Predict GENES. Select right drug. Select right dose. Develop new drugs. Non-Response Response Adverse Reaction

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1 Predict Select right drug Select right dose Develop new drugs GENES Non-Response Response Adverse Reaction

2 Phase I: Each Chromosome Has Many Genes Chromosome 12 ~ 1,300 Genes

3 Examples of Personalized Medicines FDA Label Changes- Current and Planned: 6-Mercaptopurine: polymorphism in enzyme (TPMT) serious adverse effect (bone marrow suppression) Allopurinol: polymorphism in enzyme (TPMT) serious adverse effect (bone marrow suppression) Warfarin: polymorphism in enzyme (CYP2C9) serious adverse effect (bleeding) Irinotecan: polymorphism in enzyme (UGT1A1) serious adverse effect (neutropenia, profound diarrhea) Tamoxifen: polymorphism in enzyme (CYP2D6) may lead to nonresponse in women with breast cancer

4 Examples of Personalized Medicines: Pharmacogenomic Tests The Roche AmpliChip CYP450 The world s first pharmacogenetic microarray-based test approved for clinical use by FDA and in EU. Drug Response Genes Drug Metabolism Enzyme Genes Celera Diagnostics HIV genotyping Hepatitis Genotyping FDA Guidances Pharmacogenomic Guidance Pharmacogenetic Test Guidance

5 Some Progress But Much Research is ngoing DISCVERY PRE-CLINICAL DRUG DEVELPMENT TH ER A US PEU E TI C RESEARCH CLINICAL TRIALS

6 NIH- Pharmacogenetics Research Network Pharmacogenetics of Membrane Transporters

7 Membrane Transporters in Human Genome FIC1 ENT1 ENT2 MRP1-4 MDR1 MDR3 BSEP DAT NET GAT1 SERT Hydrophobic Anions Hydrophobic Cations, Lipids Neurotransmitters CTR1 Phospholipids Cu ++ Nucleosides VMAT1 VAChT ligopeptides Nucleosides PEPT1 PEPT2 rganic Cations Fe +++ CNT1 CNT2 NRAMP1 DMT1 IREG1 CT1 CT2 CT3 ABC Family SLC Family

8 Variation in Drug Response is Determined by Variation in Drug Levels Drug Response Liver Intestine Transporters Transporters Drug Levels In Blood Transporters Drug Levels in Target Kidney Heart Lung Brain Tumor Transporters Transporters Transporters Transporters

9 rganic Cation Transporter, CT1 NH N N H NH NH2 Metformin HN N S Cimetidine N H NCN N H Liver H N MPP + MPTP H Pindolol N H + N CH 3 N CH 3

10 Two Stories About CT1 CT1 Polymorphisms: Determinant of Variation in Drug Response Metformin CT1: Determinant of Anti-Cancer Drug Specificity xaliplatin

11 Transporter Does Genetic Variation in Membrane Transporters Contribute to Variation in Drug Response? Human Genetic Studies Patients Identify Mutant/ Polymorphic Gene Mechanism Pharmacogenetic Studies Patients Cellular Studies Discover Genetic Variants

12 Resequence Gene Regions of CT1: Exons n = DNA Samples EXNS DNA Samples African Americans: Asian Americans: European Americans: Mexican Americans: 10-50

13 Genetic Variants in CT1 Identified in 247 DNA Samples from Ethnically Diverse Populations Non-synonymous SNP Amino acid deletion 3% African Americans African Americans 26% Caucasian Americans 40% Asian Americans 24%

14 Variant Transporters Are Constructed By Site Directed Mutagenesis and Then Expressed In ocytes or HEK293 Cells mrna mrna Xenopus laevis oocytes Transporter HEK293 cells Non-synonymous SNP Amino acid deletion

15 CT1 Has Many Reduced Function Variants Shu et al., PNAS, 2003

16 Metformin: A Clinically Important Drug Anti-diabetic agent, potential therapy for various diseases related to obesity. N NH N H NH NH2 METFRMIN IS A SUBSTRATE F CT1

17 Two Stories About CT1 CT1 Polymorphisms: Determinant of Variation in Drug Response Metformin CT1: Determinant of Anti-Cancer Drug Specificity xaliplatin

18 Cancer Therapy: Current Status Cancer: 2nd Killer in US Half a million death per year Expected: 1st killer next decade Anticancer therapy: Low cure rate High toxicity Poor quality of life Change in the US Death Rates by Cause, 1950 & Rate Per 100, Heart Diseases 53.3 Cerebrovascular Diseases Source: US Mortality Public Use Data Tape 2003, National Center for Health Statistics, Centers for Disease Control and Prevention, Pneumonia/ Influenza Cancer

