INTRODUCTION

The accurate prediction of human pharmacokinetics using preclinical data is essential at the drug discovery stage because it provides important insights into drug candidate selection and first-in-human study design. Clearance (CL) is a key determinant of human pharmacokinetics, and several approaches, such as in vitro–in vivo extrapolation (IVIVE) and multispecies allometric (MA) scaling, have been widely used to predict human CL. Furthermore, the proportion of low-CL compounds in drug discovery portfolios has increased in the pharmaceutical industry to approximately 30%.1, 2 This is largely attributable to the tendency to extract metabolically stable compounds during high-throughput absorption, distribution, metabolism, and excretion (ADME) screening to achieve adequate exposure at a lower dosage in humans.3 In other words, as a result of the selection of low intrinsic CL (CLint) compounds, it has become increasingly common that IVIVE cannot be applied because of the low turnover of the parent compound in microsomal or hepatocyte stability assays. In these in vitro assays, it is difficult to estimate the in vitro CLint of low-CLint compounds because of the limited incubation time for which enzymatic activity can be maintained.

Chimeric mice with humanized livers (PXB-mice) were generated from urokinase-type plasminogen activator-cDNA/severe combined immunodeficiency mice injected with human hepatocytes.4 A wide range of drug-metabolizing enzymes and transporters were expressed in the PXB-mice liver, this is considering the fact that greater than 80% of mouse hepatocytes are replaced by human hepatocytes.5, 6 Therefore, PXB-mice represent a useful animal model for predicting human CL.7 Furthermore, the single-species allometric scaling (PXB-SSS) approach has been reported to provide high predictive accuracy8, 9 and to be applicable to compounds that undergo hepatic organic anion-transporting polypeptide-mediated transport10 and compounds with long half-lives.11 However, the predictive accuracy has not been investigated for low-CLint compounds with no significant turnover of the parent compound in human hepatocyte assays.

This study evaluated the usefulness of PXB-mouse methods for predicting the human CL of low-CLint compounds compared with the conventional MA scaling approach. Furthermore, we proposed a novel physiologically based scaling (PXB-PBS) approach that assumes that in vivo CLint per hepatocyte is equal between humans and PXB-mice based on the concept that mouse-derived hepatocytes in PXB-mice are mostly replaced by human hepatocytes. Whereas the PXB-SSS approach is an empirical, simple equation using body weight and exponents, the PXB-PBS approach has physiological significance and aids in understanding the prediction of CL using PXB-mice, which can lead to improved confidence in its application in drug discovery. In addition, the PXB-PBS approach can potentially provide more accurate CL predictions than the PXB-SSS approach because it can incorporate more detailed information, such as physiological parameters and compound-dependent pharmacokinetic parameters.12

METHODS Definition of low-CLint compounds

A total of 16 commercially available compounds were selected, including those expected to have low CLint based on clinical data (<5 ml/min/kg13) and those metabolized by cytochrome P450 (CYP) and non-CYP enzymes. Because most of these compounds were metabolized in the liver, this study assumed that total CL (CLt) is equal to hepatic CL. An in vitro metabolic stability assay using cryopreserved human hepatocytes purchased from BioIVT (ZOL, 10-donor mixed gender pool, Baltimore, MD, USA) was conducted to confirm whether the compounds exhibited low turnover. Tested compounds at a final concentration of 0.1 or 1 µmol/l were incubated at a cell density of 0.5 million cells/ml for 4 h with human hepatocytes suspended in Williams’ E medium containing 0.125% bovine serum albumin, 15 mmol/l HEPES, and 2 mmol/l GlutaMAX-1. Low-CLint compounds in this study were defined as those that did not display significant turnover (<20%14; Table 1). By contrast, compounds that exhibited significant turnover in human hepatocyte assays were classified as moderate- to high-CLint compounds in this study. The details of chemicals and reagents used in this study and the detailed procedures of the human hepatocyte assay are provided in the Supporting information.

