Drug Product Performance, In Vivo: Bioavailability and Bioequivalence PDF/PPT

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Drug Product Performance, In Vivo:
Bioavailability and Bioequivalence

M.Pharmacy II Semester,

Department of Pharmaceutics,



 Drug product performance

 Purpose of bioavailability studies

 Relative and Absolute availability

 Methods for Assessing Bioavailability

 Bioequivalence studies

 Design and Evaluation of bioequivalence studies

 Study designs

 Evaluation of the data

 Bioequivalence example

 Study submission and drug review process

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 Drug product performance, in vivo, may be defined as the release of the
drug substance from the drug product leading to bioavailability of the
drug substance.

 Bioavailability studies are drug product performance studies used to define
the effect of changes in the physicochemical properties of the drug
substance, the formulation of the drug, and the manufacture process of
the drug product (dosage form).

 Drug product performance studies are used in the development of new and
generic drug products.

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 Bioavailability is one aspect of drug product quality that links the in vivo
performance of a new drug product to the original formulation that was
used in clinical safety and efficacy studies.

 Bioequivalence studies are drug product performance tests that compare
the bioavailability of the same active pharmaceutical ingredient from one
drug product (test) to a second drug product (reference).

 Bioavailability and bioequivalence can be considered as measures of the
drug product performance in vivo.

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Bioequivalence Studies in New Drug Development (NDA)

 During drug development, bioequivalence studies are used to compare

(a) early and late clinical trial formulations;

(b) formulations used in clinical trials and stability studies, if different;

(c) clinical trial formulations and to-be-marketed drug products, if different; and

(d) product strength equivalence, as appropriate.

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Bioequivalence Studies in Generic Drug Development (ANDA)

 Comparative drug product performance studies are important in the development
of generic drug products.

 A generic drug product is a multisource drug product that has been approved by
the FDA as a therapeutic equivalent to the reference listed drug product (usually
the brand or innovator drug product) and has proven equivalent drug product

 Clinical safety and efficacy studies are not generally performed on generic drug

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 Since the formulation and method of manufacture of a drug product can affect
the bioavailability and stability of the drug, the generic drug manufacturer must
demonstrate that the generic drug product is pharmaceutically equivalent,
bioequivalent, and therapeutically equivalent to the comparator brand-name drug

 Drug product performance comparison for oral generic drug products is usually
measured by in vivo bioequivalence studies in normal healthy adult subjects
under fasted and fed conditions.

 Drug product performance comparisons in vitro may also include comparative
drug dissolution/release profiles.

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 Bioavailability studies are performed for both approved active drug ingredients
and therapeutic moieties not yet approved for marketing by the FDA.

 Bioavailability is defined as the rate and extent to which the active ingredient
or active moiety is absorbed from a drug product and becomes available at the
site of action.

 Bioavailability data provide an estimate of the fraction of drug absorbed from
the formulation, and provide information about the pharmacokinetics of the

 Relative bioavailability studies compare two drug product formulations.

 A bioequivalence study is a specialized type of relative bioavailability study.

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 Bioequivalence is defined as the absence of a significant difference in the rate
and extent to which the active ingredient or active moiety becomes available at
the site of drug action when administered at the same molar dose under similar
conditions in an appropriately designed study.

 Bioavailability and bioequivalence data play pivotal roles in regulatory
submissions for marketing approval of new and generic drugs throughout the

 Each regulatory agency has developed its own unique system of guidelines
advising new and generic drug applicants on how to conduct acceptable
bioavailability and bioequivalence studies to support marketing approval.

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 Bioequivalence studies are used to compare the bioavailability of the same
drug (same salt or ester) from various drug products.

 Bioavailability and bioequivalence can be considered as performance
measures of the drug product in vivo.

 If the drug products are pharmaceutically equivalent, bioequivalent, and
therapeutically equivalent, then the clinical efficacy and the safety profile of
these drug products are assumed to be similar and may be substituted for
each other.

