Chapter 15. Drug Product Performance, In Vivo: Bioavailability and Bioequivalence

Drug product performance,1in vivo, may be defined as the release of the drug substance from the drug product leading to bioavailability of the drug substance. The assessment of drug product performance is important since bioavailability is related to the pharmacodynamic response and related adverse events. Thus, performance tests relate the quality of a drug product to clinical safety and efficacy. 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). 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.

Drug product performance studies are used in the development of new and generic drug products. The initial safety and efficacy clinical studies during new drug development may use a simple formulation such as a hard gelatin capsule containing only the active ingredient diluted with lactose. If the new drug demonstrates appropriate human efficacy and safety, a to-be-marketed drug product (eg, compressed tablet) may be developed. Since the initial safety and efficacy studies were performed using a different formulation (ie, hard gelatin capsule), the pharmaceutical manufacturer must demonstrate that the to-be-marketed drug product demonstrates equivalent drug product performance to the original formulation (Fig. 15-1). Equivalent drug product performance is generally demonstrated by an in vivo bioequivalence study in normal healthy volunteers. Under certain conditions, equivalent drug product performance may be demonstrated in vitro using comparative dissolution profiles (see Chapter 14).

As stated above, the marketed drug product that is approved by the FDA may not be the same formulation that was used in the original safety and efficacy clinical studies. After the drug product is approved by the FDA and marketed, the manufacturer may perform changes to the formulation. These changes to the marketed drug product are known as postapproval changes (see also Chapter 16). These postapproval changes, often termed SUPAC (scale-up and postapproval change), could include a change in the supplier of the active ingredient, a change in the formulation, a change in the manufacturing process and/or a change in the manufacturing site.2,3 In each case, the manufacturer must demonstrate that drug product performance did not change and is the same for the drug product manufactured before and after the SUPAC change. As shown in Fig. 15-1, drug product performance may be determined in vivo by bioequivalence studies or in vitro by comparative drug release/dissolution profiles.

Comparative drug product performance studies are important in the development of generic drug products (Fig. 15-2). A generic drug product is a multisource drug product2 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 performance. Clinical safety and efficacy studies are not generally performed on generic drug products. 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 product. 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. Similar to the brand-name drug product manufacturer, the generic drug manufacturer may make changes after FDA approval in the formulation, in the source of the active pharmaceutical ingredient, manufacturing process, or other changes.3 For any postapproval change, the manufacturer must demonstrate that the change did not alter the performance of the drug product.

1 A glossary of important terms appears at the end of this chapter.

2Multisource drug products are drug products that contains the same active drug substance in the same dosage form and are marketed by more than one pharmaceutical manufacturer.

3 SUPAC changes are discussed in Chapter 16.

Bioavailability studies are performed for both approved active drug ingredients and therapeutic moieties not yet approved for marketing by the FDA. New formulations of active drug ingredients must be approved by the FDA before marketing. In approving a drug product for marketing, the FDA ensures that the drug product is safe and effective for its labeled indications for use. Moreover, the drug product must meet all applicable standards of identity, strength, quality, and purity. To ensure that these standards are met, the FDA requires bioavailability/pharmacokinetic studies and, where necessary, bioequivalence studies for all drug products (FDA Guidance, 2003).

Invitro and/or in vivo bioequivalence studies must be performed on a drug formulation proposed for marketing as a generic drug product. The essential pharmacokinetics of the active drug ingredient or therapeutic moiety must be characterized. Essential pharmacokinetic parameters, including the rate and extent of systemic absorption (ie, bioavailability), elimination half-life, and rates of excretion and metabolism, should be established after single-dose and possibly multiple-dose administration. Data from these in vivo bioavailability studies are important to establish recommended dosage regimens and to support drug labeling.

In vivo bioavailability studies are also performed for new formulations4 of active drug ingredients or therapeutic moieties that have full NDA approval and are approved for marketing. The purpose of these studies is to determine the bioavailability and to characterize the pharmacokinetics of the new formulation, new dosage form, or new salt or ester relative to a reference formulation.

In summary, clinical studies are used to determine the safety and efficacy of drug products. 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 manufacture process of the drug product (dosage form). 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 (as defined above), then the clinical efficacy and the safety profile of these drug products are assumed to be similar and may be substituted for each other.

4 A manufacturer may design a new formulation such as an extended release drug product that contains an active drug previously approved as a different dosage form (eg, immediate release drug product)

The area under the drug concentration–time curve (AUC) is used as a measure of the total amount of unaltered drug that reaches the systemic circulation. The AUC is dependent on the total quantity of available drug, FD0, divided by the elimination rate constant, k, and the apparent volume of distribution, VD. F is the fraction of the dose absorbed. After IV administration, F is equal to unity (1.0 or 100%), because the entire dose enters the systemic circulation. The drug is considered to be completely available after IV administration. After oral or other extravascular route of administration of a drug, F may vary from a value of 0 (no drug absorption) to 1 (complete drug absorption).

Relative Availability

Relative (apparent) availability is the availability of the drug from a drug product as compared to a recognized standard. The fraction of dose systemically available from an oral drug product is difficult to ascertain. The availability of drug in the formulation is compared to the availability of drug in a standard dosage formulation, usually a solution of the pure drug evaluated in a crossover study. The relative availability of two drug products given at the same dosage level and by the same route of administration can be obtained using the following equation:

where drug product B is the recognized reference standard. This fraction may be multiplied by 100 to give percent relative availability.

When different doses are administered, a correction for the size of the dose is made, as in the following equation:

Urinary drug excretion data may also be used to measure relative availability, as long as the total amount of intact drug excreted in the urine is collected. The percent relative availability using urinary excretion data can be determined as follows:

where [Du]∞ is the total amount of drug excreted in the urine.

Relative bioavailability may exceed the value of 1 or 100% as compared to the reference drug product.

Absolute Availability

The absolute availability of drug is the systemic availability of a drug after extravascular administration (eg, oral, rectal, transdermal, subcutaneous) compared to IV dosing. The absolute availability of a drug is generally measured by comparing the respective AUCs after extravascular and IV administration. This measurement may be performed as long as VD and k are independent of the route of administration. Absolute availability after oral drug administration using plasma data can be determined as follows:

Absolute availability, F, may be expressed as a fraction or as a percent by multiplying F × 100. Absolute availability using urinary drug excretion datà can be determined by the following:

The absolute bioavailability is also equal to F, the fraction of the dose that is bioavailable. Absolute availability is sometimes expressed as a percent, ie, F = 1, or 100%. For drugs given intravascularly, such as by IV bolus injection, F = 1 because all of the drug is completely absorbed. For all extravascular routes of administration, such as the oral route (PO), the absolute bioavailability, F may not exceed 100% (F >1). F is usually determined by Equation 15.4 or 15.5, where PO is the oral route or any other extravascular route of drug administration.

The bioavailability of a new investigational drug was studied in 12 volunteers. Each volunteer received either a single oral tablet containing 200 mg of the drug, 5 mL of a pure aqueous solution containing 200 mg of the drug, or a single IV bolus injection containing 50 mg of the drug. Plasma samples were obtained periodically up to 48 hours after the dose and assayed for drug concentration. The average AUC values (0–48 hours) are given in the table below. From these data, calculate (a) the relative bioavailability of the drug from the tablet compared to the oral solution and (b) the absolute bioavailability of the drug from the tablet.

Solution

The relative bioavailability of the drug from the tablet is estimated using Equation 15.1. No adjustment for dose is necessary.

The relative bioavailability of the drug from the tablet is 1.04, or 104%, compared to the solution. In this study, the difference in drug bioavailability between tablet and solution was not statistically significant. It is possible for the relative bioavailability to be greater than 100%.

The absolute drug bioavailability from the tablet is calculated using Equation 15.4 and adjusting for the dose.

Because F, the fraction of dose absorbed from the tablet, is less than 1, the drug is not completely absorbed systemically, as a result of either poor absorption or metabolism by first-pass effect. The relative bioavailability of the drug from the tablet is approximately 100% when compared to the oral solution.

Results from bioequivalence studies may show that the relative bioavailability of the test oral product is greater than, equal to, or less than 100% compared to the reference oral drug product. However, the results from these bioequivalence studies should not be misinterpreted to imply that the absolute bioavailability of the drug from the oral drug products is also 100% unless the oral formulation was compared to an intravenous injection of the drug.

Direct and indirect methods may be used to assess drug bioavailability. The in vivo bioavailability of a drug product is demonstrated by the rate and extent of drug absorption, as determined by comparison of measured parameters, eg, concentration of the active drug ingredient in the blood, cumulative urinary excretion rates, or pharmacological effects. For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action. The design of the bioavailability study depends on the objectives of the study, the ability to analyze the drug (and metabolites) in biological fluids, the pharmacodynamics of the drug substance, the route of drug administration, and the nature of the drug product. Pharmacokinetic and/or pharmacodynamic parameters as well as clinical observations and in vitro studies may be used to determine drug bioavailability from a drug product (Table 15-1).

Plasma Drug Concentration

Measurement of drug concentrations in blood, plasma, or serum after drug administration is the most direct and objective way to determine systemic drug bioavailability. By appropriate blood sampling, an accurate description of the plasma drug concentration–time profile of the therapeutically active drug substance(s) can be obtained using a validated drug assay.

• tmax: 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 (Fig. 15-3). Drug absorption still continues after tmax is reached, but at a slower rate. When comparing drug products, tmax can be used as an approximate indication of drug absorption rate. The value for tmax will become smaller (indicating less time required to reach peak plasma concentration) as the absorption rate for the drug becomes more rapid. Units for tmax are units of time (eg, hours, minutes).
  Figure 15-3Graphic Jump Location

  

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  Plasma drug concentration–time curve.

• Cmax: The peak plasma drug concentration, Cmax, represents the maximum plasma drug concentration obtained after oral administration of drug. For many drugs, a relationship is found between the pharmacodynamic drug effect and the plasma drug concentration. Cmax provides indications that the drug is sufficiently systemically absorbed to provide a therapeutic response. In addition, Cmax provides warning of possibly toxic levels of drug. The units of Cmax are concentration units (eg, mg/mL, ng/mL). Although not a unit for rate, Cmax is often used in bioequivalence studies as a surrogate measure for the rate of drug bioavailability.
• AUC: The area under the plasma level–time curve, AUC, is a measurement of the extent of drug bioavailability (see Fig. 15-3). The AUC reflects the total amount of active drug that reaches the systemic circulation. 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, and VD = volume of distribution. The AUC is independent of the route of administration and processes of drug elimination as long as the elimination processes do not change. The AUC can be determined by a numerical integration procedure, such as the trapezoidal rule method. The units for AUC are concentration × time (eg, μg h/mL).

For many drugs, the AUC is directly proportional to dose. For example, if a single dose of a drug is increased from 250 to 1000 mg, the AUC will also show a fourfold increase (Figs. 15-4 and 15-5).

In some cases, the AUC is not directly proportional to the administered dose for all dosage levels. For example, as the dosage of drug is increased, one of the pathways for drug elimination may become saturated (Fig. 15-6). Drug elimination includes the processes of metabolism and excretion. Drug metabolism is an enzyme-dependent process. For drugs such as salicylate and phenytoin, continued increase of the dose causes saturation of one of the enzyme pathways for drug metabolism and consequent prolongation of the elimination half-life. The AUC thus increases disproportionally to the increase in dose, because a smaller amount of drug is being eliminated (ie, more drug is retained). When the AUC is not directly proportional to the dose, bioavailability of the drug is difficult to evaluate because drug kinetics may be dose dependent. Conversely, absorption may also become saturated resulting in lower-than-expected changes in AUC.

Urinary Drug Excretion Data

Urinary drug excretion data is an indirect method for estimating bioavailability. 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 (see Chapter 3).

• The cumulative amount of drug excreted in the urine, , is related directly to the total amount of drug absorbed. Experimentally, urine samples are collected periodically after administration of a drug product. Each urine specimen is analyzed for free drug using a specific assay. A graph is constructed that relates the cumulative drug excreted to the collection-time interval (Fig. 15-7).

The relationship between the cumulative amount of drug excreted in the urine and the plasma level–time curve is shown in Fig.15-6. When the drug is almost completely eliminated (point C), the plasma concentration approaches zero and the maximum amount of drug excreted in the urine, , is obtained.

• dDu/dt: The rate of drug excretion. Because most drugs are eliminated by a first-order rate process, the rate of drug excretion is dependent on the first-order elimination rate constant, k and the concentration of drug in the plasma, Cp. In Fig. 15-8, 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. Thus, a graph comparing the rate of drug excretion with respect to time should be similar in shape to the plasma level–time curve for that drug (Fig. 15-9).
  Figure 15-8Graphic Jump Location

  

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  Corresponding plots relating the plasma level–time curve and the cumulative urinary drug excretion.

  Figure 15-9Graphic Jump Location

  

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  Corresponding plots relating the plasma level–time curve and the rate of urinary drug excretion.

• t∞: The total time for the drug to be excreted. In Figs. 15-8 and 15-9, 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.

Acute Pharmacodynamic Effect

In some cases, the quantitative measurement of a drug in plasma or urine lacks an assay with sufficient accuracy and/or reproducibility. For locally acting, nonsystemically absorbed drug products, such as topical corticosteroids, plasma drug concentrations may not reflect the bioavailability of the drug at the site of action. An acute pharmacodynamic effect, such as an effect on forced expiratory volume, FEV1 (inhaled bronchodilators), or skin blanching (topical corticosteroids) can be used as an index of drug bioavailability. 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 to permit a reasonable estimate for a time period at least three times the half-life of the drug (Gardner, 1977). This approach may be particularly applicable to dosage forms that are not intended to deliver the active moiety to the bloodstream for systemic distribution.

The use of an acute pharmacodynamic effect to determine bioavailability generally requires demonstration of a dose–response curve (Fig. 15-10 and Chapter 19). Bioavailability is determined by characterization of the dose–response curve. For bioequivalence determination, pharmacodynamic 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 (Fig. 15-11). The onset time and duration of the pharmacokinetic effect may also be included in the analysis of the data. The use of pharmacodynamic endpoints5 for the determination of bioavailability and bioequivalence is much more variable than the measurement of plasma or urine drug concentrations.

Figure 15-11 shows an acute pharmacologic effect that is measured periodically after a single oral dose. The effect curve is similar to Fig. 1-2 in Chapter 1.

Clinical Observations

Well-controlled clinical trials in humans establish the safety and effectiveness of drug products and may be used to determine bioavailability. However, the clinical trials approach is the least accurate, least sensitive to bioavailability differences, and most variable. The highly variable clinical responses require the use of a large patient population which increases the study costs and requires a longer time to complete compared to the other approaches for determination of bioequivalence. The FDA considers this approach only when analytical methods and pharmacodynamic methods are not available to permit use of one of the approaches described above. Comparative clinical studies have been used to establish bioequivalence for topical antifungal drug products (eg, ketoconazole) and for topical acne preparations. For dosage forms intended to deliver the active moiety to the bloodstream for systemic distribution, this approach may be considered acceptable only when analytical methods cannot be developed to permit use of one of the other approaches.

In Vitro Studies

Comparative drug release/dissolution studies under certain conditions may give an indication of drug bioavailability and bioequivalence. Ideally, the in vitro drug dissolution rate should correlate with in vivo drug bioavailability (see Chapters 7 and 14 on in vivo–in vitro correlation, IVIVC). Comparative dissolution studies are often performed on several test formulations of the same drug during drug development. 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. Under certain conditions, comparative dissolution profiles of higher and lower dose strengths of a solid oral drug product such as an immediate-release tablet, are used to obtain a waiver (biowaiver) from performing an in vivo bioequivalence study on the lower-dose strength drug product.6

The FDA may also use in vitro approaches other than comparative dissolution for establishing bioequivalence. The use of the Biopharmaceutics Classification System (BCS) is discussed in a separate section in this chapter. The use of in vitro biomarkers and in vitro binding studies has been proposed to establish bioequivalence. For example, cholestyramine resin is a basic quaternary ammonium anion-exchange resin that is hydrophilic, insoluble in water, and not absorbed in the gastrointestinal tract. The bioequivalence of cholestyramine resin is performed by equilibrium and kinetic binding studies of the resin to bile acid salts (www.fda.gov/cder/guidance/cholesty.pdf).

5 A pharmacodynamic endpoint is an acute pharmacologic effect that is directly related to the drug's activity that can be measured quantitatively. An example is changes in FEV1 (forced expiratory volume) that can be measured after the administration of a bronchodilator such as albuterol inhalation.

6 FDA Guidance for Industry: Bioavailability and Bioequivalence: Studies for Orally Administered Drug Products—General Considerations, March, 2003.

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. However, variable clinical responses among individuals that are unrelated to bioavailability may also be due to differences in the pharmacodynamics of the drug. Differences in pharmacodynamics, ie, the relationship between the drug and the receptor site, may be due to differences in receptor sensitivity to the drug. Various factors affecting pharmacodynamic drug behavior may include age, drug tolerance, drug interactions, and unknown pathophysiologic factors.

The bioavailability of a drug may be more reproducible among fasted individuals in controlled studies who take the drug on an empty stomach. When the drug is used on a daily basis, however, the nature of an individual's diet and lifestyle may affect the plasma drug levels because of variable absorption in the presence of food or even a change in the metabolic clearance of the drug. Feldman and associates (1982) reported that patients on a high-carbohydrate diet have a much longer elimination half-life of theophylline, due to the reduced metabolic clearance of the drug (t1/2, 18.1 hours), compared to patients on normal diets (t1/2 = 6.76 hours). Previous studies demonstrated that the theophylline drug product was completely bioavailable. The higher plasma drug concentration resulting from a carbohydrate diet may subject the patient to a higher risk of drug intoxication with theophylline. The effect of food on the availability of theophylline has been reported by the FDA concerning the risk of higher theophylline plasma concentrations from a 24-hour sustained-release drug product taken with food. Although most bioavailability drug studies use fasted volunteers, the diet of patients actually using the drug product may increase, decrease, or have no effect on the bioavailability of the drug (Hendles et al, 1984).

Bases for Determining Bioequivalence

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

In a few cases, 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 and appropriately reflected in the labeling and/or the rate of absorption is not detrimental to the safety and effectiveness of the drug product.

Drug Products with Possible Bioavailability and Bioequivalence Problems

Lack of bioequivalence may be suspected when evidence from well-controlled clinical trials or controlled observations in patients of various marketed drug products do not give comparable therapeutic effects. These drug products need to be evaluated either in vitro (eg, drug dissolution/release test) or in vivo (eg, bioequivalence study) to determine if the drug product has a bioavailability problem (see also US Code of Federal Regulations, 21 CFR 320.33).

In addition, during the development of a drug product, certain biopharmaceutical properties of the active drug substance or the formulation of the drug product may indicate that the drug may have variable bioavailability and/or a bioequivalence problem. Some of these biopharmaceutic indicators include:

• The active drug ingredient has low solubility in water (eg, less than 5 mg/mL).
• The dissolution rate of one or more such products is slow (eg, <50% in 30 minutes when tested with a general method specified by the FDA).
• The particle size and/or surface area of the active drug ingredient is critical in determining its bioavailability.
• Certain structural forms of the active drug ingredient (eg, polymorphic forms, solvates, complexes, and crystal modifications) dissolve poorly, thus affecting absorption.
• Drug products that have a high ratio of excipients to active ingredients (eg, >5:1).
• Specific inactive ingredients (eg, hydrophilic or hydrophobic excipients and lubricants) either may be required for absorption of the active drug or may interfere with such absorption.
• The active drug ingredient, or its precursor is absorbed mostly in a particular segment of the GI tract or is absorbed from a localized site.
• The degree of absorption of the therapeutic moiety or its precursor is poor (eg, <50%, ordinarily in comparison to an intravenous dose), even when it is administered in pure form (eg, in solution).
• There is rapid metabolism of the therapeutic moiety in the intestinal wall or liver during the absorption process (first-pass metabolism), so that the rate of absorption is unusually important in the therapeutic effect and/or toxicity of the drug product.
• The therapeutic moiety is rapidly metabolized or excreted, so that rapid dissolution and absorption are required for effectiveness.
• The active drug ingredient or therapeutic moiety is unstable in specific portions of the GI tract and requires special coatings or formulations (eg, buffers, enteric coatings, etc) to ensure adequate absorption.
• The drug product is subject to dose-dependent kinetics in or near the therapeutic range, and the rate and extent of absorption are important in establishing bioequivalence.

Objective

All scientific studies should have clearly stated objectives. The main objective for a bioequivalence study is that the drug bioavailability from test and reference products are not statistically different when administered to patients or subjects at the same molar dose under similar experimental conditions.

Study Considerations

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.

Since bioequivalence studies are performed to compare the bioavailability of the generic drug product to the brand-name product, the statistical techniques should be of sufficient sensitivity to detect differences in rate and extent of absorption that are not attributable to subject variability. Once bioequivalence is established, it is likely that both the generic and brand-name dosage forms will produce the same therapeutic effect. The FDA publishes guidances for bioequivalence studies (www.fda.gov/cder/guidance; see also 21 CFR 320.25). Sponsors may also request a meeting with the FDA to review the study design for a specific drug product. Pharmacokinetic parameters, pharmacodynamic parameters, clinical observations and/or in vitro studies may be used to determine drug bioavailability from a drug product.

The design and evaluation of well-controlled bioequivalence studies require cooperative input from pharmacokineticists, statisticians, clinicians, bioanalytical chemists, and others. For some generic drugs, the FDA offers general guidelines for conducting these studies. For example, Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Design is available from the FDA; the publication addresses three specific aspects, including (1) logarithmic transformation of pharmacokinetic data, (2) sequence effect, and (3) outlier consideration. However, even with the availability of such guidelines, the principal investigator should prepare a detailed protocol for the study. Some of the elements of a protocol for an in vivo bioavailability study are listed in Table 15-2. Bioavailability studies for controlled-release dosage forms are discussed in Chapter 17.

For bioequivalence studies, the test and reference drug formulations must contain the pharmaceutically equivalent drug in the same dose strength and in similar dosage forms (eg, immediate release or controlled release), and it must be given by the same route of administration. Both a single-dose and/or a multiple-dose (steady-state) study may be required. Before beginning the study, the Institutional Review Board (IRB) of the clinical facility in which the study is to be performed must approve the study. The IRB is composed of both professional and lay persons with diverse backgrounds who have clinical experience and expertise as well as sensitivity to ethical issues and community attitudes. The IRB is responsible for all ethical issues including safeguarding the rights and welfare of human subjects.

The basic guiding principle in performing studies is do not do unnecessary human research. Generally, the study is performed in normal, healthy male and female volunteers who have given informed consent to be in the study. Critically ill patients are not included in an in vivo bioavailability study unless the attending physician determines that there is a potential benefit to the patient. The number of subjects in the study will depend on the expected intersubject and intrasubject variability. Patient selection is made according to certain established criteria for inclusion in, or exclusion from, the study. For example, the study might exclude any volunteers who have known allergies to the drug, are overweight, or have taken any medication within a specified period (often 1 week) prior to the study. Moderate smokers may be included in these studies. The subjects generally fast for 10 to 12 hours (overnight) prior to drug administration and may continue to fast for a 2- to 4-hour period after dosing.

Reference Listed Drug, RLD

For bioequivalence studies, 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 drug7 as the standard drug product to which all generic versions must be shown to be bioequivalent. The FDA hopes to avoid possible significant variations among generic drugs and their brand-name counterparts. Such variations could result if generic drugs were compared to different reference listed drugs.

