Applications of CYP450 Testing in the Clinical Setting

2.1.1 Psychotropic Agents

Various psychotropic agents are metabolised at least in part by CYP2D6. Depending on the pharmacokinetic (PK) and the pharmacodynamic (PD) properties of the drug, the impact of the polymorphism will be more or less pronounced. UMs may require higher doses to achieve therapeutic drug levels and effects, whereas PMs might be at increased risk of toxicity. The association between CYP2D6 PM phenotype and psychotropic agent toxicity (antidepressants, antipsychotics) has been described [19, 20]. PMs were twice as frequent amongst patients presenting ADRs (44 vs 21 %) [19]. Patients treated with CYP2D6 substrates (tricyclics, thioridazine, perphenazine, haloperidol, risperidone) experienced a higher rate of ADRs that was in turn associated with the CYP2D6 genotype (PM > IM > EM > UM) [20]. The costs associated with the treatment of the metabolic extremes (PM and UM) were US$4,000 to 6,000 per year higher than those of EM and IM on the same treatments, and the length of hospitalisation was also longer in PMs [20]. Kirchheiner et al. systematically analysed all pharmacogenetic data available on the impact of genetic polymorphisms on the positive effects of and adverse reactions to 36 antidepressants and 38 antipsychotics. CYP2D6 polymorphism was found to be relevant for 14 out of 36 antidepressants, requiring at least doubling of the dose in EMs in comparison to PMs, and for one third of the assessed antipsychotics [21].

2.1.2 Opioids

The influence of CYP2D6 polymorphisms on the analgesic response and therapeutic outcome to a number of prodrug opioids is now established. The opioids codeine, tramadol and oxycodone have to be activated by CYP2D6 into their active moieties, morphine [34, 35], O-desmethyl-tramadol (M1) [36] and oxymorphone [37, 38], respectively. The impact of CYP2D6 polymorphisms have been assessed in terms of PK and PD consequences.

In PMs for CYP2D6, reduced or absent metabolite formation and reduced analgesic effects have been observed after codeine [39], tramadol [40] and oxycodone administration [37, 38]. A cohort study showed that children having ineffective pain treatment of sickle cell crisis with codeine were more likely to have reduced CYP2D6 activity [39]. After major abdominal surgery, non-response rates to tramadol were fourfold higher amongst PMs than other CYP2D6 genotypes in a randomised prospective study [40]. In healthy volunteers, PMs experienced no analgesic effect after oxycodone administration in the experimental pain setting [38].

Conversely the CYP2D6 UM phenotype has been associated with quicker analgesic effects but higher mu-opioid-related toxicity after tramadol [41, 42] or oxycodone administration [38, 43]. The risk of codeine use in paediatrics and breastfed neonates in association with UM genotype has been reported with four fatal cases and serious toxicity cases [44, 45, 46, 47, 48, 49]. The FDA released a warning in August 2007 that codeine use by nursing mothers may increase toxicity in infants [50], and more recently about the risk of death after the use of codeine in infants for tonsillectomy/adenoidectomy in August 2012 [51]. In February 2013, the FDA stated that a new Boxed Warning and Contraindication were to be added to the drug label of codeine-containing products [52].

Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for codeine therapy in the context of CYP2D6 phenotype were recently published [53]. CPIC was established in order to provide drug-dosing guidelines based on an individual’s genotype. These peer-reviewed gene–drug guidelines are published and updated periodically on the PharmGKB website [54]. The authors recommendation is to avoid codeine use in UM and PM for CYP2D6 and consider alternative analgesics such as morphine. The classification of recommendation is labelled as “strong”. The Dutch Pharmacogenetics Working Group Guideline edited the same recommendation for codeine, tramadol and oxycodone [28].

CYP activity is modulated by inhibitors mimicking a genetic deficiency (PM phenotype) (Table 2). CYP2D6 inhibitors such as quinidine have been associated with a reduction of codeine, tramadol and oxycodone analgesic effects [34, 36, 38]. Importantly, drug interactions modulating the activity of the enzymes involved in the other pathways of elimination have to be taken into account. Indeed, they have the ability to modify the therapeutic index of a drug or its pharmacological properties, especially in the context of a genetic polymorphism. The co-administration of oxycodone with the CYP3A inhibitor ketoconazole (Table 2) has been associated with a dramatic increase in oxycodone efficacy (experimental pain setting) and toxicity, with this effect being even more pronounced in the UM for CYP2D6 [38]. Similarly a CYP3A inhibitor co-administrated with codeine induced a life-threatening opioid intoxication in a CYP2D6 UM [55].

