JOURNAL OF Pharmaceutical Sciences December 1967 volume 56, number 12 ___ Review Article __ Pharmacology and Toxicology of Toad Venom By K. K. CHEN and ALENA KOVARiKOVA CONTENTS speare made the witch throw a toad into the hell-broth. It has been long believed that han- HISTORY.. 1535 dling of toads causes warts. In a small area of CHEMICALINGREDIEXTSOF TOADPOISOX. 1536 Pharmacology of Bufadienolides. 1536 the state of Illinois, folklore has 300 references Bufagins. .... 1536 to warts of toad origin, particularly their cures (2). Bufotoxins. ... .. .. 1538 These superstitions can of course be disproved Clinical Trials... .... 1539 by laboratory experience. Houssay (3) op- Catecholamines. .. .. 1539 Epinephrine.. .. .. .. 1539 erated on 15,000 toads for endocrine studies and Norephinephrine.. .. .. .. .. 1539 we handled more than 10,000 toads for the Indolealkylamines..... 1539 collection of their poisons-with no ill effects. Noncardiotonic Sterols. .. 1540 In form of a votive animal, the toad is associated Cholesterol. ... .. .. ... .. .. 1540 with the uterus and various gynecological dis- Provitamin D. .. .. 1540 -v-Sitosterol .......... ... .. .. 1540 eases (4) in Central Europe. Bronze toads of Miscellaneous Substances.. 1540 the Perm culture of Kortheastern Russia were USE OF THE VENOMTO THE TOAD. 1540 among the archaeological findings dating from Protection from Enemies. 1540 the middle of the 1st century A.D. Significance to Behavior. 1540 ="ATURALTOLERANCETO CARDIACGLYCO- Toad medicine has been advocated all over SIDESANDAGLYCONES. 1540 the world. For many years the Chinese have RESPONSEOFOTHERTISSUESTODRUGS. 1541 used a preparation of toad venom, ch'an su, REFERENCES. 1541 for the treatment of canker sores, toothache, HISTORY sinusitis, and local inflammations (5). During the 15th century a European physician wrote a FOR CENTURIES the toad has been known to book, "De Vcnenis," in which he mentioned that produce a poisonous secretion. The Roman the toad's blood was of value in the treatment of women used the toad, although unsuccessfully, difficult breathing (6). Pharmacopeias and as a murdering agent for their unfaithful hus- dispensatories of early dates gave the dried toad bands (1). William Shakespeare, born about a prominent place among healing agents (7, 8). 400 years ago, pictured the toad as wearing a The South American Indians have made use of the precious jewel in its head.In Macbeth, Shake- skin secretion of the toad as arrow poison. These arrows are shot into game or enemies from blow- Received from the School of Medicine, Indiana University, tubes. Indianapolis, IN 46202 This investigation was supported in part by grant HE- The English word "toad" is applied to several 07714 from the U. S. Public Health Service, Bethesda, Md., and an Indiana Heart Association grant. tailless amphibians, but the various species under 1535 1536 Journal of Pharmaceutical Sciences discussion belong to the genus Bufo of the to Cn. They have a free secondary hydroxy Bufonidae family. The members of this genus group at Ca, and are known as bufagins cor- all have a pair of well-defined skin glands behind responding to aglycones of plants. The struc- the eyes, which secrete venom and are called tural formulas shown in Fig. 1 represent the "parotid" glands.The latter, however, have better known substances which are chemically no relation to salivary glands. The longitudinal bufadienolides (23) and synonymous with bufa- axis of the toad's parotid glands is parallel with gins. Because different species of toads do Hot the body axis, although that of exceptional synthesize identical bufadienolides, prefixes have species may be perpendicular to it-B. pelto- been employed for distinction. The Japanese cephaius and B. asper (9, 10). Additional glands toad apparently can produce all of them (24). may appear on arms and legs, e.g., B. alvarius In our laboratory gamabufagin and gamabufo- (11). The venom can be expressed from the talin are used interchangeably. parotid gland between the thumb and index N one of the bufadienolides in the toad venom finger, and into a glass evaporating dish held over conjugates with a carbohydrate to form a glyco- the toad's head. The toad has warts over the side, but a few couple with suberylarginine to dorsal part of the body, and their secretion is become bufotoxins. The first bufotoxin was difficult to obtain and mayor may not be identical isolated from the European common toad (25). with the parotid secretion. Vulpian (12, 13) dis- To distinguish it from other bufotoxins, Wieland's covered that the toad venom had a digitalis-like compound has been renamed vulgarobufotoxin action and that the toad's heart was relatively (26), and a structure of 14-suberylarginine bufo- immune to its own poison. The histology of talin has been proposed (22-27). Among the parotid glands and warts of European, South and amphibians, Bufo is the only genus elaborating North American toads has been