Improvement of Malayan cobra (Naja naja sputatrix) antivenin

A low molecular weight toxic fraction was isolated from venom of the Malayan cobra (Naja naja sputatrix) by Sephadex G-50 gel filtration chromatography. The fraction accounted for almost 100% of the venom lethality. Antisera were prepared by immunizing rabbits with the low molecular weight toxic fraction, the glutaraldehyde-treated low molecular weight toxic fraction and the glutaraldehyde-treated, sea snake neurotoxin-enriched low molecular weight toxic fraction, respectively. Only the serum of rabbits immunized with the glutaraldehyde-treated, neurotoxin-enriched fraction gave effective protection against high doses of Malayan cobra venom. This antiserum is thus a potent Malayan cobra antivenin. One milliliter of this antiserum was able to neutralize 1.5 mg of Malayan cobra venom. It is thus 4 – 8 times more potent than the commercially available Malayan cobra antivenins produced by immunizing horses with whole Malayan cobra venom.     

The antipyretic effect of flurbiprofen.

Flurbiprofen, like its predecessor ibuprofen, possesses antipyretic properties in the endotoxin-fevered rabbit. Comparison of flurbiprofen and ibuprofen at varying dosages, reveals that flurbiprofen is at least 15 times more potent in this species. In goats, flurbiprofen is more potent than in rabbits. Flurbiprofen inhibited the pyrogenic effect of intravenously administered leucocytic pyrogen, but it did not alter the release of leucocytic pyrogen from peritoneal exudate cells in vitro. Moreover, flurbiprofen did not inhibit endotoxin-induced release of endogenous pyrogen in vivo. Incubation of leucocytic pyrogen with flurbiprofen did not alter its pyrogenic poteny. These results suggest that the antipyretic activity of flurbiprofen is due neither to interference with endogenous pyrogen synthesis and release, nor to inactivation of circulating endogenous pyrogen.     

Histamine release,formation of prostaglandin-like activity (SRS-C) and mast cell degranulation by the direct lytic factor (DLF) and phospholipase A of cobra venom

Cobra venom, alone and in combination, on mast cell degranulation, histamine release and formation of prostaglandin-like activity (SRS-C) was studied in perfused guinea-pig lungs and in mast cell-containing rat peritoneal cell suspensions. For comparison, the effect of equivalent doses of whole cobra venom was investigated. 1. Cobra venom caused mast cell degranulation, histamine release and SRS-C formation in both systems. For comparable effects much higher doses had to be used in guine-pig lungs than in rat peritoneal cell suspensions. 2. Phase A showed little degranulation of mast cells in both systems, a limited histamine release in rat peritoneal cell suspensions and none in perfused guinea-pig lungs. It caused a considerable SRS-C formation in both, lung tissue and peritoneal cell suspensions. 3. DLF caused histamine release, SRS-C formation and mast cell degranulation in both systems; in rat peritoneal cell suspensions it acted almost as strong as equivalent doses of cobra venom, in guinea pig lungs it was much less active. 4. In rat peritoneal cell suspensions the effects of DLF and phase A in combination did not exceed the sum of their single effects. In guinea-pig lungs these two substances interacted in a potentiating synergism. It is concluded that DLF is the main cytotoxic principle of cobra venom, whereas ph-ase A alone is not cytotoxic. The difference in the synergism of DLF and ph-ase A between rat peritoneal cells and guinea-pig lungs may be due to two different actions of DLF and species differences as regards sensitivity against these actions.     

The effects and properties of sodium nucleinate as a pyrogen working-standard. 9. Pyrogen detection with epinephrine-skin-, dactinomycin- and LAL-tests. The suitability of sodium nucleinate as a pyrogen standard]

Sodium nucleinate (NN) as well as bacterial lipopolysaccharide (LPS) can be detected by epinephrine-skin, dactinomycin and LAL tests. In the quantitative determination of two pyrogen standards for rabbit tests, consisting of NN, a smaller value was found by LAL test for the standard of greatest pyrogenic effect than for that less pyrogenically effective in rabbits. A standard consisting of NN can be used for the pyrogen test in rabbits. But in the future, if necessary a standard consisting of endotoxin will be used, due to its better comparability of results obtained by LAL and rabbit tests.     

