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Abstract Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer, on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels. Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described. Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC50 value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC50 value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability. Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability.

Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer , on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels., Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described., Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC 50 value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC 50 value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability., Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability., Competing Interests: Competing interests: The authors declare that they have no competing interests.

CTK 01512–2 toxin is a recombinant peptide of the Phα1β 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. [ABSTRACT FROM AUTHOR]

Preclinical evidence suggests the potential of Phα1β, 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 Phα1β 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 Phα1β 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 Phα1β, 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 Phα1β and Morphine possesses a differential profile of central processing in the pain state.

Abstract Background: Phoneutria nigriventer venom contains Phα1β. 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 Phα1β 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 Phα1β 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 Phα1β, 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 Phα1β 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 Phα1β 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 Phα1β 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.

The incoming signals from injured sensory neurons upon peripheral inflammation are processed in the dorsal horn of spinal cord, where glial cells accumulate and play a critical role in initiating allodynia (increased pain in response to light-touch). However, how painful stimuli in the periphery engage glial reactivity in the spinal cord remains unclear. Here, we found that a hind paw inflammation induced by CFA produces robust morphological changes in spinal astrocytes and microglia compatible with the reactive phenotype. Strikingly, we discovered that a single intrathecal injection with venom peptides that inhibit calcium channels reversed all the glial pathological features of the peripheral inflammation. These effects were more apparent in rats treated with the Phα1β spider toxin (non-specific calcium channel antagonist) than ω-MVIIA cone snail toxin (selective N-type calcium channel antagonist). These data reveal for the first time a venom peptide acting on glial structural remodeling in vivo. We, therefore, suggest that calcium-dependent plasticity is an essential trigger for glial cells to initiate reactivity, which may represent a new target for the antinociceptive effects of Phα1β and ω-MVIIA toxins in inflammatory pain conditions.

The incoming signals from injured sensory neurons upon peripheral inflammation are processed in the dorsal horn of spinal cord, where glial cells accumulate and play a critical role in initiating allodynia (increased pain in response to light-touch). However, how painful stimuli in the periphery engage glial reactivity in the spinal cord remains unclear. Here, we found that a hind paw inflammation induced by CFA produces robust morphological changes in spinal astrocytes and microglia compatible with the reactive phenotype. Strikingly, we discovered that a single intrathecal injection with venom peptides that inhibit calcium channels reversed all the glial pathological features of the peripheral inflammation. These effects were more apparent in rats treated with the Phα1β spider toxin (non-specific calcium channel antagonist) than ω-MVIIA cone snail toxin (selective N-type calcium channel antagonist). These data reveal for the first time a venom peptide acting on glial structural remodeling in vivo. We, therefore, suggest that calcium-dependent plasticity is an essential trigger for glial cells to initiate reactivity, which may represent a new target for the antinociceptive effects of Phα1β and ω-MVIIA toxins in inflammatory pain conditions. [ABSTRACT FROM AUTHOR]

Phα1β peptide isolated from the venom of the Phoneutria nigriventer spider has shown higher analgesic action in pre‐clinical studies than ω‐conotoxin MVIIA peptide used to treat severe chronic pain. In view of the great potential for the development of a new Phα1β‐based drug, a Phα1β recombinant form (CTK 01512‐2) has been studied for efficacy and safety. The aim of this study was to evaluate cytotoxic, genotoxic and mutagenic effects of a Phα1β recombinant form and compare it with native Phα1β and ω‐conotoxin MVIIA. Cytotoxicity was evaluated using the MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) colourimetric assay in L929 mouse fibroblast cells (0.5‐10.0 μmol/L). Genotoxic and mutagenic activities were analysed using the alkaline comet assay in peripheral blood and spinal cord, and the micronucleus test in bone marrow from Wistar rats treated by intrathecal injection of CTK 01512‐2 (200, 500 and 1000 pmol/site), native Phα1β (500 pmol/site) and ω‐conotoxin MVIIA (200 pmol/site). CTK 01512‐2 decreased the cell viability of the L929, showing IC50 of 3.3 ± 0.1 µmol/L, while the Phα1β and ω‐conotoxin MVIIA did not show cytotoxicity (IC50 > 5.0 µmol/L). Native and recombinant Phα1β forms induced DNA damage in the spinal cord, but not in peripheral blood. CTK 01512‐2 at 1000 pmol/site increased the micronucleus frequency suggesting mutagenic effects. In conclusion, the recombinant form has cytotoxic, genotoxic and mutagenic effects, evidenced in doses five times above the therapeutic dose. [ABSTRACT FROM AUTHOR]

Aims Extensive evidence supports a role for voltage-gated calcium channels (VGCC) and TRPV1 receptors in pain transmission and modulation. We investigated the profile of analgesic interaction between Phα1β toxin (a VGCC blocker) and SB366791 (selective TRPV1 antagonist) in a model of acute pain induced by capsaicin. Changes in body temperature induced by combination regimens were also evaluated. Main methods Isobolographic approach with a fixed dose-ratio of combined drugs was used to determine whether antinociceptive interaction of Phα1β and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. Key findings Phα1β and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (α) of 0.07 for Phα1β and SB366791 when these drugs were injected together intraplantarly, which indicates that in vivo interaction between these drugs is greater than additive interaction. Synergism also occurred when intraplantar SB366791 was administered simultaneously with intrathecal Phα1β (interaction index α = 0.06) suggesting a 15 fold rise in potency on the analgesic effect of these drugs when they are added together. It was observed no significant alterations in body temperature of animals treated with this combination regimen. Significance Our data reveal that Phα1β toxin potentiates in 15 fold the antinociceptive action of the TRPV1 blocker SB366791. Therefore, lower doses of these drugs are required to achieve antinociceptive effects when these agents are given in combination. [ABSTRACT FROM AUTHOR]

The native Phα1β – a Voltage-Gated Calcium Channel (VGCC) blocker – and its Recombinant Version – were both tested in rodent pain models with an intraplantar injections of capsaicin or formalin, a chronic constriction injury, and melanoma cancer related pain. The formalin nociceptive behaviour in the neurogenic phase was not affected by the toxin pre-treatments, while in the inflammatory phase, Phα1β and the Recombinant form caused a significant reduction. The nociception that was triggered by capsaicin, an agonist of the TRPV1 vanilloid receptor, was totally blocked by 100 pmol/site, i.t. of Phα1β or the recombinant version. For the neuropathic pain that was induced by a chronic constriction injury of the sciatic nerve, Phα1β and its Recombinant reduced the allodynia that was induced by the CCI procedure in the rats and the hypersensitivity lasted for 4 h. Fourteen days after the inoculation of the B16-F10 melanoma cells in the mice, a marked hyperalgesia was induced in the melanoma cancer pain model. Phα1β and the Recombinant form reduced the hyperalgesia with a full reversion at 100 pmol/site i.t. The inhibitory effects of the nociception that was induced by native Phα1β and the Recombinant in the studied pain models were not statistically different and they developed with no side effects. [ABSTRACT FROM AUTHOR]

Phα1β, a purified peptide from the venom of the spider Phoneutria nigriventer, and its recombinant form CTK 01512-2 are voltage-dependent calcium channel (CaV) blockers of types N, R, P/Q, and L with a preference for type N. These peptides show analgesic action in different pain models in rats. The aim of this study was to evaluate the acute intrathecal toxicity of the native and recombinant Phα1β toxin in Wistar rats. Clinical signs, serum biochemistry, organ weight, and histopathological alterations were evaluated in male and/or female rats. Dyspnea was observed in males, hyporesponsiveness in females, and Straub tail and tremors in both genders. There were no significant differences in male organ weight, although significant differences in the female relative weight of the adrenal glands and spleen have been observed; these values are within the normal range. Serum biochemical data revealed a significant reduction within the physiological limits of species related to urea, ALT, AST, and FA. Hepatic and renal congestion were observed for toxin groups. In renal tissue, glomerular infiltrates were observed with increased glomerular space. These histological alterations were presented in focal areas and in mild degree. Therefore, Phα1β and CTK 01512-2 presented a good safety profile with transient toxicity clinical signals in doses higher than used to obtain the analgesic effect.

Cell-specific alternative splicing of Cacna1b pre-mRNA generates functionally distinct voltage-gated Ca V 2.2 channels. Ca V 2.2 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 Phα1β, derived from the spider Phoneutria nigriventer on Ca V 2.2 channel isoforms of dorsal root ganglia (Ca V 2.2 e37a and Ca V 2.2 e37b). Both PnTx3-4 and Phα1β are known to have analgesic effects in rodent models of pain and to inhibit Ca V 2.2 channels. Ca V 2.2 e37a and Ca V 2.2 e37b isoforms expressed in a mammalian cell line were inhibited by PnTx3-4 and Phα1β with similar potency and with similar timecourse, although Ca V 2.2 e37a currents were slightly, but consistently more sensitive to toxin inhibition compared to Ca V 2.2 e37b. The inhibitory effects of PnTx3-4 and Phα1β on Ca V 2.2-e37a and Ca V 2.2-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 Phα1β on both major splice isoforms in dorsal root ganglia constribute to understanding the analgesic actions of these ω-phonetoxins., Competing Interests: Conflict of interest Authors declare no conflict of interest

Abstract: This study investigated the effects of Phα1β, pregabalin and diclofenac using an animal model of fibromyalgia (FM). Repeated administration of reserpine (0.25 mg/kg sc) once daily for three consecutive days significantly decreased thermal hyperalgesia, mechanical allodynia, and dopamine and serotonin content in the brain on the 4th day. Phα1β and pregabalin treatment completely reverted the mechanical allodynia and thermal hyperalgesia induced by reserpine treatment on the 4th day, but diclofenac was ineffective. Reserpine treatment significantly increased the immobility time in the forced swim test, which is indicative of depression in the animals. Phα1β, but not pregabalin, reduced the immobility time (56%), suggesting that Phα1β may control persistent pathological pain in FM. [Copyright &y& Elsevier]

Abstract: Phα1β toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer, with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1β when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1β on Ca2+ transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1β reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca2+ channel blocker) was effective only when administered intrathecally. Phα1β, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca2+ transients in DRG neurons. The simultaneous administration of Phα1β and SB366791 inhibited the capsaicin-induced Ca2+ transients that were additive suggesting that they act through different targets. Moreover, Phα1β did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1β may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors. [Copyright &y& Elsevier]

Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ω-conotoxin MVIIA and Phα1β, in models of inflammatory and neuropathic pain induced with complete Freund's adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Phα1β reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ω-conotoxin MVIIA. Phα1β and MVIIA also inhibited capsaicin-induced Ca influx in DRG neurons. The inhibitory effect of Phα1β on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ω-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Phα1β, a reversible inhibitor of VSCCs with a preference for N-type Ca channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Phα1β in the treatment of chronic pain. [ABSTRACT FROM AUTHOR]

Opioids are the first-line treatment for cancer pain. Incomplete pain relief and the high rate of adverse effects of these compounds bring a need to combine them with other drugs acting on different targets. We here evaluate the antinociceptive interaction and adverse events of methadone combined with recombinant Phα1β, an analgesic toxin from Phoneutria nigriventer. Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw. von Frey filaments measured the paw-withdrawal threshold after administration of methadone, Phα1β, and their combination. The degree of interaction was evaluated using isobolographic analysis. Spontaneous performance and forced motor performance were assessed with the open-field and rotarod tests, respectively. Intestinal function was evaluated by the distance traveled by charcoal and opioid tolerance was induced by daily morphine injections. Co-administration of Phα1β with methadone synergistically reverses the melanoma-induced mechanical hypersensitivity. No motor alterations were observed but mild alterations on intestinal function after treatment with the combination that was also capable of restoring morphine analgesia in the tail-flick test after an opioid-induced tolerance. Combinatorial treatment with Phα1β and methadone produces synergistic analgesic potentiation with potential implications to pain treatment even under opioid tolerance conditions. [ABSTRACT FROM AUTHOR]

Abnormal calcium influx and glutamatergic excitotoxicity have been extensively associated with neuronal death in Huntington's disease (HD), a genetic movement disorder. Currently, there is no effective treatment for this fatal condition. The neurotoxin Phα1β has demonstrated therapeutic effects as a calcium channel blocker, for example during pain control. However, little is known about its neuroprotective effect in HD. Herein, we investigated if Phα1β is effective in inhibiting neuronal cell death in the BACHD mouse model for HD. We performed intrastriatal injection of Phα1β in WT and BACHD mice. No side effects or unusual behaviors were observed upon Phα1β administration. Using three different motor behavior tests, we observed that injection of the toxin in BACHD mice greatly improved the animals' motor-force as seen in the Wire-hang test, and also the locomotor performance, according to the Open field test. NeuN labeling for mature neuron detection revealed that Phα1β toxin promoted neuronal preservation in the striatum and cortex, when injected locally. Intrastriatal injection of Phα1β was not able to preserve neurons from the spinal cord and also not revert muscle atrophy in BACHD mice. Finally, we observed that Phα1β might, at least in part, exert its protective effect by decreasing L-glutamate, measured in cerebrospinal fluid. Our data provide evidence of a novel neuroprotector effect of Phα1β, paving a path for the development of new approaches to treat HD motor symptoms. [ABSTRACT FROM AUTHOR]

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 Phα1β 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: Phα1β 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 Phα1β 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 Phα1β 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. [ABSTRACT FROM AUTHOR]

Background: Phoneutria nigriventer venom contains Phα1β. 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 Phα1β 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 Phα1β 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 Phα1β, 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 Phα1β 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 Phα1β 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 Phα1β 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., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Phα1β, a purified peptide from the venom of the spider Phoneutria nigriventer, and its recombinant form CTK 01512-2 are voltage-dependent calcium channel (CaV) blockers of types N, R, P/Q, and L with a preference for type N. These peptides show analgesic action in different pain models in rats. The aim of this study was to evaluate the acute intrathecal toxicity of the native and recombinant Phα1β toxin in Wistar rats. Clinical signs, serum biochemistry, organ weight, and histopathological alterations were evaluated in male and/or female rats. Dyspnea was observed in males, hyporesponsiveness in females, and Straub tail and tremors in both genders. There were no significant differences in male organ weight, although significant differences in the female relative weight of the adrenal glands and spleen have been observed; these values are within the normal range. Serum biochemical data revealed a significant reduction within the physiological limits of species related to urea, ALT, AST, and FA. Hepatic and renal congestion were observed for toxin groups. In renal tissue, glomerular infiltrates were observed with increased glomerular space. These histological alterations were presented in focal areas and in mild degree. Therefore, Phα1β and CTK 01512-2 presented a good safety profile with transient toxicity clinical signals in doses higher than used to obtain the analgesic effect. [ABSTRACT FROM AUTHOR]