Dúvidas denucci@gdenucci.com Arquivo Farmacogenômica Site www.gdenucci.com

Pacientes com o mesmo diagnóstico Risco de toxicidade alto Diminuir a dose ou usar outro medicamento Resposta preditiva boa para o medicamento Resposta preditiva ruim ou ausente Use outro medicamento

Ausência de Benefício/100 Tratamentos estabelecidos com alta eficácia e % dos pacientes beneficiados e não beneficiados com o tratamento Eventos (%) Ausência de Benefício/100 Ensaio Droga Benefício/100 Placebo Tratad Hope APTC FTT 4S EPIC CURE Ramipril Aspirina Trombolíticos Simvastatina Abciximab Clopidogrel 17.8 14 11.5 28 12.8 14 10 9.6 19 8.3 9.3 3.8 4 1.9 9 4.5 2.2 96.2 96 98.1 91 95.5 97.8

Enzimas metabolisadoras Variabilidade na eficácia ou toxicidade Farmacogenética Alvos do medicamento Transporta-dores Enzimas metabolisadoras Farmacodinâmica Farmacocinética Variabilidade na eficácia ou toxicidade

Farmagenômica Farmacogenômica Distribuição Absorção Excreção Afinidade do receptor pela droga Droga atuando em produtos gênicos Curr Probl Cardiol, May 2003

The concept of pharmacogenetics. Pharmacogenomics: Challenges and Opportunities - © 2006 American College of Physicians - Ann Intern Med. 2006;145:749-757.

Two types of variability in drug action. Pharmacogenomics: Challenges and Opportunities - © 2006 American College of Physicians - Ann Intern Med. 2006;145:749-757.

PD and PK pathways of HMGCo A reductase inhibitors (Statins). Cholesterol and lipoprotein transport: genes involved in mediating statin effects on hepatic cholesterol metabolism and consequent effects on plasma lipoprotein transport. Statins inhibit endogenous cholesterol production by competitive inhibition of HMG-CoA reductase (HMGCR), the enzyme that catalyzes conversion of HMG-CoA to mevalonate, an early rate-limiting step in cholesterol synthesis. This pathway delineates genes involved in statin pharmacogenomics, including genes involved in mediating the PD effects of statins on plasma lipoprotein metabolism and those involved in the PKs effects of the drug transport and metabolism. Note the effects of inhibition of HMG-CoA reductase on major aspects of hepatic cholesterol metabolism and selected gene products that can modulate the effects of statins on metabolism and transport of plasma lipoproteins. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

PD and PK pathways of HMGCo A reductase inhibitors (Statins). PKs of Statins: representation of the superset of all genes involved in the transport, metabolism, and clearance of statin class drugs. This figure depicts a generalized view of the PKs of statins, representing the superset of all genes with a reported influence on statin transport and metabolism. Statins are dosed orally and enter the systemic circulation through intestinal cells both passively and by active transport via the ABC and SLC gene family transporters. The major organs of metabolism and elimination include the liver and, to a lesser extent, the kidney. Metabolism is catalyzed by enzymes of the CYP and UGT gene family. The main pathway of elimination is ABC-transporter-mediated biliary excretion. The more hydrophilic compounds (e.g., pravastatin) require active transport into the liver, are less metabolized by the CYP family, and exhibit more pronounced active renal excretion, whereas the less hydrophilic compounds are transported by passive diffusion and are better substrates for both CYP enzymes and transporters involved in biliary excretion. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

The home page of PharmGKB provides a straightforward schema for understanding pharmacogenomics. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Diastolic blood pressure response to metoprolol in hypertensive patients is predicted by ADRB1 diplotype. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Flow chart for the functional evaluation of genes with replicated associations. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Whole-genome approach to identify genes that predict survival. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Impact of germline TPMT genotype on incidence of toxicity (upper). The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

