Use of Pharmacogenetics in Bioequivalence Studies to Reduce Sample Size: an Example with Mirtazapine and CYP2D6

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ORIGINAL ARTICLE Use of pharmacogenetics in bioequivalence studies to reduce sample size: an example with mirtazapine and CYP2D6

N Gonza´lez-Vacarezza1, F Abad-Santos2, A Carcas-Sansuan3, P Dorado1,EPen˜ as-LLedo´ 1, F Este´ vez-Carrizo4 and A LLerena1

In bioequivalence studies, intra-individual variability (CVw) is critical in determining sample size. In particular, highly variable drugs may require enrolment of a greater number of subjects. We hypothesize that a strategy to reduce pharmacokinetic CVw, and hence sample size and costs, would be to include subjects with decreased metabolic enzyme capacity for the drug under study. Therefore, two mirtazapine studies, two-way, two-period crossover design (n ¼ 68) were re-analysed to calculate the total CVw and the CVws in three different CYP2D6 genotype groups (0, 1 and X2 active genes). The results showed that a 29.2 or 15.3% sample size reduction would have been possible if the recruitment had been of individuals carrying just 0 or 0 plus 1 CYP2D6 active genes, due to the lower CVw. This suggests that there may be a role for pharmacogenetics in the design of bioequivalence studies to reduce sample size and costs, thus introducing a new paradigm for the biopharmaceutical evaluation of drug products.

The Pharmacogenomics Journal (2013) 13, 452--455; doi:10.1038/tpj.2012.29; published online 26 June 2012 Keywords: CYP2D6; genotypes; intra-individual; variability; bioequivalence; cost

INTRODUCTION bioavailability of 50%, mainly caused by a relevant first-pass In bioequivalence studies of highly variable drugs, intra-individual metabolism. CYP2D6 contributes to about 25% of total clearance variability (CV ) is usually much greater than in other studies, of mirtazapine in subjects with only one active allele and up to w 6 which makes it necessary to substantially increase the number of 55% in the genetically defined ultrarapid metabolizers. Also, it is subjects so as to attain an adequate statistical power. Consistently, known that genetic polymorphisms influence pharmacokinetic a conventional two-way, two-period crossover study with 24 parameters of ( þ )-mirtazapine, with an increase of 79% in the subjects has been found that would not be sufficient to area under the concentration--time curve (AUC) for poor demonstrate bioequivalence.1 Then, to be able to compare the metabolizers (PMs; subjects with 0 CYP2D6 active genes) versus 7 bioavailable dose fraction between two drug products (reference extensive metabolizers (subjects with one or more active genes). and test) with reasonable accuracy and precision, it is important To the best of our knowledge, there has just been a bioequiva- that the subjects’ clearance remains steady through both periods lence study of tacrolimus showing that CVw differed across 8 (Figure 1). In other words, subjects with high CVw in drug CYP3A5 genotypes. Therefore, we re-analysed two studies of bioavailability, could show differences in drug plasma concentra- mirtazapine to evaluate for the first time whether there were tions between drug formulations due to their inherent between- differences between the total CVw and the CVws according to 9 period changes in metabolism, and not fully related to differences different CYP2D6 genotypes (0, 1 and X2 active genes). in the absorption of formulations under comparison.2 Thus, it can be expected that the inclusion of subjects with reduced metabolic enzyme capacity for the drug under study might decrease MATERIALS AND METHODS pharmacokinetic CVw and hence sample size and costs. Detailed information of the methods used in these two studies has been About 25% of the most frequently prescribed drugs are mainly described elsewhere.9 A summary of the methods is presented below. metabolised by CYP2D6 such as mirtazapine. The CYP2D6 gene that codes for CYP2D6 is highly polymorphic, with alleles producing absent catalytic activity.3 In all, 5--10% of Caucasian Study design subjects carry just CYP2D6 inactive gene copies and therefore Bioequivalence studies were carried out in a standard two-period, two- have a genetically determined null enzyme capacity 4,5 and about sequence, crossover randomized design, executed by two centres in 30% may present just one active copy related to decreased Madrid (Spain): the ‘La Paz’ and ‘La Princesa’ University Hospitals. Subjects enzyme capacity. As a consequence, drugs with relevant CYP2D6 received a single 30 mg oral dose of each mirtazapine formulation (Test metabolism, such as mirtazapine, appear as interesting probes to and Reference) in each study period, with a washout period of at least 20 assess the potential effect of genotypes on CVw. days. The Reference formulation used in both Centres was Rexer, Mirtazapine, a noradrenergic and specific serotonergic anti- Laboratorios Organon (Madrid, Spain). Test formulations were from depressant, is a CYP2D6 and CYP3A4 substrate with an absolute Laboratorios Normon SA (Madrid, Spain) and from Laboratorios Alter SA

