Study on requirements of bioequivalence for registration of pharmaceutical products in USA, Europe and Canada

Abstract

The present study was aimed to study the requirements of bioequivalence for the registration of pharmaceutical products in the USA, Europe and Canada. Before going into bioequivalence studies it is essential for the pharmaceutical industry to study the guidelines of bioequivalence for the respective country where the industry wants to market its products and thus enter into generic market. This study reviews the requirements of bioequivalence with study parameters such as study design, fasting or fed state studies, volunteers recruitment, study dose, sampling points, analytical method validation parameters, moieties to be measured in plasma, pharmacokinetic parameters, criteria for bioequivalence, GCP requirements etc, which are needed for the pharmaceutical industry to carry out bioequivalence studies and to file ANDA. Test products and reference products are needed for this study. Test products are usually manufactured by a sponsor and reference products are provided by the government laboratories of the respective countries. Sampling points also vary with respect to the regulatory guidelines of these countries. All these countries follow ICH GCP guidelines. The criterion of bioequivalence for these countries is 90% CI 80–125% for C_max, AUC_t, AUC_0–∞.

1. Introduction

The concepts of bioavailability (BA) and bioequivalence (BE) have gained considerable importance during the last three decades because of their application to new brand-name drugs, as well as to generic drugs. During this period, regulatory authorities also started developing and formulating the regulatory requirements for the approval of generic drug products. Consequently, tremendous advances have been made in the application of assessment approaches to these scientific concepts. BA and BE have become the cornerstones for the approval of brand-name and generic drugs globally and have been utilized for brand-name drugs to reduce the cost of development. It is encouraging to know that there are continuing efforts by regulatory authorities and the scientific community, both nationally and internationally, to understand and develop more efficient and scientifically valid approaches to the assessment of BE of various dosage forms including some of the tough complex special dosage forms. Because of the importance of generic drugs in healthcare, it is imperative that the pharmaceutical quality and in vivo performance of generic drugs be reliably assessed (Midha and McKay, 2009). Because generic drugs would be interchanged with innovator products in the market place, it must be demonstrated that the safety and efficacy of generics are comparable to the safety and efficacy of the corresponding innovator drugs. Assessment of “interchangeability” between the generic and the innovator product is carried out by a study of in vivo equivalence or bioequivalence (Meredith, 2003).

2. Bioequivalence study requirements for the registration of the pharmaceutical product in the USA

Requirements of bioequivalence study for the registration of orally administrated drug products are detailed in 21 CFR 320. (CFR: Code of Federal Regulations) (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?, 2013).

2.1. Experimental design

A randomized, balanced, single-dose, two-treatment (fed vs. fasting), two period, two sequence crossover design is used for studying the effects of food on the bioavailability of either an immediate-release or a modified release drug product. The test product and the reference listed drug product should be administered under fed conditions. An adequate washout period should separate the two treatments. The formulation to be tested should be administered on an empty stomach (fasting condition) in one period and following a test meal (fed condition) in the other period. A similar, two-treatment, two-period, two sequence crossover design for a fed bioequivalence study is often recommended except that the treatment should consist of both test and reference formulations administered following a test meal (fed condition). An adequate washout period should separate the two treatments in food effect bioavailability and fed bioequivalence studies. For immediate release solid/suspension dosage forms, usually a non-replicate, randomized, single dose, two treatment, two period, two sequence crossover design is performed.

2.2. Sample size and dropouts

A minimum number of 12 evaluable subjects should be included in any bioequivalence study. When an average bioequivalence approach is selected using either non-replicated or replicated designs, methods appropriate to the study design should be used to estimate sample sizes. The number of subjects for bioequivalence studies based on either the population or individual bioequivalence approach can be estimated by simulation if analytical approaches for estimation are not available. Sponsors should enter a sufficient number of subjects in the study to allow for dropouts. Because replacement of subjects during the study could complicate the statistical model and analysis; dropouts generally should not be replaced. Sponsors who wish to replace dropouts during the study should indicate this intention in the protocol. The protocol should also state whether samples from replacement subjects, if not used, will be assayed. If the dropout rate is high and sponsors wish to add more subjects, a modification of the statistical analysis may be recommended. Additional subjects should not be included after data analysis unless the trial was designed from the beginning as a sequential or group sequential design (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?, 2013).

2.3. Food-effect bioavailability and fed bioequivalence studies

Food effect bioavailability studies are usually conducted for new drugs and drug products during the investigational new drug (IND) period to assess the effects of food on the rate and extent of absorption of a drug when the drug product is administered shortly after a meal (fed conditions), as compared to administration under fasting conditions. Fed bioequivalence studies, on the other hand, are conducted for abbreviated new drug applications (ANDAs) to demonstrate their bioequivalence to the reference listed drug (RLD) under fed conditions (Meredith, 2003; http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?, 2013).

