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Pharmacogenetic Perspectives in Improving Pharmacokinetic Profiles For Efficient Bioequivalence Trials With Highly Variable Drugs: A Review
Pharmacogenetic Perspectives in Improving Pharmacokinetic Profiles For Efficient Bioequivalence Trials With Highly Variable Drugs: A Review
Pharmacogenetic Perspectives in Improving Pharmacokinetic Profiles For Efficient Bioequivalence Trials With Highly Variable Drugs: A Review
Conducting bioequivalence trials under traditional crossover study designs without exposing a large num-
ber of healthy volunteers to demonstrate two highly variable (%coefficient of variability greater than
30) test/reference (branded) drug products in different formulations to meet the standard 90% confi-
dence interval criteria of relevant pharmacokinetic metrics between 0.80 and 1.25 and to maintain the
consumer risk smaller than 5% has been a challenging task. Genetic polymorphisms encoding key drug-
metabolizing enzymes can significantly influence absorption, distribution, metabolism and elimination
of many highly variable generic drugs after administration. This article briefly reviews the case studies
and examples of utilizing pharmacogenetic screening approaches in the recent literature to alleviate the
resources and ethical burden of recruiting larger numbers of subjects in bioequivalence trials needed to
perform pharmacokinetic studies for formulations of highly variable drug products without widening the
bioequivalence acceptance limits.
First draft submitted: 10 February 2020; Accepted for publication: 5 June 2020; Published online:
30 July 2020
Keywords: bioequivalence • generics • genetic polymorphisms • highly variable drugs • intra-individual variability •
pharmacogenetic • pharmacokinetics
The innovative pharmaceutical companies are granted exclusive marketing rights for carrying a new medicine for a
certain time period after approval by regulatory agencies. After a limited period is concluded, other drug companies
could file an abbreviated new drug application for generic approval to generate their own generic versions via
bioequivalence studies where pharmacodynamics/pharmacokinetic related metrics of the test/reference (branded)
formulations such as area under the concentration (AUC)-time curve and peak plasma concentration (Cmax ) after
drug administration are expected to follow a 90% CI for the average ratios of the systematic exposure measures to
fall within the constraint of 80–125% acceptance limits [1–3]. Commonly, standard bioequivalence trials are carried
out in healthy volunteers as a two-way crossover, randomized design for subjects to take both test and reference
(branded) formulations in consequent periods. For generic drug products with intrasubject variability smaller than
30%, the difference between formulations was basically the determinant of the fulfillment of bioequivalence criteria,
but for those with increasing intrasubject coefficient of variability (ICV) over 30%, ICV instead of formulation
factor became decisive [4]. The larger ICV leads to the wider estimated CI, and it becomes very difficult to remain
within the predetermined bioequivalence limits, despite the fact that an approved medicine with higher intrasubject
variability characteristic normally possess relatively wider therapeutic index. As indicated in Figure 1, examples
exist where a highly variable test product failed to demonstrate bioequivalence when compared with itself in a
bioequivalence study using the standard design/sample size [5]. Therefore, variability is one of the fundamental
10.4155/ipk-2020-0002
C 2020 Pai-Jung Huang, Yunsheng Hsieh, Yan-Wen Huang, Li Int. J. Pharmacokinet. (2020) IPK02 eISSN 2053-0854
Ding, Chong Liu, Bo Shen, Shu-Fen Zhang, Shi-Wei Wu
Review Huang, Hsieh, Huang et al.
detriments of bioequivalence studies on determining whether two dosage forms of the same chemical compound
behave interchangeable in clinical practice. For example, if the intra-individual variability values for AUC and Cmax
measured from a repeat study of the reference formulation of progesterone, a female hormone for the regulation of
ovulation and menstruation, from a few postmenopausal females are over 60 and 95%, respectively, then a generic
company might require dosing in projected 300 postmenopausal women to achieve adequate statistical power for
a standard two-period crossover bioequivalence study.
