Review of Pharmacogenetic Testing Today Gwen McMillin, PhD ARUP Laboratories University of Utah Salt Lake City, Utah
A customer says to the pharmacist: "Why does my medication have 40 side effects?" The pharmacist replies: Because that's all we've documented so far."
Drugs are not perfect! Spear, et al. Trends Mol Medicine 7:201-206, 2001
What is pharmacogenetics (PGx)? = Pharmacology + Genetics Genetic predisposition to drug effects Individualized pharmacotherapy Revolutionize medicine The right drug at the right dose, the first time
Is PGx really new?!? 1931: phenylthiocarbamide (Science) 1943: atropine (PNAS) QuickTime and a GIF decompressor are needed to see this picture. 1956: first textbook on biochemical individuality 1962: first textbook on pharmacogenetics 1960 s + : isoniazid, warfarin, succinylcholine, nortriptyline, phenytoin, procainamide, etc.
The new weapon is the gene
Genetic differences among patients Compliance PatientÕs response to drug Interactions with concomitant drugs Diagnosis Dose
PGx contribution to therapeutics Pre-therapeutic Drug and dose selection Drug discovery Drug development QuickTime and a Sorenson Video decompressor are needed to see this picture. Post-therapeutic Troubleshooting adverse drug reactions Future drug and dose selection
Factors influencing PGx success Genotype-phenotype relationship Genotype-based dosing guidelines Severe consequences of mis-dosing Frequency of variants high enough to justify testing Availability of testing
PGx Targets
PK and PD involves many genes Absorption = PGx targets! Target organs Binding proteins Circulation Other compartments Metabolism Elimination
PGx targets in clinical use today TPMT - azathioprine, 6-mercaptopurine UGT1A1 - irinotecan CYPs - many, many drugs CYP2D6 CYP2C19 CYP2C9 VKORC1 and CYP2C9 - warfarin
Less common PGx targets NATs - isoniazid, caffeine, sulfonamides, procainamide, clonazepam, carcinogens, etc. GSTs - chemotherapy, carcinogens, etc. DPD - 5-fluorouracil MTHFR - methotrexate EGFR - gefitinib ADRB - albuteral D2Ds - antipsychotics ABCB1 - immunosuppressants, digoxin, etc.
Anatomy of a gene Coding Region Promoter Exons Intron DNA Start Transcription RNA transcript Processing AAAAAAAAA mrna
PGx method examples QuickTime and a Sorenson Video decompressor are needed to see this picture. PCR-RFLP Allele-specific PCR FRET probes Signal amplification Microsphere arrays Solid surface arrays Pyrosequencing Electrophoretic sequencing Nanoparticles
TPMT Thiopurine s-methyltransferase
TPMT Phase II enzyme that methylates sulfur groups No known physiological substrate Required for inactivation of 6-MP, azathioprine Deficiency associated with high risk for potentially fatal hematopoietic toxicity Frequency of partial deficiency ~ 1:10 Frequency of total deficiency ~ 1:300
TPMT-mediated metabolism Evans, D.A.P. Genetic Factors in Drug Therapy, 1993
6-MP metabolite concentrations correlate with genotypes Dervieux et al. Clin Chem 51(11):2074-84, 2005
TPMT genetics TPMT gene located on chromosome 6p22.3 11 alleles characterized *1 - wild type *2 - G238C (Ala to Pro) *3A - G460A (Ala to Thr) *3C - A719G (Tyr to Cys) account for ~95% of alleles in Caucasian, Black, and Asian populations Krynetski and Evans, Pharmacology 61:136-146, 2000
TPMT testing options Phenotyping Enzyme activity Metabolic ratios Genotyping Identifies patients at risk for toxicity related to 6-mercaptopurine and azathioprine Guides dosing Partial deficiency: 50-75% of standard dose Total deficiency: 5-10% of standard dose