The reference drug product should be administered by the same route as the comparison formulations unless an alternative route or additional route is needed to answer specific pharmacokinetic questions. For example, if an active drug is poorly bioavailable after oral administration, the drug may be compared to an oral solution or an intravenous injection. For bioequivalence studies on a proposed generic drug product the reference standard is the reference listed drug (RLD), which is listed in Approved Drug Products with Therapeutic Equivalence Evaluations—the Orange Book (www.fda.gov/cder/ob/default.htm), and the proposed generic drug product is often referred to as the “test” drug product. The RLD is generally a formulation currently marketed with a fully approved NDA for which there are valid scientific safety and efficacy data. The RLD is usually the innovator's or original manufacturer's brand-name product and is administered according to the dosage recommendations in the labeling.

Before beginning an in vivo bioequivalence study, the total content of the active drug substance in the test product (generally the generic product) must be within 5% of that of the reference product. Moreover, in vitro comparative dissolution or drug-release studies under various specified conditions are usually performed for both test and reference products before performing the in vivo bioequivalence study.

Modified-Release Formulations

The purpose of an in vivo bioavailability study involving an extended-release drug product is to determine if (1) the drug product meets the controlled-release claims made for it, (2) the bioavailability profile established for the drug product rules out the occurrence of any dose-dumping, (3) the drug product's steady-state performance is equivalent to that of a currently marketed non-extended-release formulation, and (4) the drug product's formulation provides consistent pharmacokinetic performance between individual dosage units. A comparison bioavailability study is used for the development of a new extended-release drug product in which the reference drug product may be either a solution or suspension of the active ingredient or a currently marketed non-controlled-release drug product such as a tablet or capsule. For example, the bioavailability of a non-controlled-release (immediate-release) drug product given at a dose of 25 mg every 8 hours is compared to an extended-release product containing 75 mg of the same drug given once daily.

For a bioequivalence study of a new generic extended-release drug product, the reference drug product is the currently marketed-extended release drug product listed as the RLD in the Orange Book and is administered according to the dosage recommendations in the approved labeling.

Combination Drug Products

Generally, the purpose of an in vivo bioavailability study involving a combination drug product containing more than one active drug substance is to determine if the rate and extent of absorption of each active drug ingredient or therapeutic moiety in the combination drug product is equivalent to the rate and extent of absorption of each active drug ingredient or therapeutic moiety administered concurrently in separate single-ingredient preparations. The reference material in such a bioavailability study should be two or more currently marketed, single-ingredient drug products, each of which contains one of the active drug ingredients in the combination drug product. The FDA may, for valid scientific reasons, specify that the reference material be a combination drug product that is the subject of an approved NDA.

For a bioequivalence study of a new generic combination drug product, the reference drug product is the currently marketed extended-release drug product listed as the RLD in the Orange Book and is administered according to the dosage recommendations in the approved labeling. All active moieties are measured and bioequivalence is determined for each.

Analytical Methods

Analytical methods used in an in vivo bioavailability, bioequivalence, or pharmacodynamic studies must be validated for accuracy and sufficient sensitivity. The actual concentration of the active drug ingredient or therapeutic moiety, or its active metabolite(s), must be measured with appropriate precision in body fluids or excretory products. For bioavailability and bioequivalence studies, both the parent drug and its major active metabolites are generally measured. For bioequivalence studies, the parent drug is measured. Measurement of the active metabolite is important for very high hepatic clearance (first-pass metabolism) drugs when the parent drug concentrations are too low to be reliable.

The analytical method for measurement of the drug must be validated for accuracy, precision, sensitivity, specificity, and robustness. The use of more than one analytical method during a bioequivalence study may not be valid, because different methods may yield different values. Data should be presented in both tabulated and graphic form for evaluation. The plasma drug concentration–time curve for each drug product and each subject should be available.

7 The reference listed drug, RLD, is listed in the Orange Book, Approved Drug Products with Therapeutic Equivalence Evaluations. www.accessdata.fda.gov/scripts/cder/ob/default.cfm.

For many drug products, the FDA, Division of Bioequivalence, Office of Generic Drugs, provides guidance for the performance of in vitro dissolution and in vivo bioequivalence studies.8 Generally, two bioequivalence studies are required for solid oral dosage forms, including (1) a fasting study and (2) a food intervention study. For extended-release capsules containing beads (pellets) that might be poured on a semi-solid food such as applesauce, an additional “sprinkle” bioequivalence study is required. Other study designs such as a multiple-dose (steady-state) bioequivalence study have been proposed by the FDA. Proper study design and statistical evolution are important considerations for the determination of bioequivalence. Some of the designs listed above are summarized here.

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 required for all immediate-release and modified-release oral dosage forms. Both male and female subjects may be used in the study. Blood sampling is performed just before (zero time) the dose and at appropriate intervals after the dose to obtain an adequate description of the plasma drug concentration–time profile. 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 normally given to the subject for at least 1 week prior to the study. In some cases, a parallel design may be more appropriate for certain drug products, containing a drug with a very long elimination half-life. A replicate design may be used for a drug product containing a drug that has high intrasubject variability.

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.

Meals that are high in total calories and fat content are more likely to affect the GI physiology and thereby result in a larger effect on the bioavailability of a drug substance or drug product. The test meal is a high-fat (approximately 50% of total caloric content of the meal) and high-calorie (approximately 800–1000 calories) meal. A typical test meal is two eggs fried in butter, two strips of bacon, two slices of toast with butter, 4 oz of brown potatoes, and 8 oz of milk. This test meal derives approximately 150, 250, and 500 to 600 calories from protein, carbohydrate, and fat, respectively (www.fda.gov/cder/guidance/4613dft.pdf).

For bioequivalence studies, drug bioavailability from both the test and reference products should be affected similarly by food. The 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 postdose. This study is required for all modified-release dosage forms and may be required for immediate-release dosage forms if the bioavailability of the active drug ingredient is known to be affected by food (eg, ibuprofen, naproxen). For certain extended-release capsules that contain coated beads, the capsule contents are sprinkled over soft foods such as applesauce, which is taken by the fasted subject and the bioavailability of the drug is then measured. Bioavailability studies might also examine the effects of other foods and special vehicles such as apple juice.

8 The FDA provides Individual Product Bioequivalence Recommendations, available at www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm075207.htm.

Subjects who meet the inclusion and exclusion study criteria and have given informed consent are selected at random. A complete crossover design is usually employed, in which each subject receives the test drug product and the reference product. Examples of Latin-square crossoverdesigns for a bioequivalence study in human volunteers, comparing three different drug formulations (A, B, C) or four different drug formulations (A, B, C, D), are described in Tables 10.3 and 10.4. 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 (see Table 15-3). In this design, each subject is his own control, and subject-to-subject variation is reduced. Moreover, variation due to sequence, period, and treatment (formulation) are reduced, so that all patients do not receive the same drug product on the same day and in the same order. 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 subject. Thus, drug product B may be followed by drug product A, D, or C (see Table 15-4). After each subject receives a drug product, blood samples are collected at appropriate time intervals so that a valid blood drug level–time curve is obtained. The time intervals should be spaced so that the peak blood concentration, the total area under the curve, and the absorption and elimination phases of the curve may be well described.

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 study. For example, a two-sequence, two-period study would be designed as follows:

Table 15-4 shows a design for three different drug treatment groups given in a three-period study with six different sequences. 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 treatment.

Replicated Crossover Study Designs

The standard bioequivalence criteria using the standard crossover design and a reasonable number of study subjects (<80 subjects) is difficult to achieve with highly variable drugs and drug products (%CV greater than 30). Drugs with high within-subject variability generally have a wide therapeutic window and despite high variability, these products have been demonstrated to be both safe and effective. Replicate designs for highly variable drugs/products require a smaller number of subjects and therefore, do not unnecessarily expose a large number of healthy subjects to a drug when this large number of subjects is not needed for assurance of bioequivalence (Haidar et al, 2008).

Replicated crossover designs are used for the determination of individual bioequivalence, to estimate within-subject variance for both the test and reference drug products, and to provide an estimate of the subject-by-formulation interaction variance. A four-period, two-sequence, two-formulation design is shown below:

In this design, the same reference and the same test are each given twice to the same subject. Other sequences are possible. In this design, reference-to-reference and test-to-test comparisons may also be made.

Scaled Average Bioequivalence

Recently a three-sequence, three-period, two-treatment partially replicated crossover design for bioequivalence studies of highly variable drugs has been recommended by the FDA (Haidar et al, 2008). The partially replicated design allows the estimation of the within-subject variance and subject-by-formulation interaction for the reference product. The time for completion of this study is shorter than the fully replicated four-way crossover design.

Under this design, if the test product has lower variability than the reference product, the study will need a smaller number of subjects to pass the bioequivalence criteria. Scaled average bioequivalence is evaluated for both AUC and Cmax.

Nonreplicate, Parallel Study Designs

A nonreplicate, 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. It is important to balance the demographics of both groups of volunteers. Blood sample collection time should be adequate to ensure completion of gastrointestinal transit (approximately 2 to 3 days) of the drug product and absorption of the drug substance. Cmax and a suitably truncated AUC, generally to 72 hours after dose administration, can be used to characterize peak and total drug exposure, respectively. For drugs that demonstrate low intra-subject variability in distribution and clearance, an AUC truncated at 72 hours (AUC072 hours) can be used in place of AUC0t or AUC0∞. This design is not recommended for drugs that have high intrasubject variability in distribution and clearance.

Multiple-Dose (Steady-State) Study Design

A bioequivalence study may be performed using a multiple-dose 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. Each subject receives either the test or reference product separated by a “washout” period, which is the time needed for the drug to be completely eliminated from the body.

To ascertain that the subjects are at steady state, three consecutive trough concentrations (Cmin) are determined. The last morning dose is given to the subject after an overnight fast, with continual fasting for at least 2 hours following dose administration. Blood sampling is then performed similar to the single-dose study.

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

• AUC0-t —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

The data are analyzed 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% to 125% of the reference product using a 90% confidence interval. Estimation of the absorption rate constant during multiple dosing is difficult, because the residual drug from the previous dose superimposes on the dose that follows. However, the data obtained in multiple doses are useful in calculating a steady-state plasma level.

The extent of bioavailability, measured by assuming the , is dependent on clearance:

Determination of bioavailability using multiple doses reveals changes that are normally not detected in a single-dose study. For example, nonlinear pharmacokinetics may occur after multiple drug doses due to the higher plasma drug concentrations saturating an enzyme system involved in absorption or elimination of the drug. Nonlinear pharmacokinetics after multiple-dose studies may be observed by rising Cmin drug concentrations after each dosing interval. With some drugs, a drug-induced malabsorption syndrome can also alter the percentage of drug absorbed. In this case, drug bioavailability may decrease after repeated doses if the fraction of the dose absorbed (F) decreases or if the total body clearance (kVD) increases.

There are several disadvantages of using the multiple-dose crossover method for the determination of bioequivalence. (1) The study takes more time to perform, because steady-state conditions must be reached. A longer time for completion of a study leads to greater clinical costs and the possibility of a subject dropping out and not completing the study. (2) More plasma samples must be obtained from the patient to ascertain that steady state has been reached and to describe the plasma level–time curve accurately. (3) Because 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. If there is wide variation in the rate of a drug's availability, then it is possible that initial high blood levels may lead to toxicity.

Clinical Endpoint Bioequivalence Study

Study design for a clinical endpoint study generally consists of a randomized, double-blind, placebo-controlled, parallel-designed study comparing test product, reference product, and placebo product in patients. In some cases, the use of a placebo may not be included for safety reasons. The primary analysis for bioequivalence is determined by evaluating the difference between the proportion of patients in the test and reference treatment groups who are considered a “therapeutic cure” at the end of study. The superiority of the test and reference products against the placebo is also tested using the same dichotomous endpoint of “therapeutic cure.”

Determination of Bioequivalence of Drug Products in Patients Maintained on a Therapeutic Drug Regimen

A bioequivalence study may be performed in patients already maintained on the reference (brand-name) drug. Due to safety concerns, certain drugs such as clozapine, a dibenzodiazepine derivative with potent antipsychotic properties should not be given to normal healthy subjects. Instead, bioequivalence studies on clozapine should be performed in patients who have been stabilized on the highest strength (eg, 100 mg) using a multiple-dose bioequivalence study design. Patients on these or other drugs such as hormone replacement therapy and cancer chemotherapeutic drugs would be at risk if a washout period is used between drug treatments. Therefore, the patient is maintained on his or her own medication, and blood sampling is performed during a dosage interval (Fig. 15-12, reference product A). Once blood sampling is accomplished, the patient takes equal oral doses of the test drug product and the reference drug product is discontinued. Drug dosing with the test drug product continues until attainment of steady state. When steady state is reached, the plasma level–time curve for a dosage interval with the second drug product is described (Fig. 15-12, drug product B). Using the same plasma measures as before, the bioequivalence or lack of bioequivalence may be determined. The patient then continues with his or her therapy with the original drug product.

If the blood level–time curve of the second drug product is comparable to that of the reference drug product, the second product is considered to be bioequivalent. If the second drug has less bioavailability (assuming that only the extent of drug absorption is less than that of the reference drug), the resulting will be smaller than that obtained with the first drug. Usually the drug manufacturer will perform dissolution and content uniformity tests before performing a bioequivalence study. These in vitro dissolution tests will help ensure that the obtained from each drug product in vivo will not be largely different from each other. In contrast, if the extent of drug availability is greater in the second drug product, the will be higher.

Levothyroxine Sodium Oral Tablets

The bioequivalence of two synthetic levothyroxine sodium oral tablets, Levothroid and Synthroid, were evaluated in 20 hypothyroid patients (Sista and Albert, 1994). The investigation was designed as a two-way crossover study in which the patients who had been diagnosed as hypothyroid by their primary-care physician were given a single 100-μg daily dose of either Levothroid or Synthroid brand levothyroxine sodium tablets for 50 days and then switched over immediately to other treatment for 50 days. Predose blood samples were taken on days 1, 25, 48, 49, and 50 of each phase, and, on day 50, a complete blood sampling was performed. The serum from each blood sample was analyzed for total and free thyroxine (T4), total and free triiodothyronine (T3), the major metabolite of T4, and thyrotropin (TSH).

1. Why were hypothyroid patients used in this study?

2. Why were the subjects dosed for 50 days with each thyroid product?

3. Why were blood samples obtained on days 48, 49, and 50?

4. Why was T3 measured?

5. Why was TSH measured?

Solution

1. Normal healthy euthyroid subjects would be at risk if they were to take levothyroxine sodium for an extended period of time.

2. The long (50-day) daily dosing for each product was required to obtain steady-state drug levels because of the long elimination half-life of levothyroxine.

3. Serum from blood samples was taken on days 48, 49, and 50 to obtain three consecutive Cmin drug levels.

4. T3 is the active metabolite of T4.

5. The serum TSH concentration is inversely proportional to the free serum T4 concentrations and gives an indication of the pharmacodynamic activity of the active drug.

Mercaptopurine (Purinethol) Oral Tablets

Mercaptopurine (Purinethol) is a cytotoxic drug used to treat cancer and is available in a 50-mg oral tablet. The FDA recommends bioequivalence steady-state studies in patients receiving therapeutic oral doses (usually 100–200 mg/d in the average adult) or maintenance daily doses (usually 50–100 mg/d in the average adult).

Patients should be on a stable regimen using the same dosage unit (multiples of the same 50-mg strength). Plasma drug concentration–time profiles are obtained in these patients at steady state with the brand product. The proposed generic drug product is then given to these patients at the same dosage regimen until steady state is reached. Plasma drug concentration–time profiles are obtained for the generic drug product, then the patients return to the original brand medication.

Pharmacokinetic 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 (AUC0t) and to infinity (AUC0∞), tmax, and Cmax. Additionally, the elimination rate constant, k, the elimination half-life, t1/2, and other parameters may be estimated. For multiple-dose studies, pharmacokinetic analysis includes calculation for each subject of the steady-state area under the curve, (AUC∞t), tmax, Cmin, Cmax, and the percent fluctuation [100 × (Cmax – Cmin)/Cmin]. Proper statistical evaluation should be performed on the estimated pharmacokinetic parameters.

Statistical Evaluation of the Data

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 (Schuirmann, 1987; FDA Guidance, 2001).

Many statistical approaches (parametric tests) assume that the data are distributed according to a normal distribution or “bell-shaped curve” (see Appendix A). The pharmacokinetic parameters such as Cmax and AUC may not be normally distributed and the true distribution is difficult to ascertain because of the small number of subjects used in a bioequivalence study. The distribution of data that have been transformed to log values resembles more closely a normal distribution compared to the distribution of non-log-transformed data.

Two One-Sided Tests Procedure

The two one-sided tests procedure is also referred to as the confidence interval approach (Schuirmann, 1987). 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 (ie, greater than 20%) between the mean parameters.

The 90% confidence limits are estimated for the sample means. The interval estimate is based on Student's t distribution of the data. In this test, presently required by the FDA, a 90% confidence interval about the ratio of means of the two drug products must be within ±20% for measurement of the rate and extent of drug bioavailability. For most drugs, up to a 20% difference in AUC or Cmax between two formulations would have no clinical significance. The lower 90% confidence interval for the ratio of means cannot be less than 0.80, and the upper 90% confidence interval for the ratio of the means cannot be greater than 1.20. When log-transformed data are used, the 90% confidence interval is set at 80% to 125%. These confidence limits have also been termed the bioequivalenceinterval (Midha et al, 1993). The 90% confidence interval is a function of sample size and study variability, including inter- and intrasubject variability.

For a single-dose, fasting or food intervention bioequivalence study, an analysis of variance (ANOVA) is usually performed on the log-transformed AUC and Cmax values. There should be no statistical differences between the mean AUC and Cmax parameters for the test (generic) and reference drug products. In addition, the 90% confidence intervals about the ratio of the means for AUC and Cmax values of the test drug product should not be less than 0.80 (80%) nor greater than 1.25 (125%) of that of the reference product based on log-transformed data. Table 15-5 summarizes the statistical analysis for average bioequivalence. Presently, the FDA accepts only average bioequivalence estimates are used to establish bioequivalence of generic drug products.

Analysis of Variance

An analysis of variance (see ANOVA) is a statistical procedure (see Appendix A) used to test the data for differences within and between treatment and control groups. A bioequivalent product should produce no significant difference in all pharmacokinetic parameters tested. The parameters tested statistically usually include AUC0t, AUC0∞, and Cmax obtained for each treatment or dosage form. Other metrics of bioavailability have also been used to compare the bioequivalence of two or more formulations. The ANOVA may evaluate variability in subjects, treatment groups, study period, formulation, and other variables, depending on the study design. If the variability in the data is large, the difference in means for each pharmacokinetic parameter, such as AUC, may be masked, and the investigator might erroneously conclude that the two drug products are bioequivalent.

A statistical difference between the pharmacokinetic parameters obtained from two or more drug products is considered statistically significant if there is a probability of less than 1 in 20 times or 0.05 probability (p ≤ .05) that these results would have happened on the basis of chance alone. The probability, p, is used to indicate the level of statistical significance. If p < .05, the differences between the two drug products are not considered statistically significant.

To reduce the possibility of failing to detect small differences between the test products, a power test is performed to calculate the probability that the conclusion of the ANOVA is valid. The power of the test will depend on the sample size, variability of the data, and desired level of significance. Usually the power is set at 0.80 with a β = 0.2 and a level of significance of 0.05. The higher the power, the more sensitive the test and the greater the probability that the conclusion of the ANOVA is valid.

A simulated example of the results for a single-dose, fasting study is shown in Table 15-6 and in Fig. 15-13. As shown by the ANOVA, no statistical differences for the pharmacokinetic parametersAUC0t, AUC0∞, and Cmax were observed between the test product and the brand-name product. The 90% confidence limits for the mean pharmacokinetic parameters of the test product were within 0.80 to 1.25 (80%–125%) of the reference product means based on log transformation of the data. The power test for the AUC measures were above 99%, showing good precision of the data. The power test for the Cmax values was 87.9%, showing that this parameter was more variable.

Table 15-7 shows 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. 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% (ie, 74%), compared to the solution at the low-range estimate and would not be considered bioequivalent based on the AUC.

For illustrative purposes, consider a drug that has been prepared at the same dosage level in three formulations, A, B, and C. These formulations are given to a group of volunteers using a three-way, randomized crossover design. In this experimental design, all subjects receive each formulation once. From each subject, plasma drug level and urinary drug excretion data are obtained. With these data we can observe the relationship between plasma and urinary excretion parameters and drug bioavailability (Fig. 15-14). The rate of drug absorption from formulation A is more rapid than that from formulation B, because the tmax for formulation A is shorter. Because the AUC for formulation A is identical to the AUC for formulation B, the extent of bioavailability from both of these formulations is the same. Note, however, the Cmax for A is higher than that for B, because the rate of drug absorption is more rapid.

The Cmax is generally higher when the extent of drug bioavailability is greater. The rate of drug absorption from formulation C is the same as that from formulation A, but the extent of drug available is less. The Cmax for formulation C is less than that for formulation A. The decrease in Cmax for formulation C is proportional to the decrease in AUC in comparison to the drug plasma level data for formulation A. The corresponding urinary excretion data confirm these observations. These relationships are summarized in Table 15-8. The table illustrates how bioavailability parameters for plasma and urine change when only the extent and rate of bioavailability are changed, respectively. Formulation changes in a drug product may affect both the rate and extent of drug bioavailability.

The contents of New Drug Applications (NDAs) and Abbreviated New Drug Applications (ANDAs) are similar in terms of the quality of manufacture (Table 15-9). The submission for an NDA must contain safety and efficacy studies as provided by animal toxicology studies, clinical efficacy studies, and pharmacokinetic/bioavailability studies. For the generic drug manufacturer, the bioequivalence study is the pivotal study in the ANDA that replaces the animal, clinical, and pharmacokinetic studies.

An outline for the submission of a completed bioavailability to the FDA is shown in Table 15-10. 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 (ie, shown to measure precisely and accurately the plasma drug concentration). The results are analyzed both statistically and pharmacokinetically. These results, along with case reports and various data supporting the validity of the analytical method, are included in the submission. The FDA reviews the study in detail according to the outline presented in Table 15-11. 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 as shown in Fig. 15-15. If the application is incomplete, the FDA will not review the submission and the sponsor will receive a Refusal to File letter.

Waivers of Invivo Bioequivalence Studies (Biowaivers)

In some cases, in vitro dissolution testing may be used in lieu of in vivo bioequivalence studies. When the drug product is in the same dosage form but in different strengths and is proportionally similar in active and inactive ingredients, an in vivo bioequivalence study of one or more of the lower strengths can be waived based on the dissolution tests and an in vivo bioequivalence study on the highest strength. Ideally, if there is a strong correlation between dissolution of the drug and the bioavailability of the drug, then the comparative dissolution tests comparing the test product to the reference product should be sufficient to demonstrate bioequivalence. For most drug products, especially immediate-release tablets and capsules, no strong correlation exists, and the FDA requires an in vivo bioequivalence study. For oral solid dosage forms, an in vivo bioequivalence study may be required to support at least one dose strength of the product. Usually, an in vivo bioequivalence study is required for the highest dose strength. If the lower-dose-strength test product is substantially similar in active and inactive ingredients, then only a comparative in vitro dissolution between the test and brand-name formulations may be used.

