Long-term antibody response and protective effect induced by attenuated scorpion toxins: Involvement of memory plasma cells.

In Algeria, Androctonus australis hector scorpion envenomation remains a major problem of public health because of non-efficient therapy. The development of safe vaccine against scorpion venom could be one key strategy for the envenomation prevention. The irradiation of venom by γ-rays develops suitable immunogens which produced effective antivenom and safe vaccine. In this study, we investigated the ability of the irradiated toxic fraction (γ-FtoxG50) to induce long-term memory humoral response in immunized animals (mice and rabbits), by involving the long-lived plasma cells to prevent efficiently the lethality of scorpion envenomation. For this purpose, an appropriate immunization schedule was established in mice and rabbits using three (3) similar doses of γ-FtoxG50 associated with Alum adjuvant. Obtained results indicate that the long-term immunogenicity of γ-FtoxG50 is able to induce the long-term memory humoral response with a high level of specific antibodies. The long-term persistence of antibody levels could depend on bone marrow memory plasma cells. These cells produce continuously antibodies without antigen stimulus. Furthermore, an enhanced memory response was obtained post-repeated envenomation with toxic native venom that leads to improved protection of animals. Together, pre-existing protective antibodies and the activation of memory B-cells could induce a rapid neutralization of scorpion toxins and long-term protection against scorpion envenomation.

In vitro anticancer activity of hydrogen sulfide and nitric oxide alongside nickel nanoparticle and novel mutations in their genes in CRC patients.

This study was carried out to assess the impact of nickel nanoparticles (NiNPs) as well as scorpion venom on colorectal cancer (CRC) cells in the presence and/or absence of 5-fluorouracil (5-FU), hydrogen sulfide (H2S), and nitric oxide (NO) donors and to determine alterations in endothelial NO synthase (eNOS) and cystathionine γ-lyase (CSE) enzyme-producing genes in CRC patients. The IC50 of both H2S and NO donors, along with NiNPs, were determined. The CRC cells were treated for 24hrs, and the cytotoxic activities were assessed using the MTT test. Moreover, the apoptosis was determined after 24hrs and 48hrs using TUNEL assay. Furthermore, the mutations in the eNOS gene (intron 4, -786T>C and 894 G>T) and CSE gene (1364GT) were determined using direct sequencing. The IC50 values for sodium disulfide (Na2S) and sodium nitroprusside (SNP) at 24hrs treatment were found to be 5 mM and 10-6 M, respectively, while the IC50 value for 5-FU was reached after 5-days of treatment in CRC cell line. Both black and yellow scorpion venoms showed no inhibition of cell proliferation after 24hrs treatment. Furthermore, Na2S showed a significant decrease in cell proliferation and an increase in apoptosis. Moreover, a co-treatment of SNP and 5-FU resulted in inhibition of the cytotoxic effect of 5-FU, while a combination treatment of NiNPs with Na2S, SNP, and 5-FU caused highly significant cytotoxicity. Direct sequencing reveals new mutations, mainly intronic variation in eNOS gene that has not previously been described in the database. These findings indicate that H2S promotes the anticancer efficiency of 5-FU in the presence of NiNPs while NO has antiapoptotic activity in CRC cell lines.

Phase 1 Safety, Pharmacokinetics, and Fluorescence Imaging Study of Tozuleristide (BLZ-100) in Adults With Newly Diagnosed or Recurrent Gliomas.

BACKGROUND: Fluorescence-guided surgery (FGS) can improve extent of resection in gliomas. Tozuleristide (BLZ-100), a near-infrared imaging agent composed of the peptide chlorotoxin and a near-infrared fluorophore indocyanine green, is a candidate molecule for FGS of glioma and other tumor types. OBJECTIVE: To perform a phase 1 dose-escalation study to characterize the safety, pharmacokinetics, and fluorescence imaging of tozuleristide in adults with suspected glioma. METHODS: Patients received a single intravenous dose of tozuleristide 3 to 29 h before surgery. Fluorescence images of tumor and cavity in Situ before and after resection and of excised tissue ex Vivo were acquired, along with safety and pharmacokinetic measures. RESULTS: A total of 17 subjects received doses between 3 and 30 mg. No dose-limiting toxicity was observed, and no reported adverse events were considered related to tozuleristide. At doses of 9 mg and above, the terminal serum half-life for tozuleristide was approximately 30 min. Fluorescence signal was detected in both high- and low-grade glial tumors, with high-grade tumors generally showing greater fluorescence intensity compared to lower grade tumors. In high-grade tumors, signal intensity increased with increased dose levels of tozuleristide, regardless of the time of dosing relative to surgery. CONCLUSION: These results support the safety of tozuleristide at doses up to 30 mg and suggest that tozuleristide imaging may be useful for FGS of gliomas.

