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  • The slide on the left lists the major isoforms of CYP450 and their relative roles in drug metabolism (not relative amounts found in the liver) based upon the number of drugs that are known to be metabolized by that particular isozyme. CYP3A is responsible for the metabolism of the largest number of drugs followed by CYP2D6. The slide on the right summarizes the relative quantity of specific P450 families found in the liver.{Shimada} The CYP3A family is present in the largest amounts. CYP2D6 accounts for less than 2% of the total content of P450 in the liver, but as shown on the left, is responsible for the metabolism of a large fraction of drugs. A large amount of cytochrome P450 has not yet been characterized. There is tremendous variability between individuals in terms of expression of cytochrome P450 isozymes. For example, CYP2D6 is not present at all in some livers. Note: 2C on the graph on the right refers to both CYP2C9 and CYP2C19. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 1994; 270(1):414-423.
  • These are the important inhibitors of CYP3A that will make patients appear phenotypically to resemble poor metabolizers. Azole antifungal drugs, in general, are potent inhibitors of CYP3A, although fluconazole is a weak inhibitor, and inhibits CYP 3A only at high doses. All the macrolide antibiotics, except azithromycin, are also potent inhibitors of this cytochrome P450 isoform. Cimetidine is a broad, but relatively weak, inhibitor of many cytochrome P450 enzymes. Also, notice that a food, grapefruit juice, is listed as an inhibitor. The role of grapefruit juice in drug interactions will be discussed later.
  • CYP2D6 metabolizes many of the cardiovascular and neurologic drugs in use toaday. Study of CYP2D6 has led to understanding the failure of codeine to relieve pain in some patients. Codeine is actually a pro-drug that is converted to morphine. Codeine itself is much less active as an analgesic, but causes nausea and other adverse effects. The absence of cytochrome P450 2D6 in 7% of Caucasians means that these individuals cannot metabolize codeine to the active metabolite, morphine, and therefore will get little, if any, pain relief from codeine.{Caraco} However, they will experience codeine’s adverse effects, particularly if the dose is increased in the futile attempt to obtain pain relief. Thirty percent of Ethiopians studied had multiple copies of the 2D6 gene (up to 13) and increased eynzyme activity resulting in ultrarapid metabolism.{Akilillu} Ultra-rapid metabolism results in lower blood levels following a standard dose of any drug metabolized by this enzyme. Therefore these patients may have an inadequate response to standard dosages of  -blockers, narcotic analgesics, or antidepressants and may require higher dosages for clinical effectiveness. Several commonly used medications inhibit CYP2D6. These include quinidine{Branch} as well as haloperidol and some other antipsychotics.{Shin 1999},{Shin 2001} The well-described pharmacokinetic interaction between selective serotonin reputake inhibitor (SSRI) antidepressants and tricyclic antidepressants appears to be due to the fact that fluoxetine and paroxetine are both potent inhibitors of CYP2D6 {Bergstrom},{Leucht} and render patients metabolically equivalent to people who do not have the enzyme. This increases the plasma levels of tricyclic antidepressants and increases the potential for side effects. In contrast, patients co-prescribed fluoxetine or paroxetine with codeine may experience no analgesic benefit, since codeine requires CYP2D6 for metabolism to morphine. Caraco Y, Sheller J, Wood AJ. Pharmacogenetic determination of the effects of codeine and prediction of drug interactions. J Pharmacol Exp Ther 1996; 278(3):1165-1174. Bergstrom RF, Peyton AL, Lemberger L. Quantification and mechanism of the fluoxetine and tricyclic antidepressant interaction. Clin Pharmacol Ther 1992; 51(3):239-248. Leucht S, Hackl HJ, Steimer W, Angersbach D, Zimmer R. Effect of adjunctive paroxetine on serum levels and side-effects of tricyclic antidepressants in depressive inpatients. Psychopharmacology (Berl) 2000; 147(4):378-383. Aklillu E, Persson I, Bertilsson L, Johansson I, Rodrigues F, Ingelman-Sundberg M. Frequent distribution of ultrarapid metabolizers of debrisoquine in an ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles. J Pharmacol Exp Ther 1996; 278(1):441-446. Branch RA, Adedoyin A, Frye RF, Wilson JW, Romkes M. In vivo modulation of CYP enzymes by quinidine and rifampin. Clin Pharmacol Ther 2000; 68(4):401-411. Shin JG, Kane K, Flockhart DA. Potent inhibition of CYP2D6 by haloperidol metabolites: stereoselective inhibition by reduced haloperidol. Br J Clin Pharmacol 2001; 51(1):45-52. Shin JG, Soukhova N, Flockhart DA. Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6. Drug Metab Dispos 1999; 27(9):1078-1084.
