Vornorexant, a novel dual orexin 1/2 receptor antagonist with a short half-life, has been approved in Japan for the treatment of insomnia. We performed in vitro assessments to evaluate the drug-drug interaction (DDI) potential of vornorexant and its metabolite M3 mediated by CYP enzymes and transporters.Human CYP reaction phenotyping studies indicated that vornorexant and M3 are primarily metabolized by CYP3A4, and to a lesser extent by CYP3A5 and CYP2C8.Vornorexant exhibited weak reversible and time-dependent inhibition of CYP3A. Both vornorexant and M3 showed the potential to induce CYP2B6 and CYP3A4. However, these effects were not considered to have any impact on causing DDIs at these unbound systemic concentrations at the clinical doses of vornorexant.Vornorexant was a weak substrate for P-gp but not for BCRP, OATP1B1, or OATP1B3.Vornorexant and M3 exhibited weak or no inhibitory effects against P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2-K.These data suggest that while vornorexant might be an object drug of CYP3A precipitants, vornorexant is unlikely to cause clinically relevant DDIs mediated by either CYP enzymes or transporters involved in the metabolism and disposition of concomitantly administered drugs.
Muvadenant is a selective, dual A2A/A2B adenosine receptor antagonist that demonstrated anti-tumour efficacy in preclinical in vitro and in vivo models.In rats,14C-muvadenant related radioactivity was quickly and widely distributed throughout the body with highest exposure observed in metabolic/excretory and melanin containing tissues and the gastrointestinal tract and lowest in the central nervous system.Following single oral administration, 6.27% of the radioactive dose was excreted via urine and 90.3% via faeces with a total recovery of 97.6%. After single intravenous dosing, the amounts of radioactivity excreted via urine and faeces were 13.7% and 80.2%, respectively, with a total recovery of 94.6% within 7 days.Whereas the parent compound accounted for the majority of the circulating radioactivity (>50%), metabolism plays the dominant role in the elimination of muvadenant.Metabolite structures derived from in vivo profiles in rats and in vitro incubations across species revealed extensive phase 1 metabolism of muvadenant with comparable metabolic pathways in human and preclinical species.
Purpose: Pomegranate peel polyphenols (PPPs) have recognised bioactive properties, but their metabolic interactions with gut microbiota are still uncertain. Materials and methods: This investigation aimed to shed light on the metabolic regulation by PPPs and the gut microbiota's role in this process in normal SD rats. Animals were administered PPPs (300 mg/kg·bw·d) for 28 days. Results: Results showed PPPs significantly reduced rat body weight, and optimised lipid profiles. PPPs modulated the gut microbial composition by enhancing the abundance of beneficial bacterial genera (e.g. Oscillibacter, Prevotella_9) and stimulating the biosynthesis of short-chain fatty acids (SCFAs). Serum metabolomics further revealed that PPPs altered key metabolic pathways, including glycine metabolism, primary bile acid biosynthesis, and tryptophan metabolism. The correlation studies indicated that PPPs target specific gut bacterial genera (Oscillibacter, Allobaculum, Prevotella) to regulate SCFAs production and key metabolic pathways, thereby optimising the metabolic status of rats. Conclusions: These findings provide a critical scientific basis for their potential application in preventing metabolic disorders.
Smoking is a public health concern, and smoking cessation methods such as nicotine replacement therapy (NRT) are often ineffective partly because they are not personalised. Smoking behaviour is related to polymorphisms of the nicotine-metabolizing enzyme P450 2A6. Accordingly, fast metabolizers are nicotine-dependent, smoke more cigarettes and have lower quitting rates compared to slow metabolizers. Thus, inhibiting the nicotine-metabolizing enzyme and prolonging the presence of nicotine in the blood could lead to enhanced NRT and/or fewer cigarettes smoked.In this study, we examined whether Ginkgo biloba (GB), its main constituent quercetin, and 8-methoxypsoralen (8-MOP) can inhibit nicotine metabolism in vivo in mice. We administered nicotine to mice in the presence or absence of the test articles and measured serum cotinine levels, brain dopamine levels and liver CYP2A5 activity.Our results indicate that 8-MOP can inhibit nicotine metabolism in vivo by 55% and increase dopamine in the brain. Chronic administration of GB and quercetin did not detectably inhibit nicotine metabolism, however, GB increased dopamine in the brain. Our results suggest that supplements or drugs that can inhibit nicotine metabolism sufficiently may help smokers reduce the number of cigarettes smoked by prolonging the presence of nicotine in the blood and dopamine in the brain.
