Vector‐mediated release of GABA attenuates pain‐related behaviors and reduces NaV1.7 in DRG neurons

Pain is a common and debilitating accompaniment of neuropathy that occurs as a complication of diabetes. In the current study, we examined the effect of continuous release of gamma amino butyric acid (GABA), achieved by gene transfer of glutamic acid decarboxylase (GAD67) to dorsal root ganglia (DRG) in vivo using a non‐replicating herpes simplex virus (HSV)‐based vector (vG) in a rat model of painful diabetic neuropathy (PDN). Subcutaneous inoculation of vG reduced mechanical hyperalgesia, thermal hyperalgesia and cold allodynia in rats with PDN. Continuous release of GABA from vector transduced cells in vivo prevented the increase in the voltage‐gated sodium channel isoform 1.7 (Na_V1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in Na_V1.7 resulting from exposure to hyperglycemia. The effect of vector‐mediated GABA on Na_V1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABA_B receptor effect that was blocked by pertussis toxin‐(PTX) interference with Gα(_i/o) function. Taken in conjunction with our previous observation that continuous activation of delta opioid receptors by vector‐mediated release of enkephalin also prevents the increase in Na_V1.7 in DRG exposed to hyperglycemia in vitro or in vivo, the observations in this report suggest a novel common mechanism through which activation of G protein coupled receptors (GPCR) in DRG neurons regulate the phenotype of the primary afferent.     

Remote nerve injection of mu opioid receptor adeno-associated viral vector increases antinociception of intrathecal morphine.

We have shown previously that using recombinant adeno-associated viral vector (rAAV) to up-regulate mu opioid receptors (muORs) in dorsal root ganglia (DRGs) increases the potency of subcutaneous morphine. Here we report an improved method of introducing rAAV-muOR viral vectors into DRGs. Instead of injecting the rAAV-muOR gene directly into DRGs as shown before, the vector was introduced into the sciatic nerve of rats. Changes in muOR expression and antinociceptive effects of intrathecal morphine in rAAV-muOR rats were examined. Immunocytochemical studies showed that the transduced muORs were expressed in all types (ie, small, medium, and large) of DRG neurons. The expression of muORs in DRG neurons, quantified by Western blotting, was increased by 1.7-fold 4 weeks after the sciatic nerve injection. The up-regulation persisted for more than 6 months. The effects of intrathecal morphine on paw withdrawal latencies to heat were studied in rats inflamed with complete Freund's adjuvant. Compared with rats injected with rAAV containing the enhanced green fluorescent protein gene (rAAV-EGFP), the antinociceptive potency of intrathecal morphine in rAAV-muOR rats was significantly increased, and the effective dose (ED50) for morphine was 5.4-fold lower (rAAV-muOR: ED50 = 0.84 microg, confidence interval, 0.70-0.99 microg; rAAV-EGFP: ED50 = 4.50 microg, confidence interval, 3.55-5.86 microg). With minimum tissue damage and a large persistent increase in the opioid potency, remote nerve injection of rAAV-muOR to up-regulate muORs could be a useful therapeutic strategy for the treatment of chronic pain. PERSPECTIVE: Injection of adeno-associated viral vector containing the muOR gene into the sciatic nerve produces a significant up-regulation of muORs in DRGs for up to 6 months without producing any immune responses in the injected animals. This results in a 5.4-fold increase in the potency of intrathecal morphine.     

Tolerance to the antinociceptive and antiexudative effects of morphine in a murine model of peripheral inflammation

