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.

Allelochemical autotoxicity in the emergent wetland macrophyte Juncus effusus (Juncaceae).

Bioassays for allelochemical toxicity of aboveground Juncus effusus tissues were conducted with seeds and seedlings of Eleocharis obtusa and Scirpus cyperinus, two emergent sedge species (Cyperaceae) found sympatric with J. effusus, and with seeds and seedlings of J. effusus itself to evaluate potential autotoxicity. Bioassays were performed under controlled, axenic conditions with aqueous shoot extract treatments simulating in situ dissolved organic carbon concentrations. With respect to the two sedge species, neither shoot development nor seedling biomass accrual was significantly suppressed by lyophilized whole extracts from J. effusus. Although the extracts induced no significant reduction in growth of E. obtusa or S. cyperinus, biomass-specific chlorophyll a concentration was significantly reduced in E. obtusa seedlings. In contrast, seedlings of J. effusus exhibited significant reductions of biomass and chlorophyll a concentrations, and seedling shoot development was retarded in response to leachate exposure. Results of the present study suggest that J. effusus seedlings possess autotoxic sensitivity to extracts of dead, aboveground tissues of adult plants.

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.

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.

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.

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)

Potential corticotropin-releasing factor pathways in the rat brain as determined by bilateral electrolytic lesions of the central amygdaloid nucleus and the paraventricular nucleus of the hypothalamus.

The projection fields of corticotropin-releasing factor (CRF)-containing perikarya in the rat central nervous system were studied using a combination of electrolytic lesions, microdissection and radioimmunoassay. The effects of bilateral electrolytic lesions of the central nucleus of the amygdala (Ce) or the paraventricular nucleus (PVN) of the hypothalamus were measured by a sensitive and specific radioimmunoassay. Following lesions of the Ce, CRF concentrations in the locus ceruleus (LC) were significantly decreased. Following lesions of the PVN, CRF concentrations in the median eminence were markedly decreased (> 85%), with smaller but consistent reductions of CRF in the hippocampus as well. In contrast to the Ce lesions, PVN lesions resulted in increases in CRF concentrations in the LC. These results further confirm the projection of CRF-containing cells from the PVN to the median eminence, provide evidence for a PVN-hippocampal CRF pathway, and suggest that the PVN modulates CRF neurons innervating the LC. Moreover, the existence of a CRF-containing pathway from the Ce to the LC appears likely, and such a circuit may play a role in the behavioral and physiological responses to stress.

Antagonization of the behavioral activation produced by direct stimulation of forebrain dopamine receptors caused by intraaccumbens injections of neurotensin.

A number of studies have shown that intracisternal, intracerebroventricular, or direct administration of neurotensin (NT) into the nucleus accumbens (ACC) can antagonize the arousal and excitement produced by activation of the mesolimbic dopamine (DA) system of rats. This study investigated where NT acts relative to DA neurons to exert this antagonistic effect. In this study we selectively removed the majority of limbic forebrain DA terminals by bilateral administration of 6-hydroxydopamine (6-OHDA) into the anterolateral hypothalamus of desipramine-pretreated rats. The 6-OHDA-treated rats subsequently developed DA receptor supersensitivity, as evidenced by behavioral supersensitivity to L-DOPA. The L-DOPA dose employed was subthreshold for behavioral excitation in control rats. The behavioral excitation to L-DOPA in 6-OHDA-treated rats consisted of increases in sniffing and increases in locomotion and/or rearing, along with decreases in resting and sleep. Following bilateral intra-ACC injections of NT, L-DOPA-induced sniffing, rearing, and locomotion decreased significantly, and resting and sleep increased significantly. These data suggest that intra-ACC NT, acting in or proximal to the ACC, can antagonize the behavioral effects of limbic DA stimulation and that this antagonism is postsynaptic to DA neurons.

Use of conditioned taste aversion as a conflict model: effects of anxiolytic drugs.

Moderate taste aversions were induced by pairing the initial consumption of 0.25% sodium saccharin (SACC) with either 25 mg/kg i.p. l-5-hydroxytryptophan or 30 mg/kg i.p. LiCl. The expression of these moderate conditioned SACC aversions was antagonized by pretreatments (i.p. or p.o.) with benzodiazepine and non-benzodiazepine anxiolytic drugs (lorazepam, diazepam, chlordiazepoxide, oxazepam, phenobarbital, meprobamate, and chlormezanone). Chlordiazepoxide produced less or no antagonism of the expression of stronger SACC aversions induced by 50 or 75 mg/kg l-5-hydroxytryptophan or by 60 or 90 mg/kg LiCl. Nonanxiolytic drugs, including dipsogenic compounds that increased the water intake of hydrated rats (2 M NaCl i.p.; isoproterenol HCl s.c.; and histamine diphosphate s.c.), and even additional 24 hr of fluid deprivation did not antagonize the expression of moderate conditioned taste aversions, indicating that anxiolytic drugs have a very selective effect and that they do not appear to act through homeostatic drinking mechanisms. An essential feature of the taste aversion conflict model is that thirsty rats encounter only SACC. When water was conspicuously available in addition to SACC in two-bottle tests, neither chlordiazepoxide nor phenobarbital antagonized the expression of conditioned taste aversion. Thus, anxiolytic drugs do not produce amnesia for the conditioned aversion, but attenuate the ability of conditioned SACC aversion to suppress SACC consumption in one-bottle tests. The antagonism of the expression of conditioned taste aversion measured with a one-bottle testing method offers a simple, sensitive, and selective screen for anxiolytic drugs. A possible mechanism by which anxiolytics increase both suppressed as well as unsuppressed fluid consumption is discussed.

