Human S9 Fractions Research Articles

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

In vitro ADME, mouse pharmacokinetics of LD14b, and bioanalysis of a novel aβ 17β-HSD10 modulator for the treatment of Alzheimer’s disease

LD14b is an amyloid-β (Aβ) 17β-hydroxysteroid dehydrogenase type 10 (Aβ-17β-HSD10) protein-protein interaction modulator that shows promising in vitro and ex vivo activity to rescue Aβ-induced mitochondrial dysfunction, Aβ-induced toxicity, and Aβ-mediated inhibition of estradiol synthesis. The current study investigated in vitro human S9 fractions metabolic stability, apparent permeability, human and mouse plasma protein binding, in vivo pharmacokinetics, and tissue distribution in Balb/cJ mice. A fast (8-min), sensitive, reliable, and reproducible LC-MS/MS method was developed and validated over the dynamic range of 1-1000 ng/mL for the quantification of LD14b in different biological matrices (plasma, liver, kidney, brain, lungs, heart). LD14b was metabolically stable in human liver S9 fractions with 70% remaining after 90 minutes of incubation, showed intermediate apparent permeability of 3.55 × 10−06 cm/s and 6.16 × 10−06 cm/s for apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A), respectively across the Caco-2 monolayer, and was medium/highly bound to human plasma proteins (84.1%), mouse plasma proteins (85.7%), and mouse brain homogenate (95.4%). LD14b showed an in vivo predicted % absorption of 52% in Balb/cJ mice and was well-distributed to the peripheral tissues (liver, kidney, lungs, and heart) including the brain.

Identification of the Biotransformation Pathways of a Potential Oral Male Contraceptive, 11β-Methyl-19-Nortestosterone (11β-MNT) and Its Prodrugs: An In Vitro Study Highlights the Contribution of Polymorphic Intestinal UGT2B17.

11β-Methyl-19-nortestosterone dodecylcarbonate (11β-MNTDC) is a prodrug of 11β-MNT and is being considered as a promising male oral contraceptive candidate in clinical development. However, the oral administration of 11β-MNTDC exhibits an ~200-fold lower serum concentration of 11β-MNT compared to 11β-MNTDC, resulting in the poor bioavailability of 11β-MNT. To elucidate the role of the first-pass metabolism of 11β-MNT in its poor bioavailability, we determined the biotransformation products of 11β-MNT and its prodrugs in human in vitro models. 11β-MNT and its two prodrugs 11β-MNTDC and 11β-MNT undecanoate (11β-MNTU) were incubated in cryopreserved human hepatocytes (HHs) and subjected to liquid chromatography-high resolution tandem mass spectrometry analysis, which identified ten 11β-MNT biotransformation products with dehydrogenated and glucuronidation (11β-MNTG) metabolites being the major metabolites. However, 11β-MNTG formation is highly variable and prevalent in human intestinal S9 fractions. A reaction phenotyping study of 11β-MNT using thirteen recombinant UDP-glucuronosyltransferase (UGT) enzymes confirmed the major role of UGT2B17 in 11β-MNTG formation. This was further supported by a strong correlation (R2 > 0.78) between 11β-MNTG and UGT2B17 abundance in human intestinal microsomes, human liver microsomes, and HH systems. These results suggest that 11β-MNT and its prodrugs are rapidly metabolized to 11β-MNTG by the highly polymorphic intestinal UGT2B17, which may explain the poor and variable bioavailability of the drug.

New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1′s redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5–10-fold) with PK studies demonstrating efficacious doses for translation to clinic.

Metabolic characterization of the new benzimidazole synthetic opioids - nitazenes.

The recent re-emergence and the increasing popularity of nitazenes, a group of new synthetic opioids (NSO) that belong to the benzimidazole chemical class, has raised public health concerns. As a class of potential opioid analgesic agents whose development was discontinued in the 1960s due to their high potential for abuse, very little is known about their metabolism and physiologic disposition. In the current study, three nitazenes-butonitazene, isotonitazene and protonitaze were incubated in human liver microsomes (HLM), human S9 (HS9) fractions and recombinant cytochrome P450 enzymes. All three nitazenes were rapidly metabolized in both HLM and HS9 with over 95% depletion within 60min. In HLM, butonitazene, isotonitazene and protonitazene had in vitro intrinsic clearance (CLint) (µL/min/mg protein) values of 309, 221 and 216 respectively compared to 150 of verapamil, the positive control. In HS9, CLint values were 217, 139, and 150 for butonitazene, isotonitazene and protonitazene respectively compared to only 35 for testosterone, the control probe substrate. Putative metabolite identified from this study include products of hydroxylation, desethylation, dealkylation, desethylation followed by dealkylation, and desethylation followed by hydroxylation. The metabolic phenotyping showed CYP2D6, CYP2B6 and CYP2C8 and the major hepatic enzymes responsible for the metabolism of nitazenes. Within 30min of incubation, CYP2D6 depleted butonitazene (99%), isotonitazene (72%) and butonitazene (100%) significantly. The rapid metabolism of nitazenes may be an important factor in accurate and timely detections and quantitation of the unchanged drugs in human matrices following intoxication or in forensic analysis. The involvement of multiple polymorphic CYPs in their metabolism may play important roles in the susceptibility to intoxication and/or addiction, depending on the activity of the metabolites.

Abstract C129: Development of new increased potency Ref-1/APE1 targeted inhibitors that show promise for clinical applications against solid tumors

Abstract AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is over-expressed in most aggressive cancers and impacts various cancer cell signaling pathways. Among its primary functions, Ref-1’s redox activity plays a significant role in activating transcription factors (TFs) such as NFkB, HIF1a, and AP-1, among others, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop even more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of more potent lead compounds that exhibit a similar/better safety profile to APX3330. Pharmacokinetics, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, TF-driven luciferase activity, and two 3-dimensional (3D) cell killing assays (interstitial Tumor-Microenvironment (TME) on a Chip and 3D spheroid) were used for inhibitor characterization. These efforts have elucidated five compounds with improved PK, target engagement, and increased efficacy for cell killing compared to parent compound. Ligand-based WaterLOGSY NMR measurements demonstrate direct interaction of these inhibitors with Ref-1. Target inhibition was further confirmed following Ref-1 treatment via reporter assay with significant and dose-dependent decreases in TF-driven luciferase activity (3-6-fold compared to APX3330). In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly with significantly increased potency (~10-fold) compared to APX3330. APX2014, 44, and 51 also inhibited tumor cell proliferation more dramatically (1.5-3.5-fold) than cancer-associated fibroblasts from the TME. Two compounds, APX2044 and 51 outperformed the other analogs as well as the parent compound as assessed by inhibition of downstream TFs regulated by Ref-1 activity, inhibition of mitochondrial metabolism, greater potency against tumors than CAFs in monolayer and in 3D co-culture experiments. PK studies also demonstrated that efficacious doses are clinically achievable for these new inhibitors. Additionally, these findings have allowed us to develop additional analogs for further refinement. Thus, our preclinical findings identified more potent inhibitors of Ref-1 redox function (5-10-fold more than APX3330) that can be translated to the clinic. This study provides invaluable information regarding new Ref-1 analogs for advancement to in vivo and eventual IND-enabling studies and demonstrates the effectiveness of these compounds in sophisticated in vitro models in cancers that are in need of therapeutic options. Citation Format: Silpa Gampala, Hye-Ran Moon, Randall Wireman, Jacqueline Peil, Sonia Kiran, Andi Masters, Christine Bach, Whitney Smith-Kinnamen, Emma Doud, Ratan Rai, Amber L Mosley, Millie M Georgiadis, Bumsoo Han, Chi Zhang, Chafiq Hamdouchi, James Wikel, Mark R Kelley, Melissa L Fishel. Development of new increased potency Ref-1/APE1 targeted inhibitors that show promise for clinical applications against solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C129.

Physiologically-based pharmacokinetic modeling of the postbiotic supplement urolithin A predicts its bioavailability is orders of magnitudes lower than concentrations that induce toxicity, but also neuroprotective effects.

A range of health benefits are attributed to consuming urolithin A (UA), such as improved muscle health, anti-aging activity and neuroprotection, whereas few studies raise possible adverse effects at high doses, including genotoxicity and estrogenic effects. Therefore, understanding UA bioactivity and safety depends on its pharmacokinetics. However, there is no physiologically-based pharmacokinetic (PBPK) model available for UA, thus limiting reliable assessment of effects observed from in vitro experimentation. We characterized glucuronidation rates of UA by human S9 fractions. Partitioning and other physicochemical parameters were predicted using quantitative structure-activity relationship tools. Solubility and dissolution kinetics were determined experimentally. These parameters were used to construct a PBPK model, and results were compared with data from human intervention studies. We evaluated how different supplementation scenarios may influence UA plasma and tissue concentrations. Concentrations at which either toxic or beneficial effects were previously observed in vitro appear unlikely to be achieved in vivo. We established a first PBPK model for UA. It enables prediction of systemic UA concentrations and is critical for extrapolating in vitro results to in vivo uses. Results support the safety of UA, but also challenge the potential for readily achieving beneficial effects by postbiotic supplementation. This article is protected by copyright. All rights reserved.

In-vitro metabolism of LXY18, an orally available, potent blocker of AURKB relocation in mitosis

This study investigated the metabolism of LXY18, a quinolone-based compound that suppresses tumorigenesis by blocking AURKB localization. Metabolite profiling of LXY18 in liver microsomes from six species and human S9 fractions revealed that LXY18 undergoes various conserved metabolic reactions, such as N-hydroxylation, N-oxygenation, O-dealkylation, and hydrolysis, resulting in ten metabolites. These metabolites were produced through a combination of CYP450 enzymes, and non-CYP450 enzymes including CES1, and AO. Two metabolites, M1 and M2 were authenticated by chemically synthesized standards. M1 was the hydrolyzed product catalyzed by CES1 whereas M2 was a mono-N-oxidative derivative catalyzed by a CYP450 enzyme. AO was identified as the enzyme responsible for the formation of M3 with the help of AO-specific inhibitors and LXY18 analogs, 5b and 5c. M1 was the intermediate of LXY18 to produce M7, M8, M9, and M10. LXY18 potently inhibited 2C19 with an IC50 of 290 nM but had a negligible impact on the other CYP450s, indicating a low risk of drug-drug interaction. Altogether, the study provides valuable insights into the metabolic process of LXY18 and its suitability as a drug candidate. The data generated serves as a significant reference point for conducting further safety assessments and optimizing drug development.

Extrahepatic in vitro metabolism of peptides; comparison of human kidney and intestinal S9 fraction, human plasma and proximal tubule cells, using cyclosporine A, leuprorelin, and cetrorelix as model compounds

Peptide therapeutics showcase number of advantages compared to the traditional small molecule drugs, e,g. they usually have higher affinity to target and lower toxicity profiles. Endogenous peptides are mostly cleared from the body through renal clearance or proteolytic hydrolysis. As a part of drug discovery, metabolite identification is an important part in their development to identify metabolic hot spots and to further improve their stability. As the catabolism of the peptides and peptide-like drugs is often considered to be extrahepatic, the use of in vitro systems derived from these organs might be beneficial. In this study, multiple extrahepatic metabolic systems were evaluated for the applicability for peptide metabolism studies. Three peptide drugs (leuprorelin, cetrorelix, cyclosporin) were incubated in kidney and intestinal S9 fraction ( ± NADPH), fresh plasma (anticoagulants EDTA and heparin separately), and plated proximal tubule cells. Additionally, leuprorelin was also incubated with human kidney microsomes and cytosol to further investigate the NADPH-dependent metabolism detected in kidney S9 fraction. Both substrate disappearance and metabolite formation were monitored, using UPLC/HR-MS analysis of the collected samples.Overall, the largest number of metabolites was formed in the incubation with kidney S9 fraction, followed by intestinal S9, while incubations with proximal tubule cells produced lower number of metabolites All investigated peptides were stable in plasma and only a few metabolites were detected, likely because the studied peptide drugs have been optimized to be stable in plasma. Leuprorelin showed NADPH-dependent metabolite formation in kidney S9 fraction, while the metabolism of cetrorelix was more NADPH independent. As expected, formation of cytochrome P450 (CYP) catalyzed metabolism of cyclosporine was not observed with the employed extrahepatic systems. The NADPH-dependent metabolism of leuprorelin was detected also in the incubation with kidney cytosol, but not with kidney microsomes, and was thus not caused by CYPs or FMOs, but with cytosolic NADPH-dependent drug metabolizing enzymes. These enzymes could, in principle, activate the amide bond via reductive or oxidative metabolism outside the amide bond. The identity of the involved drug metabolizing enzymes in this process is still unknown.

Vicagrel is hydrolyzed by Raf kinase inhibitor protein in human intestine.