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Relative and Absolute availability

 Absolute bioavailability compares the bioavailability of the active drug in the
systemic circulation following extravascular administration with the bioavailability of
the same drug following intravenous administration.

 The absolute bioavailability is calculated as follows:


 Fabs is the fraction of the dose absorbed, expressed as a percentage;

 AUCpo is the AUC following oral administration;

 Div is the dose administered intravenously;

 AUCiv is the AUC following intravenous administration; and

 Dpo is the dose administered orally.
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 Absolute availability, Fabs, may be expressed as a fraction or as a percent by
multiplying Fabs × 100.

 A drug given by the intravenous route will have an absolute bioavailability of
100% (f = 1).

 A drug given by an extravascular route may have an Fabs = 0 (no systemic
absorption) and Fabs = 1.0 (100% systemic absorption).

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Relative Bioavailability

 Another type of comparative bioavailability assessment is provided
by a relative bioavailability study.

 In a relative bioavailability study, the systemic exposure of a drug in
a designated formulation (generally referred to as treatment A or
reference formulation) is compared with that of the same drug
administered in a reference formulation (generally referred to as
treatment B or test formulation).

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 In a relative bioavailability study, the AUCs of the two
formulations are compared as follows:


 Frel is the relative bioavailability of treatment (formulation) A,
expressed as a percentage;

 AUCA is the AUC following administration of treatment
(formulation) A;

 DA is the dose of formulation A;

 AUCB is the AUC of formulation B; and

 DB is the dose of formulation B.

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Methods for Assessing Bioavailability

➢ Direct and indirect methods to assess bioavailability.

➢ The in-vivo bioavailability of a drug product is demonstrated by the rate
and extent of drug absorption.

➢ The design of the bioavailability study depends on

 The objectives of the study

 Ability to analyze the drug (and metabolites) in biological fluids

 Pharmacodynamics of the drug substance

 Route of drug administration

 Nature of the drug product.

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Plasma Drug Concentration
➢ Measurement of drug concentrations in blood, plasma, or serum after drug


➢ Most direct and objective way to determine systemic bioavailability.

➢ By blood sampling and performing validated drug assay, plasma drug
concentration-time profile of the therapeutically active drug substances can
be obtained.

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 The time of peak plasma concentration, tmax, corresponds to the time required
to reach maximum drug concentration after drug administration.

 At tmax, peak drug absorption occurs and the rate of drug absorption exactly
equals the rate of drug elimination

 Units for tmax are units of time (eg, hours, minutes).

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 The peak plasma drug concentration, Cmax, represents the maximum
plasma drug concentration obtained after oral administration of drug.

 Cmax provides indications that the drug is sufficiently systemically
absorbed to provide a therapeutic response.

 Cmax are concentration units (eg, mg/mL, ng/mL).

 The expectation is that as the rate of drug absorption goes up, the peak or
Cmax will also be larger. If the rate of drug absorption goes down, then the
peak or Cmax is smaller.

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 AUC: The area under the plasma level–time curve

 AUC, is a measurement of the extent of drug bioavailability.

 The AUC reflects the total amount of active drug that reaches the systemic

 The AUC is the area under the drug plasma level–time curve from t = 0 to t =
∞, and is equal to the amount of unchanged drug reaching the general
circulation divided by the clearance.

 where F = fraction of dose absorbed, D0 = dose, k = elimination rate constant,
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 The AUC can be determined by a numerical integration procedure, such
as the trapezoidal rule method.

 The units for AUC are concentration × time (eg, mg·h/mL).

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Urinary Drug Excretion Data

 Urinary drug excretion data is an indirect method for estimating

 The drug must be excreted in significant quantities as unchanged drug in
the urine.

 In addition, timely urine samples must be collected and the total amount of
urinary drug excretion must be obtained.

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 DU: The cumulative amount of drug excreted in the urine,

 Du, is related directly to the total amount of drug absorbed.

