Bioequivalence accomplishments, ongoing initiatives, and remaining challenges

Bioequivalence accomplishments, ongoing initiatives, and remaining

challenges1

M. N. MARTINEZ

US Food and Drug Administration, Center

for Veterinary Medicine, Rockville, MD,

USA

Martinez, M. N. Bioequivalence accomplishments, ongoing initiatives, and

remaining challenges. J. vet. Pharmacol. Therap. 37, 2–12.

Although bioequivalence (BE) concepts date back to the late 1960s, there has

been a steady evolution in the tools applied to the assessment of product

comparability. Despite these advancements, we continue to face a multitude

of unresolved challenges. Several of these challenges are unique to veterinary

medicine due to issues such as multiple species approvals, unique dosage

forms (e.g., intramammary infusion and medicated premixes), physiological

challenges (e.g., limitations in blood volume and stress reactions), and the

need to evaluate product equivalence for products intended to release drug

over a duration of months. Thus, while in some instances, we can adopt

advancements implemented by our human health counterparts but in other

situations, we need to pioneer our own method for resolving these challenges.

The purpose of this manuscript is to provide an update on recent advances,

achievements, and ongoing initiatives associated with the assessment of prod-

uct BE in veterinary medicine. This review reflects the highlights of a presen-

tation given at the 2012 meeting of the European Association for Veterinary

Pharmacology and Toxicology.

(Paper received 13 April 2013; accepted for publication 01 June 2013)

Marilyn N. Martinez, US Food and Drug Administration, Center for Veterinary

Medicine, HFV-100, Rockville, MD 20855, USA. E-mail: marilyn.martinez@

fda.hhs.gov1This article reflects the views of the author and should not be construed to

represent FDA’s views or policies.

INTRODUCTION

An examination of the changes in the bioequivalence (BE) par-

adigm provides a fascinating study of the evolution of a scien-

tific discipline (Martinez & Hunter, 2010). While today, most

scientists agree that (with all else equal) equivalent rate and

extent of drug exposure will result in equivalent therapeutic

outcomes, this perspective was born from extensive debate

within the scientific and medical communities. In reviewing

published manuscripts from the late 1960s, we find many

examples of expressed concern about the validity of BE con-

cepts. During these early years, fundamental problems were

the paucity of insights into factors impacting in vivo product

performance and a lack of efficient tools for evaluating drug

product comparability. However, even after significant

advances were achieved, there remained concerns and skepti-

cism. I myself recall working on the evaluation of human gen-

eric drug products shortly after the enactment of Drug Price

Competition and Patent Term Restoration Act of 1984 (Public

Law 98-417; Hatch Waxman) at which time state formularies

and critics of generic drug product interchangeability exhibited

misconceptions about the implications of the confidence inter-

val approach for assessing product comparability. Skeptics

argued that an allowance of 90% confidence bounds of �20%

for rate (as estimated by peak drug concentration, Cmax) and

extent (as estimated by the area under the concentration vs.

time profile, AUC) implied that two generic products could

actually differ by �40%. Such statements reflected a lack of

understanding of the statistical underpinnings upon which BE

assessments were based.

While those days are behind us, we still face a multitude of

issues that remain to be resolved. Within the area of animal

health, we encounter not only challenges impacting human BE

assessments but also those associated with dosage forms and

physiologies unique to veterinary species. Therefore, while it is

helpful to follow many of the solutions developed by our

human health colleagues, there needs to be a willingness to

forge unique paths that address the numerous issues encoun-

tered within animal health.

With this in mind, this manuscript provides a summary of

our accomplishments, ongoing initiatives, and remaining BE

challenges.

2 Published (2013). This article is a U.S. Government work and is in the public domain in the USA.

J. vet. Pharmacol. Therap. 37, 2--12. doi: 10.1111/jvp.12063. REVIEW ARTICLE

BASIC CONCEPTS

The evaluation of product BE is a compilation of several scien-

tific disciplines:

• Pharmacokinetics (PK): the way that the body handles the

active pharmaceutical ingredient (API). This includes absorp-

tion, metabolism, distribution, and elimination (ADME).

○ PK can influence the extent to which formulation differ-

ences will impact the product comparability of in vivo

blood level profiles. For example, the more rapid the elim-

ination, the more sensitive will be the profiles to product

differences in absorption rates. The presence of saturable

elimination processes can lead to exaggerated interprod-

uct differences in AUC, especially in the presence of drug

accumulation. The PK of the drug will influence the

study design (e.g., the duration of the washout interval,

the magnitude of variability in PK characteristics, and

the ability to employ a crossover vs. a parallel study

design).

○ Absorption constraints:

• The internal environment to which the dosage form

will be exposed. In turn, this dictates the rate and

extent of product dissolution, partitioning into biologi-

cal fluids and drug solubilization, regardless of whether

the product is orally, parenterally, or topically adminis-

tered.

• The characteristics of the absorption site, which

includes membrane permeability, blood flow, absorptive

surface area, and active transport processes.

• The presence of presystemic drug metabolism, whether

it be in the liver or gut. This metabolism is often a

cause for interspecies differences in apparent bioavail-

ability and for large within- and between-subject fluc-

tuations in apparent oral bioavailability.

• Biopharmaceutics: the relationship between the physico-

chemical characteristics of the drug and the formulation

through which that drug is being delivered. Integrated into

biopharmaceutics principles are the concepts of quality by

design (QbD), critical quality attributes (CQAs), and quality

target product profile (QTPP; Annon1, 2009).