2.4. Potential mechanisms of food effects on bioavailability

Food can change the BA of a drug and can influence the BE between test and reference products. Food effects on BA can have clinically significant consequences. Food can alter BA by various means, including delay in gastric emptying, stimulation of bile flow, change in gastrointestinal pH, increase of splanchnic blood flow, change in luminal metabolism of a drug substance, and physical or chemical interaction with a dosage form or a drug substance.

2.5. Study considerations

This section provides general considerations for designing food effect BA and fed BE studies. A sponsor may propose alternative study designs and data analyses. The scientific rationale and justification for these study designs and analyses should be provided in the study protocol. Sponsors may choose to conduct additional studies for a better understanding of the drug product and to provide optimal labeling statements for dosage and administration (e.g. different meals and different times of drug intake in relation to meals). In studying modified-release dosage forms, consideration should be given to the possibility that co-administration with food can result in dose dumping, in which the complete dose may be more rapidly released from the dosage form than intended, creating a potential safety risk for the study subjects (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?, 2013).

2.6. Subject selection

Both food-effect BA and fed BE studies can be carried out in healthy volunteers drawn from the general population of 18 years of age or older and capable of giving informed consent. Studies in the patient population are also appropriate if safety concerns preclude the enrollment of healthy subjects. A sufficient number of subjects should complete the study to achieve adequate power for a statistical assessment.

2.7. Dosage strength

In general, the highest strength of a drug product intended to be marketed should be tested in food-effect BA and fed BE studies. In some cases, clinical safety concerns can prevent the use of the highest strength and warrant the use of lower strengths of the dosage form. For ANDAs, the same lot and strength used in the fasting BE study should be tested in the fed BE study. For products with multiple strengths in ANDAs, if a fed BE study has been performed on the highest strength, BE determination of one or more lower strengths can be waived based on dissolution profile comparisons.

2.8. Test meal

We recommend that food-effect BA and fed BE studies be conducted using meal conditions that are expected to provide the greatest effects on GI physiology so that systemic drug availability is maximally affected. A high-fat (approximately 50% of total caloric content of the meal) and high-calorie (approximately 800–1000 calories) meal is recommended as a test meal for food-effect BA and fed BE studies. This test meal should derive approximately 150, 250, and 500–600 calories from protein, carbohydrate, and fat, respectively. The caloric breakdown of the test meal should be provided in the study report. If the caloric breakdown of the meal is significantly different from the one described above, the sponsor should provide a scientific rationale for this difference. In new drug applications (NDAs), it is recognized that a sponsor can choose to conduct food effect BA studies using meals with different combinations of fats, carbohydrates, and proteins for exploratory or label purposes. However, one of the meals for the food-effect BA studies should be the high-fat, high-calorie test meal described above.

2.9. Administration

2.9.1. Fasted treatments

Following an overnight fast of at least 10 h, subjects should be administered the drug product with 240 mL (8 fluid ounces) of water. No food should be allowed for at least 4 h post-dose. Water can be allowed as desired except for one hour before and after drug administration. Subjects should receive standardized meals scheduled at the same time in each period of the study.

2.9.2. Fed treatments

Following an overnight fast of at least 10 h, subjects should start the recommended meal 30 min prior to administration of the drug product. Study subjects should eat this meal in 30 min or less; however, the drug product should be administered 30 min after the start of the meal. The drug product should be administered with 240 mL (8 fluid ounces) of water. No food should be allowed for at least 4 h post-dose. Water can be allowed as desired except for one hour before and after drug administration. Subjects should receive standardized meals scheduled at the same time in each period of the study.

2.10. Sample collection

For both fasted and fed treatment periods, timed samples in the biological fluid, usually plasma should be collected from the subjects to permit characterization of the complete shape of the plasma concentration–time profile for the parent drug. It may be advisable to measure other moieties in the plasma, such as active metabolites, and sponsors should refer to the guidance on bioavailability and bioequivalence studies for orally administered drug products and general considerations for recommendations on these issues. Consideration should be given to the possibility that co-administration of a dosage form with food can alter the time course of plasma drug concentrations so that fasted and fed treatments can have different sample collection times.