 When the drug is almost completely eliminated (point C), the plasma
concentration approaches zero and the maximum amount of drug excreted in the
urine, Du, is obtained.

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 dDu/dt: The rate of drug excretion.

 The rate of drug excretion is dependent on the first-order elimination rate
constant, k, and the concentration of drug in the plasma, Cp.

 The maximum rate of drug excretion, (dDu/dt)max, is at point B, whereas the
minimum rate of drug excretion is at points A and C.

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 t∞: The total time for the drug to be excreted.

 The slope of the curve segment A–B is related to the rate of drug
absorption, whereas point C is related to the total time required after
drug administration for the drug to be absorbed and completely excreted,
t = ∞.

 The t∞ is a useful parameter in bioequivalence studies that compare
several drug products.

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Bioequivalence Studies based on Pharmacodynamic
Endpoints—in vivo Pharmacodynamic (Pd) Comparison

 In some cases, the quantitative measurement of a drug in plasma is not
available or in vitro approaches are not applicable. The following criteria for a
PD endpoint study are important:

 A dose–response relationship is demonstrated.

 The PD effect of the selected dose should be at the rising phase of the dose–
response curve, as shown in Fig.

 Sufficient measurements should be taken to assure an appropriate PD response

 All PD measurement assays should be validated for specificity, accuracy,
sensitivity, and precision.

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Dose–response curves for dose versus response graphed
on a log scale

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 In this case, the acute pharmacodynamic effect is measured over a period of
time after administration of the drug product.

 Measurements of the pharmacodynamic effect should be made with
sufficient frequency for a time period at least three times the half-life of the

 This approach may be particularly applicable to dosage forms that are not
intended to deliver the active moiety to the bloodstream for systemic

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 For bioequivalence
parameters including

 The total area under the
acute pharmacodynamic
effect–time curve,

 Peak pharmacodynamic
effect, and

 Time for peak
pharmacodynamic effect
are obtained from the
pharmacodynamic effect–
time curve

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Bioequivalence Studies based on Clinical
Endpoints—clinical Endpoint Study

 The clinical endpoint study is the least accurate, least sensitive to bioavailability
differences, and most variable. A predetermined clinical endpoint is used to
evaluate comparative clinical effect in the chosen patient population.

 Clinical endpoint BE studies are recommended for those products that have
negligible systemic uptake, for which there is no identified PD measure, and for
which the site of action is local.

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In vitro Studies

 Comparative drug release/dissolution studies may give an indication of drug
bioavailability and bioequivalence.

 The in vitro drug dissolution rate should correlate with in vivo drug

 The test and reference products for which in vitro release rates form the basis
of the bioequivalence usually demonstrate Q1/Q2 sameness (qualitatively same
inactive ingredients in the quantitative same amounts).

 Comparative dissolution profiles may be considered similar if the similarity
factor (f2) is greater than 50.

 For drugs whose dissolution rate is related to the rate of systemic absorption,
the test formulation that demonstrates the most rapid rate of drug dissolution
in vitro will generally have the most rapid rate of drug bioavailability in vivo.

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Bioequivalence studies

 Differences in the predicted clinical response or an adverse event may be due to
differences in the pharmacokinetic and/or pharmacodynamic behavior of the drug
among individuals or to differences in the bioavailability of the drug from the
drug product.

 Bioequivalent drug products that have the same systemic drug bioavailability will
have the same predictable drug response.

 Bioequivalence is established if the in vivo bioavailability of a test drug product
(usually the generic product) does not differ significantly (ie, statistically not
significant) from that of the reference listed drug in the product’s rate and extent
of drug absorption.

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 Bioequivalence is determined by comparison of measured parameters (eg,
concentration of the active drug ingredient in the blood, urinary excretion rates, or
pharmacodynamic effects), when administered at the same molar dose of the active
moiety under similar experimental conditions, either single dose or multiple dose.

 A drug product that differs from the reference listed drug in its rate of absorption,
but not in its extent of absorption, may be considered bioequivalent if the
difference in the rate of absorption is intentional .

