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Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance. Supplementary Information The online version contains supplementary material available at 10.1007/s13311-021-01116-4.

Ischaemic stroke is a leading cause of death and disability. One of the major pathogenic mechanisms after ischaemia includes the switch to the glycolytic pathway, leading to tissue acidification. Carbonic anhydrase (CA) contributes to pH regulation. A new generation of CA inhibitors, AN11-740 and AN6-277 and the reference compound acetazolamide (ACTZ) were investigated in two models of brain ischaemia: in rat hippocampal acute slices exposed to severe oxygen, glucose deprivation (OGD) and in an in vivo model of focal cerebral ischaemia induced by permanent occlusion of the middle cerebral artery (pMCAo) in the rat. In vitro, the application of selective CAIs significantly delayed the appearance of anoxic depolarisation induced by OGD. In vivo, sub-chronic systemic treatment with AN11-740 and ACTZ significantly reduced the neurological deficit and decreased the infarct volume after pMCAo. CAIs counteracted neuronal loss, reduced microglia activation and partially counteracted astrocytes degeneration inducing protection from functional and tissue damage.

Anti-cancer therapy based on the repeated administration of oxaliplatin is limited by the development of a disabling neuropathic syndrome with detrimental effects on the patient’s quality of life. The lack of effective pharmacological approaches calls for the identification of innovative therapeutic strategies based on new targets. We focused our attention on the imidazoline I(1) receptor (I(1)-R) and in particular on the selective I(1)-R agonist 2-(1-([1,1′-biphenyl]-2-yl)propan-2-yl)-4,5-dihydro-1H-imidazole) (carbophenyline). The purpose of this work was the preclinical evaluation of the efficacy of carbophenyline on oxaliplatin-induced neuropathic pain in mice. Carbophenyline, acutely per os administered (0.1–10 mg kg(−1)), induced a dose-dependent anti-hyperalgesic effect that was completely blocked by the pre-treatment with the I(1)-R antagonist 3 or the I(1)/α(2) receptor antagonist efaroxan, confirming the I(1)-R-dependent mechanism. Conversely, pre-treatment with the I(2)-R antagonist BU224 did not block the anti-nociceptive effect evoked by carbophenyline. Repeated oral administrations of carbophenyline (1 mg kg(−1)) for 14 days, starting from the first day of oxaliplatin injection, counteracted the development of neuropathic pain in all behavioral tests (cold plate, Von Frey, and paw pressure tests) carried out 24 h after the last carbophenyline treatment on days 7 and 14. In the dorsal horn of the spinal cord, carbophenyline significantly decreased the oxaliplatin-induced astrocyte activation detected by immunofluorescence staining by the specific labelling with GFAP antibody. In conclusion, carbophenyline showed anti-neuropathic properties both after acute and chronic treatment with preventive effect against oxaliplatin-induced astrocyte activation in the spinal cord. Therefore, I(1)-R agonists emerge as a new class of candidates for the management of oxaliplatin-induced neuropathic pain. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-020-00873-y) contains supplementary material, which is available to authorized users.

Bacopa monnieri (L.) is widely used in Ayurvedic medicine as a neural tonic for improving intelligence and memory. Several studies highlighted its efficacy in neuropsychiatric diseases but there is no evidence regarding anhedonia. Aim of the present work was to preclinically and clinically test against anhedonia a standardized B. monnieri extract (20% bacosides). In a mouse model of a depressive-like syndrome induced by lipopolysaccharide (LPS), the daily administration of the extract (50-200 mg kg-1 , p.o.) for 1 week, dose-dependently counteracted the immobility time in Porsolt and Tail suspension tests (p < .01). At the sucrose preference test (directly related to the ability for feeling pleasure) the extract treatment (100 and 200 mg kg-1 ) counteracted the reduction of sucrose intake induced by LPS (p < .01). Moreover, B. monnieri significantly reduced cytokines, cortisol, and artemin LPS-dependent alterations in plasma while increased the brain-derived neurotrophic factor levels (p < .05). The efficacy of the same extract was tested in a clinical study in which 42 patients with significant degree of anhedonia (evaluated as Snaith-Hamilton Pleasure Scale [SHAPS] score ≥ 3) were enrolled. Patients were divided into two groups and treated with citalopram or citalopram associated with B. monnieri (300 mg bid) for 4 weeks. The Pears Sample T-test showed a significant improvement (p < .05) in relevant scales (Hamilton depression rating scale, SHAPS, and strength and difficulties questionnaire) in the extract-treated group in comparison to citalopram alone was recorded. These data suggest that B. monnieri extract may be effective for the management of anhedonia and therefore should be considered for future controlled trials.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

New compounds with a 7-amino-2-arylmethyl-thiazolo[5,4-d]pyrimidine structure were synthesized and evaluated in vitro for their affinity and/or potency at the human (h) A1, hA2A, hA2B, and hA3 adenosine receptors (ARs). Several compounds (5, 8–10, 13, 18–19) were characterized by nanomolar and subnanomolar binding affinities for the hA1 and the hA2A AR, respectively. Results of molecular docking studies supported the in vitro results. The 2-(2-fluorobenzyl)-5-(furan-2yl)-thiazolo[5,4-d]pyrimidin-7-amine derivative 18 (hA1 Ki = 1.9 nM, hA2A Ki = 0.06 nM) was evaluated for its antidepressant-like activity in in vivo studies, the forced swimming test (FST), the tail suspension test (TST), and the sucrose preference test (SPT) in mice, showing an effect comparable to that of the reference amitriptyline.

Platinum-based chemotherapy is widely used for the treatment of different tumors but is associated with serious side effects, among which neuropathic pain. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have recently been validated as therapeutic agents in neuropathic pain and as antitumor agents. We report the synthesis of new organochalcogenides bearing the benzensulfonamide moiety acting as potent inhibitors of several human CA isoforms and, in particular, against hCA II and VII endowed with potent neuropathic pain attenuating effects. Moreover, in combination with cisplatin or doxorubicin, some of the new CA inhibitors enhanced the effects of the anticancer drugs capability in counteracting breast cancer MCF7 cell viability. The concomitant anti-neuropathic pain and antiproliferative effects of the new chalcogenide-based CA inhibitors represent an innovative approach for the counteraction and management of side effects associated with clinically platinum drugs as antitumor agents.