The ICV in the human pharmacokinetics of a dosed drug can be inherently due to the nature of drug,
the formulation factors of the products, bioanalytical techniques and interaction with other physiologic and
pathophysiologic factors such as administration conditions, transporter, first-pass metabolism and so on [6–16]. A
feasible remedy is to increase the number of study participants in a study and thereby to narrow the CI. However,
the development for high variable (HV) generics will become very expensive and cumbersome. Except for recruiting
large numbers of subjects [17–22], other possible solutions such as using replicating, group sequential study designs [23–
29] and multiple dosing with nonradioactive isotopes [30], have been proposed to deal with the problems of generics
possessing larger root mean square error values of relevant pharmacokinetic metrics in formulations meeting the
bioequivalence limits. Although these approaches take a longer time to complete a replicate design study (with the
potential of increasing subject dropout rate from the trials), the sample size required for a replicate design study
can be reduced up to 50% since study subjects are each employed for twice as many periods.
Davit et al. of the US FDA [5] had reviewed 1010 bioequivalence studies of 180 drugs in 2003–2005 submissions,
of which around 31% (57 out of 180) of the surveyed drugs were highly variable and 10%, 39% and 51% of these
highly variable drugs were classified either borderline, inconsistently or consistently highly variable, respectively, as
a result of the pharmacokinetic characteristics of the drug substances. Analysis of the data revealed that extensive
first-pass metabolism variability was the most important factor over food effect and physicochemical characteristics
contributing to high variability on top of formulation performance, but also influencing the different degree of
variability on both AUC and Cmax in bioequivalence trials. It is well known that pharmacogenetic variation within
an individual could have clinical consequences modulating protein function and hence drug metabolism through
multiple mechanisms. Genetic factors seem to account widely for the activity variation of drug-metabolizing
enzymes among healthy people, potentially making drug metabolism on critical pharmacokinetic end points of test
or reference formulations highly variable. This manuscript reviews proposals and case examples published in the
literature using pharmacogenetic-based methodologies for the efficient bioequivalence evaluation of generic HV
drug products.
Table 1. Summary of bioequivalence trials for highly variable drugs using pharmacogenetic screen.
Study (year) n Drug Genotypes Pharma-cokinetic Study design Polymorphism-altering pharmacokinetic Ref.
and statistical and bioequivalence outcomes
analyses
LLerena (2013), 72 Mirtazapine CYP2D6 Cmax 30 mg two-period cross-over Around1/3 reduction in the minimum [31]
Spain AUC0-120 h studies with a 20-day number of subjects could be achieved
AUC0-∞ washout period re-analyzed by recruiting poor metabolizers
ANOVA in three different genotypes Polymorphisms on AUC variability is less
importance than Cmax in bioequivalence
Abad-Santos 70 Risperidone CYP2D6 Cmax 1 mg two-period cross-over Gender yielded less effect than genetic [32]
(2015), Spain AUC0-24 h studies with a 14-day polymorphisms on within-subject
ANOVA washout period were variability. Genotyping for clinically
reanalyzed and subjects relevant polymorphisms was
were classified into four recommended for subjects participating
different phenotype groups in bioequivalence
Chen (2018), 18 Risperidone CYP2D6 Cmax 4 mg standard 2 × 2 By controlling the genotype to reduce [33]
China AUC0-96 h cross-over with a 2-week intra-individual variability, the test
AUC0-∞ washout period formulation met regulation criteria for
ANOVA bioequivalence
Cho (2006) 24 Risperidone CYP2D6 Cmax 2 mg two-period crossover Two preparations interchangeable [34]
Korea AUC0-∞ with 1-week washout under clinical practice on the basis of
ANOVA period genotypes