Pharmacokinetics and Biodistribution of Rhopalurus junceus Scorpion Venom in Tumor-Bearing Mice after Intravenous and Oral Administration

Background: Rhopalurus junceus scorpion venom has shown potential for anticancer treatment. However, there are no scientific evidence about venom pharmacokinetic (PK) and biodistribution (BD) in tumor-bearing mice. Methods: 131I-labeled venom was administrated by intravenous (IV) and oral (PO) routes at the single dose of 12.5 mg/kg. Mice were sacrificed and blood samples, major organs, and tumor were taken at 10, 20, 40, 90, 180, 300, 480, and 1440 min. Results: For IV route, maximum peak concentration (Cmax), elimination half-lives, total body clearance (CL), distribution volume (Vd), mean residence time (MRT), and area under curve (AUC) were 21.77 ± 2.45 %Dosis•h/mL, 12.65 ± 2.1 h, 4.59 ± 0.23 mL/h, 83.80 ± 12 mL, 12.49 ± 2.71 h, and 21.77 ± 2.45 %Dosis•h/mL, respectively. For PO route, they were 0.60 ± 0.07 %Dosis•h/mL, 9.33 ± 1.35 h, 36.94 ± 4.01 mL/h, 497.33 ± 30 mL, 12.40 ± 1.87 h, and 6.89 ± 1.18 %Dosis•h/mL, respectively. PK parameters (Cmax, CL, Vd, and AUC) showed significant differences between IV and PO routes. Bioavailability was 31.6 ± 4% for PO dose. Kidney, stomach, liver, and lung for IV and stomach, kidney, spleen, and lung for PO routes showed the major uptakes for 131I-labeled venom. In tumor tissue, after the maximum uptake for both routes, there was a consistent behavior of radioactivity respect to the major organs during the first 480 min. Conclusion: The PK and BD of R. junceus venom in mice depend on the administration route. These data represent a starting point for future experiments with this scorpion venom in experimental models of cancer.

Genetic engineering and characterisation of chlorotoxin-fused gelonin for enhanced glioblastoma therapy.

Despite substantial advances in its treatment, brain cancer remains a life-threatening disease with a poor survival rate. The main challenges for the conventional chemotherapy include an insufficient efficacy of drugs and toxicity caused by their nonselective mode of action. Recently, great attention has been paid to highly potent macromolecules such as gelonin, a type 1 ribosome-inactivating protein that inhibits protein translation. However, gelonin is poorly internalised into tumour cells and cannot distinguish between cancer and normal cells. To overcome these challenges, we engineered in this study a recombinant gelonin fusion protein with chlorotoxin, known as a brain cancer-homing peptide. The gelonin-chlorotoxin (Gel-CLTX) fusion chimera, produced in Escherichia coli, possessed an equipotent N-glycosidase activity with that of unmodified gelonin and, furthermore, could be selectively internalised into U-87 MG glioma cells over noncancerous glial cells. Consequently, Gel-CLTX displayed substantial inhibition of protein translation in U-87 MG cells, which eventually led to significantly augmented tumouricidal effects. When tested against xenograft tumour-bearing mice, Gel-CLTX showed higher tumour accumulation and inhibition of tumour growth than did gelonin, with a low systemic toxicity. Taken together, our results demonstrate the feasibility of using a fusion strategy for enhanced chemotherapy of brain tumours.

New and safe formulation for scorpion immunotherapy: Comparative study between saponin and FCA adjuvants associated to attenuated venom.

