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Nz pep lecture_jan2016

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This is a set of powerpoint slides with self-assessment questions interspersed throuought on drug metabolism and pharmacogenetics. The aim is to understand the mechanism of clinically significant drug interactions, recognize potentially clinically significant genetic influences on drug efficacy and toxicity, and genetic predispositions to disease due to altered drug metabolism or transport. This resource is appropriate for medical students or graduate healthcare professionals such as nursing students.

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Nz pep lecture_jan2016

  1. 1. Drug Metabolism and Pharmacogenetics Nathalie Khoueiry Zgheib, MD. Associate Professor, Pharmacology and Toxicology Faculty of Medicine, American University of Beirut nk16@aub.edu.lb Reference: The Pharmacological Basis of Therapeutics, Goodman and Gillman, 12th Edition.
  2. 2. Drug Elimination
  3. 3. Elimination = Metabolism + Excretion  Metabolism:  Biotransformation:  Conversion of non-polar parent drugs to polar metabolites.  Inactivation: detoxification.  Activation: prodrug  active drug; toxic metabolites.  Excretion:  Loss of drug molecules (parent drug and/or metabolites) from the body.
  4. 4. Sites of elimination  Metabolism:  Major: Liver  Other: intestine, placenta, Kidney…  Excretion:  Major:  Kidney  Other:  Bile (enterohepatic circulation)  Lungs  Sweat, saliva, milk…
  5. 5. Sites of drug elimination Enterohepatic circulation: first pass effect Drug Intestine Blood Liver Bile Kidneys UrineFeces Target organ
  6. 6. Why?  Drug development  Individual variability  Drug-drug interactions  Pharmacogenomics
  7. 7. Case
  8. 8. Mr. Dawa Sayalan  Mr. Sayalan is a 55 y.o. gentleman recently diagnosed with Atrial Fibrillation. He was started on warfarin (coumadin) 5 mg on day 1, and 2.5 mg on day 2.  On the third day, Mr. Sayalan was admitted to the ER with major intracranial bleeding  He passed away 1 hour later.
  9. 9.  What went wrong?  Should we have given him a different starting dose?  What dose?
  10. 10. Drug Response Efficacy Toxicity
  11. 11. Individualized drug Response Efficacy Toxicity Mr. Sayalan
  12. 12. Individual variability  Sex  Age  Ethnicity  BMI  Liver and renal disease  Occupation, stress  Smoking, alcohol, food  Drug-drug interactions  Genetic polymorphisms
  13. 13. Drug interactions: major mechanisms  Altered drug absorption  Displacement from protein binding sites  Inhibition of metabolism  Induction of metabolism  Inhibition of excretion  Induction of excretion
  14. 14. Warfarin elimination  “…elimination is almost entirely by metabolism…”  “The cytochrome P450 enzymes involved are: 2C9, 2C19, 2C8, 2C18, 1A2 and 3A4. “  “2C9 is likely to be the principal form of human liver P-450 which modulates the in-vivo anticoagulant activity of warfarin.” www.FDA.GOV
  15. 15. List of drugs Drug Effect on CYP2C9 Warfarin blood concentration Dose optimization Amiodarone Statins Fluconazole Sulfa drug Inhibitors Increases Use a LOWER dose
  16. 16. Individual variability  Sex  Age  Ethnicity  BMI  Liver and renal disease  Occupation, stress  Smoking, alcohol, food  Drug-drug interactions  Genetic polymorphisms
  17. 17. CYP2C9 Enzyme activity
  18. 18. CYP2C9 and Warfarin Warfarin dose mg/day CYP2C9 genotype Scordo et al, Clinical Pharmacology & Therapeutics, Vol 72, 36, December 2002 50% Dose reduction
  19. 19. Drug Metabolism
  20. 20. EXCRETION Functionalization Conjugation Oxygenases Transferases Drug metabolizing enzymes
  21. 21. Sites of drug metabolism  Phase I  SER  Phase II  SER and cytoplasm
  22. 22. Drug Metabolizing Enzymes
  23. 23. Cytochrome P450 Drug Metabolizing Enzymes
  24. 24. Cytochrome P450 enzyme  Heme protein oxygenase  In its ferrous form, it can bind carbon monoxide and form a complex that absorbs light at 450 nm  Cyto: cell  Chromo: color  P: pigment  450: wavelength
  25. 25. Mueller & Mueller: 1990’s  They mixed substrates with cofactors and microsomes  Monitored change in optical density of substrate  Found out that:  Need oxygen  Need NADPH and NADP+
  26. 26. CYP Reaction Drug + O2 + NADPH + H+ Drug-OH +H2O + NADP+ Has highest affinity to oxygen & CO Has highest affinity to substrate CO CO Absorbs light at 450 wavelength Use one atom from O2 and 2 electrons to oxidize substrates
