemorrheologic agent that improves the flow properties of blood by decreasing its viscosity and improving erythrocyte flexibility. These actions increase blood flow and enhance tissue oxygenation in patients with chronic peripheral arterial disease. It is indicated for the treatment of patients with intermittent claudication on the basis of chronic occlusive arterial disease of the limbs. The usual dosage is one tablet three times a day with meals.
Pharmacokinetics— Urinary recovery data suggests virtually complete absorption of pentoxifylline from the extended-release dosage form. The absolute bioavailability at steady state (every 8 hours for 6 days) is estimated to be 19.4%, indicating extensive first-pass metabolism. Pentoxifylline undergoes both oxidation and reduction metabolism with greater than 90% of a dose being eliminated renally as metabolites. Both pentoxifylline and its reduction metabolite (1-(5-hydroxyhexyl)-3,7-dimethylxanthine (M1) and its oxidation metabolite (1-(3-carboxypropyl)-3,7-dimethylxanthine (M5) have similar pharmacological effects.
Administration of single doses of 400-mg pentoxifylline extended-release tablets results in Tmax values of 2 to 4 hours and mean Cmax values of 55 to 300 ng per mL. The apparent mean half-life of the parent drug is about 3.4 hours, demonstrating the absorption-limited elimination rate of the extended-release dosage form. After multiple dosing of pentoxifylline extended-release tablets (400 mg every 8 hours for 6 to 7 days), mean steady-state values of pentoxifylline Cmax were 189 to 248 ng per mL and Tmax was 0.9 to 2 hours. Reported mean values for M1 (400-mg extended-release tablet) from single-dose studies are as follows: Cmax, 143 to 343 ng per mL; Tmax, 3.2 hours; and t(½), 3.4 hours. Mean steady-state values for M1 from multiple-dose studies were reported as follows: Cmax, 562 to 576 ng per mL; and Tmax, 2 to 2.8 hours. For M5, the corresponding values were 943 ng per mL and 1.4 hours, respectively. Drug absorption is slower when pentoxifylline is taken with food resulting in a significant decrease in Cmax and increase in Tmax for parent drug and metabolites. However, AUC is not changed significantly.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Trental® (Hoechst Marion Roussel) 400-mg extended-release tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-treatment, two-period, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A multiple-dose, steady-state, randomized, two-treatment, two-period, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  3. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of the reference formulation when given as equal labeled doses.
Design— The study design is a single dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsors should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Female subjects must be given a pregnancy test prior to beginning the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, 16, 24, and 30 hours post-dose. Plasma or serum should be separated promptly and immediately frozen until assayed. Following a 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Pentoxifylline and two of its active metabolites 1-(5-hydroxyhexyl)-3,7-dimethylxanthine (M1) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5) should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Recommended Protocol for Conducting a Multiple-Dose, Steady-State, Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the steady-state rate and extent of absorption of a generic extended-release pentoxifylline formulation with that of the reference formulation when given as equal labeled doses.
Design— The study design is a multiple-dose, steady-state, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to the two possible dosing sequences. Before the study begins, the proposed protocols should be approved by an institutional review board.
The multiple-dose, steady-state, bioequivalence study should be performed in the same manner as directed under Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions, with the following exceptions:
Procedure— Subjects should begin a regimen in which consecutive doses of the test or reference products are given every 8 hours with 240 mL of water until steady-state blood serum and plasma levels are attained by all. After reaching steady state, subjects should be administered the final dose of the test or the reference product with 240 mL of water at the next dosing interval.
Restrictions— Item “b” should state: Meals should be scheduled at least 2 hours post-dose for all doses. After the final dose of each treatment is administered and intensive sampling begins, fasting should be continued for 4 hours. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
Blood Sampling— The pre-dose blood sampling must include at least three trough level samples on three consecutive days (collected at the same time of day) to demonstrate that steady-state blood plasma and serum levels are achieved prior to the final Phase I dose. Adequate blood samples should be collected at appropriate times during a dosing interval at a steady state to permit estimation of AUCinterdose. Additional samples should then be collected to permit estimation of the terminal elimination rate constant z. Following a 1-week washout period, subjects should begin the second phase of the study.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of pentoxifylline from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, randomized, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and Phase II, and Phase II and Phase III. An equal number of subjects should be assigned to each of the six dosing sequences.
The limited food effects study should be performed in the same manner as directed under Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the six dosing sequences possible in a three-treatment, three-period study design (minimum 18 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 400-mg extended-release pentoxifylline tablet administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 400-mg extended-release Trental® tablet administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 400-mg extended-release pentoxifylline tablet administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under nonfasting conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasting and nonfasting studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, and Cmax mean values for the test product administered under nonfasting conditions are within 20% of the respective mean values obtained for the reference product administered under nonfasting conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference product used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Drug Release Testing 724— Drug release testing should be conducted on 12 individual dosage units of batches of the test and reference products employing the biostudy lots. The potential for pH dependence of drug release from an extended-release product is well recognized. Drug release profiles should, therefore, be generated in aqueous media at the following pH ranges: 1–1.5, 4–4.5, and 6.0–6.8. See In Vitro Drug Release for Quality Control Preapproval under General Guidances. The following times should be used to determine the drug release profile.
Times: 1, 2, and 4 hours and every 2 hours until 80% of drug is released.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.