The path of phenytoin and imidapril after ingestion. This figure shows the paths that are taken by the anti-epileptic drug phenytoin and the angiotensin-converting enzyme (ACE) inhibitor imidapril in the human body. Phenytoin is absorbed into the bloodstream at the gut and circulated through the liver to the brain. It crosses the blood–brain barrier where it binds and inhibits its target, neuronal sodium channels. It is pumped back out across the blood–brain barrier into the bloodstream by multidrug resistance protein 1 (MDR1, also known as ABCB1) efflux pumps. Note that MDR1 efflux pumps are also active in the gut, where they promote drug excretion (not shown). At the liver, phenytoin is metabolized by the cytochrome P450 enzymes CYP2C9 and CYP2C19, and it is eliminated through the kidneys. Imidapril is a PRO-DRUG. After its absorption from the gut into the bloodstream it is hydroxylated in the liver to the active metabolite imidaprilat. Imidaprilat binds and inhibits ACE in the plasma. Imidaprilat is also eliminated through the kidneys. PHARMACOGENETICS GOES GENOMIC - NATURE REVIEWS -GENETICS VOLUME 4 - DECEMBER 2003

Alelos da apo e apo e2 apo e3 apo e4

Kaplan-Meier em pctes com e sem o alelo apoe4 1.00 0.95 0.90 0.85 0.80 Sem e4 N=312 ~Proporção Vivo e4-Portadores N=166 0 500 1000 1500 2000 2500 Dias após Randomização Tratamento com Placebo Curr Probl Cardiol, May 2003

Tratamento com simvastatina reduz mortalidade em 13% em pacientes não apo e4 50% em pacientes apo e4

Kaplan-Meier em pctes com e sem o alelo apoe4 1.00 0.95 0.90 0.85 0.80 Sem e4 N=301 e4-Portadores N=187 Proporção Vivo 0 500 1000 1500 2000 2500 Dias após randomização Tratamento com Simvastatina Curr Probl Cardiol, May 2003

Alelos da ACE (modulam níveis da ACE em plasma) Alelo DD – níveis 2x Alelo II – níveis x Alelo ID – níveis >x e <2x

Kaplan-Meier e genótipo da ACE 1.00 0.80 0.60 0.40 0.20 0.00 p = 0.04 Sobrevivência de transplantados ACE II (N=69) ACE ID (N=154) ACE DD (N=105) 0 6 12 18 24 30 Seguimento em Meses Curr Probl Cardiol, May 2003

Kaplan-Maier em ACE DD e uso de betabloqueadores 1.00 0.80 0.60 0.40 0.20 0.00 p = 0.007 Sobrevivência de Transplantados Beta Bloqueador (N=154) Sem Beta Bloqueador (N=105) 0 6 12 18 24 30 Seguimento em Meses Curr Probl Cardiol, May 2003

Receptor adrenérgico bAR - pacientes com ICC 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Tre-164 Proporção de sobreviventes Ile-164 Ajustado RR = 4.81, p < 0.001 0 100 200 300 400 500 600 700 800 900 1000 Dias após entrada no estudo

Sintase de óxido nítrico endotelial - ICC 1.00 0.80 0.60 0.40 0.20 0.00 Glu-Glu Asp=298 % sem eventos 0 6 12 18 24 30 36 42 48 54 Seguimento em meses

Adenosina monofosfato deaminase ATP ADP IMP AMP Adenosina AMP deaminase

Adenosina monofosfato deaminase - ICC 1.0 0.8 0.6 0.4 0.2 AMPD1 (+/-)/(-/-) Probabilidade de sobrevivência sem transplante AMPD1 (+/+) 0 5 10 15 Tempo (anos)

Tiopurina Metiltransferase Polimorfismo TPMT Metila mercaptopurina – reduz F Leucemia linfocítica aguda 10% intermediária – maior toxicidade 0.3% não tem TMPT – fatal Genotipagem essencial

UDP-glucoronosiltransferase 1A1 Irinotecan – câncer de cólon, pulmão Forma ativa inativada por glucoronidação Aumento de 4x a toxicidade Genotipagem alelo UGT1a1*28