TABLE 1. Summary of drug disposition and in vitro and in vivo parameters to predict human CLt for all compounds Compounds Disposition In vitro parameters In vivo parameters % remaining after a 4-h incubation

CLint per hepatocyte

(µl/min/1 × 106 cells)

R b a f u,p

CLt

(ml/min/kg)

Humans Humans Humans Rats PXB-mice Rats Monkeys Dogs Antipyrine P450 96 ND 1 0.990 0.690 4.60 7.86 11.5 7.22 Bosentan P450, OATP 69 12.3 0.48 0.023 0.012 8.78 16.40 20.6 1.72 Carbazeran AO 0 96.9 1 0.148 0.169 99.67 42.22 87.87 11.5 Dapsone P450, NAT 94 ND 1.04 0.381 0.280 4.40 6.87 5.08 1.21 Diazepam P450 62 3.9 0.71 0.036 0.171 42.147 61.51 17.90 46.71 Disopyramide P450 71 3.2 1.2 0.161 0.610 28.86 179.46 19 29 Doxazosin P450 11 41.5 1 0.066 0.050 34.245 30.00 15.47 11.21 Ranitidine P450, FMO 83 ND 1 1.000 0.900 131.02 99.88 40.23 10.4 Reboxetine P450 35 13.9 1 0.041 0.253 17.658 61.51 14.94 22.39 (S)-Naproxen P450, UGT 102 ND 0.55 0.007 0.008 0.556 0.41 0.83 0.04 (S)-Warfarin P450 80 ND 0.55 0.013 0.005 0.484 0.21 0.102 1.49 Tenoxicam P450 96 ND 0.67 0.015 0.030 0.2878 0.49 0.061 0.104 Theophylline P450 96 ND 0.85 0.580 0.400 4.70 1.91 1.08 1.73 Timolol P450 85 ND 0.84 0.715 0.760 130.05 137.02 13.6 –b Tolbutamide P450 89 ND 0.55 0.039 0.049 0.58 0.39 0.0456 0.142 UCN−01 –b 4 83.5 1 0.003 0.0175 0.0369 77.37 3.36 10.27 Note References are provided in Table S1. Abbreviations: CLint, intrinsic clearance; Rb, blood-to-plasma concentration ratio; fu,p, fraction unbound in plasma; CLt, total clearance; PXB-mice, chimeric mice with humanized livers; ND, not determined because of the absence of significant turnover (<20%) during a 4-h incubation in the human hepatocyte assay; OATP, organic anion-transporting polypeptide; AO, aldehyde oxidase; NAT, N-acetyltransferase; FMO, flavin-containing monooxygenase; UGT, UDP-glucuronosyltransferase. a Rb was assumed to be 1 for carbazeran, doxazosin, ranitidine, reboxetine, and UCN-01 because of a lack of data in the literature. b Not available in the literature. Data collection

In vitro and in vivo data for all compounds to predict human CL are summarized in Table 1. CLt in humans, PXB-mice, rats, monkeys, and dogs and fraction unbound in plasma (fu,p) in humans and rats were obtained from experiments or the literature. References are provided in Table S1. In vitro CLint,human per hepatocyte was calculated by correcting the compound disappearance rate in human hepatocyte assays with fraction unbound in hepatocytes and medium. Further, fu,p in humans and rats was measured by the equilibrium dialysis method using a Rapid Equilibrium Dialysis Device purchased from Thermo Fisher Scientific. The detailed methods for the determination of in vitro CLint and fu,p are provided in the Supporting information. At various sampling points, the plasma samples after intravenous administration to PXB-mice (N = 3) at a cassette dose of 0.1 mg/kg were collected. The plasma samples were extracted with acetonitrile containing the internal standard verapamil, and then the plasma concentration was determined using a liquid chromatography/tandem mass spectrometry method. The details of tandem mass spectrometry are provided in Table S2. Moreover, cassette doses of 0.1 mg/kg were administered intravenously to rats and monkeys (N = 3 each), and the plasma concentration was determined in a similar manner. CLt was calculated via noncompartmental analysis using Phoenix WinNonlin version 6.3 (Certara) based on the plasma concentration–time profiles following intravenous dosing. The sources of animals were as follows: 3 male PXB-mice weighing 18.5–21.5 g and aged 17–19 weeks at dosing were supplied by PhoenixBio Co., Ltd., and the replacement rate of mouse hepatocytes with human hepatocytes was 89%–91%; 3 male Sprague–Dawley rats weighing 242–261 g and aged 6 weeks at dosing were supplied by Charles River Laboratories Japan, Inc.; and 3 male cynomolgus monkeys weighing 2.9–3.6 kg and aged approximately 3.6–3.8 years at dosing were supplied by Hamri Co., Ltd. All in vivo experiments were approved by the Institutional Animal Care and Use Committee of Mitsubishi Tanabe Pharma Corporation (Kanagawa, Japan).