If the pre-dose concentration is ⩽5% of C_max value in that subject, the subject’s data without any adjustments can be included in all pharmacokinetic measurements and calculations. It is recommend that if the pre-dose value is greater than 5% of C_max, the subject be dropped from all bioequivalence study evaluations. The subjects who experience emesis during the course of a bioequivalence study for immediate-release products are deleted from statistical analysis if vomiting occurs at or before two times median T_max. In the case of modified-release products, the data from subjects who experience emesis any time during the labeled dosing interval can be deleted (Grabowski et al., 2012).

2.11. Moieties to be measured

The moieties to be measured in biological fluid collected in bioequivalence studies are either the active drug ingredient or its active moiety in the administered dosage form (parent drug) and, when appropriate, its active metabolite(s). This guidance recommends the following approaches for bioequivalence studies: Measurement of only the parent drug released from dosage form, rather than the metabolite, is generally recommended. The rationale from this recommendation is that the concentration–time profile of the parent drug is more sensitive to changes in formulation performance than a metabolite, which is more reflective of metabolite formation, distribution, and elimination.

2.12. Collection of biological matrix and sampling schedule

Several samples of appropriate biological matrix (blood, plasma/serum, and urine) are collected at various time intervals post-dose. The sampling schedule depends on the pharmacokinetic characteristics of the drug tested. In most cases, plasma or serum is the matrix of choice. However, if the parent drug is not metabolized and is largely excreted unchanged and can be suitably assayed in the urine, urinary drug levels may be used to assess bioequivalence, if plasma/ serum concentrations of the drug cannot be reliably measured. Sufficient numbers of samples are collected during the absorption phase to adequately define the ascending portion of the plasma drug level versus time curve. Intensive sampling is carried out around the time of the expected peak concentration. Sufficient numbers of samples should also be collected in the log–linear elimination phase of the drug so that the terminal elimination rate constant and half-life of the drug can be accurately determined. A sampling period extending to at least four to five terminal elimination half-lives of the drug or four to five of the longest half-live of the pertinent analyte (if more than one analyte) is usually sufficient. The samples are appropriately processed and stored carefully under conditions that preserve the integrity of the analyte(s) (Grabowski et al., 2012).

3. Bioequivalence study requirements for registration of pharmaceutical product in Europe

The design should be based on a reasonable knowledge of the pharmacodynamics and/or the pharmacokinetics of the active substance in question. For the pharmacokinetic basis of these studies reference is made to the recommendation “Pharmacokinetic studies in man”. The design and conduct of the study should follow European-regulations on Good Clinical Practice, including reference to an Ethics Committee.

3.1. Design

The study should be designed in such a way that the formulation effect can be distinguished from other effects. If the number of formulations to be compared is two, a two-period, two-sequence crossover design is often considered to be the design of choice. However, under certain circumstances and provided the study design and the statistical analyses are scientifically sound alternative well-established designs could be considered such as parallel design for very long half-life substances and replicate designs for substances with highly variable disposition (Garcia-Arieta et al., 2012).

3.2. Subjects

3.2.1. Number of subjects

The number of subjects to be included in the study should be based on an appropriate sample size calculation. The minimum number of subjects in a cross-over study should be 12.

3.2.2. Selection of subjects

The subject population for bioequivalence studies should be selected with the aim to permit detection of differences between pharmaceutical products. In order to reduce variability not related to differences between products, the studies should normally be performed in healthy volunteers unless the drug carries safety concerns that make this unethical. This model, in vivo healthy volunteers, is regarded adequate in most instances to detect formulation differences and the results will allow extrapolation to populations in which the reference product is approved (the elderly, children, patients with renal or liver impairment, etc.). The inclusion/exclusion criteria should be clearly stated in the protocol. In general, subjects should preferably be between 18–55 years old and of weight within the normal range according to accepted normal values for the body mass index.

3.3. Study conduct

3.3.1. Standardization

The test conditions should be standardized in order to minimize the variability of all factors involved except that of the products being tested. Therefore, it is recommended to standardize diet, fluid intake and exercise. The time of day for ingestion should be specified. As fluid intake may influence gastric passage for oral administration forms, the test and reference products should be administered with a standardized volume of fluid (at least 150 mL). All meals and fluids (257 mL) taken after the treatment should also be standardized in regard to composition and time of administration during the sampling period.

3.3.2. Sampling times

A sufficient number of samples to adequately describe the complete plasma concentration–time profile should be collected. The sampling schedule should include frequent sampling around C_max to provide a reliable estimate of peak exposure. The sampling schedule should be planned to avoid C_max being the first point of a concentration time curve. When partial AUC is to be determined, frequent early sampling is recommended with preferably at least two quantifiable samples before expected T_max. The sampling schedule should also cover the plasma concentration time curve long enough to provide a reliable estimate of the extent of exposure which is achieved if AUC_t is at least 80% of AUC_∞. At least three to four samples are needed during the terminal log–linear phase in order to reliably estimate the terminal rate constant (which is needed for a reliable estimate of AUC_∞). A sampling period longer than 72 h is not considered necessary for any immediate release formulation. Hence, for drugs with a long half-life, comparison of extent of exposure using truncated AUCs at 72 h is acceptable (Garcia-Arieta et al., 2012; Schellekens et al., 2011).

