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ADME PK

Microsomal binding assay

Use our microsomal binding assay to improve your prediction of in vivo pharmacokinetics and drug-drug interactions by correcting for the extent of binding to microsomes.

The microsomal binding assay is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Determining the extent of microsomal binding

  • Drug that is sequestered in microsomes in vitro is presumed to be unavailable for direct interaction with metabolising enzymes, just as drug that is bound to plasma proteins and tissue macromolecules in vivo is presumed to be unable to be directly acted on by drug metabolising enzymes.
  • Microsomal binding is an important factor in the prediction of in vivo pharmacokinetics from in vitro drug metabolism data.
  • It has been recognised that correcting for nonspecific binding in the in vitro microsomal stability assays can improve the accuracy of in vivo metabolic clearance prediction(2,3,4).
  • Knowledge of fuinc has also been shown to be important for the prediction of in vivo drug-drug interactions.
  • Cyprotex's Microsomal Binding assay uses equilibrium dialysis to determine the extent at which a compound binds to microsomes (fraction unbound value).
It has been recognized that nonspecific microsomal binding in the in vitro metabolic assays can significantly affect the observed kinetics of metabolism and hamper the accurate prediction of clearance, and there are now several examples where knowledge of the extent of microsomal binding can lead to a better understanding of the relationship between in vitro metabolism and in vivo pharmacokinetics.

1Austin RP, Barton P, Cockroft SL, Wenlock MC and Riley RJ (2002) Drug Metab  Dispos 30(12); 1497-1503

Protocol

Microsomal binding assay protocol

Method Equilibrium Dialysis
Test Article Concentration 3 μM (different concentrations available)
Protein Concentration 0.5 mg/mL (different concentrations available)
Number of Replicates 2
Temperature 37°C
Test Article Requirements 150 μL of 10 mM solution
Analysis method LC-MS/MS quantification (both microsome and buffer standards prepared)
Data Delivery Fraction unbound in incubation at 0.5 mg/mL
Recovery

Data

Data from Cyprotex's Microsomal Binding assay

14 compounds were screened in Cyprotex's Microsomal Binding assay using human liver microsomes (n=6 per run) on 3 separate occasions. The data are highly reproducible for a range of fraction unbound values (Figure 1 and Figure 2).

 
Figure 1
The graph shows the reproducibility of 14 compounds (n=6 replicates) within 1 run of Cyprotex's Microsomal Binding assay (error bars represent the standard deviation).
Figure 2
The graph shows the reproducibility of 14 compounds between 3 separate runs of Cyprotex's Microsomal Binding assay (error bars represent the standard deviation).

Q&A

Questions and answers on microsomal binding

Why is it important to investigate microsomal binding?

Subcellular fractions such as liver microsomes are useful in vitro models for investigation of hepatic clearance and drug-drug interactions. However, it is the unbound substrate or inhibitor concentration which is important in the prediction of in vivo pharmacokinetics as only the free drug can interact with drug metabolising enzymes in the microsomal incubations. There are now several studies where knowledge of the extent of microsomal binding has led to a better understanding of the relationship between in vitro metabolism and in vivo pharmacokinetics.

Please provide an overview of Cyprotex's Microsomal Binding assay.

The most commonly reported method for investigating the extent of binding to microsomes is equilibrium dialysis. A semi permeable membrane separates a protein containing compartment from a protein free compartment. The system is allowed to equilibrate overnight at 37°C. The test compound present in each compartment is quantified by LC-MS/MS.

The extent of binding is reported as a fraction unbound (fu) value which is calculated as detailed below;

PC = Test compound concentration in protein-containing compartment.
PF = Test compound concentration in protein-free compartment.

Why is microsomal binding important in prediction of clearance from in vitro microsomal clearance incubations?

Only free unbound compound is available to be metabolised by the enzymes present in microsomal incubations. Therefore, it is important to consider the extent of binding when performing microsomal clearance studies. Indeed, there have now been several reported examples in the literature where correcting for non-specific microsomal binding has improved the prediction of in vivo clearance (Carlile et al. 19992, Obach 19973, 19994).

