CYP Enzymes Research Articles

Characterization of the metabolic contributions of cytochrome P450 isoforms to bicyclol using the relative activity factor method

Bicyclol is a hepatoprotective agent widely used for treating chronic hepatitis and drug-induced liver injuries in clinics. The purpose of the study was to elucidate the contribution of CYP450 enzymes to the metabolism of bicyclol using the relative activity factor approach. After incubation with human liver microsomes and recombinant human liver CYP450 enzymes, the calculated contribution of CYP3A4 and 2C19 to the metabolism of bicyclol was 85.6–90.3% and 9.2–9.7%, respectively. The metabolism was interrupted in the presence of CYP3A4 and 2C19 selective inhibitors. These findings help to predict or avoid metabolic drug–drug interactions or toxicity in clinical applications of bicyclol.

There and Back Again: A Perspective on 20 Years of CYP4Z1.

Cytochrome P450 (CYP)4Z1, a highly expressed CYP gene in breast cancer, was one of the last CYPs to be identified in the human genome, some 20 years ago. CYP4 enzymes typically catalyze ω-hydroxylation and metabolize ω3 and ω6 polyunsaturated fatty acids to bioactive lipid metabolites that can influence tumor growth and metastasis. These attributes of CYP4Z1 make it an attractive target for new chemotherapeutic drug design, as a potential biomarker for selection of patients that might respond favorably to drugs and for developing enzyme inhibitors as potential therapeutic agents. This review summarizes the current state of knowledge regarding the advancing biochemistry of CYP4Z1, its role in breast cancer, and the recent synthesis of selective chemical inhibitors of the enzyme. We identify gaps that need to be filled to further advance this field and present new experimental data on recombinant CYP4Z1 expression and purification of the active catalytic form. SIGNIFICANCE STATEMENT: In breast cancer, an unmet need is the availability of highly effective therapeutic agents, especially for triple negative breast cancer. The relevance of the work summarized in this mini-review is that it identifies a new potential drug target, CYP4Z1, and discusses ways in which the gene product's catalytic activity might be modulated in order to combat this malignancy and limit its spread.

Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 μg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity

BackgroundMetabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur.MethodsVarious concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent.ResultsAll the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 – 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested.ConclusionTherefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Cardioprotective response and senescence in aged sEH null female mice exposed to LPS.

Deterioration of physiological systems, like the cardiovascular system, occurs progressively with age impacting an individual's health and increasing susceptibility to injury and disease. Cellular senescence has an underlying role in age-related alterations and can be triggered by natural aging or prematurely by stressors such as the bacterial toxin lipopolysaccharide (LPS). The metabolism of polyunsaturated fatty acids by CYP450 enzymes produces numerous bioactive lipid mediators that can be further metabolized by soluble epoxide hydrolase (sEH) into diol metabolites, often with reduced biological effects. In our study, we observed age-related cardiac differences in female mice, where young mice demonstrated resistance to LPS injury, and genetic deletion or pharmacological inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid attenuated LPS-induced cardiac dysfunction in aged female mice. Bulk RNA-sequencing analyses revealed transcriptomics differences in aged female hearts. The confirmatory analysis demonstrated changes to inflammatory and senescence gene markers such as Il-6, Mcp1, Il-1β, Nlrp3, p21, p16, SA-β-gal, and Gdf15 were attenuated in the hearts of aged female mice where sEH was deleted or inhibited. Collectively, these findings highlight the role of sEH in modulating the aging process of the heart, whereby targeting sEH is cardioprotective.NEW & NOTEWORTHY Soluble epoxide hydrolase (sEH) is an essential enzyme for converting epoxy fatty acids to their less bioactive diols. Our study suggests deletion or inhibition of sEH impacts the aging process in the hearts of female mice resulting in cardioprotection. Data indicate targeting sEH limits inflammation, preserves mitochondria, and alters cellular senescence in the aged female heart.

