Utility of Chimeric Mice with Humanized Livers for Predicting Hepatic Organic Anion-Transporting Polypeptide 1B-Mediated Clinical Drug-Drug Interactions.

The influence of transporters on the pharmacokinetics of drugs is being increasingly recognized, and drug-drug interactions (DDIs) via modulation of transporters could lead to clinical adverse events. Organic anion-transporting polypeptide 1B (OATP1B) is a liver-specific uptake transporter in humans that can transport a broad range of substrates, including statins. It is a challenge to predict OATP1B-mediated DDIs using preclinical animal models because of species differences in substrate specificity and abundance levels of transporters. PXB-mice are chimeric mice with humanized livers that are highly repopulated with human hepatocytes and have been widely used for drug metabolism and pharmacokinetics studies in drug discovery. In the present study, we measured the exposure increases [blood AUC (area under the blood/plasma concentration-time curve) and Cmax] of 10 OATP1B substrates in PXB-mice upon coadministration with rifampin, a potent OATP1B specific inhibitor. These data in PXB-mice were then compared with the observed DDIs between OATP1B substrates and single-dose rifampin in humans. Our findings suggest that the DDIs between OATP1B substrates and rifampin in PXB-mouse are comparable with the observed DDIs in the clinic. Since most OATP1B substrates are metabolized by cytochromes P450 (CYPs) and/or are substrates of P-glycoprotein (P-gp), we further validated the utility of PXB-mice to predict complex DDIs involving inhibition of OATP1B, CYPs, and P-gp using cyclosporin A (CsA) and gemfibrozil as perpetrators. Overall, the data support that the chimeric mice with humanized livers could be a useful tool for the prediction of hepatic OATP1B-mediated DDIs in humans. SIGNIFICANCE STATEMENT: The ability of PXB-mouse with humanized liver to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs) in humans was evaluated. The blood exposure increases of 10 OATP1B substrates with rifampin, an OATP1B inhibitor, in PXB-mice have a good correlation with those observed in humans. More importantly, PXB-mice can predict complex DDIs, including inhibition of OATP1B, cytochromes P450 (CYPs), and P-glycoprotein (P-gp) in humans. PXB-mice are a promising useful tool to assess OATP1B-mediated clinical DDIs.

Simultaneous measurement of mouse and human albumin in chimeric mice with humanized livers.

Background: The degree of human hepatocyte replacement in chimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reports that directly compare the remaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma. Results: A robust correlation was observed between the serum human albumin levels measured by LC-MS/MS and the estimated replacement index of PXB-mice. Conclusion: All data were shown to be specific and suitable to accurately quantify both human and mouse albumin from plasma of chimeric mice with humanized livers.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

BACKGROUND & AIMS: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. METHODS: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. RESULTS: AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. CONCLUSIONS: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Evaluation of the Utility of PXB Chimeric Mice for Predicting Human Liver Partitioning of Hepatic Organic Anion-Transporting Polypeptide Transporter Substrates.

The ability to predict human liver-to-plasma unbound partition coefficient (Kpuu) is important to estimate unbound liver concentration for drugs that are substrates of hepatic organic anion-transporting peptide (OATP) transporters with asymmetric distribution into the liver relative to plasma. Herein, we explored the utility of PXB chimeric mice with humanized liver that are highly repopulated with human hepatocytes to predict human hepatic disposition of OATP substrates, including rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide. In vitro total uptake clearance and transporter-mediated active uptake clearance in C57 mouse hepatocytes were greater than in PXB chimeric mouse hepatocytes for rosuvastatin, pravastatin, pitavastatin, and valsartan. Consistent with in vitro uptake data, enhanced hepatic uptake and resulting total systemic clearance were observed with the above four compounds in severely compromised immune-deficient (SCID) control mice compared with the PXB chimeric mice, which suggest that mouse has a stronger transporter-mediated hepatic uptake than human. In vivo liver-to-plasma Kpuu from PXB chimeric and SCID control mice were also compared, and rosuvastatin and pravastatin Kpuu in SCID mice were more than 10-fold higher than that in PXB chimeric mice, whereas pitavastatin, valsartan, and repaglinide Kpuu in SCID mice were comparable with Kpuu in PXB chimeric mice. Finally, PXB chimeric mouse liver-to-plasma Kpuu values were compared with the reported human Kpuu, and a good correlation was observed as the PXB Kpuu vales were within 3-fold of human Kpuu Our results indicate that PXB mice could be a useful tool to delineate hepatic uptake and enable prediction of human liver-to-plasma Kpuu of hepatic uptake transporter substrates. SIGNIFICANCE STATEMENT: We evaluated PXB mouse with humanized liver for its ability to predict human liver disposition of five organic anion-transporting polypeptide transporter substrates. Both in vitro and in vivo data suggest that mouse liver has a stronger transporter-mediated hepatic uptake than the humanized liver in PXB mouse. More importantly, PXB liver-to-plasma unbound partition coefficient (Kpuu) values were compared with the reported human Kpuu, and a good correlation was observed. PXB mice could be a useful tool to project human liver-to-plasma Kpuu of hepatic uptake transporter substrates.

