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Basic Pharmacokinetics REV. 97.4.22 8-1Copyright 1996 Michael C. Makoid All Rights Reserved http://kiwi.creighton.edu/pkinbook/

CHAPTER 8 Bioavailability, Bioequivalence,

and Drug Selection

Author: Rasma Chereson

Reviewer: Umesh Banakar

OBJECTIVES

1. Given sufficient data to compare an oral product with another oral product or an

IV product, the student will estimate (III) the bioavailability (compare AUCs) and

judge (VI) professional acceptance of the product with regard to bioequivalence

(evaluate (VI) AUC, and ).

2. The student will write (V) a professional consult using the above calculations.

Tp Cp( )ma x


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Bioavailability, Bioequivalence, and Drug Selection


8.1 Bioavailability, Bioequivalence and Drug Product Selection

Bioavailability and bioequivalence of drug products, and drug product selection

have emerged as critical issues in pharmacy and medicine during the last threedecades. Concern about lowering health care costs has resulted in a tremendous

increase in the use of generic drug products; currently about one half of all pre-

scriptions written are for drugs that can be substituted with a generic product (1)

Over 80% of the approximately 10,000 prescription drugs available in 1990 were

available from more than one source (2). With the increasing availability and use

of generic drug products, health care professionals are confronted with an

ever-larger array of multisource products from which they must select those that

are therapeutically equivalent.

This phenomenal growth of the generic pharmaceutical industry and the abun-

dance of multisource products have prompted some questions among many healthprofessionals and scientists regarding the therapeutic equivalency of these prod-

ucts, particularly those in certain critical therapeutic categories such as anticonvul-

sants and cardiovasculars (1, 3-5). Inherent in the currently accepted guidelines

for product substitution is the assumption that a generic drug considered to be

bioequivalent to a brand-name drug will elicit the same clinical effect. As straight-

forward as this statement regarding bioequivalence appears to be, it has generated

a great deal of controversy among scientists and professionals in the health care

field. Numerous papers in the literature indicate that there is concern that the cur-

rent standards for approval of generic drugs may not always ensure therapeutic

equivalence (6-18).

The availability of different formulations of the same drug substance given at the

same strength and in the same dosage form poses a special challenge to health care

professionals, making these issues very relevant to pharmacists in all practice set-

tings. Since pharmacists play an important role in product-selection decisions

they must have an understanding of the principles and concepts of bioavailability

and bioequivalence.

8.1.1 RELATIVE AND ABSOLUTE BIOAVAILABILITY

Bioavailability is a pharmacokinetic term that describes the rate and extent towhich the active drug ingredient is absorbed from a drug product and becomes

available at the site of drug action. Since pharmacologic response is generally

related to the concentration of drug at the receptor site, the availability of a drug

from a dosage form is a critical element of a drug products clinical efficacy. How-

ever, drug concentrations usually cannot be readily measured directly at the site of


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action. Therefore, most bioavailability studies involve the determination of drug

concentration in the blood or urine. This is based on the premise that the drug a

the site of action is in equilibrium with drug in the blood. It is therefore possible to

obtain an indirect measure of drug response by monitoring drug levels in the blood

or urine. Thus, bioavailability is concerned with how quickly and how much of adrug appears in the blood after a specific dose is administered. The bioavailability

of a drug product often determines the therapeutic efficacy of that product since it

affects the onset, intensity and duration of therapeutic response of the drug. In

most cases one is concerned with the extent of absorption of drug, (that is, the frac-

tion of the dose that actually reaches the bloodstream) since this represents the

"effective dose" of a drug. This is generally less than the amount of drug actually

administered in the dosage form. In come cases, notably those where acute condi-

tions are being treated, one is also concerned with the rate of absorption of a drug,

since rapid onset of pharmacologic action is desired. Conversely, these are

instances where a slower rate of absorption is desired, either to avoid adverse

effects or to produce a prolonged duration of action.

"Absolute" bioavailability, F, is the fraction of an administered dose which actu-

ally reaches the systemic circulation, and ranges from F = 0 (no drug absorption) to

F = 1 (complete drug absorption). Since the total amount of drug reaching the sys-

temic circulation is directly proportional to the area under the plasma drug concen-

tration as a function of time curve (AUC), F is determined by comparing the

respective AUCs of the test product and the same dose of drug administered intra-

venously. The intravenous route is the reference standard since the dose is, by def-

inition, completely available.

(EQ 8-1

(whereAUCEVandAUCIVare, respectively, the area under the plasma concentra-

tion-time curve following the extravascular and intravenous administration of a

given dose of drug. Knowledge of F is needed to determine an appropriate oral

dose of a drug relative to an IV dose.

"Relative" or Comparative bioavailability refers to the availability of a drugproduct as compared to another dosage form or product of the same drug given inthe same dose. These measurements determine the effects of formulation differ-ences on drug absorption. The relative bioavailability of product A compared toproduct B, both products containing the same dose of the same drug, is obtained bycomparing their respective AUCs.

(EQ 8-2

F AUCev

AUCiv-----------------=

RelativeBioavailabi lt yAUCA

AUCB---------------=


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where drug product B is the reference standard. When the bioavailability of a

generic product is considered, it is usually the relative bioavailability that is

referred to. A more general form of the equation results from considering the pos

sibility of different doses,

(EQ 8-3

The difference between absolute and relative bioavailability is illustrated by the

following hypothetical example. Assume that an intravenous injection (Produc

A) and two oral dosage forms (Product B and Product C), all containing the same

dose of the same drug, are given to a group of subjects in a crossover study. Fur-

thermore, suppose each product gave the values for AUC indicated in Table 8-1 onpage 4.

TABLE 8-1. Data for Absolute and Relative Bioavailability

The F for Product B and Product C is 50% (F = 0.5) and 40% (F = 0.4), respec-

tively. However, when the two oral products are compared, the relative bioavail-

ability of Product C as compared to Product B is 80%.

8.1.2 FACTORS INFLUENCING BIOAVAILABILITY

Before the therapeutic effect of an orally administered drug can be realized, the

drug must be absorbed. The systemic absorption of an orally administered drug in

a solid dosage form is comprised of three distinct steps:

1. disintegration of the drug product

2. dissolution of the drug in the fluids at the absorption site

3. transfer of drug molecule across the membrane lining the gastrointestinal tract into the systemic

circulation.

Drug Product Area Under the Curve (mcg/ml) x hr

A Intravenous injection 100

B Oral dosage form, brand or reference standard 50

C Oral dosage form, generic Product 40

ComparativeBioavailability

AUCGeneric

DoseGeneric-----------------------------

AUCBrand

DoseBr and-------------------------

-----------------------------=


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Any factor that affects any of these three steps can alter the drugs bioavailability

and thereby its therapeutic effect. While there are more than three dozen of these

factors that have been identified (19-38), the more significant ones are summarized

here.

The various factors that can influence the bioavailability of a drug can be broadly

classified as dosage form-related or patient-related. Some of these factors are

listed in Table 8-2 on page 5 and Table 8-3 on page 5, respectively.

TABLE 8-2. Bioavailability Factors related to the dosage form

TABLE 8-3. Bioavailability Factors Related to the patient

The physical and chemical characteristics of a drug as well as its formulation are

of prime importance in bioavailability because they can affect not only the absorp-

tion characteristics of the drug but also its stability. Since a drug must be dissolved

to be absorbed, its rate of dissolution from a given product must influence its rate

of absorption. This is particularly the case for sparingly soluble drugs. All the fac-

tors listed in Table 8-2 on page 5 can alter the dissolution rate of the drug, its bio-availability, and ultimately, its therapeutic performance.

