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

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1 (2010), 1 5 & 2010 Macmillan Publishers Limited. All rights reserved X/10 ORIGINAL ARTICLE Intuitive pharmacogenetics: spontaneous risperidone dosage is related to CYP2D6, CYP3A5 and ABCB1 genotypes S Mas 1,2,3, P Gassò 1,2,3, SÁlvarez 1,2,3, E Parellada 4,2,3, M Bernardo 4,5,2,3 and A Lafuente 1,2,3 1 Department of Pathological Anatomy, Pharmacology and Microbiology, University of Barcelona, Barcelona, Spain; 2 Institut d Investigacions Biomédiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 3 Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Barcelona, Spain; 4 Psychiatry service, Hospital Clinic de Barcelona, Barcelona, Spain and 5 Department of Psychiatry and Clinical Psychobiology, University of Barcelona, Barcelona, Spain Correspondence: Dr A Lafuente, Department of Pathological Anatomy, Pharmacology and Microbiology, University of Barcelona, IDIBAPS, Casanova 143, E Barcelona, Spain. amalialafuente@ub.edu The aim of this study is to evaluate whether the quantitative prescription of risperidone (dosage) is related to the patient s metabolic status. Metabolic status was defined in terms of the most relevant polymorphisms of CYP2D6 (*3, *4, *5, *6 and *1xN), CYP3A5 (*3A) and ABCB1 (G2677T) determined a posteriori and blinded to the clinicians. This prospective and observational study includes a cohort of 151 Caucasian psychiatric patients treated with risperidone. Significant differences (Kruskal Wallis test p ¼ 0.01) among the doses administered were observed to correlate (Spearman s r ¼ 1, p ¼ 0.02) with the different CYP2D6 groups. Poor metabolizers received the lowest doses and ultra rapid metabolizers the highest. No significant correlations were observed with regard to CYP3A5 and ABCB1. We find that, despite not knowing patients metabolic status, clinicians modify risperidone dosage in order to obtain the best therapeutic option. advance online publication, 21 December 2010; doi: /tpj Keywords: pharmacogenetics; risperidone; CYP2D6; CYP3A5; ABCB1; dose titration Introduction Received 6 August 2010; revised 20 October 2010; accepted 16 November 2010 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 functionally important allelic variants of CYP2D6 described to date ( 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 ( 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 extensive metabolizers (EM; with two functional copies of the gene), respectively. Intermediate metabolizers have higher median metabolic ratios than homozygous

2 2 Risperidone intuitive pharmacogenetics EM. For CYP2D6, in addition, a subgroup of ultrarapid metabolizers (UM) with extremely high enzyme activity can be identified. Risperidone is a widely used atypical AP for the treatment of schizophrenia and other psychotic disorders. It is extensively metabolized in the liver to a pharmacologically active metabolite, 9-hydroxyrisperidone, mainly by cytochrome P450 2D6 (CYP2D6) and 3A (CYP3A4/3A5). 2 4 The antipsychotic effect of risperidone is assumed to be related to the active moiety (the sum of risperidone and 9-hydroxyrisperidone), of which 9-hydroxyrisperidone constitutes the major part in plasma. 5 Many in vivo and in vitro studies have revealed that CYP2D6 is primarily involved in risperidone metabolism. 4 Polymorphic variants of CYP2D6 have been shown to influence risperidone and 9-hydroxyrisperidone levels, although not the active moiety. 6 Several reports have recently suggested that CYP3A may be substantially involved in the metabolism of risperidone. 2 A polymorphic variant, CYP3A5*3, has been shown to modulate the risperidone, 9-hydroxyrisperidone and active moiety levels. 6 Besides the P450 metabolizing enzymes, drug transporters are believed to affect risperidone pharmacokinetics. P-glycoprotein is a member of the adenosine triphosphatebinding cassette (ABC) superfamily of transport proteins, functioning as an efflux pump involved in drug absorption and elimination. 7 It has been reported that risperidone is a substrate of P-glycoprotein. 8 Although it has not been studied in humans, it has been demonstrated in rats that risperidone crosses the blood brain barrier more efficiently than 9-hydroxyrisperidone because of the effects of P-glycoprotein transport. 9 Thus, P-glycoprotein seems to pump 9-hydroxyrisperidone out of the brain more readily than it pumps risperidone out. This may explain why plasma risperidone is more toxic than comparable plasma levels of 9-hydroxyrisperidone, even though they have similar affinities for the D2 receptor. 10 A recent study showed that ABCB1 polymorphisms have a moderate effect on 9-hydroxyrisperidone and active moiety levels. 11 According to our hypothesis, in an intuitive pharmacogenetic exercise, clinicians (who are unaware of the patient s metabolic genotype) modify AP dosage via a trial and error strategy, in order to obtain the safest and most efficient treatment, and this will correspond with the patient s metabolic status. The aim of this study is to evaluate whether the quantitative prescription of risperidone (dosage) is related to the patient s metabolic status (considering the most relevant polymorphisms of CYP2D6 (*3, *4, *5, *6 and *1xN), CYP3A5 (*3A) and ABCB1 (G2677T)), keeping in mind that genotyping was performed a posteriori as a part of independent pharmacogenetic studies. Patients and methods Participants A cohort of 321 psychiatric inpatients receiving AP therapy was recruited consecutively at the Psychiatry Service of the Hospital Clínic (Barcelona, Spain) over a period of 3 years ( ). In total, 151 subjects from this cohort (diagnosed following the DSM-IV criteria) treated with risperidone participated in this retrospective and observational study. A full description of this population can be found in previous studies Sample preparation and genotyping The CYP2D6*3, *4, *5 and *6 genotypes were available for most of the patients from previous studies. 12 The newly included patients were genotyped using the methods described previously. 17 The CYP2D6 gene duplications (*1 N) were genotyped using TaqMan Copy Number Assays (assay ID Hs _cn, Applied Biosystems, Foster City, CA, USA). The CYP3A5*3 allele and the ABCB1 (G2677T) polymorphism were detected with real-time PCR by TaqMan allelic discrimination pre-designed assays from Applied Biosystems according to the manufacturer s guidelines (assay ID C _30 and C_ C_30, Applied Biosystems). Statistics Data were analyzed using SPSS14.05 (statistical analysis software, SPSS Inc., Chicago, IL, USA). Two-tailed P-values o0.05 were considered to be of statistical significance. Means and s.d. were computed for continuous variables. Variables showing no normal distribution according to the Shapiro Wilk test were compared with a non-parametric test (Mann Whitney U-test or Kruskal Wallis test) and Spearman s rank correlation coefficients. Hardy Weinberg equilibrium and linkage disequilibrium analysis were performed with the genetics package of the statistical software R (version 2.4.0, Results The demographic and clinical characteristics of the patients are summarized in Table 1. The frequencies of the various metabolic phenotypes based on genotype categories (CYP2D6 UM, EM, IM and PM; CYP3A5 EM, IM and PM) and polymorphisms (ABCB1 (G2677T)) studied are also shown in Table 1. All the polymorphisms analyzed were in Hardy Weinberg equilibrium in cases and controls (data not shown). Figure 1 shows the mean daily dosage of risperidone in the different CYP2D6 (Figure 1a) and CYP3A5 (Figure 1b) metabolic phenotypes. Significant differences among (Kruskal Wallis test P ¼ 0.01) and correlation with (Spearman s r ¼ 1, P ¼ 0.02) the dosage administered to the different CYP2D6 groups were observed, with UM receiving the highest 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 Bonferroni s testing for pairwise comparisons between groups did not remain significant, probably because of the small sizes of the PM and UM groups. Regarding CYP3A5, although a trend could be observed with higher dosages for patients with higher metabolic activity (EM 7.3±3 mg per

3 Risperidone intuitive pharmacogenetics 3 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. a For some patients, no information on tobacco use was available (36 cases missing). b PM, 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 N, plus one detrimental allele; UM, carriers of at least one duplication allele *1 N. c EM, 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 per day, n ¼ 63; heterozygote G2677T, 7.1±3, n ¼ 59; homozygote 2677T, 6.3±4, n ¼ 29), however, it proved not to be significant (Kruskal Wallis test P ¼ 0.24). Discussion 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, mainly due to the pharmacological activity of 9-hydroxyrisperidone. Although both molecules have similar affinities for the dopamine D2 receptor, plasma risperidone levels are more toxic than comparable levels of 9-hydroxyrisperidone. 9 Figure 1 Mean risperidone daily dosage (mean±standard error) administered to the different CYP2D6 1 (a) and CYP3A5 2 (b) metabolic phenotypes based on genotype categories and ABCB1 genotypes (c). 1 Poor metabolizers (PM), carriers of two detrimental alleles *3, *4, *5 or *6; intermediate metabolizers (IM), carriers of one detrimental allele *3, *4, *5 or*6; extensive metabolizers (EM), carriers of no detrimental alleles or carriers of one duplication allele, *1 N, plus one detrimental allele; ultrarapid metabolizers (UM), carriers of at least one duplication allele *1 N 2 EM, genotype *1/*1; IM *1/*3; and PM *3/*3. This could be due to differences in their rate of permeation across the blood brain barrier. It seems that 9-hydroxyrisperidone is pumped out of the brain more readily than risperidone by ABCB1 transporter. 10 Regarding CYP3A5 and ABCB1, similar results were obtained, demonstrating that

