While lethal bites by Russell's viper (Daboia russelli siamensis) pose a serious medical problem in the developing countries of Southeast Asia, the situation is further aggravated by the limited effectiveness of antivenins in snakebite treatment (Warrell, D. A., 1992, Recent Advances in Toxinology Research, 1: 121-153, Gopalakrishnakone, P. and Tan, C. K., Eds.), National University of Singapore; Warrell, D. A. 1993 Med. J. Austr. 159: 773-779), and hypersensitivity to horse proteins in some patients during scrotherapy. Treatment of snakebite, especially that of Russell's viper, would be greatly enhanced, if an antidote could be found which would overcome the aforementioned problems associated with use of commercial antivenins.
Neutralising factors towards this end have been isolated and purified from sera of various mammals and snakes, including antihaemorrhagic factors (Catanese and Kress, 1992, Biochemistry 31: 410-418; Yamakawa and Omori-Satoh, 1992, J. Biochem. 122: 583-589; Qi, Z.-Q. et al., 1994, Toxicon 32: 1459-1469), PLA2-inhibitors (Fortes-Dias et al., 1991, Toxicon 29, 997-1008; Perales et al., 1995, Eur. J. Biochem. 227: 19-26; Kogaki et al., 1989, J. Biochem. 106, 966-971; Ohkura et al., 1993, J. Biochem. 113, 413-419) and anti-myotoxic factor (Inoue et al., 1997, Biochem. Mol. Bio. Inter., 49: 529-537). The high neutralising capacity against skin haemorrhage induced by several haemorrhagic venoms seems to be a common feature among the antihaemorrhagic factors, but their neutralising activity against venom toxins has not been tested properly and hence no claim has been made so far as to the use of antihaemorrhagic factor(s) for treating snakebite.
PLA2 is a major component of snake venoms that contributes to different pathological effects of snakebite such as neurotoxicity and myotoxicity, haemostatic disturbance, haemolysis, cardiotoxicity and hypotension. Two antitoxic PLA2 inhibitors, namely the crotalus neutralising factor (CNF) and crotoxin inhibitor from crotalus serum (CICS), have been purified and characterised from the serum of a South American rattlesnake, Crotalus d. terrificus (Fortes-Dias et al., supra; Perales et al., supra). Both are oligomeric glycoproteins with molecular weights in the range of 130-160 kDa and subunit molecular masses of around 23-25 kDa, and act specifically by neutralising crotoxin, the main toxic component with potent PLA2 activity from the South American rattlesnake venom. Although CICS and CNF appear to be the most likely candidates for use as antidotes against Crotalus snake poisoning, their effectiveness against the lethal toxicity of heterologous venoms from viperid snakes like Russell's viper is yet to be tested.
Two more PLA2 inhibitors with molecular weights of 100 kDa and 75 kDa, from crotalid snakes, Trimeresurus flavoviridis and Agkistrodon b. siniticus, respectively (Kogaki et al., supra; Ohkura et al., supra), and another 90-kDa PLA2 inhibitor from an Elapidae, Naja naja kaouthia (Inoue et al., supra) have also been purified, but their capacity to inhibit the toxic effects of the venoms has not been reported. Recently, a PLA2 inhibitor named Bothrops asper myotoxin inhibitor protein (BaMIP), which is an oligomer (molecular weight 120 kDa) composed of five 23-25 kDa subunits, has been purified from the blood plasma of Bothrops asper (Lizano et al., 1997, Biochem. J. 326: 853-859). Although it is active in inhibiting the in vitro PLA2 catalytic activity and the myotoxic and oedematogenic activities of the B. asper myotoxin isoforms, its neutralising activity against the lethal or systemic action of snake venom or toxin has not been reported.
Despite the above advances, Russell's viper bite is still posing a major life-threatening health problem in the Southeast Asia region. Thousands of bites by this snake occur yearly, resulting in an annual mortality of more than 100 in Myanmar alone (Myint-Lwin et al., 1985 Lancet II, 1259-1264). Clinical studies using Russell's viper antivenins produced from different sources have indicated that one product from one area of the region is clinically much less effective for treating bites by another subspecies of Russell's viper in a different locality (Phillips et al., 1988, Quart, J. Med. 68, 691-716). A cocktail of venoms from different Russell's viper subspecies would therefore be desirable to give a broad spectrum of protective antibodies, but this has yet to happen.
