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.     

A new potent and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L-365,260

L-365,260 (3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-N'-(3-methylphenyl)urea), interacted in a stereoselective and competitive manner with guinea pig stomach gastrin and brain cholecystokinin (CCK) receptors. The affinity of L-365,260 for both gastrin (Ki = 1.9 nM) and brain CCK-B (Ki = 2.0 nM) receptors was greater than 2 orders of magnitude higher than its affinity for peripheral pancreatic CCK-A receptors or various other receptors. L-365,260 exhibited a similar high affinity for brain CCK-B receptors of rats, mice and man. A somewhat lower affinity for gastrin and brain CCK-B (IC50 = 20-40 nM) receptors was observed in dog tissues. In vivo, oral administration of L-365,260 antagonized gastrin-stimulated acid secretion in mice (ED50 = 0.03 mg/kg), rats (ED50 = 0.9 mg/kg) and guinea pigs (ED50 = 5.1 mg/kg). L-365,260 did not affect basal acid secretion and did not antagonize histamine- or carbachol-stimulated acid secretion in mice. L-365,260 represents a potentially powerful new tool for investigating gastrin and brain CCK-B receptors, and possibly their role in physiology and disease.     

In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor, >10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.     

AG-041R, a cholecystokinin-B/gastrin receptor antagonist, stimulates the repair of osteochondral defect in rabbit model

A newly synthesized compound (AG-041R), 3R-1-(2,2Diethoxyethyl)-((4methylphenyl) amino-carbonyl methyl)-3-((4methylphenyl) ureido-indoline-2-one), is a cholecystokinin-B/gastrin receptor antagonist which has stimulatory effects on the matrix synthesis of chondrocytes in vitro. In this study, we examined the effect of AG-041R on the repair of osteochondral defects (cylindrical, 4 mm diameter) in the patellar groove of the rabbit knee joint. At the time of operation, 100 microl of 1 microM of AG-041R was administered, followed by 200 microl with an osmotic pump for 14 days. Histological and biochemical evaluations were performed at 12 and 24 weeks after surgery. The histological score of the AG-041R-treated group, the quantity of glycosaminoglycan and the ratio of chondroitin sulfate in the AG-041R-treated tissue were significantly higher than in the untreated group. Moreover, the degeneration of cartilage around the defect was suppressed in the AG-041R-treated group. These findings suggest that AG-041R is effective for the repair of osteochondral defects.     

Two new opioid delta-receptor ligands: a highly selective agonist and a potent selective antagonist in in vitro isolated preparations

N,N-Diallyl derivatives of enkephalin analogues were chemically synthesized, and their biological activities were estimated in vitro isolated preparations. N,N-Diallyl-[D-Ala2, D-Leu5]-enkephalin [test compound I] at doses up to 10 microM did not inhibit the electrically-evoked contractions of guinea-pig ileum, which had been suggested to contain opioid mu- and kappa-receptors, but it significantly depressed the contractions of mouse vas deferens, which had been indicated to contain mu-, kappa- and delta-receptors, suggesting that test compound I did not act on both mu- and kappa-receptors, but acted on delta-receptors. Additionally, the Ke (equilibrium dissociation constant) values against test compound I of naloxone were approximately 30 nM and similar to those of Mr 2266, also indicating that test compound I acted as a delta agonist. Moreover, the Ke values of ICI 154129 against compound I were approximately 340 nM, strongly suggesting that test compound I acted as a delta agonist. The Ke values of bis-[N,N-diallyl-[D-Ala2, Leu5]-enkephalyl]-cystine [test compound II] against [D-Ala2, D-Leu5]-enkephalin in mouse vas deferens and morphine or ethylketocyclazocine in guinea-pig ileum were 44.9 nM and 5.00 or 11.3 microM, respectively, showing that test compound II was a potent selective opioid delta antagonist. In conclusion, among compounds synthesized, two new opioid delta-receptor ligands, one being a highly selective agonist and the other being a potent selective antagonist in in vitro isolated preparations, were found in the present study.     

