Pharmacogenetics and personalized medicine

1. Pharmacogenetics and personalized medicine Tyrosine kinase inhibitors, the P450 cytochromes, TPMT and Warfarin

2. Pharmacogenetics is generally regarded as the study or clinical testing of genetic variation that gives rise to differing drug responses Pharmacokinetics studies the absorption, activation, metabolism and excretion of drugs (what the body does to the drug) Pharmacodynamics considers the actual target response (what the drug does to the body) genetic factors are relevant to both pharmacokinetics and pharmacodynamics (from Strachan and Read)

3. There are different stages at which genetic variation can affect a patient’s response to a drug metabolism – 2 phases of drug metabolism phase 1 often produces the biologically active molecules eg: the cytochrome P450 enzymes phase 2 produces compounds that are more easily excreted target response – process or pathway targeted responds differently eg: certain tyrosine kinase inhibitors catabolism individuals differ in the rate at which they clear the and excretion - active drug this can lead to adverse drug reactions eg: TPMT (from Strachan and Read)

4. Tyrosine kinase inhibitors (TKIs) Imatinib (Gleevec/USA or Glivec/Europe, Novartis) First TKI (developed in the late 1990s) – designed to treat CML CML – 9:22 translocation in ~90% of cases creates BCR-Abl fusion gene which gives rise to a constitutively active abnormal tyrosine kinase Imatinib developed to inhibit the fusion protein works by competitively binding to the TK domain of the fusion protein and hence inhibits terminal phosphate transfer from ATP to tyrosine residues on the substrate mutations present in the TK domain confer Imatinib resistance Thr315Ile is found in 50-90% of relapsed CML patients

5. GISTs (gastrointestinal stromal tumours) are caused by mutations in c-KIT and PDGFRA allows continuous receptor activation independent of ligand binding leads to increased cell proliferation and decreased apoptosis Imatinib also binds to the TK domain of c-KIT and PDGFRA patients with c-KIT exons 9 and 11 (in EC and JM domain) mutations in PDGFRA exon 12 (in JM domain) are sensitive and respond to Imatinib treatment Patients with c-KIT exon 17 (TK2 domain) mutations in PDGFRA exons 14 and 18 (in TK1 and TK2 domain) are resistant and do not respond to Imatinib treatment

6. Sunitinib (Sutent, Pfizer) inhibits many tyrosine kinase receptors (PGDFRs, VEGFRs, RET) used in the treatment of Imatinib resistant GISTs and renal cell carcinoma Nilotinib (Tasigna, Novartis) used in the treatment of Imatinib resistant CML effective against all TK domain point mutations except Thr315Ile

7. EGFR Tyrosine Kinase Inhibitors EGFR belongs to a family of 4 transmembrane proteins (EGFR, HER2, HER3, HER4) following ligand binding EGFR forms a homo or heterodimer this activates receptor autophosphorylation of the TK domain this triggers downstream signalling of pathways which result in cell proliferation (Ras-MAPK and PI3K/Akt) Activation of EGFR is observed in the majority of epithelial cancers EGFR was the first growth factor receptor to be proposed as a target for cancer therapy

8. Two classes of EGFR inhibitors 1) Anti-EGFR monoclonal antibodies Cetuximab (Erbitux, Bristol-Myers Squibb) given by intravenously for treatment of metastatic colorectal cancer (CC) and in cancer of the head and neck with radiotherapy Panitumumab (Vectibix, Amgen) given by intravenously for treatment of metastatic colorectal cancer (CC) both drugs bind to the extracellular domain of EGFR and prevent ligand binding and receptor dimerization

9. KRAS mutations found in 40-45% of tumours from CC patients KRAS mutations generally occur at codons 12 and 13 activating mutations in the KRAS gene result in EGFR-independent activation of MAPK pathway presence of a KRAS mutation impairs response to anti-EGFR drugs CC tumours are generally screened for KRAS mutations and treatment is only recommended for mutation negative patients (BRAF mutation V600E)

10. Two classes of EGFR inhibitors 2) Small molecule reversible EGFR tyrosine kinase inhibitors Gefitinib (Iressa, AstraZeneca) used in the treatment of non-small cell lung cancer (NSCLC) Erlotinib (Tarceva, Roche) used in the treatment of non-small cell lung cancer (NSCLC) and in advanced pancreatic cancer with chemotherapy both drugs bind competitively to the tyrosine kinase domain of EGFR, inhibiting receptor autophosphorylation and downstream signalling

11. EGFR mutations found in 5-15% of tumours from NSCLC patients (25-35% of Asian patients) mutations occur in the EGFR TK domain (exons18-21) most common mutations are deletions in ex19 and L858R in ex21 these account for 80-90% of all mutations it is thought that these mutated EGFRs bind with higher affinity to Gefitinib and Erlotinib and treatment is recommended for patients with a sensitising EGFR mutation resistance mutations also occur T790M in ex20 and ex20 insertions and treatment is not recommended in these patients

12. Cytochrome P450 enzyme family 57 human genes have been identified which code for the various cytochrome P450 enzymes catalyse the oxidation of organic substances are the major enzymes involved in drug metabolism and bioactivation, accounting for ∼75% of the total metabolism CYP2D6 involved in the metabolism of up to 25% of drugs differences in metabolism observed due to CY2D6 polymorphisms eg: debrisoquine (original observations reported) codeine tamoxifen - CYP2D6 genotyping recommended prior to treatment

