Human disposition of antimony administered as antimony sodium dimercapto-succinate

48 TRANSACTIONS OF THE ROYAL SOCIETY OF

TROPICAL MEDICINE AND HYGIENE.

Vol. 58.

No.

1. January,

1964.

COMMUNICATIONS HUMAN DISPOSITION OF ANTIMONY ADMINISTERED AS ANTIMONY SODIUM DIMERCAPTO-SUCCINATE WITH AN:ANALYSIS OF ANTIMONY CONCENTRATIONIN EXCRETEDSchistosoma haematobium OVA* BY

A. R. SCHULERT Biochemistry Division, U.S. Naval Medical Research Unit No. 3, Cairo, and Biochemistry Department, Vanderbilt University, Nashville, Tenn., U.S.A. H. G. BROWNE Parasitology Department, U.S. Naval Medical Research Unit No. 3, Cairo AND

H. H. SALEM~ Department of Tropical Medicine and Parasitology University of Alexandria, It has been previously reported (SALEM et al., 1956, 1957, 1961) that antimony dimercapto-succinate preparations, when administered to patients, directly affect the ova of Schistosoma mansoni and Schistosoma haematobium. Hatching rates are low and miracidia that emerge from the eggs exhibit sluggish motility. On these grounds Salem deduced that there was an accumulation of antimony in the ova. Several studies on the distribution and excretion of these drugs using radioactive material have been conducted including that by ABDULLAH and SEIF (1962), but no previous reports have been made of the uptake of the drugs in the parasite itself. This approach, which is quite feasible in many situations, seems to have been inadequately exploited in the past. Previous studies on radioactively labelled anthelmintics include those by BRADY et al., 1945; LAWTON et al., 1945; LAZARUS and ROGERS, 1951; ESSERMAN, 1952; TERHAAR, 1957; and KNAPP et al., 1960. BRADY et al., 1945, determined the uptake of antimony in Dirofilaria immitis in dogs by the use of Sb-124 tartar emetic. Recently, BROWNE and SCHULERT(1963) have used this approach in the study of the disposition of labelled antimony sodium meso-2, 3-dimercapt0-succinate (Astiban) in infected hamsters and mice and found a concentration of antimony in ova of S. mansoni 30 times greater than that in the liver, and several thousand times greater than that in the plasma. e The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the view of the Navy Department or the naval service at large. We gratefully acknowledge the support given by Dean Abdel Rahman el Sadr, Dr. James H. Boyers and Professor William J. Darby. Clinical and laboratory assistance was provided by S. Bessa, J. Hady, M. Halal, G. Hilal, M. Mansour, M. Maurice, S. Monem, M. Younes, S. Zeid, and A. Zeind. Hoffman-La Roche Ltd. of Basle kindly prepared the Sb-124 labelled Astiban used in these investigations. This study was supported in part by Research Grant Nonr 2049 between the Office of Naval Research and Vanderbilt University, and by PL-480 contract funds.

49

A. R. SCHULERT, H. G. BROWNE AND H. H. SALEM

I n v i e w of t h e s e f i n d i n g s a n d t h e earlier o b s e r v a t i o n s of Salem, i n v e s t i g a t i o n s w e r e c a r r i e d o u t in p a t i e n t s w i t h ~urinary s c h i s t o s o m i a s i s . A l t h o u g h t h e a m o u n t of r a d i o a c t i v e a n t i m o n y g i v e n was small, it was p o s s i b l e to m e a s u r e t h e r a d i o a c t i v i t y in t h e e x c r e t e d ova. MATERIALS

AND M E T H O D S

Six patients, four males and two females, were selected on the basis of their excreting appreciable quantities of N. kaematobium ova in the urine, and admitted to the tropical diseases ward of the Alexandria University Hospital. T h e patients ranged in age from 10 to 15 years and in weight from 23.5 to 47 kg. U n d e r the supervision of one of us (H.H.S.), each was given a single Astiban dose of 13.6 mg./kg. T h e specific activity of the Sb-124 in the drug was 0.06 microcuries/mg. Sb, (i.e., 0.015 microcuries/mg. Astiban). Thus, the radioactive dose to the patient was 0.2 microcuries/kg. Blood specimens were taken at intervals of ½, 1, 8 and 24 hours, and a final specimen was taken at 8 days. Urine was collected at the following intervals: 0-2 hours, 2-8 hours, 8-24 hours, and 24-48 hours. Subsequent 24 hour urine collections were obtained from three patients 5 weeks after injection. Ova were collected from each daily urine for 8 days by the method of BROWNE and THOMAS (1963). This screening method permitted removal of pus cells, erythrocytes, and crystals from the specimens. T h e eggs were then fixed with buffered 10 per cent. formalin. Large urate crystals, whose dimensions closely approximated to those of the eggs, were finally removed from the specimens by dissolution in 10 per cent. acetic acid. T h e eggs were then washed in distilled water, centrifuged briefly, and resuspended in 10 per cent. buffered formalin. T h e final purity of the specimens was assessed by microscopic examination. T h e pretreatment egg excretion had been determined by the method of SALEM (1961). T h e absolute numbers of excreted eggs used for radio assay were determined by a filter-counting technique (BROWNE and THOMAS, in press). Egg harvests from the first day and some from the second day were counted individually. F r o m the second day onward the eggs from the six patients were combined to give adequate numbers for convenient assay. All egg samples and early blood samples were counted in a well-type scintillation counter (Nuclear Chicago model 132A). In subsequent blood sampling, slightly larger samples were taken and ashed to give greater concentration of the Sb-124 for more accurate counting. Urine samples were counted by placing 170 ml. over the crystal. In this position the efficiency of counting is only about 1/10 of that for samples in the well, but the sample size is increased by a factor of 60, so that the count rate is increased sixfold. Although all of these samples were counted directly, including the 5-week specimens, a procedure was also devised for concentrating all the Sb-124 from a 24-hour urine specimen. T h e Sb-124 was quantitatively precipitated by the addition of antimony carrier followed by ammonium sulphide at a slightly acid pH. It appears that by means of such a procedure the radioactive antimony excreted in the urine can be assayed for periods up to several months. RESULTS AND DISCUSSION T a b l e I s h o w s t h e a n t i m o n y c o n c e n t r a t i o n in p l a s m a a n d r e d b l o o d cells of t h e six p a t i e n t s . I t is n o t e d t h a t , f o l l o w i n g i n t r a m u s c u l a r injection, t h e p l a s m a level shows t h e TABLE I.

