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8. Pharmacological comparison of the alternatively spliced short and long CCK2 receptors

Author

Morton, M F, Harper, E A, Tavares, I A, and Shankley, N P

Subjects
Mice, Indoles, Dose-Response Relationship, Drug, Papers, NIH 3T3 Cells, Animals, Humans, Receptor, Cholecystokinin B, Protein Binding
Abstract

(1) The alternatively spliced, short and long cholecystokinin receptors (CCK2S and CCK2L) were expressed in NIH3T3 cells, and compared using radioligand-binding assays with identical buffer and incubation conditions. (2) As judged by a saturation analysis, the selective CCK2-receptor antagonist radioligand [3H]-JB93182 did not discriminate between the CCK2S or CCK2L receptors. (3) A global analysis of competition studies, using a range of structurally diverse, CCK-receptor selective ligands, provided further evidence that these receptor subtypes were pharmacologically indistinguishable. However, when analysed individually a number of small, yet significant differences were observed with some of the compounds. (4) These data are consistent with previous study that suggested a possible pharmacological difference between these isoforms, at least in terms of the CCK2-receptor antagonist, L-365,260. However, it would appear that the pharmacological profile of these compounds is not consistent with their affinity at the putative G1/G2 receptors previously described by Harper et al.

Published
2003

9. Pharmacological evidence for putative CCK1 receptor heterogeneity in human colon smooth muscle

Author

Morton, M F, Harper, E A, Tavares, I A, and Shankley, N P

Subjects
Aspartic Acid, Binding Sites, Diazepam, Membranes, Time Factors, Indoleacetic Acids, Colon, Gallbladder, Muscle, Smooth, Binding, Competitive, Devazepide, Models, Biological, Receptor, Cholecystokinin A, Kinetics, Radioligand Assay, Thiazoles, Organ Culture Techniques, Naphthalenesulfonates, Organ Specificity, Papers, Humans, Protease Inhibitors, Receptors, Cholecystokinin
Abstract

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established.

Published
2002

10. Characterization of the binding of [3H]-clobenpropit to histamine H3-receptors in guinea-pig cerebral cortex membranes

Author

Harper, E A, Shankley, N P, and Black, J W

Subjects
Cerebral Cortex, Guinea Pigs, Histamine Antagonists, Imidazoles, Thiourea, In Vitro Techniques, Metyrapone, Tritium, Binding, Competitive, Kinetics, Radioligand Assay, Piperidines, Papers, Animals, Receptors, Histamine H3
Abstract

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.

Published
1999

24. Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (α) from functional bioassays.

Author

Harper, E. A., Shankley, N. P., and Black, J. W.

Subjects
*HISTAMINE receptors, *BIOLOGICAL assay, *RADIOLIGAND assay, *BIOCHEMISTRY, *CORRUPT practices in pharmaceutical research
Abstract

Background and purpose:Agonist apparent affinities (pKI′) in histamine H3-receptor binding assays were higher than expected from apparent affinity values (pKapp) estimated in bioassay. Here, we investigate whether the degree of pKI′ overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pKI′ of ligands with varying intrinsic activity.Experimental approach:In the guinea-pig ileum bioassay, intrinsic activity (α) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pKapp values were estimated using the method of Furchgott. The pKL of [3H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl2, 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PKI values were determined in competition studies in both buffers.Key results:[3H]clobenpropit saturation isotherms had n H values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pKI′ values were closer to pKapp values than in buffer B(0,0,0) but were associated with n H values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pKIL) were comparable to pKapp. Differences between the pKI′ in buffer B(0,0,0) and pKIL values in buffer B(0.07,0.1,0.1) (ΔpK) were correlated with α.Conclusions and implications:H3-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pKapp) and intrinsic efficacy. The assay predicts that some ligands previously classified as H3-receptor antagonists may possess residual intrinsic efficacy.British Journal of Pharmacology (2007) 151, 128–143. doi:10.1038/sj.bjp.0707174; published online 12 March 2007 [ABSTRACT FROM AUTHOR]

