Pharmacophore-based design and discovery of (-)-meptazinol carbamates as dual modulators of cholinesterase and amyloidogenesis.

Multifunctional carbamate-type acetylcholinesterase (AChE) inhibitors with anti-amyloidogenic properties like phenserine are potential therapeutic agents for Alzheimer's disease (AD). We reported here the design of new carbamates using pharmacophore model strategy to modulate both cholinesterase and amyloidogenesis. A five-feature pharmacophore model was generated based on 25 carbamate-type training set compounds. (-)-Meptazinol carbamates that superimposed well upon the model were designed and synthesized, which exhibited nanomolar AChE inhibitory potency and good anti-amyloidogenic properties in in vitro test. The phenylcarbamate 43 was highly potent (IC50 31.6 nM) and slightly selective for AChE, and showed low acute toxicity. In enzyme kinetics assay, 43 exhibited uncompetitive inhibition and reacted by pseudo-irreversible mechanism. 43 also showed amyloid-ß (Aß) lowering effects (51.9% decrease of Aß42) superior to phenserine (31% decrease of total Aß) in SH-SY5Y-APP695 cells at 50 µM. The dual actions of 43 on cholinergic and amyloidogenic pathways indicated potential uses as symptomatic and disease-modifying agents.

Meserine, a novel carbamate AChE inhibitor, ameliorates scopolamine-induced dementia and alleviates amyloidogenesis of APP/PS1 transgenic mice.

AIMS: To investigate whether Meserine, a novel phenylcarbamate derivative of (-)-meptazinol, possesses beneficial activities against cholinergic deficiency and amyloidogenesis, the two major pathological characteristics of Alzheimer's disease (AD). METHODS: Ellman's assay and Morris water maze were used to detect acetylcholinesterase (AChE) activity and evaluate spatial learning and memory ability, respectively. Both high content screening and Western blotting were carried out to detect ß-amyloid precursor protein (APP), while RT-PCR and ELISA were conducted to detect APP-mRNA and ß-amyloid peptide (Aß). RESULTS: In scopolamine-induced dementia mice, Meserine (1 mg/kg, i.p.) significantly ameliorated spatial learning and memory deficits, which was consistent with its in vitro inhibitory ability against AChE (recombinant human AChE, IC50 = 274 ± 49 nM). Furthermore, Meserine (7.5 mg/kg) injected intraperitoneally once daily for 3 weeks lowered APP level by 28% and Aß42 level by 42% in APP/PS1 transgenic mouse cerebrum. This APP modulation action might be posttranscriptional, as Meserine reduced APP by about 30% in SH-SY5Y-APP695 cells but did not alter APP-mRNA level. And both APP and Aß42 lowering action of Meserine maintained longer than that of rivastigmine. CONCLUSION: Meserine executes dual actions against cholinergic deficiency and amyloidogenesis and provides a promising lead compound for symptomatic and modifying therapy of AD.

Bis(9)-(-)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice.

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progressive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-ß aggregation inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive amelioration. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two "water bridges" located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000 ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose-response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics.

Novel bis-(-)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property.

The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(-)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC(50) values of 9.63µM (for ZLA) and 8.64µM (for ZLB), and prevent AChE-induced amyloid-ß (Aß) aggregation with IC(50) values of 49.1µM (for ZLA) and 55.3µM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aß aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD.

Bis-(-)-nor-meptazinols as novel nanomolar cholinesterase inhibitors with high inhibitory potency on amyloid-beta aggregation.

Bis-(-)-nor-meptazinols (bis-(-)-nor-MEPs) 5 were designed and synthesized by connecting two (-)-nor-MEP monomers with alkylene linkers of different lengths via the secondary amino groups. Their acetylcholinesterase (AChE) inhibitory activities were more greatly influenced by the length of the alkylene chain than butyrylcholinesterase (BChE) inhibition. The most potent nonamethylene-tethered dimer 5h exhibited low-nanomolar IC 50 values for both ChEs, having a 10 000-fold and 1500-fold increase in inhibition of AChE and BChE compared with (-)-MEP. Molecular docking elucidated that 5h simultaneously bound to the catalytic and peripheral sites in AChE via hydrophobic interactions with Trp86 and Trp286. In comparison, it folded in the large aliphatic cavity of BChE because of the absence of peripheral site and the enlargement of the active site. Furthermore, 5h and 5i markedly prevented the AChE-induced Abeta aggregation with IC 50 values of 16.6 and 5.8 microM, similar to that of propidium (IC 50 = 12.8 microM), which suggests promising disease-modifying agents for the treatment of AD patients.

