S9 Fraction Research Articles

Metabolism of alcohol ethoxylates (AEs) in rat, hamster, and human hepatocytes and liver S9: a pilot study for metabolic stability, metabolic pathway, and metabolites identification in vitro and in silico

Alcohol ethoxylates (AEs) are a well-known class of non-ionic surfactants widely used by the personal care market. The aim of this study was to evaluate and characterize the in vitro metabolism of AEs and identify metabolites. Five selected individual homologue AEs (C8EO4, C10EO5, C12EO4, C16EO8, and C18EO3) were incubated using human, rat, and hamster liver S9 fraction and cryopreserved hepatocytes. LC–MS was used to identify metabolites following the incubation of AEs by liver S9 and hepatocytes of all three species. All AEs were metabolized in these systems with a half-life ranging from 2 to 139 min. In general, incubation of AE with human liver S9 showed a shorter half-life compared to rat liver S9. While rat hepatocytes metabolized AEs faster than human hepatocytes. Both hydrophobic alkyl chain and hydrophilic EO head group groups of AEs were found to be target sites of metabolism. Metabolites were identified that show primary hydroxylation and dehydrogenation, followed by O-dealkylation (shortening of EO head groups) and glucuronidation. Additionally, the detection of whole EO groups indicates the cleavage of the ether bond between the alkyl chain and the EO groups as a minor metabolic pathway in the current testing system. Furthermore, no difference in metabolic patterns of each individual homologue AE investigated was observed, regardless of alkyl chain length or the number of EO groups. Moreover, there is an excellent agreement between the in vitro experimental data and the metabolite profile simulations using in silico approaches (OECD QSAR Toolbox). Altogether, these data indicate fast metabolism of all AEs with a qualitatively similar metabolic pathway with some quantitative differences observed in the metabolite profiles. These metabolic studies using different species can provide important reference values for further safety evaluation.

Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

A Comparison of In Vitro Metabolic Clearance of Various Regulatory Fish Species Using Hepatic S9 Fractions.

Bioaccumulation predictions can be substantially improved by combining in vitro metabolic rate measurements derived from rainbow trout hepatocytes and/or hepatic S9 fractions with quantitative structure-activity relationship (QSAR) modeling approaches. Compared with in vivo testing guidelines Organisation for Economic Co-operation and Development (OECD) 305 and Office of Chemical Safety and Pollution Prevention (OCSPP; an office of the US Environmental Protection Agency) 850.1730, the recently adopted OECD test guidelines 319A and 319B are in vitro approaches that have the potential to provide a time- and cost-efficient, humane solution, reducing animal use while addressing uncertainties in bioaccumulation across species. The present study compares the hepatic clearance of the S9 subcellular fraction of rainbow trout, bluegill, common carp, fathead minnow, and largemouth bass, discerning potential differences in metabolism between different warm- and cold-water species. With refinements to the in vitro metabolic S9 assay for high-throughput analysis, we measured in vitro clearance rates of seven chemicals crossing multiple classes of chemistry and modes of action. We confirmed that data from rainbow trout liver S9 fraction metabolic rates can be utilized to predict rainbow trout bioconcentration factors using an in vitro to in vivo extrapolation model, as intended in the OECD 319B applicability domain per the bioaccumulation prediction. Also, we determined that OECD 319B can be applied to other species, modified according to their habitat, adaptations to feeding behavior, and environmental conditions (e.g., temperature). Once toxicokinetics for each species is better understood and appropriate models are developed, this method can be an excellent tool to determine hepatic clearance and potential bioaccumulation across species. The present study could be leveraged prior to or in place of initiating in vivo bioconcentration studies, thus optimizing selection of appropriate fish species. Environ Toxicol Chem 2024;43:1390-1405. © 2024 SETAC.

Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be differentin vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similarenzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer bepresent if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

In Vitro to In Vivo Scalars for Drug Clearance in Nonalcoholic Fatty Liver and Steatohepatitis.

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics.

In vivo and in vitro metabolite profiling of nirmatrelvir using LC-Q-ToF-MS/MS along with the in silico approaches for prediction of metabolites and their toxicity.

Nirmatrelvir (NRV), a 3C-like protease or Mpro inhibitor of SARS-CoV-2, is used for the treatment of COVID-19 in adult and paediatric patients. The present study was accomplished to investigate the comprehensive metabolic fate of NRV using in vitro and in vivo models. The in vitro models used for the study were microsomes (human liver microsomes, rat liver microsomes, mouse liver microsomes) and S9 fractions (human liver S9 fractions and rat liver S9 fractions) with the appropriate cofactors, whereas Sprague-Dawley rats were used as the in vivo models. Nirmatrelvir was administered orally to Sprague-Dawley rats, which was followed by the collection of urine, faeces and blood at pre-determined time intervals. Protein precipitation was used as the sample preparation method for all the samples. The samples were then analysed by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-ToF-MS/MS) using an Acquity BEH C18 column with 0.1% formic acid and acetonitrile as the mobile phase. Four metabolites were found to be novel, which were formed via amide hydrolysis, oxidation and hydroxylation. Furthermore, an in silico analysis was performed using Meteor Nexus software to predict the probable metabolic changes of NRV. The toxicity and mutagenicity of NRV and its metabolites were also determined using DEREK Nexus and SARAH Nexus.

Identification of Sulfur-Containing Chlorinated Paraffin Structural Analogues in Human Serum: Origination from Biotransformation or Bioaccumulation?

Chlorinated paraffins (CPs) are manufactured and used in high quantities and have diverse structural analogues. It is generally recognized that sulfur-containing structural analogues of CPs are mainly derived from sulfate-conjugated phase II metabolism. In this study, we non-targeted identified three classes of sulfur-containing CP structural analogues (CPs-S) in human serum, including 44 CP sulfates (CPs-SO4H/CPs-SO4H-OH), 14 chlorinated benzene sulfates (CBs-SO4H), and 19 CP sulfite esters (CPs-SO3/CPs-S2O6), which were generated during the production of commercial mixtures of CPs and, thus, bioaccumulated via environmental exposures. We first wrote a program to screen CPs-S, which were baseline-separated from CPs according to their polar functional groups. Then, mass spectral analyses of alkalization-acidification liquid-liquid extracts of serum samples and Orbitrap mass spectrometry analyses in the presence and absence of tetraphenylphosphonium chloride (Ph4PCl), respectively, were performed to determine the ionization forms ([M + Cl]- or [M - H]-) of CPs-S. The presence of fragment ions (SO4H-, SO3-, SO2Cl-, and HSO3-) revealed the structures of CPs-S, which were validated by their detections in commercial mixtures of CPs. The estimated total concentrations of CPs-S in the human serum samples were higher than the concentrations of medium- and long-chain CPs. The profiles of CPs-S in human serum were similar to those detected in CP commercial mixtures and rats exposed to the commercial mixtures, but CPs-S were not detected in human liver S9 fractions or rat tissues after exposure to CP standards. These results, together with the knowledge of the processes used to chemically synthesize CPs, demonstrate that CPs-S in humans originates from environmental bioaccumulation.

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.

Genotoxicity and mutagenicity assessment of electronic cigarette liquids.

