Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine...

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© 2013 S. Karger AG, Basel 1420–8008/14/0372–0058$39.50/0 Original Research Article Dement Geriatr Cogn Disord 2014;37:58–70 Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate Alzheimer’s Disease Nirmal Sonali a Manjari Tripathi b Rajesh Sagar c Thirumurthy Velpandian d Vivekanandhan Subbiah a Departments of a Neurobiochemistry, b Neurology and c Psychiatry, All India Institute of Medical Sciences, and d Department of Ocular Pharmacology and Pharmacy, Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India Key Words Acetylcholinesterase inhibitor · Alzheimer’s disease · Alzheimer therapy · Clinical pharmacology · DNA polymorphisms · Donepezil · Drug therapy · Gene polymorphism · Memantine Abstract Aim: The impact of CYP2D6 and CYP3A4 polymorphism on the steady-state plasma concen- trations and therapeutic outcome of donepezil monotherapy and combination therapy in Alz- heimer’s disease (AD) patients. Methods: A total of 38 patients for donepezil and 17 patients for donepezil and memantine therapy, aged 55 years, were recruited meeting inclusion and exclusion criteria. Polymerase chain reaction-restriction fragment length polymorphism was performed. The liquid chromatography-tandem mass spectrometry method was used for es- timation of drug levels of donepezil and memantine. Results: Significant allele frequency was observed for CYP2D6*3 polymorphism in patients on donepezil monotherapy and combina- tion therapy. Significant allele frequency for CYP2D6*4 was observed in the patients on do- nepezil monotherapy. Conclusion: CYP2D6 polymorphism, though not significant, might par- tially be involved in the plasma concentration of AD drug. © 2013 S. Karger AG, Basel Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder. It is a progressive and fatal neurodegenerative disorder manifested by cognitive and memory dete- rioration, progressive impairment in daily activities, and a variety of neuropsychiatric Accepted: February 11, 2013 Published online: October 3, 2013 Dr. Vivekanandhan Subbiah Department of Neurobiochemistry All India Institute of Medical Sciences New Delhi 110029 (India) E-Mail svivek_aiims @ yahoo.com www.karger.com/dem DOI: 10.1159/000350050 Downloaded by: Nanyang Technological Univ. 155.69.24.171 - 1/31/2017 4:09:51 AM

Transcript of Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine...

© 2013 S. Karger AG, Basel1420–8008/14/0372–0058$39.50/0

Original Research Article

Dement Geriatr Cogn Disord 2014;37:58–70

Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate Alzheimer’s Disease

Nirmal Sonali a Manjari Tripathi b Rajesh Sagar c Thirumurthy Velpandian d Vivekanandhan Subbiah a

Departments of a Neurobiochemistry, b Neurology and c Psychiatry, All India Institute of Medical Sciences, and d Department of Ocular Pharmacology and Pharmacy, Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi , India

Key Words Acetylcholinesterase inhibitor · Alzheimer’s disease · Alzheimer therapy · Clinical pharmacology · DNA polymorphisms · Donepezil · Drug therapy · Gene polymorphism · Memantine

Abstract Aim: The impact of CYP2D6 and CYP3A4 polymorphism on the steady-state plasma concen-trations and therapeutic outcome of donepezil monotherapy and combination therapy in Alz-heimer’s disease (AD) patients. Methods: A total of 38 patients for donepezil and 17 patients for donepezil and memantine therapy, aged ≥ 55 years, were recruited meeting inclusion and exclusion criteria. Polymerase chain reaction-restriction fragment length polymorphism was performed. The liquid chromatography-tandem mass spectrometry method was used for es-timation of drug levels of donepezil and memantine. Results: Significant allele frequency was observed for CYP2D6 * 3 polymorphism in patients on donepezil monotherapy and combina-tion therapy. Significant allele frequency for CYP2D6 * 4 was observed in the patients on do-nepezil monotherapy. Conclusion: CYP2D6 polymorphism, though not significant, might par-tially be involved in the plasma concentration of AD drug. © 2013 S. Karger AG, Basel

Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disorder. It is a progressive and fatal neurodegenerative disorder manifested by cognitive and memory dete-rioration, progressive impairment in daily activities, and a variety of neuropsychiatric