Is TPMT testing cost-effective? Cost savings may be realized by reduced need for hospitalization, reduced rescue therapy for leukopenia, and lower drug requirements Marra et al. J Rheumatol 2002;29:2507-12 Tavadia et al. J Am Acad Dermatol 2000;42:628-32 Veenstra et al. AAPS PharmSci 2000;2(3):1-11
UGT1A1 UDP-glucuronosyltransferase 1A1
UGTs Family of Phase II enzymes responsible for glucuronidation (linkage of glycosyl groups to lipophilic substrates) of many endogenous and exogenous substances UGT1 Hepatic isoforms: A1, A3, A4, A6, A9 Extrahepatic isoforms: A7, A8, A10 Also UGT2, 3, and 8
Irinotecan and UGT1A1 Irinotecan is glucuronidated by several UGTs Promotor UGT1A1 variants have been strongly associated with irinotecan toxicity Severe (stage 3/4) diarrhea Severe (stage 3/4) leukopenia Reduced expression of UGT1A1 leads to accumulation of active irinotecan metabolites that are thought to be responsible for toxicity
Irinotecan metabolism SN-38 active metabolite SN-38G Inactive metabolite UGT
UGT1A1 Transcription Factor IID Promoter (TA) n TAA Exons 1 2 3 4 5 On Chr 2q37 ~230 Kb WT (TA) 6 TAA (*1) Mut (TA) 7 TAA (*28) Normal Expression Normal glucuronidation Decreased Expression (70% reduction) Low glucuronidation Diarrhea Leucopenia
Allele frequency of common UGT1A1 variants 84% (TA) 7 61.3% 38.7% 47% 42.6% (TA) 6 16% CAUCASIAN ASIAN AFRICAN Beutler et al. Proc Natl Acad Sci USA,95:8170-4, 1998
Implications of variant UGT1A1 Group All Patients Prevalence. Risk of toxicity 10% Reduce dose by 1 level (25 mg/m 2 ) Patients 7/7 10% 50% Patients 6/7 40% 12.5% Patients 6/6 50% 0% Innocenti et al J Clin Onc 2004; Rouits et al Clin Canc Res 2004
CYPs Cytochrome P450 isozymes
Cytochrome P450s Involved in metabolism of >75% of drugs Many are polymorphic Variants not associated with disease Non-genetic factors also important
Drug substrate examples 2D6 Amitriptyline Carvedilol Codeine Desipramine Fluoxetine Metoprolol Ondansetron Oxycodone Paroxetine Propanolol Risperidone Thioridazine 2C9 Celecoxib Diclofenac Ibuprofen Naproxen Phenytoin Tolbutamide Warfarin 2C19 Amitriptyline Citalopram Clomipramine Cyclophosphamide Diazepam Imipramine Lansoprazole Nelfinavir Omeprazole Phenytoin
CYP variants SNPs - not just in the coding regions! Altered expression Non-functional protein: Poor metabolizers (when homozygote for certain alleles) Partially functional protein: Intermediate metabolizers Gene deletion (CYP2D6 only thus far) Gene replications (CYP2D6 only thus far)
Alleles of CYP2D6 More than 80 known SNPs INDELs Complete gene deletion More than 2 copies Clinical significance of most unclear Zanger UM,et al. Naunyn Schmiedebergs Arch Pharmacol 2004
Frequency of variant CYPs Gene ÊAllele Phenotype Ancestry Prediction for Homozygotes Caucasians Asians CYP2C9 *2 PM 8-14% < 1% *3 PM 4-16% 2-5% CYP2C19 *2 PM 13-15% 32% *3 PM < 1% 5-10% CYP2D6 *3 PM 2% < 1% *4 PM 12-22% < 1% *5 PM 2-7% 5-13% *6 PM 2% < 1% *10 IM 1-3% 33-51% *17 IM < 1% < 1% *41 IM 9-10% 3% x N UM 1-5% 0-2%
CYP2D6 phenotypes Meyer Nat Rev Genetics 5:669-76, 2004
Nortriptyline PGx PGx target(s): CYP2D6 Consequence of variants: PM: more active drug UM: less active drug Dose Adjustment (change from standard dose): PM: 42-59% IM: 50-96% EM: 119-149% UM: 254% Kirchheiner J et al. Mol Psych 9:442-73, 2004
Metabolism of imipramine imipramine desipramine CYP3A4 CYP2D6 inactive metabolites CYP2D6 inactive metabolites conjugation and/or elimination
Death of two subjects due to imipramine and desipramine metabolite accumulation during chronic therapy Impaired metabolism due to a genetically determined slow metabolizer phenotype of CYP2D6 is suggested as a possible mechanism for fatal accumulation of these TCAs Swanson et al. J Forensic Sci, 1997 42(2):335-9
