Clinical Applications of PharmacogenomicsPharmacogenomics-- Based Medicine in Pediatrics Michael D. Reed, PharmDPharmD,, FCCP, FCP Director, Rebecca D. Considine Research Institute Division of Clinical Pharmacology & Toxicology Associate Chair, Department of Pediatrics Children’s Hospital Medical Center of Akron and Professor of Pediatrics Northeast Ohio Medical University

U.S. News and World Report, 14 January 2003

Interindividual Variability in Drug Response Disease Drug Class Rate of Poor Response Asthma Beta- Beta-agonistsagonists40- 40-7575%% Hypertension Various 30% Solid Cancers Various 7070%% Depression SSRIs, tricyclics20- 20-40%40% Diabetes SulfonylureasSulfonylureas,, others 5050%% Arthritis NSAIDs, COX-2 inhibitors 30-60% Schizophrenia Various 2525--7575%%

Factors Contributing to Interindividual Variability in Drug Disposition and Action

 Age  R/thiitRace/ethnicity  Weight  Gender PERSONALIZED MEDICINE  Concomitant Diseases  Concomitant Drugs  Social factors  GENETICS Slide 8 The Four DNA Bases

Adenosine Cytosine Thymine Guanine The Human Genome Every genome is different: ~ 3 billion basepp(airs (100% ) ~ 100 new variations per individual ~ 3 million genetic variations (0.1%)  Genetic variations can be used to explain interinter-- individual differences in drug response.  Age and disease can be used to explain intraindividual differences in drug response

The Genome and Drugs  Genes “encode” proteins or enzymes.  Differences in the sequence of a gene can cause differences in enzymes.  This is why enzymes appear in different forms in individuals.  This is also whyyppp different people process one and the same drug differently. The Foundation of Pharmacogenomics: Differences in the Genetic Code Between People

 Mutation: difference in the DNA code that occurs in less than 1% of population – Often associated with rare diseases » Cystic fibrosis, sickle cell anemia, Huntington’s disease  PolymorphismPolymorphism:: difference in the DNA code that occurs in more than 1% of the pppopulation – A single polymorphism is less liklikelelyy to be the main cause of a disease – Polymorphisms often have no visible clinical impact

Genetics vs. Genomics: ““TomAtoTomAtoor TomOto”

 Pharmacogenetics – Association between a single gene and drug response variability » Gene + Medication  Response  Pharmacogenomics – The contribution of multiple genes to drug resppyonse variability » Gene1+Gene2+Gene3+…GeneX + Medication Response Current Concept of Pharmacogenomics

Roden DM et al. Ann Intern Med 2006; 145:749-57

SNP Variability in The Human Genome

 2.85 billion base pairs  ~22,000 genes  1.7% of the genome codes for protein  3.3% of the genome is as conserved as the 1.7% that codes for protein  On average 1 SNP/1.2kb  10 – 15 million SNPS that occur at > 1% frequency  ~450,000 SNPS in Multippyly Conserved Re gions  Copy number variations exist in 55--7.5%7.5% of the germline genome  Most tumor DNA sequence is identical to that of the host  44--5%5% of the genome is in areas wwithith high copy number variation Clinical Relevance  Can we predict who will derive an optimal response?  Can we predict who will have a toxicity? ––HostHost (patient) genotype determines optimal drug therapy approach – Disease (pathogen) genotype determines optimal drug therapy approach

Pharmacogenomics

DRUG DRUG DRUG METABOLIZING TARGETS TRANSPORTERS ENZYMES

PHARMACODYNAMICS PHARMACOKINETICS

Variability in Efficacy/Toxicity

Johnson JA. Trends in Genetics 2003: 660-666 Fourteen Drugs and Their Available Pharmacogenetic Tests 2014

 Abacavir  HLA *B5701  Clopidogrel  CYP2C19  Tamoxifen  CYP2D6  metformin  OATP3  Imatinib  BCRBCR--ABLABL  55--FluorouracilFluorouracil  DPYDDPYD--TYMSTYMS  Clozapine  2 SNPs in HLA-HLA-DQB1DQB1  QTQT--prolongingprolonging Drugs  FamilionTM  Irinotecan  UGT1A1  Azathioprine and Mercaptopurine  TPMT  Warfarin  CYP2C9 and VKOR  Carbamazepine  HLAHLA--B*B* 1502  Interferon  IL 28B

