Preemptive genotyping of CYP2C8 and CYP2C9 allelic variants involved in NSAIDs metabolism for sickle cell disease pain management

Abstract

Interindividual variability in analgesic effects of NSAIDs prescribed for sickle cell disease (SCD) pain is attributed to polymorphisms in the CYP2C8 and CYP2C9 enzymes. We described CYP2C8 and CYP2C9 genotype/phenotype profiles and frequency of emergency department (ED) visits for pain management in an African American SCD patient cohort. DNA from 165 unrelated patients was genotyped for seven CYP2C8 and fifteen CYP2C9 alleles using the iPLEX® ADME PGx multiplexed panel. CYP2C8 *1(0.806), *2(0.164), *3(0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild-type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%) respectively. CYP2C9 *1(0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%) respectively. Fifty-two and fifty-five subjects respectively had at least one variant CYP2C8 or CYP2C9 allele. Although the distribution of the CYP2C9 (p= 0.0515) phenotypes was marginally significantly in high and low ED users; some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure. CYP2C8 and CYP2C9 preemptive genotyping could potentially enable clinicians to identify patients with impaired metabolic phenotypes.

Introduction

Sickle cell disease (SCD) is one of the most common genetic blood disorders worldwide that affects predominantly people of African ancestry [1]. The hallmark of SCD is the occurrence of painful vasoocclusive episodes (VOCs) that can start as early as six months of age in pediatric patients and continue to occur unpredictably throughout adult life. Severe VOC pain is associated with acute chest syndrome, organ failure and frequent visits to the emergency department (ED) for parenteral opioid treatment [2]. Prodromal signs of VOC pain are treated with a weak opioid such as codeine but more commonly with nonsteroidal anti-inflammatory drugs (NSAIDs) because of their anti-inflammatory, analgesic and anti-pyretic effects [3, 4]. However, many individuals with SCD display variable response to NSAID treatment and some even fail to achieve adequate analgesia with standard doses of NSAIDs [2, 3].

NSAIDs (e.g., ibuprofen, diclofenac, ketoprofen, naproxen, flurbiprofen, meloxicam, piroxicam and tenoxicam) are metabolized by two enzymes of the cytochrome P450 superfamily, mainly the CYP2C8 and CYP2C9 [5, 6]. Polymorphisms in these two genes have been associated with decreased enzyme activity and alteration of NSAIDs pharmacokinetic parameters [6]. Both the CYP2C8 and CYP2C9 enzymes are highly polymorphic and various allelic variants reported. More than sixteen alleles and over 60 variants have been characterized for the CYP2C8 and CYP2C9 enzymes respectively (http://www.cypalleles.ki.se/). Allelic variants impacts the metabolic activity of the CYP450 enzymes; and previous determinations of enzymatic activity and expression of most CYP450 drug metabolizing enzymes revealed four distinct metabolic phenotypes: ultrarapid metabolizers (UMs), extensive metabolizers (EMs), intermediate metabolizers (IMs) and poor metabolizers (PMs) [5,6]. Poor metabolizers are compound heterozygous for different inactivating alleles or homozygous for an inactivating variant and may display variation in the severity of functional enzyme deficiencies. Intermediate metabolizers carry one functional allele and one nonfunctional allele but may demonstrate a wide range of enzymatic activity. Extensive metabolizers have two functional alleles. Ultra-rapid metabolizers carry multiple copies of functional alleles. Current NSAIDs dosing strategy in patients with SCD is based on the assumption that the individual patient is an extensive metabolizer. However, accumulated evidence indicates association between decreased or loss of function CYP2C8 and CYP2C9 alleles with suboptimal therapeutic response and adverse effects of NSAIDs [5 - 8]. For SCD patients, suboptimal therapeutic may possibly be linked with higher likelihood of being admitted to hospital for either analgesic drug failure. To date however, relatively few studies have attempted to bridge the concept of pharmacogenetic variability as a determinant of interindividual response to NSAID therapy in SCD patients [9 -12]. In this study, we determined the frequency of pharmacologically relevant allelic variants of the CYP2C8 and CYP2C9 enzymes in a SCD patient cohort and correlate metabolic phenotypes with frequency of ED visits.