Electronic cigarettes (e-cigarettes) are often advertised as a safer alternative to traditional smoking. However, recent data suggest they may not be as safe as previously believed. This study aims to evaluate the genotoxicity and mutagenicity of e-cigarette liquids. We randomly selected eight varieties of e-cigarette liquids from the local market in Jeddah, Saudi Arabia. We evaluated their genotoxicity using the Genotoxicity SOS-Chromo Test™ Kit. In this investigation, a rat liver S9 fraction was utilized to emulate liver metabolic function to measure any chemical substance's mutagenic potential. The SOS-Chromo Test was performed by recording the β-galactosidase and alkaline phosphatase activity with and without the metabolic activation enzyme (S-9). All samples, except for the first two dilutions of sample 2, were nongenotoxic in the absence of the S9 activation enzyme, according to the genotoxicity analysis. However, when tested in the presence of the S9 enzyme, samples 2, 4, and 7 exhibited mutagenic activity at varying concentrations. Contrary to common belief, e-cigarettes are not safe. The present investigation confirms the presence of both toxicants and carcinogens in some e-cigarette liquids. This exposure could increase users' risk of various health complications.

Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

Interspecies comparison of metabolism of two novel prototype PFAS

As a result of proposed global restrictions and regulations on current-use per-and polyfluoroalkyl substances (PFAS), research on possible alternatives is highly required. In this study, phase I in vitro metabolism of two novel prototype PFAS in human and rat was investigated. These prototype chemicals are intended to be safer-by-design and expected to mineralize completely, and thus be less persistent in the environment compared to the PFAS available on the market. Following incubation with rat liver S9 (RL-S9) fractions, two main metabolites per initial substance were produced, namely an alcohol and a short-chain carboxylic acid. While with human liver S9 (HL-S9) fractions, only the short-chain carboxylic acid was detected. Beyond these major metabolites, two and five additional metabolites were identified at very low levels by non-targeted screening for the ether- and thioether-linked prototype chemicals, respectively. Overall, complete mineralization during the in vitro hepatic metabolism of these novel PFAS by HL-S9 and RL-S9 fractions was not observed. The reaction kinetics of the surfactants was determined by using the metabolite formation, rather than the substrate depletion approach. With rat liver enzymes, the formation rates of primary metabolite alcohols were at least two orders of magnitude higher than those of secondary metabolite carboxylic acids. When incubating with human liver enzymes, the formation rates of single metabolite carboxylic acids, were similar or smaller than those experienced in rat. It also indicates that the overall metabolic rate and clearance of surfactants are significantly higher in rat liver than in human liver. The maximum formation rate of the thioether congener exceeded 10-fold that of the ether in humans but were similar in rats. Overall, the results suggest that metabolism of the prototype chemicals followed a similar trend to those reported in studies of fluorotelomer alcohols.

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.

EVALUATION OF THE CYTOTOXICITY AND GENOTOXICOLOGICAL SAFETY PROFILE OF BIOACTIVE SILVER(I) COMPLEXES WITH AMINOADAMANTANE LIGANDS

Silver(I) complexes of amantadine (atdH), memantine (mtnH) and rimantadine (rtdH), named Ag-atd, Ag-mtn and Ag-rtd, respectively, were recently synthesized and described in the literature as promising antibacterial agents. In the present study, the cytotoxicity of such complexes was evaluated against cultures of primary epidermal keratinocytes (HaCaT) and murine melanoma tumor cells (B16-F10), and mutagenicity was studied by the Ames test to investigate their abilities to induce gene mutations. The Ames test was performed using Salmonella Typhimurium strains (TA98, TA100, TA102 and TA97a) capable of detecting frameshift and base pair substitution gene mutations, in experiments with and without metabolic activation (microsomal fraction S9). This study revealed significant cytotoxic activity against tumor cells and selectivity of Ag-atd and Ag-rtd complexes when compared to non-tumor human keratinocyte cells. Moreover, the Ag(I) complexes did not induce a significant growth in the number of revertant colonies when comparing with the negative control, both in the experiments without and with metabolic activation, indicating absence of mutagenic activity. The results are encouraging and collaborate in the genotoxicological investigations necessary for understanding the interaction and ability of the silver complexes to induce mutations and contribute to ensure their uses as future antibacterial or antitumor agents.

