L17-Methods used in drug metabolism studies Flashcards
what are the main cells of liver responsible for metabolism
parenchymal cells
what are the gold standard for liver studies and why?
primary hepatocytes
retain in vivo-like functions
limitations of using hepatocytes
- limited liver cell sources availible
- cryopreservation can cause loss of enzyme function
culturing:
Long-term culture leads to dedifferentiation, losing functionality.
what helps maintain function with hepatocytes ?
co-culture with non-parenchymal cells
- 3D culture with ECM sandwich mimics in vivo conditions
what are microsomes?
fragments of cell membranes containing enzymes like CYPs and UGTs essential for drug metabolism
+ve of microsomes
-cyropreservation - cna be frozen without loosing enzyme function - long term storage
pooled (multiple doners) vs induvidual (single doner)
- microsomes can easiliy be derived from any organ
why might activity levels vary in bank of human liver microsomes?
- genetic variation (SNP)
- lifetime exposure to inducers
- lifestyle differences
what does the liver S9 fraction contain ?
- cytosolic and microsomal fractions
(similar phase 2 and 2 enzymes as hepatocytes)
benefit of S9 fractions
more representivive compared with microsomes and cytosolic fractions
what is useful for investigating the contributions of individual rCYPs to metabolism ?
heterologous recominant systems
what is required for P450 expression?
- full length cDNA for the isoform of interest availible
- NADPH-P450 reductase - cytochrome B5 must be resent for activity
transient expression explain
- disapears over time
- cDNA in nucleus but doesnt achive genomic integration
- gene not reproduced
- short period of expression
SHORT TERM EXPERIMENTS
describe stable expression
- expression maintained throughout cell lineage
- genomic integration of cDNA
- gene inherited through mitosis
- daughter cells express product
GOOD FOR LONG TERM EXPERIMENTS
whats required for optimising P450 expression in e coli?
- modify cDNA N-terminal sequence
- change the amino acid after the start (Met) to Ala
- increase AT content at the 5’ end
- add histidine (his) tag at the 3’ end for easy purification
what are the outcomes of E. coli optimisation ?
- active enzyme levels are very variable
- removing part of n-terminus can make P450 enzyme soluble
- helps improve expression and funciton in e. coli
benefits of yeast as a P450 expression sytem 1
- natural (endogenous) P450 oxidoreductase
- some stains can express human oxidoreducatase
- can perform posttranslational modifications
benefits of yeast as a P450 expression sytem 2
- contains membrane organelles (human cells - good for enzyme folding)
- can isolate P450-containing microsomes for further use
- cheap and scaleable
issues with P450 expression in E.coli
- no internal membrane system
- p450s have nothing to anchor into
issues with P450 expression in yeast
- yeast already express own P450s
- difficult to isolate / investigate
benefits of insect cells compared to e coli or yeast
- carry out more complex post - translational modifications than bacteria or yeast
- cotrasnfect P450 and oxidoreductase
- HIGH LEVEL , transient expression
- allows microsome isolation
- Supersomes = ready-to-use, commercial microsomes from these cells
issues with inset cells
- baculoviral vestors are technically demanding to work with
- higher cost longer duration
What non-P450 can readily be expressed in E.coli ?
sulfotransferases, GST
what non-p450 metabolic enzyme more difficult to be expressed ?
UGT and FMO but like P450s now expressed in
insect cells using baculovirus
usefulness and benefits of p450 expression in mammalian cells
- effects of xenobiotics on cell survival
- assesment of mutagenicity
- more human relevant
- ensures proper protien folding , postranslational modification and localisation
- similar control pathways e.g. DNA repair cell cycle control gene reg
what are COS cells?
- monkey kidney cells that have little endogenous P450 but adequate P450 oxidoreductase
how do you get P450 expression in COS cells?
- cloning cDNA into vector with strong promoter and transfecting cells
- (his process allows scientists to insert a plasmid (a small DNA) into COS cells, and thanks to the SV40 system, the plasmid can copy itself inside those cells. It’s useful for studying how substances or genes behave, including in toxicological studies (studies on how toxins affect the body).)
what is a major limitation for toxicological studies? + y?
Transient expression a
major limitation for
toxicological studies
- researchers often need to observe the effects of a substance over time.
how are human lymphoblastoid cells kept growing?
stimulated to enter the G1 phase of cell cycle- immortal
how is tocicity investigated with human lymphblastoid cells?
(epstein barr virus)
- transfect with P450 cDNA
- treat with compound
- isolate metbolites
- investigate toxicity and mutagenicity
why and how do isoform-specific inhibitors block specific CYP enzymes?
- Added to human liver microsomes and drug substrates
-Helps study how drugs are metabolized by inhibiting particular enzymes
Examples of Isoform-Specific Inhibitors
Furafylline: Inhibits CYP1A2
Sulfaphenazole: Inhibits CYP2C9
Quinidine: Inhibits CYP2D6
Troleandomycin: Inhibits CYP3A4
how are antibodies used to block enzyme activity?
- Polyclonal or monoclonal antibodies can affect the activity of specific CYP isoforms
-Antibodies are raised against specific isoforms to block their activity
what is the antibody inhibition process ?
- preincubate the antibody with human liver microsomes
- add substrate (drug being tested )
HELPS STUDY the effect of inhibiting specific isoforms in drug metabolism
inhibition studies, correlation analysis human microsom ebank or recombinant p450?
- Different systems have
different advantages and
limitations – using a
combination is
recommended
pg 22 draw
describe high-throughput CYP screening
- use of flurogenic substrate
- high - throughput
- less labour intensive vs HPLC/LCMS
- could give initial idea of which CYPs are involved to study further using marker reactions/ probe substrates
24 look at
explain animal models to study drug metabolism
most common - rodents
benefits of whole body metabolim + toxicity
- wide gene variation
- some human CYPs have no animla eq - some drugs toxic there some not
pg 26
explain hepatic cell lines
- derived from liver tumour tissure or primary hepatocytes in vitro
- immortalized (non-tumour) cell lines created with viral oncogenes or telomerase
- easier to maintain and culture for experiments
- HepG2 is most commonly studied human hepatoma cell line
limiations of hepatic cell lines in drug met studies
- cancer cells need mroe energy to grow- change how they break down drugs
- no reccomended when accurate drug metabolism cruicial
- stable/transient expression of CYP/drug metabolizing enzymes can be used for specific experiments
what are in cilico computational methods used for?
- predicit regioselectivity
-predicit metabolites
-predicitinteractions of drugs with metabolising enzymes - predict toxicological effects of metabolites
