ADT Exam 3

  1. Biological factors contibuting to drug response
    • sex
    • age
    • disease
    • physiology
    • hormonal status
  2. Environmental factors contributing to drug response
    • drug treatment
    • food
    • recreational drugs
    • chemical exposure
  3. Genetic polymorphisms contributing to drug response
    • drug metabolizing enzymes
    • transporters
    • transcription factors
    • drug receptors
  4. Metabolism results in
    • detoxification and elimination
    • activation of prodrug
    • formation of toxic metabolite
  5. Enzymes catalyzing metabolite formation typically show
    wide inter-individual variability in protein expression and/or catalytic activity
  6. Phase I Biotransformation
    • slow
    • oxidation
    • reduction
    • hydrolysis
  7. Phase II biotransformation
    • fast
    • glucuronidation
    • sulfation
    • glutathione conjugation
  8. location of most phase I reactions
  9. location of phase II reactions
    mitochondria and microsomes
  10. CYP450
    • major route of Phase I metabolism
    • heme-thiolate containing family of proteins in ER
    • 57 forms identified and characterized in the human
    • broad overlapping substrate specificity
    • distinct catalytic activity and regulation
  11. basic reaction catalyzed by CYP450
    • monoxygenation
    • one atom of O is incorporated into a substrate and the other is reduced to water with NADPH
  12. Alteration of P450 Activity
    • enzyme induction
    • enzyme inhibition
    • genetic polymorphism
  13. Does not appear to be inducible
  14. Inducing agents
    • rifampin
    • penobarbital
    • phenytoin
    • carbamazepine
  15. Inducing agents decrease effects of
    • birth control
    • cyclosporin
    • warfarin
    • protease inhibitors
  16. fasting and alcohol cause
    increased effect of acetaminophen
  17. types of inhibition
    • competitive
    • non-competitive
    • mechanistic (suicide)
    • complex
  18. competitive inhibition
    • drugs compete for active site of enzyme
    • reversible
  19. uncompetitive inhibition
    drug binds to P450 (outside of active site) then alters binding ability
  20. mechanism based inhibition
    • production of covalent adduct that eliminates the p450
    • irreversible
  21. complex inhibition
    • drug/metabolite forms tight complex with enzyme that may be titrated away
    • reversible
  22. increased pharmacodynamic effect of warfarin seen with
    CYP2C9 inhibitors (diclofenac)
  23. increased toxic effect seen with terfenedine and cisapride along with
    • cyp3A inhibitors
    • ketoconazole
  24. increased side effects of PIs seen with
    • CYP3A inhibitors
    • ketoconazole
  25. ___ % of caucasian population deficient in CYP2D6
  26. Poor metabolizers have
    two inactive forms (alleles) of the gene
  27. some drugs that may not work with CYP2D6 PM
    • codeine
    • tramadol
    • tamoxifen
  28. approaches to study phenotype
    • invitro system/expression of genetic variants
    • analysis of tissue samples
    • in vivo phenotyping (volunteers, patients)
  29. Most relevant for clinical phenotyping
    in vivo phenotyping of volunteers and patients
  30. most sensitivity and resolution
    in vitro systems/ expression of genetic variants
  31. Phenotype
    any observable or measureable morphologic, analytic biochemical, physiological or behavioral characteristics of an organism
