Nuria M. Vivas

Assessing the (a)symmetry of concentration-effect curves: empirical versus mechanistic models

Modeling the shape of concentration-effect curves is of prime importance in pharmacology. Geometric descriptors characterizing these curves (the upper and lower asymptotes, the mid-point, the mid-point slope, and the point of inflection) are used for drug comparison or for assessing the change in agonist function after a system modification. The symmetry or asymmetry around the mid-point of a concentration-effect curve is a fundamental property that, regretfully, is often overlooked because, generally, models yielding exclusively symmetric curves are used. In the present review, empirical and mechanistic models are examined in their ability to fit experimental data. The geometric parameters of a survey of empirical models, the Hill equation, a logistic variant that we call the modified Hill equation, the Richards function, and the Gompertz model are determined. To analyze the relationship between asymmetry and mechanism, some examples from the ionic channel field, in an increasing degree of complexity, are used. It is shown that asymmetry arises from ionic channels with multiple binding sites that are partly occupied. The operational model of agonism is discussed both in its empirical general formulation and including the signal transduction mechanisms through G-protein-coupled receptors. It is shown that asymmetry results from systems where receptor distribution is allowed. Developed mathematical models are compared for describing experimental data on alpha-adrenoceptors. The existence or not of a relationship between the shape of the curves and receptor reserve is discussed.

Synthesis and evaluation of tacrine–Huperzine a hybrids as acetylcholinesterase inhibitors of potential interest for the treatment of alzheimer’s disease

Seventeen polycyclic compounds related to tacrine and huperzine A have been prepared as racemic mixtures and tested as acetylcholinesterase (AChE) inhibitors. The conjunctive pharmacomodulation of huperzine A (carbobicyclic substructure) and tacrine (4-aminoquinoline substructure) led to compound 7jy, 2.5 times less active than tacrine as AChE inhibitor, but much more active than its (Z)-stereoisomer (7iy). Derivatives 7dy and 7ey, lacking the ethylidene substituent, showed to be more active than tacrine. Many other structural modifications of 7jy led to less active compounds. Compounds 7dy and 7ey also showed to be much more active than tacrine in reversing the partial neuromuscular blockade induced by d-tubocurarine.

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues

The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.

Synthesis and evaluation of tacrine-related compounds for the treatment of Alzheimer's disease

Abstract A number of polycyclic compounds related to tacrine have been prepared by condensation of ortho-aminobenzonitriles and 2-aminocyclopentenecarbonitrile with several C2v-symmetric diketones under AlCl3 or ZnCl2 catalysis. Monocondensation products 8 together with syn- and anti-dicondensation products 9 and 10, respectively, were formed in different proportions depending mainly on the starting diketone. These compounds were separated by column chromatography, fully characterized by spectroscopic and elemental analyses and tested as acetylcholinesterase (AchE) inhibitors. Syn- and anti-compounds 9 and 10, derived from diketones 7y and 7z, have significant anti-AchE activity although compounds 8 and derivatives of diketones 7v, 7w and 7x were inactive in the range of concentrations studied. Compound 9ay was the most potent of the group, being 4.4-fold less active than tacrine as anti-AchE in biochemical assays, but only slightly less potent in biological studies and 3-fold less toxic. Compound 9ay was also able to reverse cognitive deficits in middle-aged rats.

Characterisation of the anticholinesterase activity of two new tacrine-huperzine A hybrids

The effects of two tacrine-huperzine A hybrids, (+/-)-huprine Y and (+/-)-huprine Z, have been evaluated. Bovine and human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) inhibition were assayed by Ellmans method. The two huprines were more active than both tacrine and (-)-huperzine A as inhibitors of both human and bovine AChE, and they acted as mixed-type AChE inhibitors. Moreover, (+/-)-huprine Y exhibited a tight binding character seen in the experiments of reversibility of bovine AChE inhibitory activity. In addition, both compounds were more active toward AChE than toward BChE. Also, the selectivity for the human AChE was greater than for the bovine enzyme. In ex vivo studies performed in mice, both drugs showed a clear inhibitory activity of brain AChE, 20 min after i.p. injection, (+/-)-huprine Y being more potent than (+/-)-huprine Z [ID(50) 1.09 (0.39-2.98) vs. 5.77 (3.29-10.30) micromol/kg]. The time-course study of the inhibitory effect displayed a t(1/2) of 1 h for the two compounds. These results show that these drugs are two potent, central AChE inhibitors of potential interest in the treatment of AD.

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of 4-amino-2, 3-diaryl-5, 6, 7, 8-tetrahydrofuro(and thieno)[2, 3-b]-quinolines, and 4-amino-5, 6, 7, 8, 9-pentahydro-2, 3-diphenylcyclohepta[e]furo(and thieno)-[2, 3-b]pyridines.

The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities of a series of 4‐amino‐2, 3‐diaryl‐5, 6, 7, 8‐tetrahydrofuro[2, 3‐b]quinolines (10—12)/4‐amino‐5, 6, 7, 8‐tetrahydro‐2, 3‐diphenylthieno[2, 3‐b]quinoline (14) and 4‐amino‐5, 6, 7, 8, 9‐pentahydro‐2, 3‐diphenylcyclohepta[e]furo[2, 3‐b]pyridine (13)/4‐amino‐5, 6, 7, 8, 9‐pentahydro‐2, 3‐phenylcyclohepta[e]thieno[2, 3‐b]pyridine (15) are described. These compounds are tacrine (THA) analogues which have been prepared either from readily available 2‐amino‐3‐cyano‐4, 5‐diarylfurans (16—18) or from 2‐amino‐3‐cyano‐4, 5‐diphenylthiophene (19), via Friedländer condensation with cyclohexanone or cycloheptanone. These compounds are competitive inhibitors for acetylcholinesterase, the more potent being compound (13) which is three‐fold less active than tacrine. The butyrylcholinesterase inhibition activity is significant only in compounds 10 and133, which are ten‐fold less active than tacrine. It is found that the products 11 and 12 strongly inhibit acetylcholinesterase, and show excellent selectivity regarding butyrylcholinesterase.

α1‐Adrenoceptor vasoconstriction in the tail artery during ageing

We have studied the α1‐adrenoceptor‐mediated responses in intact tail artery rings from 3–4 and 20–22 months old Sprague‐Dawley rats, focusing on possible endothelial alterations. The influence of nitric oxide released by the endothelium, the number of α1‐adrenoceptors and the functional receptor reserve were evaluated to determine their contribution to the contractile response mediated by this receptor. The state of the endothelial layer was assessed by confocal microscopy. Noradrenaline (1 nM–100 μM) induced concentration‐dependent vasoconstriction. The maximum contractions to noradrenaline (P<0.05) and to 75 mM KCl (P<0.01) were higher in young than in old animals. The density (Bmax) of α1‐adrenoceptors and the dissociation constant (KD) obtained in [3H]‐prazosin binding experiments were unchanged by age. The apparent affinity (pKA) and the percentage of functional receptors (qx100) remaining after phenoxybenzamine (0.03 μM) were similar in both age groups. After partial α1‐adrenoceptor inactivation with phenoxybenzamine, NG‐nitro‐L‐arginine methylester (30 μM) significantly potentiated the E/[A] curve to noradrenaline in young rats. However, only responses to 0.1 to 1 μM noradrenaline were significantly potentiated in old animals. In addition, 94% of the vessels from young, but only 52% from old rats were relaxed by 80–100% of the noradrenaline (0.03 μM) contraction, with 1 μM acetylcholine. No modifications in the area (μm2) or in the number of endothelial nuclei (per mm2) were observed between age groups. An elongation of the nuclei of endothelial cells was observed in the old animals. These data suggest that the noradrenaline‐induced contraction is decreased in old rats probably due to differences in either the contractile machinery or postreceptor mechanisms. These alterations may be accompanied by an impairment of the release or production of NO from endothelial cells.

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.

D2 dopamine receptors and modulation of spontaneous acetylcholine (ACh) release from rat striatal synaptosomes

1 The effect of two D3/2 dopamine receptor agonists, LY‐171555 (quinpirole) and 7‐hydroxy‐N,N‐di‐n‐propyl‐2‐aminotetralin (7‐OH‐DPAT) on spontaneous [3H]‐acetylcholine ([3H]‐ACh) release were investigated in rat striatal synaptosomes. 2 Quinpirole and 7‐OH‐DPAT inhibited in a concentration‐dependent manner the basal efflux of [3H]‐ACh with similar Emax (maximal inhibitory effect) values (29.95±2.91% and 33.19±1.21%, respectively). Significant differences were obtained between the pEC50 (−log of molar concentration) of quinpirole (7.87±0.12) and 7‐OH‐DPAT (7.21±0.17; P<0.01). 3 Different concentrations (0.3–10 nM) of haloperidol (D2/3 dopamine receptor antagonist) shifted to the right the concentration‐response curves elicited by quinpirole and 7‐OH‐DPAT, without modifications in the Emax. 4 Slopes of a Schild plot obtained with haloperidol in the presence of quinpirole and 7‐OH‐DPAT were not signficantly different from unity (0.85±0.05 and 1.17±0.11, respectively) and consequently haloperidol interacted with a homogeneous receptor population. The pKB values of haloperidol obtained from Schild regression were 9.96±0.15 (in presence of quinpirole) and 9.90±0.09 (in presence of 7‐OH‐DPAT). 5 Specific binding of [3H]‐YM‐09151‐2 to membranes of striatal synaptosomes and cells expressing D2 and D3 dopamine receptors was inhibited by haloperidol. Analysis of competition curves revealed the existence of a single population of receptors. There were no differences between the estimated pKi (−log of molar concentration) values for synaptosomes (8.96±0.02) and cells expressing D2 receptors (8.81±0.05), but the pKi value from cells expressing D3 dopamine receptors differed significantly (8.48±0.06; P<0.01). 6 In conclusion, the data obtained in the present study indicate that quinpirole and 7‐OH‐DPAT, two D3/2 dopamine receptor agonists, inhibit the spontaneous [3H]‐ACh efflux and this effect is competitively antagonized by haloperidol and probably mediated through dopamine D2 receptors.

Endothelial modulation of α1-adrenoceptor contractile responses in the tail artery of spontaneously hypertensive rats

1 Vascular contraction induced by phenylephrine was studied in tail artery rings from spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY) with particular focus on the role of endothelium. The influence of receptor reserve and the density of α1‐adrenoceptors on the possible differences observed were also analysed. 2 Phenylephrine (0.01–100 μm) induced concentration‐dependent vasoconstrictions. The maximum response (α, P < 0.001) was greater but the pEC50 (P < 0.05) smaller in rings from SHR than from WKY rats irrespective of the presence or absence of endothelium. 3 Removal of endothelial cells resulted in a decrease of the maximum contraction with no modification in the pEC50 in arteries from both WKY and SHR. 4 The density of α1‐adrenoceptors (Bmax) and the dissociation constant (KD) were found to be the same for preparations from SHR and WKY rats in [3H]‐prazosin binding experiments. 5 The apparent affinity (pKA) determined by the nested hyperbolic method and the operational model was similar in tail arteries from the two rat strains, irrespective of the presence or absence of endothelium. However, in endothelium‐denuded rings, the pKA value was enhanced when compared with intact rings, in both SHR and WKY rats. 6 In rings from hypertensive rats, the operational parameter maximum possible effect (Em) was greater and the agonist efficacy (τ) was smaller than in rings from normotensive rats. When the endothelium was removed log τ and Em diminished in preparations from both rat strains. 7 In summary, the increased maximum responsiveness to phenylephrine in rings from SHR could be due to enhancement in Em. The log τ values indicate a deterioration in the transduction of the stimulus provided by the agonist in tail arteries from hypertensive animals. This study also suggests that the absence of endothelium modifies the α1‐adrenoceptor‐mediated vasocontriction probably by altering the transduction signalling mechanisms. The importance of analysing the degree of endothelium functionality when comparing results from different groups of rats is stated.