Inhibitor Report for: HuprineX

Huprine X (HX) is a synthetic anticholinesterasic compound that exerts a potent inhibitory action on acetylcholinesterase (AChE) activity, an agonist effect on cholinergic receptors, neuroprotective activity in different neurotoxicity models in vivo and in vitro and cognition enhancing effects in non-transgenic (C57BL/6) and transgenic (3xTg-AD, APPswe) mice. In this study, we assessed the ability of HX (0.8 mg/kg, 21 days) to prevent the damage induced by kainic acid (KA; 28 mg/kg) regarding apoptosis, glia reactivity and neurogenesis in mouse brain. KA administration significantly modified the levels of pAkt1, Bcl2, pGSK3beta, p25/p35, increased the glial cell markers and reduced the neurogenesis process. We also observed that pre-treatment with HX significantly reduced the p25/p35 ratio and increased synaptophysin levels, which suggests a protective effect against apoptosis and an improvement of neuroplasticity. The increase in GFAP (88%) and Iba-1 (72%) induced by KA was totally prevented by HX pre-treatment, underlying a relevant anti-inflammatory action of the anticholinesterasic drug. Our findings highlight the potential of HX, in particular, and of AChEIs, in general, to treat a number of diseases that course with both cognitive deficits and chronic inflammatory processes.

Background: Different studies have established that cholinergic neurodegeneration could be a major pathological feature of Alzheimer's disease (AD). Thus, enhancement of the central cholinergic neurotransmission has been regarded as one of the most promising strategies for the symptomatic treatment of AD, mainly by means of reversible acetylcholinesterase inhibitors (AChEIs). The cognitive-enhancing properties of both huprine X, a new AChEI, and the structurally related huperzine A, as well as their effects on the regulation of several neurochemical processes related to AD have been studied in triple transgenic mice (3xTg-AD). Methods: Seven-month-old homozygous 3xTg-AD male mice, which received chronic intraperitoneal treatment with either saline, huprine X (0.12 micromol.kg(-1)) or huperzine A (0.8 micromol.kg(-1)) were subjected to a battery of behavioural tests after 3 weeks of treatment and thereafter the brains were dissected to study the neurochemical effects induced by the two AChEIs. Results: Treatments with huprine X and huperzine A improved learning and memory in the Morris water maze and some indicators of emotionality without inducing important adverse effects. Moreover, huprine X and huperzine A activate protein kinase C/mitogen-activated protein kinase pathway signalling, alpha-secretases (ADAM 10 and TACE) and increase the fraction of phospho-glycogen synthase kinase 3-beta. Conclusion: Results obtained herein using a sample of 3xTg-AD animals strongly suggest that the treatment with the two AChEIs not only improves the cognitive performance of the animals but also induces some neurochemical changes that could contribute to the beneficial effects observed.

Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.

Huprine X (HX) is a synthetic anticholinesterasic compound that exerts a potent inhibitory action on acetylcholinesterase (AChE) activity, an agonist effect on cholinergic receptors, neuroprotective activity in different neurotoxicity models in vivo and in vitro and cognition enhancing effects in non-transgenic (C57BL/6) and transgenic (3xTg-AD, APPswe) mice. In this study, we assessed the ability of HX (0.8 mg/kg, 21 days) to prevent the damage induced by kainic acid (KA; 28 mg/kg) regarding apoptosis, glia reactivity and neurogenesis in mouse brain. KA administration significantly modified the levels of pAkt1, Bcl2, pGSK3beta, p25/p35, increased the glial cell markers and reduced the neurogenesis process. We also observed that pre-treatment with HX significantly reduced the p25/p35 ratio and increased synaptophysin levels, which suggests a protective effect against apoptosis and an improvement of neuroplasticity. The increase in GFAP (88%) and Iba-1 (72%) induced by KA was totally prevented by HX pre-treatment, underlying a relevant anti-inflammatory action of the anticholinesterasic drug. Our findings highlight the potential of HX, in particular, and of AChEIs, in general, to treat a number of diseases that course with both cognitive deficits and chronic inflammatory processes.

