Reversible Cholinesterase Inhibitor Research Articles

Development and validation of high-performance liquid chromatography method for the simultaneous quantification of rivastigmine hydrogen tartrate and asiaticoside co-loaded in niosomes: A Box–Behnken design approach

Rivastigmine hydrogen tartrate (RHT), a reversible cholinesterase inhibitor, is considered as the first-line therapy for mild to moderate Alzheimer’s disease. Asiaticoside (AS), a pentacyclic triterpenoid saponin, is well known as cognitive enhancer due to its antioxidant effect. Based on the hypothesis of their synergistic therapeutic potential, RHT and AS were co-encapsulated in niosomal formulation. A simple, precise, and accurate high-performance liquid chromatography method was developed for simultaneous quantitative analysis. The chromatographic parameters were optimized by Box-Behnken experimental design. The separation was performed on a reversed-phase Phenomenex C18 (150 mm × 4.6 mm, 5 μm) column at 30 °C under the UV detection of 210 nm. The optimized mobile phase consisted of a mixture of 20 mM potassium dihydrogen phosphate buffer (pH 2.6) and acetonitrile (72:28 % v/v) under the isocratic mode at the flow rate of 0.9 mL/min. The developed method was fully validated under the ICH guidelines and could be successfully applied for simultaneous quantitative analysis of RHT and AS in niosomal formulation.

Conjugates of amiridine and thiouracil derivatives as effective inhibitors of butyrylcholinesterase with the potential to block β-amyloid aggregation.

New amiridine-thiouracil conjugates with different substituents in the pyrimidine fragment (R = CH3 , CF2 Н, CF3 , (CF2 )2 H) and different spacer lengths (n = 1-3) were synthesized. The conjugates rather weakly inhibit acetylcholinesterase (AChE) and exhibit high inhibitory activity (IC50 up to 0.752 ± 0.021 µM) and selectivity to butyrylcholinesterase (BChE), which increases with spacer elongation; the lead compounds are 11c, 12c, and 13c. The conjugates are mixed-type reversible inhibitors of both cholinesterases and practically do not inhibit the structurally related off-target enzyme carboxylesterase. The results of molecular docking to AChE and BChE are consistent with the experiment on enzyme inhibition and explain the structure-activity relationships, including the rather low anti-AChE activity and the high anti-BChE activity of long-chain conjugates. The lead compounds displace propidium from the AChE peripheral anion site (PAS) at the level of the reference compound donepezil, which agrees with the mixed-type mechanism of AChE inhibition and the main mode of binding of conjugates in the active site of AChE due to the interaction of the pyrimidine moiety with the PAS. This indicates the ability of the studied conjugates to block AChE-induced aggregation of β-amyloid, thereby exerting a disease-modifying effect. According to computer calculations, all synthesized conjugates have an ADME profile acceptable for drugs.

Addition of Neostigmine and Atropine to Conventional Management of Postdural Puncture Headache in Patients Undergoing Lower Abdominal Surgeries

Abstract Background Postdural puncture headache (PDPH) is a complication of spinal anesthesia or lumbar puncture and is an unpleasant experience for the patient as well as the anesthetist. It is thought to result from meningeal traction related to low cerebrospinal fluid (CSF) pressure or cerebral vasodilation as an indirect effect of decreased CSF pressure. Neostigmine is a reversible inhibitor of the enzyme cholinesterase, which results in an increased concentration of the neurotransmitter acetylcholine. It produces cholinergic-mediated analgesia and has been used as an adjunct analgesic postoperatively. Neostigmine can pass through the choroid plexus but not the blood–brain barrier, the central effect of both neostigmine and atropine influence both CSF secretion and cerebral vascular tone which are the primary pathophysiological changes in PDPH. Objective Assess the effectiveness of postdural anesthesia with addition of Neostigmine and Atropine versus conventional postdural anesthesia. Patients and Methods A 90 patients were included in our prospective randomized controlled double-blind study that was conducted at Ain Shams University Hospitals for 4 months on patients who fulfilled the inclusion criteria: patients with ASA Grade I & II, aged from (18 to 40) years old and scheduled for cesarean section. All patients aged >18 or <40, body weight >50, ASA III, IV, patients with uncontrolled chronic diseases such as hypertension, cardiac, renal, hepatic, cerebral diseases and peripheral vascular diseases, patients with history of PDPH, chronic headache, cluster headache, migraine, convulsions, cerebrovascular accident, signs of meningismus, preeclampsia, eclampsia coagulopathy, and previous neurological diseases and patients with history of bronchial asthma were excluded. Patients were divided into 2 groups: Group I (experimental group): neostigmine and atropine and Group II (placebo group): Saline placebo. Results The results of this study showed that incidence of postoperative headache was significantly lower among the intervention group, furthermore, VAS was significantly lower in the experimental arm versus placebo one. However, there was no significant difference regarding demographic, vital signs, and need for post-operative analgesia. The presented study showed that there is statistically non-significant difference between the studied groups regarding age, weight, and height and body mass index. There is statistically non-significant difference between the studied groups regarding duration of surgery. Conclusion After demonstrating many management options for post dural puncture headache (PDPH), in this study, we tried to prove the value of the use of neostigmine as a new management option that looks promising to alleviate the symptoms of such distressing condition. The current study results proved that the drug reduced the number of cases that experienced (PDPH) after spinal anesthesia for lower segment caesarean section, and how it prevent &control headache. A combination of neostigmine and atropine are effective in managing PDPH by reducing the associated VAS score and preventing persistence of headache.

Targeting α7-nAChR by galantamine mitigates reserpine-induced fibromyalgia-like symptoms in rats: Involvement of cAMP/PKA, PI3K/AKT, and M1/M2 microglia polarization

Fibromyalgia (FM) is a pain disorder marked by generalized musculoskeletal pain accompanied by depression, fatigue, and sleep disturbances. Galantamine (Gal) is a positive allosteric modulator of neuronal nicotinic acetylcholine receptors (nAChRs) and a reversible inhibitor of cholinesterase. The current study aimed to explore the therapeutic potential of Gal against reserpine (Res)-induced FM-like condition along with investigating the α7-nAChR's role in Gal-mediated effects. Rats were injected with Res (1 mg/kg/day; sc) for 3 successive days then Gal (5 mg/kg/day; ip) was given alone and with the α7-nAChR blocker methyllycaconitine (3 mg/kg/day; ip), for the subsequent 5 days. Galantamine alleviated Res-induced histopathological changes and monoamines depletion in rats' spinal cord. It also exerted analgesic effect along with ameliorating Res-induced depression and motor-incoordination as confirmed by behavioral tests. Moreover, Gal produced anti-inflammatory effect through modulating AKT1/AKT2 and shifting M1/M2 macrophage polarization. The neuroprotective effects of Gal were mediated through activating cAMP/PKA and PI3K/AKT pathways in α7-nAChR-dependent manner. Thus, Gal can ameliorate Res-induced FM-like symptoms and mitigate the associated monoamines depletion, neuroinflammation, oxidative stress, apoptosis, and neurodegeneration through α7-nAChR stimulation, with the involvement of cAMP/PKA, PI3K/AKT, and M1/M2 macrophage polarization.

Reversible Cholinesterase Inhibitor Loaed Chitosan Based Nanoparticle

Memantine hydrochloride is a is a reversible cholinesterase inhibitor used in the treatment of Alzheimer’s disease, low-moderate affinity, uncompetitive n-methyl-d-aspartate (NMDA) receptor antagonist, with strong voltage dependency and rapid blocking/unblocking kineticsThe present study was explore the potential of mamentine loaded nanoparticle.with varying quantity of chitosan byionotropic gelation method.The effect of chitosan quantity on the particle size was studied by varying stirring time and stirring speed. The particle morphology can be modulated by selecting the agitation speed as well as drug polymer ratio In the present study.The evaluation parameters like Zeta potential, Entrapment efficiency and Poly dispersity index,of optimized formulation CN15 was found to be 364.2±3.37, -8.46, 79.9±0.2 sand 0.283± 0.048 respectively Determination of percentage yield and loading efficiency,in vitro drug release was also found optimum en 99-100%.

Conjugates of Tacrine and Salicylic Acid Derivatives as New Promising Multitarget Agents for Alzheimer's Disease.

A series of previously synthesized conjugates of tacrine and salicylamide was extended by varying the structure of the salicylamide fragment and using salicylic aldehyde to synthesize salicylimine derivatives. The hybrids exhibited broad-spectrum biological activity. All new conjugates were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The structure of the salicylamide moiety exerted little effect on anticholinesterase activity, but AChE inhibition increased with spacer elongation. The most active conjugates were salicylimine derivatives: IC50 values of the lead compound 10c were 0.0826 µM (AChE) and 0.0156 µM (BChE), with weak inhibition of the off-target carboxylesterase. The hybrids were mixed-type reversible inhibitors of both cholinesterases and displayed dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking, which, along with experimental results on propidium iodide displacement, suggested their potential to block AChE-induced β-amyloid aggregation. All conjugates inhibited Aβ42 self-aggregation in the thioflavin test, and inhibition increased with spacer elongation. Salicylimine 10c and salicylamide 5c with (CH2)8 spacers were the lead compounds for inhibiting Aβ42 self-aggregation, which was corroborated by molecular docking to Aβ42. ABTS•+-scavenging activity was highest for salicylamides 5a-c, intermediate for salicylimines 10a-c, low for F-containing salicylamides 7, and non-existent for methoxybenzoylamides 6 and difluoromethoxybenzoylamides 8. In the FRAP antioxidant (AO) assay, the test compounds displayed little or no activity. Quantum chemical analysis and molecular dynamics (MD) simulations with QM/MM potentials explained the AO structure-activity relationships. All conjugates were effective chelators of Cu2+, Fe2+, and Zn2+, with molar compound/metal (Cu2+) ratios of 2:1 (5b) and ~1:1 (10b). Conjugates exerted comparable or lower cytotoxicity than tacrine on mouse hepatocytes and had favorable predicted intestinal absorption and blood-brain barrier permeability. The overall results indicate that the synthesized conjugates are promising new multifunctional agents for the potential treatment of AD.

Therapeutic and medicinal effects of snowdrop (Galanthus spp.) in Alzheimer's disease: A review.

Genus Galanthus (Amaryllidaceae) is an early spring flowering bulbous plant. Galanthus species contain alkaloids that have shown pharmacological activity. Galanthamine is an alkaloid that was extracted from Galanthus and other Amaryllidaceae. Owing to its acetylcholinesterase (AChE) inhibitory activity, galanthamine is used and marketed to treat Alzheimer's disease (AD). The aim of the present study, while introducing the botanical and pharmacological characteristics and various aspects of the medicinal plant Galanthus, is to emphasize the effect of this plant in the treatment of AD. In this web-based study in 2021, articles indexed in scientific databases in English language, including ISI Web of Knowledge, PubMed, Scopus, MedLib, Medknow, SID, ISC, and also articles and e-books published in Springer, Elsevier, John Wiley and Sons, and Taylor and Francis were evaluated from 1990 to 2021, using the following keywords: "Galanthus" "galanthamine," "Alzheimer's disease." Amaryllidaceae-type alkaloids possess an anticholinesterase activity. The most studied Galanthus alkaloid, galanthamine, is a long-acting, selective, reversible, competitive inhibitor of AChE and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine (ACh). Owing to its AChE inhibitory activity, galanthamine is used to treat certain stages of AD. Galantamine can act as a parasympathomimetic agent, especially as a reversible cholinesterase inhibitor. Galantamine is not structurally associated with other AChE inhibitors. Hence, its proposed mechanism of action involves the reversible inhibition of AChE, preventing hydrolysis of ACh that results in an increased concentration of ACh at cholinergic synapses.

Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands.

Seven pyridoxal dioxime quaternary salts (1-7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43-76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (Ki = 100-400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293).

Efficacy of donepezil for the treatment of oxaliplatin-induced peripheral neuropathy: DONEPEZOX, a protocol of a proof of concept, randomised, triple-blinded and multicentre trial

BackgroundThe use of oxaliplatin in digestive tract cancers could induce severe peripheral neuropathy (OIPN) decreasing the quality of life of patients and survivors. There is currently, no univocal treatment for these peripheral neuropathies. Donepezil, a reversible inhibitor of cholinesterase, used to treat Alzheimer’s disease and dementia, is reported to have a good safety profile in humans, and preclinical data have provided initial evidence of its effectiveness in diminishing neuropathic symptoms and related comorbidities in OIPN animal models.MethodsThe DONEPEZOX trial will be a proof-of-concept, randomised, triple-blinded, and multicentre study. It will be the first clinical trial evaluating the efficacy and safety of donepezil for the management of OIPN. Adult cancer survivors with OIPN that report sensory neuropathy according to QLQ-CIPN20 sensory score (equivalence of a grade ≥ 2), at least 6 months after the end of an oxaliplatin-based chemotherapy will be included. Eighty patients will be randomly assigned to receive either donepezil or placebo over 16 weeks of treatment. The primary endpoint will be the rate of responders (neuropathic grade decreases according to the QLQ-CIPN20 sensory score) in the donepezil arm. The severity of OIPN will be assessed by the QLQ-CIPN20 sensory scale before and after 16 weeks of treatment. The comparison versus the placebo arm will be a secondary objective. The other secondary endpoints will be tolerance to donepezil, the severity and features of OIPN in each arm before and after treatment, related-comorbidities and quality of life. Fleming’s one-stage design will be used for sample size estimation. This design yields a type I error rate of 0.0417 and power of 91% for a responder rate of at least 30% in donepezil arm. A total of 80 randomized patients is planned.DiscussionThis study will allow, in the case of positive results, to initiate a phase 3 randomized and placebo-controlled (primary endpoint) clinical study to assess the therapeutic interest of donepezil to treat OIPN.Trial registrationNCT05254639, clincialtrials.gov, Registered 24 February 2022.

Bioavailability Study of a Transdermal Patch Formulation of Rivastigmine Compared with Exelon in Healthy Subjects.

Rivastigmine is a reversible cholinesterase inhibitor indicated for the treatment of all stages of Alzheimer's disease (AD). Transdermal patch formulation allows smooth and continuous drug delivery. Its tolerability, efficacy and convenience of use increase treatment compliance. This study was designed to evaluate the bioavailability and to assess the bioequivalence of two rivastigmine transdermal patches at steady state (RIV-TDS Test Product versus Exelon Marketed Reference Product), with a release rate of 13.3mg/24h, after multiple patch applications. As secondary objectives, safety, patch adhesion and skin irritation were evaluated. This was an open-label, randomized, balanced, two-period, two-sequence, cross-over study of healthy adults (n=31). The treatment period consisted of two 5-day study periods during which consecutive daily application of the investigational patches with a release rate of 13.3mg/24h rivastigmine took place. Serial blood samples were collected to measure plasma concentrations. Adhesion and skin irritation assessments were performed after application of patches. Point estimates and 90% confidence intervals of pharmacokinetic parameters at steady state, viz. area under the plasma concentration versus time curve from dosing time to the end of the dosing interval τ (profile day) at steady state [AUC0-τ,ss] (97.4; 88.8-106.9), maximum plasma concentration within the dosing interval τ (profile day) at steady state [Cmax,ss] (99.6; 90.4-109.7) and trough plasma concentration at the end of the dosing interval τ (profile day) at steady state [Cτ,ss] (96.8; 86.2-108.9), demonstrated that both patches were bioequivalent. Evaluation of patch adhesion showed better skin adherence for RIV-TDS as well as dermal response scores (skin tolerability after removal). For both products, bioequivalence was shown andsystemic tolerability was in accordance with the safety profile of the drug substance. The trial is registered in ClinicalTrials.gov: NCT03573050 and EudraCT: 2018-000968-28.

Ipidacrine (Axamon), A Reversible Cholinesterase Inhibitor, Improves Erectile Function in Male Rats With Diabetes Mellitus-Induced Erectile Dysfunction.

ABSTRACTSBackgroundManagement of diabetes mellitus-induced erectile dysfunction (DMED) is challenging because of its insufficient responses to phosphodiesterase type 5 inhibitors.AimTo compare the effects of ipidacrine, a reversible cholinesterase inhibitor, and sildenafil on DMED in a rat model of streptozotocin (STZ)-induced diabetes.MethodsErectile dysfunction (ED) caused by STZ-induced diabetes mellitus was modeled in adult male Wistar rats, which were randomized to 4 groups: untreated diabetic rats, sildenafil (5 mg/kg), ipidacrine (3.6 mg/kg) and ipidacrine (6.7 mg/kg). The test drug (ipidacrine), comparator (sildenafil) or control substance (1% starch solution) were administered orally for 5 days or 14 days. Erectile function was assessed by the change in the maximum intracavernous pressure (ICPmax) following cavernous nerve electrical stimulation. The mean arterial pressure (MAP) was recorded, and the ICPmax/MAP ratio was calculated. Sexual behavior, cholinesterase activity and blood testosterone level tests assessed.Main Outcome MeasureThe quantitative value of ICPmax/MAP 14 days after the start of administration of the test drug and the comparison drug.ResultsAnimals with STZ-induced diabetes mellitus showed a significant decrease in ICPmax and ICPmax/MAP ratio compared to the intact control group. When ipidacrine was administered to rats with DMED for 14 days, an increase in these indicators was noted. It was proved that ipidacrine at a dose of 6.7 mg/kg has noninferiority compared to sildenafil on the DMED model. Significant increase in ICPmax compared to STZ-control after electrostimulation of the cavernous nerve was recorded following administration of ipidacrine at a dose of 6.7 mg/kg (P < .05) and sildenafil at a dose 5 mg/kg (P < .05). Neither the test drug, nor the comparator were associated with increase in testosterone levels in blood; as well both drugs did not promote activation of sexual behavior.Clinical ImplicationsIpidacrine may be considered as an effective therapy for DMED but needs to be verified in human investigations.Strengths & LimitationsThe role of ipidacrine, was firstly demonstrated in rats with DMED. However, the results were obtained in animal experiments, and will be further tested in the study of receptor interactions and the determination of cellular targets.ConclusionThis is the first study to show that administration of ipidacrine, the reversible cholinesterase inhibitor, improved erectile function in diabetic rats and these results may be beneficial in further studies using ipidacrine for treatment of DMED, particularly in non-responders to PDE5 inhibitors.Bykov V, Gushchina E, Morozov S, et al. Ipidacrine (Axamon), A Reversible Cholinesterase Inhibitor, Improves Erectile Function in Male Rats With Diabetes Mellitus-Induced Erectile Dysfunction. Sex Med 2022;10:100477.

Optimized Rivastigmine Nanoparticles Coated with Eudragit for Intranasal Application to Brain Delivery: Evaluation and Nasal Ciliotoxicity Studies.

Rivastigmine, a reversible cholinesterase inhibitor, is frequently indicated in the management of demented conditions associated with Alzheimer disease. The major hurdle of delivering this drug through the oral route is its poor bioavailability, which prompted the development of novel delivery approaches for improved efficacy. Due to numerous beneficial properties associated with nanocarriers in the drug delivery system, rivastigmine nanoparticles were fabricated to be administer through the intranasal route. During the development of the nanoparticles, preliminary optimization of processing and formulation parameters was done by the design of an experimental approach. The drug–polymer ratio, stirrer speed, and crosslinking time were fixed as independent variables, to analyze the effect on the entrapment efficiency (% EE) and in vitro drug release of the drug. The formulation (D8) obtained from 23 full factorial designs was further coated using Eudragit EPO to extend the release pattern of the entrapped drug. Furthermore, the 1:1 ratio of core to polymer depicted spherical particle size of ~175 nm, % EE of 64.83%, 97.59% cumulative drug release, and higher flux (40.39 ± 3.52 µg.h/cm2). Finally, the intranasal ciliotoxicity study on sheep nasal mucosa revealed that the exposure of developed nanoparticles was similar to the negative control group, while destruction of normal architecture was noticed in the positive control test group. Overall, from the in vitro results it could be summarized that the optimization of nanoparticles’ formulation of rivastigmine for intranasal application would be retained at the application site for a prolonged duration to release the entrapped drug without producing any local toxicity at the mucosal region.

Design of a Reversible Cholinesterase Inhibitor Mamentine HCL Nanogel: Formulation and In-vitro Evaluation

Memantine hydrochloride is a is a reversible cholinesterase inhibitor used in the treatment of Alzheimer’s disease, low-moderate affinity, uncompetitive n-methyl-d-aspartate (NMDA) receptor antagonist, with strong voltage dependency and rapid blocking/unblocking kinetics. The present study was explore the potential of thermosensitive nanogel of mamentine loaded nanoparticle. In situ gel choosing due to restrict unwanted exposure in blood and other healthy tissues, thus eliminate hemolytic side effects of the drug and offer easy administration in vivo. Nanoparticle prepared by ionic gelation method and further the dried nanoparticle incorporates with in situ gel. The in situ gel prepared by cold method using the solutions of Poloxamer-188 and Carbopol-934. The Transmission electron microscopy showed the spherical particles with smooth surface which was in conformity with the SEM and Zetasizer data for particle size. The pH of the formulations was found to be satisfactory and was in the range of 6.8±0.039 -7.4±0.053 and also mucoadhesive strength was show in table. The mucoadhesive strength of all formulations was varies from 2398±0.0004 to 4945±0.0002 dynes/cm2. In-vitro diffusion study of the in situ gel (N1-N8) was performed using modified Franz diffusion cell with dialysis membrane in phosphate buffer pH 6.5 for a period of 24 hours. The in vitro release study were fitted into various kinetic models viz zero order, first order, higuchi model and korsmeyer peppas equation. Stability studies for optimized formulations were carried out at 4.0 ± 0.5°C and 37 ± 0.5ºC for a period of four weeks. There was no significant variation found in physical appearance, average particle size and % drug content of the in situ nanogel N2. No visible changes in the appearance of the gel formulation were observed at the end of the storage period.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

Combined Pre- and Posttreatment of Paraoxon Exposure.

Aims: Organophosphates (OPCs), useful agents as pesticides, also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators is unsatisfactory. Experimental data indicate that superior therapeutic results can be obtained when reversible cholinesterase inhibitors are administered before OPC exposure. Comparing the protective efficacy of five such cholinesterase inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, or K-27), we observed best protection for the experimental oxime K-27. The present study was undertaken in order to determine if additional administration of K-27 immediately after OPC (paraoxon) exposure can improve the outcome. Methods: Therapeutic efficacy was assessed in rats by determining the relative risk of death (RR) by Cox survival analysis over a period of 48 h. Animals that received only pretreatment and paraoxon were compared with those that had received pretreatment and paraoxon followed by K-27 immediately after paraoxon exposure. Results: Best protection from paraoxon-induced mortality was observed after pretreatment with physostigmine (RR = 0.30) and K-27 (RR = 0.34). Both substances were significantly more efficacious than tacrine (RR = 0.67), ranitidine (RR = 0.72), and pyridostigmine (RR = 0.76), which were less efficacious but still significantly reduced the RR compared to the no-treatment group (paraoxon only). Additional administration of K-27 immediately after paraoxon exposure (posttreatment) did not further reduce mortality. Statistical analysis between pretreatment before paraoxon exposure alone and pretreatment plus K-27 posttreatment did not show any significant difference for any of the pretreatment regimens. Conclusions: Best outcome is achieved if physostigmine or K-27 are administered prophylactically before exposure to sublethal paraoxon dosages. Therapeutic outcome is not further improved by additional oxime therapy immediately thereafter.

Structural isomers of saligenin-based β2-agonists: synthesis and insight into the reaction mechanism.

Salmeterol and albuterol are well-known β2-adenoreceptor agonists widely used in the treatment of inflammatory respiratory diseases, such as bronchial asthma and chronic obstructive pulmonary disease. Here we report the preparation of structural isomers of salmeterol and albuterol, which can be obtained from the same starting material as the corresponding β2-agonists, depending on the synthetic approach employed. Using 1D and various 2D NMR measurements, we determined that the structure of prepared isomers holds the β-aryl-β-aminoethanol moiety, in contrast to the α-aryl-β-aminoethanol moiety found in salmeterol and albuterol. We investigated the reaction of β-halohydrin and amines responsible for the formation of β-aryl-β-amino alcohol - both experimentally and using computational methods. The structure of β-halohydrin with the methyl salicylate moiety imposes the course of the reaction. The solvent plays a relevant, yet ambiguous role in the direction of the reaction, while the strength of the base influences the reaction yield and isomer ratio in a more evident way. Using computational methods, we have shown that the most probable reaction intermediate responsible for the formation of the unexpected isomer is the corresponding para-quinone methide, which can be formed due to phenol present in the methyl salicylate moiety. After successful preparation of albuterol and salmeterol isomers, we tested their inhibition potency to human acetylcholinesterase (AChE) and usual and atypical butyrylcholinesterase (BChE). Kinetic studies revealed that both isomers are low-potency reversible inhibitors of human cholinesterases.