1CICAB Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz, Spain; 2Service of Clinical Pharmacology, Hospital Universitario de la Princesa, Instituto Teo´ﬁlo Hernando, Instituto de Investigacio´n Sanitaria Princesa (IP), Madrid, Spain; 3Clinical Pharmacology Service, Hospital Universitario La Paz, Pharmacology Department, School of Medicine, Universidad Auto´ noma de Madrid, Madrid, Spain and 4Centre for Biomedical Sciences, University of Montevideo, Montevideo, Uruguay. Correspondence: Dr A Llerena, CICAB, Clinical Research Centre, Extremadura University Hospital and Medical School, Badajoz 06080, Spain. E-mail: [email protected] Received 27 February 2012; revised 10 May 2012; accepted 23 May 2012; published online 26 June 2012 Use of pharmacogenetics in bioequivalence studies N Gonza´lez-Vacarezza et al 453 AUC F × D Cl F = × MSE − T = T × R ≅ T CVW (%) 100 e 1 (I) F × D 2 AUCR ClT R FR ⎛ ⎞ CVW × + 2 N ≥ 2 ×⎜ ⎟ (t /2,N−2 t ,N−2) (II) Figure 1. AUC ratio for oral administration. AUC, area under the ⎝ 0.2 ⎠ concentration-time curve; F, bioavailable dose fraction; D, dose; Cl, clearance; T, Test drug product; R, Reference drug product. Figure 2. (I) Formula for CVw (%) determination. MSE, mean squared error. (II) Formula for sample size estimation in bioequivalence studies. (Madrid, Spain) for the ‘La Paz’ Hospital and ‘La Princesa’ Hospital, respectively.