3.3.3. Fasting or fed conditions

In cases where information is required in both the fed and fasted states, it is preferable to conduct a four-period single dose crossover design study (both products fed and fasted) rather than conducting two separate bioequivalence studies in fed and fasted states, respectively. In a four-period crossover design study, the food effect on test and reference products can be evaluated which is not the case when conducting two separate two-period, two-sequence single dose crossover design studies under fasting and fed conditions, respectively. In addition to the bioequivalence evaluation of test/reference in fasting and in fed state, the food effect can be presented for test and reference, i.e. the ratio food/fasting and 90% confidence interval for test and reference, respectively. The meal should be a “standardized non high-fat meal” (about 650 kcal with about 30% of calories derived from fat). The composition of the meal should be described with regard to protein, carbohydrate and fat content (specified in grams, calories and relative caloric content (%).

3.4. Reference and test product

Test products in an application for a generic product are normally compared with the corresponding dosage form of an innovator medicinal product (reference product). The choice of reference product should be justified by the applicant. For an abridged application claiming essential similarity to a reference product, application to numerous associate countries based on bioequivalence with a reference product from one associate country can be made. Such an application can be considered acceptable unless there is a significant difference between the reference products originating from the same manufacturer (or its subsidiaries/licensees), in terms of the qualitative and quantitative composition in excipients. Concerned associate countries may request information from the first associate country on the reference product, namely on the composition, manufacturing process and finished product specification (Schellekens et al., 2011).

4. Bioequivalence study requirements for registration of pharmaceutical product in Canada

Selection of subjects for a study

A certain minimum number of subjects should be used, and, if a subject withdraws or must be removed from the study, then an explanation for the withdrawal or removal must be included in the study documentation.

4.1. Choice of subjects

Drugs with uncomplicated characteristics can usually be tested in normal, healthy volunteers. The investigators should ensure that female volunteers are not pregnant or likely to become pregnant during the study. Confirmation should be obtained by urine tests just before the first and last doses of the study. In some instances, studies may be required to ascertain bioavailability in patients or subjects with special characteristics – for example, for drugs to be used in the treatment of conditions accompanied by altered absorption or distribution, or for drugs to be used in special age groups such as children or the elderly (Shram, 2012).

4.2. Considerations in subject characteristics

An important objective in the selection of subjects is to reduce the intra-subject variability in pharmacokinetics that may be attributable to certain characteristics of the subject. Subjects should be assigned in such a way that the study design is balanced for any factors that are suspected to contribute to variability.

4.2.1. Age

Subjects should be between the age of legal majority and the age of onset of age associated changes in organic function. This description typically coincides with an age range of 18–55 years, inclusive.

4.2.2. Height/weight ratio

The ratio for healthy volunteer subjects should be within 15% of the normal range – for example, as given in current Ciba-Geigy or Metropolitan Life Insurance tables.

4.2.3. Health

The health of the volunteers must be determined by the supervising physician through a medical examination and review of results of routine tests of liver, kidney, and hematological functions. An electrocardiogram should be included in the study documentation if the drug has a cardiac effect. Aberrant laboratory values should be rechecked and a summary must be presented along with the physician’s opinion. Psychological characteristics should also be assessed by the physician in order to exclude patients unlikely to comply with study restrictions or unlikely to complete the study. Subjects who have been previously treated for gastrointestinal problems (such as ulcers), or convulsive, depressive, or hepatic disorders, and in whom there is a risk of a recurrence during the study period, should be excluded.

4.3. Number of subjects

The number of subjects to be used in the cross-over study should be estimated by considering the standards that must be passed and the drug products being compared. The probability that a study of a given size will pass the standards depends on the expected mean difference between the test and reference formulations of both AUC_t and C_max, and the anticipated intra-subject coefficient of variation (CV) of both AUC_t and C_max. For drugs with uncomplicated characteristics, the intra-subject CV is generally less than 20%; however, as a result of sampling, or if the study is poorly run, the intra-subject CV can be higher. The minimum number of subjects is 12, but a larger number is often required (Shram, 2012).