Examples of Drug Metabolism Pharmacogenomics

New Engl J Med 2003; 348: 529-37 Examples of Drug Metabolism Pharmacogenomics

New Engl J Med 2003;348:529-37

Clinically Important DrugDrug--DrugDrug Interactions: The Importance of Hepatic Cytochrome 450 Isozyme Activity Cytochrome PolyPoly-- Substrate Inhibitors Inducers Isozyme morphism

1A2 Caffeine, clozapine, Amiodarone Charcoal grill cyclobenzaprine, estradiol, Cimetidine Cigarette smoke fluvoxamine, haloperidol, Fluvoxamine Cruciferous phenacetin, propranolol, R-R- Mexiletine vegetables warfarin, tacrine, TCA Quinolones Omeprazole demethylation (amitrip, clomip, Ticlopidine Rifampin imip), theopylline, zileuton 2C9 Yes Celecoxib, diclofenac, fluoxetine, Azoles Rifampin fluvastatin, irbesartan, ibuprofen, Chloramphenicol losartan, naproxen, phenytoin, INH piroxicam, SS--warfarin,warfarin, SSRI’s sulfamethexazole, tolbutamide, Statins tamoxifen Sulfaphenazole 2C19 Yes Citalopram, cyclophosphamide, Cimetidine Carbamazepine (?) 33--5%5% diazepam, imipramine, naproxen, Fluoxetine Prednisone Rifampin (?) Cauc PM propranolol, proton pump Is, Fluoxamine phenytoin, R-R-warfarin,warfarin, SS-- Ketoconazole 1515--20%20% mephenytoin, tolbutamide Paroxetine Asian PM PPI’s Ticlopidine Clinically Important DrugDrug--DrugDrug Interactions: The Importance of Hepatic Cytochrome 450 Isozyme Activity, Continued

Cytochrome PolyPoly-- Substrate Inhibitors Inducers Isozyme morphism

2D6 Yes Amphetaprine, beta blockers , Celecoxib Rifampin (?) 55--10%10% captopril, chlorpromazine, codeine, Cimetidine debrisoquine, d-methorphan, Fluoxetine/ Cauc PM ecanide, flecanide, fluoxetine, Norfluoxetine fluvoxamine, imipramine, Paroxetine metaprolol, mexiletine, Quinidine norfluoxetine, nortriptyline, Sertraline ondansetron, paroxetine, propranolol, respiridone, tamoxifen, TCA’s (part), thioridazine, timolol, tramadol, venlafaxine 2E1 No Acetaminophen, dapsone, Ethanol Rifampin theophylline (?) INH

Clinically Important DrugDrug--DrugDrug Interactions: The Importance of Hepatic Cytochrome 450 Isozyme Activity, Continued

Cytochrome PolyPoly-- Substrate Inhibitors Inducers Isozyme morphism

3A3/3A4 Possible Alfentanyl, alprazolam , Azoles Barbiturates amlodepine, astemizole, Ciprofloxacin Carbamazepine atorvastatin, buspirone, Fluoxetine Glucocorticoids chlorpheniramine, cisapride, Fluvoxamine Phenobarbital clarithromycin, cocaine, Macrolides Phenylbutazone cyclosporine, diazepam, diltiazem, Naringenin Phenytoin erythromycin, estradiol, fentanyl, Nefazadone Rifampin haloperidol (part), hydrocortisone, Protease itraconazole, lidocaine, loratadine, inhibitors lovastatin, midazolam, nifedipine, Sertraline omeprazole, ondansetron (part), TAO progesterone, quinidine, (R)(S) warfarin,simvastatin, statins (not pravastatin), tacrolimus, tamoxifen, taxol, TCA demethylation, terfenadine, trazodone, triazolam, verapamil CYP2D6 Polymorphisms  CYP2D6 is responsible for the metabolism of a number of different drugs – Antidepressants, antipsychotics, analgesics, cardiovascular drugs  Over 100 polymorphisms in CYP2D6 have been identified  Based on these polymorphisms, patients are phenotypically classified as: – Ultrarapid metabolizers (UMs) – Extensive metabolizers (EMs) – Poor metabolizers (PMs)

CYP2D6 Phenotypes

NEJM 2003; 348:529

Roden DM et al. Ann Intern Med 2006; 145:749-57 Incidence of CYP2D6 UM Genotype  American  4.3%  Turkish  8.7%  African American  4.9%  Ethiopian  29%  Saudi Arabian  21%  Columbian  1.7%

Bernard et al. Oncologist 2006;11:126-35

CYP2D6 Polymorphisms and Psychiatric Drug Response

 Increased rate of ad verse effects in poor metabolizers due to increased plasma concentrations of drug: – Fluoxetine (Prozac ) death in child attributed to CYP2D6 poor metabolizer genotype – Side effects of antipsychotic drugsdrugs occur more frequently in CYP2D6 poor metabolizers – CYP2D6 poor metabolizerswith severe mental illness had more adverse drug reactions, increased cost of care, and longer hospital stays Strattera® ((AtomoxetineAtomoxetine))  Treatment of attention deficit hyperactivity disorder – CYP2D6 poor metabolizers have 1010--foldfold higher plasma concentrations to a given dose of STRATTERA compared with extensive metabolizers ––AApproximately 7% of Caucasians are poor metabolizers – Higher blood levels in poor metabolizers may lead to a higher rate of some adverse effects of STRATTERA

Metabolic Disposition of Selected Opioid Analgesics Opioid Agent Metabolic Disposition Codeine CYP2D6, CYP3A4, UGT2B7 Dihydrocodeine CYP2D6, CYP3A4, UGT2B7 Fentanyl CYP3A4 Hydrocodone CYP2D6, 3A4,UGT, other CYPs Hydromorphone UGT1A3 & 2B7,DyHydroReduc Methadone CYP3A4, CYP2B6 Morphine UGT1A3, UGT2B7 Nalexone UGT2B7 and other UGTs Oxymorphone UGT2B7, UGT1A3 Tramadol CYP2D6, CYP2B6, CYP3A4 CYP2D6 and Codeine  Codeine requires activation by CYP2D6 in ordtder to exert titl its analgesi ifftc effect  Due to genetic polymorphisms, 22--10%10% of the population cannot metabolize codeine and are resistant to the analgesic effects  Interindividual variability exists in the adequacy of pain relief when uniform doses of codeine are given

Pharmacogenomics and the BreastBreast--FedFed Infant

 FullFull--termterm health male infant – Vaginal delivery – D7: intermittent periods of difficulty in BF and lethargy – D11: pediatric office visits; regained birth weight – D12: grey skin, milk intake fallen – D13: the baby was found dead – Unremarkable autopsy: infant morphine 70mg/ml (0(0--2.22.2 typical)  Mother – APAP 500 ml/codeine 30mg pp episiotomy pain – 2 tabs q 12h decreased 1 tab q12 day 2 for somnolence and conconsstipationtipation – Continued tablets for 2 weeks – D10: maternal milk (stored) morphine 87mg/ml (typical 1 .9 -20.5 mg/ml – Maternal CYP2D6*2x2 duplication: UM  Explanation – Mother ultra rapid metabolizerphenotype – Infant with physiologically impaired CYP capacity – Question of concurrent unrecognizedunrecognized infant pathology

Koren G, et al, Lancet 2006;368(#9536):704 Int J Clin Pharm 2011;33:33-43

Examples of Drug Metabolism Pharmacogenomics

New Engl J Med 2003;348:529-37 Warfarin and CYP2C9  Widely prescribed anticoagulant drug used to prevent blood clots  Narrow range between efficacy and toxicity  Large variability in the dose required to achieve therapeutic anticoagulation – Doses vary 1010--foldfold between people  CYP2C9 is the enzyme responsible for the metblitabolism of warfifarin  SNPs exist in CYP2C9 gene that decrease the activity of the CYP2C9 metabolizing enzyme

CYP2C9 Pharmacogenetics

Scordo et al., Clin Pharmacol Ther 72:702-10,2002 VKOR and Warfarin  Warfarin works by inhibiting Vitamin K Epoxide Reductase (VKOR)  VKOR helps recycle vitamin K which is important in proper functioning of clotting factors  By inhibiting VKOR, warfarin alters the vitamin K cycle and results in the production of inactive clotting factors  Polymorphisms exist in the gene for VKOR (VKORC1)