One of the more important factors that affects the dissolution rate of slowly dis-

solving substances is the surface area of the dissolving solid (39). Peak blood lev-

els occurred much faster with the smaller particles than the larger ones, primarily

Physicochemical properties of the drug Formulation and manufacturing variables

Particle size

Crystalline structure

Degree of hydration of crystal

Salt or ester form

Amount of disintegrant

Amount of lubricant

Special coatings

Nature of diluent

Compression force

Physiologic factors Interactions with other substances

Variations in absorption power along GI tract

Variations in pH of GI fluids

Gastric emptying rate

Intestinal motility

Perfusion of GI tract

Presystemic and first-pass metabolism

Age, sex, weight

Disease states

Food

Fluid volume

Other drugs


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as a result of their faster dissolution rate. Particle size can also have a significan

effect on AUC(40). Serum levels of phenytoin after administration of equal doses

containing micronized (formulation G) and conventional (formulation F) drug

were measured. Based on the AUC, almost twice as much phenytoin was

absorbed after the micronized preparation (40).

There are numerous reports of the effects of formulation and processing variables

on the dissolution of active ingredients from drug products; an apparently iner

ingredient may affect drug absorption. For example, magnesium stearate, a lubri-

cant, commonly used in tablet and capsule formulations, is water-insoluble and

water-repellent. Its hydrophobic nature tends to retard drug dissolution by pre-

venting contact between the solid drug and the aqueous GI fluids. Thus, increas-

ing the amount of magnesium stearate in the formulation results in a slower

dissolution rate of the drug, and decreased bioavailability(34) .

The nature of the dosage form itself may have an effect on drug absorption charac-teristics. The major pharmaceutical dosage forms for oral use are listed in Table 8-

4 on page 6 in order of decreasing bioavailability of their active ingredients. The

decreasing bioavailability is related to the number of steps involved in the absorp-

tion process following administration. The greater the number of steps a product

must undergo before the final absorption step, the slower is the availability and the

greater is the potential for bioavailability differences to occur. Thus, solutions

(elixirs, syrups, or simple solutions) generally result in faster and more complete

absorption of drug, since a dissolution step is not required. Enteric-coated tablets

on the other hand, do not even begin to release the drug until the tablets empty

from the stomach, resulting in poor and erratic bioavailability.

TABLE 8-4. Bioavailability and oral Dosage Forms

Bioavailability studies with pentobarbital from various dosage forms show the

absorption rate of pentobarbital after administration in various oral dosage formsdecreased in the following order: aqueous solution > aqueous suspension of the

free acid > capsule of the sodium salt > tablet of the free acid (41).

In addition to the dosage form-related factors identified above, bioavailability may

also be affected by a variety of physiologic and clinical factors related to the

patient (Table 8-3 on page 5). Considerable inter-subject differences in the bio-

Fastest availability

Slowest availability

Solutions

Suspensions

Capsules

Tablets

Coated tablets

Controlled-release formulations


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availability of some drugs have been observed. These can often be attributed to

individual variations in such factors as GI motility, disease state and concomi-

tantly-administered food or drugs.

One example of the myriad of physiologic factors that can affect the bioavailabil-ity of an orally-administered drug is a patients gastric emptying rate. Since the

proximal small intestine is the optimum site for drug absorption, a change in the

stomach emptying rate is likely to alter the rate, and possibly the extent, of drug

absorption. Any factor that slows the gastric emptying rate may thus prolong the

onset time for drug action and reduce the therapeutic efficacy of drugs that are pri-

marily absorbed from the small intestine. In addition, a delay in gastric emptying

could result in extensive decomposition and reduced bioavailability of drugs that

are unstable in the acidic media of the stomach (e.g. penicillins and erythromycin)

Differences in stomach emptying among individuals have been implicated as a

major cause of variations in the bioavailability of some drugs, particularly thosewith acid-resistant enteric coatings. In a study (42), after the administration of 1.5

g acetaminophen to 14 patients, the maximum plasma concentration ranged from

7.4 to 37 mcg/ml, and the time to reach the maximum concentration ranged from

30 to 180 minutes. Both these parameters of bioavailability were linearly related

to the gastric emptying half-life found in each patient.

There are numerous factors that affect gastric emptying rate (Table 8-5 on page 8)

(43). Factors such as a patients emotional state, certain drugs, type of food

ingested and even a patients posture can alter the time course and extent of drug

absorption.


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TABLE 8-5. Factors influencing Gastric Emptying Rate

Since drugs are generally administered to patients who are ill, it is important to

consider the effects of the disease process on the bioavailability of the drug. Dis-

ease states, particularly those involving the GI tract, such as celiac disease, Crohns

disease, achlorhydria, and hypermotility syndromes can certainly alter the absorp-

tion of a drug (32). In addition, some diseases concerning the cardiovascular sys-tem and the liver may also alter circulating drug levels after oral dosing.

Drugs are frequently taken with food, and patients often use mealtimes to remind

them to take their medications. However, food can have a significant effect on the

bioavailability of drugs. The influence of food on drug absorption has been recog-

FACTOR

INFLUENCE ON GASTRIC

EMPTYING RATE

Increased viscosity of stomach contents decreased

Body position

lying on left side decreased

Emotional state

stress

depression

anxiety

increased or decreased

decreased

increased

Activity, exercise decreased

Type of meal

fatty acids, fats

carbohydratesamino acids

decreased

decreaseddecreased

pH of stomach contents

decreased

increased

decreased

increased

Disease states

gastric ulcers

Crohns disease

hypothyroidism

hyperthyroidism

decreased

decreased

decreased

increased

Drugs

atropine

propantheline

narcotic analgesics

amitriptyline

metoclopramide

decreased

decreased

decreased

decreased

increased


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nized for some time, and several reviews have been published on the influence of

food on drug bioavailability (30-32, 36, 44). Food may influence drug absorption

indirectly, through physiological changes in the GI tract produced by the food

and/or directly, through physical or chemical interactions between the drug mole-

cules and food components. When food is ingested, stomach emptying is delayedgastric secretions are increased, stomach pH is altered, and splanchnic blood flow

may increase. These may all affect bioavailability of drugs. Food may also inter-

act directly with drugs, either chemically (e.g. chelation) or physically, by adsorb-

ing the drug or acting as a barrier to absorption. In general, gastrointestina

absorption of drugs is favored by an empty stomach, but the nature of drug-food

interactions is complex and unpredictable; drug absorption may be reduced

delayed, enhanced or unaffected by the presence of food. Table 8-6 on page 9

summarizes some of the studies that have indicated the effect of food on the bio-

availability of a variety of drugs.

TABLE 8-6. Effect of Food on Drug Absorption

The effect of food and type of diet on the bioavailability of erythromycin is shown

in a study by Welling (45). The absorption of the antibiotic is significantly

reduced when it is administered with food compared with its absorption under fast-

ing conditions. This reduced absorption is primarily a result of degradation of the

acid-labile erythromycin due to prolonged retention in the stomach.

Delayed absorption due to food has been demonstrated in the case of cephradine ina study by Mischler (46). Similar results have been observed with other oral ceph-

alosporins.

Some drugs demonstrate enhanced bioavailability in the presence of food. This

has been attributed to a variety of factors, including improved compound solubility

and more time for dissolution because of delayed gastric emptying. In the case of

Reduced Absorption Delayed Absorption Increased Absorption

Ampicillin

Aspirin

Atenolol

Captopril

Erythromycin

Ethanol

Hydrochlorothiazide

Penicillins

Tetracyclines (most)

Acetaminophen

Aspirin

Cephalosporins (most)

Diclofenac

Digoxin

Furosemide

Nitrofurantoin

Sulfadiazine

Sulfisoxazole

Chlorothiazide

Diazepam

Griseofulvin

Hydralazine

Labetalol

Metoprolol

Nitrofurantoin

Propranolol

Riboflavin

Source: Ref. 32


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highly metabolized agents, such as propranolol and metoprolol, the enhanced

availability may be due to increased splanchnic blood flow causing reduced

first-pass clearance. The circulating levels of these drugs dosed under fasting and

non-fasting conditions have been presented in a study by Melander (47).