4.4. Drop-outs and withdrawal of subjects from a study

The subjects must be available, without compulsion, for all legs of the study. It is recommended that the number of subjects should be sufficient to allow for possible drop-outs or withdrawals. Reasons for withdrawal (e.g., adverse drug reaction) must be reported and the subject’s plasma (or blood, or serum) level data provided. The results of all samples that were measured in subjects who were withdrawn from the study must be included in the report. If subjects drop out of the study for personal reasons, the subject’s blood samples do not have to be assayed. Reimbursement policy should be specified prior to initiating the study and should be in agreement with medical research council (MRC) or similar guidelines.

4.5. Study design and environment

The design of a bioavailability study should minimize variability that is not attributable to the drug per se and should eliminate bias as much as possible. The guidance in this section serves for the usual case. Other designs may be permissible after consultation with Health Canada (HC) before the study is initiated. The basic design to be used is a two-period cross-over, in which each subject is given the test and reference formulations. In cases where more than two formulations are under study, or are studied under different conditions, each volunteer should receive all treatments in a restricted randomized design. However, when the number of treatments result in a study that is longer than a month, a balanced incomplete block design may be considered.

4.6. Standardization

Every effort should be made to standardize the study conditions in every phase of the study – for example, exercise, diet, smoking, and alcohol use. It is preferable to use non-smokers; where smokers are included, they must be so identified. Volunteers should not take any other drug, including alcoholic beverages and over-the counter (OTC) drugs, for an appropriate interval before-as well as during-the study. In the event of emergency, the use of any drug must be reported (dose and time of administration). The decision whether to include or exclude the results from a subject who has varied from the established protocol should be made before statistical analysis commences (Shram, 2012; Daley-Yates al., 2009).

4.7. Blinding

If possible, the study should be conducted in such a way that the subject is not aware of which product is administered. Furthermore, the person checking for adverse reactions and the person conducting the analysis of samples must not know which product was administered. Other individuals involved in the administration of the drugs, the surveillance of the patients, or the analysis of plasma (or blood, or serum) data should not know which product was administered.

4.8. Administration of food and fluid

The administration of food and fluid should be controlled carefully. Normally, subjects should fast for 10 h before drug administration. A fast means that no food or solids are to be consumed, although alcohol-free and xanthine-free clear fluids are permissible the night prior to the study. On the morning of the study, up to 250 mL of water may be permitted up to two hours before drug administration. The dose should be taken with water of a standard volume (e.g., 150 mL) and at a standard temperature. Two hours after drug administration, 250 mL of xanthine-free fluids are permitted. Four hours after drug administration, a standard meal may be taken. All meals should be standardized and repeated on each study day. If the oral preparation is being compared with an intravenous (IV) dose, the food and fluid restrictions noted above should also apply to the IV dose. Some drugs are given with food to reduce gastrointestinal side effects. Studies of such drugs should include studies with standard meals. The nature of the test meal to be administered in the part of the study where the formulation is given in the presence of food-should be determined based on the physicochemical and pharmacokinetic characteristics of the drug and its formulation. The purpose is to select a test meal that has the greatest potential to demonstrate altered bioavailability. The meal should be given within a predetermined, constant time of administration of the drug. The appropriate choice of the meal’s timing and its contents should be chosen carefully.

4.9. Sampling times

The duration of blood or urine sampling in a study should be sufficient to account for at least 80% of the known area under curve (AUC) to area under curve to infinity (AUC_∞). This period is usually at least three times the terminal half-life of the drug. To permit calculation of the relevant pharmacokinetic parameters, from 12 to 18 samples should be collected per subject per dose. An account of inter-subject variability should be used in the placement and number of samples. The exact times at which the samples are taken must be recorded and spaced such that the following information can be estimated accurately (Walker, 2006).

4.10. Sampling of blood or urine

Under normal circumstances, blood should be the biological fluid sampled to measure the concentrations of the drug. In most cases the drug may be measured in serum or plasma; however, in some cases, whole blood may be more appropriate for analysis. If the concentrations in the blood are too minute to be detected and a substantial amount (>40%) of the drug is eliminated unchanged in the urine, then the urine may serve as the biological fluid to be sampled. When urine is collected at the study center, the volume of each sample must be measured immediately after collection and included in the report. Urine should be collected over no less than three times the terminal elimination half-life. For a 24 h study, sampling times of 0–2, 2–4, 4–8, 8–12, and 12–24 h are usually appropriate. Quantitative creatinine determinations of each urine sample are also required. Sometimes the concentration of drug in a fluid other than blood or urine may correlate better with effect. Nevertheless, the drug must first be absorbed prior to distribution to the other fluids such as the cerebrospinal fluid, bronchial secretions, and so on. Thus, for bioavailability estimations, blood is still to be sampled and assayed (Walker, 2006).