13. Cytochrome P450 2D6 (CYP2D6) pharmacogenetics The frequency distribution of the ratio of debrisoquine to its metabolite 4-hydroxydebrisoquine in 1011 Swedish subjects. PM, poor metabolizer; EM, extensive metabolizer; UM, ultrarapid metabolizer

14. Adverse drug reactions Thiopurine methyltransferase (TPMT) involved in the metabolism of the immunosupressant drugs azathioprine and 6-mercaptopurine (used in ALL, organ transplantation, autoimmune disease etc) in individuals deficiency in TPMT activity, thiopurine metabolism proceeds by other pathways, one of which produces active thiopurine which is toxic to the bone marrow at high concentrations 10% of individuals are heterozygous for low-activity variants of TPMT 0.3% of individuals are homozygous for low-activity variants of TPMT 3 variants account for 90% of the low-activity alleles homozygous individuals require lower doses of the drugs (6-10% of standard dose) or risk bone marrow toxicity and suppression

15. Thiopurine S-methyltransferase (TPMT) pharmacogenetics The frequency distribution shows the level of red blood cell (RBC) TPMT activity in 298 randomly selected Caucasian blood donors. Presumed genotypes for the TPMT genetic polymorphism are also shown. TPMTL and TPMTH (low and high, respectively) were allele designations used before the molecular basis for the polymorphism was established Human thiopurine S-methyltransferase (TPMT) alleles TPMT *1 is the most common allele (wild type), TPMT *3A is the most common variant allele in Caucasians, and TPMT *3C is the most common variant allele in East Asian subjects

16. Warfarin warfarin is the most widely prescribed oral anticoagulant drug in North America and Europe activity is monitored by a coagulation test to ensure an adequate yet safe dose is taken serious adverse reactions still occur levels are too high - haemorrhage levels are too low - thromobosis or embolism warfarin is a mix of S - and R-warfarin – both isoforms are active S form (3-5 x more potent) – catalysed by mainly CYP2C9 R form - catalysed by mainly by CYP1A2 and CYP3A4

17. Mechanism of action of warfarin

18. Genetic Variation 2 common CYP2C9 polymorphisms Arg144Cys(CYP2C9*2) and Ile358Leu (CYP2C9*3) associated with decreased CYP2C9 activity (5-12% of wild type) in general patients requiring a low warfarin dose (with an increased risk of haemorrhage) were shown to have 1 or more of these alleles did not explain most of the variation in final warfarin dose required the vitamin K epoxide reductase complex (VKORC1) gene was identified in 2004 10 non-coding SNPs were identified within the VKORC1 gene

19. VKORC1 haplotypes associated with high and low warfarin dose requirements were identified (2 fold difference in dose) VKORC1haplotyping explained 25% of the variance in warfarin dose CYP2C9genotyping explained 6-10% of the variance in warfarin dose other studies only type for VKORC1 -1639G>A promoter variant (or 1173C>T which is in tight linkage disequilibrium) warfarin sensitive patients requiring lower doses were shown to have the -1639AA genotype -1639G>A abolishes the E-box consensus sequence in a luciferase assay - the G allele promoter was shown to have 44% greater activity when compared with the A allele promoter there are also a number of warfarin resistant VKORC1 missense variants

20. However, typing for these variants does not explain all of the variation observed in warfarin dosing requirements and it is known that many other CYP enzymes are involved in the catabolism of warfarin FDA now recommends genotyping for CYP2C9 and VKORC1 before warfarin use however, many clinicians are sceptical about the value of genotyping as it does not fully predict response other studies are underway to identify other significant variants (functional candidate approaches, GWAS etc)

21. All of these cases are examples of personalised medicine using genetic information However the introduction of low cost whole genome sequencing will provide individual genotypic information However before this personalized genetic information can be used to direct treatment we need to determine the functional significance and biology of any particular genetic variant

22. References Strachan, T., Read, A. 2010. Human Molecular Genetics 4th Edition. Miettinen, M., Lasota, J. 2006. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch. Pathol. Lab Med. 130:1466-1478. Swords R. et al. 2009. Nilotinib: optimal therapy for patients with chronic myeloid leukemia and resistance or intolerance to imatinib. Drug Des. Devel. Ther. 3:89-101. Ciardiello, F., Tortora, G. 2008. EGFR Antagonists in Cancer Treatment. New Eng. J. Med. 358:1160-1174. Wang, L. 2010. Pharmacogenomics; a systems approach. Wiley Interdisciplinary Reviews: Systems Biology and Medicine 2(1):3-22. Dong, X-W., et al. 2010. Thiopurine S-methyltransferase polymorphisms and thiopurine toxicity in treatment of inflammatory bowel disease. World. J. Gastroenterol. 16(25):3187–3195. The International Warfarin Pharmacogenetics Consortium. 2009. Estimation of the Warfarin Dose with Clinical and Pharmacogenetic Data. New Eng. J. Med. 360:753–764. Rieder, M.J., et al. 2005 Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Dose New Engl. J. Med. 352:2285-2293. Limdi, N.A., et al. 2008. VKORC1 polymorphisms, haplotypes and haplotype groups on warfarin dose among African–Americans and European–Americans. Pharmacogenomics 9(10):1445-1458. Guttmacher, A., et al. 2010. Personalized genomic information: preparing for the future of genetic medicine. Nature Reviews Genetics 11:161-165.