pharmacogenetics polymorphism and efficacy of drugs

Student ID No. 50904989 MT5004

Pharmacogenetics, Polymorphisms and Therapeutic efficacy

IntroductionTherapeutics would have been a lot easier if the response to the dose of drugs were always the same. But in practice, there exists an inter-individual variability in the response exhibited for same dose of a drug (Fromm, 1997). Inter-individual variable response is a major issue affecting pharmacotherapy. Inter-individual variable drug response can be attributed to genetic polymorphism in drug metabolizing enzymes or in drug transporters. (Marin, 2009). This variability may explain the efficacy of a drug in a given patient as well as its adverse side effects. (Marin, F.2009). The most commonly occurring genetic variation that affects drug action is single nucleotide polymorphism (SNP) (Pfost, 2000) (refer fig 1).

Fig 1. Showing Single Nucleotide polymorphism

Image taken from http://en.wikipedia.org/wiki/Single-ucleotide_polymorphism#.22SNP.22

Genetic polymorphism can be defined as a monogenic trait, which occurs in the population in at least two genotypes and phenotypes, neither of which has a frequency of less than 1%. (Fromm, 1997)

In 1980’s, Frank Gonzalez discovered that the basis for poor debrisoquine metabolism, which affected 8% of Caucasian population, was defective cytochrome P450 2D6 enzyme. Later many molecular defects have been linked to variations in cytochrome P450 which is a drug metabolizing enzyme. (Maxwell, 2006)

Variability produced by genetic polymorphism can affect genes which encode for disposition, metabolism, transporters, or targets of the drug which has the potential to alter the individual response (Marin, F.2009) thus explaining the efficacy, toxicity or the kinetics of the drugs. (Robert et al., 2005). E.g.

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http://en.wikipedia.org/wiki/Single-ucleotide_polymorphism#.22SNP.22


increased incidence of neurotoxicity was observed in individuals who were receiving succinylcholine as isoniazid therapy because they were slow acetylators. (Maxwell, 2006)

Study of how inheritance of genetic variations influences an individual's response to drugs is called as Pharmacogenetics. (AMEYAW, 2006) .Term ‘pharmacogenetics’ was given by Vogel in 1959(Pfost, 2000).

Pharmacogenetics involves the use of DNA analysis techniques to list out the variations within the human genome specially SNPs –so that the new or existing therapeutic agents can be used with maximal efficacy and minimal toxicity. (Pfost, 2000).Pharmacogenetics is of utmost importance for drugs which have a narrow therapeutic range between effectiveness and toxicity. (AMEYAW, 2006)

The knowledge of genetic polymorphisms that affect drug efficacy and toxicity will enable physicians to optimize medication based on the patient’s genetic constitution, which will result in fewer adverse reactions. (Pfost, 2000)

Pharmacogenetics as a processThere are three major phases in pharmacogenetics process:(1) Discovering SNP: It involves identification and listing of SNPs in the human genome and out of these SNP’s some affect drug response or cause toxicity.(2) Clinical trails are designed and conducted to perform genetic analysis so that clinical association between SNP and variation in drug response could be established.

(3) SNP diagnostics: the SNP-based diagnostic tests are conducted to ascertain whether or not a patient gives the desired response to a particular drug. (Pfost,2000)

In this write up, important genetic polymorphisms of cytochrome P450 enzymes, esp. of CYP2C9 and of CYP2C19 and their therapeutic implication are discussed in detail.

GENETIC POLYMORPHISM OF CYTOCHROME P450 ENZYMESThe cytochrome P450 (CYP) are drug metabolizing enzymes important in Phase I metabolism .The human genome comprises of 57 functional CYP genes and 58 pseudogenes which are divided into 14 sub families (Zhou, 2009).in humans, of the total drug oxidation occurring 90% is attributed to CYP1A2 (4%), 2A6 (2%), 2C9 (10%), 2C19 (2%), 2E1 (2%), 2D6 (30%), and 3A4 (50%) (Zhou, 2009) .