Functional roles and localization of hydrolases in the Japanese mitten crab Eriocheir japonica

The Japanese mitten crab Eriocheir japonica inhabits rivers throughout Japan and is being cultivated for food. To conduct aquaculture efficiently, it is crucial to comprehend the physiological functions of the target organisms. However, there is a lack of fundamental information on Japanese mitten crabs. In this study, hydrolases were extracted from the midgut glands of Japanese mitten crabs and their metabolic activities were analyzed. An enzyme with hydrolytic activity was discovered within the cytosol of the midgut gland. Western blot analysis also revealed that the Japanese mitten crab contains a hydrolase with cross-reactivity to human carboxylesterase 1 (hCES1) antibodies. The substrate specificity of the S9 fraction of the midgut gland was investigated and, interestingly, it was revealed that it reacts well with indomethacin phenyl ester and fluorescein diacetate, which are substrates of hCES2, not substrates of hCES1. Furthermore, this enzyme was observed to metabolize the ester derivative of astaxanthin, which is a red pigment inherent to the Japanese mitten crab. These findings underscore the significance the midgut gland in the Japanese mitten crab as an important organ for metabolizing both endogenous and exogenous ester–type compounds.

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.

Novel Independent Trans- and Cis-Genetic Variants Associated with CYP2D6 Expression and Activity in Human Livers.

Cytochrome P450 2D6 (CYP2D6) is a critical hepatic drug-metabolizing enzyme in humans, responsible for metabolizing approximately 20%-25% of commonly used medications such as codeine, desipramine, fluvoxamine, paroxetine, and tamoxifen. The CYP2D6 gene is highly polymorphic, resulting in substantial interindividual variability in its catalytic function and the pharmacokinetics and therapeutic outcomes of its substrate drugs. Although many functional CYP2D6 variants have been discovered and validated, a significant portion of the variability in the expression and activity of CYP2D6 remains unexplained. In this study, we performed a genome-wide association study (GWAS) to identify novel variants associated with CYP2D6 protein expression in individual human livers, followed by a conditional analysis to control for the effect of functional CYP2D6 star alleles. We also examined their impact on hepatic CYP2D6 activity. Genotyping on a genome-wide scale was achieved using the Illumina Multi-Ethnic Genotyping Array (MEGA). A data-independent acquisition (DIA)-based proteomics method was used to quantify CYP2D6 protein concentrations. CYP2D6 activity was determined by measuring the dextromethorphan O-demethylation in individual human liver s9 fractions. The GWAS identified 44 single nuclear polymorphisms (SNPs) that are significantly associated with CYP2D6 protein expressions with a P value threshold of 5.0 × 10-7 After the conditional analysis, five SNPs, including the cis-variants rs1807493 and rs1062753 and the trans-variants rs4073010, rs729559, and rs80274432, emerged as independent variants significantly correlated with hepatic CYP2D6 protein expressions. Notably, four of these SNPs, except for rs80274432, also exhibited a significant association with CYP2D6 activities in human livers, suggesting their potential as novel and independent cis- and trans-variants regulating CYP2D6. SIGNIFICANT STATEMENT: Using individual human livers, we identified four novel cis- and trans-pQTLs/aQTLs (protein quantitative trait loci/activity quantitative trait loci) of Cytochrome P450 2D6 (CYP2D6) that are independent from known functional CYP2D6 star alleles. This study connects the CYP2D6 gene expression and activity, enhancing our understanding of the genetic variants associated with CYP2D6 protein expression and activity, potentially advancing our insight into the interindividual variability in CYP2D6 substrate medication response.