Chemotherapy-induced neuropathy (CIN) is a major dose-limiting side effect of anticancer therapy that can compel therapy discontinuation. Inadequate analgesic efficacy of current pharmacological approaches requires the identification of innovative therapeutics and, hence, the purpose of this study is to conduct a preclinical evaluation of the efficacy of DDD-028, a versatile pentacyclic pyridoindole derivative, against paclitaxel-induced neuropathic pain. In two separate experiments, DDD-028 was administered per os acutely (1–25 mg kg−1) or repeatedly (10 mg kg−1) in paclitaxel-treated rats. The response to mechanical noxious stimulus (paw pressure) as well as to non-noxious mechanical (von Frey) and thermal (cold plate) stimuli was investigated. Acute administration of DDD-028 induced a dose-dependent anti-neuropathic pain effect in all tests performed. Further, repeated daily treatment for 18 consecutive days (starting the first day of paclitaxel administration) significantly reduced the development of pain over time without the development of tolerance to the anti-hyperalgesic effect. Ex vivo analysis showed that DDD-028 was able to reduce oxidative damage of dorsal root ganglia as evidenced by the increase in the level of carbonylated proteins and the decrease in catalase activity. In the lumbar spinal cord, periaqueductal gray matter, thalamus, and somatosensory cortex 1, DDD-28 significantly prevented the activation of microglia and astrocytes. The pharmacodynamic study revealed that the pain-relieving effects of DDD-028 were fully blocked by both the non-selective nicotinic receptor (nAChR) antagonist mecamylamine and by the selective α7 nAChR antagonist methyllycaconitine. In conclusion, DDD-028 was active in reducing paclitaxel-induced neuropathic pain after single or repeated administrations without tolerance development and displaying a double symptomatic and neuroprotective profile. DDD-028 could represent a valuable candidate for the treatment of CIN. Supplementary Information The online version contains supplementary material available at 10.1007/s13311-021-01069-8.

Objectives The creation of a new valid preclinical model of articular pain by the intra-articular (i.a.) injection of mucilages for the screening of new treatments against arthritis. Methods A single intra-articular injection (20 μl) of mucilages (from Althaea officinalis roots and Linum usitatissimun seeds) or vegetal components (Amorphophallus konjac gum powder and β-glucan, used as reference standard) were assessed in the rat. The pathology progression was monitored by behavioural measurements (paw pressure test, von Frey test, incapacitance test and beam balance test) and compared to that induced by the i.a. injections of monoiodioacetate (MIA) and Complete Freund's Adjuvant (CFA), well-recognized models of osteoarthritis and rheumatoid arthritis, respectively. Key findings Among all, the mucilage of L. usitatissimun showed the best pro-algic profile inducing a painful long-lasting condition. Hypersensitivity was characterized as a mixed form of inflammatory and neuropathic pain by the responsiveness to ibuprofen (100 mg/kg, p.o.) and pregabalin (30 mg/kg, p.o.). The histological evaluation of joint showed a damage that represents both MIA and CFA features. Conclusions In conclusion, a single i.a. injection of L. usitatissimun mucilage can represent a valid model to assess articular pain in the rat for the screening of new treatments against arthritis.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4β2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4β2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4β2, α7, and α3β2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4β2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3β2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 μM). T...

Peripheral neuropathies, characterized by altered nociceptive and muscular functions, are related to oxidative stress. Thioctic acid is a natural antioxidant existing as two optical isomers, but most clinically used as racemic mixture. The present study investigated the central nervous system’s changes which followed loose-ligation-derived compression of sciatic nerve, the putative neuroprotective role of thioctic acid and the pain-alleviating effect on low-back pain suffering patients. Loose ligation of the right sciatic nerve was performed in spontaneously hypertensive rats (SHR), a model of increased oxidative stress, and in normotensive Wistar-Kyoto rats (WKY). Animals with sciatic nerve ligation were left untreated or were treated intraperitoneally for 15 days with 250 μmol·kg−1·die−1 of (+/−)-thioctic acid; 125 μmol·kg−1·die−1 of (+/−)-thioctic acid; 125 μmol·kg−1·die−1 of (+)-thioctic acid lysine salt; 125 μmol·kg−1·die−1 of (−)-thioctic acid; 300 μmol·kg−1·die−1 pregabalin. Control SHR and WKY rats received the same amounts of vehicle. The clinical trial NESTIORADE (Sensory-Motor Neuropathies of the Sciatic Nerve: Comparative evaluation of the effect of racemic and dextro-rotatory forms of thioctic acid) examined 100 patients (49 males and 51 females aged 53 ± 11 years) dividing them into two equal-numbered groups, each treated daily for 60 days with 600 mg of (+/−)-thioctic acid or (+)-thioctic acid, respectively. The trial was registered prior to patient enrollment at EudraCT website (OSSC Number: 2011-000964-81). In the preclinical study, (+)-thioctic acid was more active than (+/−)- or (−)-enantiomers in relieving pain and protecting peripheral nerve as well as in reducing oxidative stress and astrogliosis in the spinal cord. Main findings of NESTIORADE clinical trial showed a greater influence on painful symptomatology, a quicker recovery and a better impact on quality of life of (+)-thioctic acid vs. (+/−)-thioctic acid. These data may have a pharmacological and pharmacoeconomical relevance and suggest that thioctic acid, above all (+)-enantiomer, could be considered for treatment of low-back pain involving neuropathy.

Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.

In the most recent decades, oxaliplatin has been used as a chemotherapeutic agent for colorectal cancer and other malignancies as well. Oxaliplatin interferes with tumor growth predominantly exerting its action in DNA synthesis inhibition by the formation of DNA-platinum adducts that, in turn, leads to cancer cell death. On the other hand, unfortunately, this interaction leads to a plethora of systemic side effects, including those affecting the peripheral and central nervous system. Oxaliplatin therapy has been associated with acute and chronic neuropathic pain that induces physicians to reduce the dose of medication or discontinue treatment. Recently, the capability of oxaliplatin to alter the genetic and epigenetic profiles of the nervous cells has been documented, and the understanding of gene expression and transcriptional changes may help to find new putative treatments for neuropathy. The present article is aimed to review the effects of oxaliplatin on genetic and epigenetic mechanisms to better understand how to ameliorate neuropathic pain in order to enhance the anti-cancer potential and improve patients’ quality of life.

This work was aimed at enhancing butamben (BTB) anesthetic efficacy by the “drug-in cyclodextrin (CD)-in deformable liposomes” strategy. In the study, phase-solubility studies with natural (α-, β-, γ-) and derivative (hydroxypropyl-α-and β-, sulfobutylether-β, methyl-β) CDs evidenced the highest BTB affinity for βCD and its derivatives and indicated methyl-βCD (RAMEB) as the best carrier. Drug-RAMEB complexes were prepared by different techniques and were characterized for solid-state and dissolution properties. The best BTB–RAMEB product was chosen for entrapment in the aqueous core of deformable liposomes containing stearylamine, either alone or with sodium cholate, as edge activators. Double-loaded (DL) liposomes, bearing the lipophilic drug (0.5% w/v) in the bilayer and its hydrophilic RAMEB complex (0.5% w/v) in the aqueous core, were compared to single-loaded (SL) liposomes bearing 1% w/v plain drug in the bilayer. All vesicles showed homogeneous dimensions (i.e., below 300 nm), high deformability, and excellent entrapment efficiency. DL-liposomes were more effective than SL ones in limiting drug leakage (&lt, 5% vs. &gt, 10% after a 3 months storage at 4 °C). In vivo experiments in rabbits proved that all liposomal formulations significantly (p &lt, 0.05) increased the intensity and duration of drug anesthetic action compared to its hydroalcoholic solution, however, DL liposomes were significantly (p &lt, 0.05) more effective than SL ones in prolonging BTB anesthetic effect, owing to the presence of the drug-RAMEB complex in the vesicle core, acting as a reservoir. DL liposomes containing both edge activators were found to have the best performance.

Epigenetic mechanisms are involved in epilepsy and chronic pain development. About that, we studied the effects of the natural histone deacetylase (HDAC) inhibitor sodium butyrate (BUT) in comparison with valproic acid (VPA) in a validated genetic model of generalized absence epilepsy and epileptogenesis. WAG/Rij rats were treated with BUT (30 mg/kg), VPA (300 mg/kg), and their combination (BUT + VPA) daily per os for 6 months. Rats were subjected at Randall–Selitto, von Frey, hot plate, and tail flick tests after 1, 3, and 6 months of treatment to evaluate hypersensitivity to noxious and non-noxiuous stimuli. Moreover, PPAR-γ (G3335 1 mg/kg), GABA-B (CGP35348 80 mg/kg), and opioid (naloxone 1 mg/kg) receptor antagonists were administrated to investigate the possible mechanisms involved in analgesic activity. The expression of NFkB, glutathione reductase, and protein oxidation (carbonylation) was also evaluated by Western blot analysis. WAG/Rij rats showed an altered pain threshold throughout the study (p < 0.001). BUT and BUT + VPA treatment reduced hypersensitivity (p < 0.01). VPA was significantly effective only after 1 month (p < 0.01). All the three receptors are involved in BUT + VPA effects (p < 0.001). BUT and BUT + VPA decreased the expression of NFkB and enhanced glutathione reductase (p < 0.01); protein oxidation (carbonylation) was reduced (p < 0.01). No effect was reported with VPA. In conclusion BUT, alone or in coadministration with VPA, is a valuable candidate for managing the epilepsy-related persistent pain.

Opioids are broad spectrum analgesics that are an integral part of the therapeutic armamentarium to combat pain in the clinical practice. Unfortunately, together with analgesia, a number of adverse effects can occur such as nausea, vomiting, constipation, gastrointestinal alterations and cognitive impairments. Naltrexone is a competitive antagonist of opioids receptors commonly used to treat opioids addiction, its oral use against agonists side effects is limited by the decrease of opioids-therapeutic efficacy and own adverse effects. The intranasal delivery of naltrexone could offer a quick and effective achievement of CNS based on extracellular mechanisms including perineural and perivascular transport. Aim of the study was to test the efficacy of intranasal low-dose naltrexone in reducing intraperitoneal morphine and oxycodone side effects in rodents. In mice, 1 g naltrexone intranasally administered 30 min before opioids, reduced cognitive impairments and motor alteration induced by 10 mg kg-1 morphine and 60 mg kg-1 oxycodone in the Passive avoidance and Rota rod tests, respectively. Moreover, naltrexone rebalanced opioids-induced reduction of the intestinal transit and latency of feces expulsion as well as food intake inhibition. Importantly, 1 g naltrexone instillation did not block analgesia as demonstrated by the Hot plate test. In rats, intranasal naltrexone counteracted the opioid-induced pica phenomenon related to emesis and increased water and palatable food intake. The effects were comparable to that achieved by metoclopramide used as reference drug. Treatments did not influence body weight. Lastly, the safety of the intranasal delivery has been checked by hematoxylin-eosin staining that did not show histological alterations of the nasal cavity. In conclusion, intranasal low-dose naltrexone counteracted morphine and oxycodone induced gastrointestinal and CNS side effects without impairing opioids analgesia. It is candidate to be a valid clinical strategy deserving deeply analysis.

Hydrochlorothiazide (HCT) is widely used in pediatrics for hypertension management. Due to the lack of pediatric commercial forms, community or hospital pharmacies generally prepare HCT extemporaneous pediatric suspensions by dispersing in water a portion of a crushed tablet or the drug powder; however, any dose or stability control is usually done on these preparations. Obtaining stable HCT solutions is very challenging, due to its low water-solubility and pH-dependent degradation. The aim of this work was to develop a stable 2 mg/mL-HCT oral pediatric solution without using co-solvents. Combined use of cyclodextrins (CD) and hydrophilic polymers was exploited to improve poor HCT solubility and stability. HPβCD and SBEβCD were selected, considering their safe toxicological profiles, while PVP resulted the best among the tested polymers. Low PVP concentrations (0.2–1.0%) improved the solubilizing efficiency of both CDs, allowing to reach the prefixed HCT concentration. Different CD-PVP concentrations were used to prepare several 2 mg/mL-HCT solutions in pH 5.5 buffer. The best stability was shown by solutions containing the highest SBEβCD concentration (25 mM), which allowed a 3-months stability at 4 °C. In vivo studies on rats showed that such formulation allowed a more pronounced and more reproducible diuretic effect than the corresponding HCT suspension.

Kappa opioid receptor (KOPr) agonists represent alternative analgesics for their low abuse potential, although relevant adverse effects have limited their clinical use. Functionally selective KOPr agonists may activate, in a pathway-specific manner, G protein-mediated signaling, that produces antinociception, over β-arrestin 2-dependent induction of p38MAPK, which preferentially contributes to adverse effects. Thus, functionally selective KOPr agonists biased toward G protein-coupled intracellular signaling over β-arrestin-2-mediated pathways may be considered candidate therapeutics possibly devoid of many of the typical adverse effects elicited by classic KOPr agonists. Nonetheless, the potential utility of functionally selective agonists at opioid receptors is still highly debated; therefore, further studies are necessary to fully understand whether it will be possible to develop more effective and safer analgesics by exploiting functional selectivity at KOPr. In the present study we investigated in vitro functional selectivity and in vivo antinociceptive effects of LOR17, a novel KOPr selective peptidic agonist that we synthesized. LOR17-mediated effects on adenylyl cyclase inhibition, ERK1/2, p38MAPK phosphorylation, and astrocyte cell proliferation were studied in HEK-293 cells expressing hKOPr, U87-MG glioblastoma cells, and primary human astrocytes; biased agonism was investigated via cAMP ELISA and β-arrestin 2 recruitment assays. Antinociception and antihypersensitivity were assessed in mice via warm-water tail-withdrawal test, intraperitoneal acid-induced writhing, and a model of oxaliplatin-induced neuropathic cold hypersensitivity. Effects of LOR17 on locomotor activity, exploratory activity, and forced-swim behavior were also assayed. We found that LOR17 is a selective, G protein biased KOPr agonist that inhibits adenylyl cyclase and activates early-phase ERK1/2 phosphorylation. Conversely to classic KOPr agonists as U50,488, LOR17 neither induces p38MAPK phosphorylation nor increases KOPr-dependent, p38MAPK-mediated cell proliferation in astrocytes. Moreover, LOR17 counteracts, in a concentration-dependent manner, U50,488-induced p38MAPK phosphorylation and astrocyte cell proliferation. Both U50,488 and LOR17 display potent antinociception in models of acute nociception, whereas LOR17 counteracts oxaliplatin-induced thermal hypersensitivity better than U50,488, and it is effective after single or repeated s.c. administration. LOR17 administered at a dose that fully alleviated oxaliplatin-induced thermal hypersensitivity did not alter motor coordination, locomotor and exploratory activities nor induced pro-depressant-like behavior. LOR17, therefore, may emerge as a novel KOPr agonist displaying functional selectivity toward G protein signaling and eliciting antinociceptive/antihypersensitivity effects in different animal models, including oxaliplatin-induced neuropathy.

Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2&ndash, 2 mg mL&minus, 1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 &mu, g i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.

A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace [3H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC50 values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC50 = 8.66 nM) and 12d (IC50 = 5.19 nM) appeared as the most effective ligands being their affinity values significantly lower than that of diazepam (IC50 = 18.52 nM). Compounds 12a-f were examined in vivo for their pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All the new synthesized compounds were able to induce a significant antianxiety effect and, among them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly prevent the memory impairment evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety effect was observed. Finally, molecular docking simulations were carried out in order to assess the binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind the BDZ binding site in a similar fashion to flumazenil.

We report a series of compounds 1-17 derived from the antiepileptic drug Sulthiame (SLT) from which both the benzenesulfonamide and the sultam moiety were retained. All compounds were tested in vitro for their inhibition activity against the human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, VII, IX and XII isoforms. Among the series, derivatives 1 and 11 showed great enhancement of both inhibition potency and selectivity towards the hCA VII isoform, when compared to the reference SLT drug. The binding mode of 11 within the hCA VII active site was deciphered by means of X-ray crystallography and revealed the sultam moiety being exposed to the rim of the active site. In vivo experiments on a model of neuropathic pain induced by oxaliplatin clearly showed 11 being an effective pain relieving agent and therefore worth of further exploitation towards the validation of the hCA VII as new target for the management of neuropathies. (C) 2021 Elsevier Masson SAS. All rights reserved., Scientific and Technical Research Council of Turkey [TUBITAK117S516]; Ege University [16-ECZ-012]; Bando di Ateneo per il Finanziamento di Progetti Competitivi per Ricercatori a Tempo Determinato (RTD) dell'Universita di Firenze - 2020-2021; Fondazione Cassa di Risparmio di Firenze [ECR2018.1001], Ozlem Akgul (O.A.) is grateful to The Scientific and Technical Research Council of Turkey [TUBITAK117S516] and Ege University [16-ECZ-012].; Fabrizio Carta (F.C.) is grateful to Bando di Ateneo per il Finanziamento di Progetti Competitivi per Ricercatori a Tempo Determinato (RTD) dell'Universita di Firenze -2020-2021 and Fondazione Cassa di Risparmio di Firenze (Grant Number ECR2018.1001) which partially funded this work.

The beneficial effects of isothiocyanate-based compounds, as well as their safety, have been shown in neuropathological disorders, such as neuropathic pain. Aim of the present work was to study the efficacy of the glucosinolate glucoraphanin (GRA) and the derived isothiocyanate sulforaphane (SFN), secondary metabolites occurring exclusively in Brassicales, on chemotherapy-induced neuropathic pain. Mice were repeatedly treated with oxaliplatin (2.4 mg kg-1 ip) for 14 days to induce neuropathic pain. GRA and SFN effects were evaluated after a single administration on Day 15 or after a daily repeated oral and subcutaneous treatment starting from the first day of oxaliplatin injection until the 14th day. Single subcutaneous and oral administrations of GRA (4.43-119.79 μmol kg-1 ) or SFN (1.33-13.31 μmol kg-1 ) reduced neuropathic pain in a dose-dependent manner. The repeated administration of GRA and SFN (respectively 13.31 and 4.43 μmol kg-1 ) prevented the chemotherapy-induced neuropathy. The co-administration of GRA and SFN in mixture with the H2 S binding molecule, haemoglobin, abolished their pain-relieving effect, which was also reverted by pretreating the animals with the selective blocker of Kv7 potassium channels, XE991. GRA and SFN reduce neuropathic pain by releasing H2 S and modulating Kv7 channels and show a protective effect on the chemotherapy-induced neuropathy.

Oxaliplatin is a key drug in the treatment of advanced metastatic colorectal cancer. Despite its beneficial effects in tumor reduction, the most prevalent side-effect of oxaliplatin treatment is a chemotherapy-induced neuropathy that frequently forces to discontinue the therapy. Indeed, along with direct damage to peripheral nerves, the chemotherapy-related neurotoxicity involves also the central nervous system (CNS) as demonstrated by pain chronicity and cognitive impairment (also known as chemobrain), a newly described pharmacological side effect. The presence of the blood brain barrier (BBB) is instrumental in preventing the entry of the drug into the CNS; here we tested the hypothesis that oxaliplatin might enter the endothelial cells of the BBB vessels and trigger a signaling pathway that induce the disassembly of the tight junctions, the critical components of the BBB integrity. By using a rat brain endothelial cell line (RBE4) we investigated the signaling pathway that ensued the entry of oxaliplatin within the cell. We found that the administration of 10 μM oxaliplatin for 8 and 16 h induced alterations of the tight junction (TJs) proteins zonula occludens-1 (ZO-1) and of F-actin, thus highlighting BBB alteration. Furthermore, we reported that intracellular oxaliplatin rapidly induced increased levels of reactive oxygen species and endoplasmic reticulum stress, assessed by the evaluation of glucose-regulated protein GRP78 expression levels. These events were accompanied by activation of caspase-3 that led to extracellular ATP release. These findings suggested a possible novel mechanism of action for oxaliplatin toxicity that could explain, at least in part, the chemotherapy-related central effects.

The hydrochlorothiazide (HCT) low solubility and permeability give rise to limited and variable bioavailability; its low stability makes it difficult to develop stable aqueous liquid formulations; its low dose makes the achievement of a homogeneous drug distribution very difficult. Thus, the aim of this study was to investigate the effectiveness of a strategy based on the development of nanostructured lipid carriers (NLC) as an innovative oral pediatric formulation of HCT with improved therapeutic efficacy. The performance of various synthetic and natural liquid lipids was examined and two different preparation methods were employed, i.e. homogenization-ultrasonication (HU) and microemulsion (ME), in order to evaluate their influence on the NLC properties in terms of size, polydispersity index, ζ-potential, entrapment efficiency, gastric stability, and drug release properties. Precirol®ATO5 was used as solid lipid and Tween®80 and Pluronic®F68 as surfactants, formerly selected in a previous study focused on the development of HCT-solid lipid nanoparticles (SLNs). The presence of Pluronic®F68 did not allow ME formation. On the contrary, using Tween®80, the ME method enabled a higher entrapment efficiency than the HU. Regardless of the preparation method, NLCs exhibited great entrapment efficiency values clearly higher than previous SLNs. Moreover, NLC-ME formulations provided a prolonged release, which lasted for 6 h. In particular, NLC-ME containing Tween®20 as Co-Surfactant showed the best performances, giving rise to a complete drug release, never achieved with previous SLN formulations, despite their successful results. In vivo studies on rats confirmed these results, displaying their best diuretic profile. Moreover, all HCT-loaded NLC formulations showed higher stability than the corresponding SLNs.

Peripheral neuropathies are chronic painful syndromes characterized by allodynia, hyperalgesia and altered nerve functionality. Nerve tissue degeneration represents the microanatomical correlate of peripheral neuropathies. Aimed to improve the therapeutic possibilities, this study investigated the hypersensitivity and the neuromorphological alterations related to the loose ligation of the sciatic nerve in rats. Effects elicited by treatment with acetyl-L-carnitine (ALCAR) in comparison to gabapentin were assessed. Axonal injury, reduction of myelin deposition and accumulation of inflammatory cells were detected in damaged nerve. A decrease of phosphorylated 200-kDa neurofilament (NFP) immunoreactivity and a redistribution in small clusters of myelin basic like-protein (MBP) were observed in ipsilateral nerves. Treatment with ALCAR (100 mg/kg intraperitoneally - i.p.) and gabapentin (70 mg/kg i.p.) administered bis in die for 14 days induced a significant pain relieving effect. ALCAR, but not gabapentin, significantly countered neuromorphological changes and increased axonal NFP immunoreactivity. These findings indicate that both ALCAR and gabapentin significantly decreased the hypersensitivity related to neuropathic lesions. The observation of the positive ALCAR effect on axonal and myelin sheath alterations in damaged nerve supports its use as neurorestorative agent against neuropathies through mechanism(s) consistent to those focused in this study.

Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM_12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells' aggressiveness.

The FOLFOX family of chemotherapy regimens are hampered by the development of a painful neuropathy. Current clinical treatments are inadequate, and furthermore, the research of innovative drugs is strongly disadvantaged by the absence of a preclinical model based on the complete mixture of FOLFOX components. The aim of this study was to set up a rat model of FOLFOX-induced neuropathy in rats, validate its predictability by reference drugs, and evaluate the effectiveness of the new anti-neuropathic compound dimiracetam. Male Sprague–Dawley rats were treated intraperitoneally with the FOLFOX components (6 mg kg−1 oxaliplatin, 50 mg kg−1 5-FU, 90 mg kg−1 leucovorin calcium salt) or oxaliplatin alone (6 mg kg−1) on days 0, 7, 14, and 21, whereas a separate group received one more injection of FOLFOX on day 28. Pain behavioural measurements (paw pressure, cold plate, and electronic Von Frey tests) and motor coordination (Rota-rod test) were assessed before and after treatments. Behavioural, motor, neurological, and autonomic parameters (open field and Irwin tests) were evaluated. FOLFOX reduced the pain threshold in response to mechanical noxious and thermal (cold) non-noxious stimuli beginning from day 14 up to day 42 comparably to oxaliplatin alone. A fifth FOLFOX injection enhanced the severity but not the duration of painful alterations. Spontaneous activity, behavioural, autonomic, and neurological functions were also affected, whereas the motor coordination was not altered. On day 22, duloxetine (15 mg kg−1, per os), morphine (10 mg kg−1, subcutaneously), or pregabalin (20 mg kg−1, per os), acutely administered, reduced the FOLFOX-dependent hypersensitivity. Repeated treatments with dimiracetam (150 mg kg−1, per os, twice daily, from day 22) significantly protected rats from FOLFOX-induced alterations of pain threshold as well as from autonomic and neurological impairments taking effect after 7 days treatment. Pregabalin repeatedly administered (20 mg kg−1, per os, twice daily, from day 22) was less effective in reducing mechanical hypersensitivity. A clinically consistent model of FOLFOX-induced neurotoxicity has been developed in rats. Dimiracetam fully reduced hypersensitivity and neurological alterations showing a relevant profile as anti-neuropathic resource.

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.

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.

Despite several studies suggested that inattention and impulsivity-compulsivity could represent two core dimensions of hoarding disorder (HD), only a small case series study investigated the effectiveness of attention-deficit-hyperactivity-disorder (ADHD) medications in HD. The aim of the present study was to target attentional and inhibitory control networks in HD patients through the ADHD medication atomoxetine, moving from a preclinical investigation on an animal model of compulsive-like behavior (marble burying test) to a clinical investigation on both medicated and unmedicated patients with a primary diagnosis of HD without ADHD. Our preclinical investigation showed that acute administration of atomoxetine significantly reduced the compulsive-like behaviours of mice in the marble burying test without affecting neither locomotor activity and coordination nor exploration behaviours. When compared, atomoxetine and fluoxetine showed similar effects on the marble burying test. However, fluoxetine impaired both locomotor and exploratory activity. In our clinical investigation 12 patients were enrolled and 11 patients completed an open trial with atomoxetine at flexible dose (40–80 mg) for 12 weeks. At the endpoint the mean UCLA Hoarding Severity Scale score decreased by 41.3% for the whole group (p = 0003). Six patients were classified as full responders (mean symptom reduction of 57.2%) and three patients as partial responders (mean symptom reduction of 27.3%). Inattentive and impulsivity symptoms showed a significant mean score reduction of 18.5% from baseline to the endpoint (F (1,9) = 20.9, p = 0.0013). Hoarding symptoms improvement was correlated to reduction of patients’ disability and increased in their global functioning. These preclinical and clinical data suggest that atomoxetine may be effective for HD and therefore should be considered for future controlled trials.

Recently, studies have focused on the antihyperalgesic activity of the A(3) adenosine receptor (A(3)AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A(3)AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A(3)AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A(3)AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca(2+)-activated K(+) (K(Ca)) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca(2+) influx via N-type voltage-dependent Ca(2+) channels, as Cl-IB-MECA–induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca(2+) currents, and its effect was inhibited by 56% in the presence of A(3)AR antagonist MRS1523, demonstrating that the majority of adenosine’s effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A(3)AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca(2+) measurements confirmed the inhibitory role of A(3)AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A(3)AR activation could be mediated through N-type Ca(2+) channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A(3)AR-based therapy as a viable approach to alleviate pain in different pathologies.

To study new target-oriented molecules that are active against rheumatoid arthritis-dependent pain, new dual inhibitors incorporating both a carbonic anhydrase (CA)-binding moiety and a cyclooxygenase inhibitor (NSAID) were tested in a rat model of rheumatoid arthritis induced by CFA intra-articular (i.a.) injection. A comparison between a repeated per os treatment and a single i.a. injection was performed. CFA (50 &micro, L) was injected in the tibiotarsal joint, and the effect of per os repeated treatment (1 mg kg&minus, 1) or single i.a injection (1 mg ml&minus, 1, 50 &micro, L) with NSAIDs-CAIs hybrid molecules, named 4 and 5, was evaluated. The molecules 4 and 5, which were administered daily for 14 days, significantly prevented CFA-induced hypersensitivity to mechanical noxious (Paw pressure test) and non-noxious stimuli (von Frey test), the postural unbalance related to spontaneous pain (Incapacitance test) and motor alterations (Beam balance test). Moreover, to study a possible localized activity, 4 and 5 were formulated in liposomes (lipo 4 and lipo 5, both 1 mg ml-1) and directly administered by a single i.a. injection seven days after CFA injection. Lipo 5 decreased the mechanical hypersensitivity to noxious and non-noxious stimuli and improved motor coordination. Oral and i.a. treatments did not rescue the joint, as shown by the histological analysis. This new class of potent molecules, which is able to inhibit at the same time CA and cyclooxygenase, shows high activity in a preclinical condition of rheumatoid arthritis, strongly suggesting a novel attractive pharmacodynamic profile.

The management of pain in patients affected by diabetic neuropathy still represents an unmet therapeutic need. Recent data highlighted the pain-relieving efficacy of glucosinolates deriving from Brassicaceae. The purpose of this study was to evaluate the anti-hyperalgesic efficacy of Eruca sativa defatted seed meal, along with its main glucosinolate, glucoerucin (GER), on diabetic neuropathic pain induced in mice by streptozotocin (STZ). The mechanism of action was also investigated. Hypersensitivity was assessed by paw pressure and cold plate tests after the acute administration of the compounds. Once bio-activated by myrosinase, both E. sativa defatted meal (1 g kg&minus, 1 p.o.) and GER (100 &micro, mol kg&minus, 1 p.o., equimolar to meal content) showed a dose-dependent pain-relieving effect in STZ-diabetic mice, but the meal was more effective than the glucosinolate. The co-administration with H2S scavengers abolished the pain relief mediated by both E. sativa meal and GER. Their effect was also prevented by selectively blocking Kv7 potassium channels. Repeated treatments with E. sativa meal did not induce tolerance to the anti-hypersensitive effect. In conclusion, E. sativa meal can be suggested as a new nutraceutical tool for pain relief in patients with diabetic neuropathy.

To examine the effects of the alpha-amylase inhibitor isoform 1 called phaseolamin, a standardized extract from white kidney beans (Phaseolus vulgaris L) was tested against the hallmarks of metabolic syndrome. The efficacy of a per os repeated treatment with P. vulgaris extract (500 mg/kg) was compared with metformin (100 mg/kg) and atorvastatin (10 mg/kg) in a model of metabolic syndrome evoked by prolonged high fat diet (HFD, week 1 to week 19) in C57BL/6 mice. Bean extract and compounds administration started after metabolic syndrome establishment (week 11). P. vulgaris extract reduced the body weight overtime, as well as effectively lowered glycaemia, triglycerides, and cholesterol. On week 19, bean extract normalized the HFD-evoked tolerance to glucose and insulin. According to the phytochemical characterization, it inhibited the alpha-amylase activity. Animals treated with the extract were rescued from motor impairments and nociceptive threshold alterations induced by HFD. Specific organs analysis revealed that P. vulgaris extract decreased hepatic steatosis and lipid peroxidation in liver. It protected the heart from HFD oxidative alterations increasing the expression of the detoxifying enzymes catalase and glutathione reductase, and normalizing NADH dehydrogenase level. The histological analysis of aorta showed a protection about the development of fatty streaks in the muscular layers. In conclusion, a prolonged treatment with the standardized extract of P. vulgaris significantly reduced several pathological features related to a metabolic syndrome-like condition, a multifactorial approach that candidates this vegetal product as a possible therapeutic option against metabolic syndrome.

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1–25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki

The development of specific and age-appropriate pediatric formulations is essential to assure that all children and their care-givers can easily access to safe and effective dosage forms. The need for developing specific pediatric medicinal products has been highlighted by the European Medicines Agency. The aim of this study was to investigate the effectiveness of combining the advantages of both cyclodextrin (CD) complexation and loading into nanostructured lipid carriers (NLC), to obtain a liquid oral pediatric formulation of hydrochlorothiazide (HCT), endowed with safety, dosage accuracy, good stability and therapeutic efficacy. Equimolar drug combinations as physical mixture (P.M.) or coground product (GR) with hydroxypropyl-&beta, cyclodextrin (HP&beta, CD) or sulfobutylether-&beta, cyclodextrin (SBE&beta, CD) were loaded into NLC, then characterized for particle size, homogeneity, Zeta potential, entrapment efficiency, gastric and storage stability. The presence of HP&beta, CD allowed higher entrapment efficacy than NLC loaded with the plain drug, and enabled, in the case of GR systems a complete and sustained drug release, attributable to the wetting and solubilising properties of HP&beta, CD toward HCT. In vivo studies on rats proved the superior therapeutic effectiveness of HCT-in HP&beta, CD-in NLC formulations compared to the corresponding free HCT-loaded NLC, thus confirming the successfulness of the proposed approach in the development of an efficacious liquid oral formulation of the drug.

We studied the effects of intestinal inflammation on pentylenetetrazole (PTZ)-induced seizures in mice and the effects thereon of some antiepileptic and anti-inflammatory treatments to establish if a link may exist. The agents tested were: alpha-lactoalbumin (ALAC), a whey protein rich in tryptophan, effective in some animal models of epilepsy and on colon/intestine inflammation, valproic acid (VPA), an effective antiepileptic drug in this seizure model, mesalazine (MSZ) an effective aminosalicylate anti-inflammatory treatment against ulcerative colitis and sodium butyrate (NaB), a short chain fatty acid (SCFA) normally produced in the intestine by gut microbiota, important in maintaining gut health and reducing gut inflammation and oxidative stress. Intestinal inflammation was induced by dextran sulfate sodium (DSS) administration for 6 days. Drug treatment was started on day 3 and lasted 11 days, when seizure susceptibility to PTZ was measured along with intestinal inflammatory markers (i.e. NF-κB, Iκ-Bα, COX-2, iNOS), histological damage, disease activity index (DAI) and SCFA concentration in stools. DSS-induced colitis increased seizure susceptibility and while all treatments were able to reduce intestinal inflammation, only ALAC and NaB exhibited significant antiepileptic properties in mice with induced colitis, while they were ineffective as antiepileptics at the same doses in control mice without colitis. Interestingly, in DSS-treated mice, VPA lost part of its antiepileptic efficacy in comparison to preventing seizures in non-DSS-treated mice while MSZ remained ineffective in both groups. Our study demonstrates that reducing intestinal inflammation through ALAC or NaB administration has specific anticonvulsant effects in PTZ-treated mice. Furthermore, it appears that intestinal inflammation may reduce the antiepileptic effects of VPA, although we confirm that it decreases seizure threshold in this group. Therefore, we suggest that intestinal inflammation may represent a valid antiepileptic target which should also be considered as a participating factor to seizure incidence in susceptible patients and also could be relevant in reducing standard antiepileptic drug efficacy.

Neuropathic pain is originated from different alterations of the nervous system. The difficulty of treatment strongly impairs quality of life of affected people. It is associated with severe, chronic sensory disturbances characterized by spontaneous pain, increased responsiveness to painful stimuli and pain perceived in response to normally non-noxious stimuli. The underlying mechanisms are complex and involve both peripheral and central nervous components. The noradrenergic system plays a pivotal role in the control of pain since its widespread distribution in the “pain matrix” representing a valuable therapeutic target. This review focused on the α 2 adrenoceptor subtype modulation as strategy for neuropathic pain relief. Drugs acting as direct α 2 adrenoceptor agonists (clonidine and dexmedetomidine) were analyzed as well as the indirect α 2 adrenoceptor modulators. The overview included norepinephrine reuptake inhibitors (reboxetine, maprotiline), serotonin/norepinephrine reuptake inhibitors (venlafaxine, milnacipran, amitriptyline, duloxetine, bicifadine) and the compounds characterized by a double pharmacodynamic mechanism combining the norepinephrine reuptake inhibition and the μ opioid agonist profile (tramadol and tapentadol). A summary of recent compounds was illustrated.

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.

A prominent role of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels has been suggested based on their expression and (dys)function in dorsal root ganglion (DRG) neurons, being likely involved in peripheral nociception. Using HCN blockers as antinociceptive drugs is prevented by the widespread distribution of these channels. However, tissue-specific expression of HCN isoforms varies significantly, HCN1 and HCN2 being considered as major players in DRG excitability. We characterized the pharmacological effect of a novel compound, MEL55A, able to block selectively HCN1/HCN2 isoforms, on DRG neuron excitability in-vitro and for its antiallodynic properties in-vivo. HEK293 cells expressing HCN1, HCN2, or HCN4 isoforms were used to verify drug selectivity. The pharmacological profile of MEL55A was tested on mouse DRG neurons by patch-clamp recordings, and in-vivo in oxaliplatin-induced neuropathy by means of thermal hypersensitivity. Results were compared to the non-isoform-selective drug, ivabradine. MEL55A showed a marked preference toward HCN1 and HCN2 isoforms expressed in HEK293, with respect to HCN4. In cultured DRG, MEL55A reduced Ih amplitude, both in basic conditions and after stimulation by forskolin, and cell excitability, its effect being quantitatively similar to that observed with ivabradine. MEL55A was able to relieve chemotherapy-induced neuropathic pain. In conclusion, selective blockade of HCN1/HCN2 channels, over HCN4 isoform, was able to modulate electrophysiological properties of DRG neurons similarly to that reported for classical Ih blockers, ivabradine, resulting in a pain-relieving activity. The availability of small molecules with selectivity toward HCN channel isoforms involved in nociception might represent a safe and effective strategy against chronic pain.

The facilitation of opioid medication is eliciting a nemetic problem since increasing overdose deaths involve prescription of opioid pain relievers. Chronic painful diseases require higher doses of opioids, progressively with the development of tolerance to the antinociceptive effect. Novel strategies for the maintenance of low dosed opioid effectiveness are necessary to relieve pain and decrease abuse, overdose, and side effects. N-Palmitoylethanolamine (PEA) is an endogenous compound able to preserve the homeostasis of the nervous system and to delay the development of morphine tolerance. In the present study, a preemptive and continuative treatment with ultramicronized PEA (30 mg/kg, daily, per os) enhanced the acute antinociceptive efficacy of morphine (10 mg/kg subcutaneously) in rats and prolonged the responsiveness to the natural opioid. Moreover, PEA-treated animals had a more rapid recovery from tolerance. Four opioid free days were enough to regain sensitivity to morphine whereas control animals needed 31 days for full recovery of tolerance. Characteristically, PEA acquired per se antinociceptive properties in tolerant animals, suggesting the possibility of an integrated morphine/PEA treatment protocol. To maintain a significant analgesia, morphine dose had to be increased from 5 up to 100 mg/kg over 17 days of daily treatment. The same pain threshold increase was achieved in animals using preemptive PEA (30 mg/kg, daily) joined to a combinatorial acute treatment with morphine (5–20 mg/kg s.c.) and PEA (30–120 mg/kg, p.o.). Representatively, on day 17, the magnitude of analgesia induced by 100 mg/kg morphine was obtained by combining 13 mg/kg of morphine with 120 mg/kg of PEA. PEA strengthens the efficacy and potency of morphine analgesia, allowing prolonged and effective pain relief with low doses. PEA is suggested in association with morphine for chronic pain therapies distinguished by low risk of side effects.

A delivery system based on poly(lactic-co-glycolic acid) polymer (PLGA) microparticles has been developed for parenteral administration of the local anesthetic prilocaine in its free base form. Both drug-free and drug-loaded microparticles, prepared by a double-emulsion-evaporation method, were characterized for mean size by Laser Diffraction Analysis, while their morphology was investigated by scanning electron microscopy. The preparation technique allowed obtainment of homogeneous microparticles of about 25 µm diameter, suitable for subcutaneous administration. The encapsulation efficiency, determined by both direct and indirect methods, was around 36-38%. Differential Scanning Calorimetry was used to characterize the solid state of the raw materials, assess drug-polymer compatibility and miscibility and highlight possible modifications of the components induced by the preparation method. In vitro release studies showed a sustained release profile, with about 80% of drug released after the first 24 h. The anesthetic effect of the formulation was evaluated in vivo on rats, according to the test of cutaneous trunci muscle reflex. Administration of prilocaine base as PLGA microparticles allowed to significantly enhance both extent (60% AUC increase) and duration (100% increase) of the anesthetic effect in the animal model, in comparison with the equivalent dose of prilocaine hydrochloride aqueous solution.

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.

Oxaliplatin treatment is associated with the development of a dose-limiting painful neuropathy impairing patient’s quality of life. Since oxidative unbalance is a relevant mechanism of oxaliplatin neurotoxicity, we assessed the potential antioxidant properties of Vitis vinifera extract in reducing oxaliplatin-induced neuropathy as a valuable therapeutic opportunity. A hydroalcoholic extract of Vitis vinifera red leaf was characterized and tested in primary rat astrocyte cells treated with oxaliplatin (100 μM). Oxaliplatin lethality in the human adenocarcinoma cell line HT-29 was evaluated in the absence and presence of the extract. In vivo, pain hypersensitivity was measured in a rat model of neuropathy induced by oxaliplatin and ex vivo molecular targets of redox balance were studied. Vitis vinifera extract (50 μg mL−1, 4 h incubation) significantly reduced the oxaliplatin-dependent superoxide anion increase and lipid peroxidation in rat astrocytes but did not interfere with the mortality elicited by oxaliplatin in HT-29 cancer cells. In oxaliplatin-treated rats, a repeated daily administration of the Vitis vinifera extract (300 mg kg−1, p.o.) significantly prevented mechanical and thermal hypersensitivity to noxious and non noxious stimuli. mRNA and protein levels of Nrf2 were normalized in spinal cord and DRGs. Moreover, in the spinal cord, the extract significantly decreased the activation of astrocytes. Vitis vinifera reduced oxidative damages and relieved pain without influencing oxaliplatin anti-cancer activity.

Ischemia of brain areas is a global health problem, causing death or long-term disability. Current pharmacological options have limited impact on ischemic damages. Recently, a relationship between hypoxia and carbonic anhydrase (CA) over-expression has been highlighted suggesting CA inhibition as a possible target. This study aimed to evaluate the pharmacological profile of sulfonamide and coumarin CA inhibitors in rats underwent permanent middle cerebral artery occlusion (pMCAO). The neurological score of pMCAO rats was dramatically reduced 24 h after occlusion. Repeated subcutaneous injections of the CA inhibitors 4 and 7 (1 mg kg(-1)) were able to increase the neurological score by 40%. Compound 7 showed the tendency to reduce the volume of hemisphere infarction. The standard CA inhibitor acetazolamide was ineffective. The properties of novel CA inhibitors to improve neurological functionalities after cerebral ischemic insult are shown. The CA involvement in cerebral hypoxic phenomena deserves deeper investigations.

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.

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.

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.

Accumulating evidence suggests that opioid analgesics can lead to paradoxical sensitization to pain when delivered in different administration patterns. Although opioid tolerance-induced hyperalgesia is largely studied, little is known about the mechanisms underlying acute ultra-low-dose morphine hyperalgesia. Activation of spinal glial cells is reported to regulate pain hypersensitivity. To elucidate the mechanism involved in acute ultra-low-dose morphine hyperalgesia, we tested whether an opioid agonist promoted the activation of spinal astrocytes and microglia and investigated the cellular pathways involved. Ultra-low-dose morphine activated spinal astrocytes with no effect on microglia. The astrocyte activation was selectively prevented by the opioid antagonist naloxone, the μ-opioid receptor (MOR) silencing and the JNK inhibitor SP600125. Morphine elevated spinal JNK1, JNK2, and c-Jun phosphorylation. Conversely, phosphorylation of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription-1 (STAT-1) was not elevated, and nuclear factor kappa B (NF-κB) levels remained unmodified. Administration of SP600125 and the N-methyl-D-aspartate (NMDA) antagonist MK801 prevented morphine hyperalgesia. Ultra-low-dose morphine increased protein kinase C (PKC) γ phosphorylation. Pretreatment with a PKC inhibitor prevented morphine hyperalgesia and JNK and c-Jun overphosphorylation, indicating PKC is a JNK upstream modulator and illustrating the presence of a pathway involving PKC, NMDA, and JNK activated by morphine. Immunofluorescence experiments indicated the neuronal localization of spinal MOR. However, JNK was not detected in MOR-expressing cells, showing the presence of a neuron-astrocyte signaling pathway. These results illustrate the selective activation of an astrocyte JNK pathway after the stimulation of neuronal MOR, which contributes to ultra-low-dose morphine hyperalgesia.

Background Tanacetum parthenium L., commonly called Feverfew, is known for anti-inflammatory and anti-migraine properties. Purpose Aimed to individuate new therapeutical strategies to control acute and persistent pain induced by different origins we tested two hydroalcoholic extracts obtained from Feverfew flowers and leaves, respectively. Study Design Extracts were characterized according to the European Pharmacopoeia monograph. Both the extracts were tested after acute per os administration in the dose range 30–1000 mg kg −1 . The anti-nociceptive properties were evaluated by the Writhing test in mice. Results The number of abdominal contractions was dose dependently reduced by the flower extract. It reduced mechanical hypersensitivity (Paw pressure test) related to the acute inflammatory phase induced by carrageenan similarly to diclofenac and ibuprofen. In the osteoarthritis model induced by intra articular injection of monoiodoacetate (MIA) the flower extract significantly increased the pain threshold peaking 30 min after treatment. Moreover, it was effective in the chronic constriction injury model of neuropathic pain showing activity similar to the anti-epileptic drug gabapentin. The flower extract activity was confirmed in rat models of chemotherapy-induced neuropathic pain. The mechanical hypersensitivity induced by repeated treatments with the anticancer drug oxaliplatin and with the antiviral dideoxycytidine was significantly reduced after a single injection of Feverfew flower extract. The leaf extract showed lesser efficacy and potency and it was devoid of any effect in carrageenan-, MIA- and chemotherapy-induced pain. Conclusion The present Feverfew flower extract behaves as a potent pain reliever in acute, inflammatory, articular and neuropathic pain. It appears as a natural strategy potentially suitable for the treatment of different kinds of pain.