From the foregoing, an antivenin drug that will neutralise the toxicity of Russell's viper venoms from all subspecies will have enormous potential as an antidote for all Russell's viper bites, irrespective of subspecies differences. Moreover, a drug having neutralising activity not only for Russell's viper venoms but also for venoms of all major snake species will be a major breakthrough.
PLA2 enzymes have been identified and purified from bovine, porcine, and human pancreas (Fleer et al., 1978, Eur. J. Biochem. 82: 261-269; Verheij et al., 1983, Biochem. Biophys. Acta 747: 93-99; Puijk et al., 1977, Biochem. Biophys. Acta 492: 254-259), and in human synovial fluid aspirates from rheumatoid and osteoarthritis patients (Parks et al., 1990, Adv. Exp. Med. Biol. 275: 55-81; Seilhames et al, 1989, J. Biochem. (Tokyo) 106: 38-42). Because of their implication in a range of diseases including rheumatoid and osteoarthritis, asthma, acute pancreatitis, septic shock, etc. (Vadas et al., 1986, Lab. Invest. 55: 391-404; Vadas et al., 1993, J. Lipid Med. 8: 1-30; Michaels et al., 1994, Biochem. Pharmacol 48: 1-10), recent research has focused on the role of PLA2-inhibitors (PLIs) as possible anti-inflammatory agents (Glaser, 1995, Advances in Pharmaco. 32: 31-66). A number of PLIs have thus been purified and characterized from a variety of sources, including plant, fungi, and bacteria (Cuellar et al., 1996, J. Nat. Prod. 59: 977-979; Matsumoto et al., 1995, J. Antibiotics 48: 106-112; Lindahl et al., 1997, Inflammation 21: 347-356). Additionally, PLIs that interact with PLA2s and inhibit their enzymatic activity, have been purified almost entirely from the sera of venomous snakes belonging to Elapidae and Crotalidae families (Thwin et al., 1988, Toxicon 36: 1471-1482; Ohkura et al., 1999, J. Biochem. (Tokyo) 125: 375-382; Okumura et al., 1998, J. Biol. Chem. 273: 19469-19473; Hains et al., 2000, J. Biol. Chem. 275: 983-991). However, very little attempt has so far been made to produce recombinant proteins that are functionally active as the native inhibitors, nor to study the therapeutic relevance of the recombinant PLA2 inhibitors. Recently, a PLI with potent antitoxic and anti-inflammatory activities has been purified for the first time by us, from the serum of the non-venomous snake Python reticulatus (Thwin et al., 1999, Toxicon 37: 1465). This PLI, initially termed PAF (Python Antitoxic Factor), and later renamed as PIP (Phospholipase Inhibitor from Python), has also been cloned, sequenced, and functionally expressed as fusion protein in Escherichia coli (Thwin et al., 2000, Biochemistry [accepted for publication]). A broad aspect of this invention concerns the potential therapeutic significance of this recombinant inhibitor protein as an antidote for snakebite and other PLA2-related inflammatory conditions.
While the recombinant inhibitor protein can be used either alone or in combination with the neutralizing antibodies to improve the efficacy of commercial antivenins, we have noted that the structural information obtained from our previous study on the inhibitor protein can be effectively utilized to identify small molecular weight short peptides that may act as a surrogate for the larger molecule and be useful as potential anti-inflammatory agent. This will improve the therapeutic usefulness, and at the same time help to reduce the untoward effects commonly associated with the use of high molecular weight homologous or heterologous complexes like the recombinant protein. Short peptides called antiflammins that are synthesized based on the region of highest homology between uteroglobin and lipocortin I, have previously been shown to inhibit PLA2 (Snyder et al., 1999, J. Pharmacol. Exp. Ther. 288: 1117-1124; Rodgers et al., 1997, J. Invest. Surgery 10: 31-36), although there are some reports suggesting that these antiflammins are devoid of PLA2 inhibitory activity (Marastoni et al., 1993, Drug Res. 43: 997-1000; Hope et al., 1991, Agents & Actions 34: 77-80). Development of such small molecular weight peptide inhibitors has been the goal of researchers at many pharmaceutical companies. In this study, a family of oligopeptides corresponding to a region of high amino acid sequence similarity between PIP (Thwin et al., 2000, GENBANK Accession No. AF 232771) and other structurally related snake serum PLIs have been synthesized and examined for their anti-inflammatory activity. Thus another aspect of this invention relates to the synthetic decapeptide that shows potent in vitro PLA2 inhibitory activity and striking anti-inflammatory effects in vivo.