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Pharmacokinetics of 125I-labelled venom from the scorpion Androctonus amoreuxi, Aud. and Sav.
Toxkon, Vol . 18, pp. 301-308. © Pagamon Press Ltd. 1980 . Printed in England.
0041-0101/80/0501-0301502.00/0
PHARMACOKINETICS OF 121 I-LABELLED VENOM FROM THE SCORPION ANDROCTONUS AMOREUXI, AUD. AND SAV. M. ISMAIL,t M. E. ABDULLAH, A. M. MoRAD and A. M. AGEEL Departments of Pharmacology and Pharmaceutics, Faculty of Pharmacy, University of Riyadh, Riyadh, Saudi Arabia (Acceptedfor publication 22 November 1979) M. ISMAIL, M. E. ABDULLAH, A. M. MoRAD and A. M. AGEEL. Pharmacokinetics of 1251labelled venom from the scorpion Androctonus amoreuxi, Aud. and Sav. Toxicon 18, 301-308, 1980.-Androctonus amoreuxi venom was radioiodinated using the chloramine T method of [1251) iodide oxidation giving a spec . act. of 12-5 pCi/mg. Gel filtration on Sephadex G-50 and cellulose acetate electrophoresis of the labelled venom showed good correlation between protein concentration and radioactivity . Labelled venom was injected i.v. into rabbits anaesthetized with urethane and blood samples were withdrawn from a common carotid artery at times from I to 270 min after venom injection for measurement of radioactivity and determination of blood glucose. Abiphasic blood level curve showing a rapid initial declining phase during the first 15 min followed by a slower declining phase was obtained . This behaviour is characteristic of a two-compartment open model. The rate constants between blood and tissues "kcT", tissues and blood "kTc" and thedisposition rate constant "kd" were estimated to be 9.8 x 10 - 2, 3.34 x 10-2 and 6-67 x 10-3, respectively . The values indicate a very rapid distribution of venom from blood to tissues with an estimated half-life of 5-6 min whereas the overall elimination half-life is 104 min. Steady-state distribution indicated that 75 % of the injected venom was in the tissue compartment after equilibrium was reached. Significant rise in blood glucose was observed when equilibrium was reached and continued throughout the equilibrium phase. Tissue distribution of labelled venom showed major radioactivity to be in liver and kidney. Much less activity was found (in decreasing order) in thyroid, lungs, heart, uterus, intestine, ovary, diaphragm and spleen . INTRODUCTION
THE SCORPION Androctonus amoreuxi is widely distributed in Egypt and neighbouring countries (BALOZET, 1971) . Its venom has potent neurotoxic and cardiotoxic properties (GHAZAL et al., 1975). An antivenin prepared against A. amoreuxi venom had protective action against the lethal and many of the pharmacological effects of the venom (ISMAIL et al., 1975). Although much work has been done on scorpion venoms and their components
(composition, amino acid sequence, pharmacological and toxicological properties ;
MIRANDA et al., 1960, 1964 a, b; ROCHAT et al., 1967, 1970 a, b ; ISMAIL et al., 1972, 1973, 1974, 1975), little work has been carried out on plasma concentration, tissue distribution
and excretion of the venoms . No attempts have been made to correlate venom-blood or tissue levels with the type and magnitude of pharmacological effect . Opinions even differ about the proper time of administering the antivenin following scorpion sting. Most investigators believe that the antivenin should be administered immediately after scorpion sting otherwise the venom will produce irreversible lesions (BALOZET, 1971). Whether this *Supported by Grunt PHRCI from the Research Center, Faculty of Pharmacy, University of Riyadh. tOn leave of absence from Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt. 301
302
M . ISMAIL, M . E . ABDULLAH, A . M . MORAD and A . M . AGEEI.
is true, or it is the venom diffusion into the tissues that limits the effectiveness of the antivenin remained to be answered . It was thought worthwhile, therefore, to study the pharmacokinetics of scorpion venom with the aim of finding a correlation between venom concentration in blood and tissues and pharmacological actions . A . amoreuxi venom was chosen as a model for this study and its ability to cause hyperglycemia was selected as the quantitative pharmacological effect . MATERIALS AND METHODS Venom obtained from mature A . amoreuxi by electrical stimulation of the telson was suspended in water, centrifuged and the supernatant freeze-dried . Dried venom was kept in a desiccator over Silica gel at -17°C, and was reconstituted by the addition of saline or 0-5 M phosphate buffer, pH 7-5, immediately before use. Radioiodination of the venom was carried out using an adaptation of the chloramine-T method of GREENWOOD et al. (1963). To 1 mCi of carrier free sodium iodide-1=3I free from reducing agents (Radiochemical Centre, Amersham, England) in 1 ml of 0-5 M phosphate buffer (pH 7-5, contained in a rubber capped vial), 20 mg of the lyophilized venom (in 1 ml 0-5 M phosphate buffer, pH 7-5) and 20 mg chloramineT (fresh solution in 0-5 ml 0-5 M phosphate buffer, pH 7-5) were added . Immediately after mixing, 1 ml of sodium metabisulphite solution (96 mg/ml in 0-05 M phosphate buffer, pH 7-5) and 2 ml carrier potassium iodide solution (0-4 g/ml in 0-05 M phosphate buffer, pH 7-5) were