Andolast acts at different cellular levels to inhibit immunoglobulin E synthesis.

The anti-asthmatic agent andolast is thought to inhibit the release of allergic mediators, but its mechanism of action is not fully understood. We investigated whether the compound inhibits immunoglobulin E (IgE) synthesis and tested the hypothesis that andolast affects immunoglobulin class switching. Interleukin (IL)-4 and the interaction of CD40 expressed on B cells with its ligand on T cells are necessary for IgE synthesis. Thus, peripheral blood mononuclear cells (PBMCs) from 40 asthmatic, 16 non-asthmatic allergic, and 9 normal donors were stimulated with IL-4 and/or anti-CD40 antibody. T cells from 9 additional allergic donors were activated with anti-CD3/CD28 antibodies to express IL-4 mRNA. After incubation in the absence or presence of test compounds, immunoglobulin concentrations were measured by enzyme immunoassay, and mRNA levels were analyzed by RT-PCR. Andolast significantly inhibited IgE synthesis by stimulated PBMCs from both asthma patients and combined allergic/normal donors. In mechanistic studies, andolast was found to act at different cellular levels. Firstly, it reduced by about 45 percent (p<0.05) the levels of IL-4 mRNA in T cells stimulated with anti-CD3/CD28. Secondly, andolast reduced by about 36 percent (p<0.05) the expression of epsilon germline transcripts in PBMCs stimulated with IL-4/anti-CD40. Thirdly, the effect of andolast on immunoglobulin synthesis was selective in that the production of IgG4 antibodies was not significantly inhibited. Our findings, while supporting the evidence that andolast is effective for the treatment of asthma, provide new insights into its mechanism of action.

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC.

Recently an innovative novel class angiotensin-AT1 antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF3COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100microl loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2microg/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT1 antagonist in rat plasma and urine.

N-terminal anthranoyl-phenylalanine derivatives as CCK1 receptor antagonists: the final approach.

Starting from our lead compound, VL-0395, an anthranilic acid based CCK1 receptor antagonist, and following the well established "step by step" lead investigation strategy, we describe the final step of the anthranilic acid N-terminal optimization. Improvements for both affinity and selectivity towards CCK1 receptors have been accomplished through introduction of the fluoro substituent at C-5 and C-7 position of the indole ring together with the appropriate configuration of the aminoacidic chiral center.

Characterization of a novel putative cognition enhancer mediating facilitation of glycine effect on strychnine-resistant sites coupled to NMDA receptor complex.

The effects of (S)-4-amino-5-[(4,4-dimethylcyclohexyl)amino]-5-oxo-pentanoic acid ((S)CR 2249), a new chemical entity selected among a series of glutamic acid derivatives, were investigated on N-methyl-D-aspartate (NMDA)-evoked release of [3H]noradrenaline from rat hippocampal slices. (S)CR 2249 facilitated glycine-mediated reversion of kynurenate antagonism at strychnine-insensitive glycine receptors coupled to the NMDA receptor. The potency of glycine (EC50 = 21.5 microM +/- 4.2) was not significantly influenced by (S)CR 2249. Nevertheless, the efficacy of the glycine effect was enhanced in a concentration-dependent manner (3-10-30 microm) by (S)CR 2249. The interaction of (S)CR 2249 with NMDA receptors was also studied with binding experiments, in which we examined the effect of (S)CR 2249 on the modulation by glutamate, glycine and spermine of [3H]dizocilpine (MK-801) binding. (S)CR 2249, increased [3H]MK-801 binding in a concentration-dependent manner and we found positive cooperative interactions between glycine and (S)CR 2249, indicating that (S)CR 2249 probably acts at a separate allosteric site to increase NMDA receptor functionality.

Antiallergic and cytoprotective activity of new N-phenylbenzamido acid derivatives.

