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BACKGROUND: RNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC. METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC. RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells. CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.
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Neoplasias de la Mama , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Metiltransferasas , Estabilidad del ARN , Proteína 1 de Unión a la Caja Y , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Metilación , Metiltransferasas/metabolismo , Metiltransferasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Proteína 1 de Unión a la Caja Y/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
BACKGROUND: RNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear. METHODS: The correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. RESULTS: RNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1). CONCLUSIONS: Our study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Gefitinib/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Recurrencia Local de Neoplasia , ARN , Receptores ErbB/genética , Proteína 1 de Unión a la Caja Y , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Metiltransferasas/genéticaRESUMEN
BACKGROUND: Endoscopic resection is increasingly used in the treatment for early gastric cancer (EGC); however, about 15% of endoscopic submucosal dissection (ESD) cases report non-curative resection. The efficacy of different remedial interventions after non-curative ESD for EGC remains controversial. This meta-analysis aimed to compare the long-term outcomes of additional surgery and non-gastrectomy treatment for EGC patients who underwent non-curative ESD. METHODS: All relevant studies published up to October 2021 were systematically searched in the PubMed, Web of Science, and Embase databases. The medical subject headings terms "early gastric cancer," "gastrectomy," "endoscopic submucosal dissection," and their related free keywords were used to search relevant articles without restrictions on regions, publication types, or languages. The Newcastle-Ottawa Quality Assessment Scale was used to evaluate the quality of the included studies. Odds ratios (ORs) with 95% confidence intervals (CIs) of 5-year overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS) and hazard ratios (HRs) with 95% CIs of OS were calculated using a random- or fixed-effects model. RESULTS: This meta-analysis included 17 retrospective cohort studies with 5880 patients, of whom 3167 underwent additional surgery and 2713 underwent non-gastrectomy. We found that patients receiving additional gastrectomy had better 5-year OS (ORâ=â3.63, 95% CIâ=â3.05-4.31), DSS (ORâ=â3.22, 95% CIâ=â2.22-4.66), and DFS (ORâ=â4.39, 95% CIâ=â1.78-10.82) outcomes than those receiving non-gastrectomy treatments. The pooled HR also showed that gastrectomy following non-curative ESD significantly improved OS (HRâ=â0.40, 95% CIâ=â0.33-0.48). In addition, elderly patients benefited from additional surgery in consideration of the 5-year OS (HRâ=â0.54, 95% CIâ=â0.41-0.72). CONCLUSIONS: Compared with non-gastrectomy treatments, additional surgery offered better long-term survival outcomes for patients with EGC who underwent non-curative ESD.
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Resección Endoscópica de la Mucosa , Neoplasias Gástricas , Anciano , Humanos , Supervivencia sin Enfermedad , Estudios Retrospectivos , Neoplasias Gástricas/cirugía , Resultado del TratamientoRESUMEN
A direct transformation of non-preactivated benzyl alcohols to benzyl stannanes and benzyl silanes was realized through Pd-catalyzed C(sp3)-O activation process. By using versatile tin and silicon sources, these reactions exhibit a broad substrate scope and a high efficiency under mild conditions, affording functionalized benzyl and allylic stannanes and benzylsilanes with high yields. The successful implementation of gram-scale stannylation/silylation as well as the one-pot Stille coupling reaction demonstrates the potential application of this method in organic synthesis. Both experimental and theoretical investigations reveal the mechanistic details of this reaction.
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Background: Sorafenib (SOR) is an oral, potent, selective, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) used as the first-line therapy for advanced hepatocellular carcinoma (HCC). Baicalin (BG) is used as adjuvant therapy for hepatitis, which accounts for the leading cause of the development of HCC, and is commonly coadministered with SOR in clinic. The purpose of the current study was to characterize the pharmacokinetic changes of SOR and the potential mechanism when SOR is administered concomitantly with BG in rats for single and multiple doses. Methods: Parallel randomized pharmacokinetic studies were performed in rats which received SOR (50 mg/kg, i.g.) alone or coadministered with BG (160 mg/kg, i.g.) for single and multiple doses (7 days). Plasma SOR levels were quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Rat liver microsomes (RLMs) which isolated from their livers were analyzed for CYP3A and SOR metabolism activities. The inhibitory effect of BG on the metabolism of SOR was also assessed in pooled human liver microsomes (HLMs). The effects of BG on the intestine absorption behaviors of SOR were assessed in the in situ single-pass rat intestinal perfusion model. Results: Coadministration with BG (160 mg/kg, i.g.) for single or multiple doses significantly increased the Cmax, AUC0-t, and AUC0-∞ of orally administered SOR by 1.68-, 1.73-, 1.70-fold and 2.02-, 1.65-, 1.66- fold in male rats and by 1.85-, 1.68-, 1.68-fold and 1.57-, 1.25-, 1.24- fold in female rats, respectively (p < 0.01 or p < 0.05). In vitro incubation assays demonstrated that there were no significant differences of K m , V max , and CL int of 1-OH MDZ and SOR N-oxide in RLMs between control and multiple doses of BG-treated groups. BG has no obvious inhibitory effects on the metabolism of SOR in HLMs. In comparison with SOR alone, combining with BG significantly increased the permeability coefficient (P eff ) and absorption rate constant (K a ) of the SOR in situ single-pass rat intestinal perfusion model. Conclusion: Notably enhanced oral bioavailability of SOR by combination with BG in rats may mainly account for BG-induced SOR absorption. A greater understanding of potential DDIs between BG and SOR in rats makes major contributions to clinical rational multidrug therapy in HCC patients. Clinical trials in humans and HCC patients need to be further confirmed in the subsequent study.
