Focusing on the 5F-MDMB-PICA, 4F-MDMB-BICA synthetic cannabinoids and their primary metabolites in analytical and pharmacological aspects.

Nowadays, more and more new synthetic cannabinoids (SCs) appearing on the illicit market present challenges to analytical, forensic, and toxicology experts. For a better understanding of the physiological effect of SCs, the key issue is studying their metabolomic and psychoactive properties. In this study, our validated targeted reversed phase UHPLC-MS/MS method was used for determination of urinary concentration of 5F-MDMB-PICA, 4F-MDMB-BICA, and their primary metabolites. The liquid-liquid extraction procedure was applied for the enrichment of SCs. The pharmacological characterization of investigated SCs were studied by radioligand competition binding and ligand stimulated [35S]GTPγS binding assays. For 5F-MDMB-PICA and 4F-MDMB-BICA, the median urinary concentrations were 0.076 and 0.312 ng/mL. For primary metabolites, the concentration range was 0.029-881.02* ng/mL for 5F-MDMB-PICA-COOH, and 0.396-4579* ng/mL for 4F-MDMB-BICA-COOH. In the polydrug aspect, the 22 urine samples were verified to be abused with 6 illicit drugs. The affinity of the metabolites to CB1R significantly decreased compared to the parent ligands. In the GTPγS functional assay, both 5F-MDMB-PICA and 4F-MDMB-BICA were acting as full agonists, while the metabolites were found as weak inverse agonists. Additionally, the G-protein stimulatory effects of the full agonist 5F-MDMB-PICA and 4F-MDMB-BICA were reduced by metabolites. These results strongly indicate the dose-dependent CB1R-mediated weak inverse agonist effects of the two butanoic acid metabolites. The obtained high concentration of main urinary metabolites of 5F-MDMB-PICA and 4F-MDMB-BICA confirmed the relevance of their routine analysis in forensic and toxicological practices. Based on in vitro binding assays, the metabolites presumably might cause a lower psychoactive effect than parent compounds.

Synthesis of tritium-labeled Lu-PSMA-617: Alternative tool for biological evaluation of radiometal-based pharmaceuticals.

This project focuses on the generation and evaluation of functional alternatives to radiometal-based pharmaceuticals supporting basic research and the in vitro developmental phase. Employing robust tritium chemistry and non-radioactive metal surrogates in two synthetic and labeling strategies resulted in ([ring-3H]Nal)PSMA-617 and ([α,ß-3H]Nal)PSMA-617. In particular, ([α,ß-3H]Nal)Lu-PSMA-617 exhibited high radiolytic as well as metal-complex stability and was compared to the clinically-established radiopharmaceutical [177Lu]Lu-PSMA-617. The cell-based assays confirmed the applicability of ([α,ß-3H]Nal)Lu-PSMA-617 as a substitute of [177Lu]Lu-PSMA-617 in pre-clinical biological settings.

Rimonabant-Based Compounds Bearing Hydrophobic Amino Acid Derivatives as Cannabinoid Receptor Subtype 1 Ligands.

In this study, 1H-pyrazole-3-carboxylic acids related to the cannabinoid type 1 (CB1) receptor antagonist rimonabant were amidated with valine or tert-leucine, and the resulting acids were further diversified as methyl esters, amides, and N-methyl amides. In vitro receptor binding and functional assays demonstrated a wide series of activities related to the CB1 receptors (CB1Rs). Compound 34 showed a high CB1R binding affinity (K i = 6.9 nM) and agonist activity (EC50 = 46 nM; E max = 135%). Radioligand binding and [35S]GTPγS binding assays also demonstrated its selectivity and specificity to CB1Rs. Moreover, in vivo experiments revealed that 34 was slightly more effective than the CB1 agonist WIN55,212-2 in the early phase of the formalin test, indicating a short duration of the analgesic effect. Interestingly, in a mouse model of zymosan-induced hindlimb edema, 34 was able to maintain the percentage of paw volume below 75% for 24 h following subcutaneous injection. After intraperitoneal administration, 34 increased the food intake of mice, suggesting potential activity on CB1Rs.

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Novel High Affinity Sigma-1 Receptor Ligands from Minimal Ensemble Docking-Based Virtual Screening.

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.

Characterization of a functional V1B vasopressin receptor in the male rat kidney: evidence for cross talk between V1B and V2 receptor signaling pathways.

