Pharmacogenomics

1. Pharmacogenomics
Dr Shinde Viraj Ashok
Junior Resident
Department of Pharmacology

2. Overview
Definitions
Genetic variations in enzymes
Role of pharmacogenomics in drug discovery and development
Issues of concern in Pharmacogenomics
Regulatory guidance
Conclusion

3. Definitions
Pharmacogenetics –
study of genetic basis for variation in drug response
 Pharmacogenomics –
makes use of genetic make up (genome) of an
individual to choose drug therapy for responders and avoid
giving such drug to non responders ( i.e tailoring of drug
therapy on the basis of individuals genotype)

4. Allele – one of two alternative forms of gene
that arise by mutation & are found at same
gentic locus
Each star (*) allele is defined as specific
sqeuence variation(s) with in gene locus e.g
single nucleotide polymorphisms
Single nucleotide polymorphism – substitution
of one DNA unit for another at a particular
site

5. Sum of allelic activity score ranges from 0 & ≥
3 and this used to define phenotypes as
poor metabolisers = 0
intermediate metabolisers = 0.5
extensive metabolisers = 1 – 2
ultra rapid metabolisers = ≥ 2
CPIC – clinical pharmacogenetics
implementation consortium

6. Pharmacogenomics Concept

7. Drug Response
 Individual Variability in Drug Therapy
 Factors Affecting Individual Drug Response
◦ Genetic Polymorphisms of Drug Targets
◦ Genetic Polymorphisms of Drug-Metabolizing Enzymes
◦ Genetic Polymorphisms of Drug Transporters
◦ Genetic Variables Affecting Adverse Drug Reactions

8. Genetic variations in enzymes
Phase I enzymes :
Biotransformation - Over 75% of prescribed
drugs
Polymorphisms of phase I enzymes
◦ Significantly affect blood levels of drugs
◦ Which in turn may alter response to many drugs

9. CYP2D6
 Metabolism - up to one quarter of all drugs
used clinically (ß blockers, antidepressants,
antipsychotics & opioid analgesics)
 Highly polymorphic over 100 alleles defined
ie, CYP2D6
◦ alleles *3, *4, *5, and *6 - non-functional;
◦ alleles *10, *17, and *41 - reduced function;
◦ alleles *1 and *2 - fully functional.

10.  Poor metabolisers(PMs) &
Intermediate metabolisers
(IMs) - likely to
experience insufficient
pain relief
 Ultra rapid metabolisers
(UMs) –
 ↑ risk for side effects
 Due to higher systemic
concentrations of morphine
 Eg - Drowsiness and
respiratory depression
Codiene
CYP2D6
Morphine
CPIC guidelines recommend –
 EMs & IMs - standard starting doses with close monitoring,
especially in IMs
 PMs and UMs - alternative agent e.g. morphine , non opoid
analgesic

11. CYP2C19
 Metabolize acidic drugs including
◦ Proton-pump inhibitors
◦ Antidepressants
◦ Antiepileptics
◦ Antiplatelet drugs
 Four clinical phenotypes - based on activity of CYP2C19
◦ Poor metabolisers(PM)
◦ Intermediate metabolisers (IM)
◦ Extensive metabolisers(EM)
◦ Ultra rapid metabolisers(UM)
 Highly polymorphic over 30 alleles defined

12.  Poor metabolisers(PM) phenotype
◦ Asians (~16%)
◦ Europeans and Africans (~2–5%)
 Carriers of reduced function CYP2C19*2
alleles taking clopidogrel are at ↑ risk for
serious adverse cardiovascular events

13. Clopidogrel - thienopyridine antiplatelet
prodrug indicated for prevention of
atherothrombotic events
85%
- Hepatic esterases
- Inactive carboxylic acid
derivative
15%
- two sequential cyp-mediated
oxidation reactions(predominantly
CYP2C19)
- active thiol metabolite - antiplatelet
activity
Genetic polymorphisms CYP2C19 gene –
↓ active metabolite formation & reduce drug’s antiplatelet
activity associated with variability in response to clopidogrel
Current clinical recommendations from Clinical
Pharmacogenetics Implementation Consortium(CPIC)
specific for acute coronary syndrome with percutaneous
coronary intervention (PCI)
 Standard starting doses are recommended in
extensive metabolisers(EMs) and ultra rapid
metabolisers (UMs)
 Use of an alternative antiplatelet agent in poor
metabolisers (PMs)and intermediate
metabolisers (IMs)Eg, prasugrel or ticagrelor