Pindolol Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Pindolol is a nonselective beta- adrenergic antagonist with intrinsic sympathomimetic activity in therapeutic dosage ranges. Pindolol has low membrane stabilizing activity. The drug is indicated for the management of hypertension. It may be used alone or concomitantly with other antihypertensive agents, particularly with a thiazide type diuretic.
In the management of hypertension, the recommended initial dose is 5 mg twice a day administered alone or in combination with other antihypertensives. The dosage may be adjusted in increments of 10 mg per day, up to a maximum dosage of 60 mg per day.
Pharmacokinetics— Following intravenous administration of pindolol, the plasma concentration-time data can be described by a biexponential equation. The disposition of pindolol after oral administration has also been reported to be monophasic with an elimination half-life of approximately 3 to 4 hours in healthy subjects or hypertensive patients with normal renal function.
Pindolol is rapidly absorbed, achieving peak plasma concentrations within 1 to 2 hours of drug administration. It has a stated bioavailability of greater than 95%. Earlier studies also reported oral bioavailability of 90% to 100%. One study, however, reported an oral absolute bioavailability of only 53%. The bioavailability of pindolol is not significantly affected by co-administration with food. Food, however, increases the rate of absorption. Pindolol has a negligible first-pass effect. The blood concentrations are linearly proportional to the administered dose in the range of 5 to 20 mg.
The lipid solubility of pindolol is considered intermediate in comparison with other beta blockers. The apparent volume of distribution has been reported to be 1.2 to 2 L per kg. Pindolol is about 40% to 60% bound to plasma proteins and is evenly distributed between plasma and red cells.
Approximately 35% to 40% of the administered dose is excreted unchanged in the urine and 60% to 65% is metabolized, primarily to hydroxy-metabolites, which are excreted as glucuronides and ether sulfates. The polar metabolites are excreted with a half-life of approximately 8 hours. About 6% to 9% of an administered intravenous dose is excreted from the bile into the feces.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Visken® (Sandoz) 10-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of pindolol from a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy nonsmoking volunteers aged 18 to 50 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered two 10-mg tablets of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.50, 0.75, 1, 1.50, 2, 3, 4, 6, 8, 10, 12, 16, and 24 hours post-dose. Plasma or serum should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Pindolol should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product versus 12 units of the reference product. The biostudy lots should be used for those product strengths tested in vivo. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: 0.1 N hydrochloric acid; 500 mL.
Apparatus 2: 50 rpm.
Times: 5, 10, and 15 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerance (Q): not less than 80% in 15 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 5-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2), provided the following conditions are met:
  1. The 5-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 10-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 5-mg tablet of the generic product meets dissolution test requirements.