Opioids are used in humans in the management of chronic osteoarticular pains, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. Our aim was to characterize morphine tolerance in a murine model of arthritis that mimics the sequence of events occurring in humans. Inflammation was induced by the intraplantar injection of complete Freund's adjuvant (CFA) and tolerance by the implantation of a 75-mg morphine pellet. We assessed the antihyperalgesic (plantar and Randall-Selitto tests), antiallodynic (Von Frey test), and antiexudative (Evans blue) effects of morphine, the mu-opioid receptor (MOR) mRNA levels in dorsal root ganglia (DRG), and MOR protein levels in DRG and plantar tissue. Inflammation induced plasma extravasation, and it significantly increased the antihyperalgesic effects of morphine (p < 0.05). Morphine pellet implantation decreased morphine potency in all tests. ED(50) values decreased 4.4 and 7.3 times in the absence and presence of inflammation in the plantar test and 2.7 and 5.3 times in the Randall-Selitto test, whereas plasma extravasation decreased 4.2 times. MOR mRNA levels in the DRG were not affected 7 days after inflammation, whereas chronic morphine administration induced a discrete increase (p < 0.05). MOR protein in the DRG or the paw was unchanged. The results show that inflammation enhances the development of tolerance to the antihyperalgesic and antiexudative effects of morphine. At the molecular level, our results suggest that these effects are not mediated by changes in MOR expression but by other changes in receptor activation/internalization.     

Chronic pain and genetic background interact and influence opioid analgesia, tolerance, and physical dependence

Opioids are commonly used in the treatment of moderate to severe pain. However, their chronic use is limited by analgesic tolerance and physical dependence. Few studies have examined how chronic pain affects the development of tolerance or dependence, and essentially no studies have looked at the role of both genetics and pain together. For these studies we used 12 strains of inbred mice. Groups of mice from each strain were tested at baseline for morphine analgesic sensitivity, mechanical nociceptive threshold, and thermal nociceptive threshold. Mice were then given morphine in a 4-day escalating morphine administration paradigm followed by reassessment of the morphine dose-response relationship. Finally, physical dependence was measured by administering naloxone. Parallel groups of mice underwent hind paw injection of complete Freund's adjuvant (CFA) to induce chronic hind paw inflammation 7 days prior to the beginning of testing. The data showed that CFA treatment tended to lower baseline ED(50) values for morphine and enhanced the degree of analgesic tolerance observed after 4 days of morphine treatment. In addition, the degree of jumping behavior indicative of physical dependence was often altered if mice had been treated with CFA. The influence of background strain was substantial for all traits measured. In silico haplotypic mapping of the tolerance and physical dependence data demonstrated that CFA pretreatment altered the pattern of the predicted associations and greatly reduced their statistical significance. We conclude that chronic inflammatory pain and genetics interact to modulate the analgesic potency of morphine, tolerance, and physical dependence.     

Effects of the bisphosphonate ibandronate on hyperalgesia,substance P,and cytokine levels in a rat model of persistent inflammatory pain

The anti‐inflammatory and analgesic properties of different bisphosphonates have been demonstrated in both animal and human studies. Ibandronate is a third‐generation bisphosphonate effective in managing different types of bone pain. In this study we investigated its effects in a standard pre‐clinical model of inflammatory pain. We evaluated the effects of a single injection of different doses (0.5, 1.0, and 2.0mg/kg i.p.) of ibandronate on inflammatory oedema and cutaneous hyperalgesia produced by the intraplantar injection of complete Freund's adjuvant (CFA) in the rat hind‐paw. In addition, we measured the effects of this drug (1.0mg/kg i.p.) on hind‐paw levels of different pro‐inflammatory mediators (PGE‐2, SP, TNF‐α, and IL‐1β). We also measured the levels of SP protein and of its mRNA in the ipsilateral dorsal root ganglia (DRG). Ibandronate proved able to reduce the inflammatory oedema, the hyperalgesia to mechanical stimulation, and the levels of SP in the inflamed tissue as measured 3 and 7 days following CFA‐injection. This drug significantly reduced the levels of TNF‐α and IL‐1β only on day 7. On the other hand, the levels of PGE‐2 in the inflamed hind‐paw were unaffected by the administration of this bisphosphonate. Finally, ibandronate blocked the overexpression of SP mRNA in DRG induced by CFA‐injection in the hind‐paw. These data help to complete the pharmacodynamic profile of ibandronate, while also suggesting an involvement of several inflammatory mediators, with special reference to substance P, in the analgesic action of this bisphosphonate.     

Transgene-mediated enkephalin release enhances the effect of morphine and evades tolerance to produce a sustained antiallodynic effect in neuropathic pain

We examined the pharmacologic characteristics of herpes simplex virus (HSV) vector-mediated expression of proenkephalin in the dorsal root ganglion in a rodent model of neuropathic pain. We found that: (i). vector-mediated enkephalin produced an antiallodynic effect that was reversed by naloxone; (ii). vector-mediated enkephalin production in animals with spinal nerve ligation prevented the induction of c-fos expression in second order sensory neurons in the dorsal horn of spinal cord; (iii). the effect of vector-mediated enkephalin enhanced the effect of morphine, reducing the ED(50) of morphine 10-fold; (iv). animals did not develop tolerance to the continued production of vector-mediated enkephalin over a period of several weeks; and, (v). vector transduction continued to provide an analgesic effect despite the induction of tolerance to morphine. This is the first demonstration of gene transfer to provide an analgesic effect in neuropathic pain. The pharmacologic analysis demonstrates that transgene-mediated expression and local release of opioid peptides produce some effects that are distinct from peptide analogues delivered pharmacologically.     

Inhibition of morphine tolerance by spinal melanocortin receptor blockade

Chronic use of morphine is accompanied by the development of morphine tolerance, which is one of the major problems associated with opiate treatment. Possible modulation of opioid effects by melanocortin receptor ligands has been recently demonstrated. Therefore, we investigated the influence of repeated intrathecal injection of a melanocortin receptor antagonist (SHU9119, JKC-363) on the development of morphine tolerance as measured by tail-flick test. It was also examined whether a single i.t. SHU9119 and JKC-363 administration could counteract the loss of analgesic potency of morphine in morphine tolerant rats. We examined also the influence of chronic morphine administration on mu-opioid receptor (MOR) and melanocortin 4 receptor (MC4-R) mRNAs in the rat spinal cord and dorsal root ganglia (DRG) during morphine tolerance. Morphine treatment (10mg/kg, i.p. twice daily) over 8 days induced tolerance as reflected by a significant reduction of withdrawal latency from 181 to 25% above baseline in the tail-flick test. Repeated co-administration of morphine and SHU9119 or JKC-363, significantly prevented the development of morphine tolerance. A single administration of an MC4-R antagonist restored morphine analgesic potency in morphine tolerant rats. Using RT-PCR we demonstrated no changes in the spinal cord but there was a decrease in MOR and increase in MC4-R gene expression in the DRG of rats tolerant to morphine. These results suggest that MC4-R may be involved in the mechanisms of opioid tolerance and antagonists of this receptor may be a possible new target in the search for strategies preventing the development of opioid tolerance.     

Role of spinal 5-HT(1A) receptors in morphine analgesia and tolerance in rats.

We here studied the involvement of spinally located 5-HT(1A) and opioid receptors, in the paradoxical effects that their activation can produce on nociception. Intrathecal (i.t.) injection of the 5-HT(1A) receptor agonist 8-hydroxy-2-[di-n-propylamino] tetralin (8-OH-DPAT) (1-10 microg) induced analgesic effects in the formalin model of tonic pain whereas in the paw pressure test, it decreased the vocalization threshold. In this latter test, i.t. 8-OH-DPAT also markedly reduced the analgesic effect of systemic morphine (5-10 mg/kg, s.c.). At 10 microg, 8-OH-DPAT totally abolished the effect of 5 mg/kg of morphine; this inhibitory effect was antagonized by pre-treatment with 0.63 mg/kg of the 5-HT(1A) antagonist WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl)-cyclohexanecarboxamide-trihydrochloride). In contrast, the i.t. injection of WAY-100635 (1-10 microg) dose-dependently potentiated the antinociceptive activity of a dose of morphine (2.5 mg/kg, s.c.). Furthermore, WAY-100635 (10 microg, i.t.) potentiated morphine analgesia in morphine-tolerant rats. These findings demonstrate that 5-HT(1A) receptor agonists can act in the spinal cord to produce both hyper- and hypo-algesic effects and play a major role in the opioid analgesia and tolerance.     

Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post‐spinal nerve ligation or post‐sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site‐specifically following L5 spinal nerve ligation. Real‐time RT—PCR, immunohistochemistry, and Western blot analysis demonstrated a down‐regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR‐positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post‐nerve injury and in the L4 and L5 spinal cord on day 35 post‐nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain‐associated behaviors and opioid analgesia.     

Effects of Cannabinoid Type 2 Receptor Agonist AM1241 on Morphine-Induced Antinociception,Acute and Chronic Tolerance,and Dependence in Mice

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Previous studies showed that cannabinoid type 2 (CB₂) receptor ligands may modulate opioid effects. However, there is no report of the effect of CB₂ receptor agonist on acute morphine tolerance and physical dependence. We therefore investigated the effect of a CB₂ receptor agonist (AM1241) on morphine-induced morphine tolerance and physical dependence in mice. Repeated coadministration of AM1241 (1 or 3mg/kg intraperitoneally) and morphine (10mg/kg subcutaneously) for 7days increased the mechanical paw withdrawal threshold in mice as measured by the von Frey filament test, and 3mg/kg AM1241 in combination with morphine increased the thermal paw withdrawal latency as measured by the hot-plate test. Combination with 3mg/kg AM1241 and morphine increased acute morphine antinociception. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced acute morphine tolerance, and 3mg/kg AM1241 reduced chronic morphine tolerance. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced naloxone-precipitated withdrawal jumping, but not diarrhea. Coadministration of AM1241 and morphine did not inhibit spontaneous locomotor activity. Pretreatment with 3mg/kg AM1241 decreased the chronic morphine-induced Iba1 expression in spinal cord. Coadministration of AM1241 (3 mg/kg) reduced the production of interleukin-1β, tumor necrosis factor-α, and interleukin-6 induced by long-term and acute morphine treatment. Our findings suggest that the coadministration of the CB₂ receptor agonist and morphine could increase morphine antinociception and reduce morphine tolerance and physical dependence in mice.Perspective:The combination of a CB₂ agonist and morphine may provide a new strategy for better treatment of acute and chronic pain and prevention of opioid tolerance and dependence. This finding may also provide a clue for the treatment of opioid tolerance and dependence in clinics.     

Antinociceptive effect of the cannabinoid agonist,WIN 55,212‐2, in the orofacial and temporomandibular formalin tests

Orofacial pain disorders are frequent in the general population and their pharmacological treatment is not always adequately resolved. Cannabinoids have demonstrated their analgesic effect in several pain conditions, both in animal models and in clinical situations. The aim of the present study was to evaluate the cannabinoid‐mediated antinociception in two inflammatory models of orofacial pain (orofacial and temporomandibular joint (TMJ) formalin test) and to compare it with a spinal inflammatory model (paw formalin test). WIN 55,212‐2 (0.5, 1 mg/kg), a synthetic cannabinoid agonist, was intraperitoneally (i.p.) administered prior to formalin and significantly reduced the nociceptive behavioural responses in these inflammatory tests. To elucidate which subtype of receptor could be involved in such effect, two selective cannabinoid antagonists were administered prior to WIN. SR141716A (1 mg/kg i.p.), the CB1 receptor‐selective antagonist, was able to prevent the cannabinoid‐induced analgesia in all three models, whereas SR144528 (1 mg/kg i.p.), the CB2 receptor‐selective antagonist, only prevented it in the paw formalin test. A comparison with the antinociceptive effects of morphine (2.5, 5, 10 mg/kg, i.p.), indomethacin (2.5, 5 mg/kg, i.p.) and ketamine (25, 50 mg/kg, i.p.) was also performed. Morphine displayed a dose‐dependent reduction of acute and inflammatory pain in all three models, whereas indomethacin and ketamine only attenuated inflammatory pain at the highest tested doses. These results indicate that the cannabinoid‐induced antinociception in the orofacial region is mediated by activation of CB1 cannabinoid receptor. Moreover WIN was as effective as morphine and more effective than indomethacin and ketamine, in oral inflammatory pain.     

The functional expression of mu opioid receptors on sensory neurons is developmentally regulated; morphine analgesia is less selective in the neonate

Opioid requirements in neonatal patients are reported to be lower than older infants and this may be a reflection of the developmental regulation of opioid receptors. In this study we have investigated the postnatal regulation of Mu opioid receptor (MOR) function in both rat lumbar dorsal root ganglion (DRG) cultures and behavioural mechanical and thermal reflex tests in rat pups. Immunostaining with MOR and selective neurofilament (NF200) antibodies was combined with calcium imaging of MOR function in cultured neonatal and adult rat dorsal root ganglion cells. Calcium imaging showed that a significantly greater number of neonatal DRG neurons expressed functional MOR compared to adult (56.5+/-3.4 versus 39.9+/-1.5%, n=8, mean+/-SEM, P<0.001). This expression is confined to the large, neurofilament positive sensory neurons, while expression in small, nociceptive, neurofilament negative neurons remains unchanged. Sensory threshold testing in rat pups showed that the analgesic potency of systemic morphine to mechanical stimulation is significantly greater in the neonate and declines with postnatal age. Morphine analgesic potency in thermal nociceptive tests did not change with postnatal age. These experiments show that the MOR expressed on large DRG neurons in neonates are functional and are subject to postnatal developmental regulation. This changing functional receptor profile is consistent with greater morphine potency in mechanical, but not thermal, sensory tests in young animals. These results have important clinical implications for the use of morphine in neonates and provide a possible explanation for the differences in morphine requirements observed in the youngest patients.     

Opioid receptor–triggered spinal mTORC1 activation contributes to morphine tolerance and hyperalgesia

The development of opioid-induced analgesic tolerance and hyperalgesia is a clinical challenge for managing chronic pain. Adaptive changes in protein translation in the nervous system are thought to promote opioid tolerance and hyperalgesia; however, how opioids drive such changes remains elusive. Here, we report that mammalian target of rapamycin (mTOR), which governs most protein translation, was activated in rat spinal dorsal horn neurons after repeated intrathecal morphine injections. Activation was triggered through μ opioid receptor and mediated by intracellular PI3K/Akt. Spinal mTOR inhibition blocked both induction and maintenance of morphine tolerance and hyperalgesia, without affecting basal pain perception or locomotor functions. These effects were attributed to the attenuation of morphine-induced increases in translation initiation activity, nascent protein synthesis, and expression of some known key tolerance-associated proteins, including neuronal NOS (nNOS), in dorsal horn. Moreover, elevating spinal mTOR activity by knocking down the mTOR-negative regulator TSC2 reduced morphine analgesia, produced pain hypersensitivity, and increased spinal nNOS expression. Our findings implicate the μ opioid receptor–triggered PI3K/Akt/mTOR pathway in promoting morphine-induced spinal protein translation changes and associated morphine tolerance and hyperalgesia. These data suggest that mTOR inhibitors could be explored for prevention and/or reduction of opioid tolerance in chronic pain management.     

Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion

Endogenous opiate peptides acting pre- and post-synaptically in the dorsal horn of spinal cord inhibit transmission of nociceptive stimuli. We transfected neurons of the dorsal root ganglion in vivo by footpad inoculation with 30 microl (3 x 10(7) p.f.u.) of a replication-incompetent (ICP4-deleted) herpes simplex virus (HSV) vector with a cassette containing a portion of the human proenkephalin gene coding for 5 met- and 1 leu-enkephalin molecules under the control of the human cytomegalovirus immediate-early promoter (HCMV IEp) inserted in the HSV thymidine kinase (tk) locus. Vector-directed expression of enkephalin produced a significant antinociceptive effect measured by the formalin footpad test, that was most prominent in the delayed ("tonic") phase 20-70 min after the administration of formalin. The magnitude of the antinociceptive effect diminished over 4 weeks after transduction, but reinoculation of the vector reestablished the analgesic effect, without evidence for the development of tolerance. The antinociceptive effect was blocked completely by intrathecal naltrexone. These results suggest that gene therapy with an enkephalin-producing herpes-based vector may prove useful in the treatment of pain.     

Naloxone blocks the formalin-induced increase of substance P in the dorsal horn

Substance P (SP) has been proposed as a mediator of nociception in the dorsal horn of the spinal cord. Activation of nociceptive pathways by stimuli such as formalin injected into the hind paw has been shown to produce an increase in the amount of immunoreactive SP in primary afferent neurons. Opiate agonist and antagonist binding in the dorsal horn has been shown to affect the SP levels and release. In order to determine the effects of opiates on SP mediated nociception in the spinal cord, anesthetized rats pretreated subcutaneously with morphine, naloxone, or saline were injected in the right hind paw with 0.4 ml of either saline of 5% formalin. After 1 h, the animals were perfused and the lumbar enlargement of the spinal cord removed. SP-like immunoreactivity (SPLI) in the dorsal horns was quantitated using immunohistochemical staining and manual photometry. The results show that formalin injection increases the SPLI in the dorsal horn after 1 h, as does pretreatment with morphine. Morphine pretreatment combined with formalin injection further increases SPLI, but not significantly higher than either treatment alone. The morphine-induced increases could be blocked by naloxone, which had no effects on saline-treated controls. Most importantly, naloxone was able to block the formalin-induced increase in SPLI, implying that endogenous opioid systems play a role in the SP increases seen during formalin-induced nociception.     

Anti-allodynic effects of peripheral delta opioid receptors in neuropathic pain

The analgesic effects of local administration of opioid agonists into peripheral tissues in alleviating pain have been well documented in both clinical and preclinical studies, although few studies have examined their effects in neuropathic pain. In this study, we investigated the anti-allodynic effects of peripherally acting delta opioid receptor (DOR) agonists in a rat model of neuropathic pain. Peripheral nerve injury (PNI) produced a time-dependent decrease in mechanical withdrawal thresholds that was attenuated by local administration into the hind paw of either morphine or the DOR agonist deltorphin II. Using Western blotting techniques, no change in DOR protein expression was detected in DRG ipsilateral to the site of injury compared to contralateral. However, an up-regulation of DOR protein was found in neuropathic DRG compared to sham, suggesting that there may be a bilateral increase in the expression of DOR following PNI. Results obtained from immunohistochemical studies confirmed up-regulation in small and large DRG neurons in neuropathic compared to sham animals. Additionally, there was an increase in DOR protein within the ipsilateral sciatic nerve of neuropathic animals compared to sham and contralateral neuropathic conditions indicating the occurrence of receptor trafficking to the site of injury. Taken together, our findings suggest that functional peripheral DORs are present in sensory neurons following PNI and validate the development of selective DOR agonists for alleviating neuropathic pain.     

Engineering an endomorphin-2 gene for use in neuropathic pain therapy

Endomorphin-2 (EM-2) is a carboxy-amidated tetrapeptide that binds the mu-opioid receptor with high affinity and is analgesic in several animal models of pain. Endomorphin peptides have been isolated from bovine and human brain, but no DNA sequences corresponding to a potential preproendomorphin gene have been identified in human genome sequence databases. In this study we designed a tripartite synthetic gene to direct production, cleavage, and amidation of EM-2, and placed the endomorphin gene expression cassette in a replication defective Herpes simplex virus (HSV) vector (vEM2). Biosynthesis of amidated endomorphin-2 peptide was quantified by radioimmunoassay and the identity confirmed by mass spectroscopy following vEM2 transduction of cultured primary dorsal root ganglion neurons. Subcutaneous inoculation of vEM2 resulted in vector delivery to dorsal root ganglion where expression of EM-2 peptide from the engineered gene was confirmed by ELISA. vEM2 delivery provided an analgesic effect in the spinal nerve ligation model of neuropathic pain measured by reduction of mechanical allodynia and thermal hyperalgesia. The analgesic effect of vEM2 was blocked by intrathecal delivery of the mu-receptor antagonist CTOP. The gene construct design described represents a broadly useful platform for biosynthesis and delivery of carboxy-amidated peptides for therapeutic and experimental purposes, and the results demonstrate that HSV-gene transfer to sensory neurons provides an effective means to achieve local biosynthesis of endomorphin peptides for the treatment of chronic pain.     

Differential behavioral effects of peripheral and systemic morphine and naloxone in a rat model of repeated acute inflammation. (Unité de Physiopharmacologie du Système Nerveux, Paris, France) Anesthesiology. 2001;94:870–875.

This study compared the behavioral effects of intraplantar and intravenous morphine and naloxone in a rat model of repeated acute carrageenan‐induced inflammation in which enhanced response to noxious stimuli result from sensitization in peripheral tissues or central sensitization. After the first carrageenan injection, intraplantar and intravenous morphine produced significant increase of vocalization thresholds to paw pressure in inflamed, but not in noninflamed paws. After the second carrageenan injection, the antinociceptive effects of intraplantar morphine were significantly reduced compared with those obtained after the first carrageenan injection, whereas effects of intravenous morphine were significantly enhanced and present in both hind paws. Intravenous naloxone demonstrated similar pronociceptive patterns after the first and second carrageenan injection. Intraplantar naloxone methiodide produced pronociceptive effects in inflamed hind paw that were significantly enhanced after the second carrageenan injection. Conclude that when the inflammation is enhanced by recurrent stimulations, the antinociceptive effects of systemic morphine are enhanced. This increase is more likely related to central than peripheral sites of action, beyond endogenous opioid system activation. Comment by Octavio Calvillo, M.D., Ph.D. There is evidence that opioid antinociception is enhanced in the presence of inflammation. In fact, there is evidence that opioids produce analgesia through peripheral opioid receptors within inflamed tissue. Primary afferent neurons are known to contain opioid receptors. It remains unclear whether the antinociceptive effects of opioids are related predominantly to central, peripheral sites of action or both. The authors compared the effects of intraplantar and intravenous morphine and naloxone in a rat model of carrageenan‐induced inflammation. In this model, hyperalgesia is due probably to both peripheral and central sensitization. The antinociceptive effects of intraplantar morphine, intravenous morphine, intraplantar naloxone, and intravenous naloxone were assessed on the vocalization thresholds to noxious pressure 3 hours after carrageenan plantar injections. Intraplantar and intravenous morphine increased the vocalization thresholds after carrageenan injection in inflamed but not on noninflamed paws. The antinociceptive effects of intraplantar morphine were reduced compared to the first response whereas the effects of intravenous morphine were significantly enhanced and present on both inflamed and noninflamed paws. Intravenous naloxone demonstrated similar pronociceptive patterns after the first and second carrageenan injection. Intraplantar naloxone produced pronociceptive effects in inflamed hind paws that were significantly enhanced after the second carrageenan injection. The authors concluded that when inflammation is enhanced by recurrent stimulation the antinociceptive effects of systemic morphine are enhanced. This is more probably related to central than to peripheral sites of action.     

Spinal DN-9, a Peptidic Multifunctional Opioid/Neuropeptide FF Agonist Produced Potent Nontolerance Forming Analgesia With Limited Side Effects

The development of multitarget opioid drugs has emerged as an attractive therapeutic strategy to eliminate opioid-related side effects. Our previous study developed a series of opioid and neuropeptide FF pharmacophore-containing chimeric peptides, including DN-9 (Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH₂), which produced potent nontolerance forming analgesia at the supraspinal level. In the present study, the antinociceptive effects of DN-9 in a series of preclinical pain models and the potential side-effects were investigated at the spinal level in mice. In the tail-flick test, intrathecal injection of DN-9 produced potent analgesia with an ED₅₀ value at 1.33 pmol, and the spinal antinociception of DN-9 was mainly mediated by μ- and κ-opioid receptors. In addition, DN-9-induced spinal antinociception was augmented by the neuropeptide FF receptors antagonist. Furthermore, DN-9 could decrease both the frequency and amplitude of sEPSCs in lamina IIo neurons of the spinal cord, which were mediated by opioid receptors. In contrast to morphine, chronic intrathecal treatments with DN-9 did not induce analgesic tolerance, c-Fos expression or microglial activation. Intrathecal injection of DN-9 showed potent analgesia with antinociceptive ED₅₀ values between .66 and 55.04 pmol in different pain models, including the formalin test, acetic acid-induced writhing test, carrageenan-induced inflammatory pain and neuropathic pain. Moreover, DN-9 did not show side effects in locomotor function and coordination, gastrointestinal transit inhibition, the cardiovascular system, and body temperature regulation at antinociceptive doses. Taken together, the present study showed DN-9 produced effective, nontolerance forming analgesia with reduced side effects at the spinal level. DN-9 might be a promising compound for developing multifunctional opioid analgesics with limited adverse effects.PerspectiveThis article presents the potent and nontolerance forming analgesia effects of DN-9 in a series of preclinical pain models with less opioid related adverse effects at the spinal level in mice. This study also demonstrates that DN-9 has translational potential into an intrathecal analgesic.