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GENETIC POLYMORPHISMS OF CYP1A2CYP1A2 is hepatic drug metabolizing enzyme e.g. caffeine, clozapine, tizanidine and ramelton. CYP1A2 is present on chromosome 15. Several variants of CYP1A2 have been found which are linked with reduced CYP1A2 expression. (Flockhart,2009)

CYP1A2 is responsible for the metabolism of caffeine. Caffeine is used to study the genetic as well as non genetic influences on the CYP1A2 activity (Flockhart,2009).

Increased in clearance was observed with CYP1A2*1F variant while other SNPs and variant allele may decrease or increase the caffeine clearance (Flockhart,2009).

There appears to be an association between CYP1A2*1F and smoking which increases clozapine clearance hence they become non- responsive for the normal dose (Flockhart,2009).

GENETIC POLYMORPHISMS OF CYP2B6CYP2B6 belongs to the pool of CYP enzymes found in liver. CYP2B6 is present on 19th chromosome (Flockhart,2009). CYP2B6 metabolizes these drugs -efavirenz, nevirapine, cyclophosphamide, bupropion, ifosfamide, thioTEPA, methadone, meperidine, artemisinin, ketamine and propofol (Flockhart, 2009).

Chemicals and drugs can influence the activity of CYP2B6 either by inducing it or inactivating it (Flockhart, 2009).

The studies conducted by researchers showed that over 100 SNP’s exists in the (Lang, et al.,2004) CYP2B6 and there exists 28 alleles but other studies showed that there exists several more DNA variations in the entire CYP2B6. Polymorphism of CYP2B6 causes partial diminished activity, non-function or increase in function of CYP2B6 genes (Flockhart, 2009).

CYP2B6 is an important enzyme in the metabolism of the Efavirenz, which is a non- nucleoside reverse transcriptase inhibitor used in HIV therapy. (Flockhart, 2009).It was clear from the clinical studies conducted by Zanger et al. showed that individuals who were homozygous for 516T (CYP2B *6/*6) genotype had higher plasma concentration of efavirenz than heterogeneous CYP2B6 *1/*6 genotypes and homozygous 516C (CYP2B6 *1/*1) ( Zanger et al.,2007). Hence, it is clear that individuals homozygous for 516T (CYP2B 6 *6) genotype are at high risk of efavirenz induced CNS toxicity (Flockhart, 2009).

GENETIC POLYMORPHISMS OF CYP2C

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Cytochrome P450 2C are of 4 types (2C8, 2C9, 2C18 and 2C19) and are expressed in liver. Among these isoforms 2C9 and 2C19 are shown to exhibit extensive polymorphism (Ishizaki, 2002).CYP2C9 is located on long arm of chromosome 10, which also contains genes for CYP2C8, 2C18 and 2C19 (Flockhart,2009).

CYP2C9 is the most abundant of all the CYP enzyme found in liver and shows a selectivity for the oxidation of small, lipophilic anions such as ibuprofen, diclofenac and gemfiborzil as substrates (Zhou, 2009).

CYP 2C9 and 2C19 metabolize important therapeutic drugs e.g. metabolism of the anticoagulant warfarin by Cytochrome P4502C9. Warfarin is an inhibitor of vitamin K dependent clotting factors. (Maxwell, 2006)

A study conducted has found, 556 SNPs in the CYP2C9 upstream sequence which influences the activity of CYP2C9. (Refer table 1) CYP2C9*2 and CYP2C9*3 are the variants which have 12% and 5% activity that of the wild type allele. Therefore, require much lower doses of warfarin, than the patients with the wild type gene. (Maxwell, 2006)

Table 1. Polymorphic CYP2C isoforms and its substrates, inhibitors and inducers.

Table taken from (Ishizaki, 2002)

Polymorphism in Cytochrome P450 2C9CYP2C9 metabolizes a wide range of therapeutic drugs, such as S-warfarin, phenytoin, tolbutamide, glipizide, torsemide, losartan, and several nonsteroidal Anti

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-inflammatory agents (refer Table 1 & 3)

Table 2. CYP2C9 allelic variants and their frequencies in various ethnic populations.

