New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom

Scorpion venoms are very complex mixtures of molecules, most of which are peptides displaying different kinds of biological activity. Indeed, these peptides specifically bind to a variety of pharmacological targets, in particular ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins modulating Na+, K+, Ca2+ and Cl(-) currents have been described in scorpion venoms. In this work, we have used several specific antibodies raised against the most lethal scorpion toxins already described to screen the Moroccan scorpion Androctonus mauretanicus mauretanicus venom in order to characterize new compounds. This immunological screening was also implemented by toxicity tests in mice and with mass spectrometry study, providing new informations on the molecular composition of this venom. In fine, we were able to determine the molecular masses of 70-80 different compounds. According to the immunological data obtained, many toxins cross-react with three sera raised against the most lethal alpha-toxins found in North African scorpion venoms, but not at all with those raised against the main beta-toxins from South and North American venoms. Some of the previously described toxins from Androctonus mauretanicus mauretanicus venom could thus be detected by combining immunological tests, toxicity in mice and molecular masses. Among these toxins, one of them, which showed a mild cross-reaction with the serum raised against AaH I (a highly potent toxin from the venom of Androctonus australis), was identified as Amm III and fully sequenced.     

Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces S-phase population in human breast cancer cells (MCF-7)

Venom of some species of scorpions induces apoptosis and arrests proliferation in cancer cells. This is an important property that can be harnessed and can lead to isolation of compounds of therapeutic importance in cancer research. Cytotoxicity was investigated using MTT reduction and confirmed with lactate dehydrogenase release following venom exposure. Apoptosis was evaluated with determination of mitochondrial membrane potential, reactive nitrogen species assay, measurement of Caspase-3 activity and DNA fragmentation analysis. To confirm that venom can inhibit DNA synthesis in proliferating breast cancer cells, immunocytochemical detection of BrdU incorporation was done. Our results demonstrated that venom of Odontobuthus doriae not only induced apoptosis but lead to the inhibition of DNA synthesis in human breast cancer cells (MCF-7). Cell viability decreased with parallel increment of LDH release in dose dependent manner after treatment with varying concentrations of venom. Moreover, venom depleted cellular antioxidants evidenced by depression of GSH and Catalases and concomitantly increased reactive nitrogen intermediates (RNI). These events were related to the depolarization of mitochondria and associated Caspase-3 activation following venom treatment in a concentration dependent manner. Finally, fragmentation of nuclear DNA following venom treatment confirmed the apoptotic property of the said venom. These results suggest that venom of O. doriae can be potential source for the isolation of effective anti-proliferative and apoptotic molecules.     

OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as alpha-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K(+) channels (vertebrate Kv1.1-Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2+/-2.7nM.     

A novel depolarizing activity of scorpion venom fraction M1 due to activation of skeletal muscle nicotinic receptors.

A depolarizing activity following interaction with nicotinic acetylcholine receptors (nAchRs) in skeletal muscle cells, was observed for the first time in the non-toxic venom fraction (M1) of the yellow scorpion Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by recording changes in [Ca2+]i. When applied, M1 (10 microg/mL) induced a transient increase of [Ca2+]i which could be blocked by a prior application of alpha-Bungarotoxin (alpha-Bg-Tx).     

Effect of leukocyte inhibitors benzydamine and cyclophosphamide, on lung injury caused by Tityus discrepans scorpion venom

We studied the effects of benzydamine (BZ) and cyclophosphamide (CP) on acute lung injury caused by Tityus discrepans venom. Mice (male, IVIC strain, ) were pretreated with either BZ or CP i.p. or saline. Envenoming ( mouse) was induced sc. Lung fraction area occupied by fibrin (FF), nuclei (NF), alveolar space (AS) and parenchyma (PF) were determined. Venom increased FF, NF and PF, significantly, and decreased AS. BZ antagonised venom's effect on FF sharply, antagonised partially effects on PF and AS, but was not able to antagonise effect on NF. CP abolished venom effects on NF, AS and PF, but was not able to antagonise the effect on FF. CP was slightly less effective than BZ in reducing FF. Fibrin deposition was associated to leukocyte sequestration. Blocking pro-inflammatory leukocyte metabolic pathways with BZ diminished FF, suggesting that neutrophil activation, inflammation and coagulation are correlated in the genesis of scorpionism acute lung injury.     