Methods

Human subjects

The study participants were randomly selected patients with SCD receiving care at the Georgia Regents University Comprehensive Sickle Cell Center clinics. The clinics are located in six towns in south-eastern Georgia. The study was approved by the Georgia Regents University Institutional Review Board. Written informed consent or assent was obtained from each patient prior to inclusion into the study. Study participants were recruited between January 2011 and January 2013. Medical records of the study participants were reviewed to abstract SCD genotypes, NSAID prescriptions, clinical and acute care utilization data.

CYP2C8 and CYP2C9 genotyping

Whole blood samples (10 ml in tubes containing EDTA) were collected from the study participants in steady state. Genomic DNA was extracted using the Puregene® DNA Purification Kit (Qiagen, CA, USA) according to the manufacturer's instructions. We used the iPLEX® ADME PGx multiplex panel (Sequenom, Inc, San Diego, CA) to genotyped seven CYP2C8 alleles (*1, *2, *3, *4, *5, *7, and *8) and 15 CYP2C9 alleles (*1, *2,*3, *4, *5, *6, *8, *9, *10, *11, *12, *13, *15, *25 and *27) across all study participants as previously described [13]. Briefly, the iPLEX® ADME PGx multiplexed panel uses Sequenom Bioscience's iPLEX biochemistry with specific ADME oligo multiplex mixes on the MassARRAY® system to simultaneously interrogate 192 biologically-relevant polymorphisms in 36 pharmacogenes. After running the reactions, mutations were detected, quantified, and genotype reports automatically created using Sequenom TYPER software (http://bioscience.sequenom.com/iplex-adme-pgx-panel). TYPER software assigns the wild-type (*1) CYP2C8 and CYP2C9 alleles in the absence of other detectable variant alleles The CYP allele designations refer to those defined by the Cytochrome P450 Allele Nomenclature Committee [14].

Statistical analysis

The primary outcome measure was genotype frequencies. The secondary end point compares CYP2C8 and CYP2C9 genotypes with the number of ED visits for VOC pain. Descriptive analyses were used for baseline demographic and clinical data and to compare allele frequencies between the study participants and published data of other populations. The CYP2C8 and CYP2C9 allele and genotype frequencies were presented as percentage of the study cohort with 95% confidence interval. The observed genotype frequencies were compared with those expected for concordance with Hardy-Weinberg equilibrium using the X² test. A p-value of less than 0.05 was deemed to represent statistical significance. All statistical analyses were performed using SPSS statistical package version 19.0.

Results

Demographics

This study elucidates the allelic variants of the CYP2C8 and CYP2D9 in a SCD cohort. A total of 165 SCD patients (82 males) were recruited. The study participants were all African Americans. Race was self-reported by the subjects. The study participants' demographic features and clinical characteristics are summarized in Table 1. The subjects ranged in age from 16 to 61 years and their body mass index (BMI) ranged from15.3 to 38.4. SCD genotype frequencies were distributed as SS (97.5%), SB Thal° (1.8%), and S-Los Angeles (0.6%) respectively. Ibuprofen, aspirin and naproxen were the NSAIDs routinely prescribed. One hundred and ten subjects (67%) were prescribed hydroxyurea. In terms of treatment of SCD, hydroxyurea is the only FDA approved drug and has been associated with decreased frequency of VOC and morbidity [2, 4].

Table 1. Cohort demographic and clinical characteristics.

		No (%) of subjects
Sex
	Male	82 (49.6%)
	Female	83 (50.4%)
Age
Median
Male:	30.5 yrs. (16 – 57)
Female:	31.7 yrs. (18 – 61)
Ethnic origin
	African-American	165 (100%)
SCD genotype
	SS	161 (97.5%)
	SB Thal°	3 (1.8%)
	S-Los Angeles	1 (0.6%)
BMI
Median
Male:	23.1 (15.3 – 35.2)
Female:	23.7 (16.5 – 38.4)
NSAIDs	Ibuprofen (Motrin, Advil)	50 (30.3%)
	Naproxen (Aleve)	10 (6.1%)
	Aspirin	5 (3%)
	Acetaminophen (Tylenol)	20 (12.1%)
Hydroxyurea therapy	Yes	110 (67%)

