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Evaluation of trypsin treatment for snakebite envenomation
SHORT COMMUNI CATIONS EVALUATION
OFTRYPSIN TREATMENT KIR SNAKEBITE ENVENOMATION HUEKHEN HUANGand C. Y. LEE
pbarmacdoeical Imtitute, cOlk&je ofMedicine,National TaiwanUnivmity, Taipci,Taiwan,RcpublicofCbi~
(Accepted
for publication 8 January 1980)
SPECIFICantivenom is the most important therapeutic agent availabk for the effective treatment of snakebite poisoning. However, there are two main disadvantages of antivenom; cost and adverse reactions. Since the toxic components of snake venoms are proteins, which can be destroyed by proteolytic enxymes, )IsluNG et uZ. (1975) have claimed that trypsin can prevent snake venom poisoning if injected locally at the site of the snakebite. Their results indicated that all the experimental animals survived, when a suitable dose of trypsin was injected locally and promptly into mice and dogs following a ‘kthal dose of Nu@ or some other elapid venoms. RUSWLL.F.E. and GUBENSEIC. F. (unpublished data; quoted from review by F.E.R. Toxicon 14,405,1976), however, faikd to con&m the effectiveness of trypsin treatment, using Crotalus and Vipm venoms instead of elapid venoms. They conclude that trypsin is of no value in altering either the lethal dose of the venom or its nezrotixing activity. Since the species of snake venoms used were different in the two experiments, it seemed of interest to reevaluate whether trypsin is of any value in snakebite poisoning. In the present study, two elapid venoms, Nub nab otra and Bungs m&cinctur, and two crotalid venoms, Agkimxbn ucutus and Crotuiiu atrox, were used. The effectiveness of trypsin in the treatment of Nuju nuja atru envenomation was also compared with that of antivenom. The venoms of Nuju nuju utru, Bungum mulCncm and Agkisbudon acutus were freshly colkcted and freeze-dried in our laboratory and Cmtalus otrox venom was purchased from Miami Serpentarium Laboratories, U.S.A. Trypsin (14,100 BAEE’ units/mg protein) was a product of Sigma Chemical Co., U.S.A. Na# naja utru antivenom (100 TUtlml) was a gift from the National Institute of Preventive Medicine, Taipei, R.O.C. The lethality of each venom was assayed in mice (NIH strain) weighing lS_22 g by S.C. injection. The LD#,was calculated by the method of LIZHFIEU) and -XON (1949). The effect of the enzyme or antivenom on the lethality of the venom was evaluated as follows: the venom dissolved in 0.1 ml saline was injected S.C.in the thigh. Then, trypsin (in 0.1 ml saline) or antivenom (0.1 ml) was injected into the same site promptly after the injection of venom. The animals that died within 48 hr were counted. At least 20 mice were used for each dose. *OneBAEE unit will pmdux a A &, of 0.001 per oh et pH 7.6 at 2532, using~N-benmyl-L~ cater (BARE) aauubatrate. *One Tanaka unit will neutralize 1 ML.D ofvenom invilm. 475
ethyl
476
Short Canmunications
In a preliminary experiment, it was found that trypsin (50 U/g) protected mice injected with 2 a of Naja naja afra venom (100% lethality reduced to 5%). An increase of the amount of trypsin to 100 U/g did not improve the effectiveness. For Bungarus multicinctus venom, however, 108 U of trypsin per g mouse was required to achieve the best protection; the lethality of 2 LDMwas reduced from 100 to 30%. Further increase of the amount of trypsin beyond 100 U/g did not show any better protection for either venom. In addition, at a dose level of 2fKlU/g, trypsin itself caused marked myonecrosis at the site of injection; lower doses of trypsin did not cause myonecrosis. As calculated from the dose-lethality curves (Fig. I), the LD%,,of Baja naja afra venom alone was 056 &g and in the presence of 100 U trypsin per g was l-8 &g. Thus, the protective ratio was 3-2 (Table 1). The protective ratios of trypsin (100 U/g) for other venoms are also listed in Table 1. Trypsin has the best protective effect against Naja naja afra venom poisoning, less effect against Bungarus mJricincacs venom and no effect on the two crotalid venoms. The protective ratio with Naja naja atra antivenom is greater than with trypsin.