Although vasopressin V1B receptor (V1BR) mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using the selective V1B agonist d[Leu4, Lys8]VP, either fluorescent or radioactive, we showed that V1BR is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1BR were very low compared with the V2 receptor (V2R). On the microdissected IMCD, d[Leu4, Lys8]VP had no effect on cAMP production but induced a dose-dependent and saturable intracellular Ca2+ concentration increase mobilization with an EC50 value in the nanomolar range. This effect involved both intracellular Ca2+ mobilization and extracellular Ca2+ influx. The selective V1B antagonist SSR149415 strongly reduced the ability of vasopressin to increase intracellular Ca2+ concentration but also cAMP, suggesting a cooperation between V1BR and V2R in IMCD cells expressing both receptors. This cooperation arises from a cross talk between second messenger cascade involving PKC rather than receptor heterodimerization, as supported by potentiation of arginine vasopressin-stimulated cAMP production in human embryonic kidney-293 cells coexpressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1B agonist triggered significant diuretic effects, in contrast with injection of selective V2 agonist. This study brings new data on the localization and signaling pathways of V1BR in the kidney, highlights a cross talk between V1BR and V2R in the IMCD, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.NEW & NOTEWORTHY Although V1BR mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using original pharmaceutical tools, this study brings new data on the localization and signaling pathways of V1BR, highlights a cross talk between V1BR and V2 receptor (V2R) in the inner medullary collecting duct, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation.

Pro-Inflammatory Cytokines: Potential Links between the Endocannabinoid System and the Kynurenine Pathway in Depression.

Substance use/abuse is one of the main causes of depressive symptoms. Cannabis and synthetic cannabinoids in particular gained significant popularity in the past years. There is an increasing amount of clinical data associating such compounds with the inflammatory component of depression, indicated by the up-regulation of pro-inflammatory cytokines. Pro-inflammatory cytokines are also well-known to regulate the enzymes of the kynurenine pathway (KP), which is responsible for metabolizing tryptophan, a precursor in serotonin synthesis. Enhanced pro-inflammatory cytokine levels may over-activate the KP, leading to tryptophan depletion and reduced serotonin levels, which can subsequently precipitate depressive symptoms. Therefore, such mechanism might represent a possible link between the endocannabinoid system (ECS) and the KP in depression, via the inflammatory and dysregulated serotonergic component of the disorder. This review will summarize the data regarding those natural and synthetic cannabinoids that increase pro-inflammatory cytokines. Furthermore, the data on such cytokines associated with KP activation will be further reviewed accordingly. The interaction of the ECS and the KP has been postulated and demonstrated in some studies previously. This review will further contribute to this yet less explored connection and propose the KP to be the missing link between cannabinoid-induced inflammation and depressive symptoms.

N,N-Dimethyltryptamine attenuates spreading depolarization and restrains neurodegeneration by sigma-1 receptor activation in the ischemic rat brain.

Dimethyltryptamine (DMT), an endogenous ligand of sigma-1 receptors (Sig-1Rs), acts against systemic hypoxia, but whether DMT may prevent cerebral ischemic injury is unexplored. Here global forebrain ischemia was created in anesthetized rats and aggravated with the induction of spreading depolarizations (SDs) and subsequent short hypoxia before reperfusion. Drugs (DMT, the selective Sig-1R agonist PRE-084, the Sig-1R antagonist NE-100, or the serotonin receptor antagonist asenapine) were administered intravenously alone or in combination while physiological variables and local field potential from the cerebral cortex was recorded. Neuroprotection and the cellular localization of Sig-1R were evaluated with immunocytochemistry. Plasma and brain DMT content was measured by 2D-LC-HRMS/MS. The affinity of drugs for cerebral Sig-1R was evaluated with a radioligand binding assay. Both DMT and PRE-084 mitigated SDs, counteracted with NE-100. Further, DMT attenuated SD when co-administered with asenapine, compared to asenapine alone. DMT reduced the number of apoptotic and ferroptotic cells and supported astrocyte survival. The binding affinity of DMT to Sig-1R matched previously reported values. Sig-1Rs were associated with the perinuclear cytoplasm of neurons, astrocytes and microglia, and with glial processes. According to these data, DMT may be considered as adjuvant pharmacological therapy in the management of acute cerebral ischemia.

Lessons on the Sigma-1 Receptor in TNBS-Induced Rat Colitis: Modulation of the UCHL-1, IL-6 Pathway.