Application of a Novel Mass Spectral Data Acquisition Approach to Lipidomic Analysis of Liver Extracts from Sitaxentan-Treated Liver-Humanized PXB Mice.

The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their entering the collision cell. By rapidly scanning the resolving quadrupole window over a specified mass range, co-eluting precursor ions are transmitted sequentially into the collision cell, where collision energies are cycled between low and elevated levels to induce fragmentation. This method of data generation provides both precursor and fragment ion information at high specificity, allowing for greater accuracy of compound identification, whether using a database, spectral libraries, or comparison to authentic standards. The value of the approach in simplifying and "de-cluttering" the spectra of co-eluting lipids is shown with examples from lipidomic profiles obtained in investigations of the composition of organic extracts of livers obtained from SCID and chimeric liver-humanized mice administered under various experimental conditions.

The metabolic fate of fenclozic acid in chimeric mice with a humanized liver.

The metabolic fate of the human hepatotoxin fenclozic acid ([2-(4-chlorophenyl)-1,3-thiazol-4-yl]acetic acid) (Myalex) was studied in normal and bile-cannulated chimeric mice with a humanized liver, following oral administration of 10 mg/kg. This in vivo animal model was investigated to assess its utility to study "human" metabolism of fenclozic acid, and in particular to explore the formation of electrophilic reactive metabolites (RMs), potentially unique to humans. Metabolism was extensive, particularly involving the carboxylic acid-containing side chain. Metabolism resulted in the formation of a large number of metabolites and involved biotransformation via both oxidative and conjugative routes. The oxidative metabolites detected included a variety of hydroxylations as well as cysteinyl-, N-acetylcysteinyl-, and cysteinylglycine metabolites. The latter resulted from the formation of glutathione adducts/conjugates providing evidence for the production of RMs. The production of other classes of RMs included acyl-glucuronides, and the biosynthesis of acyl carnitine, taurine, glutamine, and glycine conjugates via potentially reactive acyl-CoA intermediates was also demonstrated. A number of unique "human" metabolites, e.g., those providing evidence for side-chain extension, were detected in the plasma and excreta of the chimeric liver-humanized mice that were not previously characterised in, e.g., the excreta of rat and C57BL/6 mice. The different pattern of metabolism seen in these chimeric mice with a humanized liver compared to the conventional rodents may offer clues to the factors that contributed to the drug-induced liver injury seen in humans.

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein.

The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.

Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

Differential effect of troglitazone on the human bile acid transporters, MRP2 and BSEP, in the PXB hepatic chimeric mouse.

