Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene) MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

[D-Ala2, D-Leu5] Enkephalin Inhibits TLR4/NF-κB Signaling Pathway and Protects Rat Brains against Focal Ischemia-Reperfusion Injury.

BACKGROUND: Cerebral ischemia-reperfusion (I/R) injury is the main cause of acute brain injury, which is a life-threatening disease due to the lack of effective treatments. [D-Ala2, D-Leu5] enkephalin (DADLE) is a synthetic delta-opioid receptor agonist that is reported to confer neuroprotective effect; however, the underlying mechanism is still being explored. The purpose of the present study is to determine whether DADLE administrated intracerebroventricularly could attenuate the cerebral I/R injury, to determine if this is through inhibiting the toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway and therefore inhibiting neuroinflammation in an ischemic stroke model. METHODS: Rats were subjected to 120 minutes of ischemia by transient middle cerebral artery occlusion (MCAO). At 45 minutes after ischemia, DADLE or control vehicle (artificial cerebrospinal fluid, ACSF) was given to the rats intracerebroventricularly. Neurological deficit, cerebral infarct volume, and histopathological changes were assessed at 24 hours after reperfusion. Brain inflammation was assessed by measuring tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the ischemic penumbra by ELISA. The expression of TLR4 was determined by immunohistochemistry staining and western blotting. The expression of NF-κB was investigated by western blotting. RESULTS: Compared with the vehicle-treatment (ACSF), DADEL improved neurological deficit (9.6 ± 2.1 versus 13.8 ± 1.9), reduced cerebral infarct volume (18.74 ± 3.30% versus 10.57 ± 2.50%), and increased the number of normal neurons (29.72 ± 8.53% versus 51.37 ± 9.18%) after cerebral I/R injury in rats (all P < 0.05). Expressions of inflammatory molecules including TNF-α and IL-6 were highly expressed in the vehicle-treated rats, whereas treatment with DADLE downregulated these expressions (P < 0.05). Additionally, cerebral I/R injury significantly increased the TLR4 and NF-κB expression in vehicle-control group, which was markedly inhibited by DADLE (P < 0.05). CONCLUSIONS: DADLE, administrated intracerebroventricularly at 45 minutes after cerebral ischemia, significantly ameliorated I/R-induced brain damage in rats. This kind of neuroprotective effect appears to be related to the downregulation of TLR4-mediated inflammatory responses.

Biphalin-A Potent Opioid Agonist-As a Panacea for Opioid System-Dependent Pathophysiological Diseases?

Biphalin, one of the opioid agonists, is a dimeric analog of enkephalin with a high affinity for opioid receptors. Opioid receptors are widespread in the central nervous system and in peripheral neuronal and non-neuronal tissues. Hence, these receptors and their agonists, which play an important role in pain blocking, may also be involved in the regulation of other physiological functions. Biphalin was designed and synthesized in 1982 by Lipkowski as an analgesic peptide. Extensive further research in various laboratories on the antinociceptive effects of biphalin has shown its excellent properties. It has been demonstrated that biphalin exhibits an analgesic effect in acute, neuropathic, and chronic animal pain models, and is 1000 times more potent than morphine when administered intrathecally. In the course of the broad conducted research devoted primarily to the antinociceptive effect of this compound, it has been found that biphalin may also potentially participate in the regulation of other opioid system-dependent functions. Nearly 40 years of research on the properties of biphalin have shown that it may play a beneficial role as an antiviral, antiproliferative, anti-inflammatory, and neuroprotective agent, and may also affect many physiological functions. This integral review analyzes the literature on the multidirectional biological effects of biphalin and its potential in the treatment of many opioid system-dependent pathophysiological diseases.

Transplantation of Mesenchymal Stromal Cells Expressing the Human Preproenkephalin Gene Can Relieve Pain in a Rat Model of Neuropathic Pain.

Transgenic therapy for central neuralgia faces the problems of low expression and weak targeting and affects superficial but not deep neurons. In this study, we generated a lentivirus vector with human preproenkephalin gene (hPPE) expression driven by the transcriptional amplification strategy system (TAS) and established a primary bone marrow-derived mesenchymal stromal cell (BMSC) line stably expressing hPPE for transplantation into a rat model of neuropathic pain rat. The paw thermal withdrawal latency assay and paw mechanical withdrawal threshold assay showed that unlike control BMSCs and BMSCs with hPPE overexpression driven by the CMV or Synapsin 1 (SYN1) promoter, TAS-hPPE BMSCs had a robust and lasting analgesic effect. The TAS-hPPE BMSC-treated group exhibited higher expression of TAS-driven hPPE and a higher ratio of BMSCs in the midbrain, spinal cord and cortex then the CMV-hPPE BMSC- and SYN1-hPPE BMSC-treated groups. Moreover, we also observed that TAS-hPPE BMSCs displayed a greater tendency to differentiate into neurons and exhibit neuronal-like distribution than CMV-hPPE or SYN1-hPPE BMSCs. In conclusion, our study shows that the TAS improves BMSC transgenic therapy for neuropathic pain treatment.

Conjugate of Enkephalin and Temporin Peptides as a Novel Therapeutic Agent for Sepsis.

Antimicrobial peptides (AMPs) exhibit a wide spectrum of actions, ranging from a direct bactericidal effect to multifunctional activities as immune effector molecules. The aim of this study was to examine the anti-inflammatory properties of a DAL-PEG-DK5 conjugate composed of a lysine-rich derivative of amphibian temporin-1CEb (DK5) and dalargin (DAL), the synthetic Leu-enkephalin analogue. Detailed study of the endotoxin-neutralizing activity of the peptide revealed that DAL-PEG-DK5 interacts with LPS and the LPS binding protein (LBP). Moreover, DAL-PEG-DK5 prevented dimerization of TLR4 at the macrophage surface upon LPS stimulation. This inhibited activation of the NF-κB signaling pathway and markedly reduced pro-inflammatory cytokine production. Finally, we showed that aggregation of DAL-PEG-DK5 into amyloid-like structures induced by LPS neutralized the endotoxin proinflammatory activity. Consequently, DAL-PEG-DK5 reduced morbidity and mortality in vivo, in a mouse model of endotoxin-induced septic shock. Collectively, the data suggest that DAL-PEG-DK5 is a promising therapeutic compound for sepsis.

Gene therapy for pain: results of a phase I clinical trial.

OBJECTIVE: Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)-based vectors can reduce pain-related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans. METHODS: We conducted a multicenter, dose-escalation, phase I clinical trial of NP2, a replication-defective HSV-based vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ), and concurrent opiate usage. RESULTS: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle- and high-dose cohorts reported pain relief as assessed by NRS and SF-MPQ. INTERPRETATION: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation.

Acute and chronic cardioprotection by the enkephalin analogue, Eribis peptide 94, is mediated via activation of nitric oxide synthase and adenosine triphosphate-regulated potassium channels.

BACKGROUND/AIMS: Eribis peptide 94 (EP 94) is a new enkephalin derivative which potently binds to the µ- and δ-opioid receptor. In this study, we determined the effects of EP 94 and potential mechanism(s) involved in cardioprotection of the rat heart. METHODS AND RESULTS: An acute (5 and10 min into ischemia) and a chronic (24 h prior to ischemia) EP 94 administration produced a similar 30-40% reduction in infarct size/area at risk and the effects were blocked by the K(ATP) channel antagonists, HMR 1098 and 5-HD. The cardioprotective effects were blocked by a nonselective nitric oxide synthase (NOS) inhibitor (L-NAME) following acute administration and by a selective iNOS inhibitor (1400W) following chronic administration. CONCLUSION: These results suggest that EP 94 may have potential for the treatment of ischemic heart disease via a nitric oxide (NO)-K(ATP)-mediated mechanism.

Biphalin, a dimeric opioid peptide, reduces neonatal hypoxia-ischemia brain injury in mice by the activation of PI3K/Akt signaling pathway.

Previous studies have demonstrated that the activation of delta opioid receptors is neuroprotective against neonatal hypoxia-ischemia (HI) brain injury. The aim of this study was to investigate the neuroprotective effects of biphalin, a dimeric opioid peptide, in a mouse model of neonatal HI and the underlying mechanisms. On postnatal day 10, mouse pups were subjected to unilateral carotid artery ligation followed by 1 h of hypoxia (10 % O2 in N2). For treatment, biphalin (5 mg/kg, 10 mg/kg, 20 mg/kg) was administered intraperitoneally immediately after HI. The opioid antagonist naloxone or phosphatidylinositol-3-kinase inhibitor Ly294002 was administered to determine the underlying mechanisms. Infarct volume, brain edema, phosphorylated Akt and apoptosis-related proteins levels were evaluated by using a combination of 2,3,5-triphenyltetrazolium chloride staining, brain water content and Western blotting at 24 h after HI. The long-term effects of biphalin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests at 3 weeks post-HI. Biphalin (10 mg/kg) significantly reduced the infarct volume and ameliorated brain edema. Biphalin also had long-term protective effects against the loss of ipsilateral brain tissue and resulted in improvements in neurobehavioral outcomes. However, naloxone or Ly294002 abrogated the neuroprotective effects of biphalin. Furthermore, biphalin treatment significantly preserved phosphorylated Akt expression, increased Bcl-2 levels, and decreased Bax and cleaved caspase 3 levels after HI. These effects were also reversed by naloxone and Ly294002 respectively. In conclusion, biphalin protects against HI brain injury in neonatal mice, which might be through activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

Biphalin, a dimeric enkephalin analog, is under investigation as a potential, long-lasting medication of pain associated with chronic diseases, like cancer or AIDS. The role of cytokines, and splenocytes in anti-Friend leukemia virus (FLV) activity of biphalin, a synthetic opioid, and AZT was investigated in vitro. Mouse splenocytes inhibited FLV replication in Mus dunni (Dunni) cells when they were added to the cell culture. This inhibitory effect of splenocytes also was evident when cells were combined with biphalin and AZT as measured using a focus-forming assay. Under cell-free conditions, recombinant interferon gamma (IFNgamma), interleukin 2 (IL-2) and IL-4 directly inhibited the FLV reverse transcriptase (RT) activity by 27% to 36%. IFNgamma at 0.005 pg to 500 ng inhibited FLVRT activity by 61% to 80%. Acombination of 250 ng IFNgamma and 50 mug biphalin resulted in a 94% reduction of FLVRT activity, as compared with 61% inhibition by IFNgamma alone. The combination of AZT and IFNgamma, IL-2 or IL-4 also induced a stronger suppression of FLV RT activity than either cytokine or AZT used alone. In addition, cloned RT from Moloney murine leukemia virus (MMLV) was directly sensitive to inhibition by biphalin. Thus, the anti-FLV effects of splenocytes in combination with biphalin and AZT in cell culture are likely mediated to a large degree by the direct effect of cytokines. This antiviral activity of splenocytes or cytokines combined with chemotherapy, biphalin, and/or AZT, could be used as a complementary therapy to current approaches for retroviral infection and benefit acquired immunodeficiency syndrome (AIDS) patients. In conclusion, biphalin applied primarily as a new medicine for chronic pain treatment in AIDS patients may play a significant beneficial role as a component of antiviral HIV multidrug therapies.

Antinociceptive potency of enkephalins and enkephalinase inhibitors in the mouse model of colorectal distension-proof-of-concept.

Irritable bowel syndrome (IBS) is a chronic disease characterized by abdominal pain and changes in bowel habits. Patients with IBS comprise a significant portion of attendants at the outpatient clinics. Targeting intestinal opioid receptors was found successful in alleviating pain and diarrhea-two major symptoms of IBS. In this study, we aimed to evaluate a novel potential pharmacological option: the use of enkephalinase inhibitors in therapy of visceral pain occurring in the course of IBS. We thus assessed the antinociceptive efficacy of enkephalins: Leu-enkephalin and Met-enkephalin, and enkephalinase inhibitors: opiorphin and sialorphin in the mouse model of visceral pain induced by colorectal distension. Leu-enkephalin, Met-enkephalin, and sialorphin, but not opiorphin, at the dose of 1 mg/kg injected subcutaneously potently decreased the visceromotor response to colon distension as compared to control. To conclude, enkephalinase inhibitors are worth being considered as potential therapeutics in patients with chronic abdominal pain and/or changed bowel habits, that is, suffering from IBS.

Antinociceptive Effect of Intrathecal Injection of Genetically Engineered Human Bone Marrow Stem Cells Expressing the Human Proenkephalin Gene in a Rat Model of Bone Cancer Pain.

Background. This study aimed to investigate the use of human bone marrow mesenchymal stem cells (hBMSCs) genetically engineered with the human proenkephalin (hPPE) gene to treat bone cancer pain (BCP) in a rat model. Methods. Primary cultured hBMSCs were passaged and modified with hPPE, and the cell suspensions (6 × 106) were then intrathecally injected into a rat model of BCP. Paw mechanical withdrawal threshold (PMWT) was measured before and after BCP. The effects of hPPE gene transfer on hBMSC bioactivity were analyzed in vitro and in vivo. Results. No changes were observed in the surface phenotypes and differentiation of hBMSCs after gene transfer. The hPPE-hBMSC group showed improved PMWT values on the ipsilateral side of rats with BCP from day 12 postoperatively, and the analgesic effect was reversed by naloxone. The levels of proinflammatory cytokines such as IL-1ß and IL-6 were ameliorated, and leucine-enkephalin (L-EK) secretion was augmented, in the hPPE-engineered hBMSC group. Conclusion. The intrathecal administration of BMSCs modified with the hPPE gene can effectively relieve pain caused by bone cancer in rats and might be a potentially therapeutic tool for cancer-related pain in humans.

Biphalin: The Foundation of Bivalent Ligands.

Seldom in medicinal chemistry does one ligand present the ability to study two separate phenomena in a pharmacological process. The discovery of biphalin with other homodimeric ligands has given scientists a tool that not only explores how to increase the efficacy of the ligand, but also explore the possible interactions of hetero and homo dimerization of the receptors themselves. As a straight ligand, biphalin has allowed scientists to increase efficacy by direct modification of the residues to affect the message-address interactions with receptors. This led to the exploration of ligand linkers to increase efficacy and it was this modification of the linkers led to discoveries that suggested dimerization of receptor system occurs as a secondary modulation of signal transduction. Even more recently, exploration of the advances in linkers through the discovery of bitopicity seems to modulate the actual receptors to increase the binding and signal transdcution of the ligand. This is accomplished by possible slight conformational changes in the receptors before binding of the ligand located at the end of the linker. These advances were made by the work of the late Prof. Andrzej W. Lipkowski. This review gives the foundation of biphalin and in turn celebrates the contributions of Prof. Lipkowski made in this area.

Local intramuscular injection of a plasmid encoding human proenkepahlin attenuates incision pain in rats.

We investigated the antinociceptive effect of local intramuscular injection of a plasmid encoding human proenkephalin (pVAX1-hPPE) on postoperative pain in rats. Male Sprague-Dawley rats with incision-induced pain were intramuscularly injected into injured plantaris muscle with empty vector (pVAX1) or pVAX1-hPPE, respectively. Paw mechanical threshold and thermal latency in the 200µg pVAX1-hPPE treated rats were significantly higher at 6h and on 1day, and lasted until day 7 after intramuscular administration, respectively. The analgesic effects were reversed by methylnaltrexone, suggesting that the antinociceptive effect of pVAX1-hPPE was mediated through peripheral opioid receptor pathway. In contrast, incisional or pVAX1-treated rats did not significantly affect pain thresholds. These results demonstrated that single intramuscular injection of pVAX1-hPPE attenuated incision-induced pain in rats, and it is worthy of further study as a potential gene therapy for postoperative pain.

Mixed MOP/DOP agonist biphalin elicits anti-transit effect in mouse models mimicking diarrhea-predominant irritable bowel syndrome symptoms.

BACKGROUND: Opioid receptors play a crucial role in the maintenance of homeostasis in the gastrointestinal (GI) tract. The aim of this study was to characterize the effect of biphalin, a mixed MOP/DOP agonist, on mouse intestinal contractility in vitro and GI motility in vivo and in animal models mimicking symptoms of diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: The effect of biphalin on muscle contractility in vitro was characterized in the ileum and colon. The anti-transit activity of biphalin in vivo was assessed in the following tests: whole gastrointestinal transit, colonic bead expulsion, fecal pellet output and castor oil-induced diarrhea, alone and in the presence of naloxone, and MOP and DOP antagonists. RESULTS: In vitro, biphalin (10(-10)-10(-6)M) inhibited colonic and ileal smooth muscle contractions in a concentration-dependent, opioid antagonist-reversible manner. In vivo, biphalin at the dose of 5mg/kg ip prolonged the whole GI transit and inhibited colonic bead expulsion. Biphalin reversed hypermotility and exerted anti-diarrheal effect in mouse models mimicking IBS-D symptoms. CONCLUSION: Biphalin is an interesting template for novel opioid-based agents to be used in therapy of functional GI diseases.

Biphalin protects against cognitive deficits in a mouse model of mild traumatic brain injury (mTBI).

Traumatic brain injury (TBI) is often a result of traffic accidents, contact sports or battlefield explosions. A mild form of traumatic brain injury (mTBI) is frequently underestimated, as the immediate physical symptoms decrease rapidly and conventional neuroimaging studies often do not show visible evidence of brain lesions. However, cognitive impairments persist for weeks, months or even years after the incident. Endogenous opioids were documented to play a role in thmodulation of mTBI pathology, whereas exogenous opioids were shown to possess neuroprotective properties. In the present study, biphalin, a dimeric enkephalin analog, improved cognitive performance in the Morris Water Maze and Novel Object Recognition tests in a mouse weight-drop model of mTBI. The effect of a single systemic injection of 10 mg/kg biphalin immediately after trauma was reversed by naltrexone, suggesting an opioid receptor-mediated mechanism. Biphalin also reduced cortical and hippocampal neurodegeneration, as shown by silver staining. Our data indicates that opioid receptor activation by biphalin may provide neuroprotection of post-traumatic neurodegeneration processes and may protect against memory impairments.

Biphalin analogs containing ß(3)-homo-amino acids at the 4,4' positions: Synthesis and opioid activity profiles.

Biphalin, a synthetic opioid octapeptide with a palindromic sequence has high analgesic activity. Biphalin displays a strong affinity for µ and δ-opioid receptors, and a significant to κ-receptor. The paper reports the synthesis of novel analogs of biphalin containing ß(3)-homo-amino acid residues at the 4,4' positions and a hydrazine or 1,2-phenylenediamine linker. The potency and selectivity of the peptides were evaluated by a competitive receptor-binding assay in rat brain homogenate using [(3)H]DAMGO (a µ ligand) and [(3)H]DELT (a δ ligand). Analogs with ß(3)-h-p-NO2Phe in positions 4 and 4' are the most active compounds. Selectivity depends on the degree of freedom between the two pharmacophore moieties. Analogs with a hydrazine linker show noticeable binding selectivity to µ receptors (IC50(µ)=0.72nM; IC50(δ)=4.66nM), while the peptides with a 1,2-phenylenediamine linker show slight δ selectivity (IC50(µ)=10.97nM; IC50(δ)=1.99nM). Tyr-d-Ala-Gly-ß(3)-h-p-NO2PheNHNH-ß(3)-h-p-NO2Phe (1) and (Tyr-d-Ala-Gly-ß(3)-h-p-NO2PheNH)2 (2) produced greater antinociceptive effect compared to morphine after i.t. administration.

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice.

N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (mu) and delta (delta) agonists, however, has not been reported. In these experiments, selective mu and delta receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of mu agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a delta agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a delta-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective mu and delta opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.

Antinociceptive effects of dynorphin peptides in a model of inflammatory pain.

Dynorphin A (DYN) peptides, administered into the central nervous system, have produced inconsistent analgesic actions in tests using thermal stimuli. This study examined antinociceptive effects of intravenous and intraplantar DYN-(2-17) against noxious pressure in rats with Freund's adjuvant-induced unilateral hindpaw inflammation. The effects of DYN-(2-17) were compared to those of the opioid agonists morphine. (D-Ala2,N-Methyl-Phe4,Gly-ol5)-enkephalin (DAMGO) and DYN-(1-17). Intravenous DYN-(2-17) (0.188-10 mg/kg) produced dose-dependent elevations of paw pressure thresholds in inflamed and in non-inflamed paws. These effects were similar in magnitude to those of subcutaneous morphine (2 mg/kg), at doses of 0.375-1.5 mg/kg they were significantly greater on the inflamed (right) than on the non-inflamed (left) paw, and they were not reversible by intravenous naloxone (1-10 mg/kg). Intraplantar Dyn-(2-17)(0.001-0.3 mg) was ineffective, whereas both intraplantar DYN-(1-17)(0.15-0.3 mg) and DAMGO (0.008-0.016 mg) produced dose-dependent and naloxone-reversible elevations of paw pressure thresholds. The intraplantar injection of both Dyn peptides produced a transient increase in the volume of non-inflamed paws. These findings suggest that intravenous DYN-(2-17) produces possibly centrally mediated, non-opioid antinociceptive effects against noxious pressure. At certain doses these effects are more potent in inflamed than in non-inflamed paws. In contrast to the opioid peptides DYN-(1-17) and DAMGO, DYN-(2-17) does not appear to have no peripheral antinociceptive actions.

Epidural opioid analgesia in infant rats I: mechanical and heat responses.

The aim of this study was to investigate the analgesic effects of epidural opioids in neonatal rat pups. The contribution of individual opioid receptor subtypes in the spinal cord to analgesia at different developmental stages was investigated using epidural mu (morphine sulphate), delta (DPDPE) and kappa (U69593) opioid receptor agonists in neonatal rats aged postnatal day (P) 3, 10 and 21. Thresholds for flexion withdrawal reflexes to mechanical stimuli (von Frey hairs) and to noxious heating of the hind paw were low in neonates and increased with postnatal age. The analgesic action of each opioid receptor agonist followed an individual developmental pattern. In mechanical tests, all three opioid agonists were considerably more efficacious analgesics in younger animals and ED50s at P3 were always lower than at P21. In heat tests, the pattern differed. The efficacy of the kappa opioid agonist decreased with postnatal age, morphine efficacy increased over the same period and the effects of the delta agonist remained relatively unchanged. The distribution and concentration of tritiated morphine in the spinal cord following epidural administration did not alter significantly with postnatal age, suggesting that opioid access is not a major determinant of the effects reported here. It is concluded that whereas heat pain is particularly sensitive to spinal kappa opioids in neonates, mechanical sensory thresholds are generally sensitive to all spinal opioids in the newborn. The differing epidural opioid requirements compared to older subjects is likely to be due to developmental changes in spinal cord opioid receptor distribution or pharmacology.

Epidural opioid analgesia in infant rats II: responses to carrageenan and capsaicin.

The aim of this study was to investigate the analgesic effects of epidural opioids upon persistent pain sensitivity in neonatal rat pups. Two models of persistent pain were used, subcutaneous injection of carrageenan, and topical application of capsaicin cream, both to the hind paw. The contribution of individual opioid receptor subtypes in the spinal cord to analgesia were tested at different developmental stages using epidural mu (morphine sulphate), delta (DPDPE) and kappa (U69593) opioid receptor agonists in neonatal rats aged P (postnatal day) 3, 10 and 21. Rat pups at all three ages displayed a reduction in mechanical (von Frey hair) threshold following carrageenan-induced inflammation of the hind paw that was evident at 3 h and was still present 5 h after application. This effect was greatest in magnitude at P21. This response was blocked by low doses of all three agonists at all ages, relative effectiveness varying with age. Comparison with potencies in acute tests (Marsh, D., Dickenson, A., Hatch, D. and Fitzgerald, M., Epidural opioid analgesia in infant rats I: mechanical and heat responses, Pain 82 (1999) 23-32) show that opioid potency is significantly greater in the presence of carrageenan inflammation at all ages. Topical capsaicin application to the hind paw produced a significant fall in withdrawal latencies to noxious heat. Generally, epidural opioid agonists did not block this C-fibre induced sensitization except at P3, when morphine and DPDPE did prevent the fall in threshold in a dose dependent manner. The results show that newborn rat pups are capable of displaying both allodynia and hyperalgesia following experimental inflammation that is blocked by epidural mu, delta and kappa opioids. The opioid potency is enhanced compared with antinociception in acute tests. This is not observed following capsaicin hyperalgesia and is therefore not a general consequence of C fibre induced increases in central excitability but relies upon mechanisms special to inflammatory pain.