Published
2007
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27. Optimization of the in Vitro and in Vivo Properties of a Novel Series of 2,4,5-Trisubstituted Imidazoles as Potent Cholecystokinin-2 (CCK<INF>2</INF>) Antagonists

Author

Buck, I. M., Black, J. W., Cooke, T., Dunstone, D. J., Gaffen, J. D., Griffin, E. P., Harper, E. A., Hull, R. A. D., Kalindjian, S. B., Lilley, E. J., Linney, I. D., Low, C. M. R., McDonald, I. M., Pether, M. J., Roberts, S. P., Shankley, N. P., Shaxted, M. E., Steel, K. I. M., Sykes, D. A., Tozer, M. J., Watt, G. F., Walker, M. K., Wright, L., and Wright, P. T.

Abstract

The systematic optimization of the structure of a novel 2,4,5-trisubstituted imidazole-based cholecystokinin-2 (CCK2) receptor antagonist afforded analogues with nanomolar receptor affinity. These compounds were now comparable in their potency to the bicyclic heteroaromatic-based compounds 5 (JB93182) and 6 (JB95008), from which the initial examples were designed using a field-point based molecular modeling approach. They were also orally active as judged by their inhibition of pentagastrin stimulated acid secretion in conscious dogs, in contrast to the bicyclic heteroaromatic-based compounds, which were ineffective because of biliary elimination. Increasing the hydrophilicity through replacement of a particular methylene group with an ether oxygen, as in 3-{[5-(adamantan-1-yloxymethyl)-2-cyclohexyl-1H-imidazole-4-carbonyl]amino}benzoic acid (53), had little effect on the receptor affinity but significantly increased the oral potency. Comparison of the plasma pharmacokinetics and the inhibition of pentagastrin-stimulated acid output following bolus intraduodenal administration of both 53 and 6 indicated that 53 was well absorbed, had a longer half-life, and was not subject to the elimination pathways of the earlier series.

Published
2005

28. Scaffold Hopping with Molecular Field Points:  Identification of a Cholecystokinin-2 (CCK<INF>2</INF>) Receptor Pharmacophore and Its Use in the Design of a Prototypical Series of Pyrrole- and Imidazole-Based CCK<INF>2</INF> Antagonists

Author

Low, C. M. R., Buck, I. M., Cooke, T., Cushnir, J. R., Kalindjian, S. B., Kotecha, A., Pether, M. J., Shankley, N. P., Vinter, J. G., and Wright, L.

Abstract

A new molecular modeling approach has been used to derive a pharmacophore of the potent and selective cholecystokinin-2 (CCK2) receptor antagonist 5 (JB93182), based on features shared with two related series. The technique uses “field points” as simple and effective descriptions of the electrostatic and van der Waals maxima and minima surrounding a molecule equipped with XED (extended electron distribution) charges. Problems associated with the high levels of biliary elimination of 5 in vivo required us to design a compound with significantly lower molecular weight without sacrificing its nanomolar levels of in vitro activity. Two new series of compounds were designed to mimic the arrangement of field points present in the pharmacophore rather than its structural elements. In a formal sense, two of the three amides in 5 were replaced with either a simple pyrrole or imidazole, while some features thought to be essential for the high levels of in vitro activity of the parent compounds were retained and others deleted. These compounds maintained activity and selectivity for this receptor over CCK1. In addition, the reduction in molecular weight coupled with lower polarities greatly reduced levels of biliary elimination associated with 5. This makes them good lead compounds for development of drug candidates whose structures are not obviously related to those of the parents and represents the first example of scaffold hopping using molecular field points.

Published
2005

30. Nonpeptide Cholecystokinin-2 Receptor Agonists

Author

Kalindjian, S. B., Dunstone, D. J., Low, C. M. R., Pether, M. J., Roberts, S. P., Tozer, M. J., Watt, G. F., and Shankley, N. P.