The pharmacological properties of the dual opioidergic and monoaminergic tramadol [56] may also be modified as a consequence of CYP activity modulation by drug interactions and/or genetic polymorphisms. In CYP2D6 PMs, tramadol properties will move towards a monoaminergic antidepressant-like molecule devoid of opioidergic activity, whereas the opioidergic properties will be more pronounced in CYP2D6 UM. Different pharmacological effects will therefore be expected depending on the activity of CYP2D6. Some authors have summarised recommendations on the usage of opioids while administrating CYP3A and 2D6 inhibitors, and CYP3A inducers [57] (Table 2). The recommendation is to avoid the combination of oxycodone and CYP3A inhibitors as well as CYP3A inducers and to monitor patients on oxycodone with a CYP2D6 inhibitor. Regarding codeine, the authors’ recommendations are to avoid the co-administration of a CYP2D6 inhibitor or consider alternative pain treatment, and to monitor if administration is concomitant with a CYP3A inhibitor or inducer. Codeine should be avoided when a CYP3A inhibitor is co-prescribed in a highly susceptible population (such as UM for CYP2D6) [57]. The importance of these pharmacogenetic modulations of drug interactions in the clinical setting needs to be further investigated.

2.1.3 Antiarrhythmic Agents

CYP2D6 is involved in the metabolism of carvedilol, bufuralol, metoprolol, propranolol and timolol. CYP2D6 genetic polymorphism has an impact on the disposition of metoprolol, bufuralol and timolol, whereby PMs have a higher exposure (four- to sixfold), higher plasma concentrations (two- to fivefold) and a prolonged half-life (two- to threefold). The PM genotype has furthermore been associated with an increased risk of toxicity after metoprolol administration [22, 58] and some authors demonstrated that PMs had a fivefold higher risk of developing adverse effects [59]. The increased efficacy or higher risk of toxicity in PMs was furthermore demonstrated after timolol (more pronounced heart rate reduction) [60], carvedilol (reduction of the systolic blood pressure) [61], propafenone (central side effects five times more pronounced and increased QRS narrowing at a given concentration) administration [62]. After propranolol administration, an open study did not show any difference in the PK parameters or the clinical response depending on CYP2D6 phenotype [60]. The Dutch Pharmacogenetics Working Group Guideline recommended a 70–75 % reduction of metoprolol and propafenone in PMs or alternative drug selection [28]. In UMs, an alternative drug should be selected or metoprolol dose titrated to a maximum of 250 % of the normal dose. Insufficient data are available to allow calculation of dose adjustment for propafenone but the recommendation is to adjust dose to plasma concentration and record ECG or select an alternative drug.

A double-blind randomised control study assessed the PK and PD consequences of concomitant administration of carvedilol and a CYP2D6 inhibitor (fluoxetine) in cardiac failure. Fluoxetine stereospecifically inhibited (R)-(+)-carvedilol, but had no clinical impact on the side effects, heart rate and blood pressure in this pilot study [63]. In healthy volunteers receiving metoprolol, after the co-administration of the CYP2D6 inhibitor paroxetine 20 mg/day, both (S)- and (R)-metoprolol AUC increased three- and fourfold, respectively, and the half-life of both isomers increased by approximately twofold. The decreases of the heart rates and systolic blood pressures were significantly more pronounced after paroxetine administration [64]. Similarly, acute myocardial infarction patients treated with metoprolol and paroxetine had a significant metoprolol AUC increase, and a reduction of metoprolol dose was required in two patients owing to excessive bradycardia and severe orthostatic hypotension [65].

