Lecture 8/9 - Experimental Models and Methods to Evaluate Metabolism Flashcards
(13 cards)
What is the philosophy for drug development?
-less toxic and high efficiency
-minimal drug
-know all metabolites - Any metabolite accounting for 2% or more need to characterize - fda has different toxicity criteria for cancer drugs (hiv used to be in that category)
-need better methods for metabolite identification
What is an overview of metabolism evaluation?
in vitro analysis tier 1 - log D, aqueous solubility, microsome stability, plasma stability
in vitro analysis tier 2 - plasma protein binding, permeability, cytotoxicity, cyp450 inhibition
in vivo analysis tier 1 - rapid assesment of compound, exposure - auc
in vivo analysis tier 2 - pharmacokinetics
What are some reaction phenotyping assays?
elimination of parent drug over time
-formation of metabolites over time
-kinetics of the clearance and generation of metabolites
What are reaction phenotyping stduies performed based on?
metabolite formation or substrate depletion
If all metabolites of a drug are known and metabolite standards are available then what can be done?
can use that to determine whcih enzymes are repsojnsible for the formation of a metabolite
What are some toxic metabolites?
toxic metabolites; fatty, aldehydes, and carbonyls- if have reactive metabolites body conjugates it so need to see how many conjugated metabolites I have - so need to worry about reactive intermediates you do not see - isolate conjugated metabolites so put in glutathione and cysteine and if you see these adults form can find the toxic metabolites so add conjugates into media then see - if green boxes on previous slide greater than 5% bad
What do in vitro drug metabolism studies for preclinical drug development show?
metabolite stability, identification , reaction phenotyping, species comparisons
take drug and put in heaptocytes - need transporters to bring drug in
-take drug put in s9 and is a fraction of cells an does not need to transport drug into the cell - put it in and run a mass spec for med id
-reaction phenotyping
-species comparisons - mice rats and dog pig rabbit monkey and then compare to human because fad will have you pick two animals to pick one lower and one higher order (guinea pigs lower order and ferrets high order) - sheep and cows very expensive and monkey limited amount if doing biologics and need monkey - whichever animal has closest metabolism to human pick
What is the general protocol for metabolic stability studies for drugs?
species - human, rat, mouse, dog, primate, minipig guinea pig, other species availble on request
test compound conc - 1uM
protein conc - 0.5mg/mL
time points - 0,5,15,30, and 45 mins
cofactor - 1mM NADPH
final dmso conc - 0.25%
compound requirements - 50uL of 10mM DMSO solution
controls - 0uM, minus cofactor (45 mins only), positive control compounds with known activity
analysis methods - LC-MS/MS
data delivery - intrinsic clearance, standard error of intrinsic clearance, half-life
What are the approaches to developing metabolic stability?
positive control substrates - 7-ethoxycoumarin, midazolam to validate that the test system is functionally within specification
negative controls - include both time zero and zero co factor samples
number of replicates - triplicates
instrumentation - LC-MS triple quadrupole MS
What are some species differences in drug metabolizing enzymes?
-orthologs of the major dmes are found in most species; however, within a species even a single amino acid change can alter the substrate affinity of an enzyme and potentially the metabolic clearance of the compound
dogs- not NAT
guinea pigs - no ST, no N-hydroxylation
cats - poor UGT activity
rats - rapid metabolizer, CYP2c has gender differences
cynomolus monkeys - has low cyp1a2 activity
How can isolated hepatocytes be used for an in vitro metabolism study?
-they are the gold standard for in vitro metabolism studies - have a full complement of hepatic dmes
-human hepatocytes are easy to use
-fresh cells not readily available
-can be cryopreserved
-use subcellular fractions where you break the hepatocyte and in the er get the liver micorosmes; get rid of cellular fraction might eliminate the enzymes - so companies do hepatocytes versus s9
hiv protease inhibitors causes clearance of hormones - hiv women need higher birth control pills due to protease inhitbuors increasing phase ii metabolism but block other metabolism - as ww age steroids go down -when you exercise regenerate steroidal metabolism
