Indian Journal of Expcrimcntal Biology Vol. 40, Dcccmber 2002, pp. 1353- 1358

Review Article

Snake venom as therapeutic agents: From toxin to drug development

Sanjoy Kumar Pal l, Aparna Gomes2, S C Dasgupta' & Antony Gomes*

Laboratory of Toxinology & Expcrimental Pharmacodynamics . Dcpartment of Phy~ i o l ogy, Uni versity of Calculla 92. A PC Road, Kolkata 700009. India

Snake bitc injuri es and dcath arc soc io-mcdica l problcms of considcrablc magnilUdc. In India a large number or pcoplc sufTcr and die evcry year duc to snakc vcnom poisoning. Snakc vcnom, though grcatly fearcd , is a nalUral biological resourcc, containing sevcral componcnts that cou ld be of potcntialthcrapcutic valuc. Usc of snakc vcnom in different patho­physiological conditions has bcen mcntioncd in Ayurvcda, homeopathy and folk mcd icine. It is well known that snake venom is complcx mixturc of cnzymcs, pcptidcs and protcins of low molccular mass with spccific chcmica l and biological activit ics. Snakc vcnom con tains scvcral ncurotoxic. cardiotoxic. cy totox ic, nervc growth factor, lectins, disintrigrins. hucmorrhagins and many othcr diffcrcnt cnzymcs. These protcins not only innict death to animals and humans, but can also hc uscd for thc trca tmcnt or th rombosis, arthriti s. canccr and many othcr di scascs. An ovcrvicw of va ri ous snakc vcnom componcnts that havc prospects in hca lth and di seascs arc di sc usscd in thi s rcvicw.

Venomous and poisonous aninla ls are a signifi cant cause of global morbidity and mortality. In snakes, venom, used for defense, is an evo lutionary adapta­tion to immobili ze the prey . Venoms are the secretory substances of the venomous an imals, which are syn­thes ized and stored in spec ific areas of their body. Snake venoms are a unique physio log ica l product of nature as they are mixtures of different substances, which are hi ghly spec ific and have great affin ity fo r different crucial and essential functional organi za tion of ce ll s and ti ssues. Efforts are already on for the use of these natural resources as power fu l probes for el u­cidating complex biological processes of vital impor­tance. Several iso lated snake venom proteins with a known mode of action have found practical applica­ti on as pharmaceut ica l agents, diagnost ic reagents or preparative tools in haemostaseo logy, neurobiology and complement research I. Varieti es of tox i ns are of interes t in drug design, because the tox ins provide three-dimensional templates for creating small mole­cu le mimicking interes ting pharmacological proper-

. ? ll es-.

Prescnt address: I Departl11cnt or Gastrocntcrology, Sanjay Gandhi Postgraduatc Institute of Medical Sciences, Racba rcli Road. Lucknow 226 014. India. 2NcIV Drug Dcvclopmcnt Divi sion, Indian Inst itutc or Chemica l Biology. Kolkata 700032. India . .1post Gradua tc Departmcnt or Zoo logy, Maulana Azad Collcgc, Kolkata 7000 13, India. *Corrcspolldcll t author: Phonc:91 -033-350-8386 Fax : 9 1-033-35 1-9755 E-Illa i I :agomcs(!:il cllcc.ernet. in

The use of snake venom as medicine was known to man for centuries. It is over sixty years since it was first reali zed that the physiologica ll y act ive compo­nents of snake venoms might have therapeutic poten­tial 3A

. In Charak Sall/hila cobra venom has been said to be use ful in DlIshyodara and l a/odara (asc iti es) . SlIshl'llta and Vagb//Clla also mentioned similar use. Sarallghara SOli/ita menti oned the use of cobra venom in 'Sallllipatik lwom'. In the Unan i system of med icine cobra venom has been used as a ton ic, aph­rodi siac, hepatic stimulant and for rev i va l in co llapsed conditions5

. Venoms of Viper, Crota/lI s, Cobra and Lacasis are also routinely used in homeopathic medi­cine. Chinese physicians use snake venom products routinely to treat stroke and view them as effective and relatively safe6

. Natural protease inhibitors to haemorrhagins in snake venoms and their potential use in medicine have al so been reported 7

.