A series of new N-phenylbenzamido acid derivatives was synthesized and evaluated for their ability to inhibit the IgE-mediated passive cutaneous anaphylaxis in the rat (PCA), as well as for their capacity to inhibit gastric mucosal damage induced by the oral administration of absolute alcohol in the rat. Some of these new derivatives exhibit potent antiallergic and cytoprotective activity, 20-80 times higher than that of the reference, disodium cromoglycate (DSCG). Structure-activity relationships are discussed. The antiallergic activity of one of the more potent compounds of this series, i.e. 4-(1H-tetrazol-5-yl)-N-[4-(1H-tetrazol-5-yl)phenyl]benzamide (compound 44, CR 2039) was further evaluated in vivo. This compound antagonizes the bronchoconstriction induced by aerosolized ovalbumin in both anesthetized and conscious IgE sensitized guinea pigs with ID50 of 3.7 mg/animal (tracheal insufflation) and 20 mg/kg (im). Further cytoprotective effects were evaluated in gastric ulcer models induced by the acute oral administration of hypertonic sodium chloride solution or by acetic acid and by the subchronic administration of glucose in fasted animals. In the models used experimentally CR 2039 is effective, whereas DSCG seems to be devoid of any protective activity. Such a potent antiallergic and mucosal protectant could provide a new potential agent in the therapy of atopic allergic diseases.

Structure-antigastrin activity relationships of new (R)-4-benzamido-5-oxopentanoic acid derivatives.

New (R)-4-benzamido-5-oxopentanoic acid derivatives were synthesized by a stereoconservative procedure and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK-B) CCK receptors, or the binding of [3H]pentagastrin to rabbit gastric glands, as well as to inhibit, in vivo, the acid secretion induced by pentagastrin infusion in the perfused rat stomach. The parent compound of this series (lorglumide) is the first nonpeptidic, potent and selective antagonist of the CCK-A receptor. Chemical manipulations of the structure of lorglumide led to the discovery of selective antagonists of the CCK-B/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives exhibit different affinities with rabbit gastric gland cells and rat cortex membranes, suggesting that the stomach gastrin receptor (arbitrarily termed CCK-B1 receptor) is not as closely related to the CCK central receptor (termed CCK-B2) as previously hypothesized. The antigastric activity of the most potent compound of the series, i.e. (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid (compound 28, CR 2194) was further evaluated in vivo: in the first hour after administration the compound inhibits acid secretion induced by pentagastrin infusion, in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch), with ID50s (mg/kg) of 15.5 (iv) (cat), 8.7 (IV) (dog) and 24.2 (oral) (Heidenhain dog). The characteristics of CR 2194, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure, and the activity after oral administration, indicate that this compound is a useful tool in the study of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use.

Structure-antigastrin activity relationships of new spiroglumide amido acid derivatives.

A series of new spiroglumide amido acid derivatives was synthesized and evaluated for their ability to inhibit the binding of cholecystokinin (CCK) to guinea pig brain cortex (CCKB receptors) and peripheral rat pancreatic acini (CCKA receptors), as well as to inhibit in vitro the gastrin-induced Ca2+ increase in rabbit gastric parietal cells. Appropriate chemical manipulations of the structure of spiroglumide (CR 2194), i.e., (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid, led to potent and selective antagonists of CCKB/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives, as, for example, compound 54 (CR 2622), i.e., (S)-4-[[(R)-4'-[(3,5-dichlorobenzoyl)-amino]-5'-(8- azaspiro[4.5]decan-8-yl)-5'-oxo-pentanoyl]amino]-5- (1-naphthylamino)-5-oxopentanoic acid, exhibit activity 70-170 times greater than that of spiroglumide, depending upon the model used (IC50 = 2 x 10(-8) vs 140 x 10(-8) mol in binding inhibition of [3H]-N-Me-N-Leu-CCK-8 in guinea pig brain cortex and IC50 = 0.7 x 10(-8) vs 122.3 x 10(-8) mol in inhibition of gastrin-induced Ca2+ mobilization in parietal cells of rabbit, respectively). Computer-assisted conformational analysis studies were carried out in order to compare the chemical structure of both the agonist (pentagastrin) and the antagonist (54).

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 1. Mapping the central 5-HT3 receptor binding site by arylpiperazine derivatives.