Reflex activity caused by laryngoscopy and intubation is obtunded differently by meptazinol, nalbuphine and fentanyl.

BACKGROUND AND OBJECTIVE: To evaluate the different potencies of several opioids in obtunding reflex mechanisms of laryngoscopy and intubation. METHODS: Three groups of patients (each n = 25, ASA 1-2) undergoing elective plastic surgery were randomly given meptazinol (2.5 mg kg-1), nalbuphine (0.3 mg kg-1) or fentanyl (5 microg kg-1) in a blinded fashion prior to laryngoscopy and intubation. This was followed by a standardized bolus induction of a barbiturate and a muscle relaxant. The response to laryngoscopy and intubation was studied, using blood pressure, heart rate and bispectral index. RESULTS: With fentanyl, there was an increase of heart rate by 17%, and systolic blood pressure by 7% when compared to control. Bispectral index dropped an additional 8% when compared to 1 min after barbiturate induction. In the nalbuphine group there was a 16% increase in systolic blood pressure, and a 16% increase in heart rate when compared to control. Also, bispectral index increased by 18% when compared to 1 min after barbiturate injection. The group receiving meptazinol demonstrated no cardiovascular changes although bispectral index dropped by an additional 19% when compared to 1 min after barbiturate injection. CONCLUSION: Meptazinol, appears to depress cardiovascular stimulatory effects and electroencephalogram arousal induced by laryngoscopy and intubation better than nalbuphine or fentanyl.

Conformational re-analysis of (+)-meptazinol: an opioid with mixed analgesic pharmacophores.

AIM: To further investigate the analgesic pharmacophore of (+)-meptazinol. METHODS: Two different opioid pharmacophores, Pharm-I and Pharm-II, were established from structures of nine typical opiates and meperidine by using molecular modeling approaches according to their different structure activity relationship properties. They were further validated by a set of conformationally constrained arylpiperidines. Two conformers of (+)-meptazinol (Conformer-I and Conformer-II) detected in solution were then fitted into the pharmacophores, respectively, by Fit Atoms facilities available in SYBYL, a computational modeling tool kit for molecular design and analysis. RESULTS: Conformer-I fit Pharm-I from typical opiates well. However, Conformer-II fit none of these pharmacophores. Instead, it was found to be similar to another potent analgesic, benzofuro[2,3-c]pyridin-6-ol, whose pharmacophore was suggested to hold the transitional state between the two established pharmacophores. Unlike typical analgesics derived from 4-aryl piperidine (eg, meperidine) with one conformer absolutely overwhelming, the (+)-meptazinol exists in two conformers with similar amounts in solution. Furthermore, both conformers can not transform to each other freely in ordinary conditions based on our NMR results. CONCLUSION: (+)-meptazinol was suggested to be an opioid with mixed analgesic pharmacophores, which may account for the complicated pharmacological properties of meptazinol.

Investigation of the binding mode of (-)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method.

Molecular docking has been performed to investigate the binding mode of (-)-meptazinol (MEP) with acetylcholinesterase (AChE) and to screen bis-meptazinol (bis-MEP) derivatives for preferable synthetic candidates virtually. A reliable and practical docking method for investigation of AChE ligands was established by the comparison of two widely used docking programs, FlexX and GOLD. In our hands, we had more luck using GOLD than FlexX in reproducing the experimental poses of known ligands (RMSD<1.5 A). GOLD fitness values of known ligands were also in good agreement with their activities. In the present GOLD docking protocol, (-)-MEP seemed to bind with the enzyme catalytic site in an open-gate conformation through strong hydrophobic interactions and a hydrogen bond. Virtual screening of a potential candidate compound library suggested that the most promising 15 bis-MEP derivatives on the list were mainly derived from (-)-MEP with conformations of (S,S) and (SR,RS) and with a 2- to 7-carbon linkage. Although there are still no biological results to confirm the predictive power of this method, the current study could provide an alternate tool for structural optimization of (-)-MEP as new AChE inhibitors. [Figure: see text].

Structural comparisons of meptazinol with opioid analgesics.