  • Cytochrome P450 1A2 is an important drug metabolizing enzyme in the liver that metabolizes many commonly used drugs including theophylline, imipramine, propranolol, and clozapine. CYP1A2 is significantly induced in a clinically relevant manner by tobacco smoking. The clearance of theophylline, imipramine, propranolol and clozapine are all increased by smoking. Thus, people who smoke may require higher doses of some medications that are substrates of CYP1A2. In contrast, a smoker would require a decrease in theophylline dosage if, for example, smoking were discontinued and the enzyme no longer induced. This topic has been recently reviewed by Zevin and Benowitz.{Zevin} Inhibitors of CYP1A2, including some fluoroquinolone antibiotics, can increase the plasma concentrations of drugs that are metabolized by CYP1A2, with a potential for increased toxicity.{Raaska}{Grasela} Zevin S, Benowitz NL. Drug interactions with tobacco smoking. An update. Clin Pharmacokinet 1999; 36(6):425-438. Raaska K, Neuvonen PJ. Ciprofloxacin increases serum clozapine and N-desmethylclozapine: a study in patients with schizophrenia. Eur J Clin Pharmacol 2000; 56(8):585-589. Grasela TH, Jr., Dreis MW. An evaluation of the quinolone-theophylline interaction using the Food and Drug Administration spontaneous reporting system. Arch Intern Med 1992; 152(3):617-621.
  • It would be impossible to memorize all of the drug interactions that have been presented here. Fortunately there are aids to help health care providers to prevent drug interactions, such as the one shown here. This is a pocket version of a much larger CYP P450 drug interaction table. A more complete version of this card is maintained on the internet at www.drug-interactions.org . This table includes a listing of the 6 major cytochrome P450 isozymes involved in drug metabolism and the drugs that are metabolized by them. We recommend using this or another table as a quick reference for detection of potential drug interactions. If two drugs are metabolized by the same cytochrome P450 isozyme, it is very possible that competitive inhibition could lead to higher than usual levels of either or both of the drugs. If a drug is metabolized by a specific cytochrome P450 and is taken with an inhibitor or inducer of that isozyme, an interaction is also likely. The following are examples of how to use this card. Suppose your patient is taking amiodarone and you want to add a statin agent to decrease the patient’s cholesterol (follow red circles and arrows above). The card shows that amiodarone is an inhibitor of CYP2D6 and CYP3A. We also note that lovastatin and simvastatin are metabolized by CYP3A and that if given with amiodarone (which is inhibiting the enzyme) a toxic level of the statin may occur. The result may be an adverse reaction (rhabdomyolysis or liver toxicity). The best choice would be pravastain, which is not metabolized by CYP3A. Another example would be if your patient were taking an HIV protease inhibitor and wants to take St. John’s Wort (follow green squares and arrows above). According to the card, St. John’s Wort induces CYP3A4, which metabolizes most protease inhibitors. The concomitant administration of St John’s Wort with protease inhibitors could result in the induction of CYP3A4, increased metabolism, and subtherapeutic levels of the protease inhibitor. Laminated versions of this card can be ordered from the web site listed above. When on the web site, it is possible to easily obtain the reference for a given drug by clicking on the drug. The web site hyper-links to PubMed and searches for a list of the relevant publications.