CYP2C8 alleles and genotype frequencies

Table 2 showed our cohort CYP2C8 allele and genotype frequencies. The CYP2C8* 1 is considered the wild type with normal enzyme activity. The abnormal CYP2C8*2 and *3 alleles are the most prevalent alleles associated with decreased enzyme activity, but are unevenly distributed in racial and geographic populations [15]. The CYP2C8*4 through CYP2C8*14 variants alleles are rare and found in less than 1% of racial populations [16, 17]. In our cohort, we identified four CYP2C8 alleles. The CYP2C8*1 wild-type allele frequency was 0.806. Of the three variant alleles identified, the *2 occurred in the highest frequency (0.164). This was followed by CYP2C8*3 (0.018) and *4 (0.012) respectively. The CYP2C8*5, *7 and *8 rare variants found in less than 1% of populations, mainly Asians, were not detected in our cohort [18, 19].

Table 2. Allelic, genotypic and phenotype frequencies of CYP2C8 gene polymorphisms.

Allele	Genetic mutation	Enzyme activity	Number of alleles	Allelic frequency	95% CI (±)	95%CI Range
*1	Wild type	Normal	266	0.806	0.043	0.763 - 0.849
*2	Missense	Decreased	54	0.164	0.040	0.124 - 0.204
*3	Missense	Decreased	6	0.018	0.014	0.004 - 0.033
*4	Missense	Decreased	4	0.012	0.012	0.000 - 0.024
*5	Frame shift	None	0	0.000	0.000
*7	Missense	None	0	0.000	0.000
*8	Missense	Decreased	0	0.000	0.000
Genotype	Number of subjects	Observed Genotype frequency (%)	Expected Genotype frequency (%)	Predicted Phenotype
Wild type
*1/*1	110	66.7	65.0	EM
Heterozygous
*1/*2	40	24.2	26.4	IM
*1/*3	4	2.4	2.9	IM
*1/*4	2	1.2	2.0	IM
Sub total	46	27.8
Homozygous variant/compound heterozygous
*2/*2	5	3.0	2.7	PM
*2/*3	2	1.2	0.6	PM
*2/*4	2	1.2	0.4	PM
*3/*3	0	0.0	0.0	PM
*3/*4	0	0.0	0.0	PM
Sub total	9	5.4
Total	165	100	99.9

Abbreviation: CI, confidence interval

The CYP2C8*1, *2, *3, and *4 frequencies were concordant with Hardy-Weinberg equilibrium. Because of substrate–dependent functional activity of the CYP2C8 alleles and discrepancies between in vitro and in vivo data the genotype frequencies were distributed as homozygous wild-type, heterozygous and homozygous variant/compound heterozygous respectively [17]. The CYP2C8 1/*1 was the most frequent genotype related to NSAID metabolism and it corresponds to an predicted extensive metabolizer phenotype (66.7%); the CYP2C8 *1/*2, *1/*3 and *1/*4 genotypes correspond to an intermediate metabolizer phenotype and accounted for 27.8% of the study cohort; whereas the poor metabolizers (*2/*2, *3/*3 and *2/*4 genotypes) accounted for 5.4 % of cohort.

CYP2C9 allele, genotypes and phenotype frequencies

The CYP2C9 allele, genotype and predicted metabolic phenotype frequencies are summarized in Table 3. We surveyed 15 CYP2C9 alleles (*1, *2, *3, *4, *5, *6, *8, *9, *10, *11, *12, *13, *15, *25 and *27) and identified eight alleles (*1, *2, *3, *5, *6, *8, *9, *11) with the wild type *1 occurred in the highest frequency (0.824) in our cohort. The CYP2C9*8 (0.042) and *9 (0.061) were the most common variant alleles. The combined frequency for the reduced activity CYP2C9*2, *3,*5, *8, *9, *11 and the null *6 variants was 0.176. The CYP2C9 *4, *12, *13, *15, *25, and *27 alleles were not detected in our cohort. The high frequency (0.061) of the CYP2C9*9 allele made *1/*9 the most common genotype with allelic variant in the cohort (9.7%). The observed frequencies for the overall cohort were concordant with Hardy-Weinberg equilibrium. CYP2C9 phenotypes have been designated extensive (two functional alleles), intermediate (one functional allele/one dysfunctional) and poor metabolizers (two nonfunctional alleles). Based on the observed genotypes and published criteria, we assigned predicted phenotype frequencies for our study participants as follows: extensive (68.5%), intermediate (18.1%) and poor metabolizers (0.6%) respectively [20-24]. Because some of the variant CYP2C9 alleles do not have clear phenotypic consequences, the predicted metabolic phenotype for four CYP2C9 genotypes (*5/*9, *6/*8, *8/*9, and *9/*11) were indeterminate.

