Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2014/07/08/dmd.114.057935.DC1.html 1521-009X/42/9/1540–1547$25.00 DRUG METABOLISM AND DISPOSITION Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics http://dx.doi.org/10.1124/dmd.114.057935 Drug Metab Dispos 42:1540–1547, September 2014 A Useful Model Capable of Predicting the Clearance of Cytochrome P450 3A4 (CYP3A4) Substrates in Humans: Validity of CYP3A4 Transgenic Mice Lacking Their Own Cyp3a Enzymes s Tetsuya Mitsui, Takayuki Nemoto, Taiji Miyake, Shunsuke Nagao, Kotaro Ogawa, Motohiro Kato, Masaki Ishigai, and Hideyuki Yamada Preclinical Research Department, Research Division, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan (Te.M., T.N.,Ta.M., S.N., K.O., M.K., M.I.); and Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (H.Y.) Received March 13, 2014; accepted July 7, 2014 The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R2 = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates. Introduction species. Several additional modifications to simple allometry have been proposed to improve the accuracy of human clearance prediction by introducing correction factors such as the rule of exponents (Mahmood and Balian, 1996). However, other investigators have claimed that those modifications produce very little significant improvement (Nagilla and Ward, 2004). The major drawback in allometric scaling is the fact that, due to its empirical nature, this method pays no attention to species differences in metabolic and disposition processes between humans and animals. As far as physiologic approaches are concerned, in vitro–in vivo exploratory (IVIVE) prediction is a well-known method for the prediction of hepatic clearance (Houston, 1994). The IVIVE prediction consists of the following two procedures: the first step is an in vitro procedure to determine the intrinsic hepatic clearance (CLint,in vitro) using liver microsomes or hepatocytes, and the second one is the extrapolation procedure to convert CLint,in vitro into the in vivo CLh using physiologically based scaling factors and mathematical models. However, the IVIVE tends to underestimate hepatic clearance (Ito and Houston, 2005; Riley et al., 2005; Brown et al., 2007). In addition, the clearance values obtained vary markedly in both experimental and extrapolation procedures (Hallifax and Houston, 2009). The percentage of drugs for which the Prediction of the drug clearance in humans during the preclinical stage is essential for selecting drug candidates having a desired pharmacokinetic profile such as a suitable elimination half-life, bioavailability, and other parameters. In particular, hepatic clearance (CLh) is important because a number of drug candidates are metabolized in the liver. Empirical and physiologic approaches using liver microsomes and hepatocytes have been proposed to predict human CLh (Ito and Houston, 2005; Tang and Mayersohn, 2005; Zanelli et al., 2012) However, these methods cannot always give us a satisfactory prediction, and, in many cases, the CLh obtained exceeds the acceptable value, which is desired to be within a range from 0.5- to 2-fold of the actual value. In the empirical approach, allometric scaling has been proposed (Boxenbaum, 1984), and this has been widely used for the prediction of human clearance. Simple allometry is an easy to perform method because it needs only the body weight and the in vivo data that are obtained from a routine preclinical study using one or several animal dx.doi.org/10.1124/dmd.114.057935. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: ALP, alprazolam; CL, clearance; CLh, hepatic clearance; CLtot,b, systemic blood clearance; CLint,h,in vitro, in vitro intrinsic hepatic clearance; CLtot,p, systemic plasma clearance; CLr, renal clearance; CLint,h, hepatic intrinsic clearance; DMSO, dimethylsulfoxide; F, bioavailability; Fa, fraction absorbed; Fg, intestinal availability; Fh, hepatic availability; FLD, felodipine; fm,CYP3A4, fraction metabolism mediated by CYP3A4; fB, unbound fraction in blood; fu, unbound fraction; HLM, human liver microsomes; IVIVE, in vitro–in vivo exploratory; LC-MS/MS, liquid chromatography with tandem mass spectrometry; MDZ, midazolam; MLM, mouse liver microsome; NFD, nifedipine; NTL, nitrendipine; Qh, hepatic blood flow; QND, quinidine; RB, blood-to-plasma concentration ratio; RED, rapid equilibrium