Disruption of the microbiota-gut-brain axis is a defining characteristic of the α-Gal A (-/0) mouse model of Fabry disease.

Fabry disease (FD) is an X-linked metabolic disease caused by a deficiency in α-galactosidase A (α-Gal A) activity. This causes accumulation of glycosphingolipids, especially globotriaosylceramide (Gb3), in different cells and organs. Neuropathic pain and gastrointestinal (GI) symptoms, such as abdominal pain, nausea, diarrhea, constipation, and early satiety, are the most frequent symptoms reported by FD patients and severely affect their quality of life. It is generally accepted that Gb3 and lyso-Gb3 are involved in the symptoms; nevertheless, the origin of these symptoms is complex and multifactorial, and the exact mechanisms of pathogenesis are still poorly understood. Here, we used a murine model of FD, the male α-Gal A (-/0) mouse, to characterize functionality, behavior, and microbiota in an attempt to elucidate the microbiota-gut-brain axis at three different ages. We provided evidence of a diarrhea-like phenotype and visceral hypersensitivity in our FD model together with reduced locomotor activity and anxiety-like behavior. We also showed for the first time that symptomology was associated with early compositional and functional dysbiosis of the gut microbiota, paralleled by alterations in fecal short-chain fatty acid levels, which partly persisted with advancing age. Interestingly, most of the dysbiotic features suggested a disruption of gut homeostasis, possibly contributing to accelerated intestinal transit, visceral hypersensitivity, and impaired communication along the gut-brain axis.

Persulfidation of mitoKv7.4 channels contributes to the cardioprotective effects of the H2S-donor Erucin against ischemia/reperfusion injury.

BACKGROUND: Hydrogen sulfide (H2S) 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 H2S 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 H2S-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 H2S and concentration-dependently protected H9c2 cells against H2O2-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.

Environmental training is beneficial to clinical symptoms and cortical presynaptic defects in mice suffering from experimental autoimmune encephalomyelitis.

The effect of "prophylactic" environmental stimulation on clinical symptoms and presynaptic defects in mice suffering from the experimental autoimmune encephalomyelitis (EAE) at the acute stage of disease (21 ±â€¯1 days post immunization, d.p.i.) was investigated. In EAE mice raised in an enriched environment (EE), the clinical score was reduced when compared to EAE mice raised in standard environment (SE).Concomitantly, gain of weight and increased spontaneous motor activity and curiosity were observed, suggesting increased well-being in mice. Impaired glutamate exocytosis and cyclic adenosine monophosphate (cAMP) production in cortical terminals of SE-EAE mice were evident at 21 ±â€¯1 d.p.i.. Differently, the 12 mM KCl-evoked glutamate exocytosis from cortical synaptosomes of EE-EAE mice was comparable to that observed in SE and EE-control mice, but significantly higher than that in SE-EAE mice. Similarly, the 12 mM KCl-evoked cAMP production in EE-EAE mice cortical synaptosomes recovered to the level observed in SE and EE-control mice. MUNC-18 and SNAP25 contents, but not Syntaxin-1a and Synaptotagmin 1 levels, were increased in cortical synaptosomes from EE-EAE mice when compared to SE-EAE mice. Circulating IL-1ß was increased in the spinal cord, but not in the cortex, of SE-EAE mice, and it did not recover in EE-EAE mice. Inflammatory infiltrates were reduced in the cortex but not in the spinal cord of EE-EAE mice. Demyelination was observed in the spinal cord; EE significantly diminished it. We conclude that "prophylactic" EE is beneficial to synaptic derangements and preserves glutamate transmission in the cortex of EAE mice. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment".

In vitro and in vivo characterization of the bifunctional µ and δ opioid receptor ligand UFP-505.

BACKGROUND AND PURPOSE: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reducing side-effects. We have evaluated the mixed µ opioid receptor agonist/ δ opioid receptor antagonist UFP-505 in vitro and in vivo. EXPERIMENTAL APPROACH: We measured receptor density and function in single µ, δ and µ /δ receptor double expression systems. GTPγ35 S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments, we collected tissues to measure receptor densities. KEY RESULTS: UFP-505 bound to µ receptors with full agonist activity and to δ receptors as a low efficacy partial agonist At µ, but not δ receptors, UFP-505 binding recruited arrestin. Unlike morphine, UFP-505 treatment internalized µ receptors and there was some evidence for internalization of δ receptors. Similar data were obtained in a µ /δ receptor double expression system. In rats, acute UFP-505 or morphine, injected intrathecally, was antinociceptive. In tissues harvested from these experiments, µ and δ receptor density was decreased after UFP-505 but not morphine treatment, in agreement with in vitro data. Both morphine and UFP-505 induced significant tolerance. CONCLUSIONS AND IMPLICATIONS: In this study, UFP-505 behaved as a full agonist at µ receptors with variable activity at δ receptors. This bifunctional compound was antinociceptive in rats after intrathecal administration. In this model, dual targeting provided no advantages in terms of tolerance liability. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Increase of neurofilament-H protein in sensory neurons in antiretroviral neuropathy: Evidence for a neuroprotective response mediated by the RNA-binding protein HuD.

