Pain Medication DNA Insight TM

Pain Medication DNA Insight TM TESTING SUMMARY JULY 2014 AUTHORS Alok Tomar, PhD Adrian Vilalta, PhD

Pain Medication DNA Insight TM Precision Medicine and Pharmacogenetics of Pain Medications 1. Introduction The sometimes dramatic difference in an individual s response to medications has long been recognized by physicians. This extraordinary variation in patients reaction to medications has been attributed to genetic as well as non-genetic factors. However, genetic variations are generally believed to account for up to 95 percent of the observed variability in drug disposition and effects 1. Numerous clinical studies have demonstrated that the genetic variations in genes encoding drug-metabolizing enzymes, significantly contributes to inter-individual variability in drug disposition and effects. The growing understanding of genetics of drug disposition and effects has given rise to the field of pharmacogenetics also referred to as pharmacogenomics. The use of modern pharmacogenomics provides an attractive opportunity to help identify the medications and doses that are best suited for an individual patient, thus allowing for personalized or precision treatment. Drug metabolism within individuals depends on numerous factors, including environment, age, gender, nutrition, and genetics. However, the overall contribution of genetics in drug metabolism is considered relevant to the improvement of clinical outcomes and the development of pharmacogenetics as a predictive tool for drug discovery and patient care 2. Analgesics have commonly narrow therapeutic windows in which the drug provides optimum pain relief while reducing the risk of potentially severe adverse events. In addition, the basis of drug on drug interactions is often poorly understood. Therefore, treatment strategies based on the one-size-fits-all model have proven to be inadequate and potentially dangerous for patients. The potential of pharmacogenetics to help identify potential responders from non-responders to specific medications, avoid adverse events and optimizing drug dose has been recognized by the US FDA. Currently the FDA has approved pharmacogenetic information for over 100 medications including popular analgesics such as tramadol, codeine and carisoprodol 3. 2. Pain Medication Metabolism and Pharmacogenomics The proteins that metabolize or are receptors for pain medications regulate drug disposition and effects. Genetic variations in genes that encode drug metabolizing enzymes or drug receptors can result in inter-individual variations to drug response, i.e., the degree to which an individual metabolizes a drug or triggers a biological response can have a large effect in the success of the drug therapy. Therefore, genetic tests that determine gene variations can be useful in adjusting drug dosage in affected individuals and to maximize drug efficacy while minimizing drug associated adverse effects. Potential drug on drug interactions as well as effects from herbs or foods can be better understood based on the genetics of the patient. Table 1, Table 2 and Table 3 provide a partial list of substrates, inhibitors and inducers of some key enzymes involved in pain medication metabolism 4. Some important classes of analgesics and the key genes involved in the drug response are discussed below. JULY 2014 / PAGE 2 OF 15

2.1. Opioids Opioids have been used in medicine for centuries; the use of poppy opium predates recorded history. This class of compounds has been extensively used to treat acute pain as well as to alleviate severe chronic pain associated with certain terminal conditions. However, the use of opioids needs to be carefully balanced against their side effects including sedation, respiratory depression, cardiac rhythm modification and risk of addiction. Codeine Codeine is an opioid analgesic used to relieve mild to moderately severe pain 5. The hepatic CYP2D6 enzyme metabolizes inactive codeine to active morphine, which binds the mu-opioid receptor with an affinity 200-fold greater than codeine 6. The exact mechanism of codeine s analgesic effect is unknown [5]. Variants of the CYP2D6 gene that affect enzyme function have been shown to be associated with codeine metabolism and analgesic effects 7-10. Hydrocodone Hydrocodone is an opioid analgesic used to relieve moderate to moderately severe pain. The CYP2D6 enzyme is responsible for 95% of the conversion of hydrocodone to hydromorphone in liver microsomes 11. Hydromorphone has greater affinity to mu-opioid receptors than hydrocodone 6, 12, 13. Variants of the CYP2D6 gene that affect enzyme function have been shown to be associated with hydrocodone metabolism 14. JULY 2014 / PAGE 3 OF 15