Transcript

  • 1.
    • What influences drug metabolism?
    • Enzyme concentration
    • Enzyme induction
    • Drug structure effects
    • Genetic factors - Pharmacogenetics
    Drug Metabolism: Factors
  • 2. Enzyme Activity
    • Function of enzyme concentration and activity
    • If the rate of metabolism decreases
      • Increased intensity and duration of drug action
      • Increased accumulation in plasma, increased toxicity risk
    • If the rate of metabolism increases
      • Decreased intensity and duration of drug action
      • In rare cases toxicity may increase - metabolites
    • Age differences
      • Premature and newborn babies have yet to develop maximal oxidative and conjugative enzyme capabilities
        • Approaches adult levels at 1-2 months age
        • Example: newborn jaundice or neonatal hyperbilirubinemia (kernicterus) is caused by the inability to conjugate glucuronic acid with bilirubin (Heme from hemoglobin metabolism)
      • Old Age – may influence metabolism (underlying disease)
  • 3.
    • “ Enzyme induction”
      • Results from drug or chemical exposure
      • Very important source of drug-drug interactions
      • Caused by the increased rate of enzyme production
      • Often drugs can increase their own rate of metabolism
    Enzyme Activity
  • 4.
    • “ Enzyme induction” continued
      • Compounds that enhance metabolism: Phenobarbital and other barbiturates, glutethimide, phenylbutazone, meprobamate, ethanol, phenytoin, rifampin, griseofulvin, carbamazepine
      • Classical example: Phenobarbitol
        • If a patient starts phenobarbitol while taking warfarin, blood levels and dosage adjustment of warfarin will need be monitored and adjusted
        • If patient stops phenobarbitol, dosage will need to be decreased
        • Oral contraceptives are rendered ineffective by phenobarbitol and rifampin due to increased estrogen metabolism
      • Endogenous compounds can also be metabolized faster
        • Example – phenobarbitol can be used to increase conjugation of bilirubin with glucuronic acid in neonates with jaundice
      • Smokers often metabolize drugs faster due to smoke chemicals
        • Example: theophylline t 1/2 = 4.1 vs. 7.2 hours
    Enzyme Activity
  • 5.
    • “ Enzyme induction” continued
      • Two inducer categories:
        • Phenobarbitol-like inducers (P-450 enzymes)
        • Polycyclic aromatic hydrocarbon-like inducers (P-448 enzymes)
          • Selective enzyme for aromatic hydrocarbons
    • Remember: metabolism of drugs and chemicals can result in toxic metabolites from otherwise non-toxic compounds
    • Enzyme inhibition and inhibition of metabolism
      • Leads to drug accumulation and toxicity
      • Mechanisms
        • Substrate competition
        • interference with protein synthesis
        • Interference with drug metabolizing enzymes
        • Hepatotoxicity leading to decreased metabolism
        • Others
    Enzyme Activity
  • 6. Structural Factors
      • Many drugs are racemic mixtures
      • Typically one enantiomer is more bioactive
        • Receptor binding phenomenon
      • Selective metabolism: “S ubstrate stereoselectivity ”
      • Often enantiomers metabolized by different enzymes
  • 7.
    • Stereochemical aspects cont:
      • Preferential metabolic formation of a stereoisomer: “ product selectivity ”
      • When a ketone is reduced to an alcohol, one stereoisomer is preferred
      • Hydroxylation can also be stereoselective
    Structural Factors
  • 8.
    • Stereochemical aspects cont:
      • Regioselective metabolism
        • selective metabolism of one of 2 or more of the same functional groups located on a molecule
    Structural Factors
  • 9.