Studies on the relationship between the endotoxin induced fever and the antipyretic effect of reserpine in rabbits (author's transl)]

It has been well documented that fever could be mediated with endogenous pyrogen released from reticuloendothelial system(RES) by administration of bacterial endotoxin(LPS) intravenously to rabbits. On the contrary, reserpine which has various pharmacological activities by depleting catecholamines decreases the normal body temperature as well as endotoxin induced fever. In this paper, we focussed our attention on the effect of reserpine on the production of endogenous pyrogen with relation to the antipyretic effect in endotoxin fever and obtained the following results: Endogenous pyrogen could be detected by intravenous administration of LPS(0.5 micrograms/kg) during the fever. However, endogenous pyrogen was undetectable with intracisternal administration of LPS(0.01 microgram/body) which provoked long-lasting fever. Reserpine (1 mg/kg, i.v.) decreased both body temperature induced by intracisternal administration of LPS(0.01 microgram/body) or intravenous administration of LPS(0.5 microgram/kg), however the degree was more extensive in cases of LPS-induced fever. Pretreatment of rabbits with reserpine (1 mg/kg, i.v.) suppressed the fever induced by an intravenous administration of LPS(0.5 microgram/kg), but did not suppress the release of endogenous pyrogen. These data suggest that endogenous pyrogen may not be an important factor in the pathogenesis of endotoxin fever.     

Assay of pyrogens by interleukin-6 release from monocytic cell lines.

A novel in-vitro system has been developed for the detection and quantification of pyrogen in pharmaceutical products. The measured variable was evoked secretion of the pyrogenic cytokine interleukin-6 from MONO MAC 6 monocytic cells incubated with the product. The interleukin-6 was detected using a specific and sensitive ELISA developed for this purpose. The test system detected pyrogenic contamination in 3 batches of therapeutic human serum albumin which had caused adverse reactions in recipients. The contamination was not detected in conventional tests: the rabbit pyrogen test and the limulus amoebocyte lysate test.     

The effects of peptidoglycan, a pyrogenic constituent of gram-positive microorganisms, on the pharmacokinetics of rifampicin

Pharmacokinetics of rifampicin (20 mg/kg orally or i.v.) was determined in calves and rabbits. Seven days later a model pyrogen was administered i.v. to the same animals and 1 hr later the rifampicin administration was repeated. The pharmacokinetic analysis of oral rifampicin was performed using a one-compartment open model with absorption. Intravenously administered rifampicin was analysed by a two-compartment intravascular model. Injection of peptidoglycan in pyrogenic doses led to a significant increase of orally applied rifampicin serum levels in both animal species. The i.v. administration of rifampicin had the same parameters in the control and peptidoglycan experiments. Daily pretreatment of rabbits with small doses of peptidoglycan induced tolerance to the pyrogenic effect. In tolerant animals we did not observe any changes of rifampicin serum levels. Elevated temperature alone was not responsible for observed pharmacokinetic changes leading to the increase of bioavailability of oral rifampicin since another pyrogenic substance (endotoxin) had an opposite effect on pharmacokinetics of previously tested drugs.     

Central versus peripheral sites of antipyretic action of a non-steroidal anti-inflammatory agent, AD-1590, in rabbits

The site of antipyretic action of AD-1590 in the sequential process involved in the development of fever caused by bacterial pyrogen (LPS) was investigated in rabbits. AD-1590 (1 microgram/ml) did not inactivate both LPS and leucocytic pyrogen (LP) and did not affect the generation of LP in the in vitro test. AD-1590 (0.1 mg/kg i.v.) prevented the fever caused by LP as well as LPS, but did not prevent the fever by PGE2 (100 ng/rabbit) injected into the preoptic anterior hypothalamic (PO/AH) regions. A significant antipyretic effect of AD-1590 on LPS-fever was found when AD-1590 (4 micrograms/rabbit) was injected into the PO/AH regions. AD-1590 (0.4 mg/kg i.v.) did not produce anti-pyretic activity against 2,4-dinitrophenol-hyperthermia; the monoamine levels in the brain were not affected with AD-1590 (10 mg/kg p.o.). These results suggest that AD-1590, like acidic non-steroidal anti-inflammatory drugs, produces its antipyretic action through the central mechanisms.     