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Design and Evaluation of bioequivalence studies

 Objective:

The main objective for a bioequivalence study is that the drug
bioavailability from test and reference products is not statistically different when
administered to patients or subjects at the same molar dose from pharmaceutically
equivalent drug products through the same route of administration under similar
experimental conditions.

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 The basic design for a bioequivalence study is determined by

(1) The scientific questions and objectives to be answered,

(2) The nature of the reference material and the dosage form to be tested,

(3) The availability of analytical methods,

(4) The pharmacokinetics and pharmacodynamics of the drug substance,

(5) The route of drug administration, and

(6) Benefit–risk and ethical considerations with regard to testing in humans.

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Reference Listed Drug (RLD)

 For bioequivalence studies of generic products, one formulation of the
drug is chosen as a reference standard against which all other
formulations of the drug are compared.

 The FDA designates a single reference listed drug as the standard drug
product to which all generic versions must be shown to be bioequivalent.

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Regulatory Recommendations for Optimizing
Bioavailability Study Design

 Use of a randomized crossover design whenever possible

 Enrolling both male and female subjects whenever possible

 Administering single doses rather than multiple doses, as single-dose
studies are more sensitive, although multiple-dose studies may be more
suitable in some cases

 Conducting the studies under fasting and fed conditions.

 Measuring the parent drug rather than metabolites, unless the parent
cannot be reliably measured. Pre-systemically formed metabolites that
contribute meaningfully to safety and efficacy should also be measured

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 Cmax and tmax be measured to compare peak exposure and rate
of absorption.

 AUC0-t (AUC to the last measurable drug concentration) and
AUC0-∞ (AUC extrapolated to infinity) be measured to compare
total exposure or extent of drug absorption.

 Drug exposure parameters should be log-transformed before
statistical comparisons.

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Factors Influencing Bioavailability and Impact on Drug

 Physicochemical properties of the drug and formulation

 Drug stability and pH effects

 Pre-systemic and first-pass metabolism

 Prodrugs

 Food effects

 Effects of drug–drug interactions

 Efflux transporters

 Age

 Disease state
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 Two bioequivalence studies are required for solid oral dosage forms,

 Fasting study

 Food intervention study

 For extended-release capsules containing beads (pellets) “sprinkle”
bioequivalence study is recommended.

 Other study designs

 Parallel design,

 Replicate design,

 Multiple-dose (steady-state) bioequivalence studies

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Fasting study

 Bioequivalence studies are usually evaluated by a single-dose, two-period, two-
treatment, two-sequence, open-label, randomized crossover design comparing equal
doses of the test and reference products in fasted, adult, healthy subjects.

 This study is requested for all immediate-release and modified-release oral dosage

 The subjects should be in the fasting state (overnight fast of at least 10 hours) before
drug administration and should continue to fast for up to 4 hours after dosing.

 No other medication is given to the subject for at least 1 week prior to the study.

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Food Intervention Study

 Co-administration of food with an oral drug product may affect the
bioavailability of the drug.

 Food intervention or food effect studies are generally conducted using meal
conditions that are expected to provide the greatest effects on GI physiology
so that systemic drug availability is maximally affected.

 Food effects on bioavailability are generally greatest when the drug product is
administered shortly after a meal is ingested.

 The nutrient and caloric contents of the meal, the meal volume, and the meal
temperature can cause physiological changes in the GI tract in a way that
affects drug product transit time, luminal dissolution, drug permeability, and
systemic availability.

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 For bioequivalence studies for generic drugs, drug bioavailability from both the test
and reference products should be affected similarly by food.

 The usual study design uses a single-dose, randomized, two-treatment, two-period,
crossover study comparing equal doses of the test and reference products.

 Following an overnight fast of at least 10 hours, subjects are given the recommended
meal 30 minutes before dosing.

 The meal is consumed over 30 minutes, with administration of the drug product
immediately after the meal.

