Formation Of Reactive Metabolites Research Articles

PHARMACOLOGICAL APPLICATION OF THIOPHENE DERIVATIVES

Thiophene nucleus has been established as the potential entity in the largely growing chemical world of heterocyclic compounds processing promising pharmacological characteristics.The knowledge of various synthetic pathway and the diverse physicochemical parameters of such compounds draw the especial attention of medical chemist to produce combanitorial library and carry out exhaustive efforts in the search of lead molecules. Thiophene is a five membered , sulfur containing hetero aromatic ring generally used as building block in drugs. It is structural metabolism leads to the formation of reactive metabolites. This compounds are widely spread in nature & have diversified application in design of new drug molecule. Thiophene has been established as the potential entity in largely growing chemical world of heterocyclic compounds possessing a pharmacological characteristics. A series of thiophene compound can be synthesize through various routes with pharmacological activity. The simple five – membered heterocycle are thiophene, with sulphur atom. It also undergo electrophilic aromatic substitution very readily.Sulphur is the least electron donor as compare to nitrogen and oxygen.Thiophene is similar to benzene in reactivity. Thiophene is the least reactive of the three because the p orbital of the lone pair of electrons on sulphur that conjugates with the ring is a 3p orbital rather than the 2p orbital of N or O, so overlap with 2p orbitals on carbon is less good. Thiophene undergo more or less normal Friedel- Craft reactions, although the less reactive unhydrides( aceticunhydride, Ac2O ) are used instead of acid chlorides, and weaker Lewis acid than AlCl3 are preferred. The regioselectivity is the – the 2- position is more reactive than 3- position. The present review includes the properties, synthesis, and a wide spectrum of biological activities of thiophene based scaffolds, and summary of thiophene containing active pharmaceutical ingredients (APIs) available in the market. KEY WORDS :Thiophene, Medicinal Importance, Biological Importance, Reaction of thiophene

Mechanistic investigation of liver injury induced by BMS-932481, an experimental ɣ-secretase modulator.

BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury.

Message Passing Neural Networks Improve Prediction of Metabolite Authenticity.

Cytochrome P450 enzymes aid in the elimination of a preponderance of small molecule drugs, but can generate reactive metabolites that may adversely react with protein and DNA and prompt drug candidate attrition or market withdrawal. Previously developed models help understand how these enzymes modify molecule structure by predicting sites of metabolism or characterizing formation of metabolite-biomolecule adducts. However, the majority of reactive metabolites are formed by multiple metabolic steps, and understanding the progenitor molecule's network-level behavior necessitates an integrative approach that blends multiple site of metabolism and structure inference models. Our previously developed tool, XenoNet 1.0, generates metabolic networks, where nodes are molecules and weighted edges are metabolic transformations. We extend XenoNet with a bidirectional message passing neural network that integrates edge feature information and local network structure using edge-conditioned graph convolutions and jumping knowledge to predict the authenticity of inferred Phase I metabolite structures. Our model significantly outperformed prior work and algorithmic baselines on a data set of 311 networks and 6606 intermediates annotated using a chemically diverse set of 20 736 individual in vitro and in vivo reaction records accounting for 92.3% of all human Phase I metabolism in the Accelrys Metabolite Database. Cross-validated predictions resulted in area under the receiver operating characteristic curves of 88.5% and 87.6% for separating experimentally observed and unobserved metabolites at global and network levels, respectively. Further analysis verified robustness to networks of varying depth and breadth, accurate detection of metabolites, such as d,l-methamphetamine, that are experimentally observed or unobserved in different network contexts, extraction of important metabolic subnetworks, and identification of known bioactivation pathways, such as for nimesulide and terbinafine. By exploiting network structures, our approach accurately suggests unreported metabolites for experimental study and may rationalize modifications for avoiding deleterious pathways antecedent to reactive metabolite formation.