Abstract

In the course of structural explorations around a series of potent CCK2 receptor antagonists, it was noted that simple N-methylation of the indolic N−H in the parent molecule gave rise to behavior in vivo that was consistent with the compound acting as an agonist. Exploration in vitro confirmed this property, and it was shown that the agonist action could be blocked by the reference CCK2 receptor antagonist, L-365,260. Further examples of this type of modification were explored, and a common theme with regard to agonist behavior was uncovered. Some molecular modeling is also presented in an attempt to throw light on the nature of the ligand receptor interactions that may be giving rise to the differing properties of these, apparently, structurally similar molecules.

Published
2001

31. Development of Peptide 3D Structure Mimetics:  Rational Design of Novel Peptoid Cholecystokinin Receptor Antagonists

Author

Low, C. M. R., Black, J. W., Broughton, H. B., Buck, I. M., Davies, J. M. R., Dunstone, D. J., Hull, R. A. D., Kalindjian, S. B., McDonald, I. M., Pether, M. J., Shankley, N. P., and Steel, K. I. M.

Abstract

The two hormones cholecystokinin and gastrin share the same C-terminal sequence of amino acids, namely Gly29-Trp30-Met31-Asp32-Phe33-NH2. Nevertheless, this congruence has not precluded using this structure to develop selective ligands for either CCK1 or CCK2 receptors. Manipulation of the hydrophobic residues at positions 31 and 33 gave a series of CCK1 tripeptide antagonists, typified by N-t-BOC-Trp-2-Nal-Asp-2-(phenyl)ethylamide (pKB 6.8 ± 0.3). Molecular modeling was used to identify the bioactive conformation of these CCK1-selective compounds and prompted the design of new peptoid structures. We aimed to maintain the conformation of the parent series by exploiting patterns of hydrogen-bonding and π-stacking interactions present in the original molecule, rather than introducing additional covalent bonds. The prototype, N-(succinyl-d-Asp-2-phenylethylamido)-l-Trp-2-(2-naphthyl)ethylamide, was a potent and selective CCK1 antagonist (pKB 7.2 ± 0.3). Furthermore, the new series showed patterns of biological activity that mirrored those of the parent tripeptides. These compounds contain elements of both peptide primary and secondary structure and represent a novel approach to designing peptidomimetics. Interesting results were obtained from comparing models of a representative tripeptide CCK1 antagonist with a conformation of CCK30-33 that others have proposed to be responsible for its activity at the CCK2 receptor. The results suggest that CCK1 and CCK2 receptors recognize enatiomeric dispositions of the Trp30 indole, Asp32 carboxylic acid, and C-terminal phenyl groups arrayed about a common backbone configuration. This “functional chirality” may underpin the mechanism by which these closely related receptor systems bind CCK30-33 and explain patterns of selectivity observed with optical isomers of a number of peptoid and nonpeptide ligands.

Published
2000

32. Design, Synthesis, and Structure−Activity Relationships of Novel Non-Imidazole Histamine H<INF>3</INF> Receptor Antagonists

Author

Linney, I. D., Buck, I. M., Harper, E. A., Kalindjian, S. B., Pether, M. J., Shankley, N. P., Watt, G. F., and Wright, P. T.

Abstract

Novel, potent, and selective non-imidazole histamine H3 receptor antagonists have been prepared based on the low-affinity ligand dimaprit (pKI 7.32 ± 0.12, pKB 5.93 ± 0.17). Detailed structure−activity studies have revealed that N-(4-chlorobenzyl)-N-(6-pyrrolidin-1-ylhexyl)guanidine (pKI 8.38 ± 0.21, pKB 8.39 ± 0.13), 30, and N-(4-chlorobenzyl)-N-(7-pyrrolidin-1-ylheptyl)guanidine (pKI 8.78 ± 0.12, pKB 8.38 ± 0.10), 31, exhibit high affinity for the histamine H3 receptor. Antagonists 30 and 31 demonstrate significant selectivity over the other histamine, H1 and H2, receptor subtypes and a 100-fold selectivity in the σ1 binding assay. Compounds 30 and 31 are the most potent, selective non-imidazole histamine H3 receptor antagonists reported in the literature to date.

