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 ( = 27) or hepatitis C (HCV, = 30) cirrhotic versus noncirrhotic (control) livers ( = 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.

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.

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.

Background and ObjectiveUGT abundance and activity are associated with age and genetic polymorphisms. The aims of this project were to apply our previously generated quantitative UGT proteomics data i) to predict the effect of UGT2B7 ontogeny on the pharmacokinetics (PK) of morphine (an active metabolite) in breastfed neonate of a codeine‐prescribed mother, and ii) to predict age‐ and genotype‐dependent variability in the PK of MK‐7246 (MK; a UGT2B17 substrate) using physiologically‐based PK (PBPK) modeling.MethodsFirst an adult PBPK model was constructed and validated against the observed PK data following single oral dose of codeine (30 mg) and MK (1 mg) using Simcyp simulator. The simulation trial size was consisted of 10 trials of 10 subjects. The predicted adult codeine and morphine concentrations were validated by comparing literature data from adults with different CYP2D6 genotypes (i.e., poor, extensive and ultra‐rapid metabolizers, PM, EM, UM, respectively) and in breast‐feeding mother. We then predicted PK of codeine and morphine in neonates by integrating average milk intake, steady‐state milk‐to‐plasma ratio (2.5) and UGT2B7 ontogeny data into the PBPK model. Likewise, disposition of MK was predicted in children (age 0–18) and subjects carrying UGT2B17*2/*2, *1/*2 vs. reference allele (*1/*1) by integrating UGT2B17 proteomics data in the liver and intestine into the model. The models were evaluated by visual predictive checks and by comparison of predicted (mean and 5–95 % confidence intervals (CIs)) data with the observed PK parameters.ResultsThe predicted codeine mean AUC (ng/ml.hr) values in CYP2D6 PM, EM and UM adults were 211, 194 and 180 vs. 180, 191, and 192 (observed1), respectively. Similarly, the predicted morphine AUC (ng/ml.hr) values after codeine dose in adults were 0 (PM), 9.5 (EM), and 17 (UM), which were within the range of the observed data1 (Fig 1A). The normal neonatal morphine levels were closely predicted, i.e., < 2 ng/ml; however, the reported toxic levels linked to death of a neonate2 were only predicted accurately (54 vs. 70 ng/ml) when UGT2B7 activity was limited or not considered in mother and neonates. The predicted MK AUC (ng/ml.hr) also correlate the observed values in the adults (61.3 vs. 61.7; 4.8 vs. 6.1; 3.6 vs. 2.5) in case of carriers of UGT2B17 *2*2 (deletion), *1*2 and wild type *1*1 alleles3 (Fig 1B). We then predicted that MK clearance could be highly variable across age, e.g., 2 (neonates) to 85 (adults) ml/min/kg in the *1*1 individuals.DiscussionAge‐ and genotype‐dependent UGT abundance data are useful in predicting inter‐individual variability in drug metabolism. Consideration of variability in UGT2B7 mediated morphine metabolism after its formation from codeine could predict morphine toxicity, which is not fully explained by CYP2D6 variability alone2. Unprecedented variability in UGT2B17 protein abundance and its higher protein abundance in the intestine could be associated with larger age‐ and genotype‐dependent PK of its substrates as demonstrated for MK‐7246.Support or Funding InformationNIH R01 HD081299This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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