Effect of the route of administration on the neutralizing potency of antivenins

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EFFECT OF THE ROUTE OF ADMINISTRATION ON THE NEUTRALIZING POTENCY OF ANTIVENINS A. BILLINGS, BILL D . ASHLEY, EDITH B . and JOYCE C. Gosrz

WALTBR F . KOCHOLATY, THOMAS

L13DFORD

Biocheanistry Division, U .S . Army Medical Research Laboratory, Fort Knox, Kentucky, U .S .A . (Acceptedfor publication 22 September 196 A6etract-Using a variety of venons, it was demonstrated that the route of administration has a decisive influence in the demonstration of the protective effect of a given antiv~in . It is suggested that in testing the efficacy of an immune serum, both i.v, and i .p . administration should be given consideration. INTRODUCTION

A RBCENT paper [1] dealing with the immunization of rabbits with photooxidized venoms of Bothrops atrox riper and of Crotalus durissus durlssus described two distinctly different immunogenic responses ; it appeared that the y-globulin isolated from C. durissus durissttsimmunized rabbits gave a high degree of protection against this venom, while the y-globulin isolated from B. atrox riper-immunized animals afforded no protection against the latter venom. Immunization of rabbits with B. atrox riper venom utilizing different adjuvants led to the same results. In another paper [2] it was reported that rabbits immunized with photooxidized venom of Naja naja showed a very weak immunogenic response . Continuing the protection studies with mice by varying the routes of injection of immunoglobulin and of venom it became evident that protection could be obtained. Since it seemed unlikely that the y-globulins isolated from animals injected with B. atrox riper or Naja raja venom should be unique in this respect--0ne strongly hemorrhagic and necrogenic, the other a predominantly neurotoxic venom-the experiments were extended to other venom immunoglobulins. The results reported here tend to show that the route of administration is of decisive importance in the demonstration of the protective effect in the antivenin-venom interaction and that it will vary with the type of venom under study. MATERIALS AND METHODS

All y-globulins from rabbits or from goats were isolated according to CAMPBBLL et al. [3], as described previously [4]. Wyeth's Antivenin (Crotalidae) Polyvalent of equine origin Lot No. 07901 and Pentex Fraction II y-globulin from rabbit and horse respectively, were also used in the control experiments. White Swiss mice, weighing ZO-25 g, were used in the lethality tests. Naja raja kaouthia venom was purchased from the Ross Allen Institute and Micrunrs julvius fulvius venom was purchased from Calbiochem (L,ot 53064) Bothrops atrox riper and Crotalus durissus 165

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W. F. KOCHOLATY, T. A. BILLINGS, B. D. ASHLEY, E. B. LEDFORD and J. C. GOETZ

durissus venoms were donated by H. H. Flowers. The remaining venoms were obtained from snakes kept in our laboratory. Intravenous injections were given into one of the tail veins; i.p. injections were given into the lower left quadrant of the abdomen . Neutralization tests were conducted by incubating 100 mg amounts* of the immunoglobulins with varying Lnbo doses of venom for 60 min at 37° in a total volume of 1 ~0 or 1 ~25 ml . Volumes of 020 or 025 ml containing 25 mg of the immunoglobulin and appropriate amounts of venom were given to mice by i.v. injection. Protection studies were carried out by i.v. or i.p. injection of the immunoglobulins followed by the i.v. or i.p. administration of varying LDbo doses of venom. Survivors were counted 24 hr after the venom was administered . RESULTS

Naja naja In the preceding paper [2] it was reported that only one half of the sera (Group A, including Nos. 23-25, 31, 33) obtained from 10 rabbits immunized with 22 mg of photooxidized venom of Naja raja gave a weak positive precipitin test up to the sixth dilution, while the other half (Group B, including Nos. 26-30) of the sera were practically negative . Furthermore, it was shown that y-globulins isolated from sera of Group A gave almost no protective effect when injected i.v. and the mice challenged 30 min later by i.p. injection ofvenom produced results which indicated that a test of the protective effect of the y-globulins isolated from sera of Group B would be superfluous. However, as shown in Table 1, when both injections were administeréd i.v. (y-globulin followed by various venom doses 30 min later) the protective effect of the rabbit y-globulins from sera of Group A was evident. Surprisingly, protection was also obtained with the y-globulin preparation from sera of Group B.t Lv.-i.v. protection studies with rabbit immunoglobulin from sera of Group C obtained by immunization of rabbits with double the amount of photooxidized Naja naja venom could not be performed due to lack of material . TABLE 1 .

Ln w

1 2 2~5 3 4 S 6

PROTECTION OF MICE AOAINSr NUja IIaJa VENOM BY RABHIT IMMUNOGLOHULIN

Neutralization by `A' 0/3 0/3 3/3 3/3 3/3

PIOteCtlOn by `A' i.v :i.v, i.v:i.p. 0/4 0/4 2/4 4/4

3/3 3/3 3/3 2/2

PIOteCtiOII by `B' i.v :i.v . i.v:i.p. 0/5 0/5 1/5 3/4 5/S

2/4 4/4 4/4

VeIIOm controls i.v . 15/27 27/27 -

Lv :i.v . ; i.v :i.p . : i.v . igjection of 25 mg of rabbit immune serum, followed 30 min later by an i.v. or i.p . injection of the ~so indicated. Numbers are deaths to 24 hr after injection over the total number of mice used . *20 Mg amounts were used with C. durissus durissus venom. tThis would indicate that the precipitin test carried out with whole venom is of little value as a diagnostic test where the presence of a variety of antigens and antibodies will influence their mutual solubilities.

Effort of the Route of Administration on the Neutralizing Potency of Antivenins

167

Bothrops atrox asper As reported previously [1], no protection against B. atrox asper venom was obtained with a y-globulin isolated from rabbits that had been immunized with photooxidized B. atrox asper venom. When the routes of injection were varied as in the experiments described earlier, significant differences in the degree of protection were obtained . These results are summarized in Table 2. TABLE Z. PROTECTION OF MICE AGAINST BOt%1lOJlS a1rUxasper VENOM BY RAHHIT IMMUNOGLOHULIN

Lu,o 1 2 3 4 S 6 7"S 10

Neutralization* 0/4 0/4 0/4 0/4 0/4 0/4 4/4 4/4

Protection* i.v:i.v. i.v.-i.p . 0/S 1/S 0/S 1/S 0/S 3/S S/S

0/3 0/3 3/3 3/3 3/3 3/3 3/3 3/3

COntI'Ols ~1 .V.~ Venom Venom and normal alone rabbit y-globulint 3/S S/S

3/4 4/4

Numbers are deaths to 24 hr after injection over the total number of mice used. *By immunoglobulin obtained from rabbits immunized with photooxidized venom of B. afros asper. ti'entex rabbit y-globulin .

Agkistrodon piscivorus The y-globulinsobtained from rabbitsinoculated withphotooxidized venom ofAgkistrodorl piscivorus reported previously [4] were used. Differences in the protection level are evident depending upon the various routes of administration. The results summarized in Table 3 show again that an i.v.-i.v. administratio n of immunoglobulin and venom resulted in a higher Ln~ protection rate than an i.v.-i.p. (globulin and venom) administration. TABLE 3. PROTECTION OF MICE AOAINSI' .i$t[istrolioltpl.4C1VOr1lS VENOM BY RABBrT IMMUNOGLOHULIN

LDaa 1 2 3 4 S 6 7" S

Neutralization* 1.V. 0/3 0/3 0/3 1/3

Protection* i.v:i.v. i.v:i.p. 0/4 0/4 1/4 3/4 3/4 4/4

0/3 1/3 3/3

Venom alone

Venom and normal rabbit y-globulint

6/8 8/8

3/4 4/4

Numbers are deaths to 24 hr after injection over the total number of mice used. *By y-globulin obtained from rabbits immunized with photooxidized venom of A. plscirorus . tPentex rabbit y-globulin.

Crotalus atrox and Crotalus durissus durissus* When the polyvalent antivenin (Wyeth) was tested for its neutralization and protection *Neutralization and protection experiments were mported previously using C. atrox [Sl and C. durissus durissus venoms with y-globulin derived from rabbits that had been immunized with the photooxidized venoms of these reptiles, however, the amounts of y-globulin were insufficient for a complete study.