How do I calculate the in vitro unbound intrinsic clearance?

The unbound intrinsic clearance is calculated using the following equation:

How is microsomal binding important in the prediction of drug-drug interactions from in vitro inhibition data?

Just as nonspecific binding reduces the concentration of free drug available to be metabolised by an enzyme, it also reduces the concentration which is available to inhibit enzymes. Indeed, it has been demonstrated that non-specific microsomal binding can account for underestimation of inhibitor potency (i.e., overestimation of IC50 or Ki values) when dealing with lipophilic basic drugs (Tran et al. 20025; Margolis and Obach, 20036), with the potential implication being an underestimation of risk from drug-drug interactions. The risks of not considering nonspecific microsomal binding in estimating inhibitory potency may be greater when dealing with mechanism-based inhibitors, due to the relatively high microsomal concentrations that are typically used during preincubation of the inhibitor in these experiments. Therefore it is important to use the unbound concentration of inhibitor for calculation of inhibition potential. This is calculated using the following equation:

Alternatively either the IC50 or Ki values can be corrected directly in a similar manner to provide the unbound value:


What microsomal protein concentration should I perform my microsomal binding study at?

Ideally the microsomal binding assessment should be performed at the same concentration as the microsomal stability assay. However, if this is not practical then it is possible to convert from the fraction unbound at one microsomal protein concentration to the fraction unbound at another using an equation described by Austin et al. (2002)1.

Where
fu2 = fraction unbound at second predicted microsomal concentration
fu1 = fraction unbound at first observed microsomal concentration
C2 = second microsomal concentration
C1 = first microsomal concentration

The graph below displays a set of eight compounds which have been screened at three microsomal concentrations, 0.5mg/ml, 1mg/ml and 2mg/ml through Cyprotex's Microsomal Binding assay. Using the equation described above, the fraction unbound values determined at 1mg/ml and 2mg/ml were converted to a fraction unbound at 0.5mg/ml. All values in the graph, therefore, represent the fraction unbound at 0.5 mg/ml after the conversion is applied. The data are good evidence that the equation is a useful model for converting between fraction unbound values at different microsomal concentrations.

Figure 3
Graph shows the data for eight compounds screened at three microsomal concentrations in Cyprotex's Microsomal Binding assay. Fraction unbound values at 1mg/ml and 2mg/ml have been converted to a fraction unbound value at 0.5mg/ml.

How does Cyprotex's Microsomal Binding Data compare with Literature Data?

The graph below shows the comparison between Cyprotex's Microsomal Binding data and literature values. The data show remarkably good correlation especially when considering that all the incubation conditions varied considerably between the different laboratories. All fraction unbound values displayed in Figure 4 have been converted to a fraction unbound at 0.5mg/ml for comparison purposes.

Figure 4
Graph shows the comparison between literature and Cyprotex's data.

Literature data were taken from the following papers:
Obach 19973; Obach 19994; Venkatakrishnan et al . 20007; Naritomi et al . 20018

What positive control is used in the assay?

The positive control compound used for the assay is amitriptyline. Amitriptyline is highly bound to microsomes in a range of species.

References

1 Austin RP et al. (2002) Drug Metab Dispos 30(12); 1497-1503
2 Carlile DJ et al. (1999) Br J Clin Pharmacol 47(6); 625-635
3 Obach RS. (1997) Drug Metab Dispos 25(12); 1359-1369
4 Obach RS. (1999) Drug Metab Dispos 27(11); 1350-1359
5 Tran TH et al. (2002) Drug Metab Dispos 30(12); 1441-1445
6 Margolis JM and Obach RS (2003) Drug Metab Dispos 31(5); 606-611
7 Venkatakrishnan K et al. (2000) J Pharmacol Exp Ther 293(2); 343-350
8 Naritomi Y et al. (2001) Drug Metab Dispos 29(10); 1316-1324

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