Accepted: February 11, 2013 Published online: October 3, 2013

Dr. Vivekanandhan Subbiah Department of Neurobiochemistry All India Institute of Medical Sciences New Delhi 110029 (India) E-Mail svivek_aiims @ yahoo.com

www.karger.com/dem

DOI: 10.1159/000350050

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59Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

symptoms and behavioral disturbances. AD patients are currently treated with cholinesterase inhibitors, neuroprotective drugs, antidepressants, anxiolytics, antiparkinsonian drugs, anti-convulsants and neuroleptics at a given time of the disease. Donepezil is a piperidine derivative that reversibly inhibits acetylcholinesterase [1] ; it can be taken once daily and has been shown to produce less gastrointestinal side effects and weight loss than the other acetylcholinesterase inhibitors. Many of these substances are metabolized by enzymes known to be genetically variable. Drug metabolism includes phase I reactions (i.e., oxidation, reduction, hydrolysis) and phase II conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). CYP2D6 and CYP3A4/5 are involved in this study. Polymorphic variants in these genes can induce alteration in drug metabolism modifying the efficacy and safety of the prescribed drugs.

Polymorphisms of drug metabolism divide the population into at least two phenotypes, extensive (EM) and poor metabolizer (PM). The allele and genotypic frequency of phase I and phase II metabolizing enzymes has been reported in different populations and revealed wide ethnic variation. However, there are no data on the polymorphisms of drug-metabolizing enzymes in AD patients in India. Donepezil is metabolized mainly by the cytochrome p450 enzymes CYP2D6 and 3A [2] . However, some studies have reported an impact of CYP polymor-phisms on the drug levels in patients on donepezil monotherapy [3–5] . So far, no studies have found a correlation between relevant CYP2D6 and CYP3A4 genotypes in patients undergoing donepezil combination therapy and drug levels and clinical response. Thus, we have evaluated the role of CYP2D6 and CYP3A4 polymorphism on the steady-state plasma concentrations and therapeutic outcome of donepezil monotherapy and combination therapy in AD patients.

Materials and Methods

Patient Recruitment A total of 38 patients (25 male and 13 female) for donepezil and 17 patients (13 male and 4 female) for

donepezil and memantine therapy, aged ≥ 55 years, with mild to moderate AD were recruited from the outpa-tient Department of Neurology, Neuroscience Centre, All India Institute of Medical Sciences, New Delhi, India. All age-matched controls (n = 40) were collected from the hospital staff and their relatives or from unrelated attendants of patients for allele and genotypic frequency comparisons.

Standard Protocol Approvals, Registrations and Patient Consents The approval of the study was obtained from our institute ethics committee on human experimentation.

Written informed consent was obtained from each patient and controls or from the relatives in case of criti-cally disabled persons with dementia.

Inclusion Criteria for Patients The following inclusion criteria were used: (1) North Indian subjects; (2) diagnosis of probable AD

according to the NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association) criteria [6] , DSM-IV and responder or nonresponder status defined by the Mini Mental State Examination (MMSE) [7] with mild to moderate disease severity, and (3) having taken the acetylcholinesterase inhibitor donepezil (monotherapy and combi-nation therapy with the NMDA antagonist, memantine) for at least 4 weeks.

Exclusion Criteria for Patients The following exclusion criteria were used: (1) unwilling or unable to fulfill the requirements of the

study; (2) had clinically significant and unstable medical illness/had undergone medical/surgical hospital-ization within 1 month before the study; (3) known or suspected history of dementia secondary to abuse of psychoactive substances; vision and hearing sufficient for compliance with testing procedures; (4) labo-ratory values within normal limits; (5) comorbidity of primary psychiatric (i.e., schizophrenia, major depression onset before the AD onset) or neurological disorders (i.e., stroke, Parkinson’s disease, seizure disorder, or head injury with loss of consciousness within the past year), and (6) known suspected history

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60Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

of alcoholism or drug abuse. Patients suffering from diabetes mellitus, hyperlipoproteinemia, cancer, sickle cell anemia, and liver disease were also excluded from the study.

Inclusion Criteria for Controls Inclusion criteria were: (1) the healthy, normal subjects were neither related to one another nor to AD

patients; (2) we selected all age-, ethnicity- and sex-matched controls in the range of 55–90 years, and (3) the MMSE score was well above the limit for the normal cognitive level and not satisfying DSM-IV and NINCDS-ADRDA criteria for diagnosis of AD.