Abstract CT131: Effect of rivoceranib administered as 200 mg once daily on the pharmacokinetics of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 substrates

Abstract Title: Effect of Rivoceranib Administered as 200 mg Once Daily on the Pharmacokinetics of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 Substrates Background: Rivoceranib is a selective vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor with potent antitumor activity. The purpose of this study is to evaluate the effect of rivoceranib on the pharmacokinetics of various CYP substrates in healthy subjects following administration of rivoceranib 200 mg once daily (QD), which is similar to the proposed rivoceranib dosing regimen (250 mg QD) in combination with camrelizumab for the treatment of hepatocellular carcinoma. Methods: This open-label, fixed-sequence, crossover drug-drug interaction phase 1 study evaluated the impact of multiple oral doses of rivoceranib at 200 mg QD on the pharmacokinetics of single oral doses of CYP enzyme substrates included in the Cooperstown 5+1 cocktail (midazolam 2 mg [CYP3A4/5 substrate], warfarin 10 mg [CYP2C9 substrate]/vitamin K 10 mg, dextromethorphan 30 mg [CYP2D6 substrate], omeprazole 40 mg [CYP2C19 substrate], and caffeine 200 mg [CYP1A2 substrate]). On Day 1, subjects received a single oral dose of the Cooperstown cocktail. Blood samples were collected predose on Day 1 and up to 120 hours post-dose for pharmacokinetic analyses. On Days 6-15, subjects received rivoceranib at 200 mg QD. On Day 11, a single dose of the Cooperstown Cocktail was co-administered with rivoceranib at 200 mg. Each dose was administered under postprandial conditions except for Day 11 when subjects fasted. Blood samples were collected predose on Day 11 and up to 120 hours post cocktail dosing for pharmacokinetic analyses. Subjects returned on any day between Days 21-25 for safety follow-up. Results: Rivoceranib 200 mg QD increased dextromethorphan AUC0-inf by 144% and Cmax by 70%, increased midazolam AUC0-inf by 65% and Cmax by 16%, increased the warfarin AUC0-inf by 28% and Cmax by 5%, increased omeprazole AUC0-inf by 77% and Cmax by 62%, and decreased caffeine AUC0-inf by 20% and Cmax by 7%. Cooperstown Cocktail alone or coadministration of Cooperstown Cocktail and rivoceranib was generally well tolerated. Conclusions:Rivoceranib at 200 mg QD does not substantially affect the exposure of the substrates of CYP3A4/5, CYP2C9, CYP2C19, and CYP1A2. Recommendation of dose adjustment of these CYP substrates is not needed when administered with rivoceranib at 200 mg QD. Rivoceranib may increase the exposure of the substrate of CYP2D6 by approximately 100%, indicating that a dose adjustment of CYP2D6 substrates and/or close monitoring patients should be considered when administered with rivoceranib 200 mg QD. Citation Format: Seong Jang, Xiaohui (Tracey) Wei, Xianzhang Meng, Joseph Reitano, Vinoo Urity, Cheol Hee Park, Bill Strickland, Grace Lee, David Nguyen. Effect of rivoceranib administered as 200 mg once daily on the pharmacokinetics of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 substrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT131.

Insight into chemically reactive metabolites of aliphatic amine pollutants: A de novo prediction strategy and case study of sertraline

The uncommon metabolic pathways of organic pollutants are easily overlooked, potentially leading to idiosyncratic toxicity. Prediction of their biotransformation associated with the toxic effects is the very purpose that this work focuses, to develop a de novo method to mechanistically predict the reactive toxicity pathways of uncommon metabolites from start aliphatic amine molecules, which employed sertraline triggered by CYP450 enzymes as a model system, as there are growing concerns about the effects on human health posed by antidepressants in the aquatic environment. This de novo prediction strategy combines computational and experimental methods, involving DFT calculations upon sequential growth, in vitro and in vivo assays, dissecting chemically reactive mechanism relevant to toxicity, and rationalizing the fundamental factors. Significantly, desaturation and debenzylation-aromatization as the emerging metabolic pathways of sertraline have been elucidated, with the detection of DNA adducts of oxaziridine metabolite in mice, highlighting the potential reactive toxicity. Molecular orbital analysis supports the reactivity preference for toxicological-relevant C-N desaturation over N-hydroxylation of sertraline, possibly extended to several other aliphatic amines based on the Bell-Evans-Polanyi principle. It was further validated toward some other wide-concerned aliphatic amine pollutants involving atrazine, ε-caprolactam, 6PPD via in silico and in vitro assays, thereby constituting a complete path for de novo prediction from case study to general applications.