  27. 27. Copyright © 2015 McGraw-Hill Education. All rights reserved. CYPs are embedded in the phospholipid bilayer of the endoplasmic reticulum (ER). Most of the enzyme is located on the cytosolic surface of the ER. A second enzyme, the flavoprotein NADPH-cytochrome P450 oxidoreductase, transfers electrons to the CYP where it can, in the presence of O2, oxidize xenobiotic substrates, many of which are hydrophobic and dissolved in the ER. A single NADPH-CYP oxidoreductase species transfers electrons to all CYP isoforms in the ER. Each CYP contains a molecule of iron-protoporphyrin IX that functions to bind and activate O2. Substituents on the porphyrin ring are methyl (M), propionyl (P), and vinyl (V) groups. Location of CYPs in the cell. Flavoprotein
  28. 28. Favored site of O2 attack in CYP catalyzed reactions:  Terminal (Ω) carbon in long aliphatic compounds  Ω-1 carbon for relatively short side chains  Next to heterocarbon (i.e. next to O, N, S, etc…)  At the double bond in aromatic, unsaturated and heterocyclic compounds
  29. 29. CYP mediated oxidative reactions
  30. 30. RELATIVE HEPATIC CONTENT OF CYP ENZYMES The Human Cytochrome P450 (CYP) Allele Nomenclature Committee http://www.cypalleles.ki.se/ Most important CYPs involved in metabolism of xenobiotics - Gene products differ in their aa sequence and in their affinity to various compounds - Families share ≥ 40% homology - Subfamilies share ≥ 55% homology CYP2B6 2.20%CYP2C9 11.70% CYP2C8 2.20% CYP2C19 12.60% CYP1A/2 10.90% CYP3A4/5 33.90% CYP2D6 20.90% CYP2EI 5.60%
  31. 31. CYP table (Flockhart) http://medicine.iupui.edu/flockhart/table.htm Not very specific binding: “promiscuous”
  32. 32. CYP Substrates  Endogenous: - Steroids and bile acids - Very specific & fast  Exogenous: Multiple substrates to 1 isoenzyme Multiple isoenzymes to 1 substrate Multiple and non specific binding sites Non efficient slow enzymatic reactions Rate limiting step in metabolism Clinically relevant drug interactions Drug – drug interactions
  33. 33. Enzyme inhibition  Decrease in substrate metabolism  Mechanism:  Competitive inhibition, reversible (most common)  Inactivation
  34. 34. What happens if 2 drugs, A & B, that are both inactivated by CYP2C9 are given together? A. Drug A may inhibit the metabolism of drug B, hence the patient may develop toxicity from drug B. B. Drug B may inhibit the metabolism of drug A, hence the patient may develop toxicity from drug B. C. Cyp2C9 will be inactivated by drugs A & B. D. This is wrong prescribing, drugs A&B should not be given together.
  35. 35. CYP3A inhibition: Cisapride (prepulsid)  Removed from the market in 2000  Q-T interval prolongation and fatal arrhythmias  When given with CYP3A4 inhibitors  e.g. erythromycin http://www.rxlist.com/propulsid-drug.htm
  36. 36. Enzyme induction  Increase in substrate metabolism  Mechanism:  Enhance rate of synthesis (most common)  Reduce rate of degradation
  37. 37. CYP3A induction: T.M. Wilson, S. A. Kliewer 2002:1, 259-266 St. John’s wort Carbamazepine St. John’s Wort: over the counter herbal product used to treat mild depression Carbamazepine: “autoinduction”
  38. 38. Drug development experiments  In Vitro:  Using SER microsomes  In Vivo:  Giving the drug to humans
  39. 39. CYP enzymes activities assessments in vivo Probe drugs: Probe drug (Debrisoquine) CYP Metabolism (CYP2D6) Metabolite (OH-debrisoquine) URINE blood Calculate: DRR= OH-debrisoquine/OH-debrisoquine + debrisoquine (in urine, at 8 hours) or Debrisoquine/4-OH-debrisoquine metabolic ratio
  40. 40. Pharmacogenetics
  41. 41. Individual variability  Sex  Age  Race  BMI  Liver and renal disease  Occupation, stress  Smoking, alcohol, food  Drug-drug interactions  Genetic polymorphisms
  42. 42. Pharmacogenetics  Definition: Study of how genes affect the way people respond to drug therapy. Drug Disposition Response  Goal: individualize drug therapy to a person’s unique genetic makeup: “individualized medicine”
  43. 43. How common?  75% -85% of variability in drug metabolism is heritable.  Idiosyncratic reactions
  44. 44.  Genetic Polymorphism: Variation in the DNA sequence that is present at an allele frequency of 1% or greater in a population.  INDEL (Insertions, deletions)  Single Nucleotide Polymorphisms (SNPs)
  45. 45. Phenotype  Observable drug response  PM= Poor Metabolizer  IM= Intermediate Metabolizer  EM= Extensive Metabolizer  UM= Ultra Metabolizer  Healthy subjects unless challenged with inappropriate drug or drug dose.
