Cytochrome P450 Interactions

Focusing on drug metabolism safety testing

Two inter-connected considerations for IND and NDA submission are safety and drug-drug interaction (DDI) studies. In vitro metabolism studies can help identify potential DDI liabilities, and the in vitro prediction can help guide design of your clinical DDI studies. Our validated in vitro metabolism studies, tied in with our well-characterized in vitro transporter assessments, allow Absorption Systems to support your drug from safety testing for IND submission to drug-drug interaction studies for NDA submission.

Induction

Why CYP induction testing?
CYP induction results in the increase in drug metabolism (metabolic clearance) and a subsequent decrease in drug therapeutic efficacy. Absorption Systems provides services to evaluate two markers of CYP450 induction: enzyme activity and gene expressions (mRNA of the gene encoding the enzyme of interest). We use clinically relevant concentrations with human hepatocytes to evaluate induction of CYPs, including CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4/5. Cell cytotoxicity by MTS assay is also evaluated in the same wells. Induction can also be performed in multiple animal species.

How do we approach CYP Induction testing?
Freshly isolated or cryopreserved hepatocytes are incubated with test article for 3 days.

  • Enzyme activity approach: Hepatocytes are then incubated with a selective probe substrate. Appearance of metabolite is compared to that of a control treatment (no test article) via LC-MS/MS in order to assess the ability of the test article to induce a given CYP’s activity.
  • mRNA approach: mRNA is then isolated from the hepatocytes. cDNA is synthesized and used for qPCR to measure fold induction of CYP-specific mRNA. Comparison of treated cells to a control treatment (no test article) gives the article’s CYP induction potential.

Induction Testing

  • Enzyme Activity Analysis
    • Incubate with test article
    • Assay enzyme activity with probe substrate
    • LC-MS/MS Quantitate
    • Compare treated vs control
  • mRNA
    • Isolate of mRNA from hepatocytes
    • qPCR analysis
    • Compare treated vs control
  • Clinically relevant concentration range
  • Human & animal hepatocytes
  • CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4/5
  • Cell cytotoxicity by MTS assay

Inhibition

Why CYP Inhibition testing?

CYP inhibition can result in a decrease in the rate of drug metabolism, either for the inhibitor itself (if it inhibits an enzyme that also metabolizes it) or for other, potentially co-administered drugs, either of which can result in toxity. Absorption Systems provides services to evaluate enzyme inhibition via reversible or time-dependent mechanisms at a single concentration or multiple concentrations (reversible: 1C50, Ki; time-dependent: IC50 shift, KI, kinact. The main focus of enzyme inhibition is CYP due to regulatory requirements, the fact that most drugs are metabolized at least in part by CYPs, and the many examples of clinically significant (some fatal) DDIs resulting from CYP inhibition.

How do we approach CYP Inhibition testing?

We incubate the test article, at either a single concentration or 7 concentrations, with a selective probe substrate and human liver microsomes. We monitor a specific metabolite and compare its rate of appearance with that of a solvent control to assess the extent of inhibition of a given CYP by each concentration of a test article. An IC50 can be determined when 7 concentrations are assessed. For reversible inhibition, an IC50 ≤ the plasma Cmax translates to a likely clinical DDI.
Time-dependent inhibition (TDI) is evaluated by quantifying the IC50 shift after pre-incubation of test article with liver microsomes +/- NADPH. Clinical correlation for a positive TDI score can then be determined by a follow-up in vitro measurement of KI and kinact.  Comparison of KI and kinact to plasma Cmax can predict potential clinical DDIs due to TDI.

Inhibition Testing

  • Reversible
    • Single or multiple concentrations
    • Monitor appearance of specific probe metabolite
    • IC50 Determined
  • Time Dependent
    • IC50 shift (screen)
    • KI, kinact (definitive)

Phenotyping

Why CYP phenotyping testing?

CYP phenotyping identifies the individual CYP isoform(s) responsible for metabolism of a test article. This is one of the obligtions of a sponsor, to perform a thorough investigation of the pathways of elimination (including metabolism) of an NME. In addition,  knowing the isoform(s) responsible for metabolism of a test article can guide the choice of clinical DDI study design (i.e., choice of co-dosed inhibitor or inducer).  Absorption Systems provides CYP phenotyping using two approaches: individual human recombinant CYPs and human liver microsomes +/- specific inhibitors.

How do we approach CYP phenotyping?

  • Incubation of test article with human liver microsomes and specific chemical inhibitors: Disappearance of test article is compared to that of a control treatment (no inhibitor) in order to assess the contribution of a given CYP to the test article’s metabolism.
  • Incubation of test article with human recombinant CYP enzymes: Disappearance of test article is compared to that of a control treatment (non-transfected) in order to assess the contribution of a given CYP to the test article’s metabolism.

Phenotype Testing

  • Human liver microsomes +/- selective inhibitors
  • Individual human recombinant CYPs
  • Estimate in vitro intrinsic clearance