Published
2000

33. An improved in vitrobioassay for the study of 5‐HT4receptors in the human isolated large intestinal circular muscle

Author

Prins, N H, Shankley, N P, Welsh, N J, Briejer, M R, Lefebvre, R A, Akkermans, L M A, and Schuurkes, J A J

Abstract

Recently, it was demonstrated that 5‐HT induces relaxation of human colon circular muscle through activation of 5‐HT4receptors and 5‐HT7receptors. The aim of the current study was to develop a new in vitrobioassay of human colon that would facilitate the pharmacological analysis of 5‐HT responses mediated solely by 5‐HT4receptors.Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully‐defined, agonist concentration‐response curves by cumulative dosing. Under these conditions, 5‐HT induced a concentration‐dependent relaxation (pEC507.31, Hill slope 0.91).Neither methysergide (10 μM) nor granisetron (1 μM) affected the 5‐HT‐induced relaxation, suggesting that 5‐HT1, 5‐HT2, 5‐HT3, 5‐ht5, 5‐HT6or 5‐HT7receptors are not involved. The lack of effect of tetrodotoxin (0.3 μM) indicated a direct effect of 5‐HT on the smooth muscle.The selective 5‐HT4receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5‐HT‐induced relaxation (pKB9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA210.34) and depression of the 5‐HT curve. These affinity estimates are similar to those previously reported for 5‐HT4receptors.The selective 5‐HT4receptor agonists, prucalopride and R076186, induced relaxations (pEC507.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA2estimates of 9.31 and 9.21, respectively.To summarise, in KCl (80 mM)‐contracted muscle strips, 5‐HT induces relaxation through activation of a homogeneous smooth muscle 5‐HT4receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5‐HT4receptors in vitro.

Published
2000
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35. Resultant action of cimetidine in a cardiac adenylate cyclase assay: its elucidation by concentration-ratios analysis.

Author

Trist, D G, Leff, P, Black, J, Gerskowitch, V P, and Shankley, N P

Abstract

Cimetidine is widely used as a ligand for the classification of histamine H2-receptors in peripheral and central tissues. It demonstrates apparently simple competitive antagonism in isolated tissue assays and in brain adenylate cyclase assays. In this study its action in a cardiac adenylate cyclase assay was compared with its effects in the isolated guinea pig right atrial preparation. Using either histamine or impromidine, cimetidine expressed similar affinity (pKB = 6.1) in the intact tissue assay. In washed homogenates of guinea pig ventricle cimetidine appeared to antagonize competitively the stimulation by histamine of adenylate cyclase but the estimated pKB was 6.7. In contrast, the pKB for tiotidine was similar in both assays (7.7). Application of a recently developed concentration-ratios method indicated that cimetidine expressed a resultant action in the cyclase assay consisting of competitive H2-receptor antagonism and an additional inhibitory property involving a postreceptor action. The concentration-ratios analysis allowed the purely competitive component of cimetidine action to be quantified in the cyclase assay giving a pKB of 6.0.

Published
1987

41. Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays.

Author

Harper EA, Shankley NP, and Black JW

Subjects
Animals, Biological Assay, Buffers, Calcium Chloride pharmacology, Guinea Pigs, Ileum drug effects, Ileum physiology, Male, Quinolines pharmacology, Receptors, Histamine H3 analysis, Thiourea metabolism, Tritium, Imidazoles metabolism, Radioligand Assay, Receptors, Histamine H3 metabolism, Thiourea analogs & derivatives
Abstract

Background and Purpose: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity., Experimental Approach: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers., Key Results: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha., Conclusions and Implications: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.

