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Guidance for Industry: Conduct and Analysis of Bioavailability and Bioequivalence Studies - Part B: Oral Modified Release Formulations

1996-11-10
ISBN: 0-662-25235-7
Catalogue No.: H42-2/73-1996E

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Foreword

Guidance documents are meant to provide assistance to industry and health care professionals on how to comply with the policies and governing statutes and regulations. They also serve to provide review and compliance guidance to staff, thereby ensuring that mandates are implemented in a fair, consistent and effective manner.

Guidance documents are administrative instruments not having force of law and, as such, allow for flexibility in approach. Alternate approaches to the principles and practices described in this document may be acceptable provided they are supported by adequate scientific justification. Alternate approaches should be discussed in advance with the relevant program area to avoid the possible finding that applicable statutory or regulatory requirements have not been met.

As a corollary to the above, it is equally important to note that Health Canada reserves the right to request information or material, or define conditions not specifically described in this guidance, in order to allow the Department to adequately assess the safety, efficacy or quality of a therapeutic product. Health Canada is committed to ensuring that such requests are justifiable and that decisions are clearly documented.

This document should be read in conjunction with the accompanying notice and the relevant sections of other applicable guidances.

Table of Contents

  1. Introduction
  2. Types of Studies and Data Required for Approval
  3. Standards for Comparable Bioavailability (Group II Drug Products)
  4. Standards for Bioequivalence (Group III Drug Products)
  5. Planning a Bioavailability Study
  6. Selection of Subjects for a Study
  7. Study Design and Environment
  8. Test and Reference Drug Products
  9. Measurement Methodology
  10. Analysis of Data
  11. Sample Analysis for a Single-Dose Comparative
  12. Sample Analysis for a Multiple-Dose Comparative Bioavailability Study
  13. Sample Analysis for a Single-Dose Bioequivalence Study
  14. Sample Analysis for a Multiple-Dose Bioequivalence Study
  15. Glossary of Terms

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1 Introduction

This guidance document provides information about how to establish and conduct bioavailability studies for modified-release (MR) dosage forms of oral drugs that are used for systemic effects. The information contained in this guidance document deals with drugs that have uncomplicated characteristics (as described in Conduct and Analysis of Bioavailability and Bioequivalence Studies: Part A-Oral Dosage Formulations Used for Systemic Effects).

Separate guidances and standards are required for each of the circumstances in which an MR formulation might be developed. These circumstances fall into three groups:

  • Group I-includes original MR dosage forms when the drug is not marketed in either a conventional-release or MR dosage form.
  • Group II-includes first market entry MR dosage forms that are developed after a conventional-release or a different kind of MR formulation is marketed.
  • Group III-includes second and subsequent market entry MR dosage forms that are developed to be bioequivalent to marketed MR dosage forms. This guidance mainly addresses Groups II and III.

Each of the remaining sections of this guidance document deals with a particular aspect of a bioavailability study:

  • Section 2, "Types of Studies and Data Required for Approval", covers the types of studies and data required for approval.
  • Section 3, "Standards for Comparable Bioavailability", describes the standards used for determining bioavailability.
  • Section 4, "Standards for Bioequivalence", describes the standards used for determining bioequivalence.
  • Section 5, "Planning a Bioavailability Study", presents the procedures to be followed during the planning stage of the study.
  • Section 6, "Selection of Subjects for a Study", discusses methods for choosing subjects for a study.
  • Section 7, "Study Design and Environment", describes the study design and the environment in which a study should be conducted.
  • Section 8, "Test and Reference Drug Products", discusses the dosages and strengths of test and reference drug products used in a study.
  • Section 9, "Measurement Methodology", explains the measuring of active substances, or metabolites, or both over time, as well as the validation requirements for analytical methods.
  • Section 10, "Analysis of Data", describes the analysis of recorded data.
  • Section 11, "Sample Analysis for a Single-Dose Comparative Bioavailability Study", contains sample analyses and calculations of data from a single-dose study of comparable bioavailabilities for
    Group II formulations.
  • Section 12, "Sample Analysis for a Multiple-Dose Comparative Bioavailability Study", contains sample analyses and calculations of data from a multiple-dose study of comparable bioavailabilities for Group II formulations.
  • Section 13, "Sample Analysis for a Single-Dose Bioequivalence Study", contains sample analyses and calculations of data from a single-dose study of bioequivalence for Group III formulations.
  • Section 14, "Sample Analysis for a Multi-Dose Bioequivalence Study", contains sample analyses and calculations of data from a multiple-dose study of bioequivalence for Group III formulations.
  • Section 15, "Glossary of Terms", provides definitions of important terms.

Information about establishing and conducting bioavailability studies for conventional formulations of oral drugs is provided in the guidance document Conduct and Analysis of Bioavailability and Bioequivalence Studies: Part A-Oral Dosage Formulations Used for Systemic Effects.

1.1 Definition of Modified-Release Dosage Forms

Modified-release dosage forms are drug formulations that differ from conventional formulations in the rate at which the drug is released. For the purpose of these guidances, MR forms include formulations designed to meet one or more of the following objectives:

  • To delay disintegration, de-aggregation, or dissolution so that the drug's rate of degradation is altered.
  • To delay or decrease the rate of absorption so that the likelihood of gastrointestinal or other adverse effects is diminished (e.g., enteric-coated forms).
  • To provide effective drug concentrations for a longer period of time after a single dose.
  • To deliver the drug initially at a rate similar to that obtained with the conventional form, and to provide effective drug concentrations for a longer period of time.
  • To minimize fluctuations in drug concentrations during the dosing interval.
  • To provide, after single administration, multiple peaks and troughs in the serum concentration-time curves similar to those achieved after repeated dosing with the conventional formulation.

MR formulations require guidances and standards that differ from those for conventional drug formulations because:

  • a greater likelihood exists that increased inter-subject variability in bioavailability will occur, including the possibility of dose-dumping;
  • an increased risk of adverse effects such as gastrointestinal irritation may exist; depending on the site of drug release, or absorption, or both; and
  • a possibility exists that accumulation may occur when the drug is given in repeated doses at the recommended dose intervals.

2 Types of Studies and Data Required for Approval

Bioavailability data must be obtained for all modified-release forms. However, the types of studies required and the pharmacokinetic parameters to be evaluated differ depending on the product. Factors to be considered include:

  • Whether the drug product is the original entry of the chemical entity, or the first or second market entry of the modified-release formulation.
  • The extent to which the drug accumulates after repeated dosing.
  • The potential for adverse reactions-especially from drugs with a steep dose-response curve and those with potential organ toxicity.

Appropriate clinical studies must support claims for the effectiveness and safety of original and first market entry MR formulations (Groups I and II). For second and subsequent market entry MR preparations (Group III) that have been shown to be bioequivalent with the original or first market entry MR form, plasma concentrations alone may form the basis of an approval for the new product. Exemption from clinical trials may also be given for first market entry (Group II) MR products composed of certain non-prescription drugs; however, manufacturers should clear this approach with Health Canada (HC).

2.1 Delayed-Release Drug Products

In the case of enteric-coated drugs, comparative bioavailability can usually be demonstrated using the AUC and Cmax requirements for uncomplicated drug formulations, provided that the only difference between the enteric-coated formulation and the corresponding immediate-release product is a time shift in the concentration-time curve (i.e., no other modification of release occurs). Studies must be carried out using both subjects that have fasted and those that have eaten an appropriate meal at a specified time before taking the drug. The reference product for Group III is to be the innovator's enteric-coated product (or the market leader's enteric-coated product if there is no recognized innovator).

2.2 Market Entry

If the MR formulation is the original market entry of the chemical substance (Group I), selected pharmacokinetic parameters must be determined as part of demonstrating the product's efficacy and safety.

If the MR formulation is a first-entry product, and a conventional-release formulation is already marketed (Group II), then comparable bioavailability should be demonstrated. The studies should be generally pursued in the context of demonstrating the efficacy and safety of the recently-developed drug product and should use an appropriate reference formulation. The investigations should show that the product meets the standards outlined in (Section 3, "Standards for Comparable Bioavailability").

If the MR formulation is a second or subsequent market entry product
(Group III), developed while marketed MR product(s) are already available, then bioequivalence studies using an appropriate reference product must be performed. The studies should show that the product meets the standards outlined in (Section 4, "Standards for Bioequivalence"). When the standards in (Section 4) are not met, full clinical studies are required to support claims as prescribed for Group II products.

2.3 Single-Dose Studies

For the assessment of first market entry and second market entry MR formulations, studies must be performed using single-dose administration, both in subjects that have fasted and in subjects that have eaten a meal standardized to challenge the formulation. The following pharmacokinetic parameters should be calculated from the concentrations in plasma (or blood or serum):

  • AUCX
  • AUCτ
  • AUCI
  • Cmax
  • Tmax
  • λ

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2.4 Multiple-Dose Studies for Formulations Likely to Accumulate

For formulations that are likely to accumulate (i.e., AUCX / AUCI < 0.8), safety requires that steady-state studies be performed in addition to the single-dose studies. The following pharmacokinetic parameters should be calculated from the concentrations in plasma (or blood or serum):

  • AUCτ
  • Cmax
  • Tmax
  • Cpd
  • Cmin
  • fluctuation

Where the AUCX ÷ AUCI ratio cannot be reliably determined, accumulation must be assumed to occur.

3 Standards for Comparable Bioavailability (Group II Drug Products)

3.1 General Requirements

The standards described below must be met for parameters calculated from the observed concentrations, as well as those corrected for measured drug content.

The pharmacokinetic characteristics should support the claims of the manufacturer that appear on the label.

3.2 Single-Dose Studies

3.2.1 Design of Studies

A single dose study should be a comparison between a single dose of the first market entry MR formulation and the doses of the innovator's conventional formulation that the MR formulation is intended to replace. (The doses of the conventional formulation are administered according to the conventional dosing regimen.) When identical doses of conventional and MR formulations cannot be administered, a proportionality correction must be made for the calculation of relevant parameters.

One objective of these studies is to evaluate comparable bioavailability under fasting conditions. However, safety of the subjects may require that an investigation be conducted after the administration of a standardized meal to challenge the formulation. In this case, manufacturers should consult with Health Canada before undertaking the study.

Another objective of these studies is to compare the bioavailability of the test drug product observed under fed and fasting conditions. Such a comparison contributes to the appropriate labelling of the MR drug product.

These objectives can be achieved by applying one of the following design schemes:

  1. A four-period cross-over trial with four complementary sequences of four treatment conditions. In the four treatment conditions, both the test and reference drug products should be assessed in the fasting state as well as following the administration of a standardized meal to challenge the formulation.
  2. A three-period cross-over trial with three complementary sequences of treatment conditions. Both test and reference drug products should be assessed in the fasting state. In addition, the test formulation should be evaluated following the administration of a standardized meal to challenge the formulation.
  3. Two cross-over trials, both with two periods and two sequences of drug product administration. One trial should compare the test and reference formulations in the fasting state. The other trial should evaluate the test drug product, both in the fasting state and following the administration of a standardized meal to challenge the formulation.

3.2.2. Relative Mean AUC

The relative mean measured AUC of the modified-release formulation to the conventional formulation should be between 80% and 125% in the fasting state.

The AUC may be evaluated by determining AUCτ, provided that AUCT obtained by the linear trapezoidal rule is at least 80% of the extrapolated AUCI (i.e., AUCτ ÷ AUCI ≥ 0.80).

3.2.3 Relative Mean Cmax

The relative mean measured Cmax of a single dose of the modified-release formulation to the conventional formulation should not exceed 125% in the fasting state. The maximum concentration measured with a single dose of the MR formulation should be compared with the largest peak concentration recorded following repeated administration of the conventional formulation. For the conventional formulation, the same peak must be selected for all subjects.

In some cases, the intended use of the MR formulation may call for a modification of the above Cmax criterion. In such cases, manufacturers should consult with Health Canada before undertaking the study.

3.3 Steady-State Studies for Formulations Used at a Dose Interval Likely to Lead to Accumulation (AUCX / AUCI < 0.8 for the MR Product)

3.3.1 Design of Studies

In addition to the single-dose studies described in the Section 3.2, a comparison should be made between the first market entry MR formulation and equivalent doses of the conventional formulation over the dosing interval (claimed for the MR product) at steady state.

Generally, steady-state studies should be performed under fasting conditions. However, safety of the subjects may require that an investigation be conducted after the administration of an appropriate meal at a specified time before taking the drug. In this case, manufacturers should consult with Health Canada before undertaking a study.

3.3.2 Relative Mean AUC

The relative mean AUCτ at steady state of the modified-release formulation to the conventional formulation should be between 80% and 125%.

3.3.3 Relative Mean Cmax

The relative mean measured Cmax at steady state of the modified-release formulation to the largest peak concentration of the conventional formulation should not exceed 125%.

4 Standards for Bioequivalence (Group III Drug Products)

4.1 General Requirements

The standards described below must be met for parameters calculated from the observed concentrations, as well as those corrected for measured drug content.

The pharmacokinetic characteristics should support the claims of the manufacturer that appear on the label.

Other standards may be required to compare the shapes of the plasma concentration versus time curves.

4.2 Single-Dose Studies

4.2.1 Design of Studies

A second or subsequent market entry MR formulation should be compared with the Group I or II MR product with which bioequivalence is claimed. Both formulations should be administered as single doses.

The objective of these studies is to evaluate the bioequivalence of the test and reference drug products under both fasting and fed conditions. However, safety of the subjects may require that an investigation be conducted after the administration of an appropriate meal at a specified time before taking the drug. In this case, manufacturers should consult with Health Canada before undertaking the study.

This objective can be achieved by applying one of the following design schemes (A is the most informative, C the least):

  1. A four-period cross-over trial with four complementary sequences of four treatment conditions. Both the test and reference drug products should be assessed in the fasting state as well as after the administration of an appropriate meal at a specified time before taking the drug.
  2. Two cross-over trials. The first trial should compare the test and reference drug products under fasting conditions. The drugs should be administered during two periods and with two sequences of treatment conditions. The second trial should compare the test and reference formulations following the administration of an appropriate meal at a specified time before taking the drug, as well as the test formulation under fasting conditions. The trial should be conducted with three periods and three complementary sequences of drug administrations.
  3. Two cross-over trials, both with two periods and two sequences of test and reference drug product administration. One trial should be conducted in the fasting state. The other trial should be conducted after the administration of an appropriate meal at a specified time before taking the drug.

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4.2.2 Relative Mean AUC

The 90% confidence interval of the relative mean AUC of the test to reference formulation should be within 80% to 125% in the fasting state and after the administration of an appropriate meal at a specified time before taking the drug.

AUC may be evaluated by determining AUCτ, provided that AUCτ obtained by the linear trapezoidal rule is at least 80% of the extrapolated AUCI
(i.e., AUCτ ÷ AUCI ≥ 0.80).

4.2.3 Relative Mean Cmax

The relative mean measured Cmax of the test to reference formulation should be between 80% and 125% in the fasting state and after the administration of a standardized meal to challenge the formulation.

4.3 Steady-State Studies for Formulations Used at a Dose Interval Likely to Lead to Accumulation (AUCX / AUCI < 0.8 for the MR Product)

4.3.1 Design of Studies

A second or subsequent market entry MR formulation should be compared with the Group I or II MR product with which bioequivalence is claimed. Both formulations should be administered at steady state. This comparison should be conducted in addition to the single- dose studies described in (Section 4.2).

Generally, steady-state studies should be performed under fasting conditions. However, safety of the subjects may require that an investigation be conducted after the administration of an appropriate meal at a specified time before taking the drug. In this case, manufacturers should consult with Health Canada before undertaking the study.

4.3.2 Relative Mean AUCτ

The 90% confidence interval of the relative mean AUCτ of the test to reference formulation should be within 80% to 125%.

4.3.3 Relative Mean Cmax and Cmin

The relative mean measured Cmax at steady state of the test to reference formulation should be within 80% to 125%.

The relative mean measured Cmin at steady state of the test to reference formulation should not be less than 80%.

5 Planning a Bioavailability Study

This section identifies the documentation that must be prepared when a bioavailability study is planned. Descriptions of the study objectives, principal and other investigators, facilities, and ethical review boards must be included in the reports that follow or accompany each bioavailability study.

5.1 Study Objectives

The objectives of the bioavailability study should be clearly stated, together with the therapeutic rationale for, and the pharmacokinetic objectives of, the modified-release formulation. Information should be provided to justify why the drug is included in the category of modified-release drugs without complicated characteristics.

5.2 Principal and Other Investigators

The identity and duties of the individuals who are responsible for the study, and for the safety of the subjects participating in the study, must be provided. * Co-investigators, including those responsible for the clinical component of the study, the drug measurements, and the statistical analyses, must be identified. A curriculum vitae for each investigator must be obtained and appended to the study documentation.

*Guidelines on Research Involving Human Subjects, Medical Research Council of Canada, 1987.

5.3 Clinical, Laboratory, and Analytical Facilities

The location of all facilities should be identified and their suitability demonstrated. "Suitability" is determined with respect to the physical plant and the capability of a facility involved in experiments using human subjects or in the analysis of biological samples. These aspects of the facility should conform to current requirements for Good Clinical Practice or Good Laboratory Practice.*

5.4 Institutional (Ethical) Review

Documentary evidence that the study has been approved by an appropriate institutional ethical review board must be provided. The current guidelines of the Medical Research Council of Canada (MRC)** or a comparable agency should be used for such a review.

The guideline used for the ethical review should be identified in the documentary evidence for the study.

The reimbursement policy should be specified before initiating the study, and should be in agreement with MRC or similar guidelines.

* Source: Proposed Rule Obligations of Clinical Investigators, Federal Register, 43, 35210, (1978) and Code of Federal Regulations, 21, Part 58 (revised April 1988).

** Guidelines on Research Involving Human Subjects, Medical Research Council of Canada, 1987.

6 Selection of Subjects for a Study

This section describes the selection criteria for including subjects in a bioavailability study. This section also identifies how the characteristics of the subjects may affect the study.

6.1 Choice of Subjects

Modified-release dosage forms of drugs with uncomplicated characteristics can usually be tested in normal, healthy volunteers. Investigators should ensure that female volunteers are not pregnant or likely to become pregnant during the study. Confirmation should be obtained by pregnancy tests conducted just before the first and last doses of the study.

Bioavailability studies of a modified-release dosage form in healthy control volunteers may not apply to all circumstances. Where substantial differences in the absorption or disposition of a drug owing to a disease process are known, the more appropriate approach may be to study the product in patients. Examples include:

  • Preparations indicated for use in young children, where rapid clearance may invalidate the label claim of less frequent administration.
  • MR products whose sustained-release effect is dependent on pH changes in the gastrointestinal tract, where the drug is designed for use in patients likely to have achlorhydria.

