Spontaneous Risperidone Dosage Is Related to CYP2D6, CYP3A5 and ABCB1 Genotypes

Intuitive pharmacogenetics: spontaneous risperidone dosage is related to CYP2D6, CYP3A5 and ABCB1 genotypes

S Mas1,2,3, P Gasso` 1,2,3, The aim of this study is to evaluate whether the quantitative prescription of ´ 1,2,3 4,2,3 risperidone (dosage) is related to the patient’s metabolic status. Metabolic SAlvarez , E Parellada , status was defined in terms of the most relevant polymorphisms of CYP2D6 4,5,2,3 M Bernardo and (*3, *4, *5, *6 and *1xN), CYP3A5 (*3A) and ABCB1 (G2677T) determined a A Lafuente1,2,3 posteriori and blinded to the clinicians. This prospective and observational study includes a cohort of 151 Caucasian psychiatric patients treated with 1Department of Pathological Anatomy, risperidone. Significant differences (Kruskal–Wallis test p ¼ 0.01) among the Pharmacology and Microbiology, University of doses administered were observed to correlate (Spearman’s r ¼ 1, p ¼ 0.02) Barcelona, Barcelona, Spain; 2Institut d’Investigacions Biome´diques August Pi i Sunyer with the different CYP2D6 groups. Poor metabolizers received the lowest (IDIBAPS), Barcelona, Spain; 3Centro de doses and ultra rapid metabolizers the highest. No significant correlations Investigacio´n Biome´dica en Red de Salud Mental were observed with regard to CYP3A5 and ABCB1. We find that, despite not 4 (CIBERSAM), Barcelona, Spain; Psychiatry knowing patients’ metabolic status, clinicians modify risperidone dosage in service, Hospital Clinic de Barcelona, Barcelona, Spain and 5Department of Psychiatry order to obtain the best therapeutic option. and Clinical Psychobiology, University of The Pharmacogenomics Journal (2012) 12, 255–259; doi:10.1038/tpj.2010.91; Barcelona, Barcelona, Spain published online 21 December 2010

Correspondence: Keywords: pharmacogenetics; risperidone; CYP2D6; CYP3A5; ABCB1; dose titration Dr A Lafuente, Department of Pathological Anatomy, Pharmacology and Microbiology, University of Barcelona, IDIBAPS, Casanova 143, E-08036 Barcelona, Spain. E-mail: [email protected]

Introduction

One of the most important issues regarding the variability of effect of antipsychotic drugs (AP) is the consequences of genetic variation in the regulators of drug metabolism. Concerning the metabolism of AP, the main metabolic pathway for many typical AP is CYP2D6, whereas for atypical AP, the most important CYPs are CYP3A4/A5, CYP2D6 and CYP1A2. CYP2D6 represents an average of 2% of hepatic CYP content and it is one of the best-known polymorphic drug-metabolizing enzymes with over 75 function- ally important allelic variants of CYP2D6 described to date (http://www.imm.ki. se/cypalleles). The CYP3A family, including CYP3A4, CYP3A5, CYP3A7 and CYP3A43, exhibits broad substrate specificity and metabolizes more than 50% of all pharmaceutical drugs.1 The CYP3A5 protein is polymorphically expressed in the liver and intestines, and several genetic variants have been identified (http:// www.imm.ki.se/cypalleles). Individuals with deficient CYP enzyme activity are classified as poor metabolizers (PM) and carry two detrimental CYP alleles. Individuals with slightly subnormal or normal rates of metabolism are defined as either intermediate metabolizers (IM; with only one functional copy) or Received 6 August 2010; revised 20 October 2010; accepted 16 November 2010; published extensive metabolizers (EM; with two functional copies of the gene), respectively. online 21 December 2010 Intermediate metabolizers have higher median metabolic ratios than homozygous Risperidone intuitive pharmacogenetics S Mas et al 256

EM. For CYP2D6, in addition, a subgroup of ultrarapid Hospital Clı´nic (Barcelona, Spain) over a period of 3 years metabolizers (UM) with extremely high enzyme activity can (2002À2004). In total, 151 subjects from this cohort be identified. (diagnosed following the DSM-IV criteria) treated with Risperidone is a widely used atypical AP for the treatment risperidone participated in this retrospective and observa- of schizophrenia and other psychotic disorders. It is tional study. A full description of this population can be extensively metabolized in the liver to a pharmacologically found in previous studies.12–16 active metabolite, 9-hydroxyrisperidone, mainly by cyto- chrome P450 2D6 (CYP2D6) and 3A (CYP3A4/3A5).2–4 Sample preparation and genotyping The antipsychotic effect of risperidone is assumed to The CYP2D6*3, *4, *5 and *6 genotypes were available for be related to the active moiety (the sum of risperidone most of the patients from previous studies.12 The newly and 9-hydroxyrisperidone), of which 9-hydroxyrisperidone included patients were genotyped using the methods constitutes the major part in plasma.5 Many in vivo and described previously.17 in vitro studies have revealed that CYP2D6 is primarily The CYP2D6 gene duplications (*1 Â N) were genotyped involved in risperidone metabolism.4 Polymorphic variants using TaqMan Copy Number Assays (assay ID Hs00010001_cn, of CYP2D6 have been shown to influence risperidone and Applied Biosystems, Foster City, CA, USA). 9-hydroxyrisperidone levels, although not the active moiety.6 The CYP3A5*3 allele and the ABCB1 (G2677T) poly- Several reports have recently suggested that CYP3A may be morphism were detected with real-time PCR by TaqMan substantially involved in the metabolism of risperidone.2 allelic discrimination pre-designed assays from Applied Bio- A polymorphic variant, CYP3A5*3, has been shown to systems according to the manufacturer’s guidelines (assay ID modulate the risperidone, 9-hydroxyrisperidone and active C__26201809_30 and C_11711720C_30, Applied Biosystems). moiety levels.6 Besides the P450 metabolizing enzymes, drug transporters Statistics are believed to affect risperidone pharmacokinetics. Data were analyzed using SPSS14.05 (statistical analysis P-glycoprotein is a member of the adenosine triphosphate- software, SPSS Inc., Chicago, IL, USA). Two-tailed P-values binding cassette (ABC) superfamily of transport proteins, o0.05 were considered to be of statistical significance. functioning as an efflux pump involved in drug absorption Means and s.d. were computed for continuous variables. and elimination.7 It has been reported that risperidone is Variables showing no normal distribution according to the a substrate of P-glycoprotein.8 Although it has not been Shapiro–Wilk test were compared with a non-parametric test studied in humans, it has been demonstrated in rats (Mann–Whitney U-test or Kruskal–Wallis test) and Spear- that risperidone crosses the blood–brain barrier more man’s rank correlation coefficients. Hardy–Weinberg equili- efficiently than 9-hydroxyrisperidone because of the effects brium and linkage disequilibrium analysis were performed of P-glycoprotein transport.9 Thus, P-glycoprotein seems to with the genetics package of the statistical software R (version pump 9-hydroxyrisperidone out of the brain more readily 2.4.0, www.r-project.org). than it pumps risperidone out. This may explain why plasma risperidone is more toxic than comparable plasma Results levels of 9-hydroxyrisperidone, even though they have similar affinities for the D2 receptor.10 A recent study The demographic and clinical characteristics of the patients showed that ABCB1 polymorphisms have a moderate effect are summarized in Table 1. The frequencies of the various on 9-hydroxyrisperidone and active moiety levels.11 metabolic phenotypes based on genotype categories According to our hypothesis, in an intuitive pharmaco- (CYP2D6 UM, EM, IM and PM; CYP3A5 EM, IM and PM) genetic exercise, clinicians (who are unaware of the patient’s and polymorphisms (ABCB1 (G2677T)) studied are also metabolic genotype) modify AP dosage via a trial and error shown in Table 1. All the polymorphisms analyzed were in strategy, in order to obtain the safest and most efficient Hardy–Weinberg equilibrium in cases and controls (data not treatment, and this will correspond with the patient’s shown). metabolic status. The aim of this study is to evaluate Figure 1 shows the mean daily dosage of risperidone in the whether the quantitative prescription of risperidone (do- different CYP2D6 (Figure 1a) and CYP3A5 (Figure 1b) sage) is related to the patient’s metabolic status (considering metabolic phenotypes. Significant differences among (Kruskal– the most relevant polymorphisms of CYP2D6 (*3, *4, *5, *6 Wallis test P ¼ 0.01) and correlation with (Spearman’s r ¼ 1, and *1xN), CYP3A5 (*3A) and ABCB1 (G2677T)), keeping in P ¼ 0.02) the dosage administered to the different CYP2D6 mind that genotyping was performed a posteriori as a part of groups were observed, with UM receiving the highest independent pharmacogenetic studies. risperidone dosage (8.8±5 mg per day, n ¼ 8), followed by EM (7.4±3 mg per day, n ¼ 90), IM (6.5±2 mg per day, n ¼ 37) and PM (5.9±2 mg per day, n ¼ 15). Post-hoc Patients and methods Bonferroni’s testing for pairwise comparisons between groups did not remain significant, probably because of the Participants small sizes of the PM and UM groups. Regarding CYP3A5, A cohort of 321 psychiatric inpatients receiving AP therapy although a trend could be observed with higher dosages for was recruited consecutively at the Psychiatry Service of the patients with higher metabolic activity (EM 7.3±3 mg per

