Lecture 7 - Pgx - 23 Sep 2006

September 23, 2006

Pharmacogenetics Dr. Halit CANATAN Department of Pharmacology and Toxicology Faculty of Medicine, Kuwait University RM 67, Phone: 6344 E-mail: [email protected] Office hours: Saturday, Sunday & Tuesday: 12:00-14:00

Learning Objectives • What is Pharmacogenetics/Pharmacogenomics (PGx) ? • Importance of SNPs in PGx • Examples of human PGx Disorders: - Acetylation polymorphism - Difference in metabolic activity of enzyme due to mutations or duplications - Succinylcholine apnea - G6PD deficiency - Expression level of receptors

• ADRs and PGx • Major tools used in PGx • Future and challenges of PGx

References •

• • •

•

Gurwitz D, et al. Pharmacogenomics education: International Society of Pharmacogenomics recommendations for medical, pharmaceutical, and health schools deans of education. Pharmacogenomics Journal 5:221-225, 2005. Pharmacogenomics: the search for individualized therapies", ed: Licioni, Wong, Wiley-VCH, verlag Gmbh, Winheim, Germany, 2002. Evans WE, Relling MV. Moving towards individualized medicine with pharmacogenomics. Nature. 429:464-468, 2004. Frueh FW, Gurwitz D. From pharmacogenetics to personalized medicine: a vital need for educating health professionals and the community. Pharmacogenomics. 5:571-579, 2004. Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999.

What is Pharmacogenetics ?

PGx is NOT…..

1 + Rx =  2 + Rx =  3 + Rx =  Why ???

Ideal Drug ? • An ideal drug is the one effectively treats and/or prevents disease and has no adverse effects. • However, drug responses vary greatly among individuals, and a medication is rarely effective and safe in all patients.

• These differences are due to genetic as well as environmental effects on drug absorption, distribution, metabolism and excretion (pharmacokinetics) and on receptor or postreceptor sensitivity (pharmacodynamics).

• When a Doctor determines the dose of a drug, it is always a compromise between not too high and not too low for a particular patient or group of patients. • Pharmacogenetics (PGx) is the field of Pharmacology concerned with unanticipated or unusual response(s) to drugs that may have genetic basis for their action. • Unusual (idiosyncratic) drug responses must be distinguished from responses such as allergic reactions or drug overdose.

• Pharmacogenetic differences in response to drugs indicate an inherited defect.

• Pharmacogenetic factors may cause either a diminished or enhanced response to drug. • Pharmacogenetic disorders are inherited in the same manner as inborn errors of metabolism. • But, they are different than inborn errors of metabolism (e.g. phenylketonuria, PKU):

Subject lives his/her life without the disorder as long as he/she never takes the drug into body !!!

PGx: definition

Pharmacogenetics: the study of inter-individual differences in drug response due to genetic variations. • Recently another broader term, Pharmacogenomics, has been introduced.

Genome: All the genetic material in the chromosomes of a particular organism. Genome size is generally given as its total number of bps.

PGx: definition

• Pharmacogenomics: whole genome analysis of genetic determinants of drug efficacy and toxicity, including the identification of drug targets as result of such studies. • Both terms are often used as interchangeably, and they are usually abbreviated as PGx.

Single-Nucleotide Polymorphisms •

Genetic polymorphism: A variation in DNA sequence occurring with a frequency of at least 1% in the population.

• Majority of genetic variations are singlenucleotide polymorphisms (SNPs; “snips”). - SNPs are common DNA sequence variations among individuals. - Many SNPs that do not themselves change protein expression and cause disease. But they may be close on the chromosome to deleterious mutations.

- SNPs are used as markers to unearth mutations and accelerate efforts to find therapeutic drugs.

The most common sources of variation between humans are SNPs: single base differences between genome sequences. Fragments of two sequences, with eight SNPs, are shown.

