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Student ID No.

50904989 MT5004

Pharmacogenetics, Polymorphisms and Therapeutic efficacy

Introduction
Therapeutics 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.,

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2005). E.g. 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 process
There 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 ENZYMES


The 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 CYP1A2


CYP1A2 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 CYP2B6


CYP2B6 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 2C9

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CYP2C9 metabolizes a wide range of therapeutic drugs, such as S-warfarin,


phenytoin, tolbutamide, glipizide, torsemide, losartan, and several
nonsteroidal Anti
-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*.

Figure taken from (Zhou, 2009).


Role of CYP2C9
polymorphism in
Diabetes
Sulfonylureas are used to
treat type II diabetes.
They are metabolized by
hydroxylation of the

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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 Epilepsy


Phenytoin (5,5-Diphenylhydantoin) is an anticonvulsant drug. Phenytoin is
oxidized by
CYP2C9 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 Hyperlipidemia


Statins 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


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-

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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 P4502C19
In 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).

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.

Conclusion
Overall 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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