Tail Flick Research Articles

Selective MOR activity of DAPEA and Endomorphin-2 analogues containing a (R)-γ-Freidinger lactam in position two

The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the biological stability of peptides and an appropriate tool to elucidate their structure-activity relationships. The endomorphin-2 (EM-2) and [D-Ala2, des-Leu5] enkephalin amide (DAPEA) are two linear opioid tetrapeptides agonists of MOR and MOR/DOR respectively. Herein, we investigated the influence of the incorporation of (R/S)-Agl in position 2 and 3 on the biological profile of the aforementioned products in vitro and in vivo. Receptor radiolabeled displacement and functional assays were used to measure in vitro the binding affinity and receptors activation of the novel analogues. The mouse tail flick and formalin tests allowed to observe their antinociceptive effect in vivo. Data revealed that peptide A2D was able to selectively bind and activate MOR with a potent antinociceptive effect after intracerebroventricular (i.c.v.) administration, performing better than the parent compounds EM-2 and DAPEA. Molecular docking calculations helped us to understand the key role exerted by the Freidinger Agl moiety in A2D for the interaction with the MOR binding pocket.

Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling.

The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.

Phytochemical Investigation and HPTLC Fingerprinting Analysis of Selaginella bryopteris Ethanolic Plant Extract for Analgesic and Anti-inflammatory Activities in Animals

The present study was performed to determine analgesia and anti-inflammatory activities of whole plant extract of Selaginella bryopteris on animals. The extract of S. bryopteris whole plant was performed using ethanol (70%) as a solvent. The phytochemical investigation of plant extract was done to evaluated secondary metabolites present in the plant. It was further characterized using HPTLC technique to identify active constituents present in the S. bryopteris extract. The efficacy of plant extract was evaluated using the hot plate method and tail flick method against pain and carrageenan-induced paw oedema model against inflammation. Results exhibited that S. bryopteris extract possesses alkaloids, glycosides, tannins, saponins, flavonoids, carbohydrate, protein and amino acids, steroids, vitamins, gums and mucilages. HPTLC fingerprinting analysis revealed the presence of gallic acid and rutin as active constituents present in the plant extract. S. bryopteris extract showed significant analgesic and anti-inflammatory activities. Thus, it can be used as a safer alternative drug against pain and inflammation in comparison to existing non-steroidal anti-inflammatory drugs (NSAIDs).

A study to investigate the chemical potency, physical stability, and efficacy of analgesic agents over a period of two years post their expiry date

BackgroundAs per regulations, drugs have to be used within their shelf life and must be discarded after their expiry date. The present study was conducted in commonly used analgesic agents to investigate the change in the physical, chemical, and efficacy parameters of the drugs over a period of two years after their expiry date. MethodsThe drugs studied were Tab Ibuprofen, Tab Diclofenac, Tab Piroxicam, Inj Diclofenac, and Inj Piroxicam. The parameters investigated were chemical potency, physical stability, and analgesic efficacy. Testing for these parameters was carried out once before expiry to establish the baseline followed by at expiry, and then every six months for two years post expiry date. ResultsThe active ingredient in all the drugs were within the accepted range as per IP during the study period indicating that they retained their potency up to a period of two years post expiry. The analgesic efficacy as determined by the “Radiant Heat Tail flick” and “Acetic acid-induced writhing” model also showed no significant difference in the Maximum Possible Efficacy and Percent protection, indicating that the drugs retained their efficacy. There was a slight difference in the dissolution times at expiry as compared to different time periods in the case of Ibuprofen and Diclofenac, while there are was no statistical difference in the disintegration times during the study period. ConclusionThe analgesic drugs in our study retained their chemical potency, physical stability and analgesic efficacy up to a period of two years after expiry. The expiry date of a drug, as estimated currently, is not an accurate reflection of its actual shelf life.