The volume of fluid with which an orally administered dose is taken can also

affect a drugs bioavailability. Drug administration with a larger fluid volume wil

generally improve its dissolution characteristics and may also result in more rapid

stomach emptying. Thus, more efficient and more reliable drug absorption can be

expected when an oral dosage form is administered with a larger volume of fluid.

(45) .

Interactions between drugs can have a significant effect on the bioavailability of

one or both drugs. Such interactions may be direct, as in chelation of tetracycline

by polyvalent metal ions in antacids or the adsorption of digoxin by

cholestyramine resin, or indirect, as with the increased rate of acetaminophenabsorption due to the increased gastric emptying rate produced by metoclopra-

mide. Most of the reported drug-drug interactions have resulted in a reduction in

the rate and/or extent of drug absorption, the most frequent causes being complex-

ing of a drug with other substances, reduced GI motility and alterations in drug

ionization (24, 30, 32, 48, 49). Table 8-7 on page 10 summarizes the major mech-

anisms of GI drug interactions affecting bioavailability.

TABLE 8-7. Drug interactions affecting absorption

An example of a direct interaction between drugs affecting bioavailability is the

interaction between iron and tetracycline. This is a well-documented and clini-cally significant interaction which can result in a dramatic reduction in serum con-

centration of tetracycline (50).

The above potential sources of alteration in a drugs bioavailability must be kept in

mind when attempting to evaluate the relative performance of drug products on the

1. Change in gastric or intestinal pH

2. Change in gastrointestinal motility

3. Change in gastrointestinal perfusion

4. Interference with mucosal function (drug-induced malabsorption syndromes)

5. Chelation

6. Exchange resin binding

7. Aadsorption

8. Solution in poorly absorbable liquid

Source: Ref. 23


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basis of studies performed with healthy human volunteers. These studies are gen

erally performed under tightly-controlled fasting conditions in the absence of other

drugs. In practice, however, drugs are seldom taken under such ideal conditions

and the factors leading to changes in drug absorption must be taken into consider-

ation.

8.1.3 METHODS OF ASSESSING BIOAVAILABILITY

Bioavailability testing is a means of predicting the clinical efficacy of a drug; the

estimation of the bioavailability of a drug in a given dosage form is direct evidence

of the efficiency with which a dosage form performs its intended therapeutic func-

tion.

The bioavailability of a drug substance formulated into a pharmaceutical product

is fundamental to the goals of dosage form design and essential for the clinicalefficacy of the medication. Thus, bioavailability testing, which measures the rate

and extent of drug absorption, is a way to obtain evidence of the therapeutic utility

of a drug product. Bioavailability determinations are performed by drug manufac-

turers to ensure that a given drug product will get the therapeutic agent to its site of

action in an adequate concentration. Bioavailability studies are also carried out to

compare the availability of a drug substance from different dosage forms or from

the same dosage form produced by different manufacturers.

In-vivo methods One method for assessing the bioavailability of a drug product is through the dem-

onstration of a clinically significant effect. However, such clinical studies are

complex, expensive, time-consuming and require a sensitive and quantitative mea-

sure of the desired response. Further, response is often quite variable, requiring a

large test population. Practical considerations, therefore, preclude the use of this

method except in initial stages of development while proving the efficacy of a new

chemical entity.

Quantification of pharmacologic effect is another possible way to assess a drugs

bioavailability. This method is based on the assumption that a given intensity of

response is associated with a particular drug concentration at the site of action

e.g., variation of miotic response intensity can be directly related to the oral dose

of chlorpromazine. However, monitoring of pharmacologic data is often difficult

precision and reproducibility are difficult to establish, and there are only a limited

number of pharmacologic effects (e.g. heart rate, body temperature, blood sugar

levels) that are applicable to this method.

Because of these limitations, alternative methods have been developed to predict

the therapeutic potential of a drug. The current method to assess the clinical per

formance of a drug involves measurement of the drug concentrations in the blood


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or urine. In such studies a single dose of the drug product is administered to a

panel of normal, healthy adult (18- to 35-year old) subjects. Blood and/or urine

samples are collected over a period of time following administration and are ana-

lyzed for drug content. Based on the blood concentration as a function of time

and/or urinary excretion profile, inferences are drawn regarding the rate and extentof absorption of the drug. These studies are relatively easy to conduct and require

a limited number of subjects.

Blood level studies- Blood level studies are the most common type of human bioavailability studies,

and are based on the assumption that there is a direct relationship between the con-

centration of drug in blood or plasma and the concentration of drug at the site of

action. By monitoring the concentration in the blood, it is thus possible to obtain

an indirect measure of drug response. Following the administration of a single

dose of a medication, blood samples are drawn at specific time intervals and ana-

lyzed for drug content. A profile is constructed showing the concentration of drug

in blood at the specific times the samples were taken . The key parameters to note

are:

1. AUC , The area under the plasma concentration-time curve, The AUC is proportional to the

total amount of drug reaching the systemic circulation, and thus characterizes the extent of

absorption.

2. Cmax , The maximum drug concentration. The maximum concentration of drug in the plasma

is a function of both the rate and extent of absorption. Cmax will increase with an increase in

the dose, as well as with an increase in the absorption rate.

3. Tmax , The time at which the Cmax occurs. The Tmax reflects the rate of drug absorption, and

decreases as the absorption rate increases.

Bioavailability (the rate and extent of drug absorption) is generally assessed by the

determination of these three parameters.

Since the AUC is representative of, and proportional to, the total amount of drug

absorbed into the circulation, it is used to quantitate the extent of drug absorption.

The calculation of AUC has been discussed in Chapter 4. A variety of pharmacok-

inetic methods have been suggested for the calculation of absorption rates (51-56).

For clinical purposes, it is generally sufficient to determine Cmax and Tmax. If al

other factors are constant, such as the extent of absorption and rate of elimination

then Cmax is proportional to the rate of absorption and Tmax is inversely propor-

tional to the absorption rate. Thus, the faster the absorption of a drug the higherthe maximum concentration will be and the less time it will take to reach the max-

imum concentration.

Urinary Excretion Data - An alternative bioavailability study measures the cumulative amount of unchanged

drug excreted in the urine. These studies involve collection of urine samples and

the determination of the total quantity of drug excreted in the urine as a function of

0


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time. These studies are based on the premise that urinary excretion of the

unchanged drug is directly proportional to the plasma concentration of total drug.

Thus, the total quantity of drug excreted in the urine is a reflection of the quantity

of drug absorbed from the gastrointestinal tract. Consider the following example

two products, A and B, each containing 100 mg of the same drug are administeredorally. A total of 80 mg of drug is recovered in the urine from Product A, but only

40 mg is recovered from Product B. This indicates that twice as much drug was

absorbed from Product A as from Product B. (The fact that neither produc

resulted in excretion of the entire dose might be due to the existence of other routes

of elimination, e.g. metabolism).

This technique of studying bioavailability is most useful for those drugs that are

not extensively metabolized prior to urinary elimination. As a rule-of-thumb

determination of bioavailability using urinary excretion data should be conducted

only if at least 20% of a dose is excreted unchanged in the urine after an IV dose

(56). Other conditions which must be met for this method to give valid resultsinclude:

1. the fraction of drug entering the bloodstream and being excreted intact by the kidneys must

remain constant.

2. collection of the urine has to continue until all the drug has been completely excreted (five times

the half-life1).

Urinary excretion data are primarily useful for assessing extent of drug absorption

although the time course for the cumulative amount of drug excreted in the urine

can also be used to estimate the rate of absorption. In practice, these estimates are

subject to a high degree of variability, and are less reliable than those obtained

from plasma concentration-time profiles (57). Thus, urinary excretion of drug isnot recommended as a substitute for blood concentration data; rather, these studies

should be used in conjunction with blood level data for confirmatory purposes.