  32. In pharmacotherapy "phenotype" means
    any change in measured/observed parameter after drug administration
  33. Phenocopy
    mimicry of genetic defect by an environmental influence
  34. Phenotypic information after drug administration
    • plasma drug/metabolite concentrations
    • drug/metabolite in urine
    • AUC
    • C13 -CO2 marks metabolism in expired air
    • pharmacodynamic response
    • molecular/cellular functional assays
    • clinical outcomes
  35. Why do we care about phenotype?
    • predict drug response
    • right drug for right disease for right patient
    • estimate biotransformation to select proper dose
    • characterize developement of enzymes from birth to adult
  36. Options for estimating biotransformation capacity
    • pharmacokinetic studies
    • phenotyping studies
  37. Pharmacokinetic studies
    • AUC reliable estimate of clearance
    • invasive
    • ethics of non-therapeutics used in special populations (babies)
    • limitations of with sample volumes and scheduling
    • parent compound vs metabolite
  38. Phenotyping studies
    • measure of target enzyme activity
    • estimate clearance via metabolic pathway
    • relatively non-invasive (saliva, urine, breath test)
    • selection of phenotyping probe is essential
    • validation of probe as a measure of target enzyme activity is critical
  39. Characteristics of phenotyping probes
    • safe
    • non-invasive
    • measures enzyme of interest metabolic pathway
    • intrinsic activity of enzyme should be rate limiting
    • not inhibitory to other enzymes
    • target pathway should be major route in vivo
    • probe should be readily absorbed
    • metabolite rapidly excreted
    • sufficient sensitivity
    • availability
    • low cost
  40. Metabolite ratios
    • surrogate measure for enzyme activty
    • most commonly used are urinary ratios
    • simplest ratio is drug/metabolite
  41. Intent of metabolite ratios
    tailor individual doses of therapeutic drugs
  42. Phenotyping procedure
    • obtain pre-dose blood or urine samples
    • administer probe
    • collect biological sample (blood, breath, urine) or complete PK time course
    • parent drug and metabolite analysis
  43. Parent drug and metabolite analysis
    • HPLC
    • GC with UV
    • fluorescence
    • MS detection
    • IR spectra of pre and post breath sample
  44. DM metabolite ratios
    • DM/DX is used
    • >0.3 = PM
  45. Phenotyping probes for pediatrics
    • safe for all ages
    • perceived as safe by the parent
    • oral administration/non invasive (urine, breath)
    • suitable formulation
    • estimation of target enzyme activity (glucuronidation)
  46. Purpose of Pre-Clinical (in virto) studies
    • identify most active compunds
    • select appropriate animal models for toxicity studies prior to human exposure
    • determine PK and metabolism characteristics to provide rational design of clinical trials and predict outcomes
  47. Role of drug metabolism information in drug developement
    • stability
    • metabolite identification
    • interspecies comparisons
    • toxicology
    • drug-prodrug evaluation
    • pharmacokinetic variability
    • drug-drug interactions
  48. In vitro systems for studying xenobiotics from worst to best
    • cultured hepatocytes
    • liver slices
    • isolated hepatocytes/cDNA expressed enzymes
    • sucellular fractions (microsomes; HLMs)
  49. in vitro systems for studying xenobiotic induction of metabolism from worst to best
    • Liver slices
    • cultured hepatocytes/expression constructs
  50. what is needed for In vitro incubations to assess P450 activity
    • enzyme source
    • substrate
    • buffer
    • nadph
    • oxygen
    • reaction vessel
    • stop reagent
    • analytical method
  51. What is reaction phenotyping
    identification of the enzyme responsible for metabolizing a drug
  52. Why perform reaction phenotyping
    • to predict pharmacokinetic variability by identifying drugs that are metabolized by a polymorphic enzyme
    • predict potential drug interactions
  53. Experimental approaches to reaction phenotyping
    • correlation analysis
    • inhibition
    • purified P450 enzymes and/or cDNA expressed enzymes
    • kinetic analyses
  54. type of reaction phenotypine
    • western blot
    • gives total amount of protein not functional protein
  55. Correlation analysis
    • comparison of metabolite formation with rates of marker activites in the bank
    • cofactors must be present in same proportions
    • reactions must be performed under same conditions
    • more useful if only 2 forms of p450s are involved
  56. correlation analysis pitfalls
    • can be influenced by a single outlying point
    • regression line may not pass close to the origin
    • mis-interpretation of data
    • lack of independent variation
    • multiple p450s with similar kms
    • discovery of a new enzyme
  57. chemical inhibition
    • readily available and inexpensive
    • solvent choice is important
    • mechanism based inhibitors require pre-incubation
  58. concentration used for chemical inhibition is dependent on
    • ki of the inhibitory chemical
    • km for the formation of the metabolite
  59. competitive inhibition should be
    concentration dependent
  60. pitfalls of chemical inhibition
    • solvents can inhibit
    • inhibitors of one p450 can activate another
    • inappropriate concentrations of inhibitors or substrates used
    • some inhibitors are not commercially available
  61. antibody inhibition
    • inhibition of metabolite formation in the presence of specific p450 inhibitory antibodies
    • inhibit non-competitively
    • not influenced by substrate concentration
  62. potential probelms with antibody inhibition
    • difficult to obtain
    • may only partially inhibit enzyme activity
    • must be well characterized
    • high ratio of antibody:microsomal protein may produce non-speficic effects (10:1)
  63. cDNA-Expressed P450s
    • method of choice to compare allelic variants
    • can determine Km for individual enzyme
    • can determine specificity of chemical inhibitors
    • not effective in determining rate
  64. cDNA expressed P450 pitfalls
    • variable expression or accessory protein may effect activity
    • lipid composition differing from HLM may affect activity
    • comparison of recombinant P450s and HLMs require a correction factor for comparisons
  65. Kinetic analysis
    • uses HLMs
    • obtain Vmax and Km
    • gives intrinsic clearance
    • shows whether metabolite formation is catalyzed by one or more enzymes
  66. Pitfalls of kinetic analyses
    • protein concentraiton <0.5 mg/ml recommended
    • substrate consumption should be <15%
  67. intrinsic clearance
    • vmax/km
    • measure of clearance of drug via metabolism by the liver
    • can be used to model in vivo clearance values
  68. characterization of allelic variants
    • variant DNA sequence obtained
    • Variant cloned into plasmid
    • Variant transfered into expression system
    • Isolate expressed protein
    • kinetic analysis with marker substrate & compare to wt
  69. no known inducers
  70. more important as a cause of serious drug interactions
    • inhibition
    • occurs more immediately
  71. to study P450 inhibition
    • human liver microsomes
    • determine type of inhibition
    • determine ki
  72. why anticipate and minimize drug interactions?
    • minimize toxicity and loss of efficacy
    • cost in $ and time to develope a drug
Card Set
ADT Exam 3
Assessment of Phenotype