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The imidazo[1,5-a]quinoline scaffold of central benzodiazepine receptor (CBR) ligands was used as the pharmacophore in the design of bivalent ligands bearing spacers showing variable length and different physicochemical features. The newly designed compounds were synthesized along with the corresponding reference monovalent compounds bearing the corresponding spacers terminated with a tert-butoxycarbonyl group. The novel compounds were tested in binding assays with different CBR preparations such as the cerebral cortex from male CD-1 albino mice or the human recombinant α1β3γ2 and α2β3γ2 γ-aminobutyric acid type A receptors (GABA A Rs) stably expressed in mouse L(tk-) cells. The tested compounds showed IC 50 values from the sub-micromolar up to the nanomolar range with very similar inhibition constants values for the two isoforms of GABA A Rs. The similarity in the affinity between the bivalent ligands and the corresponding monovalent ones appeared to rule out any bivalent interactions of these ligands with the two isoforms of GABA A Rs. Similarly, both series were able to inhibit the binding of radiolabeled flumazenil to GABA A Rs in cortical membranes of albino CD-1 mice, but most of the tested compounds showed biphasic inhibition curves, suggesting the existence of two well-distinct populations of binding sites. Finally, some CBR ligands selected from the bivalent ligands (i.e. 6a,c) and from the reference monovalent ligands (i.e. 7a) were then tested in vivo for their potential pharmacological effects, evaluating four classical benzodiazepine actions such as anticonvulsant, anxiolytic, locomotor, and anti-amnesic activities. All the tested compounds showed anticonvulsant and anxiolytic properties with neither muscle relaxant effect nor learning and memory impairments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Hydrogen sulfide (H 2 S) is a gasotransmitter deeply involved in cardiovascular homeostasis and implicated in the myocardial protection against ischemia/reperfusion. The post-translational persulfidation of cysteine residues has been identified as the mechanism through which H 2 S regulates a plethora of biological targets. Erucin (ERU) is an isothiocyanate produced upon hydrolysis of the glucosinolate glucoerucin, presents in edible plants of Brassicaceae family, such as Eruca sativa Mill., and it has emerged as a slow and long-lasting H 2 S-donor., Aim: In this study the cardioprotective profile of ERU has been investigated and the action mechanism explored, focusing on the possible role of the recently identified mitochondrial Kv7.4 (mitoKv7.4) potassium channels., Results: Interestingly, ERU showed to release H 2 S and concentration-dependently protected H9c2 cells against H 2 O 2 -induced oxidative damage. Moreover, in in vivo model of myocardial infarct ERU showed protective effects, reducing the extension of ischemic area, the levels of troponin I and increasing the amount of total AnxA1, as well as co-related inflammatory outcomes. Conversely, the pre-treatment with XE991, a blocker of Kv7.4 channels, abolished them. In isolated cardiac mitochondria ERU exhibited the typical profile of a mitochondrial potassium channels opener, in particular, this isothiocyanate produced a mild depolarization of mitochondrial membrane potential, a reduction of calcium accumulation into the matrix and finally a flow of potassium ions. Finally, mitoKv7.4 channels were persulfidated in ERU-treated mitochondria., Conclusions: ERU modulates the cardiac mitoKv7.4 channels and this mechanism may be relevant for cardioprotective effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxaliplatin-induced neuropathy (OXAIN) is a major adverse effect of this antineoplastic drug, widely used in the treatment of colorectal cancer. Although its molecular mechanisms remain poorly understood, recent evidence suggest that maladaptive neuroplasticity and oxidative stress may participate to the development of this neuropathy. Given the role played on protein remodeling by ubiquitin-proteasome system (UPS) in response to oxidative stress and in neuropathic pain, we investigated whether oxaliplatin might cause alterations in the UPS-mediated degradation pathway, in order to identify new pharmacological tools useful in OXAIN. In a rat model of OXAIN (2.4 mg kg -1 i.p., daily for 10 days), a significant increase in chymotrypsin-(β5) like activity of the constitutive proteasome 26S was observed in the thalamus (TH) and somatosensory cortex (SSCx). In addition, the selective up-regulation of β5 and LMP7 (β5i) subunit gene expression was assessed in the SSCx. Furthermore, this study revealed that oprozomib, a selective β5 subunit proteasome inhibitor, is able to normalize the spinal prodynorphin gene expression upregulation induced by oxaliplatin, as well as to revert mechanical allodynia and thermal hyperalgesia observed in oxaliplatin-treated rats. These results underline the relevant role of UPS in the OXAIN and suggest new pharmacological targets to counteract this severe adverse effect. This preclinical study reveals the involvement of the proteasome in the oxaliplatin-induced neuropathy and adds useful information to better understand the molecular mechanism underlying this pain condition. Moreover, although further evidence is required, these findings suggest that oprozomib could be a therapeutic option to counteract chemotherapy-induced neuropathy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A novel series of 1,5-diarylpyrrol-3-sulfur derivatives (10-12) was synthesized and characterized by NMR and mass spectroscopy and x-ray diffraction. The biological activity of these compounds was evaluated in in vitro and in vivo tests to assess their COX-2 inhibitory activity along with anti-inflammatory and antinociceptive effect. Results showed that the bioisosteric transformation of previously reported alkoxyethyl ethers (9a-c) into the corresponding alkyl thioethers (10a-c) still leads to selective and active compounds being the COX-2 inhibitory activity for most of them in the low nanomolar range. The oxidation products of 10a,b were also investigated and both couple of sulfoxides (11a,b) and sulfones (12a,b) showed an appreciable COX-2 inhibitory activity. Molecular modeling studies were performed to investigate the binding mode of the representative compounds 10b, 11b, and 12b into COX-2 enzyme and to explore the potential site of metabolism of 10a and 10b due to the different in vivo efficacy. Among the developed compounds, compound 10b showed a significant in vivo anti-inflammatory and antinociceptive activity paving the way to develop novel anti-inflammatory drugs., (Copyright © 2019. Published by Elsevier Ltd.)

The development of tolerance to the antinociceptive effect is a main problem associated with the repeated administration of opioids. The progressively higher doses required to relieve pain reduce safety and exacerbate the side effects of classical opioid receptor agonists like morphine. Nociceptin/orphanin FQ (N/OFQ) and its NOP receptor constitute the fourth endogenous opioid system that is involved in the control of broad spectrum of biological functions, including pain transmission. Aim of this work was to evaluate the relevance of the N/OFQ-NOP system in morphine antinociceptive action and in the development of morphine tolerance in the rat. Continuous spinal intrathecal infusion of morphine (1-3 nmol/h) evoked analgesic effects for 5 days in wild type animals. The same doses infused in NOP(-/-) rats showed a lower analgesic efficacy, while the onset of tolerance was delayed to day 9. N/OFQ (1-3 nmol/h), continuously infused in NOP(+/+) animals, showed an analgesic profile similar to morphine. Immunohistochemical analysis of the dorsal horn of the spinal cord of morphine tolerant NOP(+/+) rats showed an increased number of Iba1- and GFAP-positive cells (microglia and astrocytes, respectively). Interestingly, microglia but not astrocyte activation was observed in NOP(-/-) morphine tolerant rat. A selective activation of astrocytes was observed in the dorsal horn of wild type N/OFQ tolerant rats. The antinociceptive effect of morphine partially depends by the N/OFQ-NOP system that participates in the development of morphine tolerance. In particular, NOP receptors are involved in morphine-induced astrocyte activation, and N/OFQ per se increases astrocyte density., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuropathic pain affects millions of people causing disability and impairing quality of life. Commonly used analgesics are generally characterized by limited therapeutic outcomes. The serotonin 5-HT 1A receptor and the α 2 adrenergic receptors are involved in central nociceptive mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation may modulate the nervous signaling altered by neuropathies, the purpose of the present research is the study of the combined activation of 5-HT 1A and α 2 receptors by rationally designed imidazoline ligands ((S)-(-)-1 and 2-5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1-1mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more than 1h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind limb weight bearing alterations. The clinically-used compound gabapentin (100mg/kg p.o.) induced a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model, the selected analogues, compounds 2-5 (1mg/kg p.o.) were effective 30min after administration. In particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was significantly prevented by the selective 5-HT 1A receptor antagonist WAY 100635 (1mg/kg intraperitoneally, i.p.) and, at a lesser extent, by the α 2 antagonist yohimbine (3mg/kg i.p.). A novel pharmacodynamic approach to the treatment of neuropathic pain is presented., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The piperazine ring of the potent nootropic drug DM235 has been decorated with H-bond donor and acceptor groups (CH 2 OH, CH 2 OMe, CH 2 OCOMe, COOEt); the aim was to insert new functional groups, suitable for further chemical manipulation. The influence of these modifications on nootropic activity was assessed by means of the mouse passive avoidance test; some of the newly synthesized molecules (alcohol 7b, acetate 8b and ester 10d) showed interesting in vivo potency. This makes it possible to use these functional groups for adding other residues, in order to increase molecular diversity, or for anchoring a biotin group, to obtain compounds useful to capture the biological target. Moreover, the new compounds will improve our knowledge of structure activity relationships of this family of drugs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The synthesis of a new series of 6-phenyl- and 6-benzylpyrazolo[1,5-a]pyrimidin-7(4H)-ones 2a-g and 3a-g, strictly related to derivatives with pyrazolobenzotriazine (PBT) and pyrazoloquinazoline (PQ) scaffold, was realized. The in vitro GABA A -receptor subtype affinity was evaluated and from preliminary pharmacological studies, compound 3g shows anxiolytic-like effect at 10-30mg/kg., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Several drugs and agents are currently used for the treatment of neuropathic pain. Among them amitriptyline, a tricyclic antidepressant drug, represent a first line treatment. Despite its well-documented clinical efficacy, amitriptyline is ineffective in some animal models of neuropathic pain. The aim of this study was to investigate into amitriptyline poor efficacy in neuropathic pain and to determine the role of c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the analgesic effects of this drug. Experiments were performed in mice with painful peripheral neuropathies due to the antiretroviral agent 2,3-dideoxycytidine (ddC), and with the partial sciatic nerve injury produced in the spared nerve injury model (SNI). In mice subjected to SNI and antiretroviral treatment, amitriptyline did not attenuate mechanical allodynia and thermal hyperalgesia. Conversely, intrathecal injection of the JNK inhibitor SP600125 prevented SNI and ddC-induced nociceptive behavior and, its inactive dose co-administrated with amitriptyline induced an antinociceptive effect. Western blotting analysis showed an upregulation of p-JNK in the lumbar spinal cord of SNI and ddC-exposed mice, that was further enhanced after amitriptyline administration. Additionally, amitriptyline further promoted astrocyte activation in neuropathic mice, as illustrated by the increased expression of glial fibrillary acidic protein (GFAP), that was attenuated by intrathecal injection of the JNK inhibitor. These data indicate astrocyte JNK activation as counteracting pathway to amitriptyline analgesic response. Targeting the JNK pathway in spinal astroglia may present an efficient way to improve the analgesic efficacy of amitriptyline in the neuropathic pain treatment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It has been proposed that depression is associated with dysfunction of hippocampal plasticity. Novel hypotheses suggest that antidepressants induce neuronal structural plasticity, although the underlying mechanisms still remain unclear. Therefore, the aim of this study was to investigate the effects of amitriptyline on levels of phosphorylated heavy neurofilament subunit (NF-H) in the hippocampus of mice exposed to acute and chronic behavioral despair paradigms. Immunoblotting experiments showed that animals exposed to the tail suspension test (TST) displayed diminished levels of pNF-H 24h after testing. Repeated administration of amitriptyline (10mg/kg i.p.) prevented this decreased hippocampal phosphorylation of NF-H. Conversely, administration of citalopram (10mg/kg i.p.) left unchanged pNF-H levels. The expression of pNF-H was also analyzed by immunofluorescence in mice exposed to the unpredictable chronic mild stress paradigm (UCMS), an experimental model of depression. Mice that developed a depressive-like behavior showed a decreased pNF-H immunostaining selectively in the hippocampal CA3 region. Chronic administration of amitriptyline reversed the despaired behavior induced by exposure to UCMS paradigm and, fully recovered pNF-H labeling to control values. Our results indicate that the cytoskeletal remodeling induced by amitriptyline in the hippocampal CA3 region might underpin its behavioral efficacy. Hippocampal alterations of the NF appeared associated with the mechanism of this antidepressant drug and may contribute to its psychotherapeutic actions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A solid lipid nanoparticle (SLN) formulation was developed with the aim of improving the oral bioavailability and the therapeutic effectiveness of glibenclamide (GLI), a poorly water-soluble drug used in the treatment of type 2 diabetes. The SLN was prepared using different lipid components (Precirol® and Compritol®) and preparation procedures. Precirol-based SLN, obtained with the emulsion of solvent evaporation technique gave the best results and was selected for drug loading. Addition of lecithin to the SLN core or PEG coating was effective in increasing the nanoparticles stability in simulated gastric solution. Both such formulations were stable after one month storage at 5±3°C, exhibited the absence of in vitro cytotoxicity, and presented a similar in vitro prolonged-release, reaching 100% release after 24h. The lecithin-containing GLI-loaded SLN formulation, selected for in vivo studies in virtue of its higher EE% than the PEG-coated formulation (70.3% vs 19.6%), showed a significantly stronger hypoglycemic effect with respect to the drug alone, in terms of both shorter onset time and longer duration of the effect. These positive results indicated that the proposed SLN approach was successful in improving GLI oral bioavailability, confirming its potential as an effective delivery system for a suitable therapy of diabetes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: One of the most discomfortable dose-limiting adverse reactions of effective drugs for the treatment of solid tumors is a peripheral neuropathy which is the main reason for dose reduction and discontinuation of the therapy. We identified oxidative stress as one target of oxaliplatin toxicity in the search of possible adjuvant therapies to prevent neuropathy and alleviate pain. Therefore, we studied an effective SOD mimetic compound, MnL4, as a possible adjuvant treatment in in vitro cellular cultures and in vivo on a rat model of oxaliplatin-induced neuropathy., Methods and Results: All rat manipulations were carried out according to the European Community guidelines for animal care. We performed experiments on SH-SY5Y, HT-29 and primary cortical rat astrocytes. Incubation with 100 µM oxaliplatin increased superoxide anion production and caspase 3/7 activity in the neuronal cell line SH-SY5Y and cortical astrocytes. MnL4 (10 µM) significantly reduced the increase in superoxide anion in both cell types, but prevented caspase 3/7 activity only in astrocytes. MnL4 reduced lipid peroxidation induced by oxaliplatin and normalized the intracellular calcium signal evoked by ATP and acetylcholine in astrocytes, preincubated with oxaliplatin. MnL4 did not interfere with the concentration- and time-dependent cytotoxic effects of oxaliplatin on the cancer cell lines HT-29 and LoVo. In vivo MnL4 reduced the response at mechanical noxious and mechanical and thermal non-noxious stimuli in oxaliplatin treated animals. Rat rota-rod performances were improved., Conclusion: Since MnL4 exerts its beneficial effects without interfering with the anticancer activity of oxaliplatin, it could be proposed as adjuvant to prevent and reduce oxaliplatin induced neuropathy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oxaliplatin and paclitaxel are considered central components in the treatment of colorectal and breast cancer, respectively. The development of neuropathy during chronic treatment represents the major dose-limiting side effect that leads to discontinuation or interruption of therapies. The management of neuropathy is a challenge to individuate innovative therapeutic strategies based on new targets and correct routes of administration. We evaluated the hypersensitivity reliever effect of different opioid receptor agonists in rat models of oxaliplatin and paclitaxel-induced neuropathy. Compounds were spinally infused by intrathecal catheter. In oxaliplatin-treated rats, 0.3 nmol morphine induced the reversion of the mechanical hypersensitivity (Paw-pressure test), nociceptin/orphanin FQ (N/OFQ; 0.3-3 nmol) significantly increased the pain threshold without reaching the values of the control animals. The N/OFQ peptide (NOP) receptor full agonist UFP-112 reverted pain threshold alterations at lower dosage (0.1 nmol) vs morphine and N/OFQ, the partial agonist UFP-113 (0.1-1 nmol) was similar to N/OFQ. The higher efficacy of morphine vs N/OFQ was highlighted also in paclitaxel-treated rats. The mechanical hypersensitivity was fully reverted by 0.1 nmol UFP-112 and UFP-113. In conclusion, intrathecal μ opioid peptide (MOP) and NOP receptor agonists relieved chemotherapy-induced neuropathic pain. The synthetic peptides showed valuable potency and efficacy suggesting the NOP system as an exploitable target., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The pharmacological management of chronic pain is a major therapeutic problem. The need of repeated treatments reduces the usefulness of classical analgesic drugs, like μ opioid receptor (MOP) agonists, characterized by tolerance development, side effects and abuse. Moreover, the pathological persistence of pain modifies nociceptive signals and pain-devoted structure activity weakening MOP agonists and making difficult the research of new active molecules. Nociceptine/orphanin FQ (N/OFQ) and its receptor (NOP) offers a peculiar opioid system able to interact with MOP receptors and made more sensitive by chronic pain conditions. The pain reliever efficacy of NOP agonists against persistent pain, mainly neuropathic pain, has been highlighted after intrathecal infusions in rats and non human primates (NHPs). The differences emerged between the effects of NOP stimulation in rodents and NHPs allow to hypothesize the relevance of NOP modulators in higher organisms strongly encouraging the development of compounds active by a systemic route. Possible applicative perspectives are (i) selective NOP agonists as such, (ii) NOP modulation as adjuvant of MOP-based treatments, or (iii) mixed non-selective agonists vs NOP and classical opioid receptors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Morphine-induced hyperalgesia is a pharmacological phenomenon often hindering its prolonged applications in the clinic. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. Extracellular signal-regulated kinase (ERK) pathway contributes to pain sensitization, and its phosphorylation under pain conditions results in the induction and maintenance of pain hypersensitivity. The present study was designed to determine whether low dose morphine treatment in mice could influence the spinal activity of ERK. The data showed that morphine (1 µg/kg) induced a marked increase in ERK phosphorylation. Intrathecal pre-treatment with a selective mitogen-activated and extracellular signal-regulated kinase (MEK) inhibitor PD98059, attenuated morphine-associated thermal hyperalgesia. Morphine exposure increased phosphorylation of c-JUN, that was prevented by the inhibition of ERK pathway. In addition, double immunofluorescence studies revealed that, p-ERK and p-c-JUN are localized on neurons of the spinal dorsal horn expressing µ receptors. These data suggest that ERK contributes to the morphine-induced hyperalgesia by regulating the activation of c-JUN., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A number of heterocycles bearing an arylpiperazinylalkyl side chain and structurally related to the previously described lead ET1 (4-amino-6-methyl-2-[3-(4-p-tolylpiperazin-1-yl)propyl]-5-vinylpyridazin-3(2H)-one) was synthesized and tested for their antinociceptive activity in Writhing Test. Many compounds, tested at doses of 20-40 mg/kg po were able to reduce the number of abdominal constrictions by more than 47% and, in same cases, the potency is comparable to lead ET1 as for 5e, 24a, 27b and 27c. The analgesia induced by the active compounds was completely prevented by pretreatment with α2-antagonist yohimbine, confirming the involvement of the adrenergic system in the mechanism of action for these new compounds., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Sorafenib is a kinase inhibitor anticancer drug whose repeated administration causes the onset of a peripheral painful neuropathy. Notably, the efficacy of common analgesic drugs is not adequate and this often leads pre-mature discontinuation of anticancer therapy. The aim of this study was to establish a rat model of sorafenib-induced neuropathic pain, and to assess the effect of the new anti-neuropathic compound dimiracetam in comparison with gabapentin, pregabalin and duloxetine., Methods: Male Sprague-Dawley rats were treated i.v. (10 mg kg(-1)), i.p. (10 and 30 mg kg(-1)) or p.o. (80 and 160 mg kg(-1)) with sorafenib once daily for 21 days. Pain behaviour measurements (cold plate, paw pressure, electronic von Frey) were performed on days 0, 7, 14 and 21., Results: Sorafenib lowered the paw-licking threshold to non-noxious cold stimuli on day 14 of all protocols evaluated. The i.p. administration resulted in greater efficacy than the other administration routes. Sorafenib treatments did not affect paw-withdrawal responses to non-noxious or to noxious mechanical stimuli. On day 14, dimiracetam (300 mg kg(-1)), gabapentin (100 mg kg(-1)), pregabalin (30 mg kg(-1)) and duloxetine (30 mg kg(-1)) were acutely administered p.o. in sorafenib i.p.-treated rats. A single oral dose of dimiracetam induced a statistically significant increase of the pain threshold 15 min after administration. Pregabalin induced a comparable effect, whereas gabapentin and duloxetine were ineffective. Repeated twice-daily administration of dimiracetam (150 mg kg(-1) p.o.), starting on the first day of i.p sorafenib administration, significantly protected rats from sorafenib-induced decrease in the paw-licking threshold., Conclusions: A rat model of sorafenib-induced hypersensitivity to cold stimulation has been established. Dimiracetam and pregabalin are effective in prevention of sorafenib-induced neuropathy in this model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Severe pain occurs in the context of many diseases and conditions and is a leading cause of disability. Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the N/OFQ peptide (NOP) receptor. This peptidergic system controls pain transmission and in particular spinally administered N/OFQ has robust antinociceptive properties. The aim of this study was to investigate the spinal antinociceptive properties of NOP peptide agonists after acute and subchronic treatment in rats. Doses unable to alter motor coordination were selected. UFP-112 (full NOP agonist) and UFP-113 (partial NOP agonist) were administered intrathecally (i.t.) by spinal catheterization. Acute injection of UFP-112 induced antinociceptive response at lower dosages (0.03-1nmol i.t.) compared to morphine and similar to N/OFQ. UFP-113 was effective in a 0.001-1nmol i.t. dose range. The antinociceptive effects of NOP ligands were no longer evident in rats knockout for the NOP gene, while those of morphine were maintained. The continuous spinal infusion (by osmotic pumps) of 0.1nmol/h UFP-112 and UFP-113 showed antinociceptive action comparable to 1-3nmol/h morphine or N/OFQ. The antinociceptive effect of morphine progressively decreased and was no longer significant after 6 days of treatment. Similar results were obtained with N/OFQ, UFP-112, and UFP-113. The acute i.t. injection of morphine in animals tolerant to N/OFQ and UFP-112 evoked analgesic effects. Neither morphine nor N/OFQ induced antinociceptive effects in morphine- and UFP-113-tolerant rats. In conclusion this study highlights the analgesic efficacy and potency of UFP-112 and UFP-113 underlining the relevance of NOP system in analgesia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known, and some appear to be involved in pain. These sulfonamides showed modest inhibition against the cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain relieving effect, a fact never observed earlier. This is the first report of rationally designed sulfonamide CA inhibitors with pain effective modulating effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Behavioral responses to food deprivation are a fundamental aspect of nervous system function in all animals. Several signaling molecules in the mammalian brain act through G proteins of the Gi/o family to mediate response to food restriction. The present study examined whether food intake changes under a condition of little stimulation to eat, such as that elicited by 4h of food deprivation, was altered by Gi/o isoform silencing induced by intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (aODN) against the α subunit of Gi1, Gi2, Gi3, Go1 and Go2. The effect of aODN pretreatments on food intake was evaluated 15, 30, 45, and 60min after food re-administration. Selective effects were noted on food intake with anti-Giα1 (3.12-25nmol), Giα3 (1.56-12.5nmol) and Goα2 (3.12-25nmol) aODN exerting increase in food consumption, while anti-Giα2 (3.12-25nmol) and Goα1 (3.12-25nmol) aODN exerting decrease in food consumption. We observed the effect of the α-subunit silencing on food consumption as soon as 15min after food readministration, that was still significant after 60min. At the highest effective doses, different for each anti-Gαi/o subunit, any treatment did not impair motor coordination, nor modified spontaneous mobility and exploratory activity. These results indicate a relevant distinction between Gαi/o subunits on feeding behavior, and suggest that Gi/o proteins are critical for the integrative modulation of normal feeding behavior. Changes in Gi/o protein activity may be associated with modifications of feeding., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We report herein the synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl, sulfamoyl acetamides and ethyl acetates that selectively inhibit cyclooxygenase-2 (COX-2) isoform. Among the newly synthesized compounds, some of them were endowed with a good activity against COX-2 and a good selectivity COX-2/COX-1 in vitro as well as a desirable analgesic activity in vivo, proving that replacement of the ester moiety with an amide group gave access to more stable derivatives, characterized by a good COX-inhibition., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fluoxetine has been shown to be effective in clinical and experimental studies of neuropathic pain. Besides to increase serotonin levels in the synaptic cleft, fluoxetine is able to block the serotonergic 5-HT2C receptor subtype, which in turn has been involved in the modulation of neuropathic pain. This study investigated the effect of repeated treatments with fluoxetine on the neuropathic nociceptive response induced by oxaliplatin and the effects of both treatments on 5-HT2C receptor mRNA expression and protein levels in the rat spinal cord (SC), rostral ventral medulla (RVM), midbrain periaqueductal gray (PAG) and amygdala (Amy). Nociception was assessed by paw-pressure, cold plate and Von Frey tests. Fluoxetine prevented mechanical hypersensitivity and pain threshold alterations induced by oxaliplatin but did not prevent the impairment in weight gain induced by this anticancer drug. Ex vivo analysis revealed that oxaliplatin increased the 5-HT2C receptor mRNA expression and protein levels in the SC and PAG. Similar effects were observed in fluoxetine-treated animals but only within the PAG. While oxaliplatin decreased the 5-HT2C mRNA expression levels in the Amy, fluoxetine increased their protein levels in this area. Fluoxetine impaired the oxaliplatin effects on the 5-HT2C receptor mRNA expression in the SC and Amy and protein levels in the SC. All treatments increased of 5-HT2C receptor mRNA expression and protein levels in the PAG. These results suggest that the effects of fluoxetine on neuropathic pain induced by oxaliplatin are associated with quantitative changes in the 5-HT2C receptors located within important areas of the nociceptive system., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A niosomal formulation, functionalized with N-palmitoylglucosamine, was developed as potential brain targeted delivery system of dynorphin-B. In fact, this endogenous neuropeptide, selective agonist of k opioid receptors, is endowed with relevant pharmacological activities on the central nervous system, including a marked antinociceptive effect, but is unable to cross the blood brain barrier (BBB), thus requiring intracerebroventricular administration. Statistical design of experiments was utilized for a systematic evaluation of the influence of variations of the relative amounts of the components of the vesicle membrane (Span 60, cholesterol and SolulanC24) on vesicle mean diameter, polydispersity index and drug entrapment efficiency, chosen as the responses to optimize. A Scheffé simplex-centroid design was used to obtain the coefficients of the postulated mathematical model. The study of the response surface plots revealed that variations of the considered factors had different effects on the selected responses. The desirability function enabled for finding the optimal mixture composition, which represented the best compromise to simultaneously optimize all the three responses. The experimental values obtained with the optimized formulation were very similar to the predicted ones, proving the validity of the proposed regression model. The optimized niosomal formulation of dynorphin-B administered intravenously to mice (100mg/kg) showed a pronounced antinociceptive effect, significantly higher (P<0.05) than that given by i.v. administration of the simple solution of the peptide at the same concentration, proving its effectiveness in enabling the peptide brain delivery. These positive results suggest that the proposed approach could be successfully extended to other neuro-active peptides exerting a strong central action, even at low doses, but unable to cross the BBB., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report herein the development, synthesis, physicochemical and pharmacological characterization of a novel class of pharmacodynamic hybrids that selectively inhibit cyclooxygenase-2 (COX-2) isoform and present suitable nitric oxide releasing properties. The replacement of the ester moiety with the amide group gave access to in vivo more stable and active derivatives that highlighted outstanding pharmacological properties. In particular, the glycine derivative proved to be extremely active in suppressing hyperalgesia and edema., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Oxaliplatin is a platinum-organic drug with antineoplastic properties used for colorectal cancer. With respect to the other platinum derivates oxaliplatin induces only a mild hematological and gastrointestinal toxicity. Its limiting side effect is its neurotoxicity, which results in a sensory neuropathy. Repeated oxaliplatin treatment in the rat led to a neuropathic pain characterized by a significant oxidative damage throughout the nervous system. The natural antioxidants silibinin and α-tocopherol reduce redox alteration and prevent pain. Starting from the "oxidative hypothesis" as a molecular basis of chemotherapy-induced neurotoxicity, we decided to explore deep inside the mechanisms of oxaliplatin neurotoxicity and search for a cellular system useful for screening antioxidant compounds that can reduce oxaliplatin neurotoxicity. Focusing on various constituents of the central nervous system, we used the neuronal-derived cell line SH-SY5Y and primary cultures of rat cortical astrocytes. Oxaliplatin significantly increased superoxide anion production and induced lipid peroxidation (malonyldialdehyde levels) and protein (carbonylated proteins) and DNA oxidation (8-OH-dG levels). Silibinin and α-tocopherol (10µM) were able to reduce the oxidative damage in both cell types. These antioxidants fully protected astrocytes from the caspase 3 apoptotic signaling activation induced by oxaliplatin. The damage prevention effects of silibinin and α-tocopherol on nervous system-derived cells did not interfere with the oxaliplatin antineoplastic in vitro mechanism as evaluated on a human colon adenocarcinoma cell line (HT29). Moreover, neither silibinin nor α-tocopherol modified the oxaliplatin-induced apoptosis in HT29 cells, suggesting a different antiapoptotic profile in normal vs tumoral cells for these antioxidant compounds. In conclusion, because data obtained in in vitro cellular models parallel the in vivo study we propose cell models to investigate oxaliplatin neurotoxicity and to screen possible therapeutic adjuvant agents., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chemotherapy-induced neuropathies are widespread disorders evoked by characteristic damage of the nervous system. Sensory alterations, as paresthesia and dysesthesia, and severe pain are disabling side effects that altered quality of life, leading to therapy discontinuation. These kind of neuropathies are extremely difficult to treat and actual therapies are generally palliative. A great deal of interest has evolved around the relevance of nicotinic receptors as target for chronic pain therapy. Selective receptor subtype modulators have been described as active in pain relief. On the other hand, the profile of nicotine as such, or delivered by tobacco smoke, is a matter of debate since the analgesic properties may be impaired by receptor desensitization and tolerance. Nicotine acute effect on nociceptive threshold was evaluated in the Chronic Constriction Injury model in comparison with neuropathies induced by chemotherapeutic agents. Fourteen days after nerve injury, intraperitoneally administered nicotine (0.5-1.5 mg/kg) reduced hypersensitivity to noxious and non-noxious stimuli. Painful neuropathic state was alternatively established by the intravenously injection of the antiviral agent dideoxycytidine (25 mg/kg). Nicotine significantly reduced antiviral-dependent alterations of the nociceptive threshold. Moreover, nicotine decreased neuropathic pain induced by repeated intraperitoneal administration of the anticancer agent oxaliplatin (2.4 mg/kg), lowering the hypersensitivity to mechanical and thermal stimuli. In conclusion, intraperitoneal nicotine administration controls neuropathic pain evoked by traumatic or toxic nervous system alterations. These results support the nAChR modulation as a possible therapeutic approach to the complex, undertreated chemotherapy-induced neuropathies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We report the synthesis and bio-pharmacological evaluation of a class of pyrrole derivatives featuring a small appendage fragment (carbaldehyde, oxime, nitrile) on the central core. Compound 1c proved to be extremely effective in vivo, showing an interesting anti-nociceptic profile that is comparable to reference compounds already marketed, hence representing a great stimulus for a further improvement of this class of molecules., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABAA receptors subtype (benzodiazepine site on GABAA-receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABAA modulators. In fact, there is much evidence that GABAA-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABAA-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA(A)) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented research towards the development of new agents for the relief of pain. Starting from our previously reported ligands at the benzodiazepine site on GABA(A) receptors showing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that compounds 4, 10(±) and 11 have a very promising antihyperalgesic profile in different animal pain models (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since it exerted its protective effect starting from the dose of 3mg/kg po, after single injection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The role of voltage-gated sodium channels in the transmission of neuropathic pain is well recognized. For instance, genetic evidence recently indicate that the human Nav1.7 sodium channel subtype plays a crucial role in the ability to perceive pain sensation and may represent an important target for analgesic/anti-hyperalgesic drugs. In this study a newly synthesized tocainide congener, named NeP1, was tested in vitro on recombinant hNav1.4 and hNav1.7 channels using patch-clamp technique and, in vivo, in two rat models of persistent neuropathic pain obtained either by chronic constriction injury of the sciatic nerve or by oxaliplatin treatment. NeP1 efficiently blocked hNav1.4 and hNav1.7 channels in a dose- and use-dependent manner, being by far more potent than tocainide. Importantly, the new compound displayed a remarkable use-dependent effect, which likely resulted from a very high affinity for inactivated compared to closed channels. In both models of neuropathic pain, NeP1 was greatly more potent than tocainide in reverting the reduction of pain threshold in vivo. In oxaliplatin-treated rats, NeP1 even produced greater and more durable anti-hyperalgesia than the reference drug tramadol. In addition, in vivo and in vitro studies suggest a better toxicological and pharmacokinetic profile for NeP1 compared to tocainide. Overall, these results indicate NeP1 as a new promising lead compound for further development in the treatment of chronic pain of neuropathic origin., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Damages to the nervous system are the primarily cause of neuropathy and chronic pain. Current pharmacological treatments for neuropathic pain are not able to prevent or revert morphological and molecular consequences of tissue injury. On the other hand, many neurotrophins, like nerve growth factor (NGF), paired off restorative effects with hyperalgesia. Interestingly, the glial cell line-derived neurotrophic factors GDNF and Artemin (ARTN) seem to support neuron survival and to normalize abnormal pain behaviour. In the present research protein levels of NGF, GDNF and ARTN were evaluated in a rat model of peripheral neuropathy, the chronic constriction injury (CCI). NGF was increased by CCI in the ipsilateral dorsal root ganglia (DRG), in the spinal cord and in the periaqueductal grey matter (PAG). On the contrary, ARTN was decreased bilaterally in DRG, spinal cord and PAG. GDNF levels decreased in ipsilateral DRG, whereas the constriction did not modify its expression in the central nervous system districts. Repeated treatments with the antihyperalgesic and neuroregenerative compound acetyl-l-carnitine (ALCAR; 100 mgkg(-1) i.p. twice daily for 15 days) was able to prevent the increase of NGF levels. In conditions of pain relief ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham animals that underwent the same ALCAR treatment, showed increased levels of ARTN both in the DRG and in the spinal cord. These data offer a new point of view on the mechanism of the antihyperalgesic as well as the neuroprotective effect of ALCAR., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Current pharmacological treatments for neuropathic pain have limited efficacy and severe side-effect limitations. St. John's Wort (SJW) is a medicinal plant, mainly used as antidepressant, with a favourable side-effect profile. We here demonstrate the ability of SJW to relieve neuropathic pain in rat models. The antihyperalgesic profile and mechanism of action of SJW and its main components were studied in two rat models of neuropathic pain: the chronic constriction injury and the repeated administration of oxaliplatin. SJW, acutely administered at low doses (30-60 mg kg(-1) p.o.), reversed mechanical hyperalgesia with a prolonged effect, being effective up to 180 min after injection. Further examinations of the SJW main components revealed that hyperforin and hypericin were responsible for the antihyperalgesic properties whereas flavonoids were ineffective. The effect of SJW on the PKC expression and activation was investigated in the periaqueductal grey (PAG) area by immunoblotting experiments. Mechanistic studies showed a robust over-expression and hyperphosphorylation of the PKCgamma (227.0+/-15.0% of control) and PKCepsilon (213.9+/-17.0) isoforms in the rat PAG area. A single oral administration of SJW produced a significant decrease of the PKCgamma (131.8+/-10.0) and PKCepsilon (105.2+/-12.0) phosphorylation in the PAG area due to the presence of hypericin. Furthermore, SJW showed a dual mechanism of action since hyperforin antinociception involves an opioid-dependent pathway. Rats undergoing treatment with SJW and purified components did not show any behavioural side effects or signs of altered locomotor activity. Our results indicate SJW as a prolonged antihyperalgesic treatment through inhibition of PKC isoforms and their phosphorylation., (2009 Elsevier Inc. All rights reserved.)

The gamma isoform of protein kinase C (PKCgamma) is an injury-activated intracellular modulator that boosts neuronal activity in algesic and neuroregenerative signalling pathways. Acetyl-L-carnitine (ALCAR), a physiological compound with role in bioenergetic functions, shows an antihyperalgesic effect and at the same time can exert neuroregenerative and neuroprotective effects. Aimed to explore the link between pain and neuroregeneration, the effect of ALCAR treatment (100 mg kg(-1) i.p. twice daily for 15 days) on PKCgamma and mitogen-activated protein kinases (MAPKs) expression has been evaluated in CCI (chronic constriction injury) rats. The sciatic nerve and the lumbar tract of the spinal cord were processed to evaluate the levels of the phosphorylated form of PKCgamma, ERK 1,2, SAP/JNK, p-38 and c-Jun; furthermore, the mRNA expression of the early genes c-Jun and c-Fos has been investigated. Fifteen days after injury, the analysis in the sciatic nerves highlighted a bilateral increase of the activated forms of PKCgamma, ERK 1,2 and SAP/JNK, whereas c-Jun showed an increase only ipsilaterally. ALCAR completely prevented mechanical hyperalgesia and provoked in the nerve a c-Jun increment only. In the lumbar tract of the spinal cord, higher levels of activated PKCgamma, ERK 1,2, p38, SAP/JNK and c-Jun proteins were detected in the ipsilateral side in respect of sham. ALCAR was able to stimulate this expression profile. At the transcriptional level c-Jun mRNA was increased in the ipsilateral side of spinal cord of CCI saline-treated rats, whereas c-Fos mRNA was unchanged. ALCAR had a stimulatory effect on both these early genes. These findings may represent a different approach in the study of the complex balance between pain and neuroregeneration and could constitute the basis for developing new disease modifying agents in the treatment of neuropathic pain.

A series of amides and sulfonamides, structurally related to DM235 (sunifiram) and MN19 (sapunifiram), derived by ring expansion or contraction, or by inversion of the exocyclic amide function, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, and with a potency similar to the parent compounds.

A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties.

Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward cyclooxygenase (COX) isozymes. Analysis of enzymatic affinity and COX-2 selectivity led us to the selection of one compound (+/-)-10b that was further tested in vitro in the human whole blood (HWB) and in vivo for its anti-inflammatory activity in mice. The affinity data have been rationalized through docking simulations.

The importance of an intracellular calcium content increase to obtain cholinergic antinociception was demonstrated. The physiological and pathological role of ryanodine receptors (RyRs), receptors involved in the mobilization of intracellular calcium stores, at the CNS level is poorly understood. The aim of the present study was, therefore, to investigate the role of supraspinal endoplasmic type 1, 2 and 3 RyR subtypes in muscarinic antinociception in conditions of acute thermal (hotplate test) and inflammatory (abdominal constriction test) pain. In the absence of isoform selective RyR antagonists, types 1, 2 and 3 RyR knockdown mice were obtained. Western blotting experiments were performed to quantify the RyR isoform protein levels in knockdown mice demonstrating a selective protein level reduction in knockdown animals. I.c.v. pretreatment with an antisense oligonucleotide (aODN) against type 1 or type 3 RyR prevented cholinergic antinociception in the hotplate test shifting to the right of the physostigmine dose-response curve. This antagonistic effect disappeared 7 days after the end of the aODN administration. Conversely, the physostigmine analgesia remained unmodified in type 2 RyR knockdown mice. Similar results were obtained in the abdominal constriction test. Mice undergoing aODN treatments showed neither alteration of animals' gross behavior nor locomotor impairment (rota-rod and hole board tests). These results elucidate the intracellular mechanism underlying muscarinic antinociception. A selective involvement of RyR1 and RyR3 in supraspinal muscarinic analgesia was demonstrated whereas RyR2 appears not to play an essential role in acute thermal and inflammatory pain.

The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA(A)/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.

Chemical modifications of dimiracetam, a bicyclic analogue of the nootropic drug piracetam, afforded a small set of novel derivatives that were investigated in in vivo models of neuropathic pain. Compounds 5, 7 and 8 displayed a very promising antihyperalgesic profile in rat models of neuropathic pain induced by both chronic constriction injury of the sciatic nerve and streptozotocin. The compounds completely reverted the reduction of pain threshold evaluated by the paw pressure test. Importantly these derivatives did not induce any behavioural impairment as evaluated by the rotarod test. These results suggest that compounds 5, 7 and 8 might represent novel and well-tolerated therapeutic agents for the relief of neuropathic pain.

A series of 2-oxopiperazine, 4-aminomethyl-, 3-amino- and 3-aminomethylpiperidine analogues of DM235 (sunifiram) and MN19 (sapunifiram), two previously reported potent cognition-enhancers, have been synthesized and tested in the mouse passive-avoidance test. The compounds display minimal effective doses in the range 0.3-10mg/kg. Although the new substances do not show improved activity when compared to the parent compounds, some useful information has been obtained to understand structure-activity relationships. In addition, the 3-aminopiperidine moiety appears to be a promising scaffold to synthesize new drugs endowed with cognition-enhancing activity.

The involvement of central endoplasmic inositol 1,4,5-trisphosphate receptors (IP3R) in muscarinic antinociception was investigated in the mouse hot plate test. Selective knockdown of type 1, 2 and 3 IP3R was obtained by means of an antisense oligonucleotide (aODN) strategy. A selective IP3R protein level reduction of approximately 30-50% produced by aODN administration for each receptor subtype investigated was demonstrated by Western blotting experiments. I.c.v. pretreatment with an aODN complementary to the sequence of the type 2 IP3R (0.1-3 nmol per mouse i.c.v.) prevented the antinociception induced by physostigmine (0.15 mg kg(-1) s.c.) and oxotremorine (60 microg kg(-1) s.c.). Similarly, an aODN against type 3 IP3R (0.1-3 nmol per mouse i.c.v.) antagonized cholinergic antinociception. A shift to the right of the physostigmine dose-response curve was obtained after anti-type 2 IP3R2 and anti-type 3 IP3R treatments. Conversely, pretreatment with an aODN complementary to the sequence of type 1 IP3R (0.1-5 nmol per mouse i.c.v.) did not modify the antinociception induced by physostigmine and oxotremorine. Mice undergoing treatment with aODNs did not show any impairment of the locomotor activity, spontaneous motility and exploratory activity as revealed by the rota-rod and hole board tests. These results indicate a selective involvement of type 2 and 3 IP3R in central muscarinic antinociception in mice.