Generally, such patients will have to be studied under controlled conditions during regular treatment with the drug. The number of samples or level of control of study conditions may therefore be limited. Study design and other important issues should be discussed with Health Canada before initiating studies undertaken in patients.

6.1.1 Blood Volume

In studies of patients where the total volume of blood samples is an issue, the normally stringent criteria governing blood sampling frequency for AUC calculations may have to be modified. Consultation with Health Canada is encouraged if such a modification is made.

6.2 Considerations of Subject Characteristics

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

  1. Age - Subjects should be between the ages of legal majority and the onset of age-associated changes in organic function. This description typically coincides with an age range of 18 to 55 years, inclusive.
  2. Height/weight ratio - The ratio for healthy volunteer subjects should be within 15% of the normal range, (e.g., the range given in current Ciba-Geigy or Metropolitan Life Insurance tables).

An electrocardiogram should be included in the study documentation if the drug has a cardiac effect. Aberrant laboratory values should be double-checked, and a summary must be presented together with the physician's opinion.

Psychological characteristics should also be assessed by the physician so that patients unlikely to comply with study restrictions or unlikely to complete the study can be excluded.

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.

6.3 Number of Subjects

A minimum of 12 subjects should be used and, if a subject withdraws or must be removed from the study, an explanation for the withdrawal or removal must be included in the study documentation.


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6.3.1 Estimating the 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 (see Sections 3 and 4) 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 for the parameters of interest; and
  • the anticipated intra-subject coefficient of variation (CV) of these parameters.

For many drugs with uncomplicated characteristics, the residual CV in the analyses of variance (see Sections 11 through 14) is generally less than 20%. However, as a result of sampling, or if the study is poorly run, the residual CV can be higher.

The minimum number of subjects is 12, but a larger number is often required.

This sample size calculation must be provided in the study protocol. To estimate the number of subjects required, refer to Conduct and Analysis of Bioavailability and Bioequivalence Studies: Part A, (Section 3.3).

6.3.2 Accounting for Drop-outs and Withdrawals

More subjects than the sample-size calculation requires should be recruited into the study. This strategy allows for possible drop-outs and withdrawals.

Two basic methods are used to account for drop-outs and withdrawals. First, a fixed number (one or two for each sequence) of subjects are added to the sample-size number. Second, a fixed number of subjects are added into the study. These subjects are designated as extras. Only if a subject drops out will the appropriate extra subject's blood samples be assayed. The method of accounting for drop-outs and withdrawals must be outlined in the protocol.

6.3.3 Additional Trials

Because of the possibility of random variation or larger-than-expected relative difference, or both, there is no guarantee that a calculated sample size will pass the standards. If the study is run with the appropriate size and the standards are not met, the sponsor may conduct an additional trial with a minimum of 12 subjects. This option, if chosen, must be stated in the study protocol. Two criteria must be met before the combination of results from trials is acceptable (see Conduct and Analysis of Bioavailability and Bioequivalence Studies: Part A):

  • The same protocol must be used (i.e., same formulations, same blood sampling times, a minimum number of 12 subjects, etc.).
  • Consistency tests must be met at an ALPHA error rate of 5%.

6.4 Drop-outs and Withdrawal of Subjects from a Study

The subjects must be available, without coercion, 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. (See Section 6.3, "Number of Subjects".)

Reasons for withdrawal (e.g., adverse drug reactions) must be reported, and the subject's plasma (or blood or serum) levels provided. The results of all samples that were measured in subjects who were withdrawn from the study must be included in the report. If a subject drops out of the study for personal reasons, the individual's blood samples do not have to be assayed.

7 Study Design and Environment

The design of a bioavailability or bioequivalence study should minimize variability that is not attributable to the drug itself and should eliminate bias to any possible extent. The guidances in this section can be used for the usual cases. Other designs may be permissible after consultation with the Health Canada before the study is initiated.

7.1 Standardization

Every effort should be made to standardize the study conditions (e.g., exercise, diet, smoking, and alcohol use) in all phases of the study. It is preferable to use non-smokers; where smokers are included, they must be so identified.

Volunteers should take no other drug, including alcoholic beverages and over-the-counter (OTC) drugs, for an appropriate interval before or during the study. In an emergency, the use of another drug must be reported (i.e., dose and time of administration). The decision to include or exclude the results from a subject who has varied from the established protocol should be made before statistical analysis commences.

7.2 Blinding

If possible, bioequivalence trials should be conducted in such a way that the subjects do not know which product (test or reference) is being administered. Individuals involved in the administration of the drugs, the surveillance of the patients, and checking for adverse reactions should not know which product was administered. Furthermore, in both bioequivalence and comparable bioavailability studies, the person conducting the analysis of samples must not know which product was administered.

7.3 Administration of Food and Fluid

The administration of food and fluid should be carefully controlled. Subjects should fast for at least 10 hours before drug administration. A fast means that no food or fluids are to be consumed, although alcohol-free and xanthine-free clear fluids are permissible the night before the study. On the morning of the study, up to 250 mL of water may be permitted up to two hours before administration of the drug. The dose should be taken with a standard volume of water (e.g., 150 mL) and at a standard temperature. Two hours after administration of the drug, 250 mL of xanthine-free fluids are permitted. Four hours after administration of the drug, a standard meal may be taken. All meals should be standardized and repeated on each study day.

Safety of the subjects may require that an investigation be conducted after the administration of an appropriate meal at a specified time before taking the drug, rather than under fasting conditions. In this case, manufacturers should consult with Health Canada before undertaking the study.

If steady-state studies are required, the food and fluid restrictions noted above should apply on the day the plasma profiles are to be obtained, as well as on the preceding evening. 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.

The nature of the test meal-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 can challenge the formulation (i.e., the meal has the greatest potential to demonstrate altered bioavailability). The meal should be given within a pre-determined, constant time of administration of the drug. The timing and contents of the meal should be chosen carefully.

7.4 Posture and Physical Activity

For most drugs, subjects should not be allowed to recline until at least two hours after ingestion of the drug. Physical activity and posture should be standardized as much as possible to limit effects on gastrointestinal blood flow and motility. The same pattern of posture and activity should be maintained for each study day.

7.5 Interval Between Study Days

The interval between study days should be long enough to permit elimination of essentially all of the previous dose from the body. The interval should be the same for all subjects and, to account for variability in elimination rate between subjects, should normally be not less than 10 times the mean terminal half-life of the drug (generally, the interval between study days should not exceed four weeks). In addition, the drugs must be administered at approximately the same time on each study day and, where possible, the same day of the week.

7.6 Sampling Times

The duration of blood or urine sampling in a study should be sufficient to account for at least 80% of the known AUC to infinity (AUCI). This period is at least three times the terminal half-life of the drug.

To permit calculation of the relevant pharmacokinetic parameters, 12 to 18 samples should be collected per subject per dose. The inter-subject variability should be taken into account 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 and precisely:

  1. peak concentration of the drug in the blood (Cmax);
  2. when appropriate, the AUCτ should be at least 80% of the calculated AUCI; and
  3. the terminal disposition rate constant of the drug.

7.6.1 Estimating the Terminal Disposition Rate Constant

The estimated terminal disposition rate constant may not be precise if only a few points are used for its estimation by linear regression. To reduce this imprecision, four or more points should be determined during the terminal log-linear phase of the curve. If urine is used as the biological sampling fluid (see Section 7.7), sufficient samples must be obtained to permit an estimate of the rate and extent of renal excretion.

7.6.2 Evidence of Steady State

For steady-state studies of drugs with uncomplicated characteristics, at least three consecutive pre-dose concentration levels (Cpd) are required to provide evidence of steady state. Generally, observations of Cpd for the test and reference products should be recorded at the same time of the day. One of these measurements could be taken based on the first sample of the study day in which a profile over the dosing interval is being established. Steady state is usually achieved when repeated doses of a formulation are administered over a period that exceeds five disposition half-lives of the modified-release form.

The number and timing of samples required during steady-state studies are dependent on:

  • the length of the dose interval;
  • timing of meals;
  • the pharmacology and the pharmacokinetics of the drug; and
  • whether the conventional or modified-release form is being studied.

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7.6.3 Other Considerations

The levels at the beginning of the dosing interval and at the end are both required. The number of samples must be sufficient to provide the information required to sustain label claims, to identify Cmax and Cmin, and to calculate AUC over the dosing interval (AUCτ).

For studies involving food consumption, additional sampling may be required during the period when modifications of the serum concentration versus time curve can be anticipated.

7.7 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 (more than 40%) of the drug is eliminated unchanged in the urine, then urine may serve as the biological fluid to be sampled.

The volume of each urine sample must be measured immediately after collection and be included in the report. Urine should be collected over no less than three times the terminal elimination half-life. For a 24-hour study, sampling times of 0 to 2, 2 to 4, 4 to 8, 8 to 12, and 12 to 24 hours are usually appropriate. Quantitative creatinine determinations on each urine sample are also required.

7.8 Handling of Samples

Samples should be processed and stored under conditions that have been shown not to cause significant degradation of the analytes. Appropriate storage conditions should be confirmed with samples from subjects who have been given the drug under study, in case there is evidence that metabolites are likely to interconvert to the parent drug.

7.9 Identification of Adverse Reactions and Side Effects

In some cases, adverse drug reactions result from factors other than the active ingredient in a formulation. The rate of dissolution or absorption, and excipients within formulations, may affect the frequency, onset, and severity of adverse drug reactions. The incidence, severity, and duration of adverse reactions and side effects observed during the study must be reported. The probability that an adverse effect is drug-induced is to be judged by the investigator.

As much as possible, the same observer and format should be used for eliciting and recording information on adverse drug reactions for all subjects. Questions concerning adverse reactions and side effects should be asked on each study day by the "blinded" observer.

For drugs with known adverse reactions and side effects-for example, metallic taste, postural hypotension, cardiac dysrhythmia-the specific questions should be raised, and observations, such as blood pressure measurement and electrocardiogram, should be performed and recorded at the time the reactions are known to occur with respect to the time of administration. In asking the questions, the interviewer should avoid leading the subject to believe that the reactions are expected or unexpected. The subject should be questioned in private.

8 Test and Reference Drug Products

8.1 Chemistry

The products must meet a Schedule B or other applicable standard of identity, quality, purity, and potency acceptable to Health Canada. The chemistry and manufacturing guidances for preclinical and new drug submissions should be consulted for an interpretation of the general technical requirements listed in sections C.08.002(2) and C.08.005(1), respectively.

8.2 Dosage and Strength

In bioavailability studies, the dose that is administered for both the modified-release and conventional-release products should be identical. When identical doses cannot be administered, a proportionality correction must be made for the calculation of relevant parameters.

In bioequivalence studies, the molar equivalent dose of each product should be used. The lots for bioavailability and bioequivalence testing should be taken from a batch that is comparable in size and is produced using the same type of equipment and procedures that are proposed for market. In other words, the lots for bioavailability and bioequivalence testing should be representative of proposed production batches.

8.2.1 Strength

For an uncomplicated drug in which the proportions of excipients to the drug and the dissolution characteristics are the same across all strengths, it is sufficient to establish the bioavailability of one strength. Whether all of the strengths of other products should be tested will depend on the extent to which the formulation differs among the various strengths.

8.2.2 Proportional Dose Claims

When a modified-release product in the form of a scored tablet possesses the claim that a portion of the tablet may be administered to provide a proportional dose, evidence must be presented to justify the claim.

8.3 Selection of Reference Product

8.3.1 New Active Substance

For a first market entry new active substance (Group I), an oral solution should be used as the reference product, when possible. The oral solution can be prepared from an intravenous solution, if one is available. In the case of insoluble drugs, the reference product should be as agreed with Health Canada.

8.3.2 Marketed Products

The first modified-release (Group II) product must be compared with the marketed conventional product.

For bioequivalence studies (Group III), the reference product must be a drug product marketed in Canada by the innovator, the licensee, or the market leader (if there is no recognized innovator). For bioequivalence studies, the reference product must be the product with which bioequivalence is claimed.

9 Measurement Methodology

Bioavailability determinations rely on the adequacy of the analytical methods used for the parent drugs and, when appropriate, the metabolites of those drugs. This section describes the attributes of such methods and the validation procedures required in reports to establish and maintain selectivity, range, precision, and accuracy.

9.1 Drug and Drug Metabolites

The determination of bioavailability is dependent on the reliable, precise, and accurate measurement of the active ingredient, or its metabolites, as a function of time. Normally, measurement of the parent compound or active ingredient will be adequate; however, in certain circumstances, the measurement of metabolites may be required. When a pro-drug is administered, the active component should be measured.

9.2 Assay Methodology

The analytical methods used to measure the drug, or its metabolites, in plasma, blood, serum, or urine must be reproducible, specific, and sufficiently sensitive, precise, and accurate. When these operating parameters have been shown to be adequate in the hands of the test laboratory, the investigators can then undertake the bioavailability study.

The principles and procedures for analytical validation described in the summary document "Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies", V.P. Shah et al (1992), Journal of Pharmaceutical Sciences 81(3) should be followed. In addition to pre-study validation, appropriate performance characteristics (accuracy, precision, quality control) should be documented for each analytical run during a study, according to the principles of Good Laboratory Practice (GLP).

9.3 Stability

For samples to maintain their stability (avoiding degradation of analytes), they should be handled according to the directions outlined in (Section 7.8, "Handling of Samples"). Validation must be included.

9.4 Limit of Quantitation (LOQ)

The analytical method chosen should be capable of assaying the analyte over the expected concentration range. A reliable lowest limit of quantitation (LOQ) should be established, based on an intra-day and inter-day coefficient of variation (CV) not greater than 20%. The limit of detection (LOD)-the lowest concentration that can be differentiated from background levels-is usually lower than the LOQ. Values between LOQ and LOD should be identified as "below quantitation limits".

9.5 Recovery

The reproducibility of the absolute recovery of drug during the sample preparation procedure should be demonstrated for low, medium, and high concentrations, based on the expected range.

9.6 Specificity

It must be demonstrated that endogenous compounds in the biologic matrix, nutrients, metabolites, and degradation products do not interfere with the assay method. In cases where a stereospecific method is used, proof of specificity must be documented. Specificity should be established using at least six independent sources of the same matrix in the species being studied.


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9.7 Standard Curves

A standard curve demonstrates the range of concentrations over which an analyte can be reliably determined in matrix, using a minimum of five concentration points. Standard curves should be run on each day of the analysis. The within-day and between-day variability in the standard curves must be reported, together with the coefficients of variation (CVs) obtained during measurement of the samples. These attributes will be used to determine the acceptability of the standard curve. The number of standards to be used will be a function of the dynamic range and nature of the concentration-detector response relationship. The standard curve should be determined using an appropriate algorithm.

9.8 Precision and Accuracy

The precision and accuracy of the assay must be determined for low, medium, and high drug concentrations in the biological matrix, based on the expected range. Inter-day and intra-day accuracy should be within 15% of the nominal value. For precision, the CV should be no greater than 15%, except at the limit of quantitation, when a value no greater than 20% is acceptable.

9.8.1 Replicate Samples

In general, single-sample analysis will suffice. When single assays are performed, 15% of the incurred samples must be randomly selected and re-assayed. (Studies in which the sample of blood is insufficient for duplicate analysis should include a pre-study verification with incurred samples.)

The second measurement is not to be averaged with the first; only the variation between samples is to be summarized and then reported separately. The purpose of re-assaying is to establish that the degree of precision obtained with incurred samples is similar to that obtained for spiked standard or quality control (QC) samples.

If the proposed method does not have the potential to give the required precision, all samples may be replicated. If this is done, the replicates would be averaged. The variation between samples should be reported separately.

9.9 Quality Control for Spiked Samples

For stable analytes, QC samples must be prepared in the fluid of interest (e.g., plasma), including concentrations at the low, middle, and high segments of the calibration range. The quality control samples must be stored with the study samples. Quality control samples are accepted for stability if they exhibit similar characteristics to those taken from volunteers.

For less stable analytes, daily or weekly QC samples may have to be prepared.

A minimum of six QC samples, composed of three concentrations in duplicate, must be blinded and analyzed with each batch of study samples for each analytical day or run.

9.10 Aberrant Values (Repeat Assays)

In most studies, some blood/plasma/serum or urine samples will require re-assay. Criteria for identifying these samples should be pre-established.

Certain aberrant values can be identified before breaking the analytical code. These values may be attributed to such factors as:

  • processing errors,
  • equipment failure,
  • obviously poor chromatography, or
  • quality control samples outside pre-defined tolerances.

Other apparently aberrant values may become evident after the analytical code is broken. In some cases, the original assay value would show poor pharmacokinetic fit (but this should be applied with caution). In other cases, there may be a need to confirm a double peak. For aberrant values that have become evident after the analytical code is broken, the submission must note the reason for the repeat assay.

When the results of a repeat assay differ from the original by more than 15%, a third analysis should be performed. When three replicate analyses indicate that one is spurious, the average of the other two should be used. The criteria that were used for selection of replicates to be included in the calculations should be stated.

10 Analysis of Data

When all measurements of samples have been completed, the collected information must be analyzed. This section discusses the data that must be recorded, the parameters, the statistical analyses that must be performed on the data, and the format that should be used to present the results in reports.

10.1 Presentation of Data

The concentrations of the drug in plasma (or blood, or serum) for each subject, sampling time, and formulation should be tabulated. Unadjusted, measured concentrations should be provided. All concentrations and sample times should be supplied, for each combination of subjects and formulations, in computer-readable form.*

Deviations from the protocol (e.g., missed samples or late collection of samples) should be clearly identified in the tables.

Two graphs should be drawn for each subject. Two graphs should also be drawn for the mean values of all subjects. In each case, one of the graphs should be linear while the other semilogarithmic. On these graphs, the drug concentrations from the reference and test formulations should be plotted against the sampling times. Natural logarithms (ln) are to be employed. Usually, the semilogarithmic graphs should display the regression lines that are employed to estimate the terminal disposition rate constant (λ ) for the two formulations.

10.2 Pharmacokinetic Parameters

The estimated pharmacokinetic parameters should be supplied, for each combination of subjects and formulations, in computer-readable form. * All parameters should be estimated from measured drug concentrations.

*See the Guidelines on the Preparation of Human Abbreviated Drug Submissions: Bioequivalence Studies Drugs Directorate, 1996

10.2.1 Single-Dose Studies

For single-dose studies, estimates of the following pharmacokinetic parameters must be tabulated for each combination of subjects and formulations:

  1. AUCτ - Area under the concentration versus time curve, measured to the last quantifiable concentration using the linear trapezoidal rule.
  2. AUCX - Area under the concentration versus time curve over the usual dosing interval, 0-X.
  3. AUCI - AUC measurement up to the last quantifiable concentration, plus an additional area extrapolated to infinity, calculated using .
  4. AUCX/AUCI - Ratio of AUC over the dosing interval to the total AUC extrapolated to infinity.
  5. AUCτ/AUCI - Ratio of AUC, measured to the last quantifiable concentration, to the total AUC extrapolated to infinity.
  6. Cmax - Maximum observed concentration.
  7. Tmax - Time at which the observed Cmax occurred.
  8. λ - Terminal disposition rate constant.