Envenoming by scorpion is a major health problem in Maghreb regions as well as in several regions of the world. Immunotherapy is the only effective treatment for scorpion stings. The immune sera are obtained from hyper-immunized animals with a formulation of venom associated to Freund's Complete Adjuvant (FCA). This formulation seems to protect against several alterations in immunized animals leading to worsening of their health due to added toxicity of native venom and FCA adjuvant. This study aims to provide a more efficient and non-toxic alternative to this formulation. Two formulations of saponin or FCA associated to irradiated venom of Androctonus australis hector (Aah) were used to compare their safety and their efficiency to better enhance the antibody titers against toxic antigens. Both of these formulations were used in immunization schedule of three months. Blood samples were collected every week, cell count, myeloperoxydase (MPO) and eosinophil peroxidase (EPO) activities and specific antibody titers were evaluated. Four months after the last immunization, rabbits were challenged with increased doses of native Aah venom. Results showed that immunization with saponin formulation induced lower inflammatory cell activation as well as reduced MPO and EPO activities compared to that using FCA. The formulation of irradiated venom with saponin seems also to be more efficient in the activation of lymphocytes resulting in higher titers of specific IgG. The immunoprotective effect evaluation showed that the formulation using saponin seems to protected animals until 3 LD50 of native venom compared to that using FCA which protected only until 2 LD50. These results indicate that saponin formulation with irradiated antigen could be more efficient and safe immunizing preparation for the production of sera against scorpion envenomation.

Inhibition of spinal MAPKs by scorpion venom peptide BmK AGAP produces a sensory-specific analgesic effect.

Background Several studies have shown that scorpion venom peptide BmK AGAP has an analgesic activity. Our previous study also demonstrated that intraplantar injection of BmK AGAP ameliorates formalin-induced spontaneous nociceptive behavior. However, the effect of intrathecal injection of BmK AGAP on nociceptive processing is poorly understood. Methods We investigated the effects of intrathecal injection of BmK AGAP on spinal nociceptive processing induced by chronic constrictive injury or formalin. Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and the von Frey filaments test. Formalin-induced spontaneous nociceptive behavior was also investigated. C-Fos expression was assessed by immunohistochemistry. Phosphorylated mitogen-activated protein kinase (p-MAPK) expression was monitored by Western blot assay. Results Intrathecal injection of BmK AGAP reduced chronic constrictive injury-induced neuropathic pain behavior and pain from formalin-induced inflammation, accompanied by decreased expression of spinal p-MAPKs and c-Fos protein. The results of combining low doses of different MAPK inhibitor (U0126, SP600125, or SB203580; 0.1 µg for each inhibitor) with a low dose of BmK AGAP (0.2 µg) suggested that BmK AGAP could potentiate the effects of MAPK inhibitors on inflammation-associated pain. Conclusion Our results demonstrate that intrathecal injection of BmK AGAP produces a sensory-specific analgesic effect via a p-MAPK-dependent mechanism.

Reactogenicity and safety assessment of an attenuated nanovaccine against scorpion envenomation: Preclinical study.

An attenuated nanovaccine (Nps - V∗) has been developed to protect humans from fatal scorpion envenomation in at-risk regions. This study was conducted to evaluate the toxicity and the local reactogenicity of the Nps - V∗ nanovaccine developed against Androctonus australis hector (Aah) venom. Assessment of the systemic inflammatory response and serum cytokine levels were evaluated in vaccinated mice with 100µg of irradiated Aah venom (V∗) encapsulated or not into polymeric calcium-alginate nanoparticles (Nps) and injected by subcutaneous (s.c) route. The local reactogenicity was evaluated by dermal Draize observations and skin tissue analysis at the injection site of vaccinated rabbits with 250 or 500µg of V∗-loaded into Nps. All animals gained weight and had normal food consumption during the study. Additionally, results showed that the nanoformulation Nps - V∗ did not cause clinical evidence of systemic toxicity in mice or rabbits, a transient edema/erythema at the injection site was only recorded as treatment-related reactogenicity. These results indicated a favorable safety profile for Nps - V∗ and supported its use in superior animal tests, then in a Phase 1 clinical trial.

Local knockdown of Nav1.6 relieves pain behaviors induced by BmK I.