F&l. me-CURVESOF Naja ~ja atraVENOM (NNAV). (0-0)V~aloac;(.-.)venomplustrypsin(100U/g).
Neurotoxins are the major toxic components of elapid venoms, which are responsible for respiratory par-a@& (LEE, 1972). The major neurotoxin from Naja naja atra venom, cobrotoxin, is composed of 62 amino acid residues and has a mol. wt of 1649 (YANG et al., 1%9). This postsynaptic toxin is readily digested by trypsin and loses its lethal character (YANG, 1965). On the other hand, Bungarus multivenom contains both the postsynaptic toxin, a-bungarotoxin, and the presynaptic toxin, /3-bungarotoxin &HANG and LEE, 1963). a-Bungarotoxin consists of 74 amino acid residues and has a mol. wt of 7983 ~cral.,19n),wficreas&~~~iscomposedof1~0oacklresidues,havinga mol. wt of 21,000 (KoNIX)ezal., 1978).The neurotoxicityof /?bungarotoxin is rather msistant to dig&on by trygein, chymotry@n and pronase (HOWARD and TRUOG 1977). The lesser ef6ectivenessoftrypsintreatment~~~mulcicinchrvvenomisapparen~duetothe resistance of &bungarotoxin to tryptic digestion. The cause of death after crotalid envenomation is more complex, chiefly due to hemorrhage, circulatory collapse, local necrotic
477
Short Communications TABLE1.~OFTRVFWNAND
ANITVENOM ON VENOMF’OLWNMG
UlltTdCd VettOm NIliollljo~
Treatment Dow Trypsin lOOU/g
Treatad
GA
0
Fvotbive ratio 3.2
(0.5Eil)’
(1.6E96)
Antivenom 0.5Tulg
(O.zE61)
(2.si-Ym)
5.0
Bungawtssu&hm
Trypain lCOU/g
(O.E.33)
0.52 (0~45-0.60)
1.8
Askf=adonm
Trypain lOOU/g
15.0 16.0 (13.39-18.32) (14~16-18~08)
1.1
cmtaiusabvx
Trypain lOOU/g
21.0 (18.92-23.3 1)
1.0
21.5 (19+t-2365)
effect, and/or disturbances in blond coagulation (JI&NEZ-PORRAS,1970; DE VIUESand COHEN. 1969). Tbe present experiment indiites that toxic components of crotalid venoms appear to be more resistant to trypsin digestion. When the time interval between venom and trypsin injection was increased to 20 min, almost no protection was observed (Table 2), in contradiction to the results of HSIUNG et al. (1975) who reported that all of the mice injected with cobra venom survived when trypsin was given within I.5min and 5040% of the mice survived even when trypsin was given 20-50 min later. As shown in Table 2, antivenom still possessed excellent protective effect even when the time delay was as long as 20 min. When trypsin (100 U/g and 200 U/g) was injected i.p. after 2 LDWof Nu&rnuja ufra venom, no protective effect was observed. These results indicate that trypsin is inferior to antivenom in the treatment of cobra venom poisoning, however it may be of some value if injected locally within 10 min after cobra-bite. It is of no value for crotalid snakebite. TABLET.-0FTWPSlN TIMEINIERVNSAFIER
Dowof
BUlKlED
Tiieaftcr
Treatment
trcar.mcnt vclnrm(min)
T@
100u/g Kmu/g o-5 TuIg
nrpsin Alltlvcllall
AT VARIOUS
Najanajaatravwo~
ii 20
Number of
dalths x20 19mJ
m
478
Short Communications
REFERENms