Hua (2018), 260 Gefitinib CYP3A4 Cmax 250 mg 2 × 2 cross-over with The activity of metabolizers was [35]
China CYP2D6 AUC0-216 h a washout period of 21 days influenced with drug exposures but not
AUC0-168 h bioequivalence results
AUC0-∞
ANOVA
Cedillo-Carvallo 70 Tolterodine CYP2D6 AUC0-t 2 mg 2 × 2 cross-over design A minimum 30% reduction in sample [36]
(2019), Mexico ANOVA with at least a 3-day size achieved by genotype-based
washout period enrichment strategy
Cedillo-Carvallo 36 Clopidogrel CYP2C19 Cmax 75 mg 2 × 2 cross-over with Selected subjects homozygous for a [37]
(2014), Mexico AUC0-36 h a 7-day washout period certain haplotype increased the
AUC0-∞ stringency of bioequivalence statistics
ANOVA
Chung (2015) 104 Voriconazo-le CYP2C19 Cmax 200 mg 2 × 2 cross-over Genotyping affected both [38]
Korea AUC0-144 h conducted after a single pharmacokinetics and intra-individual
AUC0-∞ intravenous infusion and variability
ANOVA genotype identified and
reanalyzed
Jiang (2013) 24 Citalopram CYP2C19 Cmax 20 mg, 2 × 2 cross-over with The test and reference tablets met the [39]
China AUC0-144 h a 2-week washout period regulatory bioequivalence criteria in the
AUC0-∞ selected healthy male extensive
ANOVA metabolizers
Yong Chung 29 Tacrolimus CYP3A5 Cmax 1 mg, 2 × 2 cross-over with The sample size needed for specific [40]
(2010) S. Korea AUC0-96 h a 21-day washout period genotypes to show bioequivalency can
AUC0-∞ be reduced from nongenotyping cases
ANOVA
Choi (2015) 46 Erlotinib CYP1A1 Cmax 150 mg, 2 × 2 cross-over CYP1A2 alone significantly affected [41]
Korea CYP1A2 AUC0-96 h with a 2-week washout metabolism; no corresponding
CYP3A4 ANOVA period pharmacokinetic variability reported
Peiró (2009) 18 Tenoxicam CYP2C8 Cmax 2 × 2 cross-over with a Phenotyping resulted in a reduced [42]
Spain CYP2C9 AUC0-∞ 3-week washout period pharmacokinetic variability
ANOVA
ANOVA: Analysis of variance; AUC: Area under the concentration; Cmax : Peak plasma concentration; n: Sample size.
pharmacokinetic techniques used for determining if HV test/reference drug products are interchangeable with
minimum number of study subjects are described below.
Study design
Bioequivalence studies were carried out in a standard randomized, open-label, two-treatment, two-period, two-
sequence, crossover study with a washout period of half-life-related time frames and executed by multiple centers
in Hospitals. In each period, subjects randomly received a single oral or an intravenous infusion dose of a test
or a reference drug formulation obtained from different sponsors. Serial blood samples of a certain volume for
bioanalytical assays were taken at predose, and more than 12 certain sampling time points post dose.
Bioanalytical method
Concentrations of the dosed compounds in biological fluids were quantified by validated high-performance liquid
chromatography coupled with mass spectrometry-based methods in compliance with good laboratory practices. All
biological samples were pretreated prior to injection into analytical instruments.
Genotyping method
Clinically relevant polymorphisms among drug-metabolizing enzymes including various allelic variants that might
influence intrasubject variations in the pharmacokinetic profile of individual generics in terms of efficacy and
safety profile were genotyped using the TaqMan allelic discrimination assays. Predose blood samples for genotype
assays were collected in K2EDTA anticoagulation tubes. Genomic DNA was extracted from peripheral blood
in anticoagulation tubes and stored at −80◦ C until PCR analysis for genotyping. The DNA was quantified by
spectrophotometry using validated genotyping assays. The CYP alleles were determined by allele-specific real-time
PCR instrument. Subjects with a certain allelic variant were classified into specific genotype.
CVω = e MSE − 1
Where mean squared error (MSE) is the MSE obtained from analysis of variance or estimated from the study
with replicate administration of the same formulation providing the appropriate variance term for computing CIs
for the difference between genotypes and formulation.