Gene da colinesterase plasmática Apnéia prolongada após succinilcolina 1 em 187 em Valencia 1 em 3460 europeus 1 em 25 x 106 asiáticos Complicação facilmente tratada, não há necessidade de genotipagem

Leucemia mielóide crônica Translocação no cromossoma Filadelfia Alterou localização dos genes bcr e abl bcr-abl tirosina quinase fica ativa Imatinib bloqueia especificamente Imatinib 88% resposta positiva em pctes

Câncer de mama Herceptin – ab citotóxico contra Her-2/neu. Her-2/neu aumentada em 25% pctes Herceptina funciona somente nestes pacientes

Substratos para citocromo P4502D6 b-bloqueadores – alprenolol, carvedilol, propranolol Anti-arrítmicos – flecainida, mexiletina, propafenona Neurolépticos – haloperidol, clozapina, olanzapina, risperidona Antidepresssivos – amitriptilina, clomipramina, paroxetina Antieméticos – ondansetrona, tropisetrona Outros – anfetamina, codeína, debrisoquina, dextrometorfano

Inibidores do citocromo P4502D6 Neurolépticos – clomipramina, levopromazina, haloperidol Antidepressivos – fluoxetina, paroxetina, sertralina Antieméticos - metoclopramida Anti-histamínicos – clorfeniramina, cimetidina, clemastina, difenilhidramina Outros – ritonavir, quinidina, amiodarona

Genótipo do citocromo P4502D6 Metabolisadores ultrarápidos Metabolisadores rápidos Metabolisadores lentos

Genótipo do citocromo P4502D6 Metabolisadores ultrarápidos – muito baixa em orientais (<<1%), baixa em europeus do norte (<1%), 7% em espanhóis, 29% em etíopes Metabolisadores rápidos Metabolisadores lentos – muito baixa em orientais

Tratamento de náusea e vômito in quimioterapia - Tropisetron 42 pacientes, 30% tiveram náusea e vômito CitP4502D6 – maior frequência dos demais pacientes Genotipagem recomendável – evitaria emese severe em 1/50 pacientes.

Polimorfismo genético (acetiladores rápidos e lentos) 25 20 15 Frequeência (no de indivíduos) 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 Concentração isoniazida (mg/mL)

A superfamília do citocromo P450

Diferenças Farmacogenéticas das Enzimas que metabolizam medicamentos Incidência de deficiência ou metabolizadores lentosa Enzima Substratos típicos Pseudocolinesterase plasmática Desidrogenase alcoólica CYP2C19 CYP2D6 CYP2C? Acetil-N-transferase Metiltransferase 1 in 3000 5-10% (approx. 90% em Asiáticos) 5% (approx. 20% em Asiáticos) 5 - 10% Muito raro Approx. 60% (approx. 5% em japoneses) 0.5 % Suxametônio (succinilcolina) Etanol S-Mefenitoína, omeprazole Debrisoquina, espartina, metoprolol, dextrometorfan Fenitoína Isoniazida, hidralazina, procainamida 6-Mercaptopurina a Para caucasianos