Voltage-gated sodium channels (VGSCs) in peripheral nociceptive sensory neurons are critical to transmit pain signals. BmK I purified from the venom of scorpion Buthus martensi Karsch (BmK) has been demonstrated to be the primary contributor of envenomation-associated pain. However, the role of distinct VGSCs such as Nav1.6 in the induction and maintenance of pain behaviors induced by BmK I was ambiguous. Herein, using molecular and behavioral approaches we investigated the mRNA and protein expression profiles of Nav1.6 in rat DRG after intraplantar injection of BmK I and tested the pain behaviors after knockdown of Nav1.6 in BmK I-treated rats. It was shown that during induction and maintenance of pain responses induced by BmK I, the expression of Nav1.6 in DRG was found to be significantly increased at both mRNA and protein levels. The percentage of co-localization of Nav1.6 and Isolectin B4, a molecular marker of small diameter non-peptidergic DRG neurons, was increased at the maintenance phase of pain responses. Furthermore, spontaneous pain and mechanical allodynia, but not thermal hyperalgesia induced by BmK I, were significantly alleviated after knockdown of Nav1.6. These data strongly suggest that Nav1.6 plays an indispensable role in the peripheral pain hypersensitivity induced by BmK I.

Safety and efficiency of active immunization with detoxified antigen against scorpion venom: side effect evaluation.

OBJECTIVE: The efficiency and safety of vaccine are the most important properties, however, as any medication, it can induce side effects. This prophylactic therapy could be used to prevent the lethal and pathophysiological effects induced after scorpion envenomation. METHODS: In this study, detoxified venom associated to alum adjuvant (V*alum) is used as a vaccine against scorpion venom for immunization of mice. We evaluate the safety and the inflammatory response of this vaccine. We also investigated the protective effect of this formulation against the toxicity of native Androctonus australis hector venom. RESULTS: Results showed no adverse events occurred after immunization of animals. This active immunization of animals did not cause change in vascular permeability, no edema formation in the studied organs. Furthermore, there are no IgE production in sera, nor change in the morphology of the mast cells in skin tissues. However, low inflammatory response triggered by activating the recruitment of eosinophils associated to IL-4 and IL-5 release was observed. All immunized animals are protected from the toxic effects of native venom until 6 LD50 and to 7 LD50 after the second challenge. CONCLUSION: This safe vaccine preparation seems to induce a long-term protection without any risk of deleterious inflammatory response.

Modulatory features of the novel spider toxin µ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus.

BACKGROUND AND PURPOSE: Naturally occurring dysfunction of voltage-gated sodium (NaV ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. EXPERIMENTAL APPROACH: We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model. KEY RESULTS: We identified a potent NaV inhibitor named µ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (CaV 3) currents but had no activity against the voltage-gated potassium (KV 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV 1.1-1.7). Df1a also slowed fast inactivation of NaV 1.1, NaV 1.3 and NaV 1.5 and modified the voltage-dependence of activation and inactivation of most of the NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the NaV activator OD1. CONCLUSION AND IMPLICATIONS: µ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.

Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells.

Cardiorespiratory alterations in rodents experimentally envenomed with Hadruroides lunatus scorpion venom

Abstract Background Hadruroides lunatus is the most abundant scorpion species in the Peruvian central coast, where most of the accidents involving humans are registered. In spite of its prevalence, there are only very few studies on H. lunatus envenomation. The aim of the present study was to analyze the cardiorespiratory alterations caused by H. lunatus envenomation in rodents. Methods Wistar rats injected with H. lunatus scorpion venom were submitted to electrocardiography. After euthanasia, rat lungs were collected and histopathologically analyzed. Mouse cardiomyocytes were used to perform immunofluorescence and calcium transient assays. Data were analyzed by ANOVA or Student’s t-test. The significance level was set at p< 0.05. Results It was observed that H. lunatus venom increased heart rate and caused arrhythmia, thereby impairing the heart functioning. Lungs of envenomed animals showed significant alterations, such as diffuse hemorrhage. In addition, immunofluorescence showed that H. lunatus venom was capable of binding to cardiomyocytes. Furthermore, mouse ventricular cardiomyocytes incubated with H. lunatus venom showed a significant decrease in calcium transient, confirming that H. lunatus venom exerts a toxic effect on heart. Conclusion Our results showed that H. lunatus venom is capable of inducing cardiorespiratory alterations, a typical systemic effect of scorpionism, stressing the importance of medical monitoring in envenomation cases.

Investigation of in vivo potential of scorpion venom against skin tumorigenesis in mice via targeting markers associated with cancer development.