4.11. Test and reference drug products

Drug and reference materials must be of high quality and mention must be made in the study documentation (report) of the dosage and strength of the drug and what reference product is used in the study. In bioequivalence studies, the molar equivalent dose of each product should be used. The lots for comparative bioavailability testing should be taken from a batch that is comparable in size and is produced using the same type of equipment and procedures proposed for the market. In other words, the lots for comparative bioavailability testing should be representative of proposed production batches. For an uncomplicated drug in which the proportions of excipients to the drug and the dissolution characteristics are the same, it is sufficient to establish the bioavailability of one strength. Whether all strengths of other products should be tested will depend on the extent to which the formulation differs among strengths. For some of the complicated drugs-such as those with narrow therapeutic ranges, steep dose–response characteristics, or with non-linear kinetics that would influence a single-dose study, the bioavailability of each strength of the drug should be established.

4.11.1. Selection of reference product

For a new drug substance (i.e., the first market entry), an oral solution should be used as the reference product when possible. The oral solution can be prepared from an intravenous solution, if available. In bioequivalence studies, the reference product is a drug product that has been issued a notice of compliance pursuant to section C.08.004 of the Food and Drug Regulations, and is currently marketed in Canada by the innovator, a drug product acceptable by the director. Table 1 includes documents required to file ANDA and Table 2 contains contents to be considered while preparing protocol synopsis.

Table 1.

Document requirements to file ANDA.

Title page	1. Study title
	2. Name of sponsor
	3. Name and address of the sites where the clinical and analytical aspects of the studies were carried out
	4. Name, address of the investigator(s)
	5. Name, address of the clinical investigator(s)
Table of contents
(I) Study resume	1. Name, and signature of the investigator(s)
	2. Name, and signature of the clinical investigator(s)
	3. Products’ information including names and batch numbers of the reference and test products compared and the source of the reference product
	4. Summary of bioequivalence study
	5. Summary of bioequivalence data
	6. Figure of mean plasma concentration–time profile
	7. Figure of mean cumulative urinary excretion
	8. Figure of mean urinary excretion rates
(II) Clinical study	9. Introduction
	10. Summary of the study
	11. Details of the study
	12. Demographic characteristics of the subjects
	13. Subject assignment in the study
	14. Details of clinical activity
	15. Details of a justification for, any deviations from the protocol, drop-outs, withdrawal from the study
	16. Form for theoretical sampling frequency and actual sampling times
	17. Adverse reactions report
(III) Assay methodology and validation	18. Assay method description
	19. Validation procedure
	20. Summary of validation
	21. Data on linearity of standard samples
	22. Data on inter-day precision and accuracy
	23. Data on analyte(s) stability
	24. Figure for standard curve(s) for low/high ranges
	25. Representative chromatograms demonstrating the specificity and sensitivity of the assay including chromatograms of LLOQ and blanks (Copies of all the chromatograms should not be included but must be available to be supplied upon request)
	26. Sample calculation
(IV) Pharmacokinetic parameters	27. Definition and calculations of the pharmacokinetic parameters
	28. Individual and average pharmacokinetic parameters
	29. Drug levels at each sampling time and pharmacokinetic parameters
	30. Figure of mean plasma concentration–time profile (presented as both linear–linear and log–linear graphs)
	31. Figures of individual subject plasma concentration–time profiles (presented as both linear–linear and log–linear graphs)
	32. Figure of mean accumulative urinary excretion
	33. Figures of individual subject cumulative urinary excretion
	34. Figure of mean urinary excretion rates
	35. Figures of individual subject urinary excretion rates
	36. Tables of individual subject data arranged by drug, drug/period, drug/sequence
(V) Statistical analyses	37. Details and results of statistical analysis
	38. Summary of statistical significance
	39. Summary of statistical parameters
	40. Analysis of variance
	41. Parametric and/or nonparametric 90% confidence intervals (lower limit, upper limit and point estimate)
	42. Two one-sided t-tests (lower limits, upper limits of the calculated test statistics and the tabulated t-value)
(VI) Protocol including the criteria for inclusion, exclusion or removal of subjects
(VII) Informed consent
(VIII) Appendices	43. Randomization schedule
	44. Analytical raw data
	45. Medical record and clinical reports
(IX) In vitro testing	46. Dissolution testing
	47. Dissolution assay methodology
	48. Content uniformity testing
	49. Potency determination

Table 2.

Considerations for preparing protocol synopsis (Chen, 2011).