Identification of missing CYP450 enzymes involved in paclitaxel biosynthesis and heterologous reconstitution of baccatin III

Identification of missing CYP450 enzymes involved in paclitaxel biosynthesis and heterologous reconstitution of baccatin III

In Vitro Hepatotoxicity of Routinely Used Opioids and Sedative Drugs.

A hepatocyte cell line was used to determine the hepatotoxicity of sedatives and opioids, as the hepatotoxicity of these drugs has not yet been well characterized. This might pose a threat, especially to critically ill patients, as they often receive high cumulative doses for daily analgosedation and often already have impaired liver function due to an underlying disease or complications during treatment. A well-established biosensor based on HepG2/C3A cells was used for the determination of the hepatotoxicity of commonly used sedatives and opioids in the intensive care setting (midazolam, propofol, s-ketamin, thiopental, fentanyl, remifentanil, and sufentanil). The incubation time was 2 × 3 days with clinically relevant (Cmax) and higher concentrations (C5× and C10×) of each drug in cell culture medium or human plasma. Afterward, we measured the cell count, vitality, lactate dehydrogenase (LDH), mitochondrial dehydrogenase activity, cytochrome P 450 1A2 (CYP1A2), and albumin synthesis. All tested substances reduced the viability of hepatocyte cells, but sufentanil and remifentanil showed more pronounced effects. The cell count was diminished by sufentanil in both the medium and plasma and by remifentanil only in plasma. Sufentanil and remifentanil also led to higher values of LDH in the cell culture supernatant. A reduction of mitochondrial dehydrogenase activity was seen with the use of midazolam and s-ketamine. Microalbumin synthesis was reduced in plasma after its incubation with higher concentrations of sufentanil and remifentanil. Remifentanil and s-ketamine reduced CYP1A2 activity, while propofol and thiopental increased it. Our findings suggest that none of the tested sedatives and opioids have pronounced hepatotoxicity. Sufentanil, remifentanil, and s-ketamine showed moderate hepatotoxic effects in vitro. These drugs should be given with caution to patients vulnerable to hepatotoxic drugs, e.g., patients with pre-existing liver disease or liver impairment as part of their underlying disease (e.g., hypoxic hepatitis or cholestatic liver dysfunction in sepsis). Further studies are indicated for this topic, which may use more complex cell culture models and global pharmacovigilance reports, addressing the limitation of the used cell model: HepG2/C3A cells have a lower metabolic capacity due to their low levels of CYP enzymes compared to primary hepatocytes. However, while the test model is suitable for parental substances, it is not for toxicity testing of metabolites.

Safety and pharmacokinetics of antifungal agent VT-1598 and its primary metabolite, VT-11134, in healthy adult subjects: phase 1, first-in-human, randomized, double-blind, placebo-controlled study of single-ascending oral doses of VT-1598.

VT-1598 is a novel fungal CYP51 inhibitor and 1-tetrazole-based antifungal drug candidate with improved selectivity minimizing off-target binding to and inhibition of human CYP450 enzymes. Data are presented from this first clinical study in the evaluation of the safety and pharmacokinetic (PK) of single ascending doses of 40, 80, 160, 320, and 640mg VT-1598, comprising a 160mg cohort in both fasting and fed states. Eight healthy adults per dose were randomized to receive either oral VT-1598 or placebo (3:1). Over the dose range, exposures were with relatively high variation. The maximum plasma concentrations (Cmax) for VT-1598 were 31.00-279.4ng/ml and for its primary metabolite, VT-11134, were 27.80-108.8ng/ml. The plasma area under the concentration-time curve to the last measurable concentration (AUC0-last) for VT-1598 were 116.1-4507 ng*h/ml, and for VT-11134 were 1140-7156 ng*h/ml. The dose proportionality was inconclusive based on the results of the power model. The peak concentration time (Tmax) was 4-5 h for VT-1598 and for VT-11134. Half-life was 103-126 h for VT-11134. After food intake, Cmax of VT-1598 increased by 44% (geometric mean ratio (GMR), 1.44; 90%CI [0.691, 2.19]) and AUC0-last by 126% (GMR, 2.26; 90%CI [1.09, 3.44]), while exposure of VT-11134 was decreased 23% for Cmax (GMR, 0.77; 90%CI [0.239, 1.31]) and unchanged for AUC0-last (GMR, 1.02; 90%CI [0.701, 1.33]). Neither VT-1598 nor VT-11134 were detected in urine. No serious adverse events (AEs) or AEs leading to early termination were observed. The safety and PK profiles of VT-1598 support its further clinical development.