dialysis; Tg, transgenic; WT, wild-type. 1540 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 ABSTRACT Human Clearance Prediction using CYP3A4 Transgenic Mice from Sigma-Aldrich (St. Louis, MO). All other reagents and solvents were of analytical grade and commercially available. Animals and Liver Microsomes. Male CYP3A4-Tg mice and wild-type mice (FVB mice) at the age of 6 to 8 weeks were purchased from Taconic Farms (Germantown, NY). All mice were kept in an animal facility under conditions of controlled temperature (25 6 2°C) and humidity (55% 6 15%) under a 12-hour light/dark cycle. All animal experimental procedures were preapproved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co. Ltd., which was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Frederick, MD). The liver microsomes of CYP3A4-Tg mice and WT mice were obtained from CXR Biosciences Ltd. (Dundee, UK). A pooled preparation of HLM from 15 donors was purchased from Xenotech (Lenexa, KS). In Vitro Metabolic Studies. The incubation mixture had a total volume of 800 ml and consisted of 100 mM sodium/potassium phosphate buffer (pH 7.4), 5 mM magnesium chloride, 2 mM NADPH, 1 mM substrate dissolved in dimethylsulfoxide (DMSO; final v/v concentration: 0.1%), and 0.16 mg of liver microsomal protein. Only in the case of alprazolam metabolism was 0.32 mg of microsomal protein added to the incubation mixture. After preincubation at 37°C for 5 minutes, the reaction was initiated by adding NADPH solution. After incubation at 37°C for 5, 10, 20, and 30 minutes, the reaction was stopped by adding 50 ml of ice-cold acetonitrile containing raloxifene (200 ng/ml, DMSO) as an internal standard. The reaction mixture was transferred to a 96-well filter plate (0.45 mm MultiScreen; Millipore, Billerica, MA) and centrifuged at 2000 rpm for 5 minutes to remove precipitated proteins. A portion (15 or 20 ml) of the filtrate obtained was injected into a liquid chromatography system equipped with tandem mass spectrometers (LC-MS/MS), and the target drug was determined using either analytical condition (A) or (B). Materials and Methods Chemicals. Alprazolam (ALP) and midazolam (MDZ) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Felodipine (FLD), nifedipine (NFD), nitrendipine (NTL), and quinidine (QND) hydrochloride were purchased TABLE 1 The CLint, in vitro of CYP3A4 substrates in liver microsomes prepared from human donors (HLM), wild-type mice (WT MLM), and CYP3A4-Tg mice (CYP3A4-Tg MLM), and fm,CYP3A4 in CYP3A4-Tg mouse liver microsomes Pooled liver microsomes were prepared from three CYP3A4-Tg mice and three WT mice and used for this assay. CLint, fm,CYP3A4 in vitro CYP3A4-Tg MLMa Substrate HLM WT MLM CYP3A-Tg MLM (–) Ab (+) Ab ml/min per mg protein Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine 16 1850 1110 724 1140 91 NC 815 673 281 659 155 15 2980 952 731 2070 111 ND 127 281 18 584 57 1.00 0.96 0.71 0.98 0.72 0.49 MLM, mouse liver microsomes; NC, not calculated. a (–) Ab and (+) Ab mean the values obtained by incubating the substrate in the absence and presence of anti-CYP3A4 antibody, respectively. Fig. 1. Correlation of the CLint,in vitro of CYP3A4 substrates between liver microsomes prepared from CYP3A4-Tg mice and humans (A), and between microsomes from wild-type mice and humans (B). The solid line represents a regression line empirically fitted to the data for all substrates, and dotted lines represent the levels of the 2-fold or 0.5-fold of the regression value. Each plot represents the mean of duplicate assays. ALP was omitted from Fig. 1B because the metabolic activity for this substrate was below the detection limit in wild-type mice. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 variance in the clearance predicted by IVIVE from the observed value is within 2-fold is estimated to be around 60% (Howgate et al., 2006; Lavé et al., 2009). This figure is similar to those obtained by allometric scaling (Lombardo et al., 2013), and the predictability of these methods does not appear to be sufficient. It is expected that the mice expressing human metabolic enzymes will be useful for the prediction of human hepatic clearance. Among drug metabolizing enzymes, cytochromes P450 (P450) make the greatest contribution to drug metabolism, and CYP3A4, which is the most abundant isoform in the human liver and intestine, contributes to more