AIM: To investigate the mechanism of action of a potent analgesic, (+/-)-meptazinol. METHODS: The structures of meptazinol enantiomers were compared with opioid pharmacophore and tramadol. RESULTS: Neither enantiomer of meptazinol fitted any patterns among the opioid pharmacophore and tramadol, although they did share some structural and pharmacological similarities. However, the structure superpositions implied that both enantiomers of meptazinol might share some similar analgesic mechanisms with typical opiate analgesics. CONCLUSION: Meptazinol should have a different mechanism of action to known analgesics, which would be helpful in further investigations of meptazinol in the search for non-addictive analgesics.

A novel method to calculate the extent and amount of drug transported into CSF after intranasal administration.

The aim of this paper is to establish a novel method to calculate the extent and amount of drug transported to brain after administration. The cerebrospinal fluid (CSF) was chosen as the target region. The intranasal administration of meptazinol hydrochloride (MEP) was chosen as the model administration and intravenous administration was selected as reference. According to formula transform, the extent was measured by the equation of X(A)CSF, infinity/X0 = Cl(CSF) AUC(0-->infinity)CSF/X0 and the drug amount was calculated by multiplying the dose with the extent. The drug clearance in CSF (Cl(CSF)) was calculated by a method, in which a certain volume of MEP solution was injected directly into rat cistern magna and then clearance was assessed as the reciprocal of the zeroth moment of a CSF level-time curve normalized for dose. In order to testify the accurateness of the method, 14C-sucrose was chosen as reference because of its impermeable characteristic across blood-brain barrier (BBB). It was found out that the MEP concentrations in plasma and CSF after intranasal administration did not show significant difference with those after intravenous administration. However, the extent and amount of MEP transported to CSF was significantly lower compared with those to plasma after these two administrations. In conclusion, the method can be applied to measure the extent and amount of drug transported to CSF, which would be useful to evaluate brain-targeting drug delivery.

Antinociceptive effects of meptazinol and its isomers on carrageenan-induced thermal hyperalgesia in rats.

Using the latency of paw withdrawal (PWL) from a noxious thermal stimulus as a measure of hyperalgesia, the effects of i.p. injection of meptazinol and its isomers, 112824 and 112825, on carrageenan-induced thermal hyperalgesia were studied in awaked carrageenan-inflamed rats. Peripheral inflammation was induced by intraplantar (i.pl.) injection of carrageenan (2 mg/100 microl) into one hindpaw in rats. Carrageenan produced marked inflammation (edema and erythema) and thermal hyperalgesia in the injected paws, which peaked at 3 h after injection and showed little change in magnitude for another 3 h. Injection of 0.1 mg/kg meptazinol (i.p.) at 3 h after carrageenan had no effect on the PWLs of either inflamed or non-inflamed hindpaw during the next 100 min (P>0.05, n=8). At the dosage of 1 and 10 mg/kg, meptazinol produced marked anti-nociception and anti-hyperalgesia in non-inflamed and inflamed hindpaw, respectively (P<0.05, n=8-11). The prolonging effect of meptazinol on PWL in inflamed hindpaw was more potent than that in non-inflamed hindpaw. Pre-administration of 1.5 mg/kg naloxone significantly antagonized meptazinol-induced anti-nociception and anti-hyperalgesia. Intraperitoneal injection of an isomer of meptazinol, 112825 (1.5 mg/kg), but not 112824 (1 mg/kg), markedly increased the PWL of the non-inflamed hindpaw. Nevertheless, both the isomers produced similar anti-hyperalgesic effect to that of meptazinol (P<0.05, n=8), which was completely reversed by naloxone (1.5 mg/mg). The results suggest that meptazinol and its isomers have anti-nociceptive and anti-hyperalgesic properties with the former more potent. The effects are mainly mediated by mu opioid receptors. This study provides an important clue for extending clinical utilization of meptazinol and its isomers.

Lack of sensitization during place conditioning in rats is consistent with the low abuse potential of tramadol.

Based on the recent finding that tramadol (TRAM) produces conditioned place preference (CPP) and dopamine release in the nucleus accumbens, it was suggested that the abuse liability of TRAM may be greater than hitherto assumed. We re-evaluated the effects of TRAM in CPP and behavioral sensitization, in comparison with morphine (MOR) and meptazinol (MEPT), an opioid drug with minimal abuse potential. While MOR produced CPP and very strong locomotor sensitization, TRAM and MEPT produced only CPP. It has been suggested that sensitization plays an important role in the development of addiction, hence our results suggest that the abuse potential of TRAM might resemble more that of MEPT than that of MOR, and they are consistent with the clinical picture, in that although TRAM is not completely devoid of positively reinforcing effects, reports on abuse are rare. The low propensity to induce addiction may be related to the lack of changes in the brain circuitry mediating reward and motivation, as evidenced by the lack of sensitization.