Synthesis and pharmacological evaluation of a series of condensed quinoline and pyridine derivatives bearing a N-methylpiperazine moiety attached to the 2-position of the quinoline or pyridine nucleus are described. 5-HT receptor binding studies revealed subnanomolar affinity for the 5-HT3 receptor subtype in some of the compounds under study. The most active compound (5b) displayed a Ki value about 1 order of magnitude higher than that of quipazine along with a higher selectivity. The potential 5-HT3 agonist/antagonist activity of four selected compounds was assessed in vitro on 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Compound 5j acted as a 5-HT3 agonist in this assay with an EC50 value close to that reported for quipazine, while 5b was a partial agonist with an EC50 value of about 0.25 nM, and compound 5c possessed antagonist properties with an IC50 value (approximately 8 nM) in the same range as those of previously characterized 5-HT3 receptor antagonists. Qualitative and quantitative structure-affinity relationship studies carried out by making use of theoretical molecular descriptors allowed to elucidate the role of the main pharmacophoric components and to develop a model for the interaction of the 5-HT3 ligands related to quipazine with their receptor.

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 2. Molecular basis of the intrinsic efficacy of arylpiperazine derivatives at the central 5-HT3 receptors.

Novel 5-HT3 receptor ligands were designed and synthesized with the aim of obtaining deeper insight into the molecular basis of the intrinsic efficacy of arylpiperazines interacting with the central 5-HT3 receptor. The newly synthesized compounds and some previously published compounds belonging to the same class of heteroarylpiperazines were tested for their potential ability to displace [3H]granisetron from rat cortical membranes. These 5-HT3 receptor binding studies revealed subnanomolar affinity in several of the compounds under study. The most active ligands were quipazine derivatives bearing a phenyl group in the 4-position and various oxygenated alkyl side chains in the 3-position of the quinoline nucleus. Qualitative and theoretical quantitative structure-affinity relationship studies were carried out, and the interaction model for the 5-HT3 ligands related to quipazine with their receptor, proposed in part 1 of the present work, was updated to incorporate the latest data. The potential 5-HT3 agonist/antagonist activity of 12 selected compounds was assessed in vitro on the 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Their intrinsic efficacy ranged from the 5-HT3 full agonist properties of compounds 7a and 8h, i to those of partial agonists 10a,d and antagonists 8b,d,e, and 9c, d,h,i. The comparison between these functional data and those relative to the previously described compounds suggested that in this class of 5-HT3 ligands the intrinsic efficacy is modulated in a rather subtle manner by the steric features of the heteroaryl moiety.

Protective effect of CR 1409 (cholecystokinin antagonist) on experimental pancreatitis in rats and mice.

CR 1409, a glutaramic acid derivative with competitive cholecystokinin-antagonistic activity, was administered IP and evaluated in comparison with proglumide (the model CCK-receptor antagonist), gabexate (protease inhibitor) and PGE2 (cytoprotective) on two different models of experimental pancreatitis. Acute pancreatitis was induced in mice by six IP injections of 50 micrograms/kg caerulein at hourly intervals. The drugs were administered 30 minutes before each caerulein administration. Blood samples and pancreata were collected 3 hours after the last caerulein injection. In the second experiment, pancreatitis was induced in rats by injecting 0.3 ml 6% sodium taurocholate interstitially into the pancreas. The drugs were administered twice, 30 minutes before and 3 hours after taurocholate. The animals were killed 6 hours after laparotomy and blood samples and pancreata were collected. CR 1409 exhibited on both pancreatitis models a protective effect in a dose range of 0.3-10 mg/kg. Proglumide exhibited a protective activity at higher doses (200-400 mg/kg). Gabexate and PGE2 were effective only in pancreatitis induced by taurocholate in a dose range of 30-60 mg/kg and 60-130 micrograms/kg respectively. These results, showing a high protective effect of CR 1409 on different models of acute pancreatitis, suggest an important role of CCK in the pathogenesis of pancreatitis.

Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats.

New glutaramic acid derivatives with cholecystokinin antagonistic activity were evaluated for their capacity to inhibit the satiety effect induced in the rat by intraperitoneal (i.p.) injection of cholecystokinin octapeptide (CCK-8). The most active compound, CR 1409, is about 4000 times more potent than proglumide when injected peripherally (i.p.). This compound competitively inhibits the action of CCK-8 at the receptor responsible for the satiety effect. In contrast, CR 1409, i.p. or intracerebroventricularly (i.c.v.) injected does not exhibit antagonistic effects when CCK-8 is administered i.c.v., confirming the existence of at least two different populations of CCK receptors.