added . Unbound iodine was removed by dialysis against 500 ml of 0-9 % sodium chloride solution at 4°C with six changes of the salt solution over a period of 36 hr . The dialyzed venom was adjusted to volume by the addition of 0-9% sodium chloride solution. The labelling efficiency ranged between 52 and 55 % and the spec . act. of the labelled venom was 12-5 pCi per mg . Fractionation of the labelled venom was carried out using Sephadex G-50 (Pharmacia, Uppsala, Sweden) with bed dimensions of 2-5 x 25 cm equilibrated with barbitone buffer (0-07 M, pH 8-6) . Two mg labelled venom were mixed with 8 mg non-labelled dialyzed venom in 2 ml barbitone buffer (0-07 M, pH 8-6) and applied to the column. Elution was continued with barbitone buffer and 1 ml fractions were collected at a flow rate of 15 ml per hr. The labelled venom was also fractionated using cellulose acetate electrophoresis and barbitone buffer pH 8-6 as described by GHAZAL et al. (1975) . Protein was estimated spectrophotometrically at 280 nm . Female albino rabbits weighing 2-22 kg were anaesthetized with urethane (1 -75 g/kg i .v.) . Polyethylene catheters were introduced into the left common carotid artery for the collection of serial blood samples . Two mg labelled-venom dissolved in 1 ml saline (spec. act . 12-5 pCi/mg) were injected i .v. into each rabbit via the marginal ear vein. Control rabbits were injected with 1 ml of 1 251 solution in saline (25 pCi/ml) . Two ml blood samples were withdrawn immediately before and at 1, 3, 5, 10, 15, 30, 60, 120, 180 and 270 min following injection of the venom or iodine solution. Heparin (1000 U/kg) was used as an anticoagulant . The bladder was empited before venom injection and total urine, representing the 270 min period, was collected using polyethylene tubing. At the end of the experiment the rabbits were bled to death and the thyroid gland, heart, lungs, diaphragm, liver, kidneys, spleen, intestine, uterus and ovaries were removed, blotted between filter papers and weighed. Radioactivity in organs, blood and urine was measured in biovials using a Beckman Gamma 4000 counting system with a 2 in . sodium iodide crystal (doped with thallium iodide) and connected to a Texas Instrument 700 printer. Blood glucose was estimated in 0- 1 ml samples from the venom-treated rabbits using the glucose oxidase method of ScHnHiDT (1971) and test combination kits (Boehringer, Mannheim, Germany) . RESULTS Using Sephadex G-50, protein was distributed in 36 tubes giving the profile shown in Fig . 1 . Peak protein concentration was in tubes 14-23 while peak radioactivity was in tubes 25-37 (Fig . 1) . Gel filtration of free 1751 under the same conditions revealed a single peak coming between tubes 45 and 55 . Electrophoretic separation of the iodinated and noniodinated dialyzed venom in barbitone buffer, pH 8-6, showed that the two venoms had identical electrophoretic behavior, and autoradiography showed that radioactivity was distributed in all fractions of the iodinated venom . The values obtained from measurement of blood radioactivity were normalized to 20,000 counts/min/ml at the 1 min sampling time . The mean counts/min/ml for the number of rabbits used at each sampling time was plotted against time on a semi-logarithmic graph paper. The data obtained for the labelled venom, revealed a biphasic curve characterized by a rapid initial declining phase followed by a slower terminal declining phase (Fig. 2) .
Pharmacokinetics of Scorpion Venom
FIG. 1 . GEL FILTRATION OF 125-1-LABELLED A. amoreuxi VENOM ON SEPHADEx G-50. Two mg labelled venom +8 mg non labelled dialysed venom in 2 ml barbitone buffer (0-07 M, pH 8-6) were applied to the column. The column (2-5 x 25 cm) was equilibrated and eluted with barbitone buffer (0-07 M, pH 8-6) . Flow rate 15 ml/hr; volume of fractions I ml . ~--~, Venom protein ; /--/, radioactivity .
Time, min FIG. 2. DISTRIBUTION OF RADIOACIIvrrY BETWEEN BLOOD (A-A) AND TISSUES FOLLOWING THE i.V . INJECTION OF 115I-LABELLED A. aMOreuxI vENOM.
The curve represents the average values obtained from 8 rabbits. The vertical lines represent S.E .M . Tissue levels were calculated from equation 5 in text .
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The experimental data points were fitted to a biexponential equation of the form ; Cc =Ae - ° 1+Be - 9r (1) where C, is venom concentration in blood (expressed as counts/min/ml), a and p are the apparent first-order fast and slow disposition rate constants respectively and A and B are the corresponding zero time intercepts . The parameters R and B were estimated graphically from the slope and intercept of the least square line obtained from the terminal experimental data points (Fig . 2). Parameters a and A were estimated graphically by applying the feathering technique (GIBALDI and PERRIERE, 1975). The values obtained for A, B, a and P were 16,200 counts/min/ml, 5700 counts/min/ml, 0123 min-' and 00018 min -', respectively . The apparent first-order intercompartmental rate constants, kcT and k Tc, and the apparent first-order elimination rate constant from the central compartment, kd, were calculated from equations 2-4 (GIBALDI and PERRIERE, 1975) ; kTc =
kd -
AP A -{-}- Ba B.