14. Dihydropyrimidine Dehydrogenase (DPD)
 Encoded by DPYD gene –
◦ First and rate-limiting step in pyrimidine catabolism
◦ Major elimination route for fluoropyrimidine
chemotherapy agents
 Three non-functional alleles - rare - ie,
DPYD*2A, *13 & rs67376798
 Allele *2A most commonly observed allele & is
often only variant tested in commercial
genotyping platform

15.  Example
 5-fluorouracil (active compound)(5-FU) ; capecitabine & tegafur
(oral prodrugs)
 To treat solid tumors ( colorectal and breast cancer)
 Capecitabine & tegafur - Only 1–3% administered dose of
prodrug is
 Converted to active cytotoxic metabolites, ie, 5-
fluorouridine 5'monophosphate (5-FUMP) and 5-fluoro-2'-
deoxyuridine-5'-monophosphate (5-FdUMP)
 Effectively target rapidly dividing cancer cells and inhibit
DNA synthesis
 Majority of an administered dose (~80%) is subjected to
pyrimidine catabolism via DPD & is excreted in urine

16.  Complete or partial deficiency of DPD can lead to
 ↑ Half-life of toxic metabolites F-UMP and f-dump
 ↑ risk for severe dose-dependent fluoropyrimidine
toxicities
 Eg, myelosuppression, mucositis, neurotoxicity, hand-
and-foot syndrome & diarrhoea
 CPIC guidelines recommend that
 Normal activity – standard dose
 Reduced activity – reduce initial dose 50% & titrate
based on toxicity or on pharmacokinetic test results
 Complete deficiency – different non fluoropyrimidine
anticancer drugs

17. PHASE IIENZYMES
 Conjugate endogenous molecules
◦ eg sulfuric acid, glucuronic acid & acetic acid, onto
a wide variety of substrates in order to enhance
their elimination from body
 Polymorphic phase II enzymes - ↓drug
elimination & ↑ risks for toxicities

18. Uridine5'-Diphosphoglucuronosyl Transferase1
(UGT1A1)
 Encoded by the UGT1A1 gene
 Individuals with UGT1A1*28/*28 genotype –
◦ Associated with reduced expression of UGT1A1
enzyme
◦ ↑ risk for adverse drug reactions with UGT1A1 drug
substrates
◦ Due to ↓ biliary elimination

19. Example
 Irinotecan –
 Indicated - treatment of metastatic carcinoma of colon or
rectum
 Hepatic carboxylesterase enzymes → cytotoxic metabolite,
SN-38 ( inhibits topoisomerase 1 )
 Active SN-38 metabolite is responsible for
 Majority of therapeutic action
 Dose-limiting bone marrow and gastrointestinal toxicities
 Inactivation of SN38 occurs via polymorphic UGT1A1 enzyme
and carriers of UGT1A1*28 variant –
 ↑ risk for severe life-threatening toxicities, eg, neutropenia
and diarrhea, due to ↓ clearance of SN-38 metabolites

20. CPIC guidelines
 Normal activity -*1/*1 , *1/*28 – standard starting
dose
 Reduced activity – *28/*28 - reduce starting dose by at
least one dose level

21. Thiopurine S-Methyltransferase(TPMT)
 Attaches methyl group onto aromatic & heterocyclic
sulfhydryl compounds
◦ Responsible for pharmacologic deactivation of thiopurine
drugs
 Genetic polymorphisms in gene encoding TPMT - 3
clinical TPMT activity phenotypes
◦ High
◦ Intermediate &
◦ Low activity
 Associated with differing rates of inactivation of
thiopurine drugs & altered risks for toxicities

22.  Example
 Azathioprine, 6-mercaptopurine (6-MP) & 6-thioguanine
(6- TG)
 Azathioprine (a prodrug of 6-MP) and 6-MP - treating
immunologic disorders
 6-MP and 6-TG (Anti-cancer agents )
 Activated by salvage pathway enzyme hypoxanthine-
guanine phosphoribosyltransferase (HGPRTase) to
form 6thioguanine nucleotides (TGNs)
 6thioguanine nucleotides (TGNs)- responsible for
majority of therapeutic efficacy & bone marrow
toxicity

23. CPIC guidelines
 Normal , high activity – Standard starting dose
 Intermediate activity – Start 30 – 70 % starting dose &
titrate every 2-4 weeks with close clinical monitoring of
tolerability eg TLC , LFTs
 Low activity –
 Malignant disease – drastic reduction of thiopurine
doses
 Non malignant diseases – alternative non thiopurine
immunosuppressive agents

