Identification of harmful drinking in subjects who have had their driving license suspended due to alcohol use: a retrospective Italian study.

OBJECTIVE: Early identification of Harmful Drinking (HD) is difficult, and underestimated. The aim of our retrospective study was to investigate the presence of HD in a population of subjects who had their driving license suspended due to driving under the influence of alcohol. MATERIALS AND METHODS: We retrospectively recruited 979 subjects. During the first appointment (T0), clinical and laboratory characteristics of patients were evaluated, and the AUDIT questionnaire was administered. Two groups were then defined: Harmful Drinking (HD) and non-HD, and all subjects underwent a brief interview for 5-10 minutes before being assigned to a group. RESULTS: 95.9% of our sample were identified as non-HD, whereas 4.1% of them were HD; twenty-one (2.1%) of the HD underwent a control appointment (T1), and 17 (1.7%) of them were diagnosed with alcohol use disorder (AUD); there was a statistically significant reduction in mean daily alcohol intake (p<0.009), and in the mean values of the blood markers of HD between T0 and T1 in HD. CONCLUSIONS: The present study shows that 4.1%, and 1.7% of subjects presented a diagnosis of HD and AUD, respectively, and their entry in a protocol of drinking monitoring proved beneficial in reducing alcohol intake. Thus, the implementation of strict surveillance of subjects found driving under the influence of alcohol involving a network of professional figures (from police forces to specialists in alcohol addiction treatment) may help to detect and to treat subjects with HD and AUD, and to monitor their alcohol use over time.

Cystamine-tacrine dimer: a new multi-target-directed ligand as potential therapeutic agent for Alzheimer's disease treatment.

Alzheimer's disease (AD) is the most common cause of dementia, clinically characterized by loss of memory and progressive deficits in different cognitive domains. An emerging disease-modifying approach to face the multifactorial nature of AD may be represented by the development of Multi-Target Directed Ligands (MTDLs), i.e., single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. The structure of tacrine, an acetylcholinesterase (AChE) inhibitor (AChEI), has been widely used as scaffold to provide new MTDLs. In particular, its homodimer bis(7)tacrine represents an interesting lead compound to design novel MTDLs. Thus, in the search of new rationally designed MTDLs against AD, we replaced the heptamethylene linker of bis(7)tacrine with the structure of cystamine, leading to cystamine-tacrine dimer. In this study we demonstrated that the cystamine-tacrine dimer is endowed with a lower toxicity in comparison to bis(7)tacrine, it is able to inhibit AChE, butyrylcholinesterase (BChE), self- and AChE-induced beta-amyloid aggregation in the same range of the reference compound and exerts a neuroprotective action on SH-SY5Y cell line against H(2)O(2)-induced oxidative injury. The investigation of the mechanism of neuroprotection showed that the cystamine-tacrine dimer acts by activating kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Tacrine derivatives and Alzheimer's disease.

To date, the pharmacotherapy of Alzheimer's disease (AD) has relied on acetylcholinesterase (AChE) inhibitors (AChEIs) and, more recently, an N-methyl-D-aspartate receptor (NMDAR) antagonist. AD is a multifactorial syndrome with several target proteins contributing to its etiology. "Multi-target-directed ligands" (MTDLs) have great potential for treating complex diseases such as AD because they can interact with multiple targets. The design of compounds that can hit more than one specific AD target thus represents an innovative strategy for AD treatment. Tacrine was the first AChEI introduced in therapy. Recent studies have demonstrated its ability to interact with different AD targets. Furthermore, numerous tacrine homo- and heterodimers have been developed with the aim of improving and enlarging its biological profile beyond its ability to act as an AChEI. Several tacrine hybrid derivatives have been designed and synthesized with the same goal. This review will focus on and summarize the last two years of research into the development of tacrine derivatives able to hit AD targets beyond simple AChE inhibition.

MTDL design strategy in the context of Alzheimer's disease: from lipocrine to memoquin and beyond.