Full characterization of three toxins from the Androctonus amoreuxi scorpion venom

In this study, we have characterized the immunological and pharmacological properties of the three major α-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121].The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of α-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as α-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.     

Investigating the chemical profile of regenerated scorpion (Parabuthus transvaalicus) venom in relation to metabolic cost and toxicity

We investigated the biochemical profile of regenerated venom of the scorpion Parabuthus transvaalicus in relation to its metabolic cost and toxicity. Using a closed-system respirometer, we compared oxygen consumption between milked and unmilked scorpions to determine the metabolic costs associated with the first 192 h of subsequent venom synthesis. Milked scorpions had a substantially (21%) higher mean metabolic rate than unmilked scorpions, with the largest increases in oxygen consumption occurring at approximately 120 h, 162 h, and 186 h post-milking. Lethality tests in crickets indicated that toxicity of the regenerated venom returned to normal levels within 4 d after milking. However, the chemical profile of the regenerated venom, as evaluated by FPLC and MALDI-TOF mass spectrometry, suggested that regeneration of different venom components was asynchronous. Some peptides regenerated quickly, particularly those associated with the scorpion's “prevenom,” whereas others required much or all of this time period for regeneration. This asynchrony could explain the different spikes detected in oxygen consumption of milked scorpions as various peptides and other venom components were resynthesized. These observations confirm the relatively high metabolic cost of venom regeneration and suggest that greater venom complexity can be associated with higher costs of venom production.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Statin induced myotoxicity: The lactone forms are more potent than the acid forms in human skeletal muscle cells in vitro

Statins exist in both acid and lactone forms in vivo. High plasma levels of the lactone forms have been observed in patients with statin induced myopathy. In the present study, the hypothesis that lactone forms have a higher potency of inducing myotoxicity as compared to acid forms was investigated. Primary human skeletal muscle cells were incubated with increasing concentrations of lactone and acid forms of atorvastatin, fluvastatin, pravastatin and simvastatin. Following incubation, living myotubes were quantified by fluorescence staining. Atorvastatin lactone showed a 14-fold, fluvastatin lactone a 26-fold, pravastatin lactone a 23-fold, and simvastatin lactone a 37-fold higher potency to induce myotoxicity compared to their corresponding acid forms. Thus, for the four different statins the present study shows a significantly higher potency of the lactone forms, than the respective acid forms, to induce myotoxicity in human skeletal muscle cells in vitro. These results clearly indicate the need to differentiate between acid and lactone forms in future investigation of statin myotoxicity.     

The benzodiazepine midazolam preferentially blocks inactivated Na channels in skeletal muscle fibre

Summary The effects of the benzodiazepine midazolam were studied on frog skeletal muscle fibres held under current- or voltage-clamp conditions. Midazolam induced a concentration-dependent (10^–5 mol/1 to 10^–3 mol/1) block of the action potential and of the underlying Na current. Block of the Na current occurred without any changes in its voltage dependence or in its activation and inactivation kinetics. An apparent dissociation constant of 223 mol/1 was determined for midazolam from the rested Na channels of well polarized fibres. The blocking effect of a threshold concentration (10^–5 mol/1) could be greatly enhanced (up to the complete suppression of the current) by predepolarizations, positive holding potentials or high stimulation frequencies. This apparent voltage- and frequency-dependent block (no use dependence, i.e., no activation block) could be ascribed to a blockade of inactivated Na channels. From the apparent shift towards negative potentials of the steady-state inactivation curve, a dissociation constant of 6.0 mol/1 was calculated for midazolam from the inactivated Na channels, according to the modulated-receptor model. These results show that midazolam preferentially blocks inactivated rather than rested Na channels, and suggest that this mechanism of action might contribute to the well-known myorelaxant effect of the benzodiazepines.Abbreviations MID midazolam - GABA -aminobutyric acid - HP holding potential - AP action. potential - I_Na sodium current - Pdp predepolarization Correspondence to C. O. Malecot at the above address     

