Genetically Individualized Therapies in Reducing Drug Toxicity

1. Genetically Individualized Therapies in Reducing Drug Toxicity Ya-Chun Huang December 4, 2006

2. A Major Clinical Problem — Drug Toxicity • Drug toxicity => i.e. adverse drug reactions (ADRs) • > 2 million hospitalized patients have severe ADRs annually in US even when drugs are appropriately prescribed and administered. • 6.7% of inpatients have serious ADRs and 0.32% have fatal reactions, the latter causing about 100,000 deaths per year in the USA. JAMA, April 15, 1998-Vol 279, No. 15

3. Expense of Drug-Induced Illness • The cost of drug-related morbidity and mortality in US health care system exceeds $136 billion annually. • ADRs ranked between the 4th to 6th leading causes of death in the US. Pharmacotherapy 20(3):330-335, 2000. Fharmacogenomics(2003) 4(1), 1-4

4. Problem Anticancer Drugs due to ADRs • Irinotecan (CPT-11) • Colorectal and lung cancers, pediatric solid tumors (rhabdomyosarcoma, neuroblastoma) • Diarrhea, leukopenia, anorexia, neutropenia • 5-Fluorouracil (5-FU) • Breast, head and neck, anal, stomach, colonand some skin cancers. • Leucopenia, fatigue, bone marrow suppression  and ulcers , diarrhoea, alopecia Annu. Rev. Med. 2006. 57:119–37

5. Problem Non-anticancer Drugs due to ADRs • Warfarin • Anticoagulant • Bleeding complications => 0.6% (fatal), 3.0% (major), and 9.6% (minor) • Proton pump inhibitors (PPIs) • Gastric acid–related disorders (peptic ulcer and gas­troesophageal reflux disease) • Stomach pain, upper respiratory tract infection, diarrhea • Mercaptopurine, Azathioprine, Thioguanine • Leukemia, rheumatic diseases, inflammatory bowel diseases, and solid organ transplantation • Severe, sometimes fatal, hematologic toxicity Annu. Rev. Med. 2006. 57:119–37

6. Mechanisms of Action for Drug Toxicity • Five major mechanisms • On-target toxicity • Off-target toxicity • Immune reaction • Biological activation to toxic metabolites • Idiosyncratic toxicities • Recent advances in genomics, proteomics and metabonomics should positively aid the prediction of toxicity • by enabling more accurate studies of genetic variation, such as polymorphisms in drug-metabolizing enzymes, • by further elucidating the pathways involved in drug-mediated cell toxicity Nature Reviews Drug Discovery 4, 410-420 (2005)

7. Contexts of Drug Toxicity • On-target toxicity • Based on the drug’s mechanism of interaction with its intended target. • e.g. Statins • All of the toxicities result from inhibition of the enzyme target 3-hydroxymethyl glutaryl CoA reductase in muscle instead of liver. • Off-target pharmacology • A drug’s interaction with a system other than that for which it was intended • e.g. Terfenadine & hERG channel effects • Toxicity or other adverse reaction may result from the nonspecific action on off-target pathways. The AAPS Journal 2006; 8 (1) Article 12

8. Contexts of Drug Toxicity • Biological activation to toxic metabolites • Bioactivation of drugs to reactive intermediates that bind covalently to macromolecules • e.g. Acetaminophen. => The path-ways relevant to bioactivation of acetaminophen as shown: The AAPS Journal 2006; 8 (1) Article 12

9. Contexts of Drug Toxicity • Hypersensitivity & immunological reactions • Autoimmune responses, in which the body’ s immune system begins to respond to the compounds as foreign entities. • e.g. Penicillins and other β-lactum antibiotics • Idiosyncratic Toxicities • These problems do not appear until late clinical trials or after a drug enters the market. • The major cause of drug withdrawal from the market. The AAPS Journal 2006; 8 (1) Article 12

10. Metabolic Enzymes for Toxicity • Poor metabolisers will be overdosed and be at high risk of drug toxicity, whereas ultrarapid metabolisers will be underdosed. • If the therapeutic effect depends on the formation of an active metabolite (eg, morphine from codeine). => Poor metabolisers will have no drug effect and ultrarapid metabolisers may have exaggerated drug responses. The AAPS Journal 2006; 8 (1) Article 12

