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Clinical Pharmacogenetics

Clinical Pharmacogenetics. David A Flockhart MD, PhD Chief, Division of Clinical Pharmacology Professor of Medicine, Genetics and Pharmacology Indiana University School of Medicine. Human Genome + Prescription Errors = HUGE Public Expectations of Pharmacogenetics.

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Clinical Pharmacogenetics

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  1. Clinical Pharmacogenetics David A Flockhart MD, PhD Chief, Division of Clinical Pharmacology Professor of Medicine, Genetics and Pharmacology Indiana University School of Medicine

  2. Human Genome +Prescription Errors= HUGE Public Expectations ofPharmacogenetics

  3. The 20th Century in Small Molecule Pharmacology Transporters Receptors Phosphatases Targets 2nd messengers Protein kinases GI Lumen Blood Cell

  4. Mechanisms of Inherited Genetic Variability (All are in germ line DNA or mitochondrial DNA) Single nucleotide polymorphisms (SNPs) Deletions Duplications Insertions VNTRs

  5. From: Evans WE, Relling MV. Science 286:487-491, 1999.

  6. Methods in Pharmacogenetics • Population distribution analysis with Normit plots using a valid probe to detect phenotypic polymorphism (> 1% of population) • Identification of gene and variants • Family and twin studies to confirm genetic characteristics (dominant, recessive, Mendelian, maternal etc.) • Development of a genetic test for DNA variants • Correlation between genotype and phenotype • Application in Clinical Practice

  7. Polymorphic Distribution Antimode

  8. Skewed Distribution

  9. Properties of an ideal pharmacogenetic probe to measure phenotype • Specific for the trait in question • Sensitive • Simple to carry out • Inexpensive • Easy to assay • Clinically benign

  10. Examples of Genetic Effects on Human Drug Absorption, Action and Elimination • Absorption: • Alcohol Dehydrogenase • Aldehyde Dehydrogenase • Cytochrome P450 3A5 • P-glycoprotein • Multidrug Resistance Transporter (MRP) • Action • G-protein variants • Angiotensin II receptor and Angiotensinogen variants • 2receptor • Dopamine D4 receptor • Endothelial NO synthase • 5HT4receptor

  11. Examples of Genetic Effects on Human Drug Absorption, Action and Elimination (continued) • Cytochrome P450 2A6 • Cytochrome P450 2C9 • Cytochrome P450 2C19 • Cytochrome P450 2D6 • Cytochrome P450 3A5 • Regulation of cytochrome P450 3A4 • Dihydropyridine Dehydrogenase (DPD) • UDP-Glucuronyl Transferase 1A1 (UGT 1A1) • Glutathione - S - Transferase (GST) • Thiopurine methyl transferase (TPMT) • Flavin Mono-Oxygenase 3 (FMO-3)

  12. Genetics and Drug Absorption

  13. Digoxin Transport across the GI lumen P-gp Transport ATP  ADP Passive Diffusion Enterocyte GI Lumen

  14. P-Glycoprotein Pharmacogenetics : Effect of a “wobble” (no coding change) SNP in exon 26 Fig. 3. Correlation of the exon 26 SNP with MDR-1 expression. The MDR-phenotype (expression and activity) of 21 volunteers and patients was determined by Western blot analyses. The box plot shows the distribution of MDR-1 expression clustered according to the MDR-1 genotype at the relevant exon 26 SNP. The genotype-phenotype correlation has a significance of P = 0.056 (n = 21). Eichelbaum et al. Proc Nat Acad Sci March, 2000.

  15. 0.25 mg of digoxin po at steady state Eichelbaum et al, Proc Nat Acad Sci, 2000:March

  16. Digoxin Transport across the Blood-Brain Barrier P-gp Transport ATP  ADP Passive Diffusion Brain Blood

  17. Note • Pharmacokinetic changes do not always have predictable pharmacodynamic consequences • Wobble changes may be important even though the mechanism involved is unclear

  18. Aldehyde Dehydrogenase Genetics • 10 human ALDH genes • 13 different alleles • autosomal dominant trait because of lack of catalytic activity if one subunit of the tetramer is inactive • ALDH2 deficiency results in build up of toxic acetaldehyde • Absent in up to 45% of Chinese, not at all in Caucasians or Africans

  19. Genetics and Drug Elimination

  20. Effect of CYP2C19 genotype and omeprazole on diazepam pharmacokinetics [Diazepam] PMs (nM) EMs Andersson et al, 1990. Time after infusion (hrs)

  21. Specific CYP2C19 inhibition by omeprazole Omeprazole (M) Ko JW and Flockhart DA, 1997.

  22. Lessons Learned • The environment can mimick genetic effects convincingly: tests of phenotype will always be important • Genetics is not everything, so every genetic association must be examined for potential environmental confounders

  23. Cytochrome P450 2D6 • Absent in 7% of Caucasians • Hyperactive in up to 30% of East Africans • Catalyzes primary metabolism of: • propafenone • codeine • -blockers • tricyclic antidepressants • Inhibited by: • fluoxetine • haloperidol • paroxetine • quinidine

  24. Paroxetine and CYP2D6 genotype change the plasma concentrations of endoxifen Flockhart et al. JNCI In Press, December 2003

  25. Plasma Endoxifen Concentration After 4 Months Tamoxifen TreatmentN = 80 P =0.000006

  26. CYP2D6 Alleles • 43 as of May, 2002 • 24 alleles have no activity • 6 have decreased activity • The *2 variant can have 1,2,3,4,5 or 13 copies i.e increased activity

  27. From: Dalen P, et al. Clin Pharmacol Ther 63:444-452, 1998.

  28. Oligonucleotide array for cytochrome P450 genotesting From: Flockhart DA and Webb DJ. Lancet End of Year Review for Clinical Pharmacology, 1998.