Metabolism of codeine codeine morphine CYP2D6 CYP3A4 norcodeine conjugation and/or elimination
Metabolism of codeine codeine CYP2D6 CYP2D6 CYP2D6 morphine CYP2D6 CYP3A4 norcodeine conjugation and/or elimination
Codeine intoxication associated with ultrarapid CYP2D6 metabolism Life-threatening opioid intoxication development in a patient after he was given small doses of codeine for the treatment of a cough Gasche Y et al. N Engl J Med, 2004 351:2827-31
Codeine PGx PGx target(s): CYP2D6 Consequence of variants: PM: no active drug IM: less active drug - approximately 20% lower concentrations of morphine than in EM UM: more active drug - up to 800% higher concentrations of morphine than in EM Dose Adjustment (change from standard dose): PM: Select a different drug IM: Modest decrease - 100% EM: 100 % UM: Dramatic decrease in dose or a different
Is CYP genotyping cost-effective? Genotype-based dose selection reduces costs by reducing costs associated with ADRs Clin Chem 2004 50(9):1623-33 Thromb Haemost. 2004 Sep;92(3):590-7 Identify individuals at high-risk for ADRs; give less expensive drugs to low-risk individuals Clin Chem 2005 51(2):376-85
Warfarin a specific application of CYP2C9 and VKORC1 genotyping
Warfarin (Coumadin) Most widely used anticoagulant in the world 24,289,522 prescriptions were filled in the US in 2004 Individual response is highly variable Maintenance dose may range from 1-40 mg/day Annual bleeding rate ~1 % fatal ~5 % major ~15 % minor
Warfarin Factors contributing to variability in response PK: metabolism PD: mechanism of action Other: age, gender, BMI, other drugs, clinical status Primary routes of metabolism (S)-enantiomer (3-5 times more potent): CYP2C9 (R)-enantiomer: CYP1A2, CYP3A4 Mechanism of action inhibition of vitamin K epoxide reductase (VKOR) which recycles vitamin K and subsequently activates clotting factors II, VII, IX, and X
Simplified PK and PD of warfarin warfarin NADH NAD + VKOR CYP2C9 Oxidized Vitamin K Reduced Vitamin K 7-hydroxywarfarin
Warfarin and CYP2C9 Frequency of variants ~20-30% of caucasians < 5% of asians and african americans Consequence of variants: Reduced clearance, more active drug Dose Guidance *1/*2 reduce maintenance dose by 20% *1/*3 reduce maintenance dose by 40% *2/*2 reduces warfarin clearance by 40% *2/*3 reduce maintenance dose by 70% *3/*3 reduce maintenance dose by 90%
VKOR genetics The VKOR complex, subunit 1 (VKORC1), is found on chromosome 16p12-q21 Produces a 163 amino acid protein, VKOR Variants are associated with warfarin resistance or sensitivity but are still being characterized in terms of function, linkage, impact, etc. -1639G>A - AA genotype requires lower doses 1173C>T - CC genotype requires higher doses
Genotype-based warfarin dosing Estimated to account for 55% of variability in warfarin dose selection Sqrt(Dose) = 0.628 0.0135(Age) 0.240(CYP2C9*2) 0.370 (CYP2C9*3) 0.241(VKORC1) + 0.0162(Height) Age: years CYP2C9: input 0, 1, or 2 based on #of variant alleles VKORC1: input 1 for GG, 2 for GA, and 3 for AA Height: cms Sconce et al. Blood 106(7):2329-33, 2005
Visions of the future
Future of PGx testing More clinical data Outcome studies Labeling changes Testing driven by products Wider availability of testing Personalized management teams Application-based panels QuickTime and a Sorenson Video decompressor are needed to see this picture.
Thank-you for your attention mcmillga@aruplab.com 800-242-2787, ext. 2671