Exclusion Criteria for Controls Exclusion criteria were: (1) controls taking any form of medication or having had surgery or suffered

trauma in the past 30 days were excluded from the study; (2) unwilling or unable to fulfill the requirements of the study, and (3) controls with a history of bleeding, thrombotic or cardiac disorders were also excluded from the study.

Responder/Nonresponder Assessment Criteria According to the NICE (National Institute for Health and Clinical Excellence) requirements, a responder

was defined as a patient who showed improvement or no deterioration in cognition as evaluated by means of the MMSE (National Health Service Constitution, 2009, http://www.nice.org.uk). Treatment adherence was monitored by supervised pill count after dispensing the drug.

Blood Collection In the case of both patients as well as controls, 6-ml peripheral blood samples were collected in 2%

EDTA as well as plain vials. Plasma was isolated within 2 h by centrifugation at 4 ° C and 10,000 rpm for 20 min and stored at –20 ° C for drug analysis and other tests. Peripheral blood leukocytes were also stored at –20 ° C for extracting DNA and performing the molecular tests.

Genetic Analysis Genomic DNA was extracted from 2 ml blood through the phenol-chloroform method. Polymerase chain

reaction-based restriction fragment length polymorphism assay was performed for phase I drug-metabo-lizing enzymes with the primers and restriction enzymes as already specified [8, 9] . Sequencing was carried out commercially through Xcelris, Ahmedabad, India, to confirm the restriction fragment length polymor-phism results.

Determination of Donepezil and Memantine The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for the analysis of

donepezil and memantine with minor modifications [10, 11] . For the quantification of donepezil and memantine, an isocratic mobile phase containing 70% acetonitrile with 0.1% formic acid and 30% aqueous 10 m M ammonium acetate containing 0.1% formic acid was used. The mobile phase was 0.22-μm filtered, online degassed and pumped at a rate of 0.5 ml/min. The samples were loaded into a 96-well tray and injected using autosampler kept at 20 ° C. The total LC run time was 5 min and 20 μl of the sample was subjected to quantification. Using the Turbo Ion Spray (ESI) source, the chromatographic eluents were subjected to ionization in the positive ion mode at 5,500 V. Quantification of donepezil was performed using the multiple reaction monitoring mode, based on parent/product ion transition 1 (380.5/91) and transition 2 (380.5/288.3). For memantine, quantification was performed using the multiple reaction monitoring mode, based on parent/product ion transitions 180.2/163.2. The transitions for homatropine were 276.1/142. The main instrument parameters of the mass spectrometer are as follows: source-dependent parameters were gas 1 (60 psi); gas 2 (40 psi); curtain gas (20 psi); ion spray voltage (5,500 V), and temperature (400 ° C). Compound-dependent parameters for analyte such as declustering potential, entrance potential, collision energy and cell exit potential were set at 109, 10, 61 and 15 V for tran-sition 1 and 47, 9, 35 and 13 for transition 2, respectively. For memantine, compound-dependent parameters were set at 61, 8, 21 and 8 V. Collision-activated dissociation and dwell time was kept at 8 (arbitrary value) and 100 ms. Homatropine was used as an internal standard for both donepezil and memantine.

Standard Preparation Stock solutions of donepezil and memantine were prepared in blank plasma. The stock concentration

of 1 mg/ml was used for all 3 drugs. This stock solution was appropriately diluted with blank plasma to reach

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61Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

the required concentrations ranging from low to high. The standard calibration curve was prepared by taking 20 μl of standard solution from serial dilutions and was extracted using 200 μl of extraction solvent. The above mixture was vortexed for 1 min using a cyclomixer and subjected to centrifugation at 10,000 rpm for 10 min. The clear supernatant of 100 μl was transferred to 96-well plates and subjected to quantification using the LC-MS/MS method. Stock solution (1 mg/ml) of internal standard was prepared by dissolving homatropine in 50% methanol. It was further diluted to reach 500 ng/ml in the extraction solvent containing 70% acetonitrile with 0.1% formic acid.

