Hepatocyte clearance

As mentioned in Sect. 3, it is important to establish a detailed lead profile at the beginning of a lead identification effort. Criteria vary in different lead identification or hit-to-lead groups, but generally include some or all of the following potency, functional activity, selectivity, MW, clogP, solubility, permeability, microsomal stability and/or hepatocyte clearance, and preliminary PK including oral bioavail-ability. An example of a lead profile for a kinase inhibitor project is illustrated in Table 1 [21],... 

First, if the compound was cleared mainly by hepatic metabolism in the animal species tested and if human hepatocytes in vitro suggest the same will be true in humans, then the measured hepatocyte clearance may be used in a process called in vitro/in vivo scaling (20, pp. 207-228) to provide an estimate of the human intrinsic clearance. The application of Equation 5 then gives an estimate of the human systemic clearance. Second, the animal PK parameters of CL and Vj can be subjected to allometric scaling (20, pp. 207-228) whereby the PK parameter is related to a measurable allometric variable such as body mass, body surface area, heart rate, and so forth. (21) by fitting these parameter-variable pairs for several species to an empirical power equation of the form... 

FIGURE 30.11 Intrinsic clearance in vivo versus hepatocyte clearance of 17 drugs metabolized by cytochromes P450 in the rat. The line represents the predicted correlation. Hepatocyte clearance was measured either by substrate loss ( ) or metabolite formation (I). Intrinsic clearance was normalized to a standard rat weight (SRW) of 250 grams. (Reproduced with permission from HoustonJB. Biochem Pharmacol 1994 47 1469-79.)... 

The availability of human tissues has allowed several human DMPK parameters to be evaluated in drug discovery before the compound enters into clinical studies. One such assay is the determination of human hepatocyte clearance. Since liver is the major site of metabolism for most drugs, screening compounds for metabolic stability in liver preparations is usually one of the primary screenings employed in drug... 

The chemical structure of crizotinib contains both pyridine and piperidine moieties which have measured p/fa values of 5.4 and 8.9, respectively. The compound thus exhibits pH-dependent solubility 0.034 mg/mL in water, 41 mg/mL in simulated gastric fluid (pH = 1.6), and 0.19 mg/mL in simulated intestinal fluid (pH = 6.5). Crizotinib displayed moderate human hepatocyte clearance and low-to-moderate permeability in Caco-2 cell assays. The compound also displayed good and consistent bioavailability in preclinical species, such as rat, dog, and monkey [%F 63 (rat), 65 (dog), 42 (monkey)] along with a relatively large volume of distribution (Vss about 13 L/kg) and moderate clearance values which translated to a long ti/2 (5.5-17 h). ... 

THE MAMMALIAN ASIALOGLYCOPROTEIN RECEPTOR IS INVOLVED IN CLEARANCE OF CERTAIN GLYCOPROTEINS FROM PLASMA BY HEPATOCYTES... 

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Rat hepatocyte intrinsic clearance Human microsome intrinsic clearance Rat IV clearance... 

Hepatocytes, whether freshly cultured or cryo-preserved, can provide an assessment of not only CYP metabolism but also clearance by other metabolizing enzymes and potentially the role of transporters [51]. The accuracy of the data is of course dependent on how well the proteins in the hepatocytes function after culturing or freezing. 

Organ clearances are usually based on in vivo rather than in vitro studies. Drug metabolic parameters, Vm and Km, can be estimated from in vitro hepatic enzyme and hepatocyte preparations [53], Knowledge of total clearance and fractional... 

As described above, it will be normal to assume that the dose interval is 24 hours, i.e., once-a-day dosing. Absorption can be estimated with good confidence from absorption in the rat (see Section 6.1). Clearance is the sum of the predicted hepatic, renal, biliary and extrahepatic clearance. Hepatic clearance can be derived from in vitro studies with the appropriate human system, using either microsomes or hepatocytes. We prefer to use an approach based on that described by Houston and Carlile [83], Renal clearance can be predicted allometrically (see section 6.8.1). The other two potential methods of clearance are difficult to predict. To minimize the risks, animal studies can be used to select compounds that show little or no potential for clearance by these routes. As volume can be predicted from that measured in the dog, after correction for human and dog plasma protein binding (see Section 6.2), it is possible to make predictions for all of the important parameters necessary. 

Houston, J. B., Carlile, D. J., Prediction of hepatic clearance from micro-somes, hepatocytes and liver slices, Drug Metab. Rev. 1997, 29, 891-922. 

It is also important to predict the in vivo biliary excretion clearance in humans, and for this purpose MDCK II cell lines expressing both uptake and efflux transporters may be used (Fig. 12.3) [92, 93]. It has been shown that MRP2 is expressed on the apical membrane, whereas OATP2 and 8 are expressed on the basolateral membrane after cDNA transfection (Fig. 12.3) [92, 93]. The transcellular transport across such double-transfected cells may correspond to the excretion of ligands from blood into bile across hepatocytes. Indeed, the vectorial transport from the basal to apical side was observed for pravastatin only in OATP2- and MRP2-expressing... 

Miyauchi, S., Sawada, Y., Iga, T., Hanano, M., Sugiyama, Y., Comparison of the hepatic uptake clearances of fifteen drugs with a wide range of membrane permeabilities in isolated rat hepatocytes and perfused rat livers, Pharm. Res. 1993, 10, 434-440. 

An important DMPK property of a NCE is oral bioavailability (F) of the compound in various pre-clinical species.3 The oral bioavailability of a compound is dependent on several factors including intestinal permeability (estimated by the Caco-2 assay) and hepatic clearance (estimated with an in vitro metabolic stability assay).3 30 The metabolic stability assay is typically performed by incubating test compounds in liver microsomes or hepatocytes. The results can provide estimates of in vivo stability in terms of metabolic liabilities.3 8 59 62 Several authors described this assay as an important tool for the rapid assessment of the DMPK properties of NCEs.3 6 8111819 26 44 59 62-65... 

Lau, Y.Y. et al. 2002. Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. Drug Met. Disp. 30 1446. 

Typically, microsomal fractions are used for TDI analysis, but in many cases TDI is performed in rCYPs. The ability to use rCYPs for TDI relies on the accurate ability to extrapolate the rCYP data [186]. The second requirement for successful extrapolation of rCYP data is that the clearance pathways of the inhibitor in question are understood. For example, raloxifene has been shown to be a strong MBI of CYP3A4 in microsomes and rCYPs, but in hepatocytes the most predominant pathway is glucuronadation, which would effectively mask any CYP3A4 inactivation observed in... 

In rats treated with disulfiram prior to oral dosing with 1,2-dibromoethane (Plotnik et al. 1979), there was decreased clearance of radiolabeled 1,2-dibromoethane from the body with increased concentration in tissues (liver, kidney, spleen, testis, and brain). In the liver of the disulfiram-1,2-dibromoethane group, there was preferential uptake of labeled 1,2-dibromoethane in hepatocyte nuclei, indicative of DNA binding. 

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