Background: Different studies have established that cholinergic neurodegeneration could be a major pathological feature of Alzheimer's disease (AD). Thus, enhancement of the central cholinergic neurotransmission has been regarded as one of the most promising strategies for the symptomatic treatment of AD, mainly by means of reversible acetylcholinesterase inhibitors (AChEIs). The cognitive-enhancing properties of both huprine X, a new AChEI, and the structurally related huperzine A, as well as their effects on the regulation of several neurochemical processes related to AD have been studied in triple transgenic mice (3xTg-AD). Methods: Seven-month-old homozygous 3xTg-AD male mice, which received chronic intraperitoneal treatment with either saline, huprine X (0.12 micromol.kg(-1)) or huperzine A (0.8 micromol.kg(-1)) were subjected to a battery of behavioural tests after 3 weeks of treatment and thereafter the brains were dissected to study the neurochemical effects induced by the two AChEIs. Results: Treatments with huprine X and huperzine A improved learning and memory in the Morris water maze and some indicators of emotionality without inducing important adverse effects. Moreover, huprine X and huperzine A activate protein kinase C/mitogen-activated protein kinase pathway signalling, alpha-secretases (ADAM 10 and TACE) and increase the fraction of phospho-glycogen synthase kinase 3-beta. Conclusion: Results obtained herein using a sample of 3xTg-AD animals strongly suggest that the treatment with the two AChEIs not only improves the cognitive performance of the animals but also induces some neurochemical changes that could contribute to the beneficial effects observed.

BACKGROUND: Several studies implicate acetylcholinesterase (AChE) in the pathogenesis of Alzheimer's disease (AD), raising the question of whether inhibitors of AChE also might act in a disease-modifying manner. Huprine X (HX), a reversible AChE inhibitor hybrid of tacrine and huperzine A, has shown to affect the amyloidogenic process in vitro. In this study, the aim was to investigate whether HX could affect the AD-related neuropathology in vivo in two mouse models. METHODS: Tg2576 (K670M/N671L) (APPswe) and 3xTg-AD (K670M/N671L, PS1M146V, tauP301L) mice were treated with HX (0.12 mumol/kg, i.p., 21 days) or saline at 6-7 months. Human beta-amyloid (Abeta) was measured by ELISA, synaptophysin by Western blot and alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) were analyzed by [(125)I]alpha-bungarotoxin autoradiography. RESULTS: Treatment with HX reduced insoluble Abeta1-40 (about 40%) in the hippocampus of 3xTg-AD mice, while showing no effect in APPswe mice. Additionally, HX markedly increased cortical synaptophysin levels (about 140%) and decreased (about 30%) the levels of alpha7 nAChRs in the caudate nucleus of 3xTg-AD mice, while increasing (about 10%) hippocampal alpha7 nAChRs in APPswe mice. CONCLUSION: The two mouse models react differently to HX treatment, possibly due to their differences in brain neuropathology. The modulation of Abeta and synaptophysin by HX in 3xTg-AD mice might be due to its suggested interaction with the peripheral anionic site on AChE, and/or via cholinergic mechanisms involving activation of cholinergic receptors. Our results provide further evidence that drugs targeting AChE affect some of the fundamental processes that contribute to neurodegeneration, but whether HX might act in a disease-modifying manner in AD patients remains to be proven.

The main goal of the present study was to analyse the effects of (+/-)-huprine X ((+/-)-HX) and galantamine (GAL), with potentiating action on nicotinic receptors, and huperzine A (HPA), devoid of nicotinic activity, on [3H]-acetylcholine ([3H]-ACh) release in striatal slices of rat brain. All compounds are non-covalent and reversible inhibitors of AChE. Addition of (+/-)-HX (0.01 microM), GAL (10 microM) and HPA (0.1 microM) to the superfusion medium decreased the release of the ACh neurotransmitter to a similar extent: 36%, 30% and 34%, respectively (P<0.01). This effect was reverted in the presence of atropine (ATR; 0.1 microM), which blocks the pre-synaptic muscarinic M2 receptor. After that, a wide range of concentrations of drugs, concomitantly with ATR (0.1 microM), was studied in the presence of haloperidol (HAL; 0.01 microM), a dopamine D2 antagonist. In these conditions, a dose-dependent increase of [3H]-ACh release was observed in the presence of (+/-)-HX, GAL and HPA. To test the role of nicotinic receptors in the drugs' effects on [3H]-ACh release, mecamylamine (MEC) 100 microM was used to block such receptors. MEC alone significantly decreased neurotransmitter release by 18% (P<0.05), but no change was obtained in the presence of both ATR and MEC. Under these conditions, (+/-)-HX, GAL and HPA increased the release of [3H]-ACh by 37%, 25% and 38%, respectively (P<0.01). Taking into account all of these data, the present results suggest that the effects induced by (+/-)-HX and GAL nicotinic-receptor potentiators seem to be mainly due to their ability in inhibiting acetylcholinesterase activity, but not by interaction on the nicotinic receptors.