ap
kTc
kcT = a + (3 - kTc -f- kd
(2)
(3) (4)
The values obtained for kTc, kd and kcT were 00334 min-', 000667 min- ] and 0"098 min- ', respectively . The tissue venom concentration at various time intervals was calculated from the equation; kcT Co WO , - e -° r) CT (5) where, CT is venom concentration in tissues (expressed as counts/min/ml), C,,' is blood venom concentration obtained from the zero time intercept of the blood concentration curve. The curve obtained for the tissue venom concentration showed a rapid ascending phase during the first 15 min that reached a peak within 37 min (Fig. 2). Steady state venom distribution between tissues and blood was calculated from the ratio of the tissue venom to blood venom concentrations after equilibrium distribution was reached. A ratio of 3 :1 was obtained . The data obtained from the control rabbits injected with`f were similarly plotted where a biphasic curve was obtained (Fig. 3). The pharmacokinetic parameters were calculated as described before . The values obtained for A, B, a, R, kcT , kTc and kd were 13,156 counts/ min/nil, 8844 counts/min/ml, 0084 min- ', 000048 min- ', 0052 min- 1, 0034 min- 1 and 000119 min-', respectively . Tissue levels for 1231 were calculated using equation 5. The curve obtained showed a rapid ascending phase during the first 15 min that reached a peak within 60 min (Fig. 3). The steady state equilibrium distribution of 1231 between tissues and blood was calculated and a ratio of 156 :1 was obtained . The pattern of venom tissue distribution (Fig. 4), showed that the highest uptake was in the liver, kidneys and lungs constituting about 84% of the total tissue absorbed dose as compared to 60% for the '231 group. Much lower uptake of the labelled venom was found in the heart, uterus, intestine and ovaries as compared with the ' 231-group (Fig. 4). The
Pharmacokinetics of Scorpion Venom
305
M X
E c E v
0
u g v 0
RADIOACTIVITY BETWEEN BLOOD (A-A) AND TISSUES FOLLOWING THE i.V. INJECTION OF 1271. Blood levels are the average values obtained from 8 rabbits . The vertical lines represent S.E .M. Tissue levels were calculated from equation 5 in text . FIG. 3.
DISTRIBUTION
OF
Do . 4 . TISSUE DISTRIBurION
OF 1 2 51-LABELLED
A . anloreuxi VENOM ([]) AND 1271 (0) IN RABBITS FOLLOWING i.V. ADMINISTRATION. L, liver ; K, kidney ; T, thyroid ; Lu, lung ; H, heart ; U, uterus ; 1, intestine ; D, diaphragm ; S, spleen ; O, ovary. Values are the average obtained from 8 rabbits . The vertical lines indicate S .E .M . Asterisk values are significantly different from the 1251 group . percentage radioactivity of the total injected dose present in urine 270 min following injection was 2045 ± 0-94 for the labelled venom group and 7-87 ± 0-41 for the 125 I-group
(P<0-001).
Measurement
of blood glucose
level at different sampling times showed a marked hyper-
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M . ISMAIL, M. E. ABDULLAH, A. M. MORAD and A . M. GAEEL
glycemic effect caused by the venom (Fig. 5). The rise in blood glucose started 3-5 min after venom injection, became significantly higher than the preinjection level 60 min following injection of the venom and continued to rise steadily until death of the animals (Fig. 5).
Time, min FIG. 5. EFFECT OF A . amoreu\! VENOM ON BLOOD GLUCOSE OF RABBITS.
Labelled venom was injected i.v. at time zero . Values are the average obtained from 8 rabbits. The vertical lines indicate S.E .M . Asterisk values are significantly higher than the preinjection level (at time zero). DISCUSSION
Gel filtration of the labelled venom showed radioactivity to be present in all protein fractions, though it was not directly correlated to protein concentration . This might be due to the different nature of proteins in the different venom fractions . Similarly, fractionation of the labelled venom by cellulose acetate electrophoresis using barbitone buffer, pH 8-6, revealed eight components (GHAZAL et al., 1975) and autoradiography of the electrophoretic runs showed that radioactivity was distributed in all fractions of the iodinated venom . Plotting of the blood level of the labelled venom vs time revealed a biphasic curve characteristic of a two-compartment open pharmacokinetic model. This model (shown below) consists of a central compartment (blood) and a peripheral compartment (tissues) . It is assumed that elimination takes place from the central compartment . kd
kc7 .
Urine ; -Central compartment -Peripheral compartment kTC
The pharmacokinetic analysis of the data according to this model, showed that venom uptake by the tissue compartment is rather rapid, with an estimated half-life of 5-6 min (a = 1-23 x 10-1 min -1 ). The elimination half-life was estimated to be 6-4 hr (P = 1-8 > 10- 3 min-I). Tissue venom concentrations showed a continuous rise reaching a peak level within 37 min . Calculation of the steady state distribution of the venom between blood and tissues, showed that 75 0X of the radioactivity was in the tissue compartment indicating a
Pharmacokmetics of Scorpion Venom
30 7
greater affinity of the venom to the tissues . Analysis of the data obtained from the control rabbits injected with 1251 showed a slower tissue uptake with an estimated half-life of8-3 min '1 was estimated to be 24-2 hr (R = (a = 8-4 x 10- 2 min -1). The elimination half-life of 12 4-78 x 10 - 4 min -1), a value which is approxiamtely four times longer than that obtained for the labelled venom. Tissue levels for 1251 showed an ascending phase that reached a '1 from blood to tissues as compared peak within 60 min, indicating a slower uptake of 12 '1 between blood and tissues showed 61 to the labelled venom . The distribution of 12 of the radioactivity to be in the tissue compartment as compared to 75% for the labelled venom . These results indicate that the pharmacokinetic parameters obtained are due to '1 which might be liberated from it. A factor in favor the labelled venom and not to the 12 of this assumption comes from measurement of the radioactivity in the thyroid glands of both the control and labelled venom groups where much less activity was found in the latter group. Further evidence was obtained from measurement of urinary radioactivity as a percentage ofthe total injected dose where a significantly lower value (P<0-001) was found