Fibrillogellolytic alld fibrillolytic activity of sllake vellom

The ability of some snake venom enzymes to clot fibrinogen has resulted in great therapeutic importance. These enzymes remove fibrinogen from the circulation w ithout converting it to fibrin , or causing platelet ag­gregation . Snake venom proteinases that cleave peptide bonds in the fibrinogen molecule can be divided in to three groups (i) Thrombin li ke enzymes (thrombin pro­teases), (ii ) fibrinogenolytic enzymes, (iii ) enzymes activating plasminogen8

. The venoms from three snakes have been shown to induce defibrinogenation, ancord from the venom of Ca//use/osllf([ rh odoSIOIl f([,

1354 INDIAN J EXP BIOl, DECEMBER 2002

batroxobin from the venom of SO/hrops alox lIIooj elli

and crotalase from Crolallls adallla llleLiS. A ncord and batroxob in have been in vesti gated in patients w ith stroke, deep ve in thrombos is and cerebral infarc ti on9

,

myocardial infarcti on, peripheral atrial thrombosis, priap ism and sick le ce ll cri sis. Gasmi el al. 10 reported the thromboly ti c ac ti vity o f Vipem lebelilla fibrino­genase (VIF) in a rat model of venous thrombos is. Thrombus was produced in the posteri or vena cava by injec ting human f ibrinogen and th rombin. Injec ti on of V IF induces fl ow res toration after one hour and measurement of the fibrinogen level decreases by abollt 30% after 3 hr. V IF act ion is not dependent on plasmi nogen acti vators and may act synergisti ca ll y with them, thereby providing an in triguing potential clini ca l applicati on for di sso lu tion of blood clot. Fur­ther, that several fibrinogenoly ti c enzy mes have been purified by different laboratori es, like atroxase from Crowllls afrox ll , f ibro lase from Agkisrrodoll cOlllor­frixl 2, and lebetase from Vipera lebefill a l3. At present there is growing interes t in f ibrinolytic or f ibrinoly ti c agen ts because of their possible appli cati on in throm-b · . d I ~ I ) d' . I OSIS ep lso es . . , an In expenmenta tumor meras-

. I' 16 TI .. .. f' f" b I 17 I X tasls Slut les. l e III vlfro activity 0 I ro ase . I 14 . . d . I b anc atroxase were Investi gate In a venous t lrom 0-

sis model system. These studies demonstrate the po­tentia li ty of the venom fibrinol y ti c enzymes as clini ­ca ll y lI seful th rombo lyti c agents. Zhu ef al? ) worked out the ribrinogenolytic properties of natrahagin, a proteinase from cobra venom and its effects in human platelet aggregati on. Natrahag in is oc -y f ibrinogenase with an inhi bitory effect on platelet membrane glyco­protein dependent platelet aggregat ion. The effects of green pit viper Trilll eresllrII.I' albolalHis and Trilll ere­

slims lIIacraps venom on the fibrino ly ti c system in humans were also studied. I t was round that venoms from both the vipers possess a thromb in like effect ill I' ifro but cause a defibrinati on syndrome ill Vi VO"I .

Prote ins found in venoms, especiall y of the snake fam il y Viperidae, often exert with a narrow spec i fic­ity, ac ti va ting, inact i vat ing or other convert ing effects on different components of the homeostati c and fibri­nolyt ic system respect i ve ly. Si igur el 01.22 con firmed the ex istence of both coagulants and anticoagulants of the hemostati c system in the venom of Vipera lebef­ilia. This venom contains both factor X ac ti vator and factor V activator f ibrinolyt ic enzymes. Wang ef 01. 23

reported defibrinogenating ef fects of batroxobin (Oe­f ibrase) in rats and inhibition of migration of human vascular smooth musc le cell s by the plasma of ba­troxobin treated rats in vitro . Gomes and Oe24 have

ident ified a peptide tox in from the Ind ian King Cobra Ophiop/7agus hannah venom by T LC fo llowed by HPLC. The peptide named as "Hannahpep" possessed fibrinolytic activ ity, with a possibil ity of use in th rombos is.

Cardiotollic alld autiarrythmic activity of snake venom Signi ficant contribution comes from the work of

Shermann el 01. 25 . He observed that Malayan pit V iper venom has blood thinning properties and could be effective in trea tin g stroke patients. From a study of 500 stroke patien ts, 42% who were given the snake venom drug (Ancord) w ithin 3 hr of stroke. Mayberg & Fulan 26 reported that onset regained signifi cant functioning compared to 34% who got a placebo. The ro le of ancard in pati ents w ith heparin induced thrombocytopeni a thrombosis has been clearly estab­li shed 27 . Use of ancord has also been reported sLlcces­si ve in cardi ac catheteri zation and coronary artery bypass graft ing28

. The anti thromboti c and throm­bolyt ic act ivit ies of agkisac lltaci n (Agk), a component isola ted from Agkisfrodon aCllfllS venom, were deter­mined ill vifro and ill vi vo. Agk call signi f icantly in ­hi bi t thrombus format ion and accelerates thrombol y­sis of pulmonary emboli in rats29

. Recent ly, Gomes ef 01.30 identi fies a non protei n micromolecul ar toxin (mo l. wt 260) from the Indian King Cobra venom. Thi s tox in possesses antiarry th mic properti es at microgram leve l.