than 50% of the metabolism of marketed drugs (Guengerich, 1999; Williams et al., 2004). In view of this background, we focused on transgenic mice carrying the CYP3A4 gene (CYP3A4-Tg mice) for estimating human hepatic clearance. To enhance the validity of this mouse model for the prediction of human clearance, mouse genes coding for Cyp3a enzymes (eight isoforms) were deleted from the CYP3A4-Tg mice used in this study (van Herwaarden et al., 2007). An earlier study showed that the intrinsic clearance for the metabolism of triazolam, a specific CYP3A4 substrate, by liver microsomes prepared from CYP3A4-Tg mice was comparable with that in human liver microsomes (HLM) and markedly higher than the values in microsomes from Cyp3a isoform-null mice and wild-type (WT) mice (van Waterschoot et al., 2009). Accordingly, it is to be expected that the hepatic intrinsic clearance of CYP3A4 substrates in humans can be reproduced in CYP3A4-Tg mice. To evaluate the validity of this CYP3A4-Tg mouse model in more detail, this study investigated whether the in vivo data obtained after administration of CYP3A4 substrates to CYP3A4-Tg mice can predict the hepatic clearance in humans. To this end, we investigated the correlation in the intrinsic hepatic clearances obtained from in vitro and in vivo experiments using six CYP3A4 substrates between CYP3A4-Tg mice and humans. In addition, we determined the relative contribution of CYP3A4 to the metabolism (fraction of metabolism by CYP3A4: fm,CYP3A4) as a correction factor because it is conceivable that murine enzymes other than Cyp3a isoforms significantly contribute to the metabolism of CYP3A4 substrates in CYP3A4-Tg mice. 1541 1542 Mitsui et al. TABLE 2 The unbound fraction in plasma (fu), the blood-to-plasma concentration ratio (RB), and the unbound fraction in blood (fB) of CYP3A4 substrates in wild-type mice and humans Mice Humans Substrate References fu Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine a b a 0.241 0.003 0.055 0.052 0.016 0.292 RB a 0.955 0.789 0.853 0.483 0.706 1.561 b fB fu 0.252 0.004 0.064 0.108 0.023 0.187 0.268 0.004 0.016 0.023 0.020 0.069 RBb fB 0.818 0.680 0.653 0.670 1.460 0.930 0.327 0.006 0.025 0.035 0.014 0.074 Each value represents the mean of triplicate in vitro assays using WT (FVB) mouse blood or plasma. Each value was obtained from literature references. Concentration of test compound in the buffer chamber sample Concentration of test compound in the plasma chamber sample After centrifugation, 50 ml of water was added to the filtrate, and part (15 ml) of the mixture was subjected to LC-MS/MS analysis to determine the drug. The RB and fB were calculated using eqs. 2 and 3. RB ¼ Concentration of test compound in plasma sample 1 Concentration of test compound in plasma sample 2 f B ¼ f u =RB ð3Þ Pharmacokinetic Studies in CYP3A4-Tg Mice and WT Mice. The test drugs were intravenously injected into CYP3A4-Tg mice and WT mice at the following doses: ALP, NFD, and NTR, 1 mg/kg per 10 ml vehicle; FLD, MDZ, and QND, 2 mg/kg per 10 ml vehicle. ALP, MDZ, and QND were dissolved in 5 mM HCl, and FLD, NFD, and NTR were dissolved in ethanol-20% (v/v) polyethylene glycol (1:19 v/v). Blood samples (40 ml) were collected from the orbital vein using heparinized hematocrit capillary tubes 2, 15, 30, 60, 120, 240, 420, and 1440 minutes after intravenous administration. The plasma was prepared by centrifuging the blood at 4 and 1°C at 5000 rpm for 5 minutes and frozen at 220°C until analysis. The urine was collected for 24 hours after dosing, and the urine volume was recorded. An aliquot of each urine sample was also stored at 220°C until required for analysis. Conditions of LC-MS/MS Analysis. The following conditions were used: the instrument was a Shimadzu 10AD HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with an API4000 LC-MS/MS system (AB SCIEX, Framingham, MA); the column was a Sunniest C18 (2 50 mm, 3 mm; ChromaNik Technologies, Osaka, Japan) at a column temperature of 40°C; the mobile phase was operated under gradient conditions and consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B) (Supplemental TABLE 3 Pharmacokinetic parameters of CYP3A4 substrates after a single intravenous administration to CYP3A4-Tg mice and wild-type mice Each value represents the mean of three animals. Substrate ð1Þ Blood-to-Plasma Concentration Ratio (RB). Blood