Table 1: Partial list of drugs metabolized by CYP enzymes 4. CYP2B6 CYP2C9 CYP2C19 CYP2D6 artemisinin NSAIDs: PPIs: Beta Blockers: bupropion diclofenac esomeprazole carvedilol cyclophosphamide ibuprofen lansoprazole S-metoprolol efavirenz naproxen omeprazole propafenone ifosfamide piroxicam pantoprazole timolol ketamine Oral Hypoglycemics: Anti-epileptics: Antidepressants: meperidine tolbutamide diazepam amitriptyline methadone glipizide phenytoin clomipramine nevirapine glyburide phenobarbitone desipramine propofol Angiotensin II Blockers: Others: duloxetine selegiline losartan amitriptyline fluoxetine irbesartan carisoprodol imipramine Others: citalopram paroxetine celecoxib clomipramine Antipsychotics: fluvastatin clopidogrel haloperidol phenytoin cyclophosphamide risperidone rosiglitazone imipramine thioridazine torsemide labetalol Others: valproic acid proguanil aripiprazole warfarin voriconazole atomoxetine zafirlukast codeine dextromethorphan doxepine flecainide mexiletine ondansetron oxycodone risperidone tamoxifen Tamoxifen tramadol venlafaxine JULY 2014 / PAGE 4 OF 15

Table 2: Partial list of CYP enzymes inhibitors 4. CYP2B6 CYP2C9 CYP2C19 CYP2D6 clopidogrel amiodarone cimetidine bupropion thiotepa efavirenz esomeprazole fluoxetine ticlopidine2 fluconazole2 felbamate paroxetine voriconazole isoniazid fluoxetine quinidine1 metronidazole fluvoxamine duloxetine paroxetine isoniazid amiodarone sulfamethoxazole ketoconazole cimetidine voriconazole lansoprazole aripiprazole omeprazole diphenhydramine oral contraceptives chlorpheniramine pantoprazole clomipramine ticlopidine2 doxepin voriconazole haloperidol methadone ritonavir terbinafine Table 3: Partial list of CYP enzymes inducers 4. CYP2B6 CYP2C9 CYP2C19 CYP2D6 artemisinin carbamazepine efavirenz carbamazepine nevirapine rifampin efavirenz phenobarbital ritonavir nevirapine rifampin St. John s Wort phenobarbital St. John s Wort phenytoin rifampin JULY 2014 / PAGE 5 OF 15

Oxycodone Oxycodone is an opioid analgesic used to relieve moderate to moderately severe pain 15. In one of oxycodone s two major metabolic pathways, the hepatic CYP2D6 enzyme metabolizes it to oxymorphone, which binds the mu-opioid receptors with a 40-fold greater affinity than oxycodone 16. The CYP3A enzymes mediate the other major metabolic pathway that converts oxycodone to noroxycodone, which has a weaker affinity for mu-opioid receptors than either oxycodone or oxymorphone 16. Variants of the CYP2D6 gene that affect enzyme function have been shown to be associated with oxycodone metabolism 16-19. Tramadol Tramadol is an opioid analgesic used to relieve moderate to severe pain 20. It is administered as a racemic mixture of two enantiomers, (+)-tramadol and (-)-tramadol. The hepatic CYP2D6 enzyme metabolizes (+)-tramadol to (+)-O-desmethyltramadol, which binds the mu-opioid receptor with an affinity 700-fold greater than the parent drug. However, tramadol also contributes an analgesic effect, although through different monoaminergic pathways 21. Variants of the CYP2D6 gene that affect enzyme function have been shown to be associated with tramadol metabolism 22-24 and analgesic effects 23, 25-27. Fentanyl Fentanyl (Sublimaze, Actiq, Durogesic and others) is a synthetic opioid analgesic used to relieve pain in cancer patients and as a surgical anesthetic. This drug acts primarily as a mu-opioid receptor agonist and is metabolized to an inactive metabolite by the CYP3A4 enzyme. The exact mechanism of fentanyl s analgesic effect is unknown. Variants of the OPRM1 gene, which encodes the mu-opioid receptor, have been shown to be associated with the analgesic efficacy of fentanyl. Methadone Methadone (Methadose, Diskets) is an opioid analgesic used to relieve moderate to severe pain. It is also used for maintenance treatment of opioid addiction. Methadone is a mu-opioid receptor agonist that is usually administered as a racemic mixture. The (S)-enantiomer is associated with cardio toxicity, whereas the (R)-enantiomer binds the mu-opioid receptor more strongly and is primarily responsible for the methadone s therapeutic effect 28, 29. The primary pathway of methadone metabolization involves N-demethylation by the CYP2B6 and CYP3A4 enzymes and results in formation of inactive metabolites 30. CYP2B6 preferentially demethylates (S)-methadone 31, 32. Variants of the CYP2B6 gene have been shown to be associated with methadone metabolism and QTc interval prolongation 29, 33-35. JULY 2014 / PAGE 6 OF 15