中华眼镜蛇全毒对离体大鼠窦房结和乳头肌的电生理效应

用玻璃微电极和心脏活体灌流技术,观察到中华眼镜蛇全毒(1μg/ml)在5min内使离体大鼠窦房结起搏动作电位振幅(APA)降低48.1±SD5.8mV,最大舒张期电位(MDP)衰减-52.0±3.0mV,并最好发生“非可逆性去极化”。舒张期4相去极化速率(RP4D)减慢57.1±SD3.5 mV/s,并产生不规则的类“后电位振荡”现象,导致4相去极化和复极化的紊乱,窦性周长(P)亦延长150±SD70ms。离体大鼠乳头肌经蛇毒1μg/ml灌流,3min内即呈现剧烈的负变力效应,等长峰张力(PCF)衰减了12.5±SD 2.8mm。上述结果提示,中华眼镜蛇全毒中可能含有类“心脏毒素”组分。     

Proteome and immunome of the venom of the Thai cobra, Naja kaouthia.

The proteome of the Thai cobra, Naja kaouthia, venom, revealed by two-dimensional liquid chromatography/tandem mass spectrometry, was found to consist of peptides which could be matched with 61 proteins in the database. These proteins were classified into 12 groups according to the differences in their biological activities: cardiotoxins, cobra venom factors, a cysteine-rich toxin, cytotoxins, kaouthiagin, mocarhagin, muscarinic toxin-like proteins, neurotoxins, an oxoglutarate dehydrogenase, phospholipases, serum albumin, and a weak toxin. Horse derived- anti-N. kaouthia venom hyperimmune serum currently used for the treatment of cobra ophitoxaemia reacted only to the cobra venom factors and phospholipases in the cobra holovenom by two-dimensional gel electrophoresis based-immunoblotting. The venom proteomic insight of this study should pave the way for preparing a therapeutic anti-venom of improved quality, i.e. also containing antibodies to the newly revealed toxic, but poorly immunogenic, minor venom components. It is expected that such a preparation should have a higher effectiveness than the currently used anti-venom in resuscitating cobra-bite victims.     

Cardiovascular and respiratory effects of cobra venom and a venom fraction

The cardiovascular and respiratory effect of whole cobra venom and a purified cobra venom fraction were compared in dogs and cats. Both the crude venom and the fraction affected the respiration of dogs markedly and that of cats to a lesser degree. At lethal doses respiratory failure occurred. Cobra venom greatly lowered the blood pressure and decreased the heart rate of dogs and cats. Lethal doses of the fraction produced minimal hypotension and minimal bradycardia in dogs; sublethal and lethal doses produced a similar bradycardiac response in cats.     

Further studies on the antipyretic action of polymyxin B in pyrogen-induced fever.

A study of the antipyretic effect of polymyxin B was undertaken to determine how this agent reduces fever in rabbits. It involved the effects of the drug: (1) on fever induced by exogenous pyrogenes (E. coli lipopolysaccharide, synthetic double-stranded ribonucleic acid, sodium nucleinate from yeast) and leucocytic pyrogen, (2) on the release of endogenous pyrogen in vivo and in vitro, and (3) on leucocytic and exogenous pyrogens in vitro. The results indicate that polymyxin B produces an antipyretic effect in endotoxin-induced fever primarily by an interaction of this cationic macromolecule with the anionic endotoxin molecule. Further it is likely that polymyxin B inhibits endogenous pyrogen synthesis and/or release from polymorphonuclear leucocytes.     

Liver function and pyrexia caused by a pyrogen from Escherichia coli, lysergic acid diethylamide and dinitrophenol

Rabbits with liver damaged by carbon tetrachloride do not respond with hyperthermia to a lipopolysaccharide pyrogen from Escherichia coli or dinitrophenol, but lysergic acid diethylamide develops its usual effect. Rabbits with obstructive liver damage react with hyperthermia to all three pyrogenic factors. The results support the concept of a peripheral action of pyrogen. It is suggested that the liver plays a rôle in the process of transforming the bacterial pyrogen into the endogenous pyrogen.     

