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.

The Venom of Philippine Tarantula (Theraphosidae) Contains Peptides with Pro-Oxidative and Nitrosative-Dependent Cytotoxic Activities against Breast Cancer Cells (MCF-7) In Vitro.

BACKGROUND: Breast cancer is a multifactorial disease that affects women worldwide. Its progression is likely to be executed by oxidative stress wherein elevated levels of reactive oxygen and nitrogen species drive several breast cancer pathologies. Spider venom contains various pharmacological peptides which exhibit selective activity to abnormal expression of ion channels on cancer cell surface which can confer potent anti-cancer activities against this disease. METHODS: Venom was extracted from a Philippine tarantula by electrostimulation and fractionated by reverse phase-high performance liquid chromatography (RP-HPLC). Venom fractions were collected and used for in vitro analyses such as cellular toxicity, morphological assessment, and oxidative stress levels. RESULTS: The fractionation of crude spider venom generated several peaks which were predominantly detected spectrophotometrically and colorimetrically as peptides. Treatment of MCF-7 cell line of selected spider venom peptides induced production of several endogenous radicals such as hydroxyl radicals (•OH), nitric oxide radicals (•NO), superoxide anion radicals (•O2-) and lipid peroxides via malondialdehyde (MDA) reaction, which is comparable with the scavenging effects afforded by 400 µg/mL vitamin E and L-cysteine (p<0.05). Concomitantly, the free radicals produced decrease the mitochondrial membrane potential and metabolic activity as detected by rhodamine 123 and tetrazolium dye respectively (p>0.05). This is manifested by cytotoxicity in MCF-7 cells as seen by increase in membrane blebbing, cellular detachment, caspase activity and nuclear fragmentation. CONCLUSION: These data suggest that the Philippine tarantula venom contains peptide constituents exhibiting pro-oxidative and nitrosative-dependent cytotoxic activities against MCF-7 cells and can indicate mechanistic insights to further explore its potential application as prooxidants in cancer therapy.
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Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria.

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of "tiger" spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product's diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

Phlogiellus bundokalbo spider venom: cytotoxic fractions against human lung adenocarcinoma (A549) cells

Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from Phlogiellus bundokalbo and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells. Methods: Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa FluorTM 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A2 (PLA2) activity of the cytotoxic fractions were also measured. Results: We observed and isolated six fractions from the venom of P. bundokalbo collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA2 activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA2 and cytotoxicity. Conclusion: The venom of P. bundokalbo spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.(AU)

Effect of Tarantula Cubensis Extract (Theranekron) on Peripheral Nerve Healing in an Experimental Sciatic Nerve Injury Model in Rats.

AIM: To investigate the effects of systemic application of Theranekron on peripheral nerve healing after compression type peripheral nerve injury. MATERIAL AND METHODS: Twenty-one female Wistar albino rats were randomly divided into 3 groups (n=7): Control (C), injury (I), and Theranekron (T). The right sciatic nerves of rats in the I and T groups were compressed via an aneurysm clip for 5 minutes and 0.3 ml Theranekron D6 was applied via subcutaneous administration once a week in the T group for a total period of four weeks. Nerve conduction velocity and proximal and distal latency of the rats were measured at the end of day 30. The right sciatic nerves of the rats were then removed and myelin damage grading, axon counting, fibrosis assessment, caspase-3, and NF-kB immunochemical staining were performed. The data were analysed statistically and a p value of less than 0.05 was considered to be significant. RESULTS: Axonal degeneration, vacuolization and myelin destruction were found to be markedly greater in group T. Fibrosis and caspase-3 immunoreactivity were less intense in group T. There was a statistically significant difference in the electrophysiological results of groups I and T. However, there were no statistically significant differences in axon number and NF-kB immunochemical evaluation of groups I and T. CONCLUSION: The findings of this study show that Theranekron decreases axonal and myelin damage after sciatic nerve injury and that this neuroprotective effect of Theranekron can be attributed to its anti-inflammatory effect on pro-inflammatory cytokine levels.