Table 3. Allelic, genotypicand phenotypicfrequencies of CYP2C9 gene polymorphisms.

Allele	Genetic mutation	Enzyme Activity	Number of alleles	Allelic frequency	95% CI (±)	95%CI Range
*1	Wild type	Normal	272	0.824	0.041	0.783-0.865
*2	Missense	Decreased	9	0.027	0.018	0.010-0.045
*3	Missense	Decreased	4	0.012	0.012	0.000-0.024
*4	Missense	NA	0	0.000	0.000
*5	Missense	Decreased	3	0.009	0.010	-0.001-0.019
*6	Frame shift	None	3	0.009	0.010	-0.001-0.019
*8	Missense	Decreased	14	0.042	0.022	0.021- 0.064
*9	Missense	NA	20	0.061	0.026	0.035 - 0.086
*10	Missense	NA	0	0.000	0.000
*11	Missense	Decreased	5	0.015	0.013	0.002 - 0.028
*12	Missense	Decreased	0	0.000	0.000
*13	Missense	Decreased	0	0.000	0.000
*15	Missense	NA	0	0.000	0.000
*25	Frame shift	None	0	0.000	0.000
*27	NA	NA	0	0.000	0.000
Predicted Metabolizer Phenotype	Number of subjects	Observed Genotype frequency (%)	Expected Genotype frequency (%)
Extensive metabolizer
*1/*1	113	68.5	67.9
Intermediate Metabolizer
*1/*2	8	4.8	4.5
*1/*3	3	1.8	2.0
*1/*5	2	1.2	1.5
*1/*6	2	1.2	1.5
*1/*8	11	6.7	7.0
*1/*9	16	9.7	10.0
*1/*11	4	2.4	2.5
Sub total	46	27.8
Poor metabolizer
*2/*3	1	0.6	0.1
Unknown metabolizer status
*5/*9	1	0.6	0.1
*6/*8	1	0.6	0.1
*8/*9	2	1.2	0.5
*9/*11	1	0.6	0.2
Sub total	5	3.0

Abbreviation: CI, confidence interval.

Correlation between study participants' genomic and clinical data

Table 4 describes the study participants' CYP2C8 and CYP2C9 genomic and ED clinical data. Out of a total of 152 participants with ED visit clinical records, we had 39 high ED users (>=3 ED visits per year) and 113 low ED users (<3 ED visits per year). The distribution for the CYP2C8 predicted phenotypes was not significantly different in high and low ED users (p= 0.1668). However, the distribution of predicted phenotypes was marginally significantly in high and low ED users (p= 0.0515) for the CYP2C9. Table 5 presents detailed genomic and clinical data, including NSAIDs prescriptions, number of VOC days and pain score ranges during ED visits or hospital admissions for selected participants with deficient genomic metabolic and clinical risk profiles for NSAIDs therapeutic failure. Table 6 compares our CYP2C8 and CYP2C9 data to allelic data previously reported in other African American and African ethnic populations [19-25]. There is limited published data available from other populations for several minor frequency CYP2C8 and CYP2C9 alleles making it somewhat difficult to compare our data with some African ethnic groups with high incidence of SCD.

Table 4. Distribution of CYP2C8 & CYP2C9 phenotypes and ED visits.

CYP2C8	*High	+Low	CYP2C9	High	Low
EM	23 (0.61)	83 (0.74)	EM	27 (0.71)	91 (0.81)
IM	15 (0.39)	29 (0.26)	IM	9 (0.24)	10 (0.09)

EM: extensive metabolizer; Intermediate metabolizer; ED: Emergency department; UNK: Unknown

^*(>=3 ED visits per year);

⁺(<3 ED visits per year)

Table 5. Genomic and clinical data for selected SCD participants with compromised metabolic genotypes.