Subchronic use of rivastigmine increases procognitive flexibility across multimodal behavioral tasks in healthy male rats

Rivastigmine (RVT) is a reversible inhibitor of cholinesterase approved worldwide for the treatment of cognitive dysfunctions, especially in Alzheimer’s disease. Most previous pre-clinical studies have examined the effects of RVT treatment in a wide variety of pathological research models. Nonetheless, the effects of this drug on sensorimotor gating, memory, and learning tasks in healthy subjects remains unclear. In this study, we investigate the procognitive effects of RVT treatment in healthy rats through sensorimotor gating evaluations (measured as prepulse inhibition of the acoustic startle reflex), active avoidance learning, and spatial memory learning in a radial maze. There is an increase in the amplitude of the startle reflex in RVT-treated rats compared to the control groups, whereas the latency remained constant. Sensorimotor gating values were also incremented compared to those values from controls. In active avoidance, rats treated with RVT learned faster to successfully perform the task compared to controls, but afterwards all groups exhibited virtually identical results. During the sessions in the radial maze, RVT-treated rats committed fewer errors in both the working and reference memory compared to controls. All in all, our results support the hypothesis that RVT treatment may entail procognitive effects in healthy subjects.

Influence of Process Parameters on the Characteristics of Hydrophilic Drug-Loaded Microparticles through Double Emulsion Solvent Evaporation Technique

The purpose of this study was to investigate the influence of process parameters on the characteristics of microparticles using double emulsion solvent evaporation method for encapsulation of hydrophilic drug. Donepezil hydrochloride (DPH), a reversible cholinesterase inhibitor, was selected as a model hydrophilic drug. Prior to conducting an experiment, the target particle size of microparticles was set at approximately 200 μm. The investigated process parameters include pH of outer water phase, stirring time, polymer amount, and volume of outer water phase. The results showed that DPH-loaded microparticles was successfully prepared in two steps. In the first step, the primary emulsion was prepared by dissolving DPH in distilled water before emulsifying in dichloromethane (DCM) containing different amounts of poly(butylmethacrylate-co-2-dimethylaminoethyl-methacrylate-co-methyl-methacrylate) (PBM-DM-MM) using ultrasonic probe. In the second step, the primary emulsion was emulsified in polyvinyl alcohol (PVA) solution by overhead stirrer to prepare double emulsion. After solvent evaporation, the microparticles were collected by centrifugation and washed with distilled water. Based on the statistical analysis, stirring time, polymer amount and volume of outer water phase were the main significant parameters influencing particle size of microparticles.

Immunomodulatory role of nonneuronal cholinergic signaling in myocardial injury

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Some Possibilities to Study New Prophylactics against Nerve Agents.

Nerve agents belong to the most dangerous chemical warfare agents and can be/were misused by terrorists. Effective prophylaxis and treatment is necessary to diminish their effect. General principles of prophylaxis are summarized (protection against acetylcholinesterase inhibition, detoxification, treatment "in advance" and use of different drugs). They are based on the knowledge of mechanism of action of nerve agents. Among different examinations, it is necessary to test prophylactic effectivity in vivo and compare the results with protection in vitro. Chemical and biological approaches to the development of new prophylactics would be applied simultaneously during this research. Though the number of possible prophylactics is relatively high, the only four drugs were introduced into military medical practice. At present, pyridostigmine seems to be common prophylactic antidote; prophylactics panpal (tablets with pyridostigmine, trihexyphenidyl and benactyzine), transant (transdermal patch containing HI-6) are other means introduced into different armies as prophylactics. Scavenger commercionally available is Protexia®. Future development will be focused on scavengers, and on other drugs either reversible cholinesterase inhibitors (e.g., huperzine A, gallantamine, physostigmine, acridine derivatives) or other compounds.