1. Half life is defined as the length of time required to lose 50% of the drug in the body, assuming first order elimination.

Single-dose versus

Multiple-Dose-

Most bioavailability evaluations are made on the basis of single-dose administra-

tion. The argument has been made that single doses are not representative of the

actual clinical situation, since in most instances, patients require repeated adminis-

tration of a drug. When a drug is administered repeatedly at fixed intervals, with

the dosing frequency less than five half-lives, drug will accumulate in the body and

eventually reach a plateau, or a steady-state

At steady-state, the amount of drug eliminated from the body during one dosinginterval is equal to the available dose (rate in = rate out); therefore, the area under

the curve during a dosing interval at steady-state is equal to the total area under the

curve obtained when a single dose is administered. This AUC can therefore be


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used to assess the extent of absorption of the drug, as well as its absolute and rela-

tive bioavailability.

Multiple-dose administration has several advantages over single-dose bioavailabil-

ity studies, as well as some limitations. These are summarized in Table 8-8 onpage 14 (54, 59).

TABLE 8-8. Multiple dose vs. single dose studies in bioavailability studies

When a drug obeys linear, first-order kinetics, it is possible to estimate the results

that would be obtained during multiple dosing from single-dose studies. Projec-

tion is easily made with regard to the extent of absorption, using the AUC follow-ing a single dose. Results from bioequivalence studies indicate that conclusions on

the extent of absorption as assessed by the AUC can be made equally well on the

basis of a single or multiple dose study (60). Assessing the rate of absorption dur-

ing multiple-dosing from single-dose studies has presented a greater problem.

Although a number of single-dose characteristics have been suggested as indica-

tors of rate of absorption during multiple dosing (e.g. percent peak-trough fluctua-

tion and percent peak-trough swing), results of bioequivalence studies indicate that

only the plateau time (the time during which the concentration exceeds 75% of the

maximum concentration, t 75% Cmax) and the residual concentration at the end of

the dose interval produce consistent results in assessing the rate of absorption in

single- and multiple-dose studies (54, 61).

In the case of drugs exhibiting nonlinear kinetics, establishing a linear relationship

between single- and multiple-dose bioavailability data has proven to be a difficult

task. Thus, it has been recommended that for drugs with either saturable elimina-

tion or a nonlinear first-pass effect, steady-state studies be carried out to assess

their bioavailability (62).

Advantages:

Eliminates the need to extrapolate the plasma concentration profiles to obtain the total AUCafter a single dose

Eliminates the need for a long wash-out period between doses

More closely reflects the actual clinical use of the drug

Allows blood levels to be measured at the same concentrations encountered therapeutically

Because blood levels tend to be higher than in the single-dose method, quantitative determina-

tion is easier and more reliable

Saturable pharmacokinetics, if present, can be more readily detected at steady-state

Limitations:

Requires more time to complete

More difficult and costly to conduct (requiring prolonged monitoring of subjects

Greater problems with compliance control

Greater exposure of subjects to the test drug, increasing the potential for adverse reactions


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8.1.4 STUDY DESIGN

Bioavailability studies involve the administration of the test dosage form to a panel

of subjects, after which blood and/or urine samples are collected and analyzed for

drug content. Based on the concentration profile of the drug, a judgement is maderegarding the rate and extent of absorption of the drug. Normally, the study is con

ducted in a group of healthy, male subjects who are of normal height and weight

and range in age from 18 to 35 years (6). Questions have been raised regarding the

extent to which such a population reflects the performance of a given drug product

in a actual patient population. At first glance, it would seem that bioavailability

should be determined in patients actually suffering from the disease for which the

drug is intended, or in patients representative of the age and sex of subjects who

would be using the drug. However, there are several very good reasons for using

healthy volunteers rather than patients. In bioavailability studies, it is assumed

that there are no physiologic changes in the subjects during the course of the study

If actual patients were used, this would not be a valid assumption, due to possiblechanges in the disease state. Another potential problem with using patients is tha

many patients take more than one drug. This could result in a drug-drug interac-

tion which could influence the bioavailability of the test drug. In addition, diet and

fluid volume intake, both of which can influence a drugs bioavailability are more

difficult to control in a patient population than in a panel of healthy test subjects

In general, it is more difficult with patients to have a standardized set of conditions

which are necessary for a dependable bioavailability study. However, it must be

recognized that factors that may affect a drugs performance in a patient population

may not be detected in a group of healthy subjects. Thus, it is best to conduct a

separate study in patients to determine if the disease, for which the drug is intended

to be used, alters the bioavailability of the drug.

Other important considerations in the methodology of a bioavailability study are

sample size, period of trial, and sampling. For statistical purposes, twelve subjects

are considered to be a minimum sample size. Otherwise there will not be enough

data to draw valid conclusions (63). The bioavailability testing period should be of

a sufficient length of time to ensure that drug absorption has been completed. This

length of time is at least three times the half-life of the drug; generally a period of

four to five times the half-life is used (63, 64). Blood samples should be taken

with sufficient frequency to permit an accurate determination of tmax, Cmax and

AUC.

8.1.5 IN-VITRO DISSOLUTION AND BIOAVAILABILITY

Pharmaceutical scientists have for many years been attempting to establish a corre-

lation between some physicochemical property of a dosage form and the biological


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availability of the drug from that dosage form. The term commonly used to

describe this relationship is "in-vitro/in-vivo correlation" (65). Specifically, it is

felt that if such a correlation could be established, it would be possible to use

in-vitro data to predict a drugs in-vivo bioavailability. This would drastically

reduce, or in some cases, completely eliminate the need for bioavailability testsThe desirability for this becomes clear when one considers the cost and time

involved in bioavailability studies as well as the safety issues involved in adminis-

tering drugs to healthy subjects or patients. It would certainly be preferable to be

able to substitute a quick, inexpensive in-vitro test for in-vivo bioavailability stud-

ies. This would be possible if in-vitro tests could reliably and accurately predic

drug absorption and reflect the in-vivo performance of a drug in humans.

Disintegration Tests- The early attempts to establish an indicator of drug bioavailability focused on dis-

integration as the most pertinent in-vitro parameter. The first official disintegra-

tion test appeared in the United States Pharmacopeia (USP) in 1950. However

while it is true that a solid dosage form must disintegrate before significant disso-

lution and absorption can occur, meeting the disintegration test requirement onlyinsures that the dosage form (tablet) will break up into sufficiently small particles

in a specified length of time. It does not ensure that the rate of solution of the drug

is adequate to produce suitable blood levels of the active ingredient. Therefore

while the test for tablet disintegration is very useful for quality control purposes in

manufacturing, it is a poor index of bioavailability.

Dissolution Tests- Since a drug must go into solution before it can be absorbed, and since the rate at

which a drug dissolves from a dosage form often determines its rate and/or extent

of absorption, attention has been directed at the dissolution rate. It is currently

considered to be the most sensitive in-vitro parameter most likely to correlate with

bioavailability.Official dissolution tests - There are two official USP dissolution methods: Apparatus 1, (basket method)

and Apparatus 2 (paddle method). For details of these dissolution tests, the reader

is recommended to consult USPXXII/NFXVII (66).

Dissolution tests are an extremely valuable tool in ensuring the quality of a drug

product. Generally, product-to-product variations are due to formulation factors

such as particle size differences, excessive amounts of lubricant and coatings

These factors are reactive to dissolution testing. Thus, dissolution tests are very

effective in discriminating between and within batches of drug product(s). The

dissolution test, in addition, can exclude definitively any unacceptable product.