The venom of spider Haplopelma hainanum suppresses proliferation and induces apoptosis in hepatic cancer cells by caspase activation in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Spiders and spider venoms have been used in traditional Chinese medicine to treat various ailments for more than 1000 years. For instance, several large spiders have been utilized by the Li People, who mainly live in Hainan Island of China, in their own unique traditional Chinese medicine therapy. Recent studies have indicated that spider venoms may be an important source of bioactive compounds for anti-tumor treatments. However, the specific mechanisms underlying these activities are not yet completely understood. AIM OF THE STUDY: The present study investigated how the venom of the spider Haplopelma hainanum regulate proliferation and apoptosis in HepG2 cells via the underlying molecular mechanisms. MATERIALS AND METHODS: We treated HepG2 cells with various concentrations of the spider venom (0, 10, 50, 100 and 200 µg/mL) for 48 h, and then analyzed anti-proliferation activity, apoptosis-inducing effects, mitochondrial membrane potential (Δψm) and changes in the pro-apoptotic pathway. The anti-proliferation activity was detected by the MTT assay and Western blotting. Flow cytometry was used to analyze both apoptosis and mitochondrial membrane potential. The key pro-apoptotic molecules in the caspase-3 and -9 dependent mitochondrial pathway, including Bcl2 family, were assessed through realtime PCR, Western blotting and enzymatic test. RESULTS: Obvious morphological changes induced by the spider venom included decreased cell numbers, shorter cell length and reduced cell adhesion. MTT and Western blotting demonstrated that the spider venom potently suppressed cell proliferation in a dose- and time-dependent manner with IC50 of 126.00 µg/mL for 48 h. In addition, the spider venom caused a reduction in the mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm under the participation of Bax. Finally, cytochrome c activated caspase-3 and caspase-9, and induced the apoptosis in the HepG2 cells. CONCLUSION: The results indicated that the venom of H. hainanum exhibited potent inhibition effects in HepG2 cells through suppressing proliferation, reducing the mitochondrial membrane potential, activating caspase-3 and caspase-9, and inducing the apoptosis through a mitochondrial-dependent pathway.

Antinociceptive effect of a novel armed spider peptide Tx3-5 in pathological pain models in mice.

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 µg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Partial Characterization of Venom from the Colombian Spider Phoneutria Boliviensis (Aranae:Ctenidae).

We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors.

Effects of Tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats.

INTRODUCTION: Aflatoxins are toxic fungal metabolites that have adverse effects on humans and animals. Tarantula cubensis D6 is used as a homeopathic medicine for different purposes. The present study investigates the effects of Tarantula cubensis D6 on the oxidant-antioxidant balance and some biochemical parameters against exposure to aflatoxin. METHODS: Thirty-two Sprague-Dawley female rats were used and evenly divided into four groups. Group 1 served as control. Groups 2, 3, and 4 received 200 µl/kg.bw/day Tarantula cubensis D6 (applied subcutaneously), 400 µg/kg.bw/day total aflatoxin (approximately 80% AF B1, 10% AF B2, 6 %AF G1, and 4% AF G2), and 200 µl/kg.bw/day Tarantula cubensis D6 plus 400 µg/kg.bw/day total aflatoxin, respectively, for 28 days. At the end of 28 days, blood samples and some organs (liver, kidney, brain, and spleen) were taken from all the animals. Oxidative stress markers (MDA, SOD, CAT, GSH-Px) and some biochemical parameters (glucose, triglyceride, cholesterol, BUN, creatinine, AST, ALT and ALP, total protein, albumin) were evaluated in blood samples and tissues. RESULTS: Aflatoxin caused negative changes in all oxidative stress parameters and some biochemical parameters (glucose, triglyceride, cholesterol, creatinine, AST, ALT, ALP, total protein, albumin). Administration of Tarantula cubensis D6 partly alleviated aflatoxin-induced negative changes. CONCLUSIONS: Our results indicated that Tarantula cubensis D6 partially neutralized the deleterious effects of aflatoxin.

Mapping the interaction site for the tarantula toxin hainantoxin-IV (ß-TRTX-Hn2a) in the voltage sensor module of domain II of voltage-gated sodium channels.