2.1.4 Tamoxifen

Tamoxifen is approved in the treatment of metastatic breast cancer as well as an adjuvant therapy of breast cancer in postmenopausal women. The two active metabolites of tamoxifen produced predominantly by CYP2D6 as well as CYP3A are 4-hydroxytamoxifen and endoxifen; their affinity for the oestrogen receptor is 100 times higher than tamoxifen and their potency is 30- to 100-fold greater [66, 67]. Endoxifen is considered the most clinically relevant active metabolite in terms of anticancer effect and hormone receptor blockade. Endoxifen plasma concentrations in newly diagnosed breast cancer CYP2D6 PM and IM genotyped women were 26 and 55 % of those in EMs, respectively [68]. The same was shown in CYP2D6 PMs owing to drug inhibition by paroxetine [69]. Thirty-nine and 9 % of the variability of steady-state concentrations of endoxifen and 4-hydroxytamoxifen, respectively, were explained by CYP2D6 genotypes and among PMs 93 % had endoxifen levels below IC₉₀ values [70]. PMs are therefore likely to experience therapeutic failure after tamoxifen, whereas UMs might be prone to increased toxicities [71]. Homozygous CYP2D6*4 carriers (PMs) were shown to have increased risk of breast cancer recurrence, shorter relapse-free periods and worse event-free survival rates as compared to carriers of functional alleles [72, 73, 74]. The impact of CYP2D6*10 homozygotes (IMs) on the PK and PD behaviour of tamoxifen was assessed in Asians [75, 76, 77, 78] . CYP2D6*10/*10 has lower endoxifen and 4-OH-tamoxifen concentrations, shorter median time to progression, and higher incidence of recurrence within 10 years after the operation. The FDA has recommended labelling change to indicate that postmenopausal oestrogen receptor-positive breast cancer patients taking adjuvant tamoxifen who are homozygous for CYP2D6*4 have a significant decreased relapse-free survival as compared to other genotypes. However no recommendation on the routine testing of all women on tamoxifen was made, and the impact of other deficient variants (such as CYP2D6*10) was not addressed. A matched case-control study using the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG8) demonstrated that PMs or carriers of a deficient allele had a higher likelihood of an event than EMs (OR 2.45 and 1.67, respectively) during the first 5 years of therapy [79].

In 98 Japanese breast cancer patients on 20 mg tamoxifen daily (adjuvant setting), Kiyotani et al. evaluated the impact of dose adjustment according to CYP2D6 genotype. In CYP2D6*10 heterozygotes and homozygotes, the dose was increased to 30 and 40 mg/day, respectively. The achieved endoxifen and 4-OH-tamoxifen plasma concentrations were increased to similar levels as those in the CYP2D6*1/*1 patients receiving tamoxifen 20 mg/day [80]. The dose increase did not result in increased side effects. However the dose increase did not lead to dose-proportional increase in endoxifen concentrations. Others authors demonstrated that increasing the tamoxifen dose from 20 to 40 mg/day resulted in increased endoxifen concentrations in PMs but not in EMs [81]. Another study demonstrated that increasing tamoxifen dose from 20 to 30 mg in PMs was associated with a 90 % in the endoxifen levels by day 60 and an increased antioestrogenic activity score [82].

Conflicting results have been reported regarding the association between CYP2D6 genotype and tamoxifen effects. The pitfalls of these retrospective studies (small cohorts, limited CYP2D6 allele coverage, quality of genotyping, correct genotype–phenotype assignment) were pointed out by several authors [83, 84]. Two large prospective studies (The Breast International Group BIG 1-98 trial and the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial) were unsupportive with regard to CYP2D6 genotyping in the adjuvant setting as no association was demonstrated with breast cancer recurrence [85, 86]. However these two studies have been criticised as endoxifen plasma concentrations were not measured and drug inhibition was not taken into account (Table 2). Pharmacogenetics experts asked for the retraction of the BIG 1-98 study on the basis of massive departures from Hardy–Weinberg equilibrium with the possibility of bias introduced by the CYP2D6 genotype from the tumour (somatic) and not the host genome (germline DNA) [83, 87]. Some authors have therefore stated that “until prospective adjuvant trial data are available, the current evidence is sufficient to accept the CYP2D6–tamoxifen pharmacogenetic relationship in postmenopausal women” [83]. Therefore CYP2D6 genotyping may be useful in selecting adjuvant hormonal therapy in postmenopausal women and aromatase inhibitors might be preferred in CYP2D6 PMs as recommended by the Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association. Medications decreasing CYP2D6 activity, such as antidepressants, should be avoided when prescribing tamoxifen (Table 3). This needs to be confirmed in well-conducted prospective studies with tamoxifen adjuvant therapy.