10.2.2 Steady-State Studies

For steady-state studies, estimates of the following pharmacokinetic parameters must be tabulated for each combination of subjects and formulations:

  1. Cpd - Pre-dose concentrations determined immediately before a dose at steady state. (Note: to assess whether steady state has been achieved, at least three consecutive pre-dose concentrations, generally at the same time of day, must be determined.)
  2. AUCτ - Area under the concentration versus time curve measured throughout the dosing interval, using the linear trapezoidal rule.
  3. Cmax - Maximum observed concentration.
  4. Cmin - Minimum observed concentration.
  5. Tmax - Time at which the observed Cmax occurred.
  6. (Cmax − Cmin) ÷ (AUCτ ÷ τ) × 100 - Fluctuation (expressed as a percentage) determined as the range of concentrations divided by the average steady-state concentration.

10.2.3 Additional Parameters

Additional pharmacokinetic parameters may also be presented, but the methods used to estimate them should be fully described. The means and coefficients of variation should be given for each parameter and for each formulation.


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10.3 Statistical Analysis

The analysis of variance (ANOVA) tables submitted with the study documentation (report) should include the appropriate statistical tests of all effects in the model. The output from ANOVAs appropriate to the study design and execution must be expressed with enough significant figures to permit further calculations.

Analysis of Tmax,λ , and fluctuation should be carried out on the raw scale, while calculations for AUCX, AUCτ, AUCτ, AUCI, Cmin, Cpd, and Cmax should use the logarithmic (ln) scale.

The analyses should include all data for all subjects. Supplementary analyses may also be carried out with selected points, or subjects, or both, excluded from the analyses. Such exclusions must be justified, and the reasons documented. It is rarely acceptable to exclude more than 5% of the subjects or more than 10% of the data for a single subject-formulation combination.

A summary of results should be reported on a separate page for each parameter as detailed in the samples provided in (Sections 11 and 14). The report should contain:

  1. means and CVs (across subjects) for each product;
  2. the ANOVA;
  3. in single-dose studies: AUCX, AUCτ, and Cmax ratios, Tmax, and λ or in steady-state studies: AUCτ, Cmin, and Cmax ratios and fluctuation differences for test products versus reference products;
  4. the 90% confidence interval about the mean AUCτ, AUCτ, Cmin, Cmax, and fluctuation comparisons; and
  5. a summary of comparisons for AUCX, AUCτ, AUCτ, λ , Cpd, Cmin, Cmax, and fluctuation uncorrected and corrected for measured content of the dosage forms.

11 Sample Analysis for a Single-Dose Comparative Bioavailability Study

The following tables and figures illustrate data collected and used in a sample single-dose comparative bioavailability study. An analysis of this data is also shown.

Although a comparative bioavailability study may include many formulations, the basic analysis is the same - each test formulation is compared to a reference formulation. The analysis of a single-dose comparative bioavailability study must have the following sections:

  1. A randomization scheme for the design, where all subjects randomized into the study are included and identified by code, sequence, and dates of the dosing periods for both test and reference formulations. (See Section 11.1.)
  2. A summary of drug concentrations (visual and quantitative) at each sampling time for each subject for both test and reference formulations. (See Section 11.2.)
  3. A summary of the estimates of the parameters as defined in Section 11.3 for both test and reference formulations, including the means, standard deviations, and CVs. (See Section 11.4.)
  4. A formal statistical analysis of the relevant parameters with comparisons of the test formulations to the reference formulations.
    (See Sections 11.5 through 11.9.)
  5. A summary of corrections for potency (measured content) in estimates. (See Section 11.10.)
  6. Sample concentration-time profiles that should be given for each subject. (See Section 11.11.)

All the sample statistical analyses that follow have a minimum of two formulations (test and reference) given on two dosing days or periods.

11.1 Randomization Scheme of the Design

Shown in Table 11-A is the randomization scheme for the cross-over design used in the study. In any study, all subjects who were randomized into the study must be included. Even those subjects that did not complete the study must be included and identified accordingly. Subject numbers that appear on informed consent forms and reporting forms must be given. Also, if any other subject identification code was used, it should be given here. The sequence to which the subject was randomized should be given. Finally, all dosing periods and dates must be given.

11.2 Summary of Drug Concentrations

Tables 11-B and 11-C show a list of the concentrations at each sampling time for each subject for the test and reference formulations, respectively. If any concentration is missing it should be identified, and the reason it is missing given (e.g., lost sample; sample not collected).

Although no formal statistical analysis is required at each sampling time, it is recommended that summary statistics be given at each sampling time for each formulation. It is also helpful if the limit of quantitation of the analytical method is given in this table.

Table 11-A - Randomization Scheme of the Cross-over Design for the Comparison of Test (T) Versus Reference (R) Formulations
Subject Period
Number ID Sequence Feb. 1, 1989 Feb. 8, 1989
001 A RT R T
002 B TR T R
003 C TR T R
004 D RT R T
005* E TR T -
006 F TR T R
007 G RT R T
008 H RT R T
009 I RT R T
010 J TR T R
011** K TR - -
012 L RT R T
013 M TR T R
014 N RT R T

* Subject did not appear for second period.
** Subject did not appear for either period.

Table 11-B - Drug Concentrations* (µg/mL) for the test Formulations
ID Seq. Per. Sampling Times (hours)
0.0 1.0 2.0 4.0 6.0 8.0 10.0 12.0 13.0 14.0 16.0 18.0 20.0 22.0 24.0 36.0 48.0
A RT 8 Feb. 0.4 5.2 24.4 45.8 42.0 40.1 41.5 42.0 40.7 45.1 42.6 45.5 44.7 42.5 42.3 27.1 18.1
B TR 1 Feb. 0.0 12.8 61.8 65.5 58.3 46.4 49.2 40.1 46.7 47.5 40.1 34.9 32.2 29.2 29.0 13.1 8.3
C TR 1 Feb. 0.0 21.7 49.1 57.0 53.1 45.0 41.6 44.2 47.9 53.6 56.7 61.3 60.3 60.8 66.8 36.7 22.4
D RT 8 Feb. 0.0 19.4 46.9 56.4 57.8 55.5 60.9 45.7 46.1 40.2 38.4 34.2 28.6 24.1 27.0 12.8 7.3
F TR 1 Feb. 0.0 23.7 37.0 39.2 41.9 41.4 51.3 46.1 40.2 38.8 30.3 26.7 24.5 22.2 20.4 11.8 6.5
G RT 8 Feb. 0.3 31.2 54.8 65.7 61.1 57.3 55.8 48.0 48.2 50.6 44.4 43.3 45.7 45.8 46.2 22.9 13.6
H RT 8 Feb. 0.0 23.2 29.4 32.4 43.8 41.9 42.5 38.0 36.0 31.9 24.9 24.0 23.8 22.3 21.1 10.1 6.4
I RT 8 Feb. 0.0 25.0 58.5 63.3 51.4 42.0 47.8 43.6 43.8 36.2 31.9 26.8 35.8 31.8 29.4 25.3 13.7
J TR 1 Feb. 0.0 17.1 28.0 38.1 47.9 42.1 44.8 44.7 41.6 48.2 44.3 39.4 39.3 42.0 43.6 26.3 18.0
L RT 8 Feb. 0.0 17.2 15.0 12.8 20.0 27.5 30.3 28.7 27.0 23.7 25.9 22.7 19.8 19.0 18.2 9.4 6.1
M TR 1 Feb. 0.0 4.9 1.7 36.7 62.6 54.4 47.2 43.1 37.5 37.6 34.0 30.5 27.4 22.8 23.1 11.1 6.9
N RT 8 Feb. 0.5 34.6 52.1 56.3 52.5 47.2 52.7 71.8 76.3 68.9 55.8 53.8 54.8 53.1 51.2 25.2 18.8
Mean – – 0.1 19.7 38.1 47.3 49.4 45.1 47.1 44.7 44.3 43.5 39.1 37.8 36.4 34.6 34.9 19.3 12.2
STD – – 0.2 9.1 18.6 16.2 11.7 8.1 7.9 9.9 11.7 11.6 10.4 11.7 12.8 13.8 15.0 9.0 6.0
CV – – 185g 46.1 48.8 34.2 23.6 18.0 46.8 22.2 11.9 26.7 26.6 31.1 35.2 40.0 43.1 46.4 49.0

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.

Table 11-C - Drug Concentrations* (µg/mL) for the Reference Formulation
ID Seq. Per. Sampling Times (hours)
0.0 1.0 2.0 4.0 6.0 8.0 10.0 12.0 13.0 14.0 16.0 18.0 20.0 22.0 24.0 36.0 48.0
A RT 1 Feb. 0.0 11.9 25.4 60.1 54.1 41.0 34.9 31.7 30.6 31.0 58.7 68.1 62.2 57.1 51.9 28.1 17.4
B TR 8 Feb. 0.0 78.4 53.7 40.4 31.9 29.8 24.6 20.8 27.6 55.8 53.3 46.8 39.4 36.9 36.9 16.9 12.1
C TR 8 Feb. 0.8 89.2 70.5 53.6 43.4 36.8 30.7 27.1 63.3 66.0 64.6 57.5 52.8 47.2 46.8 27.6 17.6
D RT 1 Feb. 0.0 110.7 71.6 52.0 47.2 36.8 31.9 27.7 88.5 89.7 70.5 64.4 52.4 44.4 40.8 18.8 10.5
F TR 8 Feb. 0.0 54.5 54.4 39.2 32.9 29.9 25.6 22.2 75.1 69.8 51.3 42.2 34.4 29.8 29.9 15.3 10.8
G RT 1 Feb. 0.0 33.2 72.6 64.7 47.3 40.4 33.5 28.9 26.2 24.2 67.4 63.2 60.3 51.4 50.5 21.7 12.2
H RT 1 Feb. 0.0 69.3 66.9 46.3 35.2 31.2 25.1 24.4 44.2 73.5 57.7 49.6 40.8 36.0 34.5 15.0 9.1
I RT 1 Feb. 0.0 49.3 65.1 74.6 56.8 44.6 37.2 32.1 29.2 46.7 63.4 38.0 69.7 59.2 54.6 25.1 14.6
J TR 8 Feb. 0.3 5.9 15.9 61.0 44.0 33.3 32.7 27.4 25.8 73.6 63.8 53.3 49.3 41.2 40.2 18.4 13.7
L RT 1 Feb. 0.0 44.3 83.0 57.8 47.9 39.2 33.2 29.1 27.3 31.9 64.3 59.6 56.3 48.0 46.6 20.9 13.6
M TR 8 Feb. 0.0 66.5 54.5 41.2 36.1 28.5 23.8 19.4 76.7 68.6 49.6 48.8 37.8 31.6 31.4 12.9 12.1
N RT 1 Feb. 0.0 79.1 66.5 51.4 43.5 36.1 30.7 24.8 84.6 79.0 63.9 55.8 49.1 45.7 44.0 22.5 13.6
Mean - - 0.1 57.7 58.3 53.5 43.4 35.6 30.3 26.3 49.9 59.1 60.7 53.9 50.4 44.0 42.3 20.3 13.1
STD - - 0.2 31.0 19.7 10.8 8.0 5.1 4.5 4.1 25.6 21.1 6.6 9.2 10.8 9.4 8.1 5.0 2.6
CV - - 260g 53.7 33.7 20.1 18.5 14.4 14.7 15.5 51.3 35.7 10.8 17.1 21.5 21.3 19.1 24.4 19.6

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.


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11.3 List of Parameters and Definitions

Table 11-D shows a list of the parameters used in the analysis and their definitions. If any other parameters are used, they must also be clearly defined.

Table 11-D - Parameter Definitions
Parameter Definition
Cmax Maximum observed concentration (µg/mL)
Tmax Sampling time at which Cmax occurred (h).
AUCX Area under the raw concentration versus time curve, over the dosing interval of the test formulation, calculated using the linear trapezoidal rule (µg•h/mL).
AUCτ Area under the raw concentration versus time curve calculated using the linear trapezoidal rule from time 0 to LQCT (µg•h/mL).
AUCI Area to infinity = AUCτ + CT/λ, where CT is the estimated concentration at LQCT (µg•h/mL).
(AUCT ÷ AUCI) × 100 Percent of the area measured by AUCτ relative to the extrapolated total AUC (%).
(AUCX ÷ AUCI) × 100
 Percent of the area measured by AUCX relative to the extrapolated total AUC (%).
λ Terminal disposition rate constant calculated from the points on the log-linear end of the concentration versus time curve (h−1).
TLIN Time point where log-linear elimination begins (h).
LQCT Lowest Quantifiable Concentration Time. Time at which the last concentration occurred that is above the limit of quantitation (h).
T½ Drug half-life = In2÷λ = 0.693÷λ (h).

11.4 Summaries of Parameter Estimates

Table 11-E and 11-F list, for each subject, the estimates of the parameters defined in Table 11-D for the test and reference formulations respectively. Summary statistics (arithmetic means or medians, standard deviations, and CVs) should be given for each formulation.

The AUCX/AUCI ratio is used to determine whether the drug accumulates. For this example, the mean ratio of less than 80 percent indicates that the drug accumulates. Therefore, a multiple-dose study must be run.

The AUCτ/AUCI ratio is used to determine whether the subjects were sampled for a sufficient length of time. For this example, the mean ratio is greater than 80 percent, indicating that the subjects were sufficiently sampled. If the reference is an immediate-release formulation, then Cmax and Tmax are taken from the dosing interval that shows the largest peak (Cmax).

Table 11-E - Summary of Parameters for Each Subject Given the Test Formulation
ID Seq. Per. Parameters
Cmax
(µg
/mL)		 Tmax
(h)		 AUCX
(µg•h
/mL)		 AUCτ
(µg•h
/mL)		 AUCI
(µg•h
/mL)		 AUCX
÷
AUCI
%		 AUCτ
÷
AUCI
%		 λ(h−1) TLIN
(h)		 LQCT
(h)		 T½
(h)
A RT 8 Feb. 46 4 945 1633 2166 44 75 0.0339 24 48 20.4
B TR 1 Feb. 66 4 1024 1405 1559 66 90 0.0511 24 48 13.6
C TR 1 Feb. 67 24 1228 2204 2746 45 80 0.0415 24 48 16.7
D RT 8 Feb. 61 10 1004 1363 1508 67 90 0.0499 24 48 13.9
F TR 1 Feb. 51 10 822 1125 1264 65 89 0.0472 24 48 14.7
G RT 8 Feb. 66 4 1194 1828 2100 57 87 0.0490 24 48 14.1
H RT 8 Feb. 44 6 740 1027 1149 64 89 0.0498 24 48 13.9
I RT 8 Feb. 63 4 984 1546 2038 48 76 0.0303 24 48 22.9
J TR 1 Feb. 48 14 959 1645 2158 44 76 0.0347 24 48 20.0
L RT 8 Feb. 30 10 520 778 908 57 86 0.0453 24 48 15.3
M TR 1 Feb. 63 6 818 1132 1269 65 89 0.0486 16 48 14.3
N RT 8 Feb. 76 13 1304 2026 2440 53 83 0.0423 22 48 16.4
Mean* - - 57 8 962 1476 1776 56 84 0.0436 24 48 16.3
STD - - 13 5.9 220 422 575 9 6 0.0071 2.3 0 3.1
CV - - 23 65 23 29 32 16 7 16.354 10 0 18.9

* For Tmax, TLIN, and LQCT, these are medians.

Table 11-F - Summary of Parameters for Each Subject Given the Reference Formulation
ID Seq. Per. Parameters
Cmax
(µg
/mL)		 Tmax
(h)		 AUCX
(µg•h
/mL)		 AUCτ
(µg•h
/mL)		 AUCI
(µg•h
/mL)		 AUCX
÷
AUCI
%		 AUCτ
÷
AUCI
%		 λ(h−1) TLIN
(h)		 LQCT
(h)		 T½
(h)
A RT 1 Feb. 60 4 1099 1852 2219 50 83 0.0462 12 48 15.0
B TR 8 Feb. 78 1 945 1441 1686 56 85 0.0460 12 48 15.1
C TR 8 Feb. 89 1 1218 1936 2381 51 81 0.0393 12 48 17.6
D RT 1 Feb. 111 1 1323 1855 2035 65 91 0.0565 12 48 12.3
F TR 8 Feb. 55 1 950 1378 1625 58 85 0.0416 12 48 16.7
G RT 1 Feb. 73 2 1155 1791 1995 58 90 0.0578 12 48 12.0
H RT 1 Feb. 69 1 1039 1481 1637 63 90 0.0552 12 48 12.6
I RT 1 Feb. 75 4 1236 1952 2206 56 88 0.0552 12 48 12.6
J TR 8 Feb. 61 4 1005 1549 1827 55 85 0.0452 12 48 15.3
L RT 1 Feb. 83 2 1147 1759 2010 57 88 0.0510 12 48 13.6
M TR 8 Feb. 67 1 983 1399 1652 60 85 0.0413 12 48 16.8
N RT 1 Feb. 79 1 1215 1831 2107 58 87 0.0481 12 48 14.4
Mean* - - 75 1 1109 1685 1948 57 87 0.0486 12 48 14.5
STD - - 15 1 125 219 259 4 3 0.0064 2.5 0 1.9
CV - - 20 68 11 13 13 8 3 13.222 23 0 13.3

* For Tmax, TLIN, and LQCT, these are medians.

11.5 AUCτ Analysis

The necessary information and summary for the analyses of AUCτ are shown in Tables 11-G, 11-H, and 11-I.

In this example, the AUCτ ratio passes the bioavailability criterion.

Table 11-G - AUCτ (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test
AUCτ		 Reference
AUCτ		 Relative
AUCτ (%)		 Test
ln(AUCτ)		 Reference
ln(AUCτ)
A 1633 1852 88 7.40 7.52
B 1405 1441 97 7.25 7.27
C 2204 1936 114 7.70 7.57
D 1363 1855 73 7.22 7.53
F 1125 1378 82 7.03 7.23
G 1828 1791 102 7.51 7.49
H 1027 1481 69 6.93 7.3
I 1546 1952 79 7.34 7.58
J 1645 1549 106 7.41 7.35
L 778 1759 44 6.66 7.47
M 1132 1399 81 7.03 7.24
N 2026 1831 111 7.61 7.51
Mean 1476 1685 87 7.26 7.42
STD 422 219 20 0.3 0.13
CV 29 13 23 - -
Table 11-H - AUCτ (µg•h/mL) Analysis-ANOVA for ln(AUCτ)
Source df SS MS F PR > F
Seq 1 0.0183 0.0183 0.23 0.644
Subject (Seq) 10 0.8042 0.0804 2.50 0.082
Period 1 0.0564 0.0564 1.76 0.215
Form 1 0.1285 0.1285 3.99 0.074
Residual 10 0.3216 0.0322 - -

Intra-subject CV = 100 × (MS Residual)0.5 = 100 × (0.0322)0.5 = 18 percent

Table 11-I - AUCτ (µg•h/mL) Analysis-Calculations

Difference = Test x − Reference x = 7.2604 − 7.4089 = −0.1485

SEDifference = 0.0743

AUC Ratio = 100 × eDifference = 100 × e−0.1485 = 86%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.05, 10 × SEDifference)

Lower = 100 × e(−0.1485 − 1.812 × 0.0743) = 75%

Upper = 100 × e(−0.1485 + 1.812 × 0.0743) = 99%


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11.6 Cmax Analysis

The necessary information and summary for the analyses of Cmax are shown in Tables 11-J, 11-K, and 11-L.