 The drug product is given with 240 mL (8 fluid oz) of water.

 No food is allowed for at least 4 hours post dose.

 This study is requested for all modified-release dosage forms and may be requested for
immediate-release dosage forms if the bioavailability of the active drug ingredient is
known to be affected by food (eg, ibuprofen, naproxen).

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 Extended-release capsules that contain coated beads, can be sprinkled over
soft foods such as applesauce.

 This is taken by the fasted subject and the bioavailability of the drug is then
measured for the NDA.

 For generic drug products in Abbreviated New Drug Applications (ANDAs),
this study is performed as a bioequivalence study to demonstrate that both
products, sprinkled on food, will have equivalent bioavailability.

 Bioavailability studies might also examine the effects of other foods and
special vehicles such as apple juice.

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Crossover Study Designs
 A complete crossover design, in which each subject receives the test drug product

and the reference product.

 The Latin-square design plans the clinical trial so that each subject receives each
drug product only once, with adequate time between medications for the
elimination of the drug from the body.

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Characteristics of Crossover study design:

 Each subject is his own control.

 Subject-to-subject variation is reduced.

 Variations due to sequence, period, and treatment (formulation) are reduced.

 The order in which the drug treatments are given should not stay the same in
order to prevent any bias in the data due to a residual effect from the previous

 Possible carryover effects from any particular drug product are minimized by
changing the sequence or order in which the drug products are given to the

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 Period refers to the time period in which a study is performed.

 A two-period study is a study that is performed on two different days (time periods)
separated by a washout period during which most of the drug is eliminated from the
body—generally about 10 elimination half-lives.

 A sequence refers to the number of different orders in the treatment groups in a

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Replicated Crossover Study Designs
 The standard bioequivalence criterion using the two-way crossover design does not

give an estimate of within-subject (intrasubject) variability.

 By giving the same drug product twice to the same subject, the replicate design
provides a measure for within-subject variability.

 Replicate design studies may be used for highly variable drugs and for narrow
therapeutic index drugs

 Replicate designs for highly variable drugs/products require a smaller number of
subjects and do not unnecessarily expose a large number of healthy subjects to a

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Narrow Therapeutic Index Drugs

 The FDA currently recommends that bioequivalence studies of narrow therapeutic
index drugs should employ a four-way, fully replicated, crossover study design.

 The replicated study design permits comparison of both test and reference means
and test and reference within-subject variability

 An additional test recommended in bioequivalence studies of generic narrow
therapeutic index drugs is a test for within-subject variability.

 The test determines whether within-subject variability of the test narrow therapeutic
index drug does not differ significantly from that of the reference by evaluating the
test/reference ratio of the within-subject standard deviation.

 The FDA currently recommends that all bioequivalence studies on narrow
therapeutic index drugs must pass both the reference-scaled approach and the
unscaled average bioequivalence limits of 80.00%–125.00%.

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Parallel Study Designs

 A non-replicate, parallel design is used for drug products that contain drugs that
have a long elimination half-life or drug products such as depot injections in
which the drug is slowly released over weeks or months.

 In this design, two separate groups of volunteers are used. One group will be
given the test product and the other group will be given the reference product.

 Cmax and a suitably truncated AUC, generally to 72 hours after dose
administration, can be used to characterize peak and total drug exposure,

 This design is not recommended for drugs that have high intrasubject variability in
distribution and clearance.

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Multiple-Dose (Steady-State) Study Design

 Multiple doses of the same drug are given consecutively to reach steady-state
plasma drug levels.

 The multiple-dose study is designed as a steady-state, randomized, two-
treatment, two-way, crossover study comparing equal doses of the test and
reference products in healthy adult subjects.