documented by bufagins and bufotoxins (28). Recently it has several workers (14-16). been reported that a North African grasshopper, Ugly and venomous, one species of toads, Poekilocerus bufonius, feeding on milkweeds, is B. marinas, has saved sugar crops and other capable of storing glycosides of cardenolide type cultivated fields in the Hawaiian Islands and (29). Puerto Rico by devouring the insects which do Bufagins-The action of bufagins upon the damage to the plantations. Although the toad heart follows almost the same pattern as that of is reputed to enjoy longevity, it lives in the digitalis glycosides. Perfusion by various methods laboratory from 8.5 to almost 16 years (17). with appropriate concentrations in the frog Attempts to isolate pharmacologically active results in systolic standstill. The absorption substances from the European common toad is generally poor. The positive inotropic action were initiated over a half century ago (18). is easily demonstrated in mammals, such as the Abel and Macht (8) succeeded in isolating "bufa- cat, by slow infusion of a dilute solution of gin" and epinephrine from the parotid venom of regularobufagin.Electrocardiographic changes B. marinus. This was followed by the eventual are most instructive. Figure 2 serves as an identification of bufotalin and bufotoxin from example. The prolongation of P-R interval, B. bufo bufo by Wieland and Weil (19) and cino- bradycardia, the diphasic T-wave, the nodal bufagin and cinobufotoxin from ch'an su (20). rhythm, the A-V dissociation, and the idio- Marked advances were made at the University of ventricular rhythm are obvious. The slowing of Basel (21, 22), for numerous cardioactive sub- heart rate can be abolished by atropinization but stances were isolated from ch'an su, and later not by vagotomy. The effect of bufagins on the from the venoms of other species by elegant heart is less cumulative than that of digitoxin, modern methods. It is now clear that the being more like that of ouabain. There is a low chemical composition of the venom of each order of absorption through the gastrointestinal species of Bufo differs from one another, although tract in mammals. Thus, no more than 15% there are many similarities. Most species of cinobufagin is absorbed by oral administration elaborate in addition, noncardiotonic sterols, in anesthetized cats, and 50% of bufalin (36) indolealkylamines, and several, catecholamines. by intrajejunal injection in nonanesthetized dogs. CHEMICAL INGREDIENTS OF TOAD VENOM Because the chemical structures of bufagins differ from one another, their cardiac activity Pharmacology of Bufadienolides varies. Table I shows the potencies of 27 com- The cardia active sterols are naturally the pounds. Their mean (geometric) lethal doses center of interest. In all instances the lactone (LD's) are determined in groups of 10 or more ring is 6-membered of a-pyrone type, attached etherized cats. It should be noted that bufota- / Vol. 56, No. 12, December 1967 1537 ,co co co IA IA A t OH OH OAe .OH ~ ~ ~ HO H HO H HO ~H BUFALIN BUFOTALIN TELOCINOBUFAGIN co HCOrt--P° HO.. 1 A OH ~ ~ HO H HO HO OH BUFOTALIDIN GAMABUFOTALIN ARENOBUFAGIN Fig. l-Structural [orrnu- las jor better known sub- stances which are chemi- caliybufadenolides (23) and O'CO O'CO 0 synonymous with bujagins. IA IA rt--P o o ~OAC ~ HO~ H HO OH HO H RESIBUFOGENIN MARINOBUFAGIN CINOBUFAGIN (O,~O O'CO O° IA HCO o o OAe ~ ~ HO HO H HO OH ?OH DESACETYLCINOBUFAGIN BUFOTALININ CINOBUFOTALIN plant ongm is exemplified by several species (37-39). When the means are converted to their reciprocals, they become the number of t + 1., . -I,: I.. !: LD's:per mg. as given in Table I, expressing the I • direct relationship of activity. The very high potency (toxicity) of some of these substances t • _' I ! ! I IL I 1 ---L_ L 1 1 J ' can be appreciated if one realizes that 1 Gm. of , I' i : t I'," 11" , t I I acetyl bufotalidin may have 15,600 cat LD's. The contributions of the Basel scholars make ,:~~Vr-t·:i it possible to arrive at certain trends of structure- activity relationship (30). If we consider bu- falin the simplest bufadienolide, substitution of H with 5!J-OH in telocinobufagin, or with lla- OH in gamabufagin increases the activity (Table I).A 12-keto group in arenobufagin further enhances the activity.The presence of a free 16-0H in desacetyl bufotalin reduces the toxicity, but 16-acetyl in bufotalin brings back the potency of bufalin. In other cases, 16-desacetyla- tion abolishes the cardiac effect of cinobufagin Fig. 2-Electrocardiographic changes induced by and cinobufotalin. 3-Acetylation of cinobufo- regularobujagin in a cat. Regularobujagin 'in per- talin, marinobufagin, and bufotalidin (helle- cent oj jatal dose (top to bottom): 0, 57, 60, 67, 99. brigenin) has a favorable influence on the po- tency to various degrees, but the same reaction lidin is identical with hellebrigenin, an aglycone with cinobufagin produces the opposite effect.