24. Other enzymes
G6PD
 Gene that encodes G6PD enzyme is
◦ Located on X chromosome
◦ Highly polymorphic over 180 genetic variants identified that result
in enzyme deficiency
 Glucose 6-phosphate dehydrogenase (G6PD)
◦ First & rate-limiting step in pentose phosphate pathway
◦ Supplies a significant amount of reduced NADPH in body
 Red blood cells (RBCs)-
◦ Mitochondria are absent
◦ G6PD - exclusive source of NADPH
◦ Reduced glutathione - play critical role in prevention of oxidative
damage

25.  Under normal conditions –
◦ G6PD in RBCs is able to detoxify unstable oxygen
species
◦ While working at just 2% of its theoretical
capacity
 Individuals with G6PD deficiency (WHO
classification)
Defined as less than 60% enzyme activity
are at ↑ risk for abnormal RBC destruction, ie,
haemolysis due to ↓ antioxidant capacity under
oxidative pressures

26. Classification of G6PD deficiency
(WHO Working Group, 1989).

27.  Examples
Rasburicase
• Recombinant - urate - oxidase enzyme
• Initial management of uric acid levels in
cancer patients receiving chemotherapy
Manufacturer recommends that
• Patients at high risk (individuals of african or
mediterranean ancestry) be screened prior
to initiation of therapy
• Rasburicase not be used in G6PD deficiency

28. GENETIC VARIATIONSIN TRANSPORTERS
 Plasma membrane transporters
◦ Located on epithelial cells of many tissues, eg,
intestinal, renal, and hepatic membranes,
◦ Mediate selective uptake and efflux of endogenous
compounds and xenobiotics including many drug
products
 Genetic differences in transporter genes
alter drug disposition and response may ↑ risk
for toxicities.

29. ORGANIC ANION TRANSPORTER (OATP1B1)
 Encoded by the SLCO1B1 gene –
 Transporter –
◦ Located on sinusoidal membrane (facing the blood)
of hepatocytes
◦ Responsible for hepatic uptake of mainly weakly
acidic drugs and endogenous compounds eg statins,
methotrexate & bilirubin
 40 non-synonymous variants (nsSNPs) of this
transporter - some of which result ↓
transport function

30.  Example
 Common variant rs4149056 in SLCO1B1,
 ↑ systemic exposure of simvastatin
 Identified to have single strongest association with
simvastatin-induced myopathy
 For individuals receiving simvastatin with reduced OATP1B1
function (at least one non-functional Allele),
CPIC recommends a lower simvastatin dose or an
alternative statin

31. GENETIC VARIATIONSIN IMMUNE SYSTEM
FUNCTION
 Genetic sources of variation -
Pharmacodynamic genes (drug receptors and
drug targets genes)
 Example - Polymorphism in HLA loci is
associated with a predisposition to drug
toxicity.

32. DRUG-INDUCED HYPERSENSITIVITY REACTIONS
 Worst hypersensitivity reactions are
◦ Liver injury
◦ Toxic epidermal necrosis (TEN)
◦ Stevens-johnson syndrome (SJ S)
(Drugs and/or their metabolites form antigens)
 Drug classes associated
◦ sulfonamides
◦ nonsteroidal anti-inflammatory drugs (NSAIDs),
◦ antibiotics, steroids,
◦ antiepileptic agents & methotrexate

34. Example
 Abacavir - Hypersensitivity reactions - SJS
(idiosyncratic)
◦ Consistent with allele frequencies of HLA-B*57:01
◦ Activated to carbovir triphosphate -reactive molecule -
involved in immunogenicity of abacavir
◦ Mediated by activation of cytotoxic CD8 T cells
◦ Because of importance of abacavir in therapeutics,
genetic testing of the HLA-B*57:01 biomarker
associated with abacavir hypersensitivity has been
rapidly incorporated into clinical practice

35.  Flucloxacillin hypersensitivity reactions –
◦ Lead to drug-induced liver toxicity
◦ Highly significant association was identified with
polymorphism linked to HLAB*57:01

36. IFNL3 (Il-28B)(INTERFERON LAMBDA 3)
 rs12979860 variant near IFNL3 is considered
strongest baseline predictor of a cure for
patients with HCV-1 receiving PEG-IFN-a/ RBV
 Approximately two fold greater cure rates were
observed in patients with a favourable genotype
 Favourable allele, rs12979860 variant, is
inherited most frequently in Asians (~90%), and
least frequently in Africans