In this example, the Cmax ratio passes the bioavailability criterion.

Table 11-J - Cmax (µg/mL) Analysis-Data
ID Raw Scale Log Scale
Test
Cmax		 Reference
Cmax		 Relative
Cmax (%)		 Test
ln(Cmax)		 Reference
ln(Cmax)
A 46 60 76 3.82 4.10
B 66 78 84 4.18 4.36
C 67 89 75 4.2 4.49
D 61 111 55 4.11 4.71
F 51 55 94 3.94 4.00
G 66 73 90 4.19 4.28
H 44 69 63 3.78 4.24
I 63 75 85 4.15 4.31
J 48 61 79 3.88 4.11
L 30 83 37 3.41 4.42
M 63 67 94 4.14 4.20
N 76 79 96 4.33 4.37
Mean 57 75 77 4.01 4.30
STD 13 15 18 0.26 0.19
CV 23 20 23 - -
Table 11-K - Cmax (µg/mL) Analysis-ANOVA for ln(Cmax)
Source df SS MS F PR > F
Seq 1 0.0005 0.0005 0.01 0.933
Subject (Seq) 10 0.6867 0.0687 1.84 0.175
Period 1 0.0652 0.0652 1.75 0.215
Form 1 0.4279 0.4279 11.49 0.007
Residual 10 0.3275 0.037 - -

Intra-subject CV = 19 percent

Table 11-L - Cmax (µg/mL) Analysis-Calculations

Difference = Test x − Reference x = 4.0185 − 4.2893 = −0.2708

SEDifference = 0.0799

Cmax Ratio = 100 × eDifference = 100 × e−0.2708 = 76%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.05, 10 × SEDifference)

Lower = 100 × e(−0.2708 − 1.812 × 0.0799) = 66%

Upper = 100 × e(−0.2708 + 1.812 × 0.0799) = 88%

11.7 AUCI Analysis

The necessary information and summary for the analyses of AUCI are shown in Tables 11-M and 11-N.

Table 11-M - AUCI (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test
AUCI		 Reference
AUCI		 Relative
AUCI (%)		 Test
ln(AUCI)		 Reference
ln(AUCI)
A 2166 2219 98 7.68 7.7
B 1559 1686 92 7.35 7.43
C 2746 2381 115 7.92 7.78
D 1508 2035 74 7.32 7.62
F 1264 1625 78 7.14 7.39
G 2100 1995 105 7.65 7.6
H 1149 1637 70 7.05 7.4
I 2038 2206 92 7.62 7.7
J 2158 1827 118 7.68 7.51
L 908 2010 45 6.81 7.61
M 1269 1652 77 7.15 7.41
N 2440 2107 116 7.80 7.65
Mean 1776 1948 90 7.43 7.57
STD 575 259 22 0.34 0.13
CV 32 13 25 - -
Table 11-N - AUCI (µg•h/mL) Analysis-ANOVA for ln(AUCI)
Source df SS MS F PR > F
Seq 1 0.0090 0.0090 0.08 0.777
Subject (Seq) 10 1.0623 0.1062 2.7 0.066
Period 1 0.0271 0.0271 0.69 0.426
Form 1 0.0912 0.0912 2.32 0.159
Residual 10 0.3931 0.0393 - -

Intra-subject CV = 20 percent

11.8 Tmax Analysis

The necessary information and summary for the analyses of Tmax are shown in Tables 11-O and 11-P.

Table 11-O - Tmax (h) Analysis-Data
ID Test
Tmax		 Reference
Tmax
A 4 4
B 4 1
C 24 1
D 10 1
F 10 1
G 4 2
H 6 1
I 4 4
J 14 4
L 10 2
M 6 1
N 13 1
MEDIAN 8 1
STD 5.9 1.3
Table 11-P - Tmax (h) Analysis-ANOVA
Source df SS MS F PR > F
Seq 1 20.74 20.74 1.28 0.285
Subject (Seq) 10 162.26 16.23 0.86 0.591
Period 1 34.40 34.40 1.83 0.206
Form 1 334.40 334.40 17.75 0.002
Residual 10 188.43 18.84 - -

Intra-subject CV = 77 percent


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11.9 λ Analysis

The necessary information and summary for the analyses of λ (h−1) are shown in Tables 11-Q and 11-R.

Table 11-Q - λ(h−1) Analysis-Data
ID Test
λ		 Reference
λ
A 0.0339 0.0462
B 0.0511 0.046
C 0.0415 0.0393
D 0.0499 0.0565
F 0.0472 0.0416
G 0.0490 0.0578
H 0.0498 0.0552
I 0.0303 0.0552
J 0.0347 0.0452
L 0.0453 0.051
M 0.0486 0.0413
N 0.0423 0.0481
MEDIAN 0.0436 0.0486
STD 0.0071 0.0064
CV 16.354 13.222
Table 11-R - λ (h−1) Analysis-ANOVA
Source df SS MS F PR > F
Seq 1 0.0001 0.0001 2.33 0.158
Subject (Seq) 10 0.0005 0.0001 1.79 0.186
Period 1 0.0002 0.0002 8.19 0.017
Form 1 0.0001 0.0001 3.72 0.083
Residual 10 0.0003 0.0003 - -

Intra-subject CV = 11 percent

11.10 Calculations for AUC Ratio and Cmax Ratio Estimates Corrected for Measured Content

Tables 11-S and 11-T show the method of calculating the AUC ratio and Cmax ratio estimates and their confidence limits, based on correction for measured content. Each formulation is adjusted to 100 percent of the label claim. The whole analysis need not be repeated; only the corrected estimates need be given.

Table 11-S - Estimates Based on Correction for Measured Content
  Test Formulation Reference Formulation
Lot number EX110 40905
Expiry date 06/90 05/90
Date of analysis 06/14/88 06/14/88
Measured content
(% of label claim)		 95.5 99.0
Correction factors
raw scale-multiply 1.0471 1.0101
log scale-add 0.0460 0.0100

Table 11-T - AUC Ratio and Cmax Ratio-Calculations

Based on measured contents in Table 11-S, the following factor is obtained:

ln(99.0 ÷ 95.5 ) = 0.0360

which is to be added to the estimates on log scale.

Therefore:

AUCX Ratio = 100 × e(−0.1486 + 0.0360) = 89%

Lower Limit = 100 × e(−0.1486 + 0.0360 − 1.812 × 0.0681) = 79%

Upper Limit = 100 × e(−0.1486 + 0.0360 + 1.812 × 0.0681) = 101%

AUCτ Ratio = e(−0.1485 + 0.0360) × 100% = 89%

Lower Limit = 100 × e(−0.1485 + 0.0360 − 1.812 × 0.0743) = 78%

Upper Limit = 100 × e(−0.1485 + 0.0360 + 1.812 × 0.0743) = 102%

Cmax Ratio = 100 × e(−0.2708 + 0.0360) = 79%

Lower Limit = 100 × e(−0.2708 + 0.0360 − 1.812 × 0.0799) = 68%

Upper Limit = 100 × e(−0.2708 + 0.0360 + 1.812 × 0.0799) = 99%

11.11 Concentration - Time Profiles (Subject A)

Figure 11.1 gives a plot of the concentration-time profile for subject A. Each plot must include profiles for all formulations given to that subject. Similar profiles should be given for each subject.

Figure 11.1: Concentration- Time Profile for Subject A

Concentration-Time Profile for subject A

Figure 11.2 gives a plot of the ln(concentration)-time profile for subject A. This plot must contain the regression lines from which the terminal disposition rate constants (λ ) were estimated. This line must start and end at the time points considered to be in the log-linear elimination phase. Any point that was not used to estimate the regression line must be identified.

Figure 11.2: Ln(concetration)- Time Profile for Subject A

Ln(concentration)


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Figure 11.3 shows a profile of the arithmetic means over all subjects for each sampling time.

Figure 11.3: Average Concentration-Time Profile for All Subjects

Average Concentration

Figure 11.4 shows a profile of the ln(arithmetic means) over all subjects for each sampling time.

Figure 11.4: Ln(average concentration)- Time Profile for All Subjects

Ln(average concentration)

12 Sample Analysis for a Multiple-Dose Comparative Bioavailability Study

The following tables and figures illustrate data collected and used in a sample multiple-dose comparative bioavailability study. An analysis of this data is also shown.

Although a comparative bioavailability study may include many formulations, the basic analysis is the same-each test formulation is compared to a reference formulation.

The analysis of a multiple-dose comparative bioavailability study must have the following sections:

  1. A randomization scheme for the design, where all subjects randomized into the study are included and identified by code, sequence, and dates of the dosing periods for both test and reference formulations. (See Section 12.1.)
  2. A summary of drug concentrations (visual and quantitative) at each sampling time for each subject for both test and reference formulations. (See Section 12.2.)
  3. A summary of the estimates of the parameters as defined in Section 12.3 for both test and reference formulations, including the means, standard deviations, and CVs. (See Section 12.4.)
  4. A summary of pre-dose concentrations of the test and reference formulations. (See Section 12.5.)
  5. A formal statistical analysis of the relevant parameters with comparisons of the test formulations to the reference formulations. (See Sections 12.6 through 12.9.)
  6. A summary of corrections for potency (measured content) in estimates. (See Section 12.10.)
  7. Sample concentration-time profiles that should be provided for each subject. (See Section 12.11.)

All the sample statistical analyses that follow have a minimum of two formulations (test and reference) given on two dosing days or periods.

12.1 Randomization Scheme of the Design

Shown in Table 12-A is the randomization scheme for the cross-over design used in the study. In any study, all subjects who were randomized into the study must be included. Even those subjects that did not complete the study must be included and identified accordingly. Subject numbers that appear on informed consent forms and reporting forms must be given. Also, if any other subject identification code was used, it should be given here. The sequence to which the subject was randomized should be given. Finally, all dosing periods and dates must be given.

12.2 Summary of Drug Concentrations

Tables 12-B and 12-C show a list of the concentrations at each sampling time for each subject for the test and reference formulations, respectively. If any concentration is missing, it should be identified, and the reason it is missing given (e.g., lost sample; sample not collected).

Although no formal statistical analysis is required at each sampling time, it is recommended that summary statistics be given at each sampling time for each formulation. It is also helpful if the limit of quantitation of the analytical method is given at this table.

Table 12-A - Randomization Scheme of the Cross-over Design for the Comparison of Test (T) Versus Reference (R) Formulations
Subject Period
Number ID Sequence Feb. 14, 1989 Feb. 28, 1989
001 A RT R T
002 B TR T R
003 C TR T R
004 D RT R T
005* E TR T -
006 F TR T R
007 G RT R T
008 H RT R T
009 I RT R T
010 J TR T R
011** K TR - -
012 L RT R T
013 M TR T R
014 N RT R T

* Subject did not appear for second period.
** Subject did not appear for either period.

Table 12-B - Drug Concentrations* (µg/mL) for the Test Formulation
ID Seq Per. Sampling Times (hours)
0.0 1.0 2 4.0 6.0 8.0 10.0 12.0 13.0 14.0 16.0 18.0 20.0 22.0 24 36.0 48
A RT 28 Feb. 46.4 56.5 69.5 86.3 73.7 76.0 68.8 64.9 64.9 65.5 65.8 54.7 52.7 53.5 50.1 24.4 15.9
B TR 14 Feb. 31.0 40.9 50.1 56.1 50.4 47.2 39.2 35.8 32.8 29.2 25.5 22.6 21.4 19.3 17.6 9.6 6.6
C TR 14 Feb. 51.0 57.0 54.7 55.0 67.8 70.8 81.7 71.9 72.8 69.9 58.4 69.2 65.7 51.1 53.0 30.0 19.1
D RT 28 Feb. 31.3 43.6 64.5 62.5 67.5 58.9 59.9 50.4 58.7 59.6 46.2 39.8 40.5 36.4 45.0 23.0 14.1
F TR 14 Feb. 43.2 58.6 69.1 76.2 64.5 57.5 49.9 57.8 55.2 56.1 51.2 49.6 46.6 42.9 47.6 29.5 16.2
G RT 28 Feb. 48.7 52.6 60.5 99.2 79.1 74.0 82.2 82.3 67.7 70.4 55.1 55.5 47.8 47.0 59.7 25.7 14.3
H RT 28 Feb. 35.8 46.3 65.0 69.6 68.4 59.7 50.0 43.1 39.9 43.5 36.5 33.3 37.6 33.9 38.0 24.6 12.9
I RT 28 Feb. 47.0 44.9 52.1 88.0 75.7 60.4 52.2 46.6 4.05 38.6 32.9 35.0 36.6 35.8 31.9 17.3 9.3
J TR 14 Feb. 46.7 48.8 48.4 74.9 63.9 57.8 46.0 41.0 39.6 37.1 32.4 31.3 28.6 29.7 30.8 17.1 12.0
L RT 28 Feb. 60.9 61.0 60.2 82.2 67.0 62.0 53.9 46.2 43.8 41.2 30.6 30.6 29.0 29.0 27.6 13.7 7.7
M TR 14 Feb. 37.8 50.2 56.8 63.6 55.8 43.9 42.5 37.5 35.3 33.2 31.1 33.9 28.9 25.8 24.8 20.1 7.7
N RT 28 Feb. 38.9 34.4 49.1 79.9 65.7 70.2 82.1 71.6 67.0 73.4 65.1 61.4 55.7 53.0 56.2 29.2 17.0
Mean - - 43.2 49.6 58.3 74.5 66.6 61.5 59.0 54.1 51.5 51.5 44.2 43.1 40.9 38.1 40.2 22.0 12.7
STD - - 8.7 8.0 7.6 13.6 7.9 9.9 15.8 15.3 14.4 16.1 14.5 14.5 13.1 11.3 13.6 6.5 4.1
CV - - 20.1 16.1 13.0 18.2 11.9 16.1 26.8 28.3 27.9 31.2 32.8 33.7 32.1 29.7 33.9 29.7 32.3

* Limit of quantitation is 0.2µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.


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Table 12-C - Drug Concentrations* (µg/mL) for the Reference Formulation
ID Seq Per. Sampling Times (hours)
0.0 1.0 2 4.0 6.0 8.0 10.0 12.0 13.0 14.0 16.0 18.0 20.0 22.0 24 36.0 48
A RT 14 Feb. 42.8 63.9 90.5 66.4 62.2 54.7 46.2 41.4 90.8 81.4 65.1 60.8 53.1 50.1 42.4 25.3 17.6
B TR 28 Feb. 46.0 101.2 80.9 67.1 53.8 44.7 40.2 34.8 56.3 77.9 61.6 52.2 45.9 45.1 40.4 19.4 10.1
C TR 28 Feb. 55.8 115.9 95.0 87.1 68.7 59.5 55.7 48.6 46.2 57.6 77.5 74.7 67.6 61.5 60.5 33.0 22.1
D RT 14 Feb. 36.2 48.3 96.5 67.9 59.3 50.4 39.7 33.3 81.6 92.4 67.9 50.6 47.8 41.5 38.0 18.2 10.8
F TR 28 Feb. 36.8 89.3 77.2 60.0 50.0 41.2 41.8 33.2 49.5 74.5 69.5 50.8 45.6 40.0 39.6 17.9 10.9
G RT 14 Feb. 52.1 77.8 103.2 78.7 64.1 51.3 43.2 38.5 35.6 52.5 64.3 70.7 65.0 53.7 49.2 23.3 12.8
H RT 14 Feb. 43.3 68.1 83.7 67.4 59.5 48.0 39.3 35.2 71.3 89.8 71.4 56.2 43.8 37.7 35.2 19.9 10.1
I RT 14 Feb. 46.6 87.7 102.1 89.2 64.2 53.8 47.9 40.4 36.4 48.9 67.8 69.3 66.8 57.9 54.7 27.4 13.7
J TR 28 Feb. 44.2 95.1 74.5 75.0 59.8 44.4 45.2 37.2 76.8 93.5 67.5 55.4 53.4 43.0 42.7 21.7 13.6
L RT 14 Feb. 48.5 90.1 95.8 71.4 62.4 49.4 49.7 41.9 39.4 65.1 75.5 72.5 57.0 49.5 46.6 13.9 12.3
M TR 28 Feb. 48.7 74.1 87.9 71.4 61.7 53.7 43.0 37.4 92.6 87.3 69.6 59.4 50.3 43.9 41.8 19.7 11.7
N RT 14 Feb. 43.9 102.1 93.0 73.4 58.0 50.5 42.7 40.1 44.5 57.4 66.7 68.9 63.4 57.1 55.9 36.4 17.3
Mean - - 45.4 84.5 90.0 72.9 60.3 50.1 44.6 38.5 60.1 73.2 68.7 61.8 55.0 48.4 45.6 23.0 13.6
STD - - 5.6 18.8 9.4 8.6 4.9 5.1 4.8 4.4 21.4 16.3 4.5 9.0 8.8 7.8 7.9 6.5 3.7
CV - - 12.6 22.3 10.4 11.8 8.1 10.1 10.7 11.4 35.6 22.3 6.5 14.5 16.0 16.0 17.3 28.4 27.0

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.

12.3 List of Parameters and Definitions

Table 12-D shows a list of the parameters used in the analysis and their definitions. If any other parameters are used, they must also be clearly defined.

Table 12-D - Parameter Definitions
Parameter Definition
Cmax Maximum observed concentration (µg/mL).
Cmin Minimum observed concentration (µg/mL).
Cpd Pre-dose concentration from same time of each day (µg/mL).
Tmax Sampling time at which Cmax occurred (h).
AUCτ Area under the concentration versus time curve, over the dosing interval of the test formulation, calculated using the linear trapezoidal rule (µg•h/mL).
Fluctuation (Cmax − Cmin) ÷ (AUCτ ÷ τ) × 100.

12.4 Summaries of Parameters Estimates

Tables 12-E and 12-F list, for each subject, the estimates of the parameters defined in Table 12-D for the test and reference formulations respectively. Summary statistics (arithmetic means or medians, standard deviations, and CVs) should be given for each formulation. If the reference is an immediate-release formulation, Cmax and Tmax are taken from the dosing interval of largest peak (largest Cmax).