The overexpression of P-glycoprotein (P-gp) in tumour cells and its mediated drug efflux represent key mechanisms of cancer multidrug resistance (MDR). Natural compounds are attractive candidates for reversing MDR due to their multi-target activity and hypotoxicity. This study evaluated the ability of leucoseptoside A (LeuA) to reverse P-gp-mediated MDR in MCF-7/ADR cells.The MDR reversal activity of LeuA was assessed based on changes in drug sensitivity measured using the MTT assay. P-gp expression was measured by Western blotting. The intracellular accumulation of rhodamine 123 (Rh123) and adriamycin (ADR) was determined using confocal laser scanning microscopy and flow cytometry, and P-gp ATPase activity was assessed using the Pgp-Glo™ system. The binding mode and affinity of LeuA for P-gp were predicted by molecular docking.LeuA markedly enhanced the sensitivity of MCF-7/ADR cells to ADR and paclitaxel (PTX). This mechanism was achieved by inhibiting P-gp efflux rather than altering its protein expression levels. Molecular docking analysis further revealed that LeuA competitively binds to the drug-binding pocket of P-gp with a higher binding affinity than verapamil (VRP).LeuA is a potent reversal agent for P-gp-mediated MDR, promising as a natural inhibitor for cancer therapy.
1. Postoperative cognitive dysfunction (POCD) is a major issue in elderly surgical patients. Oxycodone (Oxy) reduced POCD risks versus other anaesthetics, but its mechanism is unknown. This study probes microRNA (miR)-150-5p's possible role. 2. Preoperative and postoperative serum samples were obtained from elderly patients, categorised into the POCD and non-POCD groups after oxycodone anaesthesia. In vivo POCD mice and in vitro LPS-induced cell models were constructed, both treated with Oxy. miR-150-5p Levels were measured by RT-qPCR. ROC curve evaluated its diagnostic potential for POCD. Morris Water Maze tested spatial learning/memory in POCD mice. Flow cytometry assessed cell apoptosis. 3. At baseline, miR-150-5p and Nuclear Antigen H731 (H731) levels were similar between groups; however, at 72 h post-op, the POCD group had lower miR-150-5p and higher H731. MiR-150-5p acted as a risk factor and effective biomarker for identifying elderly patients at high risk of POCD. Oxy alleviated hippocampal injury and cognitive deficits in aged POCD mice, which were reversed by miR-150-5p knockdown but restored by H731 depletion. miR-150-5p knockdown abolished Oxy-mediated protection against apoptosis, inflammation, and oxidative stress, whereas H731 silencing rescued such effects. 4. miR-150-5p served as a diagnostic biomarker in elderly patients undergoing Oxy-under general anaesthesia.
Here we investigated the direct effect of formaldehyde, a major environmental and occupational pollutant, and cytoprotective potency of crocin as a natural compound against its toxicity in human lymphocytes, an important immune cell.For this purpose, human lymphocytes were isolated from peripheral blood and exposed to different concentrations of formaldehyde for 4 hours. Then, to investigate the protective potential of crocin, the human lymphocytes were simultaneously treated with its different concentrations in presence of formaldehyde. A set of biochemical and flow cytometric indices including cell viability, ROS production, mitochondrial membrane potential (MMP) collapse, lysosomal damage, malondialdehyde (MDA) and glutathione (GSH) levels, as indicator of oxidative stress were measured.The findings showed that formaldehyde significantly induced ROS formation, MMP collapse, lysosomal membrane destruction, lipid peroxidation and cytotoxicity. Our observations showed that simultaneous exposure of crocin with formaldehyde could not significantly reduce the toxicity parameters caused by formaldehyde.Overall, we observed that formaldehyde can cause direct toxicity via ROS formation, MMP collapse, lysosomal damages and oxidative stress in human lymphocytes and crocin with routine concentrations cannot reduce these toxicities. Therefore, reducing the toxicity of formaldehyde in lymphocytes by stronger antioxidant agents is suggested in future studies.
Cis-2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (cis-THSG), a photoisomer of the major bioactive component in Polygoni Multiflori Radix Praeparata (He-Shou-Wu), has enhanced hypolipidemic activity but is associated with idiosyncratic hepatotoxicity. Its metabolic fate under pathological conditions and key clearance enzymes remain unclear.This study aimed to compare metabolite profiles of cis-THSG in normal and hyperlipidemic rats in vivo and in vitro, identify metabolites, and characterise enzyme kinetics in rat liver microsomes (RLMs) and major CYP450 isoforms. Metabolites in plasma, urine, faeces, bile, and intestinal bacteria incubation fluids were analysed by UHPLC-Q-TOF-MS/MS. Enzymatic kinetics and CYP450 isoforms were investigated via chemical inhibition assays.A total of 27 metabolites were characterised; cis-THSG underwent rapid glucoside hydrolysis, and its aglycone underwent phase I and II reactions. Hyperlipidaemia reduced metabolites to 20 and markedly increased plasma cis-THSG and aglycone abundance. Kinetic parameters were Km 32.86 μmol/L, Vmax 0.7285 μmol/(min·mg protein), CLint 0.02217 L/(min·mg protein). Rat liver microsomal CYP isoforms analogous to human CYP3A4 and CYP2C9 mediated cis-THSG oxidative metabolism.This study provides the first comprehensive metabolite map of cis-THSG, identifies its aglycone as a potential bioactive form, and reveals a disease-related metabolic shift. CYP phenotyping offers a mechanistic basis for predicting clinical drug-drug interactions.
Quantitative analytical method was developed for six coumarin constituents in Angelicae pubescentis radix (APR), and the inhibitory effect of the herb's extract on cytochrome P450 enzymes was investigated.A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of six coumarins (imperatorin, 8-methoxypsoralen, isopimpinellin, isoimperatorin, bergapten, and cnidilin) in APR. Concurrently, the inhibitory effects of its aqueous extract on multiple P450s were evaluated in rat liver microsomes using a 'Pittsburgh cocktail' approach.The method demonstrated excellent linearity (R2 > 0.999), precision, stability, repeatability (RSD < 3.0%), and recovery (95.15-102.36%). The extract exhibited significant time-dependent inhibition of CYP1A2 (residual activity 58.2%), with weaker effects on other P450s.A robust LC-MS/MS method was successfully established. The extract of APR was identified as a potent time-dependent inhibitor of CYP1A2 in rat liver microsomes (RLMs), suggesting a potential risk for metabolism-based drug interactions in rats that warrants further investigation in human systems.
Lumateperone is an antipsychotic metabolised to four active metabolites IC200131, IC200161, IC200565 and IC201308, which are closely related to its clinical efficacy and safety.For evaluating the clinical pharmacokinetics of lumateperone and its active metabolites in Chinese subjects, a high-throughput LC-MS/MS method was developed and validated for the simultaneous quantification of lumateperone and its four metabolites in human plasma. Chromatographic separation was achieved using an alkaline mobile phase and a bidentate silane column (Zorbax Extend-C18) with two-step gradient elution, which overcame peak tailing and carryover while enabling rapid analysis. The calibration ranges were 0.100-80.0 ng/mL for lumateperone and IC200131, 0.0400-32.0 ng/mL for IC200161 and IC201308, and 0.150-30.0 ng/mL for IC200565.The validated method was successfully applied to a pharmacokinetic study of lumateperone tosylate capsules. The clinical trial was registered at the Chinese Clinical Trial Registry (identifier: CTR20242699). Under fasting conditions, the mean Cmax values were 30.6 ng/mL for lumateperone, with corresponding AUC0-t values of 72.8 h·ng/mL. Under fed conditions, food delayed absorption and decreased Cmax for all analytes.This study reports the first comprehensive pharmacokinetic profiles of lumateperone and its four active metabolites in Chinese subjects. The validated method provides a robust foundation for pharmacokinetic research of lumateperone in humans.
