ω-Phonetoxins inhibit voltage-gated calcium CaV2.2 ion channel splice isoforms of dorsal root ganglia.

Cell-specific alternative splicing of Cacna1b pre-mRNA generates functionally distinct voltage-gated CaV2.2 channels. CaV2.2 channels mediate the release of glutamate from nociceptor termini in the dorsal horn spinal cord and they are implicated in chronic pain. One alternatively spliced exon in Cacna1b, e37a, is highly expressed in dorsal root ganglia, relative to other regions of the nervous system, and it is particularly important in inflammatory hyperalgesia. Here we studied the effects of two ω-phonetoxins, PnTx3-4 and Phα1ß, derived from the spider Phoneutria nigriventer on CaV2.2 channel isoforms of dorsal root ganglia (CaV2.2 e37a and CaV2.2 e37b). Both PnTx3-4 and Phα1ß are known to have analgesic effects in rodent models of pain and to inhibit CaV2.2 channels. CaV2.2 e37a and CaV2.2 e37b isoforms expressed in a mammalian cell line were inhibited by PnTx3-4 and Phα1ß with similar potency and with similar timecourse, although CaV2.2 e37a currents were slightly, but consistently more sensitive to toxin inhibition compared to CaV2.2 e37b. The inhibitory effects of PnTx3-4 and Phα1ß on CaV2.2-e37a and CaV2.2-e37b channels were voltage-dependent, and both occlude the inhibitory effects of ω-conotoxin GVIA, consistent with a common site of action. The potency of PnTx3-4 and Phα1ß on both major splice isoforms in dorsal root ganglia constribute to understanding the analgesic actions of these ω-phonetoxins.

Preclinical study in a postoperative pain model to investigate the action of ketamine, lidocaine, and ascorbic acid in reversing fentanyl-induced, non-glutamate-dependent hyperalgesia.

Introduction: Opioid-induced hyperalgesia (OIH) is a paradoxical phenomenon in which exposure to opioids can increase sensitivity to painful stimuli. Currently, several drugs have been used in an attempt to prevent OIH. We design this study to address the effect of preemptive treatment with ketamine, lidocaine, and ascorbic acid in a rat preclinical model of perioperative opioid-induced hyperalgesia. Methods: To reproduce OIH in a model of postoperative pain, rats received successive doses of fentanyl subcutaneously and underwent an incision in the paw. In an attempt to prevent OIH, ketamine, lidocaine, and ascorbic acid were administered before treatment with fentanyl. The von Frey test and the hot-plate test were used to evaluate mechanical allodynia and thermal hyperalgesia, respectively, with a follow-up period from 1 hour up to 7 days after surgery. Spinal cord nerve terminals (synaptosomes) were used to assess glutamate release under our experimental conditions. Results: Consecutive fentanyl injections increased the postoperative pain as indicated by increased thermal hyperalgesia and allodynia 48 hours after incision. Ketamine, lidocaine, and the combination of ketamine + lidocaine were able to prevent thermal hyperalgesia but not mechanical allodynia. Ascorbic acid did not prevent the hyperalgesia induced by fentanyl. We found no correlation between spinal glutamate release and the pharmacological treatments. Conclusion: Fentanyl induced a hyperalgesic effect that last few days in a postoperative model of pain. Hyperalgesic effect was not totally inhibited by ketamine and lidocaine in rats. Increased glutamate release was not the main molecular mechanism of fentanyl-induced hyperalgesia.

Ion Channels-related Neuroprotection and Analgesia Mediated by Spider Venom Peptides.

Ion channels play critical roles in generating and propagating action potentials and in neurotransmitter release at a subset of excitatory and inhibitory synapses. Dysfunction of these channels has been linked to various health conditions, such as neurodegenerative diseases and chronic pain. Neurodegeneration is one of the underlying causes of a range of neurological pathologies, such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebral ischemia, brain injury, and retinal ischemia. Pain is a symptom that can serve as an index of the severity and activity of a disease condition, a prognostic indicator, and a criterion of treatment efficacy. Neurological disorders and pain are conditions that undeniably impact a patient's survival, health, and quality of life, with possible financial consequences. Venoms are the best-known natural source of ion channel modulators. Venom peptides are increasingly recognized as potential therapeutic tools due to their high selectivity and potency gained through millions of years of evolutionary selection pressure. Spiders have been evolving complex and diverse repertoires of peptides in their venoms with vast pharmacological activities for more than 300 million years. These include peptides that potently and selectively modulate a range of targets, such as enzymes, receptors, and ion channels. Thus, components of spider venoms hold considerable capacity as drug candidates for alleviating or reducing neurodegeneration and pain. This review aims to summarize what is known about spider toxins acting upon ion channels, providing neuroprotective and analgesic effects.

Critical Pronociceptive Role of Family 2 Voltage-Gated Calcium Channels in a Novel Mouse Model of HIV-Associated Sensory Neuropathy.

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN.

Systemic, Intrathecal, and Intracerebroventricular Antihyperalgesic Effects of the Calcium Channel Blocker CTK 01512-2 Toxin in Persistent Pain Models.

CTK 01512-2 toxin is a recombinant peptide of the Phα1ß version derived from the venom of the Phoneutria nigriventer spider. It acts as an N-type voltage-gated calcium channel (VGCC) blocker and shows a prolonged effect on preventing and reducing nociception. Herein, CTK 01512-2 was tested on two models of persistent pain, the chronic post-ischemia pain (CPIP) and the paclitaxel-induced peripheral neuropathy, to evaluate its systemic, intrathecal, and intracerebroventricular effects on mechanical hypersensitivity and thermal allodynia. Glial cell viability was also investigated using the MTT test. The results showed that CTK 01512-2 intrathecal and systemic treatments reduced the mechanical hypersensitivity induced by CPIP, mainly between 1-4 h after its administration. Additionally, intrathecal treatment reduced the CPIP-induced thermal allodynia. In its turn, the intracerebroventricular treatment showed mechanical antihyperalgesic and thermal antiallodynic effects in the paclitaxel-induced peripheral neuropathy. These data reinforce the therapeutic potential of CTK 01512-2 to treat persistent pain conditions and offer a perspective to use the systemic route. Moreover, CTK 01512-2 increased the glial cell viability in the MTT reduction assay, and it may indicate a new approach to managing chronic pain. The results found in this study help to pave new perspectives of pain relief treatments to patients affected by chronic pain.

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways.

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Isobolographic analysis reveals antinociceptive synergism between Phα1ß recombinant toxin and morphine in a model of cancer pain in C57BL/6J mice.

BACKGROUND: Phoneutria nigriventer venom contains Phα1ß. This toxin and its recombinant form have a remarkable analgesic potential that is associated with blockage of voltage-gated calcium channels and TRPA1 receptors. Although morphine is a mainstay drug to treat moderate and severe pain related to cancer, it has serious and dose-limiting side effects. Combining recombinant Phα1ß and morphine to treat pain is an interesting approach that has been gaining attention. Therefore, a quantitative and reliable method to establish the strength of the antinociceptive interaction between these two substances is necessary. The present study was designed to investigate the nature of the functional antinociceptive (analgesic) interaction between Phα1ß recombinant toxin and morphine in a model of cancer pain. METHODS: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw of C57BL/6J mice. Von Frey filaments measured the paw-withdrawal threshold after intrathecal administration of morphine, recombinant Phα1ß, and their combination. Thermal hyperalgesia was assessed using Hargreaves apparatus. The degree of interaction was evaluated using isobolographic analysis. Spontaneous and forced motor performance was assessed with the open-field and rotarod tests, respectively. RESULTS: Co-administration of recombinant Phα1ß and morphine synergistically reverses the melanoma-induced mechanical hyperalgesia. The potency of the mixture, measured as the effective dose to reach 50% of maximum possible effect (MPE) in ameliorating mechanical hyperalgesia, was about twice fold higher than expected if the interaction between morphine and recombinant Phα1ß was merely additive. Treatment with the combination at doses necessary to reach 50% of MPE caused no spontaneous nor forced motor alterations. CONCLUSION: The combinatorial use of recombinant Phα1ß and morphine allows significant and effective dose reduction of both agents, which has translational potential for opioid-sparing approaches in pain management related to cancer.

Effects of intravenous administration of recombinant Phα1ß toxin in a mouse model of fibromyalgia.

This study investigated the effects of intravenous (iv) administration of recombinant Phα1ß toxin, pregabalin, and diclofenac by the intrathecal route using an animal model fibromyalgia (FM). The reserpine administration (0.25 mg/kg s. c) once daily for three consecutive days significantly induced hyperalgesia, immobility time, and sucrose consumption in mice on the 4th day. Reserpine caused hyperalgesia on the mechanical and thermal hyperalgesia on the 4th day was reverted by recombinant Phα1ß (0.2 mg/kg iv) and pregabalin (1.25 µmol/site i. t) treatments. In contrast, diclofenac (215 nmol/site i. t) was ineffective. Recombinant Phα1ß toxin, pregabalin, and diclofenac did not affect the depressive-like behavioural effect induced by reserpine on mice during the forced swim and sucrose consumption tests. The data confirmed the analgesic effect of the recombinant Phα1ß toxin administered intravenously in a fibromyalgia mouse model.

Mapping of Brain Activity in the Analgesia Induced by Phα1ß and Morphine.

Preclinical evidence suggests the potential of Phα1ß, a toxin obtained from the venom of spider Phoneutria nigriventer, as a new analgesic drug. Molecular brain imaging techniques have afforded exciting opportunities to examine brain processes in clinical pain conditions. This paper aims to study the brain regions involved in the analgesic effects of Phα1ß compared with Morphine, in a model of acute pain induced by formalin in Sprague Dawley rats. We used 18F-fluorodeoxyglucose as a metabolic radiotracer to perform brain imaging of rats pretreated with Phα1ß or Morphine in a model of acute inflammatory pain caused by intraplantar injection of formalin. The rats' hind paw's formalin stimulation resulted in a brain metabolic increase at the bilateral motor cortex, visual cortex, somatosensory cortex, thalamus, and cingulate cortex.In rats treated with Phα1ß, selective inhibition of unilateral motor cortex and cingulate cortex was observed. Morphine treatment leads to small and selective inhibition at the bilateral amygdala striatum and accumbens. Our results indicate that the analgesic effect of Phα1ß and Morphine possesses a differential profile of central processing in the pain state.

Calcium channels blockers toxins attenuate abdominal hyperalgesia and inflammatory response associated with the cerulein-induced acute pancreatitis in rats.

Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1ß and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca2+ signals may be useful as a prophylactic treatment of pancreatitis. We explored the pathophysiological roles of three peptide toxins: Phα1ß and its recombinant form CTK 01512-2-blockers of TRPA1 receptor and HVCCs and ω-conotoxin MVIIA, a specific blocker of N-type calcium channels in cerulein-induced AP. Cerulein injection elicits AP in rats, evidenced by an increase in hyperalgesic pain, inflammatory infiltration, amylase and lipase secretion, and reactive oxygen species, TNF-α, and p65 NF-κB levels. These effects of cerulein-induced AP were abolished by Phα1ß and its recombinant form CTK 01512-2, whereas ω-conotoxin MVIIA had no effect on the induced increase in pancreatic enzyme secretion. Our results demonstrate that Phα1ß and CTK 01512-2 toxins-antagonists of HVCCs and TRPA1 receptor presented an effective response profile, in the control of nociception and inflammatory process in the AP model in rats, without causing changes in spontaneous locomotion of the rats.

Phoneutria nigriventer Tx3-3 peptide toxin reduces fibromyalgia symptoms in mice.

Fibromyalgia is characterized by the amplification of central nervous system pain with concomitant fatigue, sleep, mood disorders, depression, and anxiety. It needs extensive pharmacological therapy. In the present study, Swiss mice were treated with reserpine (0.25 mg/kg, s.c.) over three consecutive days, in order to reproduce the pathogenic process of fibromyalgia. On day 4, the administrations of the Tx3-3 toxin produced significant antinociception in the mechanical allodynia (87.16% ±12.7%) and thermal hyperalgesia (49.46% ± 10.6%) tests when compared with the PBS group. The effects produced by the classical analgesics (duloxetine 30 mg/kg, pramipexole 1 mg/kg, and pregabalin 30 mg/kg, p.o., respectively) in both of the tests also demonstrated antinociception. The administrations were able to increase the levels of the biogenic amines (5-HTP and DE) in the brain. The treatments with pramipexole and pregabalin, but not duloxetine, decreased the immobility time in the FM-induced animals that were submitted to the forced swimming test; however, the Tx3-3 toxin (87.45% ± 4.3%) showed better results. Taken together, the data has provided novel evidence of the ability of the Tx3-3 toxin to reduce painful and depressive symptoms, indicating that it may have significant potential in the treatment of FM.

Isobolographic analysis reveals antinociceptive synergism between Phα1ß recombinant toxin and morphine in a model of cancer pain in C57BL/6J mice

Background: Phoneutria nigriventer venom contains Phα1ß. This toxin and its recombinant form have a remarkable analgesic potential that is associated with blockage of voltage-gated calcium channels and TRPA1 receptors. Although morphine is a mainstay drug to treat moderate and severe pain related to cancer, it has serious and dose-limiting side effects. Combining recombinant Phα1ß and morphine to treat pain is an interesting approach that has been gaining attention. Therefore, a quantitative and reliable method to establish the strength of the antinociceptive interaction between these two substances is necessary. The present study was designed to investigate the nature of the functional antinociceptive (analgesic) interaction between Phα1ß recombinant toxin and morphine in a model of cancer pain. Methods: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw of C57BL/6J mice. Von Frey filaments measured the paw-withdrawal threshold after intrathecal administration of morphine, recombinant Phα1ß, and their combination. Thermal hyperalgesia was assessed using Hargreaves apparatus. The degree of interaction was evaluated using isobolographic analysis. Spontaneous and forced motor performance was assessed with the open-field and rotarod tests, respectively. Results: Co-administration of recombinant Phα1ß and morphine synergistically reverses the melanoma-induced mechanical hyperalgesia. The potency of the mixture, measured as the effective dose to reach 50% of maximum possible effect (MPE) in ameliorating mechanical hyperalgesia, was about twice fold higher than expected if the interaction between morphine and recombinant Phα1ß was merely additive. Treatment with the combination at doses necessary to reach 50% of MPE caused no spontaneous nor forced motor alterations. Conclusion: The combinatorial use of recombinant Phα1ß and morphine allows significant and effective dose reduction of both agents, which has translational potential for opioid-sparing approaches in pain management related to cancer.(AU)

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Phalfa1 beta toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Ph1 toxin: a review of mechanisms of action involving pain pathways

Abstract Ph1 is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Ph1 to treat chronic pain reverted opioid tolerance with a safer profile than -conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Ph1 (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Ph1 antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Isobolographic analysis reveals antinociceptive synergism between Ph1 recombinant toxin and morphine in a model of cancer pain in C57BL/6J mice

Abstract Background: Phoneutria nigriventer venom contains Ph1. This toxin and its recombinant form have a remarkable analgesic potential that is associated with blockage of voltage-gated calcium channels and TRPA1 receptors. Although morphine is a mainstay drug to treat moderate and severe pain related to cancer, it has serious and dose-limiting side effects. Combining recombinant Ph1 and morphine to treat pain is an interesting approach that has been gaining attention. Therefore, a quantitative and reliable method to establish the strength of the antinociceptive interaction between these two substances is necessary. The present study was designed to investigate the nature of the functional antinociceptive (analgesic) interaction between Ph1 recombinant toxin and morphine in a model of cancer pain. Methods: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw of C57BL/6J mice. Von Frey filaments measured the paw-withdrawal threshold after intrathecal administration of morphine, recombinant Ph1, and their combination. Thermal hyperalgesia was assessed using Hargreaves apparatus. The degree of interaction was evaluated using isobolographic analysis. Spontaneous and forced motor performance was assessed with the open-field and rotarod tests, respectively. Results: Co-administration of recombinant Ph1 and morphine synergistically reverses the melanoma-induced mechanical hyperalgesia. The potency of the mixture, measured as the effective dose to reach 50% of maximum possible effect (MPE) in ameliorating mechanical hyperalgesia, was about twice fold higher than expected if the interaction between morphine and recombinant Ph1 was merely additive. Treatment with the combination at doses necessary to reach 50% of MPE caused no spontaneous nor forced motor alterations. Conclusion: The combinatorial use of recombinant Ph1 and morphine allows significant and effective dose reduction of both agents, which has translational potential for opioid-sparing approaches in pain management related to cancer.

Phα1ß, a dual blocker of TRPA1 and Cav2.2, as an adjuvant drug in opioid therapy for postoperative pain.

Opioids are the "gold standard" treatment for postoperative pain, but these drugs also have limiting adverse effects. Thus, adjuvant drugs might be useful in opioid therapy for postoperative pain. The aim of the present study was to evaluate the effect of Phα1ß, a dual blocker of Cav2 and TRPA1 channels, on antinociceptive and adverse actions of morphine in a model of postoperative pain. Phα1ß (100-300 pmol/site) or morphine (3-10 mg/kg), alone, largely reduced postoperative nociception. However, Phα1ß (100 pmol/site) or morphine (10 mg/kg) also produced motor impairment. Lower doses of Phα1ß (30 pmol/site) or morphine (1 mg/kg), that did not have an effect alone, showed antinociceptive effect when concomitantly administrated. Moreover, co-administration of Phα1ß (30 pmol/site) with morphine (1 or 10 mg/kg) was unable to cause motor impairment. Preoperative repeated treatment with morphine increased the expression of Cav2 and TRPA1 channels in spinal cord, and caused tolerance and withdrawal syndrome, which were reversed with a single injection of Phα1ß (30 pmol/site). When injected postoperatively, escalating doses of morphine worsened postoperative hyperalgesia, induced tolerance, and withdrawal syndrome. Similarly, Phα1ß (30 pmol/site) reversed these adverse effects. Single or repeated morphine caused constipation, which was not altered by Phα1ß. Thus, a low dose of Phα1ß potentiated the analgesia, and reversed some adverse effects of morphine on operated mice, indicating the potential use of this agent as an adjuvant drug in opioid therapy for postoperative pain.

Analgesic effects of the CTK 01512-2 toxin in different models of orofacial pain in rats.

BACKGROUND: Orofacial pain is clinically challenging, having therapeutic failures and side effects. This study evaluated the antinociceptive activities of the CTK 01512-2 toxin, the TRPA1 channel antagonist, and the selective inhibitor of the N-type voltage-gated calcium channels (N-type VGCC), in different pain models. MATERIALS AND METHODS: The trigeminal ganglia were stimulated in vitro with capsaicin. The in vivo models received subcutaneous (sc) injections of formalin into the upper lip of the rats, Freund's Complete Adjuvant (FCA) into the temporomandibular joint (TMJ), and infraorbital nerve constrictions (IONC). CTK 01512-2 at concentrations of 30, 100, and 300 pmol/site, intrathecally (ith), and MVIIA at 10, 30, and 100 pmol/site in the formalin test, guided the doses for the models. The glutamate levels in the CSF of the rats that were submitted to IONC were analyzed. RESULTS: CTK 01512-2 decreased the nociceptive behavior in the inflammatory phase of the formalin test (65.94 ± 7.35%) and MVIIA in the neurogenic phase (81.23 ± 3.36%). CTK 01512-2 reduced facial grooming with FCA in the TMJ (96.7 ± 1.6%), and in the IONC neuropathy model, it decreased heat hyperalgesia (100%) and cold hyperalgesia (81.61 ± 9.02%). The levels of glutamate in the trigeminal ganglia in vitro (81.40 ± 8.59%) and in the CSF in vivo (70.0 ± 9.2%) were reduced. CONCLUSIONS: The roles of TRPA1 in pain transduction and the performance of CTK 01512-2 in the inhibition of the N-type VGCCs were reinforced. This dual activity may represent an advantage in clinical treatments.

Analgesic and side effects of intravenous recombinant Phα1ß.

BACKGROUND: Intrathecal injection of voltage-sensitive calcium channel blocker peptide toxins exerts analgesic effect in several animal models of pain. Upon intrathecal administration, recombinant Phα1ß exerts the same analgesic effects as the those of the native toxin. However, from a clinical perspective, the intrathecal administration limits the use of anesthetic drugs in patients. Therefore, this study aimed to investigate the possible antinociceptive effect of intravenous recombinant Phα1ß in rat models of neuropathic pain, as well as its side effects on motor, cardiac (heart rate and blood pressure), and biochemical parameters. METHODS: Male Wistar rats and male Balb-C mice were used in this study. Giotto Biotech® synthesized the recombinant version of Phα1ß using Escherichia coli expression. In rats, neuropathic pain was induced by chronic constriction of the sciatic nerve and paclitaxel-induced acute and chronic pain. Mechanical sensitivity was evaluated using von Frey filaments. A radiotelemeter transmitter (TA11PA-C10; Data Sciences, St. Paul, MN, USA) was placed on the left carotid of mice for investigation of cardiovascular side effects. Locomotor activity data were evaluated using the open-field paradigm, and serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were examined. RESULTS: Intravenous administration of recombinant Phα1ß toxin induced analgesia for up to 4 h, with ED50 of 0.02 (0.01-0.03) mg/kg, and reached the maximal effect (Emax = 100% antinociception) at a dose of 0.2 mg/kg. No significant changes were observed in any of the evaluated motor, cardiac or biochemical parameters. CONCLUSION: Our data suggest that intravenous administration of recombinant Phα1ß may be feasible for drug-induced analgesia, without causing any severe side effects.

Analgesic and side effects of intravenous recombinant Ph alfa 1 beta

Background: Intrathecal injection of voltage-sensitive calcium channel blocker peptide toxins exerts analgesic effect in several animal models of pain. Upon intrathecal administration, recombinant Phα1β exerts the same analgesic effects as the those of the native toxin. However, from a clinical perspective, the intrathecal administration limits the use of anesthetic drugs in patients. Therefore, this study aimed to investigate the possible antinociceptive effect of intravenous recombinant Phα1β in rat models of neuropathic pain, as well as its side effects on motor, cardiac (heart rate and blood pressure), and biochemical parameters. Methods: Male Wistar rats and male Balb-C mice were used in this study. Giotto Biotech® synthesized the recombinant version of Phα1β using Escherichia coli expression. In rats, neuropathic pain was induced by chronic constriction of the sciatic nerve and paclitaxel-induced acute and chronic pain. Mechanical sensitivity was evaluated using von Frey filaments. A radiotelemeter transmitter (TA11PA-C10; Data Sciences, St. Paul, MN, USA) was placed on the left carotid of mice for investigation of cardiovascular side effects. Locomotor activity data were evaluated using the open-field paradigm, and serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were examined. Results: Intravenous administration of recombinant Phα1β toxin induced analgesia for up to 4 h, with ED50 of 0.02 (0.01-0.03) mg/kg, and reached the maximal effect (Emax = 100% antinociception) at a dose of 0.2 mg/kg. No significant changes were observed in any of the evaluated motor, cardiac or biochemical parameters. Conclusion: Our data suggest that intravenous administration of recombinant Phα1β may be feasible for drug-induced analgesia, without causing any severe side effects.(AU)