Concentration of antimony in plasma and red blood cells (all values in y.g./ml.). Plasma RBC ½ hr.

Plasma RBC 1 hr.

Plasma RBC 8 hrs.

Plasma RBC 24 hrs.

Patient A.Y. 13 F, 47 kg. K.H. 12 M, 33 kg. A.S. 15 M, 46 kg. A.Y'. 11 M, 24 kg. E.A. 14 M, 26 kg. F.A. 10 F, 26 kg.

Average RBC/Plasma

7.50 5.57 7.11 7.56 6.35 8.28 7.06 0.3

1.51 2.22 1.87 2.06 2.15 1.86

6.35 3.95 6.11 5.78 5.27 3.97

1.95

5.24 0.4

1.98 1.74 2.41 1.79 2.33 1.84

0.25 0.18 0.30 0.16 0.11 0.21

2.02

0.20 3.2

0.70 0.56 0.84 0.56 0.61 0.62

0.23 0.41 0.17 0.08 0.05 0.12

0.65

0.18

0.29 0.20 0.62 0.29 0.25 0.22 0.32 1.9

50

A N T I M O N Y C O N C E N T R A T I O N I N EXCRETED i

Schistosoma mansoni

OVA

highest value at 1-half hour, whereas for blood cells the 1-half and 1-hour values are about equal. After 1 hour the plasma concentration falls rapidly so that the 8-hour value is only 4 per cent. of the 1 hour value, after which the decline is slight. Red blood cells follow a similar pattern with a lag period. The ratio of concentration in the red blood cells to that in the plasma gradually rises in time, as might be expected. The decrease in this ratio between 8 and 24 hours is explained on the basis that the plasma reaches its low residual value before the red cells. Table II shows the urinary excretion of each patient. It is striking that the pattern is very consistent, even for the 0-2-hour interval where the results of variation in the rate of absorption from an intramuscular depot might be expected to be most pronounced. Thus, in the six cases, the 0-2-hour excretion was 21.4 4- 1.4 per cent., and the 24-hour excretion was 36.5 =k 1.0 per cent. The excretion on the second day dropped to 1.8 -4- 0.2 per cent. of the administered dose. It appears that at this point the antimony is somewhat fixed in the body so that excretion proceeds very slowly. Measurable quantities are still excreted at 5 weeks. Further preliminary data indicate that the pattern of excretion is a function of the administered dose. For example, the fraction excreted in the first 24 hours appears to be much less when small quantities of Astiban are given. TABLE II. Urinary excretion (all values in percentage of administered dose).

Patient A.Y. K.H. A.S. A.F. E.A. F.A. Average

24F

0-2 hrs.

2-8 hrs.

8-24 hrs.

0-24 hrs.