    • Pharmacologically active metabolites
      • Function of:
        • Plasma accumulation
        • Rate of excretion (decreased renal function)
      • Metabolites no longer thought inactive
      • Many metabolites are now marketed as drugs
    Structural Factors
  • 10. Other Factors
    • Misc factors affecting metabolism
      • Dietary factors
        • Protein and carbohydrate consumption
        • Indoles in brussels sprouts, cabbage and cauliflower
        • Charcoal-broiled meats  polyaromatics induce enzymes
        • Malnutrition
        • Starvation
        • Vitamins and minerals
      • Underlying disease states
          • Hepatic cancer, cirrhosis, hepatitis
          • Hyper- or hypothyroid disease
      • Pregnancy
      • Circadian rhythm
  • 11. Pharmacogenomics
    • American Journal of Health-System Pharmacy
    • Margaret Ma, Michael Woo, Howard Mcleod
    • Vogel – “study of the role of genetics in drug response”
    • One of the most rapidly growing areas of pharmacy
    • Genetic makeup is responsible for a significant portion of drug-induced toxicity.
    • Eventually, pharmacogenomics may become a tool for individualizing drug therapy!
  • 12. Overview
    • Genetic differences
      • Man vs. Monkey vs. Rabbit vs. Rat vs. Guinea pig
      • Differences can be where in the drug metabolism occurs
        • Meta vs. para in aromatic rings and which of two aromatic rings
      • Example: Cats
        • Can’t conjugate phenols by glucuronic acid
        • Sulfate conjugate instead  ASA BAD for kitty!
      • Example: Pigs
        • Lack sulfotransferase but very efficient glucoronic acid conjugation
      • Ex: Rabbits
        • Cottontail met. hexobarbitol 10X faster than New Zealand
      • Humans: Genetic/hereditary differences account for huge differences seen in the rate of enzyme metabolism
  • 13.
    • Differences between the sexes
      • Appears to be species dependent
        • Huge difference between male and female rats
        • No differences in rabbits and mice
      • May also be a function of what drug is being metabolized
      • Sex hormones: androgens tend to increase metabolism
      • Humans
        • Examples: nicotine and aspirin
    Overview
  • 14. Genetics Review
    • Allele: Any one of a series of two or more different genes that occupy the same position (locus) on a chromosome.
    • Alleles determine genotype
    • Genotype displayed as a phenotype (eye color)
    • Two identical alleles result in a homozygous dominant or recessive trait of that gene.
      • Blue eyes, blonde hair,
    • Single nucleotide polymorphism: SNP
      • Nonsynonymous or synonymors (silent)
    • Variations in human genome often SNP’s
  • 15. Glucose-6-Phosphate Dehydrogenase
    • Early 1950’s discovery (one of the first!)
    • Anti-malarials causing hemolytic anemia in people with G6PD deficiency
    • More than 400 known variants
      • All seem to produce decreased G6PD activity.
      • Reduced GSH concentrations in RBC’s
      • Hemolytic anemia
    • Affects 400 million people worldwide.
    • Most are asymptomatic
    • Deficiency is an X-linked recessive trait
    • G6PD deficiency varies among ethnic groups
      • Most common in males of Mediterranean/African heritage
  • 16. Glucose-6-Phosphate Dehydrogenase
    • G-6-PD expressed in all body tissues
      • Carbon flow through pentose phosphate shunt
      • Production of NADPH
      • Glutathione reduction (GSSG  GSH)
    • Absence of GSH allows oxidation of Hb sulfhydroxyl groups  hemolysis
    • Now over 20 drugs known!
      • Primaquine, sulfones, sulfonamides , nitrofurans , Vitamin K analogues, cefotetan , chloramphenicol
  • 17. N-Acetyl Transferase ( NAT )
    • Phase II conjugating enzyme (Liver)
      • N- acetylation (deactivation): arylamines ( carcinogens )
      • O- acetylation (activation): hererocyclic amines
    • Most work on NAT2 locus – 27 alleles reported
    • Possible link to cancer risk?
    • Acetylation with Acetyl-CoA is either fast or slow
      • Genetic differences in NAT activity
        • Caucasions & African Americans – 40-70% Slow
        • Japanese & Canadian Eskimo – 10-20% Slow
        • Egyptians > 80% - Slow
        • Asia: Further N, less chance of Slow.
