Targeting of ADAMTS5's ancillary domain with the recombinant mAb CRB0017 ameliorates disease progression in a spontaneous murine model of osteoarthritis.

OBJECTIVE: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. DESIGN: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2 µg, CRB0017 12 µg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. RESULTS AND CONCLUSIONS: All histological scores were significantly decreased in the CRB0017 12 µg/knee group compared to vehicle, while administration of CRB0017 1.2 µg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA.

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

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

Glutamate signaling in chondrocytes and the potential involvement of NMDA receptors in cell proliferation and inflammatory gene expression.

OBJECTIVE: Increased levels of glutamate, the main excitatory neurotransmitter, are found in the synovial fluid of osteoarthritis (OA) patients. Our aim was to study glutamate signaling in chondrocytes, focusing on the composition, pharmacology, and functional role of N-methyl-d-aspartate (NMDA) glutamate receptors. METHODS: We used the human chondrocyte cell line SW1353 and, in parallel, primary rat articular chondrocytes. Glutamate release and uptake were measured by fluorimetric and radiometric methods, respectively. Gene expression was analyzed by quantitative polymerase chain reaction. NMDA receptor pharmacology was studied in binding experiments with [3H]MK-801, a specific NMDA receptor antagonist. RNA interference was used to knock-down the expression of NR1, a subunit of NMDA receptors. RESULTS: Glutamate release, sodium- and calcium-dependent glutamate uptake, and the expression of a glutamate transporter were observed in chondrocytes. NR2D was the most abundant NMDA receptor subunit in these cells. Consistent with this observation, the binding affinity of [3H]MK-801 was much lower in chondrocytes than in rat brain membranes (mean K(d) values of 700 and 2.6 nM, respectively). NR1 knock-down, as well as NMDA receptor blockade with MK-801, reduced chondrocyte proliferation. Interleukin (IL)-1beta significantly altered glutamate release and uptake (about 90% increase and 50% decrease, respectively, in SW1353 cells). Moreover, IL-1beta induced the gene expression of cytokines and enzymes involved in cartilage degradation, and MK-801 significantly inhibited this response. CONCLUSIONS: Our findings suggest that chondrocytes express a self-sufficient machinery for glutamate signaling, including a peripheral NMDA receptor with unique properties. This receptor may have a role in the inflammatory process associated with cartilage degradation, thus emerging as a potential pharmacological target in OA.

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

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

CCK1 receptor antagonist, dexloxiglumide: effects on human isolated gallbladder. Potential clinical applications.

Cholecystokinin is the main hormonal regulator of gallbladder motility. Dexloxiglumide, the active enantiomer of loxiglumide, interacts competitively with CCK1 receptors as determined in preclinical studies, such as specific radioligand binding assays or functional studies on isolated guinea pig gallbladder, where it inhibited smooth muscle cell contractions induced by cholecystokinin-octapeptide (CCK-8), the most prominent active forms of cholecystokinin. Dexloxiglumide has a potent antagonistic effect, of a competitive nature, on human gallbladder cholecystokinin type 1 receptors. In isolated human gallbladder, dexloxiglumide produced a concentration-dependent rightward shift of the cholecystokinin-octapeptide curve, without affecting its maximal response. Gallbladder motility was evaluated in clinical studies. Dexloxiglumide, orally administered to healthy volunteers at putative therapeutic doses, did not interfere with post-prandial gallbladder kinetics, despite an increase of fasting gallbladder volume. At present, dexloxiglumide is in an advanced stage of clinical research in gastroenterology. Overall, clinical observations suggest that dexloxiglumide may become an effective treatment in several gastrointestinal disorders. Moreover, the beneficial effects can be obtained without increasing the risk of gallstones formation, a potential hazard subsequent to the inhibition of gallbladder contractions and the resulting bile stasis. The potent and selective antagonist dexloxiglumide may offer a possible therapeutic tool for use not only in functional gastrointestinal disorders, such as irritable bowel syndrome, constipation, gastroesophageal reflux disease and functional dyspepsia, but also in other pathologies, such as biliary colics, pancreatic diseases and gastrointestinal tumors.