Published
2007
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42. Pharmacological evidence for putative CCK(1) receptor heterogeneity in human colon smooth muscle.

Author

Morton MF, Harper EA, Tavares IA, and Shankley NP

Subjects
Aspartic Acid pharmacology, Binding Sites, Binding, Competitive, Devazepide pharmacology, Diazepam pharmacology, Gallbladder metabolism, Humans, Indoleacetic Acids pharmacology, Kinetics, Membranes, Models, Biological, Naphthalenesulfonates pharmacology, Organ Culture Techniques, Organ Specificity, Protease Inhibitors pharmacology, Radioligand Assay, Receptor, Cholecystokinin A, Thiazoles pharmacology, Time Factors, Aspartic Acid analogs & derivatives, Colon metabolism, Muscle, Smooth metabolism, Receptors, Cholecystokinin metabolism
Abstract

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established., (British Journal of Pharmacology (2002) 136, 873-882)

Published
2002
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43. Histamine receptor assays.

Author

Shankley NP, Morton MF, and Watt GF

Subjects
Animals, Guinea Pigs, Histamine Agonists metabolism, Histamine Antagonists metabolism, In Vitro Techniques, Male, Ileum metabolism, Radioligand Assay methods, Receptors, Histamine metabolism
Abstract

This unit describes three standard in vitro bioassays for studying histamine H₁, H₂ and H₃ receptors in isolated intact tissues removed from the guinea pig. Both the H₁ and H₃ receptor assays are based on preparations of the ileum, whereas the spontaneously beating right atrium assay is used for the H₂-receptor.This unit describes three standard in vitro bioassays for studying histamine H₁, H₂ and H₃ receptors in isolated intact tissue.

Published
2001
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44. Design, synthesis, and structure-activity relationships of novel non-imidazole histamine H(3) receptor antagonists.

Author

Linney ID, Buck IM, Harper EA, Kalindjian SB, Pether MJ, Shankley NP, Watt GF, and Wright PT

Subjects
Animals, Binding, Competitive, Cerebral Cortex metabolism, Dimaprit chemistry, Dimaprit pharmacology, Drug Design, Guanidines chemistry, Guanidines metabolism, Guanidines pharmacology, Guinea Pigs, Histamine Antagonists chemistry, Histamine Antagonists metabolism, Histamine Antagonists pharmacology, Ileum drug effects, Ileum physiology, In Vitro Techniques, Ligands, Muscle Contraction drug effects, Pyrrolidines chemistry, Pyrrolidines metabolism, Pyrrolidines pharmacology, Radioligand Assay, Receptors, Histamine H1 metabolism, Receptors, Histamine H2 metabolism, Receptors, Histamine H3 metabolism, Receptors, sigma metabolism, Structure-Activity Relationship, Dimaprit analogs & derivatives, Dimaprit chemical synthesis, Guanidines chemical synthesis, Histamine Antagonists chemical synthesis, Pyrrolidines chemical synthesis, Receptors, Histamine H3 drug effects
Abstract

Novel, potent, and selective non-imidazole histamine H(3) receptor antagonists have been prepared based on the low-affinity ligand dimaprit (pK(I) 7.32 +/- 0.12, pK(B) 5.93 +/- 0.17). Detailed structure-activity studies have revealed that N-(4-chlorobenzyl)-N-(6-pyrrolidin-1-ylhexyl)guanidine (pK(I) 8.38 +/- 0.21, pK(B) 8.39 +/- 0.13), 30, and N-(4-chlorobenzyl)-N-(7-pyrrolidin-1-ylheptyl)guanidine (pK(I) 8.78 +/- 0.12, pK(B) 8.38 +/- 0.10), 31, exhibit high affinity for the histamine H(3) receptor. Antagonists 30 and 31 demonstrate significant selectivity over the other histamine, H(1) and H(2), receptor subtypes and a 100-fold selectivity in the sigma(1) binding assay. Compounds 30and 31 are the most potent, selective non-imidazole histamine H(3) receptor antagonists reported in the literature to date.