for the 125 1 group as compared to the labelled venom group. Comparison of the course of pharmacologic effect and of venom concentration in the central compartment shows that the site of action of the venom is not located in the central compartment ; otherwise, the earliest measurement of blood glucose would have been expected to yield the highest rise. Correlation of the intensity of hyperglycemia with venom concentration in the tissue compartment shows that the intensity of the pharmacologic effect was not at its maximum during the distributive phase (when tissue levels are rising) . There was an apparent delay in the onset and peak of the pharmacologic effect when compared with the peak of venom concentration in the tissue compartment . Thus an insignificant rise in blood glucose was shown at times where tissue concentration of the venom was at its maximum (37 min). On the contrary maximum hyperglycemia occurred during the elimination phase (when tissue levels are declining). This excludes the possibility that the site of action of the venom is an indistinguishable part of the tissue compartment . In such a case the pharmacologic response would be essentially the same for a given concentration of the venom during the distributive phase as during the elimination phase (LEVY et al., 1969). These findings could be explained either of two ways. Firstly, the tissue compartment could be classified into a rapidly accessible compartment and a slowly accessible compartment with the venom acting through the slowly accessible compartment . Secondly, the venom might act indireclty causing hyperglycemia through triggering the release of another substance or through transformation to an intermediate. Evidence for the first possibility was shown from difference in the peaks ofhyperglycemia in response to A. amoreuxi venom injected intraventricularly or i.v. Peak effect was reached within 30 min following intraventricular injection in comparison with 3-4 hr for the i.v. injection reflecting a permeability factor to the site of action which was suggested to be the central nervous system (ISMAIL, et al., 1977) . Evidence for the second possibility comes from analysis of the mechanism of the hyperglycemic effect of the venom which was shown to be due to the venom-induced release of tissue and medullary catecholamines which in turn act through inhibition of secretion of insulin (ISMAIL, et al., 1977) . Our results show that the venom is distributed very rapidly to the tissues with an estimated half-life of 5-6 min. This might explain the ineffectiveness of scorpion antivenin injected 15 min following the i.v. injection of scorpion venom into mice (BAL.OZET, 1971) . The literature contains no data concerning the pharmacokinetics of scorpion antivenin ; but it is expected, because of the large molecular size of the globulin fraction containing the antivenin, either to remain in the blood or to have a much slower rate of distribution to the Tox. 1813-1
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tissues . This in turn could relate the ineffectiveness of serotherapy with scorpion antivenin, injected some hours after scorpion sting, to inability ofthe antivenin to diffuse from blood to tissues rather than to irreversible lesions caused by the venom. This conclusion, however, should be taken cautiously until more direct evidence on antivenin distribution is obtained . REFERENCES
BALOZET, L . (1971) Scorpionism in the Old World. In : Venomous Animals and their Venoms, Vol . 111, Venomous Invertebrates, p. 349 (BUCHERL, W . and BUCKLEY, E., Eds.). New York : Academic Press. GHAZAL, A., ISMAIL, M., ABDEL-RAHMAN, A. A. and EL-ASMAR, M. F. (1975) Pharmacological studies of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom. Toxicon 13, 253 . GIBALDI, M. and PERRIERE, D . (1975) Pharmacokinetics. New York : Marcel Dekker . GREENWOOD, F. C., HUNTER, W. M. and GLOVER, J . S. (1963) The preparation of Jill-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114 . ISMAIL, M., OSMAN, O . H ., IBRAHIM, S. A . and EL-ASMAR, M. F . (1972) Cardiovascular and respiratory responses to the venom from the scorpion Leiurus quinquestriatus. E. Ajr. med. J. 49, 273 . ISMAIL, M., OSMAN, O. H., and EL-ASMAR, M. F . (1973) Pharmacological studies of the venom from the scorpion Buthus minax (L . Koch). Toxicon 11, 15. ISMAIL, M., KERTESZ, G., OSMAN, O. H . and SIDRA, M . S . (1974) Distribution of 1231-labelled scorpion (Leiurus quinquestriatus H & E) venom in rat tissues . Toxicon 12, 209. ISMAIL, M., GHAZAL, A., ABDEL-RAHMAN . A, A. and EL-ASMAR, M . F . (1975) Immunological studies with scorpion (Androctonus amoreuxi, Aud. & Sav.) venom . Toxicon 13, 405 . ISMAIL, M., ELKHAWAD, A. O . A. and EL-SEWEIDY, M . (1977) Hyperglycemic effect of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom in rabbits. Abstracts of XVth Congress of Pharmaceutical Sciences .. Cairo, 1977, p . 98. LEVY, G., GiBALDI, M. and JusKo, W . (1969) Multicompartment pharmacokinetic models and pharmacologic effects . J. Am. Pharm . Ass. 58, 422. MIRANDA, F., ROCHAT, H . and LISSITZKY, S . (1960) On the neurotoxin of the poison of two species of North African scorpions. Bull. Soc. Chim. Biol. 42, 379 . MIRANDA, F., RocHAT, H . and LISSrrZKY, S. (1964a) Neurotoxins of two North African species of scorpions. 1 . Purification of neurotoxins (Scorpamines) of Androctonus australis and Buthus occitanus. Toxicon 2, 51 . MIRANDA, F., RDCHAT, H. and LISSrrZKY, S . (19646) Neurotoxins of two North African species of scorpions. It. Properties of neurotoxins (Scorpamines) of Androctonus australis and Buthus occitanus . Toxicon 2,113 . RocHAT, C., ROCHAT, H., MIRANDA, F. and LlssrrZKY, S . (1967) Purification and some properties of the neurotoxin of Androctonus australis. Biochemistry 6, 578 . RocHAT, H., RocHAT, C., KUPEYAN, C . and MIRANDA, F. (1970x) Amino acid sequence of neurotoxin I of
Androctoms australis . Eur . J. Biochem . 17, 262 .
ROCHAT, H., RocHAT, C., KUPEYAN, C., MIRANDA, F. and LISSITZKY, S . (19706) Scorpion neurotoxins : a family of homologous proteins . Fedn . Eur . Biochem . Soc. 10, 349 . SCHMIDT, F. H . (1971) Methoden der Harn-und Blutzucker-bestimmung II . Blutzucker. In : Handbuch des Diabetes mellitus, p. 938 (PFEIFFER, E. F., Ed .) . Munchen : Lehmanns .
0041-0101/80/0501-0301502.00/0
PHARMACOKINETICS OF 121 I-LABELLED VENOM FROM THE SCORPION ANDROCTONUS AMOREUXI, AUD. AND SAV. M. ISMAIL,t M. E. ABDULLAH, A. M. MoRAD and A. M. AGEEL Departments of Pharmacology and Pharmaceutics, Faculty of Pharmacy, University of Riyadh, Riyadh, Saudi Arabia (Acceptedfor publication 22 November 1979) M. ISMAIL, M. E. ABDULLAH, A. M. MoRAD and A. M. AGEEL. Pharmacokinetics of 1251labelled venom from the scorpion Androctonus amoreuxi, Aud. and Sav. Toxicon 18, 301-308, 1980.-Androctonus amoreuxi venom was radioiodinated using the chloramine T method of [1251) iodide oxidation giving a spec . act. of 12-5 pCi/mg. Gel filtration on Sephadex G-50 and cellulose acetate electrophoresis of the labelled venom showed good correlation between protein concentration and radioactivity . Labelled venom was injected i.v. into rabbits anaesthetized with urethane and blood samples were withdrawn from a common carotid artery at times from I to 270 min after venom injection for measurement of radioactivity and determination of blood glucose. Abiphasic blood level curve showing a rapid initial declining phase during the first 15 min followed by a slower declining phase was obtained . This behaviour is characteristic of a two-compartment open model. The rate constants between blood and tissues "kcT", tissues and blood "kTc" and thedisposition rate constant "kd" were estimated to be 9.8 x 10 - 2, 3.34 x 10-2 and 6-67 x 10-3, respectively . The values indicate a very rapid distribution of venom from blood to tissues with an estimated half-life of 5-6 min whereas the overall elimination half-life is 104 min. Steady-state distribution indicated that 75 % of the injected venom was in the tissue compartment after equilibrium was reached. Significant rise in blood glucose was observed when equilibrium was reached and continued throughout the equilibrium phase. Tissue distribution of labelled venom showed major radioactivity to be in liver and kidney. Much less activity was found (in decreasing order) in thyroid, lungs, heart, uterus, intestine, ovary, diaphragm and spleen . INTRODUCTION
THE SCORPION Androctonus amoreuxi is widely distributed in Egypt and neighbouring countries (BALOZET, 1971) . Its venom has potent neurotoxic and cardiotoxic properties (GHAZAL et al., 1975). An antivenin prepared against A. amoreuxi venom had protective action against the lethal and many of the pharmacological effects of the venom (ISMAIL et al., 1975). Although much work has been done on scorpion venoms and their components
(composition, amino acid sequence, pharmacological and toxicological properties ;
MIRANDA et al., 1960, 1964 a, b; ROCHAT et al., 1967, 1970 a, b ; ISMAIL et al., 1972, 1973, 1974, 1975), little work has been carried out on plasma concentration, tissue distribution
and excretion of the venoms . No attempts have been made to correlate venom-blood or tissue levels with the type and magnitude of pharmacological effect . Opinions even differ about the proper time of administering the antivenin following scorpion sting. Most investigators believe that the antivenin should be administered immediately after scorpion sting otherwise the venom will produce irreversible lesions (BALOZET, 1971). Whether this *Supported by Grunt PHRCI from the Research Center, Faculty of Pharmacy, University of Riyadh. tOn leave of absence from Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt. 301
302
M . ISMAIL, M . E . ABDULLAH, A . M . MORAD and A . M . AGEEI.
is true, or it is the venom diffusion into the tissues that limits the effectiveness of the antivenin remained to be answered . It was thought worthwhile, therefore, to study the pharmacokinetics of scorpion venom with the aim of finding a correlation between venom concentration in blood and tissues and pharmacological actions . A . amoreuxi venom was chosen as a model for this study and its ability to cause hyperglycemia was selected as the quantitative pharmacological effect . MATERIALS AND METHODS Venom obtained from mature A . amoreuxi by electrical stimulation of the telson was suspended in water, centrifuged and the supernatant freeze-dried . Dried venom was kept in a desiccator over Silica gel at -17°C, and was reconstituted by the addition of saline or 0-5 M phosphate buffer, pH 7-5, immediately before use. Radioiodination of the venom was carried out using an adaptation of the chloramine-T method of GREENWOOD et al. (1963). To 1 mCi of carrier free sodium iodide-1=3I free from reducing agents (Radiochemical Centre, Amersham, England) in 1 ml of 0-5 M phosphate buffer (pH 7-5, contained in a rubber capped vial), 20 mg of the lyophilized venom (in 1 ml 0-5 M phosphate buffer, pH 7-5) and 20 mg chloramineT (fresh solution in 0-5 ml 0-5 M phosphate buffer, pH 7-5) were added . Immediately after mixing, 1 ml of sodium metabisulphite solution (96 mg/ml in 0-05 M phosphate buffer, pH 7-5) and 2 ml carrier potassium iodide solution (0-4 g/ml in 0-05 M phosphate buffer, pH 7-5) were added . Unbound iodine was removed by dialysis against 500 ml of 0-9 % sodium chloride solution at 4°C with