4 4 Risperidone intuitive pharmacogenetics Table 2 Comparison of the percentage dose recommendations published in the literature and those obtained in the present study Metabolic phenotype Present study (%) Kirchheiuer et al. (%) 19 PM IM EM UM Abbreviations: EM, extensive metabolizers; IM, intermediate metabolizers; PM, poor metabolizers; UM, ultrarapid metabolizers. both genes may be important in determining risperidone clinical outcome. In fact several authors have recently claimed that these intervene in risperidone pharmacokinetics. 6,11 In our sample, the CYP2D6 status was unknown by the clinicians, however, they corrected risperidone dosage through an intuitive pharmacogenetic process. The observed differences in dosing are modest (UM 8.8 mg per day and PM 5.9 mg per day), in comparison with the magnitude of the differences that would be predicted from pharmacokinetic parameters (that is, for a dose of 6 mg per day, the ratio of risperidone/9-hydroxyrisperidone for a UM was 0.02 and for a PM ). However, the doses for each CYP2D6 metabolic phenotype in our study are consistent with the percent dose recommendations that appear in the literature 19 (Table 2). Although the difference in dosage among CYP2D6 metabolic phenotypes is significant, there is considerable overlap between categories. This overlap reflects the complexity of the phenotypes with regard to drug pharmacokinetics. Whereas the genotype remains unchanged, the phenotype varies over time due to several factors such as drug and diet interactions, aging, smoking, hepatic or biliary disease, the course of the disease and the duration of the treatment. 18 Risperidone treatment, like that with other AP, begins at the lowest dosage to avoid adverse drug reactions. The dosage is then gradually increased, through a trial and error strategy, in order to reach efficacy paying special attention to the appearance of adverse reactions. Our results demonstrate the suitability of this dose titration strategy. However, it could be improved if pharmacogenetic testing is considered, as the effective dosage will be achieved quickly without increasing the risk of adverse reactions (CYP2D6 PM only account for 16% of patients with adverse risperidone reactions and 9% of those discontinuing risperidone treatment 20 ) reducing hospitalization costs. Moreover, dose titration is based on clinical response, and the dosage is only changed once the patient fails to respond adequately to the drug or has an adverse reaction. In the case of excessive dosing, adverse reactions may sometimes look like inadequate dosing (that is, akathisia may seem as agitation, Parkinsonism as deficit symptoms). In such cases, dose titration could prove to be economically inefficient by extending hospitalization or leading to more disability, or leading to more adverse drug effects which could cause noncompliance and relapse. To introduce CYP pharmacogenetics into clinics, it seems advisable to demonstrate that it is cost-effective. Recently, Rodríguez-Antona et al. 10 estimated the costs of PM identification by genotyping in Caucasians at about US$3500, and the costs of extra hospitalization for CYP2D6 PM patients to be about US$4900. In addition, the authors highlight the fact that psychiatric patients typically required chronic medication and that there are also indirect costs related to adverse reactions, such as productivity losses for the patients and their family members. Moreover, it is important to note that genotyping tests have the advantage of being performed once in a lifetime and that CYP variation, especially CYP2D6, can explain unexpected side effects and therapeutic failures of many other drugs (20 25% of current drugs on the market). The study of the influence of CYP genotyping on the dose titration process of other AP (that is, olanzapine and CYP1A2, CYP2D6; clozapine and CY1A2, CYP3A5) will also assist in the pharmacogenetic transition into the clinics. With respect to the CYP2D6 genotyping methods, several alternatives have been developed. The AmpliChip from Roche (Roche Diagnostics, Indianapolis, IN, USA) covers the most common alleles leading to the different phenotypes, including UM in a single analysis, but with a high cost (at least US$600). However, methods to detect PM are not complex and cheaper (starting at US$250), for example the method developed by our group and used in previous studies. 12,17 As in this study, this method could be combined with others to identify UM phenotypes, for example TaqMan Copy Number Assays from Applied Biosystems. Besides the impact of CYP status on risperidone efficacy (UM phenotypes) or toxicity (PM phenotypes), another utility of CYP genotyping is therapeutic drug monitoring for control of suspected non-compliance. Schizophrenia patients may fail to take medication, discontinue therapy prematurely, or deviate from the prescribed regimen. 21 Multiple objective methods including pill counts, prescription refill records, therapeutic drug monitoring and technical monitoring have been used to assess compliance with medication regimens, 22 but no single measure can be considered the gold standard for all types of compliance research. In the case of drug monitoring, pharmacogenetic testing, especially CYP2D6 genotyping, has been demonstrated to improve the sensitivity and specificity of conventional monitoring by identifying pseudo non-compliant patients who have low plasma concentrations as a result of greater metabolic activity. 23 In conclusion, we find evidence that, despite not knowing patients metabolic status, clinicians modify risperidone dosage in order to obtain the best therapeutic option through an intuitive pharmacogenetic process. Conflict of interest The authors declare no conflict of interest.

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