Cancer is the leading cause of morbidity and mortality all over the world in spite of the advances made in its management. In this study, we investigated the in vivo anti-tumorigenic potential of the venom obtained from a medically important scorpion species Leiurus quinquestriatus on chemically induced skin cancer in mice. Animals were divided into five groups, with 13 animals in each group. All the treatments were given topically on the shaved dorsal surface of the skin. Animals in Group 1 received vehicle only (0.2 mL acetone). Moreover, 7,12-dimethylbenz[a]anthracene (DMBA, 400 nmol per mouse) was applied to all the animals in the remaining four groups. After 1 week, different concentrations of venom (17.5 µg, 35 µg, and 52.5 µg per animal) were applied to each animal in the Groups III-V. Thirty minutes after the application of venom, croton oil was applied on the same position where venom was administered to the animals of Groups III-V. Animals in Group II were treated as the positive control (without venom) and received croton oil as in Groups III-V. The findings of this study revealed that venom extract of L. quinquestriatus inhibits DMBA + croton oil-induced mouse skin tumor incidence and tumor multiplicity. Venom treatment also decreased the expression of proinflammatory cytokines. Immunohistochemistry results showed a downregulation of the expression of molecular markers such as Ki-67, nuclear factor kappa-B, cyclooxygenase-2, B-cell lymphoma-2, and vascular endothelial growth factor, in venom-treated animals. Our findings suggest that the venom of L. quinquestriatus possesses in vivo anticancer potential and may be used in the development of anticancer molecules.

Brain natriuretic peptide suppresses pain induced by BmK I, a sodium channel-specific modulator, in rats.

BACKGROUND: A previous study found that brain natriuretic peptide (BNP) inhibited inflammatory pain via activating its receptor natriuretic peptide receptor A (NPRA) in nociceptive sensory neurons. A recent study found that functional NPRA is expressed in almost all the trigeminal ganglion (TG) neurons at membrane level suggesting a potentially important role for BNP in migraine pathophysiology. METHODS: An inflammatory pain model was produced by subcutaneous injection of BmK I, a sodium channel-specific modulator from venom of Chinese scorpion Buthus martensi Karsch. Quantitative PCR, Western Blot, and immunohistochemistry were used to detect mRNA and protein expression of BNP and NPRA in dorsal root ganglion (DRG) and dorsal horn of spinal cord. Whole-cell patch clamping experiments were conducted to record large-conductance Ca2+-activated K+ (BKCa) currents of membrane excitability of DRG neurons. Spontaneous and evoked pain behaviors were examined. RESULTS: The mRNA and protein expression of BNP and NPRA was up-regulated in DRG and dorsal horn of spinal cord after BmK I injection. The BNP and NPRA was preferentially expressed in small-sized DRG neurons among which BNP was expressed in both CGRP-positive and IB4-positive neurons while NPRA was preferentially expressed in CGRP-positive neurons. BNP increased the open probability of BKCa channels and suppressed the membrane excitability of small-sized DRG neurons. Intrathecal injection of BNP significantly inhibited BmK-induced pain behaviors including both spontaneous and evoked pain behaviors. CONCLUSIONS: These results suggested that BNP might play an important role as an endogenous pain reliever in BmK I-induced inflammatory pain condition. It is also suggested that BNP might play a similar role in other pathophysiological pain conditions including migraine.

Medical application of scorpion venom to breast cancer: A mini-review.

Breast cancer is the leading cause of mortality in women worldwide. Today, 1 in 8 women born in the United States will have an invasive cancer in their lifetime. Despite significant attempts, the prognosis of metastatic breast cancer still remains poor. This has compelled scientists to look elsewhere for better therapeutic outcomes. Recent advances in venomic studies have demonstrated some promise in cancer-related ailments. Scorpion venom, a complex cocktail of biogenic amines, proteins, peptides, mucoproteins, organic salts and neurotoxins has shown a potential therapeutic application due to its cytotoxic, apoptogenic, immunosuppressive and antiproliferative properties. This communication reviews the effects of scorpion venom components on breast cancer and their mechanisms.