Metabolism (Biotransformation) of Drugs Conjugation Reaction Endogenous Conjugant Intracellular Sites Common Substrates Drug Examples Acetylation Acetyl-CoA Cytosol -OH, -COOH, -NH2, -NR2, -SH Clonazepam, dapsone, isoniazid, sulfonamides, valproate Glutathione conjugation Reduced form of γ-Glu-Cys-Gly (the most common intracellular thiol) Cytosol and microsomes Electrophilic benzyl halides, aliphatic nitrate esters, epoxides, and quinines Acetaminophen, ethacrynic acid Gly (amino acid) conjugation Gly, Glu, others Mitochondria -COOH Benzoic and salicylic acid Glucoronidation UDPGA (uridine-5’- diphospho-α-D-glucuronic acid Microsomes Hydroxyl, amino, or sulfhydryl groups Acetaminophen, codeine, diazepam, disulfiram, ethinyl estradiol, fentanyl galantamine, lorazepam, modafinil, morphine, propanolol, paroxetine, sulfonamides Methyllation (N-, O-, and S-) CH3 from S-adeno sylmethionine (SAM) Cytosol (eg, COMT) -OH, -NH2, -SH Oxprenolol (N-), clomethiazole and isoproterenol (O-), captopril (S-) Sulfate conjugation 3’-Phosphoadenosine 5’-phosphosulfate (PAPS) Cytosol -OH, -NH2, Acetaminophen, ethinyl estradiol, methyldopa, paoxetine, steroids, triamterene Netter’s Iluustrated Pharmacology – Chapter 1 – Fig. 1.29

Cytochrome P-450 Ribbon model of CYP2C9 isozyme CYP3A 50% CYPD6 25% 5% 15% 5% Other CYP1A2 Netter’s Iluustrated Pharmacology – Chapter 1 – Fig. 1.30

Cytochrome P-450 CYP Substrate 1A2 Acetaminophen, antipyrine, caffeine, clomipramine, olanzapine, ondansetron, phenacetin, rilozole, ropinirole, tamoxifen, theophylline, warfarin 2A6 Coumarin 2B6 Artemisinin, buproprion, cyclophosphamide, S-mephobarbital, S-mephenytoin, (N-demethylation to nirvanol), propofol, selegiline, sertraline 2C8 Pioglitazone 2C9 Carvedilol, celecoxib, fluvastatin, glimepiride, hexobarbital, ibuprofen, losartan, mefenamic, meloxicam, montelukast, nateglinide, phenytoin, tolbutamide, trimethadone, sulfaphenazole, warfarin, ticrynafen, zafirlukast 2C19 Citalopram, diazepan, escitalopram, esomeprazole (S9 isomer of omeprazole), irbesartan, S-mephenytoin, naproxen, nirvanol, omeprazole, pantoprazole, proguanil, propranolol 2D6 Almotriptan, bufuralol, bupranolol, carvedilol, clomipramine, clozapine, codeine, debrisoquin, dextromethorphan, dolasetron, fluxetine (S-norfluoxetine), formoterol, galantamine, guanoxan, haloperidol, hydrocodone, 4-methoxy-amphetamine, metoprolol, mexiletine, olanzapine, oxycodone, paroxetine, phenformin, phenothiazine, propoxyphene, selegiline, (deprenyl), sparteine, thioridazine, timolol, tolterodine, tramadol, tricyclic antidepressants, type 1C antiarrhythmics (eg. encainide, flecainide, propafenone), venlafaxina Netter’s Iluustrated Pharmacology – Chapter 1 – Fig. 1.30

Cytochrome P-450 (cont.) CYP Substrate 2E1 Acetaminophen, Chlorzoxazone, enflurane, halothane, ethanol (minor pathway) 3A4 Acetaminophen, alfentanil, almotriptan, amiodarone, astemizole, beclomethasone, bexarotene, budesonide, S-bupivacaine, carbamazepine, citalopram, cocaine, cortisol, cyclosporine, dapsone, delavirdine, diazepam, dihydroergotamine, dihydropyridines, dihydropyridines, diltiazem, escitalopram, ethinyl estradiol, fentanyl, finasteride, fluticasone, galantamine, gestodone, imatinab, indinavir, itraconazole, letrozole, lidocaine, loratadine, losartan, lovastatin, macrolides, methadone, miconazole, midazolam, mifepristone (RU-486), montelukast, oxybutynin, paclitaxel, pimecrolimus, pimozide, pioglitazone, progesterone, quinidine, rabeprazole, rapamycin, repamycin, repaglinide, ritonavir, saquinavir, spironolactone, sulfamethoxazole, sufentanil, tacrolimus, tamoxifen, terfenadine, testosterone, tetrahydrocannabinol, tiagabine, triazolam, troleandomycin, verapamil, vinca alkaloids, ziprasidone, zonisamide 27 Doxercalciferol (activated) No / minimal involvement Abacavir, acyclovir, alendronate, amiloride, benazepril, cabergoline, digoxin, disoproxil, hydrochlorothiazide, linezolid, lisinopril, olmesartan, oxaliplatin, metformim, moxifloxacin, raloxifene, ribavirin, risedronate, telmisartan, tenofovir, tiludronic acid, valacyclovir, valsartan, zoledronic acid Netter’s Iluustrated Pharmacology – Chapter 1 – Fig. 1.30