Effect of three nonpeptide cholecystokinin antagonists on human isolated gallbladder.

Cholecystokinin is the most important stimulant of postprandial gallbladder contraction, and a regulator of gallbladder fasting tone. The aim of this study was to evaluate the effect of dexloxiglumide on isolated human gallbladder contraction induced by cholecystokinin-octapeptide and to compare this effect to that of lorglumide and amiglumide, two glutaramic acid analogs of dexloxiglumide. The negative logarithms of the antagonist dissociation constant (pK(B)) values were 7.00 +/- 0.14, 6.95 +/- 0.11, and 6.71 +/- 0.10 for lorglumide, dexloxiglumide, and amiglumide, respectively. Dexloxiglumide produced a concentration-dependent rightward shift of the cholecystokinin-octapeptide curve, without affecting its maximal response. A similar effect was obtained both with lorglumide and amiglumide. Moreover, the slopes for the three antagonists did not differ significantly from unity. These data show that the three molecules have a potent antagonistic effect, of a competitive nature, on gallbladder cholecystokinin type 1 receptors. It may be concluded that dexloxiglumide, lorglumide, and amiglumide exhibit a promising therapeutic profile for biliary colic and other gastrointestinal disorders in which CCK1 receptors play important physiological roles.

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

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

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

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

CR 2945: a novel CCKB receptor antagonist with anxiolytic-like activity.

The effects of CR 2945, an antranilic acid derivative member of a novel family of non-peptide CCKB receptor antagonists, have been compared with those of CAM-1028, an analogue of the CCKB receptor antagonist CI-988, L-365,260 a benzodiazepine derivative CCKB antagonist, CR 1795, an analogue of the CCKA receptor antagonist lorglumide and diazepam, a benzodiazepine receptor agonist, in several rodent screens sensitive to conventional anxiolytics. CR 2945 displayed nanomolar affinity for rat CCKB receptors and showed a selectivity ratio of about 9000 for the CCKB over the CCKA receptor. In ex-vivo binding studies, CR 2945, after i.v. and s.c. administration, inhibited the binding of [125I] (BH)-CCK8 in rat cortex homogenate with ID50s of 10.9 mg/kg and 13.5 mg/kg, respectively. In four rodent tests of anxiety (mouse black/white box, rat elevated plus-maze, rat elevated zero-maze and punished licking test in the rat) CR 2945 (0.1-10 mg/kg s.c. or orally) showed significant dose-dependent anxiolytic-like effects. The reference CCKB antagonist compounds CAM-1028 and L-365,260 showed an anxiolytic-like activity less robust than that of CR 2945 in the elevated zero-maze after s.c. administration, and these compounds were inactive in the elevated plus-maze after oral administration. The magnitude of the activity of CR 2945 was comparable to that of diazepam, but without signs of sedation and ataxia. Furthermore, a 7-day repeated treatment with CR 2945 at 10 mg/kg/day s.c. did not induce tolerance or withdrawal anxiety in rats. CR 1795 showed anxiolytic-like activity with a bell-shaped dose-response curve in the elevated zero-maze model in rats (0.1-10 mg/kg, orally and s.c.), whereas in the mouse black/white box test and in the rat elevated plus-maze test it was less effective. The results suggest that CR 2945 might be a promising alternative to the existing therapy of anxiety-related disorders.

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

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

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

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

Synthesis, biological evaluation, and quantitative receptor docking simulations of 2-[(acylamino)ethyl]-1,4-benzodiazepines as novel tifluadom-like ligands with high affinity and selectivity for kappa-opioid receptors.

The synthesis and biological evaluation of a series of 2-substituted 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a kappa-opioid agonist and a cholecystokinin-A (CCK-A) antagonist, are reported. The radioligand binding models used in these studies were [(125)I](BH)-CCK-8 in rat pancreas (CCK-A), [(3)H]-(MENLE(28,31))-cck-8 in guinea pig cerebral cortex (CCK-B), and [(3)H]U-69593 (kappa(1)), [(3)H]DAMGO (mu), and [(3)H]DADLE (delta) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for kappa-opioid receptors. In particular, the 2-thienyl derivative 7A(X = H) with a K(i) = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig kappa (kappa(1))-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure-affinity relationships in this class of receptors.