Comparative study on analgesic effect of tetrodotoxin in four acute pain models

Objective To evaluate the analgesic effect of tetrodotoxin (TTX) in four types of acute pain models and provide experimental support for its rational application. Methods Mice or rats were intramuscularly pretreated with morphine (1 mg/kg) or TTX (0, 0.5, 1, 2, 4 and 8 μg/kg) 40 min before acetic acid writhing test, formalin stimulation test, hot plate test or tail flick test. Pain response or pain threshold were recorded, and inhibition rate was calculated during the tests. The arachidonic acid of serum was determined by Elisa. Results No significant analgesic effects were observed with morphine in all four acute pain models. TTX dose-dependently reduced the number of writhing induced by acetic acid and inhibited the pain response induced by formalin during phase I and phase II, with the highest inhibition rate of more than 80.00% in two pain models. TTX showed analgesic effect in tail flick test and hot plate test, with the highest inhibition rate of 25.00% and 19.79%, respectively. Both acetic acid and formalin increased arachidonic acid in animal serum, but TTX had no significant inhibitory effect on the releasing of arachidonic acid. Conclusion TTX showed significant analgesic effect in the chemical stimulation pain models induced by acetic acid and formalin, but limited analgesic effect was observed on the physical stimulation pain model induced by heat (hot plate and hot water). TTX may produce analgesic effect by blocking the inflammatory mediators mediating pain response.

Evaluation of analgesic and anti-inflammatory properties of the Allium cepa extract in rats

Background: The onion plant (Allium cepa) is the most widely cultivated species of the genus Allium. It has antibiotic, antidiabetic, antihypercholesterolemic, antioxidant, hemostatic, antimutagenic effects. The current study is aimed to evaluate analgesic and anti-inflammatory properties of Allium cepa extracts in rat models.Methods: In this study analgesic activity in rats is tested by Eddy’s hot plate method, tail flick method. In Eddy’s hot plate method rats are individually placed on the heated plate of the analgesic meter maintained at 550C and mean reaction time was calculated at regular time intervals. In tail flick method the tail withdrawal from the heat (flicking response) is taken as the end point. Anti-inflammatory property is studied by carrageenan induced paw edema model, in which the paw volume is measured with a plethysmograph.Results: In Eddy’s hot plate method the mean reaction time increased significantly (p<0.05) with high dose of Allium cepa at 1 hour and 2 hours when compared to control. In tail flick method the mean reaction time is increased significantly (p<0.05) with high dose of Allium cepa at 1 hour and 1.5 hour when compared to control. In carrageenan induced paw edema method Allium cepa exhibited anti-inflammatory activity by significant (p<0.05) suppression of paw edema when compare to control at 1 hour after carrageenan injection to rat paw.Conclusions: Allium cepa has significant analgesic and anti-inflammatory activity in hot plate, tail flick and carrageenan induced paw edema model.

Pharmacological Screening of Substituted Benzimidazole Derivatives

In the present study some substituted benzimidazole derivatives were screened for several biological activities. The synthesized compounds were subjected to evaluation of central analgesic, anti-inflammatory, cytotoxicity, antimicrobial and antioxidant activities by radiant heat induced tail flicking, carrageenan induced rat paw edema inhibition, brine shrimp lethality bioassay, disc diffusion and DPPH free radical scavenging methods, respectively. Compounds 2a, 2c and 2d elongated the tail flicking time by 58.07-, 51.59- and 76.65%, respectively (p < 0.001) at 50 mg/kg body weight dose compared to the standard morphine (87.17%). Compounds 2b, 2c and 2d showed prominent anti-inflammatory activity at 100 mg/kg body weight dose (% of paw edema inhibition 81.75%, 79.09% and 86.69%, respectively) compared to the standard aceclofenac (87.83%). Among the synthesized benzimidazole derivatives, compounds 1a, 1b, 1c, 2a and 2d exhibited potent cytotoxicity with the IC50 values of 5.47-, 11.92-, 4.55-, 7.63- and 7.94 μg/ml, respectively. In addition, compounds 1c and 2d displayed mild to moderate zone of inhibition (8-12 mm). On the other hand, 1a and 1b showed very mild antioxidant activity with IC50 values of 12.25 × 103 μg/ml and 87.33 ×103 μg/ml. Among all the derivatives, 2c, 2d and 1c can be potential candidates for designing new analgesic, anti-inflammatory and anti-cancer agents in future.
 Dhaka Univ. J. Pharm. Sci. 20(1): 95-102, 2021 (June)

