Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Potent antinociceptive effects of TRK-820, a novel kappa-opioid receptor agonist

TRK-820, a new type of 4,5-epoxymorphinan derivative, was investigated in vivo for antinociceptive activities and its selectivity on various opioid receptors in mice. TRK-820 given s.c. or p.o. was found to be 351- and 796-fold more potent than U50,488H with acetic acid-induced abdominal constriction test. The duration of the antinociceptive effect produced by TRK-820 was longer than that produced by mu-opioid receptor agonist morphine or other kappa-opioid receptor agonists. In addition, with four other antinociceptive assays, low temperature hot plate (51 degrees C), thermal tail flick, mechanical tail pressure and tail pinch tests, TRK-820 was also found to be 68- to 328-fold more potent than U-50488H, and 41- to 349-fold more potent than morphine in producing antinociception, as comparing the weight of the different compound. However, TRK-820 was less active in inhibiting the high temperature (55 degrees C) hot plate response. The antinociceptive effects produced by TRK-820 were inhibited by nor-BNI, but not by naloxone or naltrindole (NTI) with the abdominal constriction test, indicating that the antinociception is selectively mediated by the stimulation of kappa-, but not mu- or delta-opioid receptors. Co-administration of TRK-820 with morphine slightly enhanced the antinociception induced by morphine in the mouse hot plate test. On the other hand, pentazocine significantly reduced the morphine-induced antinociception. TRK-820 produced sedation at doses, which are much higher than the doses for producing antinociception. These results indicate that the potent antinociception induced by TRK-820 is mediated via the stimulation of kappa-, but not mu- or delta-opiod receptors.     

Genetic deletion of microglial Panx1 attenuates morphine withdrawal,but not analgesic tolerance or hyperalgesia in mice

Opioids are among the most powerful analgesics for managing pain, yet their repeated use can lead to the development of severe adverse effects. In a recent study, we identified the microglial pannexin-1 channel (Panx1) as a critical substrate for opioid withdrawal. Here, we investigated whether microglial Panx1 contributes to opioid-induced hyperalgesia (OIH) and opioid analgesic tolerance using mice with a tamoxifen-inducible deletion of microglial Panx1. We determined that escalating doses of morphine resulted in thermal pain hypersensitivity in both Panx1-expressing and microglial Panx1-deficient mice. In microglial Panx1-deficient mice, we also found that acute morphine antinociception remained intact, and repeated morphine treatment at a constant dose resulted in a progressive decline in morphine antinociception and a reduction in morphine potency. This reduction in morphine antinociceptive potency was indistinguishable from that observed in Panx1-expressing mice. Notably, morphine tolerant animals displayed increased spinal microglial reactivity, but no change of microglial Panx1 expression. Collectively, our findings indicate microglial Panx1 differentially contributes to opioid withdrawal, but not the development of opioid-induced hyperalgesia or tolerance.     

Asymmetric dimethylarginine (ADMA)--a modulator of nociception in opiate tolerance and addiction?

Nitric oxide (NO) is generated from l-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The l-arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener N(G)-monomethyl-l-arginine (l-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulating neuronal activity. Humans generate approximately 300mumol ( approximately 60mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite. Increased NO production-either prior to or concurrently with opioid administration-results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the mu-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective mu-opioid agonist to activate the mu-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC(50) 0.71 microM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies. We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease mu-opiate receptor constitutive activity, resulting in alteration of the analgesic dose-response curve of morphine.     

3H][D-Ala2,NMePhe4,Gly-ol5]-enkephalin (mu-opioid) binding in beige-J mice

Tritiated [D-Ala2,NMePhe4,Gly-ol5]-enkephalin ([3H]DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on [3H]DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.     

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be 相似文献   

BiP,an endoplasmic reticulum chaperone,modulates the development of morphine antinociceptive tolerance

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock‐in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild‐type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild‐type mice. The activation of glycogen synthase kinase 3b (GSK‐3b) was associated with morphine tolerance, because an inhibitor of GSK‐3β prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK‐3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild‐type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.     

Antinociceptive activity of a novel buprenorphine analogue

HS-599 is a didehydroderivative of buprenorphine that displays high affinity and good selectivity for mu-opioid receptors. We studied its antinociceptive properties after s.c. injection in mice with the tail-flick and hot-plate tests. In the tail-flick test HS-599 (AD50 = 0.2801 micromol/kg s.c.) behaved as a full agonist and was twice as potent as buprenorphine (AD50=0.4569 micromol/kg s.c.) and 50 times more potent than morphine (AD50 = 13.3012 micromol/kg s.c.). Whereas the mu-opioid receptor antagonists naloxone (1-10 mg/kg s.c.) and naltrexone (5-15 mg/kg s.c.) antagonized HS-599 induced analgesia, the delta-opioid receptor antagonist naltrindole (20 mg/kg s.c.) and the kappa-opioid receptor antagonist nor-binaltorphimine (20 mg/kg s.c.) did not. With the hot-plate test at 50 degrees C, HS-599 (AD50 = 0.0359 micromol/kg s.c.) was a full agonist about 130 times more potent than morphine (AD50 = 4.8553 micromol/kg s.c.). With a high intensity nociceptive stimulus (55 degrees C) HS-599 (AD50 = 1.0382 micromol/kg s.c.) remained 7 times more potent than morphine (AD50 = 7.0210 micromol/kg s.c.) but never exceeded the 55% of the maximum possible effect, behaving as a partial agonist able to antagonize morphine antinociception in a dose-dependent manner. HS-599 promises to be a potent and safe new analgesic, preferentially acting at spinal level.     

The effects of pituitary adenylate cyclase-activating polypeptide on acute and chronic morphine actions in mice

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on pain sensitivity, on morphine analgesia, on morphine tolerance and withdrawal were investigated in mice. The heat-radiant tail-flick test was used to assess antinociceptive threshold. Intracerebroventricular (i.c.v.) administration of PACAP alone had no effect on pain sensitivity but in a dose of 500 ng, it significantly diminished the analgesic effect of a single dose of morphine (2.25 mg/kg, s.c.). PACAP (500 ng, i.c.v.) significantly increased the chronic tolerance to morphine and enhanced the naloxone (1 mg/kg, s.c.)-precipitated withdrawal jumping. Theophylline (1 mg/kg, i.p.) pretreatment significantly enhanced the effect of PACAP on morphine analgesia but the effects of PACAP on tolerance and withdrawal were unaffected upon theophylline administration. On the grounds of our previous studies with vasoactive intestinal polypeptide (VIP), it appears that different receptors are involved in the effects of PACAP in acute and chronic morphine actions. Our results indicate that PACAP-induced actions likely participate in acute and chronic effects of morphine and suggest a potential role of PACAP in opioid analgesia, tolerance and withdrawal.     

Antinociceptive effects of morphine and naloxone in mu-opioid receptor knockout mice transfected with the MORS196A gene

Background  

Opioid analgesics such as morphine and meperidine have been used to control moderate to severe pain for many years. However, these opioids have many side effects, including the development of tolerance and dependence after long-term use, which has limited their clinical use. We previously reported that mutations in the mu-opioid receptors (MOR) S196L and S196A rendered them responsive to the opioid antagonist naloxone without altering the agonist phenotype. In MORS196A knock-in mice, naloxone and naltrexone were antinociceptive but did not cause tolerance or physical dependence. In this study we delivery this mutated MOR gene into pain related pathway to confirm the possibility of in vivo transfecting MORS196A gene and using naloxone as a new analgesic agent.     

Interactions of galanin and opioids in nociceptive modulation in the arcuate nucleus of hypothalamus in rats

The fact that galanin, beta-endorphin and their receptors are present in the arcuate nucleus of hypothalamus (ARC), coupled with our previous observation that both beta-endorphin and galanin play antinociceptive roles in pain modulation in the ARC, made it of interest to study their interactions. The hindpaw withdrawal latency (HWL) in response to noxious thermal and mechanical stimulation was assessed by the hot-plate test and the Randall Selitto Test. We showed that the antinociceptive effect induced by intra-ARC injection of galanin was dose-dependently attenuated by the following intra-ARC injection of naloxone. Furthermore, intra-ARC administration of the selective mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) attenuated the increased HWL induced by intra-ARC injection of galanin in a dose-dependent manner, while the delta-opioid receptor antagonist naltrindole or the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) did not. Moreover, intra-ARC injection of a galanin receptor antagonist galantide attenuated intraperitoneal morphine-induced increases in HWLs. These results demonstrate that the antinociceptive effect of galanin was related to the opioid system, especially mu-opioid receptor was involved in, and that systemic morphine induced antinociception involves galanin in the ARC.     

C57BL/6J-bgJ (beige) mice: differential sensitivity in the tail flick test to centrally administered mu- and delta-opioid receptor agonists

The antinociceptive effects of two mu-opioid receptor agonists, morphine and [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO), and a selective delta-receptor agonist, [D-Pen2, L-Pen5]enkephalin (DPLPE), were determined in C57BL/6J-bgJ (beige) and control mice (CRS-CDl and C57BL/6By) using a standard tail-flick assay. The antinociceptive response of C57BL/6J-bgJ mice to intracerebro-ventricularly administered morphine and DAGO was significantly reduced compared to controls, but there was no difference in the antinociceptive response to DPLPE. These results suggest that there is a genetic deficit of mu-opioid receptor number or a genetically-induced alteration in receptor function in regions of C57BL/6J-bgJ brains involved in antinociception, that delta-opioid receptors can mediate antinociception in mice, and that the C57BL/6J-bgJ strain may offer a practical new animal model for studying the function of opioid receptor subtypes.     

Morphine withdrawal modifies antinociceptive effects of acute morphine in rats

Repeated opioid use is known to cause tolerance of antinociceptive effects. Whether opioid abstinence modifies antinociceptive effects is unknown. Here we reported that morphine withdrawal for 18 h and 4 days after repeated morphine treatment largely reduced tail-flick latencies compared with control, while the rats showed severe withdrawal syndromes. However, the latencies and withdrawal syndromes were restored to control level at 20 days withdrawal. Similarly, antinociceptive effects of acute morphine were decreased at 18 h and further decreased at 4 days but restored to control level at 20 days withdrawal. Behavioral stress that was given to the rats at 18 h withdrawal further reduced tail-flick latencies and antinociceptive effects. Conversely, the glucocorticoid receptor antagonist RU38486 increased tail-flick latencies and antinociceptive effects at 4 days withdrawal. These results suggest that morphine withdrawal could evoke behavioral stress to modify antinociceptive effects, implicating a significant influence of opioid abstinence on chronic pain treatment.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Astroglial Knockout of Glucocorticoid Receptor Attenuates Morphine Withdrawal Symptoms,but Not Antinociception and Tolerance in Mice

The development of tolerance and drug dependence limit the clinical application of opioids for the treatment of severe pain. Glucocorticoid receptors (GRs) are among molecular substrates involved in these processes. Most studies focus on the role of neuronal GR, while the involvement of GR on glial cells is not fully understood. To address this issue, we used a transgenic model of conditional GR knockout mice, targeted to connexin 30-expressing astrocytes, treated with repeated doses of morphine. We observed no difference between control mice and astrocytic GR knockouts in the development of antinociceptive tolerance. Nevertheless, when animals were subjected to precipitated withdrawal, knockouts presented some attenuated symptoms, including jumping. Taken together, our data suggest that hippocampal and spinal astrocytic GRs appear to be involved in opioid withdrawal, and drugs targeting the GR may relieve some symptoms of morphine withdrawal without influencing its antinociceptive properties.

     

Effect of naltrindole on the development of physical dependence on morphine in mice: A behavioral and biochemical study

The effect of pretreatment with a δ opioid receptor antagonist, naltrindole (NTI), on the development of physical dependence on morphine was investigated in mice. Several withdrawal signs, an increase in cortical noradrenaline (NA) turnover and a decrease in dopamine (DA) turnover in the limbic forebrain were observed following naloxone challenge in morphine-dependent mice. Pretreatment with NTI (0.3–5 mg/Kg, S.c.) during chronic morphine treatment dose-dependently suppressed the behavioral and biochemical changes after withdrawal. The blocking effects of NTI suggest that δ opioid receptors may play a significant role in modulating the development of physical dependence on morphine.     

Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous mu-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 microg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.     

Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of mu-opioid receptor

Recent studies showed that oxytocin plays an important role in nociceptive modulation in the central nervous system. The present study was undertaken to investigate the role of oxytocin in antinociception in the nucleus raphe magnus (NRM) of rats and the possible interaction between oxytocin and the opioid systems. Intra-NRM injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulation in rats. The antinociceptive effect of oxytocin was significantly attenuated by subsequent intra-NRM injection of the oxytocin antagonist 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin. Intra-NRM injection of naloxone dose-dependently antagonized the increased HWLs induced by preceding intra-NRM injection of oxytocin, indicating an involvement of opioid receptors in oxytocin-induced antinociception in the NRM of rats. Furthermore, the antinociceptive effect of oxytocin was dose-dependently attenuated by subsequent intra-NRM injection of the mu-opioid antagonist beta-funaltrexamine (beta-FNA), but not by the kappa-opioid antagonist nor-binaltorphimine (nor-BNI) or the delta-opioid antagonist naltrindole. The results demonstrated that oxytocin plays an antinociceptive role in the NRM of rats through activating the oxytocin receptor. Moreover, mu-opioid receptors, not kappa and delta receptors, are involved in the oxytocin-induced antinociception in the NRM of rats.     

The opioid peptide analogue biphalin induces less physical dependence than morphine

We compared the physical dependence liability of biphalin, a dimeric enkephalin analogue that possesses high antinociceptive activity, with that of morphine in equipotent intravenous doses. Naloxone challenge produced severe withdrawal signs after a 5-day infusion of morphine but only minor withdrawal signs after a 5-day biphalin infusion. In a cross-dependence study, biphalin did not suppress body weight loss after morphine withdrawal, but successfully suppressed weight loss after pentazocine withdrawal. These data support consideration of biphalin as a new analgesic with a novel pharmacological profile and minimum dependence liability.