19.7 23.3 17.4 23.2 26.0 21.4

11.4 10.7 16.4 9.0 10.3 12.9

2.6 2.1 4.4 2.7 3.7 1.7

36.1 33.7 38.2 34.9 40.0 36.0

1.0 2.1 1.6

2.9-+0.4

36.5--+1.0

1.8 :kO.2

21.4~1.4 l l . 8 : k l .2

1.8 2.5

35th day

36th day

0.28

0.27

0.07 0.37

0.08 0.25

Data on the ova are given in Table III, in which the amounts of antimony present in the ova from each patient in the first 24 hours are noted, as well as that for combined samples for subsequent 24-hour intervals up to 13 days. The values for the first day are compared with the plasma levels at 24 hours and are based on an estimated Wet weight of 4.2 x 10-7 g. per ovum obtained by BROWNEand SCHULERT(1963) for purified preparations of S. mansoni eggs. Since all the egg specimens on the first day, with the exception of that from patient A.Y., were free from contamination by cellular debris or crystals, it is considered that all of the antimony contained in them was concentrated in the eggs. The eggs from case A.Y. on the first day and the combined samples from days 9 to 13 were moderately to heavily contaminated with large epithelial cells, so that the possibility exists that these cells made a significant contribution to the amount of antimony in the sample. (Subsequently means have been devised to obviate this contamination). This possibility appears unlikely however, in the view of the fact that: i) concentrations of the antimony in blood and urine are three orders o~fmagnitude less than in the ova, and ii) the pattern appears similar in the contaminated and uncontaminated samples. The ratios of ova to plasma antimony concentrations in human beings ranged as shown in Table III, and on the average were somewhat less than those found for S. mansoni

51

A. R. SCHULERT, H. G. B R O W N E AND H. H. SALEM

TABLE III.

Day

Case

Antimony content of ova excreted in the urine.

Number of ova

1

N.Y.

K.H. A.S. A.F. E.A.

F.A. Total ,7

3 4 5 6 7 8 9 11. 13

2,360 22,150 13,560 560 9,100 4,600 52,300 63,300 28,000 54,500 24,700 2,240 9,660 3,590 560 330 220

Micrograms per ovum x 10-~ 4.6 2.0 1.8 41.4 3.7 4.4 3.0 1.5 2.8 1.2 2.5 2.1 1.9 3.6 16.6 70.2 49.1

Micrograms 24 hr. plasma per gramme micrograms/ mg. 110 48 43 988 88 105 71 39 67 29 65 50 45 86 395 1670 1170

0.23 0.41 0.17 0.08 0.05 0.12 0.18

Ova/Plasma x 103 0.5 0,1 0.3 12.4 1.8 0.9 0.4

from the livers and intestines of hamsters 24 hours after injection of much larger doses. Ratios for the latter ranged from 480 to 15,000. Antimony concentration in excreted ova might not be expected to be the same as that in ova retained in the tissues because at the start of treatment the latter are less mature. Other studies in animals indicate that immature eggs take up larger concentrations of antimony. It is noted that egg excretion was suppressed after the eighth day, which is generally the case following a single injection of a large dose of the drug. Assuming that the human pattern of egg excretion follows that regularly observed in animals, one would expect the eggs excreted at the end of a week to be those that were in their most immature stage of development when the drug was administered. These would be exposed to the drug for the longest period of time during their maturation in the host. It is tempting to speculate that this is the reason for the apparent increase in antimony concentration in the eggs from the eighth day onward, but the paucity of eggs, the small n u m b e r of observations and the contamination with epithelial cells, render such a conclusion hazardous at present.

CONCLUSIONS 1) Intramuscularly administered antimony sodium dimercapto-succinate is excreted via the urine to the extent of about 20 per cent. in the first 2 hours and 35 per cent. in the first 24 hours after a single dose of 13.6 mg./kg. Thereafter, excretion proceeds very slowly over a long period of time. 2) T h e ova of Schistosoma haematobium concentrate the antimony from this drug to a very marked degree. 3) These findings tend to corroborate those made earlier by one of us (Salem) which indicate that the ovum is a stage in which the parasite is attacked by the drug. T h e y also follow the pattern observed earlier in infected hamsters and mice.

52

ANTIMONY CONCENTRATIONIN EXCRETED Schistosoma mansoni OVA REFERENCES

ABDULLAH, A. ~¢ SEIF, M. (1962). Ciba Foundation Symposium, Bilharziasis, Cairo, March 1962, 287. London: J. & A. Churchill Ltd. BRADY, F. J., LAWTON, A. H., COWIE, D. B., ANDREWS, H. L., N~ss, A. T. & OGDEN, G. E. (1945). Amer. J. trop. Med., 25, 103. BROWNE, H. G. 86 SCHULERT, A. R. (1963). Fed. Proc. (April 1963). 86 ~ (1963). Unpublished report. - 86 THOMAS, J. I. (1963). J. Parasit., 49, 371. --86 - In press. ESSERMAN, H. B. "(1952). Aust. J. Sci. Res. B5(4), 485. KNAPP, S. E., HANSEN, M. F., MOSER, H. C. 86 MCFARLAND, R. H. (1960). Exp. Parasit., 9, 56. LAWTON, A. H., NESS, A. T., BRADY, F. J. 86 DEAN, B. C. (1945). Sdence, 102, 120. LAZARUS, M. 86 ROGERS, W. P. (1951). Aust. J. Sci. Res. B4(2), 163. SALEM, H. H., SHERIF, A. F. & FRIEDHEIM, E. A. H. (1956), W.H.O. Conference on bilharziasis, Brazzaville. --., FRIEDHEIM, E. A. H. 86 SHERIF, A. F. (1957). ft. Egypt. Publ. Hlth. Ass., 32, 313. - - . 86 SHERIF, A. F. (1961). Ibid., 36, 129. TERHAAR, C. J. (1957). Unpublished dissertation, Kansas State College, 97 p. -

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