from the WT mice was collected using a heparinized syringe and centrifuged for 5 minutes (4°C, 15,000 rpm) to prepare plasma. To obtain the blood-to-plasma concentration ratio (RB), the blood and plasma were separately processed as follows. The plasma (98 ml) from WT mice was mixed with a test drug (final concentration 1 mM), and incubated at 37°C for 5 minutes (designated plasma sample 1). The blood (98 ml) was also spiked with a drug and incubated in a similar way, and centrifuged at 15,000 rpm for 5 minutes (4°C). The plasma obtained was designated plasma sample 2. A portion (5 ml) of plasma samples 1 and 2 was mixed with 5 ml of ethanol and 100 ml of ice-cold acetonitrile containing raloxifene (200 ng/ml, DMSO) as an internal standard. These mixtures were transferred to a 96-well filter plate (0.45 mm MultiScreen), and centrifuged at 3000 rpm for 10 minutes. ð2Þ Vss CLtot,p CLtot,b l/kg CYP3A-Tg mice Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine WT mice Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine CLr CLh CLint,h Fha ml/min per kg 1.62 1.64 1.12 0.36 1.19 3.88 10.4 34.5 63.1 16.7 42.9 80.9 10.9 43.7 74.0 34.5 60.7 51.8 0.19 0.00 0.07 0.00 0.01 3.34 10.7 43.7 73.9 34.5 60.7 48.5 46.0 17,200 2990 431 5170 413 0.88 0.51 0.18 0.62 0.33 0.46 1.98 2.03 1.37 1.09 1.47 2.95 22.0 41.1 87.5 8.74 51.1 71.4 23.0 52.0 103 18.1 72.4 45.8 0.30 0.01 0.01 0.00 0.03 1.69 22.7 52.0 103 18.1 72.3 44.1 108 22,900 NC 193 7970 353 0.75 0.42 NC 0.80 0.20 0.51 NC, not calculated; Vss, volume of distribution at steady state. a Fh was calculated using the following equation: Fh = 1 2 CLh/Qh. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 Immunoinhibition of the CYP3A4-Mediated Reaction. Anti-CYP3A4 antibody (MAB-CYP3A4, mouse anti-human CYP3A4 monoclonal IgM; BD Biosciences, Woburn, MA) was used to estimate fm,CYP3A4 in terms of the metabolism catalyzed by the hepatic microsomes of CYP3A4-Tg mice. The highly specific nature of this antibody to CYP3A4 was reported elsewhere (Gelboin et al., 1995). Anti-CYP3A4 antibody was added to the incubation mixture before preincubation. All other conditions for incubation were identical to those given in the previous section. The amount of anti-CYP3A4 antibody needed to inhibit the CYP3A4-mediated reaction was determined using midazolam as the substrate. According to the results obtained, anti-CYP3A4 antibody twice the amount of microsomal protein (enzyme source) reduced the midazolammetabolizing activity to less than 10% of the control activity without the antibody (Supplemental Fig. 1). Based on this, an amount of anti-CYP3A4 antibody 5-fold greater than the microsomal protein was added to the incubation mixture to obtain complete inhibition of CYP3A4. Unbound Fraction. The ratio of the unbound form of test drug (unbound fraction: fu) in plasma was determined by equilibrium dialysis using a rapid equilibrium dialysis (RED) device (Thermo Fisher Scientific, Rockford, IL). For this, the plasma (200 ml) of untreated WT mice spiked with a test drug (2 mM, final concentration) was applied to the plasma chamber of the RED device, whereas 350 ml 133 mM phosphate buffer (pH 7.4) was applied to the buffer chamber. The RED device was then sealed and incubated for 4 hours at 37°C at 800 strokes/min in M-BR-022 bioshaker (TAITEC, Saitama, Japan). After incubation, 10 ml of plasma was removed from the plasma chamber, and 60 ml of phosphate buffer was removed from the buffer chamber and mixed with 60 ml of 133 mM phosphate buffer and 10 ml of blank plasma, respectively, in 96-well plates to which had been added in advance 200 ml of purified water and 10 ml of acetonitrile containing 20 nM warfarin an internal standard. The solution in the 96-well plates was transferred to a 96-well filter plate (0.45 mm MultiScreen) and centrifuged at 2000 rpm for 5 minutes to remove precipitated proteins. A portion (20 ml) of the filtrate was subjected to LC-MS/MS analysis, and the drug concentration was measured. The fu of each drug was determined in duplicate, and the fu was calculated using eq. 1. fu ¼ Ogawa et al., 2013 Edgar et al., 1985 Ogawa et al., 2013 Ogawa et al., 2013 Mikus et al., 1987; Soons and Breimer, 1991 Ogawa et al., 2013 Human Clearance Prediction using CYP3A4 Transgenic Mice 1543 TABLE 4 Pharmacokinetic parameters of CYP3A4 substrates in humans Vssa Substrate CLtot,pa CLtot,b