Protection of acetylcholinesterase by meptazinol in mice exposed to di-isopropyl fluorophosphate. Comparison with physostigmine.

The protective action of meptazinol against acute di-isopropyl fluorophosphate (DFP) intoxication has been evaluated in mice by measuring the effects on the DFP LD50 of the pretreatment of the animals with increasing doses of the drug. Meptazinol at the doses 15, 30 and 45 mg kg-1 injected 15 min before DFP caused a dose-dependent increase in the DFP LD50, resulting in protection ratios equal to 2.1, 4.8 and 9.7, respectively, in the absence of atropine and 2.5, 4.7, and 8, respectively, in the presence of atropine sulphate (17.4 mg kg-1) therapy. Under the same experimental conditions, the protective ratio of 0.1 mg kg-1 physostigmine sulphate was 2.2 and 7.3 in the absence and presence of atropine therapy, respectively. In separate experiments, the time course of acetylcholinesterase (AChE) activity recovery was evaluated in the brain and diaphragm of mice pretreated with meptazinol (30 mg kg-1) or physostigmine (0.1 mg kg-1) 15 min before poisoning with DFP (8 mg kg-1). Ten minutes after poisoning, residual AChE activity in the brain averaged 4, 47 and 15% of that in controls in animals pretreated with atropine alone, atropine plus meptazinol or atropine plus physostigmine, respectively. Twenty four hours after poisoning, brain AChE activity averaged 31 and 47% of that in controls in mice protected by meptazinol and physostigmine, respectively. The data from the diaphragm closely paralleled those from the brain. It is concluded that high doses of meptazinol exert antidotal action against acute DFP poisoning in the mouse comparable in efficacy with that of physostigmine combined with atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of meptazinol on evoked responses in rat vas deferens.

Responses of rat isolated vas deferens to electrical stimulation through field electrodes (400 mA, 1 ms duration, single shocks at 5 min intervals) were potentiated by meptazinol (10 to 300 microM) in whole tissues and also in the separated prostatic and epididymal portions. The effect was fast in onset, reproducible and easily reversed by washing. Prazosin (0.1 microM) practically abolished the response of the epididymal portion to electrical stimulation while the response of the prostatic portion was only slightly reduced (less than 20%). In the presence of prazosin, meptazinol still produced potentiation of the response of the prostatic portion. Nifedipine (2 microM) practically abolished the response of the prostatic portion to electrical stimulation while the response of the epididymal portion was only slightly reduced (less than 20%). In the presence of nifedipine, meptazinol no longer produced potentiation of the response of the epididymal portion. Exogenous ATP (5 microM to 1 mM) and phenylephrine (1 to 50 microM) produced a contractile response which was potentiated in the presence of meptazinol (100 microM) but in the presence of meptazinol (100 microM) and nifedipine (5 mM) together, potentiation of phenylephrine no longer occurred. It is suggested that potentiation by meptazinol of electrically induced responses in this tissue is due to a direct action on the smooth muscle.

On the mechanism involved in the ability of meptazinol to potentiate the effects of sympathetic nerve stimulation.

Mouse isolated vas deferens responded to field stimulation (0.1 Hz) with twitch responses which were abolished by alpha beta-methyleneadenosine 5'-triphosphate (0.5 microM) and were potentiated 2 to 3 fold by meptazinol (5-300 microM). Exogenous adenosine 5'-triphosphate (4-30 microM) also caused a twitch response but was unaffected by meptazinol (30 microM) as was the response to phenylephrine. The effect of meptazinol on the electrically-induced twitch was reproducible, fast in onset, easily reversed by washing and was still seen in the presence of prazosin (1 microM), yohimbine (1 microM), propranolol (0.1 microM), atropine (0.1 microM), physostigmine (1 microM), cocaine (1 microM) or desmethylimipramine (0.3 microM) indicating that the mechanism involved does not depend on adrenoceptors, cholinergic mechanisms or blockade of uptake. Mouse isolated atria responded to stimulation (1, 2 or 5 Hz) of their sympathetic nerves via transmural electrodes with chronotropic responses which were abolished by atenolol (5 and 50 microM) but were unaffected by alpha beta-methyleneadenosine 5'-triphosphate (0.5 microM). Meptazinol (100 microM) failed to potentiate the responses. It is suggested that meptazinol potentiates the effects of the non-adrenergic non-cholinergic transmitter thought to be involved in the response of the mouse vas deferens to electrical stimulation.

