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16 Drug Product Performance,

In Vivo: Bioavailability and


Bioequivalence
Barbara Davit, Dale Conner, and Leon Shargel

Chapter Objectives DRUG PRODUCT PERFORMANCE


»» Define bioavailability, Drug product performance,1 in vivo, may be defined as the release
bioequivalence, and drug of the drug substance from the drug product leading to bioavail-
product performance. ability of the drug substance. The assessment of drug product
»» Explain why certain drugs performance is important since bioavailability is related both to the
and drug products have low pharmacodynamic response and to adverse events. Thus, perfor-
bioavailability. mance tests relate the quality of a drug product to clinical safety
and efficacy. Bioavailability studies are drug product performance
»» Explain why first-pass effect as
studies used to define the effect of changes in the physicochemical
well as chemical instability of a
properties of the drug substance, the formulation of the drug, and
drug can result in low relative
the manufacture process of the drug product (dosage form). Drug
bioavailability.
product performance studies are used in the development of new
»» Distinguish between and generic drug products.
bioavailability and Bioavailability is one aspect of drug product quality that links
bioequivalence. the in vivo performance of a new drug product to the original for-
»» Explain why relative mulation that was used in clinical safety and efficacy studies.
bioavailability may have values Bioequivalence studies are drug product performance tests that
greater than 100%. compare the bioavailability of the same active pharmaceutical
ingredient from one drug product (test) to a second drug product
»» Explain why bioequivalence (reference). Bioavailability and bioequivalence can be considered
may be considered as a measure as measures of the drug product performance in vivo.
of drug product performance.
»» Describe various methods Bioequivalence Studies in New Drug Development (NDA)
for measuring bioavailability During drug development, bioequivalence studies are used to com-
and the advantages and pare (a) early and late clinical trial formulations; (b) formulations
disadvantages of each. used in clinical trials and stability studies, if different; (c) clinical
»» Describe the statistical criteria trial formulations and to-be-marketed drug products, if different;
for bioequivalence and 90% and (d) product strength equivalence, as appropriate. Bioequivalence
confidence intervals. study designs are used to support new formulations of previously
approved products, such as a new fixed-dose combination version
of two products approved for coadministration, or modified-release
versions of immediate-release products. Postapproval, in vivo

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

469
470    Chapter 16

»» Explain the conditions Clinical efficacy


Clinical and safety studies
under which a generic drug Active pharmaceutical
drug
ingredient (drug substance)
product manufacturer may product
PK/BA studies
request a waiver (biowaiver)
for performing an in vivo
bioequivalence study. Marketed drug product Dissolution profiles and/or
(brand) bioequivalence studies
»» Define therapeutic equivalence
and explain why bioequivalence
is only one component of the
regulatory requirements for Postapproval changes
Dissolution profiles and/or
bioequivalence studies
therapeutic equivalence.
FIGURE 16-1  Drug product performance and new drug product devel-
opment for NDAs. Drug product performance may be determined in vivo by
bioequivalence studies or in vitro by comparative drug dissolution studies.
BA = bioavailability.

bioequivalence studies may be needed to support regulatory


approval of major changes in formulation, manufacturing, or site,
in comparison to reference formulation (usually the prechange
formulation) (Fig. 16-1).
The initial safety and clinical efficacy 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. 16-1).
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 pro-
files (see Chapter 15).
As stated above, the marketed drug product that is approved
by the US Food and Drug Administration (FDA) may not be the
same formulation that was used in the original safety and clinical
efficacy studies. After the drug product is approved by the FDA
and marketed, the manufacturer may perform changes to the for-
mulation. These changes to the marketed drug product are known
as postapproval changes (see also Chapter 17). These postapproval
changes, often termed SUPAC (scale-up and postapproval change
based on several FDA guidance documents), 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
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     471

Comparative dissolution
Generic
Active pharmaceutical profiles and/or
drug
ingredient (drug substance) bioequivalence studies
product
to approved RLD

Dissolution profiles plus


Postapproval changes bioequivalence studies,
if required

FIGURE 16-2  Drug product performance and generic drug product development. Drug product performance may be deter-
mined in vivo by bioequivalence studies or in vitro by comparative drug/release dissolution studies.

change in the manufacturing site. In each case, the in vivo bioequivalence studies in normal healthy adult
manufacturer must demonstrate that drug product subjects under fasted and fed conditions. Drug product
performance did not change and is the same for the performance comparisons in vitro may also include
drug product manufactured before and after the comparative drug dissolution/release profiles. Similar
SUPAC change. As shown in Fig. 16-1, drug product to the brand-name drug product manufacturer, the
performance may be determined by in vivo bioequiv- generic drug manufacturer may make changes after
alence studies or by in vitro comparative drug release FDA approval in the formulation, in the source of the
or dissolution profiles. active pharmaceutical ingredient, manufacturing pro-
cess, or other changes. For any postapproval change,
Bioequivalence Studies in Generic Drug the manufacturer must demonstrate that the change
Development (ANDA) did not alter the performance of the drug product.
Comparative drug product performance studies are
important in the development of generic drug prod-
ucts (Fig. 16-2). A generic drug product is a multi- PURPOSE OF BIOAVAILABILITY
source drug product2 that has been approved by the AND BIOEQUIVALENCE STUDIES
FDA as a therapeutic equivalent to the reference Bioavailability and bioequivalence studies are impor-
listed drug product3 (usually the brand or innovator tant in the process of approving pharmaceutical prod-
drug product) and has proven equivalent drug product ucts for marketing. Bioavailability is defined as the
performance. Clinical safety and efficacy studies are rate and extent to which the active ingredient or active
not generally performed on generic drug products. moiety is absorbed from a drug product and becomes
Since the formulation and method of manufacture of available at the site of action (US-FDA, CDER,
a drug product can affect the bioavailability and sta- 2014a). Bioavailability data provide an estimate of the
bility of the drug, the generic drug manufacturer must fraction of drug absorbed from the formulation, and
demonstrate that the generic drug product is pharma- provide information about the pharmacokinetics of the
ceutically equivalent, bioequivalent, and therapeuti- drug. Relative bioavailability studies compare two
cally equivalent to the comparator brand-name drug drug product formulations. A bioequivalence study is
product. Drug product performance comparison for a specialized type of relative bioavailability study.
oral generic drug products is usually measured by Bioequivalence is defined as the absence of a signifi-
cant difference in the rate and extent to which the
2Multisource drug products are drug products that contain the same
active ingredient or active moiety becomes available at
active drug substance in the same dosage form and are marketed by the site of drug action when administered at the same
more than one pharmaceutical manufacturer. molar dose under similar conditions in an appropri-
3Reference listed drugs corresponding to proposed generic versions
ately designed study.
are listed by the US-FDA in its publication Approved Drug Products Bioavailability and bioequivalence data play piv-
with Therapeutic Equivalence Evaluations (Orange Book). otal roles in regulatory submissions for marketing
472    
Chapter 16

approval of new and generic drugs throughout the A drug product’s bioavailability provides an estimate
world. Each regulatory agency has developed its own of the relative fraction of the administered dose that
unique system of guidelines advising new and generic is absorbed into the systemic circulation (US-FDA,
drug applicants on how to conduct acceptable bioavail- CDER, 2014c). Determining the fraction (f) of
ability and bioequivalence studies to support marketing administered dose absorbed involves comparing the
approval. A recent survey of international bioequiva- drug product’s systemic exposure (represented by
lence guidelines showed that there are more similarities the concentration-versus-time or pharmacokinetic
than differences among approaches used by various profile) with that of a suitable reference product. For
international jurisdictions (Davit et al, 2013). In this systemically available drug products, bioavailability
chapter, discussion of the relationship between bio- is most often assessed by determining the area under
availability, bioequivalence, and drug approval require- the drug plasma concentration-versus-time profile
ments will focus on the perspective of the FDA. Where (AUC). The AUC is considered the most reliable
appropriate, the reader will be directed to references measure of a drug’s bioavailability, as it is directly
covering international jurisdictions for further reading. proportional to the total amount of unchanged drug
In summary, clinical studies are used to determine that reaches the systemic circulation (Le, 2014).
the safety and efficacy of drug products. Bioavailability Figure 16-3 shows how the drug concentration-ver-
studies are drug product performance studies used to sus-time profile is used to identify the pharmacoki-
define the effect of changes in the physicochemical netic parameters that form the basis of bioavailability
properties of the drug substance, the formulation of the and bioequivalence comparisons.
drug, and manufacture process of the drug product
(dosage form). Bioequivalence studies are used to com- Absolute Bioavailability
pare the bioavailability of the same drug (same salt or
Absolute bioavailability compares the bioavailability
ester) from various drug products. Bioavailability and
of the active drug in the systemic circulation fol-
bioequivalence can be considered as performance mea-
lowing extravascular administration with the bio-
sures of the drug product in vivo. If the drug products
availability of the same drug following intravenous
are pharmaceutically equivalent, bioequivalent, and
administration (Fig. 16-4). Intravenous drug adminis-
therapeutically equivalent (as defined by the regulatory
tration is considered 100% absorbed. The route of
agency such as the FDA), then the clinical efficacy and
extravascular administration can be inhaled, intra-
the safety profile of these drug products are assumed to
muscular, oral, rectal, subcutaneous, sublingual, topi-
be similar and may be substituted for each other.
cal, transdermal, etc. The absolute bioavailability is
the dose-corrected AUC of the extravascularly admin-
Frequently Asked Questions istered drug product divided by the AUC of the drug
»» Why are bioequivalence studies considered as drug product given intravenously. Thus, for an oral formu-
product performance studies? lation, the absolute bioavailability is calculated as
»» What are the differences between a safety/efficacy follows:
study and an in vivo bioequivalence study? How do
the study objectives differ? AUCpo ⋅ Div
Fabs =
»» What’s the difference between drug product
AUCiv ⋅ Dpo
performance and bioequivalence?
where
Fabs is the fraction of the dose absorbed, expressed as
RELATIVE AND ABSOLUTE a percentage;
AUCpo is the AUC following oral administration;
AVAILABILITY Div is the dose administered intravenously;
Regulatory agencies such as the FDA require sub- AUCiv is the AUC following intravenous administra-
mission of bioavailability data in applications to tion; and
market new drug products (US-FDA, CDER, 2014b). Dpo is the dose administered orally.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     473

350

300 Cmax

Plasma drug concentration (ng/mL) 250

200

150

100

50
tmax AUC

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Time (hours)
FIGURE 16-3  Plasma drug concentration–time curve after oral drug administration.

Absolute availability, Fabs, may be expressed as Relative Bioavailability


a fraction or as a percent by multiplying Fabs × 100. Another type of comparative bioavailability assess-
A drug given by the intravenous route will have an ment is provided by a relative bioavailability study.
absolute bioavailability of 100% (f = 1). A drug In a relative bioavailability study, the systemic expo-
given by an extravascular route may have an Fabs = 0 sure of a drug in a designated formulation (generally
(no systemic absorption) and Fabs = 1.0 (100% sys- referred to as treatment A or reference formulation)
temic absorption). is compared with that of the same drug administered

100
IV
90
Oral
80
Drug plasma concentration

70

60

50

40

30

20

10

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hours)
FIGURE 16-4  Relationship between plasma drug concentration-versus-time profiles for an intravenously administered
formulation versus an orally administered formulation. In an absolute bioavailability study, the systemic exposure profile of a drug
administered by the oral route (black curve) is compared with that of the drug administered by the intravenous route (green curve).
474    
Chapter 16

in a reference formulation (generally referred to as fixed-dose combination doublet) or concurrently


treatment B or test formulation). In a relative bio- according to an approved combination regimen
availability study, the AUCs of the two formulations (ie, two treatments). Relative bioavailability study
are compared as follows: designs are also commonly used for bridging formu-
lations during drug development, for example, to
AUC A ⋅ DB evaluate how drug systemic availability from a new
Frel = 100 ⋅
AUC B ⋅ DA premarket formulation compares with that from an
existing premarket formulation.
where
Frel is the relative bioavailability of treatment (formu- PRACTICE PROBLEM
lation) A, expressed as a percentage;
AUCA is the AUC following administration of treat- The bioavailability of a new investigational drug was
ment (formulation) A; studied in 12 volunteers. Each volunteer received
DA is the dose of formulation A; either a single oral tablet containing 200 mg of the
AUCB is the AUC of formulation B; and drug, 5 mL of a pure aqueous solution containing
DB is the dose of formulation B. 200 mg of the drug, or a single IV bolus injection
containing 50 mg of the drug. Plasma samples were
Relative bioavailability studies are frequently obtained periodically up to 48 hours after the dose
included in regulatory submissions. For example, the and assayed for drug concentration. The average AUC
FDA recommends that new drug developers rou- values (0–48 hours) are given in the table below.
tinely use an oral solution as the reference for a new From these data, calculate (a) the relative bioavail-
oral formulation, for the purpose of assessing how ability of the drug from the tablet compared to the
formulation impacts bioavailability. Other types of oral solution and (b) the absolute bioavailability of
relative bioavailability studies used in drug develop- the drug from the tablet.
ment include studies to characterize food effects and
drug–drug interactions. In a food-effect bioavail- Dose AUC Standard
ability study, oral bioavailability of the drug product Drug Product (mg) (mg · h/mL) Deviation
given with food (usually a high-fat, high-calorie
meal) is compared to oral bioavailability of the drug Oral tablet 200 89.5 19.7
product given under fasting conditions. The drug Oral solution 200 86.1 18.1
product given under fasting conditions is treated as
IV bolus 50 37.8 5.7
the reference treatment. The goal of a drug–drug injection
interaction study is to determine whether there is an
increase or decrease in bioavailability in the pres-
ence of the interacting drug. As such, the general Solution
drug–drug interaction study design compares drug The relative bioavailability of the drug from the tab-
relative bioavailability with and without (reference let is estimated in the equation below. No adjustment
treatment) the interacting drug. Relative bioavail- for the dose is necessary since the nominal doses are
ability studies are used in developing new formula- the same.
tions of existing immediate-release drug products,
such as new modified-release versions or new fixed- Relative bioavailability = 89.5 = 1.04 or 104%
86.1
dose combination formulations. In the case of a new
modified-release version, the reference product is The relative bioavailability of the drug from the tab-
the approved immediate-release product. In the case let is 1.04, or 104%, compared to the solution. In this
of a new fixed-dose combination, the reference prod- study, the difference in drug bioavailability between
uct can be the single-entity drug products adminis- tablet and solution would need to be analyzed statis-
tered either separately (ie, three treatments for a tically to determine whether the difference in drug
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     475

bioavailability is statistically significant. It is possible absorption, as determined by comparison of mea-


for the relative bioavailability to be greater than 100%. sured parameters. The FDA’s regulations (US-FDA,
In this case, the tablet formulation may have some CDER, 2014a) list the following approaches to
property or excipient that increases bioavailability. determining bioequivalence, in descending order of
The absolute drug bioavailability from the tablet accuracy, sensitivity, and reproducibility:
is calculated and adjusted for the dose.
• In vivo measurement of active moiety or moieties
89.5/200 in biological fluid (ie, a pharmacokinetic study)
F = absolute bioavailability = • In vivo pharmacodynamic (PD) comparison
37.5/50
• In vivo limited clinical comparison
= 0.592 or 59.2%
• In vitro comparison
• Any other approach deemed acceptable (by the
Because F, the fraction of dose absorbed from the
FDA)
tablet, is less than 1, the drug from the oral tablet is
not completely absorbed systemically, as a result of For drug products that are not intended to be
either poor oral absorption of the drug itself, formu- absorbed into the bloodstream, bioavailability may
lation effects that reduce oral bioavailability, or metab- be assessed by measurements intended to reflect the
olism by first-pass effect (presystemic elimination). rate and extent to which the active ingredient or
The relative bioavailability of the drug from the active moiety becomes available at the site of action.
tablet is approximately 100% when compared to the The design of the bioavailability study depends on
oral solution. the objectives of the study, the ability to analyze the
The comparison between oral solution (little to drug (and metabolites) in biological fluids, the phar-
no formulation effect) and IV administration gives macodynamics of the drug substance, the route of
information on the absorption of the drug itself when drug administration, and the nature of the drug prod-
formulation effects are virtually nonexistent. With uct. For all systemically active drugs, with a few
this knowledge, one can interpret the absolute bio- exceptions, bioequivalence should be demonstrated
availability from the tablet and know if there is an by an in vivo study based on pharmacokinetic (PK)
effect of that formulation to change bioavailability or endpoints, as this is the most sensitive, accurate, and
relative bioavailability is the same whether the tablet reproducible approach. The other approaches—PD,
formulation wasn’t even there. clinical, or in vitro—may be more appropriate for
Results from bioequivalence studies may show locally acting drugs that are not systemically absorbed,
that the relative bioavailability of the test oral product such as those administered topically or those that act
is greater than, equal to, or less than 100% compared locally within the gastrointestinal (GI) tract. These
to the reference oral drug product. However, the results latter BE approaches are considered on a case-by-case
from these bioequivalence studies should not be misin- basis (Table 16-1). Detailed examples to illustrate when
terpreted to imply that the absolute bioavailability of PD, clinical, or in vitro approaches are most suitable
the drug from the oral drug products is also 100% for establishing BE are presented below.
unless the oral formulation was compared to an intra-
venous injection (completely bioavailable) of the drug.
IN VIVO MEASUREMENT OF
ACTIVE MOIETY OR MOIETIES
METHODS FOR ASSESSING IN BIOLOGICAL FLUIDS
BIOAVAILABILITY AND
Plasma Drug Concentration
BIOEQUIVALENCE Measurement of drug concentrations in blood,
Direct and indirect methods may be used to assess plasma, or serum after drug administration is the
drug bioavailability. Bioequivalence of a drug prod- most direct and objective way to determine systemic
uct is demonstrated by the rate and extent of drug drug bioavailability. By appropriate blood sampling,
476    
Chapter 16

TABLE 16-1  Methods for Assessing For many systemically absorbed drugs, small differ-
Bioavailability and Bioequivalence ences in tmax may have little clinical effect on overall
In vivo measurement of active moiety or moieties in
drug product performance. However, for some drugs,
biological fluids such as delayed action drug products, large differ-
ences in tmax may have clinical impact.
Plasma drug concentration
  Time for peak plasma (blood) concentration (tmax)
  Peak plasma drug concentration (Cmax) Cmax: The peak plasma drug concentration,
 Area under the plasma drug concentration–time curve Cmax, represents the maximum plasma drug concen-
(AUC) tration obtained after oral administration of drug. For
Urinary drug excretion many drugs, a relationship is found between the
  Cumulative amount of drug excreted in the urine (Du) pharmacodynamic drug effect and the plasma drug
  Rate of drug excretion in the urine (dDu/dt) concentration. Cmax provides indications that the
  Time for maximum urinary excretion (t) drug is sufficiently systemically absorbed to provide
In vivo pharmacodynamic (PD) comparison a therapeutic response. In addition, Cmax provides
  Maximum pharmacodynamic effect (Emax) warning of possibly toxic levels of drug. The units of
  Time for maximum pharmacodynamic effect Cmax are concentration units (eg, mg/mL, ng/mL).
  Area under the pharmacodynamic effect–time curve
  Onset time for pharmacodynamic effect
Although not a unit for rate, Cmax is often used in
bioequivalence studies as a surrogate measure for the
Clinical endpoint study rate of drug bioavailability. So, the expectation is
 Limited, comparative, parallel clinical study using prede-
termined clinical endpoint(s) and performed in patients
that as the rate of drug absorption goes up, the peak
or Cmax will also be larger. If the rate of drug absorp-
In vitro studies tion goes down, then the peak or Cmax is smaller.
  Comparative drug dissolution, f2 similarity factor
  In vitro binding studies
 Examples: Cholestyramine resin—In vitro equilibrium AUC: The area under the plasma level–time
and kinetic binding studies curve, AUC, is a measurement of the extent of drug
bioavailability (see Fig. 16-3). The AUC reflects the
Any other approach deemed acceptable (by the FDA)
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
an accurate description of the plasma drug concen- equal to the amount of unchanged drug reaching the
tration–time profile of the therapeutically active drug general circulation divided by the clearance.
substance(s) can be obtained using a validated drug
assay. ∞
[AUC]∞0 = ∫ Cp dt (16.1)
0
tmax: The time of peak plasma concentration,
tmax, corresponds to the time required to reach maxi- FD0 FD0
[AUC]∞0 = = (16.2)
mum drug concentration after drug administration. clearance kVD
At tmax, peak drug absorption occurs and the rate of
drug absorption exactly equals the rate of drug elimi- where F = fraction of dose absorbed, D 0 = dose,
nation (Fig. 16-3). Drug absorption still continues k = elimination rate constant, and VD = volume of
after tmax is reached, but at a slower rate. When com- distribution. The AUC is independent of the route
paring drug products, tmax can be used as an approxi- of administration and processes of drug elimination
mate indication of drug absorption rate. The value as long as the elimination processes do not change.
for tmax will become smaller (indicating less time The AUC can be determined by a numerical inte-
required to reach peak plasma concentration) as the gration procedure, such as the trapezoidal rule
absorption rate for the drug becomes more rapid. method. The units for AUC are concentration × time
Units for tmax are units of time (eg, hours, minutes). (eg, mg·h/mL).
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     477