Peptide toxins often have pharmacological applications and are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a group of potential VGSC inhibitors have been reported from tarantula venoms, little is known about the mechanism of their interaction with VGSCs. In this study, we showed that hainantoxin-IV (ß-TRTX-Hn2a, HNTX-IV in brief), a 35-residue peptide from Ornithoctonus hainana venom, preferentially inhibited rNav1.2, rNav1.3 and hNav1.7 compared with rNav1.4 and hNav1.5. hNav1.7 was the most sensitive to HNTX-IV (IC50∼21nM). In contrast to many other tarantula toxins that affect VGSCs, HNTX-IV at subsaturating concentrations did not alter activation and inactivation kinetics in the physiological range of voltages, while very large depolarization above +70mV could partially activate toxin-bound hNav1.7 channel, indicating that HNTX-IV acts as a gating modifier rather than a pore blocker. Site-directed mutagenesis indicated that the toxin bound to site 4, which was located on the extracellular S3-S4 linker of hNav1.7 domain II. Mutants E753Q, D816N and E818Q of hNav1.7 decreased toxin affinity for hNav1.7 by 2.0-, 3.3- and 130-fold, respectively. In silico docking indicated that a three-toed claw substructure formed by residues with close contacts in the interface between HNTX-IV and hNav1.7 domain II stabilized the toxin-channel complex, impeding movement of the domain II voltage sensor and inhibiting hNav1.7 activation. Our data provide structural details for structure-based drug design and a useful template for the design of highly selective inhibitors of a specific subtype of VGSCs.

In vitro potential of Lycosin-I as an alternative antimicrobial drug for treatment of multidrug-resistant Acinetobacter baumannii infections.

The resistance of multidrug-resistant Acinetobacter baumannii (MDRAB) isolates to most traditional antibiotics results in huge challenges for infection therapy. We investigated the in vitro activities of both l- and d-lycosin-I against MDRAB. These two compounds displayed high antibacterial activities and rapid bactericidal effects against MDRAB. Moreover, the compounds retained their activity even at high salt (Mg(2+) or Ca(2+)) concentrations. These results demonstrate the potential of lycosin-I to be developed as a new antibiotic.

Inhibitory effects of a peptide-fusion protein (Latarcin-PAP1-Thanatin) against chikungunya virus.

Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2µg/ml, which is approximately half of the EC50 of PAP1 (23.7µg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.

Phα1ß toxin prevents capsaicin-induced nociceptive behavior and mechanical hypersensitivity without acting on TRPV1 channels.

Phα1ß 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 Phα1ß when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1ß on Ca²âº transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1ß 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 Ca²âº channel blocker) was effective only when administered intrathecally. Phα1ß, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca²âº transients in DRG neurons. The simultaneous administration of Phα1ß and SB366791 inhibited the capsaicin-induced Ca²âº transients that were additive suggesting that they act through different targets. Moreover, Phα1ß did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1ß may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

The venom of the spider Macrothele raveni induces apoptosis in the myelogenous leukemia K562 cell line.

Spider venoms are a rich source of bioactive compounds with therapeutic potential. In traditional Chinese medicine, spiders and spider venoms have been used in the treatment of various ailments. In the present study, the venom of the spider Macrothele raveni potently suppressed cell growth in the myelogenous leukemia K562 cell line in a dose and time-dependent manner with an IC(50) of 5.1 µg/mL. The venom also had a low inhibitory effect on human lymphocytes with an IC(50) of approximately 36.4 µg/mL, indicating that the venom is relatively selective for leukemic cells. Venom treated K562 cells showed typical morphological indicators of apoptosis including condensation of nuclei and fragmentation of DNA. Annexin V-FITC and propidium iodide dual staining further demonstrated that the venom had potent apoptogenic activity. Venom treatment induced caspase 3 and caspase 8 activation in K562 cells and promoted PARP cleavage. The present results indicate that the venom of the spider M. raveni potently and selectively suppresses the growth of K562 cells by inducing apoptosis via caspase 3 and caspase 8 mediated signaling pathways.

Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling.

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.

The antinociceptive efficacy of HWTX-I epidurally administered in rheumatoid arthritis rats.

Rheumatoid arthritis (RA) is one of the inflammatory kinds of arthritis in the clinical situation, and cytosolic Ca2+ overload has been proposed as one of the primary factors for many inflammatory cells activation, which lead to relative enzymes and inflammatory factors release. It is therefore accepted that Ca2+ channel blockers can protect joint injury from inflammation. In the present study we investigated the possible molecular mechanism of the antinociceptive efficacy of HWTX-I, a spider peptide toxin blocking Ca2+ channels, on the rat rheumatoid arthritis model. Our study demonstrates that HWTX-I can relieve pain in the inflammatory joints and eliminate arthrocele to some degree. Moreover, HWTX-I can also decrease the concentration of tumour necrosis factor α (TNF-α) and increase the concentration of interleukin 4(IL-4) and interleukin 10(IL-10) in rat's serum. HWTX-I can also decrease the mRNA expression level of related factors of TNF-α, interleukin 1ß (IL-1ß) and interleukin 6(IL-6) in inflammatory pathways in rheumatoid arthritis. Therefore, the present results show that the epidural administration of HWTX-I is effective in antinociception in the rat model of rheumatoid arthritis, which may act through its inhibition on certain inflammatory pathways.

Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain.

Venoms peptides have produced exceptional sources for drug development to treat pain. In this study we examined the antinociceptive and side effects of Tx3-3, a peptide toxin isolated from Phoneutria nigriventer venom, which inhibits high-voltage-dependent calcium channels (VDCC), preferentially P/Q and R-type VDCC. We tested the effects of Tx3-3 in animal models of nociceptive (tail-flick test), neuropathic (partial sciatic nerve ligation and streptozotocin-induced diabetic neuropathy), and inflammatory (intraplantar complete Freund's adjuvant) pain. In the tail-flick test, both intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of Tx3-3 in mice caused a short-lasting effect (ED(50) and 95% confidence intervals of 8.8 [4.1-18.8] and 3.7 [1.6-8.4] pmol/site for i.t. and i.c.v. injection, respectively), without impairing motor functions, at least at doses 10-30 times higher than the effective dose. By comparison, ω-conotoxin MVIIC, a P/Q and N-type VDCC blocker derived from Conus magus venom, caused significant motor impairment at doses close to efficacious dose in tail flick test. Tx3-3 showed a long-lasting antinociceptive effect in neuropathic pain models. Intrathecal injection of Tx3-3 (30 pmol/site) decreased both mechanical allodynia produced by sciatic nerve injury in mice and streptozotocin-induced allodynia in mice and rats. On the other hand, i.t. injection of Tx3-3 did not alter inflammatory pain. Taken together, our data show that Tx3-3 shows prevalent antinociceptive effects in the neuropathic pain models and does not cause adverse motor effects at antinociceptive efficacious doses, suggesting that this peptide toxin holds promise as a novel therapeutic agent for the control of neuropathic pain. The Brazilian armed spider Tx3-3, a new P/Q and R-type calcium channel blocker, effectively alleviates allodynia in animal neuropathic pain models.

Black widow spider envenomation in pregnancy.

OBJECTIVE: Little data exist regarding the optimal treatment and outcomes of pregnancies complicated by black widow spider envenomation. Our objective is to evaluate the clinical effects, medical outcomes, and treatment differences between pregnant and nonpregnant women. METHODS: This observational study is based on a review of the database maintained by the American Association of Poison Control Centers from 2003 to 2007. RESULTS: Of the 12,640 human black widow spider envenomations reported at 61 poison centers in the United States, 3194 (25.3%) involved women of reproductive age, defined as age 15-45 years of age, with 97 (3.0% of reproductive-age women) being pregnant. Comparing pregnant and nonpregnant women, there were no significant differences in recommended or administered treatments. Pregnant women were more likely than nonpregnant women (OR: 1.84, 95% CI: 1.20-2.83) to have outcomes coded as minor, moderate, or major rather than no effect. Significantly higher percentages of pregnant patients were treated at a healthcare facility where they were either released (36.1% vs. 19.9%, p < 0.001) or admitted (13.4% vs. 4.0%, p < 0.001), than nonpregnant women. There were no documented pregnancy losses. CONCLUSIONS: Black widow spider envenomation is a rare occurrence in pregnant women and the short-term outcomes appear to be favorable.

Latrotoxin-induced exocytosis in mast cells transfected with latrophilin.

α-Latrotoxin (α-LTX) is known to cause massive exocytosis from presynaptic nerve terminals. We investigated the effects of α-LTX on exocytotic release from mast cells, typical non-neuronal secretory cells. When we transfected mast cells with latrophilin, a specific receptor for α-LTX, α-LTX caused intracellular Ca(2+) to increase and led to exocytosis in the presence of extracellular Ca(2+). On the other hand, neither Ca(2+) increase nor exocytosis was observed in the absence of extracellular Ca(2+). These results indicate that α-LTX, together with latrophilin, works as a Ca(2+) ionophore. However, α-LTX had additional effects on signal transduction in mast cells. We found that inhibitors of protein kinase C (PKC) partially suppressed exocytosis. Furthermore, several soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including SNAP-23, were phosphorylated by α-LTX. These results suggest that exocytosis induced by α-LTX can be explained by (1) elevation of intracellular Ca(2+), (2) phosphorylation of SNARE proteins including SNAP-23, syntaxin-4 and VAMP-8 through PKC-dependent and -independent pathways. Our study may provide a new system to investigate the action of α-LTX and the mechanism of exocytosis in mast cells.