Typical neonicotinoids and organophosphate esters, but not their metabolites, adversely impact early human development by activating BMP4 signaling

Recent studies have highlighted the presence of potentially harmful chemicals, such as neonicotinoids (NEOs) and organophosphate esters (OPEs), in everyday items. Despite their potential threats to human health, these dangers are often overlooked. In a previous study, we discovered that NEOs and OPEs can negatively impact development, but liver metabolism can help mitigate their harmful effects. In our current research, our objective was to investigate the toxicity mechanisms associated with NEOs, OPEs, and their liver metabolites using a human embryonic stem cell-based differentiation model that mimics early embryonic development. Our transcriptomics data revealed that NEOs and OPEs significantly influenced the expression of hundreds of genes, disrupted around 100 biological processes, and affected two signaling pathways. Notably, the BMP4 signaling pathway emerged as a key player in the disruption caused by exposure to these pollutants. Both NEOs and OPEs activated BMP4 signaling, potentially impacting early embryonic development. Interestingly, we observed that treatment with a human liver S9 fraction, which mimics liver metabolism, effectively reduced the toxic effects of these pollutants. Most importantly, it reversed the adverse effects dependent on the BMP4 pathway. These findings suggest that normal liver function plays a crucial role in detoxifying environmental pollutants and provides valuable experimental insights for addressing this issue.

A multivariate data analysis approach for the investigation of in vitro derived metabolites of ACP-105 in comparison with human in vivo metabolites

Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry.The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.

Identification of the Metabolites of Both Formononetin in Rat Hepatic S9 and Ononin in Rat Urine Samples and Preliminary Network Pharmacology Evaluation of Their Main Metabolites.

Astragalus membranaceus is a traditional Chinese medicine derived from the roots of Astragalus membranaceus (Fisch.) Bge., which has the same medicinal and edible uses in China. It is also widely used in daily food, and its pharmacological effects mainly include antioxidant effects, vascular softening effects, etc. Currently, it is increasingly widely used in the prevention of hypertension, cerebral ischemia, and stroke in China. Formononetin and its glucopyranoside (ononin) are both important components of Astragalus membranaceuss and may play important roles in the treatment of cardiovascular diseases (CVDs). This study conducted metabolic studies using formononectin and its glucopyranoside (ononin), including a combination of the in vitro metabolism of Formonetin using rat liver S9 and the in vivo metabolism of ononin administered orally to rats. Five metabolites (Sm2, 7, 9, 10, and 12) were obtained from the solution incubated with formononetin and rat hepatic S9 fraction using chromatographic methods. The structures of the five metabolites were elucidated as (Sm2)6,7,4'-trihydroxy-isoflavonoid; (Sm7)7,4'-dihydroxy-isoflavonoid; (Sm9)7,8,4'-trihydroxy-isoflavonoid; (Sm10)7,8,-dihydroxy-4'-methoxy-isoflavonoid; and (Sm12)6,7-dihydroxy-4'-methoxy- isoflavonoid on the basis of UV, NMR, and MS data. Totally, 14 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis, from which the formononetin was incubated with rat hepatic S9 fraction, and the main metabolic pathways were hydroxylation, demethylation, and glycosylation. Then, 21 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis from the urine samples from SD rats to which ononin was orally administered, and the main metabolic pathways were glucuronidation, hydroxylation, demethylation, and sulfonation. The main difference between the in vitro metabolism of formononetin and the in vivo metabolism of ononin is that ononin undergoes deglycemic transformation into Formonetin in the rat intestine, while Formonetin is absorbed into the bloodstream for metabolism, and the metabolic products also produce combined metabolites during in vivo metabolism. The six metabolites obtained from the aforementioned separation indicate the primary forms of formononetin metabolism, and due to their higher contents of similar isoflavone metabolites, they are considered the main active compounds that are responsible for pharmacological effects. To investigate the metabolites of the active ingredients of formononetin in the rat liver S9 system, network pharmacology was used to evaluate the cardiovascular disease (CVD) activities of the six primary metabolites that were structurally identified. Additionally, the macromolecular docking results of six main components and two core targets (HSP90AA1 and SRC) related to CVD showed that formononetin and its main metabolites, Sm10 and Sm12, may have roles in CVD treatment due to their strong binding activities with the HSP90AA1 receptor, while the Sm7 metabolite may have a role in CVD treatment due to its strong binding activity with the SRC receptor.