This study reports the development and in vivo evaluation of a liposomal formulation of the local anaesthetic benzocaine. Multi-lamellar (MLV) and small uni-lamellar (SUV) vesicles entrapping benzocaine were prepared using 50:50 w/w phosphatidylcholine-cholesterol as lipophilic phase and 50:50 v/v ethanol-water as hydrophilic phase. Liposome size, Zeta-potential, encapsulation efficiency and skin penetration properties were determined. Drug permeation from liposomal dispersions, as such or formulated in Carbopol gel, was evaluated through artificial lipophilic membranes and excised abdominal rat skin, whereas in vivo anaesthetic effect was tested on rabbits. Interestingly, addition of the drug into the hydrophilic phase, rather than into the lipophilic one, during liposome preparation enabled an improvement of the MLV's entrapment efficiency from 29.7% to 82.3%. On the other hand, sonication conditions to obtain SUV influenced size and polydispersity index of the vesicles and reduced the entrapment efficiency by about 30%. All liposomal-benzocaine formulations showed sustained release properties and a more intense anaesthetic effect than plain drug. Permeation experiments from drug solutions in gel containing the same amount of ethanol as in the liposomal formulations made it possible to exclude a possible enhancer effect of this solvent, at least when not used in liposomal formulations. MLV with the drug added into the hydrophilic phase gave the most effective formulation, showing a permeability coefficient value 2.5 times higher than that of the plain drug and allowing a significant improvement (P<0.01) not only of intensity but also of duration of anaesthetic effect of benzocaine. These results suggest that a suitably developed liposomal formulation of benzocaine can be of actual value for improving its clinical effectiveness in topical anaesthesia.

The synthesis and the binding study of new 3-iodiopyrazolo[5,1-c][1,2,4] benzotriazine 5-oxides 8-alkyloxy substituted are reported. The replacement at position 3 with an iodine atom, with respect to substituents capable to form a three centered hydrogen bond and/or to form pi-pi stacking interaction with receptor protein, gave high affinity ligands, independently of the 8-alkyloxy substituent. High-affinity ligands were studied in mice in vivo for their pharmacological effects, considering five potential benzodiazepine actions: anxiolytic-like effects, motor coordination, anticonvulsant action, mouse learning and memory impairment, and ethanol-potentiating action. Compounds 5c and 5'c have an inverse agonist profile and for the first time is evidenced a pro-mnemonic activity. These compounds were evaluated also for their binding at Benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) subtype to evaluate their subtype selectivity.

The aim of the present paper was to investigate the role of the octamethylene spacer of methoctramine (1) on the biological profile. Thus, this spacer was incorporated into a dianiline or dipiperidine moiety to determine whether flexibility and the basicity of the inner nitrogen atoms are important determinants of potency with respect to muscarinic receptors. The most potent compound was 4, which displayed, in the functional assays, a comparable potency at muscarinic M(2) receptors with respect to 1, and, in the binding assays, a loss of potency and selectivity toward muscarinic M(1) and M(3) receptor subtypes. Both compounds were endowed with antinociceptive activity. Furthermore, in microdialysis tests in rat parietal cortex, they enhanced acetylcholine release, most likely by antagonizing presynaptic muscarinic receptor subtypes.

Previous data indicate that methylamine injection in fasted healthy mice produced a hypophagic effect dependent of neuronal K(v)1.6 channels expression and increased by alpha-aminoguanidine, an inhibitor of semicarbazide-sensitive benzylamine oxidase enzymes mainly involved in amine degradation. In the present work we have investigated: 1) the level of expression and activity of the semicarbazide-sensitive benzylamine oxidase; 2) the effect of methylamine alone and in the presence of alpha-aminoguanidine on food intake of genetic obese and type II diabetes mice (the db/db mice). Db/db mice showed higher levels of semicarbazide-sensitive benzylamine oxidase activities in adipose tissue and in plasma than their lean counterpart (db/db(+) mice). Methylamine (30-75 microg, i.c.v.) showed similar hypophagic effects in obese and lean mice consistently with the levels of neuronal K(v)1.6 found in both animal strains. Alpha-aminoguandine (50 mg/kg, i.p.) increased methylamine (i.c.v.) hypophagia in both obese and lean mice and only in obese mice when methylamine was given i.p. These results suggest a crucial role of semicarbazide-sensitive benzylamine oxidase activity in controlling methylamine hypophagia in hyperphagic diabetic mice.

Structure-activity relationships on two novel potent cognition enhancing drugs, unifiram (DM232, 1) and sunifiram (DM235, 2), are reported. Although none of the compounds synthesised reached the potency of the parent drugs, some fairly active compounds have been identified that may represent new leads to develop other cognition enhancing drugs. An interesting result of this research is the identification of two compounds (13 and 14) that are endowed with amnesing activity (the opposite of the activity of the original molecules) and are nearly equipotent to scopolamine. Moreover, two compounds of the series (5 and 6) were found endowed with analgesic activity on a rat model of neuropathic pain at the dose of 1 mg/kg.

The post-receptorial mechanism of the amnesic action of the alpha2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the alpha-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 microg per mouse i.c.v.) reversed the amnesia induced by both alpha2-agonists. Similarly, anti-Gialpha1 (6.25-12.5 microg per mouse i.c.v.), anti-Gialpha3 (3.12-12.5 microg per mouse i.c.v.), anti-Goalpha1 (12.5-25 microg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Gialpha2 (3.12-25 microg per mouse i.c.v.) and anti-Goalpha2 (12.5-25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by the alpha2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz., (Copyright 2004 IBRO)

The effect of the i.c.v. administration of antisense oligodeoxynucleotides directed against the alpha subunit of different Gi-proteins (anti-Gialpha(1), anti-Gialpha(2), anti-Gialpha(3), anti-Goalpha(1), anti-Goalpha(2)) on the amnesia induced by the H(1)-antihistamine diphenhydramine (20 mg kg(-1) s.c.) was evaluated in the mouse passive avoidance test. Pretreatment with anti-Gialpha(1) (12.5-25 microg per mouse i.c.v.) and anti-Gialpha(2) (25 microg per mouse i.c.v.), administered 24 and 18 h before test, prevented antihistamine-induced amnesia. By contrast, pretreatment with an anti-Gialpha(3) (25 microg per mouse i.c.v.), anti-Goalpha(1) (25 microg per mouse i.c.v.) and anti-Goalpha(2) (25 microg per mouse i.c.v.) did not modify the detrimental effect induced by diphenhydramine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by the Gi(1)- and Gi(2)-protein pathway in the transduction mechanism involved in the impairment of memory processes produced by the H(1)-antihistamine diphenhydramine.