SNPs & PGx • Critical SNPs can be clinically relevant and impact drug response or lead to expression or suppression of inheritable disease phenotype. • SNPs have important role in PGx due to their high prevalence rate and ease of their analysis. • Once specific SNPs are discovered, the next step is to determine whether these SNPs have phenotypic or physiological consequences.

G Protein Beta 3 Gene (GNB3) Case: A "functional" polymorphism, or only "another" of the countless SNP ? •

There is a polymorphism (C825T) in the gene encoding the G protein b3 subunit (GNB3) • This SNP is associated with some diseases such as hypertension and obesity. • GNB3 825T is associated with the occurrence of the splice variant Gb3s, which, despite a deletion of 41 amino acids, is functionally active in reconstituted systems. • There is a strong association among GNB3 825T, the occurrence of Gb3s, and enhanced signal transduction via G proteins.

• The G-protein 825T allele is associated with altered drug responses while the underlying mechanism is not fully understood. • Differential expression of transcripts from the C and T alleles could contribute to this process.

Gene structure and alternative splicing mechanism of GNB3. A, Exons 8, 9, and 10 and the intervening introns are shown. Alternative splicing of exon 9 leads to a deletion (dark gray) in Gb3s. Numbers in parentheses indicate distances in nucleotides between sequence motifs and intron / exon junctions. C/T indicates the C825T polymorphism. B, Proposed modular structure of Gb3, a propeller protein consisting of 7 WD domains. Parts that are deleted in Gb3s are indicated in black. C, Gene structure of GNB3. Boxes represent exons (numbers above). ATG and TGA indicate the translation start and stop codons, respectively. Arrows indicate the localization of several polymorphisms in GNB3. Numbers in brackets refer to the genomic localization of the respective polymorphisms. (ref: Rosskopf et al. Hypertension. 36(1):33-41, 2000).

• Conclusion: The GNB3 825 CC genotype is associated with non-response in HCV-1infected patients.

Invitation • Date: September 27, 2006 • Time: 10:30 a.m. • Location: Rm. 3-89

EXAMPLES OF HUMAN PGx DISORDERS 1. ACETYLATION POLYMORPHISM 2. DIFFERENCE IN METABOLIC ACTIVITY OF ENZYME DUE TO MUTATIONS OR DUPLICATIONS 3. SUCCINYLCHOLINE APNEA 7. G6PD DEFICIENCY 5. GENETIC MODIFICATION OF RECEPTORS

EXAMPLE 1: ACETYLATION POLYMORPHISM

• Acetylation is one of several conjugation reactions which determines the fate of drugs and other chemicals. • Rx is catalyzed by N-acetyltransferases (NAT).

• N-acetylator capacity of an individual is determined genetically and constant throughout his/her life. • Based on plasma concentration at a specific time after a fixed dose of the drug, individuals can be classified as - slow or rapid acetylators.

 

 

N

O

N

NAT2

H

H N N H H

Isoniazid

O

 

minor

O

CH3 N N H O

N-Acetylisoniazid

N

OH

N-acetylation (NAT2) Polymorphism • Peripheral Neuropathy was noticed in patients treated for tuberculosis with isoniazid, an antitubercular drug (Late 1940’s). • Genetic factors influencing isoniazid blood levels in humans was documented (1959)

PGx of Acetylation Plasma isoniazid concentrations were measured in 267 subjects six hours after an oral dose. Bimodal distribution in the rate of acetylation is due to genetic polymorphisms within NAT2 gene. (N Engl J Med. 348: 529, 2003)

Examples of drugs whose metabolism is affected by acetylation

• Isoniazid:

peripheral neuropathy

• Sulfasalazine: • Benzidine:

hemolytic anemia urinary bladder cancer

• Problem: Slow acetylation may lead to exaggerated responses and toxicity !!! • Solution: May need to lower the dose !!!

EXAMPLE 2: DIFFERENCE IN METABOLIC ACTIVITY OF ENZYME DUE TO MUTATIONS OR DUPLICATIONS

• The superfamily of cytochrome P450 (CYP) enzymes is the most important oxidative enzymatic system involved in drug metabolism.

• There may be a defect in drug oxidation by CYP2D6, a member of CYP super family, resulting in a major problem.

Genetic Classification of CYPs • Several ( at least 12) CYP Families identified • Main CYP gene family members: – – – –

CYP1 CYP2 CYP3 CYP4

• Classification is based on amino acid sequence and is lot limited to a particular species

CYP 2D6 • CYP 2D6 is responsible for metabolism of more than 25% of drugs available in the market including - antiarrhytmic drugs, - antidepressants, - neuroleptics, - beta-adrenoceptor blockers, - others eg. debrisoquine, codeine, etc. (Ref: British Journal of Pharmacology, 53:111-122)

Some drugs whose metabolism is catalyzed by CYP2D6 (Ref: British Journal of Pharmacology, 53:111-122)

Differences in metabolic activity of CYP2D6 is attributed to mutations or duplications of CYP2D6 gene Example: Patients taking antidepressant nortriptyline require different doses based on CYP2D6 genotype

- Normal CYP2D6 activity (one or more active alleles): 100-150 mg/day nortriptyline - Poor metabolizers (2 inactive alleles): 10-30 mg/day nortriptyline - Ultra-rapid metabolizers (CYPD26 duplication): 500 mg/day nortriptyline !!!

Patients taking antidepressant nortriptyline require different doses based on CYP2D6 genotype - Normal CYP 2D6 activity (one or more active alleles): (Lancet, 356:1667, 100-150 mg/day nortriptyline 2000)

- Poor metabolizers (2 inactive alleles): 10-30 mg/day nortriptyline

- Ultra-rapid metabolizers (CYP D26 duplication): 500 mg/day nortriptyline !!!

CYP2D6 and Codeine • Codeine is metabolized by CYP2D6 to morphine. H3CO

O-demethylation

O

NCH3

CYP2D6

Morphine

HO

CODEINE

• Since morphine is the active form having analgesic properties, poor CYP2D6 metabolizers will not benefit relief from codeine !

•

PM (poor metabolizer

B. IM (intermediate metabolizer) C. EM (extensive metabolizer) D. UM (Ultra Metabolizer)

Pharmacogenetic Effect of Cytochrome Genotypes (http://www.healthanddna.com/professional/pharmacogenetics.html)

• Michael died from an adverse reaction to Fluoxetine hydrochloride (Prozac). • (Prozac is the most widely prescribed antidepressant in history) • He was a slow metabolizer for CYP2D6 gene • His Doctor would have changed his medication if s/he had known !!! •

(Ref:http://www.hcroi.com/presentations/ Coleman,%20Howard%20(session%201 1.05).ppt)

Based on PGx data, FDA started to include PGx-based drug safety and efficacy drug labels !

Drug

Biomarker

Drug Label

Propafenone is used to treat arrhythmias and to maintain a normal heart rate

Genetic polymorphisms in drug metabolizing enzymes

(Ref: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999)

EXAMPLE 3: SUCCINYLCHOLINE APNEA • Succinylcholine is a rapid acting, rapid recovery neuromuscular blocking agent. SUCCINYLCHOLINE +

O

(H3C)3NH2CH2C O C

O CH2CH2

+ C O CH2CH2N(CH3)3

• Often used to produce muscular relaxation during surgery • usual paralysis lasts 2 to 6 min in patients

Succinylcholine is metabolized by pseudocholinesterase • Genetic variation is one of the major factors determining the activity of enzyme

SUCCINYLCHOLINE APNEA • Pseudocholinesterase deficiency decreases succinylcholine inactivation. • There are several variants of gene affecting metabolization of succinylcholine

• Occasionally even when conventional doses of succinylcholine are used, prolonged paralysis of the respiratory muscles results.