The multifunctional nature of Alzheimer's disease (AD) provides the logical foundation for the development of an innovative drug design strategy centered on multi-target-directed-ligands (MTDLs). In recent years, the MTDL concept has been exploited to design different ligands hitting different biological targets. Our first rationally designed MTDL was the polyamine caproctamine (1), which provided a synergistic cholinergic action against AD by antagonizing muscarinic M(2) autoreceptors and inhibiting acetylcholinesterase (AChE). Lipocrine (7) represented the next step in our research. Due to its ability to inhibit AChE catalytic and non-catalytic functions together with oxidative stress, 7 emerged as an interesting pharmacological tool for investigating the neurodegenerative mechanism underlying AD. Memoquin (9) is a quinone-bearing polyamine endowed with a unique multifunctional profile. With its development, we arrived at the proof of concept of the MTDL drug discovery approach. Experiments in vitro and in vivo confirmed its multimodal mechanisms of action and its interaction with different end-points of the neurotoxic cascade leading to AD. More recently, the MTDL approach led to carbacrine (12). In addition to the multiple activities displayed by 7, 12 displayed an interesting modulation of NMDA receptor activity. The pivotal role played by this target in AD pathogenesis suggests that 12 may be a promising new chemical entity in the MTDL gold rush.

LC determination of leuprolide component amino acids in injectable solution by phanquinone pre-column derivatization labelling procedure.

A sensitive LC method for the determination of leuprolide acetate component amino acids in injectable solution with fluorogenic pre-column derivatization has been developed. The derivatization reaction with phanquinone was optimised by a series of experiments. Histidine, arginine, serine, tryptophan, glutamic acid, tyrosine, methionine, isoleucine, leucine and phenylalanine were separated on a reversed-phase ODS column using as eluent a binary mixture of triethylammonium phosphate buffer-methanol, under gradient elution conditions. The derivatives were eluted in 30 min with good reproducibility. The hydrolysis reaction of the peptide was carried out at reflux with 12 N hydrochloric acid for 2 h 30 min. The intra-day accuracy of the entire procedure (hydrolysis, derivatization, LC separation) ranged from 80.5 to 109.5% of the nominal concentration of leuprolide acetate and the precision (%R.S.D.) was less than 5.8%; the inter-day accuracy was in the range 81.5-107.2% and corresponding R.S.D. values were less than 4.6%. The detection limits (signal-to-noise ratio=3) for the adducts are 30-800 fmol.

Design, synthesis, and biological activity of methoctramine-related polyamines as putative G(i) protein activators.

The universal template approach provided a prospect of modifying methoctramine (2) structure. Thus, polyamines 3-7 were designed in which the flexibility of the diaminohexane spacer of 2 was replaced by a bipiperidinyl moiety. In electrically stimulated guinea pig left atria, these novel polyamines, unlike prototype 2, displayed a potent intrinsic activity, which was in contrast with the muscarinic antagonism shown in binding studies by some of them (3 and 4) and was inhibited by benzalkonium chloride, an inhibitor of G(i) proteins.

Analogues of prazosin that bear a benextramine-related polyamine backbone exhibit different antagonism toward alpha1-adrenoreceptor subtypes.

Hybrid tetraaamine disulfides 4-9 were synthesized by combining the structural features of prazosin (1), a competitive alpha1-adrenoreceptor antagonist, and benextramine (2), an irreversible alpha1/alpha2-adrenoreceptor antagonist, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D). To verify the role of the disulfide moiety on the interaction with alpha1-adrenoreceptor subtypes, carbon analogues 10-15 were included in this study. All quinazolines lacking the disulfide bridge behaved, like 1, as competitive antagonists, whereas all polyamine disulfides displayed a nonhomogeneous mechanism of inhibition at the three subtypes since they were, like 2, noncompetitive antagonists at the alpha1A and alpha1B subtypes while being, unlike 2, competitive antagonists at the alpha1D. In particular, the blocking effects were characterized by a decrease of the maximal response to noradrenaline that was affected only slightly by washings. Probably the alpha1A and alpha1B subtypes bear in the binding pocket a suitable thiol function that would suffer an interchange reaction with the disulfide moiety of the antagonist and which is missing, or not accessible, in the alpha1D subtype. Polyamines 8, 9, and 14, among others, emerged as promising tools for the characterization of alpha1-adrenoreceptors, owing to their receptor subtype selectivity. Finally, the effect of nonbasic substituents on the phenyl ring of prazosin analogues 16-28 on potency and selectivity for the different subtypes can hardly be rationalized.

Analysis of the muscarinic receptor subtype mediating inhibition of the neurogenic contractions in rabbit isolated vas deferens by a series of polymethylene tetra-amines.

The pharmacological characteristics of the presynaptic muscarinic receptor subtype, which mediates inhibition of the neurogenic contractions in the prostatic portion of rabbit vas deferens, have been investigated by using a series of polymethylene tetra-amines, which were selected for their ability to differentiate among muscarinic receptor subtypes. It was found that all tetra-amines antagonized McN-A-343-induced inhibition in electrically stimulated rabbit vas deferens in a competitive manner and with affinity values (pA:(2)) ranging between 6.27+/-0.09 (spirotramine) and 8.51+/-0.02 (AM170). Competition radioligand binding studies, using native muscarinic receptors from rat tissues (M(1), cortex; M(2), heart; M(3), submaxillary gland) or from NG 108-15 cells (M(4)) and human cloned muscarinic M(1)-M(4) receptors expressed in CHO-K1 cells, were undertaken with the same tetra-amines employed in functional assays. All antagonists indicated a one-site fit. The affinity estimates (pK:(i)) of tetra-amines calculated in binding assays using native receptors were similar to those obtained using cloned receptors. Among these compounds some displayed selectivity between muscarinic receptor subtypes, indicating that they may be valuable tools in receptor characterization. Spirotramine was selective for M(1) receptors versus all other subtypes (pK:(i) native: M(1), 7.32+/-0.10; M(2), 6.50+/-0.11; M(3), 6.02+/-0.13; M(4), 6.28+/-0.16; pK:(i) cloned: M(1), 7.69+/-0.08; M(2), 6.22+/-0.14; M(3), 6.11+/-0.16; 6.35+/-0.11) whereas CC8 is highly selective for M(2) receptors versus the other subtypes (pK:(i) native: M(1), 7.50+/-0.04; M(2), 9.01+/-0.12; M(3), 6.70+/-0.08; M(4), 7.56+/-0.04; pK:(i) cloned: M(1), 7.90+/-0.20; M(2), 9.04+/-0.08; M(3), 6.40+/-0.07; M(4), 7.40+/-0.04). Furthermore, particularly relevant for this investigation were tetra-amines dipitramine and AM172 for their ability to significantly differentiate M(1) and M(4) receptors. The apparent affinity values (pA:(2)) obtained for tetra-amines in functional studies using the prostatic portion of rabbit vas deferens correlated most closely with the values (pK:(i)) obtained at either native or human recombinant muscarinic M(4) receptors. This supports the view that the muscarinic receptor mediating inhibition of neurogenic contractions of rabbit vas deferens may not belong to the M(1) type but rather appears to be of the M(4) subtype.

Hexahydrochromeno[4,3-b]pyrrole derivatives as acetylcholinesterase inhibitors.

In a search for less flexible analogues of caproctamine (1), a diamine diamide endowed with an interesting AChE affinity profile, we discovered compound 2, in which the terminal 2-methoxybenzyl groups of 1 have been incorporated into a tricyclic system. Since this compound retains good AChE inhibitory activity and its hexahydrochromeno[4,3-b]pyrrole moiety is reminiscent of the hexahydropyrrolo[2,3-b]indole of physostigmine (3), we have designed and synthesized carbamates 4-6, and their biological evaluation has been assessed in vitro against human AChE and BChE. The 6-carbamate 4 was almost as potent as physostigmine and was 60- and 550-fold more potent than the 7-carbamate 5 and the 8-carbamate 6, respectively. The two enantiomers of 4, (-)-4 and (+)-4, did not show a marked enantioselectivity. Finally, a similar time-dependent pattern of inhibition of AChE was observed for 3 and 4.

[4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]-trimethylammonium (McN-A-343)-related compounds. Effect of the butynyl chain inclusion into an aromatic unit on the potency for muscarinic receptors.

A series of derivatives of the known M1 selective muscarinic receptor agonist McN-A-343 (1) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1. The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M1 receptor agonists more potent than the prototype 1, even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 were selective agonists towards muscarinic M3 receptors, while compounds 14, 16 and 18 were selective muscarinic M2 receptor agonists. The most interesting derivative was 8, a full agonist at muscarinic M3 receptors devoid of activity at both muscarinic M1 and M2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3-8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4, 6, 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.

[Determination of carbohydrate trasferrin (CDT) in the legal medical assessment of being fit to drive]. / Il dosaggio della transferrina carboidrato carente (CDT) nella valutazione medico-legale dell'idoneità alla guida.

BACKGROUND: According to the literature carbohydrate-deficient transferrin (CDT) is thought to be the most sensitive and specific marker of alcohol abuse, but must always be combined with other laboratory tests. Until now the amount of CDT that indicates a state of chronic alcoholism has not been established. Therefore, our aim was to quantify the percentage of CDT that discriminates social drinkers from alcoholics. METHODS: A retrospective study was carried out covering a period of four months on patients who came to us after having their driving licenses suspended for drink driving: 100 male and female subjects aged between 21 and 65 years were examined. This population was compared to a control group of 50 subjects matched for age, who consumed a moderate amount of alcohol, and had never had their driving licenses suspended. RESULTS: The percentage of CDT was found by heterogenous enzyme immunoassay that involves column separation and turbidimetry. There was a notable difference in the amount of CDT between the two groups. The ANOVA and Levene tests were used for statistical analysis. CONCLUSIONS: The authors found the percentage amount of CDT that discriminates the two groups, highlighting the important role of this marker of alcohol abuse in a relevant social context.

Design, synthesis, and biological evaluation of symmetrically and unsymmetrically substituted methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists.

The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M(2) and M(3) receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M(2) receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M(2) receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.

WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors.

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6, 7-dihydro-5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]meth yl] -2-methylbenzo[b]thiophen-4(5H)-one that was reported to display an intriguing selectivity profile at alpha(1)-adrenoreceptors. This synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of (-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral building block for the synthesis of (-)-3 starting from (+)-(2R)-2, 3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9) and (1S,2S, 5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim of this project was to further investigate whether it is possible to differentiate between these compounds with respect to their affinity for alpha(1)-adrenoreceptor subtypes and the affinity for 5-HT(1A) receptors, as 1 binds with high affinity at both receptor systems. The biological profiles of reported compounds at alpha(1)-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO and HeLa cells membranes expressing the human cloned alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and (+)-4 toward 5-HT(1A) receptors was evaluated by determining the induced stimulation of [(35)S]GTPgammaS binding in cell membranes from HeLa cells transfected with human cloned 5-HT(1A) receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R configuration, as in (+)-3 and (-)-4, conferred higher affinity at alpha(1)-adrenoreceptors, whereas a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT(1A) receptors. For the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the stereoisomers displayed a similar alpha(1)-selectivity profile, that is alpha(1D) > alpha(1B) > alpha(1A), which is different from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be agonists at the 5-HT(1A) receptors, with a potency comparable to that of 5-hydroxytryptamine.

Universal template approach to drug design: polyamines as selective muscarinic receptor antagonists.

The concept that polyamines may represent a universal template in the receptor recognition process is embodied in the design of new selective muscarinic ligands. Tetraamines 4-7 and 16-20 and diamine diamides 8-15 were synthesized, and their pharmacological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig left atrium (M2) and ileum (M3) and by binding assays in rat cortex (M1), heart (M2), submaxillary gland (M3), and NG 108-15 cells (M4). It has been confirmed that appropriate substituents on the terminal nitrogens of a tetraamine template can tune both affinity and selectivity for muscarinic receptors. The novel tetraamine C-tripitramine (17) was able to discriminate significantly M1 and M2 receptors versus the other subtypes, and in addition it was 100-fold more lipophilic than the lead compound tripitramine. Compound 14 (tripinamide), in which the tetraamine backbone was transformed into a diamine diamide one, retained high affinity for muscarinic subtypes, displaying a binding affinity profile (M2 > M1 > M4 > M3) qualitatively similar to that of tripitramine. Both these ligands, owing to their improved lipophilicity relative to tripitramine and methoctramine, could serve as tools in investigating cholinergic functions in the central nervous system. Furthermore, notwithstanding the fact that the highest affinity was always associated with muscarinic M2 receptors, for the first time polyamines were shown to display high pA2 values also toward muscarinic M3 receptors.

Antagonist binding profile of the split chimeric muscarinic m2-trunc/m3-tail receptor.

Recent evidence suggests that G-protein-coupled receptors can behave as multiple subunit receptors, and can be split into parts, maintaining their binding ability. Transfection of a truncated muscarinic m2 receptor (containing transmembrane domains I-V, named m2-trunc) with a gene fragment coding for the carboxyl-terminal receptor portion of the muscarinic m3 receptor (containing transmembrane domains VI and VII, named m3-tail) results in the formation of a binding site with a high affinity for the muscarinic ligand N-[3H]methylscopolamine. In this paper we analyse the antagonist binding profile of this chimeric m2-trunc/m3-tail receptor in comparison with the wild-type muscarinic m2 and m3 receptors. While many of the substances tested had an intermediate affinity for the chimeric m2-trunc/m3-tail receptor compared with m2 and m3, some compounds were able to distinguish between the chimeric m2-trunc/m3-tail receptor on the one hand and the m2 or the m3 receptor on the other. Among them, tripitramine (a high-affinity M2 receptor antagonist) bound to the m2-trunc/m3-tail receptor with the same affinity as m2, but it bound to the m3 receptor with a 103-fold lower affinity; pirenzepine (a selective muscarinic M1 receptor antagonist) bound to the chimeric receptor with an affinity that was 12- and 3-fold higher than that of m2 and m3, respectively. The results of this study demonstrate that the chimeric m2-trunc/m3-tail receptor has a pharmacological profile distinct from that of the originating muscarinic m2 and m3 receptors.

Search for selective antagonists at alpha 1-adrenoreceptors: neutral or negative antagonism?

In this article the use of competitive antagonists as tools in receptor characterization and classification is discussed. It is pointed out that caution is required in receptor characterization because negative antagonism (inverse agonism) rather than neutral antagonism could play a relevant role. This implies that antagonists should be evaluated not only with regard to their affinity, but also with regard to their ability to affect the equilibrium between the two receptor states, namely active and inactive states. Since affinity and efficacy of a negative antagonist are system dependent the use of negative antagonists as competitive antagonists in receptor characterization may give rise to false differences in receptor subtypes. Finally, this article summarizes recent developments in the design of new alpha 1-adrenoreceptor antagonists which are structurally related to prazosin or WB 4101.

Search for alpha 1-adrenoceptor subtypes selective antagonists: design, synthesis and biological activity of cystazosin, an alpha 1D-adrenoceptor antagonist.

Two novel quinazolines (2 and 3) related to both prazosin and its open analogue 1 were synthesized, and their biological profile at alpha 1-adrenoceptor subtypes was assessed by functional assays in rat isolated tissues, namely prostatic vas deferens (alpha 1A), spleen (alpha 1B) and aorta (alpha 1D). Furthermore, the binding profile of 3 was assessed at native alpha 2 and D2 receptors, and cloned human 5-HT1A receptors, in comparison to prazosin, (+)-cyclazosin, 1 and BMY 7383. It turned out that the cystamine-bearing quinazoline 3 (cystazosin) has a reversed affinity profile relative to (+)-cyclazosin owing to a higher affinity for alpha 1D-adrenoceptors and a significantly lower affinity for the alpha 1A and alpha 1B subtypes. Furthermore, in comparison to BMY 7378, cystazosin (3) displays a much better specificity profile since it has lower affinity for D2 and 5-HT1A receptors.

In vitro characterization of tripitramine, a polymethylene tetraamine displaying high selectivity and affinity for muscarinic M2 receptors.

1. The antimuscarinic effects of tripitramine were investigated in vitro in isolated driven left (force) and spontaneously beating right (force and rate) atria as well as in the ileum of guinea-pig and rat and in the trachea and lung strip of guinea-pig and compared with the effects of methoctramine. 2. Tripitramine was a potent competitive antagonist of muscarinic M2 receptors in right and left atria. The pA2 values ranged from 9.14 to 9.85. However, in the guinea-pig and rat left atria but not in guinea-pig right atria, tripitramine at lower concentrations (3-10 nM) produced a less than proportional displacement to the right of agonist-induced responses owing to the presence of a possible saturable removal process. 3. Tripitramine was about three orders of magnitude less potent in ileal and tracheal than in atrial preparations (pA2 values ranging from 6.34 to 6.81) which makes it more potent and more selective than methoctramine. 4. Another intriguing finding was the observation that the pA2 value of 7.91 observed for tripitramine in guinea-pig lung does not correlate with that found at both muscarinic M2 and M3 receptor subtypes, which clearly indicates that the contraction of guinea-pig lung strip is not mediated by these muscarinic receptor subtypes. 5. A combination of tripitramine with atropine resulted in addition of the dose-ratios for left atria as required for two antagonists interacting competitively with the same receptor site, whereas the same combination gave a supra-additive antagonism on guinea-pig ileum which suggests that tripitramine interacts with a second interdependent site. 6. Tripitramine was more specific than methoctramine since, in addition to muscarinic receptors, it inhibited only frog rectus abdominis muscular (pIC50 value of 6.14) and rat duodenum neuronal (pIC50 value of 4.87) nicotinic receptors among receptor systems investigated, namely alpha 1-, alpha 2-, and beta 1-adrenoceptors, H1- and H2-histamine receptors, and muscular and neuronal nicotinic receptors.

The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists.

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.