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DDI

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Understanding your drug’s potential for drug-drug interactions (DDIs) is crucial for determining safety and efficacy. 

Drug-drug interactions can lead to unexpected changes in metabolism or transporter efflux/uptake, often with serious or fatal consequences.  IONTOX, LLC offers the full suite of in vitro assays described in the FDA guidance for drug interaction studies (FDA, 2012).  This includes the following:

Drug metabolism studies assessing Phase 1 enzymes in human microsomes or primary human hepatocytes. The CYP enzymes assessed are CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A4.  Other CYP enzymes are available upon request (e.g., CYP2A6, CYP2J2, CYP4A11, CYP4F2, CYP2E1).  LC/MS/MS is used to determine % of parent compound remaining.

The graph illustrates the metabolism of midazolam, which is selectively metabolized by CYP3A4. 

 

 

 

Drug metabolism studies assessing inhibition of Phase 2 enzymes in human UGT Supersomes™. The UGT enzymes assessed include UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15.  Inhibition of UGT enzymes is performed with industry standard probes.

The graph titled UGT1A$ illustrates the inhibition of UGT1A4 by diclofenac.  Diclofenac is one of the positive controls in this assay as inhibits a number of the UGT isoforms.

 

 

 

Drug metabolism studies assessing non-Phase 1 and non-Phase 2 enzymes in recombinant Supersomes™ (e.g., monoamine oxidase [MAO], xanthine oxidase [XO], flavin monooxygenase [FMO] and alcohol/aldehyde dehydrogenases). LC/MS/MS is used to determine % of parent compound remaining.

 

The graph titled ” Metabolism of Selegiline Over Time” illustrates the metabolism of selegiline.  Selegiline is primarily metabolized by monoamine oxidase B, but at high concentrations is also metabolized by monoamine oxidase A.

CYP enzyme inhibition in human microsomes. The CYP enzymes assessed are CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6 AND CYP3A4.  Other CYP enzymes are available upon request (e.g., CYP2A6, CYP2J2, CYP4A11, CYP4F2, CYP2E1).  Specific industry standard probes specific for each CYP enzyme are used to determine inhibition (IC50).

 

 

CYP enzyme induction in human primary hepatocytes via two methods. The first available method is qRT-PCR to determine changes in the expression of CYP mRNA.  Additionally, the activity of specific CYP enzymes can be assessed using industry standard probes (assessed by LC/MS/MS) that are specific for each CYP enzyme.  The CYP enzymes assessed are CYP1A2, CYP2B6 and CYP3A4.  Other CYP enzymes are available upon request.

The graph titled “CYPA2” illustrates the induction of CYP1A2 human primary hepatocytes in response to the respective positive controls.  Omeprazole is the positive control for CYP1A2, while phenobarbital is the positive control for CYP2B6 and rifampicin is the positive control for CYP3A4.

 

Transporter studies to assess efflux/uptake. The transporters that can be assessed are P‑gp, BCRP, OATP1B1, 926 OATP1B3, OAT1, OAT3, and OCT2.  Other transporters (e.g., BSEP, MRP) may be included as well.  Specific industry standard probes for each transporter are used to determine inhibition/interaction as a substrate.  This work is performed with in collaboration with IONTOX partner Qualyst.

Plasma protein binding studies to assess the binding affinity of the test compound to blood plasma proteins, using either human or rat serum. SPME filters are incubated with the plasma and test material for 30 minutes. The SPME filters are then incubated with internal standards.  LC-MS/MS is then used to assess the concentration of test compound on the SPME filters and percent bound (%B) is calculated.

The graph to the left illustrates the plasma protein binding (% bound) of a control and two blinded compounds.

 

 

References

Food and Drug Administration (2012) Guidance for Industry.  Drug Interaction Studies — Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations.  https://www.fda.gov/downloads/drugs/guidances/ucm292362.pdf