11. How do Species and Human Interindividual Variations Affect Toxicity? For the two polymorphisms • WT/WT : Homozygous for the wild-type allele • WT/V : Heterozygous for one wild-type and one variant (V) allele • V/V : Heterozygous for two variant alleles N Engl J Med 2003;348:6

12. Clinically important genetic polymorphisms of drug metabolism in ADRs DPD TPMT UGT1A1 Lancet 2000; 356: 1667–71

13. Two Ways for Altered Enzyme Activity • Genetic testing • Detect the presence or absence of, or change in a particular gene or chromosome. • Directly: by analysing the chromosomes or DNA of an individual; indirectly: by examining the products of their DNA, such as RNA or proteins. • Direct or indirect tests for a gene sequence or gene product are applied to test for response to enzyme activity. • Chemical testing • Detect catalyst amount Annu. Rev. Med. 2006. 57:119–37

14. Which Better – Genetic or Chemical Testing • Genetic testing • Genetic factors are estimated to account for 15%–30% of interindividual differences in drug metabolism and response, but for certain drugs or classes of drugs, genetic factors are of utmost importance and can account for up to 95% of interindividual variability in drug • Of 27 drugs frequently cited in ADR studies, 59% are metabolized by at least one enzyme with a variant allele associated with decreased drug metabolism • The best-recognized examples (TPMT, CYP2D6 )are genetic polymorphisms of drug-metabolizing enzymes, which affect about 30% of all drugs. • Genetic factors play a role in the pathogenesis of predictable ADRs. Annu. Rev. Med. 2006. 57:119–37

15. How/What do you Do for SNPs? Annu. Rev. Med. 2006. 57:119–37

16. Examples of Genetic Tests FDA Approved on the Market FDA suggest through package labeling that certain genetic tests be performed for patient receptivity before dispensing. • TPMT • One of the two ‘valid biomarkers’ for pharmacogenetics and pharmacogenomics in the 2003 FDA ‘Draft Guidance for Pharmacogenomic Data Submission’ • UGTA1 • The recent FDA approval of UGT1A1 pharmacogenetic testing for irinotecan chemotherapy followed a strategic plan aimed at developing standards of specialized genetic testing methodologies (pharmacogenetics) to provide a safe effective treatment. Clin Cancer Res 2006;12(14) July 15, 200

17. Microarray Gene Chips • Microarrays (Gene Chips) are used to detect the presence of specific genetic sequences in any sample. • Amplichip CYP450: The first commercial clinical test platform www.virtualsciencefair.org/.../why_gen.html

18. TPMT and UGTA1 Genetic Testings • Decreased TPMT enzyme activity in 80% to 95% of patients has been attributed to three of nine variant alleles: TPMT*2, TPMT*3A prevalent in Caucasians, and TPMT*3C prevalent in Asian, African, and African-American populations. • TPMT-deficient children having acute lymphoblastic leukemia • A variant in the promoter of UGT1A1 gene, UGT1A1*28 allele, has been extensively studied, and pharmacogenetic relationships between the variant and ADR to irinotecan have been reported. • Genetic variations of the UGT1A1 gene is the most important hereditary factor to predict severe ADR to irinotecan. Drug Meta Rev. 37:565–574, 2005; Clin Cancer Res 2006;12(14), 2006

19. SNPs and Toxicity and Drugs JAMA, April 15, 1998-Vol 279, No. 15

20. Cost per Genetic Testing • More than 4,000 diseases are known to be genetic and heritable. • Tests cost from $50 for a panel of cystic fibrosis mutations to $3,100 for testing one of those genes associated with a high risk of breast cancer. • The $400 TPMT genetic test in the treatment of Crohn's disease, ulcerative colitis, and irritable bowel syndrome. The drugs are fatal in 1 in 300 cases. • The $150 genetic test UGT1A1, a test for the metabolism of irinotecan. Irinotecan can cause dangerous or lethal reactions in up to 30 percent of recipients. Oncogene (2006) 25, 1629–1638

21. Purpose/Goal of Genetic Testing • Reducing risk • Genomic information may allow more accurate prediction of an individual’s drug response and selection of the appropriate drug dosage to achieve the optimal therapeutic response, avoid therapeutic failure, and minimize side effects and toxicity. • Make drug better • Saving expensive cost • A small investment in testing today can prevent or mitigate human suffering, while saving on health care costs . • One $3,100 breast cancer-screening test on a woman with a family history of the disease can potentially save $100,000 in the cost of caring for her after a diagnosis Oncogene (2006) 25, 1629–1638

22. Is Genetic Testing for 5-FU Toxicity Good by A Clinical Trial? • 5-Fluorouracil (5-FU): anticancer drug • Similar to TPMT, Dihydropyrimidine dehydrogenase (DPD) deficiency constitutes an inborn error in pyrimidine metabolism associated with thymineuraciluria in pediatric patients and an increased risk of toxicity in cancer patients receiving 5-FU treatment. • Using the way that is the same as TPMT way to judge whether genetic testing for 5-FU toxicity is feasible. J. Clin. Invest. 1996. 98:610- 615.

