Pharmacogenomics for the anti-platelet drug

Transcription

1 Pharmacogenomics for the anti-platelet drug Su-Jun Lee, PhD Dept. of Pharmacology, Pharmacogenomics Research Center, Inje University College of Medicine, Busan

2 Conflict of interest disclosure None Committee of Scientific Affairs Committee of Scientific Affairs

3 Everyone is Unique Pharmacogenomics (PGx): to study the genetic basis to understand individual variations in drug response Pharmacogenomics (PGx) has a great potential for personalized medicine

4 Human Platelets - Small irregularly shaped cell fragment from fragmentation of megakaryocytes - A natural source of growth factors (PDGF, TGF-beta, etc) - Major contributor to the maintenance of hemostasis. - Multiple pathways and receptor sites (P2Y12, P2Y1, TxA2, etc) Drugs used to alter platelet function Oral agents: aspirin, clopidogrel, cilostazol, ticlopidine, ticagrelor, prasugrel IV agents: abciximab, eptifibatide, tirofiban.

5 Platelet inhibition by clopidogrel is highly variable Ischemic risk Bleeeding risk Aggmax 10% %IPA 20% J Am Coll Cardiol. 2007;50:1132-7

6 Association Between Clopidogrel Resistance and Cardiovascular Risk Bleeding events from 4 th quartile Circulation, 2004; 109,

7 Pharmacogenomics for the anti-platelet drug Background - Platelet inhibition by antiplatelet agents is highly variable. - Patients with reduced inhibition have increased risk for cardiovascular events. Goal - To identify factors contributing to variable responses from antiplatelet drugs

8 Candidate Genes: Liver, Platelet, Endothelium Clopidogrel, Prasugrel, Ticagrelor, Ticlopidine PGE2-EP3 R-Gi PGE2-EP2 R-Gs PGI2 R-Gs cilostazol 3A4, 2C19 camp PDE3 PDE3 ( ) ADP - P2Y12 Gi Gi Platelet camp down camp down VASP Cox-1,2 ARA PGH2 TXA2 Syn TXA2 Aspirin ADP P2Y1 Gq Gq Ca++ up Ca++ up CYP2C19 CYP3A4 CYP4A/4F PON-1, etc. + - Coagulation factors Endothelium PGI2 PGE2 Liver vwf PGI2 syn PGE2 syn NO NOS3 Degradation Activation of GlIIa/IIb on platelets Abciximab Eptifibatide Tirofiban Blood Platelet aggregation CLOT & Thrombosis

9 Strategies for Identification of Genetic Defects Isolate gene from individuals Sequence all Intron/Exon junctions after PCR Promoter Strategy 1 Amplify & sequence phenotyped subjects Compare vs normal group Strategy 2 Amplify & sequence normal population Compare vs NCBI Ref seq

10 Background of CYP2C19 Genetic Polymorphisms Metabolizes 15% of clinically useful drugs (e.g., PPIs, Diazepam, Citalopram, Clopidogrel, etc) 15-30% incidence of poor metabolizers of CYP2C19 in Asians vs. 3-6% incidence of PMs in Caucasians CYP2C19 PM: diminished effectiveness of clopidogrel CYP2C19*2 (Splicing variant), CYP2C19*3 (Stop codon) CYP2C19*17 (Promoter variant, increased activity) 17-20% in Caucasians vs % in Asians Nomenclature: CYP2C19*1, CYP2C19.1, CYP2C19 *1/*3, CYP2C19*2/*17

11 Effect of CYP2C19 Genotype on Clopidogrel Outcomes in MI Patients (n=532) PM: CYP2C19*2/*2, *2/*3, *3/*3 Circ Cardiovasc Genet, 6,