  29. Lessons from CYP Pharmacogenetics • Multiple genetic tests of one gene may be needed to accurately predict phenotype • Gene duplication in the germline exists • All SNPs are not “tag” SNPs

  30. Genetic alterations in Phase 2 enzymes with clinical consequences UGT1A1 NAT-2SULT1A1COMTTPMT

  31. UDP Glucuronyl Transferase 1A1 • Responsible for Gilbert’s Bilirubinemia • absent in ~15% of Caucasians • < 5% Asians • > 50% of Africans • > 50% of Hispanics • Decreased activity in hypoglycemic and malnourished conditions, so Gilbert’s hyperbilirubinemia is “revealed” by these conditions.

  32. 45 40 35 50 P=0.007 45 30 40 35.7 Objective response (%) 25 35 30 15 25 % grade 4/5 neutropenia 16.3 20 15 8.6 10 0 5 0 6/6 6/7 7/7 UGT1A1 genotype UGT1A1 TA repeat genotype alters irinotecan neutropenic/activity 41.9 P=0.045 33.8 20 14.3 10 5 6/6 6/7 7/7 UGT1A1 genotype N=524 McLeod H. et al, 2003.

  33. N-Acetylation PolymorphismNAT-2 • Late 1940’s : Peripheral Neuropathy noted in patients treated for tuberculosis. • 1959 : Genetic factors influencing isoniazid blood levels in humans. Trans Conf Chemother Tuberc 1959: 8, 52–56.

  34. NAT-2 substrates(All have been used as probes) • Caffeine • Dapsone • Hydralazine • Isoniazid • Procainamide

  35. Incidence of the Slow Acetylator NAT-2 phenotype • 50% among Caucasians • 50% among Africans • 20% among Egyptians • 15% among Chinese • 10% among Japanese

  36. Onset of Positive ANA Syndrome with Procainamide. Woosley RL, et al. N Engl J Med 298:1157-1159, 1978.

  37. Clinical relevance of the NAT-2 polymorphism • Higher isoniazid levels, greater neuropathy in slow acetylators • Faster ANA appearance with procainamide in slow acetylators • Hydralazine-induced lupuserythematosus is much less common in rapid than slow acetylators

  38. Thiopurine Methyl Transferase • Homozygous mutants are 0.2% of Caucasian Populations • Heterozygotes are ~ 10% • Homozygous wild type is 90% • Metabolism of Azathioprine • 6-Mercaptopurine

  39. Thiopurine Methyl Transferase Deficiency From: Weinshilboum et al. JPET;222:174-81. 1982

  40. Effect of TPMT genotype on duration of Azathioprine therapy. From: Macleod et al: Ann Int Med 1998;

  41. Examples of Human Receptors shown to be genetically polymorphic with possible alterations in clinical phenotype • G-proteins • Angiotensin II receptor and angiotensinogen • Angiotensin converting enzyme • 2 receptor • Dopamine D4 receptor • Endothelial NO synthase • 5HT4 receptor

  42. Functional consequences of Gly389 polymorphism

  43. The Case-control Study Bengtsson et al. Circulation 2001, 104: 187-190

  44. Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Ser Gly Gly Arg Gly Arg Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Gly Arg Gly Gly Arg Gly Arg Gly Arg Gly Gly Gly Gly Arg Gly Haplotypes Diplotypes Ying-Hong Wang PhD, Indiana University School of Medicine

  45. Observed b1AR Haplotypes in Caucasians and African American Women (WISE study) Terra et al. Clin. Pharmacol. Ther. 71:70 (2002)

  46. Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Ser Gly Gly Arg Gly Arg Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Gly Arg Gly Gly Arg Gly Arg Gly Arg Gly Gly Gly Gly Arg Gly Haplotypes Diplotypes Ying-Hong Wang PhD, Indiana University School of Medicine

  47. Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Ser Gly Gly Arg Gly Arg Gly SR/SR SR/SG SR/GR Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Gly Arg Gly Gly Arg SG/GR Gly Arg Gly Arg Gly Gly Gly Gly Arg Gly Of 9 theoretical diplotypes, only 4 were present in the study population Haplotypes Diplotypes Ying-Hong Wang PhD, Indiana University School of Medicine

  48. Diplotype Predicts Beta-blocker Effect Johnson et al. Clin Pharmacol & Ther. 2003,74:44-52.

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