The present experiments were developed to analyze the direct and/or potentiation effect of (+/-)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quin oline hydrochloride ((+/-)huprine X) on nicotinic receptors using a synaptosomal superfusion method. (+/-)Huprine X (1 microM, 10 microM) increased [(3)H]-ACh release only at 10 microM (46%; P < 0.001) in basal, but not in stimulated, conditions. This effect was completely reverted by mecamylamine (100 microM; MEC). Potentiation of evoked-[3H]-ACh release induced by ACh (1 microM) and by galantamine (GAL) 0.4 microM and physostigmine (PHY) 10 microM (55% and 50%, respectively; P < 0.001), two well-known allosteric compounds, corroborate that the present experimental approach is a suitable method to study potentiation effects on nicotinic receptors in the central nervous system nerve terminals. (+/-)Huprine X potentiated the evoked-[3H]-ACh release induced by ACh (1 microM) by 166% and 90% (P < 0.001) at 10 microM and 30 microM, respectively, and this effect was completely blocked by MEC (100 microM). In the presence of different ACh concentrations, (+/-)huprine X 10 microM potentiated evoked-[3H]-ACh release at low ACh concentrations, while a decrease in neurotransmitter release was observed at high ACh concentrations. The highest potentiation effect was obtained at the ACh/(+/-)huprine X concentration ratio of 1:10, and this potentiation was observed at as low a (+/-)huprine X concentration as 0.1 microM (P < 0.05). While the results suggest that huprine may enhance the potency or effectiveness of ACh by an effect involving nicotinic receptors we cannot completely discard that the results could be explained by acetylcholine esterase inhibition.

Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.

The interaction of rac-12-amine-3-clor-6,7,10,11-tetrahydro-9-ethyl-7-11-methanecyclo-octane[b]quino line ((+/-)huprine X) with M(1) and M(2) receptors has been studied in rat brain. Specific binding of [(3)H]pirenzepine or [(3)H]quinuclinidylbenzylate to hippocampus preparations was inhibited by (+/-)huprine X. This drug displayed a greater affinity for M(1) (K(i)=0.338+/-0.41 microM) than M(2) (K(i)=4.66+/-0.32 microM) receptors. In functional studies, (+/-)huprine X (1 microM) increased the release of [(3)H]dopamine in cortical synaptosomes, and this effect was partially reverted by atropine and mecamylamine, suggesting an agonistic effect on both M(1) and nicotinic receptors. The inhibitory effect of (+/-)huprine X (10 microM) on [(3)H]acetylcholine release and the subsequent reversion by atropine suggests that the drug also has an agonist effect on M(2) receptors. The present results demonstrate that this acetylcholinesterase inhibitor has an ample cholinergic profile, which suggests a potential source of interest of (+/-)huprine X in Alzheimer's disease therapy.

The effects of the tacrine-huperzine A hybrid acetylcholinesterase inhibitors, (+/-)-12-amino-3-chloro-9-methyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride ((+/-)-huprine Y) and (+/-)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride ((+/-)-huprine X), were tested on spontaneous synaptic activity by measuring the amplitude, the rise time, the rate of rise, the half-width and the area or the electrical charge of the miniature endplate potentials (m.e.p.ps) recorded extracellularly on Torpedo electric organ fragments. (+/-)-Huprine Y and (+/-)-huprine X at a concentration of 500 nM increased all the m.e.p.p. variables analyzed. The effect of (+/-)-huprine Y was smaller than that of (+/-)-huprine X for all the variables except for the rate of rise where there was no significant difference. The effects of these drugs were also tested on nicotinic receptors by analyzing the currents elicited by acetylcholine (100 microM) in Xenopus laevis oocytes, transplanted with membranes from Torpedo electric organ. Both drugs inhibited the currents in a reversible manner, (+/-)-huprine Y (IC(50)=452 nM) being more effective than (+/-)-huprine X (IC(50)=4865 nM). The Hill coefficient was 0.5 for both drugs. The inhibition of the nicotinic receptor was voltage-dependent and decreased at depolarizing potentials, and there was no significant difference in the effects between (+/-)-huprine Y and (+/-)-huprine X at concentrations near to their IC(50) values. At depolarizing potentials between -20 and +15 mV, these drugs did not have any detectable effect on the blockade of the nicotinic receptor. Both huprines increased the desensitization of the nicotinic receptors since the current closed quickly in the presence of the drugs, and there was no significant difference in this effect between (+/-)-huprine Y (500 nM) and (+/-)-huprine X (5 microM). We conclude that (+/-)-huprine Y and (+/-)-huprine X increase the level of acetylcholine in the synaptic cleft more effectively than tacrine. The interaction of (+/-)-huprine X with nicotinic receptors is weaker than that of (+/-)-huprine Y, suggesting that (+/-)-huprine X would be more specific to maintain the extracellular acetylcholine concentration.

Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.