Mohab M. Ibrahim

Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS.

We designed AM1241, a selective CB2 cannabinoid receptor agonist, and used it to test the hypothesis that CB2 receptor activation would reverse the sensory hypersensitivity observed in neuropathic pain states. AM1241 exhibits high affinity and selectivity for CB2 receptors. It also exhibits high potency in vivo. AM1241 dose-dependently reversed tactile and thermal hypersensitivity produced by ligation of the L5 and L6 spinal nerves in rats. These effects were selectively antagonized by a CB2 but not by a CB1 receptor antagonist, suggesting that they were produced by actions of AM1241 at CB2 receptors. AM1241 was also active in blocking spinal nerve ligation-induced tactile and thermal hypersensitivity in mice lacking CB1 receptors (CB1-/- mice), confirming that AM1241 reverses sensory hypersensitivity independent of actions at CB1 receptors. These findings demonstrate a mechanism leading to the inhibition of pain, one that targets receptors localized exclusively outside the CNS. Further, they suggest the potential use of CB2 receptor-selective agonists for treatment of human neuropathic pain, a condition currently without consistently effective therapies. CB2 receptor-selective agonist medications are predicted to be without the CNS side effects that limit the effectiveness of currently available medications.

CB2 cannabinoid receptor-mediated peripheral antinociception

&NA; Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence implicates the CB1 receptor in the production of antinociception. However, the capacity of CB2 receptors, which are located outside the central nervous system (CNS), to produce antinociception is not known. Using AM1241, a CB2 receptor‐selective agonist, we demonstrate that CB2 receptors produce antinociception to thermal stimuli. Injection of AM1241 in the hindpaw produced antinociception to a stimulus applied to the same paw. Injection of an equivalent dose of AM1241 into the paw contralateral to the side of testing did not. The antinociceptive actions of AM1241 were blocked by the CB2 receptor‐selective antagonist AM630, but not by the CB1 receptor‐selective antagonist AM251. AM1241 also produced antinociception when injected systemically (intraperitoneally). The antinociceptive actions of systemic AM1241 were blocked by injection of AM630 into the paw where the thermal stimulus was applied, but not the contralateral paw. These findings demonstrate the local, peripheral nature of CB2 cannabinoid antinociception. AM1241 did not produce the CNS cannabinoid effects of hypothermia, catalepsy, inhibition of activity or impaired ambulation, while this tetrad of effects was produced by the mixed CB1/CB2 receptor agonist WIN55,212‐2. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB2 receptors. CB2 receptor agonists may have promise clinically for the treatment of pain without CNS cannabinoid side effects.

Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors.

Background Cannabinoid receptor agonists inhibit inflammatory hyperalgesia in animal models. Nonselective cannabinoid receptor agonists also produce central nervous system (CNS) side effects. Agonists selective for CB2 cannabinoid receptors, which are not found in the CNS, do not produce the CNS effects typical of nonselective cannabinoid receptor agonists but do inhibit acute nociception. The authors used the CB2 receptor–selective agonist AM1241 to test the hypothesis that selective activation of peripheral CB2 receptors inhibits inflammatory hyperalgesia. Methods Rats were injected in the hind paw with carrageenan or capsaicin. Paw withdrawal latencies were measured using a focused thermal stimulus. The effects of peripheral CB2 receptor activation were determined by using local injection of AM1241. CB2 receptor mediation of the actions of AM1241 was shown by using the CB2 receptor–selective antagonist AM630 and the CB1 receptor–selective antagonist AM251. Results AM1241 fully reversed carrageenan-induced inflammatory thermal hyperalgesia when injected into the inflamed paw. In contrast, AM1241 injected into the contralateral paw had no effect, showing that its effects were local. AM1241 also reversed the local edema produced by hind paw carrageenan injection. The effects of AM1241 were reversed by the CB2 receptor–selective antagonist AM630, but not by the CB1 receptor–selective antagonist AM251. AM1241 also inhibited flinching and thermal hyperalgesia produced by hind paw capsaicin injection. Conclusions Local, peripheral CB2 receptor activation inhibits inflammation and inflammatory hyperalgesia. These results suggest that peripheral CB2 receptors may be an appropriate target for eliciting relief of inflammatory pain without the CNS effects of nonselective cannabinoid receptor agonists.

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats

&NA; Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA‐mediated central sensitization by the actions of glutamate and by the non‐opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L5/L6) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L5 and L6 spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S2 spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S2 spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S2 ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A(1–17) and of MK‐801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S2 ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.

CB2 cannabinoid receptor agonists: pain relief without psychoactive effects?

Cannabinoid receptor agonists significantly diminish pain responses in animal models; however, they exhibit only modest analgesic effects in humans. The relative lack of efficacy in man may be because of the dose limitations imposed by psychoactive side effects. Cannabinoid agonists that selectively target CB(2) (peripheral) cannabinoid receptors should be free of psychoactive effects, perhaps allowing for more effective dosing. CB(2) receptor activation inhibits acute, inflammatory and neuropathic pain responses in animal models. In preclinical studies, CB(2) receptor agonists do not produce central nervous system effects. Therefore, they show promise for the treatment of acute and chronic pain without psychoactive effects.

CB2 cannabinoid receptor mediation of antinociception

Abstract Management of acute pain remains a significant clinical problem. In preclinical studies, CB2 cannabinoid receptor‐selective agonists inhibit nociception without producing central nervous system side effects. The CB2 receptor‐selective agonist AM1241 produces antinociceptive effects that are antagonized by CB2, but not CB1, receptor‐selective antagonists, suggesting that activation of CB2 receptors results in antinociception. However, it has not been possible to definitively demonstrate that these effects are mediated by CB2 receptors, because we have lacked the pharmacological tools to confirm the in vivo receptor selectivity of the antagonists used. Further, recent evidence for cannabinoid‐like receptors beyond CB1 and CB2 raises the possibility that AM1241 exerts its antinociceptive effects at uncharacterized CB2‐like receptors that are also inhibited by AM630. The experiments reported here further test the hypothesis that CB2 receptor activation inhibits nociception. They evaluated the antinociceptive actions of AM1241 and the less‐selective CB2 receptor agonist WIN55,212‐2 in wild‐type Symbol mice and in mice with genetic disruption of the CB2 receptor (Symbol mice). AM1241 inhibited thermal nociception in Symbol mice, but had no effect in Symbol littermates. WIN55,212‐2 produced equivalent antinociception in Symbol and Symbol mice, while its antinociceptive effects were reduced in Symbol compared to Symbol mice. The effects of morphine were not altered in Symbol compared to Symbol mice. These data strongly suggest that AM1241 produces antinociception in vivo by activating CB2 cannabinoid receptors. Further, they confirm the potential therapeutic relevance of CB2 cannabinoid receptors for the treatment of acute pain. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available.

Regular exercise reverses sensory hypersensitivity in a rat neuropathic pain model: Role of endogenous opioids

Background:Exercise is often prescribed as a therapy for chronic pain. Short-term exercise briefly increases the production of endogenous analgesics, leading to transient antinociception. In limited studies, exercise produced sustained increases in endogenous opioids, sustained analgesia, or diminished measures of chronic pain. This study tests the hypothesis that regular aerobic exercise leads to sustained reversal of neuropathic pain by activating endogenous opioid-mediated pain modulatory systems. Methods:After baseline measurements, the L5 and L6 spinal nerves of male Sprague–Dawley rats were tightly ligated. Animals were randomized to sedentary or 5-week treadmill exercise–trained groups. Thermal and tactile sensitivities were assessed 23 h after exercise, using paw withdrawal thresholds to von Frey filaments and withdrawal latencies to noxious heat. Opioid receptor antagonists were administered by subcutaneous, intrathecal, or intracerebroventricular injection. Opioid peptides were quantified using immunohistochemistry with densitometry. Results:Exercise training ameliorated thermal and tactile hypersensitivity in spinal nerve–ligated animals within 3 weeks. Sensory hypersensitivity returned 5 days after discontinuation of exercise training. The effects of exercise were reversed by using systemically or intracerebroventricularly administered opioid receptor antagonists and prevented by continuous infusion of naltrexone. Exercise increased &bgr;-endorphin and met-enkephalin content in the rostral ventromedial medulla and the mid-brain periaqueductal gray area. Conclusions:Regular moderate aerobic exercise reversed signs of neuropathic pain and increased endogenous opioid content in brainstem regions important in pain modulation. Exercise effects were reversed by opioid receptor antagonists. These results suggest that exercise-induced reversal of neuropathic pain results from an up-regulation of endogenous opioids.

Inhibition of pain responses by activation of CB2 cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.

Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antiserum

The co-administration of morphine at spinal (i.th.) and supraspinal (i.c.v.) sites to the same rat produces antinociceptive synergy, a phenomenon which may underlie the clinical analgesic utility of this drug. In animals with peripheral nerve injury, however, the antinociceptive potency and efficacy of i.th. morphine is significantly decreased. Here, the possible loss of spinal/supraspinal morphine antinociceptive synergy and relationship to elevation of spinal dynorphin content was studied. Ligation of lumbar spinal nerves resulted in elevated dynorphin in the ipsilateral lumbar and sacral spinal cord. In sham-operated rats supraspinal/spinal co-administration of morphine produced synergistic antinociception which was unaffected by i.th. MK-801 or dynorphin A((1-17)) antiserum. In nerve-injured rats, i.th. morphine was inactive against tactile allodynia and showed diminished in potency against acute nociception without supraspinal/spinal antinociceptive synergy. Antiserum to dynorphin A((1-17)) or the non-competitive NMDA antagonist MK-801 increased the antinociceptive potency of i.th. morphine, restored supraspinal/spinal morphine antinociceptive synergy and elicited a dose-related i.th. morphine antiallodynic action. These agents did not demonstrate antinociceptive or antiallodynic activity alone and did not alter morphine actions in sham-operated animals. The loss of spinal/supraspinal antinociceptive synergy and lack of antiallodynic activity of spinal morphine appear to be due to the elevation across multiple spinal segments of dynorphin following nerve injury. Pathological actions of elevated dynorphin may directly or indirectly modulate the NMDA receptor, result in a loss of supraspinal/spinal morphine synergy and may thus account for the decreased clinical analgesic efficacy of morphine in peripheral neuropathies.

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti.

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 µg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 µg. After a blood meal on day five, sugar-fed females accumulated 120-140 µg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 µg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 µg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 µg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.