In vivo pharmacological characterisation of bilastine, a potent and selective histamine H1 receptor antagonist

OBJECTIVE: We set out to establish the in vivo histamine H(1) receptor antagonistic (antihistaminic) and antiallergic properties of bilastine. METHODS: In vivo antihistaminic activity experiments consisted of measurement of: inhibition of increase in capillary permeability and reduction in microvascular extravasation and bronchospasm in rats and guinea pigs induced by histamine and other inflammatory mediators; and protection against lethality induced by histamine and other inflammatory mediators in rats. In vivo antiallergic activity experiments consisted of measurement of passive and active cutaneous anaphylactic reactions as well as type III and type IV allergic reactions in sensitised rodents. RESULTS: In the in vivo antihistaminic activity experiments, bilastine was shown to have a positive effect, similar to that of cetirizine and more potent than that of fexofenadine. The results of the in vivo antiallergic activity experiments showed that the properties of bilastine in this setting are similar to those observed for cetirizine and superior to fexofenadine in the model of passive cutaneous anaphylactic reaction. When active cutaneous anaphylactic reaction experiments were conducted, bilastine showed significant activity, less potent than that observed with cetirizine but superior to that of fexofenadine. Evaluation of the type III allergic reaction showed that of the antihistamines only bilastine was able to inhibit oedema in sensitised mice, although its effect in this respect was much less potent than that observed with dexamethasone. In terms of the type IV allergic reaction, neither bilastine, cetirizine nor fexofenadine significantly modified the effect caused by oxazolone. CONCLUSIONS: The results of our in vivo preclinical studies corroborate those obtained from previously conducted in vitro experiments of bilastine, and provide evidence that bilastine possesses antihistaminic as well as antiallergic properties, with similar potency to cetirizine and superior potency to fexofenadine.     

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.     

Polymyxin B is a selective and potent antagonist of calmodulin

Polymyxin B, a cyclic peptide antibiotic, is considered to be a rather selective antagonist of protein kinase C. This drug is therefore widely used to evaluate the involvement of protein kinase C in cellular processes. In the present study, we investigated the effects of polymyxin B on the activity of calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase in vitro. The drug potently inhibited this enzyme (IC50 80 nM in the presence of 500 microM Ca2+), while about 200-fold higher concentrations were required to inhibit protein kinase C to the same extent. Phosphodiesterase inhibition was competitive with respect to Ca2+ and calmodulin. Evidence for the formation of a complex between polymyxin B and calmodulin was obtained by polyacrylamide gel electrophoresis under non-denaturing conditions, and by affinity chromatography of calmodulin on polymyxin B-agarose. We therefore suggest that, at least in vitro, polymyxin B is a potent and selective inhibitor of calmodulin.     

In vitro phamacology of MK-996, a new potent and selective angiotensin II (AT1) receptor antagonist

MK-996 (N-((4′-((5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl) (1,1′-biphenyl)-2-yl) sulfonylbenzamide) interacted in a competitive manner with rabbit aortic angiotensin II (All) receptors as determined by Scatchard analysis of specific binding of [¹²⁵l]-Sar¹lle⁸-All. MK-996 also exhibited high affinity at All receptors in several tissues from different animal species (K_i = 0.1–0.4 nM). In vitro functional assays utilizing All-induced aldosterone release in rat adrenal cortical cells demonstrated further that MK-996 acts as a competitive, high affinity antagonist of All (pA₂ = 10.3) and lacks agonist activity. MK-996 also potently inhibited All-induced contractile response in isolated rabbit aorta and pulmonary artery with a reduction in maximal response. The specificity of MK-996 for All receptors was demonstrated by its lack of activity (IC₅₀> 1 μM) in several other receptor binding assays and its inability to affect in vitro functional responses produced by other agonists. MK-996 demonstrated a very high selectivity for the AT₁ compared to AT₂ receptor subtype (AT₂ IC₅₀ ≥ 2 μM). Direct binding studies using [³H]-MK-996 in rat adrenal indicated specific binding of [³H]-MK-996 is saturable and of high affinity (K_d = 0.47 nM). The specific [³H]-MK-996 binding in rat adrenal represents binding to pharmacologically relevant AT₁ receptors as demonstrated by the similar K_i values for various All agonists and antagonists in inhibiting specific ³H-MK-996 and [¹²⁵l]-All binding to AT₁ receptors. Dissociation rate studies of specific [³H]-MK-996 binding indicated a t1/2 of 103 min. This slow dissociation may account for the reduction in maximal responses to All in MK-996 treated isolated blood vessels.     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.     

5'-Guanidinonaltrindole, a highly selective and potent kappa-opioid receptor antagonist

5'-Guanidinonaltrindole (GNTI) possesses 5-fold greater opioid antagonist potency (K(e)=0.04 nM) and an order of magnitude greater selectivity (selectivity ratios >500) than the prototypical kappa-opioid receptor antagonist, norbinaltorphimine, in smooth muscle preparations. Binding and functional studies conducted on cloned human opioid receptors expressed in Chinese hamster ovarian (CHO) cells afforded pA(2) values that were comparable to the smooth muscle data. In view of the high selectivity and potency of GNTI, it is a potentially valuable pharmacological tool for opioid research.     

