METABOLIC STABILITY IN VITRO ASSAYS

Will the test compound / drug compound remain circulating in plasma within the body?

As mentioned earlier the liver is the major drug metabolizing organ for the large majority of pharmaceutical drugs.  For this reason, the in vitro models used to investigate drug metabolism often focus on hepatocytes or subcellular fractions of the liver such as microsomes, cytosol, S9 or mitochondria where concentrations of particular enzymes are high.

Microsomes contain Phase I oxidative enzymes including CYP enzymes. 

Liver microsomes are subcellular fractions which contain membrane bound drug metabolizing enzymes.  These do not have an intact cell membrane.

The assay uses subcellular fractions of liver, microsomes, to investigate the metabolic fate of compounds. Liver microsomes consist mainly of endoplasmatic reticulum and contain many drug-metabolizing enzymes, including cytochrome P450s (CYPs), flavin  monooxygenases, carboxylesterases, and epoxide hydrolase.

Liver microsomes are available commercially as frozen preparations that are usually prepared in bulk with pooled livers from sacrificed mice, rat or human cadavers (corpse/dead bodies).

Addition of relevant co-factor to the incubation is necessary (NADPH and UDPGA are added.  These are cofactors which are needed to initiate the Phase I metabolic reactions)

The use of species-specific microsomes can be used to enable an understanding of interspecies differences. [for example along with HLM (Human liver microsomes) – MLM (mouse liver microsomes) and RLM (rat liver microsomes) are used]

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Most often, metabolic stability of compounds are assessed at a single concentration (typically 10 μM) at t = 0 and at t = 60 min. Stability of compounds are tested in human (other species available) liver microsomes.

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S9 fraction is the post-mitochondrial supernatant fraction.  It contains both cytosolic and microsomal enzymes.  This is to identify if cytosolic enzymes are responsible for the formation of a metabolite. 

The advantage of using S9 fraction for in vitro screening is that it contains a wide variety of both phase I and phase II enzymes. 

Addition of relevant co-factor to the incubation is necessary [cofactors such as UDPGA and PAPS to investigate Phase II metabolic pathways.]

Hepatocytes are more representative of the in vivo situation because they contain a cell membrane and do not require additional co-factors.  Hepatocytes contain full complement of enzymes for both Phase I and Phase II metabolism.

The use of species-specific cryopreserved hepatocytes can be used to enable an understanding of interspecies differences in metabolism.

CYP inhibition studies: 

·         CYPs constitute a superfamily of heme enzymes

·         CYPs play a major role in the metabolism of a wide array of xenobiotics including drugs, chemical carcinogens, insecticides, petroleum products, and other environmental pollutants.

·         Although the liver is the primary organ for drug metabolism, extrahepatic tissues such as lung, kidney and intestine, also play an important role for detoxification or biotransformation of xenobiotics. Each tissue has a unique P450 isozyme distribution and regulatory mechanism for cytochrome P450 gene (CYP) expression.

v  In vitro cytochrome P450 inhibition data are useful in designing strategies for investigating clinical Drug-Drug interaction Studies

v  Assessment of the potential of a compound to inhibit a specific cytochrome P450 enzyme is important as co-administration of compounds may result in one or both inhibiting the other’s metabolism. This may affect plasma levels in vivo and potentially lead to adverse drug reactions or toxicity

v  CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 are few important CYP450 enzymes.  Identifying which of these enzymes are responsible for drug metabolism is important.

v  All of these cytochrome P450 inhibition reactions are incubated separately for each isoform.

v  CYP450 3A4 is of paramount importance, because it is the most abundant P450 in the human liver and is known to metabolize the majority of drugs whose biotransformation is known.