Role of Aryl Hydrocarbon Receptor and Oxidative Stress in the Regioselective Toxicities of Hydroxychrysenes in Embryonic Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are environmental contaminants that can be created through oxidation of parent PAHs. Previous studies have found that 2-hydroxychrysene (2-OHCHR) caused anemia in embryonic Japanese medaka whereas 6-hydroxychrysene (6-OHCHR) did not, an example of regioselective toxicity. Anemia was prevented by cytochrome P450 (CYP) inhibition, which reduced the formation of the potential oxidatively active metabolite, 1,2-catechol, from 2-OHCHR. 2-OHCHR has also been found to be a four-fold more potent aryl hydrocarbon receptor (AhR) agonist compared with 6-OHCHR. These findings led us to hypothesize that AhR activation and/or oxidative stress play an important role in 2-OHCHR toxicity. Although treatments with the AhR agonists polychlorinated biphenyl (PCB)126 and 2-methoxychrysene (2-MeOCHR) did not cause significant toxicity, pretreatments with the AhR antagonist, CH-223191, reduced anemia by 97.2 ± 0.84% and mortality by 96.6 ± 0.69%. Aryl hydrocarbon receptor inhibition by the antagonist was confirmed by significant reductions (91.0 ± 9.94%) in induced ethoxyresorufin-O-deethylase activity. Thiobarbituric acid reactive substances concentrations were 32.9 ± 3.56% higher (p < 0.05) in 2-OHCHR treatments at 100 hours postfertilization compared with controls. Staining 2-OHCHR-treated embryos with the reactive oxygen species (ROS) scavenger 2',7'-dichlorofluorescin diacetate revealed 32.6 ± 2.69% of 2-OHCHR-treated embryos exhibiting high concentrations of ROS in caudal tissues, which is a site for embryonic hematopoiesis in medaka. Pretreatment with antioxidants, N-acetylcysteine (NAC)or vitamin E (Vit E) significantly reduced 2-OHCHR-induced anemia (NAC: 80.7 ± 1.12% and Vit E: 99.1 ± 0.43%) and mortality (NAC: 67.1 ± 1.69% and Vit E: 98.9 ± 0.66%). These results indicate that AhR may mediate 2-OHCHR toxicity through canonical signaling by up-regulating CYP1, enhancing the formation of reactive metabolites of 2-OHCHR that generate ROS within caudal hematopoietic tissues, potentially disrupting hematopoiesis, leading to anemia and subsequent mortality. Environ Toxicol Chem 2023;42:698-706. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Mechanistic aspects of reactive metabolite formation in clomethiazole catalyzed biotransformation by cytochrome P450 enzymes.

Clomethiazole (CLM), a sedative and anticonvulsant drug, is commonly employed for the treatment of alcohol withdrawal syndromebecause it suppresses cytochrome P450 (P450) activity associated with the generation of free radicals and liver damage. The catalyzed biotransformation of thiazole-containing drugs by P450 is known to afford reactive metabolites. These metabolites can alter the biological functions of macromolecules and result in toxicity and adverse drug interactions. Multitargeted molecular modeling and quantum chemical DFT calculations were performed to explore the binding modes and molecular mechanisms underlying the mechanism-based inactivation (MBI) of P450 by CLM. The mechanistic details associated with reactive metabolite formation from further metabolic processes were extensively assessed. Seven possible routes were proposed for CLM-P450 biotransformation including CLM hydroxylation, sulfoxidation, N-oxidation, CN epoxidation (oxaziridine formation), and CC epoxidation. The results revealed a degree of preference for the C-N epoxidation pathway because of the low energy requirements of its rate-determining step (8.74 and 10.07 kcal mol-1 for LS and HS states, respectively). A kinetic competition for the CLM-methyl hydroxylation pathway was detected because the H-abstraction energy barrier was relatively comparable to the thermodynamically prevailing oxaziridine formation rate-determining step (12.58 and 14.52 kcal mol-1 for quartet and doublet states, respectively). Our studies assessed the mechanisms of covalent nucleophilic epoxide adduct formation through nucleophilic addition, hydrolysis of epoxidation products, and nonenzymatic degradation. CLM was shown to display P450-inhibitory activity by forming covalent adducts rather than further metabolization to reactive metabolites. The outcomes of molecular docking allowed assessing the binding profile of CLM with three human P450 isozymes, namely, CYP2E1, CYP3A4, and CYP2D6.