Inflammatory Bowel Disease (IBD) is an autoimmune ailment of the gastrointestinal (GI) tract, which is characterized by enhanced activation of proinflammatory cytokines. It is suggested that the sigma-1 receptor (σ1R) confers anti-inflammatory effects. As the exact pathogenesis of IBD is still unknown and treatment options are limited, we aimed to investigate the effects of σ1R in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis. To this end, male Wistar-Harlan rats were used to model colitic inflammation through the administration of TNBS. To investigate the effects of σ1R, Fluvoxamine (FLV, σ1R agonist) and BD1063 (σ1R antagonist) were applied via intracolonic administration to the animals once a day for three days. Our radioligand binding studies indicated the existence of σ1Rs as [3H](+)-pentazocine binding sites, and FLV treatment increased the reduced σ1R maximum binding capacity in TNBS-induced colitis. Furthermore, FLV significantly attenuated the colonic damage, the effect of which was abolished by the administration of BD1063. Additionally, FLV potentially increased the expression of ubiquitin C-terminal hydrolase ligase-1 (UCHL-1) and the levels of endothelial nitric oxide synthase (eNOS), and decreased the levels of interleukin-6 (IL-6) and inducible NOS (iNOS) expression. In summary, our study offers evidence for the anti-inflammatory potential of FLV and σ1R in experimental colitis, and our results present a promising approach to the development of new σ1R-targeted treatment options against IBD.

Long-term systemic administration of kynurenic acid brain region specifically elevates the abundance of functional CB1 receptors in rats.

Kynurenic acid (KYNA) is one of the most significant metabolite of the kynurenine pathway both in terms of functional and potential therapeutic value. It is an N-methyl-D-aspartate (NMDA) receptor antagonist, but it can also activate the G-protein coupled receptor 35 (GPR35), which shares several structural and functional properties with cannabinoid receptors. Previously our group demonstrated that systemic chronic KYNA treatment altered opioid receptor G-protein activity. Opioid receptors also overlap in many features with cannabinoid receptors. Thus, our aim was to examine the direct in vitro and systemic, chronic in vivo effect of KYNA on type 1 cannabinoid receptor (CB1R) binding and G-protein activity. Based on competition and [35S]GTPγS G-protein binding assays in rat brain, KYNA alone did not show significant binding towards the CB1R, nor did it alter CB1R ligand binding and agonist activity in vitro. When rats were chronically treated with KYNA (single daily, i.p., 128 mg/kg for 9 days), the KYNA plasma and cerebrospinal fluid levels significantly increased compared to vehicle treated group. Furthermore, in G-protein binding assays, in the whole brain the amount of G-proteins in basal and in maximum activity coupled to the CB1R also increased due to the treatment. At the same time, the overall stimulatory properties of the receptor remained unaltered in vehicle and KYNA treated samples. Similar observations were made in rat hippocampus, but not in the cortex and brainstem. In saturation binding assays the density of CB1Rs in rat whole brain and hippocampus were also significantly enhanced after the same treatment, without significantly affecting ligand binding affinity. Thus, KYNA indirectly and brain region specifically increases the abundance of functional CB1Rs, without modifying the overall binding and activity of the receptor. Supposedly, this can be a compensatory mechanism on the part of the endocannabinoid system induced by the long-term KYNA exposure.

Synthesis, biochemical, pharmacological characterization and in silico profile modelling of highly potent opioid orvinol and thevinol derivatives.

Morphine and its derivatives play inevitably important role in the µ-opioid receptor (MOR) targeted antinociception. A structure-activity relationship study is presented for novel and known orvinol and thevinol derivatives with varying 3-O, 6-O, 17-N and 20-alkyl substitutions starting from agonists, antagonists and partial agonists. In vitro competition binding experiments with [3H]DAMGO showed low subnanomolar affinity to MOR. Generally, 6-O-demethylation increased the affinity toward MOR and decreased the efficacy changing the pharmacological profile in some cases. In vivo tests in osteoarthritis inflammation model showed significant antiallodynic effects of thevinol derivatives while orvinol derivatives did not. The pharmacological character was modelled by computational docking to both active and inactive state models of MOR. Docking energy difference for the two states separates agonists and antagonists well while partial agonists overlapped with them. An interaction pattern of the ligands, involving the interacting receptor atoms, showed more efficient separation of the pharmacological profiles. In rats, thevinol derivatives showed antiallodynic effect in vivo. The orvinol derivatives, except for 6-O-desmethyl-dihydroetorfin (2c), did not show antiallodynic effect.

Steroidal ferrocenes as potential enzyme inhibitors of the estrogen biosynthesis.