EXAMPLE 4: G6PD Deficiency • G6PD is present in all human cells but is particularly important to red blood cells (RBCs) • It is required to make NADPH in RBCs. • It is also required to make glutathione. • Glutathione and NADPH both help protect red blood cells against oxidative damage. • Thus, when G6PD is defective, oxidative damage to red blood cells readily occurs, and they break open as a result. This event is called hemolysis, and multiple hemolyses in a short time span constitute an episode of hemolytic anemia.

• The Pentose Phosphate Pathway. Note the importance of G6PD in the production of reduced G-SH, ribose, and NADPH (adapted from: Yoshida and Beutler, 1986, pg.8). - NADP+ = nicotinamide adenine dinucleotide phosphate - NADPH = reduced nicotinamide adenine dinucleotide phosphate - GS-SG = oxidized glutathione - G-SH = reduced glutathione

G6PD Deficiency • The most common enzyme deficiency ! • Worldwide 400 million (?) patients !!! • Patients with G6PD deficiency are at increased risk of developing hemolytic anemia when given oxidant drugs, such as antimalarials (eg, chloroquine, primaquine), aspirin, probenecid, and vitamin K. (http://www.merck.com/mrkshared/mmanual/section22/chapter301/301a.jsp)

Compounds reported in the literature that may induce hemolysis in G6PD deficient individuals (from: Avery, 1980; Koda-Kimble, 1978). ANALGESICS/ANTIPYRETICS

-

acetanilid acetophenetidin (phenacetin) amidopyrine (aminopyrine) antipyrine aspirin phenacetin probenicid pyramidone ANTIMALARIALS chloroquine hydroxychloroquine mepacrine (quinacrine) pamaquine pentaquine primaquine quinine quinocide CARDIOVASCULAR DRUGS procainamide quinidine SULFONAMIDES/SULFONES dapsone sulfacetamide sulfamethoxypyrimidine sulfanilamide sulfapyridine sulfasalazine sulfisoxazole

-

MISCELLANEOUS alpha-methyldopa ascorbic acid dimercaprol (BAL) hydralazine mestranol methylene blue nalidixic acid naphthalene niridazole phenylhydrazine pyridium quinine toluidine blue trinitrotoluene urate oxidase vitamin K (water soluble)

-

CYTOTOXIC/ANTIBACTERIAL chloramphenicol co-trimoxazole furazolidone furmethonol nalidixic acid neoarsphenamine nitrofurantoin nitrofurazone PAS para-aminosalicylic acid

EXAMPLE 5: GENETIC MODIFICATION OF RECEPTORS • Effectiveness of drugs is determined by their binding to receptors in addition to serum levels. • For example, Familial hypercholesterolemia (FH) is a disease in which the ability to synthesize receptors for low-density lipoprotein (LDL) is impaired. • LDL receptors (LDLRs) are needed for hepatic uptake of LDL.

EXAMPLE 5: GENETIC MODIFICATION OF RECEPTORS • Patients with FH have very high levels of circulating LDL. • HMG-CoA reductase inhibitors (important class of drug for lowering circulating cholesterol levels; Atorvastatin, Lovastatin, etc.) function largely by increasing number of hepatic LDLRs. • These drugs are useless to FH patients since they lack the genetic material needed for LDLR !!! (HMG : 3-Hydroxy-3-MethylGlutaryl)

ADVERSE DRUG REACTIONS (ADRs) & PGx • ADRs are responsible for: - 6.7% of hospitalization (2.2 million/yr) - 0.32 % of mortality (100,000 deaths/yr) • 6th leading cause of death (in the USA) • Although ADRs may normally occur as result of higher levels of drugs, genetic variation may increase an individual’s risk of developing ADR. • Severity of ADRs as well as response to a given medication vary widely among individuals and are determined by genetic make-up.

• PGx provides a tool to profile an individual using her/his unique genetic features. • Based on these information, physicians can develop a personalized (customized) therapy regimen for each patients or group of patients with similar genetic background. • This approach will optimize therapeutic outcome whereas it will also minimize ADRs.