Characterization of antigastrin activity in vivo of CR 2194, a new R-4-benzamido-5-oxo-pentanoic acid derivative.

The antigastrinic activity, in vivo, of CR 2194 (R-4-(3-chlorobenzamido)-5-(8-azaspiro[4.5]decan-8-yl) -5-oxo pentanoic acid) was assessed in various animal species. CR 2194 antagonized pentagastrin-stimulated gastric acid secretion in the rat (ID50 = 11 mg/kg i.v.), dog (ID50 = 5.9 mk/kg i.v. or 28.8 mg/kg os) and cat (ID50 = 15.5 mg/kg i.v.). CR 2194, in the cat, inhibited both competitively and non-competitively the gastric acid secretion stimulated with increased doses of pentagastrin, with a pA2 of 4.89. In the rat and in the dog the antagonism seemed to be non-competitive and the respective pD'2 calculated were 4.54 and 4.42. The interaction of CR 2194 with the gastrin receptors appeared reversible, as demonstrated by the return to normal values of the acid output after the conclusion of the i.v. infusion, during pentagastrin continuous stimulation in the dog. The antigastrin activity was specific: CR 2194 was unable to antagonize the gastric acid secretion stimulated by carbachol or histamine in the rat up to the dose of 100 mg/kg. CR 2194 was effective to antagonize the gastric acid secretion stimulated by gastrin release after meal ingestion in the Heidenhain pouch dog model. The ID50 calculated was 2.89 mg/kg after oral administration. All these characteristics make CR 2194 an important compound in the investigation of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use.

Cytosolic Ca2+ evaluation in rabbit parietal cells: a novel method to screen gastrin receptor antagonists.

We have evaluated the application of the fura-2 method to detect cytosolic Ca2+ increase in gastric cells expressing CCKB/gastrin receptors, in order to screen gastrin receptor antagonists, as an alternative to functional studies. We have characterized the receptors on parietal cell suspension from rabbit gastric mucosa and validated the method using both the CCKB and CCKA receptor agonists and antagonists. Human gastrin I (gastrin) (0.1 nM-4 microM) and sulfated cholecystokinin 26-33 (CCK-8) (0.01 nM-2 microM) dose-dependently augmented cytosolic Ca2+. The efficacies of the two agonists were similar, but the potency of CCK-8 (EC50 1.03 nM) was about 10-fold greater than that of gastrin (11 nM). Response to a submaximal dose of gastrin (50 nM) was dose-dependently blocked by the CCKB-receptor antagonists CAM-1028 (4-[[2-[[3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-[[[1,7,7-trimethylbicyclo[2, 2,1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino-4-oxo -[1 S-1 alpha, 2 beta [S'(S')4 alpha]]-butanoate-N-methyl-D-glucamine) (IC50 1.9 nM), L-365,260 (3 R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1, 4-benzodiazepin-3-yl)-N'-(3-methylphenyl)urea) (IC50 10 nM) and spiroglumide ((R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan -8-yl)-5-oxopentanoic acid) (IC50 2 microM). The results were in agreement with those obtained from binding studies in guinea-pig cortical membranes. The model was employed to optimize the synthesis of a new class of spiroglumide analogues which led to a new molecule, (S)-4-¿(R)-4'-(3,5-dichlorobenzoylamino)-5'-(8-azaspiro[4.5] decan-8-yl)-5'-oxo)-pentanoylamino-5-(1-naphthylamino)-5-oxo pentanoic acid (CR 2622), whose potency was about 100-fold greater than that of spiroglumide. CR 2622, as well as the other CCKB receptor antagonists tested, exhibited no effect on basal [Ca2+]i. The simplicity and the reproducibility of this method suggest that it is a useful model to screen gastrin and antigastrin activity in parallel or as an alternative to binding studies.

Characterization of antisecretory and antiulcer activity of CR 2945, a new potent and selective gastrin/CCK(B) receptor antagonist.

