Tail-flick Test Research Articles

Development of a Selective Peptide κ-Opioid Receptor Antagonist by Late-Stage Functionalization with Cysteine Staples.

The κ-opioid receptor (KOR) is an attractive target for the development of novel drugs. KOR agonists are potentially safer pain medications, whereas KOR antagonists are promising drug candidates for the treatment of neuropsychiatric disorders. Hitherto, the vast majority of selective drug leads that have been developed for KOR are small molecules. In this study, novel peptide probes were designed by using an endogenous dynorphin A1-13 sequence as a template for peptide stapling via late-stage cysteine functionalization. Leveraging this strategy, we developed a stable and potent KOR antagonist, CSD-CH2(1,8)-NH2, with approximately 1000-fold improved selectivity for KOR over μ- and δ-opioid receptors. Its potent competitive KOR antagonism was verified in KOR-expressing cells, peripheral dorsal root ganglion neurons, and using the tail-flick and rotarod tests in mice. This work highlights the value of cysteine stapling to develop selective peptide probes to modulate central KOR function, as innovative peptide drug candidates for the treatment of KOR-related illnesses.

Toll-like Receptor 2-Melatonin Feedback Loop Regulates the Activation of Spinal NLRP3 Inflammasome in Morphine-Tolerant Rats.

Morphine is amongst the most effective analgesics available for the management of severe pain. However, prolonged morphine treatment leads to analgesic tolerance which limits its clinical usage. Previous studies have demonstrated that melatonin ameliorates morphine tolerance by reducing neuroinflammation. However, little is known about the relationship between Toll like receptor 2 (TLR2) and neuroinflammation in morphine tolerance. The aim of this study was to explore the role of TLR2 in morphine tolerance and its connections with melatonin and Nod-like receptor protein 3 (NLRP3) inflammasome. Sprague-Dawley rats were treated with morphine for 7 days and tail-flick latency test was performed to identify the induction of analgesic tolerance. The roles of TLR2 in microglia activation and morphine tolerance were assessed pharmacologically, and the possible interactions between melatonin, TLR2 and NLRP3 inflammasome were investigated. Morphine tolerance was accompanied by increased TLR2 expression and NLRP3 inflammasome activation in spinal cord. whereas melatonin level was down-regulated. Chronic melatonin administration resulted in a reduced TLR2 expression and NLRP3 inflammasome activation. Moreover, the analgesic effect of morphine was partially restored. Inhibition of TLR2 suppressed the microglia and NLRP3 inflammasome activation, as well as restored the spinal melatonin level while attenuated the development of morphine tolerance. Furthermore, the inhibition of microglia activation ameliorated morphine tolerance via inhibiting TLR2-NLRP3 inflammasome signaling in spinal cord. In this study, we directly demonstrate a TLR2-melatonin negative feedback loop regulating microglia and NLRP3 inflammasome activation during the development of morphine tolerance.

Himalayan Pyracantha crenulata (D.Don) M.Roem. leaf and fruit extracts alleviate algesia through COX-2 and Mu-opioid receptor mediated pathways

Ethnopharmacological relevancePyracantha crenulata (D.Don) M.Roem., a plant of high nutritional and medicinal value is traditionally employed for its analgesic property in joint and body pain in the Kumaun region of Western Himalaya. Aim of the studyTo validate the traditional claims for analgesic property of Pyracantha crenulata. MethodsHydroethanolic extract of P. crenulata leaves and fruits were tested for their analgesic potential in rodent models for algesia by tail immersion test, tail flick test, Eddy's hot plate model, and formalin induced paw irritation test in Wistar rats. Molecular docking and dynamics studies were also performed to understand the possible mechanisms. ResultsBoth P. crenulata fruit extract and leaf extract exhibited significant amelioration in all the tested experimental models of algesia acting through central and peripheral mechanisms. The efficacy in reducing nociception was found comparable to diclofenac that was used as a reference standard. Molecular docking and dynamic simulation studies further established binding affinity of gallic acid (confirmed to be present in P. crenulata leaf extract through HPTLC profiling) with cyclooxygenase-2 (COX-2) and mu-opioid receptors, suggesting the modulatory effect of these extracts on COX-2 and mu-opioid receptors in combating algesia. ConclusionP. crenulata extracts produce analgesic effects plausibly through COX-2 and mu-opioid receptor mediated pathways.