Alltineoplastic activity of sllake venom Snake venom has been used to develop newer

drugs to combat vari ous di seases including cancer. Ca lmette el 01.3 in vesti gated the LI se of cobra venom in the trea tment of cancer in mice. Match 3 1. 32 showed that cobra venom, in ex tremely minute doses pro­duced analgesic effects. This led to th e poss ibil i ty of therapeutic use of the cobra venom in arthriti s and cancer. Phospholipase A2 was isolated from BOfhrops

Il ewweidii venom which produced cytotox ic act iv ity on B 16 FlO melanoma ce ll33 Basavarjappa ef a l . 3~ and Rudrammaji ef 0/.

35 showed that Indian cobra Naja naja venom was cyto tox ic to Ehrli ch asci tes tu­mour cell s. Cytotox ic P4 was isolaled from Naja Ili­

g ricollis venom, wh ich produced cy totox ic effec t on Wehi-B leukemia ce1l36

. VRCTC-3 10, an animal de­ri ved anticancer agent, from snake venom a combina­ti on product of crotoxin and cardi otox in, possessing anti tumour acti v ity in vivo. A phase I study was per­formed to evaluate the max imum tolerated dose (MTO), safety profile and phannacok ineti c data o f

PAL el. a/.: SNAKE VE OM AS THERAPEUTIC AGE TS 1355

VRTC-31O with PLA2 acti vity and inhibitory effec ts . I d ' II I' 37· , 9 T agalllst luman an mUrIne tumour ce Illes . . wo

purified venom toxins have been combined to produce I . . d 40 4 lB' d h· h f t liS ul1lque pro uct · . . eSI e ti S, anot er actor

was isolated from Naja naja atra venom which posessed a cytotoxin without phospholipase acti vi ty and had a selecti ve cytotox ic action on human cancer ce ll lines42

. Cobra venom factor treatment was intro­duced in latent feline leukemic virus immune cats. After one to three weeks an increase in viral antigen in marrow myelomonocyti c ce ll s, a circulating im­mune complex was noted by Kraut et a1. 43

. The inter­acti on between tumour cc ll s and microvasculature including the adhesion of tumour ce ll to endothelium and extra ce llular matrix as well as their migratory ac ti vity are prerequisites for metastasis to occur. In thi s connection, it has been observed that thrombin is capable of enhanc ing ill vifro tumour meta'stasis po­tential in terms of adhesive properti es and migratory response. Rhodopsin an arg-gly-asp containing anti­platelet snake venom peptide served as an inhibitory agent in the prevention o f thrombin enhanced metas­tasi 44 . Hevnandez et 0 1.45 reported that fraction of CrowIIl .s· dIlrissIIs terrificIIs venom, cro tamine and crotoxin complex A and B when treated for spontane­ous sarcoma cell s of female rats, induced both tumour regression are increase in animals survival time. In contrast to these findi ngs Shi an-Lin er al.46 failed to obta in sati sfactory resul ts in tumour growth control with the venom of Naja lI([ja. Iwaguchi ef al. 47 studi ed ill Fivo and ill Fitm effects o f cy totoxin 1 and II from Noja lIoja venom on normal and tumour cell lines. No antitumour effects we re ohserved ill vi Fo . However, ill \Iifm , they noti ced higher cy toly ti c effects on tumour cells than on normal ce ll s. /11 Fif ro and ill \'i \'() ex­peri ment carri ed out to eva I uate the effec ts o f Naja lI aj (/ sialllellsis venom and that o f crotox in complex A and B from CrowlIls d llrissIIs ferri/lc IIs venom on tumour ce ll vi z. human metastati c breast adenoca rci­noma, murine sarcoma I RO, Ehli ch asc ites tumour and breas t carcinoma, fail ed to demonstrate any antitu ­moUl' ellec ts4X

• A nother famil y o f snake venom anti ­coagulants known as di sintrigrin s are current ly being tested as anti tumour agents because they al so interfere with the functi on o f intregrin on tumour cell s. M ark­land eI al. ·19 has tested ,7 disintrigrin from southern copperhead venom for act iv ity aga inst human mam­mary tumour in a mouse model. Disint ri grin inhibits tumour growth and also angiogenes is and metastasis, probabl y because they prevent the normal functi on o f intregrin on endothelium('. Salmos in is a snake venom

derived novel disintregin antagoni zed platelet aggre­gation. Both the metastatic tumour growth and solids tumour growth th at developed in mice were effec­tively suppressed by slamos in50

. The anticancer acti v­ity o f Indian krait BIIllgIlms caemleus venom has been evaluated against Ehrlich ascites carc inoma (EAC) in Balb C mice. This venom, at vari ous do ·e levels inhibited tumour ce ll growth, both ill Fiva and ill vitro. Thi s cytotoxic activity of venom was al so observed by MTT assay and confirmed by 31-1 thymidine incorporation. Superoxide dismutase was also influenced by venom51

. Recently Markland et al. 52 isolated a nove l snake venom disintrigrin th at inhi b­its human ovari an cancer dissemination in ovacar 5 cell line and angiogenesis in an orthoped ic nude mouse model.

