in vitro DMPK Flashcards
how is oral absorption predicted (lipinski rule of 5)
Lipinski’s rule of 5
1. MW<500
2. cLogP < 5 (lipophilicity measure)
3. hydrogen donors < 5
4. N/O atoms < 10 (no more than 10 H bond acceptors)
95% of drugs do not break more than two rules
how is absorption modelled in vitro, vivo and situ.
in vitro:
1. artificial membranes like PAMPA.
2. epithelial monolayers (MDCK/CACO-2)
3. affinity against efflux transporters (cell free ATPase assays/cell based with recombinant transporter expression)
in situ:
chamber isolated perfused rat gut (compound administered to whole intestine looks at disappearance from gut and appearance into blood)
in vivo:
hepatic portal vein sampling with radiolabelled compound with p glycoprotein knockout mice. (oral and iv administration needed)
what does PAMPA estimate
(synthetic phospholipid membrane)
passive transcellular permeability. (hydrophobic membrane with no transporters)
determine apparent permeability in cm/s
lipophilicity plays major role in passive diffusion.
polar surface area, molecular volume, hydrogen bonding also affect passive permeability.
does not account for intake and efflux transporters.
why are MDCK AND CACO cell lines important
mimic intestinal cells to understand process of oral drug absorption.
(contains enzymes and transporters.
permeability can be measured for both directions)
what are characteristics of CACO2 cells
Caco-2 cells grown (harder than pampa).
absorption quantified by HPLC or MS.
originated from human colonic cancer cells that mimic small intestine.
cells placed on semi-permeable membrane.
must calculate standard curves as there is variability in cell lines in terms of expression of transporters.
what can in vitro absorption assays with Caco2 cells determine
mechanism of absorption
(paracellular passive diffusion, transcellular passive diffusion , efflux, active accumulation/against conc. gradient)
measure drug levels to basolateral side from apical side
measure drug levels to apical side from basolateral side
if A to B = B to A passive ba/ab=1
if A to B > B to A active uptake into blood ba/ab<1
if A to B < B to A effluxed. ba/ab >1
What are some characteristics of MDCK cell lines
they are canine kidney cells
takes 3 days to grow
can be genetically altered to express one transporter of interest
how can plasma protein binding be measured and why is it important
fresh plasma in filter devices or using dialysis
determines levels of unbound drug in circulation (drug available to target. also determines clearance)
how is Blood brain barrier permeability measured
transfected MDCK cell lines
(MDR1 or BCRP genes)
what are examples of in vitro renal models
- transfected cell lines (MDCK/HEK transfected with uptake and efflux transporters)
- primary cell cultures (proximal tubule cells)
- kidney slices
- isolated perfused kidney (intact organ, expensive viable for 2 hours)
what are microsomes
fragments of smooth ER with ribosomes. they contain all membrane bound proteins of the cell. cofactors like NADPH can be used to initiate metabolism simulations in vitro.
what are benefits of using hepatocytes
contain all metabolising enzymes of the liver (microsomal and cytosolic)
can be used to asses metabolic stability and to identify metabolites. (assessed metabolism and metabolites)
how is intrinsic clearance (metabolic clearance) measured
substrate disappearance is measured at set enzyme and substrate concentrations.
substrate dissaperance to time (x) graph
natural log graph
slope is rate of metabolism
vmax/Km (initial rate/substrate conc)
what is the difference between first order and zero order kinetics
first: metabolism dependent on one substrate conc (at start of metabolite production)
zero: metabolism dependent on enzyme conc. (towards end of metabolite production)
what are some requirements for ivive
- major clearance pathway must be metabolic
- metabolism must be first order
- substrate cant inhibit product
- disappearance of substrate must be detectable.