Similar functional roles of the Orexin-1 and Orexin-2 receptors within the dentate gyrus area of the hippocampus in the stress-induced antinociceptive responses in the acute pain model in the rat

Studies establish that the brain's Orexin system is involved in pain modulation. Orexin-1 and orexin-2 receptors (OX1 and OX2r, respectively) are essential in responsiveness to stressful stimuli. Some evidence indicates that the hippocampus's dentate gyrus (DG) potentially modulates pain and stress. The present study examined the involvement of OX1 and OX2 receptors within the DG in response to acute pain after exposure to forced swim stress (FSS). Five to seven days post-stereotaxic surgery, the baseline tail-flick latency (TFL) was taken from the animal, then rats unilaterally received through an implanted cannula either different doses of OX1r antagonist (SB334867; 1, 3, 10, and 30 nmol), OX2r antagonist (TCS OX2 29; 1, 3, 10 and 30 nmol), or vehicle (0.5 μl solution of 12% DMSO). After 5 min, rats were exposed to the FSS for six minutes. Subsequently, the tail-flick test was conducted, and the TFLs were measured at the 60-min time set intervals. Results indicated that FSS produces antinociceptive responses in the tail-flick test. Two-way ANOVA analysis showed that Microinjection of OX1r and OX2r antagonists into the DG region of the brain reduced FSS-induced analgesia in the tail-flick test. The decrement effects of these two antagonists were almost the same. Additionally, results showed that the role of both receptors was the same in modulating stress-induced analgesia (SIA). These findings show that the orexin system in the hippocampal DG region might be partially involved in the SIA in acute pain.

Teneligliptin Co-Infusion Alleviates Morphine Tolerance by Inhibition of Spinal Microglial Cell Activation in Streptozotocin-Induced Diabetic Rats.

Morphine (MOR) is a commonly prescribed drug for the treatment of moderate to severe diabetic neuropathic pain (DNP). However, long-term MOR treatment is limited by morphine analgesic tolerance (MAT). The activation of microglial cells and the release of glia-derived proinflammatory cytokines are known to play an important role in the development of MAT. In this study, we aimed to investigate the effects of the dipeptidyl peptidase-4 inhibitor (DPP-4i) teneligliptin (TEN) on MOR-induced microglial cell activation and MAT in DNP rats. DNP was induced in four groups of male Wistar rats through a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg, freshly dissolved in 5 mmol/L citrate buffer, pH 4.5). Sham rats were administered with the vehicle. Seven days after STZ injection, all rats were implanted with an intrathecal (i.t) catheter connected to a mini-osmotic pump, divided into five groups, and infused with the following combinations: sham + saline (1 µL/h, i.t), DNP + saline (1 µL/h, i.t), DNP + MOR (15 µg/h, i.t), DNP + TEN (2 µg/h, i.t), and DNP + MOR (15 µg/h, i.t) + TEN (2 µg/h, i.t) for 7 days at a rate of 1 μL/h. The MAT was confirmed through the measurement of mechanical paw withdrawal threshold and tail-flick tests. The mRNA expression of neuroprotective proteins nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the dorsal horn was evaluated by quantitative PCR (qPCR). Microglial cell activation and mononucleate cell infiltration in the spinal cord dorsal horn were assessed by immunofluorescence assay (IFA) and Western blotting (WB). The results showed that co-infusion of TEN with MOR significantly attenuated MAT in DNP rats through the restoration of neuroprotective proteins Nrf2 and HO-1 and suppression of microglial cell activation in the dorsal horn. Though TEN at a dose of 2 μg has mild antinociceptive effects, it is highly effective in limiting MAT.

The antipsychotic olanzapine reduces memory deficits and neuronal abnormalities in a male rat model of Autism