19 Anti Cancer Drug Therapy: Two Major Challenges Drug resistance Decrease of intracellular drug levels Mutation of drug targets Lack of tumor specificity Anti-cancer drug targets are present in normal tissues and tumors

20 Platinum-based Anticancer Agents Most active anticancer agents Testicular cancer (Cisplatin, cure rate > 90%) varian cancer, head and neck cancer Multiple drugs approved H N 3 Pt H N 3 Cisplatin C l C l H 3 N C Pt H 3 N C Carboplatin H 2 N N H 2 Pt xaliplatin C C H 3 N H 3 N Pt N H 2 Pt H 2 N Nedaplatin (Japan) Lobaplatin (China) Heptaplatin (South Korea) H 2 N N H 2 Pt In treatment regimens for 50% of cancer patients

21 Platinum Drugs Have Different Anticancer Spectra Cisplatin Carboplatin xaliplatin: Similar Anticancer Spectrum Colorectal Cancer Question: What gives platinum compounds their tumor specificity?

22 Platinum Drugs Share a Common Mechanism of Action R 1 H R 2 H Intra-strand crosslinks R 1 H R 2 H Inter-strand crosslinks

23 Platinum Uptake Mechanisms May Be Very Specific Reduced platinum uptake is the most common observation in platinum-resistant cells? CTs?

24 CT1 and Platinum Drug Cytotoxicity Percent of cell growth Cisplatin MCK 20 hct Concentration of cisplatin (µm) Percent of cell growth MCK hct1 Carboplatin Concentration of carboplatin (µm) Percent of cell growth MCK hct1 xaliplatin Concentration of oxaliplatin (µm) Zhang S, et al. Cancer Research, 2006, 66(17):

25 CT1 and Platinum-DNA Adduct Formation Platinum-DNA adduct formation after oxaliplatin incubation 0.05 Platinum-DNA adducts (pmol platinum / µg DNA) hct1 MCK Genomic DNA associated platinum was determined by ICP-MS Zhang S, et al. Cancer Research, 2006, 66(17):

26 CT2 and xaliplatin Percent of cell growth xaliplatin Cytotoxicity 120 MCK 100 hct Concentration of oxaliplatin (µm) Zhang S, et al. Cancer Research, 2006, 66(17): Platinum uptake (pmol / mg protein hr) Platinum-DNA adducts (pmol platinum / µg DNA) xaliplatin Uptake Control Cimetidine treated hct2 MCK Platinum-DNA adduct Control Cimetidine treated hct2 MCK

27 Expression of CTs in Colon Cancer RT-PCR study: hct1 hct2 HEK- CT2 LS180 DLD SW620 HCT116 HT29 RK Normal colon tissue Colon Cancer Samples GAPDH Zhang S, et al. Cancer Research, 2006, 66(17):

28 CT Inhibitor Reduces xaliplatin Cytotoxicity in Colon Cancer Cell Lines IC 50 values (µm) RK With CT Inhibitor LS180 With CT Inhibitor 0 oxaliplatin cisplatin 2 0 oxaliplatin cisplatin Control 1.5 mm cimetidine treated Similar observations were obtained in all the six colon cancer cell lines Zhang S, et al. Cancer Research, 2006, 66(17):

29 CT1 Polymorphism Non-synonymous SNP Variant S14F R61C G220V G401S V408M 420del G465R Total Extracellular Amino acid deletion frequency Highest frequecy 3.1 (AA) 7.2 (CA) 0.5 (AA) 1.1 (CA) 41 (CA) 19 (CA) 4 (CA) AA: African American; CA: Caucasian Cytoplasm Shu Y, et al. PNAS 13;100(10):

30 xaliplatin and CT1 Polymorphisms Platinum-DNA adduct formation xaliplatin cytotoxicity Platinum-DNA adducts (pmols / ug DNA) Mock Ref S14F R61C G220V G401S V408M 420Del G465R Patients with variant CT1 may have an unfavorable response to oxaliplatin IC 50 of oxaliplatin (µm) mock Ref S14F R61C G220V G401S V408M 420Del G465R

31 Conclusions And Future: Personalized Medicines Predict Drug Response Based n Genetic Variation (e.g., metformin, oxaliplatin) Gene Card Tissue Specific Drug Targeting Using Influx Transporters (e.g., platinums and CTs) Pharmaceutical Scientists Should Play A Major Role

32 They Did It! Shu Zhang Yan Shu

33 Thanks To: Laboratory Members Chaline Brown Rich Castro Claire Brett Ryan wen Genomics Core Pui Kwok Bioinformatics Core Tom Ferrin Conrad Huang Susan Johns Doug Stryke Collaborators Stephen J. Lippard (MIT) Katherine Lovejoy (MIT) Xin Chen Joe W. Gray Anna Lapuk