37. POLYGENIC EFFECTS
Polygenic influences – combinatorial
effect of multiple genes on drug response, may
more accurately describe individual
differences with respect to clinical outcomes

38. Example
CYP2C9 & VKORC1 on warfarin
 Allele CYP2C9*2 –
◦ Leads to reduced metabolism of CYP2C9
substrates,
◦ Including a 30–40% reduction in S-warfarin
metabolism
 Allele CYP2C9*3 –
◦ Lowered affinity for many CYP2C9 substrates &
◦ More marked 80–90% reduction in S-warfarin
metabolism

39.  Most important consequences of VKORC1
polymorphism (VKORC1-1639G>A)
◦ Reduced expression of VKORC1 in liver
◦ ↑ sensitivity to warfarin
◦ ↑ risk for excessive anticoagulation following standard
warfarin dosages
 VKORC1-1639G>A polymorphism occurrence-
◦ Asian populations (~90%) &
◦ Africans (~10%) Gene-based dosing may help - Optimize warfarin therapy
management and minimize risks for adverse drug reactions

40. Application of Pharmacogenomics
in drug development
Drugtarget and pharmacogenomics
 Drug discovery starts with identification of a potential target at
which drug can act
 Target can be an
 Enzyme in a vital pathway
 Receptor
 Transporter
 Protein in signal transduction or
 Any protein produced in a pathological condition
 After sequencing of human genome, number of drug targets was
estimated to be around 8000, out of which 4990 could be actually
acted upon - 2329 for antibodies & 794 for drug proteins

41. Examples: Drug Target
 C-KIT expression in GIST –Imatinib
 CCR5 -Chemokine C-C motif receptor on
human T-cell – Maraviroc
 EGFR expression - Erlotinib
 HER2/neu expression – Trastuzumab

42. Pharmacogenomics and clinical trials
 Incorporation of pharmacogenomic testing
with clinical trials has multiple advantages
 Two most important concerns for new drug
development are efficacy and safety

43.  Availability of sophisticated pharmacogenetic
tools - Attrition rate can be significantly
reduced (reduction in loss of financial
resources for drug development)
 Drug metabolized by polymorphic enzymes
◦ Can be identified during preclinical studies with in
vitro methods
◦ Decision regarding continuation of trial can be
made

44. Prediction of efficacy of drug
 Drugs designed with pharmacogenomic
support have predetermined efficacy status
 Chance of a drug failing in preclinical &
clinical studies due to absence of efficacy is
minimized

45. Example,
Drug trastuzumab –
 Anti-HER2 monoclonal antibody against
metastatic breast cancer
 Found to be effective only in women over
expressing HER2 protein during early clinical
trials
 In subsequent trials - studies were done only on
women found to be over expressing HER2 protein
 Approval for marketing - before starting
therapy testing for HER2 over expression must
be done

46.  Pharmacogenomics – Used to identify target
population that would benefit most from drug
Example –
 Association between polymorphisms in
◦ apolipoprotein E (APOE)
◦ cholesteryl ester transfer protein(CETP)
◦ stromelysin-1 &
◦ ß-fibrinogen with progression of atherosclerosis,
cardiovascular events & death
 People with such polymorphisms derived
maximum benefit from HMG-CoA inhibitors, as
compared to those without polymorphisms

47. Prediction of safety of drug
 During a clinical trial –
◦ Occurrence of a serious adverse event could
jeopardize drug status
◦ Such an event would culminate in termination of
clinical trial
 Drug toxicity
◦ Mainly due to ↑ plasma levels of drug
◦ Result of poor metabolizing capacity owing to
genetic polymorphisms

48.  Availability of high throughput genotyping
methods, pharmacogenetic testing can be
incorporated into inclusion criteria for selecting
a subject for trial
 Poor metabolizers -
◦ Tend to attain higher plasma concentration of drug
◦ Higher incidence of toxicity
 If poor metabolizers are avoided in study
occurrence of serious adverse events - reduced

49. Issues of concern in
Pharmacogenomics
 Practical application in routine patient care is
at present limited due to pre requirement of
multiple drug specific genotyping screening
which involves huge cost
 Will lead to ‘discrimination’ in medical therapy
provided to a patient – treatment will be
based which in turn correlated with ethnic
and racial factors

