1,4-Benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonists.

A series of 1,4-benzodiazepines, N-1-substituted with an N-isopropyl-N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.

A centralized global automation group in a decentralized organization.

In the latter part of the 1990s, many companies have worked to foster a 'matrix' style culture through several changes in organizational structure. This type of culture facilitates communication and development of new technology across organizational and global boundaries. At Glaxo Wellcome, this matrix culture is reflected in an automation strategy that relies on both centralized and decentralized resources. The Group Development Operations Information Systems Robotics Team is a centralized resource providing development, support, integration, and training in laboratory automation across businesses in the Development organization. The matrix culture still presents challenges with respect to communication and managing the development of technology. A current challenge for our team is to go beyond our recognized role as a technology resource and actually to influence automation strategies across the global Development organization. We shall provide an overview of our role as a centralized resource, our team strategy, examples of current and past successes and failures, and future directions.

Sulfated cholecystokinin (26-33) induces mild taste aversion conditioning in rats when administered by three different routes.

We investigated the ability of sulfated cholecystokinin (26-33) (CCK-8) and cholecystokinin (30-33) (CCK-4) to induce taste aversion or avoidance conditioning (TAC) in a one-bottle testing paradigm after either intravenous (i.v.), intracerebroventricular (i.c.v.), or intraperitoneal (i.p.) administration. Significant TAC was induced by i.p. administration of CCK-8 at 0.1 but not at 0.025, 0.5, or 1.0 micromol/kg; the TAC was not robust and, in this case, not even dose related. I.p. administration of CCK-4 at 0.05, 0.1, 0.5, or 1.0 micromol/kg did not induce TAC, replicating other studies from our lab. Mild but significant TAC was also induced by i.v. administration of CCK-8 (at 0.025 and 1.0 but not 0.1 or 0.5 micromol/kg) but not by i.v. administration of CCK-4 (at 0.05, 0.1, 0.5, or 1.0 micromol/kg). Finally, mild but significant TAC was induced by i.c.v. (i.e., lateral ventricular) administration of CCK-8 (at 0.0015 but not at 0.015 micromol/brain) but not by i.c.v. administration of CCK-4 (at 0.005 or 0.010 micromol/brain). Because CCK-4 failed to induce TAC, CCK-8 apparently induced TAC via all three routes by an action at a CCK(a), not CCK(B), receptor mechanism. Because i.c.v. or i.v. administrations of CCK-8 were not more efficacious than i.p. administration, the taste avoidance induced by i.p. administration of CCK-8 was not so mild simply because it failed to reach a critical central locus adequately or because it failed to be delivered at a critical speed (i.e., via i.v. injections). We demonstrate that CCK-8 can induce mild TAC at either peripheral or central sites and suggest that these effects of CCK-8 may be independent and may be a sign of salience but not necessarily of toxicosis.

Optimization of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepines as potent, orally active CCK-A agonists.

We previously described a series of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepine CCK-A agonists exemplified by compound 1 (GW 5823), which is the first reported binding selective CCK-A full agonist demonstrating oral efficacy in a rat feeding model. In this report we describe analogs of compound 1 designed to explore changes to the C3 and N1 pharmacophores and their effect on agonist activity and receptor selectivity. Agonist efficacy in this series was affected by stereoelectronic factors within the C3 moiety. Binding affinity for the CCK-A vs CCK-B receptor showed little dependence on the structure of the C3 moiety but was affected by the nature of the second substituent at C3. Structure-activity relationships at the N1-anilidoacetamide "trigger" moiety within the C3 indazole series were also investigated. Both agonist efficacy and binding affinity within this series were modulated by variation of substituents on the N1-anilidoacetamide moiety. Evaluation of several analogs in an vivo mouse gallbladder emptying assay revealed compound 1 to be the most potent and efficacious of all the analogs tested. The pharmacokinetic and pharmacodynamic profile of 1 in rats is also discussed.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity (II): Optimization of the C3 amino substituent.

Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3 phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was retained with a variety of substituted ureas and amide analogs. Three compounds were identified which were orally active in the mouse gallbladder emptying assay (MGBE). The 2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative (29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding assays. GW7854 was a full agonist (EC50 = 60 nM) for calcium mobilization on CHO K1 cells expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA2 = 9.1) on CHO K1 cells expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist which is orally active in two in vivo models of CCK-A-mediated agonist activity.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger".