2.2.1 Clopidogrel

Clopidogrel is a prodrug whose in vivo metabolite binds to the platelet P2Y₁₂ receptor causing irreversible blockade. Approximately 85 % of the parent drug is inactivated by human carboxylesterase 1, whereas the remainder is transformed to the intermediate, inactive oxo-clopidogrel by CYP2C19, CYP1A2 and CYP2B6. The CYP3A, CYP2B6, CYP2C9 and CYP2C19 are responsible for the second-step production of the active metabolite [94]. Many studies have shown that carriers of certain variant alleles exhibit a reduced capacity to produce the active metabolite and are therefore at increased risk of adverse cardiovascular events [92]. In 2010, the FDA added a Boxed Warning to the clopidogrel label to emphasis that patients who are PM for CYP2C19 are at an increased risk of adverse cardiovascular outcomes because of a reduced effectiveness of clopidogrel [95]. Bauer et al. [96] recently conducted a systematic review and meta-analysis to examine the association between CYP2C19 genotype and the clinical efficacy of clopidogrel, in a total of 15 studies. The presence of at least one LOF allele was associated with an increased risk of stent thrombosis (summary OR 1.77; 95 % CI 1.31–2.40; P < 0.001). However, the authors did not find an association between CYP2C19 polymorphism (LOF or GOF allele) and the clinical efficacy of clopidogrel used to prevent major adverse cardiovascular events (OR 1.11; 95 % CI 0.89–1.39; P = 0.36 for carriers of at least one LOF allele, and OR 0.93; 95 % CI 0.75–1.14; P = 0.48 for carriers of a GOF allele). In contrast, in a more recent meta-analysis that included 16 studies, the presence of at least one LOF allele was associated with a significant increase in adverse clinical events which were defined as a composite endpoint of death, myocardial infection, stent thrombosis or ischaemic stroke (OR 1.42; 95 % CI 1.13–1.78) [97]. Evidence supports an effect of CYP2C19 genotype on protection from major adverse cardiovascular outcomes for acute coronary syndrome/percutaneous coronary intervention, but not for lower-risk conditions [98]. Therefore, despite the large number of studies published, guiding clopidogrel dosing on the basis of CYP2C19 genotype is still a matter of debate [99] (Table 3).

2.2.2 Proton Pump Inhibitors

Proton pump inhibitors are used to treat acid-related disorders such as gastro-oesophageal reflux disease or oesophagitis/gastritis. Furthermore, PPIs are the treatment of choice for Helicobacter pylori infection, in combination with antibiotics. They undergo hepatic metabolism via the CYP450 pathways and the isoforms CYP2C19 and CYP3A4 in particular [100]. As a result of the metabolic pathways of PPIs, CYP2C19 polymorphism has an impact on their PK behaviour and clinical efficacy. The PK properties of PPIs (AUC, C _max and clearance) have been shown to be significantly different between CYP2C19 PMs and EMs. The ratios of the mean AUC values in PMs versus EMs for omeprazole, pantoprazole, lansoprazole and rabeprazole were 6.3, 6, 4.3 and 1.8, respectively [101]. These differences in terms of PK properties translate into differences in their clinical effects. It has been shown that the mean intragastric pH values were higher in PMs than in EMs for both omeprazole and lansoprazole [102]. A meta-analysis, which included 20 studies, showed a significant difference in H. pylori eradication rates between wild-type individuals and carriers of at least one LOF allele (OR 2.26; 95 % CI 1.58–2.96; P < 0.0001) when all PPI-based therapies were combined. The difference was greater when wild-type homozygous and variant homozygous patients were compared (OR 2.79; 95 % CI 1.77–4.41; P < 0.0001). When individual agents were analysed separately, a significant difference was observed for omeprazole and lansoprazole, whereas no difference between all genotypes was observed for rabeprazole [103]. This is probably due to the metabolism of rabeprazole which involves a non-enzymatic reduction [101].

2.2.3 Psychotropic Agents

CYP2C19 is one of the major metabolic pathways for several antidepressants, including imipramine, clomipramine, citalopram, sertraline, fluoxetine, venlafaxine and moclobemide [104]. Imipramine efficacy and adverse effects are associated with the sum of the plasma concentrations of imipramine and its active metabolite desipramine. The formation of the latter is mediated via CYP2C19. Some authors reported an increase in the imipramine/desipramine concentration in CYP2C19 PMs, potentially resulting in an increased frequency of adverse effects. For this reason, a dose reduction of 70 % of the recommended dose or the selection of an alternative drug is warranted [105].