Eplerenone: a new selective aldosterone receptor antagonist

Eplerenone is a specific aldosterone receptor antagonist that has been shown to have antihypertensive efficacy and to reduce end-organ manifestations in experimental animal models. Studies in humans have confirmed the blood pressure-lowering efficacy of this agent, as well as providing evidence of benefit in congestive heart failure. The side-effect profile indicates that the specificity of eplerenone for the mineralocorticoid receptor is responsible for the lower incidence of sex hormone- related side-effects than have been seen with other mineralocorticoid receptor blockers. Hyperkalemia is most frequently observed with eplerenone among patients with severe impairment of renal function and in diabetics with microalbuminuria, especially in combination with angiotensin-converting enzyme inhibitors. This agent appears to be a promising and effective new addition for the treatment of hypertension and heart failure with few of the side-effects that have plagued earlier drugs of this class.     

Buprenorphine is a potent kappa-opioid receptor antagonist in pigeons and mice

Buprenorphine was studied for its antagonist activity against the specific kappa-opioid agonist U-50,488H in pigeons responding under a multiple schedule of grain presentation and in mice in an antinociception test. U-50,488H decreased rates of responding of pigeons over the dose range (2.5-20 mg/kg i.m.). In the presence of 0.32 mg/kg of buprenorphine, the U-50,488H dose-effect curve was shifted to the right approximately two-fold. Buprenorphine alone (0.01-0.08 mg/kg s.c.) inhibited in mice the abdominal stretching induced by i.p. acetic acid. beta-Funaltrexamine pretreatment blocked the mu-like agonist analgesic effect of buprenorphine and revealed an antagonist action of buprenorphine against 2.5 mg/kg of U-50,488H over the same dose range that it produced antinociception at the mu-receptor. Thus, buprenorphine is a potent kappa-opioid receptor antagonist, producing the kappa-antagonist activity over the same dose range that it produces its mu-mediated partial agonist activity.     

Characterization of the binding of [3H]L-365,260: a new potent and selective brain cholecystokinin (CCK-B) and gastrin receptor antagonist radioligand

[3H]L-365,260, [(3R-(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-N'-(3-methylphenyl)urea], a new potent and selective nonpeptide brain cholecystokinin (CCK-B) and gastrin receptor antagonist, bound saturably and reversibly to guinea pig brain membranes. Scatchard analysis indicated a single class of high affinity (Kd = 2.3 nM) binding sites. The binding of [3H]L-365,260 was stereospecific, because unlabeled L-365,260 (an R-enantiomer) was approximately 100 times more potent than its S-enantiomer in displacing binding. The relative potencies of various CCK/gastrin-related peptides and nonpeptide peripheral CCK-A antagonists in displacing [3H]L-365,260 brain binding correlated with their potencies in displacing the binding of 125I-CCK to brain receptors but not their potencies in displacing the peripherally selective CCK-A ligand [3H]L-364,718 from pancreatic receptors. The regional distribution of [3H]L-365,260 binding in various brain areas correlated with 125I-CCK binding. Specific [3H]L-365,260 binding to guinea pig brain membranes was reduced by omission of NaCl but was not affected by omission of MgCl2 or addition of guanosine 5'-(beta-gamma-imido)triphosphate or various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. [3H]L-365,260 also bound in a specific manner to guinea pig gastric glands but only negligibly to guinea pig or rat pancreas. The binding of [3H]L-365,260 to gastric glands was inhibited by CCK/gastrin antagonists with potencies similar to those for inhibition of 125I-gastrin binding in this tissue. Collectively, the data indicates that [3H]L-365,260 represents a new potent nonpeptide antagonist radioligand suitable for the study of brain CCK-B and gastrin receptors.     

Bulbocapnine is not a selective DA1 receptor antagonist

Antagonist activity of bulbocapnine on DA1 versus DA2 dopamine receptors was studied simultaneously in a dog under pentobarbitone anaesthesia and without phenoxybenzamine pretreatment. Fenoldopam (SK&F 82526) injected into the renal artery was the DA1 agonist and dipropyl dopamine or piribedil injected into the femoral artery were the DA2 agonists. Both at 0.5 and 1 mg kg-1 doses intravenous bulbocapnine caused nearly equal inhibition of DA1 and DA2 dopamine receptor-mediated responses. Our results show that under these experimental conditions bulbocapnine is not a selective DA1 dopamine antagonist.     

The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist.

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors.(ABSTRACT TRUNCATED AT 250 WORDS)