Published
2000
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45. From histamine to imidazolylalkyl-sulfonamides: the design of a novel series of histamine H3-receptor antagonists.

Author

Tozer MJ, Harper EA, Kalindjian SB, Pether MJ, Shankley NP, and Watt GF

Subjects
Animals, Guinea Pigs, Kinetics, Models, Chemical, Drug Design, Histamine Antagonists chemical synthesis, Imidazoles chemical synthesis, Receptors, Histamine H3 chemistry, Sulfonamides chemical synthesis
Abstract

Histamine was converted to a selective histamine H3-receptor antagonist by capping the primary amine with 2-naphthalenesulfonyl chloride. Higher receptor affinity and lower variability in the data from the various bioassays were achieved with the 2-naphthalensulfonamides of histamine homologues.

Published
1999
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46. International Union of Pharmacology. XIII. Classification of histamine receptors.

Author

Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R, and Haas HL

Subjects
Amino Acid Sequence, Animals, Humans, Molecular Sequence Data, Protein Conformation, Receptors, Histamine chemistry, Receptors, Histamine metabolism, Sequence Homology, Amino Acid, Signal Transduction, Receptors, Histamine classification
Published
1997

47. Pharmacological analysis of the CCKB/gastrin receptors mediating pentagastrin-stimulated gastric acid secretion in the isolated stomach of the immature rat.

Author

Hills DM, Gerskowitch VP, Roberts SP, Welsh NJ, Shankley NP, and Black JW

Subjects
Animals, Binding, Competitive drug effects, Enterochromaffin Cells drug effects, Enterochromaffin Cells metabolism, Famotidine pharmacology, Gastric Mucosa drug effects, Histamine H2 Antagonists pharmacology, In Vitro Techniques, Kinetics, Models, Biological, Rats, Rats, Wistar, Receptors, Cholecystokinin antagonists & inhibitors, Stimulation, Chemical, Gastric Acid metabolism, Gastric Mucosa metabolism, Histamine pharmacology, Pentagastrin pharmacology, Receptors, Cholecystokinin drug effects
Abstract

1. The CCKB/gastrin receptors mediating pentagastrin stimulation of gastric acid secretion by histamine release and by direct stimulation of oxyntic cells have been characterized in the immature rat isolated stomach assay. This was achieved by estimating antagonist affinity values for competitive antagonists from three distinct chemical classes (L-365,260, PD134,308 and JB93190) in the absence and presence of a high concentration of the histamine H2-receptor antagonist, famotidine (30 microM). 2. Pentagastrin produced concentration-dependent stimulation of gastric acid secretion in the absence and presence of famotidine. Famotidine depressed the maximum secretory response to pentagastrin although the degree of depression varied between experimental replicates (25-60%). This variation was attributed to the histamine-release mediated component of acid secretion, as judged by the consistency of the maximum responses obtained in the presence, but not absence, of famotidine. 3. All three CCKB/gastrin receptor antagonists behaved as surmountable antagonists in the absence and presence of famotidine. JB93190 (pKB approximately 9.1, approximately 8.9, in the absence and presence of famotidine, respectively) was approximately 30 fold more potent than either L-365,260 (pKB approximately 7.4, approximately 7.1) or PD134,308 (pKB approximately 7.6, approximately 7.4). 4. It was assumed that the famotidine treatment converted pentagastrin-stimulated acid secretion from a combination of an indirect action due to the release of histamine and a direct action on the oxyntic cell to solely a direct action on the oxyntic cell. A simple mathematical model of this two-receptor system was developed. The direct and indirect components were assumed to sum to produce the total response to pentagastrin obtained in the absence of famotidine. It was found that this model could account quantitatively for the behaviour of the three antagonists without invoking a difference in antagonist affinity for the CCKB/gastrin receptors mediating the direct and indirect actions of pentagastrin. However, a conclusion of receptor homogeneity has to be qualified because the model was also used to generate simulations which indicated that the analysis could only detect antagonist affinity differences of greater than one log-unit between enterochromaffin-like (ECL) and oxyntic cell CCKB/gastrin receptor populations.