l/kg Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine CLra CLint,h Fhb References 3.24 6730 381 207 1530 64.6 0.95 0.21 0.65 0.72 0.41 0.80 Greenblatt and Wright, 1993; Venkatakrishnan et al., 2005 Edgar et al., 1985 Thummel et al., 1996; Ogawa et al., 2013 Palma-Aguirre et al., 1989; Rashid et al., 1995 Mikus et al., 1987 Ueda et al., 1976; Brøsen et al., 1990 CLh ml/min per kg 1.25 3.20 0.78 0.94 5.40 2.70 1.05 11.1 4.74 3.90 18.7 4.70 1.28 16.4 7.26 5.82 12.8 5.05 0.26 0.00 0.00 0.00 0.51 0.91 1.03 16.4 7.26 5.82 12.3 4.14 Vss, volume of distribution at steady state. a Each value was quoted from the reference indicated. b Fh was calculated using the following equation Fh = 1 2 CLh/Qh. Y ¼ a expð 2 Kel XÞ ð4Þ where a is a constant and Kel is a rate constant for the disappearance of test drugs. The CLint,in vitro was calculated according to eq. 5. CLint;in vitro ¼ Kel ½incubation volumeðmlÞ ½enzyme amount ðmg proteinÞ ð5Þ a ¼ ð1 þ 4RN DN Þ1=2 ð12Þ RN ¼ f B CLint;h =Qh ð13Þ Results In Vitro Metabolism of CYP3A4 Substrates. Six CYP3A4 substrates—ALP, FLD, MDZ, NFD, NTR, and QND—were incubated with NADPH-fortified HLM as well as microsomes prepared from WT and CYP3A4-Tg mice. The kinetic parameters obtained are shown in Table 1. The CLint,in vitro values for six substrates in CYP3A4-Tg mice were comparable with or greater than those in HLM, whereas five substrates out of six exhibited a lower CLint,in vitro value in WT mice The contribution of CYP3A4 to the in vitro metabolism of a substrate was expressed as fm,CYP3A4. The fm,CYP3A4 for each substrate was calculated using eq. 6. fm;CYP3A4 ¼ 1 CLint;in vitro with Anti CYP3A4 antibody CLint;in vitro without Anti CYP3A4 antibody ð6Þ Determination of In Vivo Pharmacokinetic Parameters. The pharmacokinetic parameters for the CYP3A4-Tg mice were calculated by a noncompartmental analysis using Phoenix WinNonlin version 6.1 software (Pharsight Corp., Mountain View, CA). The systemic plasma clearance (CLtot,p), the systemic blood clearance (CLtot,b), and the hepatic blood clearance (CLh) were determined using eqs. 7–9: CLtot;p ¼ Dose ∕ AUCinf ð7Þ CLtot;b ¼ CLtot;p ∕ RB ð8Þ CLh ¼ CLtot;b 2 CLr ð9Þ The renal clearance (CLr) was determined by dividing the amount of unchanged drug excreted in the urine over 24 hours by the area under the blood concentration-time curve (AUC0–24). The distribution volume at steady state (Vss) was calculated by multiplying the mean resident time (MRTinf) by the CLtot,p. The hepatic availability (Fh) was determined using eq. 10. The hepatic blood flow rates (Qh) in mice and humans were obtained from a reference (Davies and Morris, 1993): 90 (mice) and 20.7 (humans) ml/min per kg and used in the calculation. Fh ¼ 1 2 CLh ∕ Qh ð10Þ The hepatic intrinsic clearance (CLint,h) of a CYP3A4 substrate in CYP3A4Tg mice and humans was calculated by applying Fh, Qh, fB, and the dispersion number (DN = 0.17) in eqs. 11–13 for a dispersion model: Fh ¼ 4a ð1 þ aÞ2 exp½ða 1Þ=2DN ð1 aÞ2 exp½ ða þ 1Þ=2DN ð11Þ Fig. 2. Correlation for the CLtot,b (A) and CLh (B) of intravenously administered CYP3A4 substrates between CYP3A4-Tg mice and humans. The solid line represents a regression line empirically fitted to the data for all substrates. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 Tables 1-1 and 1-2); the flow rate was 0.2 ml/min; ionization mode was used with electrospray, and the detected ion was a positive or negative ion fragmented from a precursor ion formed in the first mass spectrometry (Supplemental Tables 2-1 and 2-2). Determination of CLint,in vitro and fm,CYP3A4. The rate of disappearance of a test drug from the incubation mixture was calculated by fitting the timedependent loss of substrate to a monoexponential decay curve defined by eq. 4. 