23. Efficacy and Toxicity of 5-FU • 5-fluorouracil (5-FU): A valuable weapon in the battle against cancer, such as colorectal, gastric, pancreatic, head, neck, breast, ovarian, and cervical cancers. • Some patients, however, are genetically predisposed to adverse reactions to 5-FU.

24. Symptoms of Side Effects of 5-FU • Stomatitis, diarrhoea, dermatitis, nausea, hair loss, fever, leukopenia, thrombocytopenia, myelosuppression, neurotoxicity, and cardiotoxicity. • In some instances, these side effects will culminate in death.

25. Prevalence of ADRs Caused by 5-FU • Women appear to be more susceptible than men. • 1/3 of all patients with a toxicity 4 after 5-FU treatment are carriers of the Exon-14 Skipper Mutation (DPYD*2) • Doctors initiate treatment with 5-FU for approx. 100,000 patients annually. • 45 % of 600 patients suffer severe to life-threatening side effects, including death.

26. 5-FU and DPD Enzyme • DPD which is the initial and rate-limiting enzyme in the three-step metabolic pathway leading to the catabolism of the pyrimidine bases uracil and thymine. • DPD, the key enzyme that degrades the structurally related pyrimidine antimetabolite 5-FU, is known to be a principal factor in clinical responses to 5-FU. J. Clin. Invest. 1996. 98:610- 615.

27. Mechanism of 5-FU Toxicity • The initial and rate-limiting enzyme in the catabolism of 5-FU is DPD, catalysing the reduction of 5-FU into DHFU. • FdUMP is the cytotoxic product resulting from a multi-step 5-FU activation route. • FdUMP inhibits the enzyme thymidylate synthase (TS), which leads to intracellular accumulation of dUMP and depletion of deoxy-thymidine-monophosphate dTMP. • This causes arrest of DNA synthesis. • DPD Deficiency will cause toxicity J. Clin. Invest. 1996. 98:610- 615.

28. Genetic Testing for DPD Deficiency • A DPD deficiency is increasingly being recognized as an important pharmacogenetic disorder in the etiology of severe 5-FU-associated toxicity. • Genetic deficiency of DPD enzyme results in an error in pyrimidine metabolism associated with thymine-uraciluria and an increased risk of toxicity in cancer patients receiving 5-FU chemotherapy. • Polymorphisms of DPD and the thymidilate synthase genes in relation to severe toxicity of treatment with 5-FU. • Among these polymorphisms, the exon 14-skipping mutation (DPYD*2)appears to be the most prominent genetic change related to severe DPD deficiency

29. Prevalence of DPD Deficiency • Approximately 5% of women in one breast cancer population were found to be DPD-deficient. • 3% to 5% of individuals have reduced DPD activity, which is associated with severe and sometimes life-threatening 5-FU toxicity in cancer patients. • 90% of individuals heterozygous for a mutant DPDallele has a DPD activity below the threshold value indicative of increased risk of 5-FU toxicity Clinical Cancer Research 2004. 10, 7100–7107

30. DPD Activity and Genotype Clinical Cancer Research 2004. 10, 7100–7107

31. Obtaining Data on Variant Alleles of DPD • The identification of variant alleles associated with DPD deficiency by a high-throughput denaturing HPLC (DHPLC) method . • A reliable method for the investigation of large samples in an acceptable cost and time range. • This technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. Analytical Biochemistry 306, 63–73 (2002)

32. Linking 5-FU Identified in ADR Studies to Variant Alleles Clin Cancer Res 2006;12(14), 2006

33. DPD — Clinical Testing • Over 20 different mutations are known in the DPD which could be associated with a loss of enzyme function. • Conducting a population study using a German cohort to determine the frequency of DPD defects in the German population and to detect new toxicity-associated mutations. • DHPLC: develop a sensitive and efficient screening of tumor patients to identify patients with mutations in the DPD gene which might be related to 5-FU toxicity. Analytical Biochemistry 306, 63–73 (2002)

