endomorphin-2 and Neuralgia

Substance P (SP) is associated with pain and inflammatory processes and is released from terminals of specific sensory nerves. This undecapeptide that mediates its effects through the neurokinin type 1 (NK1) receptor, is rapidly degraded in vivo to smaller fragments. The heptapeptide SP(1-7) with a hitherto unknown receptor, is a major bioactive fragment and displays often opposite actions to those induced by SP. Hence, SP(1-7) elicits anti-nociceptive and anti-hyperalgesic effects. These observations have attracted a substantial interest and in this mini-review the efforts to transform the heptapeptide SP(1-7) into more drug-like small-molecule SP(1-7) peptidomimetics as a potential new class of analgesics are summarized. Structure-activity relationship studies and subsequent amidation of the C-terminal and truncations from the N-terminal of the heptapeptide delivered the bioactive dipeptide amide Gln- Phe-NH2 showing a high affinity at the SP(1-7) binding site. Similarly, endomorphin-2, an endogenous opioid ligand containing a C-terminal carboxamide group, demonstrated a high affinity at the SP(1-7) binding site. Endomorphin-2 subjected to truncations yielded the potent dipeptide amide Phe-Phe-NH2. Structural optimization of the latter furnished more drug-like high affinity ligands and among those a constrained cis-3-phenylpyrrolidine derivative that after peripheral administration produced a significant anti-allodynic effect in a mouse SNI model of neuropathic pain. This SP(1-7) peptidomimetic was as effective as SP(1-7) in alleviating mechanical allodynia in mice. Although, additional structural modifications are needed to achieve compounds exhibiting high/fair bioavailability after oral administration, the examples presented herein demonstrate that the bioactive peptides SP(1-7) and endomorphin-2 can be converted into low molecular weight compounds that are able to mimic the in vivo actions of the heptapeptide SP(1-7).

Topics: Analgesics; Animals; Binding Sites; Dipeptides; Humans; Hyperalgesia; Neuralgia; Oligopeptides; Peptide Fragments; Peptidomimetics; Protein Conformation; Structure-Activity Relationship; Substance P

Spinal analgesic effects of endomorphin-1 and endomorphin-2 were studied during acute, inflammatory, and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5-10 micrograms i.t.), as well as their analogues, increased the tail-flick and the paw pressure latencies. In a model of inflammatory pain, the formalin-induced behavior was attenuated by endomorphins; however, the effect studied was not dose-dependent and was less pronounced in comparison with that evoked by morphine. On the other hand, in rats with a sciatic nerve injury (crush), endomorphins antagonized allodynia in a dose-dependent manner, whereas morphine was found to be ineffective in a similar dose range. Endomorphins also exhibited an antinociceptive potency in rats tolerant to morphine. In conclusion, our results show a powerful analgesic action of endomorphins at the spinal level. The most interesting finding is a strong effect of endomorphins in neuropathic pain, which opens up a possibility of using these compounds in pain therapy.

Topics: Analgesics, Opioid; Animals; Inflammation; Neuralgia; Oligopeptides; Pain; Pain Threshold; Rats; Spinal Cord

The present study was undertaken to further investigate the spinal anti-allodynic effects of endomorphins (EMs) and their C-terminal hydrazide modified analogs EM-1-NHNH

Topics: Analgesics, Opioid; Animals; Azides; Disease Models, Animal; Hyperalgesia; Injections, Spinal; Male; Mice; Narcotic Antagonists; Neuralgia; Oligopeptides; Pain Measurement; Receptors, Opioid, mu

The spared nerve injury (SNI) model is a new animal model that can mimic several characteristics of clinical neuropathic pain. Opioids are recommended as treatment of neuropathic pain. Therefore, the present study was conducted to investigate the antinociceptive effects of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given centrally and peripherally in the SNI model of neuropathic pain in mice.. The SNI model was made in mice by sparing the sural nerve intact, when the other 2 of 3 terminal branches of the sciatic nerve (common peroneal and tibial nerves) were tightly ligated and cut. Von Frey monofilaments were used to measure the SNI-induced mechanical allodynia-like behavior. The antiallodynic effects of EM-1 and EM-2 were determined after central and peripheral administration in the SNI model of neuropathic pain. Also, the specific opioid receptor antagonists were used to determine the opioid mechanisms of EMs involved in neuropathic pain. Values were expressed as the mean ± standard deviation.. Our results showed that the SNI mice developed prolonged mechanical allodynia-like behavior in ipsilateral paw after surgery, with the withdrawal threshold value being 0.061 ± 0.02 g after 14 days. EM-1 and EM-2 produced significant antiallodynic effects in ipsilateral paw after intracerebroventricular (i.c.v.) administration, more effective than that of morphine. The peak withdrawal thresholds of 10 nmol EM-1 and EM-2 determined at 5 minutes after injection were 0.92 ± 0.36 and 0.87 ± 0.33 g, respectively, higher than that of morphine (0.46 ± 0.20 g). Moreover, both EMs (10 nmol, i.c.v.) exerted significant antiallodynic effects in the contralateral paw, whereas no significant antinociceptive activity was seen after i.c.v. administration of morphine with equimolar dose. It was noteworthy that EM-1 and EM-2 produced antinociception through distinct μ1- and μ2-opioid receptor subtypes, and the EM-2-induced antiallodynia contained an additional component that was mediated by the release of endogenous dynorphin A, acting on κ-opioid receptor. In addition, the antiallodynic activities of peripheral administration of EM-1, EM-2, and morphine were also investigated. Intraplantar, but not subcutaneous administration of EM-1 and EM-2 also exhibited potent antinociception, establishing the peripheral and local effects. Both μ1- and μ2-opioid receptor subtypes, but not the δ- or κ-opioid receptors were involved in the peripheral antiallodynia of EMs.. The present investigation demonstrated that both EM-1 and EM-2 given centrally and peripherally produced potent antiallodynic activities in SNI mice, and differential opioid mechanisms were involved.

Topics: Analgesics, Opioid; Animals; Disease Models, Animal; Hyperalgesia; Male; Mice; Neuralgia; Oligopeptides; Pain Measurement; Peroneal Nerve; Sural Nerve; Tibial Nerve; Treatment Outcome

Activation of the mu-opioid receptor provides the gold standard for pain relief, but most opioids used clinically have adverse effects that have contributed to an epidemic of overdose deaths. We recently characterized mu-opioid receptor selective endomorphin (EM) analogs that provide potent antinociception with reduction or absence of a number of side effects of traditionally prescribed opioids including abuse liability, respiratory depression, motor impairment, tolerance, and inflammation. The current study explores the effectiveness of these EM analogs relative to morphine in four major pain models by intrathecal as well as intravenous administration in male Sprague Dawley rats and subcutaneous administration in male CD-1 mice. In the spared nerve injury model of neuropathic pain, mechanical allodynia and mechanical hyperalgesia were assessed with von Frey and Randall-Selitto tests, respectively. In the paw incision model of postoperative pain, von Frey testing was used to assess mechanical allodynia and thermal hyperalgesia was evaluated with Hargreaves testing. In the Complete Freund's Adjuvant model of inflammatory pain, thermal hyperalgesia was assessed using Hargreaves testing. In CD-1 mice, visceral pain was assessed with the acetic acid writhing test. In all cases, EM analogs had equal or greater potency and longer duration of action relative to morphine. The data suggest that EM analogs, particularly analog 4 (ZH853), could provide effective therapy for a diverse spectrum of pain conditions with low risk of adverse side effects compared with currently used opioids such as morphine.. Novel EM analogs show equal or greater potency and effectiveness relative to morphine in multiple pain models. Together with substantially reduced side effects, including abuse liability, the compounds show promise for addressing the critical need for effective pain relief as well as reducing the opioid overdose epidemic.

Topics: Analgesics, Opioid; Animals; Disease Models, Animal; Hyperalgesia; Inflammation; Injections, Intravenous; Injections, Spinal; Male; Mice; Morphine; Neuralgia; Nociceptive Pain; Oligopeptides; Pain, Postoperative; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Visceral Pain

Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the neural mechanisms underlying CNP remain unclear. There is increasing evidence implicating the involvement of spinal endomorphin-2 (EM2) in neuropathic pain. In this study, we used a vincristine-evoked rat CNP model displaying mechanical allodynia and central sensitization, and observed a significant decrease in the expression of spinal EM2 in CNP. Also, while intrathecal administration of exogenous EM2 attenuated allodynia and central sensitization, the mu-opioid receptor antagonist β-funaltrexamine facilitated these events. We found that the reduction in spinal EM2 was mediated by increased activity of dipeptidylpeptidase IV, possibly as a consequence of chemotherapy-induced oxidative stress. Taken together, our findings suggest that a decrease in spinal EM2 expression causes the loss of endogenous analgesia and leads to enhanced pain sensation in CNP.

Topics: Animals; Antineoplastic Agents, Phytogenic; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Electrophysiology; Fluorescent Antibody Technique; Hyperalgesia; Immunoblotting; Immunoenzyme Techniques; Injections, Spinal; Male; Neuralgia; Oligopeptides; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Spinal Cord; Vincristine

To construct a transgene expressing human endomorphin-2 by linking the signal peptide of mouse nerve growth factor (PN) to a human endomorphin-2 DNA sequence containing a short linker recognized by the protease FURIN and test the analgesic effect of endomorphin-2 on neuropathic pain.. The transgene was inserted into the cosmid pAxCAwt to generate PN-EM-2-pAxCAwt. The recombinant adenovirus Ad-PNEM2 was packaged and propagated in HEK293 cells. After the Ad-PNEM2-infected NIH3T3 cells had been cultured, protein expression was examined by immunofluorescence and ELISA. A CCI rat model was constructed and the Ad-PNEM2 was administered intrathecally. The rats' pain thresholds (PWL) were measured and the presence of endomorphin-2 in the cerebrospinal fluid was confirmed through ELISA.. The Ad-PNEM2 expressed endomorphin-2 smoothly and abundantly in NIH3T3 cells at a significantly higher rate than the viral control (P<0.01) or blank control (P<0.01). The expressed endomorphin-2 was mainly observed in the cytoplasm. The concentration of endomorphin-2 in the cerebrospinal fluid increased 1 day after injection and peaked between 7 and 14 days after injection. After injection, PWL approached normal levels in the operated study group. No significant change was observed in the control groups. There was a significant correlation between PWL and endomorphin-2 level (r = 0.944, P<0.001).. The constructed human endomorphin-2 transgene was expressed effectively, and endomorphin-2 expressed by the recombinant adenovirus altered the threshold to thermal stimulus and showed good analgesic effect.

Topics: Adenoviridae; Animals; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Injections, Spinal; Male; Mice; Nerve Growth Factor; Neuralgia; NIH 3T3 Cells; Oligopeptides; Pain Measurement; Pain Threshold; Protein Sorting Signals; Rats; Rats, Sprague-Dawley; Reaction Time; Time Factors; Transfection

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.

Topics: Animals; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Ganglia, Spinal; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; Hyperalgesia; Ligation; Male; Mass Spectrometry; Neuralgia; Oligopeptides; Pain Measurement; Pain Threshold; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Spinal Nerves

Nerve injury often leads to chronic, sometimes excruciating, pain. The mechanisms contributing to this syndrome include neurochemical plasticity in neurons involved in the earliest stages of pain transmission. Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) is an endogenous morphine-like substance that binds to the mu-opioid receptor with high affinity and selectivity. Endomorphin-2-like immunoreactivity (LI) is present in the superficial layers of the dorsal horn in the spinal cord and in primary afferents, suggesting a role for this peptide in pain transmission. To determine whether spinal endomorphin-2-LI is altered in an animal model of chronic pain, the left sciatic nerve of Swiss Webster and ICR mice was ligated in a modified Seltzer model of nerve injury. Changes in endomorphin-2-LI were assessed by immunocytochemistry at 2, 4 and 14 days after nerve injury. The side of the spinal cord ipsilateral to the nerve injury exhibited a dramatic decrease in endomorphin-2-LI relative to the contralateral side and to control animals. The change was restricted to the medial dorsal horn in the lumbar segments innervated by the sciatic nerve. Substance P-LI showed a small decrease, while calcitonin gene-related peptide-LI was unchanged. Both thermal hyperalgesia, as evidenced by significantly decreased paw withdrawal latencies, and decreased endomorphin-2-LI were observed within 2 days of injury and were most pronounced at 2 weeks after injury. The decrease in endomorphin-2-LI during the development of chronic pain is consistent with the loss of an inhibitory influence on pain transmission. These results provide the first evidence that reduction of an endogenous opioid in primary afferents is associated with injury-induced chronic pain.

Topics: Animals; Calcitonin Gene-Related Peptide; Chronic Disease; Down-Regulation; Functional Laterality; Hyperalgesia; Immunohistochemistry; Ligation; Male; Mice; Mice, Inbred ICR; Nerve Crush; Neuralgia; Oligopeptides; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Posterior Horn Cells; Reaction Time; Sciatic Nerve; Substance P

Orphanin FQ (OFQ) and endomorphins (EM) are newly characterized members of opioid peptide family. OFQ has been shown to antagonize morphine analgesia at supraspinal level, whereas endomorphins are highly selective endogenous ligands for mu receptor, showing analgesic effect at both spinal and supraspinal level. OFQ and EM-2 (EM2) immunoreactivity (ir) was measured by radioimmunoassay in nociception-related brain areas of rats subjected to L5/L6 spinal nerve ligation, using sham operated rats as control. It was found that: (1) the content of EM2-ir of spinal nerve ligated rats showed a significant increase (778%) in periaqueductal gray (PAG), and a significant decrease (43%) in striatum, compared with the control group. (2) a significant increase of the content of OFQ-ir was found in amygdala (+841%) and PAG (+459%), respectively in spinal nerve ligated rats. High pressure liquid chromatography showed that the EM2-ir and OFQ-ir were both heterogeneous with the major part eluting at the position of EM2 and OFQ standard, respectively. These results suggest that spinal nerve ligation induces significant changes in the content of EM2-ir and OFQ-ir in some discrete brain areas, which may play a role in nociceptive modulation.

Topics: Amygdala; Animals; Brain; Brain Chemistry; Disease Models, Animal; Female; Neostriatum; Neuralgia; Nociceptin; Oligopeptides; Opioid Peptides; Periaqueductal Gray; Peripheral Nervous System Diseases; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Spinal Nerves