• Statistics: the analysis and interpretation of the PK parame-

ters based upon a set of assumptions and criteria. The statis-

tical approaches used for determining product BE also

dictates many attributes of the study design, such as subject

number and the overall dosing scheme (e.g., extended period

crossover designs) that may be appropriate addressing spe-

cific sets of concerns (e.g., reference products presenting with

highly variable blood level profiles, methods for identifying

unequal carryover of PK or PD effects from one period to the

next).

• Clinical pharmacology: the allowable difference between test

and reference means (as they pertain to the rate and extent

of drug exposure) that insures that the products will be

therapeutically indistinguishable within the target patient

population. Inherent in this approach is an assumed absence

of any patient-by-formulation interaction (Hauck & Ander-

son, 1994). When dealing with a generic drug product, leg-

ally prescribed constraints on the allowable differences are

usually imposed (e.g., confidence bounds of 0.80–1.25).

However, if a drug has a narrow therapeutic window or

when a drug sponsor has right of reference to the underly-

ing safety and effectiveness data, the allowable differences

can be modified as appropriate. This distinction between

generic and pioneer products is contained within the con-

cepts of prescribability vs. switchability (Hauck and Ander-

son, 1994).

ACCOMPLISHMENTS

International harmonization

The presence of a global marketplace necessitates international

harmonization of the study requirements for demonstrating in

vivo blood level BE. Through the efforts of the Veterinary Inter-

national Conference on Harmonization (VICH), an Expert

Working Group (EWG) was convened and a draft in vivo blood

level BE guideline was developed. The VICH is a trilateral (EU-

Japan-USA) program aimed at harmonizing technical require-

ments for veterinary product registration. The EWG is

appointed to draft recommendations by the VICH Steering

Committee. Each EWG will normally consist of six topic experts

– one representing each VICH full member. Once finalized,

VICH guidelines represent a harmonization in guidance by the

European Union, the United States, and Japan, and may find

even wider acceptance across the various other jurisdictions.

For additional information on the VICH, please refer to http://

www.vichsec.org.

The VICH BE guideline is slated to progress to Step 4 of

the eight-step VICH process in the near future (for informa-

tion on the VICH step process, please refer to http://www.

vichsec.org/en/process.htm). Despite this success, there remain

numerous challenges not addressed within this guideline.

These include:

• biowaivers;

• biosimilars;

• pharmacological or clinical endpoint studies;

• human food safety;

• in vitro dissolution studies;

• study designs for those products where blood drug concen-

trations may not be indicative of drug concentrations at the

site of action. This can include products such as topically

active formulations, intramammary products, intravenous

administration of complex drug delivery systems where the

drug is release directly at the site of action.

Identifying potential solutions to existing BE challenges

In 2009, the plan for a BE initiative was presented at the Tri-

ennial meeting of the European Association of Veterinary Phar-

Published (2013). This article is a U.S. Government work and is in the public domain in the USA.

BE accomplishments and ongoing initiatives 3

macology and Toxicology (EAVPT). This initiative, sponsored

by the American Academy of Veterinary Pharmacology and

Therapeutics (AAVPT), the European College of Veterinary

Pharmacology and Toxicology (ECVPT), and the EAVPT, pro-

vided the support necessary to accomplish the following goals:

• Publication of a stimuli article describing some of the BE

challenges encountered by veterinary medicine (Martinez &

Hunter, 2010).

• The convening of an AAVPT/EAVPT/ECVPT cosponsored

webinar to review basic BE concepts. This was accomplished

in May, 2010.

• The convening of a BE Workshop as a platform for dialog on

the challenges encountered when evaluating product BE for

veterinary pharmaceuticals. This event occurred on June,

2010 (http://aavpt.affiniscape.com/associations/12658/files/

BEWorkshopfinalagenda6-4-10.pdf.

• Publication of a workshop summary report (Martinez et al.,

2011).

• Development of white papers by working groups represent-

ing experts from across the globe. These white papers were

published in a 2012 Supplemental Issue of the Journal of

Veterinary Pharmacology and Therapeutics (JVPT; Fig. 1).

As a result of this initiative, innovative solutions were pro-

posed and the groundwork established for identifying creative

paths forward on these complex issues.

UNRESOLVED CHALLENGES AND ONGOING

INITIATIVES

Some of the remaining unresolved challenges, many of which

were topics of manuscripts included in the JVPT 212 BE Sup-

plement, were reviewed at the 2012 EAVPT Triennial meeting.

These included the following topics are discussed below.

Biowaivers

The term biowaiver is applied to a regulatory drug approval

process where the efficacy and safety part of the dossier (appli-

cation) are approved based on evidence of equivalence other

than through in vivo equivalence testing. The issue of biowai-

vers has garnished a tremendous amount of attention within

both human and veterinary medicine. For products associated

with negligible systemic absorption, BE assessments have been

largely limited to clinical endpoint trials. However, given the

limited discriminative capability of such trials, the question is

whether or not alternative methods for assessing product com-

parability may not be more sensitive to potential differences in

in vivo product performance.

To expand upon this point, consider the hypothetical situation

described in Fig. 2 where we compare the clinical response to

two different formulations of an orally administered, locally act-

ing, antiparasitic agent. In this example, due to formulation dif-

ferences in their respective in vivo drug release characteristics

different amounts of a locally acting antiparasitic agent are deliv-

ered to the site of action by formulations A and B. In this exam-

ple, the local exposure associated with formulation A = 4 units

while that of formulation B = 8 units. If a clinical endpoint study

were conducted on animals infected with a wild-type strain of

the pathogen, then the percent therapeutic success (Y-axis)

achieved with each of two formulations could be indistinguish-

able and the two products would be declared bioequivalent.