Citalopram is metabolised primarily by CYP2C19 and CYP3A4, and to a lesser extent by CYP2D6 [106]. Differences in the PK properties of citalopram and escitalopram have been reported in CYP2C19 PMs and IMs [88]. However, owing to the large therapeutic window of these antidepressants, this does not appear to result in differences in their side effects. Therefore, a dose adjustment is not considered necessary. However, a recent publication reported a case of escitalopram-induced serotonin syndrome due to CYP2C19 and CYP2D6 genetic polymorphisms and drug–drug interactions with CYP2C19 and CYP3A4 inhibitors [105] (Table 2). Results from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, which included more than 1,000 patients, showed that the *2 variant allele was associated with lower odds of tolerance of citalopram (OR 0.60; 95 % CI 0.39–0.91; P = 0.02). Indeed, PMs may achieve high drug serum levels, which in some patients may be associated with lower drug tolerance. In the subset of subjects who were tolerant of citalopram treatment, remission was highest for CYP2C19 PMs, suggesting that PMs who are able to tolerate citalopram may have favourable treatment outcomes [107].

For sertraline, significant differences in PK parameters have been reported in CYP2C19 PMs, requiring a 50 % dose reduction in this population of patients [105].

2.3.1 Vitamin K Antagonists

CYP2C9 is the major route of metabolism of vitamin K antagonists such as warfarin, acenocoumarol and phenprocoumone, drugs commonly used for thrombotic complications (deep vein thrombosis, atrial fibrillation, myocardial infarct). Warfarin is metabolised by several cytochromes P450 but CYP2C9 is the major metabolic pathway. The first correlation between polymorphisms of CYP2C9, warfarin doses and the risk of bleeding was observed in 1999 [114]. In this study, patients with the CYP2C9*2 and CYP2C9*3 alleles required lower doses of warfarin. This observation has since been reported in many clinical studies [115, 116, 117] and seems to be especially noticeable at the beginning of the anticoagulant treatment [118, 119, 120]. Stabilisation of treatment is also a parameter that was found to be influenced by polymorphisms of CYP2C9. Among carriers of the CYP2C9*3 allele, the time to reach treatment stability with warfarin or acenocoumarol is longer [121, 122]. Once the dosage is stabilised, it appears that CYP2C9 polymorphisms influence the effect of oral anticoagulation to a lesser extent [123, 124]. In some studies, it has also been reported that the occurrence of supratherapeutic INRs was higher in patient carriers of these polymorphisms [122, 125]. The CYP2C9*2 allele seems to have less influence on the dose of acenocoumarol or phenprocoumon compared to warfarin [121, 122, 126, 127]. However, some studies do show a need for decreased doses of acenocoumarol in CYP2C9*2 carriers [128, 129]. Numerous guidelines have been published on the use of pharmacogenomic tests in dosing of warfarin [130]. Two online algorithms are recommended that both provide reliable and very similar results [54, 131].

2.3.2 NSAIDs

In vitro studies have demonstrated that at least 18 NSAIDs (namely aceclofenac, aspirin, azapropazone, celecoxib, diclofenac, etodolac, flurbiprofen, ibuprofen, indomethacin, lornoxicam, mefenamic acid, meloxicam, naproxen, phenylbutazone, piroxicam, suprofen, tenoxicam and valdecoxib) are metabolised, mainly, or at least in part, by CYP2C9 [132]. To date, the PK or PD consequences of CYP2C9 polymorphisms have been studied for several NSAIDs (celecoxib, diclofenac, flurbiprofen, ibuprofen, lornoxicam, piroxicam and tenoxicam), with the PD studies focusing mainly on adverse effects rather than on efficacy. Results of these different studies are often controversial. A retrospective trial (n = 218) assessed the association of polymorphic CYP2C9 alleles and the risk of acute gastrointestinal (GI) bleeding in subjects receiving NSAIDs. The frequency of CYP2C9 polymorphic alleles increased in individuals with NSAIDs-induced acute gastric bleeding. The OR for bleeding was 2.5 for heterozygous and 3.7 for homozygous carriers of mutated alleles when compared with non-bleeding subjects. Unexpectedly, this observed risk was associated with the CYP2C9*2 allele [133]. Another study (n = 26) identified a significantly higher frequency of CYP2C9*1/*3 and CYP2C9*1/*2 carriers in documented NSAID-related gastro-duodenal bleeding versus a matched control group. The presence of CYP2C9*3 was associated with a significant higher risk of bleeding (OR 7.3) even though no CYP2C9*3/*3 carriers were included in the study [134]. A more recent study with a bigger sample (n = 188) also concluded that there was an increased risk of GI bleeding when treated with NSAIDs that was linked to the presence of the CYP2C9*3 allele [135]. However, two other small (n = 23 and n = 26) retrospective studies found no relationship between NSAID-induced gastric ulceration and CYP2C9 genotype. Again, no homozygote (*3/*3) patient was included in the study [136, 137]. Even though risk of GI bleeding is clearly linked to the dose of NSAID and that PK data show a greater exposition to these drugs in carriers of the CYP2C9*2 and/or CYP2C9*3 alleles, the actual in vivo evidence on augmented risk of GI bleeding is controversial and there is therefore no recommendation of dosage adaptation in mutated patients.