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04±1.99μM, 434.60±12.46nM/min/mg protein, and 21.69±0.28ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24±0.04mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.

Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA.

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

LC/Q-TOF MS and LC/QQQ MS based bioanalysis of a new ferrocene derivative as a potential anticancer lead with promising drug-like characteristics

Pyrazolopyrimidine ring present in various approved drugs is reported to target the tyrosine kinase receptor. A new pyrazolopyrimidine ferrocene derivative, which targets tumor pyruvate kinase M2 showed an impressive antiproliferative profile against human oral squamous cell carcinoma cell line CAL27 assessed using Alamar blue assay. In line with the lead optimization process, the molecule was studied for physicochemical properties where a bioanalytical method has been developed in plasma on liquid chromatography-mass spectrometry and validated following the USFDA bioanalytical method validation guideline. Plasma stability and plasma protein binding potential of the molecule have been evaluated. All the major metabolites of the compound have been identified through in vitro metabolite study employing rat liver microsome, human liver microsome, and human S9 fractions. The in silico toxicity profile of the metabolites was assessed using ProTox II software. Log P, Log D, and pKa of the molecule were found to be 4.5, 5, and 12, respectively. The molecule was found to be quite stable in plasma and have a moderate affinity towards plasma proteins (about 75 % binding). Four major metabolites have been identified and characterized by UHPLCQ-TOF-MS. The metabolites were found to have a moderate safety profile. The validated bioanalytical method and the metabolic pathway will be useful for future clinical studies and to assess the safety profile of the molecule. The finding of this study may also be useful in analyzing the desired drug-like properties through bioanalysis while designing new chemical entities based on metallocenes.

High resolution mass spectrometry-driven metabolite profiling of baricitinib to report its unknown metabolites and step-by-step reaction mechanism of metabolism.

Metabolite profiling is an integral part of the drug development process for selecting candidates with high therapeutic efficacy and low risk. Baricitinib (BARI) was approved in 2018 by the US Food and Drug Administration to treat rheumatoid arthritis. According to the available literature, no systematic study has been reported on the metabolite profiling of BARI. The biotransformation pathway of the drug has also not been established until recently. This study aims to identify BARI metabolites generated in in vitro matrices. The in vitro metabolism study was carried out using rat liver microsome, human liver microsomes, and human S9 fraction. Ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (U-HPLC-Q/TOF) and ultra-high-performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS/MS) were used to identify and characterize the metabolites of BARI. The in silico toxicity of BARI and its metabolite was studied using ProTox-II toxicity predictor software. A total of five new metabolites have been identified amongst which two (M1 and M2) were detected on both U-HPLC/LTQ-Orbitrap-MS/MS and U-HPLC-Q/TOF and two additional metabolites (M4 and M5) were detected on U-HPLC/LTQ-Orbitrap-MS/MS. Moreover, one metabolite (M3) was only detected on LC-QTOF. The major metabolic changes were found to be N-dealkylation, demethylation, hydroxylation, and hydrolysis. Metabolites M3 and M4 were found to have the potential for carcinogenicity. The novelty of the study can be justified by the unavailability of any previous research on in vitro metabolite profiling of BARI. Furthermore, this is the first time the biotransformation pathway of BARI and the toxicity potential of its metabolites have been reported.

In vitro and in vivo pharmacological profile of OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan

Tolvaptan is an orally active vasopressin V2 receptor antagonist and used for the treatment of volume overload in some disease as an aquaretic. Tolvaptan sodium phosphate (OPC-61815) is a pro-drug of tolvaptan that was designed to improve water solubility and enable intravenous use. The conversion of OPC-61815 to tolvaptan was evaluated for in vitro and in vivo pharmacokinetic studies. The pharmacodynamics of OPC-61815 were evaluated for in vitro receptor binding affinity, in vivo aquaretic and anti-edematous action. The solubility of OPC-61815 in water at 25 °C was 72.4 mg/mL and more than 100,000 times the solubility of tolvaptan. OPC-61815 was hydrolyzed to tolvaptan by human tissue S9 fractions and main enzyme of hydrolysis was alkaline phosphatase. After intravenous administration of OPC-61815 to rats and dogs, tolvaptan was detected in plasma within 5 min and the bioavailability of tolvaptan was 57.7% and 50.9%, respectively. Binding affinity of OPC-61815 for the human V2 receptor was 1/14 weaker than that of tolvaptan. OPC-61815 exerted dose-dependent aquaretic action in rats and dogs and a corresponding anti-edematous action in rat edema models. These results suggest that OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan, is an effective aquaretic by converting to tolvaptan after intravenous administration.