Sample Preparation Twenty microliters of plasma samples were extracted using 200 μl extraction solvent, vortexed for 1 min

using a cyclomixer and subjected to centrifugation at 10,000 rpm for 10 min. Extraction solvent consisted of 70% acetonitrile containing 0.1% formic acid and 500 ng/ml homatropine (internal standard). The clear super-natant of 100 μl was transferred to 96-well plates and subjected to quantification using the LC-MS/MS method.

Statistical Analysis Statistical analysis was carried out using Stata 9.0 (College Station, Tex., USA). Data are presented as

number (percentage) or mean ± SD/median (range) as appropriate. Demographic and clinical characteristics of patients between the drug groups were compared using the Fisher exact test (categorical variable) and one-way ANOVA (continuous variable). The difference in allele and genotypic frequency was compared between AD patients and controls using the χ 2 test. The effect of each of the gene polymorphism on AD patients compared to controls was found out using logistic regression analysis. The results were reported as OR (95% CI). The difference in median drug levels and MMSE score among the 0, 1 and 2 mutant alleles in each of the genotypes was tested using the Kruskal-Wallis test. p < 0.05 was considered statistically significant.

Results

Demographic and Clinical Characteristics Patients with AD enrolled in this study are summarized in table 1 . A total of 55 patients

(38 male and 17 female) with AD (38 donepezil monotherapy and 17 donepezil and memantine combination therapy) were recruited. The mean age at onset and standard deviation of each therapy group was 64.28 ± 7.90 and 64.58 ± 11.07 years, respectively. The change in MMSE score was not found to be significant.

Genotypes Polymerase chain reaction-restriction fragment length polymorphism was performed for

CYP2D6 * 2 (2850C>T), CYP2D6 * 3 (2549A>del), CYP2D6 * 4 (1846G>A), CYP2D6 * 10 (100C>T), CYP2D6 * 17 (1023C>T), CYP3A4 * 4 (1389A>G), CYP3A4 * 5 (15820C>G) and CYP3A4 * 18 (293T>C) phase I drug-metabolizing enzymes. Allele and genotype distribution was found between AD patients and controls. All these polymorphisms were polymorphic in our study and were seen to be in Hardy-Weinberg equilibrium (cutoff p < 0.01). The genotypic and allelic frequencies of these variants for the donepezil monotherapy and combination therapy with memantine between AD patients and controls are given in tables 2 and 3 . Regarding the CYP2D6 polymorphism in patients on donepezil monotherapy, 6 (15.7%) patients carried 0 mutant alleles, 9 (23.7%) carried 1 mutant allele and 23 (60.5%) carried 2 mutant alleles. Considering the CYP3A4 polymorphism in the same group, 2 (5.3%) patients carried 0 mutant alleles, 9 (23.5%) carried 1 mutant allele and 27 (57.9%) carried 2 mutant alleles. In the done-pezil and memantine combination therapy group, no patients had 0 mutant alleles for the CYP2D6 polymorphism, 5 (29.4%) had 1 mutant allele and 12 (70.6%) carried 2 mutant alleles. Similarly, no patients carried 0 mutant alleles for the CYP3A4 polymorphism, 4 (23.5%) patients were found to have 1 mutant allele and 13 (41.2%) carried 2 mutant alleles.

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62Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

Correlation of Drug Levels and Genotypes The patients were classified as responders and nonresponders according to the MMSE

scores. Nonresponders had higher concentration-to-dose (C/D) median plasma drug levels than responders and hence we wanted to confirm that it was due to the combined effect of the gene or due to a single gene variation. Thus the patients were classified according to the genotypes in monotherapy and combination therapy of donepezil and correlated with the drug levels. Clinical outcome of the drugs was demonstrated by means of the MMSE score (MMSE score at 4 weeks – MMSE score at 16 week). A higher negative score indicates better clinical response to the drug. Patients were divided into 3 groups by the number of mutant

Demographic characteristics Donepezil Donepezil and memantine

Age at onset, years 64.28 ± 7.90(n = 38)

64.58 ± 11.07(n = 17)

Male/female 25/13 13/4MMSE score at baseline (4 weeks) 19.42 ± 5.40

(n = 38)19.35 ± 5.96(n = 17)

MMSE score (16 weeks) 20.51 ± 5.84(n = 35)

18.92 ± 5.32(n = 14)

Weight, kg 75.44 ± 9.35(n = 38)

78.29 ± 7.48(n = 17)

Systolic pressure, mm Hg 127.83 ± 11.33(n = 38)

125 ± 7.97(n = 17)

Diaystolic pressure, mm Hg 83.72 ± 6.63(n = 38)

81.6 ± 9.24(n = 17)

Values are mean ± SD.

Polymorphism Patients(n = 38)

Controls(n = 40)

pvalue

OR (95% CI)

CYP2D6*22850C>T

C = 0.64T = 0.35

C = 0.65T = 0.35

0.952 0.97 (0.48 – 1.98)

CYP2D6*32549A>del

A = 0.55Adel = 0.44

A = 0.80Adel =0.20

0.0009* 0.30 (0.14 – 0.66)

CYP2D6*41846G>A

G = 0.78A = 0.21

G = 0.58A = 0.43

0.001* 2.7 (1.29 – 6.03)

CYP2D6*10100C>T

C= 0.73T = 0.26

C = 0.63T = 0.37

0.13 1.68 (0.80 – 3.53)

CYP2D6*171023C>T

C = 0.57T = 0.42

C = 0.61T = 0.39

0.66 0.86 (0.43 – 1.73)

CYP3A4*41389A>G

A = 0.51G = 0.48

A = 0.59G = 0.41

0.35 0.74 (0.37 – 1.46)

CYP3A4*515820C>G

C = 0.52G = 0.39

C = 0.66G = 0.34

0.25 0.67 (0.33 – 1.38)

CYP3A4*18293T>C

T = 0.85C = 0.14

T = 0.74C = 0.26

0.06 2.10 (0.87 – 5.24)

* p < 0.05 was considered significant.

Table 1. Demographic and clinical characteristics

Table 2. Allele frequency of phase I drug-metabolizing enzymes in patients on donepezil monotherapy and controls

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63Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

Polymorphism Patients(n = 17)

Controls(n = 40)

pvalue

OR (95% CI)

CYP2D6*22850C>T

C = 0.74T = 0.26

C = 0.65T = 0.35

0.37 1.49 (0.57 – 4.14)

CYP2D6*32549A>del

A = 0.62Adel = 0.38

A = 0.80Adel =0.20

0.04* 0.40 (0.15 – 1.08)

CYP2D6*41846G>A

G = 0.62A = 0.38

G = 0.58A = 0.43

0.67 1.18 (0.48 – 2.9)

CYP2D6*10100C>T

C= 0.71T = 0.29

C = 0.63T = 0.38

0.42 1.44 (0.56 – 3.84)

CYP2D6*171023C>T

C = 0.56T = 0.44

C = 0.61T = 0.39

0.59 0.80 (0.32 – 1.97)

CYP3A4*41389A>G

A = 0.82G = 0.18

A = 0.59G = 0.41

0.17 1.66 (0.75 – 3.74)

CYP3A4*515820C>G

C = 0.56G = 0.44

C = 0.66G = 0.34

0.29 0.64 (0.26 – 1.60)

CYP3A4*18293T>C

T = 0.62C = 0.26

T = 0.74C = 0.26

0.69 0.83 (0.30 – 2.40)

* p < 0.05 was considered significant.

Table 4. CYP2D6 and CYP3A4 frequency, C/D donepezil and MMSE change for monotherapy

Mutant allele Number(n = 38)

Frequency,%

C/D donepezilmedian (range)

pvalue

MMSE changemedian (range)

pvalue

CYP2D6 (*2, *3, *4, *10, *17)Homozygous EM 6 15.7 0.36 (0.16 – 1.31) 0.53 0 (–2 to 3) 0.39Heterozygous EM 9 23.7 0.25 (0.15 – 1.00) 0 (–6 to 4)PM 23 60.5 0.29 (0.08 – 0.811) –2 (–10 to 4)

CYP3A4 (*4, *5,*18)Homozygous EM 2 5.3 0.20 (0.13 – 0.27) 0.03* –2 (–4 to 0) 0.68Heterozygous EM 9 23.5 0.38 (0.5 – 0.70) –2.5 (–5 to 2)PM 27 57.9 0.28 (0.08 – 1.32) –1.5 (–10 to 4)

* p < 0.05 was considered significant.