The effect of DDT and dieldrin on skeletal muscle fibres

The effects of the chlorinated hydrocarbon insecticides, DDT and dieldrin, on the electrical activity of skeletal muscle fibres of the clawed toad, Xenopus laevis, were investigated by means of intracellular microelectrodes. Both compounds caused an increase in the amplitude of the negative afterpotential and a slowing-down of the falling phase of the action potential, without affecting the resting potential. Neither DDT nor dieldrin caused repetitive activity. It is concluded that the increase in the duration of the action potential, measured at a potential level of ?50 mV, may be responsible for part of the effects of these insecticides on the mechanical response of skeletal muscle.     

Characterization of peptide components in the venom of the scorpion Liocheles australasiae (Hemiscorpiidae).

Scorpion venoms are composed of a number of neurotoxic peptides. A variety of toxins have been isolated from the venoms of scorpions of the family Buthidae, however, little interest has been paid to non-Buthidae scorpions. In this study, we examined the toxicity of the venom of Liocheles australasiae (Hemiscorpiidae) to mice and crickets, and characterized the peptide components by HPLC and mass spectrometry. Over 200 components were detected in the L. australasiae venom by LC/MS analysis, with components of molecular masses ranging from 500 to 5000 Da being particularly abundant. A number of peptides contained two to four disulfide bridges, which was estimated based on the mass difference after derivatization of Cys residues. A peptide having a monoisotopic molecular mass of 7781.6 Da and four disulfide bridges was isolated from the venom. The peptide has a primary structure similar in terms of the position of eight Cys residues to those observed in several peptides found from scorpions, ticks and insects, although biological roles of these peptides are unknown.     

The neutralizing capacity of Androctonus crassicauda antivenom against Mesobuthus eupeus scorpion venom

Scorpion envenomations are considerable health problem in tropical and subtropical regions. There are approximately 1500 species of scorpions worldwide. The number of dangerous species in the Buthidae family is significantly higher than in other families of scorpions. Mesobuthus eupeus is a member of Mesobuthus genus, Buthidae family. In this study, the potency and para-specific activities of Androctonus crassicauda antivenom were investigated against M. eupeus scorpion venom. The median lethal dose of M. eupeus and A. crassicauda scorpion venoms were found to be 0.18mg/kg, 15.45mug/kg by i.c.v injection route. The antivenom showed neutralization effect against the venoms of M. eupeus. One milliliter of A. crassicauda antivenom neutralizes 464LD(50) of M. eupeus and 940LD(50)A. crassicauda venom on mice. Western blotting demonstrated immunologic reaction with the venoms. The monovalent antivenom has immunoactivity and neutralizing capacity to the scorpion venoms. This study indicates that the antivenom produced by Refik Saydam Hygiene Center could be used for the treatment of M. eupeus stings in Turkey.     

Effects of prenatal exposure to Tityus bahiensis scorpion venom on rat offspring development

Scorpion envenoming is a public health problem. In Brazil, the scorpion Tityus serrulatus is considered the most dangerous, but a large number of exposures also occur with Tityus bahiensis. There are quite a few studies in literature about the toxic effects of this venom but it is not known if the venom causes malformations or behavioral defects to the offspring of mothers exposed to the venom during pregnancy. The objective of this work was to determine, in rats, the possible toxic effects of T. bahiensis venom on offspring when injected into rats during different periods of fetal development. Rats were assigned to one of three groups: one control group and two experimental groups that were subcutaneously injected with venom (2.5 mg/kg) on the 10th (GD10) or on 16th day (GD16) of gestation. Pups were evaluated for changes in physical and behavioral development. GD10 treatment group offspring showed an increase in body weight gain, earlier ear unfolding, incisor tooth eruption and vaginal opening. A decrease in the time of palmar grasp and surface-righting reflexes was observed only for males. In GD16 treatment group, earlier ear unfolding, incisor tooth eruption, and delay in eye opening were observed in the offspring. In female pups a decrease in weight gain and in time for palmar grasp reflex, and an increase in time for negative geotaxis were observed. In male pups a delay in the testis descent, decrease in the time of palmar grasp, increase in the time of negative geotaxis reflex and in the general and locomotor activities could be noticed. Therefore, we concluded that a moderate dose of scorpion venom administered to pregnant rats was able to elicit alterations in physical and behavioral development in the offspring during the postnatal period.     

