Cannabinoid CB_(2) receptors and spinal microglia are implicated in tingenone-mediated antinociception in mice

Objective:To investigate the antinociceptive effect of tingenone on inflammatory pain,as well as and the involvement of the cannabinoid receptors type 2(CB2)and spinal microglia in this process.Methods:Male Swiss mice were subjected to inflammatory pain induced by intraplantar injection of carrageenan.The nociceptive threshold was measured by von Frey filaments test.Tingenone was administered orally 60 min before carrageenan injection.To evaluate the involvement of CB2 receptor,endocannabinoids,and microglia,AM630(a CB2 receptor antagonist),MAFP(an inhibitor of an enzyme that hydrolyses endocannabinoids),and minocycline(a microglial inhibitor)were given intrathecally 20 min before tingenone administration.In addition,an immunofluorescence assay was used to evaluate CB2 receptor and CD11 B(a microglial marker)expression in the spinal cord dorsal horn.Results:Tingenone significantly reduced carrageenan-induced hyperalgesia,which was reversed by pretreatment with AM630.MAFP and minocycline potentiated and prolonged the tingenoneinduced antinociception.CD11 B expression was increased in the spinal cord dorsal horn of mice with inflammatory pain pretreated with tingenone,which was reduced by AM630,MAFP,and minocycline.Conclusions:CB2 receptors and endocannabinoids participate in the tingenone-induced antinociception which may involve the inhibition of microglia at spinal level.

Effect of Peptidase Inhibitors on Dynorphin A (1-17) or (1-13)-Induced Antinociception and Toxicity at Spinal Level

Our group has earlier demonstrated that three enzymes sensitive to peptidase inhibitors (PIs), amastatin (A)-, captopril (C)-, and phosphoramidon (P), played an important role in inactivation of enkephalins at the spinal level. Dynorphin-converting enzyme (DCE) hydrolyzes dynorphin (Dyn) A (1-17) or Dyn A (1-13) mainly at the Arg6-Arg7 bond. Dynorphin A and its derived peptides interact with opioid and glutamate receptors at their N- and C-terminals, respectively. The purpose of the present study was to evaluate the antinociceptive potency and toxicity of intrathecal administered Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or the DCE inhibitor p-hydroxymercuribenzoate (PHMB). The effect of these PIs on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in mouse vas deferens was also investigated. The inhibitory potency of Dyn A (1-17) on electrically-evoked contractions in mouse vas deferens under pretreatment with ACP was higher than that with AC, AP, or CP. Pretreatment with ACP augmented Dyn A (1-17) or (1-13)-induced antinociception by approximately 50- or 30-fold with no sign of allodynia when administered intrathecally at low doses. Pretreatment with ACP and PHMB induced neuropathy. These findings showed that intrathecal administration of low-dose Dyn A (1-17) or DynA (1-13) increased antinociception under pretreatment with ACP, but without signs of allodynia in rat.

Antinociceptive Effect of Ethanolic Extract of Codiaeum variegatum Leaves in Mice

Background: Codiaeum variegatum, sometimes called garden croton, is a tropical plant in the Euphorbiaceae family. Historically used to cure various conditions, including intestinal infections, fever, ulcers, wounds, and gonorrhea. This work aimed to investigate the antinociceptive effects of ethanolic extract of Codiaeum variegatum leaves (EECV) in animal models. Methods: Five different pain models—the hot plate, tail immersion, acetic acid-induced writhing, formalin, and glutamate-induced nociception tests—were utilized to assess the antinociceptive activity in mice. The traditional drugs such as diclofenac sodium (10 mg/kg, i.p.) and morphine sulphate (5 mg/kg). EECV was administered orally at varying doses of 100, 200, and 300 mg/kg (0.1 mL/mouse), while the control group was given deionized water. Results: The current study found that all mouse models of heat- and chemical-induced pain had robust EECV reflections of their antinociceptive properties (*p Conclusions: The current finding offers a fresh perspective on the ethanolic extract of Codiaeum variegatum leaves’ antinociceptive properties in mice. This plant’s phytochemical analysis revealed the presence of triterpenoids, sterols, alkaloids, flavonoids, and general glycosides, all of which may have antinociceptive properties. More research on the mechanism of action and associated pharmacological studies, such as in vivo analysis, medication formulation, and clinical trials, is strongly advised.

