Genetics and Genomics: Influence on Individualization of Medication Regimes

Genetics and Genomics: Influence on Individualization of Medication Regimes Joseph S Bertino Jr., Pharm.D., FCCP Schenectady, NY USA

Goals and Objectives To discuss pharmacogenetics and pharmacogenomics Discuss methods of measuring pharmacogenetic activity in vivo Outline pharmacogenetic mediated differences in pharmacokinetics and response Discuss specific drug dosing and prediction of drug interactions using pharmacogenetics

Important Definitions Pharmacodynamics is an expression of pharmacokinetics (i.e., drug effect) Pharmacokinetics is an expression of pharmacogenetics (i.e. ADME) Pharmacogenomics Study of hereditary basis for differences in a population s response to a drug Pharmacogenetics (effect of genetics and environment) includes: CYP450 s, Conjugative reactions and Transporters

Important Facts Human genome = 22 chromosome pairs and 1 pair of sex chromosomes Functional unit of the genome = gene 2% of genes code for proteins, remainder is structural for DNA Entire genome = 3 billion DNA pairs, with ~30,000 protein coding genes

Important Facts SNPs (single nucletide polymorphisms) account for sequence variation between people (1SNP/1000 base pairs of DNA) 1% of SNPs affect protein coding region of DNA sequence SNPs can change the way protein is made

Example of Single Nucleotide Polymorphism (SNP) As a Result of a Mutation

Important terms Alleles are the DNA code on a gene, different alleles determine different characteristics. An allele is one member of a pair that makes up a gene 2 alleles that are the same are homozygous 2 alleles that are different are heterozygous Genetic polymorphism: At least 1% of the population is genetically different from the rest

Example of Heterozygous Alleles

CYP - P450 for all mammalian species 2 C - family (17-14 human) - subfamily (42 in humans) 9 - enzyme/gene (55 genes, 29 pseudogenes in humans *1*2: -Allele pattern *1 = wild type or most common allele with most activity

Genetic Polymorphism Example

Genetic Polymorphism Results in Variability in Drug Response and Dosing Drug Metabolizing Enzymes Drug Transporters Drug Receptors Each of these can show genetic polymorphism which can affect drug response and drug dosing

Variability in Activity Variability in the activity of drug metabolizing enzymes is seen From person to person Within the same person (perhaps due to environmental factors) Transporters show variable activity based on genetics and environment Receptors show variable activity based on genetics and environment

Drug Metabolism Enzymes

Drug Metabolism Enzymes (DMEs) Drug metabolism throughout the body Adapt the organism to the environment (plant animal warfare) Activate drugs Detoxify substances and activate nontoxic substances into toxic substances

Genetic Polymorphism of DMEs Activity of an enzyme can vary from person to person based on genetics Enzyme activity can be Normal (EM or extensive metabolizer) Increased (RM or rapid metabolizer) Decreased (IM or intermediate metabolizer) Not present (PM or poor metabolizer) People with no enzyme activity have alternative methods of drug metabolism, however this is less effective

Genetics Alone Doesn t Determine Activity of Drug Metabolizing Enzymes Environmental factors also are important in determining activity of drug metabolizing enzymes Individuals can genetically be extensive metabolizers of drugs but environmental factors can make them intermediate or poor metabolizers (this is phenoconversion)

Effect of Pharmacogenetics Other exogenous (environmental) factors will affect drug metabolizing and transporter activity (Phenotype) Diet -Infx diseases/cytokines Drugs -Sex (yes, but? important) Pollution -Menstrual cycle (rare) Concurrent other diseases Age (1 st year of life)

Phase I reactions: The CYP system

Cytochrome P450 Enzymes Phase I oxidative enzyme system Present in various organs in the body Liver is the major source Intestines Monocytes Macrophages Lymphocytes Brain Lungs Kidneys

Common Phase I (CYPs) Drug Metabolizing Enzymes: Genetic Polymorphism CYP2B6 CYP2C9 CYP2C19 CYP2D6 CYP3A5 Each of these Phase I enzymes exhibits genetic polymorphism but they are also affected by exogenous factors

DME activity can vary between people (intersubject) and within (intrasubject) an individual CYP1A2 Intersubject = 20% Intrasubject 20% CYP2D6 Intrasubject = 12-136% (median 35%) Intersubject = Gene dose effect CYP3A Intrasubject =10% Intersubject = 2 fold difference (some authors suggest 10 fold) CYP2C19 Intrasubject = 18.5% (EMs) Intersubject = Gene dose effect

Ethnic Differences in Drug Metabolism (CYP2D6 example) More activity Less Activity

People have different activity of enzymes and require different doses of the same drug (CYP2D6 example)

Phase II (conjugative) enzymes (can be polymorphic) UDP-Glucuronosyltransferase [UGTs] (glucuronidation) UGT1 and UGT2 families Glutathione S-Transferase [GST] Sulfontransferase (Sulfation) [SULT] Methyltransferase (Methylation) [MT] Epoxide Hydrolases (Oxidation) [EH] NAT

Drug Transporters PGP: P-glycoprotein, ATP dependent efflux transporter (keep drugs out) MDR: Gene family that codes for PGPs, multidrug resistance genes (MDR1 only) ABC transporter (ATP binding cassette) OATP: Organic Anion Transport Protein OCTP: Organic Cation Transport Protein OATP and OCTP are influx transporters (move drugs in) These transporters can show genetic polymorphism and be affected by exogenous factors

Drug Receptors (can have genetic polymorphism) Continuing identification of various genotypes for drug receptors VKORC1 for vitamin K (warfarin) Beta receptors Opioid receptors Benzodiazepine receptors Receptors have genetic polymorphism and cause patients to respond to drugs differently. They could be affected by exogenous factors also

How do I measure it?