Table 12-E - Summary of Parameters for Each Subject Given the Test Formulation
ID Seq. Period Parameters
Cmax
(µg/mL)		 Cmin
(µg/mL)		 Tmax
(h)		 AUCτ
(µg•h/mL)		 FL**
(%)
A RT 28 Feb. 86 46 4 1558 61
B TR 14 Feb. 56 18 4 843 110
C TR 14 Feb. 82 51 10 1542 48
D RT 28 Feb. 68 31 6 1248 70
F TR 14 Feb. 76 43 4 1334 60
G RT 28 Feb. 99 47 4 1604 78
H RT 28 Feb. 70 33 4 1148 76
I RT 28 Feb. 88 32 4 1180 114
J TR 14 Feb. 75 29 4 1062 105
L RT 28 Feb. 82 28 4 1153 114
M TR 14 Feb. 64 25 4 971 96
N RT 28 Feb. 82 34 10 1534 75
Mean* - - 77 35 4 1265 84
STD - - 12 10 2 251 23
CV - - 15 29 45 20 27

* For Tmax, this is the median.
** Fluctuation.

Table 12-F - Summary of Parameters for Each Subject Given the Reference Formulation
ID Seq. Period Parameters
Cmax
(µg/mL)		 Cmin
(µg/mL)		 Tmax
(h)		 AUCτ
(µg•h/mL)		 FL**
(%)
A RE 14 Feb. 91 41 2 1456 81
B ER 28 Feb. 101 35 1 1333 120
C ER 28 Feb. 116 46 1 1657 101
D RE 14 Feb. 97 33 2 1370 111
F ER 28 Feb. 89 33 1 1272 106
G RE 14 Feb. 103 36 2 1462 111
H RE 14 Feb. 84 34 2 1351 88
I RE 14 Feb. 102 36 2 1523 104
J ER 28 Feb. 95 37 1 1432 97
L RE 14 Feb. 96 39 2 1479 91
M ER 28 Feb. 88 37 2 1458 83
N RE 14 Feb. 102 40 1 1472 101
Mean* - - 97 37 2 1439 99
STD - - 9 4 1 100 12
CV - - 9 10 33 7 12

* For Tmax, this is the median.
** Fluctuation.

12.5 Pre-dose Concentrations

Tables 12-G and 12-H list the pre-dose concentrations for each subject on each day of the study for the test and reference formulations respectively.

These tables are used to check for both compliance and whether steady-state concentrations had been reached during the study. Intra-subject statistics should be calculated for the last three pre-dose concentrations.

Table 12-G - Pre-dose Concentrations (µg/mL) for Each Subject Given the Test Formulation
ID Day Intra-Subject
Statistics*
3 4 5 6 7 8 Mean STD CV
A 18 37 37 41 46 50 46 5 10
B 8 33 54 44 31 18 31 13 42
C 22 60 43 45 51 53 50 4 8
D 7 31 31 48 31 45 41 9 22
F 7 6 14 41 43 8 44 4 8
G 14 49 44 36 49 60 48 12 25
H 6 28 31 34 36 38 36 2 5
I 14 20 68 24 47 32 34 12 34
J 18 35 48 37 47 31 38 8 21
L 6 31 34 35 61 28 41 18 43
M 7 20 49 31 38 25 31 6 21
N 19 61 42 39 39 56 45 10 22
Mean 12 34 41 38 43 40 - - -
STD 6 16 14 6 9 14 - - -
CV 49 47 33 17 20 34 - - -

* Based on concentrations from days 6, 7, and 8.


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Table 12-H - Pre-dose Concentrations (µg/mL) for Each Subject Given the Reference Formulation
ID Day Intra-Subject
Statistics*
3 4 5 6 7 8 Mean STD CV
A 17 45 53 47 43 42 44 2 5
B 12 41 43 38 46 40 42 4 10
C 18 54 55 63 56 61 60 4 6
D 11 36 39 32 36 38 35 3 9
F 11 31 32 35 37 40 37 3 7
G 12 43 47 47 52 49 49 3 5
H 9 36 40 31 34 35 34 2 6
I 15 49 67 46 47 55 49 5 10
J 14 39 45 39 44 43 42 2 6
L 14 48 45 46 49 47 47 1 3
M 12 33 42 39 49 32 43 5 12
N 14 44 40 41 44 56 47 8 17
Mean 13 42 46 42 45 46 - - -
STD 3 7 9 9 6 8 - - -
CV 20 17 20 21 14 17 - - -

* Based on concentrations from days 6, 7, and 8.

A repeated measures analysis of the last three pre-dose concentrations should be provided. An example of this analysis is shown in Table 12-I. Of main importance in this analysis is the time and time×form interaction. The error term to test time is the time×ID (Seq). The error term to test time×form is time×form×ID (Seq). Should either the time or time×form effects be significant, the study may not have been at steady state for one or both formulations

Table 12-I - Repeated Measures Analysis of ln(Cpd)
Source df SS MS F PR > F
Seq 1 0.01500416 0.01500416 0.12 0.7313
ID (Seq) 10 1.20256303 0.12025630 2.11 0.1278
Period 1 0.04556284 0.04556284 0.80 0.3926
Form 1 0.22620312 0.22620312 3.96 0.0745
ID×Form (Seq) 10 0.57066567 0.0570666 - -
Time 2 0.11182757 0.05591378 1.60 0.2274
Time×Seq 2 0.13927056 0.06963528 1.99 0.1631
Time×ID (Seq) 20 0.70058566 0.03502928 - -
Time×Period 2 0.07672565 0.03836282 1.24 0.3110
Time×Form 2 0.06390201 0.03195101 1.03 0.3746
Time×Form×ID (Seq) 20 0.61937447 0.03096872 - -
Corrected Total 71 3.74093818 - - -

12.6 AUCτ Analysis

Tables 12-J, 12-K, and 12-L provide the complete analysis required for AUCτ. Table 12-J lists the AUCτ estimates on the raw scale and the log scale. Also given is the test AUCτ as a percentage of the reference AUCτ. Summary statistics are calculated for each variable.

Table 12-J - AUCτ (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test
AUCτ		 Reference
AUCτ		 Relative
AUCτ (%)		 Test
ln(AUCτ)		 Reference
ln(AUCτ)
A 1558 1456 107 7.35 7.28
B 843 1333 63 6.74 7.19
C 1542 1657 93 7.34 7.41
D 1248 1370 91 7.13 7.22
F 1334 1272 105 7.20 7.15
G 1604 1462 110 7.38 7.29
H 1148 1351 85 7.05 7.21
I 1180 1523 77 7.07 7.33
J 1062 1432 74 6.97 7.27
L 1153 1479 78 7.05 7.30
M 971 1458 67 6.88 7.28
N 1534 1472 104 7.34 7.29
Mean 1265 1439 88 7.12 7.27
STD 251 100 16 0.21 0.07
CV 20 7 18 - -

Table 12-K gives the analysis of variance (ANOVA) for the cross-over design model for ln(AUCτ). This analysis gives the appropriate intra-subject variance estimate, MS (Residual), for the calculation of the 90% confidence interval. Any significant effects in the model, other than Subject (Seq), should be investigated. The intra-subject and inter-subject CVs should also be calculated.

Table 12-K - AUCτ (µg•h/mL) Analysis-ANOVA for ln(AUCτ)
Source df SS MS F PR > F
Seq 1 0.0495 0.0495 1.85 0.204
Subject (Seq) 10 0.2685 0.0269 1.66 0.217
Period 1 0.0362 0.0362 2.24 0.165
Form 1 0.1467 0.1467 9.09 0.013
Residual 10 0.1614 0.0161 - -

Intra-subject CV = 100 × (MS Residual)0.5 = 100 × (0.0161)0.5 = 13 percent

The AUCτ ratio estimate and its 90% confidence interval are derived in the calculations shown in Table 12-L. If this study had a balanced design (i.e., an equal number of subjects per sequence) the difference would simply be the difference in the arithmetic means of the ln(AUC)s. Since the study was not balanced, the least-squares mean estimate for each formulation is used to form this difference, together with the appropriate standard error.

Table 12-L - AUCτ (µg•h/mL) Analysis-Calculations

Difference = Test x − Reference x = 7.1095 − 7.2681 = −0.1586

SEDifference = 0.0526

AUCτ Ratio = 100 × eDifference = 100 × e−0.1586 = 85%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.025, 10× SEDifference)

Lower = 100 × e(−0.1586 − 1.812 × 0.0526) = 78%

Upper = 100 × e(−0.1586 + 1.812 × 0.0526) = 94%

12.7 Cmax Analysis

The necessary information and summary for the analyses of Cmax are shown in Tables 12-M, 12-N, and 12-O.

For this example, the Cmax ratio passes the bioavailability criterion.

Table 12-M - Cmax (µg/mL) Analysis-Data
ID Raw Scale Log Scale
Test
Cmax		 Reference
Cmax		 Relative
Cmax (%)		 Test
ln(Cmax)		 Reference
ln(Cmax)
A 86 91 95 4.46 4.51
B 56 101 55 4.03 4.62
C 82 116 70 4.40 4.75
D 68 97 70 4.21 4.57
F 76 89 85 4.33 4.49
G 99 103 96 4.60 4.64
H 70 84 83 4.24 4.43
I 88 102 86 4.48 4.63
J 75 95 79 4.32 4.55
L 82 96 86 4.41 4.56
M 64 88 72 4.15 4.48
N 82 102 80 4.41 4.63
Mean 77 97 80 4.34 4.57
STD 12 9 12 0.16 0.09
CV 15 9 14 - -


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Table 12-N - Cmax (µg/mL) Analysis-ANOVA for ln(Cmax)
Source df SS MS F PR > F
Seq 1 0.0287 0.0287 1.44 0.258
Subject (Seq) 10 0.1198 0.0200 2.27 0.106
Period 1 0.0412 0.412 4.68 0.056
Form 1 0.3591 0.3591 4.09 > 0.001
Residual 10 0.0879 0.0088 - -

Intra-subject CV = 9 percent

Table 12-O - Cmax Analysis-Calculations

Cmax Ratio = 100 × eDifference = 100 × e(4.3235 − 4.5717) = 78%

12.8 Cmin Analysis

The necessary information and summary for the analyses of Cmin are shown in Tables 12-P, 12-Q, and 12-R.

Table 12-P - Cmin (µg/mL) Analysis-Data
ID Raw Scale Log Scale
Test
Cmin		 Reference
Cmin		 Relative
Cmin (%)		 Test
ln(Cmin)		 Reference
ln(Cmin)
A 46 41 112 3.84 3.72
B 18 35 51 2.87 3.55
C 51 46 110 3.93 3.83
D 31 33 94 3.44 3.51
F 43 33 129 3.76 3.50
G 47 36 132 3.85 3.57
H 33 35 97 3.51 3.54
I 32 36 88 3.46 3.59
J 29 37 77 3.35 3.62
L 28 39 70 3.32 3.67
M 25 37 66 3.21 3.62
N 34 40 86 3.54 3.69
Mean 35 37 93 3.51 3.62
STD 10 4 25 0.31 0.10
CV 29 10 27 - -
Table 12-Q - Cmin (µg/mL) Analysis-ANOVA for ln(Cmin)
Source df SS MS S PR > F
Seq 1 0.0245 0.0245 0.37 0.559
ID (Seq) 0 0.6694 0.0669 1.62 0.228
Period 1 0.0334 0.0334 0.81 0.389
Form 1 0.0902 0.0902 2.19 0.17
Residual 0 0.4120 0.0412 - -

Intra-subject CV = 20 percent

Table 12-R - Cmin Analysis-Calculations

Cmin Ratio = 100 × eDifference = 100 × e(3.4948 − 3.6192) = 88%

12.9 Fluctuation Analysis

The necessary information and summary for the analyses of Fluctuation are provided in Tables 12-S and 12-T.

Table 12-S - Fluctuation (%) Analysis-Data
ID Test
Fluctuation		 Reference
Fluctuation
A 61 81
B 110 120
C 48 101
D 70 111
F 60 106
G 78 111
H 76 88
I 114 103
J 105 97
L 114 91
M 96 83
N 75 101
Mean 84 99
STD 23 12
CV 27 12
Table 12-T - Fluctuation Analysis-ANOVA for Fluctuation
Source df SS MS F PR > F
Seq 1 12.8 12.8 0.03 0.863
Subject (Seq) 10 3879.3 388.0 1.13 0.427
Period 1 18.9 18.9 0.05 0.819
Form 1 1484.5 1484.5 4.31 0.065
Residual 10 3440.5 344.1 - -

Intra-subject CV = 20 percent

12.10 Calculations for AUCτ Ratio, Cmax Ratio, and Cmin Ratio Estimates Corrected for Measured Content

The whole analysis need not be repeated; only the corrected estimates need be given.

Table 12-U - Estimates Based on Correction for Measured Content
  Test Formulation Reference Formulation
Lot number EX110 40905
Expiry date 06/90 05/90
Date of analysis 06/14/88 06/14/88
Measured content (% of label claim) 95.5 99
Correction factors
raw scale-multiply 1.0471 1.0101
log scale-add 0.0460 0.0100

Table 12-V - AUCτ Ratio, Cmax, Ratio and Cmin Ratio-Calculations

Based on measured contents in Table 12-U, the following factor is obtained:

ln(99.0 ÷ 95.5) = 0.0360

which is to be added to the estimates on the log scale.

Therefore:

AUCτ Ratio = e(−0.1586 + 0.0360) × 100 = 93%

90% Confidence Limits

Lower Limit = 100 × e(−0.1586 + 0.0360 − 1.812 × 0.0526) = 80%

Upper Limit = 100 × e(−0.1586 + 0.0360 + 1.812 × 0.0526) = 97%

Cmax Ratio = 100 × e(−0.2482 + 0.0360) = 81%

Cmin Ratio = 100 × e(−0.1244 + 0.0360) = 92%


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12.11 Concentration-Time Profiles (Subject A)

Figure 12.1 shows a plot of the concentration-time profile for subject A. Each plot must include profiles for all formulations given to that subject. Similar profiles should be given for each subject.

Figure 12.1: Concentration-Time Profile for Subject A

Figure 12.1: Concentration-Time Profile for Subject A

Figure 12.2 gives a plot of the ln(concentration)-time profile for subject A. This plot must contain the regression lines from which the terminal disposition rate constants (λ) were estimated. This line must start and end at the time points considered to be in the log-linear elimination phase. Any point that was not used to estimate the regression line must be identified.

Figure 12.2: Ln(concentration)- Time Profile for Subject A

Figure 12.2: Ln(concentration)-Time Profile for Subject A

Figure 12.3 shows a profile of the arithmetic means over all subjects for each sampling time.

Figure 12.3: Average Concentration - Time Profile for All Subjects

Figure 12.3: Average Concentration-Time Profile for All Subjects

Figure 12.4 shows a profile of the ln(arithmetic means) over all subjects for each sampling time.

Figure 12.4: LN(average concentration) - Time Profile for All Subjects

Figure 12.4: Ln(average concentration)-Time Profile for All Subjects

13 Sample Analysis for a Single-Dose Bioequivalence Study

The following tables and figures illustrate data collected and used in a sample single-dose bioequivalence study. An analysis of this data is also shown.

Although a bioequivalence study may include many formulations, the basic analysis is the same-each test formulation is compared to a reference formulation.

The analysis of a single-dose comparative bioequivalence study must have the following sections:

  1. A randomization scheme for the design, where all subjects randomized into the study are included and identified by code, sequence, and dates of the dosing periods. (See Section 13.1.)
  2. A summary of drug concentrations (visual and quantitative) at each sampling time for each subject for the test and reference formulations being compared. (See Section 13.2.)
  3. A summary of the estimates of the parameters as defined in Section 13.3 for the test and reference formulations being compared, including the means, standard deviations, and CVs. (See Section 13.4.)
  4. A formal statistical analysis of the relevant parameters with comparisons of the test formulations to the reference formulations. (See Sections 13.5 through 13.9.)
  5. A summary of corrections for potency (measured content) in estimates. (See Section 13.10.)
  6. Sample concentration-time profiles that should be given for each subject. (See Section 13.11.)

All the sample statistical analyses that follow have a minimum of two formulations (test and reference) given on two dosing days or periods.


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13.1 Randomization Scheme of the Design

Shown in Table 13-A is the randomization scheme of the cross-over design used in the study. In any study, all subjects who were randomized into the study must be included. Even those subjects that did not complete the study must be included and identified accordingly. Subject numbers that appear on informed consent forms and reporting forms must be given. Also, if any other subject identification code was used, it should be given here. The sequence to which the subject was randomized should be given. Finally, all dosing periods and dates must be given.

13.2 Summary of Drug Concentrations

Tables 13-B and 13-C show a list of the concentrations at each sampling time for each subject for the test and reference formulations, respectively. If any concentration is missing, it should be identified, and the reason it is missing given (e.g., lost sample; sample not collected).

Although no formal statistical analysis is required at each sampling time, it is recommended that summary statistics be given at each sampling time for each formulation. It is also helpful if the limit of quantitation of the analytical method is given in this table, along with the measured potency for the formulation.

Table 13-A - Randomization Scheme of the Cross-over Design for the Comparison of Test (T) Versus Reference (R) Formulations
Subject Period
Number ID Sequence Aug. 23, 1995 Aug. 30, 1995
001 1 TR T R
002 2 RT R T
003 3 RT R T
004 4 TR T R
005* 5 TR T -
006 6 RT R T
007 7 TR T R
008 8 RT R T
009 9 RT R T
010 10 TR T R
011** 11 TR - -
012 12 TR T R
013 13 TR T R
014 14 RT R T

* Subject did not appear for second period.
** Subject did not appear for either period.

