Rat Tail-flick Test Research Articles

The Effect of Different Training Modes on Serum Apelin and Pain Threshold in Morphine-Dependent Rats

Background: Apelin has recently been identified as an analgesic agent and a novel neuropeptide. On the other hand, it has been shown that exercise can lead to reduced pain in morphine-dependent patients. Objectives: Therefore, the aim of this study was to evaluate apelin and pain threshold changes in healthy and morphine-dependent rats in response to two exercise paradigms. Materials and Methods: In this study, 30 healthy and 30 morphine-dependent rats were used. Morphine-dependent and healthy rats were divided into six groups: 1, a control (healthy) group; 2, a healthy endurance group; 3, a healthy strength-training group; 4, an addicted control group; 5, an addicted endurance group; 6, an addicted strength-training group. Then, the training groups performed aerobic and strength training for eight weeks. After the training program, the tail flick and formalin tests were used to assess pain. Apelin was also measured by ELISA. Results: Regardless of the type of exercise, exercise significantly increased the apelin serum levels in healthy rats. The apelin levels significantly increased in the morphine-dependent rats compared with the healthy control group. Endurance, unlike strength training, significantly increased apelin in the serum compared to the addicted control group. The training led to pain relief in the morphinedependent rats and returned it to the healthy control group level. The Pearson correlation showed a reverse significant correlation between the serum apelin level and the tail flick test in the morphine-dependent rats. Conclusions: The results showed that endurance training reduced pain by increasing apelin in morphine-dependent rats. Therefore, it is suggested that this type of training be considered for the morphine-dependent patients for pain relief.

Sex hormones regulate cerebral drug metabolism via brain miRNAs: down-regulation of brain CYP2D by androgens reduces the analgesic effects of tramadol.

Brain cytochrome P450 2D (CYP2D) metabolises exogenous neurotoxins, endogenous substances and neurotransmitters. Brain CYP2D can be regulated in an organ-specific manner, but the possible regulatory mechanisms are poorly understood. We investigated the involvement of miRNAs in the selective regulation of brain CYP2D by testosterone and the corresponding alteration of the pharmacological profiles of tramadol by testosterone. The regulation of CYP2D and brain-enriched miRNAs by testosterone was investigated using SH-SY5Y cells, U251 cells, and HepG2 cells as well as orchiectomized growth hormone receptor knockout (GHR-KO) mice and rats. Concentration-time curves of tramadol in rat brain were determined using a microdialysis technique. The analgesic action of tramadol was assessed by the tail-flick test in rats. miR-101 and miR-128-2 bound the 3'-untranslated region of the CYP2D6 mRNA and decreased its level. Testosterone decreased CYP2D6 catalytic function via the up-regulation of miR-101 and miR-128-2 in SH-SY5Y and U251 cells, but not in HepG2 cells. Orchiectomy decreased the levels of miR-101 and miR-128-2 in the hippocampus of male GHR-KO mice, indicating that androgens regulate miRNAs directly, not via the alteration of growth hormone secretion patterns. Changes in the pharmacokinetic and pharmacodynamic profiles of tramadol by orchiectomy was attenuated by either testosterone supplementation or a specific brain CYP2D inhibitor. The selective regulation of brain CYP2D via brain-enriched miRNAs, following changes in androgen levels, such as in testosterone therapy, androgen deprivation therapy and/or ageing may alter the response to centrally active substances.

A new potent analgesic agent with reduced liability to produce morphine tolerance

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.

IEM-1460 and Spermine Potentiate the Analgesic Actions of Fentanyl and Analgin in Rats

Intramuscular administration of the minimum effective doses (MED) of the central analgesics fentanyl and Analgin [translator’s note: metamizole], the NMDA receptor polyamine site agonist spermine, and IEM-1460, an AMPA receptor blocker and agonist of the NMDA receptor polyamine site, induced maximal analgesic effects in the tailflick test in rats. Combined intramuscular administration of Analgin and IEM-1460 and spermine at threshold doses inactive when given alone (1/5 MED) allowed the MED of Analgin to be reduced by a factor of 120 when given in combination with IEM-1460 and by a factor of 10 when given in combination with spermine. Combined intramuscular administration of fentanyl with IEM-1460 and spermine at these threshold doses decreased the MED of fentanyl by a factor of 150 when used with IEM-1460 and a factor of 15 when used with spermine.

