M.V. Clos

Huprine X and huperzine A improve cognition and regulate some neurochemical processes related with Alzheimer's disease in triple transgenic mice (3xTg-AD).

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 µmol·kg–1) or huperzine A (0.8 µmol·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, α-secretases (ADAM 10 and TACE) and increase the fraction of phospho-glycogen synthase kinase 3-β. 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.

Interaction of a new potent anticholinesterasic compound (+/-)huprine X with muscarinic receptors in rat brain.

The interaction of rac-12-amine-3-clor-6,7,10,11-tetrahydro-9-ethyl-7-11-methanecyclo-octane[b]quinoline ((+/-)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 Alzheimers disease therapy.

Potentiation effects of (±)huprine X, a new acetylcholinesterase inhibitor, on nicotinic receptors in rat cortical synaptosomes

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]quinoline 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.

Presynaptic effect of 7-OH-DPAT on evoked [3H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D(2)-like dopamine receptor agonist) on [3H]-acetylcholine ([3H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D(2) dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 microM) inhibited the evoked [3H]-ACh release induced by K(+) 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K(+) the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1-100 microM) and TEA (1 and 5 mM) significantly enhanced [3H]-ACh release, showing 69.32%+/-7.60% (P<0.001) and 52.27%+/-5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 microM) inhibited the release induced by potassium channel-blockers approximately 25-27%. Quinine (0.1-1 microM) did not alter [3H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 microM evoked [3H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 microM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D(2) dopamine receptor inhibits the [3H]-ACh release by increasing K(+) conductance, as high K(+) concentrations abolished the inhibitory control of 7-OH-DPAT on [3H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D(2) dopamine-receptor activation seems to not involve changes in intracellular Ca(2+).

Role of calcium on the modulation of spontaneous acetylcholine efflux by the D2 dopamine receptor subtype in rat striatal synaptosomes

The role of calcium in the modulation of spontaneous [3H]acetylcholine ([3H]ACh) efflux through presynaptic D2 dopamine hetero-receptors was investigated in rat striatal synaptosomes. The kinetic studies of [3H]ACh efflux in the presence or absence of Ca2+ were carried out in nonstimulating conditions. When Ca2+ was omitted from the superfusion medium, a notable and significant (P<0.001) decrease of tritium efflux (39%) was obtained. While [3H]ACh efflux was insensitive to tetrodotoxin (TTX) 1 microM, cadmium (10 microM), a nonselective antagonist of calcium channels, significantly reduced the tritium efflux by 24% (P<0.001), while the L-type calcium antagonist, nifedipine, (30 microM) inhibited the tritium efflux by only 10% (P<0.02). 2-(4-Fenylpiperidine)cyclohexanol (vesamicol), an inhibitor of the vesicular [3H]ACh carrier, significantly depressed the spontaneous tritium efflux in the presence of Ca2+ (60%; P<0.001) and in a low-calcium medium (20%; P<0.001). Although 1 microM of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT) inhibited spontaneous [3H]ACh efflux in the presence of calcium, this dopaminergic agonist did not modify the neurotransmitter release in either the low-Ca2+ medium or in the presence of vesamicol. These results suggest that the spontaneous [3H]ACh efflux is a process involving a Ca2+-dependent component (39%), sensitive to calcium channel-blockers and vesamicol, in rat striatal synaptosomes. In addition, activation of the D2 dopamine hetero-receptor only modulates the calcium-dependent component of spontaneous [3H]ACh efflux.

Effects of (±)-huprine Y and (±)-huprine Z, two new anticholinesterasic drugs, on muscarinic receptors

The cholinergic profile of (+/-)-huprine Y and (+/-)-huprine Z on muscarinic receptors has been determined. Displacement of [3H]-pirenzepine and [3H]-QNB plus pirenzepine was performed in rat hippocampus. Both compounds showed a higher degree of affinity to M1 muscarinic receptors (P < 0.01) than to M2 muscarinic receptors. To determine the M1 agonist or antagonist role of the two huprines, studies of inositol phosphates (IP) production were performed. Both huprines significantly stimulated IP accumulation in a concentration-dependent manner. The reversion of this effect by different antagonists showed that M1 muscarinic receptors were activated by (+/-)-huprine Y and (+/-)-huprine Z, but some other mechanisms, such as alpha1-adrenoceptors or nicotinic receptors, were involved.

Synthesis, biological profiling and mechanistic studies of 4-aminoquinoline-based heterodimeric compounds with dual trypanocidal-antiplasmodial activity.

Dual submicromolar trypanocidal-antiplasmodial compounds have been identified by screening and chemical synthesis of 4-aminoquinoline-based heterodimeric compounds of three different structural classes. In Trypanosoma brucei, inhibition of the enzyme trypanothione reductase seems to be involved in the potent trypanocidal activity of these heterodimers, although it is probably not the main biological target. Regarding antiplasmodial activity, the heterodimers seem to share the mode of action of the antimalarial drug chloroquine, which involves inhibition of the haem detoxification process. Interestingly, all of these heterodimers display good brain permeabilities, thereby being potentially useful for late stage human African trypanosomiasis. Future optimization of these compounds should focus mainly on decreasing cytotoxicity and acetylcholinesterase inhibitory activity.

Effect of acetylcholinesterase inhibitors on AChE-induced PrP106-126 aggregation.

Transmissible spongiform encephalopaties are caused by an extracellular surface protein, the scrapie prion protein (PrPsc), which is an aberrant form of normal and functional cellular PrP (PrPc). The pathological hallmarks of these diseases are the accumulation and deposition of PrPsc in the form of amyloid fibrils in the central nervous system (Tateishi et al., 1988), similar to amyloid-beta (Abeta) protein in Alzheimers disease (AD). In some patients, Abeta and prion pathology can coexist (Hainfellner et al., 1998), and a common spatial pattern of protein deposition has been described (Armstrong et al., 2001). In addition, it is well-known that acetylcholinesterase (AChE) colocalizes with Abeta deposits of brains in AD patients and accelerates assembly of Abeta peptides through the peripheral site of the enzyme (Inestrosa et al., 1996). The aim of the present study was to analyze time course and concentration dependence of the AChE proaggregating effect on synthetic peptide-spanning residues 106-126 of human PrP (PrP106-126) and the reversion of this effect by different AChE inhibitors (AChEIs).

Expanding the multipotent profile of huprine-tacrine heterodimers as disease-modifying anti-Alzheimer agents.

Background: Multifactorial diseases such as Alzheimer’s disease (AD) should be more efficiently tackled by drugs which hit multiple biological targets involved in their pathogenesis. We have recently developed a new family of huprine-tacrine heterodimers, rationally designed to hit multiple targets involved upstream and downstream in the neurotoxic cascade of AD, namely β-amyloid aggregation and formation as well as acetylcholinesterase catalytic activity. Objective: In this study, the aim was to expand the pharmacological profiling of huprine-tacrine heterodimers investigating their effect on muscarinic M1 receptors as well as their neuroprotective effects against an oxidative insult. Methods: Sprague-Dawley rat hippocampus homogenates were used to assess the specific binding of two selected compounds in competition with 1 nM [3H]pirenzepine (for M1 receptors) or 0.8 nM [3H]quinuclidinyl benzilate (for M2 receptors). For neuroprotection studies, SHSY5Y cell cultures were subjected to 250 µM hydrogen peroxide insult with or without preincubation with some huprine-tacrine heterodimers. Results: A low nanomolar affinity and M1/M2 selectivity has been found for the selected compounds. Huprine-tacrine heterodimers are not neurotoxic to SHSY5Y cells at a range of concentrations from 1 to 0.001 µM, and some of them can protect cells from the oxidative damage produced by hydrogen peroxide at concentrations as low as 0.001 µM. Conclusion: Even though it remains to be determined if these compounds act as agonists at M1 receptors, as it is the case of the parent huprine Y, their low nanomolar M1 affinity and neuroprotective effects expand their multitarget profile and increase their interest as disease-modifying anti-Alzheimer agents.

Neuroprotective Effects of the Multitarget Agent AVCRI104P3 in Brain of Middle-Aged Mice

Molecular factors involved in neuroprotection are key in the design of novel multitarget drugs in aging and neurodegeneration. AVCRI104P3 is a huprine derivative that exhibits potent inhibitory effects on human AChE, BuChE, and BACE-1 activities, as well as on AChE-induced and self-induced Aβ aggregation. More recently, cognitive protection and anxiolytic-like effects have also been reported in mice treated with this compound. Now, we have assessed the ability of AVCRI104P3 (0.43 mg/kg, 21 days) to modulate the levels of some proteins involved in the anti-apoptotic/apoptotic processes (pAkt1, Bcl2, pGSK3β, p25/p35), inflammation (GFAP and Iba1) and neurogenesis in C57BL/6 mice. The effects of AVCRI104P3 on AChE-R/AChE-S isoforms have been also determined. We have observed that chronic treatment of C57BL/6 male mice with AVCRI104P3 results in neuroprotective effects, increasing significantly the levels of pAkt1 and pGSK3β in the hippocampus and Bcl2 in both hippocampus and cortex, but slightly decreasing synaptophysin levels. Astrogliosis and neurogenic markers GFAP and DCX remained unchanged after AVCRI104P3 treatment, whereas microgliosis was found to be significantly decreased pointing out the involvement of this compound in inflammatory processes. These results suggest that the neuroprotective mechanisms that are behind the cognitive and anxiolytic effects of AVCRI104P3 could be partly related to the potentiation of some anti-apoptotic and anti-inflammatory proteins and support the potential of AVCRI104P3 for the treatment of brain dysfunction associated with aging and/or dementia.