# of subject	CYP2C8 Genotype	CYP2C8 Phenotype	CYP2C9 Genotype	CYP2C9 Phenotype	Pain range*	# of VOC days	ED Visit	Hosp. Adm.	NSAID, number of times prescribed & place	Hu therapy
1	*1/*1	EM	*1/*8	IM	0-8	3	1	2	Ibuprofen 800mg ×14, tylenol 500mg × 10, ibuprofen 800 ×17	Y
2	*1/*3	IM	*1/*2	IM	0-7	1	0	1	naproxen 500 × 39	N
3	*1/*3	IM	*1/*2	IM	0-9	67	0	5	N/A	Y
4	*1/*1	EM	*1/*8	IM	0-9	8	2	1	Tylenol 250 ×10, ibuprofen 800mg × 8 (hosp), toradol 30mg IV ×5 (hosp)	Y
5	*1/*1	EM	*1/*8	IM	N/A	N/A	3	4	ASA unknown dose × 26	Y
6	*1/*3	IM	*1/*2	IM	0-5	1	2	5	Toradol 75 mg iv (hosp), acetaminophen 875 (hosp), goody pm × 64	Y
7	*2/*3	PM	*1/*2	IM	0-9	1	1	3	Motrin 800 × 2	Y
8	*1/*1	EM	*6/*8	UNK (PM?)		16	6	2	Motrin 800 × 4 (hosp), toradol 60 IM (hosp), theraflu unknown dose × 45, motrin 800 ×72, tylenol 500 × 14	Y
9	*1/*2	IM	*1/*8	IM	0-9	10	0	0	Ibuprofen 800mg × 24, tylenol 500 mg × 4	N
10	*1/*1	EM	*1/*8	IM	N/A	1	0	0	Tylenol 500mg × 1	N
11	*2/*3	PM	*1/*2	IM	0-7	37	3	1	Ibuprofen 200mg × 85	Y
12	*1/*1	EM	*8/*9	UNK	0-9	0	0	1	N/A	N
13	*1/*2	IM	*1/*6	IM	0-4	4	1	2	N/A	N
14	*1/*1	EM	*1/*8	IM	0-8	0	0	1	N/A	Y
15	*1/*1	EM	*1/*8	IM	0-1	4	0	0	Excedrin unknown dose × 17, advil unknown dose × 1	N
16	*1/*2	IM	*1/*3	IM	0-9	N/A	1	0	N/A	N
17	*1/*2	IM	*1/*8	IM	N/A	N/A	1	0	N/A	Y
18	*1/*3	IM	*1/*2	IM	N/A	N/A	0	2	N/A	N
19	*1/*1	IM	*8/*9	UNK	N/A	N/A	0	0	N/A	Y
20	*1/*2	IM	*1/*8	IM	N/A	N/A	4	0	N/A	Y
21	*1/*1	IM	*1/*3	IM	N/A	0	4	10	N/A	Y
22	*1/*2	IM	*1/*8	IM	N/A	N/A	0	0	N/A	Y
23	*1/*1	EM	*1/*8	IM	N/A	N/A	0	15	Ibuprofen 800 × 11	N
24	*1/*4	IM	*1/*2	IM	0-9	3	8	12	N/A	Y
25	*1/*1	EM	*2/*3	PM	N/A	N/A	3	3	N/A	Y

ADM: admission; EM; Extensive metabolizer; IM: Intermediate metabolizer;PM: Poor metabolizer; Hosp: hospital; ED: Emergency department; HU: hydroxyurea; N/A: Not available; UNK: Unknown; Y: Yes; N: No;

^*Pain range during ED or Hospital admission.

Table 6. CYP2C9 & CYP2C8 frequencies in previously studied populations.