For example, an immediate-release tablet is available in 200-mg, 100-mg, and 50-mg strengths. The 100- and 50-mg-strength tablets are made the same way as the highest-strength tablet. A human bioequivalence study is performed on the highest or 200-mg strength. Comparative in vitro dissolution studies are performed on the 100-mg and 50-mg dose strengths. If these drug products have no known bioavailability problems, are well absorbed systemically, are well correlated with in vitro dissolution, and have a large margin of safety, then arguments for not performing an in vivo bioavailability study may be valid. Methods for correlation of in vitro dissolution of the drug with in vivo drug bioavailability are discussed in Chapters 14 and 17. The manufacturer does not need to perform additional in vivo bioequivalence studies on the lower-strength products if the products meet all in vitro criteria.

Dissolution Profile Comparison

Comparative dissolution profiles are used as (1) the basis for formulation development of bioequivalent drug products and proceeding to the pivotal in vivo bioequivalence study; (2) comparative dissolution profiles are used for demonstrating the equivalence of a change in the formulation of a drug product after the drug product has been approved for marketing (see SUPAC in Chapter 16); and (3) the basis of a biowaiver of a lower-strength drug product that is dose proportional in active and inactive ingredients to the higher-strength drug product.

A model-independent mathematical method was developed by Moore and Flanner (1996) to compare dissolution profiles using two factors, f1 and f2. The factor f2, known as the similarity factor, measures the closeness between the two profiles:

where n is the number of time points, R1 is the dissolution value of the reference product at time t, and T1 is the dissolution value of the test product batch at time t.

The reference may be the original drug product before a formulation change (prechange) and the test may be the drug product after the formulation was changed (postchange). Alternatively, the reference may be the higher-strength drug product and the test may be the lower-strength drug product. The f2 comparison is the focus of several FDA guidances and is of regulatory interest in knowing the similarity of the two dissolution curves. When the two profiles are identical, f2 = 100. An average difference of 10% at all measured time points results in an f2 value of 50 (Shah et al, 1998). The FDA has set a public standard for f2 value between 50 and 100 to indicate similarity between two dissolution profiles.

In some cases, two generic drug products may have dissimilar dissolution profiles and still be bioequivalent in vivo. For example, Polli et al (1997) have shown that slow-, medium-, and fast-dissolving formulations of metoprolol tartrate tablets were bioequivalent. Furthermore, bioequivalent modified-release drug products may have different drug release mechanisms and therefore different dissolution profiles. For example, for theophylline extended-release capsules, the United States Pharmacopeia (USP) lists 10 individual drug release tests for products labeled for dosing every 12 hours. However, only generic drug products that are FDA approved as bioequivalent drug products and listed in the current edition of the Orange Book may be substituted for each other.

A theoretical basis for correlating in vitro drug dissolution with in vivo bioavailability was developed by Amidon et al (1995). This approach is based on the aqueous solubility of the drug and the permeation of the drug through the gastrointestinal tract. The classification system is based on Fick's first law applied to a membrane:

where Jw is the drug flux (mass/area/time) through the intestinal wall at any position and time, Pw is the permeability of the membrane, and Cw is the drug concentration at the intestinal membrane surface.

This approach assumes that no other components in the formulation affect the membrane permeability and/or intestinal transport. Using this approach, Amidon et al (1995) studied the solubility and permeability characteristics of various representative drugs and obtained a biopharmaceutic drug classification (Table 15-12) for predicting the in vitro drug dissolution of immediate-release solid oral drug products with in vivo absorption.

The FDA may waive the requirement for performing an in vivo bioavailability or bioequivalence study for certain immediate-release solid oral drug products that meet very specific criteria, namely, the permeability, solubility, and dissolution of the drug. These characteristics include the in vitro dissolution, of the drug product in various media, drug permeability information, and assuming ideal behavior of the drug product, drug dissolution, and absorption in the GI tract. For regulatory purposes, drugs are classified according to the Biopharmaceutics Classification System (BCS) in accordance the solubility, permeability, and dissolution characteristics of the drug (FDA Guidance for Industry, 2000; Amidon et al, 1995).

Solubility

An objective of the BCS approach is to determine the equilibrium solubility of a drug under approximate physiologic conditions. For this purpose, determination of pH–solubility profiles over a pH range of 1 to 8 is suggested. The solubility class is determined by calculating what volume of an aqueous medium is sufficient to dissolve the highest anticipated dose strength. A drug substance is considered highly soluble when the highest dose strength is soluble in 250 mL or less of aqueous medium over the pH range 1 to 8. The volume estimate of 250 mL is derived from typical bioequivalence study protocols that prescribe administration of a drug product to fasting human volunteers with a glass (8 oz) of water.

Permeability

Studies of the extent of absorption in humans, or intestinal permeability methods, can be used to determine the permeability class membership of a drug. To be classified as highly permeable, a test drug should have an extent of absorption >90% in humans. Supportive information on permeability characteristics of the drug substance should also be derived from its physical–chemical properties (eg, octanol: water partition coefficient).

Some methods to determine the permeability of a drug from the gastrointestinal tract include: (1) in vivo intestinal perfusion studies in humans; (2) in vivo or in situ intestinal perfusion studies in animals; (3) in vitro permeation experiments using excised human or animal intestinal tissues; and (4) in vitro permeation experiments across a monolayer of cultured human intestinal cells. When using these methods, the experimental permeability data should correlate with the known extent-of-absorption data in humans.

After oral drug administration, in vivo permeability can be affected by the effects of efflux and absorptive transporters in the gastrointestinal tract, by food, and possibly by the various excipients present in the formulation.

Dissolution

The dissolution class is based on the in vitro dissolution rate of an immediate-release drug product under specified test conditions and is intended to indicate rapid in vivo dissolution in relation to the average rate of gastric emptying in humans under fasting conditions. An immediate-release drug product is considered rapidly dissolving when not less than 85% of the label amount of drug substance dissolves within 30 minutes using USP Apparatus I (see Chapter 14) at 100 rpm or Apparatus II at 50 rpm in a volume of 900 mL or less in each of the following media: (1) acidic media such as 0.1 N HCl or Simulated Gastric Fluid USP without enzymes, (2) a pH 4.5 buffer, and (3) a pH 6.8 buffer or Simulated Intestinal Fluid USP without enzymes.

Biopharmaceutics Drug Disposition Classification System

The major aspects of BCS are the consideration of solubility and permeation. According to BCS, permeability in vivo is considered high when the active drug is systemically absorbed ≥90%. Wu and Benet (2005) and Benet et al (2008) have proposed modification of the BCS system known as the Biopharmaceutics Drug Disposition Classification System (BDDCS) which takes into account drug metabolism (hepatic clearance) and transporters in the gastrointestinal tract for drugs that are orally administered. For BCS 1 drugs (ie, high solubility and high permeability), transporter effects will be minimal. However, BCS 2 drugs (low solubility and high permeability), transporter effects are more important. These investigators suggest that the BCS should be modified on the basis of the extent of drug metabolism, overall drug disposition, including routes of drug elimination and the effects of efflux, and absorptive transporters on oral drug absorption.

Drug Products for Which Bioavailability or Bioequivalence May Be Self-Evident

The best measure of a drug product's performance is to determine the in vivo bioavailability of the drug. For some well-characterized drug products and for certain drug products in which bioavailability is self-evident (eg, sterile solutions for injection), in vivo bioavailability studies may be unnecessary or unimportant to the achievement of the product's intended purposes. The FDA will waive the requirement for submission of in vivo evidence demonstrating the bioavailability of the drug product if the product meets one of the following criteria (US Code of Federal Regulations, 21 CFR 320.22). However, there may be specific requirements for certain drug products, and the appropriate FDA division should be consulted.

1. The drug product (a) is a solution intended solely for intravenous administration and (b) contains an active drug ingredient or therapeutic moiety combined with the same solvent and in the same concentration as in an intravenous solution that is the subject of an approved, full NDA.

2. The drug product is a topically applied preparation (eg, a cream, ointment, or gel intended for local therapeutic effect). The FDA has released guidances for the performance of bioequivalence studies on topical corticosteroids and antifungal agents. The FDA is also considering performing dermatopharmacokinetic (DPK) studies on other topical drug products. In addition, in vitro drug release and diffusion studies may be required.

3. The drug product is in an oral dosage form that is not intended to be absorbed (eg, an antacid or a radiopaque medium). Specific in vitro bioequivalence studies may be required by the FDA. For example, the bioequivalence of cholestyramine resin is demonstrated in vitro by the binding of bile acids to the resin.

4. The drug product meets both of the following conditions:

   1. It is administered by inhalation as a gas or vapor (eg, as a medicinal or as an inhalation anesthetic).

   2. It contains an active drug ingredient or therapeutic moiety in the same dosage form as a drug product that is the subject of an approved, full NDA.

5. The drug product meets all of the following conditions:

   1. It is an oral solution, elixir, syrup, tincture, or similar other solubilized form.

   2. It contains an active drug ingredient or therapeutic moiety in the same concentration as a drug product that is the subject of an approved, full NDA.

   3. It contains no inactive ingredient that is known to significantly affect absorption of the active drug ingredient or therapeutic moiety.

Biologics, or biotechnology-derived drugs, in contrast to drugs that are chemically synthesized, are derived from living sources such as humans, animals, or microorganisms. Many biologics are complex mixtures that are not easily identified or characterized and are manufactured using biotechnology or are purified from natural sources. Other biological drugs, such as insulin and growth hormone, are proteins derived by biotechnology and have been well characterized.

Presently, there is no FDA regulatory pathway to establish the bioequivalence of a biotechnology-derived drug product, sometimes referred to as generic biologics or biosimilar drug products. Scientifically, there are advocates for and against the feasibility for the manufacture of generic biotechnology-derived drug products (generic biologics) that are bioequivalent to the innovator or brand-drug product.

Those opposed to the development of generic biologics have claimed that generic manufacturers do not have the ability to fully characterize the active ingredient(s), that immunogenicity-related impurities may be present in the product, and that the manufacture of a biologic drug product is process dependent.

Biosimilarity versus Interchangeability

The “Drug Price Competition and Patent Term Restoration Act of 1984,” also known as “The Hatch-Waxman Act,” established an abbreviated regulatory process for generic drug products. Under the Hatch-Waxman Act, applicants for an Abbreviated New Drug Application (ANDA) must show the bioequivalence of a generic drug, but do not need to conduct a full panel of clinical trials to demonstrate safety and efficacy of the drug. Generic drugs are not evaluated or differentiated based on their class or type and, once approved, can be substituted for brand name drugs automatically. In 2010, the U.S. Congress passed landmark healthcare reform legislation, known as the “Patient Protection and Affordable Care Act,” which reshaped the biopharmaceutical industry. This legislation contains provisions that establish, for the first time ever, an abbreviated regulatory approval pathway for generic versions of biological medicines (i.e., biosimilars). The new legislation establishes two distinct categories of biosimilar products: 1) biological products that are “biosimilar” to a reference biological product, and 2) biological products that are “interchangeable” with the reference product. A “biosimilar” product is defined as a biological product that “is highly similar to the reference product notwithstanding minor differences in clinically inactive components” and for which “there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity and potency of the product.” A biological product is “interchangeable” with a reference biological product if (i) it meets the criteria for being biosimilar to the reference product, and (ii) it can be expected to produce the same clinical result as the reference product in any given patient, and (iii) the risk in terms of safety or diminished efficacy in alternating or switching between use of the biological and reference product is not greater than the risk of using the reference product without such alteration or switch.

FDA Guidance Documents

The legislation makes clear that the FDA will play a central role in defining the specific criteria needed to demonstrate biosimilarity for a given class of biological. In deference to the FDA's expertise in this area, the legislation specifically states that the FDA can issue guidance documents with respect to the approval of a biosimilar product. The guidance can be general or specific in nature, and the public must be provided with an opportunity to comment.