The potential inhibitory effect of diverse triazolyl-ferrocene steroids on key enzymes of the estrogen biosynthesis was investigated. Test compounds were synthesized via copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes using our efficient methodology published previously. Inhibition of human aromatase, steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities was investigated with in vitro radiosubstrate incubations. Some of the test compounds were found to be potent inhibitors of the STS. A compound bearing ferrocenyl side chain on the C-2 displayed a reversible inhibition, whereas C-16 and C-17 derivatives displayed competitive irreversible binding mechanism toward the enzyme. 17α-Triazolyl-ferrocene derivatives of 17ß-estradiol exerted outstanding inhibitory effect and experiments demonstrated a key role of the ferrocenyl moiety in the enhanced binding affinity. Submicromolar IC50 and Ki parameters enroll these compounds to the group of the most effective STS inhibitors published so far. STS inhibitory potential of the steroidal ferrocenes may lead to the development of novel compounds able to suppress in situ biosynthesis of 17ß-estradiol in target tissues.

Kynurenines and the Endocannabinoid System in Schizophrenia: Common Points and Potential Interactions.

Schizophrenia, which affects around 1% of the world's population, has been described as a complex set of symptoms triggered by multiple factors. However, the exact background mechanisms remain to be explored, whereas therapeutic agents with excellent effectivity and safety profiles have yet to be developed. Kynurenines and the endocannabinoid system (ECS) play significant roles in both the development and manifestation of schizophrenia, which have been extensively studied and reviewed previously. Accordingly, kynurenines and the ECS share multiple features and mechanisms in schizophrenia, which have yet to be reviewed. Thus, the present study focuses on the main common points and potential interactions between kynurenines and the ECS in schizophrenia, which include (i) the regulation of glutamatergic/dopaminergic/γ-aminobutyric acidergic neurotransmission, (ii) their presence in astrocytes, and (iii) their role in inflammatory mechanisms. Additionally, promising pharmaceutical approaches involving the kynurenine pathway and the ECS will be reviewed herein.

Discovery of Orexant and Anorexant Agents with Indazole Scaffold Endowed with Peripheral Antiedema Activity.

The endocannabinoid system represents an integrated neuronal network involved in the control of several organisms' functions, such as feeding behavior. A series of hybrids of 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (mimonabant), a well-known inverse agonist of the type-1 cannabinoid receptor (CB1), once used as an antiobesity drug, and the N-(2S)-substitutes of 1-[(4-fluorophenyl)methyl]indazole-3-carboxamide with 1-amino-3-methyl-1-oxobutane (AB-Fubinaca), 1-amino-3,3-dimethyl-1-oxobutane (ADB-Fubinaca), and 3-methylbutanoate (AMB-Fubinaca), endowed with potent agonistic activity towards cannabinoid receptors CB1 and CB2 were in solution as C-terminal amides, acids, methyl esters and N-methyl amides. These compounds have been studied by binding assays to cannabinoid receptors and by functional receptor assays, using rat brain membranes in vitro. The most active among them as an agonist, (S)-1-(2,4-dichlorobenzyl)-N-(3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl)-1H-indazole-3-carboxamide (LONI11), and an antagonist, (S)-2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3-methylbutanoic acid (LONI4), were tested in vivo in mic, to evaluate their ability to stimulate or suppress feeding behavior after intraperitoneal (i.p.) administration. For a LONI11 formalin test and a tail flick test after an administration by the subcutaneous (s.c.) and intracerebroventricular (i.c.v.) routes, respectively, were also carried out in vivo in mice to investigate the antinociceptive property at the central and peripheral levesl. We observed a significant orexant effect for LONI11 and an intense anorexant effect for (S)-methyl 2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (LONI2) and LONI4. In zymosan-induced edema and hyperalgesia, LONI11 reduced the percent of paw volume increase and paw latency after s.c. administration, also suggesting a possible peripheral anti-inflammatory activity.

Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors.

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB1 and CB2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [35S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB2, whereas 19 mainly CB1) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 µg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties.

Effects of RVD-hemopressin (α) on feeding and body weight after standard or cafeteria diet in rats.

