Measurement of the affinity of a drug to microsomal allows the accurate estimation of in vivo pharmacokinetics and drug-drug interactions by correcting the experimental clearance with the extent of binding to microsomes. Creative Biolabs provides kinetic measurements using liver microsomes to predict in vivo metabolic clearance of compounds in humans.

In recent years, it has been recognized that nonspecific microsomal binding in the in vitro metabolic assays can significantly influence the observed kinetics of metabolism and hamper the accurate prediction of clearance. This is because only the unbound substrate is free to interact with drug metabolizing enzymes in microsomes. In case of this, illuminating the extent of microsomal binding can lead to much better understanding of the relationship between in vitro drug metabolism data and in vivo pharmacokinetics, and can be useful in investigation of hepatic clearance and drug-drug interactions.

Creative Biolabs provides a high throughput microsomal binding assay to deliver a value of free fraction of compound in the microsomal incubation (fumic). We use liver microsomes on 3 separate occasions to ensure the data are highly reproducible. Our microsomal binding assay is fully developed to use a 96-well equilibrium dialysis unit for quickly and accurately determining the unbound fraction of drugs. The results are calculated by LC-MS/MS.

Calculation of fumic

fumic is evaluated by the following formula:

Microsomal Binding PF: Test compound concentration in protein-free compartment.
PC: Test compound concentration in protein-containing compartment.

Microsomal Binding and Intrinsic Clearance

It has been noticed that evaluating the extent of nonspecific binding in the in vitro microsomal assays can improve the accuracy of in vivo metabolic clearance prediction. This is because only unbound compound is available to be metabolized by the enzymes present in microsomal incubations. Therefore, the intrinsic clearance rate (CLint) need to be adjusted by fumic using the following formula:

Microsomal Binding CLobs: Observed clearance rate in the microsomal incubation experiment
Using measured values of fumic, it could be of more general utility in the prediction of clearance.

Microsomal Binding and Drug-Drug Interactions

Knowledge of fumic is also important in the prediction of in vivo drug-drug interactions. Nonspecific microsomal binding can reduce the concentration which is available to inhibit the enzymes and leads to underestimation of inhibitor potency when dealing with lipophilic basic drugs. Therefore, inhibition constants (Ki) need to be converted to unbound Ki values by the following formula:

Unbound Ki = Ki×fumic

Species Available

We routinely measure and compare fumic using the following species:

  • Mouse, rat, rabbit, dog, monkey, hamster, guinea pig, mini pig and human

For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

  1. Austin RP, Barton P, Cockroft SL, et al. (2002) “The influence of nonspecific microsomal binding on apparent intrinsic clearance, and its prediction from physicochemical properties.” Drug Metab Dispos 30(12):1497-1503.

For Research Use Only.



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