Contribution of Uptake and Efflux Transporters to Oral Pharmacokinetics of Furosemide.

Furosemide is a widely used diuretic for treating excessive fluid accumulation caused by disease conditions like heart failure and liver cirrhosis. Furosemide tablet formulation exhibits variable pharmacokinetics (PK) with bioavailability ranging from 10 to almost 100%. To explain the variable absorption, we integrated the physicochemical, in vitro dissolution, permeability, distribution, and the elimination parameters of furosemide in a physiologically-based pharmacokinetic (PBPK) model. Although the intravenous PBPK model reasonably described the observed in vivo PK data, the reported low passive permeability failed to capture the observed data after oral administration. To mechanistically justify this discrepancy, we hypothesized that transporter-mediated uptake contributes to the oral absorption of furosemide in conjunction with passive permeability. Our in vitro results confirmed that furosemide is a substrate of intestinal breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), and organic anion transporting polypeptide 2B1 (OATP2B1), but it is not a substrate of P-glycoprotein (P-gp) and MRP2. We then estimated the net transporter-mediated intestinal uptake and integrated it into the PBPK model under both fasting and fed conditions. Our in vitro data and PBPK model suggest that the absorption of furosemide is permeability-limited, and OATP2B1 and MRP4 are important for its permeability across intestinal membrane. Further, as furosemide has been proposed as a probe substrate of renal organic anion transporters (OATs) for assessing clinical drug-drug interactions (DDIs) during drug development, the confounding effects of intestinal transporters identified in this study on furosemide PK should be considered in the clinical transporter DDI studies.

Age- and Genotype-Dependent Variability in the Protein Abundance and Activity of Six Major Uridine Diphosphate-Glucuronosyltransferases in Human Liver.

The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by ∼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine.

Abundance of Phase 1 and 2 Drug-Metabolizing Enzymes in Alcoholic and Hepatitis C Cirrhotic Livers: A Quantitative Targeted Proteomics Study.

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic (n = 27) or hepatitis C (HCV, n = 30) cirrhotic versus noncirrhotic (control) livers (n = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.