Limitations of

dissolution tests-

There are, however, problems with in-vitro dissolution testing which should be

noted - problems which make correlation with in- vivo availability difficult. The

first is related to instrument variance and the absence of a standard method. The

tests described in the USP are but a few of the large number of dissolution methods

proposed to predict bioavailability. Since the dissolution rate of a dosage form is

dependent on the methodology used in the dissolution test, changes in the appara-


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tus, dissolution medium, etc., can dramatically modify the results. Table 8-9 on

page 17 lists some of the factors related to the dissolution testing device that can

affect the dissolution rate of the drug.

TABLE 8-9. Device factors affecting dissolution

Another significant problem is related to the difference between the in-vitro and

in-vivo environments in which dissolution occurs. In-vitro studies are generally

carried out under controlled conditions in one, or perhaps two, standardized sol-

vents. The in-vivo environment (the gastrointestinal tract), on the other hand, is a

continuously changing, complex environment. There are many variables which

can affect the dissolution rate of a drug in the gastrointestinal tract, including pH

enzyme secretions, surface tension, motility, presence of other substances and

absorption surfaces (68). Thus, drugs frequently dissolve in the body at rates quitedifferent from those observed in an in-vitro test situation. Most of the official dis-

solution tests tend to be acceleration dissolution tests which bear limited or no

relationship with in-vivo dissolution.

Adding to the complexity of correlating dissolution with in-vivo absorption are

factors such as drug-drug interactions, age, food effects, health, genetic back-

ground, biorhythm and physical activity (32, 69). All these factors may have an

effect on the rate and extent of absorption of a drug. Thus, the in-vivo environ

ment is far more complex, variable, and unpredictable than any in-vitro test envi-

ronment, making in-vitro / in-vivo correlations very difficult. A simple dissolution

test in a standardized vehicle cannot reflect the in vivo absorption of a drug across apopulation (70).

Parameters used- Proper selection of the in-vitro and in-vivo parameters to be correlated is critical

in achieving a meaningful correlation. The in-vitro parameter should be selected

that has the greatest effect on the absorption characteristics of the drug (71). There

are several approaches to establishing a correlation between the dissolution of a

1. Degree of agitation

2. Size and shape of container

3. Composition of dissolution medium

pH

ionic strength

viscosity

surface tension

4. Temperature of dissolution medium

5. Volume of dissolution medium

6. Evaporation7. Hydrodynamics (flow pattern)

Source: Ref. 67


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drug in in- vitro and the bioavailability of a drug in-vivo. The in-vitro - in-vivo

correlative methods used most often are of the single-point type where the dissolu-

tion rate (expressed as the percent of drug dissolved in a given time, or the time

required for a given percent of the drug to dissolve) is correlated to a certain

parameter of the bioavailability. Examples of in-vivo parameters used includeCmax, AUC, time to reach half-maximal plasma concentration, the average

plasma concentration after 0.5 or 1 hour, maximum urinary excretion rate, and

cumulative percent excreted in urine after a given time (71- 78). According to

Wagner, the best in-vitro variable to use is the time for 50 percent of the drug to

dissolve, and the best variable from in-vivo data to use is the time for 50 percent of

the drug to be absorbed (79).

Ideally, one would hope to find a linear relationship between some measurement

of the dissolution test and some measurement based on bioavailability studies

Unfortunately, most attempts to accomplish this objective have failed.

8.1.6 IN-VITRO / IN-VIVO CORRELATION STUDIES-

There have been many attempts to establish in-vitro / in-vivo correlations for a

large variety of drugs. Some of these studies have been summarized by Welling

Banakar, and Abdou (71, 80-82).

While there are many published examples of satisfactory correlations between

absorption parameters and in-vitro dissolution tests, most studies have resulted in

poor, or moderate, in-vitro - in-vivo correlations, often involving agreement with

only one of the critical bioavailability parameters. Moreover, the positive correla-tions that have been found generally apply only to the specific formulation studied

There have been instances where the dissolution rates or various formulations of

the same drug have been significantly different, yet little or no difference was

observed in their bioavailability parameters (83-85). There have also been cases

where a drug has failed to meet compendia dissolution standards but has demon-

strated adequate bioavailability (86). Welling states: "To the writers knowledge

there have been no studies that have accurately correlated in- vitro and in-vivo data

to the point that the use of upper and lower limits for in-vitro dissolution parame-

ters can be confidently used to predict in-vivo behavior and, therefore, to replace

in-vivo testing" (71).

Even if an in-vitro test could be designed that would accurately reflect the dissolu-

tion process in the gastrointestinal tract, dissolution is only one of many factors

that affect a drugs bioavailability. For example, saturable presystemic metabolism

may affect the extent of drug absorption, but this would not be predicted by an


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in-vitro test. Dissolution studies also would not predict poor bioavailability due to

instability in gastric fluid or complexation with another drug or food component.

Thus, the ultimate evaluation a drug products performance under the conditions

expected in clinical therapy must be an in-vivo test; a dissolution test is unlikely toentirely replace bioavailability testing (70, 87, 88). In-vitro methods are importan

in the development and optimization of dosage forms while in-vivo tests are essen-

tial in obtaining information on the behavior of medication in living organisms.

One cannot be substituted for the other (69).


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8.2 Bioequivalence

Definitions With the phenomenal increase in the availability of generic drugs in recent years,

the issues of bioavailability and bioequivalence have received increasing attentionIn order for a drug product to be interchangeable with the pioneer (innovator or

brand name) product, it must be both pharmaceutically equivalent and bioequiva-

lent to it. According to the FDA, "pharmaceutical equivalents" are drug products

that contain identical active ingredients and are identical in strength or concentra-

tion, dosage form, and route of administration (89). However, pharmaceutical

equivalents do not necessarily contain the same inactive ingredients; various man-

ufacturers dosage forms may differ in color, flavor, shape, and excipients. The

terms "pharmaceutical equivalents" and "chemical equivalents" are often used

interchangeably.

"Bioequivalence" is a comparison of the bioavailability of two or more drug prod-ucts. Thus, two products or formulations containing the same active ingredient are

bioequivalent if their rates and extents of absorption are the same. When a new

formulation of an existing drug is developed, its bioavailability is generally evalu-

ated relative to the standard formulation of the originator. Indeed, a bioequiva-

lence trial against the standard formulation is the key feature of an Abbreviated

New Drug Application (ANDA) submitted to the Food and Drug Administration

by a manufacturer who wishes to produce a generic drug. For a generic drug to be

considered bioequivalent to a pioneer product, there must be no statistical differ-

ences (as specified in the accepted criteria) between their plasma concentra-

tion-time profiles. Because two products rarely exhibit absolutely identica

profiles, some degree of difference must be considered acceptable, as will be dis-cussed later.

Since the concentration of a drug in blood is used as an assessment of its clinical

performance, inherent in the demonstration that two preparations containing

equivalent amounts of the same drug produce similar concentrations of the drug

entity in blood is the assumption that they will elicit equivalent drug responses.

Thus, two products that are deemed to be bioequivalent are also assumed to be

therapeutically equivalent, and therefore interchangeable. This principle is funda-

mental to the concept of bioequivalence and is the basic premise on which it is

founded.

In general, the FDA considers two products to be "therapeutic equivalents" if they

each meet the following criteria (90):

1. they are pharmaceutical equivalents,

2. they are bioequivalent (demonstrated either by a bioavailability measurement or an in vitro stan

dard),


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3. they are in compliance with compendial standards for strength, quality, purity and identity,

4. they are adequately labelled, and

5. they have been manufactured in compliance with Good Manufacturing Practices as established

by the FDA.