In vitro–in vivo extrapolation In vitro CLint,human per hepatocyte was converted to scaled CLint,human using human scaling factors, such as liver weight (LW), body weight (BW), and hepatocellularity,15 which are presented in Table 2, according to Equation 1. CLt,human was calculated from scaled CLint,human via a dispersion model (Equation 2) using hepatic blood flow (Qh),15 which is presented in Table 2, and fraction unbound in blood (fu,b) was calculated using the human blood-to-plasma concentration ratio (Rb) obtained from the literature. (1) (2) TABLE 2. Scaling factors for the IVIVE and PXB-PBS approaches Species

Q h

(ml/min/kg)

Liver weight

(g)

Body weight

(kg)

Hepatocellularity

(1 × 106 cells/g liver)

Humans 20 1470 70 120 PXB-mice 91.3 1.977 0.02 168 Abbreviations: IVIVE, in vitro–in vivo extrapolation; PXB-mice, chimeric mice with humanized livers; PXB-PBS, physiologically based scaling using PXB-mice; Qh, hepatic blood flow.

In Equation 2, ,16 and .

MA scaling from nonclinical animal species CLt,human was predicted using the rule of exponent (ROE)17 and fu corrected intercept method (FCIM).18 According to ROE, CLt values in rats, monkeys, and dogs were plotted against BW on a log–log scale as simple allometry (SA), and then CLt,human was predicted by an allometric equation (Equation 3, 4, or 5) that was based on the exponent values obtained from SA (Equation 3). SA was used when the exponents of SA ranged from 0.55 to 0.70. Maximum life-span potential (MLP) correction was applied when SA was 0.71–1.0; that is, the product of MLP and CLt for each animal was plotted as a function of BW on a log–log scale using Equation 4. When SA greater than or equal to 1.0, brain weight (BrW) correction was applied; that is, the product of BrW and CLt for each animal was plotted in a similar manner using Equation 5. In these equations, a, b, and c were the coefficients of the allometric equations, and x, y, and z were the exponents. BW, BrW, and MLP were set at 0.25 kg, 0.00174 kg, and 4.4 years, respectively, for rats; 3.75 kg, 0.0424 kg, and 18.5 years, respectively, for monkeys; and 12 kg, 0.0754 kg, and 20.5 years, respectively, for dogs.19 MLP in years was calculated as a function of BW and BrW for each animal according to Equation 6. (3) (4) (5) (6) According to FCIM, CLt,human was predicted using Equation 7. In this equation, a was the intercept obtained from the log–log plot of CLt versus BW, and Rfu,p was the ratio of fu,p for rats and humans. (7) PXB-SSS CLt,human was predicted by the PXB-SSS approach using Equation 8.9 BW was set at 70 and 0.02 kg for humans and PXB-mice, respectively. (8) PXB-PBS CLt,human was predicted using the PXB-PBS approach following several steps (Figure 1). Based on the scaling factors of PXB-mice presented in Table 2, scaled CLint,PXB was calculated from CLt,PXB via a dispersion model (Equation 2) using Qh and fu,b, and then in vivo CLint,PXB per hepatocyte was calculated (Equation 1). The estimation or measurement methods of LW and hepatocellularity in PXB-mice, which were used as scaling factors, are provided in the Supporting information. Based on the concept that mouse-derived hepatocytes in PXB-mice are largely replaced by human hepatocytes, in vivo CLint per hepatocyte was assumed to be equal between humans and PXB-mice (Equation 9). The same assumption was applied for fu,p and Rb.7 Based on the assumption that Qh is equal in normal and PXB-mice,7 the Qh values presented in Table 2 were used.20 The inverse conversion was performed by a dispersion model using human scaling factors, and then CLt,human was estimated. For compounds for which CLt,PXB exceeds Qh, CLt was set at 90% of Qh.7(9)

Scheme for predicting human clearance (CL) for low intrinsic CL compounds with no significant turnover in human hepatocyte assays. PXB-mice, chimeric mice with humanized livers that were repopulated with human hepatocytes. PXB-SSS, prediction method based on single-species allometric scaling using PXB-mice. PXB-PBS, prediction method based on physiologically based scaling using PXB-mice. CLt, CLint, and BW, total clearance, intrinsic clearance, and body weight, respectively

