Human liver microsomes (HLM) and human liver S9 fractions (HLS9) are commonly used to study drug metabolism in vitro. However, a quantitative comparison of HLM and HLS9 proteomes is lacking, resulting in the arbitrary selection of one hepatic preparation over another and in difficulties with data interpretation. In this study, we applied a label-free global absolute quantitative proteomics method to the analysis of HLS9 and the corresponding HLM prepared from 102 individual human livers. A total of 3137 proteins were absolutely quantified, and 3087 of those were determined in both HLM and HLS9. Protein concentrations were highly correlated between the two hepatic preparations (R 5 0.87, P < 0.0001). We reported the concentrations of 98 drug-metabolizing enzymes (DMEs) and 51 transporters, and demonstrated significant differences between their abundances in HLM and HLS9. We also revealed the proteinprotein correlations among these DMEs and transporters and the sex effect on the HLM and HLS9 proteomes. Additionally, HLM and HLS9 displayed distinct expression patterns for protein markers of cytosol and various cellular organelles. Moreover, we evaluated the interindividual variability of three housekeeping proteins, and identified five proteins with low variation across individuals that have the potential to serve as new internal controls for western blot experiments. In summary, these results will lead to better understanding of data obtained from HLM and HLS9 and assist in in vitro-in vivo extrapolations. Knowing the differences between HLM and HLS9 also allows us to make better-informed decisions when choosing between these two hepatic preparations for in vitro drug metabolism studies. SIGNIFICANCE STATEMENTThis investigation revealed significant differences in protein concentrations of drug-metabolizing enzymes and transporters between human liver microsomes and S9 fractions. We also determined the protein-protein correlations among the drugmetabolizing enzymes and transporters and the sex effect on the proteomes of these two hepatic preparations. The results will help interpret data obtained from these two preparations and allow us to make more informed decisions when choosing between human liver microsomes and S9 fractions for in vitro drug metabolism studies.

Nucleoside and nucleotide analogs are an essential class of antivirals for COVID-19 treatment. Several nucleoside/nucleotide analogs have shown promising effects against SARS-CoV-2 in vitro ; however, their in vivo efficacy is limited. Nucleoside/nucleotide analogs are often formed as ester prodrugs to improve pharmacokinetics (PK) performance. After entering cells, the prodrugs undergo several enzymatic metabolism steps to form the active metabolite triphosphate nucleoside (TP-Nuc); prodrug activation is therefore associated with the abundance and catalytic activity of the corresponding activating enzymes. Having the activation of nucleoside/nucleotide prodrugs occur at the target site of action, such as the lung, is critical for anti-SARS-CoV-2 efficacy. Herein, we conducted an absolute quantitative proteomics study to determine the expression of relevant activating enzymes in human organs related to the PK and antiviral efficacy of nucleoside/nucleotide prodrugs, including the lung, liver, intestine, and kidney. The protein levels of prodrug-activating enzymes differed significantly among the tissues. Using catalytic activity values reported previously for individual enzymes, we calculated prodrug activation profiles in these tissues. The prodrugs evaluated in this study include nine McGuigan phosphoramidate prodrugs, two cyclic monophosphate prodrugs, two l -valyl ester prodrugs, and one octanoate prodrug. Our analysis showed that most orally administered nucleoside/nucleotide prodrugs were primarily activated in the liver, suggesting that parenteral delivery routes such as inhalation and intravenous infusion could be better options when these antiviral prodrugs are used to treat COVID-19. The results also indicated that the l -valyl ester prodrug design can plausibly improve drug bioavailability and enhance effects against SARS-CoV-2 intestinal infections. This study further revealed that an octanoate prodrug could provide a long-acting antiviral effect targeting SARS-CoV-2 infections in the lung. Finally, our molecular docking analysis suggested several prodrug forms of favipiravir and GS-441524 that are likely to exhibit favorable PK features over existing prodrug forms. In sum, this study revealed the activation mechanisms of various nucleoside/nucleotide prodrugs relevant to COVID-19 treatment in different organs and shed light on the development of more effective anti-COVID-19 prodrugs.

A previous in vitro study demonstrated the CES1 genetic variant, G143E (rs71647871), significantly impaired enalapril activation. Two previous clinical studies examined the impact of G143E on single-dose enalapril PK (10 mg); however, the results were inconclusive. A prospective, multi-dose, pharmacokinetics and pharmacodynamics (PK/PD) study was conducted to determine the impact of the CES1 G143E variant on enalapril steady-state PK and PD in healthy volunteers. Methods: Study participants were stratified to G143E non-carriers (n = 15) and G143E carriers (n = 6). All the carriers were G143E heterozygotes. Study subjects received enalapril 10 mg daily for seven consecutive days prior to a 72 hour PK/PD study. Plasma concentrations of enalapril and its active metabolite enalaprilat were quantified by an established liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.Results: The CES1 G143E carriers had 30.9% lower enalaprilat C max (P = 0.03) compared to the non-carriers (38.01 vs. 55.01 ng/mL). The carrier group had 27.5% lower AUC 0-∞ (P = 0.02) of plasma enalaprilat compared to the non-carriers (374.29 vs. ng*h/mL). The carriers also had a 32.3% lower enalaprilat-to-enalapril AUC 0-∞ ratio (P = 0.003) relative to the non-carriers. The average maximum reduction of systolic blood pressure in the non-carrier group was approximately 12.4% at the end of the study compared to the baseline (P = 0.001). No statistically significant blood pressure reduction was observed in the G143E carriers. Conclusions:The CES1 loss-of-function G143E variant significantly impaired enalapril activation and its systolic blood pressure-lowering effect in healthy volunteers.angiotensin-converting enzyme (ACE) inhibitors, carboxylesterase 1 (CES1), enalapril, pharmacogenetics, pharmacokinetics Principal investigator statement: The authors confirm that the Principal Investigator for this paper is Hao-Jie Zhu and that he had direct clinical responsibility for patients.ClinicalTrials.gov Identifier: NCT03051282

Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study. Conclusions Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods.