Carboxylesterase Inhibitor Research Articles

Stress indicators in conservative tissues of Humboldt penguin under captivity

Humboldt penguins (Spheniscus humboldti) from the Barcelona zoo (n=9) were followed to assess their physiological stress status using conservative protocols. Corticosterone levels were measured in feathers and plasma as indicative of chronic and acute physiological stress, respectively. Other markers: B-esterases, potentially indicative of xenobiotic exposure were measured in plasma of these same individuals and reported for the first time in this species. The sensitivity to chemicals of environmental concern, employed as plastic additives, was assessed in vitro with plasma of this species using the inhibition of carboxylesterase (CE) and acetylcholinesterase enzymatic measurements. Among the tested additives, the organophosphorus flame retardants displayed the highest in vitro inhibitory potential on basal CE activity, suggesting their potential utility as biomarkers of this particular chemical class. Additionally, enzymatic measurements in plasma are determined for the first time in Humboldt penguin and can be regarded as baseline values for a potential field monitoring application.

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

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

Phytochemical screening and toxicity assessment of compounds isolated from the leaves of Mangifera indica L. for the control of Spodoptera litura (Lepidoptera; Noctuidae)

This study aimed to analyze the phytochemical composition and antioxidative capabilities of mango leaves (Mangifera indica L., Nam Dok Mai), indigenous to Sa Kaeo Province, Thailand. Various solvents with differing polarities, including n-hexane, DCM, ethyl acetate, and MeOH, were utilized for leaf extraction. The findings revealed the existence of eight groups of phytochemical compounds: alkaloids, flavonoids, coumarins, saponins, tannins, terpenoids, steroids, and cardiac glycosides. The MeOH crude extract exhibited the highest concentration of total phenolic compounds at 409.88 ±0.02 mg GAE/g. Furthermore, the MeOH crude extract demonstrated the strongest antioxidant activity, with an IC50 value of 0.52 ±0.02 μg/ml, as determined by the DPPH method. High-performance liquid chromatography (HPLC) was employed to identify gallic acid and mangiferin in the MeOH crude extract. Laboratory tests were conducted using the topical application method to evaluate the toxicity of the M. indica leaf crude extract on 2nd instar Spodoptera litura larvae. The MeOH crude extract exhibited high efficacy, with an LD50 value of 10.58 µg per larvae within 24 hours. Gallic acid and mangiferin were identified as the primary active ingredients, with LCD50 values of 1.19 µg per larvae and 1.90 µg per larvae, respectively, within 24 hours. Additionally, the impact on detoxification enzymes (24 hours post-treatment) was assessed in surviving 2nd instar S. litura larvae using the topical application method. The MeOH extract treatment resulted in 1.31-fold inhibition of carboxylesterase (CE), 1.31-fold inhibition of glutathione-S-transferase (GST), and 1.32-fold inhibition of acetylcholinesterase (AChE).

In Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe.

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the "on-off" fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.

Rational Design of a Highly Sensitive Carboxylesterase Probe and Its Application in High-Throughput Screening for Uncovering Carboxylesterase Inhibitors.

Tracking carboxylesterases (CESs) through noninvasive and dynamic imaging is of great significance for diagnosing and treating CES-related metabolic diseases. Herein, three BODIPY-based fluorescent probes with a pyridine unit quaternarized via an acetoxybenzyl group were designed and synthesized to detect CESs based on the photoinduced electron transfer process. Notably, among these probes, BDPN2-CES exhibited a remarkable 182-fold fluorescence enhancement for CESs within 10 min. Moreover, BDPN2-CES successfully enabled real-time imaging of endogenous CES variations in living cells. Using BDPN2-CES, a visual high-throughput screening method for CES inhibitors was established, culminating in the discovery of an efficient inhibitor, WZU-13, sourced from a chemical library. These findings suggest that BDPN2-CES could provide a new avenue for diagnosing CES-related diseases, and WZU-13 emerges as a promising therapeutic candidate for CES-overexpression pathological processes.

Combining Experimental and Computational Methods to Produce Conjugates of Anticholinesterase and Antioxidant Pharmacophores with Linker Chemistries Affecting Biological Activities Related to Treatment of Alzheimer's Disease.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, the most active was 17d IC50 15.1 ± 0.2 nM) and butyrylcholinesterase (BChE, the most active was 18d: IC50 5.96 ± 0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated the potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced β-amyloid (Aβ) aggregation. Hybrids also exhibited the inhibition of Aβ self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as the inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability, suggesting the CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for the potential treatment of AD.

Drug-Drug Interaction Between Cannabidiol, Cyclosporine, and Mycophenolate Mofetil: A Case Report

Kidney transplantation remains the optimal therapy for many patients with end-stage kidney disease (ESKD). Chronic pain is one of the most common and distressing symptoms among patients with ESKD, and its treatment is a complex and challenging task to accomplish. The benefits of cannabidiol (CBD) in chronic pain treatment have been reported recently. Cannabidiol is metabolized by cytochrome P450, mainly CYP3A4 and CYP2C19, and can also undergo direct conjugation via UDP-glucuronosyltransferase enzymes, with a growing body of evidence suggesting it is also a potent inhibitor or inducer of these pathways. Cannabidiol was also found to be a potent inhibitor of carboxylesterases in vitro. Because cytochrome P450 enzymes and carboxylesterases are also responsible for the clearance and activation of immunosuppressants, respectively, drug-drug interactions are likely to occur. Here, we report a pharmacokinetic drug interaction between CBD and cyclosporine and mycophenolate mofetil in a patient with ESKD with a kidney transplantation. It is thus crucial to take into account these interactions and monitor drug levels to avoid drug toxicity or a lack of efficacy. This study is in accordance with the guidelines of the Declaration of Helsinki and the Declaration of Istanbul.

An ultrasensitive and selective near-infrared fluorescent probe for tracking carboxylesterases with large Stokes shift in living cells and mice

Carboxylesterases (CEs) play great role in CEs-related diseases and drug metabolism. Selectively monitoring its activity is important to explore its role in CEs-related diseases and drug combination. Herein, a new “turn-on” near-infrared (NIR) fluorescent probe (CHY-1) was reported with large Stokes shift (145 nm) for CEs detection. Dicyanoisophorone-based derivative was chosen as NIR fluorophore and 4-bromobutyrate was the identifying group. What’s more, CHY-1 exhibited ultra-sensitivity (LOD ∼ 9.2 × 10−5 U/mL), high selectivity against Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE) and Chymotrypsin for CEs fluorescence detection under physiological pH and temperature. Furthermore, CHY-1 showed little effect on cell viability at high concentration and featured good optical imaging character for the slight change of CEs activity induced by 5-Fu (5-Fluorouridine, anti-tumor drug) and CEs inhibitor in living cells. Moreover, CHY-1 was also used to detect the activity and distribution of CEs in mice. Taken together, CHY-1 had widely applicable value in the diagnosis of CEs-related diseases and drug combination.

Insecticidal Effect of the Entomopathogenic Fungus Lecanicillium araneicola HK-1 in Aphis craccivora (Hemiptera: Aphididae).

Aphis craccivora (Hemiptera: Aphididae) is an important pest affecting various crops worldwide. However, only few studies have been conducted on the infection of A. craccivora by Lecanicillium and related insecticidal mechanisms. We investigated the infection process of A. craccivora by Lecanicillium araneicola HK-1 using fluorescence microscopy and scanning electron microscopy (SEM), and our results indicated that the conidia of strain HK-1 easily attached to the feet and dorsum of A. craccivora. The activities of chitinase and extracellular protease were induced in the aphid after treatment with HK-1. A bioassay on A. craccivora showed that the median lethal concentration (LC50) of the fungus crude extract was 24.00 mg mL-1 for 24 h of treatment. Additionally, the results showed that the crude extract disrupted the enzyme system of A. craccivora, inducing the inhibition of carboxylesterase (CarE) and the induction of glutathione S-transferase (GST) and acetylcholinesterase (AChE). Combining these results with those of a gas chromatography-mass spectrometry (GC-MS) analysis, it is suggested that p-cymene, hymecromone, 9,12-octadecadienoic acid (Z, Z) methyl ester, and 9,12-octadecadienoic acid (Z, Z) may be connected to the insecticidal effects we observed. This study provides a theoretical basis for the use of L. araneicola HK-1 as a potential biological control agent.

Organophospate-Induced Pathology: Mechanisms of Development, Principles of Therapy and Features of Experimental Studies

Organophosphates (OP) are one of the most common neurotoxic xenobiotics. In acute OP poisoning, as a result of suppression of synaptic acetylcholinesterase (AChE) activity, a cholinergic syndrome develops, which can transform into status epilepticus. Within a few days after acute poisoning, the so-called an intermediate syndrome can develop, which is associated with prolonged inhibition of AChE, desensitization of nicotinic receptors, and functional degradation of synapses and muscle fibers. In 10–20 days after a single acute or repeated subacute poisoning, OP-induced delayed polyneuropathy (OPIDN) can develop – a neurodegenerative disease, the signs of which are ataxia, loss of function of the distal sensory and motor axons of peripheral nerves. The occurrence of a neuropsychiatric disorder (NPD) caused by chronic exposure to relatively low-toxicity organophosphorus compounds is usually not associated with acute poisoning; symptoms include cognitive impairment, chronic fatigue, and extrapyramidal symptoms. The list of possible diseases or pathological conditions (syndromes) that develop as a result of acute, subacute or chronic effects of OP on the human body has expanded in recent years due a number of known neurodegenerative diseases (Alzheimer’s, Parkinson’s, multiple sclerosis, etc.). The aging of the body in general and the aging of the brain in particular are considered in the review from the point of view of the consequences of OP poisoning, which can serve as a nonspecific trigger of aging and related neurodegenerative diseases. Gulf syndrome is not a consequence of OP intoxication, but is also of interest and is considered in the context of OP-induced pathology, since its etiology and pathogenesis are associated with the exposure to cholinesterase inhibitors. The review presents data indicating the important role of the vascular endothelium in the development of OP-induced pathology; The first suggestions were made by clinicians in the late 1980s, and the first experimental data were obtained in the early 2000s. The principles of therapy for acute poisoning are outlined, taking into account experimental data from recent years. Some methods for studying OP in experiments in vitro, ex vivo and in vivo with laboratory animals, including the use of carboxylesterase inhibitors, are presented. The most important part of in vivo investigations has been and remains the search for new biomarkers to assess the effectiveness of adjuvant and regenerative therapies.

Elucidation of Carboxylesterase Mediated Pharmacokinetic Interactions between Irinotecan and Oroxylin A in Rats via Physiologically Based Pharmacokinetic Modeling.

Our previous screening studies identified Oroxylin A (OXA) as a strong inhibitor on the carboxyolesterase mediated hydrolysis of irinotecan to SN-38. The current study employed a whole-body physiologically based pharmacokinetic (PBPK) modeling approach to investigate the underlying mechanisms of the carboxylesterase-mediated pharmacokinetics interactions between irinotecan and OXA in rats. Firstly, rats received irinotecan intravenous treatment at 35μmol/kg without or with oral OXA pretreatment (2800μmol/kg) daily for 5days. On day 5, blood and tissues were collected for analyses of irinotecan/SN-38 concentrations and carboxylesterase expression. In addition, effects of OXA on the enzyme kinetics of irinotecan hydrolysis and unbound fractions of irinotecan and SN-38 in rat plasma, liver and intestine were also determined. Finally, a PBPK model that integrated the physiological parameters, enzyme kinetics, and physicochemical properties of irinotecan and OXA was developed. Our PBPK model could accurately predict the pharmacokinetic profiles of irinotecan/SN-38, with AUC0-6h and Cmax values within ±27% of observed values. When OXA was included as a carboxylesterase inhibitor, the model could also predict the irinotecan/SN-38 plasma concentrations within twofold of those observed. In addition, the PBPK model indicated inhibition of carboxylesterase-mediated hydrolysis of irinotecan in the intestinal mucosa as the major underlying mechanism for the pharmacokinetics interactions between irinotecan and OXA. A whole-body PBPK model was successfully developed to not only predict the impact of oral OXA pretreatment on the pharmacokinetics profiles of irinotecan but also reveal its inhibition on the intestinal carboxylesterase as the major underlying mechanism.

Identification of a novel carboxylesterase dominantly expressed in the booklouse, Liposcelis bostrychophila

Booklice are tiny insect pests commonly found indoors and characterized by high proliferation ability. Booklice contamination in stored foods causes profound food loss and possibly allergic reactions after ingestion; thus, booklice are known as stored-food pests. Therefore, the safe extermination of booklice would solve food loss and allergic concerns. Organophosphates (OPs) are used as harmless insecticides against various pests; nonetheless, cases of OP poisoning are reported worldwide. Therefore, considering protecting stored-food products, OP doses should be minimal. It is generally known that carboxylesterase-mediated inactivation of OPs decreases the efficacy of OPs, suggesting that an inhibitor of carboxylesterase could be a synergist in reducing the effective dose of OP. Herein, I report the molecular cloning of a novel carboxylesterase dominantly expressed in a representative species of indoor booklice, Liposcelis bostrychophila, designated Liposcelis bostrychophila carboxylesterase 1 (LBCE1). The recombinant protein of LBCE1 was shown to have an elastase activity. Furthermore, using the 3D-structural model of LBCE1, its possible binding to OP insecticides was predicted in silico. These results suggest that a specific LBCE1 inhibitor may serve as a synergist to better control booklice.

Enzyme-targeted near-infrared fluorescent probe for organophosphorus pesticide residue detection.

Pesticide residues significantly affect food safety and harmhuman health. In this work, a series of near-infrared fluorescent probes were designed and developed by acylating the hydroxyl group of the hemicyanine skeleton with aquenching moiety for monitoring the presence of organophosphorus pesticides in food and live cells. The carboxylic ester bond on the probe was hydrolyzed catalyticallyin the presence of carboxylesterase and thereby the fluorophore was releasedwith near-infrared emission. Notably, the proposed probe 1 exhibited excellent sensitivity against organophosphorus based on the carboxylesterase inhibition mechanism and the detection limit for isocarbophos achieved 0.1734μg/L in the fresh vegetable sample. More importantly, probe 1 allowed for situ visualization of organophosphorus in live cells and bacteria, meaning great potential for tracking the organophosphorus in biological systems. Consequently, this study presents a promising strategy for tracking pesticide residues in food and biological systems.

Biotransformation and brain distribution of the anti-COVID-19 drug molnupiravir and herb-drug pharmacokinetic interactions between the herbal extract Scutellaria formula-NRICM101

The aim of this study was to explore the effects of herbal drug pharmacokinetic interactions on the biotransformation of molnupiravir and its metabolite β-D-N4-hydroxycytidine (NHC) in the blood and brain. To investigate the biotransformation mechanism, a carboxylesterase inhibitor, bis(4-nitrophenyl)phosphate (BNPP), was administered. Not only molnupiravir but also the herbal medicine Scutellaria formula-NRICM101 is potentially affected by coadministration with molnupiravir. However, the herb-drug interaction between molnupiravir and the Scutellaria formula-NRICM101 has not yet been investigated. We hypothesized that the complex bioactive herbal ingredients in the extract of the Scutellaria formula-NRICM101, the biotransformation and penetration of the bloodbrain barrier of molnupiravir are altered by inhibition of carboxylesterase. To monitor the analytes, ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLCMS/MS) coupled with the microdialysis method was developed. Based on the dose transfer from humans to rats, a dose of molnupiravir (100 mg/kg, i.v.), molnupiravir (100 mg/kg, i.v.) + BNPP (50 mg/kg, i.v.), and molnupiravir (100 mg/kg, i.v.) + the Scutellaria formula-NRICM101 extract (1.27 g/kg, per day, for 5 consecutive days) were administered. The results showed that molnupiravir was rapidly metabolized to NHC and penetrated into the brain striatum. However, when concomitant with BNPP, NHC was suppressed, and molnupiravir was enhanced. The blood-to-brain penetration ratios were 2% and 6%, respectively. In summary, the extract of the Scutellaria formula-NRICM101 provides a pharmacological effect similar to that of the carboxylesterase inhibitor to suppress NHC in the blood, and the brain penetration ratio was increased, but the concentration is also higher than the effective concentration in the blood and brain.

Continuously increased generation of ROS in human plasma after cardiac arrest as determined by Amplex Red oxidation

Oxidative stress is believed to be a major cause of injury after cardiac arrest (CA). While the effects of ROS generated within tissues have been extensively investigated, the potential of plasma-generated ROS in contributing to CA pathology has not been examined. We utilized Amplex Red (AR) to measure the real time-generation of ROS in isolated plasma from human CA patients. We first used post-CA rat plasma to identify interfering factors for AR oxidation, and then applied this knowledge to analyze human plasma samples, accounting for the identified confounders. We found significantly increased AR oxidation rates lasting for 4 h in post-CA rat plasma compared to baseline. AR oxidation was unchanged with removal of horseradish peroxidase or addition of catalase. However, adding carboxylesterase inhibitors significantly decreased AR oxidation in rat plasma, which implicated increased carboxylesterase activity, not ROS leading to increased AR oxidation. AR oxidation rates were also significantly increased in human CA patient plasma compared to control and this increase persisted even with carboxylesterase inhibition, suggesting continuously increased ROS-generation within plasma post-CA in humans. The increased ROS generation may be one major source of injury post-CA that may be mitigated with antioxidative therapeutic strategies that can manage the ROS systemically generated in plasma over time. KEY POLICY HIGHLIGHTS We examined the potential of plasma as a source of ROS generation post-cardiac arrest Rat cardiac arrest was used to guide the application of Amplex Red in human plasma ROS generation in plasma is significantly increased after cardiac arrest in humans Scavenging excessive ROS in post-resuscitation plasma may improve outcomes of patients

Sensory irritation to methylparaben is caused by its low metabolism in the skin.

Sensitive skin is a well- known skin condition showing sensory irritation to daily used products such as cosmetics or pharmaceuticals, possibly containing sensory irritants. Methylparaben (MP), widely used as a preservative, is a representative sensory irritant and hydrolyzed in the skin. We aimed to clarify the relationship between MP sensory irritation and MP hydrolysis. First, we investigated the percutaneous penetration and hydrolysis of MP by using an ex vivo pig skin system and confirmed that topically applied MP was immediately hydrolyzed to p-hydroxybenzoic acid (PHBA). We next evaluated whether MP or PHBA causes sensory irritation using a well-used stinging test in human skin and found that MP, but not PHBA, induced irritation. Additionally, MP, but not PHBA, increased intracellular calcium in cultured TRPA1-expressed HEK293 cells, supporting the stimulatory activity of MP. Five and 10 individuals with sensitive and non-sensitive skin, respectively, were selected by a questionnaire and stinging test. In their biopsied skin samples, MP hydrolytic activity was significantly lower in sensitive than non-sensitive skin. Finally, we examined the activity of carboxylesterase (CES), which promptly hydrolyzes MP to PHBA. By using specific inhibitors of CES and CES2, we found that CES1 was responsible for MP metabolism. Our study suggests that low skin metabolism of topical agents is one of the causes of skin sensory irritation and resultant sensitive skin.

Characterisation of AMB-FUBINACA metabolism and CB1-mediated activity of its acid metabolite

PurposeAMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB1) was investigated to determine the activity of the metabolite.MethodsThe effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ9-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB1.ResultsAMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ9-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB1 as compared to the acid metabolite.ConclusionsCES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.

New Multifunctional Agents for Potential Alzheimer's Disease Treatment Based on Tacrine Conjugates with 2-Arylhydrazinylidene-1,3-Diketones.

Alzheimer's disease (AD) is considered a modern epidemic because of its increasing prevalence worldwide and serious medico-social consequences, including the economic burden of treatment and patient care. The development of new effective therapeutic agents for AD is one of the most urgent and challenging tasks. To address this need, we used an aminoalkylene linker to combine the well-known anticholinesterase drug tacrine with antioxidant 2-tolylhydrazinylidene-1,3-diketones to create 3 groups of hybrid compounds as new multifunctional agents with the potential for AD treatment. Lead compounds of the new conjugates effectively inhibited acetylcholinesterase (AChE, IC50 0.24-0.34 µM) and butyrylcholinesterase (BChE, IC50 0.036-0.0745 µM), with weak inhibition of off-target carboxylesterase. Anti-AChE activity increased with elongation of the alkylene spacer, in agreement with molecular docking, which showed compounds binding to both the catalytic active site and peripheral anionic site (PAS) of AChE, consistent with mixed type reversible inhibition. PAS binding along with effective propidium displacement suggest the potential of the hybrids to block AChE-induced β-amyloid aggregation, a disease-modifying effect. All of the conjugates demonstrated metal chelating ability for Cu2+, Fe2+, and Zn2+, as well as high antiradical activity in the ABTS test. Non-fluorinated hybrid compounds 6 and 7 also showed Fe3+ reducing activity in the FRAP test. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters acceptable for potential lead compounds at the early stages of anti-AD drug development.

An Unusual Case of High 6-Monoacetylmorphine and Low Morphine in Urine: Heroin Abuse or Analytical Error?

A 59-year-old male with a past medical history of chronic low back pain, opioid use disorder, and type 2 diabetes mellitus presented to an outpatient clinic for pain contract–related urine drug screen testing. Current medications included metformin, oxycodone, and Suboxone® (buprenorphine and naloxone combination). The patient stated that 5 days prior to the visit, he stopped taking Suboxone due to complaints of nausea and vomiting. Urine toxicology test results are summarized in Table 1. ... ... Suboxone is an opioid abuse management medication that is formulated with buprenorphine and naloxone. Naloxone has limited bioavailability when administered sublingually and should be barely detectable in urine. However, when Suboxone is taken parenterally, naloxone enters the blood stream and is largely excreted in urine. In this case, the presence of a high concentration of naloxone in urine with a high ratio to buprenorphine suggested parenteral administration (1). Heroin is readily metabolized into 6-MAM and then to morphine. Typically, the 6-MAM-to-morphine ratio is <0.26, so it is uncommon to see a high positive 6-MAM with very low morphine concentrations (2). The calculated 6-MAM-to-morphine ratio in this sample was 7.27. Conversion from morphine to 6-MAM due to contaminants in enzyme hydrolysis or acid hydrolysis used during sample preparation in urine samples with extremely high morphine (>100 000 μg/L) has been reported (3). In our case, morphine was very low, so this was unlikely, but to rule out any analytical cause, the positive urine sample was sent to a reference laboratory, which confirmed our findings. The fact that 2 laboratories using different mass spectrometry–based methods showed detectable 6-MAM supported heroin use, instead of a false positive due to an interference. The unusually low morphine result in some heroin users has been reported elsewhere and may be mostly attributed to inhibitors of carboxylesterases from the heroin synthesis process (2) or explained by polymorphism in the esterase enzymes metabolizing 6-MAM to morphine (4). The physician also requested serum fentanyl testing by mass spectrometry 5 days after the original urine sample collection (fentanyl was not part of our general urine drug screen), which came back positive for fentanyl and norfentanyl. In addition to the unexpected positive amphetamine result, all of this confirmed that this patient suffered from polysubstance abuse.

Carbon nanomaterial functionalization with pesticide-detoxifying carboxylesterase

Four carbon materials, spent coffee-ground biochar, carbon black, short CNTs, and nitrogen-doped few-layer graphene (N-graphene) were tested for their functionalization with a commercial carboxylesterase. Their robustness to variations in time and key physicochemical parameters (temperature and pH) was analysed. In general, carbon nanomaterials showed better performance than biochar, both in terms of binding capacity and resilience in harsh conditions, at statistically significant levels. Among the tested materials, functionalized N-graphene also showed the highest level of inhibition of carboxylesterase by pesticide exposure. Therefore, N-graphene was selected for biotechnological application of pesticide scavenging toxicity in T. thermophila, a ciliate bioindicator of water quality. While immobilization of the enzyme was not effective in the case of carbaryl, a methyl carbamate, in the case of the organophosphorus dichlorvos, a 1- or 30-min contact time with a water solution containing 5 times the LC100 - 0.5 mM - allowed 50% and 100% rescue of ciliate survival, respectively. These results suggest that functionalization with carboxylesterase may be of additional benefit compared to bare carbon in water clean-up procedures, especially for highly hydrophilic pesticides such as dichlorvos.