  46. 46. Genotype  Simple:  Wild type TT = Gene*1/*1  Hetero TC = Gene*1/*n  Mutant type CC = Gene*n/*n ? Ethnicity  How determine Wild type? ? Many SNPs  Haplotypes ? Many genes
  47. 47. Distinct difference Polymorphic distribution Non Distinct difference Monomorphic distribution Phenotypes
  48. 48. CYP2D6  1960’s  Patient treated with nortryptilline had 30-40 fold increase in nortryptilline plasma concentration  CYP2D6 ? metabolizer
  49. 49. CYP2D6 common polymorphisms
  50. 50. CYP2D6 and antidepressants dose dose
  51. 51. Tamoxifen metabolic pathway Tamoxifen 4-OH Tamoxifen N-desmethyl Tamoxifen CYP2D6 CYP2D6 Endoxifen Active metabolites 30-100 folds more potent than tamoxifen Roberta Ferraldeschi et al, pharmaceuticals (2009)
  52. 52. You perform CYP2D6 genotyping on breast cancer patients who are maintained on tamoxifen. You are doing this because: A. CYP2D6 slow metabolizers may have a poorer response to tamoxifen. B. CYP2D6 slow metabolizers may have a higher plasma concentration of endoxifen. C. CYP2D6 ultra fast metabolizers may have a higher risk of disease recurrence. D. CYP2D6 ultra fast metabolizers may have a higher plasma concentration of tamoxifen.
  53. 53. Tamoxifen activation by CYP2D6 Goetz et al, Clinical Pharmacology & Therapeutics, Vol 83, # 1, January 2008
  54. 54. Bertilsson et al, Clinical Pharmacology and therapeutics, 51, 1992
  55. 55. Non CYP drug metabolizing enzymes
  56. 56. Drug Metabolizing Enzymes
  57. 57. Flavin-Containing Monooxygenases (FMOs)  6 families, FMO3 most abundant in liver  Minor contributors to drug metabolism  Benign metabolites  No induction or inhibition  Genetic deficiency in FMO3  accumulation of trimethylamine N-oxide (TMAO) Fish odor syndrome.
  58. 58. Hydrolytic enzymes  Carboxylesterases (CES)  Both detoxification and activation of drugs  Epoxide hydrolases (EH)  Deactivate epoxides generated by CYPs
  59. 59. Epoxide deactivation Toxic reactions: Adducts with DNA and proteins E.G. Antiepileptics: - Valproic acid and carbamazepine are usually given together -Carbamazepine: An epoxide is formed from CYP metabolism -Valproic acid inhibits EH -There is hence a potential increase in toxicity from carbamazepine epoxide
  60. 60. Phase II conjugating enzymes or transferases  Synthetic:  Result in a high molecular mass metabolite.  Glucuronidation: especially high Molecular Wt  biliary excretion  Usually terminate the biological activity of drugs.  All in cytosol of the cell, except glucuronidation.  Catalytic rates faster than CYPs.  Need a cofactor.
  61. 61. Glucuronidation: UDP-glucuronosyltransferases O O H O H OO H CO2H P O P O O HO O H O CH2 O N NH O O O O H O H O H CO2H O R + RO H or R3N UGT UDP-α-D-glucuronic acid O O H O H O H CO2H N + R R R O -glucuronide N+-glucuronide Phase I Hydroxylation N-Dealkylation
  62. 62. Crigler-Najjar syndrome Type I
  63. 63. Glucuronidation: UDP-glucuronosyltransferases (UGT1 & UGT2)  UGT1A1: involved in bilirubin metabolism  Genetic polymorphism in the reading frame of UGT1A1  Hyperbilirubinemia and jaundice  No enzyme: Crigler-Najjar syndrome Type I  Little enzyme: Crigler-Najjar syndrome Type II  Genetic polymorphism in promoter region of UGT1A1  Gilbert’s syndrome  10% of population  High serum bilirubin  Predisposed to toxicity from for example: irinotecan  Glucuronides are excreted by the kidneys or actively transported into the bile.