Advocates for the manufacture of generic biologics argue that bioequivalent biotechnology-derived drug products can be made on a case-by-case basis. Currently, manufacturers of marketed biotechnology drugs may seek to make changes in the manufacturing process used to make a particular product for a variety of reasons, including improvement of product quality, yield, and manufacturing efficiency. These manufacturers have developed improvements in production methods, process and control test methods, and test methods for product characterization. Currently, there are several manufacturers of recombinant human growth hormone (somatotropin), but none of these products has been considered as a generic alternative or therapeutic equivalent.

The manufacture of generic drug products derived from biotechnology-derived drugs should be possible. For example, a biologic manufacturer institutes a change in its manufacturing process before FDA approval of its product but after completion of a pivotal clinical study. The FDA may not require the manufacturer to perform additional clinical studies to demonstrate that the resulting product is still safe, pure, and potent. Such manufacturing process changes, implemented before or after product approval, have included changes implemented during expansion from pilot-scale to full-scale production, the move of production facilities from one legal entity to another legal entity, and the implementation of changes in different stages of the manufacturing process such as fermentation, purification, and formulation. The manufacturer may be able to demonstrate product comparability between a biologic product made after a manufacturing change (“new” product) and a product made before implementation of the change (“old” product) through different types of analytical and functional testing, with or without preclinical animal testing. The FDA may determine that two products are comparable if the results of the comparability testing demonstrate that the manufacturing change does not affect safety, identity, purity, or potency (FDA Guidance, 1996). The FDA currently requires that manufacturers should carefully assess manufacturing changes and evaluate the product resulting from these changes for comparability to the preexisting product. Determinations of product comparability may be based on chemical, physical, and biologic assays and, in some cases, other nonclinical data.

It is important to note that the FDA uses such terms as comparable and similar for approval of manufacturing changes of biologic drug products (FDA Guidance, 1996). In contrast, the FDA uses the term bioequivalence for approval of manufacturing changes of drug products that contain chemically derived active ingredients. Advocates for the manufacturer of generic biologics feel that the science and technology for the manufacture of certain bioequivalent biologic drug products are already available. Moreover, if the innovator manufacturer of a marketed biologic drug product can perform a manufacturing change and demonstrate the comparability of the “new” to the “old” marketed biologic drug product, then a generic manufacturer should be able to use similar techniques to demonstrate bioequivalence of the generic drug product.

Bioequivalence of different formulations of the same drug substance involves equivalence with respect to rate and extent of systemic drug absorption. Clinical interpretation is important in evaluating the results of a bioequivalence study. A small difference between drug products, even if statistically significant, may produce very little difference in therapeutic response. Generally, two formulations whose rate and extent of absorption differ by 20% or less are considered bioequivalent. The Report by the Bioequivalence Task Force (1988) considered that differences of less than 20% in AUC and Cmax between drug products are “unlikely to be clinically significant in patients.” The Task Force further stated that “clinical studies of effectiveness have difficulty detecting differences in doses of even 50% to 100%.” Therefore, normal variation is observed in medical practice and plasma drug levels may vary among individuals greater than 20%.

According to Westlake (1972), a small, statistically significant difference in drug bioavailability from two or more dosage forms may be detected if the study is well controlled and the number of subjects is sufficiently large. When the therapeutic objectives of the drug are considered, an equivalent clinical response should be obtained from the comparison dosage forms if the plasma drug concentrations remain above the minimum effective concentration (MEC) for an appropriate interval and do not reach the minimum toxic concentration (MTC). Therefore, the investigator must consider whether any statistical difference in bioavailability would alter clinical efficiency.

Special populations, such as the elderly or patients on drug therapy, are generally not used for bioequivalence studies. Normal, healthy volunteers are preferred for bioequivalence studies, because these subjects are less at risk and may more easily endure the discomforts of the study, such as blood sampling. Furthermore, the objective of these studies is to evaluate the bioavailability of the drug from the dosage form, and use of healthy subjects should minimize both inter- and intrasubject variability. It is theoretically possible that the excipients in one of the dosage forms tested may pose a problem in a patient who uses the generic dosage form.

For the manufacture of a dosage form, specifications are set to provide uniformity of dosage forms. With proper specifications, quality control procedures should minimize product-to-product variability by different manufacturers and lot-to-lot variability with a single manufacturer (see Chapter 16).

9For a few drug products, a high intrasubject variability (>30% CV) may be observed for which the bioavailability response changes for the same drug product each time the drug is dosed in the same subject.

The general bioequivalence study designs and evaluation, such as the comparison of AUC, Cmax, and tmax, may be used for systemically absorbed drugs and conventional oral dosage forms. However, for certain drugs and dosage forms, systemic bioavailability and bioequivalence are difficult to ascertain (Table 15-13). Drugs and drug products (eg, cyclosporine, chlorpromazine, verapamil, isosorbide dinitrate, sulindac) are considered to be highly variable if the intrasubject variability in bioavailability parameters is greater than 30% by analysis of variancecoefficient of variation (Shah et al, 1996). The number of subjects required to demonstrate bioequivalence for these drug products may be excessive, requiring more than 60 subjects to meet current FDA bioequivalence criteria. The intrasubject variability may be due to the drug itself or to the drug formulation or to both. The FDA has held public forums to determine whether the current bioequivalence guidelines need to be changed for these highly variable drugs (Shah et al, 1996).

For drugs with very long elimination half-lives or a complex elimination phase, a complete plasma drug concentration–time curve (ie, three elimination half-lives or an AUC representing 90% of the total AUC) may be difficult to obtain for a bioequivalence study using a crossover design. For these drugs, a truncated (shortened) plasma drug concentration–time curve (0–72 hours) may be more practical. The use of a truncated plasma drug concentration–time curve allows for the measurement of peak absorption and decreases the time and cost for performing the bioequivalence study.

Many drugs are stereoisomers, and each isomer may give a different pharmacodynamic response and may have a different rate of biotransformation. The bioavailability of the individual isomers may be difficult to measure because of problems in analysis. Some drugs have active metabolites, which should be quantitated as well as the parent drug. Drugs such as thioridazine and selegilene have two active metabolites. The question for such drugs is whether bioequivalence should be proven by matching the bioavailability of both metabolites and the parent drug. Assuming both biotransformation pathways follow first-order reaction kinetics, then the metabolites should be in constant ratio to the parent drug. Genetic variation in metabolism may present a bioequivalence problem. For example, the acetylation of procainamide to N-acetylprocainamide demonstrates genetic polymorphism, with two groups of subjects consisting of rapid acetylators and slow acetylators. To decrease intersubject variability, a bioequivalence study may be performed on only one phenotype, such as the rapid acetylators.

Some drugs (eg, benzocaine, hydrocortisone, anti-infectives, antacids) are intended for local effect and formulated as topical ointments, oral suspensions, or rectal suppositories. These drugs should not have significant systemic bioavailability from the site of administration. The bioequivalence determination for drugs that are not absorbed systemically from the site of application can be difficult to assess. For these nonsystemic-absorbable drugs, a “surrogate” marker is needed for bioequivalence determination (Table 15-14). For example, the acid-neutralizing capacity of an oral antacid and the binding of bile acids to cholestyramine resin have been used as surrogate markers in lieu of in vivo bioequivalence studies.

Various drug delivery systems and newer dosage forms are designed to deliver the drug by a nonoral route, which may produce only partial systemic bioavailability. For the treatment of asthma, inhalation of the drug (eg, albuterol, beclomethasone dipropionate) has been used to maximize drug in the respiratory passages and to decrease systemic side effects. Drugs such as nitroglycerin given transdermally may differ in release rates, in the amount of drug in the transdermal delivery system, and in the surface area of the skin to which the transdermal delivery system is applied. Thus, the determination of bioequivalence among different manufacturers of transdermal delivery systems for the same active drug is difficult. Dermatokinetics are pharmacokinetic studies that investigate drug uptake into skin layers after topical drug administration. The drug is applied topically, the skin is peeled at various time periods after the dose, using transparent tape, and the drug concentrations in the skin are measured.

Drugs such as potassium supplements are given orally and may not produce the usual bioavailability parameters of AUC, Cmax, and tmax. For these drugs, more indirect methods must be used to ascertain bioequivalence. For example, urinary potassium excretion parameters are more appropriate for the measurement of bioavailability of potassium supplements. However, for certain hormonal replacement drugs (eg, levothyroxine), the steady-state hormone concentration in hypothyroid individuals, the thyroidal-stimulating hormone level, and pharmacodynamic endpoints may also be appropriate to measure.

Drug product selection and generic drug product substitution are major responsibilities for physicians, pharmacists, and others who prescribe, dispense, or purchase drugs. To facilitate such decisions, the FDA publishes annually, in print and on the Internet, Approved Drug Products with Therapeutic Equivalence Evaluations, also known as the Orange Book (www.fda.gov/cder/ob/default.htm). The Orange Book identifies drug products approved on the basis of safety and effectiveness by the FDA and contains therapeutic equivalence evaluations for approved multisource prescription drug products. These evaluations serve as public information and advice to state health agencies, prescribers, and pharmacists to promote public education in the area of drug product selection and to foster containment of healthcare costs.

To contain drug costs, most states have adopted generic substitution laws to allow pharmacists to dispense a generic drug product for a brand-name drug product that has been prescribed. Some states have adopted a positive formulary, which lists therapeutically equivalent or interchangeable drug products that pharmacists may dispense. Other states use a negative formulary, which lists drug products that are not therapeutically equivalent, and/or the interchange of which is prohibited. If the drug is not in the negative formulary, the unlisted generic drug products are assumed to be therapeutically equivalent and may be interchanged.

Approved Drug Products with Therapeutic Equivalence Evaluations (Orange Book)

The Orange Book contains therapeutic equivalence evaluations for approved drug products made by various manufacturers. These marketed drug products are evaluated according to specific criteria. The evaluation codes used for these drugs are listed in Table 15-15. The drug products are divided into two major categories: “A” codes apply to drug products considered to be therapeutically equivalent to other pharmaceutically equivalent products, and “B” codes apply to drug products that the FDA at this time does not consider to be therapeutically equivalent to other pharmaceutically equivalent products. A list of therapeutic-equivalence-related terms and their definitions is also given in the monograph. According to the FDA, evaluations do not mandate that drugs be purchased, prescribed, or dispensed, but provide public information and advice. The FDA evaluation of the drug products should be used as a guide only, with the practitioner exercising professional care and judgment.

The concept of therapeutic equivalence as used to develop the Orange Book applies only to drug products containing the same active ingredient(s) and does not encompass a comparison of different therapeutic agents used for the same condition (eg, propoxyphene hydrochloride versus pentazocine hydrochloride for the treatment of pain). Any drug product in the Orange Book that is repackaged and/or distributed by other than the application holder is considered to be therapeutically equivalent to the application holder's drug product even if the application holder's drug product is single source or coded as nonequivalent (eg, BN). Also, distributors or repackagers of an application holder's drug product are considered to have the same code as the application holder. Therapeutic equivalence determinations are not made for unapproved, off-label indications. With this limitation, however, the FDA believes that products classified as therapeutically equivalent can be substituted with the full expectation that the substituted product will produce the same clinical effect and safety profile as the prescribed product (www.fda.gov/cder/ob/default.htm).

Professional care and judgment should be exercised in using the Orange Book. Evaluations of therapeutic equivalence for prescription drugs are based on scientific and medical evaluations by the FDA. Products evaluated as therapeutically equivalent can be expected, in the judgment of the FDA, to have equivalent clinical effect and no difference in their potential for adverse effects when used under the conditions of their labeling. However, these products may differ in other characteristics such as shape, scoring configuration, release mechanisms, packaging, excipients (including colors, flavors, preservatives), expiration date/time, and, in some instances, labeling. If products with such differences are substituted for each other, there is a potential for patient confusion due to differences in color or shape of tablets, inability to provide a given dose using a partial tablet if the proper scoring configuration is not available, or decreased patient acceptance of certain products because of flavor. There may also be better stability of one product over another under adverse storage conditions or allergic reactions in rare cases due to a coloring or a preservative ingredient, as well as differences in cost to the patient.