Table 13-B - Drug Concentrations* (µg/mL) for the Test Formulation
ID Seq Per. Sampling Times (hours)
0.0 1.0 2.0 3.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 48.0
1 TR 23 Aug. 0.0 0.0 0.0 16.9 48 138 136 106 110 141 146 100 65.2 45.1 27.7 13.5 8.2 9.37
2 RT 30 Aug. 0.0 0.0 0.0 7.87 31.6 63.6 53.9 63.3 99.3 118 111 82 49.3 34.6 13.9 8.54 0.0 5.89
3 RT 30 Aug. 0.0 0.0 20.1 114 153 118 73 67.8 99.1 134 148 106 107 72.7 31.5 14.3 9.95 0.0
4 TR 23 Aug. 0.0 0.0 8.4 67.4 102 140 88.6 106 95.4 94.5 92.3 72.5 55.5 28.1 12.1 5.45 0.0 0.0
6 RT 30 Aug. 0.0 0.0 0.0 24.1 59 96.7 94.8 76.5 97 109 116 89.5 99.3 61.4 31.7 13 7.14 0.0
7 TR 23 Aug. 0.0 0.0 0.0 13.8 33.4 55.4 50.5 62 88.1 90.7 71 57.5 51.7 35.8 24.8 13.2 8.93 0.0
8 RT 30 Aug. 0.0 0.0 0.0 35 62.2 60.7 45.8 39.4 41.2 37 35.9 25.5 22.5 16.3 10.5 5.56 0.0 0.0
9 RT 30 Aug. 0.0 0.0 9.87 36.1 84.7 129 128 130 180 172 153 91.7 71.1 48.4 21 11.8 6.56 0.0
10 TR 23 Aug. 0.0 0.0 5.71 32.5 63.9 72.4 59.5 41.5 43.1 75 86.3 82.4 89 62 41.2 22.8 16 7.84
12 TR 23 Aug. 0.0 0.0 30.7 121 43.5 110 89.7 91.5 104 90 82.5 60 67 50.8 29 24.5 36.9 17.7
13 TR 23 Aug. 0.0 0.0 14 75.5 92.5 94.9 63.9 50.1 70 73.2 61.3 48.1 39.9 29.1 15.6 9.03 0.0 0.0
14 RT 30 Aug. 0.0 0.0 0.0 23.7 47.9 47 31.7 19.6 39.2 51 63.4 42.1 39.6 34.7 17.9 10.2 7.5 0.0
Mean - - 0.0 0.0 7.4 47.3 68.5 93.8 76.3 71.1 88.9 98.8 97.4 71.5 63.2 43.3 23.1 12.7 8.43 3.4
STD - - 0.0 0.0 9.93 38.4 34.9 33.5 32.2 32.3 38.8 38.6 38.1 24.9 25.4 16.5 9.47 5.92 10.3 5.71
CV - - - - 134 81.2 50.9 35.7 42.2 45.4 43.6 39 39.1 34.9 40.2 38.1 41 46.7 122 168

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used. Measured content is 96%.

Table 13-C - Drug Concentrations* (µg/mL) for the Reference Formulation
ID Seq Per. Sampling Times (hours)
0.0 1.0 2.0 3.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 48.0
1 TR 30
Aug.		 0.0 0.0 0.0 0.0 54.2 133 85.2 87.7 131 143 104 83.6 53.4 40.9 19.6 9.3 0.0 0.0
2 RT 23
Aug.		 0.0 0.0 0.0 0.0 18 83.7 53.2 35 49.2 71.8 117 81.1 66.4 35.8 18.4 7.84 14.2 7.69
3 RT 23
Aug.		 0.0 0.0 0.0 15.9 112 210 100 93 178 211 243 145 107 64.1 30.5 15.5 11 0.0
4 TR 30
Aug.		 0.0 0.0 0.0 0.0 8.32 130 94 63.8 66.5 53.5 37 20.8 18.7 8.14 0.0 0.0 0.0 0.0
6 RT 23
Aug.		 0.0 0.0 0.0 0.0 42 104 65.7 48.7 92.3 129 160 126 116 66.5 26.7 13.3 8.8 0.0
7 TR 30
Aug.		 0.0 0.0 0.0 0.0 31.6 114 89.6 69 107 130 118 79.8 85 55.7 30.5 12.3 7.48 0.0
8 RT 23
Aug.		 0.0 0.0 0.0 0.0 0.0 46.5 32.4 19 27.2 35.9 37 33 28.4 19.6 11.6 8.43 5.58 0.0
9 RT 23
Aug.		 0.0 0.0 0.0 9.93 158 149 112 147 227 198 151 98.6 63.9 38.1 19.7 8.84 6.12 5.59
10 TR 30
Aug.		 0.0 0.0 0.0 0.0 13.4 124 90.4 47.9 41.4 53 89.5 114 143 105 53.7 30.1 19 9.63
12 TR 30
Aug.		 0.0 35.3 118 133 131 113 82.4 68.7 80.6 101 85.6 76.4 70.5 63.2 19.5 8.06 14.2 17.5
13 TR 30
Aug.		 0.0 0.0 0.0 0.0 0.0 59.3 49.3 34 65.7 81.1 83.4 66 71.6 53.3 26.8 17.4 10.4 8.23
14 RT 23
Aug.		 0.0 0.0 0.0 0.0 14.4 106 68.1 35.1 71.8 75.5 74.6 67.4 56.5 47.1 25.9 16.1 8.59 0.0
Mean - - 0 2.94 9.81 13.2 48.6 114 76.9 62.4 94.8 107 108 82.7 73.3 49.8 23.6 12.3 8.78 4.07
STD - - 0 102 34 37.9 54.7 42.4 23.4 35 58.6 56.6 56.8 35.5 35.4 24.9 12.8 7.35 5.59 5.74
CV - - - 346 346 287 113 37.1 30.5 56 61.8 52.9 52.5 43 48.2 50.1 54.3 59.9 63.6 14.1

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used. Measured content is 96%.

13.3 List of Parameters and Definitions

Table 13-D shows a list of the parameters used in the analysis and their definitions. If any other parameters are used, they must also be clearly defined.

Table 13-D - Parameter Definitions
Parameter Definition
Cmax Maximum observed concentration (µg/mL).
Tmax Sampling time at which Cmax occurred (h).
AUCX Area under the raw concentration versus time curve, over the dosing interval of the test formulation, calculated using the linear trapezoidal rule (µg•h/mL).
AUCτ Area under the raw concentration versus time curve calculated using the linear trapezoidal rule from time
0 to LQCT (µg•h/mL).
AUCI Area to infinity = AUCτ + CT/λ, where CT is the estimated concentration at LQCT (λg h/mL).
(AUCT ÷ AUCI) × 100 Percent of the area measured by AUCτ relative to the extrapolated total AUC (%).
(AUCX ÷ AUCI) × 100 Percent of the area measured by AUCX relative to the extrapolated total AUC (%).
λ Terminal disposition rate constant calculated from the points on the log-linear end of the concentration
versus time curve (h−1).
TLIN Time point where log-linear elimination begins (h).
LQCT Lowest Quantifiable Concentration Time. Time at which the last concentration occurred that is above the limit of quantitation (h).
T½ Drug half-life = In2÷λ = 0.693÷λ (h).

13.4 Summaries of Parameter Estimates

Tables 13-E and 13-F list, for each subject, the estimates of the parameters defined in Table 13-D for the test and reference formulations respectively. Summary statistics (arithmetic means or medians, standard deviations, and CVs) should be given for each formulation.

The AUCX/AUCI ratio is used to determine whether the drug accumulates. For this example, the mean ratio of less than 80 percent indicates that the drug accumulates. Therefore, a multiple-dose study must be run.

The AUCτ/AUCI ratio is used to determine whether the subjects were sampled for a sufficient length of time. For this example the mean ratio is greater than 80 percent, indicating that the subjects were sufficiently sampled.


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Table 13-E - Summary of Parameters for Each Subject Given the Test Formulation
ID Seq Per. Parameters
Cmax
(µg
/mL)		 Tmax
(h)		 AUCX
(µg•h
/mL)		 AUCτ
(µg•h
/mL)		 AUCI
(µg•h
/mL)		 AUCX
÷
AUCI
(%)		 AUCτ
÷
AUCI
(%)		 λ
(h−1)		 TLIN
(h)		 LQCT
(h)		 T½
(h)
1 TR 23 Aug. 146 16.00 2321 2884 3001 77 96 0.0631 32 48.00 10.98
2 RT 30 Aug. 118 14.00 1612 1922 1977 82 97 0.1544 24.00 36 4.49
3 RT 30 Aug. 153 4.00 2433 3204 3257 75 98 0.0991 32.00 48 6.99
4 TR 23 Aug. 140 6.00 1958 2422 2242 86 99 0.1967 24.00 36.00 3.52
6 RT 30 Aug. 116 16.00 1967 2604 2641 74 99 0.1837 28.00 40.00 5.77
7 TR 30 Aug. 91 14.00 1303 1720 1793 73 96 0.1201 28.00 40.00 5.77
8 RT 30 Aug. 62 4.00 832 995 1047 79 95 0.1157 24.00 36.00 5.99
9 RT 30 Aug. 180 12.00 2619 3100 3138 83 99 0.1640 28.00 40.00 4.23
10 TR 23 Aug. 89 24.00 1484 2293 2367 63 97 0.1007 32.00 48.00 6.88
12 TR 23 Aug. 121 3.00 1809 2653 3431 53 77 0.0264 32.00 48.00 26.21
13 TR 23 Aug. 95 6.00 1388 1665 1739 80 96 0.1270 24.00 40.00 5.37
14 RT 30 Aug. 63 16.00 910 1256 1309 70 96 0.1290 28.00 40.00 5.37
Mean* - - 115 13.00 1720 2211 2331 75 95 0.1233 28.00 40.00 7.47
STD - - 36 6.57 572 713 782 9 6 0.0485 3.41 5.21 6.23
CV - - 32 58.37 33 32 34 13 6 39.32 12.18 12.61 83.3

* For Tmax, TLIN, and LQCT, these are the medians.

Table 13-F - Summary of Parameters for Each Subject Given the Reference Formulation
ID Seq Per. Parameters
Cmax
(µg
/mL)		 Tmax
(h)		 AUCX
(µg•h
/mL)		 AUCτ
(µg•h
/mL)		 AUCI
(µg•h
/mL)		 AUCX
÷
AUCI
(%)		 AUCτ
÷
AUCI
(%)		 λ
(h−1)		 TLIN
(h)		 LQCT
(h)		 T½
(h)
1 TR 30 Aug. 143 14.00 1993 2361 2429 82 97 0.1495 24.00 36.00 4.64
2 RT 23 Aug. 117 16.00 1420 1917 2113 67 91 0.0398 32.00 48.00 17.40
3 RT 23 Aug. 243 16.00 3292 3969 4035 82 98 0.1490 28.00 40.00 4.65
4 TR 30 Aug. 130 6.00 1060 1114 1193 89 93 0.1162 16.00 28.00 5.96
6 RT 23 Aug. 160 16.00 2159 2833 2879 75 98 0.1692 28.00 40.00 4.10
7 TR 30 Aug. 130 14.00 1911 2490 2530 76 98 0.1732 28.00 40.00 4.00
8 RT 23 Aug. 47 6.00 622 848 902 69 94 0.1022 28.00 40.00 6.78
9 RT 23 Aug. 227 12.00 2889 3342 3406 85 98 0.0722 32.00 48.00 9.59
10 TR 30 Aug. 143 24.00 1746 2941 3029 58 97 0.1042 32.00 48.00 6.65
12 TR 30 Aug. 133 3.00 2078 2737 1153 180 238 −0.010 32.00 48.00 −71.6
13 TR 30 Aug. 83 16.00 1236 1865 1965 63 95 0.0734 32.00 48.00 9.44
14 RT 23 Aug. 106 6.00 1341 1827 1890 71 97 0.1394 28.00 40.00 4.97
Mean* - - 138 14.00 1812 2354 2294 83 108 0.1066 28.00 40.00 0.55
STD - - 54 6.05 755 896 953 32 41 0.0551 4.66 6.27 23.04
CV - - 39 48.74 42 38 52 39 38 51.75 16.44 14.92 4220

* For Tmax, TLIN, and LQCT, these are the medians.

13.5 AUCτ Analysis

Tables 13-G, 13-H, and 13-I provide the complete analysis required for AUCτ. Table 13-G lists the AUCτ estimates on the raw scale and the log scale. Also given is the test AUCτ as a percentage of the reference AUCτ. Summary statistics are provided for each variable.

Table 13-G - AUCτ (µg·h/mL) Analysis-Data
ID Raw Scale Log Scale
Test AUCτ Reference
AUCτ		 Relative
AUCτ (%)		 Test
ln(AUCτ)		 Reference
ln(AUCτ)
1 2884.06 2360.72 122.17 7.97 7.77
2 1922.05 1916.80 100.27 7.56 7.56
3 3204.37 3968.85 80.74 8.07 8.29
4 2242.25 1113.89 201.30 7.72 7.02
6 2604.06 2832.96 91.92 7.86 7.95
7 1720.14 2489.54 69.09 7.45 7.82
8 995.12 848.23 117.32 6.90 6.74
9 3099.54 3342.32 92.74 8.04 8.11
10 2292.55 2941.48 77.94 7.74 7.99
12 2652.71 2737.45 96.90 7.88 7.91
13 1665.04 1865.01 89.28 7.42 7.53
14 1255.93 1827.23 68.73 7.14 7.51
Mean 962 1109 87 6.84 7.01
STD 220 125 18 0.25 0.11
CV 23 11 21 - -

Table 13-H gives the analysis of variance (ANOVA) for the cross-over design model for ln(AUCτ). This analysis gives the appropriate intra-subject variance estimate, MS (Residual), for the calculation of the 90% confidence interval. Any significant effects in the model, other than Subject (Seq), should be investigated. The intra-subject CV should also be calculated.

Table 13-H - AUCτ (µg·h/mL) Analysis-ANOVA for ln(AUCτ)
Source df SS MS F PR > F
Seq 1 0.00912 0.00912 0.02874 0.86877
Subject (Seq) 10 3.17243 0.31724 6.99908 0.00248
Period 1 0.02173 0.02173 0.47941 0.50445
Form 1 0.00844 0.00844 0.18618 0.67527
Residual 10 0.45326 0.04533 . .

Intra-subject CV − 100 × (MS Residual)0.5 = 100 × (0.0453)0.5 = 21 percent

The AUCτ ratio estimate and its 90% confidence interval are derived in the calculations shown in Table 13-I. Since this study is a balanced design
(i.e., equal number of subjects per sequence) then the difference is simply the difference in the arithmetic means of the ln(AUC)s. When the study is unbalanced, the lease-squares means estimate for each formulation and the standard error of the difference in least-squares means are used. For this example, the AUCτ ratio passes the bioequivalence standard since the lower and upper limits fall within 80% to 125%.

Table 13-I - AUCτ (µg·h/mL) Analysis-Calculations

Difference = Test x − Reference x = 7.6455 − 7.6830 = −0.0375

SEDifference = 0.0869

AUC Ratio = 100 × eDifference = 100 × e−0.0375 = 96%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.05, 10 × SEDifference)

Lower = 100 × e(−0.0375 − 1.812 × 0.0869) = 82%

Upper = 100 × e(−0.0375 + 1.812 × 0.0869) = 113%

13.6 Cmax Analysis

The necessary information and summary for the analyses of Cmax are shown in Tables
13-J, 13-K, and 13-L.

Table 13-J - Cmax (µg/mL) Analysis-Data
ID Raw Scale Log Scale
Test Cmax Reference
Cmax		 Relative
Cmax (%)		 Test
ln(Cmax)		 Reference
ln(Cmax)
1 146.26 143.1 102.21 4.99 4.96
2 118.45 116.60 101.59 4.77 4.76
3 153.47 242.74 63.22 5.03 5.49
4 140.08 130.38 107.44 4.94 4.87
6 116.39 160.06 72.72 4.76 5.08
7 90.74 130.38 69.60 4.51 4.87
8 62.21 46.53 133.69 4.13 3.84
9 180.25 226.84 79.46 5.19 5.42
10 88.99 143.10 62.19 4.49 4.96
12 120.51 132.50 90.95 4.79 4.89
13 94.86 83.42 113.71 4.55 4.42
14 63.45 106.00 59.86 4.15 4.66
Mean 114.64 138.47 88.05 4.69 4.85
STD 36.31 54.25 23.82 0.34 0.43
CV 31.67 39.18 27.05 - -


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Table 13-K - Cmax (µg/mL) Analysis-ANOVA for ln(Cmax)
Source df SS MS F PR > F
Seq 1 0.00009 0.00009 0.00032 0.98605
Subject (Seq) 10 2.91103 0.29110 7.55217 0.00183
Period 1 0.01058 0.00158 0.27454 0.61172
Form 1 0.15428 0.15428 4.00253 0.07331
Residual 10 0.38546 0.03855 . .

Intra-subject CV = 20 percent

Table 13-L - Cmax (µg/mL) Analysis-Calculations

Difference = Test x − Reference x = 4.6924 − 4.8527 = −0.1604

SEDifference = 0.0802

Cmax Ratio = 100 × eDifference = 100 × e−0.1604 = 85%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.05, 10 × SEDifference)

Lower = 100 × e(−0.1604 − 1.812 × 0.0802) = 74%

Upper = 100 × e(−0.1604 + 1.812 × 0.0802) = 99%

13.7 AUCI Analysis

The necessary information and summary for the analyses of AUCI are shown in Tables 13-M and 13-N.

Table 13-M - AUCI (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test AUCI Reference
AUCI		 Relative
AUCI (%)		 Test
ln(AUCI)		 Reference
ln(AUCI)
1 3001.16 2429.22 123.54 8.01 7.80
2 1976.50 2113.50 93.52 7.59 7.66
3 3257.08 4034.81 80.72 8.09 8.30
4 2270.34 1193.06 190.29 7.73 7.08
6 2641.31 2878.64 91.76 7.88 7.97
7 1793.06 2530.14 70.87 7.49 7.84
8 1047.44 902.05 116.12 6.95 6.80
9 3137.79 3405.78 92.13 8.05 8.13
10 2366.76 3029.45 78.13 7.77 8.02
12 3431.12 1152.57 297.69 8.14 7.05
13 1738.86 1964.50 88.51 7.46 7.58
14 1308.74 1890.16 69.24 7.18 7.54
Mean 2330.85 2293.66 116.04 7.69 7.65
STD 782.04 952.50 66.00 0.37 0.46
CV 33.55 41.53 56.87 - -
Table 13-N - AUCI (µg•h/mL) Analysis-ANOVA for ln(AUCI)
Source df SS MS F PR > F
Seq 1 0.00140 0.00140 0.00487 0.94572
Subject (Seq) 10 2.88134 0.28813 3.31638 0.03602
Period 1 0.15039 0.15039 1.73094 0.21765
Form 1 0.01331 0.01331 0.15321 0.70370
Residual 10 0.86882 0.08688 . .

Intra-subject CV = 20 percent

13.8 Tmax Analysis

The necessary information and summary for the analyses of Tmax are shown in Tables 13-O and 13-P.

Table 13-O - Tmax (h) Analysis-Data
ID Test
Tmax		 Reference
Tmax		 Difference
Tmax
1 16.00 15.00 2.00
2 14.00 16.00 −2.00
3 4.00 16.00 −12.00
4 6.00 6.00 0.00
6 16.00 16.00 0.00
7 14.00 14.00 0.00
8 4.00 6.00 −2.00
9 12.00 12.00 0.00
10 24.00 24.00 0.00
12 3.00 3.00 0.00
13 6.00 16.00 −10.00
14 16.00 6.00 10.00
Mean 11.25 12.42 −1.17
STD 6.57 6.05 5.56
Table 13-P - Tmax (h) Analysis-ANOVA
Source df SS MS F PR > F
Seq 1 2.667 2.6667 0.03784 0.84965
ID (Seq) 10 704.667 70.4667 4.15324 0.01723
Period 1 0.167 0.1667 0.00982 0.92301
Form 1 8.167 8.1667 0.48134 0.50361
Residual 10 169.667 16.9667 . .