Aluminium phosphide (AlP) is a chemical compound that is used as a pesticide for suicidal purposes and can cause death, and it poses a challenge to health in some countries. AlP in the presence of stomach acid leads to the release of phosphine, which inhibits mitochondrial function and reduces ATP levels. This poisoning has devastating effects on the front line of damage on the cardiovascular and gastrointestinal systems, causing symptoms such as vomiting, cardiac arrhythmias, and heart failure.A focused search was performed utilising the terms 'aluminium phosphide,' 'phosphine,' 'antidote,' 'case report,' and 'treatment' within the scientific databases Scopus, Web of Science, and PubMed. Studies have shown that supportive treatment, decontamination, and antioxidant therapy can help reduce toxic effects.Many studies have been conducted in vitro and in clinical models to identify specific antidotes, providing valuable insight into the management of poisoning. Given the lack of a particular antidote, new therapeutic approaches offer promising solutions for managing this poisoning.New knowledge, including artificial intelligence, can help prevent and manage diseases and treatments. However, further efforts are needed to develop more effective treatments and prevent the misuse of this substance.
1. This study aims to investigate the effects of orally administered paeoniflorin (PF), a major bioactive compound from Paeonia lactiflora, on cytochrome P450 (CYP450) enzyme activity in rats to assess potential drug-drug interactions (DDIs). Following PF treatment, rats received a mixed probe drug solution containing phenacetin (PHE), tolbutamide (TOL), omeprazole, and metoprolol, and plasma concentrations of the substrates and metabolites were quantified using ultra-high-performance liquid chromatography-tandem mass spectrometry. The metabolic ratio, calculated as the ratio of metabolite to parent drug area under the curve (AUC), was used to evaluate CYP activity.2. Results showed that AUCAcetaminophen/AUCPHE values were significantly elevated in PF-treated groups, indicating induction of CYP1A2, whereas AUCOHTOL/AUCTOL values were significantly reduced, suggesting inhibition of CYP2C11. No significant effects were observed on CYP2C or CYP2D2 activities.3. These findings indicate that PF can modulate CYP1A2 and CYP2C11 activities under the experimental conditions tested, as assessed using PHE and TOL as functional markers. However, the magnitude of these effects was modest and observed only at relatively high PF doses (50 and 100 mg/kg). Given the inherent species differences between rat and human CYP enzymes, direct extrapolation of these findings to human CYP1A2 and CYP2C11 requires further validation in human-relevant models.
1. Triple-negative breast cancer (TNBC) is a highly aggressive subtype with few available therapeutic strategies. Berberine Hydrochloride (BBR) has shown anticancer potential but its mechanisms in TNBC remain unclear. 2. We used cell viability assays, immunohistochemistry, western blotting, and metabolic assays to investigate the effects of BBR on TNBC cells. 3. BBR markedly reduced the viability of TNBC cell lines and increased apoptosis compared with untreated controls. Relative to adjacent non-tumor tissues, TNBC tumor samples showed higher levels of total p65, p65 K310 acetylation, and p65 S536 phosphorylation; the same pattern appeared in TNBC cell lines compared with normal breast epithelial cells. In TNBC cells, both BBR and NAM increased p65 K310 acetylation while reducing S536 phosphorylation relative to vehicle treatment. SIRT2 expression was elevated in TNBC tumors and cell lines compared with their controls, whereas BBR decreased SIRT2 levels. BBR also induced stronger oxidative stress than controls and caused mitochondrial dysfunction, including membrane potential loss and reduced oxygen consumption and acidification rates. 4. BBR induces apoptosis in TNBC cells and is associated with p65 acetylation/phosphorylation changes, SIRT2 downregulation, and mitochondrial dysfunction. This study provides a novel mechanistic basis for BBR as a potential therapeutic agent for TNBC.
This study explores a dual-action approach for hypertension management based on a co-formulated suspension containing Candesartan cilexetil (CC) - Bisoprolol fumarate (BF) system, combining FTIR characterisation with computational modelling to evaluate molecular compatibility, binding behaviour, and pharmacokinetic properties.Direct in situ FTIR-ATR spectral analysis confirmed the successful interfacial wetting and co-dispersion of both drugs within the surfactant matrix, driven by robust hydrophobic interactions and cooperative hydrogen bonding. Hirshfeld surface analysis revealed complementary intermolecular interaction patterns between BF and CC, with BF exhibiting flexible hydrogen-bond networks and CC showing directional hydrogen bonding, π-π stacking, and electrostatic interactions.These distinct yet complementary profiles suggest favourable molecular packing and potential co-formulation. Molecular docking further demonstrated balanced binding of the dual system to both β1 and AT1 receptors, with binding energies of -5.19 and -4.88 kcal/mol, respectively. Furthermore, ADMET results indicate poor gastrointestinal absorption and low oral bioavailability, mainly due to high lipophilicity, which may limit in vivo efficacy, although the compound shows an overall acceptable safety profile.In summary, while the CC-BF system appears promising as a dual-target antihypertensive strategy, further formulation improvements and experimental in vitro and in vivo validation are required to confirm its therapeutic potential.