Lafoensia pacari alleviates intestinal damage by modulating cyclooxygenase-2:In silico and in vivo evaluation in a colitis model

BACKGROUND Inflammatory bowel diseases(IBD)are a worldwide health problem and mainly affect young people,consequently affecting the workforce.Available treatments are often associated with side effects,and new therapeutic options are needed.For centuries,plants have represented important substrates in the field of drug development.Lafoensia pacari(L.pacari)is a plant whose pharmaceutical potential has been described,and may have biological activity relevant to the treatment of IBD symptoms.AIM To investigate the activity of keto-alcoholic extracts of L.pacari with respect to ameliorating the inflammatory and nociceptive symptoms of acute experimental colitis in mice.METHODS Keto-alcoholic extracts of L.pacari leaves and bark were administered to male andfemale Swiss mice weighing 25 g to 30 g(n=8 male mice and n=8 female mice).The effect of these extracts was observed in an acetic acid-induced acute experimental model of colitis with regard to antinociception/analgesia and inflammatory tissue damage.Recorded macroscopic indices included the Wallace score and the colon weight obtained using a precision scale.Mechanical hyperalgesia was determined using an electronic analgesimeter.Behavior related to overt pain was determined by quantifying the number of writhing instances within 20 min of administration of acetic acid.Molecular docking was performed using human and murine cyclooxygenase-2(COX-2)with 3 flavonoids(ellagic acid,kaempferol,and quercetin)on the AutoDock Vina software.Analysis of variance followed by Tukey’s posttest was used with P<0.05 indicating significance.RESULTS In this murine model of colitis,administration of extracts from L.pacari ameliorated acetic acidinduced writhing and colitis-associated inflammatory pain.These improvements may be attributable to the reduction in edema,inflammation(e.g.,ulcers,hyperemia,and bowel wall damage),and the intensity of abdominal hyperalgesia.The keto-alcoholic extracts of L.pacari leaves and bark administered at a dose of either 100 mg/kg or 300 mg/kg significantly reduced the number of writhing events when compared to the negative control(P<0.05).Additionally,extracts of L.pacari bark also performed better than Dipyrone.Leaf extracts administered at 10 mg/kg,30 mg/kg,and 100 mg/kg and bark extracts administered at 30 mg/kg significantly reduced or prevented the development of edema in the colon of treated mice,while mesalazine did not.Moreover,using molecular docking,we observed that the flavonoids present in L.pacari extracts bind to COX-2,an event not unique to ellagic acid.CONCLUSION The results of this study demonstrate a potential novel application of L.pacari extracts for the reduction of inflammation and promotion of antinociception/analgesia as demonstrated by our findings in a murine model of colitis.These findings were also corroborated by in silico analyses,and suggest that L.pacari extracts may be a promising therapeutic agent in the treatment of IBD.

Antinociceptive Effect of Methanol Extract of Diospyros malabarica (Desr.) Kostel Leaves in Mice