Interactions of neurotensin with dopamine-containing neurons in the central nervous system.

1. Neurotensin (NT) is a tridecapeptide that fulfills many of the requisite criteria for neurotransmitter status in the mammalian central nervous system. 2. Neuroanatomical studies have revealed that DA and NT neurons are proximal in several limbic areas and that NT receptors are present on both DA perikarya and certain DA nerve terminals. 3. When injected intraventricularly or directly into the nucleus accumbens (ACC), NT produces effects similar to antipsychotic drugs, e.g. blockade of the hyperactivity produced by d-amphetamine or cocaine. 4. Chronic administration of antipsychotic drugs such as haloperidol produces increases in NT concentration in the ACC and caudate nucleus. Atypical antipsychotic drugs produce increases in NT concentration only in the ACC. 5. In certain drug-free schizophrenic patients, the concentration of NT in cerebrospinal fluid is reduced. 6. It appears that NT may modulate the activity of DA neurons in the mesolimbicocortical DA system.

Cholecystokinin octapeptide and lithium produce different effects on feeding and taste aversion learning.

The strengths of taste aversion induced by sulphated cholecystokinin 26-33 (CCK-8; 1,2,4 and 8 micrograms/kg IP) and lithium chloride (LiCl; 7.5, 15, 30 and 60 mg/kg IP) were determined in order to assess the relative aversiveness of the two compounds. All doses of LiCl induced strong aversion, but only the highest dose of CCK-8 induced aversion, which was mild. Effects of CCK-8 and LiCl on food intake were then compared in the hour (hr) following 8 hr of food deprivation; rats were on this food deprivation schedule for a relatively long time (78 days) throughout testing. All doses of CCK-8 reduced food intake significantly. Most doses of LiCl either did not affect or significantly increased food intake. Although 60 mg/kg LiCl did not affect food intake when administered 15 or 30 min before food presentation, it significantly increased food intake when administered 1, 2 or 3 hr before food presentation. Overeating of solid food may be an illness-induced behavior. Although a very high dose of LiCl (120 mg/kg) decreased food intake markedly, the rats were obviously distressed, not satiated. Failure of CCK-8 to affect feeding behavior like LiCl is indirect evidence that the reduction of food intake by CCK-8 is not merely the result of aversiveness, but is an extremely potent and specific behavioral effect.

Evidence that taste aversion learning induced by l-5-hydroxytryptophan is mediated peripherally.

Rats learned to avoid a saccharin solution if their initial consumption of it was followed by intraperitoneal (IP) administration of 25 mg/kg l-5-hydroxytryptophan (l-5-HTP); this taste aversion learning did not occur in rats pretreated with 50 mg/kg (IP) of the aromatic l-amino acid decarboxylase inhibitor RO 4-4602 (benserazide). RO 4-4602 antagonized the l-5-HTP-induced elevation of 5-hydroxytryptamine (5-HT) in the mesentery but significantly increased the l-5-HTP-induced elevation of 5-HT in the brain. These results indicate that l-5-HTP-induced taste aversion is correlated with peripheral, but not central, elevation of 5-HT.

Thyrotropin-releasing hormone and amphetamine produce different patterns of behavioral excitation in rats.

We compared the arousal and hyperactivity produced by intraperitoneal (i.p.) injections of thyrotropin-releasing hormone (TRH, pGlu-His-Pri-NH2; 10, 20, 30 and 60 mg/kg) and 0.3 and 2 mg/kg d-amphetamine (low and moderate amph., respectively) by measuring the occurrence of discrete behavioral items with a behavioral sampling and scoring method. To minimize extraneous variables affecting activity, rats were caged singly inside isolated observation chambers and tested in the daytime after a 2.5 h period of habituation. Under these conditions, vehicle (0.9% NaCl)-treated rats were inactive and either rested or slept through 80% of all time samples taken in the hour after injection. Both TRH and amph. produced significant arousal from sleeping, but TRH, at all doses tested, produced less arousal than moderate amph. and a pattern of behavioral responses which differed from both low and moderate amph. Moderate amph. produced marked increase in forward locomotion and rearing, but low amph. and TRH did not. Both TRH and low amph. increased grooming (perhaps simply by increasing wakefulness), but TRH failed to increase sniffing, a cardinal feature of ampha.-induced excitement. Unlike amph., TRH produced wet-dog shakes, piloerection, tail elevation and teeth chattering. Both mod. amph. and TRH significantly produced increased activity when compared to controls as assessed with photocell counts, though the amph. effect was more robust. The lack of arousal after i.p. injections of thyroid-stimulating hormone (10 I.U./kg) or melanocyte-stimulating hormone release-inhibiting factor (Pro-Leu-Gly-NH2; 60 mg/kg) is evidence that TRH-induced arousal is neither mediated by activation of the pituitary-thyroid axis nor by a non-specific effect of tripeptides generally.

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.