The prevalence of autism spectrum disorder (ASD), a neurodevelopmental condition that impacts social interaction and sensory processing, is rising. Valproic acid (VPA) exposure during pregnancy causes autistic-like traits in offspring. Olanzapine (OLZ), an atypical antipsychotic, is used to treat ASD. We assessed the impact of OLZ on behavior, neuromorphology, and nitric oxide (NO) levels in the hippocampus using prenatal VPA treatment in rats. It is commonly known that ASD patients exhibit sensory abnormalities. As such, we utilized the tail flick test to validate the ASD model. In the novel object recognition test (NORT), VPA exposure reduces the discrimination index (DI) in the first introduction to the novel object. Moreover, OLZ and vehicle-treated rats perform differently in the second exposition to the DI of the novel object, suggesting that OLZ reverses VPA-induced deficits in recognition memory. The latency to find the hidden platform in the Morris water maze test of memory and learning improves in VPA-exposed rats after OLZ administration, indicating that OLZ improves spatial memory in these rats. Administration of prenatal VPA induces neuronal hypotrophy and reduces spine density in pyramidal neurons of the CA1 region of the hippocampus. Treatment with OLZ corrects the neuromorphological changes brought on by VPA. In the CA1 region of the hippocampus, VPA treatment increases the number of neurons, which normalizes with OLZ treatment. OLZ increases the NO levels in the dorsal hippocampus in control rats. In rats exposed to VPA, the second-generation antipsychotic OLZ reduces memory-related and neuroplastic alterations. The current findings support the use of OLZ in this illness and further validate the use of prenatal VPA as a model of ASD.

Essential oil from leaves of Croton blanchetianus Baill does not present acute oral toxicity, has antigenotoxic action and reduces neurogenic and inflammatory nociception in mice

Ethnopharmacological relevanceCroton blanchetianus Baill., popularly known as “marmeleiro preto”, is an endemic plant from Brazil, being found mainly in the Northeast region. In traditional medicine, the use of medicines based on the leaves of this plant has been reported for the treatment of inflammatory processes, pain, urethral pain, gastrointestinal disorders, rheumatism and headache.Aim of the study: The present work describes the chemical characterization, as well as toxicological evaluation and antinociceptive activity of an essential oil of C. blanchetianus leaves (EOCb). Materials and methodsThe chemical constituents of the oil were identified by gas chromatography coupled to mass spectrometry (GC-MS). In vitro hemolytic activity was tested using mouse blood. Acute toxicity in mice was assessed by the oral or intraperitoneal administration of a single dose of 2000 mg/kg b.w. EOCb (1000 and 2000 mg/kg) was also evaluated for genotoxicity and antigenotoxicity in vivo using the micronucleus test. The antinociceptive activity of EOCb (25, 50 and 100 mg/kg) was evaluated through the abdominal writhing, formalin and tail flick tests. ResultsThe chemical characterization indicated as major components α-pinene (21.23%), β-phelandrene (13.92%), terpinolene (13.01%) and germacrene D (10.89%). EOCb did not cause hemolysis and was also neither toxic nor genotoxic, while protected the animals' bone marrow cells from damage caused by cyclophosphamide in oral treatment. However, all animals died after 15 min of intraperitoneal treatment. There was a reduction in the number of abdominal contortions (69.43–89.41%) as well as in licks in the first (38.77–84.47%) and second (59.75–90.74%) phases of the formalin test. In the latter case, the effects were reduced by naloxone and glibenclamide, indicating action via the opioid system and blockage of K+ channels. The latency time in the tail flick test also increased significantly. ConclusionIn conclusion, ingestion of EOCb proved to be safe when administered orally; however, it was lethal intraperitoneally. Additionally, EOCb protected mouse blood cell DNA against the action of cyclophosphamide and showed an antinociceptive effect via the opioid system and dependent on K+ channels.

Analgesic effect of a bradykinin antagonist – a 1,4-benzodiazepine-2-one derivative