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.

Estradiol, Estrone and Ethinyl Estradiol Metabolism Studied by High Resolution LC-MS/MS Using Stable Isotope Labeling and Trapping of Reactive Metabolites.

Biotransformation reactions that xenobiotics undergo during their metabolism are crucial for their proper excretion from the body, but can also be a source of toxicity, especially in the case of reactive metabolite formation. Unstable, reactive metabolites are capable of covalent binding to proteins, and have often been linked to liver damage and other undesired side effects. A common technique to assess the formation of reactive metabolites employs trapping them in vitro with glutathione and characterizing the resulting adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Some endogenous compounds, however, can interfere with xenobiotic metabolites of interest, making the analysis more difficult. This study demonstrates the usefulness of isotope-labeled compounds to detect and elucidate the structures of both stable metabolites and trapped adducts of three estrogen analogs using an untargeted LC-MS/MS workflow. The metabolism of estradiol, estrone and ethinyl estradiol was investigated. Unlabeled and deuterated versions of these three compounds were incubated with human or rat liver microsomes in the presence of two different trapping agents, namely glutathione and N-acetylcysteine. The detection of closely eluting deuterated peaks allowed us to confirm the formation of several known metabolites, as well as many previously uncharacterized ones. The structure of each adduct was elucidated by the detailed analysis of high-resolution MS/MS spectra for elucidating fragmentation pathways with accurate mass measurements. The use of isotopic labeling was crucial in helping confirm many metabolites and adduct structures, as well as removing endogenous interferences.

Non-innocuous Consequences of Metabolic Oxidation of Alkyls on Arenes.

Reactive metabolite (RM) formation is widely accepted as playing a pivotal role in causing adverse idiosyncratic drug reactions, with most attention paid to drug-induced liver injury. Mechanisms of RM formation are determined by the drug's properties in relation to human enzymes transforming the drug. This Perspective focuses on enzymatic oxidation of alkyl groups on aromatics leading to quinone methides and benzylic alcohol sulfates as RMs, a topic that has not received very much attention. Unlike previous overviews, we will include in our Perspective several fulvene-like methides such as 3-methyleneindole. We also speculate that a few older drugs may form non-reported methides of this class. In addition, we report a few guiding DFT calculations of changes in free energy on going from a benzylic alcohol to the corresponding methide. Particularly facile reactions of 2-aminothiazole-5-methanol and 4-aminobenzyl alcohol are noted.

Role of Protein Damage Inflicted by Dopamine Metabolites in Parkinson's Disease: Evidence, Tools, and Outlook.

Dopamine is an important neurotransmitter that plays a critical role in motivational salience and motor coordination. However, dysregulated dopamine metabolism can result in the formation of reactive electrophilic metabolites which generate covalent adducts with proteins. Such protein damage can impair native protein function and lead to neurotoxicity, ultimately contributing to Parkinson's disease etiology. In this Review, the role of dopamine-induced protein damage in Parkinson's disease is discussed, highlighting the novel chemical tools utilized to drive this effort forward. Continued innovation of methodologies which enable detection, quantification, and functional response elucidation of dopamine-derived protein adducts is critical for advancing this field. Work in this area improves foundational knowledge of the molecular mechanisms that contribute to dopamine-mediated Parkinson's disease progression, potentially assisting with future development of therapeutic interventions.

Formation of reactive metabolites of benzbromarone in humanized-liver mice

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1′-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1′,6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.