Background: Diospyros malabarica (Desr.) Kostel, a small to medium-sized tree in the Ebenaceae family, is known as “Deshi Gab” in Bangladesh. Fever, diabetes, snake bite, diarrhea, biliousness, and ulcer ailments are all treated with the herb. This study’s goal was to examine in mouse models the antinociceptive properties of methanol extract of Diospyros malabarica leaves (MEDM). Methods: For the purpose of determining the antinociceptive activity in mice, five distinct pain models including hot plate, tail immersion, acetic acid-induced writhing, formalin and glutamate-induced nociception tests were used. The conventional medications were morphine sulphate (5 mg/kg, intraperitoneally) and diclofenac sodium (10 mg/kg, intraperitoneally). While the control group was expecting deionized water, MEDM was given orally at dosages of 200, 400, and 600 mg/kg (0.1 mL/mouse, orally). Results: According to the current research, MEDM strongly reflected the antinociceptive activity of all mouse models of chemical and heat-induced pain (*p < 0.05). 400 and 600 mg/kg demonstrated a considerable (*p < 0.05) ability to prolong the reaction of latency to pain in opposition to thermally produced nociception in hot plate and tail immersion tests. Inhibition levels in the acetic acid-induced writhing test were 11.57%, 37.77%, and 51.83%, respectively. The extract suppressed 20.78%, 45.48%, and 56.93% of licking during the initial stages of formalin-induced nociception. In the late phase, the extract showed higher rates of licking than the control group (13.14%, 50.28%, and 66.85%). The glutamate-induced nociception test was significantly (*p < 0.05) prevented by the plant extract. Compared to the control, it demonstrated an inhibition of licking of 22.85%, 47.32%, and 63.42%, respectively. Conclusions: It is evident that the plant extract has exceptional analgesic properties. To determine the precise processes behind antinociceptive effect and to identify the substances that produce this activity, more research is required. The study’s findings also support the long-standing use of MEDM in painful conditions.

THE ROLE OF BRAIN-STEM DISCENDING INHIBITORY SYSTEM IN THE ANTINOCICEPTIVE EFFECT ELICITED BY MUSCLE SPINDLE AFFERENTS

Objective To analyse the antinociceptive effect of muscle spindle afferents and the involved mechanism.Methods The single unit of wide dynamic range neurons in the spinal cord dorsal horn were recorded extracelluarly.The effects of muscle spindle afferents elicited by intravenous administration of succinylcholine on nociceptive responses (C fibres evoked responses,C responses) of WDR neurons were observed before and after bilateral lesions of ventrolateral periaqueduct gray .And the effects of muscle spindle afferents on the spontaneous discharge of the tail flick related cell in the rostral ventro medial medulla and on the spontaneous discharge of the PAG neurons were observed.Results The C responses of WDR neurons were significantly inhibited by muscle spindle afferents,and the inhibitory effects were reduced by bilateral lesions of ventrolateral PAG.The spontaneous discharge of the off cell in the RVM was excited while the on cell was inhibited by intravenous administration of Sch.The spontaneous discharge of the PAG neurons were excited by muscle spindle afferents.Conclusion Muscle spindle afferents show a distinct effect of antinociception.PAG RVM descending inhibitory system may play an important role in this nociceptive modulative mechanism.

THE ROLE OF RED NUCLEUS IN THE MODULATION OF SPINAL NOCICEPTIVE TRANSMISSION AND IN NOCICEPTION ELICITED BY MUSCLE SPINDLE AFFERENTS

Objective To analyse the antinociceptive effect of red nucleus (RN) and its role in the antinociceptive effect of muscle spindle afferents. Methods The single units of RN or wide dynamic range (WDR) neuron in the spinal cord dorsal horn were extracelluarly recorded. The effects of RN stimulation on nociceptive responses (C fibers evoked responses, C responses) of WDR neurons were observed. The influence of muscle spindle afferents elicited by intravenous administration of succinylcholine (Sch) on the spontaneous discharge of RN neurons and on C responses of WDR neurons were observed. The effect of muscle spindle afferents on C responses of WDR neurons after unilateral lesions of RN was also observed. Results Electrical stimulation of the RN produced a significantly inhibitory effect on the nociceptive responses of WDR neurons. RN neurons were excited by muscle spindle afferents. Muscle spindle afferents significantly inhibited C response of WDR neurons and this inhibitory effect was reduced by lesions of RN. Conclusion RN neurons have a significant antinociceptive effect and might be involved in the antinociceptive effects elicited by muscle spindle afferents.