The antigastrinic, antisecretory and antiulcer activities of CR 2945, (R)-1-naphthalenepropanoic acid,beta-[2-[[2-(8-azaspiro[4.5]dec-8-yl-carbonyl)-4,6-dimethylph enyl] amino]-2-oxoethyl], were investigated in vitro and in vivo in rats and cats. Its activities were compared with those of two gastrin/CCK(B) receptor antagonists, L-365,260 (3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin -3-yl)-N'-(3-methylphenyl)urea and CAM-1028 (4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[[1,7,7-trimethylbicyclo [2.2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino -4-oxo-[1S-1alpha,2beta[S'(S')4alpha]]-butanoate -N-methyl-D-glucamine), of the histamine H2 receptor antagonist, ranitidine, and the proton pump inhibitor, omeprazole. Cytosolic Ca2+ elevation in rabbit parietal cells induced by gastrin (50 nM) was blocked by CR 2945 with an IC50 value of 5.9 nM. CAM-1028 and L-365,260 showed similar activity. CR 2945 antagonized pentagastrin-stimulated gastric acid secretion in rats (ED50 = 1.3 mg kg(-1) i.v. and 2.7 mg kg(-1) i.d.) and cats (1.6 mg kg(-1) i.v.). CR 2945 was slightly less potent than the reference compounds after i.v. administration, whereas after intraduodenal (i.d.) administration, it was more potent than both ranitidine and omeprazole. In the rat, the gastrin antagonism exhibited by CR 2945 was reversible and competitive, with a pA2 value of 7.33. CR 2945 had specific antigastrin activity, as it was unable to antagonize the gastric acid secretion stimulated by histamine or carbachol in rats up to the dose of 30 mg kg(-1). CR 2945 was about as efficacious as ranitidine against the indomethacin- and ethanol-induced gastric ulcers and the cysteamine-induced duodenal ulcer in rats. On the contrary, L-365,260 was only slightly effective. These results suggest that CR 2945 might be a promising compound for the therapy of acid-related disorders, and that its clinical use could help clarify the therapeutic potential of gastrin/CCK(B) receptor antagonists in the gut.

CR 2039, a new bis-(1H-tetrazol-5-yl)phenylbenzamide derivative with potential for the topical treatment of asthma.

The pharmacological activity of CR 2039 (4-(1H-tetrazol-5-yl)-N-(4-[1H-tetrazol-5-yl]phenylbenzam ide)) a newly discovered antiallergic compound is described. CR 2039 administered i.m. or i.v. inhibited rat passive cutaneous anaphylaxis (PCA) with an ED50 of 0.1 mg/kg and a potency about 15 times higher than that of disodium cromoglycate (DSCG). CR 2039 i.m., by aerosol or as dry powder insufflation, gave dose-related significant protection against IgE-dependent bronchial anaphylaxis induced by aerosolized antigen in anesthetized guinea-pigs. In conscious guinea-pigs CR 2039 given i.m. delayed dose dependently (ED50, 17 mg/kg) the onset of bronchoconstriction induced by aerosolized antigen, while DSCG was ineffective up to 100 mg/kg. The protection was accompanied by significant inhibition of the vascular permeability provoked by antigen challenge in all airway segments except trachea. CR 2039 (10-100 mg/kg i.v.) inhibited the microvascular permeability changes in a model of allergic conjunctivitis in sensitized guinea-pigs. CR 2039 inhibited dose dependently guinea-pig lung cAMP-phosphodiesterase with an IC50 of 50 microM.

Cholecystokinin (CCK) increases GABA release in the rat anterior nucleus accumbens via CCK(B) receptors located on glutamatergic interneurons.

The effects of cholecystokinin sulfate octapeptide (CCK-8S) on [3H]gamma-aminobutyric acid (GABA) release have been studied in the anterior side of the rat nucleus accumbens on tissue punches exposed in superfusion to 30 mM KCl. CCK-8S in a concentration dependent manner (10-3000 nM) increased K+-evoked [3H]GABA release (EC50=192 nM). The increase caused by 1 microM CCK-8S ranged from 37% to 42%. CR 2945, (beta-[2-[[2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylp henyl]-amino]-2-oxoethyl]-(R)-1-naphthalenepropanoic acid), a potent and selective nonpeptidergic CCK(B) antagonist, concentration-dependently blocked CCK-8S effect (IC50=2.16 nM). CCK-8S-induced increase in [3H]GABA overflow was completely blocked by 1 microM tetrodotoxin. Both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine (MK-801) antagonized the CCK-8S effect. By contrast, (+)-bicuculline, a GABA(A) receptor antagonist, was completely ineffective. Phaclofen, a selective GABA(B) antagonist, increased K+-evoked [3H]GABA release but did not affect the facilitative effect of CCK-8S. Moreover, tetrodotoxin failed to block AMPA-evoked [3H]GABA release but completely prevented the effect of NMDA (Mg2+ free conditions). The data presented suggest that CCK(B) receptors modulating [3H]GABA release from anterior accumbal punches may not be present on GABAergic terminals but could be located on glutamatergic interneurons.

Development and validation of a bioanalytical method for the determination of the cholecystokinin type-1 (CCK(1)) receptor antagonist dexloxiglumide in human plasma.

A sensitive bioanalytical method for the measurement of dexloxiglumide, a new selective and potent cholecystokinin type-1 (CCK(1)) receptor antagonist, in plasma, is reported. The method is based on reversed-phase liquid chromatography with ultraviolet absorption detection. Samples are extracted under acidic conditions into an organic solvent, and following evaporation, reconstitution and centrifugation stages, the supernatant is injected on to an ODS column with detection at 244 nm. The method has been validated over the concentration range 0.2-20 microgram/ml, 0.2 microgram/ml being the lower limit of quantification. The overall precision and accuracy (expressed as relative error) of the method was less than 6.1 and 2.3%, respectively. Dexloxigulmide was shown to be stable in plasma when stored at -20 degrees C for at least 200 days. The method is suitable for studying the pharmacokinetics of dexloxiglumide in man.

Pharmacokinetics of dexloxiglumide after administration of single and repeat oral escalating doses in healthy young males.

OBJECTIVE: To assess the pharmacokinetics, safety and tolerability of dexloxiglumide, a new CCK1 receptor antagonist currently under development for the treatment of the constipation-predominant irritable bowel syndrome. SUBJECTS AND METHODS: Twelve volunteers were enrolled in the present study and received orally 100, 200 and 400 mg of dexloxiglumide as tablets as a single dose followed by repeated t.i.d. doses for 7 days according to a randomized, double-blind, double-dummy complete crossover design. Plasma and urine were collected before drug administration and up to 72 h after dosing. Dexloxiglumide plasma and urinary concentration, determined using validated HPLC methods with UV detection, were used for the pharmacokinetic analysis by standard noncompartmental methods. In addition, dexloxiglumide safety and tolerability were evaluated throughout the study period by performing standard laboratory tests, by recording vital signs and ECGs and by monitoring the occurrence and severity of adverse events. RESULTS: After a single oral administration, dexloxiglumide was rapidly bioavailable with mean t(max) ranging from 0.9 - 1.6 h at all doses. The mean peak plasma concentrations (Cmax) were 1.7+/-0.6, 5.4+/-1.7, and 11.9+/-4.7 microg/ml, and the mean area under the plasma concentration-time curves (AUC) were 4.4+/-3.3, 8.6+/-3.6, and 18.3+/-5.9 microg x h/ml at the 3 doses, respectively. Apparent plasma clearance (CL/F) was 30.8+/-13.9, 27.2+/-10.6, and 21.1+/-8.6 l/h at the 3 doses, respectively. The apparent elimination half-life from plasma (t1/2) ranged from 2.6 - 3.3 h at the 3 doses. The excretion of unchanged dexloxiglumide in 0 - 72 h urine accounted for approximately 1% of the administered dose and this was true for all doses. Dexloxiglumide renal clearance (CLR) averaged 0.4+/-0.4, 0.4+/-0.2, and 0.3+/-0.3 l/h for the 3 doses, respectively. After the last dose of the repeated dosing period dexloxiglumide Cmax occurred at 1.1 - 1.6 h after drug administration and averaged 2.4+/-1.3, 7.1+/-2.9, and 15.3+/-2.7 microg/ml for the 3 doses, respectively. The AUC values averaged 5.9+/-3.0, 16.0+/-8.8, and 50.8+/-38.1 microg x h/ml, respectively. The area under the plasma concentration-time curve calculated at steady state within a dosing interval (AUCss) averaged 4.6+/-1.6, 11.3+/-3.6, and 28.4+/-8.2 microg x h/ml, whereas CL/F averaged 20.3+/-8.3, 16.3+/-9.0, and 10.3+/-5.0 l/h at the 3 doses, respectively. Dexloxiglumide t1/2 could not be accurately calculated due to the high inter-subject variability and to sustained dexloxiglumide plasma concentrations that precluded the identification ofthe terminal phase of the plasma concentration-time profiles. However, it appeared that dexloxiglumide t1/2 was considerably prolonged at the dose of 400 mg. CLR averaged 0.4+/-0.4, 0.3+/-0.3, and 0.3+/-0.1 l/h for the 3 doses, respectively. After a single dose, the plasma pharmacokinetics of dexloxiglumide were dose-independent in the dose range 100 - 400 mg. After repeated dose the pharmacokinetics of dexloxiglumide were virtually dose-independent in the dose range 100 - 200 mg. A slight deviation from linear pharmacokinetics was found with a dose of 400 mg. Dexloxiglumide plasma pharmacokinetics were also time-independent in the dose range 100 - 200 mg with a deviation from expectation based on the superimposition principle with a dose of 400 mg. Dexloxiglumide urinary excretion and renal clearance were both dose- and time-independent in the dose range 100 - 400 mg. The safety and tolerability of dexloxiglumide administered to healthy young males was good up to the maximum investigated dose of 400 mg both after single and after repeated doses. CONCLUSIONS: The safety and pharmacokinetic profile of dexloxiglumide when the drug is administered as single and repeated doses in the dose range 100 - 400 mg provides the rationale for the choice of the treatment schedule (200 mg t.i.d.) for the efficacy trials in patients with (constipation-predominant) irritable bowel syndrome.