Table taken from (Ishizaki, 2002).

Table 3. drugs that are substrates of CYP2C9

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Table taken from (Zhou, 2009)

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CYP2C9 allelic variants and the frequencies have been listed from various ethnic populations (refer Table 2). Polymorphisms in the exon 3, exon 7and exon 8 of gene CYP2C9 was determined for different ethnicities of world.(refer table 2.) (Ishizaki, 2002).

Studies have shown the possible mediation of CYP2C9 in the metabolism of drugs but its variant alleles have lower affinity to hydroxylate the therapeutic agents than the wild type. (Ishizaki, 2002).

The pharmacokinetics of glipizide and phenytoin was studied by Kidd et al. in an individual who was not suffering from any of the known defects of CYP2C19 alleles with Leu359 mutation in CYP2C9*3 allele. (Kidd, 1999) The individual’s oral clearances of the two drugs was found to be extremely low while comparing with those of the Ile359 (CYP2C9*1) homozygous individuals. (Kidd, 1999) Moreover, an extremely low blood glucose level or severe hypoglycemia was observed in homozygous Leu359pateints who took glipizide. (Kidd, 1999).

The genetic polymorphism as well as mutation (Ile359→ Leu359) in CYP2C9 is relevant in the pharmacokinetic variability of phenytoin and determines the impaired metabolism of anticonvulsants. (Ishizaki, 2002).

Patients who were homozygous for the genotype CYP2C9*3 and those who were heterozygous CYP2C9*1/*3 genotype had drastically reduced clearance of (S)-warfarin. The reduction in clearance was 60%- 90% (refer graph 1.) (Takahashi, 2001), indicating the need for adjusted dosage of warfarin for CYP2C9 genotype. Polymorphism of CYP2C9 also influences the risk of bleeding (Ishizaki, 2002).

Variant allele CYP2C9*2 causes a loss of 20–30% of the enzymatic activity of CYP2C9 toward S-naproxen, while *3 mutation reduces Vmax of CYP2C9 by 70%. Reduction in the enzyme activity is attributed to conformational changes preventing substrates to bind to enzyme (Zhou, 2009).

Graph 1. Showing % reduction in clearance of the various drugs in individuals carrying variant allele for CYP2C heterozygous 1*/3* and homozygous 1*/1* & 3*/3*.

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Figure taken from (Zhou, 2009).Role of CYP2C9 polymorphism in DiabetesSulfonylureas are used to treat type II diabetes. They are metabolized by hydroxylation of the aromatic ring. CYP2C9 is the main enzyme responsible for the hydroxylation. Since CYP2C9 is the principle enzyme in the metabolism of sulfonylureas, mutant allele CYP2C9*3 has been clearly linked to impaired clearance of sulfonylureas (Kirchheiner, J., et al.,2002).

Heterozygous and homozygous individuals for CYP2C9*3 have a clear 2- and 6- times increased AUC of sulfonylureas than the wild-type (Zhou, 2009).

Role of CYP2C9 polymorphism in EpilepsyPhenytoin (5,5-Diphenylhydantoin) is an anticonvulsant drug. Phenytoin is oxidized byCYP2C9 and to a minor extent by CYP2C19. In patients having variant allele CYP2C9*3/*3 or 6* 4- to 5-times increase in phenytoin AUCs was observed

(Kidd et al.,1999).. White patients on normal dose who had plasma concentrations within the therapeutic range, if at least one CYP2C9*2 or *3 allele present it lowered their mean dose requirement by one third (Zhou, 2009).

Role of CYP2C9 polymorphism in HyperlipidemiaStatins like fluvastatin are the commonly used HMG-CoA reductase inhibitors used to lower cholesterol. Statins are metabolized by hydroxylation by CYP2C9 and also by CYP3A4. Statins interact with CYP3A inhibitor causing drug interaction. Fluvastatin inhibits CYP2C9 at lower concentrations both in vitro and in vivo (Fischer et al., 1999).