CHANG.C. C. and ti. C. Y. (1%3) Isolation of nemotoxins from the venom of Bungarus rnulricinehu and their modes of nettmmllsCUlarbIocking ectitm. Ardrs int. Phumwldyll. 144.24 I. DE VRIES.A. and CDHW. I. (1%9) Heatorrhqk and blood coagnIatkm disturbing action of snake venoms. In: ~AdvoncrinBlood~,p.~(pollcr,L.,Ed.).Londoa:churchiU. HOWARD. B. D. and TkuoO. R (1977) ReIatinnship hctwcen the nemotoxicity and phoaphohpase A activity of &btmgarotoxin. Biuchmdy 16,122. HSRJN~X Y. L., THOU,J. C., I-IOU.Y. T., Du. T. C., CHOU,H. L. and Lr,C. Y. (1975) ExpetimentnI studies on curing clapid bite with trypain. m sin. 1.3%. J-I’ORRAS. J. M. (1970) Biorhcmistry of snake venoms. Clin. Toxiad. 3.389. KONDO.K., NMITA, K. and Dx. C. Y. (1978) ChcmkaI properbcs and amino acid axnpositkm of fl,-bungan+ toxin from the venue of Bungatus rsn&ka~. J. Biochm. (Tokyo) 83.91. LLE.C. Y. (1972) Chemistry and phmmacolugy of poIyp@de toxins in snake venoms. Ann. Rev. IYmmacol. 12,265. I.XKXI~, J. T. and W~XON. F. (lW9) A simplified method of evalnating dose-effect experiments. /. Pharmmz. ap. 7h.w. %, 99. ~.D..N~~K.,IW~~~~~.S.,SAMU1~~.Y.andLEE.C.Y.(1972)Purification,propertiesaadaminoacid ~3nettce of &ungarotoxin from the venom of Bungwus tnub&c~. HoppeScyWs Z. physiol. chin. 353 YANG,C. C. (1965) Enzytnk hydroIysis and chemicaI modification of cobrotoxin. Toxicon 3.19. YANG,C. C., YANO.H. J. and HUM. J. S. (1969) The aminn acid sqnence of cobrotoxin. Biochbn. Biophys. Acta lEl8.65.
OFTRYPSIN TREATMENT KIR SNAKEBITE ENVENOMATION HUEKHEN HUANGand C. Y. LEE
pbarmacdoeical Imtitute, cOlk&je ofMedicine,National TaiwanUnivmity, Taipci,Taiwan,RcpublicofCbi~
(Accepted
for publication 8 January 1980)
SPECIFICantivenom is the most important therapeutic agent availabk for the effective treatment of snakebite poisoning. However, there are two main disadvantages of antivenom; cost and adverse reactions. Since the toxic components of snake venoms are proteins, which can be destroyed by proteolytic enxymes, )IsluNG et uZ. (1975) have claimed that trypsin can prevent snake venom poisoning if injected locally at the site of the snakebite. Their results indicated that all the experimental animals survived, when a suitable dose of trypsin was injected locally and promptly into mice and dogs following a ‘kthal dose of Nu@ or some other elapid venoms. RUSWLL.F.E. and GUBENSEIC. F. (unpublished data; quoted from review by F.E.R. Toxicon 14,405,1976), however, faikd to con&m the effectiveness of trypsin treatment, using Crotalus and Vipm venoms instead of elapid venoms. They conclude that trypsin is of no value in altering either the lethal dose of the venom or its nezrotixing activity. Since the species of snake venoms used were different in the two experiments, it seemed of interest to reevaluate whether trypsin is of any value in snakebite poisoning. In the present study, two elapid venoms, Nub nab otra and Bungs m&cinctur, and two crotalid venoms, Agkimxbn ucutus and Crotuiiu atrox, were used. The effectiveness of trypsin in the treatment of Nuju nuja atru envenomation was also compared with that of antivenom. The venoms of Nuju nuju utru, Bungum mulCncm and Agkisbudon acutus were freshly colkcted and freeze-dried in our laboratory and Cmtalus otrox venom was purchased from Miami Serpentarium Laboratories, U.S.A. Trypsin (14,100 BAEE’ units/mg protein) was a product of Sigma Chemical Co., U.S.A. Na# naja utru antivenom (100 TUtlml) was a gift from the National