Background The first intimations of bioequivalence problems with multi-source drug products

were given by early investigations of the availability of vitamins, aspirin, tetracy-

cline, and tolbutamide (91-97). In 1974, after an extensive review of the bioavail-

ability of drugs, Koch-Weser concluded that " . . . among drugs thus far tested

bioinequivalence of different drug products has been far more common than

bioequivalence" (98). Of particular note were the studies involving digoxin; the

findings of these investigations sparked the discussion about bioequivalence

assessment that still continues today. Significant differences were seen in the bio-

availability of digoxin not only between products supplied by different companies,

but also between lots obtained from the same manufacturer (99). Because of thenarrow therapeutic range for this drug, and because the drug is utilized in the treat-

ment of cardiac patients, these findings generated a great deal of concern.

Similar reports of bioinequivalence and therapeutic inequivalence appeared for

other drugs as well, including phenytoin, phenylbutazone, chloramphenicol, tolb-

utamide and thyroid (6). The clinical significance of these reported differences in

bioavailability relates to the therapeutic index of the drug, the dose of the drug and

the nature of the disease. In 1973 the Ad Hoc Committee on Drug Product Selec-

tion of the American Pharmaceutical Association published a list of drugs with a

potential for therapeutic inequivalence based on reported evidence of bioinequiva-

lence (100). The drugs fall in three categories: "high," "moderate," or "low risk"based on the clinical implications (Table 8-10 on page 22).


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TABLE 8-10. Drugs with various risk potential for inequivalence

The concern about the bioinequivalence of some drugs led to the establishment in1974 of the Drug Bioequivalence Study Panel of the Office of Technology Assess-

ment (OTA). The objective was to ensure that drug products of the same physica

and chemical composition would produce similar therapeutic effects. Among the

11 recommendations of the Panel was the conclusion that not all chemical equiva-

lents were interchangeable, but the goal of interchangeability was achievable for

most oral drug products (101). The Report recommended that a system should be

organized as rapidly as possible to generate an official list of interchangeable drug

products. The OTA Report, as well as the growing awareness within the scientific

and regulatory communities of bioavailability problems with marketed drug prod-

ucts, focused the attention of the FDA on bioequivalence and bioavailability prob-

lems and issues.

High Risk Potential Moderate Risk Potential Low or Negligible Risk

Potential

aminophylline

aspirin (when used in high dose

levels)

bishydroxycoumarin

digoxin

dipheylhydantoin (phenytoin)

para-aminosalicylic acid

prednisolone

prednisone

quinidine

warfarin

amphetamines

(sustained-release)

ampicillin

chloramphenicol

chlorpromazine

digitoxin

erythromycin

griseofulvin

oxytetracycline

penicillin G (buffered)

pentobarbital

phenylbutazone

phenacetin

potassium chloride (solid dosage

forms)

salicylamide

secobarbital

sulfadiazine

tetracycline

tolbutamide

acetaminophen

codeine

ferrous sulfate

hydrochlorothiazide

ephedrine

isoniazid

meprobamate

penicillin VK

sulfisoxazole


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8.2.1 BIOEQUIVALENCE REGULATIONS

In 1977, the FDA implemented a series of bioavailability and bioequivalence regu-

lations which formed the basis of subsequent discussion, if not controversy, of

therapeutic equivalency of drug products (102). The regulations are divided intotwo separate regulations; Subpart B - Procedures for Determining the Bioavail-

ability of Drug Products and Subpart C - Bioequivalence Requirements. While

Table 11 summarizes the key provisions of the bioavailability regulations, those

for bioequivalence requirements are summarized in Table 8-11 on page 23.

TABLE 8-11. Key provisions for bioavailabilty regulations

Criteria for establishing

a bioequivalence

requirement -

The 1977 Bioequivalence regulations set forth the following criteria and evidence

supporting the establishment of a bioequivalence requirement for a given drug

product:

1. Evidence from well-controlled clinical trials or controlled observations in patients that such

products do not give comparable therapeutic effects.2. Evidence from well-controlled bioequivalence studies that such products are not bioequivalent

drug products.

3. Evidence that the drug products exhibit a narrow therapeutic ratio, (e.g., there is less than a

two-fold difference in the median lethal dose (LD50) and median effective dose (ED50) value

or have less than a two-fold difference in the minimum toxic concentration and minimum effec-

tive concentrations in the blood), and safe and effective use of the drug product requires careful

dosage titration and patient monitoring.

4. Competent medical determination that a lack of bioequivalence would have a serious adverse

effect in the treatment or prevention of a serious disease or condition.

5. Physicochemical evidence of any of the following:

a. The active drug ingredient has a low solubility in water--e.g., less than 5 mg/ml.

b. The dissolution rate of one or more such products is slow--e.g., less than 50 percent in

thirty minutes when tested with a general method specified by an official compendium or the

FDA.

c. The particle size and/or surface area of the active drug ingredient is critical in determining

bioavailability.

1. Defines bioavailability in terms of both the rate and extent of drug absorption.

2. Describes procedures for determining the bioavailability of drug products.

3. Sets forth requirements for submission ofin vivo bioavailability data.

4. Sets forth criteria for waiver of human in vivo bioavailability studies.5. Provides general guidelines for the conduct ofin vivo bioavailability studies.

6. Imposes a requirement for filing an Investigational New Drug Application.

Source: Ref. 103


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d. Polymorphs, solvates, complexes, and such, exist that could contribute to poor dissolution

and may affect absorption.

e. There is a high excipient/active drug ratio present in the drug product--e.g., greater than 5

to 1.

f. The presence of specific inactive ingredients (e.g. hydrophilic or hydrophobic excipients)

that either may be required for absorption of the active drug or may interfere with such absorp-

tion.

6. Pharmacokinetic evidence of any of the following:

a. The drug is absorbed in large part in a particular segment of the gastrointestinal tract or is

absorbed from a localized site.

b. Poor absorption of the drug, even when it is administered as a solution--e.g., less than 50

percent compared to an intravenous dose.

c. The drug undergoes first-pass metabolism in the intestinal wall or liver.

d. The drug is rapidly metabolized or excreted, requiring rapid dissolution and absorption for

effectiveness.

e. The drug is unstable in specific portions of the gastrointestinal tract, requiring special

coatings and formulations--e.g., enteric coatings, buffers, film coatings--to ensure adequate

absorption.

f. The drug follows nonlinear kinetics in or near the therapeutic range, and the rate and

extent of absorption are both important to bioequivalence.

Types of Bioequivalence

Requirements

In the event that a drug meets one or more of the above six criteria, a bioequiva-

lence requirement is established. The requirement could be either an in-vivo or an

in-vitro investigation, as specified by the FDA. The types of bioequivalence

requirements include the following:

1. An in-vivo test in humans.2. An in-vivo test in animals that has been correlated with human in- vivo data.

3. An in-vivo test in animals that has not been correlated with human in- vivo data.

4. An in-vitro bioequivalence standard, i.e., an in-vitro test that has been correlated with human

in-vivo bioavailability data.

5. A currently available in-vitro test (usually a dissolution rate test) that has not been correlated

with human in-vivo bioavailability data.

The regulations state that in-vivo testing in humans would generally be required if

there is well-documented evidence that pharmaceutical equivalents intended to be

used interchangeably meet one of the first three criteria used to establish a

bioequivalence requirement:

1. The drug products do not give comparable therapeutic effects.

2. The drug products are not bioequivalent.

3. The drug products exhibit a narrow therapeutic ratio (as described above), and safe and effec-

tive use of the product requires careful dosage titration and patient monitoring.


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Criteria for waiver of

evidence of in-vivo

bioavailability -

Although a human in-vivo test is considered to be preferable to other approaches

for the most accurate determination of bioequivalence, there is a provision in the

1977 regulations for waiver of an in-vivo bioequivalence study under certain cir-

cumstances. For some drug products, the in-vivo bioavailability of the drug may

be self-evident or unimportant to the achievement of the products intended pur-poses. The FDA will waive the requirement for submission of in-vivo evidence of

bioavailability or bioequivalence if the drug product meets one of the following

criteria:

1. The drug product is a solution intended solely for intravenous administration, and contains the

active drug ingredient in the same solvent and concentration as an intravenous solution that is

the subject of an approved full New Drug Application (NDA).