The aims of this study were to assess the utility of the PXB mouse model of a chimeric human/mouse liver in studying human-specific effects of an important human hepatotoxic drug, the PPARγ agonist, troglitazone. When given orally by gavage for 7 days, at dose levels of 300 and 600 ppm, troglitazone induced specific changes in the human hepatocytes of the chimeric liver without an effect on the murine hepatic portions. The human hepatocytes, in the vehicle-treated PXB mouse, showed an accumulation of electron-dense lipid droplets that appeared as clear vacuoles under the light microscope in H&E-stained sections. Following dosing with troglitazone, there was a loss of the large lipid droplets in the human hepatocytes, a decrease in the amount of lipid as observed in frozen sections of liver stained by Oil-red-O, and a decrease in the expression of two bile acid transporters, BSEP and MRP2. None of these changes were observed in the murine remnants of the chimeric liver. No changes were observed in the expression of three CYPs, CYP 3A2, CYP 1A1, and CYP 2B1, in either the human or murine hepatocytes, even though the baseline expression of the enzymes differed significantly between the two hepatocyte species with the mouse hepatocytes consistently showing increased expression of the protein of all three enzymes. This study has shown that the human hepatocytes, in the PXB chimeric mouse liver, retain an essentially normal phenotype in the mouse liver and, the albeit limited CYP enzymes studied show a more human, rather than a murine, expression pattern. In line with this conclusion, the study has shown a differential response of the human versus the mouse hepatocytes, and the effects observed are highly suggestive of a differential handling of the compound by the two hepatocyte species although the exact reasons are not as yet clear. The PXB chimeric mouse system therefore holds the clear potential to explore human hepatic-specific features, such as metabolism, prior to dosing human subjects, and as such should have considerable utility in drug discovery and development.

Pharmacokinetics and metabolism of midazolam in chimeric mice with humanised livers.

The pharmacokinetics and biotransformation of midazolam were investigated following single oral doses of 0.1, 1 and 10 mg/kg to chimeric mice with humanised livers (PXB mice) and to severe combined immunodeficient (SCID) mice used as controls. Pharmacokinetic analysis, on whole blood, revealed rapid absorption of the administered midazolam with a higher C(max) in PXB compared to SCID. The exposure to 1'-hydroxymidazolam was approximately 14-fold greater than to midazolam in the SCID mice and close to equivalent in the PXB mice. The metabolism of midazolam in SCID mice was faster than in the PXB mice such that pharmacokinetic data for midazolam in SCID mice could not be generated from the lowest dose in these animals. Both oxidative and conjugative metabolic pathways were identified in the PXB mice. All the major circulating metabolites observed in humans; 1'-hydroxymidazolam, 4'-hydroxymidazolam, 1',4'-dihydroxymidazolam and 1'-hydroxymidazolam glucuronide, were detected in the blood of PXB mice. However, 4'-hydroxymidazolam and the 1'-hydroxymidazolam glucuronide were not detected in blood samples obtained from SCID mice. The midazolam metabolite profile in the PXB mouse was similar to that previously reported for human suggesting that the PXB mouse model can provide a model system for predicting circulating human metabolites.

Evaluation of the pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone in mice with humanized livers.

The pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone were investigated following daily oral dosing, at 300 and 600 mg/kg, for 7 days to control (SCID) and chimeric (PXB) mice with humanized livers. Clinical chemistry revealed no consistent pattern of changes associated with troglitazone treatment in the PXB mouse. Human MRP2 but not mouse mrp2 was down-regulated following troglitazone treatment. Pharmacokinetic analysis revealed similar T(max) values for troglitazone in both mouse groups, a mono- and bi-phasic elimination phase in PXB and SCID mice, respectively, but a 3- to 5- and 2- to 5-fold higher C(max) and AUC, respectively, in PXB mice. Oxidative and conjugative metabolic pathways were identified, with the sulfate being the predominant metabolite in PXB compared to SCID mice (4- to 13-fold increase in liver and blood, respectively). The glucuronide conjugate was predominant in SCID mice. There was no evidence of glutathione conjugation. The primary oxidative pathways were mono- and di-oxidations which may also be attributed to quinone or hydroquinone derivatives. Several metabolites were observed in PXB mice only. As the troglitazone metabolic profiles in the PXB mouse were similar to reported human data the PXB mouse model can provide a useful first insight into circulating human metabolites of xenobiotics metabolized in the liver.

Effect of hepatitis C virus infection on the mRNA expression of drug transporters and cytochrome p450 enzymes in chimeric mice with humanized liver.

The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.

GH enhances proliferation of human hepatocytes grafted into immunodeficient mice with damaged liver.

