A Novel Ophthalmic Solution Containing Glicopro® Complex for the Treatment of Patients with Dry Eye Disease: Results from a Pilot Study.

(1) Background: Dry eye disease (DED) is a multifactorial ocular surface disease characterized by an imbalance in ocular surface homeostasis, and tear substitutes constitute the first line of treatment. The present study aimed to evaluate the changes in the signs and symptoms of patients with DED treated with a novel tear substitute containing the GlicoPro® complex. (2) Methods: Patients with DED not successfully responding to other tear substitutes were enrolled and treated with a novel ophthalmic solution (two drops four times daily). Patients were examined before starting the study treatment (T0) and after 30 (T1) and 60 (T2) days of treatment by means of Keratograph for the evaluation of the following: (i) tear meniscus height (TMH); (ii) noninvasive Keratograph break-up time (NIKBUT); (iii) bulbar redness; and (iv) infrared meibography. The SANDE questionnaire was administered to assess ocular discomfort symptoms. Analysis of the tear content of proenkephalin and Met/Leu-enkephalin was also performed. (3) Results: At T2, a significant improvement in NIKBUT first, average, and class, TMH, and SANDE score was found. The tear content of proenkephalins was significantly higher at T1, whereas processed active Met/Leu-enkephalins increased at both T1 and T2. (4) Conclusions: Our novel tear substitute based on GlicoPro® resulted in a significant improvement in ocular discomfort symptoms, tear volume, and stability in the patients treated. The increase in active peptides processed in tears may represent the pathophysiological substrate underlying this finding.

Ultramicronized N-palmitoylethanolamine Contributes to Morphine Efficacy Against Neuropathic Pain: Implication of Mast Cells and Glia.

BACKGROUND: In the current management of neuropathic pain, in addition to antidepressants and anticonvulsants, the use of opioids is wide, despite their related and well-known issues. OBJECTIVE: N-palmitoylethanolamine (PEA), a natural fatty-acid ethanolamide whose anti-inflammatory, neuroprotective, immune-modulating and anti-hyperalgesic activities are known, represents a promising candidate to modulate and/or potentiate the action of opioids. METHODS: This study was designed to evaluate if the preemptive and morphine concomitant administration of ultramicronized PEA, according to fixed or increasing doses of both compounds, delays the onset of morphine tolerance and improves its analgesic efficacy in the chronic constriction injury (CCI) model of neuropathic pain in rats. RESULTS: Behavioral experiments showed that the preemptive and co-administration of ultramicronized PEA significantly decreased the effective dose of morphine and delayed the onset of morphine tolerance. The activation of spinal microglia and astrocytes, commonly occurring both on opioid treatment and neuropathic pain, was investigated through GFAP and Iba-1 immunofluorescence. Both biomarkers were found to be increased in CCI untreated or morphine treated animals in a PEA-sensitive manner. The increased density of endoneural mast cells within the sciatic nerve of morphine-treated and untreated CCI rats was significantly reduced by ultramicronized PEA. The decrease of mast cell degranulation, evaluated in terms of reduced plasma levels of histamine and N-methyl-histamine metabolite, was mainly observed at intermediate-high doses of ultramicronized PEA, with or without morphine. CONCLUSION: Overall, these results show that the administration of ultramicronized PEA in CCI rats according to the study design fully fulfilled the hypotheses of this study.

Erucin Exerts Cardioprotective Effects on Ischemia/Reperfusion Injury through the Modulation of mitoKATP Channels.

Modulation of mitochondrial K channels represents a pharmacological strategy to promote cardioprotective effects. Isothiocyanates emerge as molecules capable of releasing hydrogen sulfide (H2S), an endogenous pleiotropic gasotransmitter responsible for anti-ischemic cardioprotective effects also through the involvement of mitoK channels. Erucin (ERU) is a natural isothiocyanate resulting from the enzymatic hydrolysis of glucosinolates (GSLs) present in Eruca sativa Mill. seeds, an edible plant of the Brassicaceae family. In this experimental work, the specific involvement of mitoKATP channels in the cardioprotective effect induced by ERU was evaluated in detail. An in vivo preclinical model of acute myocardial infarction was reproduced in rats to evaluate the cardioprotective effect of ERU. Diazoxide was used as a reference compound for the modulation of potassium fluxes and 5-hydroxydecanoic acid (5HD) as a selective blocker of KATP channels. Specific investigations on isolated cardiac mitochondria were carried out to evaluate the involvement of mitoKATP channels. The results obtained showed ERU cardioprotective effects against ischemia/reperfusion (I/R) damage through the involvement of mitoKATP channels and the consequent depolarizing effect, which in turn reduced calcium entry and preserved mitochondrial integrity.

