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.

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.

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident.

Protective role of osteopontin in endodontic infection.

Endodontic infections are polymicrobial infections resulting in bone destruction and tooth loss. The host response to these infections is complex, including both innate and adaptive mechanisms. Osteopontin (OPN), a secreted, integrin-binding protein, functions in the regulation of immune responses and enhancement of leucocyte migration. We have assessed the role of OPN in the host response to endodontic infection using a well-characterized mouse model. Periapical bone loss associated with endodontic infection was significantly more severe in OPN-deficient mice compared with wild-type 3 weeks after infection, and was associated with increased areas of inflammation. Expression of cytokines associated with bone loss, interleukin-1alpha (IL-1alpha) and RANKL, was increased 3 days after infection. There was little effect of OPN deficiency on the adaptive immune response to these infections, as there was no effect of genotype on the ratio of bacteria-specific immunoglobulin G1 and G2a in the serum of infected mice. Furthermore, there was no difference in the expression of cytokines associated with T helper type 1/type2 balance: IL-12, IL-10 and interferon-gamma. In infected tissues, neutrophil infiltration into the lesion area was slightly increased in OPN-deficient animals 3 days after infection: this was confirmed by a significant increase in expression of neutrophil elastase in OPN-deficient samples at this time-point. We conclude that OPN has a protective effect on polymicrobial infection, at least partially because of alterations in phagocyte recruitment and/or persistence at the sites of infection, and that this molecule has a potential therapeutic role in polymicrobial infections.

Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation.

Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

Promoter-independent regulation of vimentin expression in mammary epithelial cells by val(12)ras and TGFbeta.

The 1,029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val(12)ras. These cell lines respond to TGFbeta by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFbeta. In vitro, vimentin expression is induced in all the cell lines by TGFbeta treatment, whereas cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFbeta is mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFbeta treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half-life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFbeta, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.