Assessment of the Carcinogenic Potential of Pretomanid in Transgenic Tg.rasH2 Mice.

Pretomanid is a nitroimidazooxazine antimycobacterial drug that was approved as part of a three-drug oral regimen, consisting of bedaquiline, pretomanid, and linezolid, for 6-months treatment of adults with pulmonary extensively drug-resistant tuberculosis or with complicated forms of multidrug-resistant tuberculosis by the food and drug administration in the United States and regulatory bodies in over 10 other countries. Nitroaromatic compounds as a class carry a risk of genotoxicity and potential carcinogenicity based on reactive metabolite formation. A battery of good laboratory practice genotoxicity studies on pretomanid indicated that the compound was not genotoxic, however its hydroxy imidazole metabolite (M50) was genotoxic in the Ames assay. To assess the in vivo carcinogenic potential of pretomanid, hemizygous Tg.rasH2 mice were administered pretomanid once daily by oral gavage for 26 weeks. Male mice were given pretomanid in vehicle at doses of 0, 5, 15 and 40 mg/kg/day and female mice were given pretomanid in vehicle at doses of 0, 10, 30 and 80 mg/kg/day. Positive control mice of both sexes received intraperitoneal injections of urethane at 1000 mg/kg on Days 1, 3 and 5. There were no pretomanid-related early deaths, tumors, non-neoplastic microscopic findings, or gross necropsy findings at any dose level. The positive control gave the anticipated response of lung tumors. Oral administration of pretomanid to mice produced plasma exposure to the parent compound (high dose AUC of pretomanid 3 times the clinical AUC at the maximum recommended human dose) and exposure to the M50 metabolite (less than 10% of pretomanid) at all dose levels in both sexes. These data show that pretomanid was not carcinogenic in a transgenic mouse model at systemic exposures greater than human therapeutic exposures.

Multiwell photocatalytic microreactor device integrating drug biotransformation modeling and sample preparation on a MALDI target

Metabolic conversion of drugs to chemically reactive products, known as bioactivation, is considered to underlie the adverse drug reactions such as idiosyncratic hepatotoxicity. As bioactivation of most drugs involves phase I oxidation, UV-induced TiO2 photocatalytic oxidation (UV/TiO2-PCO) can be used as a fast, simple and inexpensive non-enzymatic method of oxidative metabolism simulation allowing for rapid preliminary screening of candidate drugs for reactive metabolite formation. We developed and evaluated a novel prototype 96-well on-target UV/TiO2-photocatalytic microreactor setup (PCμR96) that integrates drug metabolism simulation and sample preparation directly on a MALDI target. PCμR96 allows to sequentially perform UV/TiO2-PCO of xenobiotics, GSH or protein adduct formation, protein digestion and sample concentration, followed by surface- or matrix-assisted laser desorption ionization (SALDI/MALDI) mass spectrometry (MS). Amodiaquine (AQ), a well-studied antimalarial drug that is known to undergo oxidative biotransformation, was selected as a model compound. Oxidation products of AQ and their protein adducts were obtained using PCμR96 and identified by SALDI and MALDI MS/MS analysis. The achieved AQ oxidation products were in good agreement with the known AQ metabolites produced in biological systems. Two of the products were proved to be reactive towards cysteyl thiols of human globin.

Elucidating the structure and cytochrome P450-mediated mechanism for novel metabolites of GDC-0575 in rats