Institute of Preventive Medicine, Taipei, R.O.C. The lethality of each venom was assayed in mice (NIH strain) weighing lS_22 g by S.C. injection. The LD#,was calculated by the method of LIZHFIEU) and -XON (1949). The effect of the enzyme or antivenom on the lethality of the venom was evaluated as follows: the venom dissolved in 0.1 ml saline was injected S.C.in the thigh. Then, trypsin (in 0.1 ml saline) or antivenom (0.1 ml) was injected into the same site promptly after the injection of venom. The animals that died within 48 hr were counted. At least 20 mice were used for each dose. *OneBAEE unit will pmdux a A &, of 0.001 per oh et pH 7.6 at 2532, using~N-benmyl-L~ cater (BARE) aauubatrate. *One Tanaka unit will neutralize 1 ML.D ofvenom invilm. 475
ethyl
476
Short Canmunications
In a preliminary experiment, it was found that trypsin (50 U/g) protected mice injected with 2 a of Naja naja afra venom (100% lethality reduced to 5%). An increase of the amount of trypsin to 100 U/g did not improve the effectiveness. For Bungarus multicinctus venom, however, 108 U of trypsin per g mouse was required to achieve the best protection; the lethality of 2 LDMwas reduced from 100 to 30%. Further increase of the amount of trypsin beyond 100 U/g did not show any better protection for either venom. In addition, at a dose level of 2fKlU/g, trypsin itself caused marked myonecrosis at the site of injection; lower doses of trypsin did not cause myonecrosis. As calculated from the dose-lethality curves (Fig. I), the LD%,,of Baja naja afra venom alone was 056 &g and in the presence of 100 U trypsin per g was l-8 &g. Thus, the protective ratio was 3-2 (Table 1). The protective ratios of trypsin (100 U/g) for other venoms are also listed in Table 1. Trypsin has the best protective effect against Naja naja afra venom poisoning, less effect against Bungarus mJricincacs venom and no effect on the two crotalid venoms. The protective ratio with Naja naja atra antivenom is greater than with trypsin.
F&l. me-CURVESOF Naja ~ja atraVENOM (NNAV). (0-0)V~aloac;(.-.)venomplustrypsin(100U/g).
Neurotoxins are the major toxic components of elapid venoms, which are responsible for respiratory par-a@& (LEE, 1972). The major neurotoxin from Naja naja atra venom, cobrotoxin, is composed of 62 amino acid residues and has a mol. wt of 1649 (YANG et al., 1%9). This postsynaptic toxin is readily digested by trypsin and loses its lethal character (YANG, 1965). On the other hand, Bungarus multivenom contains both the postsynaptic toxin, a-bungarotoxin, and the presynaptic toxin, /3-bungarotoxin &HANG and LEE, 1963). a-Bungarotoxin consists of 74 amino acid residues and has a mol. wt of 7983 ~cral.,19n),wficreas&~~~iscomposedof1~0oacklresidues,havinga mol. wt of 21,000 (KoNIX)ezal., 1978).The neurotoxicityof /?bungarotoxin is rather msistant to dig&on by trygein, chymotry@n and pronase (HOWARD and TRUOG 1977). The lesser ef6ectivenessoftrypsintreatment~~~mulcicinchrvvenomisapparen~duetothe resistance of &bungarotoxin to tryptic digestion. The cause of death after crotalid envenomation is more complex, chiefly due to hemorrhage, circulatory collapse, local necrotic
477