Published
1996
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48. Analysis of the action of idazoxan calls into question the reliability of the rat isolated small mesenteric artery assay as a predictor for alpha 1-adrenoceptor-mediated pressor activity.

Author

Van der Graaf PH, Shankley NP, and Black JW

Subjects
Adrenergic alpha-Agonists pharmacology, Animals, Clonidine analogs & derivatives, Clonidine pharmacology, Dose-Response Relationship, Drug, Male, Muscle Contraction drug effects, Rats, Rats, Wistar, Reproducibility of Results, Adrenergic alpha-Antagonists pharmacology, Aorta, Thoracic drug effects, Biological Assay methods, Idazoxan pharmacology, Mesenteric Arteries drug effects, Prazosin pharmacology
Abstract

We have studied the effects of idazoxan in rat aorta and small mesenteric artery. In the aorta, idazoxan behaved as a partial agonist (pKA = 6.30). Prazosin produced rightward shift (pA2 = 9.88) and steepening of the idazoxan curve. In contrast, idazoxan had no effect of basal tension in the mesenteric artery, but shifted the noradrenaline curve to the right in a parallel manner (pA2 = 6.12). The selective alpha 1-adrenoceptor agonist, indanidine, also behaved as a partial agonist in the aorta and produced no significant contractions of the small mesenteric artery. Since idazoxan and indanidine have been reported to raise blood pressure in the pithed rat via an action at vascular alpha 1-adrenoceptors, these results call into question the reliability of the small mesenteric artery assay as a predictor for alpha 1-adrenoceptor-mediated pressor activity in vivo.

Published
1996
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49. Analysis of variation in L-365,260 competition curves in radioligand binding assays.

Author

Harper EA, Roberts SP, Shankley NP, and Black JW

Subjects
Animals, Binding, Competitive drug effects, Cerebral Cortex cytology, Cerebral Cortex drug effects, Cerebral Cortex metabolism, Cholecystokinin metabolism, Gastric Mucosa cytology, Gastric Mucosa drug effects, Guinea Pigs, In Vitro Techniques, Iodine Radioisotopes, Male, Mice, Radioligand Assay, Rats, Rats, Wistar, Succinimides, Benzodiazepinones pharmacology, Phenylurea Compounds, Receptors, Cholecystokinin antagonists & inhibitors
Abstract

1. For several years, we have used the cholecystokinin (CCK)B/gastrin receptor selective antagonist, L-365,260, as a reference compound in a variety of studies in CCKB/gastrin receptor radioligand binding assays. Here, we have analysed the competition curve data sets obtained between L-365,260 and [125I]-BH-CCK8S in guinea-pig gastric gland and mouse and rat cerebral cortex preparations. 2. Competition curves obtained for L-365,260 in the mouse cortex assay were not different from rectangular hyperbolae (slope = 1.01 +/- 0.02) implying the presence of a single population of binding sites (pKI = 8.41 +/- 0.01; data from 47 experiments, slope constrained to unity). However, in the rat cortex and guinea-pig gastric gland assays, the mean slope of the competition curves was significantly less than one and the mean apparent pKI significantly lower than that obtained in the mouse cortex (slope = 0.85 +/- 0.03, 0.90 +/- 0.03; apparent pKI = 7.98 +/- 0.05, 8.07 +/- 0.05; 48 and 45 experiments, in rat and guinea-pig, respectively). The distribution of the individual pKI and slope estimates of the competition curves in these two assays was consistent with expectations for the variable expression (in terms of absolute number and proportion) of two binding sites. The two sites were characterized by pKI values for L-365,260 of 8.50 +/- 0.04 and 8.48 +/- 0.04 for the high affinity site and 7.32 +/- 0.04 and 7.22 +/- 0.06 for the low affinity site in guinea-pig and rat, respectively. 3. The affinity estimates for L-365,260, although obtained on different tissues, are consistent with data obtained from the analysis of L-365,260 antagonism of pentagastrin-stimulated responses in mouse and rat stomach (acid secretion) and guinea-pig gastric muscle (isotonic contraction) assays. To this extent, these data suggest the existence of two CCKB/gastrin receptor subtypes.