Table 5. CYP2D6 and CYP3A4 frequency, C/D donepezil and MMSE change for combination therapy

Mutant allele Number(n = 17)

Frequency,%

C/D donepezilmedian (range)

pvalue

MMSE changemedian (range)

pvalue

CYP2D6 (*2, *3, *4, *10, *17)Heterozygous EM 5 29.4 0.35 (0.14 – 0.62) 0.29 0 (–8 to 6) 0.78PM 12 70.6 0.51 (0.24 – 1.20) –1 (–4 to 5)

CYP3A4 (*4, *5,*18)Heterozygous EM 4 23.5 0.45 (0.26 – 1.00) 0.52 –3 (–8 to 5) 0.21PM 13 41.2 0.56 (0.14 – 1.20) –1 (–3 to 6)

Table 3. Allele frequency of phase I drug-metabolizing enzymes in patients on donepezil and memantine combination therapy and controls

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64Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

alleles. Each polymorphism has 3 groups wherein subjects having 0, 1 or 2 mutant alleles are homozygous EM, heterozygous EM or PM, respectively ( tables 4 , 5 ). These classifications were done according to Varisaldi et al. [3] .

Donepezil Concentrations in the Monotherapy and Combination Therapy Groups Considering CYP2D6 and CYP3A4 Polymorphisms Donepezil concentration in the plasma ranged from 0.082 to 1.32 ng/ml for the donepezil

monotherapy group (10 mg/day), and from 0.146 to 1.20 ng/ml for the donepezil combi-nation therapy group. The drug level for 0 mutant alleles was found to be 0.36 (0.16–1.31) with a poor clinical response to donepezil for the CYP2D6 polymorphism. But the drug levels were comparatively higher for 1 mutant allele with a better clinical response, and this was found to be statistically significant for patients with the CYP3A4 polymorphism on donepezil monotherapy ( fig. 1 , 2 ).

The drug levels for 2 mutant alleles were found to be higher for patients on donepezil combination therapy with memantine, but patients showed a better clinical response with the CYP2D6 polymorphism as compared to the CYP3A4 polymorphism ( fig. 3 , 4 ). However, this was not found to be statistically significant.

Discussion

In the present study, a significant allele frequency was observed for the CYP2D6 * 3 poly-morphism in patients on donepezil monotherapy [A>del = 0.45 (patients) and 0.20 (controls)] and donepezil and memantine combination therapy [A>del = 0.38 (patients) and 0.20 (controls)]. Significant allele frequency for CYP2D6 * 4 (p = 0.001) polymorphisms was observed for the patients on donepezil monotherapy. The mutant allele frequency among controls for CYP2D6 * 3 and * 4 was found to be 0.08 and 0.17, respectively. Even though a higher frequency (0.14) was found for CYP2D6 * 2 and * 10 in our study, this was not statistically significant among our AD patient and control population. However, these data are distinct from South India where a genotype study of 447 residents of 4 South Indian states revealed a polymorphic distribution that differed somewhat from that seen in Caucasians, those of African descent, and Far East Asians [12] . The high-functioning alleles, * 1 and * 2, were most prevalent in these states, while the frequency of the intermediate * 10 allele was found to be 10%, whereas it was 5% in Africans and up to 70% in Far East Asians. The PM alleles, * 3, * 4, and * 5, were present at low frequencies, leading to the suggestion that PM rates in the South Indian states are below 5%. PM in North India has been reported to occur at a frequency of 3% [13] in contrast to South India. In one of the studies from north Delhi on schizophrenia, the frequency of CYP2D6 * 4 in the general population was G = 0.881; A = 0.119, comparable to the report of a Spanish popu-lation but notably different from reports of other populations (German, Saudi Arabian, Zimba-bwean, Ethiopians and Chinese [14] ). Further, a study from North India on Parkinson’s disease patients demonstrated a significant allele association of CYP2D6 * 4 with Parkinson’s disease patients when compared with controls (28 and 15.4%, respectively) [15] . Consistent with earlier studies in Caucasian and Japanese populations [16–18] , the present study has shown significance of AD in cases carrying the CYP2D6 * 4 (1846G>A) polymorphism, present at the intron 3/exon 4 boundary. Interestingly, the frequency of the variant allele (A) of this poly-morphism, which accounts for 70–90% of all PMs in Caucasians (0.20–0.25), was relatively less in our control population (0.17) but still higher when compared to African, African-American (0.06–0.07) and other Asian (Oriental) populations (0.01) [19] . The intestinal epithelium and liver contain the most abundant member of the CYP family, namely CYP3A, and this enzyme is responsible for the metabolism of more than half of the therapeutic drugs [20–

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65Dement Geriatr Cogn Disord 2014;37:58–70

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Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

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Fig. 1. Relationship between the CYP2D6 genotype and C/D plasma concentration for subjects on donepezil monotherapy. Horizontal lines represent median values.