Mechanistic insight into biotransformation of novel triazine-based flame retardant 1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione by human cytochrome P450s

The escalating focus on the environmental occurrence and toxicology of emerging pollutants underscores the imperative need for a profound exploration of their metabolic transformations mediated by human CYP450 enzymes. Such investigations have the potential to unravel the intricate metabolite profiles, substantially altering the toxicological outcomes. In this study, we integrated the computational simulations with in vitro metabolism experiments to investigate the metabolic activity and mechanism of an emerging pollutant, 1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione (TDBP-TAZTO), catalyzed by human CYP450s. The results highlight the important contributions of CYP2E1, 3A4 and 2C9 to the biotransformation of TDBP-TAZTO, leading to the identification of four distinct metabolites. The effective binding conformations governing biotransformation reactions of TDBP-TAZTO within active CYP450s are unveiled. Structural instability of primary hydroxyTDBP-TAZTO products suggests three potential outcomes: (1) generation of an alcohol metabolite through successive debromination and reduction reactions, (2) formation of a dihydroxylated metabolite through secondary hydroxylation by CYP450, and (3) production of an N-dealkylated metabolite via decomposition and isomerization reactions in the aqueous environment. The formation of a desaturated debrominated metabolite may arise from H-abstraction and barrier-free Br release during the primary oxidation, potentially competing with the generation of hydroxyTDBP-TAZTO. These findings provide detailed mechanistic insight into TDBP-TAZTO biotransformation by CYP450s, which can enrich our understanding of the metabolic fate and associated health risk of this chemical.

Impact of bariatric surgery on cytochrome P 450 enzyme activity.

Bariatric surgeries are becoming more prevalent as obesity rates continue to rise. Being that it is an effective weight-loss procedure, it can induce significant anatomical, physiological, and metabolic alterations, which affect the pharmacokinetics of various medications. Cytochrome (CYP) P450 is a group of enzymes that are primarily responsible for metabolizing most medications. Bariatric surgery may affect CYP activity and consequently alter metabolism of various medications, and the resulting weight loss may influence the metabolism of various drugs. This study investigates the impact of bariatric surgery on which CYP enzymes are affected and their effects medications. Authors of this study did an extensive literature review and research in databases including PubMed and EMBASE. The evidence was gathered for medication efficacy influenced by enzyme fluctuations to advocate for further studies for patients that undergo bariatric surgery. The search was limited to English-language results and is deemed up to date as of September 2023. There are numerous studies that indicated alterations of the CYP enzyme activity, which affects the pharmacokinetics of medications used to treat acute and chronic conditions after bariatric surgery. There are various mechanisms involved in CYP enzyme activity leading to fluctuations and the clearance of medications and subsequently compromising the efficacy and safety of these agents. It is imperative to conduct more prospective randomized control studies with longer duration to guide clinicians on how to manage medications with various CYP activity for patients' post-bariatric surgery.