168

W. F. KOCHOLATY, T. A. BILLINGS, B. D. ASHLEY, E. B. LEDFO1tD and J. C. GOETZ

properties against C. atrox venom in mice using a procedure similar to that described for venom, similarly as were venoms described above, comparable results were obtained using the different routes of administration (Table 4). T.~la 4.

Lnw

1 2 3 4 S 7~5 8 9 10

PnozECnoN OF MICE AC)AIIdSI'

Neutralization i.v. i.p. 0/4 0/4 0/4 0/4 4/4 4/4 4/4

0/4 2/4 4/4 4/4

CrotaiYS atrOx VENOM WITH WYErFI'S ANIIVENIN

Protection i.v .i.v . i.v:i.p. 0/4 0/4 3/4 4/4

0/4 3/4 4/4 4/4

Venom only i.v . i.p. 0/4 4/4

Venom and IIOrmal globulin* i.v :i.p .

4/8 6/8

4/8 6/8

Numbers are deaths to 24 hr after injection over the total number of mice used . *Pentex horse y-globulin.

Similar conditions prevailed when Wyeth's antivenin was examined for its neutralization and protection properties against C. dwissus durissus venom (Table 5). In this case, however, the protection values for the different routes of administration are reversed when compared with the values for C. atrox and correspond to the peculiar i.v.-i.p. lethality of this venom. Tnal.g 5.

Lnw

1 2 3 4 S 6 8 10 12 14 16 18 20 22 24

PltozecrloN szwmg wlzFl Crotalus durisstrs durissus VENOM IN MICE WrrH WYETH'S ~NZIVENnv*

Neutralization i .v. i.p . 0/4 0/4 0/4 1/4 4/4

0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 2/4 7/8 4/4 4/4

Protection i.v :i.v. i.v:i.p . 0/4 1/4 3/4 3/4 4/4 4/4 4/4

0/4 0/4 0/4 1/4 0/4 1/4 5/8 4/4 4/4

Venom alone i.v. i.p. 12/16 16/16

11/12 12/12

Venom and normal rabbit y-globulint i.p . i.v:i.v . 3/4 4/4

Numbers are deaths to 24 hr after injection over the total number of mixused. *Due to the high neutralization ability of this serum only S mg of antivenin ware used per test. tPantex rabbit y-globulin.

2/4 4/4

Effect of the Route of Administration on the Neutralizing Potency of Antivenins

169

Micrurus fulvius fulvius A recent report [6] dealt with the production of an immune serum in rabbits against the venom of the North American coral snake. These experiments were extended to goats to provide a greater amount of the immune serum. Table 6 shows the results obtained using different routes of administration and of the protective effect produced by goat y-globulin. The results of the studies are summarized in Table 7. The "neutralization and protection capacities" were calculated according to RF.HD and MuErrcx [7]. While such a TABLE

6.

PROTECTION STUDIE4 AGAINST MlCrulIL4 fülvius flllVlüS VENOM IN MICE BY GOAT GLOBULIN* LD w

1 2 2~5 3 4 5 6 7 7~5 8 10 12

Neutralization i .v . i.p . 0/4 0/3 4/4 4/4

Protxtion* i.v. i.p,

0/4 0/4 1/4 1/4 2/3 4/4

0/4 1/4 3/3 3/3

Venom control i.v . i.p.

1/4 0/5 0/3 3/4 4/4

4/4 4/4

2/8 7/8

Numbers are deaths to 24 hr after injection over the total number of mice used . *25 mg goat y-globulin for each test .

TABLE 7. "NEUTRALIZATION AND PROTECTION CAPACITIES" OF VARIOUS IMMUNOGLOHULINS USING DIFFERENT ROUTES OF INJECTION

N. naja N. naja A B

Venom: Test : Neutralization Neutralization ; Protection Protection Toxicity :

LD bo/20-25g mouse

Protection x LD w Protection x LD w (for 25 mg y-globulin)

B. atrox asper

i.v . i.p. i.v.-i.v . i.v:i.p .

2~2 4~0 0

3~0 4~6 2~0

6~8 6~9 2~5

i.v. i.p.

6 6

6 6

28 64

i.v:i.v, i.v:i.p,

24 0

28 12

193 160

A . piscivorus

4~0 5~0 3~5

C. d. C. atrox* durissus

M. f. fulviust

7~8 4~0 6~7 4~5

7~4 18~2 4~6 9~6

5~6 8~8 6~3 4~4

46 109

60 98

28 15

8~5 15

184 273

402 441

129 144

54 66

All neutralization and protection values pertain to 25 mg of y-globulin except C. durissus durissus where 5 mg was used . (A) y-Globulin isolated from rabbit sera Nos. 23-25, 31, 33. (B) y-Globulin isolated from rabbit sera Nos. 2fr30. *Wyeth's antivenin. tGoat y-globulin. $Lack of y-globulin prevented completion of these tests.

170

W. F. KOCHOLATY, T . A . BILLINGS, B. D. ASHLEY, E. B . LEDFORD and J . C. GOETZ

calculation is uncommon, and can at best present an approximation, it is revealing inasmuch as it demonstrates the differences obtainable by varying the route of administration . Lack of rabbit y-globulin, antivenin prevented the completion of several neutralization experiments. DISCUSSION

The neutralization potency of an immune serum is sometimes expressed in LDau of venom neutralized per ml of serum, and is determined by incubation of the components under standard conditions of time and temperature followed by injection of the material into mice of standard weight . Depending on the route of administration, the neutralizing ability of the serum is then expressed in the appropriate 1.Dbo of venom i.v. or i.p. As indicated in Table 7, this can lead to results varying as much as 100 per cent. The question arises as to most appropriate means for testing an immune serum. The same disparity prevails in the protection tests. Actually, neither way affords a realistic approach to the effect of venom in an actual snake bite . Envenomation rarely occurs i.p., perhaps in rare cases i.v., but usually what could be termed intramuscularly or (perhaps with small snakes) intradermally, with extremities the primary locus of envenomation . Thus, the `neutralization' tests when conducted i .v. or i.p. simply afford a convenient and reproducible route of injection, as contrasted with an intramuscular or intradermal injection that would simulate snake bite to a closer degree . While the common denominator derived from these investigations would appear to be for some venoms (B. atrox, M. fulvius, C. atrox, C. durissus) the amount of venom neutralized by a given amount of immune serum irrespective of the route of injection, this does not hold true for other venoms (Agkistrodon piscivorus, Naja naja) . With the single exception of C. durissus durissus, the degree of protection via the i.v. route is consistently higher than the i.p. administration . The variety of physical, chemical and physiologically active components contained in snake venom and their wide range in molecular weight would make for great differences in the kinetics of drug absorption and penetration to the site of action, depending upon the route of administration . It would then seem, that in order to arrive at a rational appraisal of the efficacy of immune serum, both i.v. and i.p. administration should be given consideration, at least as long as one is uncertain of the chemical nature, pharmacological activity and toxicity of the variety of components contained in the venoms . REFERENCES Kocaotn~rY, W., Ba .ur+cs, T . A ., ÀStit~,v, B. D., LEDFORD, E. B . and GoErz, J . C., Immunogenic response of the venoms of fer-de-lance, Bothrops atrox asper, and la cascabella, Crotalus durissus durissus, following photooxidative detoxification. Toxicon S, 153, 1968 . 12] Kocxoi.trrr, W., LâDFORD, E . B ., Bu,tuvas, T . A ., Gosrz, J . C . and ASHLEY, B. D ., Immunization studies with Naja raja venom detoxified by photooxidation . Toxicon 5, 159, 1968. [3] CaMesELt, D . H ., G~ttvEY, J . S ., CnEe~x, N . E. and SUSSDORF, D . H . : Methods in Immunology . New York : W. A . Begjamin, 1963 . [4] Kocxoc..~rrY, W. and ASHLEY, B . D ., Detoxification of Russell's viper (Vipera russellü) and water moccasin (Agkistrodon plscivorus) venoms by photooxidation. Toxicon 3, 187, 1966. [5] KocaourY, W., Detoxification of Crotalus atrox venom by photooxidation in the presence of methylene blue . Toxicon 3, 175, 1966. [6l KOCHOLATY, W . F ., As}u.EY, B. D . and Bn.Lnvcs, T . A ., An immune serum against the North American coral snake (M/crurus fulvius fulvlus) . Toxicoe, S, 43, 1967. Ill REEn, L. J. and MuExcH, H., A simple method of estimating fifty per cent end points. Am. J. Hyg. 21, 493, 1938.