six changes of the salt solution over a period of 36 hr . The dialyzed venom was adjusted to volume by the addition of 0-9% sodium chloride solution. The labelling efficiency ranged between 52 and 55 % and the spec . act. of the labelled venom was 12-5 pCi per mg . Fractionation of the labelled venom was carried out using Sephadex G-50 (Pharmacia, Uppsala, Sweden) with bed dimensions of 2-5 x 25 cm equilibrated with barbitone buffer (0-07 M, pH 8-6) . Two mg labelled venom were mixed with 8 mg non-labelled dialyzed venom in 2 ml barbitone buffer (0-07 M, pH 8-6) and applied to the column. Elution was continued with barbitone buffer and 1 ml fractions were collected at a flow rate of 15 ml per hr. The labelled venom was also fractionated using cellulose acetate electrophoresis and barbitone buffer pH 8-6 as described by GHAZAL et al. (1975) . Protein was estimated spectrophotometrically at 280 nm . Female albino rabbits weighing 2-22 kg were anaesthetized with urethane (1 -75 g/kg i .v.) . Polyethylene catheters were introduced into the left common carotid artery for the collection of serial blood samples . Two mg labelled-venom dissolved in 1 ml saline (spec. act . 12-5 pCi/mg) were injected i .v. into each rabbit via the marginal ear vein. Control rabbits were injected with 1 ml of 1 251 solution in saline (25 pCi/ml) . Two ml blood samples were withdrawn immediately before and at 1, 3, 5, 10, 15, 30, 60, 120, 180 and 270 min following injection of the venom or iodine solution. Heparin (1000 U/kg) was used as an anticoagulant . The bladder was empited before venom injection and total urine, representing the 270 min period, was collected using polyethylene tubing. At the end of the experiment the rabbits were bled to death and the thyroid gland, heart, lungs, diaphragm, liver, kidneys, spleen, intestine, uterus and ovaries were removed, blotted between filter papers and weighed. Radioactivity in organs, blood and urine was measured in biovials using a Beckman Gamma 4000 counting system with a 2 in . sodium iodide crystal (doped with thallium iodide) and connected to a Texas Instrument 700 printer. Blood glucose was estimated in 0- 1 ml samples from the venom-treated rabbits using the glucose oxidase method of ScHnHiDT (1971) and test combination kits (Boehringer, Mannheim, Germany) . RESULTS Using Sephadex G-50, protein was distributed in 36 tubes giving the profile shown in Fig . 1 . Peak protein concentration was in tubes 14-23 while peak radioactivity was in tubes 25-37 (Fig . 1) . Gel filtration of free 1751 under the same conditions revealed a single peak coming between tubes 45 and 55 . Electrophoretic separation of the iodinated and noniodinated dialyzed venom in barbitone buffer, pH 8-6, showed that the two venoms had identical electrophoretic behavior, and autoradiography showed that radioactivity was distributed in all fractions of the iodinated venom . The values obtained from measurement of blood radioactivity were normalized to 20,000 counts/min/ml at the 1 min sampling time . The mean counts/min/ml for the number of rabbits used at each sampling time was plotted against time on a semi-logarithmic graph paper. The data obtained for the labelled venom, revealed a biphasic curve characterized by a rapid initial declining phase followed by a slower terminal declining phase (Fig. 2) .
Pharmacokinetics of Scorpion Venom
FIG. 1 . GEL FILTRATION OF 125-1-LABELLED A. amoreuxi VENOM ON SEPHADEx G-50. Two mg labelled venom +8 mg non labelled dialysed venom in 2 ml barbitone buffer (0-07 M, pH 8-6) were applied to the column. The column (2-5 x 25 cm) was equilibrated and eluted with barbitone buffer (0-07 M, pH 8-6) . Flow rate 15 ml/hr; volume of fractions I ml . ~--~, Venom protein ; /--/, radioactivity .
Time, min FIG. 2. DISTRIBUTION OF RADIOACIIvrrY BETWEEN BLOOD (A-A) AND TISSUES FOLLOWING THE i.V . INJECTION OF 115I-LABELLED A. aMOreuxI vENOM.
The curve represents the average values obtained from 8 rabbits. The vertical lines represent S.E .M . Tissue levels were calculated from equation 5 in text .
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The experimental data points were fitted to a biexponential equation of the form ; Cc =Ae - ° 1+Be - 9r (1) where C, is venom concentration in blood (expressed as counts/min/ml), a and p are the apparent first-order fast and slow disposition rate constants respectively and A and B are the corresponding zero time intercepts . The parameters R and B were estimated graphically from the slope and intercept of the least square line obtained from the terminal experimental data points (Fig . 2). Parameters a and A were estimated graphically by applying the feathering technique (GIBALDI and PERRIERE, 1975). The values obtained for A, B, a and P were 16,200 counts/min/ml, 5700 counts/min/ml, 0123 min-' and 00018 min -', respectively . The apparent first-order intercompartmental rate constants, kcT and k Tc, and the apparent first-order elimination rate constant from the central compartment, kd, were calculated from equations 2-4 (GIBALDI and PERRIERE, 1975) ; kTc =
kd -
AP A -{-}- Ba B.