Nucleoside reverse transcriptase inhibitors (NRTIs) are key components of HIV/AIDS treatment to reduce viral load. However, antiretroviral toxic neuropathy has become a common peripheral neuropathy among HIV/AIDS patients leading to discontinuation of antiretroviral therapy, for which the underlying pathogenesis is uncertain. This study examines the role of neurofilament (NF) proteins in the spinal dorsal horn, DRG and sciatic nerve after NRTI neurotoxicity in mice treated with zalcitabine (2',3'-dideoxycitidine; ddC). ddC administration up-regulated NF-M and pNF-H proteins with no effect on NF-L. The increase of pNF-H levels was counteracted by the silencing of HuD, an RNA binding protein involved in neuronal development and differentiation. Sciatic nerve sections of ddC exposed mice showed an increased axonal caliber, concomitantly to a pNF-H up-regulation. Both events were prevented by HuD silencing. pNF-H and HuD colocalize in DRG and spinal dorsal horn axons. However, the capability of HuD to bind NF mRNA was not demonstrated, indicating the presence of an indirect mechanism of control of NF expression by HuD. RNA immunoprecipitation experiments showed the capability of HuD to bind the BDNF mRNA and the administration of an anti-BDNF antibody prevented pNF-H increase. These data indicate the presence of a HuD - BDNF - NF-H pathway activated as a regenerative response to the axonal damage induced by ddC treatment to counteract the antiretroviral neurotoxicity. Since analgesics clinically used to treat neuropathic pain are ineffective on antiretroviral neuropathy, a neuroregenerative strategy might represent a new therapeutic opportunity to counteract neurotoxicity and avoid discontinuation or abandon of NRTI therapy.

Development of solid lipid nanoparticles as carriers for improving oral bioavailability of glibenclamide.

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.

Spinal RyR2 pathway regulated by the RNA-binding protein HuD induces pain hypersensitivity in antiretroviral neuropathy.

The antiretroviral toxic neuropathy, a distal sensory polyneuropathy associated with antiretroviral treatment, is a frequently occurring neurological complication during treatment of patients with AIDS and often leads to discontinuation of antiretroviral therapy. The mechanisms by which antiretroviral drugs contribute to the development of neuropathic pain are not known. Using drugs that reduce intracellular calcium ions (Ca(2+)), we investigated the hypothesis that altered cytosolic Ca(2+) concentration contributes to the 2',3'-dideoxycytidine (ddC)-evoked painful neuropathy. Administration of ddC induced mechanical and cold allodynia, which were abolished by intrathecal administration of TMB-8, a blocker of Ca(2+) release from intracellular stores, and by ryanodine, a RyR antagonist. Treatment with the IP3R antagonist heparin prevented mechanical allodynia with no effect on thermal response. To further clarify the pathway involved, we investigated the role of HuD, a RNA binding protein involved in neuronal function. HuD silencing reverted both mechanical and cold allodynia inducing, a phenotype comparable to that of ryanodine-exposed mice. HuD binding to the RyR2 mRNA, the most abundant RyR isoform in the spinal cord, was demonstrated and RyR2 silencing prevented the ddC-induced neuropathic pain. A positive regulation of gene expression on CaMKIIα by HuD was also observed, but sequestration of CaMKIIα had no effect on ddC-induced allodynia. The present findings identify a spinal RyR2 pathway activated in response to ddC administration, involving the binding activity on RyR2 mRNA by HuD. We propose the modulation of the RyR2 pathway as a therapeutic perspective in the management of antiretroviral painful neuropathy.

The RNA-binding protein HuD promotes spinal GAP43 overexpression in antiretroviral-induced neuropathy.

