The initial rate of catalysis by CYP450, or any enzyme, is directly proportional to the enzyme concentration (assuming the enzyme concentration is much lower than the substrate concentration). As long as you measure the initial rate (before the reaction slows down due to depletion of the substrate or product inhibition), the reaction rate will be proportional to the CYP450 concentration.

You should normalize the initial reaction rate of different samples by dividing the rate by the total protein concentration (all proteins, not just CYP450) to adjust for differences in the amount of microsomes present in each sample.

To the colleague at University of Puerto Rico:

Replying to your concerns with regard to wether you deal with induced or inhibited CYP, and relationships between CYP content and its activity, the following parameters we used to use in Xenotox, Inc (http://www.xenobiovir.com/):

1) Determining if the drug is an inhibitor of CYPs by conducting In vitro studies You should evaluate an investigational drug’s potential to inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A in both a reversible manner (i.e., reversible inhibition) and time-dependent manner (i.e., time-dependent inhibition (TDI)), and thereafter calculate the ratio of intrinsic clearance values of a probe substrate for an enzymatic pathway in the absence and in the presence of the interacting drug.

2) Determining if the investigational drug is an inducer of CYPs. By conducting in vitro, you should evaluate the potential of an investigational drug to induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4. Initially, conduct experiments to evaluate CYP1A2, CYP2B6, and CYP3A4 ONLY. If no induction of CYP3A4 enzymes is observed, evaluating the induction potential of CYP2C enzymes is not necessary because both CYP3A4 and CYP2C enzymes are induced via activation of the pregnane X receptor (PXR). If the investigational drug induces CYP3A4, please evaluate the potential of the investigational drug to induce CYP2C. However, a negative in vivo study with a CYP3A sensitive substrate can be used to rule out induction potential of an investigational drug on CYP2C enzymes, as long as the potential of CYP3A inhibition by the drug and its metabolite(s) can be excluded.

3. Data Analysis and Interpretation. If working with human samples, the induction results should be evaluated separately for each donor. If the result from at least one donor exceeds the pre-defined threshold, the sponsor should consider the drug to have induction potential and conduct a follow-up evaluation. Several basic methods can assess the potential of an investigational drug to induce metabolizing enzymes Thus utilize fold-change method: The sponsor can examine the fold-change in CYP enzyme mRNA levels when incubated with the investigational drug by using a cutoff determined from known positive and negative controls to calibrate the system. For example, a drug is interpreted as an inducer if: (1) it increased mRNA expression of a CYP enzyme in a concentration-dependent manner; and (2) the fold change of CYP mRNA expression relative to the vehicle control is ≥ 2-fold at the expected hepatic concentrations of the drug. Expected drug concentrations in the liver can be calculated by assuming a certain fold of Imax. The induction potential should not be ruled out for an investigational drug that increases CYP enzyme mRNA less than 2-fold the of vehicle control, if the increase is more than 20% of the response of the positive control. Further evaluation is recommended when there is an inconclusive finding. In this case, please consider nonbinding recommendations, such as calculate the percent of the response to the positive control (the following equation should be used: % of positive control = (mRNA fold increase of test drug treated cells - 1) × 100/ (mRNA fold increase of positive control – 1). Otherwise, you might deal with correlation methods: The sponsor may use correlation methods predict the magnitude of induction effect (e.g., AUC ratio of index substrate in the presence and absence of inducers) of an investigational drug according to a calibration curve of relative induction scores (RIS) or Imax,u/EC50 for a set of known inducers of the same CYP. If the predicted magnitude is more than a predefined cut-off (e.g., AUC ratio ≤ 0.8), a drug is considered have induction potential in vivo. Sometimes, Emax or EC50 cannot be estimated due to an incomplete in vitro induction profile. An alternative correlation approach may be used if the method is validated. Calculate Imax,u / EC50 values Emax is the maximum induction effect determined in vitro. EC50 is the concentration causing half-maximal effect determined in vitro. When static mechanistic models or dynamic mechanistic models are used for predicting DDIs caused by enzyme induction, the models should include the induction mechanism only (i.e., the model should not include concurrent inhibition predictions for an investigational drug that is hypothesized to be both an inducer and inhibitor) to assess the potential of an investigational drug to induce metabolizing enzymes.

4) When evaluating whether an investigational drug is an inhibitor of multiple CYP enzymes, tyou can prioritize in vivo DDI evaluations for various CYP enzymes with sensitive index substrates of respective pathways (see the January 2020 FDA guidance for industry Clinical Drug Interaction Studies — Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions) preferably using the in vitro inhibition parameters obtained in the same study.

5. Finally, just directly addressing your question, if induction potential is assessed via incubation of hepatocytes in culture with a test article to and is reported as the fold-change in CYP enzyme mRNA levels or the fold-change in enzyme activity, using selective probe substrates. Although both endpoints are accepted by regulatory authorities, it is mentioned that in case of activity studies, induction may be masked by concomitant inhibition. Therefore, the general practice is to perform both read-outs for a complete study design.

All the best,

Ilya Tsyrlov, MD, PhD

Have you conducted any time and protein linearity assay? If yes, and if your assay condition is within the linear range concerning time and protein concentration, it may be safe to assume that the activity is proportionate to enzyme content.

If I'm interpreting your query correctly, you were asking if simply determining the quantity of P450 by measuring its amount (CO difference spectrum?) determines its activity, the answer is NO. Otherwise, its activity is determined by the conversion of the substrate you are interested in measuring to the product according to the protocols previously suggested above. The AMOUNT of P450 protein then must be estimated by its spectral determination in an unpurified preparation and can be directly related to its protein concentration in a purified preparation. Specificity is an issue in a non-purified preparation, so you'd have to relate the induction to a specific substrate: a fatty acid, steroid, drug, carcinogen, etc. In other words, you could say that TOTAL cytochrome P450 level may be induced as measured by specific content (concentration/mg) but NOT for a specific substrate, unless you measure its rate of conversion. I hope this clarifies the issue.