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Osimertinib is a novel oral, potent, and irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for treatment of advanced T790M mutation-positive advanced non-small cell lung cancer, which is commonly combined with ginsenoside Rg3 in clinic to enhance the efficacy and minimize adverse reactions. In the present study, a highly sensitive UPLC-MS/MS method was established and validated for analysis of osimertinib in rat plasma according to US FDA guideline. Separation was performed on a C18 (2.1 × 50 mm, 2.6 µm) column using a gradient elution of ammonium formate (10 mM) with 0.1% formic acid buffer (A) and ACN (B) at a flow rate of 0.2 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization in the MRM mode. The method was validated over a concentration range of 1-400 ng/mL for osimertinib. The intra- and interday accuracy and precision values were within ±15%. No significant degradation occurred under the experimental conditions in stability assays. There was a further investigation on the effects of multiple doses of ginsenoside Rg3 on the pharmacokinetics of osimertinib in rats for the first time. The results implied that osimertinib exhibited a slow absorption and moderate-rate elimination in rats following oral administration. Coadministeration with ginsenoside Rg3 (5 mg/kg, 7 days, i.g.) may have no effect on the pharmacokinetics of osimertinib in rats. The results provide a reference for the clinical concomitant medications of Rg3 and osimertinib.
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Ribonucleic acid N6 -methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen-induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N6 -methyladenosine and acetaminophen-induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N6 -methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4-800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1-20 ng/mL for N6 -methyladenosine. It was successfully applied to the determination of N6 -methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen-induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen-induced hepatotoxicity, which will facilitate the elucidation of its mechanism.
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Adenosina/análisis , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Citidina/análisis , Guanosina/análisis , Hígado/metabolismo , ARN Mensajero/química , Uridina/análisis , Acetaminofén , Adenosina/análogos & derivados , Animales , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Masculino , Ratones , Espectrometría de Masas en TándemRESUMEN
Despite its narrow therapeutic window and large interindividual variability, cyclosporine A (CsA) is the first-line therapy following organ transplantation. Metabolized mainly by CYP3A and being a substrate of P-glycoprotein (P-gp), CsA is susceptible to drug-drug interactions. Baicalin (BG) is a drug used for adjuvant therapy of hepatitis in traditional Chinese medicine. Since its aglycone baicalein (B) inhibits CYP3A and P-gP, co-administration might affect CsA pharmacokinetics. This study investigated the effect of BG on CsA pharmacokinetics. In a two-period study, 16 healthy volunteers received a single 200 mg oral CsA dose alone (reference period) or in combination with 500 mg BG (test period). Pharmacokinetic evaluation of CsA was carried out using non-compartmental analysis (NCA) and population pharmacokinetics (popPK). Treatments were compared using the standard bioequivalence method. Based on NCA, 90% CIs of AUC and C max test-to-reference ratios were within bioequivalence boundaries. In the popPK analysis, a two-compartment model (clearance/F 62.8 L/h, central and peripheral volume of distribution/F 254 L and 388 L) with transit compartments for absorption appropriately described CsA concentrations. No clinically relevant effect of 500 mg BG co-administration on CsA pharmacokinetics was identified and both treatments were well tolerated.