Tolerance effects of non-steroidal anti-inflammatory drugs microinjected into central amygdala,periaqueductal grey,and nucleus raphe Possible cellular mechanism

Pain is a sensation related to potential or actual damage in some tissue of the body. The mainstay of medical pain therapy remains drugs that have been around for decades, like non-steroidal anti-inflammatory drugs （NSAIDs）, or opiates. However, adverse effects of opiates, particularly tolerance, limit their clinical use. Several lines of investigations have shown that systemic （intraperitoneal） administration of NSAIDs induces antinociception with some effects of tolerance. In this review, we report that repeated microinjection of NSAIDs analgin, clodifen, ketorolac and xefocam into the central nucleus of amygdala, the midbrain periaqueductal grey matter and nucleus raphe magnus in the following 4 days result in progressively less antinociception compared to the saline control testing in the tail-flick reflex and hot plate latency tests. Hence, tolerance develops to these drugs and cross-tolerance to morphine in male rats. These findings strongly support the suggestion of endogenous opioid involvement in NSAIDs antinociception and tolerance in the descending pain-control system. Moreover, the periaqueductal grey-rostral ventro-medial part of medulla circuit should be viewed as a pain-modulation system. These data are important for human medicine. In particular, cross-tolerance between non-opioid and opioid analgesics should be important in the clinical setting.

Connections between 5-HT-containing terminals and 5-HT_(2A) recep tor and γ-aminobutyric acid or glycine co-existed neurons in the rat medullary dorsal horn

Objective: To investigatetheconnectionsbetweenserotonin(5-HT)-containingterminalsand5-HT 2A receptor(5-HT 2A R)/γ-aminobutyricacid(GABA)or5-HT 2A R/glycineco-existedneuronsin theratmedullarydorsalhorn(MDH).Meth ods:Immunofluorescencehistochemicaltriple-stainingfor5-HT,5-HT 2A R,GABAor glycine.Results:5-HT-im-munoreactivefibersandterminalswerechieflylocatedinthesuperficiallaminae(laminaeⅠandⅡ)of theMDH.Neurons exhibiting5-HT 2A R-,GABA-or glycine-immunoreactivitiesweremainlyobservedin the superficiallaminae.Some5-HT 2A R-immunopositiveneuronsalsoexhibitedGABA-or glycine-immunoreactivities.5-HT-containingterminalsmade closecontactswith5-HT 2A R/GABAor5-HT 2A R/glycineco-existedneurons.Conclusion:5-HT 2A R/GABAor5-HT 2A R/glycineco-existinsomeof theneuronsinthesuperficiallaminaeof theMDH.5-HT-immunoreactiveterminalsformclose connectionswith5-HT 2A R/GABAor5-HT 2A R/glycineco-existedneurons.

THE ROLE OF NUCLEUS RAPHE MAGNUS IN THE ANTINOCICEPTIVE EFFECT OF MUSCLE SPINDLE AFFERENTS IN THE RAT

Objective To investigate the role of NRM in the antinociceptive effect of muscle spindle afferents,the influence of NRM lesion on the inhibitory effect of muscle spindle afferents on the nociceptive responses of wide dynamic range (WDR) neurons and the effects of the muscle spindle afferents on the NRM neuronal activities were observed. Methods The single units of WDR neurons in the spinal dorsal horn were recorded extracellularly, and the inhibitory effects of activating muscle spindle afferents by intravenous administration of succinylcholine (SCH) on the C fibers evoked responses (C responses) of WDR neurons were tested before and after lesion of NRM.The effects of the muscle spindle afferents activated by administrating SCH on the single NRM neurons were also examined.Results ①It was found that the C responses of WDR neurons were significantly inhibited by intravenously administration of SCH, and the inhibitory effect was reduced after lesion of NRM;②The activities of most of the NRM neurons could be changed significantly by administrating SCH. According to their responses, NRM neurons could be classified into three types:excitatory, inhibitory and non responsive neurons, and the responses were dose dependent. Conclusion These results suggest that the muscle spindle afferents evoked by SCH may activate the NRM neurons, which plays an important role in the antinociception of muscle spindle afferents.

The Primary Motor Cortex Stimulation Attenuates Cold Allodynia in a Chronic Peripheral Neuropathic Pain Condition in <i>Rattus norvegicus</i>

Background: The primary motor cortex (M1) stimulation (MCS) is a useful tool for attenuation of the peripheral neuropathic pain in patients with pharmacologically refractory pain. Furthermore, that neurological procedure may also cause antinociception in rodents with neuropathic pain. Cold allodynia is a frequent clinical finding in patients with neuropathic pain, then, we evaluated if an adapted model of neuropathy induced by chronic constriction injury (CCI) of the ischiadicus nervus (sciatic nerve) produces cold allodynia in an animal model of chronic pain. In addition, we also investigated the effect of the electrical stimulation of the M1 on chronic neuropathic pain condition in laboratory animals. Methods: Male Wistar rats were used. An adapted model of peripheral mononeuropathy induced by CCI was carried out by placing a single loose ligature around the right sciatic nerve. The acetone test was used to evaluate the cold allodynia in CCI or Sham (without ligature) rats. The MCS (M1) was performed at low-frequency (20 μA, 100 Hz) during 15 s by deep brain stimulation (DBS-Thomas Recording device) 21 days after CCI or Sham procedures. The cold allodynia was measured before and immediately after the neurostimulation of M1 in the following time-window: 0, 15 and 30 min after MCS. Results: Cold allodynia threshold increased in animals with chronic neuropathic pain submitted to the acetone test 21 days after the CCI surgery. The M1-stimulation by DBS procedure decreased the cold allodynia immediately and until 30 min after M1-stimulation in rats with chronic neuropathic pain. Conclusion: The current proposal for a CCI model by a single loose ligature of the sciatic nerve can be employed as an experimental model of chronic neuropathic pain in rats submitted to peripheral nervous system injury. The M1-stimulation produced antinociception in rats with chronic neuropathic pain. Thus, we reinforced that the MCS decreases cold allodynia in laboratory animals submitted to persistent sciatic nerve constriction and can be a more reasonable procedure for the treatment of chronic intractable neuropathic pain.

Nitridergic Modulation of the Antinociceptive Activity of Rosuvastatin in Mice

Statins, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase enzyme inhibitors, are lipid-lowering drugs, often used in the treatment of cardiovascular diseases (hyperlipidemia, atherosclerosis). It has been shown that statins have antiinflammatory effects independent of their lipid-lowering effects and these anti-inflammatory effects inhibit the inflammation and pain process. This study evaluated the antinociceptive and anti-inflammatory effects of rosuvastatin using the acetic acid writhing, the formalin hind paw, the orofacial formalin and the hot plate tests. The following experimental group were used: control, acute (1 day) and chronic (3 days) after oral gavage with rosuvastatin (3, 10, 30, 100 and 300 mg/kg). Rosuvastatin produced a dose-dependent antinociception, with different potency, in all the tests. Additionally, nitric oxide synthase inhibitors (Abbreviationsand aminoguanidine) were used to assess the nitric oxide participation on this induced rosuvastatin antinociception. The data demonstrated the antinociceptive and anti-inflammatory activity of rosuvastatin in algesiometer models of tonic or phasic pain. These activities seem to be induced by modulation of iNOS expression, a result that may be relevant in the pharmacological treatment of human pain where rosuvastatin and nitric oxide synthase inhibitors must be used.

Serotonin Influences the Endogenous Opiate Peptides in the Rat Spinal Cord to Participates in Pain Modulation

Spinal cord is a necessary pathway that transfers the body nociceptive inputs to the brain. Endogenous opiate peptides have been proven to participate in the nociceptive process at spinal level. It has reported that serotonin (5-HT, 5-hydroxytryptamine) in spinal cord plays a role in pan modulation, which can be blocked by opiate receptor antagonists. The present study was designed to investigate the interaction between 5-HT and endogenous opiate peptides at rat spinal level effecting on pain modulation. The results showed that 1) pain stimulation increased not only leucine-enkephalin (L-Ek), β-endorphin (β-Ep) and dynorphin A1-13 (DynA1-13) concentrations but also 5-HT and 5-hydorxyindoleace acid (5-HIAA, the 5-HT main metabolic product) concentrations in spinal cord significantly;2) 5-HT could increase L-Ek, β-Ep and DynA1-13 concentrations in spinal cord in a dose-dependent manner, whereas cypotolamine (a 5-HT receptor antagonist) decreased L-Ek, β-Ep and DynA1-13 concentrations in spinal cord. The data suggested that 5-HT antinociceptive role might be involved in the endogenous opiate peptide system through 5-HT receptors at spinal level.

Role of opioid receptor heterodimerization in pain modulation and tolerance development

Protein to protein interactions leading to homo/heteromerization of receptor is well documented in literature. These interactions leading to dimeric/oligomers formation of receptors are known to modulate their function, particularly in case of G-protein coupled receptors. The opioid receptor heteromers having changed pharmacological properties than the constituent protomers provides preferences for novel drug targets that could lead to potential analgesicactivity devoid of tolerance and physical dependence. Heterodimerization of opioid receptors appears to generate novel binding properties with improved specificity and lack of side effects. Further the molecules which can interact simultaneously to both the protomers of the heteromer, or to both the binding sites(orthosteric and allosteric) of a receptor protein could be potential therapeutic molecules. This review highlights the recent advancements in exploring the plausible role of heteromerization of opioid receptors in induction of tolerance free antinociception.

Evaluation of Antinociceptive and Anti-Inflammatory Effects of Synthetic O-Prenylated Phenolic Derivatives

This work describes the synthesis, antinociceptive and anti-inflammatory effect of O-prenyl derivatives: O-prenyl derivatives 2-(3-methylbut-2-enyloxy)acetophenone (L1) and 3-methoxy-4-(3-methylbut-2-enyloxy)benzaldehyde (V1). Treatment with L1- or V1-produced antinociceptive effect on classical pain models: acetic acid abdominal contortions and formalin test (first and second phases), and in hot plate or tale flick models in mice without changing the locomotors performance, and anti-oedematogenic or anti-inflammatory effect in vivo: carrageenan-induced paw oedema and peritonitis in mice. In addition, L1 and V1 derivatives reduced nitric oxide production on RAW 264.7 cells stimulated with lipopolysaccharide without changing the cell viability. Taken together, our results show for the first time that L1 and V1 can produce antinociception and modulate inflammatory response when administered in mice.

Antinociceptive activity of Ricinus communis L.leaves

Objective:To evaluate the antinociceptive activity of the methanol extract of Ricinus communis leaves(MRCL).Methods:Antinociceptive activity was evaluated using acetic acid induced writhing test,formalin induced paw licking and tail immersion method in mice at doses of 100,125 and 130 mg/kg bw.Results:The results indicated that MRCL exhibited considerable antinociceptive activity against three classical models of pain in mice.Preliminary phytochemical analysis suggested the presence of saponin,steroids and alkaloids.Conclusions:It can be concluded that MRCL possesses antinociceptive potential that may be due to saponin,steroids and alkaloids in it.

In vivo screening of essential oils of Skimmia laureola leaves for antinociceptive and antipyretic activity

Objective:To study the screening of essential oils of Skimmia laureola leaves(SLO)for acute toxicity,antinociceptive,antipyretic and anticonvulsant activities in various animal models.Methods:SLO were extracted using modified Clevenger type apparatus.Acute toxicity test was used in mice to observe its safety level.Antinociceptive activity of SLO was evaluated in acetic acid induced writhing and hot plate tests.Yeast induced hyperthermic mice and pentylenetetrazole induced convulsive mice were used for the assessment of its antipyretic and anticonvulsant profile respectively.Results:Substantial safety was observed for SLO in acute toxicity test.SLO showed a high significant activity in acetic acid induced writhing test in a dose dependent manner with maximum pain attenuation of 68.48%at 200 mg/kg i.p.However,it did not produce any relief in thermal induced pain at test doses.When challenged against pyrexia evoked by yeast,SLO manifested marked amelioration in hyperthermic mice,dose dependently.Maximum anti-hyperthermic activity(75%)was observed at 200 mg/kg i.p.after 4 h of drug administration.Nevertheless,SLO had no effect on seizures control and mortality caused by pentylenetetrazole.Conclusions:In vivo studies of SLO showed prominent antinociceptive and antipyretic activities with ample safety profile and thus provided pharmacological base for the traditional uses of the plant in various painful conditions and pyrexia.Additional detail studies are required to ascertain its clinical application.

Mechanisms of antinociceptive effects of ouabain in combination with neostigmine in the rat

BACKGROUND： It has been previously shown that intrathecal administration of either ouabain or neosdgmine can produce antinociceptive effects. Moreover, ouabain and neostigmine are differently associated with acetylcholine. OBJECTIVE： It has been hypothesized that intrathecal administration of ouabain, in combination with neostigmine, can produce antinociceptive synergistic effects. Atropine, as a competitive antagonist, was pre-injected to verify the mechanisms of action. DESIGN, TIME AND SETTING： This study was a randomized, controlled, animal experiment, performed at the State Key Laboratory of Oncology in Southern China between May 2006 and February 2007. MATERIALS： A total of 102 healthy, adult, Sprague Dawley rats were included. Ouabain and neostigmine （Sigma, USA）, as well as atropine （Tanabe Seiyaku, Japan）, were also used. METHODS： Varied doses of ouabain, neostigmine, and a combination of the two were intrathecally injected into rats. Six rats were allotted for each dose group. Intrathecal pretreatment with atropine was tested 10 minutes prior to intrathecal administration of neostigmine or the combination of ouabain and neostigmine. MAIN OUTCOME MEASURES： Tail-flick tests were performed to measure tail-flick latency （seconds） prior to and after administration. The response in the tail-flick test was expressed as the percentage of maximum possible effect （% MPE）, where % MPE = [tail-flick latency after administration （seconds） -mean baseline value for tail-flick latency]/[ 10 seconds - the mean baseline value for tail-flick latency （seconds）] x 100%. RESULTS： Rat spinal intrathecal administration of either ouabain or neostigmine alone produced antinociceptive effects in a dose-dependent manner. Intrathecally administration of neostigmine （0.05, 0.1, 0.3 μg ） in combination with ouabain （1 μ g ） produced enhanced antinociceptive effects, with a % MPE of 29%, 78%, and 95%, respectively （P 〈 0.05）. Intrathecally administration of 0.3μg neostigmine （% MPE： 45%）, in combination with 1 μ g ouabain （% MPE： 27%） produced potent antinociceptive effects （% MPE： 95%）. Intrathecally pre-injected atropine antagonized the antinociceptive effects of neostigmine （3 μg）, or a combination of ouabain （1 μg） and neostigmine （0.3 μg） （P 〈 0.01）. CONCLUSION： Rat spinal intrathecal administration of either ouabain or neostigmine alone produced dose-dependent andnociceptive effects. Ouabain enhanced the antinociceptive effects of neostigmine. Atropine antagonized the antinociceptive effects of neostigmine or the combination of ouabain and neostigmine. This occurs possibly due to the fact that atropine is a competitive antagonist of the muscarinic acetylcboline receptors.

Combinative Approaches of Chemistry, Pharmacology and Toxicology for the Optimal Pharmaceutical Preparation of an Anti-arthritic Chinese Medicine Formulation QFJBT

Objective Qing Fu Juan Bi Tang(QFJBT)is an anti-arthritic Chinese medicine formula consisting of five herbs:Aconiti Lateralis Radix Praeparata(Fu Zi,附子),Sinomenii Caulis(Qing Feng Teng,青风藤),Astragali Radix(Huang Qi,黄芪),Paeoniae Radix Alba(Bai Shao,白芍)and Moutan Cortex(Mu Dan Pi,牡丹皮),which have well-established histories of use for treatment of rheumatic and arthritic diseases.We intended to establish the optimized and standardized pharmaceutical procedures and manufacturing processes for the pilot production of QFJBT to develop it as a novel botanical drug product for treatment of rheumatoid arthritis(RA).Methods The combinative approaches of chemical assessment,toxicological and pharmacological evaluation were explored to define the pharmaceutical preparation of QFJBT.Results The optimized and standardized pharmaceutical procedures and manufacturing processes for the pilot production of QFJBT were established in terms of greatest chemical contents of bioactive constituents,potent anti-inflammatory and antinociceptive activities,and favorable safety profile.Quality analysis of the pilot product of QFJBT by high-performance liquid chromatography(HPLC)demonstrated that the chromatographic fingerprint profiles of three batches of QFJBT were basically identical and the contents of four characteristic and bioactive markers were relatively consistent.General toxicological studies showed a favorable safety profile of QFJBT.The maximum tolerated single dose of QFJBT was determined in both sexes of rats to be 33.63 g/kg body weight which is equivalent to 346 times of clinical dose.In the chronic oral toxicity study,the results of laboratory investigation showed that QFJBT at doses of 3.89,6.80 and 9.72 g/kg body weight(equivalent to 40,70 and 100-fold clinical doses,respectively)caused no changes in all hematological parameters and blood biochemical parameters of rats.No mortality or specific toxic responses were observed in animals after three months of repeated dosing with QFJBT.Conclusion The optimized and standardized pharmaceutical and manufacturing processes for the production of QFJBT have been successfully screened and identified through established rigorous in-process controls.

DN-9: a multifunctional agonist of opioid and neuropeptide FF receptors that produces nontoleranceforming analgesia via peripheral opioid receptors in inflammatory and neuropathic pain models

OBJECTIVE Considerable effort has recently been directed at developing multifunctional opioid drugs as an alternative strategy to minimize the unwanted side effects of opioid analgesics.We recently developed a novel multifunctional agonist for opioid and neuropeptide FF(NPFF) receptors named DN-9.The present study was conducted to evaluate the pharmacological activities of DN-9 after peripheral administration.METHODS Antinociceptive activities of subcutaneous DN-9 were investigated in mouse models of acute inflammatory and neuropathic pain.Furthermore,the side-effects of DN-9 were evaluated after peripheral injection in rotarod,antinociceptive tolerance,abuse and gastrointestinal transit tests.RESULTS Subcutaneous DN-9 dose-dependently produced antinociception via peripheral mu-and kappa-opioid receptors,independent of delta-opioid and NPFF receptors,in the tail-flick assay.Similarly,a dose-dependent antinociceptive effect of DN-9 was mediated via peripheral opioid receptors in other inflammatory and neuropathic pain models.Repeated treatment with DN-9 produced antinociceptive effects without a loss of potency in various models of acute,inflammatory and neuropathic pain.DN-9 maintained potent analgesia in morphine-tolerant mice.The gastrointestinal motility inhibition and abuse properties of DN-9 were significantly reduced after subcutaneous injection compared to morphine.DN-9 did not significantly influence the motor coordination of mice.CONCLUSION Subcutaneous administration of DN-9 produces potent analgesic activities with minimal side effects.These data strengthen the therapeutic potential of peripherally acting opioids with multifunctional agonistic properties that are active in a broad range of experimental pain models after peripheral delivery.