Racial & Ethnic group	CYP2C9 alleles frequency %											Ref.
	*1	*2	*3	*4	*5	*6	*8	*9	*11	*12	*13
African Americans (n=165)	0.824	0.027	0.012	0.000	0.009	0.009	0.042	0.061	0.015	0.000	0.000	This study
African American (n=300)	0.867	0.028	0.020	0.000	0.015	0.010	0.047	-	0.013	-	0.000	[19]
Ghanaian (n=204)	-	0.000	0.000	0.000	0.000	-	-	-	0.020	-	-	[21]
Beninese (n=111)	0.955	0.000	0.000	0.000	0.018	-	-	-	0.027	-	-	[22]
Mozambican (n=206)	-	0.000	0.010	-	0.019	0.000	0.146	-	0.024	-	-	[23]
Africans (n=250)	0.810	0.120	0.000	-	0.010	0.012	0.040	0.095	0.022	0.020	-	[20]
	CYP2C8 alleles frequency %
	*1	*2	*3	*4	*5	*7	*8	*9	*10	*11	*12
African American (n=165)	0.806	0.164	0.018	0.012	0.000	0.000	0.000	-	-	-	-	This study
African American (n=500)	0.878	0.100	0.010	0.012	0.000	0.000	0.000	0.000	0.000	-	0.000	[24]
Mozambican (n =360)	0.160	0.048	0.000	0.005	0.000	-	-	-	-	-	-	[25]
Ghanaian (= 204)	-	0.170	0.000	0.000	-	-	-	-	-	-	-	[21]

(-) Allele not screened in study

Discussion

To the best of our knowledge, this study describes the combined CYP2C8 and CYP2C9 allelic frequencies, genotypes and predicted phenotypes for the first time in an African American SCD patient cohort. Our study identified twenty five individuals with combined impaired CYP2C8 and CYP2C9 genotypes characterized mainly as intermediate and poor metabolizers. Interindividual variation in drug response is greatest in intermediate metabolizer phenotypic group where it is difficult to determine unequivocally the quantitative/percentage value of altered functionality of allelic variants, except for null alleles [26-28]. Perhaps more significantly, included in this phenotypic group are the six individuals with the delirious CYP2C8*3 and CYP2C9*2 allelic combination associated with major ibuprofen clearance impairment and analgesic failure [28]. To the best of our knowledge, no prior studies have identified specific African American subjects with these two genes allelic combination or assess their implications for NSAID analgesic failure.

We did not observe great variations in the frequency of null and reduced function alleles in the CYP2C8 and CYP2C9 genes to ranges reported in other African American populations. Interestingly, two previous genotyping studies reported slightly higher frequencies of mutant gene deletion alleles of the CYP3A5 and CYP2D6 in SCD patients compared to healthy African Americans [9, 12]. These deficient alleles are associated with the poor metabolizer phenotype and impaired functionality and have also been associated with failure of codeine treatment for VOC in children on hydroxyurea; and linked with higher likelihood of pediatric and adult SCD patients being admitted to the ED for parenteral opioid management [10, 11]. Although the distribution of CYP2C9 predicted phenotypes was marginally significantly in high and low ED users in our study, it was unclear in the above referenced studies whether the individuals at risk for frequent ED visits also had deficient CYP2C8 and CYP2C9 genotypes. Nonetheless, in our cohort, ten out of twelve of the combined alleles of the CYP2C8 and CYP2C9 enzymes identified contribute to deficient metabolic genotypes. Fifty-two subjects had at least one variant CYP2C9 allele (*2, *3,*5, *6, *8, *9 and *11) associated with either the intermediate metabolizer, poor, or indeterminate phenotype. Fifty-five subjects had at least one CYP2C8 variant allele (*2, * 3, *and *4) that contributes to impaired metabolic genotypes, and nine subjects were homozygous or compound heterozygous for deficient metabolic genotypes as determined in previous pharmacokinetic and pharmacodynamics studies [26-34].