ap
kTc
kcT = a + (3 - kTc -f- kd
(2)
(3) (4)
The values obtained for kTc, kd and kcT were 00334 min-', 000667 min- ] and 0"098 min- ', respectively . The tissue venom concentration at various time intervals was calculated from the equation; kcT Co WO , - e -° r) CT (5) where, CT is venom concentration in tissues (expressed as counts/min/ml), C,,' is blood venom concentration obtained from the zero time intercept of the blood concentration curve. The curve obtained for the tissue venom concentration showed a rapid ascending phase during the first 15 min that reached a peak within 37 min (Fig. 2). Steady state venom distribution between tissues and blood was calculated from the ratio of the tissue venom to blood venom concentrations after equilibrium distribution was reached. A ratio of 3 :1 was obtained . The data obtained from the control rabbits injected with`f were similarly plotted where a biphasic curve was obtained (Fig. 3). The pharmacokinetic parameters were calculated as described before . The values obtained for A, B, a, R, kcT , kTc and kd were 13,156 counts/ min/nil, 8844 counts/min/ml, 0084 min- ', 000048 min- ', 0052 min- 1, 0034 min- 1 and 000119 min-', respectively . Tissue levels for 1231 were calculated using equation 5. The curve obtained showed a rapid ascending phase during the first 15 min that reached a peak within 60 min (Fig. 3). The steady state equilibrium distribution of 1231 between tissues and blood was calculated and a ratio of 156 :1 was obtained . The pattern of venom tissue distribution (Fig. 4), showed that the highest uptake was in the liver, kidneys and lungs constituting about 84% of the total tissue absorbed dose as compared to 60% for the '231 group. Much lower uptake of the labelled venom was found in the heart, uterus, intestine and ovaries as compared with the ' 231-group (Fig. 4). The
Pharmacokinetics of Scorpion Venom
305
M X
E c E v
0
u g v 0
RADIOACTIVITY BETWEEN BLOOD (A-A) AND TISSUES FOLLOWING THE i.V. INJECTION OF 1271. Blood levels are the average values obtained from 8 rabbits . The vertical lines represent S.E .M. Tissue levels were calculated from equation 5 in text . FIG. 3.
DISTRIBUTION
OF
Do . 4 . TISSUE DISTRIBurION
OF 1 2 51-LABELLED
A . anloreuxi VENOM ([]) AND 1271 (0) IN RABBITS FOLLOWING i.V. ADMINISTRATION. L, liver ; K, kidney ; T, thyroid ; Lu, lung ; H, heart ; U, uterus ; 1, intestine ; D, diaphragm ; S, spleen ; O, ovary. Values are the average obtained from 8 rabbits . The vertical lines indicate S .E .M . Asterisk values are significantly different from the 1251 group . percentage radioactivity of the total injected dose present in urine 270 min following injection was 2045 ± 0-94 for the labelled venom group and 7-87 ± 0-41 for the 125 I-group
(P<0-001).
Measurement
of blood glucose
level at different sampling times showed a marked hyper-
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M . ISMAIL, M. E. ABDULLAH, A. M. MORAD and A . M. GAEEL
glycemic effect caused by the venom (Fig. 5). The rise in blood glucose started 3-5 min after venom injection, became significantly higher than the preinjection level 60 min following injection of the venom and continued to rise steadily until death of the animals (Fig. 5).
Time, min FIG. 5. EFFECT OF A . amoreu\! VENOM ON BLOOD GLUCOSE OF RABBITS.
Labelled venom was injected i.v. at time zero . Values are the average obtained from 8 rabbits. The vertical lines indicate S.E .M . Asterisk values are significantly higher than the preinjection level (at time zero). DISCUSSION
Gel filtration of the labelled venom showed radioactivity to be present in all protein fractions, though it was not directly correlated to protein concentration . This might be due to the different nature of proteins in the different venom fractions . Similarly, fractionation of the labelled venom by cellulose acetate electrophoresis using barbitone buffer, pH 8-6, revealed eight components (GHAZAL et al., 1975) and autoradiography of the electrophoretic runs showed that radioactivity was distributed in all fractions of the iodinated venom . Plotting of the blood level of the labelled venom vs time revealed a biphasic curve characteristic of a two-compartment open pharmacokinetic model. This model (shown below) consists of a central compartment (blood) and a peripheral compartment (tissues) . It is assumed that elimination takes place from the central compartment . kd
kc7 .
Urine ; -Central compartment -Peripheral compartment kTC
The pharmacokinetic analysis of the data according to this model, showed that venom uptake by the tissue compartment is rather rapid, with an estimated half-life of 5-6 min (a = 1-23 x 10-1 min -1 ). The elimination half-life was estimated to be 6-4 hr (P = 1-8 > 10- 3 min-I). Tissue venom concentrations showed a continuous rise reaching a peak level within 37 min . Calculation of the steady state distribution of the venom between blood and tissues, showed that 75 0X of the radioactivity was in the tissue compartment indicating a
Pharmacokmetics of Scorpion Venom
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greater affinity of the venom to the tissues . Analysis of the data obtained from the control rabbits injected with 1251 showed a slower tissue uptake with an estimated half-life of8-3 min '1 was estimated to be 24-2 hr (R = (a = 8-4 x 10- 2 min -1). The elimination half-life of 12 4-78 x 10 - 4 min -1), a value which is approxiamtely four times longer than that obtained for the labelled venom. Tissue levels for 1251 showed an ascending phase that reached a '1 from blood to tissues as compared peak within 60 min, indicating a slower uptake of 12 '1 between blood and tissues showed 61 to the labelled venom . The distribution of 12 of the radioactivity to be in the tissue compartment as compared to 75% for the labelled venom . These results indicate that the pharmacokinetic parameters obtained are due to '1 which might be liberated from it. A factor in favor the labelled venom and not to the 12 of this assumption comes from measurement of the radioactivity in the thyroid glands of both the control and labelled venom groups where much less activity was found in the latter group. Further evidence was obtained from measurement of urinary radioactivity as a percentage ofthe total injected dose where a significantly lower