Fig. 2. Relationship between the CYP2D6 genotype and C/D plasma concentration for subjects on donepezil combination therapy. Horizontal lines represent median values.

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66Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

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Fig. 3. Relationship between the CYP3A4 genotype and C/D plasma concentration for subjects on donepezil monotherapy. Horizontal lines represent median values.

Fig. 4. Relationship between the CYP3A4 genotype and C/D plasma concentration for subjects on donepezil combination therapy. Horizontal lines represent median values.

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67Dement Geriatr Cogn Disord 2014;37:58–70

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Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

22] . In addition, this enzyme may undergo induction (rifamycins) and inhibition (calcium channel blockers) depending on the drug administration, which may account for its poor or higher metabolic activity. It plays important roles in the metabolism of a wide variety of drugs such as antidiabetics, antiarrhythmics, antihistamines and synthetic estrogens [23] . Variant CYP3A4 alleles in the population may contribute to interindividual variability in CYP3A4 activity. Among Asian subjects, a number of allelic variations in the CYP3A4 gene are known to affect catalytic activity including CYP3A4 * 4, CYP3A4 * 5 and CYP3A4 * 18 [9, 24–26] . In our study, no significant allele frequency was observed for the CYP3A4 polymorphism, even though a genotypic association for CYP3A4 * 4 and * 5 between patients and controls was found. This is in accordance with a genotype and phenotype study done in a North Indian population which demonstrated the absence of CYP3A in North Indians on the basis of frequency distri-bution of the urinary 6β-OH-CS/CS ratio and the absence of the variant alleles CYP3A4 * 2, * 4, * 5, * 6 and * 10, which are common in other ethnic groups [27] . Incidences of these mutations in Chinese subjects are rare [9] .

Drug Metabolizers and Plasma Concentration In this study, only highly selective patients with mild to moderate AD were enrolled.

Patients were treated for 3 months, and response to the treatment was defined conserva-tively according to the NICE criteria as patients who show improvement or no deterioration in cognition and improvement in functional status (The National Health Service Constitution). The genotypes were correlated with the drug levels according to MMSE classification and according to the genotypes (0, 1, or 2 mutant alleles) [4, 28] . In this study, a large interindi-vidual variability in the C/D ratio of donepezil monotherapy was observed, and this vari-ability was significantly correlated with the CYP3A4 genotypes for patients on donepezil monotherapy. Responders and nonresponders having heterozygous genotypes of CYP3A4 ( * 4, * 5) [0.29 (responders), 0.57 (nonresponders); 0.36 (responders), 0.61 (nonresponders), respectively] and being on donepezil monotherapy were found to have significantly high drug levels. Nonresponders among those with CYP3A4 had significantly higher drug levels as compared to the responders. Very few mutant types but a higher amount of heterozygotes were found among the controls and patient group. It was observed that it is a very rare variant which occurs <0.5% in the Indian population and could therefore represent a rare single gene defect, rather than an actual polymorphism. This explains that in spite of being a nonre-sponder there are factors other than gene which play a major role in the metabolism of the drugs. The responders and nonresponders having heterozygous genotypes of CYP3A4 * 4 and being on donepezil combination therapy were found to have significantly higher plasma levels. This is in contrast to those on rivastigmine monotherapy and combination therapy, among the responders and nonresponders [29]. According to the genotype classification, considering the C/D ratio of donepezil, subjects who are PMs for CYP3A4 (0.38) were found to have significantly higher plasma levels with a poor clinical response as compared to homo-zygous or heterozygous EMs. For the subjects on donepezil combination therapy, hetero-zygous EM and PM subjects had almost similar drug levels. However, EM subjects were found to have better clinical response to the drug than PMs. In contrast, PMs on rivastigmine mono-therapy had better clinical response than EM subjects even though the drug level was compar-atively lower [29] . The interindividual variation in the drug response could be due to interin-dividual differences in the expression of CYP3A4 and CYP3A5 and the drug transporter P-glycoprotein [30] . However, genetic variants identified in the CYP3A4 and CYP3A5 genes have only a limited impact on the CYP3A-mediated drug metabolism [31] . CYP2D6 is not only expressed in the liver but also in the central nervous system [32] . Its role in the biotransfor-mation of endogenous compounds involved in the pathogenesis of neurodegenerative diseases has been hypothesized. This CYP2D6 enzyme has been suspected to metabolize