12 Gap between Discovering and Functional Information

13 Discovery of New CYP2C19 Genetic Variants Site Position Amino Subject number (n) Frequency Nucleotide change acid wt/wt mt/wt mt/mt (%) 5UTR -2720T>C cagtaaagctattt(t/c)atatgatacta UTR -2305G>A tgaactggga(g/a)ttgaaaaac UTR -2040C>T taaagagagcaa(c/t)caagcttatctt UTR -1418C>T gaacaaata(c/t)gatgatatct UTR -1312C>G aatgtcagctc(c/g)cgttaaggtcta UTR -889T>G cagaataactaa(t/g)gtttggaagttg UTR -806C>T ctgttctcaaag(c/t)atctctgatgta UTR -559T>C tattgaagata(t/c)atgagttatgtta UTR -529G>C atttcatgtttag(g/c)ctgctgtattttta UTR -98T>C gattggccactt(t/c)atccatcaaaga Exon 1 99C>T P33P ccctcctggccc(c/t)actcctctccca Intron 2 IVS2-23A>G gatctccctcct(a/g)gtttcgtttctc Exon 4 636G>A W212X aagcaccccctg(g/a)atccaggtaagg Exon 5 681G>A splice site attatttccc(g/a)ggaacccata Exon 5 766G>A* D256N caagaatcgatg(g/a)acatcaacaacc Intron 5 IVS5-113 T>G tttttctagtac(t/g)atactttacagt Intron 5 IVS5-51 C>G atttactgtcat(c/g)aaatatgctgtt Exon C>T V330V gattgaacgtgt(c/t)gttggcagaaac Exon A>G I331V attgaacgtgtc(a/g)ttggcagaaacc Exon G>A V394M ttccctcacttct(g/a)tgctacatgac Exon G>A E405K cccaaccca(g/a)agatgttt Exon A>C silent ggatgaaggtgg(a/c)aattttaagaaa *17 *3 *2

14 Haplotype of CYP2C19 SNPs Position (*17) IVS (*3) 681 (*2) IVS5-113 IVS Functional comparisons of variant proteins with the WT Common allele Variant allele Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4 Haplotype 6 Haplotype 7 Haplotype 8 Haplotype 9 Haplotype 10 Haplotype 11 Haplotype 12 Haplotype 13 T C G A C T C T C G T G C T T C G C T C C T A G G A G A T G C G C T A G G A G A A C Frequency S-mephenytoin 4 -hydroxylation (nmol/min/nmol P450) Wild-type 400 µm ** ** P227L D256N V394M * E405K S-mephenytoin 4 -hydroxylation (nmol/min/nmol P450) S-mephenytoin (µm) V394M Wild-type E405K D256N Expression in E.coli & Purification of P450 proteins & Quantitation of P450s Absorbance Absorbance Wild-type P450 D256N P450 V394M P450 E405K P450 P227L Cells only Wavelength (nm) Wavelength (nm) Wavelength (nm) 5-hydroxyomeprazole (nmol/min/nmol P450) Wild-type 200 µm ** ** P227L D256N V394M E405K 5-hydroxyomeprazole 5-hydroxyomeprazole activity (nmol/min/nmol (pmol protduct/min/pmol P450) Omeprazole(uM) Omeprazole (µm) Wild-type V394M E405K P227L D256N

15 Central Role of P2Y12 Receptor Signaling Collagen Inflammation Shear Epinephrine TxA 2 Amplification Thrombin Platelet Memb. ARA release X Aspirin TxA 2 ADP Amplification X Ticlopidine Clopidogrel Prasugrel Coagulation Procoagulant surface P2Y 12 Receptor Activation Granule Secretion ADP Inflammation P-selectin CD-40L Expression camp down GPIIb/IIIa Activation Platelet Aggregation Modified from Gurbel PA et al. Rev Cardiovasc Med. 2006;7:S20-S28.

16 Visit 2 ADP-induced platelet aggregation (%) Maximal ADP-induced Platelet Aggregation r = ADP-induced platelet aggregation (%) Visit 1

17 Summary of Polymorphisms in P2Y12 Gene Site Position Nucleotide change Number of subjects wt/wt wt/mt mt/mt Allele frequency (95% CI ) (%) Location 5 -UTR 48204C>T gttatctata(t/c)gcattctttt ( ) UTR 47903A>G* cactaaaaat(a/g)caaaaattag ( ) UTR 47884A>G* gccaggcatg(a/g)tggcaggcac ( ) UTR 47583G>A cagcctgggc(g/a)acagagcgag ( ) UTR 47404T>C taccaggaag(t/c)ccgctgacac ( ) UTR 47305A>T tccctggtaa(a/t)tctgattctt ( ) UTR 46737C>T gtagctggaa(c/t)taaagacatg ( ) UTR 46437C>A cagcaaaatt(c/a)atatatttta ( ) UTR 3589T>C* tgctctgcac(t/c)gtgaaaacca ( ) UTR 3435G>A ccagggaggt(g/a)tgtttctgca ( ) UTR 3001G>A* gtaaactatc(g/a)caagaacaaa ( ) UTR 2646T>C caaagttaaa(c/t)aactgtaaat ( ) UTR 2455C>T* ttggaatctg(c/t)taatttataa ( ) IVS C>T ataaaaatta(t/c)aattaaaata ( ) IVS G>A* cttatctctg(g/a)tgaaataaaa ( ) IVS T>C agattacaaa(c/t)gtcatttcaa ( ) IVS insA tcacttgtta(-/a)gttttcatta ( ) IVS T>C gagtaatttt(c/t)catcaatttc ( ) Exon 3 18C>T ccgtcgacaa(c/t)ctcacctctg ( ) Exon 3 36G>T ctgcgcctgg(t/g)aacaccagtc ( ) P2Y12 Gene C>T A>G A>G G>A T>C A>T C>T C137T G721A C1209T T742C Ins799A C18T ATG G36T * Newly identified variant TAA C>A -3589T>C -2455C>T -2646T>C -3001G>A -3435G>A Exon Both V1 and V2 forms were screened for the P2Y12 variants

18 C>T A>G A>G G>A T>C A>T C>T C>A T>C G>A T>C C>T 137 C>T 721 G>A 742 T>C 799 insa 1209 T>C 18 C>T 36 G>T Linkage Disequilibrium (LD) Map of P2Y12 SNPs 2294 bp bp 1736 bp ATG TAA P2Y12 Gene 526 bp E bp 1600 bp E2 E3

19 Distribution of P2Y12 Haplotypes and Their Influences on ADP-induced Platelet Aggregation Haplotype Code Haplotype Sequence Frequency (No. of Haplotype) Mean Aggregation (%) P H1 AAGTC 0.19 (15) 66 ± 8.1 Ref H2 AAGTT 0.21 (17) 73 ± H3 AAGCC 0.18 (15) 74 ± * H4 GTATC 0.14 (11) 68 ± H5 GAATC 0.12 (10) 64 ± H6 AAATC 0.11 (8) 66 ± Throm Res ,

20 Rofecoxib: increased risk of cardiovascular diseases Cardiovascular diseases, Heart attack, stroke

21 Unexpected Biomarker in Refecoxib-mediated Cardiovascular Events >120-fold higher blood level of 20-HETE 20-HETE: >730 papers in PubMed ( ) Liu J et al. PNAS 2010;107:

22 Inter-individual variations in 20-HETE levels in humans J Cardiovasc Pharmacol, 56, , 2010 Clin Chem, 50, , 2004

23 20-HETE? stimulus receptor 20-HETE 5.6-EET 8,9-EET 11,12-EET 14,15-ETT seh 11,12- DHET UGT Hydroxylase CYP4A11 CYP4F2 CYP4F3 CYP4V2 Epoxygenase CYP2C9 CYP2C19 CYP2J2 CYP PLC/DAG 5,12,15- HETEglucuronide 20-HETEglucuronide AAglucuronide UGT Arachidonic acid (ARA) COX PGG2 COX-1/2 PGH2 Activation of PLA2 12-LOX 5-LOX 15-LOX 12-HPETE 5-HPETE 15-HETE UGT 12-HETE 5-LOX 5-LOX LTA4 Leukotrienes LTC4 synthase LTA4 hydrolase 15-LOX LTD4 LTC4 LTB4 5-HPETE, 15-HETE 5-LOX ETT LXA4 UGT LXB4 LTE4 LTF4 LTB4- glucuronide PGIS PGDS PGES (mpges-1) PGF2α reductase TXA2S IP PGI2 PGD2 PGE2 UGT PGE2- PGF2 α FP TXA2 TP PGA3 glucuronide 6-kete-PGF1α (inactive) PGJ2 DP 9α1 11α-PGF2α TXB2 (inactive) 15-d-PGJ2 EP1 EP2 EP3 EP4 Vasodilation Inhibit platelet aggregation Smooth muscle contraction Inhibits platelet aggregation Vasodilation Hyperalgesia Immunomodulation Smooth muscle contraction Bronchoconstriction Vasoconstriction Platelet activation

24 Research background, hypothesis, and goals Background 20-HETE is a cardio toxic ARA metabolite. There is inter-individual variation in 20-HETE levels. Factors affecting this variation are not determined clearly yet. Hypotheses Genetic polymorphisms in UGT genes may affect 20-HETE glucuronidation, which may, at least in part, explain the 20-HETE variation in blood. Goals To determine which UGTs are responsible for 20-HETE-glucuronidation To study genetic influences of the UGTs on 20-HETE glucuronidation

25 20-HETE Glucuronide formation Intensity (Metabolite Area / IS Area) Major UGTs responsible for the 20-HETE-glucuronide A1 1A3 1A4 1A6 1A7 1A8 1A9 1A10 2B4 2B7 2B15 2B17 UGT isoforms

26 20-HETE glucuronidation by human liver microsomes (n=44)

27 Protein and Genotype analysis for UGT2B7, 1A9, and 1A3 Enzyme Gene Variants Region Reference Number Function UGT1A3 140T>C Exon 1 rs (*2) Decrease 31T>C Exon 1 rs (*3) Decrease 133C>T Exon 1 rs (*4) Increase UGT1A9-118T9>T10 Promotor rs (*22) Increase 1399 C>T Intron 1 rs (*3) Increase UGT2B7 211G>T Exon 1 rs (*71S) Decrease 802C>T Exon 2 rs (*2) Decrease

28 20-HETE Glucuronide formation (pmol/min/mg) 20-HETE Glucuronide formation (pmol/min/mg) Correlation between UGT genotype and 20-HETE glucuronidation among human liver microsomes 1500 r 2 = 0.328, P< r 2 = 0.309, P< Relative protein expression level of UGT2B Relative protein expression level of UGT1A9 UGT2B7 802C/C UGT2B7 802T/T UGT2B7 802C/T UGT1A9-118T 9 /T 9 UGT1A9-118T 10 /T 10 UGT1A9-118T 9 /T 10

29 Genetic Effects of UGT1A9 and UGT2B7 on 20-HETE Glucuronidation UGT1A9 Genotype -118T 9 >T C>T T 9 / T 9 C/C T 9 / T 10 C/T T 10 / T 10 T/T UGT2B7 genotype 802C>T n C/C 0 0 C/T ± 171 T/T C/C ± 267 C/T ± 110 T/T ± 330 C/C ± 247 C/T ± 301 T/T ± HETE glucuronidation (pmol/min/mg) Mean ± S.D. P value <0.0001

30 stimulus receptor 19,20- HETE 5.6-EET 8,9-EET 11,12-EET 14,15-ETT seh UGT? Hydroxylase CYP4A11 CYP4F2 CYP4F3 Epoxygenase CYP2C9 CYP2C19 CYP2J2 Activation of PLC/DAG CYP 20-HETEglucuronide AAglucuronide UGT Arachidonic acid (ARA) COX-1/2 Activation of PLA2 12-LOX 5-LOX Phospholipids 12-HPETE 5-HPETE 12-HETE 5-LOX LTA4 Leukotrienes LTC4 synthase LTA4 hydrolase 15-LOX LTD4 LTC4 LTB4 UGT LTE4 LTF4 LTB4- glucuronide 11,12- DHET PGG2 COX-1/2 15-LOX 15-HETE UGT 5-LOX 5-HPETE, 15-HETE 5-LOX ETT PGH2 5,12,15- HETEglucuronide LXA4 LXB4 PGIS PGDS PGES (mpges-1) PGF2α reductase TXAS IP PGI2 PGD2 PGE2 UGT PGE2- PGF2 α FP TXA2 TP PGA3 glucuronide 6-kete-PGF1α (inactive) PGJ2 DP 9α1 11α-PGF2α TXB2 (inactive) 15-d-PGJ2 EP1 EP2 EP3 EP4 Vasodilation Inhibit platelet aggregation Smooth muscle contraction Inhibits platelet aggregation Vasodilation Hyperalgesia Immunomodulation Smooth muscle contraction Bronchoconstriction Vasoconstriction Platelet activation

31 Identification of P450s in human Platelets Background ARA metabolite, 20-HETE, was detected in human platelets (1995), however, no studies for identification of P450s responsible for the ARA metabolism yet. Goal To identify P450s in human platelets (20-HETE, TXA2) To study functional role of the identified P450s

32 No-RT Liver Platelets Expression of P450s in human Platelets Platelets Liver Gene CYP1A1 CYP1A1 CYP1A2 CYP2C8 CYP2C9 CYP2C19 CYP2J2 CYP2U1 CYP2U1 CYP2J2 CYP2D6 CYP3A4 CYP3A5 CYP4A11 CYP4A11 CYP4F2 CYP5A1 GAPDH mrna Expression Profiles CYP4F2 GAPDH Western-blot Analysis

33 14,15-EET formation Intensity (Metabolite Area / IS Area ) 20-HETE formation Intensity (Metabolite Area / IS Area ) 8,9-EET formation Intensity (Metabolite Area / IS Area ) 11,12-EET formation Intensity (Metabolite Area / IS Area ) P450-dependent ARA metabolites produced in platelets * 6 4 * ** 2 *** 0 ARA ( M) ODYA ( M) ARA ( M) ODYA ( M) * 4 ** 2 0 ARA ( M) ODYA ( M) ** 10 *** 0 ARA ( M) ODYA ( M)

34 Genetic findings and functional studies of PGI2 synthase (CYP8A1) Wild-type Variant WT MT Overlap

35 Development of a multiplex and cost-effective genotype test toward more personalized medicine for the clopidogrel

36 Vast Amount of NGS Data Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing Exome sequencing

37 Global Efforts Human Genome Project ( ) 1000 Genomes Project ( ) Genomic Medicine RESEARCH CLINCAL CARE How to break the bottle neck? How to link the Science of Medicine to the Practice of Medicine? Is Personalized Medicine Dream or Reality?

38 Defining the meaning of DNA variation? (CSH meeting in New York with Dr. Watson, Nov. 2010) In a 1992 interview, James Watson stated that completing the human genome s DNA sequence would be a big job, requiring more than 10 years. But, understanding those instructions (the DNA sequence) may consume many hundreds of years

39 Intens. [a.u.] Prediction of Antiplatelet Drug Response Gene Expression Regulation Blood Physiology Contribution of Clinical Outcomes Genetic Polymorphism m/z ARA Metabolomics Drug DMPK ADR

40 Acknowledgements WooYoung Kim, MS (CYP2C19) IlSun Jung, MS (P2Y12) Yazun Zarrarr, PhD (20-HETE, Platelet) SunAh Cho, MS (PGI2, TXA2) Prof. HoSook Kim, Prof. Dong Hyun Kim, Prof. Jae-Gook Shin

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