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Can Interactions Between Therapeutic Proteins and Small Molecule Drugs Be Evaluated In Vitro?

The mechanisms of clearance for small molecule drugs and therapeutic proteins are fundamentally distinct and therefore therapeutic proteins are generally not expected to cause pharmacokinetic based drug-drug interactions (DDI) with co-administered small molecule drugs (read Anti-CD28 Monoclonal Antibody-stimulated Cytokines Released from Blood Suppress CYP1A2, CYP2B6 and CYP3A4 in Humwan Hepatocytes In Vitro). However, there are examples where therapeutic proteins alter the clearance of small molecule drugs. The clearance of small molecule drugs can also be impaired by inflammatory disease, such as rheumatoid arthritis. In both cases, the clearance of the small molecule is changed by cytokines suppressing drug-metabolizing enzymes. Certain therapeutic proteins, infection or inflammation stimulate cytokine release. Elevated levels of cytokines can suppress drug-metabolizing enzymes or transporters. This can lead to altered clearance of small molecule drugs and thereby increase exposure and potentially result in toxicity, loss of efficacy, or magnified pharmacological effects.

Therapeutic proteins can suppress drug-metabolizing enzymes by acting directly on hepatocytes or they can cause a systemic cytokine release from peripheral blood mononuclear cells (PBMCs). The extent of direct suppression of drug-metabolizing enzymes by cytokines or cytokine-modulating therapeutic proteins can be measured in vitro by treatment of hepatocyte cell co-cultures. The effects of individual or combinations of proinflammatory cytokines on the suppression of drug-metabolizing enzymes have been evaluated in vitro. However, therapeutic proteins that stimulate the release of cytokines from PBMCs cannot be adequately evaluated by direct treatment of cultured cells in vitro.

To evaluate the effect of cytokine released from PBMCs on drug-metabolizing enzymes, SEKISUI XenoTech and Dr. Maciej Czerwinski developed and patented (8,846,576 and 10,001,471) a method where whole blood is incubated with the therapeutic protein ex vivo. Following the incubation, plasma from the treated blood is added to hepatocyte/Kupffer cell co-cultures. To evaluate the direct effect, the therapeutic protein can be applied to the co-cultures directly. After the culture period, changes in CYP450 expression can be determined by measurement of mRNA or activity of the enzymes can be measured with selective CYP450 probe substrates and monitoring the formation of the CYP specific metabolites (view list of validated methods).

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About the Authors

Greg Loewen is the Director of Technical Support at SEKISUI XenoTech. He has worked with XenoTech since 2000 in all aspects of drug metabolism, drug transporter and bioanalysis as an analyst, study director and group leader before shifting to Business Development team. In his current role, he is responsible for scientific discussions with clients to evaluate projects and define outcomes. Greg holds a BS in Chemistry from the University of Montana.
Maciej Czerwinski currently serves as Director of Consulting at SEKISUI XenoTech. Maciej received his PhD from the University of Maryland at Baltimore Medical School in the Department of Pathology. He has been with SEKISUI XenoTech since 1999 and guides the development of products and services for the in vitro analysis of drug safety. Maciej designed a patented method to analyze the in vitro cytokine-mediated drug-drug interactions between biologics and small molecule drugs. Dr. Czerwinski is also the inventor of our patented CryostaX single-freeze pooled hepatocytes.

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