1. Lead (Pb) and cadmium (Cd) induce liver damage mainly via oxidative stress. Total saponins from ginseng stems and leaves (TSGSL) possess antioxidant properties, but their protective effect against Pb-Cd co-exposure remains unclear.2. This study aimed to investigate the hepatoprotective effect and mechanism of TSGSL in acute Pb-Cd co-exposed mice.3. Male KM mice received TSGSL (30 or 60 mg/kg, orally) once daily for 14 days, then a single acute exposure to Pb (150 mg/kg) and Cd (15 mg/kg). Liver function, oxidative stress markers, histopathology, pro-inflammatory cytokines, and Keap1/Nrf2 pathway gene mRNA were assessed.4. TSGSL dose-dependently alleviated Pb-Cd-induced liver injury. At 60 mg/kg, it reduced ALT by 46.0% and AST by 43.3%, decreased hepatic MDA by 50.0%, increased hepatic T-SOD, CAT and GSH-PX by 36.9%, 69.5% and 36.7%, respectively, and lowered pro-inflammatory cytokines (e.g. TNF-α by 51.7% and IL-1β by 46.0%). Histopathological damage was ameliorated. TSGSL upregulated mRNA expression of Keap1/Nrf2-related genes. The 30 mg/kg dose showed weaker effects.5. TSGSL protects against acute Pb-Cd-induced oxidative liver damage in mice, potentially via transcriptional upregulation of the Keap1/Nrf2 pathway and enhancement of antioxidant capacity. Further studies are needed to investigate chronic Pb-Cd co-exposure and gut microbiota involvement.
Radix Bupleuri (RB)-induced hepatotoxicity and herb-drug interactions have raised clinical concerns, yet the role of its intestinal metabolites, saikogenins (SGs), in modulating human UDP-glucuronosyltransferase (UGT) activity remains poorly understood.Using 11 recombinant human UGT isoforms and 4-methylumbelliferone as the probe substrate, we demonstrate that the parent saikosaponins (SSA, SSC, SSD) produced no potent, isoform-selective inhibition of any of the tested UGT isoforms at 100 μM, whereas the deglycosylated aglycones SGA and SGD selectively and potently inhibited UGT2B7 and UGT2B15 (IC50: 0.24-1.88 μM; Ki: 0.14-1.34 μM) through competitive mechanisms.Molecular docking revealed that deglycosylation removes steric hindrance from the sugar moiety, enabling the aglycone backbone to penetrate the catalytic pocket, where the most potent metabolite SGD anchored the conserved catalytic histidine of UGT2B7 (HIS481); inhibition of UGT2B15 instead arose from deep occupancy of the hydrophobic pocket.In vitro-in vivo extrapolation yielded an R-value of 1.36 for SGD-UGT2B7, exceeding the regulatory threshold (R ≥ 1.1), indicating a clinically significant risk when RB preparations are co-administered with narrow-therapeutic-index UGT2B7 substrates such as morphine or zidovudine.
Decitabine (DEC) is rapidly deaminated by cytidine deaminase (CDA); tetrahydrouridine (THU), a CDA inhibitor, reduces degradation and increases DEC exposure. This is clinically relevant in sickle cell disease (SCD), where DEC increases foetal haemoglobin in erythrocytes.The effect of THU pre-treatment on the tissue distribution of radiolabelled decitabine ([14C]-DEC) was studied in mouse by quantitative whole-body autoradiography and area under the concentration curve (AUC) as exposure metric.The decitabine-related exposure was highest in haematolymphoid tissues, particularly the bone marrow, followed by gastrointestinal tissues across all dosing groups, with up to 115‑ and 18‑fold higher levels than in blood, respectively.Furthermore, THU pre-treatment increased the exposure in haematolymphoid tissues, gastrointestinal tract and reproductive organs by up to 4.8-, 6.2- and 5.3-fold, respectively.In conclusion, THU pre-treatment enhances decitabine-related exposure in target tissues like bone marrow, supporting pre-dosing of THU prior to DEC treatment of SCD.