Intra-subject CV = 35 percent
100 × (MS residual)0.5 ÷ MEAN Tmax = (100 × (16.9667)0.5 ÷ (11.25 + 12.43)) ÷ 2 = 35

13.9 λ Analysis

The necessary information and summary for analyses of λ(h−1) are shown in Tables 13-Q and 13-R.

Table 13-Q - λ (h−1) Analysis-Data
ID Test
λ 		Reference
λ 		Difference
λ
1 0.0631 0.1495 −0.0864
2 0.1544 0.0398 0.1145
3 0.0991 0.1490 −0.0499
4 0.1967 0.1162 0.0804
6 0.1837 0.1692 0.0145
7 0.1201 0.1732 −0.0532
8 0.1157 0.1022 0.0135
9 0.1640 0.0722 0.0917
10 0.1007 0.1042 −0.0035
12 0.0264 −0.0097 0.0361
13 0.1270 0.0734 0.0536
14 0.1290 0.1394 −0.0104
Mean 0.1233 0.1066 0.0167
STD 0.0485 0.0551 0.0619
CV 39.3183 51.7452 369.4071
Table 13-R - λ (h−1) Analysis-ANOVA
Source df SS MS F PR > F
Seq 1 0.003192 0.0031924 0.91030 0.36253
ID (Seq) 10 0.035070 0.0035070 1.73974 0.19802
Period 1 0.000898 0.0008985 0.44572 0.51948
Residual 10 0.020158 0.0020158 . .

Intra-subject CV = 39 percent


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13.10 Calculations for AUC Ratio and Cmax Ratio Estimates Corrected for Measured Content

Please refer to Section 11.10.

13.11 Concentration-Time Profiles (Subject 1)

Figure 13.1 gives a plot of the concentration-time profile for subject 1. Each plot must include profiles for all formulations given to that subject. Similar profiles should be given for each subject.

Figure 13.1: Concentration - Time Profile for Subject 1

Figure 13.1: Concentration-Time Profile for Subject 1

Figure 13.2 gives a plot of the ln(concentration)-time profile for subject 1. This plot must contain the regression lines from which the terminal disposition rate constants (λ) were estimated. This line must start and end at the time points considered to be in the log-linear elimination phase. Any point that was not used to estimate the regression line must be identified.

Figure 13.2: Ln(concentration)-Time Profile for Subject 1

Figure 13.2: Ln(concentration)-Time Profile for Subject 1

Figure 13.3 shows a profile of the arithmetic means over all subjects for each sampling
time.

Figure 13.3: Average Concentration-Time Profile for All Subjects

Figure 13.3: Average Concentration-Time Profile for All Subjects

Figure 13.4 shows a profile of the ln(arithmetic means) over all subjects for each sampling time.

Figure 13.4: Ln(average concentration)-Time Profile for All Subjects

Figure 13.4: Ln(average concentration)-Time Profile for All Subjects

14 Sample Analysis for a Multiple-Dose Bioequivalence Study

The following tables and figures illustrate data collected and used in a sample multi-dose comparative bioavailability study. An analysis of this data is also shown.

Although a comparative bioavailability study may include many formulations, the basic analysis is the same-each test formulation is compared to a reference formulation.

The analysis of a multi-dose comparative bioavailability study must have the following sections:

  1. A randomization scheme for the design, where all subjects randomized into the study are included and identified by code, sequence, and dates of the dosing periods for both test and reference formulations. (See Section 14.1.)
  2. A summary of drug concentrations (visual and quantitative) at each sampling time for each subject for both the test and reference formulations. (See Section 14.2.)
  3. A summary of the estimates of the parameters as defined in Section 14.3 for both test and reference formulations, including the means, standard deviations, and CVs. (See Section 14.4.)
  4. A summary of pre-dose concentrations of the test and reference formulations. (See Section 14.5.)
  5. A formal statistical analysis of the relevant parameters with comparisons of the test formulations to the reference formulations. (See Sections 14.6 through 14.9.)
  6. A summary of corrections for potency (measured content) in estimates. (See Section 14.10.)
  7. Sample concentration-time profiles that should be provided for each subject. (See Section 14.11.)

All the sample statistical analyses that follow have a minimum of two formulations (test and reference) given on two dosing days or periods.


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14.1 Randomization Scheme of the Design

Shown in Table 14-A is the randomization scheme for the cross-over design used in the study. In any study, all subjects who were randomized into the study must be included. Even those subjects that did not complete the study must be included and identified accordingly. Subject numbers that appear on informed consent forms and reporting forms must be given. Also, if any other subject identification code was used, it should be given here. The sequence to which the subject was randomized should be given. Finally, all dosing periods and dates must be given.

14.2 Summary of Drug Concentrations

Tables 14-B and 14-C show a list of the concentrations at each sampling time for each subject for the test and reference formulations, respectively. If any concentration is missing, it should be identified, and the reason it is missing given (e.g., lost sample; sample not collected).

Although no formal statistical analysis is required at each sampling time, it is recommended that summary statistics be given at each sampling time for each formulation. It is also helpful if the limit of quantitation of the analytical method is given at this table.

Table 14-A - Randomization Scheme of the Cross-over Design for the Comparison of Test (T) Versus Reference (R) Formulations
Subject Period
Number ID Sequence Sept. 3, 1995 Sept. 10, 1995
001 1 TR T R
002* 2 RT R -
003** 3 RT - -
004 4 RT R T
005 5 RT R T
006 6 TR T R
007 7 RT R T
008 8 TR T R
009** 9 TR - -
010 10 RT R T
011* 11 TR T -
012 12 TR T R
013 13 TR T R
014 14 RT R T
015 15 TR T R
016 16 RT R T
017 17 RT R T
018 18 TR T R
019 19 TR T R
020 20 RT R T

* Subject did not appear for second period.
** Subject did not appear for either period.

Table 14-B - Drug Concentrations* (µg/mL) for the Test Formulation
ID Seq Per. Sampling Times (hours)
0 1.0 2.0 3.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 20.0 24.0
1 TR 03 Sep. 162.0 177.0 163.0 162.0 178.0 267.0 227.0 207.0 219.0 255.0 272.0 194.0 169.0
4 RT 10 Sep. 245.0 258.0 225.0 227.0 214.0 258.0 276.0 243.0 188.0 184.0 179.0 142.0 192.0
5 RT 10 Sep. 128.0 123.0 126.0 126.0 135.0 283.0 217.0 146.0 219.0 170.0 160.0 145.0 86.3
6 TR 03 Sep. 113.0 81.6 92.0 111.0 135.0 225.0 231.0 144.0 132.0 105.0 110.0 98.2 85.8
7 RT 10 Sep. 105.0 124.0 109.0 124.0 124.0 245.0 182.0 135.0 117.0 137.0 161.0 114.0 129.0
8 TR 03 Sep. 49.6 49.5 81.6 106.0 125.0 188.0 138.0 102.0 113.0 123.0 98.0 81.4 65.2
10 RT 10 Sep. 121 148.0 173.0 239.0 242.0 327.0 235.0 166.0 193.0 239.0 260.0 144.0 130.0
12 TR 03 Sep. 81.9 82.2 84.5 91.3 112.0 162.0 147.0 110.0 113.0 119.0 117.0 81.2 75.8
13 TR 03 Sep. 45.7 51.2 73.0 73.7 99.1 128.0 105.0 71.1 59.9 69.2 65.7 55.8 42.4
14 RT 10 Sep. 202.0 323.0 240.0 285.0 386.0 420.0 316.0 244.0 258.0 198.0 188.0 134.0 142.0
15 TR 03 Sep. 101.0 99.1 96.3 108.0 117.0 218.0 222.0 163.0 161.0 175.0 173.0 122.0 92.1
16 RT 10 Sep. 135.0 124.0 125.0 142.0 160.0 316.0 216.0 175.0 202.0 236.0 212.0 163.0 94.2
17 RT 10 Sep. 74.9 72.8 69.6 65.1 133.0 76.9 118.0 101.0 112.0 128.0 119.0 104.0 99.4
18 TR 03 Sep. 62.8 55.9 56.8 67.8 82.5 144.0 115.0 97.4 102.0 99.4 84.0 52.6 30.6
19 TR 03 Sep. 117 119.0 97.2 108.0 112.0 160.0 125.0 127.0 162.0 151.0 123.0 67.0 46.4
20 RT 10 Sep. 40.9 35.1 55.1 64.7 90.4 119.0 95.6 75.3 68.8 71.4 75.1 64.2 46.8
Mean - - 112 120.0 117.0 131.0 153.0 221.0 185.0 144.0 151.0 154.0 150.0 110.0 95.4
STD - - 56.2 77.9 56.3 66.0 75.7 90.1 66.4 53.5 57.7 57.9 62.1 41.6 46.5
CV - - 50.4 64.8 48.3 50.3 49.5 40.8 35.8 37.1 38.1 37.7 41.5 37.8 48.7

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.


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Table 14-C - Drug Concentrations* (µg/mL) for the Reference Formulation
ID Seq Per. Sampling Times (hours)
0 1.0 2.0 3.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 20.0 24.0
1 TR 10 Sep. 200.0 199.0 172.0 169.0 153.0 139.0 153.0 250.0 167.0 189.0 181.0 163.0 194.0
4 RT 03 Sep. 295.0 302.0 289.0 263.0 232.0 199.0 216.0 251.0 246.0 226.0 193.0 137.0 235.0
5 RT 03 Sep. 125.0 124.0 111.0 101.0 100.0 306.0 201.0 131.0 96.5 99.6 93.1 109.0 163.0
6 TR 10 Sep. 162.0 155.0 145.0 124.0 124.0 296.0 318.0 212.0 175.0 216.0 334.0 172.0 152.0
7 RT 03 Sep. 190.0 144.0 183.0 155.0 156.0 233.0 208.0 171.0 188.0 145.0 172.0 123.0 138.0
8 TR 10 Sep. 109.0 102.0 73.1 57.20 44.1 50.6 78.4 77.0 88.8 154.0 108.0 111.0 87.5
10 RT 03 Sep. 128.0 141.0 118.0 95.3 96.5 357.0 245.0 162.0 162.0 217.0 189.0 166.0 122.0
12 TR 10 Sep. 144.0 237.0 131.0 127.0 182.0 217.0 170.0 133.0 102.0 102.0 119.0 107.0 112.0
13 TR 10 Sep. 72.1 82.6 60.8 60.4 41.4 75.3 116.0 81.8 93.1 97.4 98.4 84.3 62.4
14 RT 03 Sep. 211.0 209.0 208.0 217.0 246.0 328.0 258.0 212.0 282.0 258.0 288.0 191.0 209.0
15 TR 10 Sep. 231.0 230.0 205.0 186.0 151.0 198.0 271.0 188.0 161.0 169.0 144.0 121.0 124.0
16 RT 03 Sep. 79.3 74.8 63.9 60.6 50.4 204.0 122.0 190.0 175.0 156.0 176.0 113.0 80.8
17 RT 03 Sep. 138.0 126.0 112.0 101.0 93.7 149.0 131.0 107.0 111.0 136.0 122.0 145.0 136.0
18 TR 10 Sep. 74.6 76.2 71.0 58.3 51.0 98.5 131.0 91.2 72.3 63.1 64.4 51.3 83.7
19 TR 10 Sep. 111.0 99.9 87.7 80.8 68.2 127.0 162.0 117.0 160.0 182.0 159.0 91.4 71.3
20 RT 03 Sep. 74.2 69.2 62.5 63.0 43.2 122.0 83.6 61.4 61.3 70.3 68.7 77.6 64.4
Mean - - 147.0 148.0 131.0 120.0 114.0 194.0 179.0 149.0 146.0 155.0 157.0 123.0 127.0
STD - - 64.4 69.0 65.7 62.6 66.8 92.4 69.8 57.0 61.8 58.0 73.5 37.9 52.9
CV - - 43.9 46.5 50.2 52.2 58.3 47.7 39.0 38.3 42.3 37.5 46.9 30.9 41.6

* Limit of quantitation is 0.2 µg/mL. Any concentration below this limit is reported as Below Quantitation Limit (BQL) except at time 0 and times before first observed concentration. However, in the calculation of summary statistics a zero is used.

14.3 List of Parameters and Definitions

Table 14-D shows a list of the parameters used in the analysis and their definitions. If any other parameters are used, they must also be clearly defined.

Table 14-D - Parameter Definitions
Parameter Definition
Cmax Maximum observed concentration (µg/mL).
Cmin Minimum observed concentration (µg/mL).
Cpd Pre-dose concentration from same time of each day (µg/mL).
Tmax Sampling time at which Cmax occurred (h).
AUCτ Area under the concentration versus time curve, over the dosing interval of the test formulation, calculated using the linear trapezoidal rule (µg•h/mL).
Fluctuation (Cmax − Cmin) ÷ (AUCτ ÷ τ) × 100.

14.4 Summaries of Parameter Estimates

Tables 14-E and 14-F list, for each subject, the estimates of the parameters defined in Table 14-D for the test and reference formulations respectively. Summary statistics (arithmetic means or medians, standard deviations, and CVs) should be given for each formulation.

Table 14-E - Summary of Parameters for Each Subject Given the Test Formulation
ID Seq. Period Parameters
Cmax
(µg/mL)		 Cmin
(µg/mL)		 Tmax
(h)		 AUCτ
(µg•h/mL)		 FL**
(%)
1 TR 03 Sep. 272 16.00 162 5128 52
4 RT 10 Sep. 276 8.00 142 4941 65
5 RT 10 Sep. 283 6.00 86 3945 120
6 TR 03 Sep. 231 8.00 82 3111 115
7 RT 10 Sep. 245 6.00 105 3425 98
8 TR 03 Sep. 188 6.00 50 2527 132
10 RT 10 Sep. 327 6.00 121 4916 101
12 TR 03 Sep. 162 6.00 76 2600 79
13 TR 03 Sep. 128 6.00 42 1741 118
14 RT 10 Sep. 420 6.00 134 5786 119
15 TR 03 Sep. 222 8.00 92 3598 87
16 RT 10 Sep. 316 6.00 94 4464 119
17 RT 10 Sep. 133 4.00 65 2491 65
18 TR 03 Sep. 144 6.00 31 1976 138
19 TR 03 Sep. 162 12.00 46 2728 101
20 RT 10 Sep. 119 6.00 35 1748 116
Mean* - - 227 6.00 85 3445 102
STD - - 86 291 40 1293 25
CV - - 38 40.13 47 38 25

* For Tmax, this is the median.
** Fluctuation.

Table 14-F - Summary of Parameters for Each Subject Given the Reference Formulation
ID Seq. Period Parameters
Cmax
(µg/mL)		 Cmin
(µg/mL)		 Tmax
(h)		 AUCτ
(µg•h/mL)		 FL**
(%)
1 TR 10 Sep. 250 10.00 139 4249 63
4 RT 03 Sep. 302 1.00 137 5223 76
5 RT 03 Sep. 306 6.00 93 3262 157
6 TR 10 Sep. 334 16.00 124 5117 98
7 RT 03 Sep. 233 6.00 123 3982 66
8 TR 10 Sep. 154 14.00 44 2195 120
10 RT 10 Sep. 357 6 95 4326 145
12 TR 10 Sep. 237 1.00 102 3296 99
13 TR 10 Sep. 116 8.00 41 1985 90
14 RT 03 Sep. 328 6.00 191 5827 56
15 TR 10 Sep. 271 8.00 121 4099 88
16 RT 03 Sep. 204 6.00 50 3149 117
17 RT 03 Sep. 149 6.00 94 3036 44
18 TR 10 Sep. 131 8.00 51 1798 107
19 TR 10 Sep. 182 14.00 68 2908 94
20 RT 03 Sep. 122 6.00 43 1739 109
Mean* - - 230 7.63 95 3512 96
STD - - 81 4.19 43 1255 31
CV - - 35 54.99 46 36 32

* For Tmax, this is the median.
** Fluctuation.

14.5 Pre-dose Concentrations

Tables 14-G and 14-H list the pre-dose concentrations for each subject on each day of the study for the test and reference formulations respectively.

These tables are used to check for both compliance and whether steady-state concentrations had been reached during the study. Intra-subject statistics should be calculated for the last three pre-dose concentrations.


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Table 14-G - Pre-dose Concentrations (µg/mL) for Each Subject Given the Test Formulation
ID Day Intra-Subject Statistics*
4 5 6 7 8 Mean STD CV
1 175 156 147 157 164 156 9 5
4 195 234 181 238 186 202 32 16
5 93 118 142 124 84 117 30 26
6 119 103 91 110 83 95 14 15
7 121 110 102 102 125 110 13 12
8 76 56 50 48 63 54 8 16
10 89 106 89 117 126 111 19 17
12 83 81 75 80 74 76 3 4
13 47 61 42 44 41 43 2 4
14 247 193 144 196 138 159 32 20
15 69 69 63 98 89 84 18 22
16 220 84 112 131 92 112 20 18
17 90 89 76 73 97 82 13 16
18 38 25 38 61 30 43 16 38
19 96 99 101 114 45 87 37 42
20 44 46 47 40 45 44 4 9
Mean 113 102 94 108 93 - - -
STD 64 54 43 55 45 - - -
CV 57 53 45 50 49 - - -

* Based on concentrations from days 6, 7, and 8.

Table 14-H - Pre-dose Concentrations (µg/mL) for Each Subject Given the Reference Formulation
ID Day Intra-Subject Statistics*
4 5 6 7 8 Mean STD CV
1 172 147 154 189 183 175 19 11
4 217 230 240 278 222 247 29 12
5 157 215 162 118 154 145 23 16
6 252 180 163 153 143 153 10 7
7 181 195 173 179 130 161 27 17
8 116 74 57 103 83 81 23 29
10 160 115 82 121 115 106 21 20
12 112 134 117 136 106 120 15 13
13 74 50 56 68 59 61 6 10
14 145 155 141 199 197 179 33 18
15 172 70 101 218 117 145 63 44
16 137 61 85 75 76 79 6 7
17 114 127 145 130 128 134 9 7
18 29 41 41 70 79 63 20 32
19 122 71 86 105 67 86 19 22
20 45 50 49 70 61 60 11 18
Mean 138 120 116 138 120 - - -
STD 58 63 56 61 50 - - -
CV 42 53 48 44 42 - - -

* Based on concentrations from days 6, 7, and 8.

A repeated measures analysis of the last three pre-dose concentrations should be provided. An example of this analysis is shown in Table 14-I. Of main importance in this analysis is the time and time×form interaction. Should either the time or time×form effects be significant, the study may not have been at steady state for one or both formulations. For this example, although the time term is significant, the significance is due to a higher value for seventh day Cpd. Since there is no increase in the means for the three Cpd, steady state is assumed.