Preparation of antibodies to king cobra (Ophiophagus hannah) venom hemorrhagin and investigation of their cross-reactivity

Hannahtoxin, the major hemorrhagin purified from king cobra (Ophiophagus hannah) venom, elicits hemorrhages in rabbits but not in mice. Two antisera against hannahtoxin were prepared: one raised against purified hannahtoxin, while the other was raised against glutaraldehyde cross-linked and detoxified hannahtoxin. The antisera were refined by pepsin digestion and ammonium sulfate precipitation. They are of approximately equal potency in their ability to neutralize the hemorrhagic activity of king cobra venom in rabbits. The antisera did not form a precipitin line with venom of snakes of the Viperidae family nor neutralize hemorrhages elicited in mice by any of these venoms. However, when the hemorrhagic activity was assayed in rabbits, both antisera were able to abolish the hemorrhages elicited by all of the venoms tested. These results suggest that hannahtoxin displays few epitopes in common with hemorrhagins of viperid venoms, except those involved in the neutralization of hemorrhagic activity in rabbits. The epitopes of viperid venom hemorrhagins involved in the neutralization reaction in rabbits are different from those in mice.     

一种水解血小板膜糖蛋白GPIb的眼镜蛇毒组分的分离纯化与鉴定

目的　建立从中华眼镜蛇毒中分离、纯化可水解血小板膜糖蛋白Ｉｂ（ｐｌａｔｅｌｅｔｍｅｍｂｒａｎｅｇｌｙｃｏｐｒｏｔｅｉｎＩｂ,ＧＰＩｂ）组分的方法。方法　肝素亲和层析,分子筛层析,ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳ ＰＡＧＥ）。结果　经Ｈｅｐａｒｉｎ ＳｅｈｐａｒｏｓｅＣＬ ６Ｂ亲和层析及ＳｅｐｈａｄｅｘＧ １５０分子筛层析,从中华眼镜蛇毒纯化出可降解纤维蛋白原和ＧＰＩｂ的活性组分,回收率为０－８８％,ＳＤＳ ＰＡＧＥ证实此组分为相对分子量约４７１００的单肽链蛋白。结论　此方法成功地从中华眼镜蛇毒中纯化出了水解ＧＰＩｂ的组分,具有高效、简便的特点。     

Synergism between phospholipase A and various peptides and SH-reagents in causing haemolysis

Summary The haemolytic action on washed guinea-pig red cells of the following substances has been studied: the direct lytic factor (DLF) of cobra venom, melittin and an apamin-containing fraction of bee venom, anaphylatoxin (AT), angiotensin, vasopressin, saponin, p-chloro-mercuribenzoate (p-CMB) and N-ethylmaleimide (NEM). Further the synergism of these substances with phospholipase A in causing haemolysis has been investigated.In regard to the lytic effects, the substances studied can be classified as follows. 1. Substances which react with SH-groups, either by means of -S-S- bonds (DLF, apamin-fraction, AT, vasopressin) or by other structures (p-CMB, NEM) produce weak or no direct haemolysis, but strongly potentiate haemolysis caused by phospholipase A. Their effect is increased by Ca⁺⁺, inhibited by EDTA, and strongly dependent on temperature (as far as has been investigated). 2. Angiotensin, a peptide without disulfide groups, is not haemolytic, neither directly nor in combination with phospholipase A. Saponin, which does not react with SH-groups, also does not show potentiated haemolysis with phospholipase A in spite of being haemolytic itself. 3. Melittin, though not containing disulfide structures, does produce potentiated haemolysis with phospholipase A, even at concentrations which are not lytic when acting alone.It is concluded that more than one mechanism of potentiating phospholipase A haemolysis exists. One possibility is the reaction of potentiating agents with SH-groups of membrane constituents (enzymes?) of the red cells. This mechanism applies to p-CMB, NEM and to disulfide-containing peptides. It is independent of detergent effects. Another mechanism may be membrane changes due to a lowering of surface tension such as that produced by melittin. It seems doubtful, however, whether this is the only molecular property responsible for the potentiation, as the detergent saponin does not have such an effect. Possibly melittin, in addition to having detergent effects interferes with the same membrane properties which are altered by the SH-reactants.

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Abbreviations used DLF direct lytic factor (cobra venom) - p-CMB p-chloro-mercuri-benzoate - NEM N-ethyl-maleimide - EDTA ethylene-diaminetetra-acetic acid - AT anaphylatoxin Our thanks are due to Dr. K. A. Forster of Mack, Illertissen, for generous supply of bee venom, to Dr. E. Stürmer, Sandoz, Basel, for vasopressin, to Mr. W. Nüsse, Department of Physiology at this Institute, who kindly estimated the surface tension of solutions, and to Mrs. Elke Lufft for skilful technical assistance.     