50.  Rare genotypes(orphan genotypes) – deprived
of health care – may not enjoy insurance
cover
 Pharmaceutical companies – will not be willing
to invest in developing drugs for groups with
less common genotype – creation of
‘therapeutic orphans’

51. Pharmacogenomics in India
 Department of Biotechnology, Ministry of
Science and Technology, New Delhi
 Human Genetics and Genome Analysis
Programme.
 Human Genome Diversity Project

52. Regulatory Guidance
 FDA: Guidance for Industry -
Pharmacogenomic Data Submissions
 EMEA (european medical agency)

53. Conlclusion
 Therapeutic response in many disease
processes is genotype-specific and
multifactorial
 Tailoring medication regimens based on
patient genomes maximizes efficacy and
compliance while avoiding adverse effects and
drug-drug interactions

54. References
 Katzungs basic and clinical pharmacology 13th
edition
 Role of pharmacogenomics in drug discovery and
development Department of Pharmacology,
JIPMER, Puducherry, India, A. Surendiran, S.C.
Pradhan, C. Adithan
 India, Department of Biotechnology, Ministry of
Science and Technology, (2013) List of Ongoing
Projects as on 30/05/2013

56. Examples: Drug Metabolism
 CYP2C19 and CYP2D6 Variants – Poor vs extensive metabolizers
 N-acetyltransferase - slow and fast acetylators
 Deficiency of dihydropyrimidine dehydrogenase (DPD) activity
– Capecitabine
 Glucose- phosphate dehydrogenase (G6PD) deficiency
Rasburicase
 Thiopurine methyltransferase deficiency or lower activity –
Azathioprine
 Homozygous UGT1A*28 allele - Irinotecan

58. Phenotyping and Genotyping
Phenotyping
◦ Dose subjects with a compound or compounds that are metabolized
to a product exclusively by the enzyme systems in question.
◦ Collect plasma or urine samples
» Single time point
» Over a period of time
◦ Analyze for model compound and metabolite
◦ Ratio of concentrations of compound and its metabolite is used to
measure metabolic capacity for a specific P450.
• Genotyping
◦ Collect blood (> 1 ml)
◦ Isolate DNA from nucleated blood cells.
◦ Amplify number of copies of DNA by the Polymerase Chain
◦ Reaction (PCR).
◦ Genotype by sequencing or probing.

59. Pharmacogenomics in Drug
Development
 DNA samples taken for ADME genotyping in drug development
Routine if one enzyme is known as the predominant route of metabolism.
 Compounds with narrow safety margin
◦ Reduce risk of developing concentration-dependent side effects when treated
with standard doses
◦ Exclude poor metabolizers (if the parent drug is predominantly biologically
active)
◦ Exclude ultra-rapid metabolizers (if metabolite is predominantly biologically
active)
 Compounds with wide therapeutic window
◦ Dose adjustments based on pharmacogenomic tests.
◦ Increase opportunity for regulatory approval on subpopulation.
◦ Less important if compound and metabolite have similar activity.
 Troubleshooting
◦ Retrospective analysis in subjects with side effects or lack of therapeutic
effect.
◦ Prediction of ethnic variation explaining profiles in different populations.

60. Labeling Regulations
 “If evidence is available to support the
safety and effectiveness of the drug only
in selected subgroups of the larger
population with a disease, the labeling shall
describe the evidence and identify specific
tests needed for selection and monitoring
of patients who need the drug.”

61. Examples of Pharmacogenomic
Information in Drug Label

63. Pegylated interferon with ribavirin –
 PEG-IFN-a/ RBV regimens - associated with
many side effects and poor response,
 Clinical decisions of whether to initiate therapy
are largely based on likelihood of sustained
virologic response(SVR)
 Europeans homozygous for favourable genotype
(IFNL3 rs12979860/rs12979860; SVR: 69%) are
more likely to achieve SVR compared with the
unfavourable genotype (IFNL3
reference/reference or reference/rs12979860;
SVR: 33% and 27%, respectively)

64.  Vitamin K epoxide reductase complex subunit
1 (VKORC1) encoded by VKORC1 gene -
Target gene of anticoagulant warfarin
 Rare genetic variants in coding region of
VKORC1 may lead to bleeding disorders eg,
multiple coagulation factor deficiency type
2A, or warfarin resistance

65.  ICH Topic E15: Definitions for genomic
biomarkers, pharmacogenomics,
pharmacogenetics,genomic data and sample
coding categories To ensure consistency in
the terminology used by the different
regions