FDA evaluation of therapeutic equivalence in no way relieves practitioners of their professional responsibilities in prescribing and dispensing such products with due care and with appropriate information to individual patients. In those circumstances where the characteristics of a specific product, other than its active ingredient, are important in the therapy of a particular patient, the physician's specification of that product is appropriate. Pharmacists must also be familiar with the expiration dates/times and labeling directions for storage of the different products, particularly for reconstituted products, to assure that patients are properly advised when one product is substituted for another.

In Table 15-16, AB1 products are bioequivalent to each other and can be substituted. AB2 products are bioequivalent to each other and can be substituted. However, an AB1 product cannot be substituted for an AB2 product. The BX product should not be used.

10Bioavailability: Bioavailability means 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. For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action.

Bioequivalence requirement: A requirement imposed by the FDA for in vitro and/or in vivo testing of specified drug products, which must be satisfied as a condition for marketing.

Bioequivalent drug products: This term describes pharmaceutical equivalent or pharmaceutical alternative products that display comparable bioavailability when studied under similar experimental conditions. For systemically absorbed drugs, the test (generic) and reference listed drug (brand name) shall be considered bioequivalent if: (1) the rate and extent of absorption of the test drug do not show a significant difference from the rate and extent of absorption of the reference drug when administered at the same molar dose of the therapeutic ingredient under similar experimental conditions in either a single dose or multiple doses or (2) the extent of absorption of the test drug does not show a significant difference from the extent of absorption of the reference drug when administered at the same molar dose of the therapeutic ingredient under similar experimental conditions in either a single dose or multiple doses and the difference from the reference drug in the rate of absorption of the drug is intentional, is reflected in its proposed labeling, is not essential to the attainment of effective body drug concentrations on chronic use, and is considered medically insignificant for the drug.

• When the above methods are not applicable (eg, for drug products that are not intended to be absorbed into the bloodstream), other in vivo or in vitro test methods to demonstrate bioequivalence may be appropriate. Bioequivalence may sometimes be demonstrated using an in vitro bioequivalence standard, especially when such an in vitro test has been correlated with human in vivo bioavailability data. In other situations, bioequivalence may sometimes be demonstrated through comparative clinical trials or pharmacodynamic studies.
• Bioequivalent drug products may contain different inactive ingredients, provided the manufacturer identifies the differences and provides information that the differences do not affect the safety or efficacy of the product.

Brand name: The trade name of the drug. This name is privately owned by the manufacturer or distributor and is used to distinguish the specific drug product from competitor's products (eg, Tylenol, McNeil Laboratories).

Chemical name: The name used by organic chemists to indicate the chemical structure of the drug (eg, N-acetyl-p-aminophenol).

Abbreviated New Drug Application (ANDA): Drug manufacturers must file an ANDA for approval to market a generic drug product. The generic manufacturer is not required to perform clinical efficacy studies or nonclinical toxicology studies for the ANDA.

Drug product: The finished dosage form (eg, tablet, capsule, or solution) that contains the active drug ingredient, generally, but not necessarily, in association with inactive ingredients.

Drug product selection: The process of choosing or selecting the drug product in a specified dosage form.

Drug substance: A drug substance is the active pharmaceutical ingredient (API) or component in the drug product that furnishes the pharmacodynamic activity.

Equivalence: Relationship in terms of bioavailability, therapeutic response, or a set of established standards of one drug product to another.

Generic name: The established, nonproprietary, or common name of the active drug in a drug product (eg, acetaminophen).

Generic substitution: The process of dispensing a different brand or an unbranded drug product in place of the prescribed drug product. The substituted drug product contains the same active ingredient or therapeutic moiety as the same salt or ester in the same dosage form but is made by a different manufacturer. For example, a prescription for Motrin brand of ibuprofen might be dispensed by the pharmacist as Advil brand of ibuprofen or as a nonbranded generic ibuprofen if generic substitution is permitted and desired by the physician.

Pharmaceutical alternatives: Drug products that contain the same therapeutic moiety but as different salts, esters, or complexes. For example, tetracycline phosphate or tetracycline hydrochloride equivalent to 250-mg tetracycline base are considered pharmaceutical alternatives. Different dosage forms and strengths within a product line by a single manufacturer are pharmaceutical alternatives (eg, an extended-release dosage form and a standard immediate-release dosage form of the same active ingredient). The FDA currently considers a tablet and capsule containing the same active ingredient in the same dosage strength as pharmaceutical alternatives.

Pharmaceutical equivalents: Drug products in identical dosage forms that contain the same active ingredient(s), ie, the same salt or ester, are of the same dosage form, use the same route of administration, and are identical in strength or concentration (eg, chlordiazepoxide hydrochloride, 5-mg capsules). Pharmaceutically equivalent drug products are formulated to contain the same amount of active ingredient in the same dosage form and to meet the same or compendial or other applicable standards (ie, strength, quality, purity, and identity), but they may differ in characteristics such as shape, scoring configuration, release mechanisms, packaging, excipients (including colors, flavors, preservatives), expiration time, and, within certain limits, labeling. When applicable, pharmaceutical equivalents must meet the same content uniformity, disintegration times, and/or dissolution rates. Modified-release dosage forms that require a reservoir or overage or certain dosage forms such as prefilled syringes in which residual volume may vary must deliver identical amounts of active drug ingredient over an identical dosing period.

Pharmaceutical substitution: The process of dispensing a pharmaceutical alternative for the prescribed drug product. For example, ampicillin suspension is dispensed in place of ampicillin capsules, or tetracycline hydrochloride is dispensed in place of tetracycline phosphate. Pharmaceutical substitution generally requires the physician's approval.

Reference listed drug: The reference listed drug (RLD) is identified by the FDA as the drug product on which an applicant relies when seeking approval of an Abbreviated New Drug Application (ANDA). The RLD is generally the brand-name drug that has a full New Drug Application (NDA). The FDA designates a single reference listed drug as the standard to which all generic versions must be shown to be bioequivalent. The FDA hopes to avoid possible significant variations among generic drugs and their brand-name counterparts. Such variations could result if generic drugs were compared to different reference listed drugs.

Drug Product Performance: 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 and leading to a pharmacodynamic response. Bioequivalence studies are drug product performance tests.

Therapeutic alternatives: Drug products containing different active ingredients that are indicated for the same therapeutic or clinical objectives. Active ingredients in therapeutic alternatives are from the same pharmacologic class and are expected to have the same therapeutic effect when administered to patients for such condition of use. For example, ibuprofen is given instead of aspirin; cimetidine may be given instead of ranitidine.

Therapeutic equivalents: Drug products are considered to be therapeutic equivalents only if they are pharmaceutical equivalents and if they can be expected to have the same clinical effect and safety profile when administered to patients under the conditions specified in the labeling. The FDA classifies as therapeutically equivalent those products that meet the following general criteria: (1) they are approved as safe and effective; (2) they are pharmaceutical equivalents in that they (a) contain identical amounts of the same active drug ingredient in the same dosage form and route of administration, and (b) meet compendial or other applicable standards of strength, quality, purity, and identity; (3) they are bioequivalent in that (a) they do not present a known or potential bioequivalence problem, and they meet an acceptable in vitro standard, or (b) if they do present such a known or potential problem, they are shown to meet an appropriate bioequivalence standard; (4) they are adequately labeled; and (5) they are manufactured in compliance with Current Good Manufacturing Practice regulations. The FDA believes that products classified as therapeutically equivalent can be substituted with the full expectation that the substituted product will produce the same clinical effect and safety profile as the prescribed product.

Therapeutic substitution: The process of dispensing a therapeutic alternative in place of the prescribed drug product. For example, amoxicillin is dispensed instead of ampicillin or ibuprofen is dispensed instead of naproxen. Therapeutic substitution can also occur when one NDA-approved drug is substituted for the same drug which has been approved by a different NDA, eg, the substitution of Nicoderm (nicotine transdermal system) for Nicotrol (nicotine transdermal system).

10The definitions are from Approved Drug Products with Therapeutic Equivalence Evaluations (Orange Book). [www.fda.gov/Drugs/InformationOnDrugs/ucm129662.htm,], Code of Federal Regulations, 21 CFR 320, and other sources.

Drug product performance may be defined as the release of the drug substance from the drug product leading to bioavailability of the drug substance. Bioequivalence is a measure of comparative drug product performance and relates the quality of a drug product to clinical safety and efficacy. The absolute availability of drug is the systemic availability of a drug after extravascular administration (eg, oral, rectal, transdermal, subcutaneous) compared to IV dosing, whereas relative bioavailability compares the bioavailability of a drug from two or more drug products. The most direct method to assess drug bioavailability is to determine the rate and extent of systemic drug absorption by measurement of the active drug concentrations in plasma. The main pharmacokinetic parameters, Cmax and AUC are used to determine bioequivalence. However, other pharmacokinetic parameters such as tmax and elimination t½ should also be assessed. The most common statistical design for bioequivalence studies is the two-way, crossover design in normal healthy volunteers. Bioequivalence is generally determined if the 90% confidence intervals for Cmax and AUC fall within 80% to 125% of the reference listed drug based on log transformation of the data. 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. The Biopharmaceutics Classification System (BCS) is based on the solubility, permeability, and dissolution characteristics of the drug. However, systemic drug bioavailability may also be affected by transporters in the GI tract, hepatic clearance, GI transit and motility, and the contents of the GI tract.

Drug product selection and generic substitution are important responsibilities of the pharmacist. A listing of approved drug products of generic drug products that may be safely substituted is available in Approved Drug Products with Therapeutic Equivalence Evaluations (Orange Book).

1. An antibiotic was formulated into two different oral dosage forms, A and B. Biopharmaceutic studies revealed different antibiotic blood level curves for each drug product (Fig. 15-16). Each drug product was given in the same dose as the other. Explain how the various possible formulation factors could have caused the differences in blood levels. Give examples where possible. How would the corresponding urinary drug excretion curves relate to the plasma level–time curves?

   Figure 15-16Graphic Jump Location

   

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   Blood level curves for two different oral dosage forms of a hypothetical antibiotic.

2. Assume that you have just made a new formulation of acetaminophen. Design a protocol to compare your drug product against the acetaminophen drug products on the market. What criteria would you use for proof of bioequivalence for your new formulation? How would you determine if the acetaminophen was completely (100%) systemically absorbed?

3. The data in Table 15-17 represent the average findings in antibiotic plasma samples taken from 10 humans (average weight 70 kg), tabulated in a four-way crossover design.

   Table Graphic Jump Location

   Table 15-17 Comparison of Plasma Concentrations of Antibiotic, as Related to Dosage Form and Time

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   Table 15-17 Comparison of Plasma Concentrations of Antibiotic, as Related to Dosage Form and Time

   	Plasma ConcentratioN (μg/mL)
   Time after Dose (h)	IV Solution (2 mg/kg)	Oral Solution (10 mg/kg)	Oral Tablet (10 mg/kg)	Oral Capsule (10 mg/kg)
   0.5	5.94	23.4	13.2	18.7
   1.0	5.30	26.6	18.0	21.3
   1.5	4.72	25.2	19.0	20.1
   2.0	4.21	22.8	18.3	18.2
   3.0	3.34	18.2	15.4	14.6
   4.0	2.66	14.5	12.5	11.6
   6.0	1.68	9.14	7.92	7.31
   8.0	1.06	5.77	5.00	4.61
   10.0	0.67	3.64	3.16	2.91
   12.0	0.42	2.30	1.99	1.83
   	29.0	145.0	116.0	116.0

   1. Which of the four drug products in Table 15-17 would be preferred as a reference standard for the determination of relative bioavailability? Why?