Short Communications TABLE1.~OFTRVFWNAND
ANITVENOM ON VENOMF’OLWNMG
UlltTdCd VettOm NIliollljo~
Treatment Dow Trypsin lOOU/g
Treatad
GA
0
Fvotbive ratio 3.2
(0.5Eil)’
(1.6E96)
Antivenom 0.5Tulg
(O.zE61)
(2.si-Ym)
5.0
Bungawtssu&hm
Trypain lCOU/g
(O.E.33)
0.52 (0~45-0.60)
1.8
Askf=adonm
Trypain lOOU/g
15.0 16.0 (13.39-18.32) (14~16-18~08)
1.1
cmtaiusabvx
Trypain lOOU/g
21.0 (18.92-23.3 1)
1.0
21.5 (19+t-2365)
effect, and/or disturbances in blond coagulation (JI&NEZ-PORRAS,1970; DE VIUESand COHEN. 1969). Tbe present experiment indiites that toxic components of crotalid venoms appear to be more resistant to trypsin digestion. When the time interval between venom and trypsin injection was increased to 20 min, almost no protection was observed (Table 2), in contradiction to the results of HSIUNG et al. (1975) who reported that all of the mice injected with cobra venom survived when trypsin was given within I.5min and 5040% of the mice survived even when trypsin was given 20-50 min later. As shown in Table 2, antivenom still possessed excellent protective effect even when the time delay was as long as 20 min. When trypsin (100 U/g and 200 U/g) was injected i.p. after 2 LDWof Nu&rnuja ufra venom, no protective effect was observed. These results indicate that trypsin is inferior to antivenom in the treatment of cobra venom poisoning, however it may be of some value if injected locally within 10 min after cobra-bite. It is of no value for crotalid snakebite. TABLET.-0FTWPSlN TIMEINIERVNSAFIER
Dowof
BUlKlED
Tiieaftcr
Treatment
trcar.mcnt vclnrm(min)
T@
100u/g Kmu/g o-5 TuIg
nrpsin Alltlvcllall
AT VARIOUS
Najanajaatravwo~
ii 20
Number of
dalths x20 19mJ
m
478
Short Communications
REFERENms
CHANG.C. C. and ti. C. Y. (1%3) Isolation of nemotoxins from the venom of Bungarus rnulricinehu and their modes of nettmmllsCUlarbIocking ectitm. Ardrs int. Phumwldyll. 144.24 I. DE VRIES.A. and CDHW. I. (1%9) Heatorrhqk and blood coagnIatkm disturbing action of snake venoms. In: ~AdvoncrinBlood~,p.~(pollcr,L.,Ed.).Londoa:churchiU. HOWARD. B. D. and TkuoO. R (1977) ReIatinnship hctwcen the nemotoxicity and phoaphohpase A activity of &btmgarotoxin. Biuchmdy 16,122. HSRJN~X Y. L., THOU,J. C., I-IOU.Y. T., Du. T. C., CHOU,H. L. and Lr,C. Y. (1975) ExpetimentnI studies on curing clapid bite with trypain. m sin. 1.3%. J-I’ORRAS. J. M. (1970) Biorhcmistry of snake venoms. Clin. Toxiad. 3.389. KONDO.K., NMITA, K. and Dx. C. Y. (1978) ChcmkaI properbcs and amino acid axnpositkm of fl,-bungan+ toxin from the venue of Bungatus rsn&ka~. J. Biochm. (Tokyo) 83.91. LLE.C. Y. (1972) Chemistry and phmmacolugy of poIyp@de toxins in snake venoms. Ann. Rev. IYmmacol. 12,265. I.XKXI~, J. T. and W~XON. F. (lW9) A simplified method of evalnating dose-effect experiments. /. Pharmmz. ap. 7h.w. %, 99. ~.D..N~~K.,IW~~~~~.S.,SAMU1~~.Y.andLEE.C.Y.(1972)Purification,propertiesaadaminoacid ~3nettce of &ungarotoxin from the venom of Bungwus tnub&c~. HoppeScyWs Z. physiol. chin. 353 YANG,C. C. (1965) Enzytnk hydroIysis and chemicaI modification of cobrotoxin. Toxicon 3.19. YANG,C. C., YANO.H. J. and HUM. J. S. (1969) The aminn acid sqnence of cobrotoxin. Biochbn. Biophys. Acta lEl8.65.