Enabling Noninvasive Systemic Delivery of the Kv1.3-Blocking Peptide HsTX1[R14A] via the Buccal Mucosa.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, a well-recognized therapeutic target for autoimmune diseases. To overcome the poor oral absorption and consequent need for regular injections, the potential of the buccal mucosa for systemic delivery of HsTX1[R14A] was investigated. For in vitro studies, FITC-HsTX1[R14A] and HsTX1[R14A], in solution or formulated in a mucoadhesive chitosan-based gel (3%, w/v) with or without cetrimide (5%, w/w), were applied to porcine buccal epithelium mounted between Ussing chambers and buccal mucosal permeation assessed. HsTX1[R14A] was also administered to Swiss outbred mice at a dose of 10 mg/kg in the same formulations. In vitro, administration of FITC-HsTX1[R14A] and HsTX1[R14A] in the chitosan gel containing cetrimide resulted in detectable buccal permeation with 0.75% and 0.58%, respectively, of the applied dose appearing in the receptor chamber over 5 h. After buccal administration to mice, HsTX1[R14A] was detected in plasma, with the presence of cetrimide in the gel further enhancing plasma exposure, with area under the plasma concentration-time curve values of 77.9 ± 9.7 and 31.0 ± 2.3 nM·h, respectively. The buccal mucosa is a promising alternative administration route for the systemic delivery of HsTX1[R14A] for the treatment of autoimmune diseases.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.

Pulmonary Delivery of the Kv1.3-Blocking Peptide HsTX1[R14A] for the Treatment of Autoimmune Diseases.

HsTX1[R14A] is a potent and selective Kv1.3 channel blocker peptide with the potential to treat autoimmune diseases. Given the typically poor oral bioavailability of peptides, we evaluated pulmonary administration of HsTX1[R14A] in rats as an alternative route for systemic delivery. Plasma concentrations of HsTX1[R14A] were measured by liquid chromatography coupled with tandem mass spectrometry in rats receiving intratracheal administration of HsTX1[R14A] in solution (1-4 mg/kg) or a mannitol-based powder (1 mg/kg) and compared with plasma concentrations after intravenous administration (2 mg/kg). HsTX1[R14A] stability in rat plasma and lung tissue was also determined. HsTX1[R14A] was more stable in plasma than in lung homogenate, with more than 90% of the HsTX1[R14A] remaining intact after 5 h, compared with 40.5% remaining in lung homogenate. The terminal elimination half-life, total clearance, and volume of distribution of HsTX1[R14A] after intravenous administration were 79.6 ± 6.5 min, 8.3 ± 0.6 mL/min/kg, and 949.8 ± 71.0 mL/kg, respectively (mean ± SD). After intratracheal administration, HsTX1[R14A] in solution and dry powder was absorbed to a similar degree, with absolute bioavailability values of 39.2 ± 5.2% and 44.5 ± 12.5%, respectively. This study demonstrated that pulmonary administration is a promising alternative for systemically delivering HsTX1[R14A] for treating autoimmune diseases.

A Combined Approach Employing Chlorotoxin-Nanovectors and Low Dose Radiation To Reach Infiltrating Tumor Niches in Glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive form of glioma, with life expectancy of around 2 years after diagnosis, due to recidivism and to the blood-brain barrier (BBB) limiting the amount of drugs which reach the residual malignant cells, thus contributing to the failure of chemotherapies. To bypass the obstacles imposed by the BBB, we investigated the use of nanotechnologies combined with radiotherapy, as a potential therapeutic strategy for GBM. We used poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PNP) conjugated to chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells. Silver nanoparticles were entrapped inside the functionalized nanoparticles (Ag-PNP-CTX), to allow detection and quantification of the cellular uptake by confocal microscopy, both in vitro and in vivo. In vitro experiments performed with different human glioblastoma cell lines showed higher cytoplasmic uptake of Ag-PNP-CTX, with respect to nonfunctionalized nanoparticles. In vivo experiments showed that Ag-NP-CTX efficiently targets the tumor, but are scarcely effective in crossing the blood brain barrier in the healthy brain, where dispersed metastatic cells are present. We show here that single whole brain X-ray irradiation, performed 20 h before nanoparticle injection, enhances the expression of the CTX targets, MMP-2 and ClC-3, and, through BBB permeabilization, potently increases the amount of internalized Ag-PNP-CTX even in dispersed cells, and generated an efficient antitumor synergistic effect able to inhibit in vivo tumor growth. Notably, the application of Ag-PNP-CTX to irradiated tumor cells decreases the extracellular activity of MMP-2. By targeting dispersed GBM cells and reducing MMP-2 activity, the combined use of CTX-nanovectors with radiotherapy may represent a promising therapeutic approach toward GBM.