        • Eskimo & Asians often Fast
      • SLOW : more likely to show toxicity or adverse reactions to drugs
      • FAST: more likely to show an inadequate therapeutic response to standard doses of drugs
    Isoniazid and Hydralazine are key drugs linked to this enzyme system
  • 18.
    • Example: Isoniazid used for tuberculosis
      • SLOW: t 1/2 = 140-200 minutes
        • Higher plasma accumulation and higher cure rate
        • More adverse side effects and drug-drug interactions
        • Example of drug interaction: phenytoin use with isoniazid
          • Isoniazid inhibits phenytoin metabolism leading to accumulation of high and toxic plasma levels of phenytoin
      • Fast – t 1/2 = 45-80 minutes
        • Lower plasma accumulation and lower cure rate
        • More associated liver damage and hepatitis with isoniazid due to the more rapid formation of more acetylhydrazine
    N-Acetyl Transferase ( NAT )
  • 19.
    • Inducers and Inhibitors:
      • An overwhelming subject  information overload!
      • Primary method to eliminate drugs
      • CYP mainly the liver; also GI epithelium and other tissues
      • Pharmacogenetic factors  large number CYP isoenzymes
        • Most arise due to single nucelotide differences or polymorphisms (SNP) in genes encoding drug metabolism enzymes
        • May result in altered activity, altered stability of the enzyme, or introduction of a premature stop codon leading to a truncated protein
        • SNP errors can lead to mis-splicing of genes, complete gene deletion or gene amplification
        • Changes can lead to drug accumulation (toxicity), increased rates of drug elimination, and changes in activity / toxicity profiles due to altered formation of active metabolites
    Cytochrome P450
  • 20.
    • Overview continued:
      • At LEAST 50 (57) isoenzymes, grouped based on their a.a. sequences
      • Example: CYP3A4: Cytochrome P450, family “3”, subfamily “A” and the 4 th enzyme in the subfamily
      • Most CYP-450 enzymes involved in drug metabolism belong to the three distinct families, CYP1, CYP2 and CYP3 (50% of all drugs)
        • Some drugs processed by several CYP450 isoenzymes
    Cytochrome P450
  • 21. CYP450 Shimada T et al. J Pharmacol Exp Ther 1994;270(1):414. CYP3A CYP2D6 CYP2C CYP1A2 CYP2E1 Relative Importance of P450s in Drug Metabolism CYP3A CYP2C CYP1A2 CYP2E1 ? CYP2D6 Relative Quantities of P450s in Liver
  • 22. CYP1A1 Multiple PAH CYP2A6 Liver aflatoxins CYP2B6 Liver nicotine CYP2C8 Liver taxol CYP2E1 Liver, GI tract ethanol, benzene CYP3A4 Liver, small intest. paracetamol Isoenzyme Organ Typical substrate Cytochrome P450 Summary
  • 23. CYP3A Family
    • Predominant subfamily of CYP enzymes
    • Expressed primarily in liver & small intestine
    • Involves metabolism of
      • HIV protease inhibitors
      • Benzodiazepines
      • Calcium channel blockers
      • HMG CoA Reductase inhibitors - Statins
      • Antineoplastic drugs
      • Nonsedating antihistamines
      • Immunosupressants
    • Variation creates efficacy & toxicity differences
    • Common types: CYP3A4, CYP3A5, & CYP3A7
  • 24.
    • CYP3A4
      • ~ 50% of drug/corticosteroid metabolism
      • Major contributor of first-pass metabolism
      • Individual variance as much as 50-fold
    • CYP3A5
      • Present in only 10 – 30% of livers tested
      • May play a significant role in CYP3A metabolism
      • Important contributor to racial CYP variation
      • Accounts for at least 50% of CYP3A in those with the wild type allele (CYP3A5*1)
      • People with at least one wild type allele express large amounts of CYP3A5  2.5 x increase in midazolam (Versed) met.