1544 Mitsui et al. compared with HLM. In microsomes from WT mice, no elimination of ALP was observed due to the low metabolic activity. In parallel with these observations, a good correlation (R2 = 0.853) was obtained between the CLint,in vitro in HLM and the micrsomes from CYP3A4-Tg mice, and the CLint,in vitro values of all substrates were plotted and found to range from 0.5- to 2-fold the regression value (Fig. 1A). Although the CLint,in vitro between HLM and the microsomes from WT mice also exhibited a high correlation coefficient (R2 = 0.877), a plot of QND did not lie within a range between 0.5- and 2-fold the regression value (Fig. 1B). In addition, a plot for ALP was omitted from Fig. 1B because the metabolic activity of this substrate was too low to be determined in wild-type mice. These pieces of evidence suggest that artificially expressed CYP3A4 but not murine peculiar Cyp3a mainly contributes to an improved correlation in CLint, h among a series of substrates exhibiting high (FLD) and low (ALP) clearances. The fm,CYP3A4, an index of the relative contribution of CYP3A4 to the metabolism of a particular drug in CYP3A4-Tg mice, was estimated by comparing the CLint,in vitro values obtained with and without anti-CYP3A4 antibody (Table 1). The result showed that fm,CYP3A4 varied from 0.49 (QND) to 1.0 (ALP). The unbound fraction in plasma or blood and the blood-to-plasma concentration ratio in FVB (WT) mice and humans are shown in Table 2. Correlation in the In Vivo Pharmacokinetics between CYP3A4-Tg Mice and Humans, and between WT Mice and Humans. Based on the plasma concentration-time profile after intravenous administration to CYP3A4-Tg mice (Supplemental Fig. 2) and WT mice, the pharmacokinetic parameters of CYP3A4 substrates were calculated (Table 3). To examine a correlation between CYP3A4-Tg mice or WT mice and humans, the human pharmacokinetic parameters published in the literature are shown in Table 4. The ratios of renal clearance (CLr) to total clearance (CLtot,b) of all substrates were estimated to be 10% or less, suggesting that hepatic metabolism plays a major role in the elimination of these drugs in CYP3A4-Tg mice. Figure 2 shows a correlation between CYP3A4-Tg mice and humans in terms of CLtot,b and CLh. As can be seen in the figure, a poor correlation for both parameters was observed between humans and CYP3A4-Tg mice (R2 = 0.155 and 0.180). Therefore, as far as the CLh is concerned, it does not appear that the human features can be predicted from those in CYP3A4-Tg mice. We then calculated the in vivo CLint,h of CYP3A4 substrates from the in vivo CLh using a dispersion model, and compared the CLint,h TABLE 5 Accuracy of the predicted human CLh of six CYP3A4 substrates based on the correlation of CLint,h between human and Tg mice compared with observed values CLh Substrate Accuracy Observed Predicted ml/min per kg Alprazolam Felodipine Midazolam Nifedipine Nitrendipine Quinidine 1.03 16.4 7.26 5.82 12.3 4.14 % 2.01 17.3 10.7 2.98 11.2 2.65 195 106 147 51 92 64 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 Fig. 3. Correlation for the CLint,h of CYP3A4 substrates between humans and CYP3A4-Tg mice or wild-type mice. The CLint,h in CYP3A4-Tg mice was calculated (A) without and (B) with multiplying by fm,CYP3A4. The CLint,h in wild-type mice was obtained without any correction (C). The solid line represents a regression line empirically (power) fitted to the data for all substrates, and the dotted lines represent 2-fold or 1/2-fold the regression value. MDZ was omitted from Fig. 3C because its CLint,h could not be calculated. Human Clearance Prediction using CYP3A4 Transgenic Mice logCLint;h;human ¼ 1:207 log ðCLint;h;CYP3A4Tg mice fm;CYP3A4 Þ 2 1:191 ð14Þ Prediction of Human CLh and Its Validity. The human CLint,h values of the six CYP3A4 substrates were simulated by applying CLint,h,CYP3A4-Tg mice and fm,CYP3A4 obtained from CYP3A4-Tg mice experiments to eq. 14; these were then converted to human CLh using eqs. 10, 11, 12, and 13. The predicted CLh obtained for each substrate and the difference (%) from the observed value are shown in Table 5. The predicted values for all CYP3A4 substrates did not exceed the real CLh by 2-fold or 0.5-fold. In contrast, a trial using WT mice for the prediction of CLint,h in humans exhibited large differences between the predicted and observed values for ALP (4.5-fold) and NFD (0.2-fold) (Supplemental Table 3). Discussion This study aimed to develop a more accurate method for the prediction of human CLh than the presently available methods, such as allometric scaling and IVIVE. To date, the main focus for obtaining human CLh predictions has been the theoretical in vivo approach, although only a few empirical approaches, such as animal scale-up, have been reported. We expected that a humanized mouse model would be a better tool for predicting human CLh, and we focused on CYP3A4Tg mice because CYP3A4 is an enzyme contributing to the metabolism of over 