Semi-synthesis of novel buprenorphine derivatives and their anti-nociceptive properties and dependency potential

Novel 1,2,3-triazole-tethered N-norbuprenorphine derivatives with an OMe or OH group at the C3 position were synthesized alongside with evaluation of their analgesic properties. The analgesic activities of the resulting library were investigated via tail flick test in mice. Our results indicated that 10b and 10e were as effective as the starting compounds 8 and 9 with ED50 equal to 16.59 and 19.44 mg/kg, respectively. To investigate the effect of a methyl group at C3 on biological properties, the most active compounds were O-demethylated and their anti-nociceptive effects were assessed. The new O-demethylated derivatives (11b and 11e) showed better analgesic properties than the parent compounds with ED50 of 14.73 and 15.80 mg/kg, respectively. Naloxone prevented the analgesic effect of the synthesized compounds, indicating that the opioid receptors are highly involved in the anti-nociceptive effects. The potential dependency effects of the most potent derivatives were studied by condition place preference test in mice and compared with morphine and buprenorphine. Interestingly, 10b, 10e, 11b, and 11e did not show any dependency effect, similar to buprenorphine.

Vitamin D and rosuvastatin obliterate peripheral neuropathy in a type-2 diabetes model through modulating Notch1, Wnt-10α, TGF-β and NRF-1 crosstalk

AimsVitamin D and rosuvastatin are well-known drugs that mediate beneficial effects in treating type-2 diabetes (T2D) complications; however, their anti-neuropathic potential is debatable. Hence, our study investigates their neurotherapeutic potential and the possible underlying mechanisms using a T2D-associated neuropathy rat model. Main methodsDiabetic peripheral neuropathy (DPN) was induced with 8 weeks of administration of a high fat fructose diet followed by a single i.p. injection of streptozotocin (35 mg/kg). Six weeks later, DPN developed and rats were divided into five groups; viz., control, untreated DPN, DPN treated with vitamin D (cholecalciferol, 3500 IU/kg/week), DPN treated with rosuvastatin (10 mg/kg/day), or DPN treated with combination vitamin D and rosuvastatin. We determined their anti-neuropathic effects on small nerves (tail flick test); large nerves (electrophysiological and histological examination); neuronal inflammation (TNF-α and IL-18); apoptosis (caspase-3 activity and Bcl-2); mitochondrial function (NRF-1, TFAM, mtDNA, and ATP); and NICD1, Wnt-10α/β-catenin, and TGF-β/Smad-7 pathways. Key findingsTwo-month treatment with vitamin D and/or rosuvastatin regenerated neuronal function and architecture and abated neuronal inflammation and apoptosis. This was verified by the inhibition of the neuronal content of TNF-α, IL-18, and caspase-3 activity, while augmenting Bcl-2 content in the sciatic nerve. These treatments inhibited the protein expressions of NICD1, Wnt-10α, β-catenin, and TGF-β; increased the sciatic nerve content of Smad-7; and enhanced mitochondrial biogenesis and function. SignificanceVitamin D and/or rosuvastatin alleviated diabetes-induced neuropathy by suppressing Notch1 and Wnt-10α/β-catenin; modulating TGF-β/Smad-7 signaling pathways; and enhancing mitochondrial function, which lessened neuronal degeneration, demyelination, and fibrosis.

Phytochemical profiling, in vitro and in vivo anti-inflammatory, analgesic and antipyretic potential of Sesuvium sesuvioides (Fenzl) Verdc. (Aizoaceae).

Sesuvium sesuvioides (Fenzl) Verdc is traditionally used in the treatment of inflammatory diseases such as arthritis and gout The aim of present study was to assess the possible anti-inflammatory, analgesic and antipyretic potential of the methanol extract of Sesuvium sesuvioides (SsCr) to prove scientifically its folklore use in the inflammatory diseases and to screen its total antioxidant capacity by multiple methods and phytocompounds by GC-MS. The preliminary phytochemical studies showed the presence of phenols, flavonoids, glycosides, coumarin, terpenoids, saponins, fats and carbohydrates in crude extract. The total phenolic contents (27.31 ± 0.28mg GAE/g) and total flavonoids (3.58 ± 0.12 mgRE/g) values were observed. The antioxidant capacity of SsCr showed significant DPPH, ABTS, CUPRAC, FRAP, PBD and metal chelating results. GC-MS analysis displayed the phytoconstituents with anti-inflammatory potentials such as 2-methoxy-4-vinylphenol, vanillin, umbelliferone, methyl ferulate, palmitoleic acid, methyl palmitate and phytol. SsCr presented noteworthy HRBC membrane stability with maximum inhibition of cell hemolysis (47.79%). In carrageenan-induced hind paw edema assay result showed dose-dependent anti-inflammatory action. SsCr presented significant (p < 0.05) analgesic activity in hot-plate and tail flicking tests similarly it also showed the noteworthy inhibition in pain latency against formalin induced analgesia at 1st and 2nd phases. SsCr reduced the acetic acid-induced writhes at different doses (250, 500 and 750mg). Results of antipyretic activity of SsCr extract were significant at 500 and 750mg. The results of in vitro and in vivo experimental studies verified the anti-inflammatory, analgesic and antipyretic potential of Sesuvium sesuvioides and supported the folklore uses of this plant.

Evaluation of Analgesic, Antidiarrheal and Hypoglycemic Activities of &lt;i&gt;Wendlandia paniculata&lt;/i&gt; (Roxb.) DC Leaves Extract using Mice Model

The leaves of Wendlandia paniculata (Roxb.) DC is a rare folk medicine among tribal people even though there is no scientific literature available regarding its pharmacological potentials. This study aimed to evaluate the effects of crude methanol extract of W. paniculata (Roxb.) DC. Leaves in mice model. The in vivo glucose-lowering capacity of methanolic crude extract was analyzed utilizing the well-known tail tipping method. The central and peripheral analgesic activity was evaluated using the ‘tail flick' and writhing assay respectively. The anti-diarrheal activity was evaluated using the method of castor oil-induced diarrhea in mice. As a result, oral administration (400 mg/ kg) of the extract resulted significant (P&lt;0.001) delay in response to pain and inhibition of acetic acid-induced writhing response. The results were comparable to the respective standards morphine (2 mg/kg) and diclofenac (50 mg/kg). Likewise, maximum reduction (p&lt;0.05) of blood glucose (32.3%) was observed 180 min after oral intake(400 mg/kg) of the extract compared to the 47% of the standard glibenclamide. Moreover, a reduction of diarrheal feces was observed 45.83% (p&lt;0.001) compared to standard loperamide 70.83%. The in vivo bioassays confirmed that the crude methanol extract of leaves of W. paniculata possesses significant analgesic (central, peripheral) and antidiarrheal activity with mild glucose-lowering activity. Isolation of bioactive compounds is needed to justify the molecule responsible for the activity.

Donepezil prevents morphine tolerance by regulating N-methyl-d-aspartate receptor, protein kinase C and CaM-dependent kinase II expression in rats

Current studies have indicated that donepezil as a cholinesterase inhibitor can attenuate morphine-induced tolerance. The present study aimed to evaluate the possible role of N-methyl-d-aspartate receptors (NMDARs), protein kinase C (PKC) and CaM-dependent kinase II (CaMKII) pathways in this effect. Female Wistar rats received daily morphine (10 mg/kg, i.p.) alone or in combination with donepezil (1.5 or 2 mg/kg, gavaged) for 14 days. The analgesic effect was assessed by Von-frey, hotplate and tail flick test. On the 15th day, the periaqueductal gray (PAG) and lumbar spinal cord of rats were dissected. Then, protein levels of NMDAR-NR1, NR2B, PKCγ and CaMKIIα were tested using Western blot method. The results showed that morphine tolerance was seen after 8–10 days of injection compared with control group, while daily co-administration of donepezil with morphine prolonged the occurrence of analgesic tolerance. Western blot showed that morphine significantly increased NR1, PKCγ and CaMKIIα expressions in PAG, and significantly increased PKCγ and CaMKIIα in spinal cord. In contrast, donepezil downregulated NR1 and PKCγ in PAG, and downregulated PKCγ and CaMKIIα in spinal cord. Moreover, donepezil alone activates NR1 and NR2B in spinal cord, which needs to be further studied. Thus, the present results suggest that the attenuation effects of donepezil on morphine tolerance are possibly mediated by preventing morphine-induced upregulations in NR1, PKCγ and CaMKIIα expressions.

Evaluation of anti-nociceptive, anti-inflammatory and hepatoprotective effects of methanol extract of Mazus pumilus (Burm. f.) Steenis (Mazaceae) herb

Purpose: This study was designed to investigate the anti-nociceptive, anti-inflammatory and hepatoprotective activities of the methanol extract of Mazus pumilus (Mazaceae) herb.&#x0D; Methods: Anti-nociceptive activity was determined using hot plate, tail flick and acetic acid-induced writing methods. Carrageenan-induced rat paw edema (0.1 mL of 1 %) model was used for the assessment of anti-inflammatory activity. The methanol extract was administered orally at three different doses (150, 300 and 600 mg/kg) to three separate groups in all the experiments. Diclofenac sodium (50 mg/kg) was used as standard drug while control group received DMSO (1 %, 10 mL/kg). The hepatocurative effect of methanol extract of M. pumilus (400 mg/kg) was determined in isoniazid (50 mg/kg) and rifampicin (100 mg/kg) induced liver injury. Silymarin (100 mg/kg) was used as standard drug for comparison. The control group received distilled water (10 mL/kg). Preliminary phytochemical screening was also carried out.&#x0D; Results: The methanol extract of M. pumilus significantly (p &lt; 0.05) augmented latency time and reduced the number of writhes in the pain models at all doses used for the assessment of antinociceptive actions. The anti-inflammatory activity of different doses of extract was evaluated by measuring the reduction in the size of the paw. A significant (p &lt; 0.05) hepatocurative effect was observed when administered after anti-tuberculosis drugs. Histopathological analysis of the liver tissues also revealed restored hepatocellular architecture.&#x0D; Conclusion: The results demonstrate the anti-nociceptive, anti-inflammatory and hepatoprotective effects of the methanol extract of M. pumilus, thus substantiating the ethnomedical claims associated with the herb.

ELAEOCARPUS SERRATUS L. EXHIBITS POTENTIAL ANALGESIC AND ANTIDIARRHEAL ACTIVITIES IN MICE MODEL

Background. Elaeocarpus serratus L. (Family: Elaeocarpaceae) is a tropical fruit tree, traditionally used in the treatments of poisoning, diarrhea, arthritis, and other diseases. Objectives. The current study was performed to conduct the analgesic, antidiarrheal, and hypoglycemic activity of E. serratus in mice model using methanolic bark crude extract. Methods. To assess the peripheral and central analgesic activity, the acetic acid-induced writhing and tail immersion methods were respectively used. The castor-oil mediated antidiarrheal method was used to assess the antidiarrheal activity whereas, the tail tipping technique was conducted to determine the hypoglycemic activity of the plant extract. Results. In the peripheral analgesic assay, the methanolic bark crude extract of E. serratus significantly inhibits the number of writing 69.77% (200 mg/kg) and 73.26% (400 mg/kg) respectively (p&lt;0.05) which was strongly comparable with standard NSAID drug diclofenac sodium 75.58% (p&lt;0.05). Similarly, it shown a significant tail flicking response for 30 minutes, 60 minutes and 90 minutes of central analgesic activity assay. In antidiarrheal activity assay, the E. serratus substantially reduced the number of diarrheal feces 64.26% (200 mg/kg, p&lt;0.05) and 78.57% (400 mg/kg, p&lt;0.05) which was also comparable with the positive control loperamide. The hypoglycemic activity of E. serratus extract was not convincing. Conclusions. Our investigation demonstrated the significant analgesic and antidiarrheal activities of methanolic bark extract of E. serratus (200 and 400 mg/kg) in mice model.

Inhibition of Heat shock protein 90 in the Spinal Cord Enhances Opioid Anti-Nociception by Disabling an Inhibitory GABA Circuit

One strategy to enhance the clinical treatment of chronic and acute pain is the development of dose-reductive compounds that, when co-administered with palliatives, can reduce the dose of opioid required for treatment while retaining analgesic efficacy and decreasing side-effects. We previously showed that intrathecal (IT) administration of the heat shock protein 90 (Hsp90) inhibitor 17-AAG potentiated the efficacy of morphine induced antinociception while reducing or not changing opioid side effects. Proteomic analysis of the translational alterations induced by IT 17-AAG administration revealed an upregulation of GABA transporter 2 (GAT-2). To elucidate the role of GAT-2 in mediating the enhancement of morphine antinociception, 17-AAG treated mice were administered a GAT inhibitor, Nipecotic Acid (NA). NA effectively blocked the 17-AAG enhancement of antinociception compared to controls, while not changing responses in controls, suggesting that GAT-2 mediates 17-AAG's antinociceptive enhancement. We hypothesized that an inhibitory GABA circuit is present in the spinal cord which reduces opioid antinociception and is disabled by Hsp90. To test this, mice were treated with IT GABA-A or GABA-B agonists prior to systemic morphine administration. The GABA-A agonist reversed 17-AAG enhanced antinociception much like NA. Intriguingly, the GABA-B agonist reversed 17-AAG antinociception in males only, suggesting a GABAergic sex-specific difference. To further test the inhibitory GABA circuit hypothesis, naive mice (no 17-AAG) were injected with IT GABA-A or GABA-B antagonists prior to morphine and tail flick. Males showed enhanced morphine antinociception with either antagonist. However, neither drug had an impact on female mice, again suggesting mechanistic sex differences. These results taken together suggest that IT 17-AAG potentiates morphine-induced antinociception via upregulation of GAT-2, relieving an inhibitory GABAergic brake on opioid antinociception. This study provides mechanistic insight into how Hsp90 inhibitors can be used to boost opioid antinociception and may have identified a novel inhibitory circuit of opioid antinociception. This study was funded by an Arizona Biomedical Research Commission New Investigator Award #ADHS18-198875 and institutional funds from the University of Arizona.

The Hsp90 Isoform‐Selective Inhibitor KUNG65 Enhances Opioid Anti‐Nociception while Reducing Tolerance

Opioids are the gold standard for pain management, however, adverse side effects like respiratory depression and addiction limit their viability. One approach to improve opioid therapy focuses on a dose-reduction strategy by amplifying opioid analgesia without boosting side effects, so that less opioid can be given. To this end, we've focused on Heat shock protein 90 (Hsp90), a central protein regulator in the cell; ongoing studies from our lab have shown that inhibition of Hsp90 in the spinal cord improves morphine anti-nociceptive potency by 2-3 fold in acute and chronic pain while reducing tolerance, rescuing established tolerance, and not changing reward and constipation, enabling a dose-reduction strategy. However, our results also showed that non-selective Hsp90 inhibitors given systemically (intravenous, IV) have the opposite effect, blocking opioid pain relief. Seeking a means to surmount this roadblock, we determined which Hsp90 isoforms regulate opioid pain relief in brain and spinal cord in mice using selective small molecule inhibitors and CRISPR/Cas9 gene editing. We found that Hsp90α alone regulated opioid signaling and pain relief in the brain. However, in the spinal cord, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all regulated opioid signaling and pain relief. This led to our hypothesis that targeting spinal cord Hsp90 with isoform-selective inhibitors given IV could boost opioid pain relief without altering side effects, enabling a dose-reduction strategy. We tested this hypothesis with the novel Grp94-selective inhibitor KUNG65, given at a 1 mg/kg dose IV in male and female CD-1 mice, followed by a 24 hr treatment time, then analysis of opioid anti-nociception and side effects. We found that systemic (IV) KUNG65 treatment resulted in a 1.9 fold increase in morphine potency to relieve tail flick pain, consistent with our earlier studies injecting inhibitor directly into spinal cord. We also found that KUNG65 boosted morphine potency in paw incision pain, with a 2.2 fold increase. Additionally, we found that KUNG65 injection could rescue established morphine tolerance in the tail flick assay, again as we found for direct spinal cord injection. These results support our hypothesis that isoform-selective Hsp90 inhibitors can selectively engage Hsp90 in the spinal cord when given systemically, resulting in improved opioid anti-nociception and side effects. These results strongly suggest that Hsp90 isoform-selective inhibitors could be a powerful new tool to improve opioid therapy through a dose-reduction strategy, and further show that this effect can be achieved through a translationally relevant dosing route.

Inhibition of Spinal Cord Hsp90 Enhances Src Kinase Signaling to Increase Opioid Anti‐Nociception

Chronic pain remains a huge social and economic burden on the United States and around the world. With few safe treatment options for these patients, many physicians turn to opioid analgesics such as morphine. These medications are not without serious side effects including constipation, respiratory depression, and addiction, which has led to an opioid addiction crisis affecting over 10 million Americans each year. This crisis emphasizes the need to find new ways to improve opioid therapy and decrease side effects like addiction. One such research area is the signal transduction cascades evoked by opioid receptor activation, which could be modulated to enhance their effects on pain management while decreasing negative side effects. Previously, our lab discovered that the molecular chaperone protein Heat shock protein 90 (Hsp90) plays a key role in mu opioid receptor (MOR) signal transduction. When inhibiting Hsp90 in the spinal cord through intrathecal administration of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) in mice we observed a significant increase in morphine anti-nociception, which could enable an opioid dose-reduction strategy. A preliminary proteomic evaluation in 17-AAG treated mouse spinal cord suggested that the inhibitory protein Src-kinase inhibitor 1 was downregulated by 17-AAG, thus suggesting that Src kinase signaling might be upregulated. To test this hypothesis, male and female CD-1 mice were treated with 0.5 nmol 17-AAG or vehicle intrathecally. After 24 hours, mice were treated with 10 nmol of the Src kinase inhibitor Src-I1 or vehicle intrathecally, then after 10 minutes, mice were administered 3.2 mg/kg morphine and a tail flick assay was performed. The enhanced anti-nociceptive effects seen with 17-AAG were completely abolished in groups that received the Src inhibitor, suggesting that Hsp90 inhibition enhances Src kinase signaling to lead to enhanced opioid pain relief. Interestingly, the group that did not receive 17-AAG but did receive Src-I1 also showed a decrease in the anti-nociceptive effects of morphine, thus suggesting a role for Src in the opioid receptor pathway at baseline without Hsp90 inhibitor treatment. Next, mice were treated with only Src-I1 or vehicle and then given morphine or vehicle, showing that Src-I1 administration had no effect on pain by itself. We also tested a post-surgical paw incision pain model to test if Src regulates opioid anti-nociception in other pain types. Similar results were seen with a complete loss of the enhanced anti-nociception of 17-AAG as well as a decrease in the anti-nociceptive effects of morphine with Src inhibitor treatment. Lastly, initial analysis by Western Blotting confirmed what we observed behaviorally. Groups treated with 17-AAG and the opioid agonist DAMGO had a significant increase in phosphorylated Src compared to groups treated with vehicle alone, thus supporting our hypothesis that Hsp90 inhibition in the spinal cord enhances opioid pain relief by upregulation of Src signaling. This has helped elucidate a key molecular mechanism by which Hsp90 regulates opioid signaling and leads to enhanced opioid antinociception, creating a potential target for future efforts to improve opioid treatment.

GPR171 Agonist, MS15203, Effectively Enhances Morphine Antinociception Without Modulating Detrimental Side Effects

Opioid drugs are limited in clinical effectiveness because of their propensity for tolerance, addiction, withdrawal, and respiratory depression. Nonetheless, this drug class is still readily prescribed for acute and chronic pain, often to the long-term detriment of patients. Safer pain therapeutics are desperately needed. One novel avenue of drug development is to search for a target capable of enhancing opioid antinociception without enhancing negative side effects. This would allow for increased opioid effectiveness with lower doses, thus minimizing negative side effects and postponing dose escalation. The previous orphan receptor, GPR171, is one such target. Our recent work showed that an agonist for GPR171, MS15203 enhances morphine antinociception in two rodent acute pain assays (McDermott et al., 2019). Combining these two drugs could potentially lead to greater antinociception with lower opioid doses. The next step in vetting MS15203 as a novel pain therapeutic is to determine its effect on negative opioid side effects. In this current study we utilized three rodent behavioral assays to assess MS15203 in tolerance development, opioid withdrawal, and reward liability. Additionally, we used immunohistochemistry to assess ex vivo MS15203-induced dopamine activation in the ventral tegmental area (VTA). Results from a hot plate dose-response show that MS15203 has no significant effect on morphine tolerance development in both males and females. However, MS15203 significantly decreases morphine tolerance in females on the tail flick test, suggesting a sex-specific and spinal-specific effect. Results from a naloxone precipitated withdrawal assay show that MS15203 does not significantly alter morphine withdrawal. Results from a conditioned place preference assay, show that MS15203 alone causes no significant place preference, and has no significant modulatory effect when paired with morphine. Similarly, MS15203, causes no significant VTA activation alone, and has no significant modulatory effect on morphine-induced activation. Taken together these results present GPR171 agonist MS15203 as an enhancer of morphine antinociception, and an overall neutral agent in morphine tolerance, withdrawal, and reward. Future studies should assess MS15203 on respiratory depression.

Discovery of a New Analgesic Peptide, Leptucin, from the Iranian Scorpion, Hemiscorpius lepturus.

Hemiscorpius lepturus scorpion stings do not induce considerable pain based on epidemiological surveys conducted in the southwest part of Iran. Accordingly, this study was aimed to identify the analgesic molecule in H. lepturus venom by analyzing a cDNA library of the scorpion venom gland looking for sequences having homology with known animal venom analgesic peptides. The analgesic molecule is a cysteine rich peptide of 55 amino acids. the synthetic peptide was deprotected and refolded. RP-HPLC, Ellman’s, and DLS assays confirmed the refolding accuracy. Circular dichroism (CD) showed helix and beta sheet contents. This peptide, called leptucin, demonstrated 95% analgesic activity at the dose of 0.48 mg/kg in hot plate assay. Leptucin at the doses of 0.32, 0.48, and 0.64 mg/kg showed 100% activity in thermal tail flick test. No hemolysis or cytotoxicity was observed at 8 and 16 μg. Histopathology evaluations indicated no hepatotoxicity, nephrotoxicity, and cardiotoxicity. We thus report that leptucin is the analgesic agent of H. lepturus venom. Regarding the high in vivo efficacy of leptucin and the fact it shows no observable toxicity, it could be suggested as a drug lead in a preclinical study of acute pain as well as the study of its mechanism of action.

ASSESSMENT OF THE ANTINOCICEPTIVE EFFECTS OF CANNABINOID RECEPTOR AGONISTS SER 601 AND L-759,633 IN AN ACUTE PAIN MODEL IN RATS

A poorly managed pain is associated with many negative results. These results disturb patients, their families and societies. Although opioid agents have extraordinary analgesic efficacy, they may cause serious adverse consequences. For medication of pain, there is a medical need to discover possible alternatives to opioids. In animal models of acute pain synthetic cannabinoid receptor agonists demonstrated analgesic effects. Selective activation of the cannabinoid 2 receptor in the rodent models does not cause psychotropic effects therefore CB2R agonists are an appealing target for the medication of pain and other pathologies. In this study we examined the antinociceptive activity of cannabinoid receptor 2 (CB2) agonists SER 601 and L-759,633 in different doses in rat models of acute pain using the hot plate and tail flick tests. These two agents exhibited dose-dependent antinociceptive effects in acute pain model. The analgesic effects of the administrated doses of L-759,633 and SER 601 reached their peak at 60 min. In the tail flick test, the antinociceptive effects of 3, 6 and 12 mg / kg SER601 in the acute pain were more than the L-759,633 groups at the same doses.