CYP inhibition





Cytochrome P450 comprise a superfamily of heme-containing mixed function oxygenases expressed in many mammalian tissues but found at the highest level in liver. These hemoproteins play an important role in metabolism of a wide variety of xenobiotics and endogenous compounds. The human CYP genes have been  characterized and classi?ed into various families and  subfamilies based on their structures. The gene families CYP1, CYP2, and CYP3 are currently thought to be the main CYP enzymem responsible for the oxidative metabolizm of drugs or xenobiotics. Inhibition of cytochrome P450-mediated metabolism is often the mechanisms for drug-drug interactions. A drug that inhibits a specific drug-metabolizing enzyme can decrease the metabolic clearance of a coadministered drug that is a substrate of the inhibited pathway. A consequence of decreased metabolic clearance is elevated blood concentrations of the co-administered drug, which may cause adverse effects or enhanced therapeutic effects. On the other hand, the inhibited metabolic pathway could also lead to decreased formation of an active metabolite of the co-administered drug, resulting in decreased efficacy of that drug.
The potential for enzyme inhibition is assessed by performing in vitro inhibition studies using cDNA-expressed enzymes or human liver microsomes.

Method: The assays for cytochrome P450 inhibition facilitate the identification of drug candidates with lower potential for drug-drug interactions. In vitro experiments conducted to determine whether a drug inhibits a specific CYP enzyme involve incubation of the drug with probe substrates for the CYP enzymes. Recombinant cytochrom P450 isoforms employs the assay: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 with various probe substrates enabling fluorescence detection or more specific LC/MS/MS analysis. The protocol uses a single substrate concentration near the apparent Km and multiple test article concentrations. An IC50 is determined as the point where 50 % inhibition of enzyme catalytic activity occurs.


CYP3A4 inhibition assay (Recombinant CYP3A4, substrate - BFC, 7-Benzyloxy-trifloromethylcoumarin)

          
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