Comprehensive metabolite identification study of arterolane using hydrophilic interaction liquid chromatography with quadrupole-time-of-flight mass spectrometry.

In 2012, arterolane (ART) in combination with piperaquine received approval in India for the treatment of plasmodium-induced malaria; however, to date, a detailed metabolite identification study of ART has not been reported. Being polar in nature, ART shows early elution on reversed-phase columns which might be the rate-limiting factor of its systematic analytical studies. We have utilized hydrophilic interaction liquid chromatography (HILIC) to separate in vitro and in vivo metabolites of ART. The possible sites of metabolism were predicted by XenoSite software to obtain an initial assessment. In vitro studies were conducted by incubating the drug with liver microsomes such as human, rat and human S9 fractions. Later, in vivo studies were performed to check the metabolites in urine, faeces and plasma. The samples were pooled and subjected to the protein precipitation method before analysis by liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/QTOFMS). We have observed 15 metabolites in this study which were phase I metabolites formed due to hydroxylation, dihydroxylation, peroxide bond scission and oxidation. Here, we report 11 metabolites of ART for the first time. The metabolic pathways and plausible structures were proposed according to accurate mass measurements and its MS/MS data. The present study comprehensively reports the in vitro and in vivo metabolism of ART mentioning 11 novel metabolites. Here, extensive use of HILIC has helped to efficiently separate various metabolites. These findings would help prospects of ART disposition and its congeners.

Species-dependent hepatic and intestinal metabolism of selective estrogen receptor degrader LSZ102 by sulfation and glucuronidation

1. LSZ102 is an orally bioavailable selective estrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass. 2. To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase enzymes potentially involved in metabolism of LSZ102 were characterized. 3. Sulfate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolized by several human sulfotransferases expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulfation in human but glucuronidation in rat. 4. As LSZ102 is metabolized by a number of different sulfotransferases, drug-drug interactions resulting from the inhibition of one sulfotransferase are unlikely. 5. Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulfate M5, glucuronide M4, and secondary glucuronide/sulfate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection.

ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection.

Abstract 1452: Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models

Abstract Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from > 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.

Avenanthramide Metabotype from Whole-Grain Oat Intake is Influenced by Faecalibacterium prausnitzii in Healthy Adults.

Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0hours vs. 4.0-4.5hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.

Characterization of Formononetin Sulfonation in SULT1A3 Overexpressing HKE293 Cells: Involvement of Multidrug Resistance-Associated Protein 4 in Excretion of Sulfate.

Formononetin is one of the main active compounds of traditional Chinese herbal medicine Astragalus membranaceus. However, disposition of formononetin via sulfonation pathway remains undefined. Here, expression-activity correlation was performed to identify the contributing of SULT1A3 to formononetin metabolism. Then the sulfonation of formononetin and excretion of its sulfate were investigated in SULT1A3 overexpressing human embryonic kidney 293 cells (or HKE-SULT1A3 cells) with significant expression of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). As a result, formononetin sulfonation was significantly correlated with SULT1A3 protein levels (r = 0.728; p < 0.05) in a bank of individual human intestine S9 fractions (n = 9). HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate metabolite. Sulfate formation of formononetin in HEK-SULT1A3 cell lysate followed the Michaelis-Menten kinetics (Vmax = 13.94 pmol/min/mg and Km = 6.17 μM). Reduced activity of MRP4 by MK-571 caused significant decrease in the excretion rate (79.1%–94.6%) and efflux clearance (85.3%–98.0%) of formononetin sulfate, whereas the BCRP specific inhibitor Ko143 had no effect. Furthermore, silencing of MRP4 led to obvious decrease in sulfate excretion rates (>32.8%) and efflux clearance (>50.6%). It was worth noting that the fraction of dose metabolized (fmet), an indicator of the extent of drug sulfonation, was also decreased (maximal 26.7%) with the knockdown of MRP4. In conclusion, SULT1A3 was of great significance in determining sulfonation of formononetin. HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate. MRP4 mainly contributed to cellular excretion of formononetin sulfate and further mediated the intracellular sulfonation of formononetin.