Published
1996
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50. Analysis of the variation in the action of L-365,260 at CCKB/gastrin receptors in rat, guinea-pig and mouse isolated gastric tissue assays.

Author

Roberts SP, Harper EA, Watt GF, Gerskowitch VP, Hull RA, Shankley NP, and Black JW

Subjects
Animals, Binding, Competitive drug effects, Famotidine pharmacology, Guinea Pigs, Histamine pharmacology, Histamine H2 Antagonists pharmacology, In Vitro Techniques, Mice, Muscle, Smooth drug effects, Muscle, Smooth metabolism, Pentagastrin pharmacokinetics, Rats, Rats, Wistar, Receptors, Cholecystokinin metabolism, Species Specificity, Stomach drug effects, Benzodiazepinones pharmacology, Gastric Mucosa metabolism, Phenylurea Compounds, Receptors, Cholecystokinin antagonists & inhibitors
Abstract

1. Since L-365,260 was first described as a selective antagonist at cholecystokinin (CCK)B/gastrin receptors, we have used it periodically as a reference compound in isolated tissue assays of guinea-pig gastric muscle and lumen-perfused stomachs from mouse and immature rat. L-365,260 behaved as a surmountable antagonist and produced parallel rightward shifts of pentagastrin concentration-effect curves' in each of the replicate experiments. The experiments were performed by several different experimenters in the same laboratories over a five year period. 2. In the isolated, lumen-perfused, immature rat stomach assay, L-365,260 behaved as a simple competitive antagonist (Schild plot slope = 1.00 +/- 0.10, pKB = 7.54 +/- 0.03 from a global analysis of the data) acting at a homogeneous population of receptors in five separate, highly-reproducible, experiments. In contrast, the replicate data sets obtained from the interaction in the isolated, lumen-perfused mouse stomach and guinea-pig gastric muscle assays, over the same period, were not consistent with the presence of a single receptor population. The guinea-pig gastric muscle data were relatively reproducible between experiments but some individual Schild plot slopes and the slope estimated from a global analysis of all the data were significantly less than unity (slope = 0.80 +/- 0.07, pA2 = 8.56 +/- 0.05 from the global analysis). The data obtained in the mouse stomach were significantly more variable than that obtained in the same assay, during the same period, from the interaction between histamine and the H2-receptor antagonist, famotidine. The individual Schild plot slopes ranged from being very flat (0.20) to being not significantly different from unity (1.23) and the pA2 values ranged from 7.68 to 8.70. 3. Overall, the data could be accounted for by assuming the variable expression of two receptor subtypes across the assays. The rat stomach appeared to express a single receptor characterized by a low affinity constant for L-365,260 (pKB approximately 7.5). The guinea-pig gastric muscle and mouse stomach data could be explained by the presence of this receptor and a second one characterized by a high affinity constant for L-365,260 (pKB approximately 8.6). The activity of the two proposed receptor subtypes was consistent between experiments in the guinea-pig and the high affinity receptor appeared to be predominant. In contrast, the mouse stomach data could only be simulated by assuming that the proportion and absolute number of each subtype varied significantly between the replicate experiments. 4. The L-365,260 affinity estimates at the inferred receptor subtypes were indistinguishable from those obtained in a corresponding analysis of the behaviour of L-365,260 in CCKB/gastrin receptor radioligand binding experiments in guinea-pig gastric gland and mouse and rat cerebral cortex preparations.

Published
1996
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