Nucleoside reverse transcriptase inhibitors (NRTIs) are known to produce painful neuropathies and to enhance states of pain hypersensitivity produced by HIV-1 infection in patients with AIDS leading to discontinuation of antiretroviral therapy, thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current study, we tested the hypothesis that HuD, an RNA binding protein known to be an essential promoter of neuronal differentiation and survival, might be involved in the response to NRTI-induced neuropathy. Antiretroviral neuropathy was induced by a single intraperitoneal administration of 2',3'-dideoxycytidine (ddC) in mice. HuD was physiologically expressed in the cytoplasm of the soma and in axons of neurons within DRG and spinal cord and was considerably overexpressed following ddC treatment. ddC up-regulated spinal GAP43 protein, a marker of neuroregeneration, and this increase was counteracted by HuD silencing. GAP43 and HuD colocalize in DRG and spinal dorsal horn (SDH) axons and administration of an anti-GAP43 antibody aggravated the ddC-induced axonal damage. The administration of a protein kinase C (PKC) inhibitor or the PKCγ silencing prevented both HuD and GAP43 increased expression. Conversely, treatment with the PKC activator PDBu potentiated HuD and GAP43 overexpression, demonstrating the presence of a spinal PKC-dependent HuD-GAP43 pathway activated by ddC. These results indicated that HuD recruitment and GAP43 protein increase are mechanistically linked events involved in the response to antiretroviral-induced neurodegenerative processes.

PKC-mediated HuD-GAP43 pathway activation in a mouse model of antiretroviral painful neuropathy.

Patients treated with nucleoside reverse transcriptase inhibitors (NRTIs) develop painful neuropathies that lead to discontinuation of antiretroviral therapy thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathy are not known. In order to elucidate the mechanisms underlying this drug-induced neuropathy, we have characterized cellular events in the central nervous system following antiretroviral treatment. Systemic administration of the antiretroviral agent, 2',3'-dideoxycytidine (ddC) considerably increased the expression and phosphorylation of protein kinase C (PKC) γ and ɛ, enzymes highly involved in pain processes, within periaqueductal grey matter (PAG), and, to a lesser extent, within thalamus and prefrontal cortex. These events appeared in coincidence with thermal and mechanical allodynia, but PKC blockade did not prevent the antiretroviral-induced pain hypersensitivity, ruling out a major involvement of PKC in the ddC-induced nociceptive behaviour. An increased expression of GAP43, a marker of neuroregeneration, and decreased levels of ATF3, a marker of neuroregeneration, were detected in all brain areas. ddC treatment also increased the expression of HuD, a RNA-binding protein target of PKC known to stabilize GAP43 mRNA. Pharmacological blockade of PKC prevented HuD and GAP43 overexpression. Silencing of both PKCγ and HuD reduced GAP43 levels in control mice and prevented the ddC-induced GAP43 enhanced expression. Present findings illustrate the presence of a supraspinal PKC-mediated HuD-GAP43 pathway activated by ddC. Based on our results, we speculate that antiretroviral drugs may recruit the HuD-GAP43 pathway, potentially contributing to a response to the antiretroviral neuronal toxicity.

Development and characterization of functionalized niosomes for brain targeting of dynorphin-B.

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.

The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

St. John's wort reversal of meningeal nociception: a natural therapeutic perspective for migraine pain.

Despite a number of antimigraine drugs belonging to different pharmacological classes are available, there is a huge unmet need for better migraine pharmacotherapy. We here demonstrated the capability of Hypericum perforatum, popularly called St. John's wort (SJW), to relieve meningeal nociception in an animal model induced by administration of the nitric oxide (NO) donors glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). GTN and SNP produced a delayed meningeal inflammation, as showed by the upregulation of interleukin (IL)-1ß and inducible NO synthase (iNOS), and a prolonged cold allodynia and heat hyperalgesia with a time-course consistent with NO-induced migraine attacks. A single oral administration of a SJW dried extract (5mg/kg p.o.) counteracted the nociceptive behaviour and the overexpression of IL-1ß and iNOS. To clarify the cellular pathways involved, the expression of protein kinase C (PKC) and downstream effectors was detected. NO donors increased expression and phosphorylation of PKCγ, PKCɛ and transcription factors, such as nuclear factor (NF)-κB, cyclic AMP response element binding protein (CREB), Signal Transducer and Activator of Transcription (STAT)-1. All these molecular events were prevented by SJW and hypericin, a SJW main component. In conclusion, SJW counteracted the NO donor-induced pain hypersensitivity and meningeal activation by blocking PKC-mediated pathways involving NF-κB, CREB, STAT1. These results might suggest SJW as an innovative and safe perspective for migraine pain.

Oxaliplatin-induced oxidative stress in nervous system-derived cellular models: could it correlate with in vivo neuropathy?

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.

Palmitoylethanolamide is a disease-modifying agent in peripheral neuropathy: pain relief and neuroprotection share a PPAR-alpha-mediated mechanism.

Neuropathic syndromes which are evoked by lesions to the peripheral or central nervous system are extremely difficult to treat, and available drugs rarely joint an antihyperalgesic with a neurorestorative effect. N-Palmitoylethanolamine (PEA) exerts antinociceptive effects in several animal models and inhibits peripheral inflammation in rodents. Aimed to evaluate the antineuropathic properties of PEA, a damage of the sciatic nerve was induced in mice by chronic constriction injury (CCI) and a subcutaneous daily treatment with 30 mg kg(-1) PEA was performed. On the day 14, PEA prevented pain threshold alterations. Histological studies highlighted that CCI induced oedema and an important infiltrate of CD86 positive cells in the sciatic nerve. Moreover, osmicated preparations revealed a decrease in axon diameter and myelin thickness. Repeated treatments with PEA reduced the presence of oedema and macrophage infiltrate, and a significant higher myelin sheath, axonal diameter, and a number of fibers were observable. In PPAR- α null mice PEA treatment failed to induce pain relief as well as to rescue the peripheral nerve from inflammation and structural derangement. These results strongly suggest that PEA, via a PPAR- α -mediated mechanism, can directly intervene in the nervous tissue alterations responsible for pain, starting to prevent macrophage infiltration.

St. John's wort relieves pain in an animal model of migraine.

BACKGROUND: Despite the substantial improvement that antimigraine drugs brought to migraineurs, there is the need for a long-acting and better tolerated migraine treatment than actual pharmacotherapy. St. John's wort (SJW), a medicinal plant endowed with a favourable tolerability profile, showed numerous bioactivities. We here investigated the pain-relieving property of SJW and its main components, hypericin and flavonoids, in a mouse model induced by nitric oxide (NO) donors administration. METHODS: The NO donors nitroglycerin and sodium nitroprusside (SNP) induced allodynia (cold plate test) and hyperalgesia (hot plate test). Western blotting experiments were performed to detect c-Fos and protein kinase C (PKC) expression within periaqueductal grey matter (PAG). RESULTS: A single oral administration of an SJW dried extract (5 mg/kg p.o.) produced a prolonged relief from pain hypersensitivity. Similarly, preventive SJW administration increased the latency to the induction of hyperalgesia and reduced the duration of the painful symptomatology. Among SJW main components, hypericin showed a similar profile of activity, whereas flavonoids were devoid of any antihyperalgesic effect. To clarify the cellular pathways involved in the SJW mechanism of action, we examined the effects induced by the herbal drug on PKC. NO donors' administration upregulated and increased phosphorylation of PKCγ and PKCε isoforms within PAG that was prevented by SJW treatment. The absence of behavioural side effects or altered animals' locomotor activity by SJW was demonstrated. CONCLUSIONS: These results suggest SJW as a safe therapeutic perspective for migraine pain, and indicate PKC as an innovative target for antimigraine therapy.

Influence of ring size on the cognition-enhancing activity of DM235 and MN19, two potent nootropic drugs.

A series of analogs of DM235 and MN19, characterized by rings with different size, have been prepared and evaluated for their nootropic activity in the mouse passive-avoidance test. It was found that the optimal ring size for the analogs of DM235, showing endocyclic both amidic groups, is 6 or 7 atoms. For the compounds structurally related to MN19, carrying an exocyclic amide group, the piperidine ring is the moiety which gives the most interesting compounds.

Enlarging the NSAIDs family: ether, ester and acid derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.

New "drug-in cyclodextrin-in deformable liposomes" formulations to improve the therapeutic efficacy of local anaesthetics.

The combined approach of cyclodextrin complexation and entrapment in liposomes was investigated to develop a topical formulation of local anaesthetics. For both benzocaine (BZC) and butamben (BTM), hydroxypropyl-beta-cyclodextrin (HPbetaCD) was a better partner than betaCD; drug-HPbetaCD coevaporated products showed the best solubility and dissolution properties, and were selected for loading into liposomes. Addition of stearylamine to the phosphatidylcholine-cholesterol mixture of the vesicle bilayer allowed obtainment of deformable liposomes with improved permeation and in vivo drug anaesthetic effect (P<0.05). Double-loaded deformable liposomes were obtained by adding the drug-HPbetaCD complex at its maximum aqueous solubility in the vesicles hydrophilic phase, and the remaining amount up to 1% as free drug in the lipophilic phase. The properties of double-loaded liposomes were compared with those of classic single-loaded ones, obtained by adding 1% free drug in the aqueous or lipophilic phase of the vesicles. Size, charge, morphology and entrapment efficiency of the different batches were investigated, respectively, by light scattering, confocal laser scanning microscopy and dialysis, while their therapeutic efficacy was evaluated in vivo on rabbits. For both drugs, double-loaded liposomes, exploiting the favourable effects of drug-CD complexation, allowed a significant (P<0.05) enhancement of intensity and duration of anaesthetic effect with respect to those single-loaded.

Effects of a new potent analog of tocainide on hNav1.7 sodium channels and in vivo neuropathic pain models.

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.