Alltiparaiytic activity ojsllake vel/Olll Venoms of several snakes are known to cause mus­

cular paral ys is. Subsequently several neurotox ic com­ponents that inhibit neuromuscular transmiss ion by attack ing different targets have been isolated. euro­tox ins from snake venoms have been utili sed in dif­ferent pharmacolog ical and biochemical studies o f nicotini c acetylcholine receptors (nAChRs) in the neurone and neuromuscular junction. The use of 125 1 labelled snake venom toxin to identify ACh R and thei r antibodies ultimately permitted both the purifi ca­ti on and characterisati on o f AchR and an understand ­ing of the patholog ical mechanism impairing neu ro­muscular transmission in myasthenia gravis (M G). A nti -AChR antibodies have been demonstrated to be the pr incipl e agents in the pathogenesis of MG, and their determinati on allowed dcfinite diagnos is of MG53

. Cl inical trial s are now considered t ~ evaluate the effec ts o f Notcx in , a snake venom phospholi pase that attacks mOlor nerve terminal s and muscular cells , in the treatment o f ptos is. It \,vas found that the sa tel­lite ce ll s between the musc le ce ll s contain mostly norl11a l mitochondria. When skeleta l muscle is dam­aged by notex in , satellite ce ll s divide to form re­placemel\ t musc le ce ll s and because ce ll regencration is accompanied by mitochondria regenerati on, the mitochondria in ncw musc le ce ll arc in much bettcr shapc than in the o ld ce ll s. As thc ptos is is a result of musc le we;ikness in the small eas il y access ible levator musc le o f the eye lid , it is an ideal candidate fur

. I (, notcx ln tlerapy .

A llti arthritic activity ojsllake vel/o/JI Just as proteases con trol the acti vati on and des truc­

ti on o f hormones and other bio log icall y important

1356 I DI AN J EXP BIOl, DECEMB ER 2002

proteins, they are also regul ated by another cl ass of molecules known as the protease inhibitors. These substances, many of which are themsel ves proteins, bind to protease enzy mes in such way as to prevent them from reacting with and clearing the bonds of hormones and other pro teins when they are no longer needed. Interest in venoms as potenti al source of anti­infl ammatory substances gained much attention after the di scovery of a purified protein component from cobra vcnom, which temporaril y depleted the termin al C3-C9 component of complement cascade. It was fo und that cobra venom factor pretreatment of rabbits prevented the inducti on of ex perimental immune complex a rthriti s5~. Snake venom has been used to elucidate the pathophysiology of several experimental model of arthriti s. Because fibrin is commonl y found in large amount in arthriti c joint and because fibri­nolys is induction with phenfo rmin has ameli orated signs and sy mptoms in human patients with rheuma­toid arthriti s, it was postulated th at fibrin plays a ro le in the acute and chroni c arthriti s process. Intraperito­nea l inj ection of aq ueous suspension of peptidog lycan polysaccharide complex induced chroni c arthriti s in rat. Pretreatment of rat with cobra venom factor de­laycd the acute in fla mmati on observed in thi s modcl for 3 days, but had no effccts on the subsequent de­velopment of chron ic arth ri tis.).) . It has been fo und that sma ll dose of cobra venom fac tor depleted rat C3 for about 3 days and delayed the onset of adju vant arthri­tis about 3 days if given within 9 days of adju vant admini stration. It is al so inhibited the maximum in ­fl ammatory response if gi ven 14 days after adju van I. These observati ons suggc. t that the third component of compl ement pl ayed an essential role in adju vant arthriti s 56. Ford e l 0 1.57 defibrin ated rabbit with arvin , an cx tract fro m the venom of Malays ian pit viper Cal­loselasllla rh odosfollla in an attempt to suppress the acu te and chroni c infl ammatory response in ex peri ­mental immune arthriti s. No inhi bitory effect on the arthriti c process was observed. Later, Stanzler and Miller5R observed that systemic deco mplementati on with cobra venom factor had no anti-infl ammatory effect in an immune complex model of rabbit arthriti s.