Abstract 3325: HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice

Abstract Purpose: This study is to characterize newly identified KRAS G12D small molecule inhibitors HBW-012-D and HBW-012-E, both showing improved oral bioavailability than MRTX1133, the known clinical compound with poor bioavailability. Method: Through our medicinal chemistry efforts, we have aimed to design novel small-molecule KRAS G12D inhibitors that are orally bioavailable. The compounds were screened via pERK and cell viability (CTG) assays in multiple cell types. The optimized compounds were further evaluated based on their pharmacokinetic (PK) properties and toxicity. The anti-tumor efficacy of the leads was confirmed in the GP2D (colorectal carcinoma with KRAS G12D mutation) mouse xenograft tumor model. Result: As shown in the table, HBW-012-D and HBW-012-E have significantly better potency and PK characteristics than MRTX1133. In the mouse GP2D model, comparing head-to-head with the same vehicle and protocol (50 mg/kg, twice daily, orally), MRTX1133 hardly inhibited tumor growth, whereas HBW-012-D inhibited tumor growth well (58 % tumor growth inhibition), and HBW-012-E not only completely inhibited tumor growth but also further regressed tumor size by around 40%. Preliminary toxicity tests showed that HBW-012-D and HBW-012-E are safe: weak inhibition of CYP450 enzymes, negative mini-Ames test, excellent kinase selectivity, and no obvious abnormalities in the 14-day rat or mouse toxicity test. Conclusion: New KRAS G12D inhibitors, HBW-012-D and HBW-012-E, were identified. Their preclinical profile, including potency, efficacy, and PK data, are all superior to MRTX1133, and their safety meets the criteria of clinical development. They show potential as a preferred oral treatment option for patients with tumors mediated by the KRAS G12D mutation, such as pancreatic cancer that still lacks effective treatments. in vitro data IC 50 (nM) MRTX1133 HBW-012-D HBW-012-E AGS (G12D, stomach, pERK) 1 0.8 0.46 AsPC-1 (G12D, pancreas, 3D CTG) 10.88 3.09 GP2D (G12D, colon, 3D CTG) 1.38 0.67 0.69 MKN-1 (WT, stomach, 3D CTG) &amp;gt;3000 Rat PK @ 100 mg/kg oral Cmax (ng/mL) 9 82 1444 AUC0~t (mg.h/mL) 20 258 5724 Citation Format: Ning Lee, Yingfu Li, Guanfeng Liu, Jiang Li, Junfeng Ren. HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3325.

Current Progress on the Influence Human Genetics Has on the Efficacy of Tyrosine Kinase Inhibitors Used to Treat Chronic Myeloid Leukemia.

The use of tyrosine kinase inhibitors (TKIs) has become the mainstay of treatment in patients suffering from chronic myeloid leukemia (CML), an adult leukemia caused by a reciprocal translocation between chromosomes 9 and 22, which createsan oncogene resulting in a myeloproliferative neoplasm. These drugs function by inhibiting the ATP-binding site on the fusion oncoprotein andsubsequently halting proliferative activity. The goal of this work is to investigate the current state of research into genetic factors that influence the efficacy of four FDA-approved TKIs used to treat CML. This overview attempts to identify genetic criteria that could be considered when choosing one drug over the others and to identify where more research is needed. Our results suggest thatthe usual liver enzymes impacting patient response may not be a major factor affecting the efficacy of imatinib, nilotinib, and bosutinib, and yet, that is where most of the past research has focused. More research is warranted on the impact that human polymorphisms of the CYP enzymes have on dasatinib. The impact of polymorphisms in UGT1A1 should be investigated thoroughly in other TKIs, not only nilotinib.The role of influx and efflux transporters has been inconsistent thus far, possibly due to failures to account for the multiple proteins that can transport TKIs and the impact that tumors have on transporter expression. Because physicians cannot currently use a patient's genetic profile to better target their treatment withTKIs, it is critical that more research be conducted on auxiliary pathways or off-target binding effects to generate new leads for further study. Hopefully, new avenues of research will help explain treatment failures and improve patient outcomes.

Tramadol and M1 Bioavailability Induced by Metamizole Co-Administration in Donkeys (Equus asinus).

Our objective was to assess the pharmacokinetic characteristics of metamizole when administered together with tramadol in a single intravenous dose to donkeys. Ten male animals received 10 mg∙kg-1 of dipyrone associated with 2 mg∙kg-1 of tramadol (T2M10) and 25 mg∙kg-1 of dipyrone with 2 mg∙kg-1 of tramadol (T2M25). Venous blood samples were taken from groups to determine the pharmacokinetics after drug administration, using initial brief intervals that were followed by extended periods until 48 h. Restlessness and ataxia were observed in two animals in the T2M25 group. Analysis revealed prolonged detectability of tramadol, 4-methylamine antipyrine, 4-aminoantipyrine (up to 24 h), and O-desmethyltramadol (up to 12 h) after administration. Although metamizole and its metabolites showed no significant pharmacokinetic changes, tramadol and O-desmethyltramadol exhibited altered profiles, likely because of competition for the active sites of CYP450 enzymes. Importantly, the co-administration of metamizole increased the bioavailability of tramadol and O-desmethyltramadol in a dose-dependent manner, highlighting their potential interactions and emphasizing the need for further dose optimization in donkey analgesic therapies. In conclusion, metamizole co-administered with tramadol interferes with metabolism and this interference can change the frequency of drug administration and its analgesic efficacy.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5–1000 μM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1–100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 μM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 μM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 μM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

Cyy-287, a novel pyrimidine-2,4-diamine derivative, efficiently mitigates inflammatory responses, fibrosis, and lipid synthesis in obesity-induced cardiac and hepatic dysfunction.