  64. 64. Patients with type II Crigler-Najjar syndrome may be treated with phenobarbital. This is explained by the fact that: A. Phenobarbital competes with bilirubin on UGT1A1 binding site. B. Phenobarbital inhibits UGT1A1 enzyme. C. Phenobarbital induces UGT1A1 enzyme. D. Phenobarbital inhibits the metabolism of bilirubin.
  65. 65. Sulfation: Sulfotransferases (SULTs) (PAPS, 3’-phosphoadenosine- 5’-phosphosulfate) R OH R O S OH O O H H NH2 N NN N OH O H H HO O P OH O O S OH O O +
  66. 66. Sulfation: Sulfotransferases (SULTs)  SULT1A1 most important  Generation of chemically reactive metabolites  cancer risk
  67. 67. Glutathione conjugation: Glutathione –S-transferases (GSTs)  Transfer of reduced glutathione= GSH
  68. 68. Glutathione conjugation: Glutathione –S-transferases (GSTs)  2 subfamilies:  Microsomal: metabolism of endogenous substances  Cytosolic: metabolism of exogenous substances  GSTA, GSTM, GSTO, GSTP, GSTS, GSTT, and GSTZ.  Involved in detoxification  Common GSTM1*0 (Null genotype) associated with cancer.  GST abundant in tumor cells  drug development:  Combine a cancer drug that is metabolized by GST with GST inhibitors  better anticancer effect
  69. 69. Acetylation: -COCH3 A r NH2 R SH R O H R NH2 + A r N CH3 O H Acetyl transferase CoA S O R N O CH3H R O O CH3 R S O CH3
  70. 70. N-Acetylation N-acetyltransferases (NAT)  NAT1 and NAT2  Are the most polymorphic of all drug metabolizing enzymes
  71. 71. Nat 2 Polymorphism Slow acetylators Slow acetylators: Higher INH concentration 5 -15% neurologic toxicity From INH 50% Whites 17% Japanese Drayer et al, Clinical Pharmacology and therapeutics, 1977
  72. 72. Methylation (MT)  High substrate specificity  Lead to more lipid soluble products  Many:  N-Methyltransferases (NMT)  Catechol-O-methyltrasnferases (COMT)  Phenol-O-methyltransferses (POMT)  Thiopurine S-methyltransferases (TPMT)  Thiol methyltransferases (TMT)
  73. 73. Treatment with 6-mercaptopurine (6-MP) in children with acute lymphoblastic leukemia (ALL) Screen for TPMT*3A polymorphism:  DECREASE dose Weinshillboum et al, Annual review of pharmacology and toxicology, 1999 1/300 whites 1/2500 asians
  74. 74. Other Drug Metabolizing Enzymes Alcohol  Aldehyde  Acetic acid 1 2 1 2 - Genetic polymorphism in Japanese - Inhibited by “Antabuse” and metronidazole (Flaggyl)
  75. 75. Pharmacogenetics  Definition: how genes affect the way people respond to drug therapy. Drug Disposition Response Target -Enzymes -Receptors Elimination -DMEs -Transporters
  76. 76. Drug metabolism Drug targets CYP2D6 TPMT Tamoxifen 6-Mercapto Purine ALOX5 5-lipoxygenase inhibitors NAT2 INH CYP2C9 VKORC1 Anticoagulants Pharmacogenetics ADRB2 B agonists UGT1A1 Irinotecan * * * * * * Clinical diagnostic tests are available EGFR * Drug elimination Transporters HER 2 * FDA TABLE: http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm
  77. 77. Drug Transporters
  78. 78. Roles of membrane transporters in pharmacokinetic pathways: 1. influx of: essential nutrients and ions 2. efflux of: cellular waste, environmental toxins…
  79. 79. Drug transporters: 2 superfamilies http://nutrigene.4t.com/humanabc.htm  ABC  ATP Binding Cassette  7 families  ABCA  ABCG  P-glycoprotein = ABCB1=MDR1  SLC  Solute Carrier  43 families e.g. SLC6A4 for serotonin (SSRIs)
  80. 80. ABC: export of drugs from the healthy tissue and facilitation of elimination from the body  any decreased activity of these transporters may be associated with a decrease in drug clearance and hence potentially more efficacy but at the expense of higher toxicity. SLC: vastly different specificities or functional roles and transport is usually bidirectional
  81. 81. MDR1 3 most common polymorphisms Gene GENE rs number Base pair change Type aa change MAF caucasian Effect on protein ABCB1 MDR1 rs1045642 C3435T Exon Ile1145Ile 0.46 Decreased expression rs2032582 G2677T G2677A Exon Ala893Ser Ala893Thr 0.40 Decreased expression rs1128503 C1236T Exon Gly411Gly 0.5 Unclear
  82. 82. What comes to your mind if you discover that a patient you are treating with the antiepileptic phenytoin for epilepsy is homozygous for MDR1 C3435T variant allele? A. Stop phenytoin as the “brain levels” of phenytoin may be too low. B. Add an MDR1 inhibitor so that phenytoin’s blood level increases. C. Monitor treatment as the patient may have a better than expected response to phenytoin. D. Honestly, NOTHING comes to my mind!