O'her possible 'herapeutic actioll of snake vellOIll The use of snake venom as an analges ic agent is

we ll documented59. The anti noc icepti ve acti vity of the

venom was dose and ti mc dependent and persisted aftcr neutrali zation with a spec ifi c antivenin . Mor­phine enhanced the analges ic effect of Crofal/us durissus rer(lficus venom and naloxone antagoni zed

thi s effect, suggesting an endorphin-li ke acti vity for the factors60

. Chen and Robinson6 1 dcmonstrated an ant i nocicepti ve effect in mice with cobrotox in , a frac­ti on obtained fro m the venom of Naja naj a afro and a similar effect has also been reported for snake venom neurotox ins admini strated orally or by inj ecti on into mice62

. A novel analgesic tox in (h3nnalgesic) was isolated fro m the venom of King Cobra Ophiophaglls hal/I/ah. The tox in did not increase the convul sion threshold in the dose range of 8-64ng/g in the max i­mal electroshock se izure tes ts in mice. The neurotox in produced analgesia in the dose range of 16- 32ng/g (ip) without causing any neurological or muscul ar dcficit63

. Two venom based medications, cobrox in and nylox in were marketed fo r the treatment of pai n, arthriti s and other di sorders but were banned by the US Food and Drug admini strati on ill 1970 because of in fec ti venessfH

. The Indi an King Cobra Opliiopliaglls hannah venom induces immunomodu latory and hae­mopoieti c stimulant acti vit/ 5

. Recentl y from the venom of Vipera rtlsselli , a heat srable compound has been identi fied whi ch was found to produce card io-

. d I ' . . I I I 66 . I respiratory 1110 u all on In anima moce s Wit 1 a pos-sible app licati on in cardi oresp iratory related path­phys iolog ical conditi on.

Conclusion It may be concluded th at in fo rmati on is now ava il ­

abl e to establ ish th at snake venom tox in may serve as a starting materi al fo r drug design ' 0 co mbat several pathophysiologica l problems such as cardiovascular di sorders, neuro log ical problems and cancer therapy. Very few clini ca l studies are avai lable and there is a need for ex tensive research programs to clini ca ll y work out the above-mentioned areas, with a view to develop newer drugs to combat human suffe ring and death globally.

Acknowledgement Thi s rev iew has been dedicated to our beloved

teacher late Prof. S C Lahiri , Kolkata, since thi s re­view was hi s ori ginal thought th at we tri ed to ful fil.

References I Siockcr K, Snakc vcnom protcins affccting hcmostasis and

fibrin olys is. in , Medical lise of snake \ enOll1 p r01eins. editcd by K Stockcr (C RC Prcss, Boca Raton) 1999 , 97.

2 Harvcy A L, Bradlcy K N, Cochran S A. Rowan E G, Pra lt J A, Lrcldt J A & Jcrusa linsky D A, Wh o.t can tox ins lellus fo r drug di scovery? Toxicon , 36 ( 1998) 1635 .

3 Ca lmclla A, Sacnz A & Costil l , Effects du vcnirnclc cobra sur les grcffes canccrcll ses cl su r Ic cancer spo nlanc (adcnu carcinoma) de la souri s, C R A cad Sci, 197 (1933) 205 .

PAL el. al. : SNAKE VE OM AS TH ERAPEUTIC AGE TS 1357

4 Macht D I, New development in the pharmaco logy and therapeutics of cobra venom, Trw/s Alii Th er Soc, 40 ( 1940) 62.

5 Debnath P K, ChalLi rvedi G N, Bhall ac harya S K & Upad­haya Y N, Comparative study of so me pharmaco logica l ac­ti ons of crude and shodhita cobra ve nom, 1 Res 11111 Med, 7 (1972) 54.

6 Senior K, Taking the bite out of snake venom, Th e Lal/cel ,

353 (1999) 1946. 7 Perez J C & Sanchez E E, Natural protease inhibitor to hem­

orrhagins in snake ve nom and their potential use in medeine, Toxicol/, 37 (1999) 703 .

8 Siigur J & Siigur E, The direc t acting fibrin (ogen)o lytic en­zy mes from snake venoms, 1 Toxicol Toxil/ i?eI' ie IV, I I ( 1992)9 1.

9 Kim J S, Yoon S S, Kwon S U, Ha J H, Suh E J & Chi H S, Treatment of acute cerebral infarcti on with arginine esterase: A controlled study with heparin . Cerebrol 'asc Dis, II (200 I) 25 1.

10 Gasmi A, chabchoub A. Guerma 7. i S, Karoui H. Elayeb M & Dell agi K. Further characteri z;lli on and thrombolyti c acti vit y in 3 rat mode l of a fibrinogenase from Vipem lehelil/ a

venom, ThrO/I/ !J Res, 86 (1997) 233 . II Willis T W & Tu A T, Purifi cation and biochemical charac­

teri sation of atroxase, a nonheJ11orrh agic fi brinolyti c protease fro m western diamondback ralliesnake ve nom. lJiochelllisl/T.

27 ( 19R8) 4769. 12 Ege n N 13 , Russe ll F E, Sammons D W, Humphreys R C,

GU3n A L & Markland F S Jr, Isolation by preparati ve isoelectri c focusing of a direct acting fibrinolytic enzyme from the veno m of A~kisl rodol/ cOlllonrix cOlllonrix (South­ern copperhead). Toxicol/ , 25 ( 19R7) 11 89.