2. The drug product is a topically applied preparation intended for local therapeutic effect.

3. The drug product is an oral dosage form that is not intended to be absorbed, e.g., an antacid.

4. The drug product is administered by inhalation and contains the active drug ingredient in the

same dosage form as a drug product that is the subject of an approved full NDA.

5. The drug product is an oral solution, elixir, syrup, tincture or other similar soluble form, thatcontains an active drug ingredient in the same concentration as a drug product that is the subject

of an approved full NDA and contains no inactive ingredient that is known to significantly

affect absorption of the active drug ingredient.

6. The drug product is a solid oral dosage form (other than enteric-coated or controlled-release)

that has been determined to be effective for at least one indication in a Drug Efficacy Study

Implementation (DESI) notice and is not included in the FDA list of drugs for which in vivo

bioequivalence testing is required.

7. The drug product is a parenteral drug product that is determined to be effective for at least one

indication in a DESI notice and shown to be identical in both active and inactive ingredients for

mulation, with a drug product that is currently approved in an NDA. (Excluded from the waiver

provision are parenteral suspensions and sodium phenytoin powder for injection.)

According to the regulations, the bioavailability of certain drug products may be

demonstrated by evidence obtained in-vitro in lieu of in-vivo data. Thus, the FDA

also permits waiver of the in-vivo requirements if a drug product meets one of the

following criteria:

1. The drug product is one for which only an in-vitro bioequivalence requirement has been

approved by the FDA.

2. The drug product is in the same dosage form, but in a different strength, and is proportionally

similar in its active and inactive ingredients to another drug product made by the same manufac

turer and the following conditions are met:

a. the bioavailability of this other product has been demonstrated

b. both drug products meet an appropriate in-vitro test approved by the FDA

c. the applicant submits evidence showing that both drug products are proportionally similar

in their active an inactive ingredients.

3. The drug product is shown to meet an in-vitro test that assures bioavailability, i.e., an in-vitro

test that has been correlated with in-vivo data.


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4. The drug product is a reformulated product that is identical, except for color, flavor, or preser-

vative, to another drug product made by the same manufacturer, and both of the following con-

ditions are met:

a. the bioavailability of the other product has been demonstrated.

b. both drug products meet an appropriate in vitro test approved by the FDA.

5. The drug product contains the same active ingredient and is in the same strength and dosage

form as a drug product that is the subject of an approved full NDA or Abbreviated New Drug

Application (ANDA) and both drug products meet an appropriate in-vitro test that has been

approved by the FDA.

Although the above list of criteria for waiver of an in-vivo bioavailability study is

quite lengthy, currently virtually all new tablet or capsule formulations from which

measurable amounts of drug or metabolites are absorbed into the systemic circula-

tion require a human bioequivalence study for approval (104).

TABLE 8-12. Key Provisions for bioequivalence requirements

8.2.2 STUDY DESIGN

A single-dose bioequivalency study is generally performed in normal, healthy

adult volunteers. The subject population should be selected carefully, so that prod

uct formulations, and not intersubject variations, will be the only significant deter-

minants of bioequivalence (105). A minimum of 12 subjects is recommended

although 18 to 24 subjects are used to increase the data base for statistical analysis.

The test and the reference products are usually administered to the subjects in the

fasting state (overnight fast for at least 10 hours, plus 2 to 4 hours after administra-

tion of the dose), unless some other approach is more appropriate for valid scien-

tific reasons. These subjects should not take any other medication for one week

prior to the study or during the study. The bioavailability is determined by the col-lection of either blood samples or urine samples over a period of time and mea-

surement of the concentration of drug present in the samples.

Generally, a crossover study design is used. Using this method, both the test and

the reference products are compared in each subject, so that inter-subject variables

1. Defines procedures for establishing a bioequivalence requirement.

2. Sets forth criteria to establish a bioequivalence requirement.

3. Describes types of bioequivalence requirements.

4. Sets forth requirement for in-vitro batch testing and certification.

5. Describes requirements for marketing a drug product subject to a bioequivalence requirement.

6. Sets forth requirements for in-vivo testing of a drug product not meeting an in-vitro bioequiv-

alence standard.

Source: Ref. 103


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such as age, weight, differences in metabolism, etc., are minimized. Each subjec

thus acts as his own control. Also, with this design, subjects daily variations are

distributed equally among all dosage forms or drug products being tested.

The subjects are randomly selected for each group and the sequence of drugadministration is randomly assigned. The administration of each product is fol-

lowed by a sufficiently long period of time to ensure complete elimination of the

drug (washout period) before the next administration. The washout period should

be a minimum of 10 half-lives of the administered drug (106). A waiting period of

one week between administration is usually an adequate washout period of most

drugs.

With a drug requiring a washout period of one week, a typical randomized two-

way crossover bioequivalency study is shown in Table 8-13 on page 27.

TABLE 8-13. Two way cross over design

a 10 subjects per group

Assuming that the in-vivo performances of the two formulations are to be com-

pared by examining their blood level profiles, one must be certain that an adequate

number of blood samples are taken. Blood samples should be drawn with suffi

cient frequency to provide an accurate characterization of the drug concentra-

tion-time profile from which tmax, Cmax and AUC can be determined. Typically

a total of 10 to 15 sampling times might be required (107). Moreover, all samples

should be taken at the same time for both the test and the reference product to per-

mit proper statistical analysis.

Additional features which contribute to good study design include:

1. All drug samples obtained for the test and reference preparations should be analyzed by the

same method.2. Identical test conditions must be used for the two groups of subjects. For example, the types of

foods, fluid intake, physical activity, and posture should all be rigidly controlled in the study.

3. The physical characteristics of the subjects (such as age, height, weight, and health) should be

standardized.

Treatment

Groupa Week 1 Week 2

I

II

A

B

B

A


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Several important questions have been raised specifically regarding the design of

the bioequivalence tests. One of these deals with the selection of the appropriate

reference standard, since this is a critical component of a protocol (6, 108). Nor-

mally, the reference product is that available from the innovator company holding

the New Drug Application. However, in cases where there may be some questionas to the bioavailability of such a product, the study may utilize a solution of the

drug instead of or in addition to the marketed product. The use of a solution can

of course, result in some difficulty in interpretation of the data: a solid dosage

form, when compared to a solution, will usually exhibit a lower Cmax and a longer

tmax. The clinical significance of these differences may be difficult to assess.

In some instances, the FDA must designate a specific product as the reference

standard from among two or more possible products; e.g., Proventil tablets, 4 mg(Schering), not Ventolin tablets 4 mg (Allen and Hanburys), is the referenceproduct in bioequivalence studies of albuterol sulfate conventional tablets (108).

Advantages of Multiple-

dose vs. single dose

studies:

Another important question is whether the bioequivalence trial should comparesingle doses of the formulations or if it should compare "steady-state" conditionsreached after multiple dosing. It would seem that multiple dosing would be thelogical choice for drugs intended for long-term use since this would give a morerealistic comparison in view of the way in which the drug is normally adminis-tered. Other advantages of conducting a multiple-dose study over a single-dosestudy include (54, 59):

1. Multiple-dosing eliminates the long washout periods required between single-dose administra-

tions. The switch-over from one formulation to the other can take place in steady state.

2. Single-dose studies may pose problems of sufficiently long sampling periods in order to get

reliable estimates of terminal half-life, which is needed for correct calculation of the total AUC

3. Multiple-dose studies yield higher concentrations of drug in the blood, making accurate mea-

surement easier. In addition, since drug concentrations need to be measured only over a single

dosing interval at steady state, the need to measure lower concentrations during a disposition

phase is avoided.

4. Multiple-dosing studies can be conducted in patients, rather than healthy volunteers, allowing

the use of higher doses.