Contents	Description required
Trial title and protocol number/code	Provide the title and protocol number/code of the trial. The version number of the protocol should also be provided
Background and rationale	A brief, concise introduction into the clinical problem and previous treatments and developments, i.e., pertinent data from previous preclinical/clinical pharmacology studies and therapeutic exploratory studies. Information on the new drug. Reasoning and justification for the proposed new approach
Trial objectives	Statement of the precise goal(s) of the trial (may be subdivided into primary and secondary objectives) which may including testing of the null hypothesis (Ho), i.e., testing a new drug population/ indication etc., as applicable
Study design and duration	1. The statement of study design should include the method of randomization, blinding and the comparative agent, if applicable
	2. A “Brief outline of the study conduct” should be included, if applicable
	3. The design of the study should be able to support any claims related to the proposed study
	4Total study duration (anticipated starting/finishing dates)
	5. Duration for each subject including post treatment period etc.
Sample size	Rationale and calculation for sample size requirement, anticipated drop-out rate etc. The sample determination may include Ho testing and desired power of the study.
Patient population	Description of specific characteristics of the trial participants (e.g. diseases/stage/indication/condition/treatment etc.) as applicable and of diagnostic criteria and assessment
Inclusion and exclusion criteria	Enumeration of conditions determining participation in the proposed clinical trial
Drug formulation	Brief description of the study drug(s) and formulation to be used in the clinical trial also include disclosure of the formulation intended to be marketed and/or any bridging studies which may be necessary, planned, initiated and/or already performed if different formulations have been used during clinical development
Dosage regimen	Rationale for dose selection. Description of the schedule(s) for using the study drug(s) including escalations/maintenance/reductions/discontinuation, as applicable. Description of other supportive measures and dose modifications for specific adverse events (anticipated toxicities), as applicable
Washout Period	Description for pre-dose, during and post-trial, as applicable
Pre-study screening and baseline evaluation	Description of the process of clinical validation for participation in the clinical trial, including methodology/schedule of events
Treatment/assessment visits	Schedule of all events/visits/procedures during the clinical trial
Concomitant medication	Enumeration and description of all disallowed/allowed drug/medications, in addition to the study drugs
Rescue medication and risk management	Description of available supportive measures/antidotes/medications/dosages/procedures (including follow-up) used to help reverse untoward effects or lack of efficacy resulting from any applications of drug(s)/procedures in connection with the clinical trial. This should include any risks, for example, dose dumping from slow release formulations or immunogenicity
Premature withdrawal/discontinuation criteria	Enumeration of all conditions/criteria and management for drug/patient’s withdrawal or Premature discontinuation, including voluntary withdrawal by subject without prejudice to future treatment by the physician
Efficacy Variables and Analysis	Description and validation of primary endpoint(s), i.e. responses/changes from baseline over time in relation to clinical trial events. Description and validation of related secondary endpoint(s) following from clinical trial events
Safety variables and analysis	Monitoring/assessing adverse drug reactions/adverse events/toxicities/clinical laboratory parameters etc. In relation to clinical trial events
Statistical analysis	1. Analysis of trial parameters (primary/secondary endpoints), population, demographics, as applicable
	2. Efficacy analysis methods and results of efficacy end-point analysis
	3. Safety analysis methods and results of safety end-point analysis
	4. Exploratory end-point analysis: evaluation effect(s) (or lack of effects) of relevant biochemical/pharmacological etc. Parameters, as applicable
	5. Pharmacokinetic endpoint analysis, as applicable. interim analysis and role of data safety monitoring board, as applicable

5. Format and content of the report on bioequivalence studies to be submitted

The report of a bioequivalence study should give the complete documentation of its protocol, conduct and evaluation complying with the declaration of Helsinki and Good Clinical Practice (GCP) rules. Studies must be approved by an independent ethics committee or institutional review board. The responsible investigator(s) should sign for their respective section of the report. Name and affiliations of the responsible investigator(s), site of the study and period of its execution should be stated. The names and batch numbers of the pharmaceutical products used in the study as well as the composition(s) of the test product should be given. The analytical validation report should be attached. Results of in vitro dissolution tests should be provided. In addition, the applicant should submit a signed statement confirming the identity of the test product with the pharmaceutical product, which is submitted for registration. All results should be presented clearly. The procedure for calculating the parameters used (e.g., AUC) from the raw data should be stated. Deletion of data should be justified. If results are calculated using a pharmacokinetic model (although not preferred), the model and computing procedure used should be justified. Individual plasma concentration/time curves should be drawn on a linear/linear scale. All individual data and results should be given, including those of eventually dropped-out subjects. Drop-out and withdrawal of subjects should be reported and accounted for. Test results of representative samples should be included. The statistical report should be sufficiently detailed, so as to enable the statistical analyses to be repeated if necessary. If the statistical methods applied deviate from those specified in the trial protocol, the reasons for the deviations should be stated. The following is a proposed format and contents of an in vivo bioequivalence study submission and accompanying in vitro data (Mugglestone, 2012) (Table 3).

