Microsomal Binding Assay

Creative Bioarray provides Microsomal Binding Assay to help customers accurately understand the relationship between in vitro drug metabolism data and in vivo pharmacokinetics, predicting hepatic clearance and drug-drug interactions.

Why do we need Microsomal Binding Assay?

• In vitro metabolism tests with non-specific microsomal binding can considerably impact the measured metabolic kinetics and make accurate clearance prediction difficult. Only unbound substrates can freely interact with drug-metabolizing enzymes in microsomes.
• Elucidating the extent of microsomal binding can provide a better understanding of the relationship between in vitro drug metabolism data and in vivo pharmacokinetics. Furthermore, it can also be used to study hepatic clearance and drug-drug interactions.
• The Microsomal Binding assay using logP for basic compounds and logD_7.4 for acidic and neutral compounds is the best method to predict the hepatic clearance of drugs and drug-drug interactions. This model can estimate the extent of binding to any given concentration of microsomes purely in terms of lipophilicity and ionization.

Brief Protocol

• Microsomal Incubations

1) The liver microsomes were incubated with the compounds in phosphate buffer (pH 7.4) at a constant temperature of 37°C.
2) After this equilibration period, the metabolic reactions were initiated by the addition of β-NADPH solution.
3) An amount of the above mixture was taken and quenched at different consecutive time points by adding methanol.
4) The quenched liquid was centrifuged, the precipitate removed, and quantified using HPLC/MS.
5) The kinetic data were analyzed using a linear fit of the natural logarithm of the ratio of the compound peak area to the internal standard peak area against time. Furthermore, calculate the CL_int value.

• Measurement of Microsomal Binding

1) The extent of binding of compounds to microsomes was determined using equilibrium dialysis.
2) Hepatic microsomes were added to one side of the dialysis chamber to suspend them in a pH7.4 phosphate buffer, and DMSO was added. A pH 7.4 phosphate buffer was used as the dialysis solution on the other side of the dialysis chamber.
3) After sealing and rotating in a 37°C water bath for 18 h, DMSO was added to the dialysate.
4) Quantification of the substances in both chambers was done using HPLC/MS.

• Measurement of LogD_7.4

Partitioning of compounds between 1-octanol and 0.02 M phosphate buffer, pH 7.4, at 20°C was determined using a standard shake flask method. HPLC analyzed samples with MS quantitation of both layers of the partition mixture.

• Measuring fu_inc

The microsomal binding assessment should ideally be performed at the same concentrations as the microsomal stability assay. However, if this is impractical, then the equation described by Austin et al.（2002） can be used to convert from the fraction unbound at one microsomal protein concentration to the fraction unbound at another.

Where

fu_inc: the fraction of unbound drug in incubation
fu₂: the free fraction at a second microsomal concentration;
fu₁: the free fraction at a first microsomal concentration;
C₁: first microsomal concentration;
C₂: the second microsomal concentration.

Quotation and ordering

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References

1. Austin, Rupert P., et al. "The influence of non-specific microsomal binding on apparent intrinsic clearance, and its prediction from physicochemical properties." Drug Metabolism and Disposition 30.12 (2002): 1497-1503.
2. Watanabe, Reiko, et al. "Predicting fraction unbound in human plasma from chemical structure: improved accuracy in the low value ranges." Molecular pharmaceutics 15.11 (2018): 5302-5311.

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For research use only. Not for any other purpose.