Piroxicam Capsules—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Piroxicam is a nonsteroidal anti-inflammatory drug (NSAID). It is indicated for acute or long-term use in the relief of signs and symptoms of osteoarthritis and rheumatoid arthritis. It is recommended that a single 20-mg dose be given daily for these conditions. The daily dose may be divided into two 10-mg capsules.
Like other nonsteroidal anti-inflammatory drugs, piroxicam inhibits prostaglandin synthesis by blocking cyclooxygenase; it has no effect on lipooxygenase. Piroxicam has been reported to have a greater effect than other nonsteroidal anti-inflammatory drugs in inhibiting rheumatoid factor production, in increasing phytohemagglutinin response, and in increasing the percentage of suppressor T-cells in peripheral blood. The significance of these effects on its anti-inflammatory and antiarthritic action is not clear.
Piroxicam should not be used in patients with the syndrome comprised of bronchospasm, nasal polyps, and angioedema precipitated by aspirin or other NSAIDs. The major adverse reaction associated with piroxicam is GI irritation.
Pharmacokinetics— Following oral administration, piroxicam is rapidly absorbed. Peak plasma levels occur in 2 to 5 hours with a terminal elimination half-life ranging between 30 and 60 hours. Two or more peaks are usually observed in the piroxicam plasma concentration-time profile between 3 and 12 hours after dosing. Enterohepatic recirculation of piroxicam has been cited as the possible explanation for these multiple peaks. However, a preliminary study in healthy volunteers did not reveal any biliary secretion of piroxicam. The prolonged half-life of piroxicam results in the maintenance of relatively stable plasma concentrations throughout the day on a once daily regimen. With repeated daily doses of 20 mg, steady-state plasma levels of 3 to 5 µg per mL are attained in 7 to 12 days.
Piroxicam is extensively bound (99%) to plasma proteins. Despite very high plasma binding, piroxicam readily penetrates into the synovial fluid of patients with rheumatoid arthritis, osteoarthritis, and reactive synovitis, where piroxicam concentrations are about 40% of those in plasma. This concentration difference of unbound piroxicam across the synovial membrane may be explained by ion trapping of piroxicam in plasma, assuming a pH for synovial fluid significantly lower than 7.4. The very high plasma binding of piroxicam limits its distribution mainly to extracellular space. As a result, the Vd of piroxicam is small, usually about 0.14 L per kg, which is a value typical for most NSAIDs.
Piroxicam is eliminated mainly by biotransformation with less than 5% of the dose excreted unchanged in urine. Hydroxylation and subsequent conjugation with glucuronic acid is the major metabolic pathway in man. This piroxicam metabolite has little or no anti-inflammatory activity.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Feldene® (Pfizer Co.) 20-mg capsules.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single dose (20-mg) of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 24, 36, 48, 72, 96, 120, 144, and 168 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 2-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Piroxicam should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of piroxicam from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 2-week washout period between Phase I and Phase II, and Phase II and Phase III. An equal number of subjects should be assigned to each of the 6 dosing sequences.
The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design (minimum 12 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 20-mg capsule administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 20-mg Feldene® capsule (Pfizer) administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 20-mg capsule administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 15, 30, 45, and 60 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 10-mg capsule of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 10-mg capsule is proportionally similar in both active and inactive ingredients to the firm's 20-mg capsule, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 10-mg capsule of the generic product meets dissolution test requirements.