      • More frequently expressed in non-caucasions
        • 30% - Caucasions, Japanese, Mexicans
        • 40% - Chinese
        • 60% - African Americans, SE Asians, Pacific Is., SW Native Am.
    • People with mutations in both 4 & 5: show lack of efficacy!
    CYP3A Family For the CYP3A family, think: INCREASE
  • 25. CYP3A Inhibitors
    • Antifungals
      • Ketoconazole
      • Itraconazole
      • Fluconazole
    • Cimetidine
    • Macrolide antibiotics
      • Clarithromycin
      • Erythromycin
      • Troleandomycin
    • Grapefruit juice
  • 26. CYP3A Inducers
    • Carbamazepine
    • Rifampin
    • Rifabutin
    • Ritonavir
    • St. John’s wort
  • 27. CYP2D6
    • Metabolizes 25 – 30% of clinically “key” medications
      • Dextromethorphan
      • Beta-blockers
      • Antiarrythmics
      • Anti-depressants
      • Antipsychotics
      • Morphine derivatives – codeine, oxycodone, etc.
      • Others
    • Most genetic variation (75 variants so far)
    • Linked more commonly to slow/poor metabolizers
      • 1% - Asians
      • 2-5% - African Americans
      • 6-10% Caucasions
    • Slower on average
    • Lower frequency of nonfunctional alleles
    • Higher frequency of reduced activity alleles
  • 28. Diversity of CYP2D6 Metabolism of the drug debrisoquine (antihypertensive)
  • 29. CYP2D6
    • Absent in 7% of Caucasians, 1 – 2% non-Caucasians
    • Hyperactive in up to 30% of East Africans
    • Catalyzes primary metabolism of:
      • Codeine
      • Many  -blockers
      • Many tricyclic antidepressants
    • Inhibited by:
      • Fluoxetine
      • Haloperidol
      • Paroxetine
      • Quinidine
    Aklillu E et al. J Pharmacol Exp Ther 1996;278(1):441– 446
  • 30. CYP2C9
    • Linked to impaired metabolism
      • Phenytoin
      • S-Warfarin
      • Tolbutamide (diabetes)
      • Losartan (antihypertensive)
      • NSAID’s including COX-2
    • Biggest problems: warfarin and phenytoin
      • Poor metabolism – increased effects!
      • Warfarin  bleeding out
    • Absent in 1% Caucasians and African-Americans
    • Inhibited by Fluconazole
  • 31. CYP2C19
    • Mutations mostly lead to slow metabolizers
    • Responsible for metabolism of relatively few drugs
    • Important drugs affected:
      • S-mephenytoin
      • Proton-pump inhibitors (omeprazole - Prilosec)
      • Diazepam - Valium
      • Propanolol – (  -blocker)
      • Imipramine – Tofranil (antidepressant)
      • Amitryptiline – Elavil (antidepressant)
    • Absent in 20 – 30% of Asians, 3–5% Caucasians
    • Inhibited by:
      • Omeprazole
      • Isoniazid
      • Ketoconazole
  • 32. CYP1A2
    • Induced by smoking tobacco
    • Catalyzes primary metabolism of:
      • Theophylline
      • Imipramine
      • Propranolol
      • Clozapine
    • Inhibited by:
      • Many fluoroquinolone antibiotics
      • Fluvoxamine
      • Cimetidine
  • 33.  
  • 34.  
  • 35. Reasons for In vitro Assays
    • Speed
    • Expense
    • Ability to select specific enzymes
    • Ability to control reaction conditions
    • Differences in human versus animal
    • isozymes
  • 36. New Technology - AmpliChip
    • July 2003 – Roche Pharm.
    • CYP2D6 & CYP2C19
    • $350 - $400
    • Roughly 10% of Caucasians and 20% of Asians are poor metabolizers
    • 100,000 Deaths in US alone
    • 25 million people affected