Table 3.

Comparative assessment of study parameters between USA, Europe and Canada (Borgherini, 2003; Nation, 1994; Galgatte et al., 2013).

Sr. no.	Parameters		USA	Europe	Canada
1	Study design		Non-replicated, randomized, crossover studies	Non-replicated, randomized, crossover studies	Non-replicated, randomized, crossover studies
2	Fasting/fed state studies		Fasting and fed	Fasting	Fasting
3	Volunteers		Sufficient to achieve adequate power	>12 (Min 80% power of acceptance criteria)	Min 80% power of acceptance criteria
4	Study dose	Test	Made by the manufacturer	Made by the manufacturer	Made by the manufacturer
		Reference	Reference listed drug in USA	European reference product	Canadian reference product
5	Sampling points		12–18 samples, more samples should be collected at Tmax, to be continued up to 3 or more half lives	At least 2 samples before expected Tmax, 3–4 terminal log–linear phase	12–18 samples per subject/dose
6	Analytical method validation parameters		Accuracy, precision, selectivity, sensitivity, reproducibility, calibration curve, LLOQ and stability	Accuracy, precision, repeatability, intermediate precision specificity, detection. limit of quantitation, linearity range	Stability, L.O.Q, specificity, recovery, standard curves, precision and accuracy
7	Moieties to be measured in plasma		Active drug/metabolites if applicable	Active drug/metabolites if applicable	Active drug/metabolites if applicable
8	Pharmacokinetic parameters		Cmax, Tmax, AUC0–t and AUC0–∞, t1/2, λz	Cmax, Tmax, AUC0–t and AUC0–∞, t1/2, λz	Cmax, Tmax, AUC0–t and AUC0–∞, t1/2, λz
9	Criteria for bioequivalence		90% CI 80.00–125.00% for Cmax, AUCt, AUC0–∞	90% CI 80.00–125.00 for Cmax, AUCt, AUC0–∞ (for highly variable drugs 75–133%)	90% CI 80–125% for Cmax, AUCt, AUC0–∞
10	GCP requirements		ICH GCP guidelines	ICH GCP guidelines	ICH GCP guidelines

5.1. United States

The FDA considers two products bioequivalent if the 90% CI of the relative mean C_max, AUC_0–t and AUC_0–∞ of the test (e.g. generic formulation) to reference (e.g. innovator brand formulation) should be within 80.00–125.00% in the fasting state. Although there are a few exceptions, generally a bioequivalent comparison of Test to Reference formulations also requires administration after an appropriate meal at a specified time before taking the drug, a so called “fed” or “food-effect” study. A food-effect study requires the same statistical evaluation as the fasting study.

5.2. Europe

According to European regulations, European medicines agency (EMEA-CPMP), Note for guidance on the investigation of bioavailability and bioequivalence, London, July 2001 CPMP/EWP/QWP/1401/98 two medicinal products are bioequivalent if they are pharmaceutically equivalent or pharmaceutical alternatives and if their bioavailability after administration in the same molar dose are similar to such a degree that their effects, with respect to both efficacy and safety, will be essentially the same. This is considered demonstrated if the 90% confidence intervals (90% CI) of the transformed natural log ratios, between the two preparations, of C_max and AUC lie in the range of 0.80–1.25.

5.3. Canada

Health Products and Food Branch guidance document provides information about how to establish and conduct bioequivalence studies for conventional formulations of oral drugs that are used for systemic effects. This guidance takes into account a variety of physicochemical, pharmacokinetic, and clinical characteristics of different drugs and drug products, in addition to the availability of methods for measuring a drug and its metabolites. The 90% confidence interval of the relative mean AUC_t and C_max of the test to reference product should be within 80–125%.

6. Conclusion

Comprehensive assessment of study parameters between USA, Europe and Canada revealed that these three countries follow ICH GCP guidelines. Moieties to be measured in plasma pharmacokinetic parameters and criteria for bioequivalence are the same. Due to harmonization of regulatory requirements for bioequivalence studies, major changes in policies and procedures concerning the determination of bioavailability and bioequivalence took place. Therefore in the near future one may expect appropriate choice of a clinically relevant bioequivalence range based on therapeutic ranges, absorption profile and ultimately experimental design to resolve the issue of intra and inter subject variability.