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

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

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

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

Methylazoxymethanol-induced microencephaly: persistent increase of cortical somatostatin-like immunoreactivity.

Treatment of pregnant rats with methylazoxymethanol (MAM) at day 15 of gestation induces a marked microencephaly in the offspring, characterized by impaired formation of interneurons in the areas affected. We have measured somatostatin immunoreactivity in the cortex, striatum and hippocampus of the offspring of pregnant rats treated with graded doses of MAM. A long-lasting increase in the cortical levels of this peptide was found, suggesting that somatostatinergic interneurons might be selectively spared by administration of the cytotoxic agent at this gestational age.

Cerebral extract from morphine-tolerant rats shows antiopiate properties in guinea pig ileum bioassay.

The existence of an endogenous antiopiate system which counteracts endogenous opiate effects has been proposed. The present study set out to seek substance/s with morphine-antagonist activity in the brain and serum of morphine-tolerant rats. Cerebral extracts were partly purified on Sephadex G 25 and serum was ultrafiltered through membranes with pore diameter smaller than 0.005 micron. On the guinea pig ileum myenteric plexus longitudinal muscle a fraction of the cerebral extract and the serum ultrafiltrate in toto did increase electrically induced contractions, and antagonized the depressant effect of morphine. The serum ultrafiltrate also enhanced longitudinal smooth muscle tone. Preliminary findings suggest that levels of endogenous morphine-antagonist substance/s are higher in morphine-tolerant rats than in controls. Only cerebral extract, not serum ultrafiltrate, inhibited [3H]-naloxone binding to cerebral opiate receptors. In the guinea pig bioassay both the cerebral extract and serum ultrafiltrate antagonized, to some extent, the inhibition elicited by morphine, norepinephrine and adenosine. These observations support the existence of endogenous compound/s which may be functional antagonist/s of opiates and play a role in the development of tolerance and dependence.

Binding studies of dermorphin and its L-form on rat brain opioid receptors.

It is well known dermorphin is a potent and long-acting opioid peptide while its synthetic L-form is almost completely devoid of biological activity. We investigated whether the L-Ala2 residue might affect the affinity of the compound for opioid receptors or make [L-Ala2] dermorphin more sensitive to metabolic degradation. Dermorphin and [L-Ala2] dermorphin were assayed in [3H]naloxone binding to opioid receptors in rat brain preparations in the absence and presence of peptidase inhibitors bestatin, captopril and thiorphan. The synthetic [L-Ala2] dermorphin showed very low affinity for the opioid receptors. This was only slightly increased in the presence of the peptidase inhibitor bestatin, alone and in combination with captopril and thiorphan. The low affinity of [L-Ala2] dermorphin was not improved even when the binding assay was carried out at 0 degrees C. We suggest that the D-Ala2 residue is essential for the binding of dermorphin to the opioid receptors as well as for its pharmacological activity.

Central pharmacological activities and opiate receptor binding studies of some dermorphin analogs.

A series of dermorphin-like compounds were injected intracerebroventricularly in the rat to assess in vivo their effects on intestinal motility and analgesia. In vitro they were tested by binding assay using 3H-naloxone as radioligand or by guinea pig ileum bioassay. The synthetic peptides were less potent than dermorphin in inhibiting intestinal transit and in producing analgesia, or even inactive up to doses 30 times the dermorphin ED50. This reduction in pharmacological activity was coupled with a decrease in binding potency. The 3H-naloxone binding studies in the absence or presence of Na+ indicated that Na+ reduced the interaction of dermorphin and its analogs with brain opiate receptors. Only the dibenzyl derivative was slightly affected by sodium, suggesting a dual action for this peptide, as confirmed by preliminary data from guinea pig ileum bioassay.