1. GDC-0575 is an ATP-competitive small-molecule inhibitor of ChK1 that is being developed by Genentech for the treatment of various human malignancies. 2. In a radiolabeled mass balance study of GDC-0575 in rats, two novel metabolites, named M12 (-71 Da,) and M17 (+288 Da), were detected as abundant circulating metabolites. 3. Subsequent mass spectrometry and nuclear magnetic resonance analysis showed that M12 was a cyclized metabolite of GDC-0575, whereas M17 was its heterodimer to the parent. We further determined that M12 was mainly generated by cytochrome P450 (Cyp) 2d2. 4. We proposed the potential mechanism was initiated by the oxidation on the pyrrole ring and subsequent cyclisation of the free primary amine onto C-3 of the pyrrole ring. This was followed by expulsion of cyclopropylcarboxamide and a loss of water to form intermediate I, which can be further oxidised to form M12, or dimerise with another molecule of GDC-0575 as nucleophile to form M17. 5. To verify this hypothesis, we attempted to trap the intermediate I with glutathione (GSH) trapping assay and the GSH conjugate on the pyrrole ring was identified. This suggests the oxidation on the pyrrole led to reactive metabolite formation and supported this proposed mechanism.

Molecular Mechanisms of Action of Selected Substances Involved in the Reduction of Benzo[a]pyrene-Induced Oxidative Stress.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) primarily formed by burning of fossil fuels, wood and other organic materials. BaP as group I carcinogen shows mutagenic and carcinogenic effects. One of the important mechanisms of action of (BaP) is its free radical activity, the effect of which is the induction of oxidative stress in cells. BaP induces oxidative stress through the production of reactive oxygen species (ROS), disturbances of the activity of antioxidant enzymes, and the reduction of the level of non-enzymatic antioxidants as well as of cytokine production. Chemical compounds, such as vitamin E, curcumin, quercetin, catechin, cyanidin, kuromanin, berberine, resveratrol, baicalein, myricetin, catechin hydrate, hesperetin, rhaponticin, as well as taurine, atorvastatin, diallyl sulfide, and those contained in green and white tea, lower the oxidative stress induced by BaP. They regulate the expression of genes involved in oxidative stress and inflammation, and therefore can reduce the level of ROS. These substances remove ROS and reduce the level of lipid and protein peroxidation, reduce formation of adducts with DNA, increase the level of enzymatic and non-enzymatic antioxidants and reduce the level of pro-inflammatory cytokines. BaP can undergo chemical modification in the living cells, which results in more reactive metabolites formation. Some of protective substances have the ability to reduce BaP metabolism, and in particular reduce the induction of cytochrome (CYP P450), which reduces the formation of oxidative metabolites, and therefore decreases ROS production. The aim of this review is to discuss the oxidative properties of BaP, and describe protective activities of selected chemicals against BaP activity based on of the latest publications.

In-line formation and identification of toxic reductive metabolites of aristolochic acid using electrochemistry mass spectrometry coupling

Small-molecule metabolism has been extensively studied in the past decades, notably driven by the development of new pharmaceutical ingredients. The understanding of metabolism is critical to the anticipation of reactive metabolite formation in vivo that is often associated with toxicity. Electrochemistry has been proposed to simulate the oxidoreductive metabolism reaction catalyzed by cytochrome P450, a family of microsomal enzymes strongly involved in xenobiotic metabolism. The implementation of an electrochemical cell online with mass spectrometry allows for the fast formation and identification of the reaction end products. This study discusses the ability of the synthetic electrochemical approach to simulate a complex lactamization reaction that involves the formation of reactive metabolites. Aristolochic acid I was used as a model molecule to evaluate the ability of electrochemical simulation to generate nitroso, hydroxylamine, N-hydroxylactam, lactam, and nitrenium ion metabolites.

A cysteine trapping assay for risk assessment of reactive acyl CoA metabolites

Some drugs with carboxylic acid moieties can potentially cause rare but severe hepatotoxicity. The reactive chemical species generated by drug metabolism are thought to be one reason for this event. Although the phase II conjugation metabolism of carboxylic acids generally renders a compound more polar and inactive, it is also responsible for the formation of reactive metabolites. This study aimed to provide a new approach towards the risk assessment of carboxylic acids in the aspect of reactive acyl CoA metabolites. Although acyl CoA metabolites have been concerned, it is difficult to detect them because of their instability. We investigated the trapping agents for acyl CoA metabolites. We found that cysteine is a good trapping agent and developed an assay method for the reactivity of acyl CoA metabolites. We evaluated 17 drugs with carboxylic acid moieties, all drugs concerned with hepatotoxicity displayed reactive potential. With consideration of the exposure of each parent drug, the correlation between drug labels and the calculated risk of carboxylic drugs was improved. These evaluations can be conducted without radiochemical reagents or the authentic standards of metabolites. We believe that the method will be beneficial for drug discovery.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