  83. 83. Resistance to phenytoin in epilepsy with the CC genotype Epilepsia, 50(1):1-23,2009
  84. 84. Increased Efflux-- ABC transporters Increased Efflux-- ABC transporters Decreased Uptake-- Solute carriers Decreased Uptake-- Solute carriers Mechanisms of resistance to anti-cancer drugs Reduced apoptosis Altered cell cycle checkpoints and/or growth pathways Increased metabolism of drugs Increased or altered targets Increased repair of damage Compartmentalization Reduced apoptosis Altered cell cycle checkpoints and/or growth pathways Increased metabolism of drugs Increased or altered targets Increased repair of damage Compartmentalization
  85. 85. Role of P-glycoprotein (ABCB1/MDR1) in cancer  Approximately 50% of human cancers express P-glycoprotein at levels sufficient to confer MDR  Cancers which acquire expression of P-gp following treatment of the patient include: leukemias, myeloma, lymphomas, breast, ovarian cancer  Preliminary results with P-gp inhibitors suggest improved response to chemotherapy in some of these patients Gottesman, M, USNCI
  86. 86. Individualization of Drug Response Efficacy Toxicity 1. Frequency and significance of the disease 2. Frequency and significance of the polymorphism 3. Therapeutic index of the drug
  87. 87. PM= Poor Metabolizer IM= Intermediate Metabolizer EM= Extensive Metabolizer Toxicity Therapeutic effect No response Time IM EM
  88. 88. Current limitations & future challenges of pharmacogenetics  Genotyping techniques  Clinical utility:  Evidence from well-powered trials  Privacy  Confidentiality: employment and insurability  Acceptance and education  Cost  Candidate SNP approach vs. large throughput genotyping
  89. 89. SNPs that change clinical outcome SNPs that change drug response SNPs that change pharmacokinetics SNPs that change activity in vitro Non-conservative amino acid changes Non-synonymous SNPs in exons Exon-based changes All SNPs Hierarchy of Pharmacogenetic Information from Single Nucleotide Polymorphisms (SNPs) drug disposition October 15, 2008: 18, 000, 000 human SNPs November 23,2010: 37,824,422 human SNPs December 15,2011: 60,481,170 human SNPs December 12,2012: 63,222,718 human SNPs September 16,2013: 73,349,282human SNPs 2 1605 CYP2C9
  90. 90. Genotyping techniques: microarrays data Relationship of SNPs with predicted warfarin therapeutic dose Blood, April 15, 2008, V111 N8
  91. 91. ACCE (CDC) http://www.cdc.gov/genomics/gtesting/acce/acce_proj.htm Analytic sensitivity & specificity Lab quality control Assay robustness Improvement of HEALTH OUTCOME Informed consent Impact on family members Stigmatization Prevalence and ability to detect the phenotype = Predictive value
  92. 92. Clinical utility questions: 1. What is the impact of a positive (negative) test on patient care? 2. Is there an effective remedy, acceptable action, or other measurable benefit? 3. Is there general access to that remedy or action? 4. Is the best being tested to a socially vulnerable population? 5. What quality measures are in place? 6. What are the financial costs associated with testing? 7. What are the economic benefits associated with actions resulting from testing? 8. What facilities/personnel are available or easily put in place? 9. What educational material has been developed and validated and which of these are available? 10. Are there informed consent requirements? 11. What methods exist for long term monitoring? 12. What guidelines have been developed for evaluating program performance? http://www.cdc.gov/genomics/gtesting/acce/acce_proj.htm

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