The Personalized Medicine

1. The Personalized Medicine By Mr.G.Thirupugal M.Sc., Microbial Gene Technology Department of Microbial Technology School of Biological Sciences Madurai Kamaraj University

2. THERE IS AN OLD SAYING The only difference between a medicine and a poison is the Dose Drug tailoring, dosage in accordance with weight, known diet and drug interactions, effectiveness of drugs varies among individuals Trial and Error Method

3. CURRENT PHARMACY PRACTICE • Finding the right medication is often a lengthy process wherein • Valuable treatment time is lost • Adverse reactions to drugs • Possibly killing 100,000 American patients, making it the fourth to sixth leading cause of death in the US • And over 2 million additional people have serious reactions to medication. The current system of one size fits all medicine clearly isn’t working!

4. PATIENT RESPONSE TO MEDICINE VARIES “One Size Does Not Fit All …” “I have hypertension which is not being controlled. My doctor prescribes a drug for hypertension, we wait 3-4 months to find it’s not working and then try another one. In 18 months, I’ve tried 6 new medications and I’m fast losing confidence in this hit or miss approach and in my physician”.

5. Reasons Why Drugs Are Pulled off the Market • Rare, Unpredictable Problems • More Toxic than Expected • Safer Options Are Not Available • Dangerous Combinations • Improper Use • When Other Risk Management Options Fail

6. The Solution is ……. PHARMACOGENETICS

7. PHARMACOGENETICS • What is it? • Is it important? • How is it currently being applied? • How will it likely be applied in the future?

8. Pharmacogenetics vs. Pharmacogenomics Pharmacogenetics Study of variability in drug response determined by single genes Pharmacogenomics Study of variability in drug response determined by multiple genes within the genome

9. Pharmacogenetics: Importance? • 20-40% of patients benefit from an approved drug • 70-80% of drug candidates fail in clinical trials • Many approved drugs removed from the market due to adverse drug effects • 1961-1992: 131 approved drugs removed from market because of severe side effects

10. ADR’s: Importance?

11. Pharmacogenetics - Why Now ? • Changing Healthcare Environment • Increasing emphasis on evidence-based medicine (risk/benefit) • Increasing demand for safer, novel therapies • Increasing application to clinical practice • Increasing expectation from policy makers e.g. DH. Nuffield, EMEA, FDA • Technological advances • DNA handling, robotics, miniaturization, results analysis • Genetic maps and markers • Human Genome Project • Single Nucleotide Polymorphism (SNP) profiles

12. DNA----->RNA---> Protein • Drug targets • If the mutated protein in the patient’s body is not the • therapeutic target of the drug ==> no effect • Drug metabolism • Slow metabolism ==> Build up of extremely high level of drug in the body ==> Toxic effect • Fast metabolism ==> elimination of the drug before it achieves the desired effect People differ in their genetic make-up and consequently in their reaction to drugs

13. Pharmacodynamics And Pharmacokinetics Breast Cancer • Abnormally high amounts of HER2 protein in 30% of patients • Herceptin binds to HER2 slowing tumour growth, 70% of patients do not respond Autoimmune disorders, childhood leukemia • Azathiprine degraded by TMPT enzyme • 0.5% Caucasians do not produce functional TMPT • Toxic levels of drug lead to acute bone marrow failure Pain relief==> Codeine converted into Morphine by product of CYP2D6

14. Pharmacogenetics Why at a recommended prescribed dosage, is a drug efficacious in most Not efficacious in others Harmful in a few

15. Pharmacogenomics To Deliver ‘Right Medicine, Right Dose, to Right Patient’ Physician Dx; clinical info

16. Pharmacogenetics Find Genetic variation responsible Prescribe drugs in accordance with patient’s genotype PERSONALIZED MEDICINE

17. Personalized Medicine? • Concept started with Karl Landsteiner • A, B, AB, O blood groups • Pharmacogenetic and Pharmaceutical Industries fuel concept • “The era of personalized medicine” • Alternative to “blockbuster” or “one size fits all” agents • Potential impact on health care costs • Potential impact on health care provision

18. Aims of Personalized Medicine • Synthesizing drugs based on genetic variation in drug response • Determining the efficacy of existing pharmaceuticals and determining individual predisposition to adverse drug reactions

19. The Process ofPersonalized Medicine 1. Locate genetic polymorphism 2. Connect with differential drug response 3.Prescribe drugs accordingly

20. Single Nucleotide Polymorphisms (SNPs) Most genetic variations are attributable to SNPs Easily detected by high throughput technologies