Role of spinal CXCL1 (GROα) in opioid tolerance: a human-to-rodent translational study.

The pivotal role of glial activation and up-regulated inflammatory mediators in the opioid tolerance has been confirmed in rodents but not yet in humans. Here, the authors investigated the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients; and to determine if up-regulated chemokines could modify opioid tolerance in rats. Cerebrospinal fluid samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The cerebrospinal fluid levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, and CX3CL1 were assayed. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 on morphine-induced acute antinociception and analgesic tolerance. CXCL1 level in cerebrospinal fluid was significantly up-regulated in the opioid-tolerant group (n = 30, 18.8 pg/ml vs. 13.2 pg/ml, P = 0.02) and was positively correlated (r = 0.49, P < 0.01) with opioid dosage. In rat experiment, after induction of tolerance by morphine infusion, the spinal cord CXCL1 messenger RNA was up-regulated to 32.5 ± 11.9-fold. Although CXCL1 infusion alone did not affect baseline tail-flick latency, the analgesic efficacy of a single intraperitoneal injection of morphine dropped significantly on day 1 to day 3 after intrathecal infusion of CXCL1. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by coadministration of CXCL1 and attenuated by coadministration of CXCL1-neutralizing antibody or CXCR2 antagonist. CXCL1 is up-regulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 signaling. Therefore, the CXCL1/CXCR2 signal pathway may be a novel target for the treatment of opioid tolerance.

The antinociceptive effect of stimulating the retrosplenial cortex in the rat tail-flick test but not in the formalin test involves the rostral anterior cingulate cortex

The stimulation of the retrosplenial cortex (RSC) is antinociceptive in the rat tail-flick and formalin tests. The rat RSC is caudal to and send projections to the ipsilateral anterior cingulate cortex (ACC), which is also involved in pain processing. This study demonstrated that pre-treating the rostral (rACC), but not the caudal ACC with CoCl2 (1mM), or the rACC ablation increased the duration of the antinociceptive effect evoked by a 15-s period of electrical stimulation (AC, 60Hz, 20μA) of the RSC in the rat tail-flick. Injecting the GABA-A antagonist bicuculline (50ng/0.25μL), but not the GABA-B antagonist phaclofen (300ng/0.25μL) into the rACC also increased the duration of the stimulation-induced antinociception from the RSC. In contrast, the effects of rACC stimulation persisted after the injection of CoCl2 (1mM) into the RSC. The injection of CoCl2 into the rACC did not change the nociceptive behavior of rats during phase 1 of the formalin response but reduced licking response duration during phase 2. This effect was similar in sham or stimulated animals at the RSC. We conclude that the antinociceptive effect of stimulating the RSC in the rat tail-flick test is modulated by the rACC involving GABA-A receptors in this cortex. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC.