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68Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

toxins implicated in the pathogenesis of Parkinson’s disease, and polymorphic variation at its locus has been associated with idiopathic Parkinson’s disease [33–35] . The CYP2D6 polymor-phism has been investigated in a number of central nervous system pathologies, including AD. Earlier reports have suggested either an increased frequency of CYP2D6 * 4 in patients with the Lewy body variant of AD [36, 37] or a decreased frequency of CYP2D6 * 4 in male patients with familiar AD [38] . Many studies have found an association and claimed that it plays a significant role in explaining the variability in the response to donepezil. In this study, among the nonresponders on donepezil therapy, heterozygotes had significantly higher drug levels than homozygotes. However, we could not find any significance for the patients on donepezil combination therapy. According to the genotype classification, it was found that homozygous EMs have higher drug levels with a poor clinical response (though not statistically significant) than heterozygous EMs or PMs on donepezil monotherapy. Statistical analyses demonstrated that the significant role of the CYP2D6 polymorphism in influencing the clinical response to donepezil was independent of the age, sex, and MMSE score at baseline. In a recent study, no effect of CYP2D6 * 10 and APOE polymorphisms on both steady-state plasma concentrations (Cp) and clinical response of donepezil in patients with mild-to-moderate AD was found [39] . Even if CYP2D6 pharmacogenetics have been claimed to play a significant role in explaining variability in response to donepezil, only 2 studies reported an impact of CYP2D6 polymor-phisms on the clinical outcome of donepezil in patients with AD. In a study by Varsaldi et al. [3] , the authors included 42 patients with probable AD who were treated for 3 months, mostly 31 patients (74%) with 5 mg daily and 11 patients (26%) with 10 mg daily. Moreover, the response to therapy was evaluated according to changes in the MMSE and Clinician Interview-Based Impression of Change Plus Caregiver Input scores. The other study [5] , which included the same patients, confirmed the results in a somewhat large population. Heterozygous EMs in the study by Magliulo et al. [5] had a better clinical response than homozygous EMs as measured by change in the MMSE score (p = 0.03). EM subjects had the lowest median C/D ratio and PMs had the highest. However, only 2 patients with these genotypes were included in the study, and the data are thus very uncertain due to the small sample size. Consistent with the latter study, the present study also showed better response for heterozygous EM and PM subjects although it was not found to be statistically significant for the donepezil mono-therapy. Though PM subjects were not reported in the study done by Magliulo et al. [5] , we observed 60% of PM subjects for the CYP2D6 polymorphism. Earlier findings have suggested that coadministration of CYP3A4-interacting drugs is unlikely to affect the C/D ratio of done-pezil or the therapeutic outcome to a clinically significant extent [3, 40, 41] . While there are studies on the impact and clinical outcome of donepezil, there are no studies yet on the impact of genetic polymorphism in patients on donepezil combination therapy. One limitation of this study was the small sample size due to the strict inclusion and exclusion criteria. For the subjects on donepezil combination therapy, it was found that PMs have a higher plasma C/D concentration with a better clinical response to the drug though it was found to be only slightly significant (p = 0.29). The poor response to the drug in spite of having a higher plasma C/D concentration could be due to various factors, such as the quanta of neurons affected, other genes responsible for the mutation in drug targets, socioeconomic factors, nutritional status as well as subclinical comorbid conditions. In conclusion, the CYP2D6 polymorphism though not significantly might partially be involved in the plasma concentration of AD drug.

Disclosure Statement

The authors report no conflict of interest.

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69Dement Geriatr Cogn Disord 2014;37:58–70

DOI: 10.1159/000350050

Sonali et al.: Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate AD

www.karger.com/dem© 2013 S. Karger AG, Basel

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