13 Gasmi A, Karou i M, Benlastar Z, Karou i H. EIAyeb M & Dellaga K, Purifi cat ion and characteri sation of a fibrino­ge n3se from Vijiem lebelil/(/ vc nom. Toxicol/ , 29 ( 199 1) 827 .

14 Markland F S, Fibri (ogen)oly ti c en7.y mes fro m snake venom, in , Haelllos/asis allli al/illlal I'e l/Oll/S. editcd byPrickle H & Markland F S (Marcel Dekker, New York) 1988, 149.

15 Stocker K. Composition of snake venoms, in , M edical lise (~r sl/ake I'el/Oll/ pJ'Oleil/s. edited by K F Stocker (Boca Raton: CRC Press) 1990, 33.

16 Markland F S, Antitumour ac ti on of crotalase, a de fibrin oge­nating snake venom enzy me, Selllil/ Throlllb Hellwsl. 12 ( 1986) 284-290.

17 Gaddis R. Ahmed , Tennant K, Markland R, Mecca T & Lacz J, 11/ vilro and il/ l'il'O thrombol yti c effi ciency of a fibri ­nolytic enzymes iso lated from the sou thern copperhead snake, FASElJ l , 3 ( 1989) 619.

18 Markland F S, Friedrichs G S, Pewill S R & Lucchesi B R, Thrombo lyti c effect of recombinalll fibrolase or APSAC in a ca nine model of carOlid art ery thrombos is, CirclIllIliol/, 90 (1994) 2448.

19 Willi s T W, Tu A T & Mill er C W. Th rombolys is with a snake venom protease in a rat model of vcnous thrombos is, Till'OlIIb Res , 53 (1989) 19.

20 Zhu Z G & Wu S G. Fibrinogenolyti c properti cs of nal ra­hagin (a protein ) from cobra ve nom and it s effect on human platelet aggregation . ZhO//~g ll o Y(W Li Xlle Baa. 20 ( 1999) 944.

2 1 Rojuckaria P. Intragu mtornchai T, Sallapiboon R, Mu an­pas itporn C, Pakmance N, Khow 0 & Swasdikul D, The effcts of green pit viper TrilllereslIl'IIs albolabris and

TrilllereSlIl'IIs lIIacrops venom on the fibrinolytic systcm in human , Toxicol/ , 37 ( 1999) 743.

22 Si igur E, Toni smagi K, Trummal K, Samel M, Vija H, Subbi J & Si igur J, Factor X activator from Vipem lebelil/a snake venom molec ular characteri za ti on and substrate spec ifi cty. Biochilll Biophys Ac({/, 156R (200 I) 90.

23 Wang D S, Han3moto M, Fang F, Ohba M, Ishii M, Kinura F, Higaki E & Senga H, De fi brinogenating effects of ba­troxobin (dcfibrase) in r:1Is and inhibition of mi grati on of human vascu lar smooth mu sc le cell s by the plasma of ba­troxobin treated rats ill vilro, AlheJ'OscleJ'Osis, 156 (200 I) 73.

24 Gomes A & De P, Hannahpep: A novel fibr inolytic peptide from the Indian King Cobra Ophiol'haglls tWl/l/ ah venom. Biochelllic Biol'hys Res COIIIIII, 266 ( 1999) 49 1.

25 Sherm<lnn D G, Atkinson R P, Chippendale T, Lev in K A. Ng K, Futrell , Hsu C Y & Levy D E, Intravenous ancord STAT study, a randomi 7.ed cOl1lrolledtrial , stroke trcatment with ancord trial , l AMA, 283 (2000) 2395.

26 Mayberg M R & Furlan A. Ancrod is snake vc nom an anti­dOle for snake, lAM A, 283 (2000) 2440.

27 Carter C J. New development in acute anticoagul ant therapy what improves over trad itional heparin are on the horizon. PO.\'lgrad M ed, 99 ( 1996) 129.

28 Lathan L 0 & Staggcrs S L, Ancrod: the usc of snake ve nom in the treatment of patients wit h heparin-induced thrombocy­topenia and throm bos is undergoi ng coronary artery by pass grarting: nursin g manage ment, /-lear! LIIII~ , 25 ( 1996) 45 1.

29 Li S. Ji H. Cheng X, Li Bx & Ng T B, Antithrombolytic and thrombolyt ic activities of Agkisaculaci n. a snake venom pro­teinase in experimcntal an imal. , Cel/ Phal'llJ(Jcol, 35 (2000) 179.

30 Gomes A, Saha Archita. Biswas A K & Dasgupta S C. Oc­currence of non protein mi cromolec ul ar tox in from the In­di an king cobra Ol'hiol'haglls hal/I/ah ve nom. (u npu blished data).

