Ahmet Dogrul

Topical cannabinoid enhances topical morphine antinociception

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail‐flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212‐2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212‐2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212‐2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212‐2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212‐2 involve CB1 receptors. Combination of topical WIN 55, 212‐2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212‐2 indicates that WIN 55, 212‐2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212‐2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain.

Spinal 5-HT7 receptors play an important role in the antinociceptive and antihyperalgesic effects of tramadol and its metabolite, O-Desmethyltramadol, via activation of descending serotonergic pathways.

Background:Tramadol is an analgesic drug, and its mechanism of action is believed to be mediated by the &mgr;-opioid receptor. A further action of tramadol has been identified as blocking the reuptake of serotonin (5-HT). One of the most recently identified subtypes of 5-HT receptor is the 5-HT7 receptor. Thus, the authors aimed to examine the potential role of serotonergic descending bulbospinal pathways and spinal 5-HT7 receptors compared with that of the 5-HT2A and 5-HT3 receptors in the antinociceptive and antihyperalgesic effects of tramadol and its major active metabolite O-desmethyltramadol (M1) on phasic and postoperative pain models. Methods:Nociception was assessed by the radiant heat tail-flick and plantar incision test in male Balb-C mice (25-30 g). The serotonergic pathways were lesioned with an intrathecal injection of 5,7-dihydroxytryptamine. The selective 5-HT7, 5-HT2, and 5-HT3 antagonists; SB-269970 and SB-258719; ketanserin and ondansetron were given intrathecally. Results:Systemically administered tramadol and M1 produced antinociceptive and antihyperalgesic effects. The antinociceptive effects of both tramadol and M1 were significantly diminished in 5-HT-lesioned mice. Intrathecal injection of SB-269970 (10 &mgr;g) and SB-258719 (20 &mgr;g) blocked both tramadol- and M1-induced antinociceptive and antihyperalgesic effects. Ketanserin (20 &mgr;g) and ondansetron (20 &mgr;g) were unable to reverse the antinociceptive and antihyperalgesic effects of tramadol and M1. Conclusions:These findings suggest that the descending serotonergic pathways and spinal 5-HT7 receptors play a crucial role in the antinociceptive and antihyperalgesic effects of tramadol and M1.

Topical cannabinoid antinociception: synergy with spinal sites

Analgesic effects of cannabimimetic compounds have been known to be related to their central effects. Cannabinoid receptors also exist in the periphery but their role in pain perception has been remained to be clarified. Therefore, we assessed topical antinociceptive effects of WIN 55, 212‐2, a mixed CB1 and CB2 receptors agonist, in mice using tail‐flick test. Immersion of the tail of mouse into the WIN 55, 212‐2 solution produced dose‐dependent antinociception. This antinociceptive activity was limited to the portion of the tail exposed to WIN 55, 212‐2. The antinociceptive response was dependent on duration of exposure to WIN 55, 212‐2 solution. The topical antinociceptive effects of WIN 55, 212‐2 were dose dependently blocked by topical pretreatment of CB1 receptor‐selective antagonist, AM 251. Thus, topical antinociceptive action of WIN 55, 212‐2 involve CB1 receptors. Intrathecal (i.th.) administration of WIN 55, 212‐2 produced a dose‐dependent antinociceptive effect. Interestingly, ineffective i.th. doses of WIN 55, 212‐2 produced a marked antinociception when combined with topical application of WIN 55, 212‐2 and topical antinociceptive effect was potentiated. The dose–response curve of i.th. WIN 55, 212‐2 was shifted to the left 15‐fold by topical WIN 55, 212‐2. This finding suggests that there is an antinociceptive synergy between peripheral and spinal sites of cannabinoid action and it also implicates that local activaton of cannabinoid system may regulate pain initiation in cutaneous tissue. Our findings support that cannabinoid system participates in buffering the emerging pain signals at the peripheral sites in addition to their spinal and supraspinal sites of action. In addition, an antinociceptive synergy between topical and spinal cannabinoid actions exists. These results also indicate that topically administered cannabinoid agonists may reduce pain without the dysphoric side effects and abuse potential of centrally acting cannabimimetic drugs.

The interaction between IL-1β and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice

&NA; It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin‐1&bgr; (IL‐1&bgr;), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL‐1&bgr;, intracerebroventricularly (i.c.v.), on morphine‐induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail‐flick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose‐dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL‐1&bgr; at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL‐1&bgr; at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL‐1&bgr; at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL‐1&bgr;, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL‐1&bgr; at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL‐1&bgr;.

Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and antinociceptive tolerance.