Searching for Novel Sources of Hydrogen Sulfide Donors: Chemical Profiling of Polycarpa aurata Extract and Evaluation of the Anti-Inflammatory Effects.

Hydrogen sulfide (H2S) is a signaling molecule endogenously produced within mammals' cells that plays an important role in inflammation, exerting anti-inflammatory effects. In this view, the research has shown a growing interest in identifying natural H2S donors. Herein, for the first time, the potential of marine extract as a source of H2S-releasing agents has been explored. Different fractions obtained by the Indonesian ascidian Polycarpa aurata were evaluated for their ability to release H2S in solution. The main components of the most active fraction were then characterized by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and NMR spectroscopy. The ability of this fraction to release H2S was evaluated in a cell-free assay and J774 macrophages by a fluorimetric method, and its anti-inflammatory activity was evaluated in vitro and in vivo by using carrageenan-induced mouse paw edema. The anti-inflammatory effects were assessed by inhibiting the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and interleukin-6 (IL-6), coupled with a reduction in nitric oxide (NO) and IL-6 levels. Thus, this study defines the first example of a marine source able to inhibit inflammatory responses in vivo through the release of H2S.

Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

Anti-Hyperalgesic Efficacy of Acetyl L-Carnitine (ALCAR) Against Visceral Pain Induced by Colitis: Involvement of Glia in the Enteric and Central Nervous System.

The management of abdominal pain in patients affected by inflammatory bowel diseases (IBDs) still represents a problem because of the lack of effective treatments. Acetyl L-carnitine (ALCAR) has proved useful in the treatment of different types of chronic pain with excellent tolerability. The present work aimed at evaluating the anti-hyperalgesic efficacy of ALCAR in a model of persistent visceral pain associated with colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS) injection. Two different protocols were applied. In the preventive protocol, ALCAR was administered daily starting 14 days to 24 h before the delivery of DNBS. In the interventive protocol, ALCAR was daily administered starting the same day of DNBS injection, and the treatment was continued for 14 days. In both cases, ALCAR significantly reduced the establishment of visceral hyperalgesia in DNBS-treated animals, though the interventive protocol showed a greater efficacy than the preventive one. The interventive protocol partially reduced colon damage in rats, counteracting enteric glia and spinal astrocyte activation resulting from colitis, as analyzed by immunofluorescence. On the other hand, the preventive protocol effectively protected enteric neurons from the inflammatory insult. These findings suggest the putative usefulness of ALCAR as a food supplement for patients suffering from IBDs.

DDD-028: A potent, neuroprotective, non-opioid compound for the treatment of diabetic neuropathy.

Diabetic neuropathy (DN) is a painful, chronic ailment that affects a large segment of diabetic population worldwide. Current medications such as pregabalin or duloxetine treat only the pain symptom associated with DN, but not the underlying nerve damage. DDD-028 (1) is a small molecule that displays potent pain-relieving activity in streptozotocin (STZ)-induced rodent model of DN. Combined with other studies indicating that DDD-028 suppresses astrogliosis and nerve damage induced by the anti-cancer drug, paclitaxel, the present study suggests that DDD-028 would be useful as a disease modifying therapeutic in the treatment of DN. The 3-dimensional structure of DDD-028 was confirmed by single crystal X-ray crystallography.

Discovery of a novel series of potent carbonic anhydrase inhibitors with selective affinity for µ Opioid receptor for Safer and long-lasting analgesia.

In this study, we investigated the development of dual-targeted ligands that bind to both µ-opioid receptor (MOR) and carbonic anhydrase (CA) enzymes, using fentanyl structure as a template. We synthesized and evaluated 21 novel compounds with dual-targeted affinity identifying the lead candidate compound 8, showing selective affinity for MOR and potent inhibition of several cytosolic CA isoforms. By means of repeated treatment of 3 daily administrations for 17 days, fentanyl (0.1 mg/kg, subcutaneously) led to tolerance development, pain threshold alterations and withdrawal symptoms in CD-1 mice, as well as astrocyte and microglia activation in the dorsal horn of the lumbar spinal cord. In contrast, compound 8 (0.32 mg/kg s.c.) maintained stable during days its analgesic effect at the higher dose tested with fewer withdrawal symptoms, allodynia development and glial cells activation. Our results suggest that targeting both MOR and CA enzymes can lead to the development of new class of potent analgesic agents with fewer side effects and reduced tolerance development. Further studies are needed to explore the potential mechanisms underlying these effects and to further optimize the therapeutic potential of these compounds.

Neurofilament light chain in plasma as a sensitive diagnostic biomarker of peripheral neurotoxicity: In Vivo mouse studies with oxaliplatin and paclitaxel - NeuroDeRisk project.

Identifying compounds that are neurotoxic either toward the central or the peripheral nervous systems (CNS or PNS) would greatly benefit early stages of drug development by derisking liabilities and selecting safe compounds. Unfortunately, so far assays mostly rely on histopathology findings often identified after repeated-dose toxicity studies in animals. The European NeuroDeRisk project aimed to provide comprehensive tools to identify compounds likely inducing neurotoxicity. As part of this project, the present work aimed to identify diagnostic non-invasive biomarkers of PNS toxicity in mice. We used two neurotoxic drugs in vivo to correlate functional, histopathological and biological findings. CD1 male mice received repeated injections of oxaliplatin or paclitaxel followed by an assessment of drug exposure in CNS/PNS tissues. Functional signs of PNS toxicity were assessed using electronic von Frey and cold paw immersion tests (oxaliplatin), and functional observational battery, rotarod and cold plate tests (paclitaxel). Plasma concentrations of neurofilament light chain (NF-L) and vascular endothelial growth factor A (VEGF-A) were measured, and histopathological evaluations were performed on a comprehensive list of CNS and PNS tissues. Functional PNS toxicity was observed only in oxaliplatin-treated mice. Histopathological findings were observed dose-dependently only in paclitaxel groups. While no changes of VEGF-A concentrations was recorded, NF-L concentrations were increased only in paclitaxel-treated animals as early as 7 days after the onset of drug administration. These results show that plasma NF-L changes correlated with microscopic changes in the PNS, thus strongly suggesting that NF-L could be a sensitive and specific biomarker of PNS toxicity in mice.

Broad-spectrum neuroprotection exerted by DDD-028 in a mouse model of chemotherapy-induced neuropathy.

ABSTRACT: Neurotoxicity of chemotherapeutics involves peculiar alterations in the structure and function, including abnormal nerve signal transmission, of both the peripheral and central nervous system. The lack of effective pharmacological approaches to prevent chemotherapy-induced neurotoxicity necessitates the identification of innovative therapies. Recent evidence suggests that repeated treatment with the pentacyclic pyridoindole derivative DDD-028 can exert both pain-relieving and glial modulatory effects in mice with paclitaxel-induced neuropathy. This work is aimed at assessing whether DDD-028 is a disease-modifying agent by protecting the peripheral nervous tissues from chemotherapy-induced damage. Neuropathy was induced in animals by paclitaxel injection (2.0 mg kg -1 i.p). DDD-028 (10 mg kg -1 ) and the reference drug, pregabalin (30 mg kg -1 ), were administered per os daily starting concomitantly with the first injection of paclitaxel and continuing 10 days after the end of paclitaxel treatment. The behavioural tests confirmed the antihyperalgesic efficacy of DDD-028 on paclitaxel-induced neuropathic pain. Furthermore, the electrophysiological analysis revealed the capacity of DDD-028 to restore near-normal sensory nerve conduction in paclitaxel-treated animals. Histopathology evidence indicated that DDD-028 was able to counteract effectively paclitaxel-induced peripheral neurotoxicity by protecting against the loss of intraepidermal nerve fibers, restoring physiological levels of neurofilament in nerve tissue and plasma, and preventing morphological alterations occurring in the sciatic nerves and dorsal root ganglia. Overall, DDD-028 is more effective than pregabalin in preventing chemotherapy-induced neurotoxicity. Thus, based on its potent antihyperalgesic and neuroprotective efficacy, DDD-028 seems to be a viable prophylactic medication to limit the development of neuropathies consequent to chemotherapy.

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Protective and Pain-Killer Effects of AMC3, a Novel N-Formyl Peptide Receptors (FPRs) Modulator, in Experimental Models of Rheumatoid Arthritis.

Rheumatoid arthritis is an autoimmune disorder that causes chronic joint pain, swelling, and movement impairment, resulting from prolonged inflammation-induced cartilage and bone degradation. The pathogenesis of RA, which is still unclear, makes diagnosis and treatment difficult and calls for new therapeutic strategies to cure the disease. Recent research has identified FPRs as a promising druggable target, with AMC3, a novel agonist, showing preclinical efficacy in vitro and in vivo. In vitro, AMC3 (1-30 µM) exhibited significant antioxidant effects in IL-1ß (10 ng/mL)-treated chondrocytes for 24 h. AMC3 displayed a protective effect by downregulating the mRNA expression of several pro-inflammatory and pro-algic genes (iNOS, COX-2, and VEGF-A), while upregulating genes essential for structural integrity (MMP-13, ADAMTS-4, and COLIAI). In vivo, AMC3 (10 mg kg-1) prevented hypersensitivity and restored postural balance in CFA-injected rats after 14 days. AMC3 attenuated joint alterations, reduced joint inflammatory infiltrate, pannus formation, and cartilage erosion. Chronic AMC3 administration reduced transcriptional changes of genes causing excitotoxicity and pain (EAATs and CCL2) and prevented morphological changes in astrocytes, including cell body hypertrophy, processes length, and thickness, caused by CFA in the spinal cord. This study demonstrates the usefulness of AMC3 and establishes the groundwork for further research.

Antinociceptive Action of Thymoquinone-Loaded Liposomes in an In Vivo Model of Tendinopathy.

Tendinopathies represent about 45% of musculoskeletal lesions and they are a big burden in clinics characterized by activity-related pain, focal tendon tenderness and intra-tendinous imaging changes. Many approaches have been proposed for tendinopathies' management (e.g., nonsteroidal anti-inflammatory drugs, corticosteroids, eccentric exercises, laser therapy), unfortunately with very little support of efficacy or serious side effects, thus making the identification of new treatments fundamental. The aim of the study was to test the protective and pain reliever effect of thymoquinone (TQ)-loaded formulations in a rat model of tendinopathy induced by carrageenan intra-tendon injection (20 µL of carrageenan 0.8% on day 1). Conventional (LP-TQ) and hyaluronic acid (HA)-coated TQ liposomes (HA-LP-TQ) were characterized and subjected to in vitro release and stability studies at 4 °C. Then, TQ and liposomes were peri-tendon injected (20 µL) on days 1, 3, 5, 7 and 10 to evaluate their antinociceptive profile using mechanical noxious and non-noxious stimuli (paw pressure and von Frey tests), spontaneous pain (incapacitance test) and motor alterations (Rota rod test). Liposomes containing 2 mg/mL of TQ and covered with HA (HA-LP-TQ2) reduced the development of spontaneous nociception and hypersensitivity for a long-lasting effect more than the other formulations. The anti-hypersensitivity effect matched with the histopathological evaluation. In conclusion, the use of TQ encapsulated in HA-LP liposomes is suggested as a new treatment for tendinopathies.

Development of Lyophilised Eudragit® Retard Nanoparticles for the Sustained Release of Clozapine via Intranasal Administration.

Clozapine (CZP) is the only effective drug in schizophrenia resistant to typical antipsychotics. However, existing dosage forms (oral or orodispersible tablets, suspensions or intramuscular injection) show challenging limitations. After oral administration, CZP has low bioavailability due to a large first-pass effect, while the i.m. route is often painful, with low patient compliance and requiring specialised personnel. Moreover, CZP has a very low aqueous solubility. This study proposes the intranasal route as an alternative route of administration for CZP, through its encapsulation in polymeric nanoparticles (NPs) based on Eudragit® RS100 and RL100 copolymers. Slow-release polymeric NPs with dimensions around 400-500 nm were formulated to reside and release CZP in the nasal cavity, where it can be absorbed through the nasal mucosa and reach the systemic circulation. CZP-EUD-NPs showed a controlled release of CZP for up to 8 h. Furthermore, to reduce mucociliary clearance and increase the residence time of NPs in the nasal cavity to improve drug bioavailability, mucoadhesive NPs were formulated. This study shows that the NPs already exhibited strong electrostatic interactions with mucin at time zero due to the presence of the positive charge of the used copolymers. Furthermore, to improve the solubility, diffusion and adsorption of CZPs and the storage stability of the formulation, it was lyophilised using 5% (w/v) HP-ß-CD as a cryoprotectant. It ensured the preservation of the NPs' size, PDI and charge upon reconstitution. Moreover, physicochemical characterisation studies of solid-state NPs were performed. Finally, toxicity studies were performed in vitro on MDCKII cells and primary human olfactory mucosa cells and in vivo on the nasal mucosa of CD-1 mice. The latter showed non-toxicity of B-EUD-NPs and mild CZP-EUD-NP-induced tissue abnormalities.

The Antinociceptive Activity of (E)-3-(thiophen-2-yl)- N -(p-tolyl)acrylamide in Mice Is Reduced by (E)-3-(furan-2-yl)- N -methyl- N -(p-tolyl)acrylamide Through Opposing Modulatory Mechanisms at the α7 Nicotinic Acetylcholine Receptor.

BACKGROUND: The primary objective of this study was to characterize the pharmacological and behavioral activity of 2 novel compounds, DM497 [(E)-3-(thiophen-2-yl)- N -(p-tolyl)acrylamide] and DM490 [(E)-3-(furan-2-yl)- N -methyl- N -(p-tolyl)acrylamide], structural derivatives of PAM-2, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor (nAChR). METHODS: A mouse model of oxaliplatin-induced neuropathic pain (2.4 mg/kg, 10 injections) was used to test the pain-relieving properties of DM497 and DM490. To assess possible mechanisms of action, the activity of these compounds was determined at heterologously expressed α7 and α9α10 nAChRs, and voltage-gated N-type calcium channel (Ca V 2.2) using electrophysiological techniques. RESULTS: Cold plate tests indicated that 10 mg/kg DM497 was able to decrease neuropathic pain in mice induced by the chemotherapeutic agent oxaliplatin. In contrast, DM490 induced neither pro- nor antinociceptive activity but inhibited DM497's effect at equivalent dose (30 mg/kg). These effects are not a product of changes in motor coordination or locomotor activity. At α7 nAChRs, DM497 potentiated whereas DM490 inhibited its activity. In addition, DM490 antagonized the α9α10 nAChR with >8-fold higher potency than that for DM497. In contrast, DM497 and DM490 had minimal inhibitory activity at the Ca V 2.2 channel. Considering that DM497 did not increase the mouse exploratory activity, an indirect anxiolytic mechanism was not responsible for the observed antineuropathic effect. CONCLUSIONS: The antinociceptive activity of DM497 and the concomitant inhibitory effect of DM490 are mediated by opposing modulatory mechanisms on the α7 nAChR, whereas the involvement of other possible nociception targets such as the α9α10 nAChR and Ca V 2.2 channel can be ruled out.

Ellagitannins and triterpenoids extracts of Anogeissus leiocarpus stem bark extracts: Protective effects against osteoarthritis.

Osteoarthritis (OA) is a complex joint disease characterized by persistent pain. Unfortunately, current pharmacological therapies are unsatisfactory and characterized by side effects, reason why new strategies are needed. We tested the efficacy of different classes of compounds, ellagitannins and olean-type triterpenoids, contained in Anogeissus leiocarpus extract (Combretaceae family) in comparison to ellagitannins of Castanea sativa extract in a rat model of osteoarthritis induced by the intra-articular injection of sodium monoiodoacetate (MIA). The decoction of stem bark of A. leiocarpus AL-DEC-TOT (300 mg/kg; 4.8% triterpenoids; 11.0% tannins), the butanol extract AL-BuOH-EXT (120 mg/kg; triterpenoids 20.9%; tannins 6.4%) and its correlated aqueous residue AL-Res-H2 O (300 mg/kg; triterpenoids 0.7%; tannins 8.7%) and the decoction of C. sativa, CS-DEC-TOT, (240 mg/kg; triterpenoids 0.65%; tannins 10.8%) were orally administered for two weeks starting from the day of the damage. Behavioural tests highlighted that all stem bark extracts of A. leiocarpus counteracted hypersensitivity development, reduced spontaneous pain, and improved motor skills. Histologically, AL-DEC-TOT, AL-BuOH-EXT and AL-Res-H2 O were effective in preventing joint alterations. In conclusion, all the extracts were effective demonstrating that both olean-type triterpenoid and ellagitannin fractions have anti-hypersensitivity and restorative properties running the stem bark extracts of A. leiocarpus as a candidate in the treatment of OA.

Ultramicronized N-Palmitoylethanolamine Regulates Mast Cell-Astrocyte Crosstalk: A New Potential Mechanism Underlying the Inhibition of Morphine Tolerance.

Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 µM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 µM, 18 h), as evaluated by ß-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1ß, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.