However, if an animal were now infected with a less susceptible

pathogen strain, the clinical response to the formulations would

be markedly different. This outcome is consistent with the work

of Holford and Sheiner (1981) where they showed that when

the response is in excess of 80% its maximum, the pharmacody-

namic endpoint will be insensitive to further changes in drug

concentrations. A similar scenario may be encountered when

attempting to derive a correlation between surrogate vs. targeted

clinical responses, between two events originating through dif-

INTRODUCTION

• Introduc on to the bioequivalence theme issue: MN Mar nez & RP Hunter

DATA ANALYSIS• Assessing product bioequivalence for extended-release formula ons and drugs with long half-lives. R. Gehring & MN Mar nez•

Su onEs ma ng product bioequivalence for highly variable veterinary drugs. R Claxton, J Cook, L Endrenyi, A Lucas, MN Mar nez, & SC

• Pharmacokine cs and pharmacodynamics of stereoisomeric drugs with par cular reference to bioequivalence determina ons. P Lees,RP Hunter, PT Reeves & PL Toutain

STUDY DESIGN

• Demonstra ng bioequivalence using clinical endpoint studies. E Bermingham, JRE Del Cas llo, C Lainesse, K Pasloske, & S Radecki

• Should licking behavior be considered in the bioavailability evalua on of transdermal products? PL Toutain, S. Modric, A Bousquet-Mélou, JM Sallovitz, & C Lanusse

• Considera ons for extrapola ng in vivo bioequivalence data across species and routes. S Modric, E Bermingham, M Heit, C Lainesse &C Thompson

• Establishing bioequivalence of veterinary premixes (Type A medicated ar cles). RP Hunter, P Lees, D Concordet, & PL Toutain

• Challenges associated with the demonstra on of bioequivalence of intramammary products in ruminants. C. Lainesse, R Gehring, KPasloske, G Smith, S Soback, S Wagner, & T Whi em

BIOWAIVERS

• The scien fic basis for establishing solubility criteria for veterinary species. MN Mar nez & R Fahmy

• Drug solubility classifica on in the dog. MN Mar nez & MG Papich

• Drug solubility classifica on in the bovine. MN Mar nez & MD Apley

• How do you define equivalence of the API of biomass products? RP Hunter, E. Deridder, A. Lucas,KO Smedley & DW Yordy

• Challenges obtaining a biowaiver for topical veterinary dosage forms. R Baynes, J Riviere,T Franz, N Monteiro-Rivere, P Lehman, M Peyrou, & PL Toutain Special Issue:

Bioequivalence, April, 2012, Volume 35, Supplement S1

Fig. 1. Contents of the JVPT supplemental

issue on product BE. BE, bioequivalence.


4 M. N. Martinez

fering mechanisms of action, or when assessing the relationship

between drug exposure and a toxic effect.

Thus, the question is whether or not a paradigm can be estab-

lished to provide greater scientific rigor to our BE assessments in

those situations when blood level BE studies are not feasible.

One possible solution could include a ‘totality-of-the-evidence’

approach, such that described by Yu (2008) and by the 2009

ICH Q8R2 document (Annon1, 2009). This approach incorpo-

rates the concept of quality by design (QbD), which has been

defined as ‘a systematic approach to development that begins

with predefined objectives and emphasizes product and process

understanding and process control, based on sound science and

quality risk management’ (ICH Q8). The foundation of the QbD

approach is a scientific understanding that supports the estab-

lishment of a design space, product specifications, and manufac-

turing controls. To accomplish this goal, the relationships

between formulation and manufacturing process variables

(including drug substance, excipient attributes, and process

parameters) need to be understood and the sources of variability

identified. It is through this understanding that the product

design space is determined. The design space is the multidimen-

sional combination of factors that have been demonstrated to

provide the desirable product quality. It represents an optimized

set of conditions whereby the formulation and process variables

give rise to a product that will have the desired in vivo perfor-

mance in the targeted patient population.

Accordingly, the QbD approach also necessitates a character-

ization of the physicochemical properties of the API. Physical

properties of the API include (Yu, 2008):

• Physical description

○ particle size, shape, and distribution;

○ polymorphism;

○ aqueous solubility as function of pH;

○ hygroscopicity;

○ melting points.

• Chemical properties include

○ pKa;

○ chemical stability in solid state and in solution;

○ photolytic and oxidative stability.

• Biological properties

○ partition coefficient;

○ membrane permeability and/or oral bioavailability.

Once the API has been characterized and the in vivo and in

vitro product performance characteristics have been identified,

a design space can be described in terms of ranges of material

attributes and process parameters within which product qual-

ity and performance attributes can be assured. Operation

within the design space will result in a product meeting the

defined quality and performance (Annon1, 2009).

The QbD paradigm provides a springboard for the develop-

ment of an approach to support the in vitro assessment of drug

products that are not systemically absorbed. This approach

involves a characterization of similarity based upon (Yu et al.,

2003):

Q1: Qualitative similarity – that is, the same components.

Q2: Quantitative similarity – that is, the same amounts of

the same components.

Q3: Structural similarity – that is, the same amounts of the

same components arranged in the same way.