21. Step 1. Identify SNPs in Genes Relevant to Drug Efficacy or Tox Human Genome 2,900,000,000 Billion total base pairs 10,000,000 Total single nucleotide polymorphisms (SNP) 300,000 Variant haplotypes 10,000 Haplotypes in pharmacologically-relevant genes

22. Single Nucleotide Polymorphisms (SNPs) • Sequenced DNA of 10-50 subjects • Use computer alignment to detect variation • If variation >1% of population: SNP • Confirm SNP by assaying for it in an ethnically diverse panel of DNA and observe occurance in different populations • Catalogue different alleles • Attempt to identify those that influence gene expression or product: focus on promoter, exons, transcript procession regions, regulatory sequences

23. Step 2. Retrospectively, Find SNPs Associated With Response Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient12 Good response No response No response Good response No response No response Good response Good response Good response Good response No response No response ATGCTTCCCTTTTAAA ATTGTTCCCTTTTAAA ATTGTTGCCTTTTAAA ATGGTTGCCTTTTAAA ATAGTTGCCTTTTAAT ATAGTTGCCTTTTAAT ATGATTGCCTTTTAAA ATGATTGGCTTTTAAA ATGTTTCGCTTTTAAA ATGTTTTGCTTTTAAA ATTTTTTGCTTTTAAA ATCTTTTGCTTTTAAA

24. Step 3. Prospectively, Determine If Those SNPs Affect Therapeutic Outcome G G G G G G G G G G G G G G G G Treat G G G G G G G G G G 25% cure 50% cure Determine statistical significance (the probability that such a difference is due to random chance)

25. SNP Diagnostics Molecular Mechanisms Differential hybridization Allele-specific nucleotide incorporation a.k.a. primer extension and single-base extension (SBE) Allele-specific DNA cleavage Assay Environment Solid supports (such as oligonucleotide chips) Homogenous solution Combination of the two environments

26. DNA CHIP

27. Other Methods of Detecting Polymorphisms SNP plentiful but not very informative Other options Haplotyping Expression Profiling

28. Haplotype SNPs that travel in groups, operate together to cause a certain drug response, usually within one gene Exist because certain polymorphisms tend to be linked Discovered by sequencing DNA Reduced complexity of genetic analysis Only a few common haplotypes in population Each person has only two haplotypes (since each person has only two copies of any particular gene) Predict activity of gene more precisely

29. 0 1 0 1 0 1 0 1 0 1 Gene SNPs 0 1 0 0 1 1 0 1 1 0 Haplotypes Gene Haplotypes Chromosome locus of gene Exons Promoters SNPs Causative Site Haplotypes are a code for defining and tracking the isoforms of a gene

30. Responders Non-responders Toxic responders Genetically Based Optimization of Drug Dosing

31. Non-responders Toxic responders Genetically Based Optimization of Drug Dosing

32. Drug Metabolisms Absorption Distribution Metabolism Elimination Pharmacokinetic Bioavailability

33. Environmental factors Absorption Distribution Target Interaction Drug Response Drug factors Biotransformation Excretion Genetic factors Variations In Drug Response

34. Typical drug metabolism • Entrance into the body • Distribution • Drug cell interactions • Drug metabolism • Excretion

35. Typical drug metabolism Oral Intravenous Ingestion Distribution [Drug Metabolism] Absorption [Drug Metabolism] [Drug Metabolism] Drug-cell interaction Distribution Drug Metabolism Drug-cell interaction Drug Metabolism Excretion Excretion

36. Bioavailability of Drugs Uptake of orally administered drug proceeds after the stomach passage via the small intestine. In the liver, a series of metabolic transformation occurs

37. Drug Modification/Metabolism • Conjugation • Glucuronic acid • Glycine • Sulfates • Acetylation • Methylation • Mercapturic acid synthesis (not common) • Can produce inactive metabolites, more toxic compound or active metabolites

38. Conjugation • Phenols, alcohols, carboxylic acids, compounds with amino or sulfhydryl groups generally form glucuronides • Aromatic acids form glycine conjugates • Phenols, alcohols or aromatic amines can undergo sulfate conjugation with donor being 3’-phospho-adenosine-5-phosphosulfate (PAPS) Glucuronyl transferase UDP-glucuronate + R-OH RO-glucuronide +UDP Glycine ATP + CoA Benzoate Benzoyl-CoA Hippurate

