Human Liver S9 Research Articles

Impact of Genetic Polymorphisms and Drug-Drug Interactions Mediated by Carboxylesterase1 on Remimazolam Deactivation.

Remimazolam (Byfavo®), a recent FDA-approved ester-linked benzodiazepine, offers advantages in sedation, such as rapid onset and predictable duration, making it suitable for broad anesthesia applications. Its favorable pharmacological profile is primarily attributed to rapid hydrolysis, the primary metabolism pathway for its deactivation. Thus, understanding remimazolam hydrolysis determinants is essential for optimizing its clinical use. This study aimed to identify the enzyme(s) and tissue(s) responsible for remimazolam hydrolysis and to evaluate the influence of genetic polymorphisms and drug-drug interactions (DDIs) on its hydrolysis in the human liver. An initial incubation study with remimazolam and phosphate buffer saline (PBS), human serum, and the S9 fractions of human liver and intestine demonstrated that remimazolam was exclusively hydrolyzed by human liver S9 fractions. Subsequent incubation studies utilizing a Carboxylesterase inhibitor (Bis-para-nitrophenylphosphate, BNPP), recombinant human Carboxylesterase1 (CES1) and Carboxylesterase 2 (CES2) confirmed that remimazolam is specifically hydrolyzed by CES1 in human liver. Furthermore, in vitro studies with wild-type CES1 and CES1 variants transfected cells revealed that certain genetic polymorphisms significantly impair remimazolam deactivation. Notably, the impact of CES1 G143E was verified using individual human liver samples. Moreover, our evaluation of the DDIs between remimazolam and several other substrates/inhibitors of CES1-including simvastatin, enalapril, clopidogrel and sacubitril- found that clopidogrel significantly inhibited remimazolam hydrolysis at clinically relevant concentrations, with CES1 genetic variants potentially influencing the interactions. In summary, CES1 genetic variants and its interacting drugs are crucial factors contributing to interindividual variability in remimazolam hepatic hydrolysis, holding the potential to serve as biomarkers for optimizing remimazolam use. Significance Statement This investigation demonstrates that remimazolam is deactivated by CES1 in the human liver, with CES1 genetic variants and DDIs significantly influencing its metabolism. These findings emphasize the need to consider CES1 genetic variability and potential DDIs in remimazolam use, especially in personalized pharmacotherapy to achieve optimal anesthetic outcomes.

Characterizing the Metabolism of Tire Rubber-Derived p-Phenylenediamine Quinones to Identify Potential Exposure Biomarkers in Humans.

There is growing evidence of the frequent detection of tire rubber-derived contaminants p-phenylenediamine-derived quinones (PPD-Qs) (e.g., highly toxic N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q)) in the environment and biota and the adverse impact on organisms. Hence, a better understanding of their biotransformation/metabolism in humans is essential. However, relevant data are lacking owing to recent discoveries. Herein, the biotransformation patterns of 6PPD-Q and other five commonly detected PPD-Qs were characterized via combined in vitro assay and maternal cord blood screening monitoring. Rapid metabolism was found for each PPD-Q incubated with human liver S9 fraction and microsomes, resulting in the formation of abundant phase I and phase II metabolites. The subsequent screening for potential PPD-Q metabolites in blood samples showed the presence of suspect metabolites. Three detected metabolites were confirmed by matching the mass spectra and retention times of in vitro metabolites. N-Dealkylated, carboxy, carbonyl, and reductive metabolites and glucose, cysteine, and methionine conjugates were observed for the first time. The semiquantitative concentrations of metabolites were higher than those of the parent PPD-Qs, and several metabolites such as carboxy products were proposed as candidate biomarkers of PPD-Q exposure to humans. 6PPD-Q and N,N'-diphenyl-p-phenylenediamine quinone were detected in maternal and/or cord whole blood samples for the first time. This study holds great importance in elucidating the potential risks and health effects of PPD-Qs.