2.2. Synthetics The synthetic pain killers are broadly classified as synthesized compounds that are not naturally occurring. Carisoprodol Carisoprodol (Soma, Sanoma and Cariosoma) is a synthetic congener of meprobamate, with centrally acting muscle relaxant effects 36. Carisoprodol is metabolized to an active metabolite meprobamate with anxiolytic effects, by CYP2C19 enzyme 37-39. Variants of the CYP2C19 gene that lead to reduced enzyme function may result in reduced metabolism of carisoprodol thus standard doses may lead to higher than normal plasma concentrations 37-39. The mechanism of action of carisoprodol is not completely understood, but use of the drug can lead to adverse effects such as tachycardia and dizziness 40. 2.3. Non-steroidal anti-inflammatories Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of drugs widely used for their analgesic, anti-pyretic and anti-inflammotory properties. Members of this class of medications include ibuprofen, celecoxib, and aspirin amongst others. NSAIDs act through the inhibition of enzymes with cyclooxygenase (COX) activity; mostly COX-1 and/or COX-2 41. Celecoxib Celecoxib (Celebrex) is a NSAID, indicated for osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis in patients two years and older, ankylosing spondylitis, acute pain and primary dysmenorrheal 42. The mechanism of action of celecoxib that results in its anti-inflammatory and pain-relieving property of the drug results from selective inhibition of prostaglandin (PG) G/H syntase-2 (PTGS2) gene that causes inhibition of PG synthesis 43. PTGS2 enzyme has COX activity and therefore celebrex is also referred to as COX-2 selective inhibitor and this subclass of NSAIDs are also referred to as coxibs 43. Celecoxib is primarily metabolized by CYP2C9 enzyme 43. Variants of the CYP2C9 gene that affect enzyme function are associated with the risk of celecoxib-induced gastrointestinal bleeding or cardio toxicity. 2.4. Anti-metabolites Drugs classified as anti-metabolites are capable of blocking the cellular metabolism. Methotrexate is an anti-folate compound which blocks the enzyme dihydrofolate reductase, which is important for folic acid metabolism and synthesis of DNA and RNA. Therefore, Methotrexate targets rapidly dividing cells, such as cancer cells, bone marrow cells, and skin cells. Methotrexate Methotrexate (Trexall, Rheumatrex) is a drug used in the treatment of lymphoma and leukemia, as well as uterine, breast, skin, ovarian and other cancers. Methotrexate is also used to treat very severe and disabling psoriasis or in hematopoetic stem cell transplantation to prevent graft-versus-host disease; low doses of the drug are used to treat rheumatoid arthritis. Some patients taking Methotrexate may experience many and/or severe side effects, which are often referred to as Methotrexate toxicity 44. Reduced function variants of the MTHFR (5, 10-methylenetetrahydrofolate reductase) gene, which is important for folate metabolism, have been shown to be associated with Methotrexate toxicity in patients with rheumatoid arthritis. JULY 2014 / PAGE 7 OF 15

3. Genetic Variation and Drug Metabolism Selected genes important for pharmacogenomics of pain medication are discussed below, with emphasis in genetic variations that result in altered drug disposition and response. 3.1. Cytochrome P450 (CYP) The CYP superfamily is one of the most important group of enzymes involved in the oxidation of therapeutic drugs, xenobiotics and endogenous compounds 2. The CYP enzymes in humans are encoded by approximately 57 genes and can be divided into families, subfamilies and polypeptides 45. The CYP enzymes and the genes encoding them are designated with the abbreviation CYP, followed by a number indicating the gene family, a capital letter indicating the subfamily, and another numeral for the individual gene. Alleles are identified by CYP gene name followed by an asterisk and a specific allele name, denoted by Arabic numerals (e.g., CYP2D6*1) 46, 47. { others ~8% CYP2B6 2-4% CYP2C19 5% CYP1A2 5% CYP3A4 40-45% CYP2C9 10% CYP2D6 20-30% Figure 1. Relative contribution to drug metabolism by CYP type. Many CYP isoforms are expressed polymorphically because of mutations in the CYP genes. The CYP2D6, CYP2C9 and CYP2C19 genes are particularly polymorphic 48. Many of these polymorphisms have functional significance, resulting in altered enzyme activity or complete loss of enzyme expression 48. The variants in CYP enzymes are therefore important determinants of drug effectiveness and adverse drug reactions 48. Individuals can be classified into distinct metabolizer classes based on the CYP variants in their genome. For example, individuals can be classified based on their CYP2D6 enzyme activity into four metabolizer groups: Ultrarapid (UM, higher than normal enzyme activity), Extensive (EM, normal enzyme activity), Intermediate (IM, intermediate enzyme activity) and Poor (PM, low or no enzyme activity). JULY 2014 / PAGE 8 OF 15