While the assessment of Q1 and Q2 may appear to be rela-

tively straightforward, the characterization of Q3 is more com-

plicated. For example, Q3 includes arrangement of matter and

the state of aggregation. Q3 considerations may include parti-

cle size distribution of a suspension, API polymorphic form and

crystalline habit. Although Q2 implies Q3 for a solution, for

suspensions, creams, ointments, and gels, the physical state

can be impacted by a variety of factors, including the physical

state of the starting materials. In this regard, Noonan (2009)

discussed vancomycin capsules, which act locally in the gut to

treat Clostridium difficile infection and Staphylococcus aureus

enterocolitis in people. He showed an example of how products

meeting the criteria for Q1 and Q2 may not meet the criteria

for Q3, citing differences in molecular weights distribution

across several batches of polyethyleneglycol (PEG) 600. Such

differences could lead to inequivalent product performance,

underscoring the importance of Q3 sameness when evaluating

product performance.

The combination of Q1, Q2, Q3, when evaluated in conjunc-

tion with a battery of in vitro release tests, will likely provide a

more sensitive metric to small changes in product performance

than might otherwise be possible in a typical clinical endpoint

BE trial. Thus, this is a paradigm worthy of further consider-

ation within the scientific community.

Highly soluble drugs

A description of drug solubility is an important starting point

for identifying the variables that can influence in vivo product

performance. Within the human drug arena, the Biopharma-

ceutics Classification System (BCS) has gained tremendous sup-

port as a prognostic tool for identifying the relationship

between product formulation and in vivo product performance

Effect of change in pathogen susceptibility on clinical endpoint decision

0

10

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40

50

60

70

80

90

100

0 2 4 6 8 10 12 14Exposure

Res

pons

e (p

erce

nt s

ucce

ss)

Less susceptiblewild-type responders

FORM A

FORM B

Fig. 2. Changes in response outcome, and therefore conclusions of clin-

ical BE, if there is a shift in the targeted drug exposure (e.g., pathogen

susceptibility) shifts. BE, bioequivalence.



(Amidon et al., 1995). The BCS classifies compounds on the

basis of their solubility and permeability. Given the availability

of high throughput tools for defining human drug solubility

and intestinal permeability, classification can be readily accom-

plished. This in turn has opened the opportunity in human

drugs for the US FDA Center for Drug Evaluation and Research

(CDER), the European Medicines Agency (EMA), and Health

Canada (HC) to employ the BCS as an instrument for identify-

ing compounds eligible for biowaivers (Annon2, 2000; An-

non3, 2008; Annon4, 2012).

Complications emerge when trying to define drug solubility

and permeability in animals. In this regard, a frequently asked

question is: If solubility is an inherent physicochemical prop-

erty of a molecule, why should there be differences in drug sol-

ubility in humans vs. veterinary species? The answer to this

question involves several factors:

• Dose:

○ As veterinary drugs are usually administered on a milli-

gram per kilogram basis, the dose needs to be evaluated

from the perspective of the size of the animal. However,

it is not known if gastrointestinal (GI) fluid volumes scale

in proportion to body weight. This complicates efforts to

establish a relationship between maximum dose and drug

solubility.

○ For some veterinary drugs, the product is administered ad

libitum in feed. Therefore, solubility criteria need to be

evaluated from the perspective of the grazing behavior of

the animal species.

• Volume: pronounced interspecies diversity exists in the phys-

iology of the GI tract. This complicates the paradigm of

assessing solubility at a well-defined dose and some pre-

scribed fluid volume.

• The pH and composition of the GI fluids is highly species-

specific (Martinez et al., 2002) and the range of pH values to

be considered should be tailored accordingly. Additionally,

for certain species (e.g., ruminants), the gastric fluids con-

tain natural surfactants and there is no normal physiological

state comparable to that of a fasted human (Martinez &

Apley, 2012). Furthermore, it may be of value to consider

the potential influence of GI ionic composition when consid-

ering the possibility of ‘common ion effect’ on the solubility

of certain salts (Martinez & Fahmy, 2012). Based upon a

review written by Dressman et al., 1998, the factors that

need to be considered when defining interspecies differences

in drug solubility include:

○ surfactants in gastric juice;

○ viscosity of the luminal fluids;

○ flow rate and fluid turnover rate;

○ GI pH and buffering capability;

○ bile acid composition.

• From a solubility perspective, body temperature itself can dif-

fer across animal species. Whether or not these slight differ-

ences in normal healthy body temperature will influence

drug solubility is a point to consider.

The US Pharmacoepia (USP) has assumed an active role in

assisting the veterinary community in its efforts to address this

challenging issue. This early effort culminated in the publica-

tion of the 2004 Stimuli article (Martinez et al., 2004), which

focused on the application of human BCS principles to the dog.

Within that stimuli article, it was noted that although it might

be feasible to estimate a maximum soluble dose (if the relation-

ship between body size and gastric fluid volume could be deter-

mined), an assessment of drug permeability would be very

difficult to obtain due to the absence of a validated canine

in vitro system. Thus, with respect to permeability, diverse GI

physiologies render it difficult to establish a single, species-inde-

pendent prediction of how a dissolved drug will penetrate the

GI mucosal membranes.

Upon eliminating the permeability parameter from our delib-

erations, this initiative was transformed into one whose goal

was to define ‘fully soluble’ based upon existing human drug

criteria for ‘highly soluble’: that is, the ability to dissolve the

highest dose strength within a set volume of fluid and across a

pH range of 1–7.5 [US FDA (Annon2, 2000)] or 1–6.8 [EMA

(Annon3, 2008) and HC (Annon4, 2012)]. Accordingly, the

2012 USP Stimuli article (Apley et al., 2012) focused on defin-

ing ‘fully soluble’ in a ruminant (cattle) and in a monogastric

species (dog). However, as veterinary drugs are generally

administered on a milligram per kilogram basis, there needed to

be a well-defined fluid volume criterion based upon the

relationship between body size and GI fluid volume. To date,

particularly in the case of dogs whose body weights can range

from 2 kg to over 70 kg, there are no published data that corre-

late GI fluid volume to body weight across the various canine

breeds. Some discrepancies also exist on the fluid volume of the

bovine rumen. The stimuli article not only was an effort to

define the appropriate media composition but also fluid volumes

appropriate for rendering an estimate of drug solubility.

This Stimuli article provided the background for discussions

at the 2012 USP Veterinary Workshop (Annon5, 2012). At

that workshop, the decision was to further limit the focus of

the initiative to species-specific test conditions for estimating

the maximum drug solubility (expressed in mg/mL). In other

words, the issues of dose and GI fluid volumes were now

outside the scope of this USP initiative. In so doing, the focus

evolved into one that is limited to a description of species-spe-

cific biorelevant conditions for estimating the solubility of an

API. Currently, a manuscript summarizing the workshop

outcomes and a corresponding third stimuli article methods on

testing drug solubility in veterinary species are in preparation.

The ultimate goal of this initiative is to develop a USP Gen-

eral Chapter on solubility assessments in veterinary species.

While the first publication will focus on drugs intended for use

in dogs or bovine, subsequently expansion of this chapter is

anticipated to include additional animal species.

Multiple species approvals

Approval of a generic formulation is contingent on the submis-

sion of data necessary to support the acquisition of all indica-


6 M. N. Martinez

tions included in the innovator’s product label. This translates

to the need for the generic drug sponsor to confirm product BE

in every major target animal species included on the approved

(pioneer) product label (Lainesse, 2012). Because of the burden

imposed by such requirements, questions have been raised as

to whether or not a finding of in vivo BE in one target animal

species can be extrapolated to other target animal species.

Unfortunately, within our current toolset, such extrapolations

are not feasible or appropriate for reasons described below.

Orally administered medications. As described in the discussion

on drug solubility, interspecies differences in GI physiology can

impact in vivo product performance. In addition to those

conditions that will influence drug solubility, other species-

specific physiological characteristics that can influence product

relative bioavailability include:

• GI transit time;

• absorptive surface area;

• diet and food effects;

• gastric crushing force;

• potential differences in site of drug absorption.

An example of how these differences can influence in vivo

product performance is exemplified by a comparison of the per-

cent ciprofloxacin absorbed following administration of an

identical formulation of a solid oral dosage form in dogs vs.

human (modeling generated with GastroPlus version 8, soft-

ware Simulations Plus, Inc., Lancaster, CA, USA). The data

used in these modeling examples were collected from an ongo-

ing collaborative study between the University of Maryland

School of Pharmacy and the US FDA Center for Veterinary

Medicine. Figure 3a,b reflect the location of ciprofloxacin

absorption across the human (a) and canine (b) GI tracts.

Upon comparing these two figures, we see several important

interspecies differences in drug absorption kinetics. Firstly, the

majority of the absorption occurred in the duodenum of

the dog as compared to the jejunum and ileum of the human.

The absorption phase was effectively completed as the drug

passed through the jejunum of the dog but continued through

the ascending colon of the human. The drug was nearly 100%

bioavailable in the dog while only 77.3% the administered dose

was absorbed in the human.

When considering these observed differences, one can predict

that a formulation that retards ciprofloxacin release will have

a greater impact on canine systemic exposure as compared to

that likely to occur in humans. In this regard, studies compar-

ing human and canine ciprofloxacin drug absorption for an

oral solution and two formulations (fast and slow release) are

near completion and will be submitted for publication. From a

regulatory perspective, species-by-formulation interactions for

oral dosage forms (i.e., species differences in the expression of

formulation effects) have been observed (M.N. Martinez,

personal unpublished observation), thus supporting the posi-

tion that it will be difficult to scientifically justify the use of

interspecies extrapolations to support decisions of product BE.

Parenteral administered medications. Interspecies differences in

the relative bioavailability of parenterally administered

products have been shown to occur (Lifschitz et al., 1999;

Martinez et al., 2001). Potential reasons for such differences

include (Martinez, 2011):

• Hot melt viscosity

○ differences in injection site physiology;

○ fluid composition;

○ fluid volumes;

○ lymphatic and vascular composition of the injection site;

○ muscle movements;

○ inflammatory responses;

○ differences in injected volumes.

(a)

(b)

Compartmental Absorption

Compartmental Absorption

85

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45

Am

ount

(mg)

Am

ount

(mg)

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00

000010020030040050060070080090100110120130140150160170180190200210220230240

0.0%S

tomach

Duodenum

Jejunum 1

Jejunum 2

lleum 1

lleum 2

lleum 3

Caecum

Asc C

olon

Total

Stom

ach

Duodenum

Jejunum 1

Jejunum 2

lleum 1

lleum 2

lleum 3

Caecum

Asc C

olon

Total

8.6%

0.0%

37.4%

4.2%0.1% 0.1% 0.1% 0.1%

99.9%

0.2%

57.8%

23.8%

16.2%

11.4%7.7%

5.3%

1.1%3.2%

77.3%

Fig. 3. Modeled location of ciprofloxacin absorption in a normal (a)

human and (b) canine subject.



Moreover, differences in drug elimination rate can influence

the degree to which inequivalence in drug absoption rate will

impact peak drug concentrations (Cmax). This point is illus-

trated in the simulated example provided in Fig. 4 where the

interproduct ratios of Cmax values are shown to depend upon

the prevailing elimination rate constant (Ke). In brief, when

the absorption rate constant (Ka) approaches (or is less than)

Ke, differences in drug absorption will have a far greater effect

on comparative Cmax values as compared to situations when

the Ke is much smaller than Ka (that is, the drug products are

very rapidly absorbed relative to the drug elimination rate

constant).

In Fig. 4a, when Ke (the elimination rate constant) equaled

0.1/h, a Cmax ratio of 1.22 was determined when comparing

profiles generated with a Ka value of 0.15/h vs. Ka = 0.25/h.

Conversely, Fig. 4b shows that with a reduction in the Ke

(i.e., a slower elimination rate constant), the same difference

in Ka values (as was used in generating Fig. 4a) now resulted

in only a 6% difference in Cmax (Cmax ratio = 1.06). If we

translate outcome to what might occur if this were linked

with an assessment of in vivo BE across two target animal

species with differing rates of drug elimination, the products

could fail to meet the criteria for one animal species but not

for the other.

Biosimilars

A biosimilar is a biological product that is highly similar to an

already approved biological product, notwithstanding minor

differences in clinically inactive components, and for which

there are no clinically meaningful differences between the bio-

similar and the approved biological product in terms of the

safety, purity, and potency. Biological products are therapies

used to treat diseases and health conditions. Unlike most pre-

scription drugs prepared through chemical processes, biological

products generally are prepared from human and/or animal

materials (Annon6, 2012).

In February, 2012, the FDA, the Center for (human) Drug

Evaluation and Research (CDER) and the Center for (human)

Biologics Evaluation and Research (CBER), issued three draft

guidances on the assessment of ‘biosimilar’ products (Annon7,

2012; Annon8, 2012; Annon9, 2012):

• Quality Considerations in Demonstrating Biosimilarity to a

Reference Protein Product.

• Scientific Considerations in Demonstrating Biosimilarity to a

Reference Product.

• Biosimilars: Questions and Answers Regarding Implementa-

tion of the Biologics Price Competition and Innovation Act

of 2009.

These draft guidances describe a risk-based ‘totality-of-the-

evidence’ approach for evaluating the data and information

submitted in support of a determination of biosimilarity

between proposed vs. marketed reference product.

Within the framework of human medicine, biosimilar prod-

ucts intended for marketing within the U.S. are the subject of a

new drug application (NDA) submitted under section 505(b)(2)

of the U.S. Food, Drug, and Cosmetic Act (the FD&C Act).

Under Section 505(b)(2), comparability of the biosimilar prod-

uct to the listed reference product can include a comparison to

structure, function, animal toxicity, human PK, and pharma-

codynamics (PD), clinical immunogenicity, as well as original

clinical safety and effectiveness data. Thus, the legal framework

of Section 505(b)(2) allows for the submission of an NDA that

contains full reports of investigations of safety and effective-

ness, as needed, but also contains components of the applica-

tion where at least some of the information required for

approval comes from studies not conducted by or for the appli-

cant and for which the applicant has not determined a right of

reference (i.e., a ‘generic-like’ application).

Within the U.S. regulatory control over human drug, regula-

tions are provided under both the Federal Food Drug and Cos-

metic Act (FD&C) and the Public Health Service Act (PHSA). To

be considered a ‘biosimilar’ drug under section 351(k) of the US

PHSA, the application must contain, among other things, infor-

mation demonstrating that ‘the biological product is biosimilar

to a reference product’ based upon data derived from:

• analytical studies that demonstrate that the biological prod-

uct is highly similar to the reference product notwithstand-

ing minor differences in clinically inactive components;

0

10

20

30

40

50

60

Con

cent

ratio

n

Ke = 0.1 Cmax .25/.15 = 1.22

ka = 0.25

ka = 0.2

ka = 0.15

0102030405060708090

100

0 10 20 30 40 50 60

Con

cent

ratio

n

Time

0 10 20 30 40 50 60Time

Ke = 0.01 Cmax .25/.15 = 1.06

ka = 0.25

ka = 0.2

ka = 0.15

(a)

(b)

Fig. 4. Influence of changing the absorption rate constant, Ka from

0.15/h to 0.25/h when the Ke (elimination rate constant) is (a) 0.1/h

and (b) 0.01/h. In both sets of simulations, dose and percent of dose

absorbed did not vary and a one compartment open body model was

employed to generate the simulated profiles.


8 M. N. Martinez

• animal studies (including the assessment of toxicity); and

• a clinical study or studies (including the assessment of

immunogenicity, and PK or PD) that are sufficient to dem-

onstrate safety, purity, and potency in one or more appropri-

ate conditions of use for which the reference product is

licensed and intended to be used and for which licensure is

sought.

The Agency has the discretion to determine that an element

described above is unnecessary. Any aspect of the proposed

product that differ from the listed drug must be supported by

adequate data and information to show that the differences do

not affect the safety and effectiveness of the proposed product.

Of potential interest are some additional thoughts and concerns

regarding biosimilar products from a European perspective that

were recently published Declerck and Simoens (2012).

While the legal tools needed to support the U.S. regulatory

approval of human biosimilar products are contained within

the FD&C Act, no comparable regulations are available for vet-

erinary drug products. Accordingly, absent from within the

regulatory framework of U.S. FDA is the legal pathway for

approving biosimilar veterinary formulations.