Ranitidine Hydrochloride Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Ranitidine hydrochloride is a histamine H2-receptor antagonist, which inhibits competitively and reversibly the interaction of histamine with H2-receptors. It reduces gastric acid secretion elicited by histamine and gastrin and stimulated by betazole and food. Ranitidine hydrochloride also inhibits basal and nocturnal acid secretion.
Ranitidine hydrochloride is indicated in active and maintenance therapies of duodenal ulcer. It is also used in treatments of active, benign gastric ulcer, pathological hypersecretory conditions, and gastroesophageal reflux disease. Serum concentrations of 36 to 94 ng per mL of ranitidine hydrochloride have been shown to inhibit 50% of stimulated gastric acid secretion. The recommended oral dosage of ranitidine hydrochloride tablets for active duodenal ulcer is 150 mg twice a day, or 300 mg at bedtime.
Pharmacokinetics— An absolute oral bioavailability of 50% has been reported for 150-mg ranitidine hydrochloride tablets. Mean values of 60% and 52% have also been reported as the absolute bioavailability of two other formulations of 150- and 100-mg ranitidine hydrochloride tablets, respectively.
A linear relationship has been found between the dose and the area under the serum concentration-time curve following the administration of oral doses of 100, 150, 250, and 400 mg of ranitidine hydrochloride tablets.
A secondary peak in the plasma concentration-time curve has been reported for ranitidine hydrochloride following oral administration of 100- and 150-mg tablets in fasting studies. The mean values reported for the peaks were 1.5 hours for the first peak and 3.9 hours for the second.
The reported elimination half-life of ranitidine hydrochloride is 2.5 to 3.0 hours. Approximately 30% of ranitidine hydrochloride is excreted unchanged in urine within 24 hours following the administration of an oral dose. The major urinary metabolite of ranitidine hydrochloride in man is N-oxide, which represents less than 4% of the dose. S-oxide (1%) and desmethyl ranitidine (1%) are the other metabolites of ranitidine hydrochloride.
Multiple-dose studies in which ranitidine hydrochloride was administered in doses of 150 and 200 mg twice daily for 28 days showed that ranitidine hydrochloride did not accumulate in the body, and the values of the pharmacokinetic parameters were similar for both single- and multiple-dose studies.
Administration of food or antacid reportedly does not change the absorption of ranitidine hydrochloride tablets significantly. However, concomitant administration of high doses of antacids with the 150-mg tablets reduced its absorption.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Zantac® (Glaxo) 300-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation of ranitidine hydrochloride tablet with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers 18 to 50 years of age and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study and between Phase I and Phase II. Nursing mothers should be excluded from the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 300-mg tablet dose of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration, when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.33, 0.50, 0.67, 1, 1.33, 1.50, 1.67, 2, 2.50, 3, 3.50, 4, 5, 6, 8, 10, 12, 16, and 24 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Analytical Methods— Ranitidine hydrochloride should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, and accuracy and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units and should be retained for a period of not less than 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following method and tolerances are currently recommended for this product:
Medium: deaerated water; 900 mL.
Apparatus 2: 50 rpm.
Times: 10, 20, 30, and 45 minutes.
Procedure— Use USP if available, or other validated method (see Validation of Compendial Methods 1225).
Tolerances (Q): not less than 80% is dissolved in 45 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test 905— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 150-mg strength of the generic product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 150-mg tablet is proportionally similar in both active and inactive ingredients to the 300-mg tablet, which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 150-mg tablet of the generic product meets the dissolution testing requirements.