50% of currently prescribed drugs (Guengerich, 1999). In this study, we initially compared the CLh between humans and CYP3A4-Tg mice, but the correlation obtained was quite poor (R2 = 0.180; Fig. 2B). This seems to be due to the fact that CLh depends on the hepatic blood flow, which is peculiar to each animal species, and this parameter exhibits a clearance due to the metabolism and disposition of the bound as well as the unbound form of the drug. It is presumed that the poor correlation of CLh between humans and CYP3A4-Tg mice is attributed, at least partially, to a species difference Fig. 4. Correlation between the corrected CLint,h of CYP3A4 substrates by fm,CYP3A4 in CYP3A4-Tg mice and human pharmacokinetic parameters of FaFg (A), Fh (B), and F (C). The solid line represents a regression line empirically fitted to the data for all substrates. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2016 between CYP3A4-Tg mice and humans, and WT mice and humans. As a power regression analysis exhibited an excellent correlation (R2 = 0.951) between CYP3A4-Tg mice and humans, the CLint,h values of five out of six CYP3A4 substrates were plotted within 2-fold range (Fig. 3A). When the CLint,h in CYP3A4-Tg mice was corrected using fm,CYP3A4, all substrates were plotted within a 2-fold range of the regression value, whereas the value of the regression coefficient remained unchanged (Fig. 3B). In the case of linear regression analysis, excellent correlations were obtained independently of the fm,CYP3A4 correction (R2 = 0.977 and 0.993). However, the CLinh,h values of ALP (5.3-fold), MDZ (3.0-fold), and QND (2.4-fold) without an fm,CYP3A4 correction, and ALP (5.8-fold) and MDZ (2.2-fold) with a correction exceeded the range of 2-fold (Supplemental Fig. 3, A and B). Although the CLint,h between WT mice and humans also showed a good correlation (R2 = 0.820), only three out of five CYP3A4 substrates (60%)—that is, FLD, NTR, and QND—were found to be within a 2-fold range (Fig. 3C). In the case of WT mice, MDZ was removed from the regression analysis because its CLh was calculated to be 103 ml/min per kg, which exceeded the blood flow in the mouse liver (90 ml/min per kg) (Davies and Morris, 1993). These results showed that the predictability of human CLint,h could be improved by using the CLint,h obtained from CYP3A4-Tg mice corrected by fm,CYP3A4. We obtained eq. 14 of the regression line: 1545 1546 Mitsui et al. F ¼ Fa Fg Fh ð15Þ Human Fh can be estimated by inserting a predicted human CLint,h into eqs. 11–13. Regarding Fa Fg, it can also be predicted from the CLint,h obtained from CYP3A4-Tg mice experiments because it has been suggested that there is an inverse correlation between CLint,h and Fa Fg in CYP3A4-dependent metabolism in humans (Kato et al., 2003). We initially used literature data about human F for six CYP3A4 substrates, and calculated the human Fa Fg using eq. 15, and F and Fh (Table 4) were calculated using eq. 10 (see Supplemental Table 4 for obtained Fa Fg values). We then compared CLint,h and Fa Fg in humans, but a clear correlation was not observed (R2 = 0.327) (Supplemental Fig. 4). When the correlation between Fa Fg in humans and CLint,h in CYP3A4-Tg mice was subsequently analyzed, a poor correlation was also observed (Fig. 4A), despite the excellent correlations obtained between CLint,h multiplied by fm,CYP3A4 and both Fh and F (Fig. 4, B and C). One of the reasons for this seems to be the high FaFg value of FLD in spite of its high CLint,h value. The Fg and Fh values of FLD can be changed along with a change in hepatic blood flow (Qh). This is because the CLh of FLD is as much as the Qh (CLh = 16.4 ml/min per kg), and Fh and Fg depend on Qh (eq. 10) and Fh (eq. 15), respectively. FLD and NFD, calcium channel blockers, exhibit vasodilator effect on the liver blood vessels, resulting in an increase in Qh (Galetin et al., 2008), and FLD causes the effect rapidly (Bengtsson-Hasselgren et al., 1990). The Qh is known to range from 17.1 to 25.5 under normal physiologic conditions (Kato et al., 2003). Although our study set the Qh at 20.7 for all drugs, the following possibility cannot be excluded that the Qh is higher than the value used in the case of FLD, and this leads to an overestimation of Fg. If the Qh of FLD is changed to 25.5, the FaFg of FLD is calculated to be 0.45, and the correlation in Fig. 4A is