ADR Test for CYP2C9 • CYP2C9 Metabolizes warfarin • Warfarin associated incidence of hemorrhages 0.8% fatal, 4.9% major, 15% minor • Poor Metabolizers at 2-4 X greater risk for hemorrhages • 5-10% of population - Poor Metabolizers • Genetic testing prior to the administration of warfarin will reduce the incidence of adverse bleeding event

•

12 CYPC29 alleles identified. Patients with CYP2C9*2 and CYP2C9*3 alleles have lower mean daily warfarin doses and a greater risk of bleeding. Testing for gene variants could potentially alter clinical management in patients commencing warfarin.

TOOLS FOR PGx • Completion of human genome project

TOOLS FOR PGx • Medical Bioinformatics: to correlate genetic information with biological activity

• Microarray Technology (DNA chip

Technology): - In the past, gene expression analysis was very laborious and difficult. - Using microarray DNA chips, thousands of genes, even whole genomes, can be analyzed in a short time !.

FUTURE OF PGx: Moving away from “one-size-fits all” therapeutics • It is hoped that within the next decade: - researchers will be able to correlate DNA variants with individual responses to medical treatments, - and identify particular subgroups of patients, and eventually develop drugs customized for those populations.

Top 10 PGx Tests 1) CYP 2D6 (*) 2) TPMT 3) CYP 2C9 4) CYP 2C19 (**) 5) NAT (*) 6) CYP 3A5 7) UGT1A1 8) MDR1/P-glycoprotein 9) CYP 2B6 10) MTHFR

September 1, 2004: First DNA Chip based test for two CYP 450 genes, CYP2D6 and CYP2C19, is approved by European Committee. December 27, 2004: U.S. Food and Drug Administration (FDA) has granted regulatory clearance for the Affymetrix GeneChip(R) System 3000Dx (GCS 3000Dx), an instrumentation system to analyze in vitro diagnostic microarrays.

AmpliChip CYP450 Array

PGx test for irinotecan, an antineoplastic drug.

CHALLENGES OF PGx • Vast amount of data, differences due to race and ethnic origins • Requirement to rely on sophisticated and highly advanced technology • Requires financial support, team work and dedicated staff • Ethical issues

ETHICAL ISSUES AND PGx • Patient privacy: Individuals may suffer stigmatization and discrimination as a result of PGx testing: - Testing for a number of different polymorphisms are associated with a particular individual or even a an ethnic group may be stigmatized !.

ETHICAL ISSUES AND PGx - If labeled as nonresponder or high risk group, they may be denied certain treatments and may suffer from insurance or employment discrimination. • Currently, no country has regulations specifically relating to PGx

HIRING PROCESS

FIRING PROCESS

Pharmacogenetic Goal To help healthcare providers select more accurately the medicines most likely to help, and least likely to cause serious side effects for patients.

“If it were not for the

great variability among individuals, medicine might as well be a science and not an art.” W. Osler, 1892

SUMMARY • Understand what is PGx and its importance • PGX syndromes are different than: - allergic rxs or drug overdose - inborn errors of metabolism • SNPs! Why they are important in PGx ? • Remember examples of PGx syndromes • Tools, challenges, Ethics of PGx • It is real ! Be ready for future !

“Do not be surprised if in the next year or two, (this kind of) DNA testing will be considered as a necessary step before writing a prescription”. Dr. Francis Collins The Director National Human Genome Research Institute

Dr. Halit CANATAN, VMD, MSc, PhD Department of Pharmacology and Toxicology Faculty of Medicine, Kuwait University RM 67, Phone: 6344

E-mail: [email protected] Saturday, Sunday & Tuesday: 12:00-14:00

Drug List : Pharmacogenetics

• Isoniazid • Nortriptyline • Codeine • Succinylcholine • Warfarin