Phoneutria Nigriventer Research Articles

Antinociceptive synergism upon the joint use of methadone and Phα1β in a model of cancer-related pain in C57BL/6J mice

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. AimsWe here evaluate the antinociceptive interaction and adverse events of methadone combined with recombinant Phα1β, an analgesic toxin from Phoneutria nigriventer. Main methodsMelanoma 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. Key findingsCo-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. SignificanceCombinatorial treatment with Phα1β and methadone produces synergistic analgesic potentiation with potential implications to pain treatment even under opioid tolerance conditions.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

Isobolographic analysis reveals antinociceptive synergism between Phα1β recombinant toxin and morphine in a model of cancer pain in C57BL/6J mice.

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.

Spider venom components decrease glioblastoma cell migration and invasion through RhoA-ROCK and Na+/K+-ATPase \u03b22: potential molecular entities to treat invasive brain cancer

BackgroundGlioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) involvement.MethodsHuman (NG97) GB cells were treated with twelve subfractions (SFs—obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test.ResultsTwo (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2.ConclusionAll components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

DDRE-19. SPIDER VENOMS AS SOURCES OF MOLECULES FOR NEW CANCER TREATMENTS: THE EFFECTS OF THE SNX-482 PEPTIDE ON MACROPHAGE MODULATION

Abstract Malignant primary brain tumors remain among the most difficult cancers to treat. In malignant tissues, macrophages are accumulated in a high infiltration being known as tumor-associated macrophages (TAMs). These cells are associated with poor prognostics in many types of cancer. Studies in our group demonstrated that the venom of Phoneutria nigriventer (PnV), a wandering spider from South America, has reduced the development of glioblastoma (GBM) in a murine model, inducing a large infiltrate of TAMs. Subsequently, in vitro results demonstrated that PnV activates macrophages, increasing the ability to kill tumor cells. The aim of this study was to analyze the effects of the peptide SNX-482 presented in the venom of Hysterocrates gigas in macrophages for further correlation with the PnV. Macrophages were differentiated from bone marrow precursors collected from male C57BL6 mice and differentiated for 7 days with M-CSF. These cells were used for polarization and coculture with T cells and analyzed by flow cytometry. PCR Array was also performed (QIAGEN) for the analysis of gene expression. The results showed that SNX-482 could activate macrophages in a not dose-dependent response. There was an increase in the main activation markers (CD40, CD80, CD86, CD68, CD83, and MHCII). The polarization indicated that the peptide potentiated the proinflammatory effect of M1 macrophages (increased MHCII and iNOS). The screening of 86 cancer-related genes showed that the Ccr4, Pdcd1, Gzmb, and IFN-γ genes had an increase in their expression. Furthermore, we developed in C57BL/6 mouse a pre-clinical model of intracranial glioblastoma using the Gl261 cell line. The results showed an applied-easy-safe model that could alter the gene expression of cancer markers. Taken together, all the results contributed to increasing the knowledge about the peptide SNX-482 and the model for further pre-clinical assays of glioblastoma, making a great advance in the development of new treatments.

EXTH-42. SPIDER VENOM COMPONENTS DECREASE GLIOBLASTOMA CELL MIGRATION AND INVASION THROUGH RHOA-ROCK AND NA+/K+-ATPASE β2: POTENTIAL MOLECULAR ENTITIES TO TREAT INVASIVE BRAIN CANCER

Abstract Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells. The involvement of RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) was also evaluated. Human (NG97) GB cells were treated with twelve subfractions (SFs - obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound heling and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Two (SF1 and SF11) of twelve SFs decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2. All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway. Support: FAPESP #2015/04194-0; CNPq #431465/2016–9.

EXTH-28. MOLECULAR CHARACTERIZATION OF HUMAN GLIOMAS AND IDENTIFICATION OF NEOPLASMS RESPONSIBLE FOR NEW BIOPHARMACEUTICALS OBTAINED FROM ANIMAL VENOM: A TRANSLATIONAL APPROACH

Abstract Gliomas correspond to approximately 80% of primary malignant brain tumors in adults. Associate histopathological classification to the identification of the molecular profile of these tumors is a great strategy to predict the tumor’s behavior and its responsiveness to the treatments. Studies have shown the potential of using biopharmaceuticals against cancer. Our research group has recently found two main subfractions isolated from Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) that can act decreasing the number of migrating cells during transwell assay. Therefore, the present study aimed to characterize, as regards molecular identity, the responsiveness of glioma samples collected from patients to the molecules purified from the PnV, in order to improve diagnostic and prognostic predictions, as well as contribute to the development of new drugs. After consent of the patients, samples of the tumors clinically diagnosed as glioma were collected during the surgical procedure to cultivation, that were performed until 10 passages to establish a lineage before use. To analyze its responsiveness to PnV in a migration assay, cultured human glioblastoma (1-JA63) cells were treated with PnV-isolated (HPLC) subfractions (SF1 and SF11 at 1 µg/ml) for 12 and 48 h (control stayed in IMDM) in a transwell system. SFs (mainly SF1) treatment induced a significant reduction in the number of migrating cells after 48 hours of exposure, in comparison to the control (untreated). Ongoing trials continue the establishment of another tumor samples of gliomas and migration tests with PnV subfractions will be carried out, to compare their effects in relation to other degrees of the disease also correlating the tumor response to the drug with the molecular profile. The results will contribute to the development of a potential individualized therapy also adding information about the correlation between the molecular identity of the tumor and its prognosis.

PnPP-19 Peptide as a Novel Drug Candidate for Topical Glaucoma Therapy Through Nitric Oxide Release.

PurposeEvaluation of PnPP-19 safety and efficacy in reducing the intraocular pressure (IOP) of animals with healthy (normotensive) and ocular hypertensive eyes. PnPP-19 is a synthetic peptide designed from Phoneutria nigriventer spider toxin PnTx2-6.MethodsToxicity tests used chicken chorioallantoic membranes. Electroretinograms (ERGs) were recorded before and after administration of different doses of PnPP-19 on the eyes of Wistar rats. Histological sections of corneas and retinas were prepared. The efficacy of PnPP-19 in reducing IOP was evaluated for normotensive and ocular hypertensive animals using a tonometer. Ocular hypertension was induced in the right eye through injection of hyaluronic acid (HA) into the anterior chamber. ERG was recorded before and after glaucoma induction. The eyes were enucleated, and the corneas and retinas were histologically evaluated.ResultsPnPP-19 showed no toxicity, being safe for ocular application. A single topical instillation of one eye drop of the peptide solution was able to reduce IOP, both in healthy and ocular hypertensive rats, for 24 hours, without eliciting any apparent toxicity. PnPP-19 is a nitric oxide inducer and the results suggest that it may improve the conventional outflow of aqueous humor (AH), preventing the progression of optic nerve degeneration.ConclusionsPnPP-19 has great potential to emerge as a promising drug for the treatment of ocular hypertension.Translational RelevanceWe regard our findings as exciting progress in translational glaucoma research, combining drug discovery, natural product research, and pharmacology, which may contribute to the establishment of new therapies for the treatment of this disease.

VEGF/VEGFR-2 system exerts neuroprotection against Phoneutria nigriventer spider envenomation through PI3K-AKT-dependent pathway

This study was undertaken to elucidate why VEGF/VEGFR-2 is elevated in the hippocampus of rats injected with Phoneutria nigriventer spider venom (PNV). PNV delays Na+ channels inactivation; blocks Ca2+ and K+ channels, increases glutamate release, causes blood-brain breakdown (BBBb), brain edema and severe excitotoxicity. Analytical FT-IR spectroscopy showed profound alteration in molecular biochemical state, with evidences for VEGFR-2 (KDR/Flk-1) signaling mediation. By blocking VEGF/VEGFR-2 binding via pre-treatment with itraconazole we demonstrated that animals' condition was deteriorated soon at 1–2 h post-PNV exposure concurrently with decreased expression of VEGF, BBB-associated proteins, ZO-1, β-catenin, laminin, P-gp (P-glycoprotein), Neu-N (neuron's viability marker) and MAPKphosphorylated-p38, while phosphorylated-ERK and Src pathways were increased. At 5 h and coinciding with incipient signs of animals' recuperation, the proteins associated with protection (HIF-1α, VEGF, VEGFR-1, VEGFR-2, Neu-N, occludin, β-catenin, laminin, P-gp efflux protein, phosphorylated-p38) increased thus indicating p38 pathway activation together with paracellular route strengthening. However, the BBB transcellular trafficking and caspase-3 increased (pro-apoptotic pathway activation). At 24 h, the transcellular route reestablished physiological state but the pro-survival pathway PI3K/(p-Akt) dropped in animals underwent VEGF/VEGFR-2 binding inhibition, whereas it was significantly activated at matched interval in PNV group without prior itraconazole; these results demonstrate impaired VEGF′ survival effects at 24 h. The inhibition of VEGF/VEGFR-2 binding identified 5 h as turning point at which multi-level dynamic interplay was elicited to reverse hippocampal damage. Collectively, the data confirmed VEGFR-2 signaling via serine-threonine kinase Akt as neuroprotective pathway against PNV-induced damage. Further studies are needed to elucidate mechanisms underlying PNV effects.

PNPP-19, A synthetic peptide designed from Phoneutria nigriventer spider toxin is effective for glaucoma treatment in rats

PNPP-19, A synthetic peptide designed from Phoneutria nigriventer spider toxin is effective for glaucoma treatment in rats

PnAn13, an antinociceptive synthetic peptide inspired in the Phoneutria nigriventer toxin PnTx4(6-1) (δ-Ctenitoxin-Pn1a).

Animal venoms are an almost inexhaustible source for promising molecules with biological activity and the venom of Phoneutria nigriventer spider is a good example of this. Among several other toxins obtained from this venom, PnTx4(6–1), also called δ-Ctenitoxin-Pn1a, was isolated and initially described as an insect toxin that binds to the site 3 of sodium channels in cockroach nerve cord synaptosomes (Periplaneta americana) and slows down sodium current inactivation in isolated axons of this animal. This toxin did not cause any apparent toxicity to mice when intracerebroventricularly injected (30 μg). Subsequently, it was demonstrated that PnTx4(6–1) has an antinociceptive effect in three different pain models: inflammatory, induced by carrageenan; nociceptive, induced by prostaglandin E2 and neuropathic, induced by sciatic nerve constriction. Using diverse antagonists from receptors, it was shown that the cannabinoid system, via the CB1 receptor, and the opioid system, through the μ and δ receptors, are both involved in the antinociceptive effect of PnTx4(6–1). In the present work, it was synthesized a peptide, named PnAn13, based on the amino acid sequence of PnTx4(6–1) in order to try to reproduce or increase the analgesic effect of the toxin. As it was seen for the toxin, PnAn13 had antinociceptive activity, when intrathecally injected, and this effect involved the cannabinoid and opioid systems. In addition, when it was evaluated the peripheral effect of PnAn13, via intraplantar administration, this peptide was able to reverse the hyperalgesic threshold, evoked by prostaglandin E2. Therefore, using different pharmacological tools, it was shown the participation of cannabinoid and opioid systems in this effect.

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG−/− mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature.

Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown.Methods:Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences.Results:The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences.Conclusions:Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage.

Background: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides.Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified.Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation.Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

Recombinant peptide derived from the venom the Phoneutria nigriventer spider relieves nociception by nerve deafferentation

The avulsion of nerve roots of the brachial plexus that is commonly seen in motorcycle accidents is a type of neuropathy due to deafferentation. This type of pain is clinically challenging since therapeutical protocols fail or have severe side effects. Thus, it is proposed to evaluate the antinociceptive activity of the recombinant CTK 01512-2 peptide that is derived from the venom of the Phoneutria nigriventer spider, as a future new therapeutical option. The neuropathic pain was surgically induced by avulsion of the upper brachial plexus trunk in groups of male Wistar rats and after 17 days, they were treated intrathecally with morphine, ziconotide, and CTK 01512–2. Behavioral tests were performed to evaluate mechanical and thermal hyperalgesia, cold allodynia, the functional activity of the front paw, and exploratory locomotion after the treatments. The peripheral blood samples were collected 6 h after the treatments and a comet assay was performed. The spinal cord was removed for the lipoperoxidation dosing of the membranes. The cerebrospinal fluid was analyzed for the dosage of glutamate. The recombinant peptide showed an antinociceptive effect when compared to the other drugs, without affecting the locomotor activity of the animals. Mechanical and thermal hyperalgesia, as well as cold allodynia, were reduced in the first hours of treatment. The levels of glutamate and the damage by membrane lipoperoxidation were shown to be improved, and genotoxicity was not demonstrated. In a scenario of therapeutical failures in the treatment of this type of pain, CTK 01512–2 was shown as a new effective alternative protocol. However, further testing is required to determine pharmacokinetics.

Immunodetection of toxins in historesin-embedded sections of Phoneutria nigriventer venom glands using laser confocal scanning microscopy

In the last decades, main advances were achieved in the identification, structural and pharmacological characterization of Phoneutria nigriventer toxins. However, studies on the venom-producing apparatus are rare. Presently, we applied immunolabeling to historesin-embedded cross-sections of P. nigriventer venom glands. Toxins and toxin-secreting cells were successfully located in situ, using laser confocal scanning microscopy. The methodological strategy was successful and may be applied in future studies on venom glands and other secreting tissues, in general.

Transcriptome analysis of the spider Phoneutria pertyi venom glands reveals novel venom components for the genus Phoneutria

Phoneutria nigriventer spider venom has been studied for more than 40 years and several components with pharmacological potential have been described in it. However, studies on venoms from other species of the Phoneutria genus are scarce. In this work, a conventional cDNA library from the species Phoneutria pertyi venom glands was constructed, aiming to identify novel putative cysteine-rich peptide toxins for the genus Phoneutria. 296 unique sequences were identified and 51 sequences corresponded to putative cysteine-rich peptide toxins. Besides cysteine-rich peptide toxins, other putative venom components such as protease inhibitors, defensins and serine proteinases were identified. Furthermore, by manual curation of the sequences with no match at UniProt, we were able to identify glycine-rich proteins (GRP), a class of venom component never described in Phoneutria genus. This work describes the first complete sequences of toxins from the venom of P. pertyi and reveals that, despite most of the retrieved toxins show a high identity to toxins identified in Phoneutria genus, novel putative toxins remains to be described.

Phoneutria nigriventer spider toxin PnTx2-1 (δ-Ctenitoxin-Pn1a) is a modulator of sodium channel gating

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called δ-ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current.

PnPP-19 Peptide Restores Erectile Function in Hypertensive and Diabetic Animals Through Intravenous and Topical Administration

IntroductionWith the aim of overcoming the high toxicity of PnTx2-6 (or δ-CNTX-Pn2a), a toxin from the venom of the armed spider (Phoneutria nigriventer), the 19-aminoacid peptide, PnPP-19 (P nigriventer potentiator peptide), was synthesized based on molecular modeling studies of PnTx2-6. PnPP-19 improved the erectile function of normotensive rats and mice, without eliciting side effects, and no signs of toxicity were observed. In addition, PnPP-19 was able to potentiate the effect of sildenafil. AimTo evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. MethodsCorpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (10−8 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123–radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure ratio in STZ-diabetic rats after PnPP-19 topical administration. Main Outcome MeasuresPnPP-19 may become a new drug able to fill the gap in the pharmacologic treatment of erectile dysfunction, especially for hypertensive and diabetic individuals ResultsPnPP-19 potentiated corpus cavernosum relaxation, in both control and SHR rats. SHR-cavernosal tissue treated with PnPP-19 (1–32 Hz) reached the same relaxation levels as control Wistar rats (16 and 32 Hz). PnPP-19 treatment improved cavernosal tissue relaxation in STZ-diabetic mice and rats. PnPP-19 enhanced cGMP levels in STZ-diabetic mice corpus cavernosum strips. After topical or intravenous administration in rats, 123I-PnPP-19 was mainly recruited to the penis. When topically administered (400 μg/rat), PnPP-19 restores erectile function in STZ-diabetic rats, also improving it in healthy rats by increasing the intracavernosal pressure/main arterial pressure ratio. PnPP-19 exhibited an additive effect when co-administered with sildenafil, showing a novel mode of action regardless of phosphodiesterase type 5 inhibition. Clinical ImplicationsPnPP-19 seems to be an indicated drug to be tested to treat ED in diabetic and hypertensive patients. Strength & LimitationsPnPP-19, although active by topical application and showing safety to human beings (not shown), has low permeability, about 10% of the applied dose. ConclusionOur results showed that PnPP-19 may emerge as a potent new drug that can be topically administered, becoming a promising alternative for erectile dysfunction treatment.Nunes da Silva C, Pedrosa Nunes K, De Marco Almeida F, et al. PnPP-19 Peptide Restores Erectile Function In Hypertensive And Diabetic Animals Through Intravenous And Topical Administration. J Sex Med 2019;16:365–374.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system.

ABSTRACTBackground: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.