Selegiline Hydrochloride Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Selegiline hydrochloride is an irreversible inhibitor of monoamine oxidase (MAO) with greater affinity for MAO Type B (predominant form in the brain) compared to Type A (predominant intestinal form). Type A MAO is not inhibited by selegiline at therapeutic doses; therefore, the ability of MAO Type A (GI tract and liver) to prevent hypertensive effects from exogenous amines absorbed from the gut is not compromised. Selegiline can be used safely without dietary restrictions and with concomitant drug use only at doses that selectively inhibit MAO Type B ( 10 mg per day). In addition to its inhibitory actions on MAO Type B, selegiline may also prevent indirect acting sympathomimetics from displacing norepinephrine from adrenergic neurons. By blocking the metabolism of dopamine in the brain, selegiline increases the amount of dopamine available to dopamine receptors that might be beneficial to treatment of Parkinsonism. Selegiline is indicated as an adjunct in the management of Parkinsonian patients being treated with levodopa and carbidopa who exhibit deterioration in the quality of their response to this therapy. Dosing is 10 mg per day; in an effort to minimize adverse effects, such as nausea and dizziness, that may follow a single 10-mg dose, divided doses of 5 mg are administered at breakfast and lunch.
Pharmacokinetics— Much of the original pharmacokinetic data reported for selegiline were obtained after administration of 14C-selegiline. The following results represent the total 14C-label (parent drug plus metabolites): 1) After oral administration of 5 mg 14C-selegiline, Cmax ranged from 33 to 45 ng per mL with a Tmax of 0.5 to 2 hours. 2) The elimination half-life of 14C-selegiline from plasma has been approximated at 39 hours (range, 16 to 69 hours). Of the three principal metabolites of selegiline—desmethylselegiline (DES), methamphetamine (MA), and amphetamine (A)—only DES is thought to contribute to the overall pharmacological effect. Due to a large volume of distribution and extensive first-pass metabolism, selegiline plasma concentrations after a 10-mg dose are difficult to detect. In a single-dose pilot study (N = 4) using this assay, the mean plasma elimination half-lives of selegiline and DES were 1.9 hours and 2.2 hours, respectively. The effects of food on selegiline absorption have not been reported.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Eldepryl® (Somerset) 5-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10% of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses (two 5-mg tablets, total dose 10-mg) of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses (two 5-mg tablets, total dose 10 mg) of the test and reference products when administered immediately following a high-fat breakfast.8
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of the reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 2-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 50 years and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered two 5-mg tablets (10 mg total dose) of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken, except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0 hours) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, 24, 36, 48, 72, and 96 hours post-dose. Plasma or serum should be separated promptly and immediately frozen until assayed. Following a 2-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Selegiline and its three metabolites desmethylselegiline (DES), methamphetamine (MA), and amphetamine (A) should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances. Pivotal statistical criteria should be applied to the three selegiline metabolites, desmethylselegiline (DES), methamphetamine (MA), and amphetamine (A). Results should also be reported for selegiline.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of selegiline from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, randomized three-treatment, three-period, six-sequence crossover with a 2-week washout period between Phase I and Phase II, and Phase II and Phase III. An equal number of subjects should be assigned to each of the six dosing sequences.
The limited food effects study should be performed in the same manner as directed under Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the six dosing sequences possible in a three-treatment, three-period study design (minimum 18 subjects). Each subject will receive the following treatments:
TREATMENT 1: Generic selegiline hydrochloride product, (two 5-mg tablets, total dose 10 mg) administered after a high-fat breakfast.
TREATMENT 2: Reference product (two 5-mg Eldepryl® tablets, total dose 10 mg) administered after a high-fat breakfast.
TREATMENT 3: Generic selegiline hydrochloride product (two 5-mg tablets, total dose 10 mg) administered under fasting conditions.
Following a 10-hour fast, the subjects receiving the treatments under nonfasting conditions should be served the indicated high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast, then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used as in the fasting and nonfasting studies. No food should be allowed for at least 4 hours post-dose, with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, and Cmax mean values for the test product administered under nonfasting conditions are within 20% of the respective mean values obtained for the reference product administered under nonfasting conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples, refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test and reference products employing the biostudy lots. The current official USP dissolution method should be followed, and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 5, 10, 20, and 30 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test 905— Content uniformity testing on the test and reference product lots should be performed as described in USP.