Inflammation and metabolic disorders are important factors in the occurrence and development of obesity complications. In this study, we investigated the protective effect and underlying mechanism of a novel pyrimidine-2,4-diamine derivative, Cyy-287, on mice fed a high-fat diet (HFD). The mice were randomly separated into four groups (n ≥ 7): control (regular diet), HFD, HFD with Cyy-287 (5 mg/kg), and HFD with Cyy-287 (20 mg/kg) following HFD feeding for 10 weeks. After a 10-week administration, ALT and AST enzymes, echocardiography, immunohistochemical (IHC), Western blot (WB), Masson and Sirius Red staining were used to evaluate functional and morphological changes to the heart and liver. Microsomes from the mouse liver were extracted to quantify the total amount of CYP450 enzymes after drug treatment. Cyy-287 decreased the levels of serum glucose, LDL, TC, ALT, and AST activities in HFD-treated mice. However, Cyy-287 administration increased ejection fraction (EF) and fractional shortening (FS) index of the heart. Cyy-287 inhibited histopathological changes in the heart and liver; decreased inflammatory activity; significantly diminished p38 mitogen-activated protein kinase (MAPK), the nuclear factor-kappa B (NF-κB) axis, and sterol regulatory element-binding protein-1c (SREBP-1c); and upregulated the AMP-activated protein kinase (AMPK) pathway in HFD-treated mice. Cyy-287 restored the content of hepatic CYP450 enzymes. These findings demonstrated that Cyy-287 protected heart and liver cells from obesity-induced damage by inhibiting inflammation, fibrosis, and lipid synthesis.

Effect of Donor Age and Liver Steatosis on Potential of Decellularized Liver Matrices to be used as a Platform for iPSC-Hepatocyte Culture.

Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization.

Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis.

The cytochrome P450 enzyme CYP121A1 endogenously catalyzes the formation of a carbon-carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of 20 CYP enzymes in Mtb, CYP121A1 continues to garner significant interest as a potential drug target. The accompanying reports the use of 19F NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that were theorized to stabilize a static active site water network. The active site residue Asn-85, whose hydrogen bonds with the diketopiperazine ring of cYY contributes to a contiguous active site water network in the absence of cYY, was mutated to a serine (N85S) and to a glutamine (N85Q). These conservative changes in the hydrogen bond donor side chain result in inactivation of the enzyme. Moreover, the N85S mutation induces reverse type-I binding as measured by absorbance difference spectra. NMR spectra monitoring the ligand-adaptive FG-loop and the active site Trp-182 side chain confirm that disruption of the active site water network also significantly alters the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrate that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. These findings implicate a slowly exchanging water network as a critical factor in CYP121A1 function and a likely contributor to the unusual rigidity of the structure.

A Preliminary Evaluation on the Antifungal Efficacy of VT-1161 against Persister Candida albicans Cells in Vulvovaginal Candidiasis.

Persister cells are a small fraction of the microbial population that survive lethal concentrations of antimicrobial agents. Candida albicans causes vaginal candidiasis, including recurrent vulvovaginal candidiasis, and may survive common antifungal treatments. The triazole VT-1161 is an antifungal agent that specifically targets fungal CYP51, as opposed to the human CYP enzyme. This work illustrates a new role of VT-1161 in eradicating the biofilm created from the persister cells of a primary biofilm of a clinical vaginal isolate of C. albicans. Antifungal activity was determined by the minimum inhibitory concentration (MIC), and the primary biofilm was treated with amphotericin B to obtain persister cells that were able to form a new biofilm. Results obtained using the new azole VT-1161 showed that VT-1161 not only eradicated a secondary biofilm formed from the persister-derived biofilm and counteracted the adhesion of C. albicans in vitro to human cells but also ameliorated C. albicans-induced infection in vivo in Galleria mellonella larvae, suggesting that it could be proposed as an alternative therapeutic strategy for the treatment of recurrent candidiasis.