Quantitative autoradiographic distribution of meptazinol-sensitive binding sites in rat brain.

1. Meptazinol is an interesting opioid-producing naloxone-reversible analgesia with few cardiovascular and respiratory effects. Recent studies indicate that mu 1 opioid receptors mediate meptazinol analgesia. Using a computerized autoradiographic subtraction technique, we have examined the regional distribution of meptazinol-sensitive [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) binding and compared this with the distribution of mu 1 binding determined by competition with low [D-Ala2,D-Leu5]enkephalin (DADL) concentrations. 2. Meptazinol and DADL lowered [3H]DAGO to similar extents in most brain regions studied. The greatest levels of inhibition were observed in the periaqueductal gray, interpeduncular nucleus, thalamus, hypothalamus, and hippocampus. Low levels of inhibition were found in the temporal and frontal cortex. The correlation between the inhibition of [3H]DAGO binding by meptazinol and that by DADL was high (r = 0.83), consistent with the binding of meptazinol to mu 1 sites.

Antagonism of morphine analgesia by CCK-8-S does not extend to all assays nor all opiate analgesics.

The conditions under which CCK-8-S may block opiate-induced analgesia were examined in detail. A U-shaped dose-response relationship was observed for the ability of CCK-8-S to attenuate (by approximately 50%, at most) morphine-induced tail flick analgesia. The analgesic effects of morphine in the hot plate or acetic acid-induced stretching tests were not altered by CCK-8-S at doses that antagonized morphine in the tail flick test. Tail flick latency elevations induced by meptazinol, a putative mu-1 receptor agonist, were also attenuated by CCK-8-S according to a U-shaped dose-response relationship, but those induced by U-50,488, a kappa agonist, were not antagonized by CCK-8-S doses that attenuated morphine analgesia. Thus, the ability of CCK-8-S to antagonize opiate analgesia does not follow a conventional dose-response relationship, does not extend to all tests of analgesia and may not extend to all opioid drugs. Analgesia mediated by the mu-1 opioid receptor subtype may be more amenable to antagonism by CCK-8-S than that mediated by the kappa receptor subtype.

Protection against diisopropylfluorophosphate intoxication by meptazinol.

The protective action of meptazinol against diisopropylfluorophosphate (DFP) was evaluated in mice which were not receiving any other therapy and in preparations of electric eel AChE and horse serum BuChE. Meptazinol injected subcutaneously in mice produced a dose-dependent reduction in the mortality resulting from a LD99.1 (8 mg/kg sc) of DFP administered later. The effectiveness of protection was inversely correlated to the time between meptazinol and DFP administrations. Under these conditions, the ED50s (95% confidence limits) of meptazinol given 15, 30, and 60 min before poisoning were 7.2 (6.4-8.1), 15.8 (13.7-18.2), and 28 (23.5-33.3) mg/kg, respectively, while full protection (100% of survivors) was obtained with 15, 30, and 60 mg/kg drug doses, respectively. Meptazinol was completely ineffective against DFP-induced lethality when administered 3 min after the poison. The protective ratio of 30 mg/kg meptazinol injected 15 min before DFP was 5.0. Pretreatment of mice with 15, 30, and 60 mg/kg meptazinol 15 min before DFP (8 mg/kg) increased brain AChE activity in DFP-treated mice from 5 +/- 0.5% to 16.2 +/- 2.5%, 42.5 +/- 4%, and 81.2 +/- 4% of control values, respectively, while it failed to increase plasma BuChE activity. Finally, concentrations of meptazinol ranging between 0.1 and 10 microM were found to afford complete protection of eel AChE against irreversible inhibition by 40 microM DFP. By contrast, horse serum BuChE was not protected against the same inhibitor by concentrations of meptazinol up to 1 mM. It is concluded that protection against DFP intoxication by meptazinol is most probably due to its protective action toward AChE.