Investigation of anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract

Ethnopharmacological relevanceStahlianthus involucratus (Zingiberaceae) has long been used in traditional medicine to treat inflammation, pain, and fever. However, no pharmacological study of this plant has been reported to confirm these activities. The aim of this study was to investigate the anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract (SiE) in animal models. Materials and methodsAnti-inflammatory activity of SiE was investigated in rats using ethyl phenylpropiolate (EPP)-induced ear edema, carrageenan- and arachidonic acid (AA)-induced hind paw edema, and cotton pellet-induced granuloma formation models. Acetic acid-induced writhing response in mice and tail-flick test in rats as well as yeast-induced hyperthermia in rats were used to investigate the antinociceptive and antipyretic activities, respectively. ResultsSiE significantly inhibited EPP-induced ear edema, carrageenan- and AA-induced hind paw edema. Its inhibitory effect in carrageenan-induced hind paw edema seemed to be in a dose-dependent manner. In cotton pellet-induced granuloma formation, SiE showed suppressive effects on granuloma formation but not on body weight gain and dry thymus weight. It could normalize serum alkaline phosphatase activity to nearly normal level. SiE also possessed a significant inhibitory effect, which seemed to be dose-dependent, on acetic acid-induced writhing response, whereas only at the highest dose of SiE could significantly increase test reaction time at all time-points in tail-flick test. However, no antipyretic activity was observed. ConclusionsThese results suggest that SiE possesses anti-inflammatory and antinociceptive, but not antipyretic, activities. This study therefore rationalizes the traditional use of SiE for the treatment of inflammation and pain.

Improvement of Lidocaine Local Anesthetic Action Using Lallemantia royleana Seed Mucilage as an Excipient.

Lallemantia royleana (Balangu) is a well known Iranian medicinal plant that its seed mucilage has many applications in modern pharmacology. Plant mucilage traditionally was used as a gel supplement, and natural matrix for sustained release of drugs. But it seems that these compounds are not a simple additive and also have many undiscovered pharmacological properties. In this research, the anesthetic action of gel prepared from Balangu mucilage alone and its mixture with lidocaine hydrochloride are compared with the effect of commercial 2% lidocaine gel by rat tail flick test. Mucilage of Balangu seed alone showed analgesic effect. Duration and potency of anesthesia induced by gel containing mucilage alone (0.01 g/mL) were identical to commercial 2% lidocaine gel. But, local anesthetic potency and duration of gel made from 2% lidocaine-mucilage gel mixture was significantly higher than commercial 2% lidocaine gel. The gel prepared from mucilage causes a good analgesia with unknown mechanism. Besides, mixture of Balangu mucilage prepared gel with lidocaine improves lidocaine anesthesia. The increase in potency of lidocaine action results from mucilage dermal penetration enhancing effects; and longer anesthetic duration of this mixture are related to the capability of mucilage based gel for sustained drug release.

Cebranopadol: A Novel Potent Analgesic Nociceptin/Orphanin FQ Peptide and Opioid Receptor Agonist

Cebranopadol (trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine) is a novel analgesic nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonist [Ki (nM)/EC50 (nM)/relative efficacy (%): human NOP receptor 0.9/13.0/89; human mu-opioid peptide (MOP) receptor 0.7/1.2/104; human kappa-opioid peptide receptor 2.6/17/67; human delta-opioid peptide receptor 18/110/105]. Cebranopadol exhibits highly potent and efficacious antinociceptive and antihypersensitive effects in several rat models of acute and chronic pain (tail-flick, rheumatoid arthritis, bone cancer, spinal nerve ligation, diabetic neuropathy) with ED50 values of 0.5-5.6 µg/kg after intravenous and 25.1 µg/kg after oral administration. In comparison with selective MOP receptor agonists, cebranopadol was more potent in models of chronic neuropathic than acute nociceptive pain. Cebranopadol's duration of action is long (up to 7 hours after intravenous 12 µg/kg; >9 hours after oral 55 µg/kg in the rat tail-flick test). The antihypersensitive activity of cebranopadol in the spinal nerve ligation model was partially reversed by pretreatment with the selective NOP receptor antagonist J-113397[1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] or the opioid receptor antagonist naloxone, indicating that both NOP and opioid receptor agonism are involved in this activity. Development of analgesic tolerance in the chronic constriction injury model was clearly delayed compared with that from an equianalgesic dose of morphine (complete tolerance on day 26 versus day 11, respectively). Unlike morphine, cebranopadol did not disrupt motor coordination and respiration at doses within and exceeding the analgesic dose range. Cebranopadol, by its combination of agonism at NOP and opioid receptors, affords highly potent and efficacious analgesia in various pain models with a favorable side effect profile.

Evaluation of analgesic activity of Terminalia arjuna (Roxb.) Wight and Arn bark: A tribal claim.

Background:Plants occupy an important place in folk medicine all over the world for centuries and indigenous communities have developed their own specific knowledge on plant resources, uses, management, and conservation. Research interest and activities in the area of ethno medicine have increased tremendously in the last decade. Currently, scientists are evincing keen interest in the scientific evaluation of ethno medical claims. Bark powder of Arjuna (Terminalia arjuna [Roxb.] Wight and Arn) is used by tribals for the management of some painful conditions.Aim:To evaluate analgesic activity of T. arjuna bark in rodents.Materials and Methods:For evaluation of analgesic activity, different experimental models, that is, the acetic acid-induced writhing syndrome in mice, formaldehyde-induced paw licking response and tail flick test in rats were designed. Experiments were carried out at two-dose levels, that is, therapeutically equivalent dose (TED) and TED × 2. Animals were divided into three groups (six animals in each group), first group serving as a control group, second and third group labeled as test drug group.Results:Test drug at both the doses significantly decreased the writhing syndrome in comparison to control the group. In comparison to control the group, incidences of formalin-induced paw licking were reduced in test drug groups in both early and late phases of pain. In tail flick response, threshold was significantly increased in both test drug groups at every time intervals.Conclusion:Study showed that stem bark of T. arjuna possesses analgesic activity in all experimental models.

Soluble guanylyl cyclase activators increase the expression of tolerance to morphine analgesic effect.

It is aimed to investigate the effects of guanylyl cyclase activation and inhibition on acute morphine antinociception and the development of tolerance to its effect. Nitric oxide-soluble guanylyl cyclase signal transduction cascade suggested to play an important role in the development of tolerance to antinociceptive effects of morphine. Nociception was evaluated by tail flick and hot plate tests in male Wistar rats. The analgesic effects of intraperitoneal protoporphyrin IX (PPIX; an activator of soluble guanylyl cyclase), 3-morpholinosydnonimine hydrochloride (SIN-1; NO donor and activator of guanylyl cyclase), S-Nitroso-N-acetylpenicillamine (SNAP; an activator of guanylyl cyclase), 3,3-Bis (amino ethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18; NO donor activating guanylyl cyclase) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of guanylyl cyclase) alone or in combination with subcutaneous morphine injection were evaluated. Their effects on morphine tolerance development were evaluated by giving these agents 20 minutes prior to twice daily morphine injection during tolerance development for 5 days. On day 6, the expression of morphine tolerance was determined. PPIX, SIN-1, SNAP and NOC-18 significantly increased expression of morphine tolerance while ODQ decreased. These data suggested that sGC activators have a significant role in tolerance to the analgesic effect of morphine (Tab. 1, Fig. 4, Ref. 29).

Role of fumaric acid in anti-inflammatory and analgesic activities of a Fumaria indica extracts.

Aim:The aim was to test whether the ethanolic extract of Fumaria indica (FI) possesses anti-inflammatory and analgesic activities, and fumaric acid (FA) could be one of its bioactive constituent involved in such activities of the extract.Materials and Methods:For anti-inflammatory activity, carrageenan-induced edema and cotton pellet induced granuloma tests in rats and for analgesic activity rat tail flick test and hot plate and acetic acid writhing tests in mice were used. All tests were performed after seven daily oral doses of the FI extract (100, 200, and 400 mg/kg/day) and pure FA (1.25, 2.50, and 5.00 mg/kg/day).Results:Anti-inflammatory activities of FI and FA were observed in carrageenan-induced edema and cotton pallet granuloma even after their lowest tested doses. No analgesic activity of lowest tested dose of FA was observed in the acetic acid writhing test, but likewise, all tested dose levels of FI, higher tested dose levels of FA were also possess significant analgesic activity in this test. Further, significant analgesic activities of both FI and FA in hot plate and tale flick tests were observed after all their tested doses.Conclusions:These observations are in agreement with our working hypothesis on the connection of FA in mode(s) of action(s) of FI, and reaffirm the conviction that FI could be an herbal alternative against fibromyalgia and other pathologies often associate with, or caused by, inflammatory processes.

Peripherally Acting Mediators of Pain and Analgesia Potentiate the Central Analgesic Effects of Fentanyl and Analgin (Dipyrone) in Rats

Intramuscular administration of the central analgesics fentanyl and Analgin (dipyrone) along with the pain mediators cholecystokinin (CCK), glutamate, ATP, and adrenaline, and the analgesia mediator adenosine, which have weak penetration into the CNS, induced maximal analgesic effects at minimally effective doses in the tailflick test in rats. The minimal effective doses of Analgin and fentanyl decreased by factors of 50‐220 in response to combined i.m. administration of each of the analgesics with CCK, glutamate, ATP, adrenaline, and adenosine at subthreshold does which were ineffective when given alone. Intragastric administration of lidocaine and subdiaphragmatic gastric vagotomy completely blocked the analgesic effects of these combinations. These data lead to the conclusion that peripherally acting pain and analgesia mediators given systemically potentiate the central analgesic actions of Analgin and fentanyl via stimulation of the chemoreceptors of vagal afferents in the gastric mucosa.

Spinal mediation of descending pain inhibitory mechanisms activated by 2/100-Hz electroacupuncture in the rat tail-flick test

Abstract Electroacupuncture (EA) has been widely used for the management of chronic pain, but the mechanism of EA-induced analgesia is not yet fully understood. The present study evaluated the effectiveness of intrathecal antagonists of serotonergic (methysergide), α1- (WB4101) and α2- (idazoxan) adrenoceptors, opioid (naloxone), muscarinic (atropine), GABAA (bicuculline) and GABAB (phaclofen) receptors in blocking 2/100-Hz electroacupuncture-induced analgesia (EAIA) in the rat tail-flick test. Rats were taken for determination of baseline tail-flick latency (TFL). Vehicle or drug was then injected intrathecally in a volum of 10 μl and EA was applied bilaterally to the Zusanli and Sanyinjiao acupoints under light isoflurane anesthesia. TFL was measured within 30 s after the end of stimulation and at 10-min intervals for up to 60 min. Twenty minutes of 2/100 Hz EA significantly increased TFL. The EAIA was completely inhibited by intrathecal methysergide (30 μg), significantly reduced in intensity and duration by intrathecal idazoxan (50 μg), naloxone (20 μg) or phaclofen (20 μg), and reduced in duration by intrathecal WB4101 (10 μg), atropine (20 μg) or bicuculline (3 μg). The intensity of the 2/100 Hz-EAIA depends on serotonergic and α2-noradrenergic descending mechanisms, and involves spinal opioid and GABAB modulation. The duration of 2/100 Hz-EAIA depends on both α1- and α2-noradrenergic descending mechanisms, and involves spinal opioid, muscarinic cholinergic, and GABAA and GABAB modulation.

Analgesic Effect of Methanol Leaf Extract of &lt;i&gt;Alstonia Boonei&lt;/i&gt; De Wild (Apocynaceae)

Purpose: To evaluate the possible analgesic properties of the methanol leaf extract of Alstonia boonei (De Wild, Apocynaceae) a locally available plant used in traditional medicine for the management of pain and other conditions.Methods: Alstonia boonei leaves were extracted with methanol. Rodent models were employed in screening the analgesic effect of the extract. Pain indices evaluated in hot plate and tail flick tests, formalin pain test and mouse writhing assay were mean reaction time to latent heat, time spent in licking of injected paw and abdominal writhes, respectively.Results: Oral administration of the extract caused a significant (p &lt; 0.05) dose-dependent reduction in the number of abdominal writhes (control, 84.67 ± 9.58; 100 mg/kg, 57.86 ± 3.07; 200 mg/kg, 24.40 ±3.92; and 400 mg/kg, 22.50 ± 2.53). The extract also produced significant (p &lt; 0.05) but non-dose dependent increase in elevation of pain threshold in the hot plate (ranging from 27.99 to 42.26 % inhibition) and tail flick tests (ranging from 34.73 to 81.42 % inhibition) in mice and rats, respectively. All doses of the extract used produced significant (p &lt; 0.05) inhibition of both phases of the formalininduced pain in mice, with a more pronounced anti-inflammatory effect on the late (ranging from 46.96 to74.12 %) phase than the early anti-neurogenic (ranging from 49.52 to 51.47 %) phase.Conclusion: The results obtained suggest that the analgesic effect of Alstonia boonei may be mediated via both central and peripheral mechanisms.Keywords: Alstonia boonei, Analgesia, Pain, Methanol extract.

Electroacupuncture at 2/100 Hz Activates Antinociceptive Spinal Mechanisms Different from Those Activated by Electroacupuncture at 2 and 100 Hz in Responder Rats

We examined the effects of intrathecal injection of desipramine and fluoxetine (selective inhibitors of norepinephrine and 5-HT uptake, resp.), thiorphan and neostigmine (inhibitors of enkephalinase and acetylcholinesterase, resp.), gabapentin (a GABA releaser), and vigabatrin (an inhibitor of GABA-transaminase) on the antinociception induced by 2 Hz, 100 Hz, or 2/100 Hz electroacupuncture (EA) applied bilaterally to the Zusanli (ST36) and Sanyinjiao (SP6) acupoints using the rat tail-flick test. We show that 2 Hz EA antinociception lasts longer after the administration of drugs that increase the spinal availability of norepinephrine, acetylcholine, or GABA; 100 Hz EA antinociception lasts longer after drug that increases the spinal availability of norepinephrine; 2/100 Hz EA antinociception lasts longer after drugs that increase the spinal availability of endogenous opioids or GABA. We conclude that the antinociceptive effect of 2/100 Hz EA is different from the synergistic effect of alternate stimulation at 2 and 100 Hz because the effect of the former is not changed by increasing the spinal availability of serotonin and lasts longer after the administration of vigabatrin. The combination of EA with drugs that increase the availability of spinal neurotransmitters involved in the modulation of nociceptive inputs may result in a synergistic antinociceptive effect in the rat tail-flick test.

Establishment of an Animal Model of Epidural Anesthesia and Sedative Tail-Flick Test for Evaluating Local Anesthetic Effects in Rats

The tail-flick (TF) test is the most frequently used method to measure pain levels and assess the effects of anesthesia. In this study, we performed the TF test in rats sedated via an indwelling epidural catheter and then examined the effectiveness of this method in evaluating the local anesthetic effects. First, an epidural catheter was inserted into the epidural space, and anesthetic [lidocaine (L) or lidocaine including adrenaline (AL)] or normal saline (NS) was administered. Under sedation, we measured the dose for disappearance of the TF response, time to TF response recovery, onset and regression of local anesthesia, as well as the effect of an added agent on its continuation. The time course of TF latency (% maximum possible effect) in the NS group did not change during the experiment. In the AL group, TF latency increased significantly more than baseline during the 30-min period after injection. This was also significantly higher than the latency in the NS group and the L group. In the L group, the TF latency increased significantly above baseline for 20 min after injection and was significantly higher than that in the NS group. Due to the fact that we were able to detect the effect of local anesthesia onset and regression, as well as the local anesthesia continuation action of an additive agent, in rats sedated via an indwelled epidural catheter, we consider our method to be an improvement over conventional methods.

Ligand-Specific Regulation of the Endogenous Mu-Opioid Receptor by Chronic Treatment with Mu-Opioid Peptide Agonists

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid2-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Also, the density of mu-receptors was upregulated by about 40% in the light membrane fraction, with no detectable changes in surface binding. Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic full mu-agonist peptide, DAMGO, and its chloromethyl ketone derivative, DAMCK. These results are discussed in light of the recently discovered phenomenon, that is, the “so-called biased agonism” or “functional selectivity”.

A Novel µ-Opioid Receptor Ligand with High In Vitro and In Vivo Agonist Efficacy

The aims of this study were to synthesize 14-O-Methylmorphine-6-O-sulfate (14-O-MeM6SU) and examine its opioid properties (potency, affinity, efficacy) in receptor ligand binding and isolated tissues (mouse vas deferens, MVD and rat vas deferens, RVD bioassays). The results were then compared to the parent compounds morphine-6-O-sulfate (M6SU) and morphine, as well as the �- opioid receptor (MOR) selective agonist peptide [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). An additional objective was to compare the effect of subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) administered 14-O-MeM6SU, M6SU and morphine in thermal nociception, rat tail-flick (RTF) test. In MVD, the EC50 (nM) value was 4.38 for 14-O-MeM6SU, 102.81 for M6SU, 346.63 for morphine and 238.47 for DAMGO. The effect of 14-O-MeM6SU and DAMGO was antagonized by naloxone (NAL) with Ke value 1-2.00 nM. The Emax values (%) were 99.10, 36.87, 42.51 and 96.99 for 14-O-MeM6SU, M6SU, morphine and DAMGO, respectively. In RVD 14-O-MeM6SU and DAMGO but not M6SU or morphine showed agonist activity. In binding experiments the affinity of 14-OMeM6SU, M6SU, morphine and DAMGO for MOR was 1.12, 11.48, 4.37 and 3.24 nM, respectively. The selectivity of 14-O-MeM6SU was κ/μ= 269 and δ/μ= 9. In G-protein activation experiments, 14-O-MeM6SU and DAMGO showed higher Emax values than M6SU or morphine. S.c. or i.c.v-injected 14-O-MeM6SU, M6SU and morphine produced a dose and time-dependent increase in RTF response latency. 14-O-MeM6SU was the most potent. Our results showed that introduction of 14-O-Me in M6SU increased the binding affinity, agonist potency, and most importantly, the intrinsic efficacy (Emax).

Development of prilocaine gels for enhanced local anesthetic action

Prilocaine, one of local anesthetics, has been used for regional pain relief. When applied as an ointments or creams, it is hard to expect their effects to last for long time, because they are easily removed by wetting, movement and contacting. For more comfortable and better application, we developed a prilocaine gel system using a bioadhesive polymer, carboxymethyl cellulose (CMC). For suitable bioadhesion, the bioadhesive force of various polymers was tested using an auto-peeling tester. The bioadhesive force of various types of CMC such as 100MC, 150MC and 300MC, was 0.0264, 0.0461 and 0.0824 N, at 1.5% concentration, respectively. The CMC-300MC gels showed the most suitable bioadhesive forces. The effect of drug concentration on drug release was studied from the prepared 1.5% CMC gels using a synthetic cellulose membrane at 37 ± 0.5°C. As the concentration of drug increased, the drug release increased. The effects of temperature on drug release from the 1.0% prilocaine gels were evaluated at 27, 32, 37 and 42°C. As the temperature of the drug gels increased, drug release increased. The enhancing effects of penetration enhancers such as pyrrolidones, non-ionic surfactants, fatty acids and propylene glycol derivatives were studied. Among the enhancers used, polyoxyethylene 2-oleyl ether was superior. The anesthetic effects were studied by a tail flick analgesic meter. In the rat tailflick test, 1.0% prilocaine gels containing polyoxyethylene 2-oleyl ether showed the most prolonged local analgesic effects. The results support the view that prilocaine gels with enhanced local anesthetic action could be developed using CMC bioadhesive polymer.