39. CO-NHNCOCH3 CO-NHNH2 N N Acetylation and Methylation • Derivatives of aniline are acetylated: the reaction of the amine group with acetyl-CoA catalyzed by a specific acetylase. • Norepinephrine and epinephrine by O-methylation; nicotinic acid by N-methylation. Donor is S-adenosylmethionine + CoASCOCH3 + CoASH

40. Genetic Variation Revealed by Drugs • Arylamine N-Acetyltransferase activity • E.C. 2.3.1.5 Liver P450 enzyme • Isoniazid metabolism • ‘Rapid inactivators and slow inactivators’ • Slow inactivators are homozygous for a recessive allele with lower enzyme activity • Rapid inactivators are at risk for liver damage • Slow inactivators at higher risk for lupus-like changes or polyneuritis • Rapid inactivators require larger doses for treatment of TB

41. Cytochrome P450 • The super-family of cytochrome P450 enzymes has a crucial role in the metabolism of drugs. • Almost every drug is processed by some of these enzymes.This causes a reduced bioavailability. • Cytochrome P450 enzymes show extensive structural polymorphism (differences in the coding region).

42. Cytochrome P450 Metabolisms • The cytochromes involved in the metabolism are mainly monooxygenases that evolved from the steroid and fatty acid biosynthesis. • 17 families of CYPs with about 50 is forms have been characterized in the human genome The iron is part of a HEM moiety CYP 3 A 4 Family>40% sequence-homology Allel Isoenzyme Sub-family>55% sequence-homology

43. Cytochrome P450 gene families Human Molluscs CYP450 Plants Insects Bacteria Yeasts Nematodes Fungi

44. Human cytochrome P450 family • The super-family of all cytochromes, the following families were confirmed in humans: • CYP 1-5, 7, 8, 11, 17, 19, 21, 24, 26, 27, 39, 46, 51 • Function • CYP 1, 2A, 2B, 2C, 2D, 2E, 3 metabolismus of xenobiotics • CYP 2G1, 7, 8B1, 11, 17, 19, 21, 27A1, 46, 51 steroid metabolism • CYP 2J2, 4, 5, 8A1 fatty acid metabolism • CYP 24 (vitamine D), 26 (retinoic acid), 27B1 (vitamine D),

45. Substrate Specificity Of CYPs Specific substrates of particular human CYPs CYP 1A2 Verapamil, imipramine, amitryptiline, caffeine (arylamine N-oxidation) CYP 2A6 Nicotine CYP 2B6 Cyclophosphamid CYP 2C9 Diclofenac, Naproxen, Piroxicam, Warfarin CYP 2C19 Diazepam, Omeprazole, Propanolol CYP 2D6 Amitryptiline, Captopril, Codeine, Mianserin, Chlorpromazine CYP 2E1 Dapsone, Ethanol, Halothane, Paracetamol CYP 3A4 Alprazolam, Cisapride, Terfenadine, ...

46. Cytochrome P450 polymorphisms „Every human differs (more or less) “ The phenotype can be distinguished by the actual activity or the amount of the expressed CYP enzyme. The genotype, however, is determined by the individual DNA sequence. Human: two sets of chromosomes That mean: The same genotype enables different phenotypes Depending on the metabolic activity, three major cathegories of metabolizers are separated: extensive metabolizer (normal), poor metabolizer, and ultra-rapid metabolizer (increased metabolism of xenobiotics)

47. CYP 2D6 Polymorphism

48. Polymorphisms Of Further CYPs • CYP 1A2 individual: fast, medium, and slow turnover of caffeine • CYP 2B6 missing in 3-4 % of the caucasian population • CYP 2C9 deficit in 1-3 % of the caucasian population • CYP 2C19 individuals with inactive enzyme (3-6 % of the caucasian and 15-20 % of the asian population) • CYP 2D6 poor metabolizers in 5-8 % of the european, 10 % of the caucasian, and <1% of the japanese population. Over expression (gene duplication) among parts of the african and oriental population. • CYP 3A4 only few mutations

49. Genotyping For P450 Alleles Affymetrix (US) has developped microarrays (gene chips) using immobilized synthetic copies of P450 nucleotides, that allow the identification of all clinically relevant allelic variants

50. N-Acetyltransferase And Other Drugs • Hydralazine (antihypertensive) • Sulphasalazine (antibiotic used in Crohn disease) • 4,4’-diaminodiphenylsulfone, Dapsone (anti-malarial, anti-leprosy) • Procainamide (antiarrhythmic) • Sulfapyridinine • Caffeine metabolite • 5-Acetyl-amino-6-formylamino-3-methyluracil (AMFU) to 1-methylxanthine ratio • HPLC measurement r=0.98