EFFECT OF THE DRUG PROSTIMUL ON THE INDICATORS OF ENDOGENOUS INTOXICATION AND ANTIOXIDANT PROTECTION IN SOWS

The article presents the results of studying the effect of the drug Prostimul on the indicators of endogenous intoxication and the antioxidant status of sows. It has been found that the use of Prostimul is accompanied by an increase in the indicators of enzymatic (catalase and glutathione peroxidase) and non-enzymatic (vitamins A, E and C) links of antioxidant protection, as well as a decrease in the content of transition products of free radical processes in the body of experimental animals. Since a decrease in the activity of the antioxidant system with an increase in the formation of free radicals and reactive metabolites is one of the basic mechanisms for the occurrence of various pathologies, it is possible to recommend the drug Prostimul as a means of prevention and pathogenetic therapy for the correction of free radical oxidation.

Hepatoprotective Potential of Malabar Tamarind (Garcinia gummi-gutta) Fruit-Rind Extract in Acetaminophen-Induced Toxicity Through Modulation of CYP2E1 Gene Expression in Hepatic Cells of Wistar Rats

The present study was aimed to assess the hepatoprotective effect of ethanolic extract of Garcinia gummi-gutta (GGE) fruit rind on acetaminophen-induced toxicity in a rat model and to explore the possible mechanism of action by assessing hepatic histopathology, serum bio­chemical parameters and expression of CYP2E1 gene in hepatic cells. The group I and 11 animals served as normal control and hepatotoxic control group, respectively; while group III, IV and V were administrated with GGE fruit-rind extract @ 50, 100 & 200 mg kg-1 orally, respectively, for 10 days. The group VI (positive control) received drug silymarin @ 100 mg kg-1 day-1 orally for 10 days. Hepatotoxicity was induced by single oral administration of acetaminophen (2 g kg-1) on 8th day. The results revealed that the extract produced dose-dependent hepatoprotective effect against acetaminophen-induced hepatic damage by improving the serum bio­chemical profile towards the normal range. Histopathology of liver showed that pretreatment with fruit-rind extract attenuated the toxicant-induced hepatocellular necrosis and enhanced the regeneration of hepatic cells. The hepatic expression level of CYP2E1 gene was found down-regulated in a dose dependent manner in extract-treated group as compared to the normal control group. The study revealed remarkable hepatoprotective effect of GGE in acetaminophen-induced toxicity in rats through regeneration of hepatic cells and by decreasing the formation of reactive metabolite, NAPQ1 by modulation of CYP2E1 gene expression in liver.

Synthesis and evaluation of tofacitinib analogs designed to mitigate metabolic activation

Tofacitinib (TFT), a JAK inhibitor used for the treatment of rheumatoid arthritis and other diseases, is associated with severe liver injury that is believed to be caused by its reactive aldehyde or epoxide metabolites. In this study, we synthesized six tofacitinib analogs designed to avoid the formation of reactive metabolites and evaluated their JAK3 inhibitory activity, metabolic stability, CYP3A time-dependent inhibition, and cytotoxicity. Our data indicated that purine analog 3, which showed little inhibition of CYP3A and cytotoxicity and inhibited JAK3 in the nanomolar range, could be a safer drug candidate than TFT. In addition, the results of the bioactivation study using TFT and its analogs suggest that the epoxide metabolite might contribute to TFT-induced CYP3A4 mechanism-based inhibition and hepatic toxicity.