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.

Several roles of CCKA and CCKB receptor subtypes in CCK-8-induced and LiCl-induced taste aversion conditioning.

Administration of a relatively large IP dose of sulfated cholecystokinin (26-33) (CCK-8; 1.0 mumol/kg) consistently induced moderate taste aversion conditioning (TAC) using a 20-min, one-bottle test in Long-Evans rats. Because CCK-8 has affinity for both CCKA and CCKB receptor subtypes, we wanted to determine the subtype involved in CCK-8-induced TAC. Pretreatment with the selective CCKA antagonist MK-329 (L-364, 718 or devazepide), at doses of 0.1, 1.0, or 10.0 mumol/kg, markedly antagonized (> 70%) CCK-8-induced TAC. Pretreatment with the selective CCKB antagonist L-365,260, at doses of 0.1 or 1.0 mumol/kg, partially antagonized (approximately 50%) CCK-8-induced TAC, although the highest dose of L-365,260. 10.0 mumol/kg, did not. These partial antagonistic effects of L-365,260 on CCK-8-induced TAC were replicated in our second study. In our third study, we observed that another CCKB antagonist, the dipeptoid CI-988, also partially antagonized CCK-8-induced TAC at a dose of 0.1, but not 1.0 or 10.0, mumol/kg. In our final study, pretreatments with a single dose (i.e., 10.0, but not 0.1 or 1.0, mumol/kg) of either MK-329 or L-365,260 were also shown to partially antagonize the formation of moderate TAC induced by treatment with LiCl at 708 mumol/kg. Marked antagonism of LiCl-induced TAC was also observed following pretreatment with the known anxiolytic chlordiazepoxide HCl at 7.4 mumol/kg. Considering the existing data on the induction of TAC by various CCK analogues, we consider an action of CCK-8 on peripheral CCKA, but not CCKB, receptors necessary for the induction of TAC. Our results of partial antagonism of CCK-8- and LiCl-induced TAC by L-365,260, CI-988, or MK-329 suggest, but do not prove, that both CCKA and CCKB mechanisms may be operative during TAC. Because the CCK antagonists affected TAC like chlordiazepoxide, blockade of CCKA and CCKB mechanisms may produce a mild anxiolytic effect.

Modification of receptor selectivity and functional activity in cholecystokinin peptoid ligands.

Hybrid analogs of the cholecystokinin A (CCK-A) receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (1,A-71623) and the CCK-B receptor selective antagonists PD-135118 (2) and CI-988 (3) were prepared. Incorporation of the Lys(Tac) side chain into 2 produced a moderately potent antagonist of CCK-8 in the isolated guinea pig gallbladder (GPGB). Incorporation of the Lys(Tac) side chain into 3 produced the novel agonist analog 7 (EC50 = 28 nM in the GPGB) with excellent affinity for both human CCK-A (IC50 = 12 nM) and CCK-B (IC50 = 17 nM) receptors. Analog 7 was a full agonist (EC50 = 3.5 nM) for calcium mobilization on CHO-K1 cells expressing hCCK-A receptors but a partial agonist on CHO-K1 cells expressing hCCK-B receptors, eliciting a weak agonist response (EC50 = 2800 nM) and antagonizing CCK-8-induced calcium mobilization (KB = 20 nM). Despite this unusual in vitro profile, analog 7 was a potent anorectic agent in rats (ED50 = 30 nmol/kg) following intraperitoneal administration.

The effects of anorectic and aversive agents on deprivation-induced feeding and taste aversion conditioning in rats.