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OBJECTIVES: To investigate the effects of multiple doses of baicalin (BG) on the pharmacokinetics of ciclosporin (CsA) in rats and the potential mechanisms. METHODS: Pharmacokinetic parameters of CsA were determined in male rats after administration of CsA (3 mg/kg, i.g. or i.v.) to rats in the presence and absence of BG (80 mg/kg, i.g. or i.v.) for 7 days. The livers and intestines of rats were isolated and the CYP3A and p-glycoprotein (P-gp) expression were analysed. The effect of BG on the intestinal absorptive behaviour of CsA was also investigated using in-vitro everted rat gut sac model. KEY FINDINGS: Baicalin (80 mg/kg, i.v., 7 days) had no effect on the intravenously administered CsA. However, BG (80 mg/kg, i.g., 7 days) significantly decreased the Cmax , AUC0-t and AUC0-∞ of orally administered CsA by 38, 26 and 25%, respectively (P < 0.01 or P < 0.05). Further study revealed that the expression of P-gp in intestine increased in oral multiple doses of BG-treated rats. The in-vitro everted rat gut sac model demonstrated BG (10 µm) significantly decreased the absorption of CsA (10 µm) in intestine (P < 0.05). CONCLUSIONS: Multiple doses of BG decreased the oral bioavailability of CsA in rats significantly, which may be mainly attributable to inhibition of absorption of CsA in intestine and induction of P-gp. The interaction between BG and CsA may occur when BG and CsA were co-administered for long-term use. The dosage adjustment and blood concentration monitoring of CsA may be required in clinic.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Ciclosporina/farmacocinética , Flavonoides/farmacología , Absorción Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Disponibilidad Biológica , Ciclosporina/administración & dosificación , Ciclosporina/sangre , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Flavonoides/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Corynoline (CRL), an isoquinoline alkaloid, is the major constituent derived from Corydalis bungeana Herba, which is a well-known Chinese herbal medicine widely used in many prescriptions. The purpose of this study was to comprehensively investigate the metabolism and bioactivation of CRL, and identify the CYP450 isoforms involved in reactive ortho-benzoquinone metabolites formation and evaluate its hepatotoxicity in mice. Here, high resolution and triple quadrupole mass spectrometry were used for studying the metabolism of CRL. Three metabolites (M1-M3) and four glutathione conjugates (M4-M7) of CRL ortho-benzoquinone reactive metabolite were found in vitro using rat and human liver microsomes supplemented with NADPH and glutathione. Four cysteine conjugates (M8-M11) were trapped in mice besides M1-M7. Using human recombinant CYP450 enzymes and chemical inhibitor method, we found that CYP3A4, CYP2C19, CYP2C9, and CYP2D6 were mainly involved in the bioactivation of CRL. Furthermore, CRL had no obvious hepatotoxicity and did not induce acute liver injuries in the experimental dosage (125-500 mg/kg) used in this study. However, phenomena of abnormal behaviors and low body temperature appeared in mice after drug administration, and three of them were dead. Tissue distribution study of CRL in mice showed that the main target organ of CRL was liver, then kidney, heart, and brain. CRL could traverse the blood-brain barrier, and have relative high concentration in brain. So, we surmise that toxicity effect of CRL on other organs may have occurred, and more attention should be paid on the traditional Chinese medicine contained CRL in clinic.
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Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (BZP) is a potential cardiovascular drug and exerts potent neuroprotective effect against transient and long-term ischemic stroke in rats. BZP could convert into 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) in vitro and in vivo. However, the pharmacokinetic profiles of BZP and Br-NBP still have not been evaluated. For the purpose of investigating the pharmacokinetic profiles, tissue distribution, and plasma protein binding of BZP and Br-NBP, a rapid, sensitive, and specific method based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) has been developed for determination of BZP and Br-NBP in biological samples. The results indicated that BZP and Br-NBP showed a short elimination half-life, and pharmacokinetic profile in rats (3, 6, and 12 mg/kg; i.v.) and beagle dogs (1, 2, and 4 mg/kg; i.v.gtt) were obtained after single dosing of BZP. After multiple dosing of BZP, there was no significant accumulation of BZP and Br-NBP in the plasma of rats and beagle dogs. Following i.v. single dose (6 mg/kg) of BZP to rats, BZP and Br-NBP were distributed rapidly into all tissues examined, with the highest concentrations of BZP and Br-NBP in lung and kidney, respectively. The brain distribution of Br-NBP in middle cerebral artery occlusion (MCAO) rats was more than in normal rats (P < 0.05). The plasma protein binding degree of BZP at three concentrations (8000, 20,000, and 80,000 ng/mL) from rat, beagle dog, and human plasma were 98.1-98.7, 88.9-92.7, and 74.8-83.7% respectively. In conclusion, both BZP and Br-NBP showed short half-life, good dose-linear pharmacokinetic profile, wide tissue distribution, and different degree protein binding to various species plasma. This was the first preclinical pharmacokinetic investigation of BZP and Br-NBP in both rats and beagle dogs, which provided vital guidance for further preclinical research and the subsequent clinical trials.
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Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (>600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, Km, Vmax, and CLint decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, Vmax and CLint decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, Km increased by 118.4% and CLint decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between Vmax values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Adulto , Anciano , Femenino , Genotipo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Preparaciones Farmacéuticas/metabolismo , Polimorfismo Genético , Adulto JovenRESUMEN
A rapid, sensitive and selective liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of sodium (±)-5-Bromo-2-(α-hydroxypentyl) benzoate (BZP) and its active metabolite 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) in rat plasma using potassium 2-(1-hydroxypentyl)-benzoate (PHPB) and l-3-n-butylphthalide (NBP) as internal standards (IS). Chromatographic separation was achieved on a Hypersil GOLD C18 column using a gradient elution of ammonium acetate and methanol at a flow rate of 0.2 mL/min. Good linearity was achieved within the wide concentration range of 5-10,000 ng/mL. The intra-day and inter-day precision was less than 8.71% and the accuracy was within -8.53% and 6.38% in quality control and the lower limit of quantitation samples. BZP and Br-NBP were stable during the analysis and the storage period. The method was successfully applied to pharmacokinetic studies of BZP in Sprague-Dawley rats for the first time. After a single intravenous administration of BZP at the dose of 0.75 mg/kg, the plasma concentration of BZP and Br-NBP declined rapidly and the AUC0-t of BZP was significantly greater in female rats compared to male rats (p < 0.05). The data presented in this study serve as a firm basis for further investigation of BZP in both preclinical and clinical phases.