N Engl J Med 2011;264:1146-1153 Warfarin Pharmacogenomics

 CYP2C9 SNPs account for a small amount of variability in warfarin doses (~ 10%)  VKORC1 SNPs explain a larger portion of variability in warfarin doses (~20(~20--25%)25%)  Almost 50% of variability in warfarin doses can be explained by a combination of factors: – VKORC1 SNPs – CYP2C9 SNPs ––NonNon--geneticgenetic factors (age, weight, concomitant drugs, concomitant disease states)

Personalized Medicine: Major Obstacle #1 "In a setback for the fledgling field of personalized medicine, Medicare has decided not to ppyay for g enetic tests intended to help doctors determine the best dose of the blood thinner warfarin for a particular patient." The Centers for Medicare and Medicaid Services (CMS), "in a proposed decision posted on its website...said that there was not enough evidence that use of the tests improved patients' health." CMS, however, "said it would pay for the tests as part of clinical trials to gather such evidence." While some research has suggested "that using the genetic test might allow the proper dose to be achieved more quickl y," CMS " said th ere was littl e evid ence th at d oi ng so t ransl at ed into a lower risk of blood clots or hemorrhages." The Times adds that "the Food and Drug Administration recommends, but does not require, a genetic test for patients starting on warfarin."

May 5, 2009 The New York Times (B3, Pollack) CYP2C19 Pharmacogenetics  1984: Unusual sedation in a subject receiving anticonvulsant mephenytion

 Impaired 44--hydroxylationhydroxylation of S--mephenytoinmephenytoin

 Affects 22--5%5% of Caucasians; 2020--25%25% of Asians

 Affected drugs include omeprazole, lansoprazole, pantoprazole, diazepam

 Major clinical consequence at present related to omeprazole pharmacodynamics and efficacy

CYP2C19 Genotype Frequencies

Morrison A, Levy R. Pharmacogenomics 2004;5:673-689 Furuta T, et al: Drug Metab Pharmacokin 20:153,2005

Furuta T, et al: Clin Pharm Ther 65:552,1999 Clopidogril and CYP2C19

 Clopidogril for Cardiovascular Disease – Inhibits ADPADP--stimulatedstimulated platelet activation – Irreversibly binds to plateletplatelet--specificspecific ADP receptor P2Y12 – Prodrug: Prodrug: CYP2C19 to active thiol metabolite – March 2010: FDA “black box warning” – Soon after AHA & ACC issued joint endorsement of CYP2C19 genotyping – Current Standard: dual Rx clopidogril + ASA  Despite Pronouncements , Enthusiasm Muted – Two large randomized genotype studies = no benefit – Lancet 2010;376:1320 (n = 10,285) NEJM 2010;363:1704 (n = 5059) – No significant effect on cardiovascular eventsevents——ACSACS or AF

Associations Between Allelic Variants and HIV Treatment Response

Drug Phenotype Gene  Abacavir Hypersensitivity HLA-B*5701 Lancet 2002:359,727 & 1121; PNAS 2004:101,4180

 Indinavir Jaundice UGTUGT--1A11A1 Atazanavir PNAS 2001:98,12671; ICAAC 2002; JID 2005;192,1381

 NRTI Lipoatrophy TNFTNF--α promoter, HFE AIDS 2002:16,2013; AIDS 2003:17,121; JID 2008:197, 858

 Nevirapine Hypersensitivity HLAHLA--DRB1*0101,DRB1*0101, --Cw8,Cw8, -B3505-B3505 Hepatotoxicity ABCB1 Pharmacokinetics CYP2B6

AIDS 2005:19,97; Pharmacogenet Genom 2005;15,1; Clin Inf Dis 2006;43,779 & 783; AIDS 2006:20,1621; AIDS 2007;21,264; Pharmacogenet Genom 2009;19,139 Thiopurine metabolism. Oral AZA is rapidly converted to 6-MP by a nonenzymatic process. Intiial 6-MP transformations occur along competing catabolic (XO, xanthine oxidase; TPMT) and anabolic (HPRT, hypoxanthine phosphoribosyltransferase) enzymatic pathways. Once formed, 6-thiosine 5’-monophosphate (6- TImP) is further catalyzed by inosine monophosphate dehydrogenase (IMPDH), and guanosine monophosphate synthetase (GMPS) and the di- and tri-derivatives of 6-thioguanosine 5’monophosphate (6- TGMP) formed by their respective kinases. T-TU, 6-thiouric acid.