improved (R2 = 0.769; data not shown). Although problems such this should be improved in the future, it is reasonable to expect that the human F for CYP3A4 substrates can be predicted using CLint,h in CYP3A4-Tg mice after intravenous administration. This is supported by a good correlation between human F and CLint,h in CYP3A4-Tg mice (Fig. 4C). In conclusion, our present study demonstrates the usefulness of CYP3A4-Tg mice lacking their own Cyp3a enzymes for predicting the human hepatic clearance of CYP3A4 substrates. The in vivo approach reported here appears to have better predictability than other previously reported methods for the pharmacokinetics of CYP3A4 substrates in humans. Recently, transgenic mice carrying human P450 enzymes, such as CYP2D6 and CYP2C9, and concomitantly lacking endogenous Cyp2d and Cyp2c have been produced (Scheer et al., 2012a,b). It is therefore expected that these humanized mice as well as CYP3A4-Tg mice will contribute to the more accurate prediction of the hepatic clearance of drugs in humans. Authorship Contributions Participated in research design: Mitsui, Nemoto, Miyake, Nagao, Ogawa, Kato, Ishigai. Conducted experiments: Mitsui, Nemoto, Miyake, Nagao. Contributed new reagents or analytic tools: Nemoto, Miyake, Nagao. Performed data analysis: Mitsui, Nemoto, Miyake, Nagao, Ogawa, Kato, Ishigai. Wrote or contributed to the writing of the manuscript: Mitsui, Nemoto, Miyake, Nagao, Ogawa, Kato, Ishigai, Yamada. References Bengtsson-Hasselgren B, Rönn O, Blychert LO, Edgar B, and Raner S (1990) Acute effects of felodipine and nifedipine on hepatic and forearm blood flow in healthy men. Eur J Clin Pharmacol 38:529–533. Boxenbaum H (1984) Interspecies pharmacokinetic scaling and the evolutionary-comparative paradigm. Drug Metab Rev 15:1071–1121. 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These results suggest that an evaluation using CYP3A4-Tg mice improves the prediction of the CLh values of CYP3A4 substrates in humans in drug discovery and development stages compared with empirical and IVIVE methods. However, it should be noted that there are drugs, such as nifedipine, for which the CLh cannot be satisfactorily predicted even after an evaluation using CYP3A4-Tg mice (see Table 5). The CLint,h in humans correlated even with the CLint,h values obtained from WT mice (in vitro, Fig. 1B; and in vivo, Fig. 3C). This good correlation is due to the moderate similarity of the amino acid sequence (62.6–75.3%) and the moderate similarity of the substrate specificity between human CYP3A and murine Cyp3a isoforms (van Herwaarden et al., 2007; McLaughlin et al., 2008). A good correlation has also been reported for the in vivo CLint,h of CYP3A4 substrates between humans and monkeys (Ogawa et al., 2013), and monkey CYP3A8 is highly homologous to human CYP3A4 (amino acid identity .90%: Uno et al., 2007). Our study provides further evidence that the CLint,h of CYP3A4 substrates can be predicted more precisely in CYP3A4-Tg mice than in WT mice and monkeys. As mentioned before, it is expected that the CLint,h in CYP3A4-Tg mice can be improved by correcting it with fm,CYP3A4, which shows the ratio of the CYP3A4 contribution to the net metabolism of a particular drug. However, as far as the six substrates were concerned, the correlation coefficient for CLint,h between transgenic mice and humans was almost identical before and after correction by fm,CYP3A4. However, it should be noted that this correction tended to improve the prediction accuracy by increasing the ratio of substrates, the CLint,h values of which were plotted within the range from 0.5-fold to 2-fold of the regression value, from 83 to 100%. These observations do not appear to suggest that correction by fm,CYP3A4 is unnecessary because there must be CYP3A4 substrates that are metabolized in the mouse by not only murine Cyp3a but also other murine P450s, except for Cyp3a (Perloff et al., 2000). It is reasonable to believe that correction by fm,CYP3A4 is a useful way of improving the predictability of human CLint,h for CYP3A4 substrates having lower fm,CYP3A4 values than the compounds used in our present study. The human CLint,h obtained from the experiments using CYP3A4Tg mice is expected to predict not only CLh but also the bioavailability (F) for CYP3A4 substrates in humans. 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