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.

Improving the accuracy of unbound fraction measurement of drug-protein binding by preconditioning the RED membrane inserts.

Aim: The objective of this study was to evaluate the rapid equilibrium dialysis (RED) device in protein binding assays in diluted protein matrices and to improve the accuracy of the unbound fraction (fu) measurement. Methodology: Protein binding assays of drug compounds in bovine serum albumin solutions and human plasma with different folds of dilution were performed using the RED device with and without preconditioning of the dialysis membrane inserts, and the results were compared with those using other approaches in this study. Results & conclusion: Preconditioning of the RED membrane inserts improved the fu data accuracy of drug-protein binding assay in matrices with relatively low protein contents and such impact could be compound dependent.

Assessment of Transporter-Mediated and Passive Hepatic Uptake Clearance Using Rifamycin-SV as a Pan-Inhibitor of Active Uptake.

The use of in vitro data for the quantitative prediction of transporter-mediated clearance is critical. Central to this evaluation is the use of hepatocytes, since they contain the full complement of transporters and metabolic enzymes. In general, uptake clearance (CLuptake) is evaluated by measuring the appearance of compound in the cell. Passive clearance (CLpd) is often determined by conducting parallel studies at 4 °C or by attempting to saturate uptake pathways. Both approaches have their limitations. Recent studies have proposed the use of Rifamycin-SV (RFV) as a pan-inhibitor of hepatic uptake pathways. In our studies, we confirm that transport activity of all major hepatic uptake transporters is inhibited significantly by RFV at 1 mM (OATP1B1, 1B3, and 2B1 = NTCP (80%), OCT1 (65%), OAT2 (60%)). Under these incubation conditions, we found that the free intracellular concentration of RFV is ∼175 µM and that several major CYPs and UGTs can be reversibly inhibited. Using this approach, we also determined CLuptake and CLpd of nine known OATP substrates across three different lots of human hepatocytes. The scaling factors generated for these compounds at 37 °C with RFV and 4 °C were found to be similar. The CLpd of passively permeable compounds like metoprolol and semagacestat were found to be higher at 37 °C compared to 4 °C, indicating a temperature effect on these compounds. In addition, our data also suggests that incorporation of medium concentrations into CLuptake and CLpd calculations may be critical for highly protein bound and highly lipophilic drugs. Overall, our data indicate that RFV, instead of 4 °C, can be reliably used to measure CLuptake and CLpd of drugs.

Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria.

A general method for determining bacterial uptake of compounds independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chemistry. We describe the evaluation of an LC-MS/MS assay measuring association of compounds with bacteria, using a set of over a hundred compounds (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been determined. All compounds were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity ( E. coli ligA251 Δ tolC). Testing a single compound concentration and incubation time, we found that, for equipotent compounds, LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-associated compound was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compounds, the distribution of compound within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chemical series to another. Our observations provide directions for further study of this difficult issue.

Approaches to measure protein binding of enzymatically unstable compounds in plasma.

AIM: To develop approaches to measure plasma protein binding (PPB) of enzymatically unstable compounds. METHODOLOGY: Bis-para-nitrophenyl phosphate (BNPP) was used to inhibit enzyme activity and stabilize two model compounds (diltiazem and oseltamivir) that are subject to enzyme-catalyzed hydrolysis in plasma. Protein binding of the compounds in BNPP-treated rat plasma was measured using equilibrium dialysis or ultrafiltration. CONCLUSION: PPB measurement of unstable compounds was improved by using enzyme inhibitor to stabilize the compounds in plasma during the assay. The effect of BNPP concentration on drug-protein binding appeared to be compound dependent. Given the compound's nonspecific binding to the assay device can be accounted for in the unbound fraction measurement, ultrafiltration can be a viable alternative or complementary approach for PPB assay of unstable compounds while minimizing the potential impact of enzyme inhibitor on drug-protein binding.

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.

Evaluation of sample extraction methods for minimizing hematocrit effect on whole blood analysis with volumetric absorptive microsampling.

AIM: Volumetric absorptive microsampler (VAMS) was designed to sample a fixed volume of blood regardless of the hematocrit (HCT) levels. Model compounds with a wide range of hydrophobicity were evaluated for their extraction recoveries from VAMS dried blood samples. RESULTS: For the highly hydrophobic compounds, recoveries with methanol or methanol/acetonitrile extraction were higher compared with using the aqueous mixture of methanol or acetonitrile. Extraction with methanol/acetonitrile (1:1) yielded more consistent recovery across the HCT range of 20-70% than using methanol alone, with good linearity, accuracy and precision achieved from 1 to 2000 ng/ml. CONCLUSION: An organic solvent sample preparation approach was developed to optimize extraction recovery and minimize the HCT effect on the analysis of VAMS dried blood samples.

Discovery and Characterization of a Water-Soluble Prodrug of a Dual Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV.

Benzimidazole 1 is the lead compound resulting from an antibacterial program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With the goal of improving key drug-like properties, namely, the solubility and the formulability of 1, an effort to identify prodrugs was undertaken. This has led to the discovery of a phosphate ester prodrug 2. This prodrug is rapidly cleaved to the parent drug molecule upon both oral and intravenous administration. The prodrug achieved equivalent exposure of 1 compared to dosing the parent in multiple species. The prodrug 2 has improved aqueous solubility, simplifying both intravenous and oral formulation.

Minimizing matrix effects while preserving throughput in LC-MS/MS bioanalysis.

BACKGROUND: Phospholipids are known to cause matrix effects in LC-MS analysis and are not effectively removed by one of the most common method of sample preparation: organic solvent protein precipitation. The objective of this research is to minimize phospholipid interferences chromatographically. RESULTS: In this article we examine several chromatographic approaches and highlight the method we developed that allows for the rapid gradient separation of model drug molecules from phospholipids. CONCLUSION: The new approach (which utilizes a mixture of methanol and acetonitrile as the organic mobile phase on a 2.1 × 20 mm C18 column) minimized phospholipids-related matrix effects in the analysis of plasma samples prepared by protein precipitation and is suitable for high-throughput bioanalysis in drug discovery.

Trace analysis of trimethoprim and sulfonamide, macrolide, quinolone, and tetracycline antibiotics in chlorinated drinking water using liquid chromatography electrospray tandem mass spectrometry.

A multirun analytical method has been developed and validated for trace determination of 24 antibiotics including 7 sulfonamides, 3 macrolides, 7 quinolones, 6 tetracyclines, and trimethoprim in chlorine-disinfected drinking water using a single solid-phase extraction method coupled to liquid chromatography with positive electrospray tandem mass spectrometry detection. The analytes were extracted by a hydrophilic-lipophilic balanced resin and eluted with acidified methanol (0.1% formic acid), resulting in analyte recoveries generally above 90%. The limits of quantitation were mostly below 10 ng/L in drinking water. Since the concentrated sample matrix typically caused ion suppression during electrospray ionization, the method of standard addition was used for quantitation. Chlorine residuals in drinking water can react with some antibiotics, but ascorbic acid was found to be an effective chlorine quenching agent without affecting the analysis and stability of the antibiotics in water. A preliminary occurrence study using this method revealed the presence of some antibiotics in drinking waters, including sulfamethoxazole (3.0-3.4 ng/L), macrolides (1.4-4.9 ng/L), and quinolones (1.2-4.0 ng/L).