Evaluating product BE for highly variable drugs

Efforts to identify statistical methods to evaluate the in vivo BE

of highly variable drugs have continued for more than two

decades (Shah et al., 1996). In general, a drug is considered to

be highly variable when the within-subject percent coefficient

of variation (%CV) is >30 (McGilveray et al., 1990; Blume

et al., 1995; Shah et al., 1996; Haidar et al., 2008). To date,

with the exception of its mention in the draft VICH Blood Level

BE Guideline, there are no stated policies regarding the adop-

tion of scaled statistical approach procedures for use in veteri-

nary medicine.

In an effort to address this gap, Claxton et al. (2012) devel-

oped a white paper addressing the potential application of a

scaled BE approach for use in veterinary species. It was noted

that the current algorithm used in human medicine necessi-

tates the use of an extended crossover study design (i.e., three

or four periods). Such designs can only be conducted in adult

animals whose blood volume and behavior will allow for the

capture of multiple samples without inducing undue stress or

compromising animal health.

Unfortunately, there are many situations encountered in vet-

erinary medicine where the use of an extended crossover

design is not feasible. Furthermore, there are many formula-

tions that are intended to release drug over a very long dura-

tion of time, thereby necessitating the use of a parallel (rather

than crossover) study design. Because between-subject varia-

tion is typically larger than within-subject variability, observ-

ing a %CV ≥30 for AUC or Cmax will not be an unusual event

when using a parallel study design. Therefore, the number of

animals needed to achieve the necessary power to demonstrate

product BE can be prohibitive.

As I have observed (personal observation), the current algo-

rithm for a scaled BE approach (which is based upon within-

subject variance estimates) cannot be readily translated to the

scaling of BE studies that employ a parallel design. This impor-

tant void in our BE tool chest needs to be resolved and clearly,

it will be up to the animal health community to address this

statistical challenge.

Evaluating product BE for long half-life (T½) drugs and/or dosageforms

As the objective of product BE studies is to compare the effects

of formulation on the rate and extent of drug absorption, it is

important to distinguish between rapidly absorbed compounds

that remain in the body for prolonged periods due to their

inherent PK characteristics vs. those compounds where the

extended duration of exposure is a function of product formula-

tion. Potential study design considerations associated with

these two distinctly different situations were considered by

Gehring and Martinez (2012).

For slowly depleting drugs in immediate release formula-

tions, there is a duration of sampling time that will maximize

the precision of the bioavailability comparison. In this regard,

the longer the sampling period in the postabsorptive phase, the

greater the likely variation in AUC values will be. This is due

both to the noise associated with quantification of low drug

concentrations and to the inherent variability of the elimina-

tion phase. For that reason, extending the duration of mea-

surement beyond 3–5 multiples of the T½ may increase the

risk of study failure, particularly when we consider that the

study usually employs a parallel study design.

In contrast, when the long duration of exposure is attribut-

able to formulation effects, sampling should continue for as

long as possible to characterize the formulation effects on prod-

uct performance. Unlike the previous example, in this situa-

tion, the terminal phase is dictated by the rate of drug

absorption, which is the precise formulation property being

investigated. Importantly, in contrast to BE studies on immedi-

ate release products where traditional AUC and Cmax metrics

are appropriate, this may not be the situation for some modi-

fied release formulations. Depending upon the intended charac-

teristics of the product release, it may be preferable to consider

the use of partial AUCs as an estimate of the rate of absorp-

tion.

Partial AUCs may be appropriate in situations when:

• The time to peak concentrations is critical to product perfor-

mance.

• There are multiple maxima.

• The duration of exposure (particularly as it pertains to some

targeted drug concentration) may be an important consider-

ation from a therapeutic perspective.

As a case in point, Endrenyi and Tothfalusi (2010) examined

blood concentration-time profile of the reference modified release

formulation of methylphenidate vs. a marketed generic product



for which there were several accounts of therapeutic failure in

children. That generic formulation was determined to be bio-

equivalent to the reference product based upon an assessment of

AUC and Cmax values. However, upon examination of the profile

shapes, Endrenyi and Tothfalusi observed that the test formula-

tion exhibited a delay in its release. Therefore, even though its

peak and total exposure were equivalent to the reference prod-

uct, the initial portion of the concentration-time profile differed,

and it was that early portion of the profile that was critical for

insuring comparability in terms of the onset of effect. This profile

difference would have been captured if partial AUCs rather than

Cmax were used in the BE evaluation.

While considering the scientific validity for considering the

use of partial AUCs in a BE assessment, we need to also consider

the challenge of determining the appropriate method for curve

segmentation. For example, it may be important to consider the

portion of the curve that is most closely aligned to some clinical

outcome, such as onset of effect or duration of action. However,

the precise timepoints included in the partial areas can substan-

tially alter the resulting test/reference ratios. Even more compli-

cated are those products that exhibit multiple maxima, where

justification for curve segmentation becomes even more arbi-

trary. Furthermore, due to the risk of inflating the Type I error

(failure to identify products as bioequivalent when they in fact

are equivalent), the number of parameters included in the BE

assessment need to be very carefully considered.