The present study aims to analyse the curative efficacy of fucoidan on MSG mediated changes in hepatorenal and reproductive organs.For this, 42 adult male rats were grouped into six (N = 7animals/group), and treated for 30 consecutive days with following dose regime: Control (saline water), MSG (60 mg/kg), Fucoidan-low-dose (FLD 100 mg/kg), Fucoidan-high-dose (FHD 200 mg/kg), MSG+FLD (60 mg/kg + 100mg/kg) and MSG+FHD (60 mg/kg + 200mg/kg). On day 31, animals were sacrificed, organs were collected, and subjected to sperm parameters, antioxidant enzyme activities, DNA damage and histomorphometric analysis.The findings showed that MSG caused significant decline in sperm parameters and antioxidant enzymes activity, while increasing the concentrations of oxidative stress markers. MSG administration also showed significant DNA damage in sperm, liver and kidney cells. Additionally, adverse histomorphometric changes including damage to seminiferous tubules, caput and caudal epididymal ducts were noted with decline in sperm concentration. Moreover, hepatorenal histological alterations were identified by leukocytic infiltration, cytoplasmic vacuolation in liver and shrinkage of glomerulus and tubular atrophy in the kidney. On contrary, fucoidan treatment reversed all MSG instigated alterations in dose dependent manner.These findings revealed that fucoidan mitigated MSG induced oxidative, genotoxic and histological alterations in rats, suggesting further mechanistic validation for translational relevance.
Hypertrophic scars (HS) represent a common complication arising from abnormal wound healing following skin injury. Resveratrol and microRNA (miRNA) have been implicated in the formation of HS. However, the precise mechanism by which resveratrol modulates hypertrophic scarring through miRNA regulation remains unclear.Investigating the potential functional effects and molecular mechanisms of resveratrol in HS.RT-qPCR was employed to detect the levels of miR-1290 and ADAMTS8. The targeted binding relationship between miR-1290 and ADAMTS8 was validated through dual luciferase reporter assays and RIP experiments. Cell proliferation, migration capacity, and inflammatory cytokine (IL-1β, IL-6) secretion levels were assessed using CCK-8 assays, Transwell migration assays, and ELISA.MiR-1290 was overexpressed in HS tissue and HSFB cells, whereas ADAMTS8 levels were markedly downregulated, exhibiting a negative correlation between the two. Resveratrol concentration-dependently inhibited miR-1290 levels and upregulated ADAMTS8 levels. ADAMTS8 may represent a direct target gene of miR-1290. Resveratrol may suppress the proliferation, migration, inflammatory cytokine levels, and fibrosis marker levels in HSFB cells. Overexpression of miR-1290 partially reversed the effects of resveratrol, whilst overexpression of ADAMTS8 suppressed the actions induced by miR-1290.Resveratrol may suppress the malignant phenotype of HSFB cells by regulating the miR-1290/ADAMTS8 axis.
1. Traditional Japanese medicines (Kampo medicines) are increasingly used concurrently with other medications, but evidence regarding their potential for drug-drug interactions (DDIs) is insufficient. Given the important role of cytochrome P450 3 A (CYP3A) in DDIs, this study assessed the CYP3A-mediated DDI risk of three Kampo medicines (Hochuekkito, Goreisan, and Ninjin'yoeito).2. We utilised a CYP3A-humanised mouse model to comprehensively evaluate the effects of the three Kampo medicines on CYP3A activity, including an analysis of the area under the plasma concentration-time curve (AUC) and AUC ratio (AUCR).3. None of the three Kampo medicines tested significantly affected the AUC of the CYP3A substrate triazolam or the AUCRs of triazolam metabolites.4. These results provide important preclinical evidence indicating the lack of a significant net effect on CYP3A-mediated DDIs for Hochuekkito, Goreisan, and Ninjin'yoeito under the tested repeated-dosing conditions, thereby supporting their safe and proper use.