Evaluation of predictive accuracy Observed CLt,human was compared with the predicted value for the IVIVE, PXB-SSS, PXB-PBS, ROE, and FCIM approaches to calculate the fold error. For each low-CLint compound, moderate- to high-CLint compounds, and all compounds, the percentages predicted within 2- and 3-fold ranges of the observed CLt,human were calculated. Moreover, the geometric mean of the ratio between the predicted and observed values, which was frequently used as the absolute average fold error (AAFE), was calculated according to Equation 10,21 and subsequently the predictability of each method was compared. For low-CLint compounds, the PXB-SSS, PXB-PBS, ROE, and FCIM approaches were compared, and IVIVE was additionally included in the comparison for moderate- to high-CLint compounds and all compounds. (10) Sensitivity analysis To fully understand the PXB-PBS approach, sensitivity analyses were conducted using a dataset of (S)-naproxen as a representative of low-CLint compounds and diazepam as a representative of moderate- to high-CLint compounds. The impact of parameters such as fu,p, Rb, and Qh, which included assumptions in the PXB-PBS approach in this study, on predicted CLt,human was investigated. Simulated CLt,human was defined as the CL generated by changing each parameter, and the magnitude of change from the predicted CLt,human was evaluated according to Equation 11 for an increased predicted value of CLt,human and Equation 12 for a decreased predicted value of CLt,human.22 These equations were primarily used to express the changes in predicted CLt,human on a similar magnitude for both positive and negative value and to evaluate the compounds on the same scale on a 3D plot, regardless of the observed CLt,human. Sensitivity analysis was conducted for fu,p in humans and PXB-mice within a 3-fold range of human fu,p, for Rb in humans and PXB-mice within the range of 0.5–2, for Qh in PXB-mice within the range of 90–180 ml/min/kg, and for Qh in humans within the range of 18–23 ml/min/kg, which were frequently used as physiological parameters in literature. (11) (12) RESULTS Determination of low-CLint compounds based on human hepatocyte assays

As the purpose of this study was to verify whether the prediction method using PXB-mice was useful for low-CLint compounds, we first conducted an in vitro metabolic stability assay using cryopreserved human hepatocytes to identify low-CLint compounds. The proportion of the parent compound remaining after 4 h of incubation is presented in Table 1. Antipyrine, dapsone, ranitidine, (S)-naproxen, (S)-warfarin, tenoxicam, theophylline, timolol, and tolbutamide met the criteria for low-CLint compounds; that is, they did not display significant turnover (<20%) during a 4-h incubation. Conversely, bosentan, carbazeran, diazepam, disopyramide, doxazosin, reboxetine, and UCN-01 (7-hydroxystaurosporine) were classified as moderate- to high-CLint compounds because they exhibited significant turnover. The plots of the proportion of each parent compound remaining after 4 h of incubation are summarized in Figure S1.

Prediction of human CL by IVIVE, MA, and PXB-mouse methods

For low-CLint compounds, CL was predicted using conventional MA scaling approaches and PXB-mouse methods, whereas for moderate- to high-CLint compounds, CL was predicted by all methods including IVIVE. The relationships between observed and predicted CLt,human are presented in Figure 2. The observed and predicted CLt,human and the fold errors are summarized in Table 3. To compare the predictability of each method, both the percentages predicted within 2- and 3-fold ranges of the observed value and AAFE are summarized in Table 4.

Relationships between observed and predicted total human clearance (CLt,human) for low intrinsic clearance (CLint) compounds and moderate- to high-CLint compounds. Panels (a), (b), (c), (d), and (e) presents the results of single-species allometric scaling from chimeric mice with humanized livers (PXB-mice), a physiologically based scaling using PXB-mice, in vitro–in vivo extrapolation (IVIVE), rule of exponent, and the fu corrected intercept method, respectively. Solid and dotted lines represent the unity and 3-fold error, respectively. PXB-SSS, prediction method based on single-species allometric scaling using PXB-mice; PXB-PBS, prediction method based on physiologically based scaling using PXB-mice; ROE, rule of exponent; FCIM, fu corrected intercept method

TABLE 3. Comparisons of observed and predicted CLt,human for all compounds Category of compounds Compounds Observed CLt,human (ml/min/kg)

Predicted CLt,human

(ml/min/kg)

Fold error (predicted/observed) PXB-SSS PXB-PBS IVIVE ROE FCIM PXB-SSS PXB-PBS IVIVE ROE FCIM Low-CLint compounds Antipyrine 0.64 0.59 0.71 ND 3.32 3.31 0.92 1.10 ND 5.19 5.17 Dapsone 0.48 0.60 0.67 ND 0.91 2.01 1.24 1.40 ND 1.90 4.19 Ranitidine 9.6 16.9 16.4 ND 5.4 11.1 1.76 1.70 ND 0.56 1.16 (S)-Naproxen 0.11 0.072 0.085 ND 0.051 0.189