Alkaloids in Withania somnifera (L.) Dunal Root Extract Contribute to Its Anti-Inflammatory Activity.

The anti-inflammatory properties of the medicinal plant Withania somnifera (L.) Dunal (WS) are generally related to withanolides; consistently, several strategies are under investigation to increase the concentration of these compounds in WS extracts. However, a potential toxicity of withanolides has been highlighted, thus questioning the safety of such preparations. At variance, the relative contribution of alkaloids is underrated, in spite of preliminary evidence underlining a possible pharmacological relevance. Starting from these considerations, the efficacy/safety profile of WS root extract (WSE) was compared with those of WS extracts which are enriched in alkaloids (WSA) and withanolides (WSW), respectively. MTT assay was used to evaluate cell viability. The anti-inflammatory activities of the different extracts were estimated throughout the assessment of the inhibition of lipopolysaccharide (LPS)-activated release of nitric oxide (NO) and the upregulation of iNOS and COX-2 protein in RAW 264.7 cells. Both WSA and WSW were able to reduce LPS-mediated effects in RAW 264.7 cells, suggesting that alkaloids and withanolides may contribute to the anti-inflammatory activity of WSE. A significant higher anti-inflammatory activity and a lower toxicity were observed when WSA was compared to WSW. The present results highlighted that the contribution of alkaloids to WS pharmacological effects should not be neglected. Particularly, these compounds may concur to reach a more advantageous efficacy/safety profile when WS is used for anti-inflammatory purposes.

Development of Oxygen-Bridged Pyrazole-Based Structures as Cannabinoid Receptor 1 Ligands.

In this work, the synthesis of the cannabinoid receptor 1 neutral antagonists 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole-3-carboxamide 1a and its deaza N-cyclohexyl analogue 1b has led to a deepening of the structure-activity studies of this class of compounds. A series of novel 4,5-dihydrobenzo-oxa-cycloheptapyrazoles analogues of 1a,b, derivatives 1c-j, was synthesized, and their affinity towards cannabinoid receptors was determined. Representative terms were evaluated using in vitro tests and isolated organ assays. Among the derivatives, 1d and 1e resulted in the most potent CB1 receptor ligands (KiCB1 = 35 nM and 21.70 nM, respectively). Interestingly, both in vitro tests and isolated organ assays evidenced CB1 antagonist activity for the majority of the new compounds, excluding compound 1e, which showed a CB1 partial agonist behaviour. CB1 antagonist activity of 1b was further confirmed by a mouse gastrointestinal transit assay. Significant activity of the new CB1 antagonists towards food intake was showed by preliminary acute assays, evidencing the potentiality of these new derivatives in the treatment of obesity.

The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells.

BACKGROUND: Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine's analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of µ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells. METHODS: A range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated. RESULTS: Data analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation. CONCLUSION: Present in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

Role of nucleus accumbens µ opioid receptors in the effects of morphine on ERK1/2 phosphorylation.

RATIONALE: Despite the critical role attributed to phosphorylated extracellular signal regulated kinase (pERK1/2) in the nucleus accumbens (Acb) in the actions of addictive drugs, the effects of morphine on ERK1/2 phosphorylation in this area are still controversial. OBJECTIVES: In order to investigate further this issue, we studied (1) the ability of morphine to affect ERK1/2 phosphorylation in the shell (AcbSh) and core (AcbC) of Sprague-Dawley and Wistar rats and of CD-1 and C57BL/6J mice and (2) the role of dopamine D1 and µ-opioid receptors in Sprague-Dawley rats and CD-1 mice. METHODS: The pERK1/2 expression was assessed by immunohistochemistry. RESULTS: In rats, morphine decreased AcbSh and AcbC pERK1/2 expression, whereas in mice, increased it preferentially in the AcbSh compared with the AcbC. Systemic SCH 39166 decreased pERK1/2 expression on its own in the AcbSh and AcbC of Sprague-Dawley rats and CD-1 mice; furthermore, in rats, SCH 39166 disclosed the ability of morphine to stimulate pERK1/2 expression. Systemic (rats and mice) and intra-Acb (rats) naltrexone prevented both decreases, in rats, and increases, in mice. CONCLUSIONS: These findings confirm the differential effects of morphine in rats and mice Acb and that D1 receptors exert a facilitatory role on ERK1/2 phosphorylation; furthermore, they indicate that, in rats, removal of the D1-dependent pERK1/2 expression discloses the stimulatory influence of morphine on ERK1/2 phosphorylation and that the morphine's ability to decrease pERK1/2 expression is mediated by Acb µ-opioid receptors. Future experiments may disentangle the psychopharmacological significance of the effects of morphine on pERK1/2 in the Acb.

Withania somnifera (L.) Dunal root extract alleviates formalin-induced nociception in mice: involvement of the opioidergic system.

Withania somnifera (L.) Dunal extracts (WSEs) may possess therapeutic perspectives in the treatment of inflammation and pain. We aimed to evaluate the antinociceptive property of a WSE in the formalin test and to investigate the involvement of several neurotransmitter systems in this effect. The time spent licking the formalin-injected paw was recorded in CD1 mice after pretreatment with increasing doses of WSE. Also, c-Fos spinal cord expression and the effects of different compounds were investigated under these experimental conditions. Finally, the efficacy of WSE was analyzed following an injection of glutamate. WSE reduced the antinociceptive response during the tonic but not the acute phase of the formalin test and decreased formalin-induced c-Fos expression in spinal neurons. These effects were antagonized by the opioid antagonist naltrexone, whereas GABA, cannabinoid, δ-opioid, and nitric oxide compounds were ineffective. The administration of WSE also reduced nociception and c-Fos expression induced by glutamate injection. These results showed that WSE is effective in assays of chemical-induced nociception, indicating that this plant has potential valuable properties for the treatment of specific painful conditions. The antinocicetive effects of WSE in the formalin test appeared to be specifically mediated by the opioidergic system, although the involvement of the glutamatergic system cannot be excluded.

Novel diazabicycloalkane delta opioid agonists.

Here we report the investigation of diazabicycloalkane cores as potential new scaffolds for the development of novel analogues of the previously reported diazatricyclodecane selective delta (δ) opioid agonists, as conformationally constrained homologues of the reference δ agonist (+)-4-[(αR)-α((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80). In particular, we have simplified the diazatricyclodecane motif of δ opioid agonist prototype 1a with bridged bicyclic cores. 3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane, 3,9-diazabicyclo[3.3.1]nonane, 3,9-diazabicyclo[4.2.1]nonane, and 3,10-diazabicyclo[4.3.1]decane were adopted as core motifs of the novel derivatives. The compounds were synthesized and biologically assayed as racemic (3-5) or diastereoisomeric (6,7) mixtures. All the novel compounds 3-7 showed δ agonism behaviour and remarkable affinity to δ receptors. Amongst the novel derivatives, 3,8-diazabicyclo[3.2.1]octane based compound 4 evidenced improved δ affinity and selectivity relative to SNC80.

Methoxyflavones from Stachys glutinosa with binding affinity to opioid receptors: in silico, in vitro, and in vivo studies.

Fractionation of the bioactive dichloromethane extract from the aerial parts of Stachys glutinosa led to the isolation of four flavones, xanthomicrol (1), sideritoflavone (2), 8-methoxycirsilineol (3), and eupatilin (4), along with two neo-clerodane diterpenes, roseostachenone (8) and a new compound, 3α,4α-epoxyroseostachenol (7). In order to study structure-activity relationships, two methoxyflavones [5-demethyltangeretin (5) and tangeretin (6)] were synthesized by the methoxylation of xanthomicrol. The isolated compounds (1-4, 7, and 8) as well as the xanthomicrol semisynthetic derivatives (5 and 6) were evaluated for their binding affinity to the µ and δ opioid receptors. Xanthomicrol was the most potent binder to both µ and δ receptors, with a Ki value of 0.83 and 3.6 µM, respectively. Xanthomicrol administered intraperitoneally in mice at a dose of 80 mg/kg significantly reduced morphine-induced antinociception in the tail flick test. Our results suggested that xanthomicrol is a µ opioid receptor antagonist. Docking experiments were carried out to acquire a deeper understanding about important structural aspects of binding of xanthomicrol. In summary, these data suggest that xanthomicrol is a valuable structure for further development into a potential µ opioid receptor antagonist.

Tricyclic pyrazoles part 7. Discovery of potent and selective dihydrothienocyclopentapyrazole derived CB2 ligands.

A series of dihydrothienocyclopentapyrazole-based derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The major term, the 6-methyl-1-(1,4-dichlorophenyl)-N-piperidinyl)-1,4-dihydrothieno[2',3'-4,5]cyclopenta[1,2-c]pyrazole-3-carboxamide (6a), displayed a high affinity and good selectivity for CB2 receptors (Ki values of 2.30 nM for CB2 receptor and 440 nM for CB1 receptors respectively). Subsequent analogue preparation resulted in the identification of compounds such as 6b, 6d, 6e, 6k, 6l, 6m, 6s and 6t that showed 1.3-485 fold selectivity for CB2 receptors with potencies in the 1.1-7.2 nM range. These compounds profiled as full agonists at CB2 receptor in an inhibition assay of P-ERK 1/2 up regulation in HL-60 cells.

Tricyclic pyrazoles. Part 6. Benzofuro[3,2-c]pyrazole: a versatile architecture for CB2 selective ligands.

A new series of 1H-benzofuro[3,2-c]pyrazole-3-carboxamides was synthesized. The novel compounds (15-24) were evaluated for their affinity to CB2 and CB1 cannabinoid receptors. The synthesis of the title compounds takes advantage of the acid-catalysed thermal cyclization of bicyclic hydrazone ethyl 2-(2-(2,4-dichlorophenyl)hydrazono)-2-(6-methyl-3-oxo-2,3-dihydrobenzofuran-2-yl)acetate to tricyclic ethyl 1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro[3,2-c]pyrazol-3-carboxylate. All the obtained derivatives showed high affinity to CB2 receptors. Moreover, significant selectivity for CB2 over CB1 receptors was highlighted for lead derivatives amongst the novel series. The best binding profiles were determined for homologues bearing monocyclic and bicyclic monoterpenic substituents at the carbamoyl group at 3 position of the pyrazole ring (KiCB2 < 4 nM). In particular, the isopinocampheyl-substituted derivative 22 exhibited the highest selectivity for CB2 receptors with Ki values of 3.7 and 2398 nM for CB2 and CB1 receptors, respectively. Preliminary functional assays evidenced CB2 agonism behaviour for all the assayed novel derivatives.

Differential modulation of GABA(A) receptor function by aryl pyrazoles.

Several aryl pyrazoles characterized by a different molecular structure (flexible vs constrained), but chemically related to rimonabant and AM251, were tested for their ability to modulate the function of recombinant α1ß2γ2L GABAA receptors expressed in Xenopus laevis oocytes. The effects of 6Bio-R, 14Bio-R, NESS 0327, GP1a and GP2a (0.3-30 µM) were evaluated using a two-electrode voltage-clamp technique. 6Bio-R and 14Bio-R potentiated GABA-evoked Cl(-) currents. NESS 0327, GP1a and GP2a did not affect the GABAA receptor function, but they acted as antagonists of 6Bio-R. Moreover, NESS 0327 inhibited the potentiation of the GABAA receptor function induced by rimonabant. The benzodiazepine site seems to participate in the action of these compounds. In fact, flumazenil antagonized the potentiation of the GABAA receptor induced by 6Bio-R, and NESS 0327 reduced the action of lorazepam and zolpidem. On the contrary, NESS 0327 did not antagonize the action of "classic" GABAergic modulators (propanol, anesthetics, barbiturates or steroids). In α1ß2 receptors 6Bio-R potentiated the GABAergic function, but flumazenil was still able to antagonize the potentiation induced by 6Bio-R. Aryl pyrazole derivatives activity at the GABAA receptor depends on their molecular structure. These compounds bind to both an αßγ binding site, and to an α/ß site which do not require the γ subunit and that may provide structural leads for drugs with potential anticonvulsant effects.

Prediction and prevention of the first psychotic episode: new directions and opportunities.

In the last few decades, substantial research has focused on the possibility of early detection and prevention of the first psychotic episode in young individuals at risk of developing this mental disturbance; however, unresolved clinical and ethical issues still call for further investigations. New perspectives and opportunities may come from the identification of selective psychopathological and instrumental markers linking the appearance of subtle psychotic symptoms with the clinical outcome of specific mental pathologies. Furthermore, empirically derived algorithms and risk staging models should facilitate the identification of targeted prevention therapies, possibly improving the efficacy of well-tolerated therapeutic approaches, such as psychological interventions and natural compound supplementations. To date, the collected evidence on the efficacy and tolerability of pharmacological prevention therapies raises more doubts than hopes. A very early detection of risk and appropriate symptomatic pattern classifications may provide a chance to better match prevention strategies with the development of psychosis.

Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine.

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine. In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid (µ, δ, k), cannabinoid (CB1, CB2), glutamatergic (NMDA), GABAergic (GABAA, GABAB), serotoninergic (5HT2A) and adrenergic (α2) receptors. The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABAA and moderate affinity for GABAB, NMDA and δ opioid receptors. WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABAA, GABAB, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.

Synthesis and biological evaluation of novel delta (δ) opioid receptor ligands with diazatricyclodecane skeletons.

Considering the interesting pharmacological profile of the delta (δ) selective opioid agonist compound SNC-80, conformationally constrained analogs containing two diazatricyclodecane ring systems in place of dimethylpiperazine core motif were synthesized. The compounds showed subnanomolar or low nanomolar δ opioid receptor binding affinity. Depending upon the substituents on the diazatricyclodecane ring, these compounds displayed varying selectivity for δ opioid receptor over µ and κ receptors. Amongst the novel compounds, 1Aa showed the more interesting biological profile, with higher δ affinity and selectivity compared to SNC-80. The δ receptor agonist profile and antinociceptive activity of 1Aa were confirmed using ex-vivo (isolated mouse vas deferens) and in vivo (tail flick) assays.

N-Alkyl dien- and trienamides from the roots of Otanthus maritimus with binding affinity for opioid and cannabinoid receptors.

Two new thienylheptatrienamides (1, 5) and one new neo-lignan (12), together with thirteen known compounds (2, 3, 4, 6-11, 13-16) were isolated from the roots of Otanthus maritimus. The structures of the new compounds were elucidated on the basis of extensive 1D and 2D NMR experiments as well as high resolution mass spectrometry. All the isolated amides (1-10), the known pontica epoxide (11) and the new neo-lignan (12) were evaluated for their binding affinity to the CB1 and CB2 as well as to the µ and δ opioid receptors. Some alkylamides showed moderately high binding affinity for CB2 receptors and 1-[(2E,4E,8Z)-tetradecatrienoyl]piperidine (10) resulted the most active one with a Ki value of 160 nM. As far as we know, this is the first example of a tertiary alkylamide that binds CB2 receptors with significant potency. Compounds that showed the highest affinity for cannabinoid receptors (6-8, 10) were much less potent against opioid receptors. Primary structure-activity relationship is discussed. Docking experiments were carried out with the aim to understand the key interactions of the most active compounds with CB2 receptor.

Withania somnifera prevents acquisition and expression of morphine-elicited conditioned place preference.

Previous studies have reported that some of the central effects of morphine are counteracted by the administration of the methanolic extract of the root of Indian ginseng, Withania somnifera Dunal (WSE). The present study sought to determine whether WSE affects acquisition and expression of morphine-elicited conditioned place preference (CPP) in CD-1 mice. In CPP acquisition experiments, WSE (0, 25, 50, and 100 mg/kg) was administered, during conditioning, 30 min before morphine (10 mg/kg), whereas in expression experiments, WSE (0, 25, 50, and 100 mg/kg) was administered 30 min before the postconditioning test. The results demonstrate (i) that WSE was devoid of motivational properties; (ii) that WSE (100 mg/kg) was devoid of effects on spontaneous and morphine-stimulated motor activity and on spatial memory; and (iii) that WSE (50 and 100 mg/kg) significantly prevented the acquisition and expression of CPP. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for µ-opioid and γ-aminobutyric acid B receptors. These experiments revealed a higher affinity of WSE for γ-aminobutyric acid B than for µ-opioid receptors. Overall, these results point to WSE as an interesting alternative tool, worthy of further investigation, to study opiate addiction.

Novel pyrazole derivatives as neutral CB1 antagonists with significant activity towards food intake.

In spite of rimonabant's withdrawal from the European market due to its adverse effects, interest in the development of drugs based on CB1 antagonists is revamping on the basis of the peculiar properties of this class of compounds. In particular, new strategies have been proposed for the treatment of obesity and/or related risk factors through CB1 antagonists, i.e. by the development of selectively peripherally acting agents or by the identification of neutral CB1 antagonists. New compounds based on the lead CB1 antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining neutral CB1 antagonists. Amongst the new derivatives described in this paper, the mixture of the two enantiomers (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-methyl-1H-pyrazole ((±)-5), and compound 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4-methyl-1H-pyrazole ((Z)-6), showed interesting pharmacological profiles. According to the preliminary pharmacological evaluation, these novel pyrazole derivatives showed in fact both neutral CB1 antagonism behaviour and significant in vivo activity towards food intake.

Synthesis and pharmacological evaluation of novel 4-alkyl-5-thien-2'-yl pyrazole carboxamides.

The synthesis of three series of novel 4-alkyl-5-(5'-chlorothiophen-2'-yl)-pyrazole-3-carbamoyl analogues of rimonabant with affinity for the CB1 cannabinoid receptor subtype is reported. Amongst the novel derivatives, compounds 21j, 22a, 22c, and 22f showed affinity values expressed as Ki ranging from 5.5 to 9.0 nM. Derivative 23e revealed a good CB1 affinity (K(i) = 11.7 nM) and the highest CB1 selectivity of the whole series (K(i)CB2/K(i)CB1 = 384.6). These new compounds appeared to be able to pass the blood brain barrier and to counteract the activity of cannabinoid agonist. According to the results of mice vas deferens assays, as in the case of rimonabant, derivatives 21a, 22a, and 22b showed inverse agonist activity. In contrast, as a preliminary result to be confirmed, compound 23a exhibited neutral antagonist profile. According to the data obtained through an acute animal model, selected compounds 21a, 22a, and 23a evidenced the capability to significantly reduce food intake. At specific conditions, the effect of the novel compounds were higher than that induced by rimonabant. Amongst the novel CB1 antagonist compounds, 23a may represent a useful candidate agent for the treatment of obesity and its metabolic complications, with reduced side effects relative to those instead observed with rimonabant.

Tricyclic Pyrazoles. Part 5. Novel 1,4-Dihydroindeno[1,2-c]pyrazole CB2 Ligands Using Molecular Hybridization Based on Scaffold Hopping.

In search of new selective CB2 ligands, the synthesis and preliminary biological evaluation of novel 1,4-dihydroindeno[1,2-c]pyrazole hybrids of the highly potent prototypicals 5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-N-fenchyl-1H-pyrazole-3-carboxamide 1 and 1-(2,4-dichlorophenyl)-6-methyl-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide 2 are detailed.We postulated that the introduction of those pharmacophoric elements essential for activity of 1 in the tricyclic core of 2 might provide CB2 ligands with further improved receptor selectivity and biological activity. Among the compounds, 6-chloro-7-methyl-1-(2,4-dichlorophenyl)-N-fenchyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (22) exhibited low two digit nanomolar affinity for the cannabinoid CB2R and maintained a high level of CB2-selectivity.

Effects of controlled-release formulations of atypical antipsychotics on functioning and quality of life of schizophrenic individuals.

INTRODUCTION: Controlled-release formulations of atypical antipsychotics have recently been introduced into clinical practice. Clinical studies have indicated that these new therapies induce meaningful improvements in the functioning and quality of life of schizophrenic individuals. AREAS COVERED: The present analysis makes an attempt to address the clinical relevance of these studies and their contribution to the understanding of the mechanisms of action of these new drugs. A Medline search was done using the keywords 'antipsychotic', 'plasma level', 'quality of life' and 'functioning'. EXPERT OPINION: After reviewing the literature, it seems that symptom control and side effects may play a role in modulating the functioning and quality of life of schizophrenic individuals treated with controlled-release formulations of atypical antipsychotics. The analysis also highlights that these new drugs may possess peculiarities and similarities in regulating patient functioning. However, the low number of clinical analyses that have focused on these aspects of antipsychotic therapy limits the interpretation of the results. Additional comparative clinical trials are needed to evaluate how the pharmacokinetic/pharmacodynamic properties of antipsychotic drugs may modulate the functioning and quality of life of schizophrenic individuals, as well as to establish whether new clinical benefits may come from the use of these drugs in schizophrenia therapy.