2.3.3 Sulfonylureas

Sulfonylureas, such as first-generation tolbutamide and second-generation gliclazide, glyburide (also called glibenclamide) and glimepiride, are oral hypoglycaemic agents used for the treatment of type 2 diabetes. They are mainly metabolised by CYP2C9 [138], with a contribution of CYP2C19 for several of these sulfonylureas [139, 140]. The PK properties of sulfonylureas have been evaluated in few studies. For tolbutamide, mean oral clearances were reduced for all healthy carriers of one or two *2 or *3 alleles, with a clearance of 0.15 l/h in homozygous carriers of CYP2C9*3 compared to 0.97 l/h in wild-type subjects. These PK differences did, however, not affect insulin and glucose concentrations [141]. In homozygous subjects for allele CYP2C9*3/*3, oral clearance of glyburide was reduced by more than 50 % compared to wild-type subjects and insulin serum levels at 12 h were significantly higher. Presence of the CYP2C9*2 allele had only a slight influence on these two parameters [142]. In another study, heterozygous carriers of CYP2C9*3 showed a median total area under the plasma concentration–time curve of 280 % and of 267 % for the values in CYP2C9*1/*1 carriers for glyburide and glimepiride, respectively [143]. A study conducted on type 2 diabetic patients, treated with either glimepiride, gliclazide or glipizide, showed that carriers of CYP2C9*2 or *3 alleles were more prone to hypoglycaemic attacks than wild-type subjects but this difference was only significantly associated with gliclazide [144]. Even if there is a clear PK relation between sulfonylureas and CYP2C9 polymorphisms, no adaptation of dosage is recommended because of a mild risk of hypoglycaemia and the possibility to monitor glucose plasma levels [54].

2.3.4 Angiotensin II Receptor Antagonists

Angiotensin II receptor antagonists, such as losartan, irbesartan and candesartan, are used in the treatment of hypertension and congestive heart failure. These drugs are metabolised by CYP2C9; in the case of losartan, this metabolism affords an active metabolite. Several studies showed either a lower rate of oxidation of losartan into its metabolite [145], a higher plasma AUC losartan/AUC metabolite ratio [146] or a decreased level of the losartan metabolite [147] in carriers of CYP2C9*2 and/or CYP2C9*3 alleles. In type 1 diabetic patients with nephropathy, the change in systolic blood pressure was significantly greater in non *3 carriers than in *3 carriers after 4 months [148]. For irbesartan, a study in Chinese patients showed an increased plasma concentration in the CYP2C9*3 carriers but without any impact on the hypotensive effect [149]. However, a greater reduction in blood pressure in carriers of the *1/*2 and the *1/*3 genotypes compared with wild-type subjects was observed with irbesartan in a Swedish population [150]. There is no recommendation for a dose adaptation when these angiotensin II receptor antagonists are prescribed to carriers of mutated alleles of CYP2C9.

2.3.5 Phenytoin

Phenytoin is a commonly used antiepileptic drug. It is mainly metabolised by CY2C9 with a minor contribution of CYP2C19. Patient carriers of at least one CYP2C9*2 or CYP2C9*3 allele with plasma concentrations in the therapeutic range needed approximately a third lower daily dose of phenytoin than wild-type patients (199 versus 314 mg/day, respectively) [151]. Similar results were found in Turkish [152], Japanese [153] and Taiwanese populations [154]. Regarding the evaluation of an increased toxicity of phenytoin in CYP2C9 mutated patients, a small study showed an increased incidence of cutaneous reactions among carriers of CYP2C9*3 compared to wild-type patients [155]. Another study showed an increased risk of neurological adverse reactions (defined as symptoms such as dizziness, nystagmus, ataxia, slurred speech, lethargy and mental confusion) in a Southern Indian population when they were carriers of the CYP2C9*3 allele [156]. Dosage adaptation has been recommended for phenytoin in CYP2C9 mutated patients, with a dose reduction of 25 % for *1/*2 and *1/*3 carriers and of 50 % for *2/*2, *2/*3 and *3/*3 carriers [28].