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Table 1 Demographic and clinical characteristics of the patients and frequencies of the various metabolic phenotypes based on the genotype categories (CYP2D6 UM, EM, IM and PM; CYP3A5 EM, IM, and PM) and polymorphisms studied (ABCB1 G2677T)

N 151 Age (years, mean±s.d.) 35.9±14 Male N (%) 84 (55.6) Smokers N (%)a 59 (51.3) Diagnosis N (%) Schizophrenia and related disorders 118 (78.1) Bipolar disorders 27 (17.9) Other 6 (3.9) Risperidone dosage (mg/day, mean±s.d.) 7.2±2.5 CYP2D6 N (%)b PM 15 (9.9) IM 37 (24.5) EM 91 (60.3) UM 8 (5.3)

CYP3A5 N (%)c PM 3 (2.0) IM 23 (15.5) EM 122 (82.4)

ABCB1 G2677T N (%) GG 62 (41.3) GT 59 (39.3) TT 29 (19.3)

Abbreviations: EM, extensive metabolizers; IM, intermediate metabolizers; PM, poor metabolizers; UM, ultrarapid metabolizers. aFor some patients, no information on tobacco use was available (36 cases missing). bPM, carriers of two detrimental alleles *3, *4, *5 or *6; IM, carriers of one detrimental allele *3, *4, *5 or *6; EM, carriers of no detrimental alleles or carriers of one duplication allele, *1 Â N, plus one detrimental allele; UM, carriers of at least one duplication allele *1 Â N. cEM, genotype *1/*1; IM *1/*3; and PM *3/*3. day, n ¼ 122; IM 6.8±3 mg per day, n ¼ 23; PM 5±1 mg per day, n ¼ 3), it proved not to be significant after statistical analysis (Kruskal–Wallis test P ¼ 0.42). When the ABCB1 (G2677T) polymorphism was considered (Figure 1c), a trend towards a higher dosage in patients with higher transporter activity, that is with higher pumping efflux of the drug out of the brain, was observed (homozygote G2677, 7.3±3mg Figure 1 Mean risperidone daily dosage (mean±standard error) ± per day, n ¼ 63; heterozygote G2677T, 7.1 3, n ¼ 59; homo- administered to the different CYP2D61 (a) and CYP3A52 (b) metabolic zygote 2677T, 6.3±4, n ¼ 29), however, it proved not to be phenotypes based on genotype categories and ABCB1 genotypes (c). significant (Kruskal–Wallis test P ¼ 0.24). 1Poor metabolizers (PM), carriers of two detrimental alleles *3, *4, *5or *6; intermediate metabolizers (IM), carriers of one detrimental allele *3, *4, *5or*6; extensive metabolizers (EM), carriers of no detrimental Discussion alleles or carriers of one duplication allele, *1 Â N, plus one detrimental allele; ultrarapid metabolizers (UM), carriers of at least one duplication allele *1 Â N 2EM, genotype *1/*1; IM *1/*3; and PM *3/*3. In our population we found a strong correlation between CYP2D6 status and risperidone dosage. The role of CYP2D6 in the efficacy or the toxicity of risperidone is controversial, This could be due to differences in their rate of permeation mainly due to the pharmacological activity of 9-hydroxy- across the blood–brain barrier. It seems that 9-hydroxyr- risperidone. Although both molecules have similar affinities isperidone is pumped out of the brain more readily than for the dopamine D2 receptor, plasma risperidone levels are risperidone by ABCB1 transporter.10 Regarding CYP3A5 and more toxic than comparable levels of 9-hydroxyrisperidone.9 ABCB1, similar results were obtained, demonstrating that

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Table 2 Comparison of the percentage dose recommenda- leading to more adverse drug effects which could cause non- tions published in the literature and those obtained in the compliance and relapse. present study To introduce CYP pharmacogenetics into clinics, it seems advisable to demonstrate that it is cost-effective. Recently, Metabolic Present Kirchheiuer et al. Rodrı´guez-Antona et al.10 estimated the costs of PM phenotype study (%) (%)19 identification by genotyping in Caucasians at about US$3500, and the costs of extra hospitalization for CYP2D6 PM 80 87 PM patients to be about US$4900. In addition, the authors IM 87 95 EM 100 100 highlight the fact that psychiatric patients typically required UM 119 115 chronic medication and that there are also indirect costs related to adverse reactions, such as productivity losses for Abbreviations: EM, extensive metabolizers; IM, intermediate metabolizers; PM, the patients and their family members. Moreover, it is poor metabolizers; UM, ultrarapid metabolizers. important to note that genotyping tests have the advantage of being performed ‘once in a lifetime’ and that CYP varia- both genes may be important in determining risperidone tion, especially CYP2D6, can explain unexpected side effects clinical outcome. In fact several authors have recently and therapeutic failures of many other drugs (20–25% of claimed that these intervene in risperidone pharmaco- current drugs on the market). The study of the influence kinetics.6,11 of CYP genotyping on the dose titration process of other In our sample, the CYP2D6 status was unknown by the AP (that is, olanzapine and CYP1A2, CYP2D6; clozapine and clinicians, however, they corrected risperidone dosage CY1A2, CYP3A5) will also assist in the pharmacogenetic through an intuitive pharmacogenetic process. The ob- transition into the clinics. served differences in dosing are modest (UM 8.8 mg per day With respect to the CYP2D6 genotyping methods, several and PM 5.9 mg per day), in comparison with the magnitude alternatives have been developed. The AmpliChip from of the differences that would be predicted from pharmaco- Roche (Roche Diagnostics, Indianapolis, IN, USA) covers the kinetic parameters (that is, for a dose of 6 mg per day, the most common alleles leading to the different phenotypes, ratio of risperidone/9-hydroxyrisperidone for a UM was 0.02 including UM in a single analysis, but with a high cost and for a PM 2.418). However, the doses for each CYP2D6 (at least US$600). However, methods to detect PM are not metabolic phenotype in our study are consistent with complex and cheaper (starting at US$250), for example the the percent dose recommendations that appear in the method developed by our group and used in previous literature19 (Table 2). Although the difference in dosage studies.12,17 As in this study, this method could be combined among CYP2D6 metabolic phenotypes is significant, there is with others to identify UM phenotypes, for example TaqMan considerable overlap between categories. This overlap Copy Number Assays from Applied Biosystems. reflects the complexity of the phenotypes with regard to Besides the impact of CYP status on risperidone efficacy drug pharmacokinetics. Whereas the genotype remains (UM phenotypes) or toxicity (PM phenotypes), another unchanged, the phenotype varies over time due to several utility of CYP genotyping is therapeutic drug monitoring factors such as drug and diet interactions, aging, smoking, for control of suspected non-compliance. Schizophrenia hepatic or biliary disease, the course of the disease and the patients may fail to take medication, discontinue therapy duration of the treatment.18 prematurely, or deviate from the prescribed regimen.21 Risperidone treatment, like that with other AP, begins at Multiple objective methods including pill counts, prescrip- the lowest dosage to avoid adverse drug reactions. The tion refill records, therapeutic drug monitoring and techni- dosage is then gradually increased, through a trial and error cal monitoring have been used to assess compliance with strategy, in order to reach efficacy paying special attention medication regimens,22 but no single measure can be to the appearance of adverse reactions. Our results demon- considered the gold standard for all types of compliance strate the suitability of this dose titration strategy. However, research. In the case of drug monitoring, pharmacogenetic it could be improved if pharmacogenetic testing is con- testing, especially CYP2D6 genotyping, has been demon- sidered, as the effective dosage will be achieved quickly strated to improve the sensitivity and specificity of conven- without increasing the risk of adverse reactions (CYP2D6 PM tional monitoring by identifying ‘pseudo’ non-compliant only account for 16% of patients with adverse risperidone patients who have low plasma concentrations as a result of reactions and 9% of those discontinuing risperidone treat- greater metabolic activity.23 ment20) reducing hospitalization costs. Moreover, dose In conclusion, we find evidence that, despite not knowing titration is based on clinical response, and the dosage is patients’ metabolic status, clinicians modify risperidone only changed once the patient fails to respond adequately to dosage in order to obtain the best therapeutic option the drug or has an adverse reaction. In the case of excessive through an intuitive pharmacogenetic process. dosing, adverse reactions may sometimes look like inade- quate dosing (that is, akathisia may seem as agitation, Parkinsonism as deficit symptoms). In such cases, dose Conflict of interest titration could prove to be economically inefficient by extending hospitalization or leading to more disability, or The authors declare no conflict of interest.