Palatability and variety of foods are major reasons for hedonic eating, and hence for obesity. Hemopressin, a hemoglobin α chain-derived peptide, plays antagonist/inverse agonist role on cannabinoid (CB)1 receptors, while RVD-hemopressin(α)[RVD-hp(α)], a N-terminally extended form of hemopressin, has been reported as an allosteric modulator of CB1 and CB2 receptors. We investigated the effects of 14 daily intraperitoneal injections of RVD-hp(α), in Sprague-Dawley rats fed a highly palatable cafeteria-style (CAF) diet (30% fat, 56% carbohydrate, 14% protein; 4.20 kcal/g) compared to standard laboratory chow (STD) food (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value; 3.20 kcal). Food intake, body weight and locomotor activity were recorded throughout the study. Finally, rats were sacrificed and agouti-related peptide (AgRP), neuropeptide Y (NPY), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) and fatty acid amide hydrolase (FAAH) gene expression in the hypothalamus was measured by real-time reverse transcription polymerase chain reaction. We found that CAF diet increased food intake as compared to STD diet. In both STD and CAF diet fed rats, RVD-hp(α) treatment inhibited food intake, increased locomotor activity but did not modify body weight. In vehicle injected animals, CAF as compared to STD diet increased AgRP gene expression. RVD-hp(α) treatment decreased POMC mRNA levels in both diet groups and lowered the elevated AgRP levels induced by CAF diet. RVD-hp(α) treatment plays an anorexigenic role paralleled by increased locomotor activity both in STD and CAF diet fed rats. The inhibition of feeding could be partially mediated by lowering of hypothalamic POMC and AgRP gene expression levels.

Synthesis and structure-activity relationships of 2- and/or 4-halogenated 13ß- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis.

Ring A halogenated 13α-, 13ß-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure-activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17ß-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Novel Fubinaca/Rimonabant hybrids as endocannabinoid system modulators.

The discovery of novel modulators of the cannabinoid system is a current topic in medicinal chemistry. In this paper, we report nine novel carboxamides designed as hybrids of Fubinaca family compounds and Rimonabant. These hybrids were obtained by linking the 1-benzyl-2,5-dichloroindazole-3-carboxylic acid to different amino acids bearing a hydrophobic side chain and three different C-terminus. The new chemical entities were tested in vitro to evaluate their bioactivity by means of receptor binding assays and [35S]GTPγS stimulation assays to reveal their affinity and potency. We found that all compounds were able to bind to the cannabinoid receptors in the low nanomolar range with a marked selectivity towards the CB1 cannabinoid receptor. Some of them are full agonists, whereas the others act as partial agonists. These molecules could be potentially used as anti-obesity agents, antiemetic and analgesics.

Effects of central RVD-hemopressin(α) administration on anxiety, feeding behavior and hypothalamic neuromodulators in the rat.

BACKGROUND: The endocannabinoid (eCB) system is strongly involved in the regulation of anxiety and feeding behavior. RVD-hemopressin(α) [RVD-hp(α)], a N-terminally extended form of hemopressin, is a negative allosteric modulator of the cannabinoid (CB) 1 receptor and a positive allosteric modulator of CB2 receptor which has been recently reported to exert anxiolytic/antidepressant and anorexigenic effects after peripheral administration in rats. Pharmacological evidences reported a possible link between brain hypocretin/orexin, monoamine and eCB systems, as regards appetite and emotional behavior control. Considering this, the aim of our work was to investigated the effects of RVD-hp(α) on anxiety like behavior and food intake after central administration and related it to monoamine levels and orexin-A gene expression, in the hypothalamus. METHODS: We have studied the effects of central RVD-hp(α) (10nmol) injection on anxiety-like behavior and feeding using different behavioral tests. Hypothalamic levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and gene expression of orexin-A and proopiomelanocortin (POMC) were measured by high performance liquid chromatography (HPLC) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis, respectively. RESULTS: Central RVD-hp(α) administration decreased locomotion activity and stereotypies. Moreover, RVD-hp(α) treatment inhibited anxiogenic-like behavior and food intake, NE levels and orexin-A gene expression, in the hypothalamus. CONCLUSION: Concluding, in the present study we demonstrated that central RVD-hp(α) induced anxiolytic and anorexigenic effects possibly related to reduced NE and orexin-A and POMC signaling, in the hypothalamus. These findings further support the central role of the peptide in rat brain thus representing an innovative pharmacological approach for designing new anorexigenic drugs targeting eCB system.

Synthesis and in Vitro Evaluation of Stabilized and Selective Neuromedin U-1 Receptor Agonists.

Neuromedin U (NMU) is a multifunctional neuropeptide which is characterized by a high conservation through all species. Herein, we describe the synthesis of a novel set of NMU-analogs based on the truncated NMU-8. Through combination of previously reported modifications, an elaborate structure-activity relationship study was performed aiming for the development of peptides with an increased selectivity toward NMU receptor 1 (NMUR1). Compound 7 possessed the highest NMUR1 selectivity (IC50 = 0.54 nM, selectivity ratio = 5313) together with an increased potency (EC50 = 3.7 nM), an 18% increase of the maximal effect at NMUR1, and a higher resistance against enzymatic degradation as compared to the native NMU-8. The development of a potent NMUR1 agonist with extended half-life could represent an attractive tool to further unveil the role of NMUR1 in NMU signaling.