3.1.1 AmpliChip CYP450 GeneChip^®

This is an oligonucleotide microarray hybridisation method for genotyping CYP2D6 and CYP2C19 that has been developed by Roche Molecular Systems and Affymetrix. It was the first clinically available tool based on microarray technology and was granted market approval by the FDA in 2004. A total of 15,000 oligonucleotide probes are included in the microarray that allow simultaneous and multiple allele testing for 20 alleles of CYP2D6 (*1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *15, *17, *19, *20, *29, *35, *36, *40 and *41) as well as 7 CYP2D6 duplications (*1 × N, *2 × N, *4 × N, *10 × N, *17 × N, *35 × N and *41 × N) and 3 CYP2C19 alleles (*1, *2 and *3) [160]. The test is based on five major processes: PCR amplification of purified DNA, labelling of the amplified product, hybridisation of the labelled amplified product to a microarray and staining of the bound products, scanning of the microarray and interpretation of the phenotype by software using an algorithm [161]. This test does not determine the exact number of extra copies of CYP2D6 alleles. The overall genotype call rate was 99.3 % for 403 tested samples. The performance of the AmpliChip in predicting CYP2D6 phenotype was assessed (n = 165) and an overall 80 % coherence was obtained. Phenotype prediction was optimal for PM (sensitivity and specificity 100 %) and satisfactory for EM (sensitivity 95 %, specificity 47 %) and IM (sensitivity 42 %, specificity 97 %) but discrepancies were observed for UM prediction (sensitivity 6 %, specificity 99 %) [162].

3.1.2 Luminex Tag-It Mutation Detection Kit

The detection kit marketed by Luminex uses a microsphere-based universal array genotyping platform [160]. The Luminex xTAG CYP2D6 kit was approved by the FDA to detect the following alleles: *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *15, *17, *29, *35, *41, ×N. However it does not specify if the allele is duplicated and it is not associated with phenotype prediction algorithm software. Seven alleles can be detected for CYP2C19 (*2, *3, and the rare *4, *5, *6, *7, *8), and five alleles for CYP2C9 (*2, *3, *4, *5 and *6).

3.1.3 iPLEX^® ADME

Three panels have been developed by Sequenom: PGx panel, CYP2C9/VKORC1 panel and CYP2C19 panel.

The iPLEX ADME PGx panel simultaneously analyses 192 SNPs in 36 genes such as CYP1A, 2A6, 2B6, 2C19, 2C8/9, 2D6, 2E1, 3A4/5, phase II drug-metabolising enzymes and drug transporters. Analysis is provided by the MassARRAY system.

Targeted assay panels can be created using the Assay Explorer program [163].

The iPLEX ADME CYP2C9/VKORC1 panel is a set of 36 SNPs for CYP2C9 and 9 SNPs for VKORC1. The iPLEX ADME CYP2C19 panel analyses 31 SNPs for CYP2C19.

3.1.4 INFINITI^® CYP2C19 Assay

The assay marketed by Autogenomics uses a hybridisation capture array with instrument-performed automated detection of multiple PCR products. PCR amplification is done offline while the rest of the processes are automated by the AutoGenomics INFINITI Analyzer. The assay is available in two formats: one sample per microarray chip or four samples. It identifies only the CYP2C19 *2, *3 and *17 alleles and corresponding genotype polymorphisms. The assay report lists the alleles and provides the genotype detected (wild type, mutant, heterozygote). The call rate reported by the manufacturer is greater than 90 % with no incorrect call rate [164].

3.1.5 eSensor^® Warfarin Sensitivity Test

This DNA hybridisation and electrochemical detection test is marketed by GenMark and allows detection and genotyping of CYP450 2C9 (*2 and *3) and VKORC1 (−1639G>A), with a result produced in approximately 3.5 h [165].

3.1.6 Spartan RX CYP2C19 System

This kit identifies carriers of the CYP2C19*2 allele using a cheek swab and adding sample to a cartridge then placing into a Spartan RX machine, printing a result in 1 h [166].