Tolmetin Sodium Capsules and Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Tolmetin sodium is a nonsteroidal anti-inflammatory agent freely soluble in water. Its mode of action is unknown. The drug is indicated for the relief of signs and symptoms of rheumatoid arthritis and osteoarthritis, both in the treatment of acute episodes and the long-term management of the chronic disease. In adults with rheumatoid arthritis, the recommended starting dose is 400 mg three times daily (1200 mg daily), preferably including a dose on arising and a dose at bedtime.
Pharmacokinetics— The drug is rapidly and almost completely absorbed following oral dosing, with peak plasma levels reached in 30 to 60 minutes. Tolmetin displays a biphasic elimination from plasma consisting of a rapid phase (T½, about 1 to 2 hours) followed by a slower phase (T½, about 5 hours). Essentially all of the dose is recovered in the urine within 24 hours either as an active metabolite or as conjugates of tolmetin. Peak plasma levels of about 40 to 50 µg per mL were obtained with a 400-mg oral dose. When taken immediately after a meal, peak plasma drug levels decreased by 50%, while total bioavailability was decreased by 16%. Administration with milk alone had no effect on peak plasma drug concentrations but decreased the total tolmetin bioavailability by 16%.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Tolectin® (McNeil Pharmaceutical) 400-mg capsules and 600-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10% that of the largest lot planned for full production or a minimum of 100,000 units, whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. A single-dose, randomized, two-period, two-treatment, two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. A single-dose, randomized, three-treatment, three-period, six-sequence crossover, limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
Recommended Protocols for Conducting a Single-Dose, Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of tolmetin sodium of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose, two-treatment, two-period, two-sequence crossover with a 1-week washout period between Phase I and Phase II dosing. An equal number of subjects should be randomly assigned to each of the two possible dosing sequences. Before the study begins, the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names, titles, and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study. It is recommended that a minimum of 24 subjects be used in this study. Subjects should be healthy, preferably nonsmoking, volunteers aged 18 to 55 and within 10% of ideal body weight for height and build, although within 15% of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin, 1983). Subjects should be selected on the basis of acceptable medical history, physical examination, and clinical laboratory test results. Female subjects must be given a pregnancy test prior to beginning the study. Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study. If smokers are included, they should be identified as such. Written, informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10 hours, subjects should be administered a single 400-mg dose (for capsules) or a single 600-mg dose (for tablets) of the test or reference product with 240 mL of water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1 hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4 hours after administration of the test or reference treatment. All meals should be standardized during the study, and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48 hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications, including oral contraceptives, beginning 2 weeks and no OTC medications beginning 1 week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-lose (0 hours) and at 0.17, 0.33, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, and 20 hours post-dose. Plasma should be separated promptly and immediately frozen until assayed. Following a minimum 1-week washout period, subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4 hours of the study. Subjects with a heart rate less than 45 bpm or greater than 110 bpm should have an electrocardiogram (lead II) performed and have their pulse monitored hourly. Subjects should report any unusual (symptoms observed during the study. Subjects should be periodically questioned during each phase of the study for any unsual symptoms experienced after drug administration.
Analytical Methods— Tolmetin should be assayed using a suitable method fully validated with respect to adequate sensitivity, specificity, linearity, recovery, accuracy, and precision (both within and between days). Stability of the samples under frozen conditions, at room temperature, and during freeze-thaw cycles, if appropriate, should be determined. Chromatograms of the analysis of the unknown samples, including all associated standard curve and quality control chromatograms, should be available for regulatory authorities. The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design under General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of tolmetin sodium from a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose, three-treatment, three-period, six-sequence crossover with a 1-week washout period between Phase I and Phase II, and Phase II and Phase III.
The limited food effects study (minimum 12 subjects) should be performed in the same manner as the single-dose study under fasting conditions, with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6 dosing sequences possible in a three-treatment, three-period study design. Each subject will receive the following treatments:
TREATMENT 1: Generic product, a 400-mg capsule or 600-mg tablet administered after a standard high-fat breakfast.
TREATMENT 2: Reference product, a 400-mg Tolectin® capsule or 600-mg Tolectin® tablet administered after a standard high-fat breakfast.
TREATMENT 3: Generic product, a 400-mg capsule or 600-mg tablet administered under fasting conditions.
Following a 10-hour fast, the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast. The subjects should have 30 minutes to finish the entire breakfast and then should immediately receive Treatment 1 or Treatment 2, taken with 240 mL of water. Subjects receiving the treatment under fasting conditions should receive Treatment 3, taken with 240 mL of water only. The same lots of the test and reference products should be used in the fasted and fed studies. No food should be allowed for at least 4 hours post-dose with water allowed after the first hour. Subjects should be served scheduled standardized meals throughout the study, and the same meals should be served during all phases of the study.
Statistical Analysis— In general, a comparable food effect will be assumed if the AUC0–t, AUC0–, and Cmax mean values for the test product administered under fed conditions are within 20% of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report, Side Effects, and Adverse Reactions— Subject medical histories, physical examinations and laboratory reports, and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application. Each reserve sample should consist of at least 200 dosage units or 5 times the amount needed for complete release testing, whichever is greater, and should be retained for a minimum of 5 years following approval of the application. For more information on retention of bioequivalence samples refer to 21 CFR 320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12 dosage units of the test product and the reference product, employing the biostudy lots. The current official USP dissolution method (see Dissolution 711) should be followed and should be referenced by the sponsor. The following times should be used to determine the dissolution profile:
Times: 10, 20, 30, and 45 minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit. The mean percentage dissolved, the range (highest, lowest) of dissolution, and the coefficient of variation (relative standard deviation) should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 200-mg strength of the generic tablet product may be granted pursuant to 21 CFR 320.22(d)(2) provided the following conditions are met:
  1. The 200-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 600-mg tablet which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 200-mg tablet of the generic product meets dissolution test requirements.
1  Further information about guidances can be obtained by contacting the United States Food and Drug Administration, Division of Bioequivalence, Office of Generic Drugs, 7500 Standish Place, Metro Park North, Rockville, MD 20855 [Phone: (301) 594-2290; FAX: (301) 594-0181]. Copies of the guidances can be obtained from the United States Food and Drug Administration, Center for Drug Evaluation and Research, Consumer Affairs Branch HFD-210 5600 Fishers Lane, Rockville, MD 20857 [Phone: (301) 827-4573, FAX: (301) 827-4577.]
2  This statement, prepared by the Division of Bioequivalence, Office of Generic Drugs (OGD), in consultation with the Division of Biometrics, Office of Epidemiology and Biostatistics, is an informal communication under 21 CFR 10.90 (b)(9) that represents the best judgment of the Division of Bioequivalence and the Office at this time. This statement does not necessarily represent the formal position of the Center for Drug Evaluation and Research (CDER), the Food and Drug Administration (FDA), and does not bind or obligate CDER or FDA to the views expressed.
3  Note that a more general equation can be written for any multi-compartmental model as AUC0– = FD / (VdLZ), where Vd is the volume of distribution relating drug concentration in plasma or blood to the amount of drug in the body during the terminal exponential phase, and Z is the terminal slope of the concentration-time curve.
9  Lund RE. Tables for an approximate test for outliers in linear models. Technometrics 1975; 17:473-476.
4  This statement, prepared by the FDA Division of Bioequivalence in the Office of Generic Drugs, is an informal communication under 21 CFR 10.90(b)(9) that represents the best judgment of the Division at this time. This statement does not necessarily represent the formal position of the Center for Drug Evaluation and Research, Food and Drug Administration, and does not bind or otherwise obligate the Center for Drug Evaluation and Research, Food and Drug Administration, to the views expressed.
6  Each subject should consume a standardized, high-fat breakfast consisting of the following:

1 buttered English muffin

1 fried egg

1 slice of American cheese

1 slice of Canadian bacon

1 serving of hash brown potatoes

8 fluid oz. (240 mL) of whole milk

6 fluid oz. (180 mL) of orange juice
5  The sponsoring firm is advised that an Investigational New Drug (IND) application may be required if dosing levels exceed those recommended in the official labeling. See Policy and Procedure Guide 36-92, “Submission of an `Investigational New Drug Application' to the FDA Office of Generic Drugs (OGD)” and 21 CFR 312.2 and 320.31(b)(1).
7  Glipizide (5 and 10 mg) and glyburide (1.25, 2.5, and 5 mg), the second generation sulfonylurea antidiabetic agents, are comparatively more potent than tolbutamide (250 and 500 mg) and tolazamide (100, 250, and 500 mg). Therefore, in a fasting bioequivalence study involving normal subjects, hypoglycemic events occur more frequently with glipizide and glyburide than with tolbutamide and tolazamide. In the case of glipizide, the hypoglycemic episodes in normal subjects participating in a fasting bioequivalence study were fewer when the glucose was given to subjects every 15 minutes than when it was given every 30 minutes. In a study with such a design, measurement of plasma glucose is not necessary because it will not reflect the pharmacodynamic endpoint. However, such design is preferable to the usual fasting study design to ensure the welfare of the subjects and to avoid excessive drop out rate.
8  Each subject should consume the following high-fat breakfast:

2 eggs fried in butter

2 slices of toasted white bread spread with butter

2 slices of bacon

2 oz. of hash brown potatoes

8 fluid oz. (240 mL) of whole milk

Auxiliary Information— Staff Liaison : Horacio Pappa, Ph.D.
Expert Committee : (BPC05) Biopharmaceutics05
USP29–NF24 Page 2929
Pharmacopeial Forum : Volume No. 31(1) Page 243
Phone Number : 1-301-816-8319