抗眼镜王蛇毒卵黄抗体的免疫活性及其对眼镜王蛇毒的中和作用

目的：探讨抗眼镜王蛇毒卵黄抗体的免疫活性及其在体内外对眼镜王蛇毒的中和作用。方法：采用嗜硫色谱法一步分离纯化抗眼镜王蛇毒卵黄抗体,通过间接ELISA法对制备的抗眼镜王蛇毒卵黄抗体进行免疫活性检测,采用小鼠体内外保护实验评估抗眼镜王蛇毒卵黄抗体对眼镜王蛇毒的中和作用。结果：嗜硫色谱法制备的卵黄抗体具有良好的免疫活性,并且对眼镜王蛇毒素显示较好的中和效应,在体外6mg/kg抗眼镜王蛇毒卵黄抗体可有效地中和约4LD50（约1.6mg/kg）的眼镜王蛇毒素,而在体内可有效地中和约3LD50（约1.2mg/kg）的眼镜王蛇毒素。结论：抗眼镜王蛇毒卵黄抗体对眼镜王蛇毒具有良好的中和作用,本项目为抗眼镜王蛇毒卵黄抗体的进一步研究开发提供实验依据。     

Platonin, a cyanine photosensitizing dye, inhibits pyrogen release and results in antipyresis

Intravenous injection of the supernatant fluids from human peripheral blood mononuclear cells (PBMC) incubated with lipopolysaccharide (LPS) caused fever in rabbits. The fever was in parallel with the levels of either interleukin-1 beta (IL-1 beta), IL-6, or tumor necrosis factor-alpha (TNF-alpha) in supernatant fluids. When incubating the platonin with the LPS-human PBMC, both the levels of IL-1 beta, IL-6, or TNF-alpha in supernatant fluids and the pyrogenicity of supernatant fluids were significantly suppressed. The febrile response to supernatant fluids from the LPS-stimulated PBMC was attenuated almost completely by adding anti-IL-1 beta, but not anti-IL-6 or anti-TNF-alpha, monoclonal antibody to supernatant fluids. In addition, both the fever and the increased levels of either IL-1 beta, IL-6, or TNF-alpha in rabbit serum following an intravenous administration of LPS were significantly attenuated by pretreatment with an intravenous dose of platonin. Furthermore, the fever induced by intravenous injection of IL-1 beta was reduced by pretreatment of rabbits with intravenous injection of platonin. The data indicate that platonin inhibits production of pyrogenic cytokines (in particular, IL-1 beta) from PBMC and results in antipyresis.     

Effects of phospholipase A2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A₂ from cobra and bee venom on the presynaptic membrane. Toxicon17, 477–488, 1979.—Phospholipases A₂ from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca²⁺ by Sr²⁺ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K⁺ on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.     

Cloning, characterization and phylogenetic analyses of members of three major venom families from a single specimen of Walterinnesia aegyptia.

Walterinnesia aegyptia is a monotypic elapid snake inhabiting in Africa and Mideast. Although its envenoming is known to cause rapid deaths and paralysis, structural data of its venom proteins are rather limited. Using gel filtration and reverse-phase HPLC, phospholipases A(2) (PLAs), three-fingered toxins (3FTxs), and Kunitz-type protease inhibitors (KIns) were purified from the venom of a single specimen of this species caught in northern Egypt. In addition, specific primers were designed and PCR was carried out to amplify the cDNAs encoding members of the three venom families, respectively, using total cDNA prepared from its venom glands. Complete amino acid sequences of two acidic PLAs, three short chain 3FTxs, and four KIns of this venom species were thus deduced after their cDNAs were cloned and sequenced. They are all novel sequences and match the mass data of purified proteins. For members of each toxin family, protein sequences were aligned and subjected to molecular phylogenetic analyses. The results indicated that the PLAs and a Kunitz inhibitor of W. aegyptia are most similar to those of king cobra venom, and its 3FTxs belongs to either Type I alpha-neurotoxins or weak toxins of orphan-II subtype. It is remarkable that both king cobra and W. aegyptia cause rapid deaths of the victims, and a close evolutionary relationship between them is speculated.