The estimated sample sizes for the bioequivalence study were calculated using the formula based on a multi-
plicative model with a power of 80%, a significance level of 0.05 and a bioequivalence range of 0.80–1.25, as
follows:
2
CVω 2
N≥2× × t α2 ,N−2 + tβ,N−2
0.2
where N is sample number, N-2 the degree of freedom, t the t-test value and α and β statistical errors.
tablets). The pharmacokinetic profiles of risperidone and its active metabolite, 9-hydroxy risperidone, after admin-
istration in study subjects with extensive metabolizers carrying CYP2D6*10 under fasting condition are observed
to be interchangeable. The 90% CI for the geometric mean ratio (test/reference) of logarithm-transformed Cmax ,
AUC0-t and AUC0-∞ was reported to be 91.3–118.1, 95.0–102.3 and 95.0–102.7% for risperidone, respectively,
and 86.4–116.0, 83.8–109.3 and 83.6–108.8% for its active metabolite, 9-hydroxy risperidone to meet the FDA’s
acceptance range. The authors projected that the cost could be saved by at least 69.5% by restricting the genotype
of study subjects to reduce the sample size in the study design for developing a generic risperidone product.
The association of pharmacokinetic variability and pharmacogenomics with the bioequivalence studies of gefi-
tinib, a tyrosine kinase inhibitor and lung cancer drug, provided by three sponsors was previously explored [35]. Two
hundred and sixty male volunteers enrolled were divided into seven bioequivalence studies conducted under fasting
or fed conditions to compare two 250 mg gefitinib tablets from three different generic medication manufacturers
(Test, T formulation) and AstraZeneca Plc (branded, R formulation). Of these studies, two were selected and carried
out for genetic analyses of CYP3A4, CYP3A5 and CYP2D6 alleles to explore the relationship between the PK
outcome and CYP genetic polymorphisms. The results suggested that the pharmacokinetics of gefitinib are highly
variable under fasting conditions. Three gene polymorphisms in various metabolic enzymes were investigated in
two studies and three CYP2D6 rs1058164 genotypes, C/C, C/G and G/G, were found to be active and related
with the drug exposures. For example, it was reported that a 39% reduction in the gefitinib AUC in CYP2D6 UMs
versus EMs was observed. However, these types of IMs did not alter intra-individual variability within the selected
trials and consequently the sample size for each study required to succeed bioequivalency in a standard two-period
crossover study.
Tolterodine used to treat an overactive bladder is metabolized to an active 5-hydroxymethyl tolterodine by
CYP2D6. Byeon et al. [36] conducted a standard bioequivalence study investigating the relationship between
CYP2D6 genotypes and pharmacokinetics of tolterodine. All healthy Korean volunteers receiving 2 mg tolterodine
tartrate were genotyped for CYP2D6 and divided into four different genotype groups as follows: CYP2D6*1/*2,
CYP2D6*1/*10, CYP2D6*10/*10 and CYP2D6*5/*10. The coefficient of variation for each CYP2D6 genotype
group and their corresponding sample sizes required to meet the power of an equivalence test were calculated. The
results showed that the pharmacokinetics and within-subject variability of tolterodine were significantly associated
with CYP2D6 genotypes. According to the intrasubject variation of AUC0-t , only 26, 44 subjects in EM group
and PM group, respectively, compared with around 70 subjects in the conventional study population data set were
needed to meet the regulatory bioequivalence criterion. These results suggested that a drug-metabolizing enzyme
genotype-based enrichment strategy can be implemented to minimize the sample size in bioequivalence studies of
highly variable drug/products.
and CYP2C19 were known as the primary isozymes involved in the N-demethylation of citalopram as an orally
administered antidepressant drug to its active metabolites, desmethylcitalopram. To avoid the additional variables
participants with a CYP2C19 genotype of *1/*1 and *1/*2 along with other inclusion criteria were enrolled to
take part in the study. Consequently, the test/reference formulations evaluated were pharmacokinetically equivalent
within the 90% CI of the ln-transformed values of Cmax , AUC0-t , and AUC0-∞ which met the regulatory criteria
for assuming bioequivalence in the selected healthy male subjects.