Challenges in Pharmacogenomics Pharmacogenomics: Challenges and Opportunities - © 2006 American College of Physicians - Ann Intern Med. 2006;145:749-757.

Examples of Associations between Drug Response and Genetic Variants* Pharmacogenomics: Challenges and Opportunities - © 2006 American College of Physicians - Ann Intern Med. 2006;145:749-757.

Browsing function in PharmGKB Browsing function in PharmGKB. PharmGKB allows users to browse the major classes of data (genetic variation in pharmacogenes, curated literature, drugs associated with genotype, phenotype, pathway or other information, pathways, diseases, and phenotype data files). The number of data objects in each category is displayed, and there is a full-text search capability to allow more focused searching. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Example of the PharmGKB gene variant browser: nitric oxide synthetase 3 (NOS3). is involved in the angiotensin and vascular endothelial growth factor (agents inhibiting the vascular endothelial growth factor signaling pathway have been developed as a new class of anticancer agents) pathways and the response to a number of drugs. The genomic DNA is the thick bar, with exons marked in brown. SNPs in PharmGKB are shown above the genomic DNA with a graphical indication of minor allele frequency. The location of SNPs in dbSNP and jSNP are shown below the genomic DNA. The table shows the chromosomal position, with links to the Golden Path genome browser, dbSNP, and with links to detailed information about the alleles, assays, frequencies, and individual-level data. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response - VOLUME 81 NUMBER 3 - MARCH 2007

Illustration of tagging SNPs. PHARMACOGENETICS GOES GENOMIC - NATURE REVIEWS -GENETICS VOLUME 4 - DECEMBER 2003 The diagram shows five haplotypes. Twelve single nucleotide polymorphisms (SNPs) are localized in order along the chromosome. The letters on the top indicate groups of SNPs that have perfect pairwise linkage disequilibrium (LD) with one another, and the numbers on the bottom indicate each of the 12 SNPs. SNP 9 is the causal variant, which in this simple example determines drug response: allele C results in a therapeutic response, whereas allele G results in an adverse reaction. In this example, the selection of just one SNP from each of the groups A–E would be sufficient to fully represent all of the haplotype diversity. Each haplotype can be identified by just five tagging SNPs (tSNPs), and the causal variant would be tagged even if it were not itself typed (in fact, multi-marker approaches to tSNP selection would reduce the set of tags to fewer than five, but this is ignored for simplicity). So, tSNP profiles that are highlighted predict an adverse reaction to the medicine. Normally, LD patterns are not so clear-cut and statistical methods are required to select appropriate sets of tSNPs.

Illustration of tagging SNPs. PHARMACOGENETICS GOES GENOMIC - NATURE REVIEWS -GENETICS VOLUME 4 - DECEMBER 2003 The diagram depicts the same 12 SNPs, but with different associations among them, as might happen in a different population group. Because patterns of LD are different, some patients would be misclassified if the same five tSNPs were used and interpreted in the same way; that is, using the same SNP profiles as defined in population A, haplotype profiles 1, 2 and 3 are predicted to have allele C at the causal SNP 9 (a therapeutic response), whereas haplotype profiles 4 and 5 are predicted to have na adverse response. However, because the pattern of association has changed, the new haplotypes 6 and 7 are misclassified as haplotype patterns 6 and 7 in population B.