34. Benefits if DPD Work • More patients can live • Although % individuals affected by UGT1A1- and TPMT-inactivating mutations is higher than % individuals affected by DPD inactivating mutations, DPD deficiency (3-5%) has been more frequently associated with fatal outcomes. • Save more than 3.6 million euros per year for the DPYD*2 pre-therapeutic testing. • About 160 patients have to be tested to save one patient from severeside effects after 5-FU treatment, because of the DPD mutation. => One test costs 150 eeuros => The costs of the treatment of a WHO grade 4 toxicity patient is 30,000 to 100,000 euros. • Genetic testing is available to help physicians address the need in defining an appropriate 5-FU medication regimen without delay. Nature Reviews Drug Discovery 4, 410-420 (2005)

35. Limitations • Recently, the medical profession became aware of the limitations that have greatly hindered advances in cancer therapeutics and response to treatment. • These limitations include the lack of pharmacogenomic education at medical schools, integration of pharmacogenomic knowledge into clinical practice, and recognition and responsiveness to the effect of pharmacogenetics on healthcare.

36. What Associate Drug Toxicity Make Me Want to Depend on Genetic Testing? • Can identify patients at risk of developing severe toxicity before the administration of a chemotherapeutic agent, especially in cancer therapeutics. • This approach should ultimately allow individualizing therapy through tailored dosing or using treatment modification strategies, thereby avoiding genetically altered drug metabolic pathways. • About 4% of all new medications are withdrawn because of ADRs, and failure of a newly released drug is disastrous for a pharmaceutical company, which may have spent > 1 billion dollars to develop that single product. Annu. Rev. Med. 2006. 57:119–37

37. Conclusion • Opportunities to reduce or prevent ADR or toxicity due to genetic variations in drug-metabolizing enzyme(s) are now possible as a result of increased genetic knowledge from the Human Genome Project and other pharmacogenetic studies • Performing a clinical genetic assay is unique in that it can inform as to whether some treatments will work, before a patient starts chemotherapy. • Genotyping enhances precision regarding the choice of drug, or the dose of the drug JAMA, November 14, 200] Vol 286, No. 18 JAMA, November 14, 200] Vol 286, No. 18

38. Conclusion • Although many nongenetic factors influence the effects of medications, including age, organ function, concomitant therapy, drug interactions, and the nature of the disease, there are now numerous examples of cases in which interindividual differences in drug response are due to sequence variants in genes encoding drug-metabolizing enzymes, drug transporters, or drug targets. • In the future, physicians will be able to use a medicine response profile to predict an individual’s likely response before a medicine is prescribed for .

39. Rebuttal

40. Necessary of Pharmacogenomics • Even if a gene has a large effect on a drug’s pharmacokinetics or pharmacodynamics, the presence of SNPs in that gene will not provide an unequivocal answer but, rather, will indicate the likelihood that an individual patient will show an altered drug response. • The variations can be of genetic, physiological, pathophysiological, or environmental origin, but a drug’s absorption, distribution and metabolism, and interactions with its target can be determined by genetic differences. • Unlike other factors influencing drug response, inherited determinants generally remain stable throughout a person’s lifetime. Annu. Rev. Med. 2006. 57:119–37

41. Clinical Trials and Surveillance • A major cause of attrition in the drug development pipeline is the toxicity of drugs, which is still difficult to predict early in drug discovery. • Severe ADRs such as hepatotoxicity or drug-induced arrhythmias continue to be significant problems for many new drugs during the development and post-marketing phases. • Dexfenfluramine, encainide, zomepirac, ticrynafen, benoxaprofen, terfenadine, and troglitazone were associated with major ADRs only when they had reached the market. THE LANCET • Vol 355 • April 15, 2000

42. Clinical Trials and Surveillance • Extensive safety testing in larger populations is one possible solution but together with the increasing demands for cost effectiveness evidence at the time of a drug launch significant barriers to drug development could be created. • Pharmacogenetics could overcome these difficulties by streamlining drug development, by enabling an extensive, regulated post-approval surveillance system to be developed, and by targeting medicines to patients likely to benefit and unlikely to experience adverse events. THE LANCET • Vol 355 • April 15, 2000

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