&NA; Recent studies indicate that sustained opioid administration produces increased expression of spinal dynorphin, which promotes enhanced sensitivity to non‐noxious and noxious stimuli. Such increased ‘pain’ may manifest behaviorally as a decrease in spinal antinociceptive potency. Here, the possibility of similar mechanisms in the antinociception of spinal cannabinoids was explored. Response thresholds to non‐noxious mechanical and noxious thermal stimuli were assessed. Antinociception was determined using the 52°C tail‐flick test. Mice received repeated WIN 55,212‐2, its inactive enantiomer, WIN 55,212‐3 or vehicle (i.th., bid, 5 days). WIN 55,212‐2, but not WIN 55,212‐3 or vehicle, produced a time‐related increased sensitivity to non‐noxious and noxious stimuli. WIN 55,212‐2, but not WIN 55,212‐3 or vehicle, elicited a significant increase in lumbar spinal dynorphin content at treatment day 5. Increased sensitivity to mechanical and thermal stimuli produced by WIN 55,212‐2 was reversed to baseline levels by i.th. MK‐801 or dynorphin antiserum; control serum had no effect. WIN 55,212‐2, but not WIN 55,212‐3 or vehicle, produced dose‐related antinociception and repeated administration resulted in antinociceptive tolerance. While MK‐801 and dynorphin antiserum did not alter acute antinociception produced by WIN 55,212‐2, these substances significantly blocked antinociceptive tolerance when given immediately prior to WIN 55,212‐2 challenge on day 5. Daily MK‐801 pretreatments, prior to WIN 55,212‐2 injection, also produced a significant block of antinociceptive tolerance. These data suggest that like opioids, repeated spinal administration of a cannabinoid CB1 agonist elicits abnormal pain, which results in increased expression of spinal dynorphin. Manipulations that block cannabinoid‐induced pain also block the behavioral manifestation of cannabinoid tolerance.

'Knock-down' of spinal CB1 receptors produces abnormal pain and elevates spinal dynorphin content in mice.

&NA; Recent studies demonstrate the possible existence of tonic modulatory control of nociceptive input mediated by spinal cannabinoid receptors (CB1). Accordingly, it is predicted that a reduction in the spinal CB1 receptors may enhance sensitivity to sensory stimuli and a decrease in spinal antinociceptive potency to cannabinoid agonists. An antisense oligodeoxynucleotide (ODN) specific to the CB1 receptor was used to ‘knock‐down’ CB1 receptors in the lumbar spinal cord and dorsal root ganglia by the local, repeated intrathecal (i.th.) administration of the ODN. This treatment resulted in a decrease in lumbar spinal CB1 receptor expression accompanied by a decrease in the response thresholds to both innocuous tactile and noxious thermal stimuli. The antinociceptive action of the CB1 agonist, WIN 55,212‐2, by i.th. administration was also significantly attenuated after treatment with the antisense ODN. Similar treatment using a mismatch control ODN had no effect on receptor protein or on sensory thresholds. The effects of the antisense ODN treatment on sensory thresholds were fully reversed after discontinuation of the ODN injection. The antisense ODN treated rats also showed a significant increase in lumbar spinal dynorphin A. Acute i.th. injection of MK‐801 or an antidynorphin antiserum blocked the antisense ODN‐induced tactile and thermal hypersensitivity. These data support the possibility of endogenous inhibitory cannabinoid tone to limit spinal afferent input of thermal and tactile stimuli. Lifting of this inhibitory tone through a ‘knock‐down’ of spinal CB1 receptors apparently lowers the thresholds for sensory input, as reflected by the actions of MK‐801 to block tactile and thermal hypersensitivity. The increased spinal dynorphin may act to further promote afferent outflow and abnormal pain because sequestration of spinal dynorphin with antiserum also reverses the manifestations of abnormal pain following knock‐down of CB1 receptors.

Spinal L-type calcium channel blockade abolishes opioid-induced sensory hypersensitivity and antinociceptive tolerance.

Recent studies have suggested that prolonged exposure to morphine results in the development of paradoxical, abnormal enhanced pain. It has also been suggested that this enhanced pain state may be interpreted as antinociceptive tolerance. Although the precise mechanisms that drive opioid-induced abnormal pain are not well known, considerable evidence suggests that this state may be supported by enhanced, stimulus-evoked excitatory transmission. We hypothesized that blockade of L-type calcium channels, which are critical for excitatory neurotransmitter release, would alter the development of opioid-induced hyperalgesia and antinociceptive tolerance. Male, Swiss-Webster mice received twice-daily intrathecal injections of morphine (10 &mgr;g) alone or in combination with amlodipine (10 &mgr;g) for 8 days. Mice receiving repeated morphine injections developed enhanced responses to tactile and thermal stimuli. These hypersensitivities were prevented by the coadministration of the putative selective L-type calcium channel blocker amlodipine. Moreover, mice receiving morphine for 8 days demonstrated a significant rightward shift of the morphine antinociceptive dose-response curve, indicative of antinociceptive tolerance, whereas those that also received amlodipine along with morphine did not demonstrate tolerance. These results suggest that blockade of the L-type calcium channels with amlodipine prevented opioid-induced hyperalgesia and the expression of antinociceptive tolerance to spinal morphine, presumably by reducing stimulus-induced excitatory neurotransmitter release.

The contribution of alpha-1 and alpha-2 adrenoceptors in peripheral imidazoline and adrenoceptor agonist-induced nociception.

We evaluated the effects of activation of peripheral adrenoceptors (AR) and imidazoline receptors on nociception and the contribution of &agr;-1 and &agr;-2 AR receptors in agonist-induced nociception by using the tail-flick test in mice. Clonidine (&agr;-2 AR agonist), agmatine (imidazoline receptor and &agr;-2 AR agonist), noradrenaline (mixed &agr;-1 and &agr;-2 AR agonist), phenylephrine (&agr;-1 AR agonist), or 0.9% saline was given by intradermal injection (10 &mgr;L) into the tail. The intradermal injection of clonidine (1, 3, and 10 &mgr;g) and agmatine (3, 30, and 50 &mgr;g) produced dose-dependent antinociception, whereas noradrenaline (1, 10, and 30 &mgr;g) and phenylephrine (1, 10 and 30 &mgr;g) produced dose-dependent thermal hyperalgesia. Clonidine (10 &mgr;g) and agmatine (50 &mgr;g)-induced peripheral antinociception were antagonized by pretreatment with yohimbine (2.5 mg/kg IP), a selective &agr;-2 AR antagonist, but not by prazosin (1 mg/kg IP), a selective &agr;-1 AR antagonist. Noradrenaline (30 &mgr;g) and phenylephrine (30 &mgr;g)-induced thermal hyperalgesia were antagonized by prazosin (1 mg/kg IP) but not by yohimbine (2.5 mg/kg IP). Our results suggest that local thermal hyperalgesic effects of noradrenaline and phenylephrine are linked to &agr;-1 AR and the peripheral antinociceptive action of clonidine and agmatine are linked to &agr;-2 AR.

The local antinociceptive actions of nonsteroidal antiinflammatory drugs in the mouse radiant heat tail-flick test

BACKGROUND:While many preclinical models detect the analgesic activity of nonsteroidal antiinflammatory drugs (NSAIDs), the radiant heat tail-flick response has repeatedly been insensitive to this class of drugs. As the tail-flick test involves nociceptive processing at spinal circuits with supraspinal modulation, it seems reasonable to assume that the NSAIDs should not modify strong nociceptive stimuli, since the primary site of action of NSAIDs is likely to be in the periphery. METHODS:We injected 3–300 &mgr;g of diclofenac, dipyrone, ketorolac, lysine acetyl salicylate, and sodium salicylate intradermally into mice tails and evaluated the tail-flick response to radiant heat. These results were compared with intraperitoneally injected controls. We also evaluated the ability of naloxone to reverse the observed effects. RESULTS:Intradermal injection of each NSAID produced a dose-dependent increase in tail-flick latency. Intraperitoneal NSAIDs injection produced no antinociceptive effects. Naloxone pretreatment had no effect on the antinociceptive effects of intradermal diclofenac, ketorolac, lysine acetyl salicylate, and sodium salicylate. Naloxone completely blocked the antinociceptive effects of intradermal dipyrone. CONCLUSIONS:Local, but not systemic, administration of NSAIDs produced antinociception in the tail-flick thermal assay. The endogenous opioid system contributes to the peripheral antinociceptive effects of dipyrone, but not to that of diclofenac, ketorolac, lysine asetyl salicylate, or sodium salicylate, suggesting differences in the mechanisms of action among the NSAIDs.

The effect of FAAH, MAGL, and Dual FAAH/MAGL inhibition on inflammatory and colorectal distension-induced visceral pain models in Rodents

Recent studies showed that the pharmacological inhibition of endocannabinoid degrading enzymes such as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) elicit promising analgesic effects in a variety of nociceptive models without serious side effects. However, the full spectrum of activities is not observed upon inhibition of either FAAH or MAGL enzymes alone and thus dual FAAH and MAGL inhibitors have been described. Visceral pain is strongly associated with inflammation and distension of the gut. Thus, we explored the comparable effects of FAAH, MAGL, and dual FAAH/MAGL inhibitors on inflammatory and mechanically evoked visceral pain models.