Aim. To study the analgesic effect of a new 1,4-benzodiazepine-2-one derivative (codenamed PAV-0056) in pain models in mice, its anti-inflammatory effect in experimental exudative inflammation in rats, and its potential ulcerogenic effect.Materials and methods. A 1,4-benzodiazepine-2-one derivative (codenamed PAV-0056) was orally administered in polyvinylpyrrolidone (PVP) solution to 192 CD-1 mice weighing 20–25 g and 140 Sprague – Dawley rats weighing 250–300 g. The analgesic effect of the PAV-0056 compound at a dose of 0.01, 0.1, and 1 mg / kg was studied in murine acute thermal pain models (hot plate test, hot water immersion tail-flick test), acute chemogenic pain models (formalin test), and visceral spasticity-related pain models (acetic acid-induced writhing test). The anti-inflammatory effect of PAV-0056 at doses of 0.01, 0.1, and 1 mg / kg was studied in an experimental rat model of inflammation induced by subplantar administration of bradykinin and histamine. The potential ulcerogenic effect was studied in intact rats, who were injected with PAV-0056 at doses of 1 and 50 mg / kg four times. The analgesic effect of the PAV-0056 compound was compared to that of diclofenac sodium at a dose of 10 mg / kg and tramadol at a dose of 20 mg / kg. Its anti-inflammatory and potential ulcerogenic effects were compared to those of diclofenac sodium at a dose of 10 mg / kg.Results. In the hot plate test, the PAV-0056 compound at a dose of 0.1 mg / kg increased response latency in mice by 36%, and at a dose of 1 mg / kg, it increased response latency by 46% (p < 0.05). In the tail-flick test, the PAV-0056 compound at a dose of 1 mg / kg increased response latency to heat stimulation in mice by 46% (p < 0.05). After subplantar administration of formalin, PAV-0056 at doses of 0.01–1 mg / kg had a pronounced analgesic effect, as shown by a decrease in the number of pain responses by 39–55% (p < 0.05). When mice were intraperitoneally injected with an acetic acid solution, the PAV-0056 compound at doses of 0.1 and 1 mg / kg reduced the frequency of writhings by 46 and 57%, respectively; at a dose of 0.1 mg / kg, it delayed the onset of the first writhing by 21% (p < 0.05). In experiments on rats, the PAV-0056 compound prevented the development of exudative inflammation induced by subplantar administration of bradykinin and did not have an anti-inflammatory effect in histamine-induced inflammation. PAV-0056 did not cause formation of gastric ulcers and gastric mucosal bleeding.Conclusion. A 1,4-benzodiazepine-2-one derivative, PAV-0056, has a pronounced analgesic effect in models of thermal, chemogenic, somatic, and visceral pain in a wide range of doses (0.01–1 mg / kg). Its analgesic effects are the same as those of diclofenac sodium at a dose of 10 mg / kg and tramadol at a dose of 20 mg / kg. The analgesic effect of the PAV-0056 compound is selective, depends little on suppression of inflammatory exudation, and is caused by bradykinin antagonism. This substance has low toxicity and does not damage the gastric mucosa.

Analgesic effect of neohesperidin is mediated by TRPV1 antagonism

Context: Transient receptor potential vanilloid type 1 (TRPV1) is a non-specific cation channel. It is one of the most important targets in pain research. Aims: To evaluate new TRPV1 antagonists without altering body temperature. Methods: Docking simulation was performed, and one of the candidate compounds, neohesperidin, was tested using thermal and chemical pain models in BALB/c mice. Rectal body temperature was measured using a temperature meter with a thermocouple probe detector, and the capsaicin-evoked calcium response was determined in dorsal root ganglia (DRG) neurons. Results: Docking resulted in the identification of 30 compounds able to interact with the essential amino acids required for the antagonistic activity of TRPV1. Neohesperidin was chosen for further investigations because of its good binding energy (-6.63 kcal/mol) and because its TRPV1 antagonistic activity was not tested before. This study reported for the first time that neohesperidin exerted analgesic activity through TRPV1 antagonism without altering body temperature. Its activity was comparable to the known TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). In the writhing test, acetic acid-induced abdominal cramps decreased by 66% using 30 mg/kg of neohesperidin. All tested doses of neohesperidin significantly decreased paw-licking time in the capsaicin-induced paw-licking test. A significant increase in the latency time in hot plate and tail flick tests was observed using 30 and 60 mg/kg of neohesperidin. In DRG neurons, neohesperidin reduced capsaicin-evoked calcium responses. Conclusions: Neohesperidin exerts a significant analgesic activity without altering body temperature, which could be due, at least partially, to its antagonistic activity against TRPV1.

Analgesic effect by using hot Plat and tail flick test in rats models for aqueuos moringa oleifer extract

Introduction Drugs and plants are closely related to each other through the use of traditional medicines that is mainly prepared from plants. Plants are screened for presence of bioactive components responsible for the activity. Various types of plants have been used not only as dietary supplements but also as traditional treatments for many diseases. Moringaoleifera of Moringaceae is a potential source of phytochemical ingredients claimed to have analgesic property. Objective The present study aimed to investigate the analgesic activity of Moringaoleifera extract in albino rats by using taıl flıck and hot plat test. Methods: Leaves of Moringaoleifera were collected April 2022 from Surman (Libya). And then the powder was macerated in 1 L of distilled water for 3 days and evaporated to dryness by rotary evaporator. The crude extract was administered by oral gavaging in doses (50mg/kg_100mg/kg). The study was done by using experimental models, the albino rats were divided into 4 groups, each group consisting of 3 rats. Group I: Control (dH2O given orally at 2 ml/kg); Group II: Standard (100 mg/kg of Panadol orally.); Group III, IV, (MOE 50, 100 mg/kg, respectively), the analgesic effect was evaluated by using taıl flıck and hot plat test, through the reaction time was recorded in various time intervals) 60, 90, and 120 minutes). Results: MOE significantly a high significant increase in latency response (P<0.001) in comparison to control and standrad. Conclusion: These results indicate the ability of MOE to inhibit thermally induced nociceptive processes, which is a characteristic of strong analgesics

Nicorandil/ morphine crosstalk accounts for antinociception and hepatoprotection in hepatic fibrosis in rats: Distinct roles of opioid/cGMP and NO/KATP pathways

Previous report indicated that nicorandil potentiated morphine antinociception and attenuated hepatic injury in liver fibrotic rats. Herein, the underlying mechanisms of nicorandil/morphine interaction were investigated using pharmacological, biochemical, histopathological, and molecular docking studies. Male Wistar rats were injected intraperitoneally (i.p.) with carbon tetrachloride (CCl4, 40%, 2 ml/kg) twice weekly for 5 weeks to induce hepatic fibrosis. Nicorandil (15 mg/kg/day) was administered per os (p.o.) for 14 days in presence of the blockers; glibenclamide (KATP channel blocker, 5 mg/kg, p.o.), L-NG-nitro-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 15 mg/kg, p.o.), methylene blue (MB, guanylyl cyclase inhibitor, 2 mg/kg, i.p.) and naltrexone (opioid antagonist, 20 mg/kg, i.p.). At the end of the 5th week, analgesia was evaluated using tail flick and formalin tests along with biochemical determinations of liver function tests, oxidative stress markers and histopathological examination of liver tissues. Naltrexone and MB inhibited the antinociceptive activity of the combination. Furthermore, combined nicorandil/morphine regimen attenuated the release of endogenous peptides. Docking studies revealed a possible interaction of nicorandil on µ, κ and δ opioid receptors. Nicorandil/morphine combination protected against liver damage as evident by decreased liver enzymes, liver index, hyaluronic acid, lipid peroxidation, fibrotic insults, and increased superoxide dismutase activity. Nicorandil/morphine hepatoprotection and antioxidant activity were inhibited by glibenclamide and L-NAME but not by naltrexone or MB. These findings implicate opioid activation/cGMP versus NO/KATP channels in the augmented antinociception, and hepatoprotection, respectively, of the combined therapy and implicate provoked cross talk by nicorandil and morphine on opioid receptors and cGMP signaling pathway. That said, nicorandil/morphine combination provides a potential multitargeted therapy to alleviate pain and preserve liver function.

The effects of chronic high-dose morphine on microgliosis and the microglial transcriptome in rat spinal cord.

Background: Opioids are efficacious and safe analgesic drugs in short-term use for acute pain but chronic use can lead to tolerance and dependence. Opioid-induced microglial activation may contribute to the development of tolerance and this process may differ between males and females. A link is suggested between this microglial activation and inflammation, disturbances of circadian rhythms, and neurotoxic effects. We set out to further delineate the effects of chronic morphine on pain behaviour, microglial and neuronal staining, and the transcriptome of spinal microglia, to better understand the role of microglia in the consequences of long-term high-dose opioid administration. Experimental Approach: In two experiments, we administered increasing subcutaneous doses of morphine hydrochloride or saline to male and female rats. Thermal nociception was assessed with the tail flick and hot plate tests. In Experiment I, spinal cord (SC) samples were prepared for immunohistochemical staining for microglial and neuronal markers. In Experiment II, the transcriptome of microglia from the lumbar SC was analysed. Key Results: Female and male rats had similar antinociceptive responses to morphine and developed similar antinociceptive tolerance to thermal stimuli following chronic increasing high doses of s.c. morphine. The area of microglial IBA1-staining in SC decreased after 2weeks of morphine administration in both sexes. Following morphine treatment, the differentially expressed genes identified in the microglial transcriptome included ones related to the circadian rhythm, apoptosis, and immune system processes. Conclusions: Female and male rats showed similar pain behaviour following chronic high doses of morphine. This was associated with decreased staining of spinal microglia, suggesting either decreased activation or apoptosis. High-dose morphine administration also associated with several changes in gene expression in SC microglia, e.g., those related to the circadian rhythm (Per2, Per3, Dbp). These changes should be considered in the clinical consequences of long-term high-dose administration of opioids.

Gallic acid attenuates the Chronic construction injury of sciatic nerve induced neuropathic pain in mice

Gallic acid (GA) is one of the natural flavonoids and it is known to protect the neuronal system from various endogenous neurodegenerative processes. The present study is focused on evaluating the role of GA in chronic construction injury (CCI) of sciatic nerve-induced neuropathic pain. The test compound i.e., gallic acid (20 and 40mg/kg), and reference compound i.e., pregabalin (PrG: 5mg/kg) were administered intravenously for 10 consecutive days. The sciatic nerve injury-induced neuralgic sensations were assessed with multiple test methods like acetone drop, pinprick, plantar, tail-flick, and tail pinch tests at different time points i.e., 0, 4th, 8th, 12th, and 16th days. The biochemical level changes i.e., thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide anion, calcium, myeloperoxidase (MPO), and tumor necrosis factor–α (TNF-α) were estimated in sciatic nerve samples. The administration of GA and PreG was shown a significant ameliorative effect against the CCI-induced pain response in a dose-dependent manner. Moreover, it also attenuated the tissue biochemical changes. The observed effects of GA were similar to reference drug PreG treated groups. Hence, GA has neuroprotective and pain relief effects against the CCI-induced neuralgia. It may be due to its natural free radical scavenging; regulation of inflammatory proteins; expression of antioxidant proteins, reduction of intracellular free calcium, and prevention of neuroinflammatory actions.

485-P: Proteomic Analysis Reveals Different Therapeutic Mechanisms for Diabetic Neuropathy between DPP-4 Inhibitor and SGLT2 Inhibitor

Aim: Diabetic neuropathy (DN) is the most prevalent complication with microangiopathy in diabetes. Although, DN would decrease quality of life, currently there is no radical treatment for DN. DPP-4 inhibitor (DP4i) and SGLT-2 inhibitor (SG2i) have been experimentally shown to possibly ameliorate DN. However, the difference in the mechanism between two agents has not been clarified so far. In this study, we performed proteomic analysis of the sciatic nerve (SN) and dorsal root ganglion (DRG) of Goto-Kakizaki rats (GK), a model of lean type 2 diabetes, treated with DP4i and/or SG2i. Methods: Male GK and control Wister rats (W) were used. Since 5 weeks of age, GK rats were treated with 10mg/day DP4i (Te), 10mg/ day SG2i (Ca), and DP4i + SG2i (CaTe) until 29 weeks of age. Thermal perception was evaluated by tail flick test. At the end of experiment, SN, DRG, and skin tissue were dissected. Proteomic analysis was performed with SN and DRG tissues by mass spectrometry. In the skin, fluorescent immunostaining with anti-PGP 9.5 antibody was performed for the assessment of intraepidermal nerve density (IENFD). Results: Tail flick test and IENFD were significantly decreased in GK compared to W (p<0.05), and significantly improved in Te, Ca, and CaTe (p<0.05, p<0.05 and p<0.01 vs GK, respectively). Proteomic analysis for SN showed only two pathways changed in Te, whereas seven and six pathways changed in the Ca and CaTe, respectively. Conversely, in the DRG, only two pathways changed in Ca, whereas the five and seven pathways changed in Te and CaTe, respectively. Conclusion: Our results suggest that the DP4i and SG2i act on the different sites of peripheral nervous system with different mechanism. Combining these two agents possibly have complementary effects on DPN treatment. Disclosure Y.Takeuchi: None. S.Osonoi: None. T.Sasaki: None. H.Mizukami: None. Funding Mitsubishi Tanabe Pharma Corporation

New Insights into the Opioid Analgesic Profile of cis-(-)-N-Normetazocine-derived Ligands.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the μ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Restraint stress potentiates sensitivity to the antinociceptive effect of morphine through orexin receptors in the ventral tegmental area

Orexin signaling in the ventral tegmental area (VTA) plays a critical role in stress and addictive behaviors. On the other hand, exposure to stress potentiates behavioral sensitization to drugs of abuse such as morphine. This study aimed to elucidate the role of orexin receptors within the VTA in restraint stress (RS)-induced morphine sensitization. Adult male albino Wistar rats underwent stereotaxic surgery, and two stainless steel guide cannulae were bilaterally implanted into the VTA. Different doses of SB334867 or TCS OX2 29 as orexin-1 (OX1) and orexin-2 (OX2) receptor antagonists were microinjected into the VTA five min before exposure to RS, respectively. A duration of three hours was considered for applying the RS, and 10 min after RS exposure, animals received a subcutaneous injection of an ineffective dose of morphine (1 mg/kg) for three consecutive days followed by a five-day drug/stress-free period. On the ninth day, the tail-flick test evaluated the sensitivity to the antinociceptive effects of morphine. The results demonstrated that the sole application of RS or morphine (1 mg/kg) could not induce morphine sensitization; however, concurrent application of RS and morphine could induce morphine sensitization. Besides, intra-VTA administration of OX1 R or OX2 R antagonists before paired administration of morphine and RS blocked morphine sensitization. The role of OX1 R and OX2 R in the induction of stress-induced morphine sensitization was almost identical. This study provides new insight into the role of orexin signaling in the VTA in the potentiation of morphine sensitization induced by RS and morphine co-administration.

Transcranial direct current stimulation alleviates nociceptive behavior in male rats with neuropathic pain by regulating oxidative stress and reducing neuroinflammation.

Transcranial direct curent stimulation (tDCS) and trans-spinal direct current stimulation (tsDCS) are promising therapies for pain that can alter the excitability of neuronal activity in cerebral cortex. The aim of the study is to investigate the therapeutic effects of direct current stimulation (DCS) over the spinal cord and cerebral cortex on oxidative stress and neuroinflammation in rats with chronic constriction injury (CCI).Male Wistar rats were randomly divided into four experimental groups: Sham, CCI, CCI + tDCS and CCI + tsDCS. The neuropathic pain model was induced by using the CCI model. Rats with neuropathy were treated with cathodal tDCS and tsDCS stimulations consisting of 0.5 mA for 30 min a day for 7 days from day 8 onwards. Locomotor activity was measured by open-field test and nociceptive behavior was assessed by hot-plate, tail-flick and Randall-Selitto tests. Following the behavioral experiments, total oxidant capacity (TOC), total antioxidant capacity (TAC) and proinflammatory cytokine levels were evaluated in spinal cord and cerebral cortex tissues. The CCI model induced significant mechanical and thermal hyperalgesia. Nociceptive behaviors in rats with CCI were reversed by DCS treatment. Higher TOC and lower TAC levels were detected in the spinal cord and cerebral cortex tissues of the CCI rats compared to the control. tsDCS treatment amended oxidant/antioxidant status. Moreover, tsDCS modulated the central levels of Tumor necrosis factor-α (TNF-α), interleukin 1-beta (IL-1β), IL-6 and IL-18. tsDCS stimulation showed better therapeutic effect on neuropathic pain by regulating oxidant/antioxidant levels and reducing neuroinflammation. DCS, especially at spinal level, may be a promising therapeutic strategy that can be used alone or in combination with other effective treatments for alleviating neuropathic pain.

Nano-invasomes for simultaneous topical delivery of buprenorphine and bupivacaine for dermal analgesia.

Opioid and local anaesthetic receptors are abundantly concentrated in different layers of the skin. Therefore, simultaneous targeting of these receptors can produce more potent dermal anaesthesia. Herein, we developed lipid-based nanovesicles for the co-delivery of buprenorphine and bupivacaine to efficiently target skin-concentrated pain receptors. Invasomes incorporating two drugs were prepared by ethanol injection method. Subsequently, the size, zeta potential, encapsulation efficiency, morphology, and in-vitro drug release of vesicles were characterized. Ex-vivo penetration features of vesicles were then investigated by the franz diffusion cell on the full-thickness human skin. Wherein, it was demonstrated that invasomes penetrated the skin deeper and delivered bupivacaine more effectively than buprenorphine to the target site. The superiority of invasome penetration was further evidenced by the results of ex-vivo fluorescent dye tracking. Estimation of in-vivo pain responses by the tail-flick test revealed that compared with the liposomal group, the group receiving invasomal formulation and drug-free invasomal formulation (only containing menthol) displayed increased analgesia in the initial times of 5 and 10 min. Also, no signs of oedema or erythema were observed in the Daze test in any of the rats receiving the invasome formulation. Finally, ex-vivo and in-vivo assays demonstrated efficiency in delivering both drugs into deeper layers of skin and exposing them to the located pain receptors, which improves the time of onset and the analgesic effects. Hence, this formulation appears to be a promising candidate for tremendous development in the clinical setting.

Anti-inflammatory and antinociceptive activities of Daniellia oliveri (Fabaceace) stem bark extract.

Daniellia oliveri (Rolfe) Hutch. & Dalziel (Fabaceae) is used for the treatment of inflammatory diseases and pains (chest pain, toothache and lumbago) and rheumatism. The study investigates the anti-inflammatory and antinociceptive properties of D. oliveri and possible mechanism of antiinflammatory action. Acute toxicity of the extract was evaluated in mice using the limit test. The anti-inflammatory activity was assessed in xylene-induced paw oedema and carrageenan-induced air-pouch models at doses of 50, 100 and 200mg/kg, p.o. Volume of exudate, total protein, leukocyte counts, myeloperoxidase (MPO) and concentration of cytokines (TNF-α and IL-6) were measured in the exudate of rats in the carrageenan-induced air-pouch model. Other parameters include lipid peroxidation (LPO), nitric oxide (NO) and antioxidant indices (SOD, CAT and GSH). Histopathology of the air pouch tissue was also carried out. The antinociceptive effect was assessed using acetic acid-induced writhing, tail flick and formalin tests. Locomotor activity was done in the open field test. The extract was analysed with HPLC-DAD-UV technique. The extract showed significant anti-inflammatory effect (73.68 and 75.79%, inhibition) in xylene-induced ear oedema test at the dose of 100 and 200mg/kg, respectively. In carrageenan air pouch model, the extract significantly reduced exudate volume, protein concentration, the migration of leukocytes and MPO production in the exudate. The concentrations of cytokines TNF-α (12.25±1.80pg/mL) and IL-6 (21.12pg/mL) in the exudate at the dose of 200mg/kg were reduced compared to carrageenan alone group (48.15±4.50pg/mL; 82.62pg/mL) respectively. The extract showed significant increase in the activities of CAT and SOD and GSH concentration. The histopathological assessment of the pouch lining revealed reduction of immuno-inflammatory cell influx. Nociception was significantly inhibited by the extract in acetic acid-induced writhing model and the second phase of formalin test indicating a peripheral mechanism of action. The open field test showed that D. oliveri did not alter locomotor activity. The acute toxicity study did not cause mortality or signs of toxicity at 2000mg/kg, p.o. We identified and quantified caffeic acid, p-coumaric acid, ferulic acid, rutin, apigenin-7-glucoside, quercetin and kaempferol in the extract. The results of our study showed that the stem bark extract of D. oliveri possesses anti-inflammatory and antinociceptive activities thereby supporting its traditional use in the treatment of some inflammatory and painful disorders.

Dexmedetomidine, an α2 agonist, increases the morphine analgesic effect and decreases morphine tolerance development by suppressing oxidative stress and TNF/IL-1 signalling pathway in rats.

The aim of the present study is to examine the possible effect de dexmedetomidine on the development of morphine tolerance in rats including nociception, morphine analgesia, apoptosis, oxidative stress, and tumour necrosis factor (TNF)/ interleukin-1 (IL-1) pathways. In this study, 36 Wistar Albino (225-245 g) rats were used. Animals were divided into 6 groups: saline (S), 20 mcg/kg dexmedetomidine (D), 5 mg/kg morphine (M), M + D, morphine tolerance (MT), and MT + D. The analgesic effect was measured with hot plate and tail-flick analgesia tests. After the analgesia tests, the dorsal root ganglia (DRG) tissues were excised. Oxidative stress parameters [total antioxidant status (TAS), total oxidant status (TOS)], TNF, IL-1 and apoptosis enzymes (Caspase-3, Caspase-9), were measured in DRG tissues. Dexmedetomidine showed an antinociceptive effect when given alone (p < 0.05 to p < 0.001). In addition, dexmedetomidine increased the analgesic effect of morphine (p < 0.001), and also decreased the tolerance to morphine at a significant level (p < 0.01 to p < 0.001). Moreover, it decreased oxidative stress (p < 0.001) and TNF/IL-1 levels when given as an additional drug of single-dose morphine and morphine tolerance group (p < 0.001). Furthermore, dexmedetomidine decreased Caspase-3 and Caspase-9 levels after tolerance development (p < 0.001). Dexmedetomidine has antinociceptive properties, and it increases the analgesic effect of morphine and also prevents tolerance development. These effects probably occur by the modulation of oxidative stress, inflammation and apoptosis.