The variant genotype CYP2C9*3/*3 was associated with 3-times increased concentrations of the active fluvastatin than the wild-type in healthy subjects, while individuals carrying the CYP2C9*1/*3 and *2/*3 genotypes had intermediate concentrations (Kirchheiner, et al.,2003).

Role of CYP2C9 polymorphism in Thrombotic diseases

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Warfarin is used in the treatment of thrombotic complications such as deep vein thrombosis, myocardial infarct, atrial fibrillation and stroke (Bovill et al., 2004).anticoagulant activity of Warfarin and other coumarin are due to inhibition of the enzymatic activity of vitamin K1 2,3-epoxide reductase complex 1 (VKORC1) (Goodstadt et al., 2004).

S-warfarin is metabolized by CYP2C9 by hydroxylation and has a narrow therapeutic index (Zhou,2009).

Slow hydroxylation of S-warfarin is due to mutant alleles of the CYP2C9 (Lal et al., 2006).Homozygous patients with variant CYP2C9*3 allele exhibited a reduction of 90% in the elimination of S-warfarin as compared to patients who were homozygous for the wild-type allele (Takahashi, 2001). Reduction in elimination of a low therapeutic index drug such as warfarin can cause serious clinical complications. Patients with such polymorphisms have 4-times high risk of bleeding complications, than a normal patient (Gage et al., 2008).

By pharmacogenetic testing of CYP2C9 metabolism we could identify the patients who are at a higher risk of haemorrhage (Zhou, 2009).

Cytochrome P4502C19In humans, metabolism of clinically available mephenytoin is stereospecific. (Kupfer,1984) Out of the two enantiomers of Mephenytoin, R enantiomer is metabolized by CYP3A4 and is demethylated to nirvanol while S enantiomer is hydroxylated by CYP2C19 (Kupfer,1984). Polymorphism in CYP2C19 which metabolizes S enantiomers results in two phenotypes – Extensive metabolizers (EM) and Poor metabolizers (PM) (Kupfer,1984).

Proton pump inhibitors are metabolized by CYP2C19 and a few PPI’s are omeprazole, lansoprazole, pantoprazole, and rabeprazole. (Andersson,1996). Rabeprazole showed a very small difference in its AUC values for Poor metabolizers and for Extensive metabolizers as compared to the other 3 PPI’s. Indicating no significant effect of CYP2C19 polymorphism on disposition of Rabeprazole (Andersson,1996). The most affected by CYP2C19 polymorphism was omeprazole in which PMs showed 20 times more AUC value than EMs followed by pantoprazole and iansoprazole and least affected was rabeprazole. PPIs on repeated doses showed increased acid suppression which is positive relationship between polymorphism and pharmacological effect (Ishizaki, 2002).

Study conducted by Furuta et al. (Furuta,1998) showed that genotyping of CYP2C19 helps in predicting susceptibility to and eradication of Helicobacter pylori infection and it can also enhance peptic ulcer healing by giving individualized therapy (Ishizaki, 2002).

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From the above facts, we can suggest that doing genotyping for CYP2C19 is important in terms of efficacy and toxicity of drugs for patients suffering from acid-related disease treatments.

ConclusionOverall impact of pharmacogenetics will be to improve patient-care processes, and public health. This can be achieved both by optimization of drug prescribing and by improvements in new drug development. With the knowledge of pharmacogenetics doctors will be able to avoid prescribing drugs to those patients who are unlikely to respond or who are more prone to adverse reactions.

Preventing toxicity and improving efficacy of the drugs will not only help patients but will also help save millions of pounds of health-care budget.( AMEYAW,2006)

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- Zanger , U.M., et al. ( 2007 ) Polymorphic CYP2B6: molecular mechanisms and emerging clinical significance . Pharmacogenomics 8 ( 7 ) , 743- 759 .

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pharmacogenetics polymorphism and efficacy of drugs

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