Sensitive SPE-HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples.

A high-performance liquid chromatography (HPLC)-method after solid-phase extraction (SPE) has been developed in order to determine a new angiotensin-AT1 antagonist, i.e. CR 3210 (C27H24N8; MW = 460.54), 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline in rat plasma and urine after oral administration to Sprague-Dawley rats. CR 3210 and the internal standard (IS) CR 1505 (loxiglumide), i.e. 4-[(3,4-dichlorobenzoyl)amino]-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid, were isolated from rat urine and plasma by solid-phase extraction. The procedure was optimized regarding the sorbent extraction material, the pH in the conditioning solution, the washing step, the dry time and the type of elution solvent. The separation was performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a capacity factor of 1.87 for CR 3210 and 1.10 for the internal standard. The selectivity of the method was satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 68.5 at 75 ng/ml and 80.9 at 3000 ng/ml; the mean recovery of CR 3210 from spiked rat urine was 69.9 at 75 ng/ml and 78.6 at 3000 ng/ml. The lower limit of detection (LOD) was 14 ng/ml in plasma and 22 ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 30 ng/ml, the lowest calibration standard using 500 microl rat plasma and urine. The procedures were validated according to international standards with a good reproducibility and linear response from 30 to 3000 ng/ml, for either plasma or urine. The sensitivity of the method allowed for its application to pharmacokinetic studies.

CR 2249: a new putative memory enhancer. Behavioural studies on learning and memory in rats and mice.

The effects of S-4-amino-5-[4,4-dimethylcyclohexyl)amino]-5-oxopentanoic acid (CR 2249), a new entity selected from a new series of glutamic acid derivatives, has been investigated in different paradigms for screening nootropics. CR 2249 ameliorated the memory retention deficit produced by scopolamine in step-through-type passive avoidance in rats and by electroconvulsive shock in step-down-type passive avoidance in mice. CR 2249 was also capable of improving performance in behavioural tests of learning and memory in the absence of cholinergic hypofunction or cognitive deficit. The activity was determined using different passive and active avoidance behavioural test procedures on rats. CR 2249 was active only when given 45 min before training and did not show any effect when administered immediately after the learning training or before the retention trial. No changes in the general behaviour or motor activity of the animals were observed, indicating that CR 2249 effects cannot be attributed to sensory-motor deficit. Microdialysis experiments have shown that CR 2249 significantly increased noradrenaline release in the hippocampus of freely moving rats and reduced 3,4-dihydroxyphenylglycol efflux. These effects have led us to hypothesize that CR 2249 memory effect might be mediated by a direct or indirect action on noradrenergic transmission. These behavioural results suggest that this new agent has clinical application in memory disorders.