Juliana Figueira da Silva

Phα1β toxin prevents capsaicin-induced nociceptive behavior and mechanical hypersensitivity without acting on TRPV1 channels

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.

ADP is a vasodilator component from Lasiodora sp. mygalomorph spider venom

Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹⁷⁷ residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.

Synergistic antinociceptive effect of a calcium channel blocker and a TRPV1 blocker in an acute pain model in mice

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 Ph&agr;1&bgr; 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 Ph&agr;1&bgr; and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. Key findings: Ph&agr;1&bgr; and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (&agr;) of 0.07 for Ph&agr;1&bgr; 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 Ph&agr;1&bgr; (interaction index &agr; = 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 Ph&agr;1&bgr; 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.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-d-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.

Characterization of the antinociceptive effect of PhTx3-4, a toxin from Phoneutria nigriventer, in models of thermal, chemical and incisional pain in mice.

Venom-derived peptides constitute a unique source of drug prototypes for the pain management. Many of them can modulate voltage-gated calcium channels that are central in the processing of pain sensation. PhTx3-4 is a peptide isolated from Phoneutria nigriventer venom, which blocks high voltage-activated calcium channels with low specificity, thereby leading to neuroprotection in models of ischemia in vitro. The aim of the present work was evaluating the potential of intrathecal PhTx3-4 in the reversal of different nociceptive states in mice, furthermore assessing the potential of PhTx3-4 in triggering motor side effects. We found that bellow 100 pmol/site, PhTx3-4 did not cause major motor side effects. By comparison, ω-conotoxin MVIIA and ω-conotoxin MVIIC triggered motor side effects at the doses of 10 and 100 pmol/site, respectively. Also, PhTx3-4 (30 pmol/site) caused no significant alterations in the forced locomotor activity test (rotarod) and in the exploratory activity test (versamax). In a model of inflammatory persistent pain (formalin test), PhTx3-4 reversed nociceptive behavior both pre or post-administered, although this effect was observed only at the inflammatory phase of the test and not at the neurogenic phase. Comparatively, ω-conotoxin MVIIC was effective only when post-administered in the formalin test. Nonetheless, PhTx3-4 treatment was devoid of action in acute nociceptive thermal model (hotplate test), whereas morphine showed efficacy in this test. Efficacy of PhTx3-4 in the formalin test was associated with inhibition of formalin-induced glutamate release in the cerebrospinal fluid. PhTx3-4, but not ω-conotoxin MVIIC, reversed NMDA-induced nociceptive behavior indicating a putative role of PhTx3-4 at ionotropic glutamate receptors. Finally, we observed efficacy of PhTx3-4 in ameliorating mechanical hypersensitivity induced by paw incision, a post-operative and more clinically relevant pain model. Taken together, our data show that PhTx3-4 possesses antinociceptive effect in different models of pain in mice, suggesting that this toxin may serve as drug prototype for pain control.

The effects of Phα1β, a spider toxin, calcium channel blocker, in a mouse fibromyalgia model.

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.

The Phoneutria nigriventer spider toxin, PnTx4-5-5, promotes neuronal survival by blocking NMDA receptors.

Spider toxins are recognized as useful sources of bioactive substances, showing a wide range of pharmacological effects on neurotransmission. Several spider toxins have been identified biochemically and some of them are specific glutamate receptors antagonists. Previous data indicate that PnTx4-5-5, a toxin isolated from the spider Phoneutria nigriventer, inhibits the N-methyl-d-aspartate receptor (NMDAR), with little or no effect on AMPA, kainate or GABA receptors. In agreement with these results, our findings in this study show that PnTx4-5-5 reduces the amplitude of NMDAR-mediated EPSCs in hippocampal slices. It is well established that glutamate-mediated excitotoxic neuronal cell death occurs mainly via NMDAR activation. Thus, we decided to investigate whether PnTx4-5-5 would protect against various cell death insults. For that, we used primary-cultured corticostriatal neurons from wild type (WT) mice, as well as from a mouse model of Huntingtons disease, BACHD. Our results showed that PnTx4-5-5 promotes neuroprotection of WT and BACHD neurons under the insult of high levels of glutamate. Moreover, the toxin is also able to protect WT neurons against amyloid β (Aβ) peptide toxicity. These results indicate that the toxin PnTx4-5-5 is a potential neuroprotective drug.

Data and calculus on isobolographic analysis to determine the antinociceptive interaction between calcium channel blocker and a TRPV1 blocker in acute pain model in mice

Determining antinociceptive interaction between Phα1β toxin (a voltage gated calcium channel blocker) and SB366791 (selective TRPV1 antagonist) may have both clinical and mechanistic implications for the pain management. This data in brief article is associated to the research paper “Synergistic antinociceptive effect of a calcium channel blocker and a TRPV1 blocker in an acute pain model in mice”. This material supports the isobolographic analysis performed with the above drugs and shows: data of the dose response curves of the agents given as single drug or combination regimens. Mathematics and statistical processing of dose response curves, proportion of drugs dosage to be used in the combination, calculus of theoretical additive DE20 dose as well as experimentally obtained DE20 are provided. It is also presented details of statistical comparison between theoretical and experimentally obtained DE20.

Conotoxin MVIIA improves cell viability and antioxidant system after spinal cord injury in rats

This study evaluates whether intrathecal MVIIA injection after spinal cord injury (SCI) elicits neuroprotective effects. The test rats were randomly distributed into six groups— sham, placebo, MVIIA 2.5 μM, MVIIA 5 μM, MVIIA 10 μM, and MVIIA 20 μM—and were administered the treatment four hours after SCI. After the optimal MVIIA dose (MVIIA 10 μM) was defined, the best time for application, one or four hours, was analyzed. Locomotor hind limb function and side effects were assessed. Forty-eight hours after the injury and immediately after euthanasia, spinal cord segments were removed from the test rats. Cell viability, reactive oxygen species, lipid peroxidation, and glutamate release were investigated. To examine the MVIIA mechanism of action, the gene expressions of pro-apoptotic (Bax, nNOS, and caspase-3, -8, -9, -12) and anti-apoptotic (Bcl-xl) factors in the spinal cord tissue samples were determined by real-time PCR, and the activities of antioxidant enzymes were also investigated. Application of intrathecal MVIIA 10 μM four hours after SCI prompted a neuroprotective effect: neuronal death decreased (22.46%), oxidative stress diminished, pro-apoptotic factors (Bax, nNOS, and caspase-3, -8) were expressed to a lesser extent, and mitochondrial viability as well as anti-apoptotic factor (Bcl-xl) expression increased. These results suggested that MVIIA provided neuroprotection through antioxidant effects. Indeed, superoxide dismutase (188.41%), and glutathione peroxidase (199.96%), reductase (193.86%), and transferase (175.93%) expressions increased. Therefore, intrathecal MVIIA (MVIIA 10 μM, 4 h) application has neuroprotective potential, and the possible mechanisms are related to antioxidant agent modulation and to intrinsic and extrinsic apoptotic pathways.