31 Match D I, Experimental and clinical study of cobra venom as analgesic , Proc Nal Acad Sci, (USA ) 22( 1936) 61.

32 Macht D I, Therapeuti c experi ences with cobra ve nom, /II/II II/ I M ed, II (1938) 1824.

33 Daniele J J, Bianco I D, Delgado C. Briones CD & Fidelia G D, A new PLA2 iso rorm iso lated from BOlhJ'Ol's 11 1'11-

lI 'iedie(Yaraca chica) venom with novcl kinetic and chrom a­tographi c properties, Toxicol/, 35 ( 1997) 1205.

34 Basavarajappa 13 S & Gowda T V. Comparat ive characteri sa­tion of two toxic phospholiapse A2 rrom Indian cobra (Naja I/oja I/aja) veno m. Toxicol/ , 30 ( 1992) 1227 .

35 Rudr<lmmaji L M S & Gowda Y V, Purification and charac­teri zati on of three ac idic cy totox ic phospholipase A2 from Indi an cobra Naja I/aja IlOja ve nom, ToxicolI, 36 (1998) 921 .

36 Chaim-Mat yas A, Borkow G & Ovadia M, Iso lation and characteri za tion of a cytotoxin P4 from the ve nom of Naja I/ilj r iclllli.\· I/ig ricollis preferentiall y active on tUlllou r ce lls. 13iochelll 1111 ,24 ( 199 1) 415.

37 Costa L A, Miles H A, Diez R A, Avanjo C E, Molina C M C & Cervellino J C, Phase I slUdy of VRCTC- 3 10, a purifi ed phospholipase A2 purified from snake venom inpati cnt s with refractory ca ncer saf'c ty and phannacok ineti c data , AlIli Cal/­cer DJ'IIIj.\', 8 ( 1997) 829.

3R Costa L A, Miles H. Araujo C E, Gonza les S & Vill arrubi a V G, Tumour regress ion of ad vanced carcinomas roll owi ng in ­tra and/or peri tumoural inoc ulation with VRCTC-3 10 in human: preliminary report or two cases, t llll/lilllOl'ham [{Jcol 1IIIIIIIIIWIO.wcol, 20 ( 1998) 15.

1358 INDIAN J EX P SIOL, DECEMBER 2002

39 Costa L A , Fornari M C, Berardi V E, M il es H A & Diez R A, I II vilro effect of snake phospholi pase, A2 on seru m IL­I JI,TNF JI and IL- Ira levels in human, 1111111111 101 lell. 75 (200 I ) 137.

40 Newman R A , Vida l J C, V iskati s L J, Johnson J & Etcheverry M A , VRCTC-31 0 a novel compound of purified an imal toxins separates an titumour efficacy fro m neurotox ic­it y, 111 1'1'.1'1 Nell' Drugs, II ( 1993) 151.

41 DeToll a L J, Stump K C, Russe ll R, V iskati s L J, Vidal J G, Newillan R A & Etcheve rry M A , Tox ici ty of thc nove l ani ­mals derived anti cancer agcnts, VRCTC-31 0: acute and sub­chronic studies in beag le dogs, Toxicolog.\'. 99 ( 1995) 31.

42 Zhong X Y. Liu G F & Wang Q C, Purificat ion and antican­cer acti vity of cy totox in - 14 frolll venom of Naja lIaja aIm.

ZhOllgg llO Yao Li Xlle /J(/{). 14 ( 1993 ) 279. 43 Kraut D I, Expcrilllcntal and clinical study of cobra vcnom as

analges ic, Proc Nat Acad Sc i (USA ), 22 ( 1936) 6 1. 44 Chi ang H S, Yang R S & Huang T F. Thrombin cnhances the

adhes ion and migration of human co lon adenocarcinoma cc ll via increased beta 3- integrin express ion on the tumour cell surface and their inhib ition by the snake venom peptide rhodostoillin . /Jr J Callcer, 73 ( 1996) 902.

45 Hernandez-Plata G, Rey L, Hern andez H, Val ero N, Cendali G. Ancz M F & Alvardo M , Con tri bucion :11 es tudi o sobre los ci'ec tos antineopl as icos del rata. in Proceedillg (d' COII ­

gress!! Latillo A lll er icallo del help£'lIIlogia , 3 Sao Paulo. Un ivesidade Estadual de CO lllpinas, 1993, 16.

46 Shiau Lin S Y, Su S C & Lec C Y. Comparat ivc studies on cy totox ins from the Indian cobra venom and cardiotox in from the Forill osan cobra venom. with spec ial reference to the effect on the growth of the Ehrlich asc ites tUlllor cell in mice, J Fom w.\'(1/1 M ed Assoc, 75 ( 1976) 32X.