Pharmacokinetics and Pharmacodynamics effects of CYP2C8 and CYP2C9 alleles

The influence of some CYP2C8 and CYP2C9 genotypes on analgesic response and therapeutic outcomes of a number of NSAIDs including ibuprofen and naproxen have been established in previous pharmacokinetics studies [26-28]. Ibuprofen pharmacokinetics data is strongly related to variant CYP2C8 and CYP2C9 genotypes: heterozygous and homozygous carriers of the CYP2C8*3 allele display ibuprofen metabolic clearance reduction of approximately 62% and 10% respectively when compared to individuals homozygous for the CYP2C8*1 and CYP2C9*1 genotype [25]. Metabolic clearance values in subjects heterozygous and homozygous for CYP2C9*2 but not carrying any other allelic mutations were 96% and 84% respectively when compared to individuals with CYP2C8*1 and CYP2C9 *1 wild type alleles [27]. The CYP2C9*2 allele when linked with the CYP2C8*3, translates into a major impairment on ibuprofen clearance as indicated above [28]. Individuals carrying CYP2C9*3 variant alleles display a mean reduction of clearance of approximately 65% and 17% for heterozygous and homozygous carriers, respectively [27]. In a recent study of 130 healthy individuals who received a single oral dose of 400mg ibuprofen, the oral clearance of ibuprofen was 4.43, 3.26, 2.91, 2.05, 1.83 and 1.13 1/hr for individuals with CYP2C9*1/*1, *1/*2, *1/*3, *2/*2, *2/*3 and *3/*3 genotypes, respectively [28]. The effects of the variant CYP2C9 alleles are dissimilar for all NSAIDs [29]. Studies with tenoxicam however indicated that oral clearance among carriers of CYP2C9*2 and CYP2C9*3 decreases to approximately 70% and 55% [30]; while oral clearance of meloxicam in individuals with the CYP2C9*1/*13 genotype was significantly decreased by 62% compared to individuals with the CYP2C9*1/*1 genotype [31].

Pharmacodynamics studies have also implicated CYP2C8 and CYP2C9 genotypes in gastrointestinal toxicity of NSAIDs. Martinez and colleagues found the CYP2C9*2 and *3 associated with a two-and-a-half fold increased risk of gastric bleeding episode after dosing with NSAIDs such as celecoxib, diclofenac, ibuprofen, indomethacin, lornoxicam, piroxicam or naproxen. The increased risk was attributed to the *2 allele which was detected in 23.4% of the study subjects with gastric bleeding episode compared with 13.7% of the control subjects [32]. In another study of gastrointestinal bleeding in NSAIDs users, the frequencies of the CYP2C8*3 and CYP2C9*2 alleles were higher in NSAIDs user who experienced a bleed versus those who did not experience a bleed (CYP2C8*3, odds ratio: 2.4, p<0.002; CYP2C9*2, odds ratio: 2.7, p < 0.013) [33]. Pilotto et al found that a significantly higher frequency of CYP2C9*1/*3 and *1/*2 genotypes were identified in patients with endoscopically documented NSAID-related gastroduodenal bleeding lesions compared to a matched control group. In the study described, the presence of the CYP2C9*3 allelic variant was associated with a significant high risk of bleeding (OR: 7:3) [34].

Clinical utility of CYP2C8 and CYP2C9 preemptive genotyping

Currently, data on the association of NSAIDs treatment with severe drug side-effects or analgesic failure are limited in SCD patient population [4]. Interestingly, recent epidemiologic data associates higher doses of some traditional and non-traditional NSAIDs with double risk of congestive heart failure, increased risk of peptic ulcer complications, gastrointestinal bleeding, and increased risk of major vascular events (non-fatal myocardial infarction, non-fatal stroke, or vascular death) [8, 35-38]. Though these risks fall quickly after drug cessation, nonetheless, with chronic NSAID use, these risks do not wane over years of use [37- 40]. For SCD patients however, due to the ubiquity and chronicity of pain experience, NSAIDs therapy is initiated very early in life and throughout the lifespan. Indeed, as Table 5 illustrates, NSAIDs are routinely prescribed to SCD with compromised metabolic profiles. Preemptive genotyping to identify patients with CYP2C8 and CYP2C9 intermediate and poor predicted metabolic phenotypes could potentially facilitate early prediction of NSAID treatment outcomes both in terms of efficacy and possible development of adverse events in SCD patients. Preemptive genotyping anticipates current and future medication prescription needs of patients as opposed to current practice whereby genotyping is performed only when clinically indicated [41]. With the preemptive genotyping model, a single blood sample is used to genotype specific patient populations for polymorphisms of pharmacogenetic significance in relevant drug metabolizing enzymes and transporters (DMET) with regards to specific medication classes [42]. CYP2C8 and CYP2C9 preemptive genotyping data could potentially facilitate quantification and clinical assessment of pharmacogenetic risk and longitudinal accumulation of NSAIDs risk burden in SCD patients. As depicted in Table 5, the identification of an impaired drug metabolic profile in a patient would alert the clinician that special attention should be given to NSAIDs drug response of the patient and would provide explanation for those individuals with unsatisfactory drug response or side effects profiles enabling clinicians to make distinctions between a medication compliance problem and a metabolic defect. More importantly, since the genetic make-up of an individual is virtually invariable, the determination of SCD patients' drug metabolic genotypes provides life-long applicable information for analgesic pharmacotherapy [40].