Table 1. CYP2D6 genotypes and classification according to active Subjects genes In all, 72 healthy volunteers, 36 in each study with a gender ratio of 1:1, were included. All participants were non-smokers, tested negative in a CYP2D6 active drugs-of-abuse urine screen, were not taking any concomitant medication, Genotype Group genes N and were considered healthy after a physical examination, a 12-lead electrocardiogram, urinalysis, haematological and blood chemistry analy- (*1/*1) Â 2 (II) 421 sis, and a review of their clinical history. Body mass index values were *1/*1 (II) 2 32 between 18 and 30. Participants who tested positive for pregnancy were (*1/*4) Â 2 (II) 2 1 excluded from the study. The protocols were approved by the Ethics *1/*9 (II) 2 1 *1/*4 (I) 1 18 Committees for Clinical Investigation of the ‘La Paz’ and ‘La Princesa’ *1/*5 (I) 1 2 Hospitals. *1/*6 (I) 1 4 *4/*9 (I) 1 1 Study protocol *3/*9 (I) 1 1 *4/*6 (0) 0 1 Participants were not allowed to consume alcohol, caffeine, chocolate, tea, *4/*4 (0) 0 6 or cola-type beverages for at least 24 h before each mirtazapine dose, and were not allowed to use any other drug starting 2 days before the Abbreviations: (II), two or more active genes; (I), one active gene; (0), no beginning of the study. active gene. Participants fasted from 10 h before, until 5 h after the administration of the medication, at which time a standard lunch was served. Venous blood samples were collected from each volunteer during each plicative model with a power of 80%, a significance level of 0.05, and a of the study periods from 0 to 96 h or 120 h after drug administration. bioequivalence range (0.80--1.25) using formula II in Figure 2. Physical examination, a 12-lead electrocardiogram, urinalysis, and a Statistical analyses were performed to analyse the pharmacokinetic haematological and blood chemistry analysis were performed parameters and the estimation of sample sizes for the genotyped subjects. not only before inclusion in the study, but also 2 weeks after the second These were distributed into three groups according to their number of period. CYP2D6 active genes: two or more active genes ((II)), one active gene ((I)), Analytical method. Plasma concentrations of mirtazapine were deter- no active gene ((0)). Additionally, analyses were also performed for two mined by high-performance liquid chromatography with a coupled mass additional, created groups: one consisting of the individuals with 0 or 1 spectrometry-validated method, in compliance with good laboratory active gene ((0) þ (I)), and the other of those with 1, 2, or more than 2 practices. active genes ((I) þ (II)). The group (I) þ (II) was considered because of the Genotyping method. Genomic DNA was isolated from whole blood from bimodal distribution of the phenotype groups for CYP2D6 in the Caucasian each subject using the Puregene DNA Isolation Kit (Gentra Systems, population.11 Minneapolis, MN, USA). The DNA was quantified spectrophotometrically The CYP2D6 *3, *4, *5, *6 and *7 variants were considered as non- and stored at 4 1C. The CYP2D6 (*3, *4, *6, *7 and *9) alleles were functional, whereas CYP2D6 *9 was considered active although it is a determined by allele-specific PCR. The alleles with deletion (*5) and variant with reduced activity (Table 1). duplication of CYP2D6 gene were analysed by long PCR. In accordance with standard procedures, the CYP2D6*1 variant allele was assigned as the one lacking the mutations analysed in the CYP2D6 gene. CYP2D6 RESULTS genotyping was only available for 68 of the 72 subjects.8 Genotypes were determined for 68 of the 72 subjects (36 women and 36 men) included in the studies. The CYP2D6 genetic variants Pharmacokinetic and statistical analyses and their classification into three groups based on the number of Peak plasma concentration (Cmax) and time to maximum plasma CYP2D6 active genes are detailed in Table 1. concentration (Tmax) were determined directly from raw data. The AUC0--t Table 2 gives the geometric mean, mean squared error and CVw was calculated by the trapezoidal rule, and AUC0--N was calculated as for each group. In group (II) (n ¼ 35), the CVw of AUC0Àlast, AUC0--t þ C(last)/lz, with C(last) being the last-measured concentration and lz AUC0ÀN and Cmax were 95.1%, 78.2% and 16.4% greater, the slope obtained from least-square regression of the terminal elimination respectively, than in group (0) (n ¼ 7). However, groups (I) phase. (n ¼ 26) and (0) had similar CVw values. An ANOVA was performed on log-transformed data of the pharmaco- The observed CVw differences may influence sample size - kinetic parameters of AUC0Àlast, AUC0ÀN and Cmax using SPSS version 15.0 estimation for bioequivalence studies (Table 3). As Cmax is a (SPSS, Chicago, IL USA). The ANOVA model included product, sequence, limiting factor for determining the sample size, the number of and period as fixed effects, and subject nested within sequence as random subjects could be reduced by 29.2% by recruiting only PM effect. The value of CVw was calculated using formula I in Figure 2. individuals. Similarly, a reduction of 8.3 or 15.3% could be Although this formula does not exactly determine CVw, which might be achieved by including individuals from groups (I) or (0) þ (I), estimated from a study with a replicate administration of the same respectively, as against the total group. On the contrary, taking formulation, it is the appropriate variance term for computing confidence individuals in groups (I) þ (II) or (II) would seem to lead to the intervals for the difference between formulations means.10 The sample opposite tendency, with increases in sample size of 5.6% and sizes for the bioequivalence study were estimated considering a multi- 12.5%, respectively (Table 3).

& 2013 Macmillan Publishers Limited The Pharmacogenomics Journal (2013), 452 -- 455 Use of pharmacogenetics in bioequivalence studies N Gonza´lez-Vacarezza et al 454 Table 2. Analysis of pharmacokinetic parameters for the three groups separately, according to the number of CYP2D6 active genes ((0), (I) and (II)), for the groups including individuals in groups (0)+(I) and (I)+(II) and for the total of individuals regardless of CYP2D6 genotype

(0) (N ¼ 7) (I) (N ¼ 26) (II) (N ¼ 35) (0)+(I) (N ¼ 33) (I)+(II) (N ¼ 61) Total (N ¼ 68)

LnAUC0Àlast Geometric mean (ng h mlÀ1) 899.2 757.8 669.4 785.8 705.8 723.6 MSE 0.00589 0.00711 0.0222 0.00655 0.0163 0.0150 CVw(%) 7.69 8.45 15.0 8.10 12.8 12.3 CI 95% [4.79--18.99] [6.59--11.77] [12.1--19.8] [6.50--10.79] [10.9--15.7] [10.5--14.8] %a 0 9.9 95.1 5.3 66.4 59.9

LnAUC0ÀN Geometric mean (ng h mlÀ1) 947.5 792.8 702.5 823.3 739.6 758.7 MSE 0.00696 0.00758 0.0219 0.00703 0.0174 0.0150 CVw(%) 8.36 8.72 14.9 8.40 13.2 12.3 CI 95% [5.21--20.68] [6.81--12.16] [12.0--19.7] [6.73--11.18] [11.2--16.2] [10.5--14.8] %a 0 4.3 78.2 0.48 57.9 47.1