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Acknowledgments treated with risperidone: considerations for cost-effectiveness studies. Pharmacogenomics 2009; 10: 685–699. PG was supported by a ‘Sara Borrell’ contract (CD09/00296) 11 Gunes A, Spina E, Dahl ML, Scordo MG. ABCB1 polymorphisms from the Spanish Ministry of Health, Instituto de Salud Carlos III influence steady-state plasma levels of 9-hydroxyrisperidone and risperidone active moiety. Ther Drug Monit 2008; 30: 628–633. (FIS, Fondo de Investigacion Sanitaria). We thank Rosa Abellana 12 Crescenti A, Mas S, Gasso´ P, Parellada E, Bernardo M, Lafuente A. PhD. (Dpt Public Health, University of Barcelona, Casanova 143, CYP2D6*3, *4, *5 and *6 polymorphisms and antipsychotic-induced E-08036 Barcelona, Spain) for statistical assessment, and the extrapyramidal side-effects in patients receiving AP therapy. Clin Exp Language Advisory Service at the University of Barcelona, Spain Pharmacol Physiol 2008; 35: 807–811. for manuscript revision. Funding: This study was supported by the 13 Gasso P, Mas S, Bernardo M, lvarez S, Parelllada E, Lafuente A. A Spanish Ministry of Health, Instituto de Salud Carlos III (FIS, Fondo common variant in DRD3 gene is associated with risperidone-induced de Investigacion Sanitaria P1060182U-2006), the Catalonia Ministry extrapyramidal symptoms. The Pharmacogenomics J 2009; 9: 404–410. of Innovation, Universities and Enterprise (DIUE, 2009SGR1295, 14 Gasso P, Mas S, Crescenti A, Bernardo M, Parellada E, Lafuente A. Lack 2009SGR1501) and SENY foundation. of association between antipsychotic-induced extrapyramidal symptoms and polymorphisms in dopamine metabolism and transport genes. Psychiatry Res 2010; 175: 173–175. 15 Lafuente A, Bernardo M, Mas S, Crescenti A, Aparici M, Gasso P et al. 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