   2. From which oral drug product is the drug absorbed more rapidly?

   3. What is the absolute bioavailability of the drug from the oral solution?

   4. What is the relative bioavailability of the drug from the oral tablet compared to the reference standard?

   5. From the data in Table 15-15, determine:

      1. Apparent VD

      2. Elimination t1/2

      3. First-order elimination rate constant k

      4. Total body clearance

   6. From the data above, graph the cumulative urinary excretion curves that would correspond to the plasma concentration–time curves.

4. Aphrodisia is a new drug manufactured by the Venus Drug Company. When tested in humans, the pharmacokinetics of the drug assumes a one-compartment open model with first-order absorption and first-order elimination:

   

   The drug was given in a single oral dose of 250 mg to a group of college students 21 to 29 years of age. Mean body weight was 60 kg. Samples of blood were obtained at various time intervals after the administration of the drug, and the plasma fractions were analyzed for active drug. The data are summarized in Table 15-18.

   Table Graphic Jump Location

   Table 15-18 Data Summary of Active Drug Concentration in Plasma Fractions

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   Table 15-18 Data Summary of Active Drug Concentration in Plasma Fractions

   Time (h)	Cp (μg/mL)	Time (h)	Cp (μg/mL)
   0	0	12	3.02
   1	1.88	18	1.86
   2	3.05	24	1.12
   3	3.74	36	0.40
   5	4.21	48	0.14
   7	4.08	60	0.05
   9	3.70	72	0.02

   1. The minimum effective concentration of Aphrodisia in plasma is 2.3 μg/mL. What is the onset time of this drug?

   2. The minimum effective concentration of Aphrodisia in plasma is 2.3 μg/mL. What is the duration of activity of this drug?

   3. What is the elimination half-life of Aphrodisia in college students?

   4. What is the time for peak drug concentration (tmax) of Aphrodisia?

   5. What is the peak drug concentration (Cmax)?

   6. Assuming that the drug is 100% systemically available (ie, fraction of drug absorbed equals unity), what is the AUC for Aphrodisia?

5. You wish to do a bioequivalence study on three different formulations of the same active drug. Lay out a Latin-square design for the proper sequencing of these drug products in six normal, healthy volunteers. What is the main reason for using a crossover design in a bioequivalence study? What is meant by a “random” population?

6. Four different drug products containing the same antibiotic were given to 12 volunteer adult males (age 19–28 years, average weight 73 kg) in a four-way crossover design. The volunteers fasted for 12 hours prior to taking the drug product. Urine samples were collected up to 72 hours after the administration of the drug to obtain the maximum urinary drug excretion, Du∞. The data are presented in Table 15-19.

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   Table 15-19 Urinary Drug Excretion Data Summary

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   Table 15-19 Urinary Drug Excretion Data Summary

   Drug Product	Dose (mg/kg)	Cumulative Urinary Drug Excretion (), 0–72 h (mg)
   IV solution	0.2	20
   Oral solution	4	380
   Oral tablet	4	340
   Oral capsule	4	360

   1. What is the absolute bioavailability of the drug from the tablet?

   2. What is the relative bioavailability of the capsule compared to the oral solution?

7. According to the prescribing information for cimetidine (Tagamet), following IV or IM administration, 75% of the drug is recovered from the urine after 24 hours as the parent compound. Following a single oral dose, 48% of the drug is recovered from the urine after 24 hours as the parent compound. From this information, determine what fraction of the drug is absorbed systemically from an oral dose after 24 hours.

8. Define bioequivalence requirement. Why does the FDA require a bioequivalence requirement for the manufacture of a generic drug product?

9. Why can we use the time for peak drug concentration (tmax) in a bioequivalence study for an estimate of the rate of drug absorption, rather than calculating the ka?

10. Ten male volunteers (18–26 years of age) weighing an average of 73 kg were given either four tablets each containing 250 mg of drug (drug product A) or one tablet containing 1000 mg of drug (drug product B). Blood levels of the drug were obtained and the data are summarized in Table 15-20.

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    Table 15-20 Blood Level Data Summary for Two Drug Products

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    Table 15-20 Blood Level Data Summary for Two Drug Products

    		Drug Product
    Kinetic Variable	Unit	A 4 × 250-mg Tablet	B 1000-mg Tablet	Statistic
    Time for peak drug concentration (range)	h	1.3 (0.7–1.5)	1.8 (1.5–2.2)	p < .05
    Peak concentration (range)	μg/mL	53	47	p < .05
    		(46–58)	(42–51)
    AUC (range)	μg h/mL	118	103	NS
    		(98–125)	(90–120)
    t1/2	h	3.2	3.8	NS
    		(2.5–3.8)	(2.9–4.3)

    1. State a possible reason for the difference in the time for peak drug concentration (tmax,A) after drug product A compared to the tmax,B after drug product B. (Assume that all the tablets were made from the same formulation—that is, the drug is in the same particle size, same salt form, same excipients, and same ratio of excipients to active drug.)

    2. Draw a graph relating the cumulative amount of drug excreted in urine of patients given drug product A compared to the cumulative drug excreted in urine after drug product B. Label axes.

    3. In a second study using the same 10 male volunteers, a 125-mg dose of the drug was given by IV bolus and the AUC was computed as 20 μg h/mL. Calculate the fraction of drug systemically absorbed from drug product B (1 × 1000 mg) tablet using the data in Table 15-21.

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    Table 15-21 Disintegration Times and Dissolution Rates of Tolazamide Tabletsa

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    Table 15-21 Disintegration Times and Dissolution Rates of Tolazamide Tabletsa

    Tablet	Mean Disintegration Timeb min (Range)	Percent Dissolved in 30 minc (Range)
    A	3.8 (3.0–4.0)	103.9 (100.5–106.3)
    B	2.2 (1.8–2.5)	10.9 (9.3–13.5)
    C	2.3 (2.0–2.5)	31.6 (26.4–37.2)
    D	26.5 (22.5–30.5)	29.7 (20.8–38.4)

    a N = 6.

    b By the method of USP-23.

    c Dissolution rates in pH 7.6 buffer.

    From Welling et al (1982), with permission.

11. After performing a bioequivalence test comparing a generic drug product to a brand-name drug product, it was observed that the generic drug product had greater bioavailability than the brand-name drug product.

    1. Would you approve marketing the generic drug product, claiming it was superior to the brand-name drug product?

    2. Would you expect identical pharmacodynamic responses to both drug products?

    3. What therapeutic problem might arise in using the generic drug product that might not occur when using the brand-name drug product?

12. The following study is from Welling and associates (1982):

    • Tolazamide Formulations: Four tolazamide tablet formulations were selected for this study. The tablet formulations were labeled A, B, C, and D. Disintegration and dissolution tests were performed by standard USP-23 procedures.
    • Subjects: Twenty healthy adult male volunteers between the ages of 18 and 38 (mean, 26 years) and weighing between 61.4 and 95.5 kg (mean, 74.5 kg) were selected for the study. The subjects were randomly assigned to four groups of five each. The four treatments were administered according to 4 × 4 Latin-square design. Each treatment was separated by 1-week intervals. All subjects fasted overnight before receiving the tolazamide tablet the following morning. The tablet was given with 180 mL of water. Food intake was allowed at 5 hours postdose. Blood samples (10 mL) were taken just before the dose and periodically after dosing. The serum fraction was separated from the blood and analyzed for tolazamide by high-pressure liquid chromatography.
    • Data Analysis: Serum data were analyzed by a digital computer program using a regression analysis and by the percent of drug unabsorbed by the method of Wagner and Nelson (1963) (see Chapter 7). AUC was determined by the trapezoidal rule and an analysis of variance was determined by Tukey's method.

    1. Why was a Latin-square crossover design used in this study?

    2. Why were the subjects fasted before being given the tolazamide tablets?

    3. Why did the authors use the Wagner–Nelson method rather than the Loo–Riegelman method for measuring the amount of drug absorbed?

    4. From the data in Table 15-22 only, from which tablet formulation would you expect the highest bioavailability? Why?

    5. From the data in Table 15-22, did the disintegration times correlate with the dissolution times? Why?

    6. Do the data in Table 15-22 appear to correlate with the data in Table 15-21? Why?

    7. Draw the expected cumulative urinary excretion–time curve for formulations A and B. Label axes and identify each curve.

    8. Assuming formulation A is the reference formulation, what is the relative bioavailability of formulation D?

    9. Using the data in Table 15-22 for formulation A, calculate the elimination half-life (t1/2) for tolazamide.

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    Table 15-22 Mean Tolazamide Concentrationsa in Serum

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    Table 15-22 Mean Tolazamide Concentrationsa in Serum

    	Treatment (μg/mL)
    Time (h)	A	B	C	D	Statisticb
    0	10.8 ± 7.4	1.3 ± 1.4	1.8 ± 1.9	3.5 ± 2.6
    1	20.5 ± 7.3	2.8 ± 2.8	5.4 ± 4.8	13.5 ± 6.6
    3	23.9 ± 5.3	4.4 ± 4.3	9.8 ± 5.6	20.0 ± 6.4
    4	25.4 ± 5.2	5.7 ± 4.1	13.6 ± 5.3	22.0 ± 5.4
    5	24.1 ± 6.3	6.6 ± 4.0	15.1 ± 4.7	22.6 ± 5.0
    6	19.9 ± 5.9	6.8 ± 3.4	14.3 ± 3.9	19.7 ± 4.7
    8	15.2 ± 5.5	6.6 ± 3.2	12.8 ± 4.1	14.6 ± 4.2
    12	8.8 ± 4.8	5.5 ± 3.2	9.1 ± 4.0	8.5 ± 4.1
    16	5.6 ± 3.8	4.6 ± 3.3	6.4 ± 3.9	5.4 ± 3.1
    24	2.7 ± 2.4	3.1 ± 2.6	3.1 ± 3.3	2.4 ± 1.8
    Cmax, μg/mLc	27.8 ± 5.3	7.7 ± 4.1	16.4 ± 4.4	24.0 ± 4.5
    tmax, hd	3.3 ± 0.9	7.0 ± 2.2	5.4 ± 2.0	4.0 ± 0.9
    AUC0–24, μg h/mLe	260 ± 81	112 ± 63	193 ± 70	231 ± 67

    aConcentrations ± 1 SD, n = 20.

    bFor explanation see text.

    cMaximum concentration of tolazamide in serum.

    dTime of maximum concentration.

    eArea under the 0–24-h serum tolazamide concentration curve calculated by trapezoidal rule.

    From Welling et al (1982), with permission.

13. If in vitro drug dissolution and/or release studies for an oral solid dosage form (eg, tablet) does not correlate with the bioavailability of the drug in vivo, why should the pharmaceutical manufacturer continue to perform in vitro release studies for each production batch of the solid dosage form?

14. Is it possible for two pharmaceutically equivalent solid dosage forms containing different inactive ingredients (ie, excipients) to demonstrate bioequivalence in vivo even though these drug products demonstrate differences in drug dissolution tests in vitro?

15. For bioequivalence studies, tmax, Cmax, and AUC, along with an appropriate statistical analysis, are the parameters generally used to demonstrate the bioequivalence of two similar drug products containing the same active drug.

    1. Why are the parameters tmax, Cmax, and AUC acceptable for proving that two drug products are bioequivalent?

    2. Are pharmacokinetic models needed in the evaluation of bioequivalence?

    3. Is it necessary to use a pharmacokinetic model to completely describe the plasma drug concentration–time curve for the determination of tmax, Cmax, and AUC?

    4. Why are log-transformed data used for the statistical evaluation of bioequivalence?

    5. What is an add-on study?