We compared the effects of intraperitoneally administered LiCl (0.5-2830 mumol/kg), sulfated cholecystokinin26-33 (10-1000 nmol/kg; CCK-8), nonsulfated CCK-8 (500 and 1000 nmol/kg), sulfated CCK26-29 (500 and 1000 nmol/kg), CCK30-33 (10-1000 nmol/kg) bombesin (10-1000 nmol/kg; BOM), (dl) fenfluramine HCl (0.9-37.3 mumol/kg; fenfluramine), fluoxetine HCl (2.9-86.7 mumol/kg; fluoxetine), and d-amphetamine sulfate (0.27-10.9 mumol/kg; AMPH) on both 18-hr deprivation-induced feeding and one-bottle, taste aversion conditioning in male, Long-Evans rats. Doses of LiCl > or = 177 mumol/kg (or 7.5 mg/kg) induced significant, dose-related taste aversions, but only doses of LiCl > or = 2123 mumol/kg (90 and 120 mg/kg) induced significant anorexia. CCK-8 induced marked anorexia (at doses > or = 25-50 nmol/kg), but only relatively mild taste aversions which were only statistically significant at the highest dose (1000 nmol/kg). The anorectic effects of CCK-8 at 500 and 1000 nmol/kg, but not at lower doses, lasted at least 3 hr. Sulfated CCK26-29, CCK30-33 and nonsulfated CCK-8 induced neither anorexia nor taste aversion. BOM induced marked anorexia at all doses tested, but did not induce statistically significant taste aversions. The nonpeptidal anorectic compounds, fenfluramine, fluoxetine, and AMPH, induced both dose-related anorexia and taste aversion conditioning. We focus on several issues concerning the interpretation of taste aversion conditioning. Our results challenge any simple relationship between the ability of a compound to induce taste aversion and to decrease feeding.

Multiple, small doses of cholecystokinin octapeptide are more efficacious at inducing taste aversion conditioning than single, large doses.

Using a one-bottle taste aversion conditioning paradigm, sulfated cholecystokinin(26-33) (CCK-8) has again been shown to induce taste aversion conditioning in rats. Even though the effective doses of CCK-8 are relatively high, they do not induce as strong an aversion as has been demonstrated with LiCl. This pharmacodynamic profile of CCK-8 (i.e., relatively moderate, but not strong, taste aversion induction) may result, in part, from its unusual pharmacokinetic profile. CCK-8 seems to have a plasma half-life of just several minutes, whereas LiCl has a plasma half-life of 6 h in rats. In the present study, CCK-8, CCK-4, or LiCl was administered either as single, large doses immediately following consumption of 0.2% sodium saccharin (SACC), or as 10 half-hourly injections of one-tenth the large dose. Presumably, multiple small doses extended the time CCK-8 and CCK-4 were acting in the body, even though the peak plasma concentrations were quantitatively lower than after the large, single doses. Ten injections of CCK-8 of 10 or 100 nmol/kg (11.4 or 114.3 micrograms/kg) induced significantly stronger taste aversions than single injections of the same total dose of 100 or 1000 nmol/kg (114.3 or 1143.3 micrograms/kg), whereas multiple injections of LiCl of 70.8 mumol/kg (3.0 mg/kg x 10) did not induce stronger taste aversions than single injections of 708 mumol/kg (30.0 mg/kg). Neither single nor multiple injections of CCK-4 of 1000 nmol/kg (596.7 micrograms/kg) x 1, or 100 or 1000 nmol/kg (59.7 or 596.7 micrograms/kg) x 10 induced any sign of taste aversion conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotensin blocks certain amphetamine-induced behaviours.

Bilateral injections of either neurotensin (NT; 0.3, 1 or 5 micrograms in 1 microliter artificial CSF) or haloperidol (HA; 2.5 or 5 micrograms in 1 microliter 0.3% tartaric acid) into nucleus accumbens of rats markedly diminished the forward locomotion and rearing induced by d-amphetamine (AM; 2 mg per kg, IP). Neither NT nor HA affected the insomnia or sniffing component of AM arousal. Isovolumetric intra-accumbens injections of artificial CSF or the endogenous decapeptide, luteinizing hormone-releasing hormone (LHRH; 3 micrograms), did not affect AM behaviours. Since intra-accumbens injections of NT (1 microgram) or HA (2.5 micrograms) neither altered forward locomotion or rearing observed in untreated rats placed in an open field nor a variety of reflex activities, the observed effects of NT and HA in AM-treated rats were probably not due to impaired motor function per se. In contrast, NT does not produce neuroleptic-like effects when injected into nucleus caudatus; HA (5 micrograms) blocked stereotyped sniffing, licking, biting and head bobbing observed after AM (5 mg per kg, IP), but NT (3 or 5 micrograms) did not. Since NT and dopamine are present in substantial quantities in the nucleus accumbens, NT may act in the nucleus accumbens to modulate dopaminergic function.