value (P<0-001) was found for the 125 1 group as compared to the labelled venom group. Comparison of the course of pharmacologic effect and of venom concentration in the central compartment shows that the site of action of the venom is not located in the central compartment ; otherwise, the earliest measurement of blood glucose would have been expected to yield the highest rise. Correlation of the intensity of hyperglycemia with venom concentration in the tissue compartment shows that the intensity of the pharmacologic effect was not at its maximum during the distributive phase (when tissue levels are rising) . There was an apparent delay in the onset and peak of the pharmacologic effect when compared with the peak of venom concentration in the tissue compartment . Thus an insignificant rise in blood glucose was shown at times where tissue concentration of the venom was at its maximum (37 min). On the contrary maximum hyperglycemia occurred during the elimination phase (when tissue levels are declining). This excludes the possibility that the site of action of the venom is an indistinguishable part of the tissue compartment . In such a case the pharmacologic response would be essentially the same for a given concentration of the venom during the distributive phase as during the elimination phase (LEVY et al., 1969). These findings could be explained either of two ways. Firstly, the tissue compartment could be classified into a rapidly accessible compartment and a slowly accessible compartment with the venom acting through the slowly accessible compartment . Secondly, the venom might act indireclty causing hyperglycemia through triggering the release of another substance or through transformation to an intermediate. Evidence for the first possibility was shown from difference in the peaks ofhyperglycemia in response to A. amoreuxi venom injected intraventricularly or i.v. Peak effect was reached within 30 min following intraventricular injection in comparison with 3-4 hr for the i.v. injection reflecting a permeability factor to the site of action which was suggested to be the central nervous system (ISMAIL, et al., 1977) . Evidence for the second possibility comes from analysis of the mechanism of the hyperglycemic effect of the venom which was shown to be due to the venom-induced release of tissue and medullary catecholamines which in turn act through inhibition of secretion of insulin (ISMAIL, et al., 1977) . Our results show that the venom is distributed very rapidly to the tissues with an estimated half-life of 5-6 min. This might explain the ineffectiveness of scorpion antivenin injected 15 min following the i.v. injection of scorpion venom into mice (BAL.OZET, 1971) . The literature contains no data concerning the pharmacokinetics of scorpion antivenin ; but it is expected, because of the large molecular size of the globulin fraction containing the antivenin, either to remain in the blood or to have a much slower rate of distribution to the Tox. 1813-1
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tissues . This in turn could relate the ineffectiveness of serotherapy with scorpion antivenin, injected some hours after scorpion sting, to inability ofthe antivenin to diffuse from blood to tissues rather than to irreversible lesions caused by the venom. This conclusion, however, should be taken cautiously until more direct evidence on antivenin distribution is obtained . REFERENCES
BALOZET, L . (1971) Scorpionism in the Old World. In : Venomous Animals and their Venoms, Vol . 111, Venomous Invertebrates, p. 349 (BUCHERL, W . and BUCKLEY, E., Eds.). New York : Academic Press. GHAZAL, A., ISMAIL, M., ABDEL-RAHMAN, A. A. and EL-ASMAR, M. F. (1975) Pharmacological studies of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom. Toxicon 13, 253 . GIBALDI, M. and PERRIERE, D . (1975) Pharmacokinetics. New York : Marcel Dekker . GREENWOOD, F. C., HUNTER, W. M. and GLOVER, J . S. (1963) The preparation of Jill-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114 . ISMAIL, M., OSMAN, O . H ., IBRAHIM, S. A . and EL-ASMAR, M. F . (1972) Cardiovascular and respiratory responses to the venom from the scorpion Leiurus quinquestriatus. E. Ajr. med. J. 49, 273 . ISMAIL, M., OSMAN, O. H., and EL-ASMAR, M. F . (1973) Pharmacological studies of the venom from the scorpion Buthus minax (L . Koch). Toxicon 11, 15. ISMAIL, M., KERTESZ, G., OSMAN, O. H . and SIDRA, M . S . (1974) Distribution of 1231-labelled scorpion (Leiurus quinquestriatus H & E) venom in rat tissues . Toxicon 12, 209. ISMAIL, M., GHAZAL, A., ABDEL-RAHMAN . A, A. and EL-ASMAR, M . F . (1975) Immunological studies with scorpion (Androctonus amoreuxi, Aud. & Sav.) venom . Toxicon 13, 405 . ISMAIL, M., ELKHAWAD, A. O . A. and EL-SEWEIDY, M . (1977) Hyperglycemic effect of scorpion (Androctonus amoreuxi, Aud. & Sav.) venom in rabbits. Abstracts of XVth Congress of Pharmaceutical Sciences .. Cairo, 1977, p . 98. LEVY, G., GiBALDI, M. and JusKo, W . (1969) Multicompartment pharmacokinetic models and pharmacologic effects . J. Am. Pharm . Ass. 58, 422. MIRANDA, F., ROCHAT, H . and LISSITZKY, S . (1960) On the neurotoxin of the poison of two species of North African scorpions. Bull. Soc. Chim. Biol. 42, 379 . MIRANDA, F., RocHAT, H . and LISSrrZKY, S. (1964a) Neurotoxins of two North African species of scorpions. 1 . Purification of neurotoxins (Scorpamines) of Androctonus australis and Buthus occitanus. Toxicon 2, 51 . MIRANDA, F., RDCHAT, H. and LISSrrZKY, S . (19646) Neurotoxins of two North African species of scorpions. It. Properties of neurotoxins (Scorpamines) of Androctonus australis and Buthus occitanus . Toxicon 2,113 . RocHAT, C., ROCHAT, H., MIRANDA, F. and LlssrrZKY, S . (1967) Purification and some properties of the neurotoxin of Androctonus australis. Biochemistry 6, 578 . RocHAT, H., RocHAT, C., KUPEYAN, C . and MIRANDA, F. (1970x) Amino acid sequence of neurotoxin I of
Androctoms australis . Eur . J. Biochem . 17, 262 .
ROCHAT, H., RocHAT, C., KUPEYAN, C., MIRANDA, F. and LISSITZKY, S . (19706) Scorpion neurotoxins : a family of homologous proteins . Fedn . Eur . Biochem . Soc. 10, 349 . SCHMIDT, F. H . (1971) Methoden der Harn-und Blutzucker-bestimmung II . Blutzucker. In : Handbuch des Diabetes mellitus, p. 938 (PFEIFFER, E. F., Ed .) . Munchen : Lehmanns .