Original article
Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system (fup/fumic) for refined prediction of phase I mediated metabolic hepatic clearance

https://doi.org/10.1016/j.vascn.2010.04.003Get rights and content

Abstract

At the drug discovery stage, in vivo metabolic hepatic clearance (CLhep) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma (fup) to unbound fraction in the microsomal system (fumic) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fup and fumic data using ultracentrifugation. The correlation for fup/fumic between the two methods for a set of 23 data points was very good with R2 of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CLhep was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CLhep using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fup only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration.

Introduction

Prediction of in vivo clearance in humans is an integral part of new chemical entity profiling in drug discovery and early development. Since hepatic metabolic contributions often play a major role in overall xenobiotic clearance, intrinsic hepatic metabolic clearance (CLint) is determined fairly early in drug discovery programs. These data are commonly generated by measuring drug disappearance in subcellular hepatic fractions, such as S9 and microsomes (Obach, 1999). This experimental approach is broadly implemented due to its simplicity, ease of use, and amenability to high throughput formats. Although at an early drug discovery stage the exact mechanism of in vivo clearance of compounds is often unknown, it is important to accurately predict in vivo metabolic hepatic clearance (CLhep) to allow rank-ordering compounds based on this parameter. Several physiological models are available to scale in vitro intrinsic metabolic clearance data to in vivo organ clearance (Pang and Rowland, 1977, Rane et al., 1977, Wilkinson and Shand, 1975). Hepatic blood flow, hepatic intrinsic clearance and plasma/serum protein binding are minimal experimental inputs required to allow scalability of these models. Obach, 1996, Obach, 1997, Obach, 1999 demonstrated the significance of including a measure of drug binding to microsomes as an additional important correction factor for accurate prediction of in vivo clearances. Several subsequent studies have evaluated nonspecific microsomal protein binding of xenobiotics and have confirmed its relevance in predicting in vivo clearance (Jones and Houston, 2004, Giuliano et al., 2005, Naritomi et al., 2001, Austin et al., 2002). In all published reports, with the exception of Obach (1997) and Skaggs, Foti, and Fisher (2006) the unbound fraction in plasma and microsomes was separately determined. For proper correction, these independently determined factors were entered in the scaling equations as a ratio of fraction unbound in plasma (fup) to fraction unbound in microsomal system (fumic).

Herein, a straightforward method for the direct and accurate experimental determination of this correction ratio (fup/fumic) is reported. Equilibrium dialysis was used to measure drug partitioning between plasma and microsomes, allowing direct calculation of fup/fumic by determining the ratio of total drug concentration in plasma to total drug concentration in microsomes (Eq. (1)).

At equilibrium, the unbound concentration in plasma, Cp × fup is equal to the unbound concentration in microsomal system, Cmic × fumic so that Cp × fup = Cmic × fumic. By rearranging this we get:fup/fumic=Cmic/CpWhere,

    Cmic

    is the total concentration of compound in microsomal system

    Cp

    is the total concentration of compound in plasma

The experimental procedure was optimized using a focused set of compounds, and subsequently used to measure fup/fumic for compounds spanning a wide range of plasma and microsomal binding. Results were validated by comparison to calculated fup/fumic using individually determined measurements of fup and fumic by ultracentrifugation. Finally, the impact of microsomal and protein binding on CLhep was illustrated using a proprietary compound.

Section snippets

Reagents

Radiolabeled verapamil (3H), digoxin (3H), and caffeine (14C) were purchased from Perkin Elmer Life and Analytical Sciences, Boston MA, amitriptyline (3H) was obtained from American Radiolabeled Chemicals Inc. St. Louis, MO, and midazolam was purchased from Sigma Aldrich, St. Louis, MO. Proprietary radiolabeled compounds and diclofenac were obtained from the Labeled Compound Synthesis group, Merck Research Laboratories, Rahway, NJ. All other investigational compounds were provided by the

Assay optimization

Equilibrium dialysis assay conditions were initially optimized for direct determination of fup/fumic in human plasma and liver microsomes using a set of 3 compounds (verapamil, caffeine, and M001 — a proprietary compound). These compounds were selected based on their broad range of binding ratio (fup/fumic) as determined by the ultracentrifugation method (Eq. (2)). The fup values for verapamil, caffeine, and M001 were 0.28, 0.91, and 0.05, respectively and the determined fumic were 0.85, 1, and

Discussion

Assay conditions were optimized for incubation temperature, time, and plate shaking speed. At 37 °C, extensive evaporation was observed within a few hours of incubation, thus this temperature was impractical to test the concept of direct ratio determination using an assay requiring extended incubation time. Evaporation was minimal at 25 °C thus this temperature was chosen for all subsequent studies. A platform shaker was used for sample agitation. At speeds greater than 300 rpm there was a risk of

Acknowledgement

The authors would like to thank Gloria Kwei for her support in the preparation of this manuscript.

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    Altogether, this could explain the discrepancy observed between the calculated and in vitro fuinc values for the more lipophilic drugs because no correction was made for binding to the apparatus. Furthermore, straightforward equilibrium dialysis methods for the simultaneous determination of the ratio between fraction unbound in plasma and the incubation medium (fup/fuinc) are available in the literature32,33 instead of measuring or predicting separately fup and fuinc. Despite the potential errors related to the binding to the experimental apparatus, these equilibrium dialysis methods still require experimental works to estimate the ratio fup/fuinc, and more importantly, it might be that the ratio fup/fuinc is not the most relevant correction factor of CLint for accurate IVIVEs of hepatic CL of drugs.1–4

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