Cytochromes P450 are a family of enzymes which play a major role in the metabolism of drugs. Assessment of the potential of a compound to inhibit a specific cytochrome P450 enzyme is important as co-administrations of compounds may result in one or both inhibiting the other’s metabolism. This may affect the in vivo plasma levels  and potentially lead to adverse drug reactions or toxicity.

For the cytochrome P450 Inhibition assays, KaLy-Cell uses accepted probe substrates and cryopreserved human hepatocytes.

A decrease in the formation of the metabolites compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).

Reversible Inhibition

The seven main cytochrome P450 isoforms (CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 - other isoforms are available on request) are investigated in KaLy-Cell's Cytochrome P450 Inhibition assay. Isoform-specific substrates are incubated individually and with a range of test compound concentrations (typically 0.1 - 25µM) with human hepatocytes in suspension. At the end of the incubation, the formation of metabolite is monitored by LC-MS/MS at each of the test compound concentrations. An example of an IC50 profile is shown in Figure below.

A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition). Following IC50 determination we can then determine the test compounds Ki against the appropriate isoform. This will give information about the potency and the type of inhibition (competitive, non-competitive, uncompetitive or mixed). the Ki can also be used to estimate the impact of any potential in vivo interactions.

We run a known specific positive control inhibitors for each of the isoform assays.

 

Time Dependent Inhibition

Cytochromes P450 (CYP) are a family of enzymes which play a major role in the metabolism of drugs. Inhibition of those CYP enzymes is one of the most common causes of drug-drug interactions. The mechanism of inhibition can be reversible, quasi-irreversible or irreversible.

The draft FDA guidance for drug interactions (2012) and the EMA guideline on the investigation of drug interactions (adopted in 2012) recommends to evaluate time dependent inhibition for investigational drugs.

The Cytochrome P450 Time Dependent Inhibition: kinact/KI assay determines kinetic constants of time dependent inhibition. The kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor and KI is the concentration of inhibitor which gives half the maximal rate of inactivation. The experimental conditions are determined according to the results of previously performed assays such as P450 reversible inhibition or IC50 shift. Compounds are evaluated by pre-incubating a range of 7 test compounds concentrations (in addition with a vehicle control) selected so to achieve a scale from no inactivation to maximal inactivation, for 6 differing pre-incubation times (including 0 min) with human hepatocytes in suspension. Following the pre-incubation, an aliquot of the pre-incubation is diluted with a buffer containing a specific cytochrome P450 probe substrate (at 5×Km concentration) for a specific incubation time. Following least-squares regression analysis to determine the negative slope of the logarithm of the % remaining activity (corrected for negative control) versus pre-incubation time, non-linear regression analysis of the negative slopes against inhibitor concentration enables kinact and KI to be calculated.

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