5. Usually, smaller intersubject variability is observed in steady-state studies, which may permit

the use of fewer subjects.

6. Nonlinear pharmacokinetics, if present, can be more readily detected at steady-state following

multiple-dosing.

Thus, for some drug products, multiple-dose bioequivalence studies are appropri-ate and should be performed. In fact, according to one of the conclusions of theBio- International '92 conference on the bioequivalence of highly variable drugs, amultiple-dose study is required in the case of compounds exhibiting nonlinearpharmacokinetics (110). The circumstances under which a multiple-dose studymay be required are summarized in the regulations (109):


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1. When there is a difference in the rate of absorption but not in the extent of absorption.

2. When there is excessive variability in bioavailability from subject to subject.

3. When the concentration of the active moiety in the blood resulting from a single dose is too low

for accurate determination.

4. When the drug product is a controlled-release dosage form.

On the other hand, multiple-dose bioequivalence studies are undesirable in some

respects. Healthy subjects should not be dosed with any drug for an extended

period of time (59). Multiple-dose studies are also generally more difficult to

carry out, especially with regard to ensuring subject compliance with dosing and

dietary restrictions. Therefore, most bioequivalence studies are conducted as sin-

gle-dose studies. Multiple-dose studies should be performed only when a sin

gle-dose study is not a reliable indicator of bioavailability (111).

8.2.3 ASSESSMENT OF BIOEQUIVALENCE

In order for different formulations of the same drug substance to be considered

bioequivalent, they must be equivalent with respect to the rate and extent of drug

absorption. Thus, the two predominant issues involved in the assessment of

bioequivalence are: the pharmacokinetic parameters that best characterize the rate

and extent of absorption and, the most appropriate method of statistical analysis of

the data.

Pharmacokinetic criteria With regard to the choice of the appropriate pharmacokinetic characteristics

Westlake suggests comparisons of the formulations should be made with respect to

only those parameter(s) of the blood level profile that possess some meaningful

relation to the therapeutic effect of the drug (107). Since the AUC is directly pro-portional to the amount of drug absorbed, this pharmacokinetic parameter is most

commonly used to characterize the extent of absorption, both in single- and multi-

ple- dose studies.

The choice of an appropriate pharmacokinetic characteristic for the rate of absorp-

tion is still being discussed with considerable controversy (112, 113). Although a

broad array of methods exists for calculating absorption rates (e.g. moment analy-

sis, deconvolution procedures and curve-fitting), the most commonly used param-

eters are peak concentration (Cmax) and time to peak concentration (tmax)

Although these parameters have been observed to have significant variances and

may be difficult to determine accurately, they remain the parameters generally

requested as rate characteristic by most regulatory authorities for immedi-

ate-release products (112).

Statistical criteria After a bioequivalence study is conducted and the appropriate parameters are

determined, the pharmacokinetic data must be examined according to a set of pre-

determined criteria to confirm or refute the bioequivalency of the test and refer-


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ence formulations. That is, one must determine whether the test and reference

products differ within a predefined level of statistical significance. Since the sta

tistical outcome of a bioequivalence study is the primary basis of the decision for

or against therapeutic equivalence of two products, it is critically important that the

experimental data be analyzed by an appropriate statistical test.

In the early 1970s, bioequivalence was usually determined only on the basis of

mean data. Mean AUC and Cmax values for the generic product had to be within

+20% of those of the reference (innovator) product (108). Although the 20% value

was somewhat arbitrary, it was felt that for most drugs, a 20% change in the dose

would not result in significant differences in the clinical response to drugs (114)

A relatively common misconception is that current regulatory standards still allow

this difference of 20% in the means of the pharmacokinetic variables (Cmax and

AUC) of the test and reference formulations. The FDAs statistical criteria for

approval of generic drugs now requires the application of confidence limits to the

mean data, using an analysis known as the two one-sided tests procedure (115).This change came about as a result of the conclusion of the FDA Bioequivalence

Task Force in 1986 that the use of a 90% confidence interval based on the two

one-sided t-tests approach was the best available method for evaluating bioequiva-

lence (111).

Westlake was the first to suggest the use of confidence intervals as a means of test-

ing for bioequivalence (116). Recognizing that no two products will result in iden

tical blood-level profiles, and that there will be differences in mean values between

products, Westlake pointed out that the critical issue was to determine how large

those differences could be before doubts as to therapeutic equivalence arose (107

117). A test formulation was considered to be bioequivalent to a reference formu-

lation if and . (119). By this procedure

if test and reference products were not bioequivalent (i.e. means differed by more

than 20%), there was a 5% chance of concluding that they are bioequivalent.

The current FDA guidelines are that two formulations whose rate and extent of

absorption differ by -20%/+25% or less are generally considered bioequivalent

(90). In order to verify that the -20%/+25% rule is satisfied, the two one-sided sta-

tistical tests are carried out: one test verifies that the bioavailability of the test

product is not too low and the other to show that it is not too high. The currenpractice is to carry out the two one-sided tests at the 0.05 level of significance.

Computationally, the two one-sided tests are carried out by computing a 90% con-

fidence interval. For approval of an ANDA, a generic manufacturer must show

that the 90% confidence interval for the ratio of the mean response (usually AUC

0.8AUCtest

AUCref------------------- 1.2< < 0.8

CpmaxtestCpmaxre f

-------------------- 1.2<


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and Cmax) of its product to that of the innovator is within the limits of 0.8 to 1.25.

Since these tests are carried out at the 0.05 level of significance, there is no more

than a 5% chance that they will be approved as equivalent if they differ by as much

or more than is allowed by the equivalence criteria (-20%/+25%).

Since this test requires that the 90% confidence interval of the difference between

the means be within a range of -20%/+25%, it is more stringent than simply requir-

ing the comparison of the test and reference products AUC and Cmax to be within

the 80 to 125% range. If the mean response of the generic product in the study

population is near 20% below or 25% above the innovator mean, one or both of the

confidence limits will fall outside the acceptable range and the product will fail the

bioequivalence test. Thus, the confidence interval requirement ensures that the

difference in mean values for AUC and Cmax will actually be less than -20%/

+25%. It should be pointed out that the standards vary among drugs and drug

classes. For example, antipsychotic agents may fall within a 30% variation and

antiarrhythmic agents may be allowed a 25% variation (122).

The actual differences between brand and generic products observed in bioequiva-

lence studies have been reported to be small. The FDA has stated that for

post-1962 drugs approved over a two-year period under the Waxman-Hatch bill

(1984), the mean bioavailability difference between the generic and pioneer prod-

ucts has been about 3.5% (120). In addition, 80% of the generic drugs approved

by the FDA between 1984 and 1986 differed from the innovator products by an

observed difference of only +5%. Such differences are small when compared to

other variables of drug therapy and would not be expected to produce clinically

observable differences in patient response.

8.2.4 CONTROVERSIES AND CONCERNS IN BIOEQUIVALENCE

The design, performance and evaluation of bioequivalence studies have received a

great deal of attention over the past decade from academia, the pharmaceutical

industry and regulatory agencies. A number of concerns and questions have been

raised about the conduct of bioequivalence studies as well as the guidelines and

criteria used to determine bioequivalence (112). Many of these concerns were

triggered by the passage of the Drug Price Competition and Patent Term Restora-

tion Act (The Waxman-Hatch Amendments) by Congress in 1984. This Act pro-

vided for an expedited approval by the FDA of generic drugs, thereby expanding

the potential generic market for prescription generic drugs (121). Shortly after the

passage of this Act, numerous published reports appeared in the scientific litera-

ture questioning the FDAs ability to ensure that generic drugs were equivalent to

the brand name drugs they were copying. Most of the concerns of the scientific

community centered around adequate standards for evaluation of bioequivalence


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and correlation between bioequivalence and therapeutic equivalence. Some of the

issues and co

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