Conclusion
This review summarizes all the pharmacogenetic–pharmacokinetic methods available from the literature for
bioequivalence assessment of highly variable drugs/products. The difficulty of fulfilling bioequivalence in HV
drug/products is in general the rather large of sample size using a standard two-period crossover study design.
Herein, the authors presented adopting pharmacogenetic approaches to minimize the within-subject variability
executed in a standard crossover bioequivalence study. It was well-documented that intrasubject pharmacokinetic
variability can be impacted through gene expression regulation at both an individual and population level. How-
ever, application of pharmacogenetics in bioequivalence studies has been considered relatively little up to now.
This article aims to begin with an overview on the ways as a research priority in which use of pharmacogenetic
screen is performed to reduce sample size while maintaining standard study design and bioequivalence acceptance
criteria. We believe that it would be beneficial to further widen pharmacogenetic–pharmacokinetic applications
in bioequivalence evaluation areas considering potential savings to patients in big fortunes after approval of HV
generic products.
Future perspective
The initial attempt of including pharmacokinetics-related pharmacogenetic screen in bioequivalence studies for
generic drug development was to explore whether clinical genetic variation in a subset of the population may
respond differently to the two formulations of highly variable generic products to such an extent that this would
lead to a decline in the recruitment of sample sizes required to meet adequate statistical power. Future implement of
quality by design and appropriate in vitro dissolution testing together with physiologically-based pharmacokinetic
modeling systems to reduce subject variance could certainly result in cost saving by improving the efficiency of
bioequivalence trials and reducing the clinical studies size.
The impact of transporter polymorphism and non-CYP Phase II-metabolizing enzymes [60–63] on altering drug
pharmacokinetics is evident to be of less significance compared with CYP polymorphic enzymes but it should not
be totally ignored as an extension of our knowledge in drug transporters being underway. Future advancement of
new technologies in genome sequencing and completed absorption, distribution, metabolism and elimination gene
analyses might play a significant role in investigating pharmacogenetic–pharmacokinetic–bioequivalence studies
for highly variable generics.
In the case of regulatory discrepancy, for instance, the EMA and the FDA reflect different claims toward selecting
subjects to execute bioequivalence trials [64]. The guidance from the EMA suggested: ‘the subject population for
bioequivalence studies should be selected with the aim to minimize variability’ while the guidance from the FDA
indicated: "in general, ... In vivo bioequivalence study subjects should be representative of the general population,
taking into account age, sex and race..." . There is a lack of global consensus with regard to the inclusion or exclusion
criteria of poor versus EMs as an effective pharmacogenetic approach to further minimize pharmacokinetic intra-
individual variability in support of official bioequivalence designs.
Executive summary
Background & rationale for efficient pharmacogenetic-pharmacokinetic-bioequivalence studies for highly variable
drugs
• In a similar way to employ younger healthy volunteers, pharmacogenetic screen of volunteers might minimize
the subject variability of pharmacokinetic outcomes in human and subsequently increase stringency of
bioequivalence studies.
• Clinical pharmacogenetic–pharmacokinetic–bioequivalence trials may help efficiently recruit smaller subject
numbers executing a standard 2 × 2 design without being outweighed by an increased number of periods to
avoid carryover (from preceding formulation) effects as compared with the replicate designs recommended by
regulatory agencies for highly variable drugs/products.
Existing bioequivalence studies
• Polymorphisms in genotypes influence the pharmacokinetics/bioequivalence results of highly variable
test/reference drugs.
• There is a tendency to employ subpopulations via pharmacogenetically homogeneous selection of study subjects
to allow better clinical pharmacokinetic profiles for bioequivalence outcomes.
• Key CYP enzyme activities are associated with intra-individual variation measures and the degree of sample size
reduction for the bioequivalence trials of HV test/reference formulations.
Open access
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