 The area under the curve during a dosing interval at steady state should be the
same as the area under the curve extrapolated to infinite time after a single

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 Pharmacokinetic analyses for multiple-dose studies include calculation of the
following parameters for each subject:

 AUC0-tau—Area under the curve during a dosing interval

 tmax—Time to Cmax during a dosing interval

 Cmax—Maximum drug concentration during dosing interval

 Cmin—Drug concentration at the end of a dosing interval

 Cav—The average drug concentration during a dosing interval

 Degree of fluctuation = (Cmax− Cmin)/Cmax

 Swing = (Cmax− Cmin)/Cmin

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 The data are analysed statistically using Analysis of Variance (ANOVA) on the
log-transformed AUC and Cmax.

 To establish bioequivalence, both AUC and Cmax for the test (generic) product
should be within 80%–125% of the reference product using a 90% confidence

 The extent of bioavailability, measured by assuming the [AUC]0, is dependent on

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 There are several disadvantages of using the multiple-dose crossover method
for the determination of bioequivalence:

 More time to reach steady-state condition.

 Greater clinical costs

 Possibility of a subject dropping out and not completing the study.

 More plasma samples must be obtained from the subject.

 Because Cav depends primarily on the dose of the drug and the time
interval between doses, the extent of drug systemically available is more
important than the rate of drug availability.

 Small differences in the rate of drug absorption may not be observed with
steady-state study comparisons

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Evaluation of the data

 For single-dose studies, including a fasting study or a food intervention
study, the pharmacokinetic analyses include calculation for each subject
of the area under the curve to the last quantifiable concentration (AUC) 0t
and to infinity (AUC)0 , tmax, and Cmax. Additionally, the elimination rate
constant, k, the elimination half-life, t1/2, and other parameters may be

 For multiple-dose studies, pharmacokinetic analysis includes calculation
for each subject of the steady-state area under the curve, t(AUC), tmax,
Cmin, Cmax, and the percent fluctuation [100 × (Cmax − Cmin)/Cmin].

 Proper statistical evaluation should be performed on the estimated
pharmacokinetic parameters.

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 Bioequivalence is generally determined using a comparison of population averages
of a bioequivalence metric, such as AUC and Cmax. This approach, termed average
bioequivalence, involves the calculation of a 90% confidence interval for the ratio of
averages (population geometric means) of the bioequivalence metrics for the test and
reference drug products.

 The two one-sided tests procedure is also referred to as the confidence interval
approach. This statistical method is used to demonstrate if the bioavailability of the
drug from the test formulation is too low or high in comparison to that of the
reference product. The objective of the approach is to determine if there are large
differences (i.e , greater than 20%) between the mean parameters.

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Bioequivalence example

 The results for a hypothetical bioavailability study in which three different tablet
formulations were compared to a solution of the drug given in the same dose in the
following table.

 As shown in the table, the bioavailability from all three tablet formulations was
greater than 80% of that of the solution.

 According to the ANOVA, the mean AUC values were not statistically different from
one another, nor different from that of the solution. However, the 90% confidence
interval for the AUC showed that for tablet A, the bioavailability was less than 80%
(i.e,74%), compared to the solution at the low-range estimate, and would not be
considered bioequivalent based on the AUC.

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Study submission and drug review process

 The contents of New Drug Applications (NDAs) and Abbreviated New Drug
Applications (ANDAs) are similar in terms of the quality of manufacture.

 The Generic drug manufacturer, the bioequivalence study is the pivotal study in the
ANDA that replaces the animal, clinical, and pharmacokinetic studies.

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 The investigator should be sure that the study has been properly designed, the
objectives are clearly defined, and the method of analysis has been validated.

 The results are analysed both statistically and pharmacokinetically.

 If necessary, an FDA investigator may inspect both the clinical and analytical
facilities used in the study and audit the raw data used in support of the
bioavailability study.

 For ANDA applications, the FDA Office of Generic Drugs reviews the entire ANDA
and if the application is incomplete, the FDA will not review the submission and the
sponsor will receive a Refusal to File letter

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Generic drug review process

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 Applied Biopharmaceutics and Pharmacokinetics by Shargel. L and Yu Andrew B.C,

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