The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes

Abstract

Drugs and other chemicals frequently bind nonspecifically to the constituents of an in vitro incubation mixture, particularly the enzyme source [e.g., human liver microsomes (HLM)]. Correction for nonspecific binding (NSB) is essential for the accurate calculation of the kinetic parameters K m , Cl int , and K i . Many tyrosine kinase inhibitors (TKIs) are lipophilic organic bases that are nonionized at physiologic pH. Attempts to measure the NSB of several TKIs to HLM by equilibrium dialysis proved unsuccessful, presumably due to the limited aqueous solubility of these compounds. Thus, the addition of detergents to equilibrium dialysis samples was investigated as an approach to measure the NSB of TKIs. The binding of six validation set nonionized lipophilic bases (felodipine, isradipine, loratidine, midazolam, nifedipine, and pazopanib) to HLM (0.25 mg/ml) was shown to be unaffected by the addition of CHAPS (6 mM) to the dialysis medium. This approach was subsequently applied to measurement of the binding of axitinib, dabrafenib, erlotinib, gefitinib, ibrutinib, lapatinib, nilotinib, nintedanib, regorafenib, sorafenib, and trametinib to HLM (0.25 mg/ml). As with the validation set drugs, attainment of equilibrium was demonstrated in HLM-HLM and buffer-buffer control dialysis experiments. Values of the fraction unbound to HLM ranged from 0.14 (regorafenib and sorafenib) to 0.93 (nintedanib), and were generally consistent with the known physicochemical determinants of drug NSB. The extensive NSB of many TKIs to HLM underscores the importance of correction for TKI binding to HLM and, presumably, other enzyme sources present in in vitro incubation mixtures. IntroductionIn vitro approaches, using human liver microsomes (HLMs), human hepatocytes, or recombinant proteins as the enzyme source are used widely to characterize the kinetics of drug metabolism and drug-drug interaction potential (Houston, 1994;Obach, 1999;Rostami-Hodjegan and Tucker, 2007;Miners et al., 2010). However, numerous drugs, especially lipophilic organic bases and neutral compounds, bind nonspecifically to the microsomal membrane. Importantly, nonspecific binding (NSB) reduces the concentration of the unbound drug in the incubation medium, resulting in overestimation of the measured kinetic constants K m (or S 50 ) and the inhibitor constant K i . Consequently, in vitro intrinsic clearance (determined as V max /K m ) and inhibitory drug-drug interaction potential (assessed as [I]/K i , where [I] is the inhibitor concentration) are both underpredicted (Obach, 1999;McLure et al., 2000;Margolis and Obach, 2003;Grime and Riley, 2006;Miners et al., 2006Miners et al., , 2010. Thus, it is now widely accepted that NSB should be accounted for when kinetic parameters are calculated from in vitro metabolism and inhibition studies.Tyrosine kinase inhibitors (TKIs) are an emerging group of drugs used in the treatment of many forms of cancer and some other diseases (e.g., idiopathic pulmonary fibrosis). Typically, TKIs are lipophilic (log P values . 2....

Evaluation of Carbazeran 4-Oxidation and O6-Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes

“…At the end of the incubation period, a 25-ml aliquot of the mixture from each of the buffer and the sample chamber was mixed with 475 ml of ice-cold acetonitrile containing 50 nM tolbutamide (internal standard; final concentration of 47.5 nM in 500 ml final volume). Control protein binding experiment was performed on human liver microsomes (100 mg) with drugs known to be highly bound to microsomal or plasma proteins; i.e., gefitinib (1 mM) (Burns et al, 2015) and abiraterone (1 mM). (Marbury et al, 2014).…”

A Fragment-Based Approach for the Computational Prediction of the Nonspecific Binding of Drugs to Hepatic Microsomes

“…It is clear, however, that physicochemical and chemical properties other than charge and lipophilicity determine NSB and hence fu mic . For example, we and others have reported that molecular mass, polar surface area, charge distribution, the number of hydrogen bond donors/ acceptors, and the presence of halogen atoms (especially the trifluoromethyl group) may all influence NSB (Gao et al, 2008;Li et al, 2009;McLure et al, 2011;Burns et al, 2015). Moreover, we demonstrated recently that algorithms commonly used to calculate logP gave widely discrepant values of this parameter, differing by as much as a factor of 2 (i.e., 2 log units) (Burns et al, 2015).…”

“…However, both techniques are time-consuming, requiring measurement of the drug concentration in each compartment of the dialysis apparatus or, in the case of ultrafiltration, in the reservoir and filtrate. Solubility is also a problem for highly lipophilic compounds (Burns et al, 2015). Given the broad dependence of NSB on lipophilicity and charge state, several algorithms based on these properties have been developed to predict fu mic , thereby circumventing the need for experimental methods (Austin et al, 2002;Hallifax and Houston, 2006;Sykes et al, 2006;SimCYP calculator).…”

“…For example, we and others have reported that molecular mass, polar surface area, charge distribution, the number of hydrogen bond donors/ acceptors, and the presence of halogen atoms (especially the trifluoromethyl group) may all influence NSB (Gao et al, 2008;Li et al, 2009;McLure et al, 2011;Burns et al, 2015). Moreover, we demonstrated recently that algorithms commonly used to calculate logP gave widely discrepant values of this parameter, differing by as much as a factor of 2 (i.e., 2 log units) (Burns et al, 2015). Perhaps not surprisingly, we observed that the published algorithms that use logP (or logD for acids) and charge state poorly predicted the fu mic values of a series of protein kinase inhibitors (Burns et al, 2015).…”

Physiologically Based Pharmacokinetic Modeling to Identify Physiological and Molecular Characteristics Driving Variability in Drug Exposure

“…24 Robust determination of in vitro dabrafenib transporter kinetics is precluded by limited aqueous solubility, which confounds assessment of compartmentalized drug concentrations in systems such as trans-well-based transporter assays. 25 In the absence of robust in vitro data, intestinal and hepatic transporter (transporter-mediated intrinsic clearance; CL intT ) and diffusion (passive diffusion clearance; CL PD ) kinetics were determined using the parameter estimation (PE) function in Simcyp. 26 Parameter estimations were based on in vivo data from a phase I single-dose escalation trial (GSK trial ID: 112680) involving 94 melanoma patients over six dose levels (37.5-300 mg).…”