去甲泽拉木醛的体外抗真菌作用研究

目的 研究去甲泽拉木醛的体外抗真菌作用。方法 采用微量液基稀释法测定去甲泽拉木醛与氟康唑单独应用于23株真菌的最低抑菌浓度（MIC）,以棋盘式微量液基稀释法测定两药联合抗耐药白念珠菌的协同指数（FICI）,判断两药联合抗菌效果;并通过纸片扩散实验直观验证两药联合的协同作用。最后通过CCK-8法测定去甲泽拉木醛的细胞毒性。结果 去甲泽拉木醛单用时呈现广谱的抗真菌作用,MIC范围为4~32 g/L。两药联用时,可将氟康唑的有效浓度从大于64 g/L降至0.25 g/L,FICI值介于0.129~0.254之间,两药表现出协同抗耐药白念珠菌作用。CCK-8结果显示,去甲泽拉木醛在高于MIC值4倍浓度下才展示出细胞毒性。结论 去甲泽拉木醛表现出较好的抗真菌作用,与氟康唑联合时有很好的协同效果,且毒性较低。     

特殊制剂体外群体生物等效性研究案例分析

目的：采用体外群体生物等效性评价方法评估4种特殊制剂的等效性。方法：对国内外指南中关于群体生物等效性的内容进行总结,选择混合标度法,针对不同制剂,主要考察粒径参数、给药装置等体外测量数据参数,将指南中的公式和方程运用于2种吸入制剂（吸入用布地奈德混悬液和左沙丁胺醇雾化吸入溶液）以及2种脂质纳米注射剂（阿扎胞苷注射剂和注射用白蛋白结合型紫杉醇）的等效性研究。结果：该文的体外群体生物等效性研究取得良好的实践结果,除了阿扎胞苷注射剂的受试制剂与参比制剂不等效,需要对粒径继续加以改进外,其他3种制剂有95%的把握度说明受试制剂与参比制剂等效。结论：该研究成功将指南中的公式运用在实际案例中,对特殊制剂体外群体生物等效性在制剂研发中的应用有指导意义。     

Effects of a cercarial toxin derived from venom of the scorpion Leiurus quinquestriatus on frog skeletal muscle

Cercarial toxin, a protein purified from Leiurus quinquestriatus venom and toxic to Schistosoma mansoni cercariae, was examined for its effects on frog twitch muscle fibres. In concentrations of 3.4–12 μM the toxin, after a delay of several minutes, produced a slowly developing depolarization of the resting membrane and high frequency miniature endplate potentials associated with spontaneous firing of muscle fibre action potentials and twitching. The action potential duration increased from about 3 to more than 100 msec. In advanced stages of poisoning the amplitude of action potentials decreased while oscillations on the plateau level occurred. Short electrical pulses were followed by repetitive firing of prolonged action potentials, while long stimuli elicited long-lasting action potentials with oscillations on the plateau level. Addition of d-tubocurarine blocked spontaneous sub- and suprathreshold activity. Increasing the Ca concentration of the medium markedly shortened the action potentials and stopped repetitive firing. It is concluded that cercarial toxin interferes with the ionic permeabilities of frog twitch fibres in a way similar to that reported for crude venom or one of its highly toxic fractions in frog nerve.     

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.     

口服固体制剂仿制药一致性评价体内外相关性研究进展

目前仿制药在我国医药市场占据很大规模,口服固体制剂一致性评价得到的关注最多。其中药品的体内外相关性一直是研究者关注的重点及难点,如何通过控制再评价品种与参比制剂体外某些特性（如溶出、制剂学因素等）的相似,达到两者体内生物等效,对于缩短药品的一致性评价进度,降低研究成本具有重大的意义。就目前口服固体制剂一致性评价体内外相关性的相关研究进展进行综述,以期为开展口服固体制剂一致性评价研究工作提供参考。