Dubinsky MC. Clin Gastro Hepatol 2:731, 2004.

Suggested Azathioprine Treatment Doses Based Upon Erythrocyte TPMT Activity TMT Activity ~ % of P opul ati on Azathi oprin e Dose (mg) Deficient (very low / 0.3 No Rx or by 90% absent) Intermediate 11 Dose by 15-50%

Normal or “High” 89 Standard Rx

Konstantopoulou M, et al; BMJ 330:350, 2005 Hematologic Parameters in Adults with Crohn’s Disease on Azathioprine REMISSION Parameter Yes (n=14) No (n=31) p Value WBC 5,350 (2,106) 8,918 (3,858) 0.004 Lymphocytes (%) 16. 6 (6. 8) 96(58)9.6 (5.8) 0. 005 Granulocytes (%) 73 (8.7) 81 (6.9) 0.007 Data presented as mean (+ SD) Hematologic Parameters in Adults with Ulcerative Colitis on Azathioprine REMISSION Parameter Yes (n=13) No (n=27) p Value WBC 6,045 (1,165) 8,767 (2,356) 0.003 Lymphocytes (%) 20 (5.3) 12 (5.6) 0.001 Granulocytes (%) 69 (6.1) 79 (8.9) 0.004

Data presented as mean (+ SD) Glass J, et al. Eur J Med Res 10:535, 2006.

Examples of Human Receptors Shown to be Genetically Polymorphic with Possible Alterations in Clinical Phenotype  G--proprotiteins  Angiotensin II receptor and angiotensinogen  Angiotensin converting enzyme  ΒΒ--receptorreceptor

 DiDDopamine D4 receptor  Endothelial NO synthase

 5HT4 receptor BetaBeta--blockersblockers and Hypertension (HTN)

 HTN is the most prevalent chronic disease in the US and a contributor to morbidityyy and mortality  BetaBeta--blockersblockers are first-first-lineline agent in the treatment of HTN  Marked variability in response to beta-beta-blockersblockers ––3030--60%60% of patients fail to achieve adequate blood pressure lowering with betabeta--blockersblockers  CbtCommon betaa--bloc kers use d in HTN: – Metoprolol – Atenolol

BetaBeta--11 Receptor Polymorphisms and Response to Metoprolol

Johnson JA et al. Clin Pharmacol Ther 2003; 74:44-52 BetaBeta--22 Polymorphisms and Response to Albuterol •Single 8 mg albuterol dose

•Albut erol -evokdiked increases i n

FEV1 were higher and more rapid in Arg16 homozyotes compared with Gly carriers

• Codon 16 polymorphism is a determinant of bronchodilator response to albuterol

Lima JJ et al. Clin Pharmacol Ther 1999; 65: 519-25

Lima JJ. Clin Pharmacol Ther 1999; 65:519-25

CarbamazepineCarbamazepine--InducedInduced Toxic Effects  HLAHLA--AA 3101 Genotype and Hypersensitivity Reactions – CBZCBZ--inducedinduced minor macpap exanthema in 55--10%10% Euro pts. – MP rash: minor form resolves spontaneously after QC – SJS & TEN most severe: overall 10% mora lity (TEN -30%) – FDA estimates 11--6/10,0006/10,000 Euro pts SJS risk ––HLAHLA--AA 3101 prevalence 2-2-5%5% Euro descent – Allele presence defined increased risk from 55--26%26% vs absence reduced risk from 55--3.8%3.8%  HLAHLA--BB 1502 Genotype & HAN Chinese – SJS & TEN “never” occurs in nonnon--carrierscarriers – Associat confirmed in Hong Kong, Malaysia, Thailand, India

– Recent study confirmed in Taiwanese (N Engl J Med 2011;364:126) – Overall: 5050--100100 screens for 1 identified patient

N Engl J Med 2011;364:1134 Distribution of the HLA-A*3101 Allele across the Spectrum of Clinical Phenotypes of Case Subjects wiwithth Carbamazepine- Induced Hypersensitivity and Control Subjects without Adverse Reactions to Carbamazepine. Subjects were recruited at centers collaborating with the University of Liverpool and Walton Centre for Neurology (UK) or centers affiliated with the EPIGEN consortium. The sample obtained from one patient with acute generalized exanthematous pustulosis (AGEP) was analyzed with the samples for the group of case subjects with the hypersensitivity syndrome. Since there were no observations of the HLA-A*3101 allele in the three case subjects with the hypersensitivity syndrome in the EPIGEN cohort, 0.5 was added to each value in a two-by-two contingency table to estimate an odds ratio. One patient with the Stevens–Johnson syndrome and toxic epidermal necrolysis (SJS–TEN) was of mixed European and Thai ancestry. Study weighting (indicated by different sizes of squares) refers to the proportion of subjects who were recruited from each study cohort. Diamonds indicate pooled odds ratios. The horizontal lines indicate 95% confidence intervals. The abbreviation df denotes degrees of freedom, and I2 is the percentage of total variation that is due to heterogeneity rather than chance.

N Engl J Med 2011;364:12 Lessons from CYP Pharmacogenetics

 Multippgle genetic tests of one g ene may be needed to accurately predict phenotype  Gene duplication in the germline exists  The environment in the form of Drug Interactions Can mimic a genetic change Several Potential Goals for Clinical Pharmacogenetic Testing

 The subsub--divisiondivision of common diseases into different molecular sub -types which may be more or less susceptible to specific treatments.  To evolve more logical approaches to dosage, efficacy and the prevention of adverse reactions by analyzing the genetic basis for differences in the pharmacokinetic or pharmacodynamic properties of drugs.  To identify genetic susceptibility to various common diseases that offer targets for pharmacological intervention.

Summary

 Pharmacogenomic testing is now being widely applied to some of the most widely prescribed drugs  Pharmacogenomic biomarkers require demonstration of clinical utility before widespread implementation – This has happened in very few cases to date  Clinical pharmacogenomic predictive tests must ppgprovide real value over existing predictors  Economic utility is often as important as clinical utility Pharmacogenetics Websites

 www.pharmgkb.org  The SNP consortium: http://brie2.cshl.org  The Human Genome: www.ncbi.nlm.nih.gov/genome/guide/H_sapiens.html  CYP alleles: www.imm.ki.se/CYPalleles/  Drug Interactions: www.drugwww.drug--interactions.cominteractions.com

REFERENCES 1. Rassekh SR, Ross CJ, Carleton BC, Hayden MR. Cancer pharmacogenomics in children: research initiatives and progress to date. Paediatr Drugs. 2013 Apr;15(2):71‐81. doi: 10.1007/s40272‐013‐0021‐9. Review. PubMed PMID: 23529868. 2. Hawcutt DB, Thompson B, Smyth RL, Pirmohamed M. Paediatric pharmacogenomics: an overview. Arch Dis Child. 2013 Mar;98(3):232‐7. doi: 10.1136/archdischild‐2012‐ 302852. EbEpub 2012 Nov 29. RiReview. PbMdPubMed PMID: 23196740. 3. Wall CA, Croarkin PE, Swintak C, Koplin BA. Psychiatric pharmacogenomics in pediatric psychopharmacology. Child Adolesc Psychiatr Clin N Am. 2012 Oct;21(4):773‐88. doi: 10.1016/j.chc.2012.07.001. Review. PubMed PMID: 23040901. 4. Wagner J, Leeder JS. Pediatric pharmacogenomics: a systematic assessment of ontogeny and genetic variation to guide the design of statin studies in children. Pediatr Clin North Am. 2012 Oct;59(5):1017‐37. doi: 10.1016/j.pcl.2012.07.008. Epub 2012 Aug 22. Review. PubMed PMID: 23036242. 5. Leeder JS, Kearns GL. Interpreting pharmacogenetic data in the developing neonate: the challenge of hitting a moving target. Clin Pharmacol Ther. 2012 Oct;;(92(4):434‐6. doi: 10.1038/clpt.2012.130. Epub 2012 Sep 5. Review. PubMed PMID: 22948896. 6. Becker ML. Pharmacogenomics in pediatric rheumatology. Curr Opin Rheumatol. 2012 Sep;24(5):541‐7. doi: 10.1097/BOR.0b013e3283556d13. Review. PubMed PMID: 22732686.