47 Iwaguchi T. Tachechi M & Hayashi K . Cytolytic act ivi ty of cy totox in isolated from Indian cobra venom against experi­mental tumour cell. /J iochelll 1111 , 10 ( 1985) 343 .

4/\ Baldi A , M llI'doh J, Medrano E E, Bonaparte Y P, Lusti g E S & RUlll i L , Estud ios tend ientes a detcrillinar las posib1cs pro­priedades antituillorai es dcl vcneno dc cobra y dcl complejo cro tox ina AyS. M edic illll ( 13 . A ires) 48 ( 19SS) 337.

49 Markland Jr. F S, Snake venoms, Drugs. 54 (S uppl 3) ( 1997) I. 50 Kang I C. Lee Y D & Killl D S . A nove l disintri grin sal­

mos in inhibits tumour angiogcnesis. Callcer Res, 59 ( 1999). 3754.

5 1 GOllles Aparna . l3esra Sheila & Vcdashi ro mani J R. Ant ican­cer acti vity of Indian K I'ait (Bllilgarus ('ae rulells ) venom in EAC- Balb C mice (Unpubli shed Data).

52 Markland F S. Shick K , Zhon Q. Golubkor V , Sherw in R. Ri chters V & Sposto R, A nove l snake venom disilllcgrin

that inhibit s human ovarian cancer di 5~emina ti o n and angio­genesis in an orthotopi c nude mousc model, H aelllos/(/sis. 3 1

(200 1) I X3 . 53 Ohta M , Ohta K & Hayashi K, Medica l applicat ion of snake

venom components, in Halldbook 0/ Nalllral ToxillS Vol 5.edited by A T Tu (Marcel Dekkcr, Inc NY) 199 1, P 349.

54 Henson P H & Cochrane C G, The cffcct of complemen t de­pleti on on ex perimental ti ssue injury. AIIII NY Awd Sci . 256 (1975) 426.

55 Schwab H, A llen J B, Anderl e S K , Dalldorf F, Eisenberg R & Cromarti e W I. Relationship of complement to experimen ­tal arthritis induced in rats with st reptococcal ce ll wa ll s. 1111-1IlIllIOlog.l'. 46 ( 1982) 83 .

56 Kourounak is L K , Nelson R A & Kupusta M A. The effect of a cobra vcnom facto r on complement and adju vant induccd di sease in rats, Artllrilis rh l' lIl11 , 16 ( I lJ73) 7 1.

57 Ford P M , Bell W R , Bluestone R. Gumpel J M & Webb F W. The effect of A rvin on experimen t:d immune arthrit is in rabbi ts, IJr J Exper PmllOl , 5 1 ( I ) 1970, 8 1-X6.

58 Stenzler SA & M ilicI' F, Fa i lure o f cobra venom factor to in­hibi t ant igen induced arthriti s, Arthrilis Rhelllll. 20 ( 1997), 1567.

59 Pi colo G. Giorgi R, Bernardi M M & Cury Y, Thc anlinoc iccpti ve cffect o f Cralal lls iIilrisslls lerriJiclIs snake venom is m:J inl y due to a supraspinall y intreg:Jtcci response, TO.licolI . 36 ( 199X) 223.

60 Giorgi R, Bernardi M M & Cury Y. An:J lgcs ic effect evo lved by low molecular wcight substances cx tracted from CroW/liS

iIllrisslIs lerri/lells vcnom, Toxicoll . 3 1 ( 1993) 1257. 6 1 Chen C J, New dcvelopmen t in acutc ant icoagulant therapy:

what improves over traditional heparin arc on the ho ri zon.~

Poslgmil M a l. 99 ( 1996) 129. 62 Xi ong Y , Wan g W, Pu X & Song J, Prc:l iminary study on thc

mech:lI1ism of using snake venoms to substi tute for Illorphin , Toxicull. 30 ( 1992) 567.

63 Pu X C, Wong P T H & Gopalakri shnakone P, A nove l anal­gesic toxin (Hannalgesin) from the veno lll of king cobra (Ophiopl/(/g lls hallllah ), ToxicOII . 33 ( 1995) 1425.

64 Ca ldwe ll J R, Vcnoms, coppcr and zinc in lhc treat mcnt of arth riti s, Rhelllll Dis Clill North Alii , 25 ( 1999) 9 19.

65 Dc Pallabi , Pharmaco logical :J nd toxicolog ica l studies on the Indian King Cobra (Ophioplwg lls !wllllilh) vcnoill. Ph .D. Thesis, Ca lcutta Uni ve rsit y, Kolkata , india 2000.

66 GOllles A. Roychowdhury S & Karnl..lkar S . Ileat stable pro­tein tox in act ive on cardiovascular- rc:sp iratory system, isola­tion & pUI- ifi eci frolll the venolll of VipI'm /'II ssl'lI i of Easte rn Indi:1 (unpublished data).