Food-by-formulation interactions

The impact of food on drug bioavailability is directly correlated

with the drug’s dose-to-solubility ratio. For low solubility com-

pounds, food intake will enhance its ability to dissolve in the GI

fluids, thereby increasing its oral absorption (Singh, 2005). In

the case of some lipophilic bases (such as propranolol), food

intake can further enhance oral drug bioavailability by reducing

its presystemic metabolic clearance if that compound exhibits

saturable first-pass drug loss (Melander & McLean, 1983). How-

ever, when dealing with the question of product BE, it is impor-

tant to distinguish between food-by-drug interactions (not

formulation-specific) vs. food-by-formulation interactions. From

a BE perspective, our concern is whether or not two products

demonstrated to be bioequivalent in one prandial state continue

to be BE in under the opposite prandial condition.

Examples of food-by-formulation interactions are provided

below:

• Particle size: Changing the particle size of cilostazol, a low

solubility compound was shown to have a marked impact

on oral bioavailability in healthy dogs, with the smaller

particles having substantially higher oral bioavailability as

compared to that of the larger particles (Jinno et al., 2006).

The particle sizes tested included submicron (wet-milled

median particle size = 0.26 lm), jet-milled (2.4 lm), and

hammer-milled (median particle size = 13 lm). When

evaluating the impact of food on the bioavailability of each

formulation, it was observed that while food increased the

oral bioavailability of the larger particles sizes associated

with the jet-milled and hammer-milled products, food

actually resulted in the opposite effect for wet-milled parti-

cles, leading to a slight decrease in oral bioavailability

(Table 1).

• Tablet formulation and strength: In this study, the reference

formulation of the drug nelfinavir (250 mg) was adminis-

tered as four tablets while the test formulation (625 mg)

was administered as two tablets (Kaeser et al., 2005). The

tablets were administered in four-period crossover involving

to 52 healthy male subjects under fed or fasted conditions.

The results of this study showed that food had a markedly

different impact on the oral absorption of the test and refer-

ence formulations (Table 2).

Another question frequently asked is why, in some situations,

food may have a negative effect on oral bioavailability. The

answer reflects the interaction between the physicochemical

properties of the drug molecule, its PK properties, the animal

species being considered, and the site of absorption. Negative

food effects may occur under the following conditions:

Table 1. Evaluating the impact of prandial state on the oral bioavail-

ability of three cilostazol formulations. Numbers provided are the fed/

fasted ratios when the oral bioavailability each of the three particle

sizes were examined in dogs (from Jinno et al., 2006)

Jet-milled Hammer-milled Wet-milled

AUC 3.7 � 0.7 1.8 � 0.6 0.76 � 0.04

Cmax 2.9 � 0.5 2.0 � 0.3 0.91 � 0.13

Table 2. Relative bioavailability of two nelfinavir formulations under fed or fasted conditions. The bottom portion of this table provides the test/ref-

erence ratios for AUC and Cmax when compared under fed vs. under fasted conditions (from Kaeser et al., 2005)

Ref fasted Ref fed Ref fasted/Fed Test fasted Test fed Test fasted/Fed

AUC0–inf (h�ng/mL) 5589 33 390 0.17 4077 31 580 0.13

Cmax (ng/mL) 812 3954 0.21 786 3999 0.20

Test/Ref fasted Test/Ref fed

AUC0–inf 0.73 0.95

Cmax 0.97 1.01


10 M. N. Martinez

• Food will reduce drug absorption of a soluble drug if that

compound is absorbed primarily in the upper small intestine.

This may be due to:

○ Increased volume in GI tract decreases concentration of

drug exposed to absorptive membrane.

○ Increased viscosity decreases interaction of drug and

absorptive membrane. In this regard, Radwan et al.

(2012) determined that a negative food effect is largely

attributed to increased GI fluid viscosity, leading to a

decrease of the API diffusion (D) and a decreased move-

ment of dissolved drug through the aqueous layer (possi-

bly increasing the thickness of the diffusion boundary

layer, d).○ A bile salt-induced decrease i Changing the particle size n

intermicellar ‘free’ drug fraction in the upper intestine

which, in turn, can result in a decrease in drug absorption.

• Food can decrease oral bioavailability due to its effects on

drug degradation, chelation, or first-pass drug loss.

CONCLUDING THOUGHTS

There are a multitude of differences between human and veter-

inary medicine that can influence the application of BE princi-

ples. Unique factors such as methods of drug delivery, multiple

species approvals, prandial state issues, animal physiological

characteristics, husbandry practices, dosages (mg/kg vs. mg

per person or per animal), and study design constraints all ren-

der it necessary for the veterinary community to be willing to

champion its own unique paths for addressing our scientific

challenges. The issues are highly complex, necessitating that

the animal health community secure the involvement highly

trained scientists across a variety of disciplines. This means

that we need to attract young talent who are interested in

exploring these veterinary issues. Given the greater job market

and financial incentives associated with human health, this is

not a simple task.

It also means that we need to establish productive collabora-

tions with our human health counterparts. The technological

advances are tremendous, and we need to insure that we make

full use of the available in silico and in vitro tools to address

our complex issues. The two communities can both learn from

each other, as we each explore the multifactorial set of vari-

ables influencing in vivo product performance. This understand-

ing will facilitate our ability to extrapolate across animal

species and for the human community, to both make better

use of animal models during formulation development and to

identify some of the potential complications that may occur as

a result of human pathologies.

Ultimately, the complexity of issues will only increase with

time as we move forward with the novel drugs and delivery

platforms necessary to address an evolving therapeutic land-

scape. Hopefully, we will succeed in our ability to address some

of the more basic questions raised in this manuscript so that

we can be prepared to meet the challenges of the future.

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12 M. N. Martinez

Documents

Bioequivalence accomplishments, ongoing initiatives, and remaining challenges