Area under curve (AUC)


Plasma level (mg/mL)

Dose (mg)
FIGURE 16-7  Relationship between AUC and dose when
Time metabolism (elimination) is saturable.
FIGURE 16-5  Plasma level–time curve following admin-
istration of single doses of (A) 250 mg, (B) 500 mg, and (C) When the AUC is not directly proportional to the
1000 mg of drug. dose, bioavailability of the drug is difficult to evalu-
ate because drug kinetics may be dose dependent.
For many drugs, the AUC is directly propor- Conversely, absorption may also become saturated
tional to dose. For example, if a single dose of a drug resulting in lower-than-expected changes in AUC.
is increased from 250 to 1000 mg, the AUC will also
show a fourfold increase (Figs. 16-5 and 16-6). Urinary Drug Excretion Data
In some cases, the AUC is not directly proportional Urinary drug excretion data is an indirect method for
to the administered dose for all dosage levels. For estimating bioavailability. The drug must be excreted
example, as the dosage of drug is increased, one of the in significant quantities as unchanged drug in the
pathways for drug elimination may become saturated urine. In addition, timely urine samples must be col-
(Fig. 16-7). Drug elimination includes the processes of lected and the total amount of urinary drug excretion
metabolism and excretion. Drug metabolism is an must be obtained (see Chapter 3).
enzyme-dependent process. For drugs such as salicylate
and phenytoin, continued increase of the dose causes Du∞ : The cumulative amount of drug excreted in
saturation of one of the enzyme pathways for drug the urine, Du∞ , is related directly to the total amount of
metabolism and consequent prolongation of the elimi- drug absorbed. Experimentally, urine samples are
nation half-life. The AUC thus increases disproportion- collected periodically after administration of a drug
ally to the increase in dose, because a smaller amount of product. Each urine specimen is analyzed for free
drug is being eliminated (ie, more drug is retained). drug using a specific assay. A graph is constructed
that relates the cumulative drug excreted to the col-
lection-time interval (Fig. 16-8).
Area under curve (AUC)

Cumulative amount
of drug in urine

0 250 500 750 1000 A B C


Dose (mg) Time
FIGURE 16-6  Linear relationship between AUC and dose FIGURE 16-8  Corresponding plots relating the plasma
(data from Fig. 16-5). level–time curve and the cumulative urinary drug excretion.
478    
Chapter 16

The relationship between the cumulative amount A


of drug excreted in the urine and the plasma level–
time curve is shown in Fig. 16-8. When the drug is

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

dDu/dt: The rate of drug excretion. Because most A B C


drugs are eliminated by a first-order rate process, the Time

rate of drug excretion is dependent on the first-order B


elimination rate constant, k, and the concentration of
drug in the plasma, Cp. In Fig. 16-9, the maximum rate

excretion (dDu/dt)
Rate of drug
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. 16-10). A B C
Time
t∞: The total time for the drug to be excreted. In FIGURE 16-10  Corresponding plots relating the plasma
Figs. 16-9 and 16-10, the slope of the curve segment level–time curve and the rate of urinary drug excretion.
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 com-
pletely excreted, t = ∞. The t∞ is a useful parameter BIOEQUIVALENCE STUDIES
in bioequivalence studies that compare several drug
products.
BASED ON PHARMACODYNAMIC
ENDPOINTS—IN VIVO
A PHARMACODYNAMIC (PD)
COMPARISON
Plasma level

In some cases, the quantitative measurement of a


drug in plasma is not available or in vitro approaches
are not applicable. The following criteria for a PD
endpoint study are important:
A B C
Time • A dose–response relationship is demonstrated.
• The PD effect of the selected dose should be at the
B rising phase of the dose–response curve, as shown
in Fig. 16-11.
Cumulative amount
of drug in urine

• Sufficient measurements should be taken to assure


an appropriate PD response profile.
• All PD measurement assays should be validated
for specificity, accuracy, sensitivity, and precision.

A B C
For locally acting, nonsystemically absorbed drug
Time products, such as topical corticosteroids, plasma
FIGURE 16-9  Corresponding plots relating the plasma drug concentrations may not reflect the bioavail-
level–time curve and the cumulative urinary drug excretion. ability of the drug at the site of action. An acute
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     479

Dose response 25
Peak effect
100
20
Intensity of response

15

Effect
50 10
Area under
5 the peak effect-
versus-time
ED50 value curve
0
0 0 5 10 15 20 25 30
Log dose Time (hours)
Time for peak effect
Dose response FIGURE 16-12  Acute pharmacodynamic effect–time
curve. It shows an acute pharmacologic effect that is measured
periodically after a single oral dose. The effect curve is similar
to Fig. 16-3.
Intensity of response

The use of an acute pharmacodynamic effect to


determine bioavailability generally requires demon-
stration of a dose–response curve (Fig. 16-11 and
Chapter 21). Bioavailability is determined by char-
Dose (arithmetic scale) acterization of the dose–response curve. For bio-
FIGURE 16-11  Dose–response curves. Dose–response equivalence determination, pharmacodynamic
curves for dose versus response graphed on a log or arithmetic parameters including the total area under the acute
scale. pharmacodynamic effect–time curve, peak pharma-
codynamic effect, and time for peak pharmacody-
namic effect are obtained from the pharmacodynamic
pharmacodynamic effect,4 such as an effect on forced effect–time curve (Fig. 16-12). The onset time and
expiratory volume, FEV1 (inhaled bronchodilators), duration of the pharmacokinetic effect may also be
or skin blanching (topical corticosteroids) can be included in the analysis of the data. The use of phar-
used as an index of drug bioavailability. In this case, macodynamic endpoints for the determination of
the acute pharmacodynamic effect is measured over a bioavailability and bioequivalence is much more
period of time after administration of the drug prod- variable than the measurement of plasma or urine
uct. Measurements of the pharmacodynamic effect drug concentrations. Some examples of drug prod-
should be made with sufficient frequency to permit a ucts for which bioequivalence PD endpoints are
reasonable estimate for a time period at least three recommended are listed on Table 16-2.
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 BIOEQUIVALENCE STUDIES BASED
moiety to the bloodstream for systemic distribution ON CLINICAL ENDPOINTS—
(Zou and Yu, 2014). CLINICAL ENDPOINT STUDY
The clinical endpoint study is the least accurate,
4A pharmacodynamic endpoint is an acute pharmacologic effect
least sensitive to bioavailability differences, and
that is directly related to the drug’s activity that can be measured most variable. A predetermined clinical endpoint is
quantitatively. used to evaluate comparative clinical effect in the
480    Chapter 16

TABLE 16-2  Examples of Drug Products for Which FDA Recommends That Bioequivalence Studies
Use Pharmacodynamic Endpoints
Drug Product Indication Mechanism of Action Endpoint

Acarbose tablet (if no Q1/Q2 Treatment of type 2 Inhibition of intestinal Reduction in blood glucose
sameness between test and diabetes a-glucosidase, thereby concentrations
reference) decreasing absorption of
starch and oligosaccharides

Lanthanum carbonate Reduction of serum Inhibits phosphate absorp- Reduction in urinary


tablet phosphate levels in patients tion by forming highly insol- phosphate excretion
with end-stage renal disease uble lanthanum phosphate
complexes in GI tract

Orlistat capsules Treatment of obesity Inhibition of intestinal Amount of fat excreted


lipase, thereby reducing in feces over 24 hours at
absorption of free fatty acids steady state
and monoacylglycerols

Fluticasone propionate Relief of skin itching and The application of cortico- Skin chromameter measure-
cream inflammation steroids causes blanching in ments through at least
the microvasculature of the 24 hours after application
skin (not the mechanism of
action, but quantitatively
measurable)

Albuterol sulfate metered Relaxes smooth muscle of A beta2-adrenergic agonist • E ither a bronchoprovoca-
dose inhaler airways, thus protecting tion or bronchodilatation
against bronchoconstrictor assay is suitable
challenges • For bronchoprovocation,
measure the concentra-
tion or dose of methacho-
line required to decrease
FEV1 by 20%
• For bronchodilatation,
measure the AUEC0-4 h,
AUEC0-6 h, and maximum
FEV1 through 6 hours
post-dose

Fluticasone propionate/ Treatment of asthma • F luticasone is an anti- Measure area under the
salmeterol xinafoate and chronic obstructive inflammatory cortico- FEV1-time curve at desig-
inhalation power pulmonary disease (COPD) steroid nated intervals on first day
• Salmeterol is a beta2- and last day of 4-week daily
adreneric agonist treatment period

Low-molecular-weight Anticoagulant Inactivation of Factor Xa • T o assure pharmaceutical


heparins for IV and Factor IIa in coagulation equivalence of two formu-
administration cascade lations, measure anti-Xa
and anti-IIa activities
• Demonstration of in vivo
bioequivalence is waived
because product is a true
solution

Adapted from Zou and Yu (2014).


Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     481

TABLE 16-3  Examples of Drug Products for Which FDA Recommends Bioequivalence Studies
with Clinical Endpoints
Product Study Patients Study Duration Endpoint(s)

Calcipotriene cream Plaque psoriasis 56 days Proportions of subjects in the PP population


with treatment success on PGA and clinical
success of PASI
Imiquimod cream Actinic keratosis 14 weeks Proportion of subjects in the PP population
with treatment success (100% clearance of
all AK lesions)
Ketoconazole Dandruff 28 days Proportion of subjects with treatment suc-
shampoo cess or cure, defined as a score of 0 or 1 on
the Global Evaluation Scale (erythema rating)
Miconazole nitrate Vulvovaginal candidiasis 21–30 days Proportion of patients with therapeutic cure,
vaginal cream defined as both mycological and clinical
cure, at the test-of-cure visit
Nitazoxanide tablets Diarrhea caused by Giardia 10 days Proportion of patients with a “well” clinical
lamblia response, defined as either (1) no symptoms,
no watery stool, and no more than 2 soft
stools with no hematochezia within the
past 24 hours or (2) no symptoms and no
unformed stools within the past 48 hours

Sucralfate tablets Active duodenal ulcer disease; 8 weeks Proportion of patients with ulcer healing at
patients must be Helicobacter week 8 by endoscopic examination; if more
pylori negative or continue to than one ulcer is observed at enrollment,
have the presence of an ulcer both must demonstrate healing at week 8
after appropriate H. pylori for success (“cure”)
treatment

chosen patient population. Highly variable clinical clinical studies have been used to establish bioequiv-
responses require the use of a large number of alence for topical antifungal drug products (eg, keto-
patient study subjects, which increases study costs conazole) and for topical acne preparations. For
and requires a longer time to complete compared to dosage forms intended to deliver the active moiety to
the other approaches for determination of bioequiva- the bloodstream for systemic distribution, this approach
lence. A placebo arm is usually included to demon- may be considered acceptable only when analytical
strate that the study is sufficiently sensitive to identify methods cannot be developed to permit use of one of
the clinical effect in the patient population enrolled in the other approaches. Some examples of drug prod-
the study. The FDA considers this approach only ucts where a clinical endpoint bioequivalence study
when analytical methods and pharmacodynamic is recommended (Davit and Conner, 2015) are listed
methods are not available to permit use of one of the in Table 16-3.
approaches described above. The clinical study is
usually a limited, comparative, parallel clinical study
using predetermined clinical endpoint(s).
IN VITRO STUDIES
Clinical endpoint BE studies are recommended Comparative drug release/dissolution studies under
for those products that have negligible systemic certain conditions may give an indication of drug bio-
uptake, for which there is no identified PD measure, availability and bioequivalence. Ideally, the in vitro
and for which the site of action is local. Comparative drug dissolution rate should correlate with in vivo
482    Chapter 16

drug bioavailability (see Chapter 15 on in vivo–in vitro recommended BE approaches consist of comparative
correlation, IVIVC). The test and reference products in vitro release testing and physicochemical charac-
for which in vitro release rates form the basis of the terization (US-FDA, CDER, 2012b).
bioequivalence usually demonstrate Q1/Q2 sameness
(qualitatively same inactive ingredients in the quanti-
tative same amounts). Comparative dissolution studies BIOEQUIVALENCE STUDIES BASED
are often performed on several test formulations of
ON MULTIPLE ENDPOINTS
the same drug during drug development. Comparative
dissolution profiles may be considered similar if the The FDA may recommend two or more bioequiva-
similarity factor (f2) is greater than 50 (see Chapter 15). lence studies, each based on a different approach,
For drugs whose dissolution rate is related to the rate for some drug products with complex delivery sys-
of systemic absorption, the test formulation that dem- tems or mechanisms of action. Some examples of
onstrates the most rapid rate of drug dissolution in vitro drug products that FDA requires multiple bioequiv-
will generally have the most rapid rate of drug bio- alence studies (Davit and Conner, 2015) are listed in
availability in vivo. Under certain conditions, com- Table 16-4.
parative 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 BIOEQUIVALENCE STUDIES
(biowaiver) of performing additional in vivo bioequiv-
Differences in the predicted clinical response or an
alence studies (see section on biowaivers).
adverse event may be due to differences in the phar-
macokinetic and/or pharmacodynamic behavior of
the drug among individuals or to differences in the
OTHER APPROACHES DEEMED bioavailability of the drug from the drug product.
ACCEPTABLE (BY THE FDA) Bioequivalent drug products that have the same sys-
temic drug bioavailability will have the same predict-
The FDA may also use in vitro approaches other than
able drug response. However, variable clinical
comparative dissolution for establishing bioequiva-
responses among individuals that are unrelated to
lence. The use of in vitro biomarkers and in vitro
bioavailability may also be due to differences in the
binding studies has been proposed to establish bio-
pharmacodynamics of the drug. Differences in phar-
equivalence. For example, cholestyramine resin is a
macodynamics, that is, the relationship between the
basic quaternary ammonium anion-exchange resin
drug and the receptor site, may be due to differences
that is hydrophilic, insoluble in water, and not
in receptor sensitivity to the drug (see Chapter 21).
absorbed in the gastrointestinal tract. The bioequiva-
Various factors affecting pharmacodynamic drug
lence of cholestyramine resin is performed by equi-
behavior may include age, drug tolerance, drug inter-
librium and kinetic binding studies of the resin to bile
actions, and unknown pathophysiologic factors.
acid salts (US-FDA, CDER, 2012a). For calcium
acetate tablets, which exert the therapeutic response
by binding phosphate in the GI tract, the FDA recom- Bases for Determining Bioequivalence
mends a relatively simple in vitro binding assay Bioequivalence is established if the in vivo bioavail-
based on the test/reference binding ratio over a range ability of a test drug product (usually the generic
of phosphate concentrations. Since this test is thought product) does not differ significantly (ie, statistically
to be highly reproducible, the BE acceptance crite- not significant) from that of the reference listed drug
rion is that the test/reference binding ratio should fall (usually the brand-name product approved through
within limits of 0.9–1.1 (US-FDA, CDER, 2011a). the NDA route) in the product’s rate and extent of
The FDA accepts various other in vitro approaches drug absorption. Bioequivalence is determined by
for BE assessment of proposed generic locally acting comparison of measured parameters (eg, concentra-
drug products. For the acyclovir topical ointment, tion of the active drug ingredient in the blood, urinary
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     483

TABLE 16-4  Drug Products for Which FDA Recommends Multiple Bioequivalence Approaches
Product Indicated to Treat Approach Endpoint

Diclofenac gel Osteoarthritis of the knee Clinical Pain score change from baseline

In vivo PK AUC, Cmax

Nitazoxanide oral Diarrhea caused by Giardia Clinical Proportion of patients with a “well” clinical response
lamblia

In vivo PK AUC, Cmax

Fluticasone Allergic rhinitis Clinical Total nasal symptom score (TNSS) change from
propionate nasal baseline
suspension

In vivo PK AUC, Cmax

In vitro Comparison of device performance with regard


to the amount of drug per actuation, droplet size
distribution, and plume shape

Mesalamine Ulcerative colitis In vivo PK AUC, pAUC, Cmax


DR and ER oral
formulations

In vitro Comparison of dissolution profiles in several different


media of varying pH values

Mesalamine rectal Distal ulcerative In vivo PK AUC, Cmax


enema colitis, proctitis, and
proctosigmoiditis

In vitro Dissolution profiles at pH 4.5, 6.8, 7.2 (Apparatus 2),


900 mL, 35, 50 rpm

Mesalamine Ulcerative proctitis In vivo PK AUC, Cmax


suppository

In vitro Comparison of physicochemical properties

Risperidone long- Bipolar I disorder and Steady-state AUCt , (Cmax)SS


acting injectable schizophrenia PK in patients

In vitro Comparison of the time for 50% of drug to be


released at two bracketing sampling times

Lansoprazole DR Gastroesophageal reflux In vivo PK AUC, Cmax


capsule disease

In vitro Comparison of sedimentation volume, granule


dispersion, recovery, and acid resistance, after
dispersing into apple juice and dispensing into
nasogastric tubes

Dexamethasone/ Prophylaxis against In vivo PK AUC, Cmax, in aqueous humor of cataract surgery
Tobramycin inflammation and infection patients
Ophthalmic during cataract surgery
Suspension

In vitro Microbial kill rates against specified microorganisms


484    Chapter 16

excretion rates, or pharmacodynamic effects), when parameters, pharmacodynamic parameters, clinical


administered at the same molar dose of the active observations, and/or in vitro studies may be used to
moiety under similar experimental conditions, either determine drug bioavailability from a drug product.
single dose or multiple dose. The design and evaluation of well-controlled bio-
In a few cases, a drug product that differs from equivalence studies require cooperative input from
the reference listed drug in its rate of absorption, but pharmacokineticists, statisticians, clinicians, bioanalyt-
not in its extent of absorption, may be considered ical chemists, and others. For some generic drugs, the
bioequivalent if the difference in the rate of absorption FDA offers general guidelines for conducting these
is intentional and appropriately reflected in the label- studies. For example, Statistical Procedures for
ing and/or the rate of absorption is not detrimental to Bioequivalence Studies Using a Standard Two-
the safety and effectiveness of the drug product. Treatment Crossover Design is available from the FDA
(US-FDA, CDER, 2000a); the publication addresses
three specific aspects, including (1) logarithmic trans-
DESIGN AND EVALUATION OF formation of pharmacokinetic data, (2) sequence effect,
BIOEQUIVALENCE STUDIES and (3) outlier consideration. However, even with the
availability of such guidelines, the principal investiga-
Objective
tor should prepare a detailed protocol for the study.
All scientific studies should have clearly stated Some of the elements of a protocol for an in vivo bio-
objectives. The main objective for a bioequivalence availability study are listed in Table 16-5.
study is that the drug bioavailability from test and For bioequivalence studies, the test and refer-
reference products is not statistically different when ence drug formulations must contain the same drug
administered to patients or subjects at the same in the same dose strength and in similar dosage
molar dose from pharmaceutically equivalent drug forms (eg, immediate release or controlled release),
products through the same route of administration and must be given by the same route of administra-
under similar experimental conditions. tion. Before beginning the study, the Institutional
Review Board (IRB) of the clinical facility in which
Study Considerations the study is to be performed must approve the study.
The basic design for a bioequivalence study is deter- The IRB is composed of both professional and lay
mined by (1) the scientific questions and objectives to persons with diverse backgrounds who have clinical
be answered, (2) the nature of the reference material experience and expertise as well as sensitivity to
and the dosage form to be tested, (3) the availability of ethical issues and community attitudes. The IRB is
analytical methods, (4) the pharmacokinetics and responsible for all ethical issues including safe-
pharmacodynamics of the drug substance, (5) the route guarding the rights and welfare of human subjects.
of drug administration, and (6) benefit–risk and ethical The basic guiding principle in performing studies
considerations with regard to testing in humans. is do not do unnecessary human research. Generally,
Since bioequivalence studies are performed to the study is performed in normal, healthy male and
compare the bioavailability of the test or generic female volunteers who have given informed consent to
drug product to the reference or brand-name prod- be in the study. Critically ill patients are not included
uct, the statistical techniques should be of sufficient in an in vivo bioavailability study unless the attending
sensitivity to detect differences in rate and extent of physician determines that there is a potential benefit to
absorption that are not attributable to subject vari- the patient. The number of subjects in the study will
ability. Once bioequivalence is established, it is depend on the expected intersubject and intrasubject
likely that both the generic and brand-name dosage variability. Patient selection is made according to cer-
forms will produce the same therapeutic effect. The tain established criteria for inclusion in, or exclusion
FDA publishes guidances for bioequivalence studies from, the study. For example, the study might exclude
(US-FDA, CDER, 2010a). Sponsors may also any volunteers who have known allergies to the drug,
request a meeting with the FDA to review the study are overweight, or have taken any medication within a
design for a specific drug product. Pharmacokinetic specified period (often 1 week) prior to the study.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     485

TABLE 16-5  Elements of a Bioavailability are compared. The FDA designates a single reference
Study Protocol listed drug5 as the standard drug product to which all
I. Title
generic versions must be shown to be bioequivalent.
A. Principal investigator (study director) The FDA hopes to avoid possible significant variations
B. Project/protocol number and date among generic drugs and their brand-name counter-
II. Study objective parts. Such variations could result if generic drugs
III. Study design were compared to different reference listed drugs.
A. Design
B. Drug products
The reference drug product should be administered
1. Test product(s) by the same route as the comparison formulations
2. Reference product unless an alternative route or additional route is needed
C. Dosage regimen to answer specific pharmacokinetic questions. For
D. Sample collection schedule example, if an active drug is poorly bioavailable after
E. Housing/confinement
F. Fasting/meals schedule
oral administration, the drug may be compared to an
G. Analytical methods oral solution or an intravenous injection. For bioequiva-
IV. Study population lence studies on a proposed generic drug product, the
A. Subjects reference standard is the reference listed drug (RLD),
B. Subject selection which is listed in the FDA’s Approved Drug Products
1. Medical history
2. Physical examination
with Therapeutic Equivalence Evaluations—the Orange
3. Laboratory tests Book (US-FDA, CDER, 2014d), and the proposed
C. Inclusion/exclusion criteria generic drug product is often referred to as the “test”
1. Inclusion criteria drug product. The RLD is generally a formulation cur-
2. Exclusion criteria rently marketed with a fully approved NDA for which
D. Restrictions/prohibitions
V. Clinical procedures
there are valid scientific safety and efficacy data. The
A. Dosage and drug administration RLD is usually the innovator’s or original manufactur-
B. Biological sampling schedule and handling er’s brand-name product and is administered according
procedures to the dosage recommendations in the labeling.
C. Activity of subjects Before beginning an in vivo bioequivalence study,
VI. Ethical considerations
A. Basic principles
the total content of the active drug substance in the
B. Institutional review board test product (generally the generic product) must be
C. Informed consent within 5% of that of the reference product. Moreover,
D. Indications for subject withdrawal in vitro comparative dissolution or drug-release studies
E. Adverse reactions and emergency procedures under various specified conditions are usually per-
VII. Facilities
VIII. Data analysis
formed for both test and reference products before
A. Analytical validation procedure performing the in vivo bioequivalence study.
B. Statistical treatment of data
IX. Drug accountability Regulatory Recommendations for
X. Appendix
Optimizing Bioavailability Study Design
The FDA lists a number of recommendations to con-
Moderate smokers may be included in these studies. sider in designing clinical relative bioavailability
The subjects generally fast for 10–12 hours (overnight) studies in drug development. These recommenda-
prior to drug administration and may continue to fast tions include the following:
for a 2- to 4-hour period after dosing.
• Use of a randomized crossover design whenever
possible
Reference Listed Drug (RLD)
For bioequivalence studies of generic products, one 5The reference listed drug (RLD) is listed in the Orange Book,
formulation of the drug is chosen as a reference stan- Approved Drug Products with Therapeutic Equivalence Evaluations.
dard against which all other formulations of the drug http://www.accessdata.fda.gov/scripts/cder/ob/default.cfm.
486    Chapter 16

• Enrolling both male and female subjects whenever Factors Influencing Bioavailability and
possible Impact on Drug Development
• Administering single doses rather than multiple Various factors influence bioavailability (Table 16-6).
doses, as single-dose studies are more sensitive, Some of these factors are listed below with implica-
although multiple-dose studies may be more suit- tions for formulation development and optimization
able in some cases of dosing regimens.
• Conducting the studies under fasting and fed con- Physicochemical properties of the drug and
ditions6 formulation. Formulations can be designed to
• Measuring the parent drug rather than metabolites, improve the bioavailability of poorly soluble drugs,
unless the parent cannot be reliably measured. Pre- extend the absorption phase by slowing the rate of
systemically formed metabolites that contribute release of drugs (controlled-release formulations), or
meaningfully to safety and efficacy should also be prevent dissolution in the gastric lumen for drugs
measured that are destroyed by gastric acidity (enteric-coated
In addition, the FDA recommends that Cmax and tmax formulations) (see also Chapter 15).
be measured to compare peak exposure and rate of An example of how formulation design can
absorption, and that AUC0-t (AUC to the last measur- improve bioavailability is shown by comparing the
able drug concentration) and AUC0-∞ (AUC extrapo- immunosuppressant drug cyclosporine systemic
lated to infinity) be measured to compare total exposure exposures provided by the Neoral® microemulsion
or extent of drug absorption. Drug exposure parame- formulation to those provided by the Sandimmune®
ters should be log-transformed before statistical com- formulation. The Neoral label states that, in a rela-
parisons. Further detail about the statistical tests will tive bioavailability study in renal transplant, rheuma-
be provided later in the discussion on bioequivalence toid arthritis, and psoriasis patients, the mean
study designs. cyclosporine AUC was 20%–50% greater, and the
mean cyclosporine Cmax was 40%–106% greater,
compared to following administration with
Sandimmune. In addition, the dose-normalized AUC
Frequently Asked Questions in liver transplant patients administered Neoral for
»» What are the study protocol considerations for con- 28 days was 50% greater and Cmax was 90% greater
ducting a bioequivalence study? than in those patients administered Sandimmune.
»» What is the reference listed drug (RLD), and how is
Drug stability and pH effects. Acid-labile drugs
the RLD selected?
potentially have low bioavailability, as they are sub-
»» How is a bioavailability study of a new molecular ject to acid-induced degradation in the low pH condi-
entity conducted? tions of the stomach. For such drugs to achieve
»» Why does the value for relative bioavailability some-
therapeutic plasma concentrations, it is necessary to
times exceed 1.0, whereas the value for absolute deliver them by formulations that protect against acid-
bioavailability cannot exceed 1.0 7? induced degradation, such as buffered products or
enteric-coated products. Enteric-coated formulations
are used to deliver acid-labile drugs such as didano-
sine (Damle et al, 2002), a purine nucleoside analog
indicted to treat HIV disease, and omeprazole and
6In a food-effect bioavailability study, the reference treatment
lansoprazole (Horn and Howden, 2005), which are
is the oral formulation of the drug product given on an empty proton pump inhibitors indicated to treat acid reflux.
stomach, which is compared with the same oral formulation given
with food, usually a high-fat, high-calorie meal. Presystemic and first-pass metabolism. The effects
7Fwill appear to exceed 1.0, if the absolute bioavailability is near of presystemic metabolism on oral bioavailability
100% and variability yields a result slightly higher than 1.0. is (Jagdale et al, 2009) illustrated by propranolol, a
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     487

TABLE 16-6  Factors Influencing Bioavailability and Impacting Drug Development


• Physicochemical properties of the drug and formulation
££ The active drug ingredient has low solubility in water (eg, less than 5 mg/mL)
££ The dissolution rate of the product is slow (eg, <50% in 30 min when tested with a general method specified by the FDA)
££ The particle size and 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 bioavailability
• Drug product
££ 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
• Drug stability
££ The drug (and drug product) has poor stability leading to short shelf life
££ 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
• pH effects (eg, pH within the gastrointestinal lumen)
• Surface of dosage form and time available for absorption
• Presystemic metabolism, including hepatic first-pass effect
• Food effects, for orally administered formulations
• 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
• Drug–drug interactions
• Efflux transporters (such as P-glycoprotein)
• 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
• Age
• Disease state

nonselective beta adrenergic receptor blocking agent Food effects. Food can either decrease drug bio-
used as an antihypertensive, antianginal, and anti­ availability or increase bioavailability, or have no
arrhythmic, presystemic metabolism. Propranolol is effect on bioavailability (Davit and Conner, 2008;
almost completely absorbed after oral administra- Dehaven and Conner, 2014). Food can influence
tion, but due to extensive first-pass metabolism in the bioavailability in a number of ways, such as affect-
liver, only about 25% of the parent drug reaches the ing gastrointestinal pH, gastric emptying, intestinal
systemic circulation. transit, splanchnic blood flow, and first-pass metabo-
lism. Food can also affect bioavailability by physical
Prodrugs that undergo rapid presystemic metab- or chemical interactions. Most food effects on drug
olism can be used to improve bioavailability, as illus- bioavailability are not considered clinically signifi-
trated by valacyclovir, a prodrug of the nucleoside cant, and, consequentially, most drug products are
analog antiviral compound acyclovir. Valacyclovir labeled to be administered without regard to meals.
undergoes rapid presystemic conversion to acyclovir. If the food effects on drug bioavailability are clini-
Both valacyclovir and acyclovir are effective in treat- cally significant, then the drug product labeling will
ing herpes infections. However, because acyclovir provide instructions about how to achieve the opti-
bioavailability is greatly enhanced when delivered by mal dosing regimen—either to take the drug only on
its prodrug valacyclovir, for treating herpes zoster, it an empty stomach, or only with food, depending on
is only necessary to administer Valtrex® (valacyclovir) the nature of the bioavailability effect and clinical
tablets administered once daily, compared to 5 times consequences.
daily for Zovirax® (acyclovir) capsules.
488    Chapter 16

An example of food reducing bioavailability other drugs be defined during drug development
and the implications for drug product labeling is (US-FDA, CDER, 2012c). Two examples of drug–
illustrated by didanosine, discussed earlier. As food drug interactions, one of enzyme inhibition and the
prolongs gastric emptying, this increases the length second of enzyme induction, will show how the
of time that the acid-labile didanosine will be in ability of coadministered drugs to alter systemic
contact with a low pH environment. The Videx® EC bioavailability impacts both recommendations for
label states that food reduced the didanosine Cmax optimal dosing regimens and development of new
by 46% and its AUC by 19%. Consequently, the formulations to maximize bioavailability.
Videx EC label recommends that didanosine should An example of a drug–drug interaction that
be taken on an empty stomach in order to avoid the increases bioavailability is provided by ritonavir
possibility of exposing a patient to subtherapeutic (an HIV protease inhibitor indicated for treating
plasma levels. HIV disease), which is a potent inhibitor of cyto-
Food-induced increases in drug bioavailability chrome P450 3A (CYP3A). As such, ritonavir coad-
can be either desirable or undesirable. The food ministration increases systemic bioavailability of
effect on isotretinoin (indicated to treat severe recal- drugs that are metabolized by CYP3A. For drugs
citrant nodular acne) bioavailability is used to opti- such as sedative hypnotics, antiarrhythmic, and
mize the dosing regimen. The Accutane® label ergot alkaloid preparations, large increases in sys-
states that for isotretinoin capsules, both the Cmax temic bioavailability caused by ritonavir coadmin-
and AUC were more than doubled when the drug istration can result in potentially serious and/or
product was taken with a meal compared with fasted life-threatening adverse events; thus, ritonavir
conditions. Consequently, the label recommends coadministration with these drugs is contraindi-
that isotretinoin capsules should always be taken cated. For other coadministered CYP3A substrate
with food. By contrast, in some cases, food-induced drugs for which ritonavir increases bioavailability,
increases in oral bioavailability may be associated such as antidepressants, clarithromycin, immuno-
with safety concerns. This situation is illustrated by modulators, rifabutin, and trazadone, the Norvir®
the drug efavirenz, a non-nucleoside reverse tran- labeling recommends either dose-adjustment or
scriptase inhibitor indicated to treat HIV disease. additional monitoring of the coadministered drug to
The Sustiva® label describes how coadministration maintain systemic bioavailability levels associated
of a high-fat, high-calorie meal increased the efavi- with safety and efficacy.
renz AUC and Cmax by 22% and 39%, respectively, Because ritonavir can significantly increase the
and coadministration of a lower-fat, lower-calorie bioavailability of CYP3A substrates, it has been
meal increased the efavirenz AUC and Cmax by 17% developed as a “booster” to improve systemic expo-
and 51%, respectively. Due to concern that exposure sure of HIV therapies that are CYP3A substrates
to higher efavirenz systemic bioavailability could and that have low oral bioavailability due to exten-
result in increased serious adverse events, the sive hepatic clearance (de Mendoza et al, 2006).
Sustiva® label recommends that efavirenz capsules Notably, ritonavir is formulated together with the
and tablets be taken on an empty stomach, prefera- HIV-1 protease inhibitor lopinavir in the fixed-dose
bly at bedtime. combination product Kaletra®. Ritonavir in the
Kaletra formulation inhibits the CYP3A-mediated
Effects of drug–drug interactions. Changes in metabolism of lopinavir, thereby increasing lopina-
drug bioavailability due to drug–drug interactions vir systemic bioavailability to levels that achieve
can occur via a variety of mechanisms, such as inhi- antiviral activity.
bition of metabolizing enzymes, induction of metab- Enzyme inducers coadministered with drugs
olizing enzymes, inhibitor of transporters, and can potentially lower systemic bioavailability to sub-
induction of transporters. The FDA recommends that therapeutic levels. An example is the antibacterial
interactions between an investigational new drug and drug rifampin (used in treatment of tuberculosis),
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     489

which is a potent inducer of cytochrome P-450 The FDA recommends that sponsors developing pedi-
enzymes. Coadministration of rifampin with drugs atric formulations conduct pharmacokinetic studies in
metabolized by metabolic pathways induced by the pediatric population to determine how the dosing
rifampin can result in lower bioavailability due to regimen should be adjusted to achieve the same sys-
acceleration of metabolism. The Rifadin® label temic exposure that is safe and effective in adults
states that, to maintain optimum therapeutic bio- (Chapter 23).
availability, dosages of drugs metabolized by these Systemic bioavailability of drugs can change with
enzymes may require dose adjustment when starting aging (Klotz, 2009). Impairments in the functional
or stopping concomitantly administered rifampin. reserve of multiple organs can occur with advancing
Some examples of these drugs for which rifampin age, and such impairments might affect drug metabo-
lowers systemic bioavailability to the extent that lism and pharmacokinetics. Advancing age is associ-
dose adjustment is needed include anticonvulsants, ated with changes such as decreases in liver mass and
antiarrhythmics, beta-blockers, calcium channel perfusion, changes in body composition, and decreases
blockers, fluoroquinolones, oral hypoglycemic agents, in renal function. Many of these changes result in
transplant drugs, and tricyclic antidepressants. For increased drug bioavailability. As a result, it is recom-
some drugs, such as oral contraceptives, coadmin- mended that clinicians carefully monitor dosing regi-
istration with rifampin is contraindicated due to mens and drug action in geriatric patients.
concerns that rifampin coadministration can lower
oral contraceptive systemic bioavailability to sub- Disease state. The bioavailability of drugs
therapeutic levels. eliminated primarily through renal excretory
mechanisms is likely to increase in patients with
Efflux transporters. The cardiac glycoside digoxin impaired renal function (Chapter 24). The FDA
is a substrate for P-glycoprotein, at the level of recommends that, where appropriate, drug phar-
intestinal absorption, renal tubular secretion, and macokinetics be characterized in patients with
biliary-intestinal secretion (Hughes and Crowe, 2010). varying degrees of renal impairment. The results of
Therefore, drugs that induce or inhibit P-glycoprotein such studies are used to determine how doses can
have the potential to alter digoxin bioavailability. be adjusted in patients with renal impairment in
Examples of such drugs include amiodarone, propafe- order to achieve the same systemic drug bioavail-
none, quinidine, and verapamil. As digoxin is a narrow ability as in patients with normal renal function
therapeutic index drug, small changes in bioavail- (US-FDA, CDER, 2010b). Similarly, it may be
ability can potentially result in serious adverse events advisable to conduct pharmacokinetic studies of
due to loss of efficacy (bioavailability is lower than drugs that are primarily cleared by the liver in
the therapeutic range) or life-threatening toxicity patients with varying degrees of hepatic impair-
(bioavailability exceeds the therapeutic range). ment (US-FDA, CDER, 2003a). The results of
Digoxin oral solution USP labeling instructs the prac- pharmacokinetic studies in hepatic-impaired
titioner to measure serum digoxin concentrations patients can be useful in determining whether dose
before initiating concomitant drugs, reduce the digoxin adjustments are required in such patients to achieve
dose once concomitant therapy is initiated, and con- the same systemic drug bioavailability as in
tinue to monitor digoxin serum concentrations. patients with normal liver function.
The systemic bioavailability of a drug in patients
Age. The systemic bioavailability of a drug is can differ from that in healthy normal subjects.
controlled by its absorption, distribution, metabolism, Ordinarily, sponsors conduct single- and multiple-
and elimination (ADME). In pediatric patients, growth dose pharmacokinetic studies in both healthy normal
and developmental changes in factors influencing subjects and the target patient population in early
ADME lead to drug bioavailability that can differ stage development, to characterize similarities and
from that of adult patients (US-FDA, CDER, 2014e). differences in drug systemic bioavailability.
490    Chapter 16

Analytical Methods Fasting Study


Analytical methods used in an in vivo bioavailability, Bioequivalence studies are usually evaluated by a
bioequivalence, or pharmacodynamic studies must single-dose, two-period, two-treatment, two-sequence,
be validated for accuracy and sufficient sensitivity. open-label, randomized crossover design comparing
The actual concentration of the active drug ingredi- equal doses of the test and reference products in
ent or therapeutic moiety, or its active metabolite(s), fasted, adult, healthy subjects. This study is requested
must be measured with appropriate precision in body for all immediate-release and modified-release oral
fluids or excretory products. For bioavailability and dosage forms. Both male and female subjects may be
bioequivalence studies, both the parent drug and its used in the study. Blood sampling is performed just
major active metabolites are generally measured. For before (zero time) the dose and at appropriate inter-
bioequivalence studies, the parent drug is measured. vals after the dose to obtain an adequate description of
Measurement of the active metabolite is important the plasma drug concentration–time profile. The sub-
for very high-hepatic clearance (first-pass metabo- jects should be in the fasting state (overnight fast of at
lism) drugs when the parent drug concentrations are least 10 hours) before drug administration and should
too low to be reliable. continue to fast for up to 4 hours after dosing. No other
The analytical method for measurement of the medication is normally given to the subject for at least
drug must be validated for accuracy, precision, sen- 1 week prior to the study. In some cases, a parallel
sitivity, specificity, and robustness. The use of more design may be more appropriate for certain drug prod-
than one analytical method during a bioequivalence ucts, containing a drug with a very long elimination
study may not be valid, because different methods half-life. A replicate design may be used for a drug
may yield different values. Data should be pre- product containing a drug that has high intrasubject
sented in both tabulated and graphic form for evalu- variability.
ation. The plasma drug concentration–time curve
for each drug product and each subject should be
Food Intervention Study
available.
Coadministration of food with an oral drug product
may affect the bioavailability of the drug. Food inter-
vention or food effect studies are generally con-
STUDY DESIGNS ducted using meal conditions that are expected to
For many drug products, the FDA, Division of provide the greatest effects on GI physiology so that
Bioequivalence, Office of Generic Drugs, provides systemic drug availability is maximally affected.
guidance for the performance of in vitro dissolution Food effects on bioavailability are generally greatest
and in vivo bioequivalence studies (US-FDA, when the drug product is administered shortly after
CDER, 2010a). Generally, two bioequivalence stud- a meal is ingested. The nutrient and caloric contents
ies are required for solid oral dosage forms, includ- of the meal, the meal volume, and the meal tempera-
ing (1) a fasting study and (2) a food intervention ture can cause physiological changes in the GI tract
study. For extended-release capsules containing in a way that affects drug product transit time, lumi-
beads (pellets) that might be poured on a semisolid nal dissolution, drug permeability, and systemic
food such as applesauce, an additional “sprinkle” availability.
bioequivalence study is recommended. Other study Meals that are high in total calories and fat con-
designs such as parallel design, replicate design, tent are more likely to affect the GI physiology and
and multiple-dose (steady-state) bioequivalence thereby result in a larger effect on the bioavailability
studies have been proposed by the FDA. Proper of a drug substance or drug product (US-FDA,
study design and statistical evaluation are important CDER, 2003b). In addition, the high fat meal can
considerations for the determination of bioequiva- have a significant effect on certain modified-release
lence. Some of the designs listed above are summa- drug products causing them to dose dump. The test
rized here. meal is a high-fat (approximately 50% of total caloric
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     491

content of the meal) and high-calorie (approximately drug formulations (A, B, C, D), are described in
800–1000 calories) meal. A typical test meal is two eggs Tables 16-7 and 16-8. The Latin-square design plans
fried in butter, two strips of bacon, two slices of toast the clinical trial so that each subject receives each
with butter, 4 oz of brown potatoes, and 8 oz of milk. drug product only once, with adequate time between
This test meal derives approximately 150, 250, and medications for the elimination of the drug from the
500–600 calories from protein, carbohydrate, and fat, body. In this design, each subject is his own control,
respectively (www.fda.gov/cder/guidance/4613dft.pdf). and subject-to-subject variation is reduced. Moreover,
For bioequivalence studies for generic drugs, variations due to sequence, period, and treatment
drug bioavailability from both the test and reference (formulation) are reduced, so that all patients do not
products should be affected similarly by food. The receive the same drug product on the same day and in
usual study design uses a single-dose, randomized, the same order. The order in which the drug treat-
two-treatment, two-period, crossover study compar- ments are given should not stay the same in order to
ing equal doses of the test and reference products. prevent any bias in the data due to a residual effect
Following an overnight fast of at least 10 hours, from the previous treatment. Possible carryover
subjects are given the recommended meal 30 min- effects from any particular drug product are mini-
utes before dosing. The meal is consumed over 30 mized by changing the sequence or order in which
minutes, with administration of the drug product the drug products are given to the subject. Thus, drug
immediately after the meal. The drug product is product B may be followed by drug product A, D, or
given with 240 mL (8 fluid oz) of water. No food is C (Table 16-8). After each subject receives a drug
allowed for at least 4 hours postdose. This study is product, blood samples are collected at appropriate
requested for all modified-release dosage forms and time intervals so that a valid blood drug level–time
may be requested for immediate-release dosage curve is obtained. The time intervals should be
forms if the bioavailability of the active drug ingre- spaced so that the peak blood concentration, the total
dient is known to be affected by food (eg, ibuprofen, area under the curve, and the absorption and elimina-
naproxen). According to the labeling for certain tion phases of the curve may be well described.
extended-release capsules that contain coated beads, Period refers to the time period in which a study
the capsule contents can be sprinkled over soft foods is performed. A two-period study is a study that is
such as applesauce. This is taken by the fasted sub- performed on two different days (time periods) sepa-
ject and the bioavailability of the drug is then mea- rated by a washout period during which most of the
sured for the NDA. For generic drug products in drug is eliminated from the body—generally about
Abbreviated New Drug Applications (ANDAs), this
study is performed as a bioequivalence study to dem-
onstrate that both products, sprinkled on food, will TABLE 16-7  Latin-Square Crossover Design
have equivalent bioavailability. Bioavailability stud- for a Bioequivalence Study of Three Drug
ies might also examine the effects of other foods and Products in Six Human Volunteers
special vehicles such as apple juice. Drug Product