Table 14-I - Repeated Measures Analysis of ln(Cpd)
Source df SS MS F PR > F
Seq 1 2.347 2.347 2.14 0.166
ID (Seq) 14 15.348 1.096 - -
Period 1 0.186 0.186 2.53 0.134
Form 1 1.573 1.573 21.41 < 0.001
ID×Form (Seq) 14 1.029 0.073 2.28 0.031
Time 2 0.529 0.265 6.16 0.006
Time×Seq 2 0.130 0.065 1.51 0.238
Time×ID (Seq) 28 1.201 0.043 1.34 0.216
Time×Period 2 0.029 0.014 0.45 0.643
Time×Form 2 0.047 0.023 0.74 0.488
Time×Form×ID (Seq) 28 0.889 0.032 - -
Corrected Total 95 - - - -

14.6 AUCτ Analysis

Tables 14-J, 14-K and 14-L provide the complete analysis required for AUCτ. Table 14-J lists the AUCτ estimates on the raw scale and the log scale. Also given is the test AUCτ as a percentage of the reference AUCτ. Summary statistics are calculated for each variable.

Table 14-J - AUCτ (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test
AUCτ		 Reference
AUCτ		 Relative
AUCτ (%)		 Test
ln(AUCτ)		 Reference
ln(AUCτ)
1 5214.31 4248.69 122.73 8.56 8.35
4 4940.91 5223.15 94.60 8.51 8.56
5 3945 3262.36 120.92 8.28 8.09
6 3111.42 5116.62 60.81 8.04 8.54
7 3424.75 3982.42 86.00 8.14 8.29
8 2526.8 2194.62 115.14 7.83 7.69
10 4916.19 4325.75 113.65 8.50 8.37
12 2599.77 3296.18 78.87 7.86 8.10
13 1740.8 1985.33 87.68 7.46 7.59
14 5786.03 5827.35 99.29 8.66 8.67
15 3598.41 4099.02 87.79 8.19 8.32
16 4464.02 3149.42 141.74 8.40 8.05
17 2490.9 3036.37 82.04 7.82 8.02
18 1976 1798.24 109.89 7.59 7.49
19 2728.37 2908.06 93.82 7.91 7.98
20 1747.55 1738.93 100.50 7.47 7.46
Mean 3450.7 3512.03 99.72 8.08 8.10
STD 1300.28 1254.67 20.13 0.39 0.38
CV 37.68 35.19 20.19 - -

Table 14-K gives the analysis of variance (ANOVA) for the cross-over design model for ln(AUCτ). This analysis gives the appropriate intra-subject variance estimate, MS (Residual), for the calculation of the 90% confidence interval. Any significant effects in the model, other than Subject (Seq), should be investigated. The intra-subject and inter-subject CVs should also be calculated.

Table 14-K - AUCτ (µg•h/mL) Analysis-ANOVA for ln(AUCτ)
Source df SS MS F PR > F
Seq 1 0.44521 0.44521 1.6849 0.21525
ID (Seq) 14 3.69921 0.26423 12.4240 0
Period 1 0.02422 0.02422 1.1389 0.30394
Form 1 0.00405 0.00405 0.1905 0.66917
Residual 14 0.29775 0.02127 - -

Intra-subject CV = 100 × (MS Residual)0.5 = 100 × (0.0213)0.5 = 15 percent

The AUCτ ratio estimate and its 90% confidence interval are derived in the calculations shown in Table 14-L. If this study had a balanced design (i.e., an equal number of subjects per sequence) the difference would simply be the difference in the arithmetic means of the ln(AUC)s. Since the study was not balanced, the least-squares mean estimate for each formulation is used to form this difference, together with the appropriate standard error.


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Table 14-L - AUCτ (µg•h/mL) Analysis-Calculations

Difference = Test − Reference = 8.0768 − 8.0992 = −0.0225

SEDifference = 0.0516

AUCτ Ratio = 100 × eDifference = 100 × e−0.235 = 98%

90% Confidence Limits

Lower, Upper = 100 × e(Difference ± t0.025, 14 × SEDifference)

Lower = 100 × e(−0.0225 − 1.761 × 0.0516) = 89%

Upper = 100 × e(−0.0225 + 1.761 × 0.0516) = 107%

14.7 Cmax Analysis

The necessary information and summary for the analyses of Cmax are shown in Tables 14-M, 14-N, and 14-O.

For this example, the Cmax ratio passes the bioequivalence criterion.

Table 14-M - Cmax (µg/mL) Analysis-Data
ID Raw Scale Log Scale
Test
Cmax		 Reference
Cmax		 Relative
Cmax (%)		 Test
ln(Cmax)		 Reference
ln(Cmax)
1 271.92 250.00 108.77 5.61 5.52
4 276.04 302.10 91.37 5.62 5.71
5 283.25 306.34 92.46 5.65 5.72
6 230.72 333.90 69.10 5.44 5.81
7 245.14 233.20 105.12 5.50 5.45
8 188.49 153.70 122.64 5.24 5.04
10 326.51 357.22 91.40 5.79 5.88
12 161.71 237.44 68.11 5.09 5.47
13 127.72 115.54 110.54 4.85 4.75
14 420.24 327.54 128.30 6.04 5.79
15 222.48 271.36 81.99 5.40 5.60
16 316.21 203.52 155.37 5.76 5.32
17 132.87 149.46 88.90 4.89 5.01
18 144.20 131.44 109.71 4.97 4.88
19 161.71 182.32 88.70 5.09 5.21
20 119.48 121.90 98.01 4.78 4.80
Mean 226.79 229.81 100.66 5.36 5.37
STD 85.75 81.44 22.27 0.38 0.38
CV 37.81 35.44 22.12 - -
Table 14-N - Cmax (µg/mL) Analysis-ANOVA for ln(Cmax)
Source df SS MS F PR > F
Seq 1 0.0287 0.0287 1.44 0.258
Subject (Seq) 10 0.1198 0.200 2.27 0.106
Period 1 0.412 0.12 4.68 0.056
Form 1 0.3591 0.3591 4.09 < 0.001
Residual 10 0.0879 0.0088 - -

Intra-subject CV = 15 percent

Table 14-O - Cmax Analysis-Calculations

Cmax Ratio = 100 × eDifference = 100 × e(5.3564 − 5.3724) = 98%

14.8 Cmin Analysis

The necessary information and summary for the analyses of Cmin are shown in Tables 14-P, 14-Q, and 14-R.

For this example, the Cmin ratio meets the bioequivalence criterion.

Table 14-P - Cmin (µg•h/mL) Analysis-Data
ID Raw Scale Log Scale
Test
Cmin		 Reference
Cmin		 Relative
Cmin (%)		 Test
ln(Cmin)		 Reference
ln(Cmin)
1 161.71 138.86 116.46 5.09 4.93
4 142.14 136.74 103.95 4.96 4.92
5 86.31 93.07 92.74 4.46 4.53
6 81.58 124.02 65.78 4.40 4.82
7 105.06 122.96 85.44 4.65 4.81
8 49.54 44.10 112.35 3.90 3.79
10 120.51 95.29 126.46 4.79 4.56
12 75.81 101.55 74.65 4.33 4.62
13 42.44 41.45 102.39 3.75 3.72
14 133.90 190.80 70.18 4.90 5.25
15 92.08 120.84 76.20 4.52 4.79
16 94.25 50.35 187.18 4.55 3.92
17 65.10 93.70 69.47 4.18 4.54
18 30.59 50.99 60.00 3.42 3.93
19 46.35 68.16 68.00 3.84 4.22
20 35.12 43.25 81.21 3.56 3.77
Mean 85.16 94.76 93.28 4.33 4.45
STD 39.87 43.18 32.11 .051 0.49
CV 46.82 45.57 34.42 - -
Table 14-Q - Cmin (µg/mL) Analysis-ANOVA for ln(Cmin)
Source df SS MS F PR > F
Seq 1 0.56639 0.56639 1.2684 0.279
ID (Seq) 14 6.25139 0.44653 10.0826 0.00005
Period 1 0.05514 0.05514 1.2450 0.2833
Form 1 0.10647 0.10647 2.4041 0.14332
Residual 14 0.62002 0.04429 - -

Intra-subject CV = 21 percent

Table 14-R - Cmin Analysis-Calculations

Cmin Ratio = 100 × eDifference = 100 × e(4.3303 − 4.4457) = 89%

14.9 Fluctuation Analysis

The necessary information and summary for the analyses of Fluctuation are provided in Tables 14-S and 14-T.

Table 14-S - Fluctuation (%) Analysis-Data
ID Test
Fluctuation		 Reference
Fluctuation
1 50.7266 62.7808
4 65.0407 75.9817
5 119.8089 156.8964
6 115.0424 98.4462
7 98.1654 66.436
8 131.9746 119.8609
10 100.5657 145.3209
12 79.3001 98.9452
13 117.5788 89.5699
14 118.7717 56.3165
15 86.9705 88.1303
16 119.3355 116.7225
17 65.3007 44.0705
18 137.9864 107.3772
19 101.4761 94.2171
20 115.8518 108.5523
Mean 101.4935 95.6015
STD 25.5512 30.7625
CV 25.1752 32.1779


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Table 14-T - Fluctuation Analysis-ANOVA for Fluctuation
Source df SS MS F PR > F
Seq 1 1.64 1.64 0.00125 0.97234
ID (Seq) 14 18441.42 1317.24 3.34187 0.01553
Period 1 26.62 26.62 0.06753 0.79875
Form 1 277.73 277.73 0.70460 0.41535
Residual 14 5518.3 394.16 - -

Intra-subject CV = 20 percent

14.10 Calculations for AUCτ Ratio, Cmax Ratio, and Cmin Ratio Estimates Corrected for Measured Content

Please refer to Section 12.10.

14.11 Concentration-Time Profiles (Subject 1)

Please refer to Section 12.11.

Figure 14.1 shows a plot of the concentration-time profile for subject 1. Each plot must include profiles for all formulations given to that subject. Similar profiles should be given for each subject.

Figure 14.1: Concentration- Time Profile for Subject 1

Figure 14.1 Concentration-Time Profile for Subject 1

Figure 14.2 gives a plot of the ln(concentration)-time profile for subject 1. This plot must contain the regression lines from which the terminal disposition rate constants (λ) were estimated. This line must start and end at the time points considered to be in the log-linear elimination phase. Any point that was not used to estimate the regression line must be identified.

Figure 14.2: LN(concentration)- Time Profile for Subject 1

Figure 14.2: Ln(concentration)-Time Profile for Subject 1

Figure 14.3 shows a profile of the arithmetic means over all subjects for each sampling time.

Figure 14.3: Average Concentration- Time Profile for All Subjects

Figure 14.3: Average Concentration-Time Profile for All Subjects

Figure 14.4 shows a profile of the ln(arithmetic means) over all subjects for each sampling time.

Figure 14.4: Ln(average concentration)- Time Profile for All Subjects

Figure 14.4: Ln(average concentration)-Time Profile for All Subjects

15 Glossary of Terms

Accuracy - The extent to which an experimentally determined value agrees with the true value.

Adverse Drug Reaction - An undesirable effect that is suspected to be associated with the use of a drug by a subject.

ANOVA Appropriate To the Design - The ANOVA (analysis of variance) selected to reflect the effects of the way in which the study was executed. For the two-period cross-over design these effects include: sequence, subject (within sequence), period, formulation and the residual error term which is at times referred to (as an approximation) as within-subject or intra-subject error.

AUC (Area Under the Curve) - The area under the drug (or metabolite) concentration in plasma (or serum, or whole blood) versus time curve. The AUC symbol may be qualified by a specific time (e.g., 8 hours, or AUC8), time of last quantifiable concentration (AUCτ), or infinity (AUCI). AUC is calculated from observed data at specific time points.

AUCI (AUC To Infinity) - The area obtained by extrapolating to infinity the AUCτ. This area can be calculated by adding CT/λ to AUCτ where CT is the last quantifiable concentration and λ is the terminal disposition rate constant.


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AUCτ (AUC To the Time of the Last Quantifiable Concentration) - AUCτ is calculated from the data observed at specific time points by the linear trapezoidal rule.

AUCτ (AUC Over the Steady-State Dosage Interval) - Area under the curve for one dosing interval (τ) at steady state. AUCτ is evaluated by the linear trapezoidal rule.

AUCX (AUC Over the Normal Dosing Interval) - Area under the curve during the usual dosing interval (0-X) following a single dose of the MR product. AUCX is evaluated by the linear trapezoidal rule.

AUC Ratio - The ratio of geometric means of the test and reference AUCs. It is calculated as the antilogarithm of the difference between the means of the logarithms (ln) of the test and reference AUCs. The Cmax and Cmin ratios should be similarly calculated. (See Sections 11, 12, 13 and 14.)

Balanced Cross-Over Design - A cross-over design in which subjects are randomly assigned into each sequence in equal numbers.

Bioavailability - The rate and extent to which a drug reaches the systemic circulation.

Bioequivalence - A high degree of similarity in the bioavailabilities of two pharmaceutical products (of the same galenic form) from the same molar dose, that are unlikely to produce clinically relevant differences in therapeutic effects, or adverse effects, or both.

Bioequivalent means that test and reference products containing an identical drug or drugs, after comparison in an appropriate bioavailability study, were found to meet the standards for rate and extent of absorption specified in these guidances.

Chiral Effects - Stereoselective effects on the disposition of drugs.

Cmax (Maximum Observed Concentration) - The observed maximum or peak concentration of drug (or metabolite) in plasma (or serum, or whole blood).

Cmin (Minimum Observed Concentration) - The observed minimum concentration at steady state. This observation is distinguished from the concentration measured immediately pre-dose at steady state (Cpd), although it may be quantitatively identical.

Cpd (Pre-dose Observed Concentration) - The concentration observed immediately before administering a dose at or near steady state.

CT (Last Quantifiable Concentration) - The last concentration of the drug (or metabolite) profile in plasma (or serum, or blood) that can be quantified and is equal to or larger than the limit of quantitation.

Comparable Bioavailability (for Group II Drug Products) - The similarity between the rate and extent to which the same molar doses of two drug products reach the systemic circulation. The comparison is between the first-market entry modified-release formulation and the innovator's conventional-release product that the MR formulation is intended to replace.

Conventional Formulation - A product formulated in a conventional manner for rapid disintegration or dissolution and systemic absorption.

Dose-Dumping - The unintended sudden release of large amounts of drug into the systemic circulation.

Excipient - Any ingredient, excluding the drug substances, incorporated in a formulation for the purpose of enhancing stability, usefulness, or appearance, or for facilitating preparation; for example, base, carrier, coating, colour, flavour, preservative, stabilizer, and vehicle.

First-Pass (or Pre-Systemic) Metabolism - The metabolism of an orally administered drug occurring during its first pass through certain metabolizing organs, before reaching the systemic circulation. Although the major site of first-pass metabolism is the liver, first-pass metabolism may also occur in the intestine or portal blood owing to the presence of enzymes (e.g., esterases). First-pass metabolism is usually assessed by comparing areas under the curve obtained following oral and intravenous doses of the drug.

Fluctuation (Cmax − Cmin) ÷ (AUCτ ÷ τ) ×100 - The range of steady-state concentrations divided by the average concentration (in %).

Formulation - An ingredient or mixture of specific ingredients; that is, drug substances and excipients in specific amounts, defining a given product.

Genetic Phenotype - A category or group to which a person may be assigned on the basis of differences in drug metabolism attributable to genetic characteristics (e.g., slow or fast metabolism).

HC - Health Canada.

Label - Includes any legend, word, or mark attached to, included in, belonging to, or accompanying any drug or package. (Section 2 of the Food and Drugs Act.)

Last Quantifiable Concentration (CT) - See CT.

Limit Of Detection (LOD) - The lowest concentration that can be differentiated from background levels.

Limit Of Quantitation (LOQ) - The lowest measured concentration on the standard curve having an acceptable degree of precision and accuracy. The LOQ cannot be below the lowest nominal concentration on the same standard curve.

Maximum Observed Concentration (Cmax) - See Cmax.

Measured Content of the Drug Product - The drug content of representative samples (i.e., the lots used in the bioavailability/bioequivalence study) of the test and reference drug products established as percent of label claim by an appropriate assay, such as USP.

Minimum Observed Concentration (Cmin) - See Cmin.

Modified-Release Dosage Form - A dosage form for which the drug-release characteristics of time-course or drug-release location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms.*

MRC - Medical Research Council of Canada.

90% Confidence Interval - An interval about the estimated value that provides 90% assurance that it contains the true value. The method of constructing the interval is described in Sections 11, 12, 13 and 14.

Non-Linear Kinetics - A general term referring to dose or time dependency in pharmacokinetic parameters arising from factors associated with absorption, first-pass metabolism, binding, and excretion.**

Precision - The closeness of agreement of values obtained in the analysis of replicate samples of the same specimen, usually indicated by the coefficient of variation (relative standard deviation).

Pre-dose Observed Concentration (Cpd) - See Cpd.

* Adapted from USP XXI (1985).

** Source: Ludden T.M. Non-Linear Pharmocokinetics. Clin Pharmacokinet, 1991, 20(6) 429-446

Pro-Drug - An inactive (or much less active) precursor that is transformed in vivo to the active drug.

Rate of Absorption - The rate at which a drug reaches the systemic circulation after oral administration.

Side-Effects - Drug-related effects not usually associated with the primary purpose of the therapy.

Standard Meal - A meal of known and fixed caloric content and carbohydrate, protein, fat, and fluid composition.

Suitability of Facilities - The physical plant and capability of a facility involved in experiments with human subjects or in the analysis of biological samples. Facilities should conform to requirements for Good Clinical Practice or Good Laboratory Practice.*

Terminal Disposition Rate Constant (λ) - The rate constant estimated from the slope of the terminal portion of the ln(drug concentration) versus time curve. The terminal half-life (T½) is calculated from this constant (T½=ln2÷λ). (Also known as Terminal Elimination Rate Constant.)

Terminal Elimination Rate Constant - See Terminal Disposition Rate Constant (λ).

Terminal Half-Life - See Terminal Disposition Rate Constant (λ).

Therapeutic Equivalence - Therapeutic equivalence means that a chemical equivalent of a drug product (i.e., containing the same amount of the same drug in the same dosage form) when administered to the same individuals in the same dosage regimen will provide essentially the same efficacy and toxicity.**

Time of Maximum Observed Concentration (Tmax) - The time after administration of the drug at which Cmax is observed.

* Source: Drug Bioequivalence. A Report of the Office of Technology AssessmentDrug Bioquivalence Study Panel Washington. 1974.

** Source: Proposed rule. Obligations of Clinical Investigators,. Federal Register, 43, 35210, 1978, and Code of Federal Regulations, 21, Part 58 revised April 1988.

Date Modified: 2009-09-09

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Important Notices