An in vitro evaluation on metabolism of mitragynine to 9-O-demethylmitragynine

Kratom (Mitragyna Speciosa Korth.) is an indigenous tree native to Southeast Asia whose leaves have been traditionally ingested as a tea and has seen its popularity increase in the United States. Although kratom and its constituents presently have no approved uses by the Food and Drug Administration, its major alkaloids (e.g., mitragynine) have psychoactive properties that may hold promise for the treatment of opioid cessation, pain management, and other indications. 9-O-demethylmitragynine is a major metabolite formed from mitragynine metabolism (36 % total metabolism) and displays similar pharmacologic activity. Cytochrome P450 (CYP) 3A4 has been identified as a major enzyme involved in mitragynine metabolism; however, the in vitro metabolism parameters of 9-O-demethylmitragynine formation are not well defined and a risk of potential drug interactions exists. Using human liver S9 fractions, 9-O-demethylmitragynine formation was generally linear for enzyme concentrations of 0–0.25 mg/mL and incubation times of 5–20 min. 9-O-demethylmitragynine displayed a Km 1.37 μM and Vmax of 0.0931 nmol/min/mg protein. Known CYP inhibitors and compounds that might be concomitantly used with kratom were assessed for inhibition of 9-O-demethylmitragynine formation. Ketoconazole, a CYP3A index inhibitor, demonstrated a significant effect on 9-O-demethylmitragynine formation, further implicating CYP3A4 as a major metabolic pathway. Major cannabinoids (10 μg/mL) displayed minor inhibition of 9-O-demethylmitragynine formation, while all other compounds had minimal effects. Mixtures of physiological achievable cannabinoid concentrations also displayed minor effects on 9-O-demethylmitragynine formation, making a metabolic drug interaction unlikely; however, further in vitro, in vivo, and clinical studies are necessary to fully exclude any risk.

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.

Differential genotoxicity of Polygoni Multiflori in rat and human: insights from Ames test and S9 metabolic activation system

The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.

Inhibitor of the non-structural protein 2 protease shows promising efficacy in mouse models of chikungunya

Chikungunya virus (CHIKV) is responsible for the most endemic alphavirus infections called Chikungunya. The endemicity of Chikungunya has increased over the past two decades, and it is a pathogen with pandemic potential. There is currently no approved direct-acting antiviral to treat the disease. As part of our antiviral drug discovery program focused on alphaviruses and the non-structural protein 2 protease, we discovered that J12 and J13 can inhibit CHIKV nsP2 protease and block the replication of CHIKV in cell cultures. Both compounds are metabolically stable to human liver microsomal and S9 enzymes. J13 has excellent oral bioavailability in pharmacokinetics studies in mice and ameliorated Chikungunya symptoms in preliminary efficacy studies in mice. J13 exhibited an excellent safety profile in in vitro safety pharmacology and off-target screening assays, making J13 and its analogs good candidates for drug development against Chikungunya.

Carboxylesterase 1-Based Drug-Drug Interaction Potential of Remimazolam: In-Vitro Studies and Literature Review.

The ultra-short-acting benzodiazepine remimazolam, approved for procedural sedation and general anesthesia, is inactivated by carboxylesterase 1 (CES1). Remimazolam´s involvement in CES1-mediated drug-drug interactions (DDIs) was investigated. Possible interactions of remimazolam were studied in co-exposure experiments with eleven different drugs. Further, substrates and inhibitors of CES1, identified in the literature, were evaluated for possible in-vivo inhibition using pharmacokinetic and Ki or IC50 values. Compounds with only one published inhibitory concentration and CES1 substrates lacking inhibition data were assigned conservative Ki values. In human liver homogenates and/or blood cells, remimazolam showed no significant inhibition of esmolol and landiolol metabolism, which, in turn, at up to 98 and 169 μM, respectively, did not inhibit remimazolam hydrolysis by human liver homogenates. In human liver S9 fractions, IC50 values ranged from 0.69 μM (simvastatin) and 57 μM (diltiazem) to > 100 μM (atorvastatin) and, for the remaining test items (bupropion, carvedilol, nelfinavir, nitrendipine, and telmisartan), they ranged from 126 to 658 μM. Remifentanil was ineffective even at 1250 μM. Guidance-conforming evaluation revealed no relevant drug-drug interactions with remimazolam via CES1. The algorithm-based predictions were consistent with human study data. Among CES1 inhibitors and substrates identified in the literature, only dapsone and rufinamide were found to be possible in-vivo inhibitors of remimazolam metabolism. Data and analyses suggest a very low potential of remimazolam for pharmacokinetic DDIs mediated by CES1. The theoretical approach and compiled data are not specific to remimazolam and, hence, applicable in the evaluation of other CES1 substrates.

Bioactivation of cinnamic alcohol in a reconstructed human epidermis model and evaluation of sensitizing potency of the identified metabolites.

Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer. Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis. A targeted multiple reaction monitoring mass spectrometry method was employed to study and quantify cinnamic alcohol along with eight potential phase I or phase II metabolites. The reconstructed human epidermis model, treated with cinnamic alcohol, was also analyzed with a non-targeted high-resolution mass spectrometry method to identify metabolites not included in the targeted method. Two metabolites identified with the targeted method, namely, pOH-cinnamic alcohol and pOH-cinnamic aldehyde, have not previously been identified in a metabolic in vitro system. Their reactivity toward biologically relevant nucleophiles was investigated and compared to their sensitizing potency in vivo in the murine local lymph node assay (LLNA). According to the LLNA, the pOH-cinnamic alcohol is non-sensitizing and pOH-cinnamic aldehyde is a moderate sensitizer. This makes pOH-cinnamic aldehyde less sensitizing than cinnamic aldehyde, which has been found to be a strong sensitizer in the LLNA. This difference in sensitizing potency was supported by the reactivity experiments. Cinnamic sulfate, previously proposed as a potential reactive metabolite of cinnamic alcohol, was not detected in any of the incubations. In addition, experiments examining the reactivity of cinnamic sulfate toward a model peptide revealed no evidence of adduct formation. The only additional metabolite that could be identified with the non-targeted method was a dioxolan derivative. Whether or not this metabolite, or one of its precursors, could contribute to the sensitizing potency of cinnamic alcohol would need further investigation. Cinnamic alcohol is one of the most common fragrance allergens and as it is more effective to patch test with the actual sensitizer than with the prohapten itself, it is important to identify metabolites with sensitizing potency. Further, improved knowledge of metabolic transformations occurring in the skin can improve prediction models for safety assessment of skin products.

Characterization and Metabolism of Drug Products Containing the Cocaine-Like New Psychoactive Substances Indatraline and Troparil.

With a rising demand of cocaine over the last years, it is likely that unregulated new psychoactive substances with similar effects such as indatraline ((1R,3S)-3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-1H-inden-1-amine) and troparil (Methyl (1R,2S,3S,5S)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-carboxylate) become popular as well. Both substances share a similar pharmacological profile as cocaine, while their potency is higher, and their duration of action is longer. This study investigated their metabolic fate in rat urine and incubations using pooled human liver S9 fraction (pHLS9). Indatraline formed two phase I and four phase II metabolites, with aromatic hydroxylation and glucuronidation being the main metabolic steps. All metabolites were detected in rat urine, while the parent compound was not detectable. Although low in abundance, indatraline metabolites were well identifiable due to their specific isotopic patterns caused by chlorine. Troparil formed four phase I and three phase II metabolites, with demethylation being the main metabolic step. Hydroxylation of the tropane ring, the phenyl ring, and combinations of these steps, as well as glucuronidation, were found. Phase I metabolites were detectable in rat urine and pHLS9, while phase II metabolites were only detectable in rat urine.

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.

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.

Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

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.

In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A “High-Throughput Pharmacokinetic” (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the “metabolism factor”. The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

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 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.

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.

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.

Roles of Fatty Acid Chain Length and Enzyme-Oriented Drug Controlled Release from pH-Triggering Self-Assembled Fatty Acid Conjugated Quetiapine Nanosuspensions.

Quetiapine (QTP) is a first-line antipsychotic drug, but its therapeutic druggability and patient adherence were limited due to high oral dose strength, low bioavailability and physicochemical/biopharmaceutical issues. To investigate the roles of fatty acid chain length and enzyme-oriented QTP controlled release from pH-triggering self-assembled fatty acid conjugated QTP nanosuspensions (NSPs). QTP was conjugated with different chain length fatty acids (C10-decanoic acid, C14-myristic acid, C18-stearic acid) to obtain QTP-fatty acid conjugates (QFCs: QD, QM, QS) by exploiting 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/4-dimethylaminopyridine (EDC/DMAP) conjugation chemistry. Then, the solubility, partitioning coefficient (log P), cell viability and cleavage kinetics of QFCs were evaluated. The pH-triggering self-assembled behaviors of QFCs to form QTP-fatty acid NSPs (QDN, QMN, QSN) by varying pH, QFC concentration and proton-to-QTP ratios were characterized. The morphological images, critical micelle concentration (CMC), physicochemical properties and enzyme-oriented QTP controlled release of NSPs were examined. Three QFCs were synthesized with different chain length fatty acids from QTP after desalting fumarate from QTP fumarate. The pH, QFC concentration and proton-to-quetiapine molar ratio could influence physicochemical properties and nanonization behaviors of QFCs. All three QFCs showed no effect on the viability of myoblast cells. The pH-triggering self-assembly of amphiphilic QFCs to form nanoparticles (NPs) occurred as the amine moiety of QTP was readily ionized in a strongly acidic environment (pH 1.2). Interestingly, the longer the fatty acid chain length, the lower water solubility, the higher log P (lipophilicity) and the smaller NP particle size were observed. The conversion rate of QFCs to liberate QTP by esterase in human plasma and liver S9 fractions was also inversely proportional to the fatty acid carbon chain length. Interestingly, the freeze-dried QMN showed the esterase-oriented controlled release of QTP over one month, unlike the initial burst release of QDN or the slowly delayed release pattern of QSN. A new pH-triggering self-assembled nanonization platform was developed using different chain length fatty acid conjugated QTP in low pH environment. By varying fatty acid chain length, the enzyme-oriented QTP controlled release dosage form was challenged to enhance the therapeutic effectiveness of QTP.