Study Study Study


CROSSOVER STUDY DESIGNS Subject  Period 1 Period 2 Period 3

Subjects who meet the inclusion and exclusion study 1 A B C


criteria and have given informed consent are selected 2 B C A
at random. A complete crossover design is usually
3 C A B
employed, in which each subject receives the test
drug product and the reference product. Examples of 4 A C B
Latin-square crossover designs for a bioequivalence 5 C B A
study in human volunteers, comparing three differ-
6 B A C
ent drug formulations (A, B, C) or four different
492    Chapter 16

TABLE 16-8  Latin-Square Crossover Design the same drug product twice to the same subject, the
for a Bioequivalency Study of 4 Drug Products replicate design provides a measure for within-subject
in 16 Human Volunteers variability. Replicate design studies may be used for
highly variable drugs and for narrow therapeutic
Drug Product
index drugs. In the case of highly variable drugs
Study Study Study Study (%CV greater than 30), a large number of subjects
Subject  Period 1 Period 2 Period 3 Period 4 (>80) would be needed to demonstrate bioequiva-
1 A B C D lence using the standard two-way crossover design.
Drugs with high within-subject variability gener-
2 B C D A
ally have a wide therapeutic window and despite
3 C D A B high variability, these products have been demon-
4 D A B C strated to be both safe and effective. Replicate
designs for highly variable drugs/products require a
5 A B D C
smaller number of subjects and, therefore, do not
6 B D C A unnecessarily expose a large number of healthy
7 D C A B subjects to a drug when this large number of sub-
jects is not needed for assurance of bioequivalence
8 C A B D
(Haidar et al, 2008).
9 A C B D Replicated crossover designs are used for the
10 C B D A determination of individual bioequivalence, to esti-
mate within-subject variance for both the test and
11 B D A C
reference drug products, and to provide an estimate
12 D A C B of the subject-by-formulation interaction variance. A
13 A C D B four-period, two-sequence, two-formulation design
is shown below:
14 C D B A

15 D B A C
  Period 1 Period 2 Period 3 Period 4
16 B A C D
Sequence 1 T R T R

Sequence 2 R T R T

10 elimination half-lives. A sequence refers to the where R = reference and T = treatment.

number of different orders in the treatment groups in


a study. For example, a two-sequence, two-period In this design, the same reference and the same test
study would be designed as follows: 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
  Period 1 Period 2
be made.
Sequence 1 T R

Sequence 2 R T Narrow Therapeutic Index Drugs


where R = reference and T = treatment. Narrow therapeutic index (NTI) drugs, also referred
to as critical dose drugs, are drugs in which small
changes in dose or concentration may lead to serious
Replicated Crossover Study Designs therapeutic failures or serious adverse drug reactions in
The standard bioequivalence criterion using the two- patients. Narrow therapeutic index drugs consistently
way crossover design does not give an estimate of display the following characteristics: (a) Subtherapeutic
within-subject (intrasubject) variability. By giving concentrations may lead to serious therapeutic failure;
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     493

(b) there is little separation between therapeutic and variable, provided that certain constraints are applied
toxic doses (or the associated plasma concentra- to this approach in order to maintain an acceptable
tions); (c) they are subject to therapeutic monitoring type I error rate and satisfy any public health con-
based on pharmacokinetic or pharmacodynamic cerns (Davit et al, 2012).
measures; (d) they possess low-to-moderate within-
subject variability (<30%); and (e) in clinical prac-   Period 1 Period 2 Period 3
tice, doses are generally adjusted in very small
increments (<20%). The FDA currently recommends Sequence 1 T R R
that bioequivalence studies of narrow therapeutic Sequence 2 R T R
index drugs should employ a four-way, fully repli-
Sequence 3 R R T
cated, crossover study design. The replicated study
design permits comparison of both test and reference
means and test and reference within-subject variabil- Under this design, if the test product has lower vari-
ity (Davit et al, 2013). ability than the reference product, the study will
An additional test recommended in bioequiva- need a smaller number of subjects to pass the bio-
lence studies of generic narrow therapeutic index equivalence criteria. Scaled average bioequivalence
drugs is a test for within-subject variability. The test is evaluated for both AUC and Cmax.
determines whether within-subject variability of the
test narrow therapeutic index drug does not differ Parallel Study Designs
significantly from that of the reference by evaluating A nonreplicate, parallel design is used for drug prod-
the test/reference ratio of the within-subject standard ucts that contain drugs that have a long elimination
deviation. The FDA currently recommends that all half-life or drug products such as depot injections in
bioequivalence studies on narrow therapeutic index which the drug is slowly released over weeks or
drugs must pass both the reference-scaled approach months. In this design, two separate groups of volun-
and the unscaled average bioequivalence limits of teers are used. One group will be given the test prod-
80.00%–125.00%. uct and the other group will be given the reference
product. It is important to balance the demographics
of both groups of volunteers. Blood sample collec-
Reference Scaled Average Bioequivalence
tion time should be adequate to ensure completion of
Recently a three-sequence, three-period, two-treatment gastrointestinal transit (approximately 2–3 days) of
partially replicated crossover design for bioequiva- the drug product and absorption of the drug sub-
lence studies of highly variable drugs has been recom- stance. Cmax and a suitably truncated AUC, generally
mended by the FDA (Haidar et al, 2008). The partially to 72 hours after dose administration, can be used to
replicated design allows the estimation of the within- characterize peak and total drug exposure, respec-
subject variance and subject-by-formulation interac- tively. For drugs that demonstrate low intrasubject
tion for the reference product. The time for completion variability in distribution and clearance, an AUC
of this study is shorter than the fully replicated four- truncated at 72 hours (AUC72 0 hours) can be used in
way crossover design. place of AUCt0 or AUC∞0 . This design is not recom-
This design is usually used for highly variable mended for drugs that have high intrasubject vari-
drugs with within-subject variability ≥30%. Large ability in distribution and clearance.
numbers of subjects may be needed in bioequiva-
lence studies of highly variable drugs; the FDA
implemented the reference-scaled average bioequiv- Multiple-Dose (Steady-State) Study Design
alence approach to ease regulatory burden and A bioequivalence study may be performed using a
reduce unnecessary human testing. Using this multiple-dose study design. Multiple doses of the
approach, the implied BE limits can widen to be same drug are given consecutively to reach steady-
larger than 80%–125% for drugs that are highly state plasma drug levels. The multiple-dose study is
494    
Chapter 16

designed as a steady-state, randomized, two-treat- Determination of bioavailability using multiple


ment, two-way, crossover study comparing equal doses reveals changes that are normally not detected
doses of the test and reference products in healthy in a single-dose study. For example, nonlinear phar-
adult subjects. Each subject receives either the test or macokinetics may occur after multiple drug doses
the reference product separated by a “washout” due to the higher plasma drug concentrations saturat-
period, which is the time needed for the drug to be ing an enzyme system involved in absorption or
completely eliminated from the body. elimination of the drug. Nonlinear pharmacokinetics
To ascertain that the subjects are at steady state, after multiple-dose studies may be observed by ris-
three consecutive trough concentrations (Cmin) are ing Cmin drug concentrations and AUCt after each
determined. The last morning dose is given to the sub- dosing interval. With some drugs, a drug-induced
ject after an overnight fast, with continual fasting for at malabsorption syndrome can also alter the percent-
least 2 hours following dose administration. Blood age of drug absorbed. In this case, drug bioavailabil-
sampling is then performed over one dosing interval. ity may decrease after repeated doses if the fraction
The area under the curve during a dosing interval at of the dose absorbed (F) decreases or if the total
steady state should be the same as the area under the body clearance (kVD) increases. It should be noted
curve extrapolated to infinite time after a single dose. that nonlinear PK can also be observed by high sin-
Pharmacokinetic analyses for multiple-dose gle doses of the drug.
studies include calculation of the following parame- There are several disadvantages of using the
ters for each subject: multiple-dose crossover method for the determina-
tion of bioequivalence. (1) The study takes more time
AUC0-tau—Area under the curve during a dosing
to perform, because steady-state conditions must be
interval
reached. A longer time for completion of a study
tmax—Time to Cmax during a dosing interval
leads to greater clinical costs and the possibility of a
Cmax—Maximum drug concentration during dos-
subject dropping out and not completing the study.
ing interval
(2) More plasma samples must be obtained from the
Cmin—Drug concentration at the end of a dosing
subject to ascertain that steady state has been reached
interval
and to describe the plasma level–time curve accu-
Cav—The average drug concentration during a ∞
rately. (3) Because Cav depends primarily on the dose
dosing interval
of the drug and the time interval between doses, the
Degree of fluctuation = (Cmax− Cmin)/Cmax
extent of drug systemically available is more impor-
Swing = (Cmax− Cmin)/Cmin
tant than the rate of drug availability. Small differ-
The data are analyzed statistically using analysis of ences in the rate of drug absorption may not be
variance (ANOVA) on the log-transformed AUC and observed with steady-state study comparisons
Cmax. To establish bioequivalence, both AUC and
Cmax for the test (generic) product should be within
80%–125% of the reference product using a 90% Clinical Endpoint Bioequivalence Study
confidence interval. Estimation of the absorption Study design for a clinical endpoint study generally
rate constant during multiple dosing is difficult, consists of a randomized, double-blind, placebo-
because the residual drug from the previous dose controlled, parallel-designed study comparing test
superimposes on the dose that follows. However, the product, reference product, and placebo product in
data obtained in multiple doses are useful in calcu- patients. A placebo arm is usually included to dem-
lating a steady-state plasma level. onstrate that the treatments are active (above the
The extent of bioavailability, measured by no-effect part of the effect versus dose curve, see
assuming the [AUC]∞0 , is dependent on clearance: Fig. 16-11) and the study is sufficiently sensitive to
identify the clinical effect in the patient population
FD0 enrolled in the study. In some cases, the use of a
[AUC]∞0 =
ClT placebo may not be included for safety reasons.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     495

The primary analysis for bioequivalence is deter- period is used between drug treatments. Therefore,
mined by evaluating the difference between the pro- the patient is maintained on his or her previous dose
portion of patients in the test and reference treatment of medication or an equal dose of the test product, and
groups who are considered a “therapeutic cure” at blood sampling is performed during a dosage interval
the end of study. The superiority of the test and refer- (Fig. 16-13, reference product A). Once blood sam-
ence products against the placebo is also tested using pling is accomplished, the patient takes equal oral
the same dichotomous endpoint of “therapeutic cure.” doses of the other drug product (test or reference) and
the previous drug product is discontinued. Drug dos-
ing with each drug product continues until attainment
Determination of Bioequivalence of Drug of steady state. When steady state is reached, the
Products in Patients Maintained on a plasma level–time curve for a dosage interval with the
Therapeutic Drug Regimen second drug product is described (Fig. 16-13, drug
A bioequivalence study may be performed in patients product B). Using the same plasma measures as
already maintained on the reference (brand-name) before, the bioequivalence or lack of bioequivalence
drug. Due to safety concerns, certain drugs such as may be determined. The patient then continues with
clozapine, a dibenzodiazepine derivative with potent his or her therapy with the original drug product.
antipsychotic properties, should not be given to nor- Products are given in random order: A then B, B
mal healthy subjects (US-FDA, CDER, 2011b). then A. Failure to do this might lead to a sequence
Instead, bioequivalence studies on clozapine should effect. The reference product that is tested is pro-
be performed in patients who have been stabilized on vided by the investigator from a known lot (not the
the highest strength (eg, 100 mg) using a multiple- patient’s own prescription).
dose bioequivalence study design. Patients on these Since the patients are being treated with the
or other drugs such as antipsychotics (US-FDA, reference (brand) product A, the drug concentrations
CDER, 2013a) or cancer chemotherapeutic drugs are at steady state prior to the start of the study and
(Kaur et al, 2013) would be at risk if a washout the accumulation phase is not observed. The test

60

Reference product A Test product B

50
Plasma drug concentration (ng/mL)

40

30

20

10

0
24 34 44 54 64 74
Time (hours)
FIGURE 16-13  Multiple-dose bioequivalence study in patients. Bioequivalence is determined by comparison of the steady-
state plasma drug-versus-time profile after administration of the reference drug product A to the steady-state plasma drug–time
profile after administration of the test drug product B.
496    
Chapter 16

drug product B is started and the reference drug each blood sample was analyzed for total and free
product A is stopped. The total plasma drug concen- thyroxine (T4), total and free triiodothyronine (T3),
trations are maintained. Bioequivalence is deter- the major metabolite of T4, and thyrotropin (TSH).
mined by comparison of the steady-state plasma
a. Why were hypothyroid patients used in this
drug-versus-time profile after administration of the
study?
reference drug product A to the steady-state plasma
b. Why were the subjects dosed for 50 days with
drug–time profile after administration of the test
each thyroid product?
drug product B.
c. Why were blood samples obtained on days 48,
If the blood level–time curve of the second drug
49, and 50?
product is bioequivalent, as shown by AUCt and
d. Why was T3 measured?
Cmax, to that of the reference drug product, the sec-
e. Why was TSH measured?
ond 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 Solution

the reference drug), the resulting Cav will be smaller
∞ a. Normal healthy euthyroid subjects would be at
than that obtained with the first drug. Cav is not actu-
risk if they were to take levothyroxine sodium
ally used as a direct measurement. Usually, the drug
for an extended period of time.
manufacturer will perform dissolution and content
b. The long (50-day) daily dosing for each prod-
uniformity tests before performing a bioequivalence
uct was required to obtain steady-state drug
study. These in vitro dissolution tests will help
∞ levels because of the long elimination half-life
ensure that the Cav obtained from each drug product
of levothyroxine.
in vivo will not be largely different from each other.
c. Serum from blood samples was taken on
In contrast, if the extent of drug availability is greater in
∞ days 48, 49, and 50 to obtain three consecutive
the second drug product, the Cav will be higher.
Cmin drug levels.
d. T3 is the active metabolite of T4.
CLINICAL EXAMPLE e. The serum TSH concentration is inversely
proportional to the free serum T4 concentrations
Levothyroxine Sodium Oral Tablets and gives an indication of the pharmacodynamic
A multiple dose relative bioavailability study8 of two activity of the active drug.
synthetic branded levothyroxine sodium oral tablets,
product A and product B, were evaluated in 20
euthyroid patients. The investigation was designed CLINICAL EXAMPLE
as a two-way crossover study in which the patients Mercaptopurine (Purinethol) Oral Tablets
who had been diagnosed as hypothyroid by their Mercaptopurine (Purinethol) is a cytotoxic drug used
primary-care physician were given a single 100-mg to treat cancer and is available in a 50-mg oral tablet.
daily dose of either product A or product B levothy- The FDA recommends bioequivalence steady-state
roxine sodium tablets for 50 days and then switched studies (US-FDA, CDER, 2011c) in patients receiving
over immediately to the other treatment for 50 days. therapeutic oral doses (usually 100–200 mg/d in the
Predose blood samples were taken on days 1, 25, 48, average adult) or maintenance daily doses (usually
49, and 50 of each phase, and, on day 50, a complete 50–100 mg/d in the average adult).
blood sampling was performed. The serum from Patients should be on a stable regimen using the
same dosage unit (multiples of the same 50-mg
8For the FDA-recommended bioequivalence study for strength). Plasma drug concentration–time profiles
levothyroxine sodium tablets, see FDA Guidance for Industry:
Levothyroxine Sodium Tablets—In Vivo Pharmacokinetic
are obtained in these patients at steady state with the
and Bioavailability Studies, and In Vitro Dissolution Testing, brand product. The proposed generic drug product is
December 2000. then given to these patients at the same dosage
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     497

regimen until steady state is reached. Plasma drug metrics for the test and reference drug products
concentration–time profiles are obtained for the (US-FDA, CDER, 2000a).
generic drug product; then the patients return to the Many statistical approaches (parametric tests)
original brand medication. assume that the data are distributed according to
a normal distribution or “bell-shaped curve” (see
Frequently Asked Questions Appendix A). The pharmacokinetic parameters such
»» What do sequence, washout period, and period as Cmax and AUC may not be normally distributed,
mean in a crossover bioavailability study? and the true distribution is difficult to ascertain
because of the small number of subjects used in a
»» Why does the FDA request a food intervention
bioequivalence study. The distribution of data that
(food-effect) study for new and generic drug products
before granting approval?
have been transformed to log values resembles more
closely a normal distribution compared to the distri-
»» What type of bioequivalence studies are requested bution of non-log-transformed data.
for drugs that are not systemically absorbed or for
those drugs in which the Cmax and AUC cannot be Two One-Sided Tests Procedure
measured in the plasma?
The two one-sided tests procedure is also referred to as
»» How do inter- and intrasubject variability affect the the confidence interval approach (Schuirmann, 1987).
statistical demonstration of bioequivalence for a This statistical method is used to demonstrate if the
drug product? 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
PHARMACOKINETIC EVALUATION if there are large differences (ie, greater than 20%)
between the mean parameters.
OF THE DATA The 90% confidence limits are estimated for the
For single-dose studies, including a fasting study or sample means. The interval estimate is based on
a food intervention study, the pharmacokinetic anal- Student’s t distribution of the data. In this test, pres-
yses include calculation for each subject of the area ently required by the FDA, a 90% confidence interval
under the curve to the last quantifiable concentration about the ratio of means of the two drug products
(AUCt0 ) and to infinity (AUC∞0 ), tmax, and Cmax. must be within ±20% for measurement of the rate and
Additionally, the elimination rate constant, k, the extent of drug bioavailability. For most drugs, up to a
elimination half-life, t1/2, and other parameters may 20% difference in AUC or Cmax between two formula-
be estimated. For multiple-dose studies, pharmaco- tions would have no clinical significance. The lower
kinetic analysis includes calculation for each subject 90% confidence interval for the ratio of means cannot
of the steady-state area under the curve, (AUCt∞ ), be less than 0.80, and the upper 90% confidence inter-
tmax, Cmin, Cmax, and the percent fluctuation [100 × val for the ratio of the means cannot be greater than
(Cmax − Cmin)/Cmin]. Proper statistical evaluation 1.20. When log-transformed data are used, the 90%
should be performed on the estimated pharmacoki- confidence interval is set at 80%–125%. These confi-
netic parameters. dence limits have also been termed the bioequivalence
interval (Midha et al, 1993). The 90% confidence
interval is a function of sample size and study vari-
Statistical Evaluation of the Data ability, including inter- and intrasubject variability.
Bioequivalence is generally determined using a com- For a single-dose, fasting or food intervention
parison of population averages of a bioequivalence bioequivalence study, an ANOVA is usually per-
metric, such as AUC and Cmax. This approach, termed formed on the log-transformed AUC and Cmax values.
average bioequivalence, involves the calculation of a There should be no statistical differences between the
90% confidence interval for the ratio of averages mean AUC and Cmax parameters for the test (generic)
(population geometric means) of the bioequivalence and reference drug products. In addition, the 90%
498    
Chapter 16

TABLE 16-9  Statistical Analysis for Average each pharmacokinetic parameter, such as AUC, may be
Bioequivalence masked, and the investigator might erroneously con-
• Based on log-transformed data
clude that the two drug products are bioequivalent.
• Point estimates of the mean ratios A statistical difference between the pharmacoki-
Test/reference for AUC and Cmax are between 80% and netic parameters obtained from two or more drug
125% products is considered statistically significant if there
• AUC and Cmax is a probability of less than 1 in 20 times or 0.05 prob-
90% confidence intervals (CI) must fit between 80%
and 125%
ability (p ≤ .05) that these results would have happened
• Bioequivalence criteria on the basis of chance alone. The probability, p, is
Two one-sided tests procedure used to indicate the level of statistical significance. If
• Test (T) is not significantly less than reference p < .05, the differences between the two drug products
• Reference (R) is not significantly less than test are not considered statistically significant.
• Significant difference is 20% (a = 0.05 significance
level)
To reduce the possibility of failing to detect
T/R = 80/100 = 80% small differences between the test products, a power
R/T = 80% (all data expressed as T/R, so this test is performed to calculate the probability that the
becomes 100/80 = 125%) conclusion of the ANOVA is valid. The power of
• The statistical model typically includes factors accounting the test will depend on the sample size, variability of
for the following sources of variation: sequence, subjects
nested in sequences, period, and treatment
the data, and desired level of significance. Usually, the
power is set at 0.80 with a b = 0.2 and a level of
From US-FDA, CDER (2000). significance of 0.05. The higher the power, the test is
more sensitive and the greater the probability that the
conclusion of the ANOVA is valid.
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 THE PARTIAL AUC IN
(125%) of that of the reference product based on log- BIOEQUIVALENCE ANALYSIS
transformed data. Table 16-9 summarizes the statisti-
Several new drug delivery systems have a complex
cal analysis for average bioequivalence. Presently,
approach to drug release (eg, combinations of zero-
the FDA accepts only average bioequivalence esti-
order and first-order release) that produces an unusu-
mates used to establish bioequivalence of generic
ally shaped plasma drug concentration-versus-time
drug products.
profile. The shape of this plasma drug concentration-
versus-time profile is related to the pharmacodynam-
Analysis of Variance ics of the drug.
An analysis of variance (see ANOVA) is a statistical To evaluate a generic dosage form of these new
procedure (see Appendix A) used to test the data for drug delivery systems, the FDA recommends includ-
differences within and between treatment and con- ing the partial AUC (pAUC) as a pivotal BE metric.
trol groups. A bioequivalent product should produce The pAUC is defined as the area under the plasma
no significant difference in all pharmacokinetic concentration-versus-time profile over two specified
parameters tested. The parameters tested statistically time points. The choice of sampling time points for
usually include AUCt0, AUC∞0 , and Cmax obtained calculating the pAUC is based on the pharmacokinetic/
for each treatment or dosage form. Other metrics of pharmacodynamic or efficacy/safety data for the drug
bioavailability have also been used to compare the under examination.
bioequivalence of two or more formulations. The The FDA currently expects the pAUC to be ana-
ANOVA may evaluate variability in subjects, treat- lyzed statistically when determining bioequivalence
ment groups, study period, formulation, and other of multiphasic modified-release (MR) formulations
variables, depending on the study design. If the vari- designed to achieve a rapid therapeutic response fol-
ability in the data is large, the difference in means for lowed by a sustained response. Such products are
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     499

12
Cmax

10

ng/mL
6

0
T Tmax t
0 10 20 30 40
Time (hours)
AUC0-T

FIGURE 16-14  Partial AUC analysis in a bioequivalence study. The partial AUC (pAUC) refers to the AUC between two speci-
fied, clinically relevant, time points on the drug plasma concentration-versus-time profile. The sampling time T should be selected
based on the pharmacokinetic and pharmacodynamic properties of the active ingredient.

generally formulated with both an immediate-release biphasic absorption characteristics, which result in
component and a delayed- or extended-release com- rapid initial absorption from the gastrointestinal tract
ponent. Figure 16-14 illustrates how a pAUC analy- similar to zolpidem tartrate immediate release, and
sis, based on two partial AUCs, is applied. The two then provide extended plasma concentrations beyond
partial AUCs consist of an early pAUC measure AUC0-T 3 hours of administration. As a result, patients receiv-
to compare test and reference exposure responsible for ing Ambien CR experience both rapid onset of sleep
early onset of response, and a late pAUC measure and maintenance of sleep. To ensure that a test zolpi-
AUCT-t to compare test and reference exposure respon- dem tartrate extended-release tablet provides the
sible for sustained response. The early AUC0-T is mea- same pharmacodynamic response (timing of sleep
sured beginning at sampling time 0 to a truncation onset and maintenance) when switched with the ref-
time T. The late AUCT-t is measured from the trunca- erence product, the FDA expects that, in a bioequiva-
tion time T to the last sampling point with measur- lence study comparing the two, the parameters
able drug concentration. These two metrics replace AUC0-1.5h, AUC1.5h-t, AUC0-∞, and Cmax will all pass
AUC0-t in bioequivalence evaluation. The bioequiva- bioequivalence limits of 80.00%–125.00% (US-FDA,
lence determination is based on comparison of test CDER, 2011d). The sampling time for the early and
and reference Cmax, AUC0-∞, AUC0-T , and AUCT-t. late pAUCs for the zolpidem extended-release tablet
The partial AUC (pAUC) refers to the AUC were selected based on zolpidem pharmacokinetic–
between two specified, clinically relevant, time points pharmacodynamic relationships.
on the drug plasma concentration-versus-time pro- The FDA recently posted a draft guidance for
file. The sampling time T should be selected based on industry recommending the application of three
the pharmacokinetic and pharmacodynamic proper- pAUC metrics, for bioequivalence studies of generic
ties of the active ingredient. versions of the methylphenidate multiphasic MR
tablet (US-FDA, CDER, 2014f). The reference listed
drug for this product is Concerta®, indicated for the
Examples of Partial AUC Analyses treatment of attention deficit hyperactivity disorder.
The first product to which this approach was applied The product is labeled to be administered once in the
was the zolpidem extended-release formulation. The morning, before the start of the school day, for pedi-
reference for this product, Ambien CR®, exhibits atric patients. The three pAUC metrics are proposed
500    
Chapter 16

to ensure that when patients for whom Concerta profiles following oral administration (US-FDA,
treatment is indicated switch formulations, they will CDER, 2013b). However, because the site of mesala-
experience equivalent therapeutic responses over the mine action is the colon and rectum, the FDA con-
course of the day. Thus, for an acceptable bioequiva- cluded that comparisons of AUC and Cmax alone in BE
lence study, the 90% confidence intervals of the studies would not distinguish between products with
geometric mean test/reference ratios Cmax, AUC0-T1, materially different mesalamine release profiles at the
AUCT1-T2, AUCT2-T3, and AUC∞ should fall within the sites of drug action (US-FDA, CDER, 2010c). Thus, the
limits of 80.00%–125.00%. The sampling time T1 for pAUC is used to analyze systemic mesalamine concen-
the first pAUC (AUC0-T1) is based on the time at trations over specified time intervals to determine
which 90%–95% of subjects are likely to achieve an whether mesalamine from test and reference products
early onset of response. The middle pAUC (AUCT1-T2) is available at the same rate and to the same extent at
comparison is to ensure similar drug exposures during the colon and rectum (Davit and Conner, 2015).
the remaining school hours (for pediatric patients)
after early onset of exposure. The late pAUC com-
parison (AUCT2-T3) is to ensure equivalent methyl-
BIOEQUIVALENCE EXAMPLES
phenidate exposures during the latter part of the A simulated example of the results for a single-dose,
dosing interval, corresponding to the duration of the fasting study is shown in Table 16-11 and in Fig. 16-15.
sustained response. As shown by the ANOVA, no statistical differences
The pAUC is also used as a BE metric in studies for the pharmacokinetic parameters, AUCt0 , AUC∞0 ,
comparing test and reference versions of mesalamine and Cmax, were observed between the test product and
orally administered MR formulations (Table 16-10). the brand-name product. The 90% confidence limits
Mesalamine is indicated to treat inflammatory dis- for the mean pharmacokinetic parameters of the test
eases of the colon and rectum, and is thought to act product were within 0.80–1.25 (80%–125%) of the
locally rather than systemically. Table 16-10 sum- reference product means based on log transforma-
marizes the mesalamine RLD oral MR formulations, tion of the data. The power test for the AUC mea-
associated indications, and Pauc metrics used in BE sures was above 99%, showing good precision of the
studies against each of these RLDs. Mesalamine is data. The power test for the Cmax values was 87.9%,
well absorbed, most likely throughout the small and showing that this parameter was more variable.
large intestines, with the result that it is possible to Table 16-12 shows the results for a hypothetical
measure plasma concentrations and determine PK bioavailability study in which three different tablet

TABLE 16-10  Bioequivalence Metrics for In Vivo Studies of Mesalamine Modified-Release Oral
Dosage Forms
Formulation Reference Bioequivalence Metrics

Mesalamine delayed-release capsule Delzicol® For both fasting and fed studies: Cmax, AUC8-48 h, AUC0-t

Mesalamine delayed-release tablet Asacol®

Mesalamine delayed-release tablet Asacol HD®

Mesalamine delayed-release tablet Lialda®

Mesalamine extended-release capsule Pentasa® For fasting study: Cmax, AUC0-3 h, AUC3 h-t, AUC0-t
For fed study: Cmax and AUC0-t are pivotal; AUC0-3 h and AUC0-t
are supportive

Mesalamine extended-release capsule Apriso®


Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     501

TABLE 16-11  Bioavailability Comparison of a Generic (Test) and Brand-Name (Reference) Drug
Products (Log-Normal Transformed Data)
p Values
90% Confidence for
Interval (Lower Limit, Product Power of ANOVA
Variable Units Geometric Mean % Ratio Upper Limit) Effects ANOVA %CV

    Test Reference          

Cmax ng/mL 344.79 356.81 96.6 (89.5, 112) 0.3586 0.8791 17.90%

ng · h/mL 2659.12 2674.92 99.4 (95.1, 104) 0.8172 1.0000 12.60%

AUC∞ 2708.63 2718.52 99.6 (95.4, 103) 0.8865 1.0000 12.20%

tmax h 4.29 4.24 101

Kelim 1/h 0.0961 0.0980 98.1

t1/2 h 8.47 8.33 101.7

The results were obtained from a two-way, crossover, single-dose study in 36 fasted, healthy, adult male and female volunteers. No statistical differ-
ences were observed for the mean values between test and reference products.

formulations were compared to a solution of the confidence interval for the AUC showed that for
drug given in the same dose. As shown in the table, tablet A, the bioavailability was less than 80% (ie,
the bioavailability from all three tablet formulations 74%), compared to the solution at the low-range
was greater than 80% of that of the solution. estimate, and would not be considered bioequivalent
According to the ANOVA, the mean AUC values based on the AUC.
were not statistically different from one another, nor For illustrative purposes, consider a drug that has
different from that of the solution. However, the 90% been prepared at the same dosage level in three

350

300
Plasma drug concentration (ng/mL)

250

200

150

100

50

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Time (hours)
A: Test B: Reference
FIGURE 16-15  Bioequivalence of test and reference drug products: mean plasma drug concentrations.
502    Chapter 16

TABLE 16-12  Summary of the Results of a Bioavailability Studya


90% Confidence
Dosage Form Cmax (lg/mL) tmax (h) AUC0–24 (lg h/mL) Fb Interval for AUC

Solution 16.1 ± 2.5 1.5 ± 0.85 1835 ± 235

Tablet A 10.5 ± 3.2c 2.5 ± 1.0c 1523 ± 381 81 74%–90%

Tablet B 13.7 ± 4.1 2.1 ± 0.98 1707 ± 317 93 88%–98%

Tablet C 14.8 ± 3.6 1.8 ± 0.95 1762 ± 295 96 91%–103%


aThe bioavailability of a drug from four different formulations was studied in 24 healthy, adult male subjects using a four-way Latin-square crossover
design. The results represent the mean ± standard deviation.
bOral bioavailability relative to the solution.
cp ≤ .05.

formulations, A, B, and C. These formulations are rapid than that from formulation B, because the tmax for
given to a group of volunteers using a three-way, ran- formulation A is shorter. Because the AUC for formu-
domized crossover design. In this experimental design, lation A is identical to the AUC for formulation B, the
all subjects receive each formulation once. From each extent of bioavailability from both of these formula-
subject, plasma drug level and urinary drug excretion tions is the same. Note, however, the Cmax for A is
data are obtained. With these data we can observe the higher than that for B, because the rate of drug absorp-
relationship between plasma and urinary excretion tion is more rapid.
parameters and drug bioavailability (Fig. 16-16). The The Cmax is generally higher when the extent of
rate of drug absorption from formulation A is more drug bioavailability is greater. The rate of drug absorp-
tion from formulation C is the same as that from formu-
lation A, but the extent of drug available is less. The
A Cmax for formulation C is less than that for formula-
tion A. The decrease in Cmax for formulation C is
AUCA = AUCB
proportional to the decrease in AUC in comparison
AUCC = 0.5 AUCA
to the drug plasma level data for formulation A. The
Plasma level

A B corresponding urinary excretion data confirm these


C observations. These relationships are summarized in
Table 16-13. The table illustrates how bioavailability
parameters for plasma and urine change when only the
Time extent and rate of bioavailability are changed, respec-
B
tively. Formulation changes in a drug product may
affect both the rate and extent of drug bioavailability.
Cumulative amount of drug in urine

A
STUDY SUBMISSION AND DRUG
B REVIEW PROCESS
C The contents of New Drug Applications (NDAs)
and Abbreviated New Drug Applications (ANDAs)
are similar in terms of the quality of manufacture
(Table 16-14). The submission for an NDA must
Time contain safety and efficacy studies as provided by
FIGURE 16-16  Corresponding plots relating plasma animal toxicology studies, clinical efficacy studies,
concentration and urinary excretion data. and pharmacokinetic/bioavailability studies. For the
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     503

TABLE 16-13  Relationship of Plasma Level and Urinary Excretion Parameters to Drug Bioavailability
Extent of Drug Bioavailability Decreases Rate of Drug Bioavailability Decreases

Parameter Change Parameter Change

Plasma data
tmax Same tmax Increase
Cmax Decrease Cmax Decrease
AUC Decrease AUC Same
Urine data
t∞ Same t∞ Increase

[ dDu /dt ]max a Decrease [ dDu /dt ]max a Decrease

Du∞ Decrease Du∞ Same

aMaximum rate of urinary drug excretion.

generic drug manufacturer, the bioequivalence study These results, along with case reports and various
is the pivotal study in the ANDA that replaces the data supporting the validity of the analytical method,
animal, clinical, and pharmacokinetic studies. are included in the submission. The FDA reviews the
An outline for the submission of a completed study in detail according to the outline presented in
bioavailability to the FDA is shown in Table 16-15. Table 16-16. If necessary, an FDA investigator may
The investigator should be sure that the study has inspect both the clinical and analytical facilities used
been properly designed, the objectives are clearly in the study and audit the raw data used in support of
defined, and the method of analysis has been vali- the bioavailability study. For ANDA applications,
dated (ie, shown to measure precisely and accurately the FDA Office of Generic Drugs reviews the entire
the plasma drug concentration). The results are ana- ANDA as shown in Fig. 16-17. If the application is
lyzed both statistically and pharmacokinetically. incomplete, the FDA will not review the submission
and the sponsor will receive a Refusal to File letter.
TABLE 16-14  NDA Versus ANDA Review
Process Frequently Asked Questions
»» What is the most appropriate bioequivalence design
Brand-Name Drug NDA Generic Drug ANDA
for a solid oral drug product containing a drug for
Requirements Requirements
systemic absorption?
1. Chemistry 1. Chemistry
»» What are some of the problems associated with
2. Manufacturing 2. Manufacturing clinical endpoint bioequivalence studies?
3. Controls 3. Controls

4. Labeling 4. Labeling
WAIVERS OF IN VIVO
5. Testing 5. Testing
BIOEQUIVALENCE STUDIES
6. Animal studies 6. Bioequivalence
(BIOWAIVERS)
7. Clinical studies
In some cases, in vitro dissolution testing may be used
8. Bioavailability in lieu of in vivo bioequivalence studies. When the drug
Source: Center for Drug Evaluation & Research, US Food & Drug product is in the same dosage form but in different
Administration, http://www.fda.gov. strengths and is proportionally similar in active and
504    Chapter 16

TABLE 16-15  Proposed Format and Contents of an In Vivo Bioequivalence Study Submission and
Accompanying In Vitro Data
Title page V.  Pharmacokinetic Parameters and Tests
Study title Definition and calculations
Name of sponsor Statistical tests
Name and address of clinical laboratory Drug levels at each sampling time and pharmacokinetic
Name of principal investigator(s) parameters
Name of clinical investigator Figure of mean plasma concentration–time profile
Name of analytical laboratory Figures of individual subject plasma concentration–time
Dates of clinical study (start, completion) profiles
Signature of principal investigator (and date) Figure of mean cumulative urinary excretion
Signature of clinical investigator (and date) Figures of individual subject cumulative urinary
excretion
Table of contents Figure of mean urinary excretion rates
I.  Study Résumé Fgures of individual subject urinary excretion rates
Product information Tables of individual subject data arranged by drug,
Summary of bioequivalence study drug/period, drug/sequence
Summary of bioequivalence data
Plasma VI.  Statistical Analyses
Urinary excretion Statistical considerations
Figure of mean plasma concentration–time profile Summary of statistical significance
Figure of mean cumulative urinary excretion Summary of statistical parameters
Figure of mean urinary excretion rates Analysis of variance, least squares estimates, and least
squares means
II.  Protocol and Approvals Asessment of sequence, period, and treatment effects
Protocol 90% confidence intervals for the difference between
Letter of acceptance of protocol from FDA test and reference products for the log-normal trans-
Informed consent form formed parameters of AUC0–t, AUC0–∞, and Cmax should
Letter of approval of Institutional Review Board be within 80% and 125%
List of members of Institutional Review Board
VII. Appendices
III.  Clinical Study Randomization schedule
Summary of the study Sample identification codes
Details of the study Analytical raw data
Demographic characteristics of the subjects Chromatograms of at least 20% of subjects
Subject assignment in the study Medical record and clinical reports
Mean physical characteristics of subjects arranged by Clinical facilities description
sequence Analytical facilities description
Details of clinical activity Curricula vitae of the investigators
Deviations from protocol
Vital signs of subjects VIII.  In Vitro Testing
Adverse reactions report Dissolution testing
Dissolution assay methodology
IV.  Assay Methodology and Validation Content uniformity testing
Assay method description Potency determination
Validation procedure
Summary of validation IX.  Batch Size and Formulation
Data on linearity of standard samples Batch record
Data on interday precision and accuracy Quantitative formulation
Data on intraday precision and accuracy
Figure for standard curve(s) for low/high ranges
Chromatograms of standard and quality control
samples
Sample calculation

Modified from Dighe and Adams (1991), with permission.


Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     505

TABLE 16-16  General Elements of a study on the highest strength. Ideally, if there is a strong
Biopharmaceutics Review correlation between dissolution of the drug and the
Introduction Summary and
bioavailability of the drug, then the comparative disso-
analysis of data lution tests comparing the test product to the reference
product should be sufficient to demonstrate bioequiva-
Study design Comments
lence. For most drug products, especially immediate-
Study objective(s) Deficiencies release tablets and capsules, no strong correlation
Assay description and validation Recommendation exists, and the FDA requires an in vivo bioequivalence
study. For oral solid dosage forms, an in vivo bioequiv-
alence study may be required to support at least one
inactive ingredients, an in vivo bioequivalence study of dose strength of the product. Usually, an in vivo bio-
one or more of the lower strengths can be waived based equivalence study is required for the highest dose
on the dissolution tests and an in vivo bioequivalence strength. If the lower-dose-strength test product is

Applicant

ANDA

No Refuse to File Letter


Acceptable and
Complete? Issued

Yes

Review by OGD/CDER
Chemistry/Micro
Bioequivalence Review
Review

Request for Plant


Labeling Review
Inspection

Bioequivalence Yes Chemistry/Micro/Label-


Review Acceptable? ing Review
No Acceptable? No

Bioequivalence Not Approvable


Defficiency Letter Letter
Preapproval
Inspection
Acceptable? No Approval Deferred
Pending
Satisfactory Results
Yes

ANDA APPROVED

FIGURE 16-17  Generic drug review process. (Source: Office of Generic Drugs, Center for Drug Evaluation & Research, US Food
& Drug Administration.)
506    
Chapter 16

substantially similar in active and inactive ingredients, • Acceptable in vitro dissolution should be demon-
then only a comparative in vitro dissolution between strated for the strength(s) for which the biowaiver
the test and brand-name formulations may be used. is sought.
For example, an immediate-release (IR) tablet is
The FDA does not grant biowaivers for generic
available in 200-mg, 100-mg, and 50-mg strengths.
modified-release products, but may deem non-
The 100- and 50-mg-strength tablets are made the
biostudy strength(s) BE to the corresponding biostudy
same way as the highest-strength tablet. A human
strength(s) subject to certain criteria. This policy
bioequivalence study is performed on the highest or
applies to all MR dosage forms, including but not
200-mg strength. Comparative in vitro dissolution
limited to delayed-release tablets and capsules,
studies are performed on the 100-mg and 50-mg
extended-release tablets, transdermal products, and
dose strengths. If these drug products have no known
long-acting injectables (Davit et al, 2013).
bioavailability problems, are well absorbed systemi-
cally, are well correlated with in vitro dissolution,
and have a large margin of safety, then arguments for Dissolution Profile Comparisons
not performing an in vivo bioavailability study may Comparative dissolution profiles are used as (1) the
be valid. Methods for correlation of in vitro dissolu- basis for formulation development of bioequivalent
tion of the drug with in vivo drug bioavailability are drug products and proceeding to the pivotal in vivo
discussed in Chapters 15 and 19. The manufacturer bioequivalence study (Chapter 15); (2) comparative
does not need to perform additional in vivo bio- dissolution profiles are used for demonstrating the
equivalence studies on the lower-strength products if equivalence of a change in the formulation of a drug
the products meet all in vitro criteria. product after the drug product has been approved for
marketing (see SUPAC in Chapter 17); and (3) the
Regulatory Perspective for Biowaiver basis of a biowaiver of a lower-strength drug product
The FDA permits the waiving of BE studies for that is dose proportional in active and inactive ingre-
products for which BE is self-evident. This includes dients to the higher-strength drug product.
solutions for parenteral, oral, or local use. There are A model-independent mathematical method
generally additional criteria to be met before a bio- was developed by Moore and Flanner (1996) to com-
waiver can be granted. Test and reference solutions pare dissolution profiles using two factors, f1 and f2.
intended for parenteral use should have the same The factor f2, known as the similarity factor, mea-
active and inactive ingredients in the same amounts. sures the closeness between the two profiles:
The FDA generally refers to this as qualitative (Q1) − 0.5
and quantitative (Q2) sameness. Generic drug prod-  1 n  
f2 = 50 × log 1 + ∑ ( R1 − T1 ) 
2
× 100
uct solutions that are intended for oral or topical use  n t =1  
can have different excipients than their correspond-
ing RLD products, but should not contain excipients
where n is the number of time points, R1 is the dissolu-
that could potentially cause differences in drug sub-
tion value of the reference product at time t, and T1 is
stance absorption.
the dissolution value of the test product batch at time t.
The FDA will consider granting biowaivers to
The reference may be the original drug product
non-biostudy strengths of a generic IR solid oral
before a formulation change (prechange) and the test
dosage form drug product line, provided that the fol-
may be the drug product after the formulation was
lowing three criteria are met:
changed (postchange). Alternatively, the reference
• An acceptable BE study is conducted on at least may be the higher-strength drug product and the test
one strength. may be the lower-strength drug product. The f2 com-
• The strength(s) for which the biowaiver is sought parison is the focus of several FDA guidances and is
should be proportionally similar to the strength on of regulatory interest in knowing the similarity of the
which BE was demonstrated. two dissolution curves. When the two profiles are
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     507

identical, f2 = 100. An average difference of 10% at According to the BCS, drug substances are clas-
all measured time points results in an f2 value of 50 sified as follows:
(Shah et al, 1998). The FDA has set a public stan-
• Class 1: high solubility–high permeability
dard for f2 value between 50 and 100 to indicate
• Class 2: low solubility–high permeability
similarity between two dissolution profiles (US-FDA,
• Class 3: high solubility–low permeability
CDER, 1997).
• Class 4: low solubility–low permeability
In some cases, two generic drug products may
have dissimilar dissolution profiles and still be bio- A theoretical basis for correlating in vitro drug
equivalent in vivo. For example, Polli et al (1997) dissolution with in vivo bioavailability was devel-
have shown that slow-, medium-, and fast-dissolving oped by Amidon et al (1995). This approach is based
formulations of metoprolol tartrate tablets were bio- on the aqueous solubility of the drug and the perme-
equivalent. Furthermore, bioequivalent modified- ation of the drug through the gastrointestinal tract.
release drug products may have different drug The classification system is based on Fick’s first law
release mechanisms and therefore different dissolu- applied to a membrane:
tion profiles. For example, for theophylline extended-
release capsules, the United States Pharmacopeia Jw = PwCw
(USP) lists 10 individual drug release tests for prod-
ucts labeled for dosing every 12 hours. However, where Jw is the drug flux (mass/area/time) through
only generic drug products that are FDA approved as the intestinal wall at any position and time, Pw is the
bioequivalent drug products and listed in the current permeability of the membrane, and Cw is the drug
edition of the Orange Book may be substituted for concentration at the intestinal membrane surface.
each other. This approach assumes that no other compo-
nents in the formulation affect the membrane perme-
ability and/or intestinal transport. Using this approach,
Frequently Asked Questions Amidon et al (1995) studied the solubility and per-
»» Why are preclinical animal toxicology studies and
meability characteristics of various representative
clinical efficacy drug studies in human subjects drugs and obtained a biopharmaceutic drug classifi-
not required by the FDA to approve a generic cation for predicting the in vitro drug dissolution of
drug product as a therapeutic equivalent to the IR solid oral drug products with in vivo absorption.
brand-name drug product? The FDA may waive the requirement for per-
forming an in vivo bioavailability or bioequivalence
»» Are bioequivalence studies needed for each dose
strength of an oral drug product? For example,
study for certain IR solid oral drug products that
an oral drug product is commercially available in meet very specific criteria, namely, the permeability,
200-mg, 100-mg, and 50-mg dose strengths. solubility, and dissolution of the drug. These charac-
teristics include the in vitro dissolution of the drug
product in various media, drug permeability infor-
mation, and assuming ideal behavior of the drug
THE BIOPHARMACEUTICS product, drug dissolution, and absorption in the GI
CLASSIFICATION SYSTEM (BCS) tract. For regulatory purposes, drugs are classified
according to the BCS in accordance with the solubility,
The BCS is a scientific framework for classifying
permeability, and dissolution characteristics of the
drug substances based on their aqueous solubility
drug (US-FDA, CDER, 2000b).
and intestinal permeability. When combined with the
dissolution of the drug product, the BCS takes into
account three major factors that govern the rate and Solubility
extent of drug absorption from IR solid oral dosage An objective of the BCS approach is to determine the
forms. These factors are dissolution, solubility, and equilibrium solubility of a drug under approximate
intestinal permeability. physiologic conditions. For this purpose, determination
508    Chapter 16

of pH–solubility profiles over a pH range of 1–8 is drug product is considered rapidly dissolving when not
suggested. The solubility class is determined by cal- less than 85% of the label amount of drug substance
culating what volume of an aqueous medium is suffi- dissolves within 30 minutes using USP Apparatus I
cient to dissolve the highest anticipated dose strength. (see Chapter 14) at 100 rpm or Apparatus II at 50 rpm
A drug substance is considered highly soluble when in a volume of 900 mL or less in each of the follow-
the highest dose strength is soluble in 250 mL or less of ing media: (1) acidic media such as 0.1 N HCl or
aqueous medium over the pH range 1–8. The volume simulated gastric fluid USP without enzymes, (2) a
estimate of 250 mL is derived from typical bioequiv- pH 4.5 buffer, and (3) a pH 6.8 buffer or simulated
alence study protocols that prescribe administration intestinal fluid USP without enzymes.
of a drug product to fasting human volunteers with a The FDA is in the process of revising the BCS
glass (8 oz) of water. guidance to permit biowaivers for generic formula-
tions of Class 3 drugs (Mehta, 2014). Table 16-17
Permeability summarizes the recently proposed FDA criteria to be
met for BCS biowaivers.
Studies of the extent of absorption in humans, or
intestinal permeability methods, can be used to deter-
mine the permeability class membership of a drug. Biopharmaceutics Drug Disposition
To be classified as highly permeable, a test drug Classification System
should have an extent of absorption >90% in humans.
The major aspects of BCS are the consideration of
Supportive information on permeability characteris-
solubility and permeation. According to BCS, perme-
tics of the drug substance should also be derived from
ability in vivo is considered high when the active drug
its physical–chemical properties (eg, octanol: water
is systemically absorbed ≥90%. Wu and Benet (2005)
partition coefficient).
and Benet et al (2008) have proposed modification of
Some methods to determine the permeability of a
the BCS system known as the Biopharmaceutics
drug from the gastrointestinal tract include (1) in vivo
Drug Disposition Classification System (BDDCS),
intestinal perfusion studies in humans; (2) in vivo
which takes into account drug metabolism (hepatic
or in situ intestinal perfusion studies in animals;
clearance) and transporters in the gastrointestinal
(3) in vitro permeation experiments using excised
tract for drugs that are orally administered. For BCS
human or animal intestinal tissues; and (4) in vitro
1 drugs (ie, high solubility and high permeability),
permeation experiments across a monolayer of cul-
transporter effects will be minimal. However, BCS 2
tured human intestinal cells. When using these meth-
drugs (low solubility and high permeability), trans-
ods, the experimental permeability data should
porter effects are more important. These investigators
correlate with the known extent-of-absorption data in
suggest that the BCS should be modified on the basis
humans.
of the extent of drug metabolism, overall drug dispo-
After oral drug administration, in vivo permea-
sition, including routes of drug elimination and the
bility can be affected by the effects of efflux and
effects of efflux, and absorptive transporters on oral
absorptive transporters in the gastrointestinal tract,
drug absorption.
by food, and possibly by the various excipients pres-
ent in the formulation.
Drug Products for Which Bioavailability or
Bioequivalence May Be Self-Evident
Dissolution The best measure of a drug product’s performance
The dissolution class is based on the in vitro dissolu- is to determine the in vivo bioavailability of the
tion rate of an IR drug product under specified test drug. For some well-characterized drug products
conditions and is intended to indicate rapid in vivo and for certain drug products in which bioavail-
dissolution in relation to the average rate of gastric ability is self-evident (eg, sterile solutions for
emptying in humans under fasting conditions. An IR injection), in vivo bioavailability studies may be
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     509

TABLE 16-17  Criteria Proposed by FDA for Consideration of BCS-Based Biowaivers of Immediate-
Release Generic Drug Products
BCS Class 1

Highly Soluble Oral Bioavailability Dissolution Criteria on Excipients

Highest strength, over ≥85% • ≥ 85% in 30 minutes at pH 1.0, • T est and reference should be
range of pH 1.0–6.8 4.5, 6.8 (“rapidly dissolving”) pharmaceutical equivalents
• Volume = 500 mL • Test and reference should not
• Paddles at 50 rpm, or basket at differ in amounts of excipients
100 rpm known to affect bioavailability

BCS Class 3

Highly Soluble Oral Bioavailability Dissolution Criteria on Excipients

Highest strength, over <85% • ≥ 85% in 15 minutes at pH 1.0, • T est and reference should be
range of pH 1.0–6.8 4.5, 6.8 (“very rapidly dissolving) pharmaceutical equivalents
• Volume = 500 mL • Test and reference formulations
• Paddles at 50 rpm, or basket at should be Q1 and Q2 the same
100 rpm

unnecessary or unimportant to the achievement of 3. The drug product is in an oral dosage form that
the product’s intended purposes. The FDA will is not intended to be absorbed (eg, an antacid
waive the requirement for submission of in vivo or a radiopaque medium). Specific in vitro
evidence demonstrating the bioavailability of the bioequivalence studies may be required by
drug product if the product meets one of the follow- the FDA. For example, the bioequivalence of
ing criteria (US-FDA, CDER, 2014a). However, cholestyramine resin is demonstrated in vitro
there may be specific requirements for certain drug by the binding of bile acids to the resin.
products, and the appropriate FDA division should 4. The drug product meets both of the following
be consulted. conditions:
a. It is administered by inhalation as a gas or
1. The drug product (a) is a solution intended vapor (eg, as a medicinal or as an inhalation
solely for intravenous administration and anesthetic).
(b) contains an active drug ingredient or b. It contains an active drug ingredient or
therapeutic moiety combined with the same therapeutic moiety in the same dosage form
solvent and in the same concentration as in an as a drug product that is the subject of an
intravenous solution that is the subject of an approved, full NDA.
approved, full NDA. 5. The drug product meets all of the following
2. The drug product is a topically applied prepara- conditions:
tion (eg, a cream, ointment, or gel intended for a. It is an oral solution, elixir, syrup, tincture,
local therapeutic effect). The FDA has released or similar other solubilized form.
guidances for the performance of bioequiva- b. It contains an active drug ingredient or
lence studies on topical corticosteroids and therapeutic moiety in the same concentration
antifungal agents. The FDA is also considering as a drug product that is the subject of an
performing dermatopharmacokinetic (DPK) approved, full NDA.
studies on other topical drug products. In addi- c. It contains no inactive ingredient that is
tion, in vitro drug release and diffusion studies known to significantly affect absorption of the
may be required. active drug ingredient or therapeutic moiety.
510    Chapter 16

GENERIC BIOLOGICS product and the reference product should consider


all relevant characteristics of the protein product
(BIOSIMILAR DRUG PRODUCTS) (eg, the primary, secondary, tertiary, and quater-
Biologics, or biotechnology-derived drugs, in contrast nary structure, post-translational modifications,
to drugs that are chemically synthesized, are derived and functional activity[ies]). The objective of this
from living sources such as humans, animals, or assessment is to maximize the potential for detect-
microorganisms. Many biologics are complex mix- ing differences in quality attributes between the
tures that are not easily identified or characterized and proposed biosimilar product and the reference
are manufactured using biotechnology or are purified product.
from natural sources. Other biological drugs, such as • Functional activities: Functional assays serve mul-
insulin and growth hormone, are proteins derived by tiple purposes in the characterization of protein
biotechnology and have been well characterized. products. These tests act to complement physico-
Advances in analytical sciences (both physicochemi- chemical analyses and are a quality measure of the
cal and biological) enable some protein products to be function of the protein product.
characterized extensively in terms of their physico- • Receptor binding and immunochemical proper-
chemical and biological properties. These analytical ties: When binding or immunochemical proper-
procedures have improved the ability to identify and ties are part of the activity attributed to the protein
characterize not only the desired product but also product, analytical tests should be performed to
product-related substances and product- and process- characterize the product in terms of these specific
related impurities. Advances in manufacturing science properties.
and production methods may enhance the likelihood • Impurities: The applicant should characterize,
that a product will be highly similar to another prod- identify, and quantify impurities (product and pro-
uct by better targeting the original product’s physio- cess related) in the proposed biosimilar product
chemical and functional properties. and the reference product.
The assessment of biosimilarity between a pro- • Reference product and reference standards: A
posed biosimilar product and its reference product thorough physicochemical and biological assess-
involves the robust characterization of the proposed ment of the reference product should provide a
biosimilar product, including comparative physico- base of information from which to develop the
chemical and functional studies. The FDA recom- proposed biosimilar product and justify reliance
mends the following factors that must be considered on certain existing scientific knowledge about the
in assessing whether products are highly similar reference product.
(US-FDA, CDER, 2014g). • Finished drug product: Product characterization
studies should be performed on the most down-
• Expression system: Therapeutic protein products
stream intermediate best suited for the analytical
can be produced by microbial cells (prokaryotic,
procedures used.
eukaryotic), cell lines of human or animal origin
• Stability: An appropriate physicochemical and
(eg, mammalian, avian, insect), or tissues derived
functional comparison of the stability of the pro-
from animals or plants. It is expected that the ex-
posed biosimilar product with that of the reference
pression construct for a proposed biosimilar prod-
product should be initiated including accelerated
uct will encode the same primary amino acid se-
and stress stability studies, or forced degradation
quence as its reference product.
studies.
• Manufacturing process: A comprehensive under-
standing of all steps in the manufacturing process The foundation for an assessment of biosimilarity
for the proposed biosimilar product should be es- between a proposed biosimilar product and its refer-
tablished during product development. ence product involves the robust characterization of
• Assessment of physicochemical properties: Physi- the proposed biosimilar product, including compara-
cochemical assessment of the proposed biosimilar tive physicochemical and functional studies.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     511

Biosimilarity Versus Interchangeability Biosimilars and interchangeable biotechnology-


The Patient Protection and Affordable Care Act of derived drugs will be considered on a case-by-case
2010 contains provisions that establish an abbrevi- basis. After FDA approval, the manufacturer must
ated regulatory approval pathway for generic ver- provide robust postmarketing safety monitoring as
sions of biological medicines (ie, biosimilars). The an important component in ensuring the safety and
new legislation establishes two distinct categories of effectiveness of biological products,
biosimilar products: (1) biological products that are
“biosimilar” to a reference biological product, and FDA Guidance Documents
(2) biological products that are “interchangeable” The legislation makes clear that the FDA will play a
with the reference product. central role in defining the specific criteria needed to
Biosimilar biological drug products are biologi- demonstrate biosimilarity for a given class of bio-
cal products that are highly similar to the reference logical. In deference to the FDA’s expertise in this
product notwithstanding minor differences in clini- area, the legislation specifically states that the FDA
cally inactive components. In addition, there are no can issue guidance documents with respect to the
clinically meaningful differences between the bio- approval of a biosimilar product. The guidance can
logical product and the reference product in terms of be general or specific in nature, and the public must
the safety, purity, and potency of the product. be provided with an opportunity to comment.
Interchangeable biological drug products are Advocates for the manufacture of generic bio-
biological products that are interchangeable with a logics argue that bioequivalent biotechnology-derived
reference biological product if (1) it meets the cri- drug products can be made on a case-by-case basis.
teria for being biosimilar to the reference product, Those opposed to the development of generic biolog-
(2) it can be expected to produce the same clinical ics or biosimilar drug products have claimed that
result as the reference product in any given patient, generic manufacturers do not have the ability to fully
and (3) the risk in terms of safety or diminished characterize the active ingredient(s), that immuno-
efficacy in alternating or switching between use of genicity-related impurities may be present in the
the biological and reference product is not greater product, and that the manufacture of a biologic drug
than the risk of using the reference product without product is process dependent. Several biosimilar drug
such alteration or switch. products have been approved in Europe. Currently,
FDA determination of biosimilar drug products there are several applications for biosimilar drug
is based on the totality of the evidence provided by a products under review by the FDA. In the United
sponsor to support a demonstration of biosimilarity. States, FDA regulatory approval is based on a step-
The FDA recommends that sponsors use a stepwise wise approach that includes a comparison of the
approach in their development of biosimilar prod- proposed product and the reference product with
ucts. FDA regulatory approval of a biosimilar drug respect to structure, function, animal toxicity, human
product is based on a stepwise approach includes a pharmacokinetics (PK) and pharmacodynamics (PD),
comparison of the proposed product and the refer- clinical immunogenicity, and clinical safety and
ence product including: effectiveness.
• Analytical studies that demonstrate that the bio-
logical product is highly similar to the reference
product notwithstanding minor differences in clin- CLINICAL SIGNIFICANCE OF
ically inactive components
BIOEQUIVALENCE STUDIES
• Animal studies (including the assessment of toxicity)
• Clinical study or studies (including the assessment Bioequivalence of different formulations of the same
of immunogenicity and pharmacokinetics or phar- drug substance involves equivalence with respect to
macodynamics) that are sufficient to demonstrate the rate and extent of systemic drug absorption.
safety, purity, and potency Clinical interpretation is important in evaluating the
512    Chapter 16

EXAMPLE • • •
results of a bioequivalence study. A small difference
between drug products, even if statistically signifi-
cant, may produce very little difference in therapeutic
IMPACT OF EFFLUX TRANSPORTERS ON
response. Generally, two formulations whose rate and
BIOEQUIVALENCE STUDY
extent of absorption differ by 20% or less are consid-
Digoxin is a drug that may be absorbed differently
ered bioequivalent. The Report by the Bioequivalence
in individuals that expressed the efflux gene MDR1.
Task Force (1988) considered that differences of less
than 20% in AUC and Cmax between drug products are Questions
“unlikely to be clinically significant in patients.” The • What would be the impact of such an individual
Task Force further stated that “clinical studies of recruited into a bioavailability study?
effectiveness have difficulty detecting differences in • Would a protocol with the usual crossover design
doses of even 50%–100%.” Therefore, normal varia- be able to adequately evaluate the bioequiva-
tion is observed in medical practice and plasma drug lence of a generic digoxin product with a refer-
levels may vary among individuals greater than 20%. ence? Explain why or why not.
According to Westlake (1973), a small, statisti- Solution
cally significant difference in drug bioavailability Bioequivalence studies for generic drug prod-
from two or more dosage forms may be detected if ucts compare the bioavailability of the drug from
the study is well controlled and the number of sub- the test (generic) product to the bioavailability of
jects is sufficiently large. When the therapeutic the drug from the reference (brand) product. The
objectives of the drug are considered, an equivalent study design is a two-way, crossover design in
clinical response should be obtained from the com- which each subject takes each drug product. The
parison dosage forms if the plasma drug concentra- study design usually includes males and females
tions remain above the minimum effective with different ethnic backgrounds. In addition,
concentration (MEC) for an appropriate interval and some studies include both smokers and nonsmok-
do not reach the minimum toxic concentration ers. Although there may be large intersubject vari-
(MTC). Therefore, the investigator must consider ability due to gender, environmental, and genetic
whether any statistical difference in bioavailability factors, the crossover design minimizes intrasu-
would alter clinical efficiency. bject variability by comparing the bioavailability of
Special populations, such as the elderly or test and reference products in the same individual.
patients on drug therapy, are generally not used for Thus each individual subject should have similar
bioequivalence studies. Normal, healthy volunteers drug absorption characteristics after taking the
are preferred for bioequivalence studies, because test or reference drug products.9
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 SPECIAL CONCERNS IN
dosage form, and use of healthy subjects should BIOAVAILABILITY AND
minimize both inter- and intrasubject variability. It is
theoretically possible that the excipients in one of BIOEQUIVALENCE STUDIES
the dosage forms tested may pose a problem in a The general bioequivalence study designs and
patient who uses the generic dosage form. evaluation, such as the comparison of AUC, Cmax,
For the manufacture of a dosage form, specifica- and tmax, may be used for systemically absorbed
tions are set to provide uniformity of dosage forms.
With proper specifications, quality control proce- 9For a few drug products, a high intrasubject variability (>30%
dures should minimize product-to-product variability CV) may be observed for which the bioavailability response
by different manufacturers and lot-to-lot variability changes for the same drug product each time the drug is dosed in
with a single manufacturer (see Chapter 18). the same subject.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     513

TABLE 16-18  Issues in Establishing in analysis of variance coefficient of variation (Shah


Bioavailability and Bioequivalence et al, 1996). The number of subjects required to
Drugs with high intrasubject variability
demonstrate bioequivalence for these drug products
may be excessive, requiring more than 60 subjects
Drugs with long elimination half-life to meet current FDA bioequivalence criteria. The
Biotransformation of drugs intrasubject variability may be due to the drug itself
  Stereoselective drug metabolism
or to the drug formulation or to both. The FDA has
held public forums to determine whether the cur-
  Drugs with active metabolites rent bioequivalence guidelines need to be changed
  Drugs with polymorphic metabolism for these highly variable drugs (Davit et al, 2012).
Nonbioavailable drugs (drugs intended for local effect)
For drugs with very long elimination half-lives
or a complex elimination phase, a complete plasma
 Antacids drug concentration–time curve (ie, three elimination
  Local anesthetics half-lives or an AUC representing 90% of the total
 Anti-infectives
AUC) may be difficult to obtain for a bioequivalence
study using a crossover design. For these drugs, a
  Anti-inflammatory steroids truncated (shortened) plasma drug concentration–
Dosage forms for nonoral administration time curve (0–72 hours) may be more practical. The
 Transdermal
use of a truncated plasma drug concentration–time
curve allows for the measurement of peak absorption
 Inhalation and decreases the time and cost for performing the
 Ophthalmic bioequivalence study.
 Intranasal
Many drugs are stereoisomers, and each isomer
may give a different pharmacodynamic response and
Bioavailable drugs that should not produce peak drug may have a different rate of biotransformation. The
levels
bioavailability of the individual isomers may be dif-
  Potassium supplements ficult to measure because of problems in analysis.
Endogenous drug levels Some drugs have active metabolites, which should
be quantitated as well as the parent drug. Drugs such
  Hormone replacement therapy
as thioridazine and selegilene have two active metab-
Biotechnology-derived drugs olites. The question for such drugs is whether bio-
  Erythropoietin interferon equivalence should be proven by matching the
bioavailability of both metabolites and the parent
  Protease inhibitors
drug. Assuming both biotransformation pathways
Complex drug substances follow first-order reaction kinetics, then the metabo-
  Conjugated estrogens lites should be in constant ratio to the parent drug.
Genetic variation in metabolism may present a bio-
equivalence problem. For example, the acetylation
of procainamide to N-acetylprocainamide demon-
drugs and conventional oral dosage forms. However, strates genetic polymorphism, with two groups of
for certain drugs and dosage forms, systemic bio- subjects consisting of rapid acetylators and slow
availability and bioequivalence are difficult to acetylators. To decrease intersubject variability, a
ascertain (Table 16-18). Drugs and drug products bioequivalence study may be performed on only one
(eg, cyclosporine, chlorpromazine, verapamil, iso- phenotype, such as the rapid acetylators.
sorbide dinitrate, sulindac) are considered to be Some drugs (eg, benzocaine, hydrocortisone, anti-
highly variable if the intrasubject variability in infectives, antacids) are intended for local effect and
bioavailability parameters is greater than 30% by formulated as topical ointments, oral suspensions, or
514    Chapter 16

TABLE 16-19  Possible Surrogate Markers for Bioequivalence Studies


Possible Surrogate Marker
Drug Product Drug for Bioequivalence

Metered-dose inhaler Albuterol Forced expiratory volume (FEV1)

Topical steroid Hydrocortisone Skin blanching

Anion-exchange resin Cholestyramine Binding to bile acids

Antacid Magnesium and aluminum hydroxide gel Neutralization of acid

Topical antifungal Ketoconazole Drug uptake into stratum corneum

rectal suppositories. These drugs should not have sig- more indirect methods must be used to ascertain bio-
nificant systemic bioavailability from the site of equivalence. For example, urinary potassium excretion
administration. The bioequivalence determination for parameters are more appropriate for the measurement
drugs that are not absorbed systemically from the site of bioavailability of potassium supplements. However,
of application can be difficult to assess. For these for certain hormonal replacement drugs (eg, levothy-
nonsystemic-absorbable drugs, a “surrogate” marker is roxine), the steady-state hormone concentration in
needed for bioequivalence determination (Table 16-19). hypothyroid individuals, the thyroidal-stimulating hor-
For example, the acid-neutralizing capacity of an oral mone level, and pharmacodynamic endpoints may also
antacid and the binding of bile acids to cholestyramine be appropriate to measure.
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
GENERIC SUBSTITUTION
route, which may produce only partial systemic bio- Drug product selection and generic drug product
availability. For the treatment of asthma, inhalation of substitution are major responsibilities for physicians,
the drug (eg, albuterol, beclomethasone dipropionate) pharmacists, and others who prescribe, dispense, or
has been used to maximize drug in the respiratory purchase drugs. To facilitate such decisions, the
passages and to decrease systemic side effects. Drugs FDA publishes annually, in print and on the Internet,
such as nitroglycerin given transdermally may differ Approved Drug Products with Therapeutic Equivalence
in release rates, in the amount of drug in the trans- Evaluations, also known as the Orange Book (www
dermal delivery system, and in the surface area of .fda.gov/cder/ob/default.htm). The Orange Book
the skin to which the transdermal delivery system is identifies drug products approved on the basis of
applied. Thus, the determination of bioequivalence safety and effectiveness by the FDA and contains
among different manufacturers of transdermal deliv- therapeutic equivalence evaluations for approved mul-
ery systems for the same active drug is difficult. tisource prescription drug products. These evaluations
Dermatopharmacokinetic studies investigate drug serve as public information and advice to state health
uptake into skin layers after topical drug administra- agencies, prescribers, and pharmacists to promote
tion. The drug is applied topically, the skin is peeled public education in the area of drug product selection
at various time periods after the dose, using trans- and to foster containment of healthcare costs.
parent tape, and the drug concentrations in the skin To contain drug costs, most states have adopted
are measured. generic substitution laws to allow pharmacists to
Drugs such as potassium supplements are given dispense a generic drug product for a brand-name
orally and may not produce the usual bioavailability drug product that has been prescribed. Some states
parameters of AUC, Cmax, and tmax. For these drugs, have adopted a positive formulary, which lists
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     515

therapeutically equivalent or interchangeable drug are evaluated according to specific criteria. The
products that pharmacists may dispense. Other evaluation codes used for these drugs are listed in
states use a negative formulary, which lists drug Table 16-20. The drug products are divided into two
products that are not therapeutically equivalent, and/ major categories: “A” codes apply to drug products
or the interchange of which is prohibited. If the drug considered to be therapeutically equivalent to other
is not in the negative formulary, the unlisted generic pharmaceutically equivalent products, and “B” codes
drug products are assumed to be therapeutically apply to drug products that the FDA, at this time,
equivalent and may be interchanged. does not consider to be therapeutically equivalent to
other pharmaceutically equivalent products. A list of
therapeutic-equivalence-related terms and their defi-
Approved Drug Products with Therapeutic nitions is also given in the monograph. According to
Equivalence Evaluations (Orange Book) the FDA, evaluations do not mandate that drugs be
The Orange Book contains therapeutic equivalence purchased, prescribed, or dispensed, but provide
evaluations for approved drug products made by vari- public information and advice. The FDA evaluation
ous manufacturers. These marketed drug products of the drug products should be used as a guide only,

TABLE 16-20  Therapeutic Equivalence Evaluation Codes


A Codes

Drug products considered to be therapeutically equivalent to other pharmaceutically equivalent products

AA Products in conventional dosage forms not presenting bioequivalence problems

AB Products meeting bioequivalence requirements

AN Solutions and powders for aerosolization

AO Injectable oil solutions

AP Injectable aqueous solutions

AT Topical products

B Codes

Drug products that the FDA does not consider to be therapeutically equivalent to other pharmaceutically equivalent products

B* Drug products requiring further FDA investigation and review to determine therapeutic equivalence

BC Extended-release tablets, extended-release capsules, and extended-release injectables

BD Active ingredients and dosage forms with documented bioequivalence problems

BE Delayed-release oral dosage forms

BN Products in aerosol–nebulizer drug delivery systems

BP Active ingredients and dosage forms with potential bioequivalence problems

BR Suppositories or enemas for systemic use

BS Products having drug standard deficiencies

BT Topical products with bioequivalence issues

BX Insufficient data

Adopted from Approved Drug Products with Therapeutic Equivalence Evaluations (Orange Book) (www.fda.cder/ob/default.htm), 2003.
516    Chapter 16

with the practitioner exercising professional care flavor. There may also be better stability of one
and judgment. product over another under adverse storage condi-
The concept of therapeutic equivalence as used tions or allergic reactions in rare cases due to a
to develop the Orange Book applies only to drug coloring or a preservative ingredient, as well as
products containing the same active ingredient(s) differences in cost to the patient.
and does not encompass a comparison of different FDA evaluation of therapeutic equivalence in no
therapeutic agents used for the same condition way relieves practitioners of their professional
(eg, propoxyphene hydrochloride versus pentazo- responsibilities in prescribing and dispensing such
cine hydrochloride for the treatment of pain). Any products with due care and with appropriate infor-
drug product in the Orange Book that is repack- mation to individual patients. In those circumstances
aged and/or distributed by other than the applica- where the characteristics of a specific product, other
tion holder is considered to be therapeutically than its active ingredient, are important in the ther-
equivalent to the application holder’s drug product apy of a particular patient, the physician’s specifica-
even if the application holder’s drug product is tion of that product is appropriate. Pharmacists must
single source or coded as nonequivalent (eg, BN). also be familiar with the expiration dates/times and
Also, distributors or repackagers of an application labeling directions for storage of the different prod-
holder’s drug product are considered to have the ucts, particularly for reconstituted products, to assure
same code as the application holder. Therapeutic that patients are properly advised when one product
equivalence determinations are not made for unap- is substituted for another.
proved, 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 EXAMPLE • • •
produce the same clinical effect and safety profile
INTERPRETATION OF THERAPEUTIC
as the prescribed product (www.fda.gov/cder/ob
EVALUATION CODE FOR NIFEDIPINE
/default.htm).
EXTENDED-RELEASE TABLETS
Professional care and judgment should be exer-
The FDA has approved a few drug products
cised in using the Orange Book. Evaluations of
containing the same active drug from different
therapeutic equivalence for prescription drugs are
pharmaceutical manufacturers, each of which
based on scientific and medical evaluations by the
has provided a separate New Drug Application
FDA. Products evaluated as therapeutically equiva-
(NDA) for its own product. Since no information
lent can be expected, in the judgment of the FDA,
is available to demonstrate whether the two
to have equivalent clinical effect and no difference
NDA-approved drug products are bioequivalent,
in their potential for adverse effects when used
each branded drug product becomes a separate
under the conditions of their labeling. However,
reference listed drug (Table 16-21). Generic drug
these products may differ in other characteristics
manufacturers must demonstrate to which RLD
such as shape, scoring configuration, release mech-
product is bioequivalent.
anisms, packaging, excipients (including colors,
flavors, preservatives), expiration date/time, and, in
some instances, labeling. If products with such dif-
ferences are substituted for each other, there is a
potential for patient confusion due to differences in In Table 16-21, AB1 products are bioequivalent
color or shape of tablets, inability to provide a to each other and can be substituted. AB2 products
given dose using a partial tablet if the proper scor- are bioequivalent to each other and can be substi-
ing configuration is not available, or decreased tuted. However, an AB1 product cannot be substi-
patient acceptance of certain products because of tuted for an AB2 product.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     517

TABLE 16-21  Nifedipine Extended-Release Oral Tablet


Active Dosage Form; Proprietary
TE Code RLD Ingredient Route Strength Name Applicant

AB1 Yes Nifedipine Extended 90 mg Adalat CC Bayer Healthcare


tablet release; oral

AB1 No Nifedipine Extended 90 mg Nifedipine Actavis


tablet release; oral

AB1 No Nifedipine Extended 90 mg Nifedipine Valeant Intl


tablet release; oral

AB2 Yes Nifedipine Extended 90 mg Procardia XL Pfizer


tablet release; oral

AB2 No Nifedipine Extended 90 mg Nifedipine Mylan


tablet release; oral

AB2 No Nifedipine Extended 90 mg Nifedipine Osmotica Pharm


tablet release; oral

TE = therapeutic equivalent.
Source: Approved Drug Products with Therapeutic Equivalence Evaluations (Orange Book), [www.accessdata.fda.gov/scripts/cder/ob/default.cfm].

GLOSSARY10 Bioequivalent drug products: This term describes


pharmaceutical equivalent or pharmaceutical alter-
Abbreviated New Drug Application (ANDA): Drug native products that display comparable bioavail-
manufacturers must file an ANDA for approval to mar- ability when studied under similar experimental
ket a generic drug product. The generic manufacturer conditions. For systemically absorbed drugs, the test
is not required to perform clinical efficacy studies or (generic) and reference listed drug (brand name)
nonclinical toxicology studies for the ANDA. shall be considered bioequivalent if (1) the rate and
Bioavailability: Bioavailability means the rate and extent of absorption of the test drug do not show a
extent to which the active ingredient or active moiety significant difference from the rate and extent of
is absorbed from a drug product and becomes avail- absorption of the reference drug when administered
able at the site of action. For drug products that are not at the same molar dose of the therapeutic ingredient
intended to be absorbed into the bloodstream, bioavail- under similar experimental conditions in either a
ability may be assessed by measurements intended to single dose or multiple doses or (2) the extent of
reflect the rate and extent to which the active ingredient absorption of the test drug does not show a signifi-
or active moiety becomes available at the site of action. cant difference from the extent of absorption of the
Bioequivalence requirement: A requirement imposed reference drug when administered at the same molar
by the FDA for in vitro and/or in vivo testing of speci- dose of the therapeutic ingredient under similar
fied drug products, which must be satisfied as a condi- experimental conditions in either a single dose or
tion for marketing. multiple doses and the difference from the reference
drug in the rate of absorption of the drug is inten-
10The
tional, is reflected in its proposed labeling, is not
definitions are from Approved Drug Products with
Therapeutic Equivalence Evaluations (Orange Book). [www essential to the attainment of effective body drug
.fda.gov/Drugs/InformationOnDrugs/ucm129662.htm], Code of concentrations on chronic use, and is considered
Federal Regulations, 21 CFR 320, and other sources. medically insignificant for the drug.
518    Chapter 16

When the above methods are not applicable (eg, Equivalence: Relationship in terms of bioavailabil-
for drug products that are not intended to be ity, therapeutic response, or a set of established
absorbed into the bloodstream), other in vivo or in standards of one drug product to another.
vitro test methods to demonstrate bioequivalence Generic name: The established, nonproprietary, or
may be appropriate. Bioequivalence may sometimes common name of the active drug in a drug product
be demonstrated using an in vitro bioequivalence (eg, acetaminophen).
standard, especially when such an in vitro test has Generic substitution: The process of dispensing a
been correlated with human in vivo bioavailability different brand or an unbranded drug product in
data. In other situations, bioequivalence may some- place of the prescribed drug product. The substituted
times be demonstrated through comparative clinical drug product contains the same active ingredient or
trials or pharmacodynamic studies. therapeutic moiety as the same salt or ester in the
Bioequivalent drug products may contain differ- same dosage form but is made by a different manu-
ent inactive ingredients, provided the manufacturer facturer. For example, a prescription for Motrin
identifies the differences and provides information brand of ibuprofen might be dispensed by the phar-
that the differences do not affect the safety or effi- macist as Advil brand of ibuprofen or as a non-
cacy of the product. branded generic ibuprofen if generic substitution is
Biosimilar or biosimilarity: The biological product permitted and desired by the physician.
is highly similar to the reference product notwith- Pharmaceutical alternatives: Drug products that
standing minor differences in clinically inactive contain the same therapeutic moiety but as different
components, and there are no clinically meaningful salts, esters, or complexes. For example, tetracycline
differences between the biological product and the phosphate and tetracycline hydrochloride equivalent
reference product in terms of the safety, purity, and to 250-mg tetracycline base are considered pharma-
potency of the product. ceutical alternatives. Different dosage forms and
Brand name: The trade name of the drug. This strengths within a product line by a single manufac-
name is privately owned by the manufacturer or dis- turer are pharmaceutical alternatives (eg, an extended-
tributor and is used to distinguish the specific drug release dosage form and a standard immediate-release
product from competitor’s products (eg, Tylenol, dosage form of the same active ingredient). The FDA
McNeil Laboratories). currently considers a tablet and capsule containing the
Chemical name: The name used by organic chem- same active ingredient in the same dosage strength as
ists to indicate the chemical structure of the drug pharmaceutical alternatives.
(eg, N-acetyl-p-aminophenol). Pharmaceutical equivalents: Drug products in
Drug product: The finished dosage form (eg, tablet, identical dosage forms that contain the same active
capsule, or solution) that contains the active drug ingredient(s), that is, the same salt or ester, are of the
ingredient, generally, but not necessarily, in associa- same dosage form, use the same route of administra-
tion with inactive ingredients. tion, and are identical in strength or concentration
Drug product performance: Drug product perfor- (eg, chlordiazepoxide hydrochloride, 5-mg cap-
mance, in vivo, may be defined as the release of the sules). Pharmaceutically equivalent drug products
drug substance from the drug product, leading to are formulated to contain the same amount of active
bioavailability of the drug substance and leading to a ingredient in the same dosage form and to meet the
pharmacodynamic response. Bioequivalence studies same or compendial or other applicable standards
are drug product performance tests. (ie, strength, quality, purity, and identity), but they
Drug product selection: The process of choosing or may differ in characteristics such as shape, scoring
selecting the drug product in a specified dosage form. configuration, release mechanisms, packaging,
Drug substance: A drug substance is the active excipients (including colors, flavors, preservatives),
pharmaceutical ingredient (API) or component in the expiration time, and, within certain limits, labeling.
drug product that furnishes the pharmacodynamic When applicable, pharmaceutical equivalents must
activity. meet the same content uniformity, disintegration
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     519

times, and/or dissolution rates. Modified-release as therapeutically equivalent those products that
dosage forms that require a reservoir or overage or meet the following general criteria: (1) they are
certain dosage forms such as prefilled syringes in approved as safe and effective; (2) they are pharma-
which residual volume may vary must deliver identi- ceutical equivalents in that they (a) contain identical
cal amounts of active drug ingredient over an identi- amounts of the same active drug ingredient in the
cal dosing period. same dosage form and route of administration, and
Pharmaceutical substitution: The process of dis- (b) meet compendial or other applicable standards of
pensing a pharmaceutical alternative for the prescribed strength, quality, purity, and identity; (3) they are
drug product. For example, ampicillin suspension is bioequivalent in that (a) they do not present a known
dispensed in place of ampicillin capsules, or tetracy- or potential bioequivalence problem, and they meet
cline hydrochloride is dispensed in place of tetracy- an acceptable in vitro standard, or (b) if they do pres-
cline phosphate. Pharmaceutical substitution generally ent such a known or potential problem, they are
requires the physician’s approval. shown to meet an appropriate bioequivalence stan-
Reference listed drug: The reference listed drug dard; (4) they are adequately labeled; and (5) they
(RLD) is identified by the FDA as the drug product are manufactured in compliance with Current
on which an applicant relies when seeking approval Good Manufacturing Practice regulations. The FDA
of an ANDA. The RLD is generally the brand-name believes that products classified as therapeutically
drug that has a full NDA. The FDA designates a equivalent can be substituted with the full expecta-
single RLD as the standard to which all generic ver- tion that the substituted product will produce the
sions must be shown to be bioequivalent. The FDA same clinical effect and safety profile as the pre-
hopes to avoid possible significant variations among scribed product.
generic drugs and their brand-name counterparts. Therapeutic substitution: The process of dispens-
Such variations could result if generic drugs were ing a therapeutic alternative in place of the pre-
compared to different RLDs. scribed drug product. For example, amoxicillin is
Therapeutic alternatives: Drug products contain- dispensed instead of ampicillin or ibuprofen is dis-
ing different active ingredients that are indicated for pensed instead of naproxen. Therapeutic substitution
the same therapeutic or clinical objectives. Active can also occur when one NDA-approved drug is
ingredients in therapeutic alternatives are from the substituted for the same drug that has been approved
same pharmacologic class and are expected to have by a different NDA, for example, the substitution of
the same therapeutic effect when administered to Nicoderm (nicotine transdermal system) for Nicotrol
patients for such condition of use. For example, ibu- (nicotine transdermal system).
profen is given instead of aspirin; cimetidine may be
given instead of ranitidine.
Therapeutic equivalents: Drug products are con- Frequently Asked Questions
sidered to be therapeutic equivalents only if they are »» Can pharmaceutic equivalent drug products that are
pharmaceutical equivalents and if they can be not bioequivalent have similar clinical efficacy?
expected to have the same clinical effect and safety »» What is the difference between generic substitution
profile when administered to patients under the con- and therapeutic substitution?
ditions specified in the labeling. The FDA classifies
520    Chapter 16

CHAPTER SUMMARY
Drug product performance may be defined as the healthy volunteers. Bioequivalence is generally deter-
release of the drug substance from the drug product mined if the 90% confidence intervals for Cmax and
leading to bioavailability of the drug substance. AUC fall within 80%–125% of the reference listed
Bioequivalence is a measure of comparative drug drug based on log transformation of the data. Food
product performance and relates the quality of a drug intervention or food effect studies are generally con-
product to clinical safety and efficacy. The absolute ducted using meal conditions that are expected to
availability of drug is the systemic availability of a provide the greatest effects on GI physiology so that
drug after extravascular administration (eg, oral, rectal, systemic drug availability is maximally affected. The
transdermal, subcutaneous) compared to IV dosing, Biopharmaceutics Classification System (BCS) is
whereas relative bioavailability compares the bioavail- based on the solubility, permeability, and dissolution
ability of a drug from two or more drug products. The characteristics of the drug. However, systemic drug
most direct method to assess drug bioavailability is to bioavailability may also be affected by transporters in
determine the rate and extent of systemic drug absorp- the GI tract, hepatic clearance, GI transit and motility,
tion by measurement of the active drug concentrations and the contents of the GI tract.
in plasma. The main pharmacokinetic parameters, Drug product selection and generic substitution
Cmax and AUC, are used to determine bioequivalence. are important responsibilities of the pharmacist. A
However, other pharmacokinetic parameters such as listing of approved drug products of generic drug
tmax and elimination t½ should also be assessed. The products that may be safely substituted is available
most common statistical design for bioequivalence in Approved Drug Products with Therapeutic
studies is the two-way, crossover design in normal Equivalence Evaluations (Orange Book).

LEARNING QUESTIONS
1. An antibiotic was formulated into two different Each drug product was given in the same dose
oral dosage forms, A and B. Biopharmaceutic as the other. Explain how the various possible
studies revealed different antibiotic blood level formulation factors could have caused the dif-
curves for each drug product (Fig. 16-18). ferences in blood levels. Give examples where
possible. How would the corresponding urinary
drug excretion curves relate to the plasma
level–time curves?
2. Assume that you have just made a new for-
A mulation of acetaminophen. Design a proto-
col to compare your drug product against the
Blood level

acetaminophen drug products on the market.


MEC
What criteria would you use for proof of
bioequivalence for your new formulation?
B How would you determine if the acetamino-
phen was completely (100%) systemically
absorbed?
0 1 2 3 4 5 3. The data in Table 16-22 represent the average
Time findings in antibiotic plasma samples taken
FIGURE 16-18  Blood level curves for two different oral from 10 humans (average weight 70 kg), tabu-
dosage forms of a hypothetical antibiotic. lated in a 4-way crossover design.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     521

TABLE 16-22  Comparison of Plasma Concentrations of Antibiotic, as Related to Dosage Form


and Time
Plasma Concentration (lg/mL)

IV Solution Oral Solution Oral Tablet Oral Capsule


Time after Dose (h)  (2 mg/kg) (10 mg/kg) (10 mg/kg) (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

 µg 
AUC  ×h 29.0 145.0 116.0 116.0
 mL 

a. Which of the four drug products in one-compartment open model with first-order
Table 16-22 would be preferred as a refer- absorption and first-order elimination:
ence standard for the determination of rela- DGI 
a k k
→ DBVD →

tive bioavailability? Why?
b. From which oral drug product is the drug The drug was given in a single oral dose
absorbed more rapidly? of 250 mg to a group of college students
c. What is the absolute bioavailability of the 21–29 years of age. Mean body weight was
drug from the oral solution? 60 kg. Samples of blood were obtained at various
d. What is the relative bioavailability of the time intervals after the administration of the drug,
drug from the oral tablet compared to the and the plasma fractions were analyzed for active
reference standard? drug. The data are summarized in Table 16-23.
e. From the data in Table 16-15, determine: a. The minimum effective concentration of
(i) Apparent VD Aphrodisia in plasma is 2.3 mg/mL. What is
(ii) Elimination t1/2 the onset time of this drug?
(iii) First-order elimination rate constant k b. The minimum effective concentration of
(iv) Total body clearance Aphrodisia in plasma is 2.3 mg/mL. What is
f. From the data above, graph the cumulative the duration of activity of this drug?
urinary excretion curves that would correspond c. What is the elimination half-life of Aphrodi-
to the plasma concentration–time curves. sia in college students?
4. Aphrodisia is a new drug manufactured by the d. What is the time for peak drug concentration
Venus Drug Company. When tested in humans, (tmax) of Aphrodisia?
the pharmacokinetics of the drug assumes a e. What is the peak drug concentration (Cmax)?
522    
Chapter 16

TABLE 16-23  Data Summary of Active Drug a. What is the absolute bioavailability of the
Concentration in Plasma Fractions drug from the tablet?
b. What is the relative bioavailability of the
Time (h) Cp (lg/mL) Time (h) Cp (lg/mL)
capsule compared to the oral solution?
0 0 12 3.02 7. According to the prescribing information for
1 1.88 18 1.86 cimetidine (Tagamet®), following IV or IM
administration, 75% of the drug is recovered
2 3.05 24 1.12
from the urine after 24 hours as the parent com-
3 3.74 36 0.40 pound. Following a single oral dose, 48% of the
5 4.21 48 0.14 drug is recovered from the urine after 24 hours
as the parent compound. From this information,
7 4.08 60 0.05
determine what fraction of the drug is absorbed
9 3.70 72 0.02 systemically from an oral dose after 24 hours.
8. Define bioequivalence requirement. Why does
the FDA require a bioequivalence requirement
f. Assuming that the drug is 100% sys- for the manufacture of a generic drug product?
temically available (ie, fraction of drug 9. Why can we use the time for peak drug
absorbed equals unity), what is the AUC for concentration (tmax) in a bioequivalence study
Aphrodisia? for an estimate of the rate of drug absorption,
5. You wish to do a bioequivalence study on three rather than calculating the ka?
different formulations of the same active drug. 10. Ten male volunteers (18–26 years of age)
Lay out a Latin-square design for the proper weighing an average of 73 kg were given either
sequencing of these drug products in six normal, 4 tablets each containing 250 mg of drug (drug
healthy volunteers. What is the main reason for product A) or 1 tablet containing 1000 mg of
using a crossover design in a bioequivalence drug (drug product B). Blood levels of the drug
study? What is meant by a “random” population? were obtained and the data are summarized in
6. Four different drug products containing the Table 16-25.
same antibiotic were given to 12 volunteer a. State a possible reason for the difference in
adult males (age 19–28 years, average weight the time for peak drug concentration (tmax,A)
73 kg) in a 4-way crossover design. The vol- after drug product A compared to the tmax,B
unteers fasted for 12 hours prior to taking the after drug product B. (Assume that all the
drug product. Urine samples were collected up tablets were made from the same formula-
to 72 hours after the administration of the drug tion—ie, the drug is in the same particle
to obtain the maximum urinary drug excretion, size, same salt form, same excipients, and
Du∞ . The data are presented in Table 16-24. same ratio of excipients to active drug.)
b. Draw a graph relating the cumulative
TABLE 16-24  Urinary Drug Excretion Data amount of drug excreted in urine of patients
Summary given drug product A compared to the
cumulative drug excreted in urine after drug
Cumulative Urinary
Dose Drug Excretion product B. Label axes.
Drug Product (mg/kg) 0–72 h c. In a second study using the same 10 male
volunteers, a 125-mg dose of the drug was
IV solution 0.2 20
given by IV bolus and the AUC was com-
Oral solution 4 380 puted as 20 mg·h/mL. Calculate the fraction
Oral tablet 4 340 of drug systemically absorbed from drug
product B (1 × 1000-mg) tablet using the
Oral capsule 4 360 data in Table 16-25.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     523

TABLE 16-25  Blood Level Data Summary for Two Drug Products
Drug Product

A, 4 × 250-mg B, 1000-mg
Kinetic Variable  Unit  Tablet Tablet Statistic 

Time for peak drug concentration (range) h 1.3 1.8 p < .05
(0.7–1.5) (1.5–2.2)

Peak concentration (range) mg/mL 53 47 p < .05


(46–58) (42–51)

AUC (range) mg · 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)

11. After performing a bioequivalence test com- 180 mL of water. Food intake was allowed at
paring a generic drug product to a brand-name 5 hours postdose. Blood samples (10 mL) were
drug product, it was observed that the generic taken just before the dose and periodically after
drug product had greater bioavailability than dosing. The serum fraction was separated from
the brand-name drug product. the blood and analyzed for tolazamide by high-
a. Would you approve marketing the generic pressure liquid chromatography.
drug product, claiming it was superior to the Data Analysis: Serum data were analyzed by
brand-name drug product? a digital computer program using a regression
b. Would you expect identical pharmacody- analysis and by the percent of drug unabsorbed
namic responses to both drug products? by the method of Wagner and Nelson (1963).
c. What therapeutic problem might arise in AUC was determined by the trapezoidal rule
using the generic drug product that might and an analysis of variance was determined by
not occur when using the brand-name drug Tukey’s method.
product? a. Why was a Latin-square crossover design
12. The following study is from Welling et al (1982): used in this study?
Tolazamide Formulations: Four tolazamide b. Why were the subjects fasted before being
tablet formulations were selected for this study. given the tolazamide tablets?
The tablet formulations were labeled A, B, C, c. Why did the authors use the Wagner–Nelson
and D. Disintegration and dissolution tests were method rather than the Loo–Riegelman method
performed by standard USP-23 procedures. for measuring the amount of drug absorbed?
Subjects: Twenty healthy adult male volunteers d. From the data in Table 16-26 only, from
between the ages of 18 and 38 years (mean, which tablet formulation would you expect
26 years) and weighing between 61.4 and the highest bioavailability? Why?
95.5 kg (mean, 74.5 kg) were selected for the e. From the data in Table 16-26, did the disin-
study. The subjects were randomly assigned tegration times correlate with the dissolution
to four groups of five each. The 4 treatments times? Why?
were administered according to 4 × 4 Latin- f. Do the data in Table 16-27 appear to corre-
square design. Each treatment was separated by late with the data in Table 16-26? Why?
1-week intervals. All subjects fasted overnight g. Draw the expected cumulative urinary excre-
before receiving the tolazamide tablet the tion–time curve for formulations A and B.
following morning. The tablet was given with Label axes and identify each curve.
524    
Chapter 16

TABLE 16-26  Disintegration Times and h. Assuming formulation A is the reference


Dissolution Rates of Tolazamide Tabletsa formulation, what is the relative bioavailabil-
ity of formulation D?
Mean Disinte-
gration Timeb Percent Dissolved i. Using the data in Table 16-27 for formula-
Tablet min (Range) in 30 minc (Range) tion A, calculate the elimination half-life
(t1/2) for tolazamide.
A 3.8 (3.0–4.0) 103.9 (100.5–106.3)
13. If in vitro drug dissolution and/or release stud-
B 2.2 (1.8–2.5) 10.9 (9.3–13.5) ies for an oral solid dosage form (eg, tablet)
C 2.3 (2.0–2.5) 31.6 (26.4–37.2) does not correlate with the bioavailability of
the drug in vivo, why should the pharmaceuti-
D 26.5 (22.5–30.5) 29.7 (20.8–38.4) cal manufacturer continue to perform in vitro
aN = 6. release studies for each production batch of the
bBy the method of USP-23. solid dosage form?
cDissolution rates in pH 7.6 buffer.
From Welling et al (1982), with permission.

TABLE 16-27  Mean Tolazamide Concentrationsa in Serum


Treatment (lg/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
ADCB

1 20.5 ± 7.3 2.8 ± 2.8 5.4 ± 4.8 13.5 ± 6.6


ADCB
3 23.9 ± 5.3 4.4 ± 4.3 9.8 ± 5.6 20.0 ± 6.4 AD CB

4 25.4 ± 5.2 5.7 ± 4.1 13.6 ± 5.3 22.0 ± 5.4 AD CB

5 24.1 ± 6.3 6.6 ± 4.0 15.1 ± 4.7 22.6 ± 5.0 AD CB

6 19.9 ± 5.9 6.8 ± 3.4 14.3 ± 3.9 19.7 ± 4.7 AD CB

8 15.2 ± 5.5 6.6 ± 3.2 12.8 ± 4.1 14.6 ± 4.2 ADCB


12 8.8 ± 4.8 5.5 ± 3.2 9.1 ± 4.0 8.5 ± 4.1 CADB

16 5.6 ± 3.8 4.6 ± 3.3 6.4 ± 3.9 5.4 ± 3.1


CADB
24 2.7 ± 2.4 3.1 ± 2.6 3.1 ± 3.3 2.4 ± 1.8
CBAD
Cmax, mg/mLc 27.8 ± 5.3 7.7 ± 4.1 16.4 ± 4.4 24.0 ± 4.5 AD CB
tmax, hd 3.3 ± 0.9 7.0 ± 2.2 5.4 ± 2.0 4.0 ± 0.9 BCDA
AUC0–24, mg h/mLe 260 ± 81 112 ± 63 193 ± 70 231 ± 67 AD CB

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.
Drug Product Performance, In Vivo: Bioavailability and Bioequivalence     525

14. Is it possible for two pharmaceutically equiva- acceptable for proving that two drug prod-
lent solid dosage forms containing different ucts are bioequivalent?
inactive ingredients (ie, excipients) to demon- b. Are pharmacokinetic models needed in the
strate bioequivalence in vivo even though these evaluation of bioequivalence?
drug products demonstrate differences in drug c. Is it necessary to use a pharmacokinetic
dissolution tests in vitro? model to completely describe the plasma
15. For bioequivalence studies, tmax, Cmax, and drug concentration–time curve for the deter-
AUC, along with an appropriate statistical mination of tmax, Cmax, and AUC?
analysis, are the parameters generally used to d. Why are log-transformed data used for the
demonstrate the bioequivalence of two similar statistical evaluation of bioequivalence?
drug products containing the same active drug. e. What is an add-on study?
a. Why are the parameters tmax, Cmax, and AUC

ANSWERS
Frequently Asked Questions example, for Period 1, half the subjects receive
treatment A, brand product, and the other half of the
Why are preclinical animal toxicology studies and subjects receive treatment B, generic product.
clinical efficacy drug studies in human subjects not
required by the FDA to approve a generic drug prod- Why does the FDA require a food intervention (food-
uct as a therapeutic equivalent to the brand-name effect) study for generic drug products before grant-
drug product? ing approval?
• Preclinical animal toxicology and clinical efficacy • Manufacturers are required to perform a food-in-
studies were performed on the marketed brand tervention bioavailability study on all drugs whose
drug product as part of the New Drug Application bioavailability is known to be affected by food. In
(NDA) prior to FDA approval. These studies do addition, a food-intervention bioavailability study
not have to be repeated for the generic bioequiva- is required on all modified-release products since
lent drug product. The manufacturer of the generic (1) the modified-release formulation (eg, enteric
drug product must submit an Abbreviated New coating, sustained-release coating) may be af-
Drug Application (ANDA) to the FDA, demon- fected by the presence of food and (2) modified-
strating that the generic drug product is a thera- release products have a greater potential to be af-
peutic equivalent (see definitions in Chapter 15) to fected by food due to their longer residence time
the brand drug product. in the gastrointestinal tract and changes in gastro-
intestinal motility.
What do sequence, washout period, and period mean
in a crossover bioavailability study? What type of bioequivalence studies are required for
drugs that are not systemically absorbed or for those
• The sequence is the order in which the drug prod-
drugs in which the Cmax and AUC cannot be measured
ucts (ie, treatments) are given (eg, brand product fol-
in the plasma?
lowed by generic product or vice versa). Sequence
is important to prevent any bias due to the order of • If the drug is not absorbed systemically from the
the treatments in the study. The term washout re- drug product, a surrogate marker must be used as a
fers to the time for total elimination of the dose. The measure of bioequivalence. This surrogate marker
time for washout is determined by the elimination may be a pharmacodynamic effect or, as in the
half-life of the drug. Period refers to the drug-dosing case of cholestyramine resin, the binding capacity
day on which the drug is given to the subjects. For for bile acids in vitro.
526    
Chapter 16

Learning Questions c. 8.75 hours


d. 5 hours
3. a. Oral solution: The drug is in the most bio- e. 4.21 mg/mL
available form. f. 77.98 mg h/mL
b. Oral solution: Same reason as above.
c. Absolute bioavailability 5. Drug Product

[AUC]soln /dosesoln Subject Period 1 Period 2 Week 3


=
[AUC]IV /dose IV 1 A B C

145/10 2 B C A
= = 1.0
29/2 3 C A B

4 A C B
d. Relative bioavailability
5 C B A
[AUC]tab /dose tab
= 6 B A C
[AUC]soln /dosesoln
6. a.  Absolute bioavailability
116/10
= = 0.80 ∞
145/10 Du,PO /dose PO 340/4
= ∞ =
Du,IV /dose IV 20/0.2

e. (1)  Cp0 = 6.67 µ g/mL
= 0.85 or 85%
(by extrapolation of IV curve)
b. Relative bioavailability
2000 µ g/kg
VD = = 300 mL/kg
6.67 µ g/mL ∞
Ducap /dose cap 360/4
= ∞ =
(2)  t1/2 = 3.01 h Dusoln /dosesol 380/4
(3)  k = 0.23 h−1
= 0.947 or 94.7%
(4)  ClT = kVD = 69 m/kg·h
7. The fraction of drug absorbed systemically is
4. Plot the data on both rectangular and semi-
the absolute bioavailability.
log graph paper. The following answers were
obtained from estimates from the plotted Fraction of drug absorbed
plasma level–time curves. More exact answers % of dose excreted after PO
may be obtained mathematically by substitution =
% of dose excreted after IV
into the proper formulas.
a. 1.37 hours 48%
= = 0.64
b. 13.6 hours 75%

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