Extensive metabolizer (normal) Cp, max Plasma level of drug AUC Poor metabolizer Time (arrows show repeated doses) Figure 1. Variation in CYP activities has an impact on drug pharmacokinetics (adapted from Guengerich, 2006 9 Clinical studies have demonstrated that individuals that were PMs for CYP2D6 metabolized drugs and taking codeine had very low systemic exposure to the active compound morphine compared to EMs 8, 9. Therefore, PMs may experience little to no pain relief from codeine 9, 10. In contrast, UMs are at high risk of severe toxicity because of above average systemic exposure to the active compound morphine 49 and may experience adverse reactions, such as respiratory depression, respiratory arrest, shock and/or cardiac arrest 7. Infants who are breastfed by mothers who are UMs and taking codeine are also at increased risk of morphine overdose, which can result in opioid toxicity in infants. Importantly, there are also differences in the ethnic distribution of CYP metabolizer status (Table 4). JULY 2014 / PAGE 9 OF 15

Table 4: Ethnic distribution of predicted CYP2D6, CYP2C19 and CYP2C9 metabolizer status. CYP Status African American Caucasian East Asian Hispanic 2D6 Poor 2-8% 5-10% <2% 3-10% 2D6 Intermediate ~30% 10-17% 50-60% ND 2D6 Extensive 60-70% 70-80% 40-50% ND 2D6 Ultrarapid ~5% 3-10% <1% 0-5% 2C19 Poor ~3.2% ~2% ~12% ND 2C19 Intermediate ~31.2% ~24% ~46.3% ND 2C19 Extensive ~58.3% ~36% ~41.7% ND 2C19 Ultrarapid ND 33% ND ND 2C9 Poor ~2% ~9% ~2% ~6% 2C9 Intermediate ~23% ~26% 12-15% 24-29% 2C9 Extensive ~75% ~66% ~86% 68-70% 3.2. MTHFR The T allele of the rs1801133 marker (C677T variant) in the MTHFR gene, which is important for folate metabolism, has been shown to be associated with methotrexate toxicity in patients with rheumatoid arthritis. The T allele results in an amino acid change that leads to reduced enzyme activity. Homozygotes for the T allele have approximately 30% of the expected MTHFR enzyme activity, and heterozygotes have approximately 65% activity, compared to the most common genotype, C allele homozygotes. Reduced MTHFR enzyme activity may result in reduced elimination of methotrexate, thus resulting in higher than expected methotrexate plasma concentrations and increasing the likelihood of methotrexate toxicity 50. 3.3. OPRM1 The mu-opioid receptors are a class of opioid receptors that preferentially bind beta-endorphin and enkephalins. The OPRM1A>G allele leads to reduced OPRM1 expression in the brain and decreased opioid receptor signaling efficiency in the pain-relevant brain region [51]. Just as in the case of CYP enzymes, there are differences in the allele distribution for OPRM1. Specifically, The G allele of the rs1799971 marker has an allelic frequency of 0.8% in people from Sub-Saharan Africa, 8.2-17% in Caucasians and 48.9% in Asians 51. JULY 2014 / PAGE 10 OF 15

4. Conclusion The growing body of clinical data indicates that understanding of a patient s genotype can be useful in deciding the best course of drug treatment. Pharmacogenetics can be a powerful tool in the physician s arsenal to help identify the optimum pain medication and dosage for a specific patient thus reducing the risk of either inadequate pain management or drug-related adverse events. In addition, knowledge of the patient s genotype can help identify potential complications arising from the use of other medications, herbal supplements and even food. These benefits are being recognized by FDA and are reflected in the growing list of medications for which pharmacogenomic information is required in the drug label. In summary, the use of genomic information has the potential for improving the utility, efficacy and safety of pain management. JULY 2014 / PAGE 11 OF 15

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