Genotyping Investigation of DME and Transporter Activity investigation of genetic codes: variant alleles PCR-based test using DNA from peripheral leukocytes or any DNA source (100% specific, 80% sensitive) Phenotyping investigation of the manifestation of genetic code differences (due to drugs, environment, diet) pharmacologic test using a safe, enzyme specific drug marker that can be easily quantitated (urine, blood, etc)..a Probe

Is DNA work really glamouous?

How Does Pharmacogenetics Affect Drug Pharmacokinetics and Drug Response?

Exposure to Standard Dosing Venlafaxine, an SSRI, racemic mixture Both R and S isomer metabolized by CYP2D6 (polymorphic) Many CYP2D6 reduced activity or null alleles Who would get the bigger exposure from a dose?

Venlafaxine and CYP2D6 Eap et al, Pharmacogenetics 2003;13:39-47

Venlafaxine + Quinidine and CYP2D6 Drug Interaction, Changing the Phenotype 4-12 fold increase No increase Eap et al, Pharmacogenetics 2003;13:39-47

Warfarin

Gage B. CPSC Advisory Committee Meeting, November 14, 2005

Dosing variability of warfarin in anticoagulation clinic More than 15 fold Reynolds KK et al. Personalized Medicine 2007;4:11-31 Valdes R, Jr. Pharmacogenetics in Warfarin Therapy:The Future of New Models

Frequency of CYP2C9 alleles Two common variant alleles of *2 and *3 with reduced DME activity Over 30% of European and Caucasian populations have one or both of *2 or *3 alleles, with allele frequencies of 0.1 and 0.08, respectively Extremely rare in Asian and African American populations, with over 95% of these populations expressing the wildtype (*1 or EM allele)

Daily maintenance Dose (mg) What is the Relationship between CYP2C9 Genotype and Warfarin Dose? 6 N=185, median time=543 days, (14-4,032 days) 5 4 3 2 1 0 *1/*1 *1/*2 *2/*2 *1/*3 *2/*3 *3/*3 Frequency 69% 15% 2.2% 10% 1.6% 2.7% Dose 100% 87% 72% 59% 42% 28% Higashi et al, JAMA 2002;287:1690-8 Huang SM. CPSC Advisory Committee Meeting, November 14, 2005

The VKORC1 receptor is where warfarin binds to cause anticoagulation Alleles are named A and G G allele is least active, warfarin binds less to receptors from individuals with GG allele patters A allele pattern binds warfarin the best Thus, individuals can be GG, GA or AA allele pattern AA should need smaller doses to obtain drug effect

Frequency of VKORC1 allele (AA most active) The allele frequency for the G allele was 0.57 and for A was 0.43 in Caucasians (n=297) Frequencies of AA, AG, and GG genotypes Caucasians (N=92): 14%, 47%, 39% Chinese (N=95): 82%, 18%, 0% Thus we would expect that individuals with the AA allele pattern to need the smallest dose of warfarin Sconce et al. Blood 2005;106:2329-2333 Yuan et al. Human Mol Genetics 2005;14:1745-1751

Effect of VKORC1 allele pattern on Warfarin Dose Sconce

Daily Maintenance Dose (mg) Effect of VKORC1 and CYP2C9 (-1639 G>A) on Warfarin Dose 6 5 4 3 2 A/A G/A G/G 19 % 56% 25 % 1 0 *1/*1 *1/*2 *1/*3 *2/*2 *2/*3, *3/*3 Sconce et al, Blood 2005;106:2329-33 Huang SM. CPSC Advisory Committee Meeting, November 14, 2005

Pharmacogenetics and the Variability in Warfarin Dose Requirement Age, Sex, Drugs, Body wt, Race, Diet, Others Unknown Unknown ~40% Unfortunately CYP2C9 and VKORC1 genotyping explain only 40-50% of the variability in warfarin dosing CYP2C9 & VKORC1

CYP2C19 and Proton Pump Inhibitors (PPIs)

PPIs and CYP2C19 Many PPIs are metabolized primarily by CYP2C19 which is a polymorphic enzyme Observational data suggest that in CYP2C19 PMs, short course (5-7 days) of PPIs with antibiotics sufficient for H. pylori treatment CYP2C19 has many alleles, *1 normal, *17 increased activity, others null (no activity) Does CYP2C19 status affect PPI PK?

Effect of CYP2C19 status on rabeprazole PK Sugimoto et al. CPT 2004;76:290-301

Rabeprazole induced ph in CYP2C19 genotypes Sugimoto et al. CPT 2004;76:290-301

Thiopurine Methyltransferase (TPMT) and Azathioprine

TPMT Azathioprine and 6-mercaptopurine are used in the treatment of ALL in children These drugs are metabolized by TPMT TPMT activity is under genetic and environmental control

TPMT and Azathioprine Eichelbaum et al. Ann Rev med 2006;119-137

Toxicity of 6MP/Azathioprine based on genetic makeup Eichelbaum et al. Ann Rev med 2006;119-137

Use of Individualized Dosing of 6MP Based on Genetics Eichelbaum et al. Ann Rev med 2006;119-137

How is pharmacogenomics being used in treating patients? 5 drugs where genetic testing is suggested: Warfarin (CYP2C9 and VKORC1) Atomoxetine (CYP2D6) 6MP or azathioprine (TPMT) Tamoxifen (CYP2D6) Abacavir (HLA-B*5701 for hypersensitivity)

Pharmacogenomics and Pharmacogenetics: Practical applications Pharmacists should be in the forefront of genomics and genetics of drugs For now, useful for thinking about drug interactions, what drug to pick to treat, and explanation of toxicities The future is VAST and bright don t miss the ride

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