LnCmax Geometric mean (ng mlÀ1) 80.1 81.1 70.4 80.8 74.7 75.3 MSE 0.0447 0.0481 0.0602 0.0450 0.0554 0.0532 CVw(%) 21.4 22.2 24.9 21.5 23.9 23.4 CI 95% [13.3--55.6] [17.3--31.2] [20.0--33.2] [17.1--28.8] [20.2--29.3] [19.9--28.4] %a 0 3.7 16.4 0.47 11.7 9.3

Abbreviations: CI 95%, 95% confidence interval for CVw; Cmax, maximum concentration; CVw (%), intra-subject coefficient of variation; (0), subjects with no CYP2D6 active gene; (I), subjects with one CYP2D6 active gene; (II), subjects with two or more CYP2D6 active genes; LnAUC0Àlast, log-transformed area under the concentration-time curve from 0 to the last quantifiable concentration; LnAUC0ÀN, log-transformed AUC0ÀN; MSE, mean squared error. aPercentage increment with respect to group (0) CVw(%).

inter-period variability in their own metabolism. In the present Table 3. Estimation of sample size of bioequivalence studies for the study of mirtazapine, recruiting just PMs would give a reduction of three groups separately, according to number of CYP2D6 active genes about 29.2% in the number of subjects required. ((0), (I) and (II)), for the groups including individuals in groups (0)+(I) The present data are given as an example of how it might be and (I)+(II) and for the total of individuals regardless of CYP2D6 possible to reduce the pharmacokinetic CVw, and consequently genotype sample size and costs, by including subjects with decreased metabolic enzyme capacity. For instance, if one assumes an T/R (0) (I) (II) (0)+(I) (I)+(II) Total average total cost of the study per participant of 4000 euros, and theoretical geometric mean ratios of 1.05 and 1.10, then the LnAUC0Àlast 1.05 4 4 13 4 10 9 inclusion of only CYP2D6 PMs would save up to 36 000 and 84 000 1.10 9 9 30 9 22 20 euros, respectively. Similarly, the inclusion of individuals with 0 LnAUC0ÀN 1.05 4 4 13 4 10 9 1.10 10 9 29 9 23 20 and 1 active CYP2D6 gene would save up to 20 000 and 44 000 LnCmax 1.05 23 30 36 27 34 32 euros, respectively. 1.10 51 66 81 61 76 72 Nevertheless, the most important limitation to implementing such a strategy based on the study of CYP2D6 PMs is the low Abbreviations: Cmax, maximum concentration; EM, extensive metabolizer prevalence of this group. Therefore, according to the present data, subjects (I)+(II); LnAUC , log-transformed area under the concentration- 0Àlast it would be possible for mirtazapine studies to also recruit subjects time curve from 0 to the last quantifiable concentration; LnAUC N, log- 0À with one active copy ((0) þ (I) group), because groups (0) and (I) transformed AUC0ÀN. Sample size estimated with the formula II indicated in Figure 2, for have similar values of CVw, and show lower variability in Test:Reference ratios of 1.05 (or 0.95) and 1.10 (or 0.90). (0), poor pharmacokinetic parameters over time than group (II). metabolizer subjects; (I), subjects with one active gene; (II), subjects with Regulatory agencies indicate that bioequivalence studies must two active genes. be conducted in homogeneous but representative samples of the general population, ensuring external validity of the results. This is clearly essential for the analysis of drug safety and efficacy to There were no statistically significant differences between ensure that the results can be generalized to the whole groups in body mass index (P ¼ 0.064), sex (P ¼ 0.305), age population. However, as the main purpose of bioequivalence (P ¼ 0.402), or sequence assignments (P ¼ 0.588). studies is to assess differences among drug products’ bioavailabilities, decreasing intra-individual variability might increase the accuracy of determinations of drug-related factors. DISCUSSION Subjects with absent CYP2D6 activity (PMs or group (0)) have lower pharmacokinetic CVw than groups (I) þ (II), (II) and the total CONFLICT OF INTEREST group. However, no statistically significant differences were found The authors declare no conflict of interest. due to the low number of individuals in this group. PMs would have less drug pharmacokinetic variability between periods, due to the lack of elimination via the CYP2D6 metabolic pathway. ACKNOWLEDGEMENTS Furthermore, this group more accurately reflects differences This work was partially supported by a scholarship of the National Agency for between the bioavailabilities of drug products, due to the lesser Research and Innovation of Uruguay, by the Instituto de Salud Carlos III, and EU

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& 2013 Macmillan Publishers Limited The Pharmacogenomics Journal (2013), 452 -- 455