Ultimately, pharmacogenetic screening for SCD patients' CYP2C8 and CYP2C9 genotypes would be useful only if it will facilitate development of NSAIDs dosing algorithms similar to those developed for warfarin and tamoxifen dosing with the CYP2C9/VKORC1 and CYP2D6 genes respectively [43-45]. However, this would require additional functionality studies of novel CYP2C8 and CYP2C9 variant alleles, as well as the effect of SCD on CYP2C8 and CYP2C9 enzyme expressions and the role of environment factors. Perhaps more immediate is the need for appropriate PK and PD studies to determine the effects of allelic variants present in the SCD population. As shown in Tables 2 and 3, the CYP2C8*2 and CYP2C9*5,*6,*8, *9 and *11 variant alleles are common in populations with genetic susceptibility for SCD, mostly populations of African ancestry. However, while the relationships between the CYP2C8*3, CYP2C9*2 and *3 alleles and metabolic indexes of several NSAIDs are well delineated, to the best of our knowledge, the pharmacokinetic effects of the CYP2C8*2 and CYP2C9*5,*6,*8, *9 and *11 alleles on NSAIDs metabolism has not been evaluated in African American populations. This lack of pharmacokinetics data not only precluded us from assigning predicted metabolic phenotype for four genotypes (i.e., *5/*9, *6/*8, *8/*9, *9/*11), but also represents a crucial knowledge gap in the use of genetic genotype to inform pharmacogenetic prescribing practice for NSAIDs dosing or for other drugs metabolized by the CYP2C8 (i.e., amiodarone, fluvastatin, simvastatin, verapamil, montelukast, amodiaquine and chloroquine, morphine and methadone) and the CYP2C9 (i.e., losartan, tolbutamide and torsemide) enzymes often prescribed for SCD patients in various parts of the world.

Our study has limitations. Though we used a multiplex genotyping panel to determine CYP2C8 and CYP2C9 genotypes, the assignment of predicted metabolic profiles in our cohort was based on allelic combinations and associated activity levels reported in the literature. Ultimately, pharmacokinetics study remains the gold standard for discerning SCD patients' metabolic phenotypes and for reporting individuals' analgesic drug response profile for specific substrates. Another study limitation is that with the iPLEX ADME PGx panel, the CYP2C9*3 and *18 are indistinguishable haplotypes. Consequently, we arbitrarily reported the CYP2C9*3 allele frequency in our cohort. Additionally, the cross-sectional nature of our study precluded reporting of daily or annual NSAIDs usage by genotypes, pain scores and analgesic response which we reserve for another project. These limitations notwithstanding, to the best of our knowledge, no study attempts to bridge the concept of pharmacogenetic variability as a determinant of interindividual response to NSAID therapy in SCD patients.

Conclusions

In summary, our study determined allele frequencies and genotypes in the CYP2C8 and CYP2C9 enzymes in a SCD patient cohort. Ten of twelve of the combined alleles of the CYP2C8 and CYP2C9 enzymes identified in our cohort contribute to deficient metabolic genotypes with fifty-two subjects having at least one variant CYP2C9 allele associated with either the intermediate metabolizer, poor, or indeterminate phenotype; while fifty-five subjects had at least one CYP2C8 variant allele that contributes to impaired metabolic genotypes. Several of the impaired function variant alleles are being reported for the first time among SCD patients. The CYP2C8 and CYP2C9 variant alleles play a significant role in the analgesic effects and toxicity of NSAIDs. These drugs used to treat VOC pain at the prodromal stage in SCD patients are associated with vascular risks, adverse effects on the gastrointestinal tract, and possibly frequent ED visits for analgesic failure [7, 8]. Our study highlights the concept of pharmacogenetic variability as a determinant of interindividual response to NSAIDs therapy in SCD patients. CYP2C8 and CYP2C9 preemptive genotyping could potentially enable clinicians to identify patients with impaired metabolic phenotypes. In tandem with preemptive genotyping, additional and appropriate pharmacokinetics and pharmacodynamics studies are required to potentially enable clinicians to tailor NSAIDs dosing accordingly to achieve optimal analgesic response.