We investigated effects of human (h) GH on the proliferation of h-hepatocytes that had been engrafted in the liver of albumin enhancer/promoter driven-urokinase plasminogen activator transgenic/severe combined immunodeficiency disease (uPA/SCID) mice (chimeric mice). The h-hepatocytes therein were considered to be deficient in GH, because hGH receptor (hGHR) is unresponsive to mouse GH. Actually, hIGF-1 was undetectable in chimeric mouse sera. The uPA/SCID mice were transplanted with h-hepatocytes from a 6-year (6Y)-old donor, and were injected with recombinant hGH (rhGH). rhGH stimulated the repopulation speed of h-hepatocytes; and up-regulated hIGF-1, human signal transducers and activators of transcription (hSTAT) 3, and cell cycle regulatory genes such as human forkhead box M1, human cell division cycle 25A, and human cyclin D1. To confirm the reproducibility of these effects of rhGH, similar experiments were run using h-hepatocytes from a 46-year (46Y)-old donor. rhGH similarly enhanced their repopulation speed and up-regulated the expression of the above-tested genes, especially hIGF-1 and hSTAT1. The extent of the enhancement by rhGH was much less than that in 6Y-hepatocyte-chimeric mice most probably due to the difference in GHR expression levels between the two donors. In conclusion, this study clearly demonstrated that rhGH stimulates the proliferation of h-hepatocytes in vivo.

Effects of low protein intake on the development of the remaining kidney in subtotally nephrectomized immature rats: apoptosis and epidermal growth factor.

Effects of low protein intake on the development of the remaining kidney in subtotally (5/6) nephrectomized immature rats were examined. Three week-old weaning rats were kept on a diet containing either 12% (Lp rats) or 18% (Np rats) protein for 4 or 8 weeks after subtotal nephrectomy. Blood urea nitrogen (BUN) concentration was determined at 2, 4, 6, and 8 weeks after 5/6 nephrectomy. At 4 or 8 weeks after the operation, glomerular sclerosis and tubulointerstitial damage were assessed by a standard semiquantitative analysis and were expressed as the glomerular sclerosis index (GSI) and interstitial fibrosis score (IFS), respectively. The localization of DNA fragmented cells in the kidney was examined by the terminal deoxynucleotidyl transferase (TdT) -mediated d-UTP-biotin nick end labeling (TUNEL) method and the localization of the epidermal growth factor (EGF) by immunohistochemical methods. BUN concentration was significantly lower in the Lp rats compared with that in the Np rats. Both 4 and 8 weeks after subtotal nephrectomy, GSI and incidence of TUNEL positive cells in the distal tubules were significantly lower in the Lp rats than in the Np rats. Four weeks after the operation, IFS was significantly lower in the Lp rats than in the Np rats. Four and 8 weeks after the operation, EGF positive cells in the distal tubules were more observed in the Lp rats than in the Np rats. These findings reveal that protein restriction is effective in preventing renal tubular scarring in immature rats and that EGF is involved in the process of this prevention.

Effects of maternal uninephrectomy on the development of fetal rat kidney: apoptosis and the expression of oncogenes.

The present study was designed to explore whether maternal uninephrectomy affects development of the collecting ducts in fetal kidney. Localization of DNA fragmented cells in the kidney of fetal rats from uninephrectomized mothers were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) method. Immunohistochemistry was used to examine the localizations of bcl-2 gene products. The gene expressions for bcl-2, p53, and WT1 mRNAs were examined by using the semi-quantitative reverse transcript-polymerase chain reaction. TUNEL positive cells were more numerous in the medullary collecting ducts of the fetuses from uninephrectomized mothers than in those of the fetuses from sham-operated ones. The expressions of bcl-2, p53, and WT1 mRNAs were lower in the fetuses from uninephrectomized mothers than in the fetuses from sham-operated ones. Cells in the medullary collecting ducts showed positive reactions to anti-bcl-2 gene products antibody with the reactions being weaker in the fetuses from uninephrectomized mothers. These results showed that maternal uninephrectomy accelerated the development of fetal rat kidney and it was associated with the lowered the expression of bcl-2 in fetal rat kidney.

Perinatal development of the rat kidney: apoptosis and epidermal growth factor.

Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney.