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Metabolism of dextrosulphenidol in several animal species
Metabolism of Dextrosulphenidol in Several Animal Species* By EVAN W. McCHESNEY, RAYMOND F. KOSS, JAMES M. SHEKOSKY, a n d WILLIAM H. DEITZ The in vivo metabolism of dextrosulphenidol has been studied using various test organisms. Differences are noted and comparisons are made as to the effect of biological variation on the absorption, distribution, and excretion of the drug.
a more detailed study of its metabolism. This procedure consists essentially of ethyl acetate extraction, alkaline hydrolysis, oxidation with periodate a t pH i . 5 , and final estimation of @methylsulfonylbenzaldehyde as an alkali salt of its p-nitrophenylhydrazone. Data on the absorption, tissue distribution, and excretion of DES in rat, dog, cat, rabbit, and man are presented in this paper.
NVESTIGATION of the metabolism of dextroIsulphenidol (DES) has thus far been limited by the lack of adequate chemical assay methods, the data which are presently available having been obtained by microbiological assay (tube dilution techniques). Using these methods, Shaffer, et al. (l), have shown DES to be well absorbed by chicks when administered orally; nevertheless, concentrations which were inhibitory to the test organism remained in the intestinal tract for several hours after a dose of 100 mg./Kg. With doses of 400 mg./Kg., serum levels of 31-62 mcg./ml. were maintained for as long as three to six hours, and levels of 4-8 mcg./ml. were detected as late as twelve hours postmedication. Drug levels in the bile were higher and more prolonged than those in the plasma. Using similar methods, one of us (W. H. D.) has studied the absorption of DES in dogs and man (2). Single doses of 55 and 150 mg./Kg. have been given to mongrel dogs: the former dose produced peak serum levels of 50 mcg./ml. a t two and four hours, and by twelve hours postmedication this level had fallen to 3 mcg./ml. The latter dose gave a four-hour level of 100 mcg./ml. and a twelve-hour level of 7 mcg./ml. Dogs also received and tolerated well doses of 50, 40, or 80 mg./Kg. twice daily for twenty-one days The initial doses resulted in two-hour serum levels of 6,10,and 22 mcg.,/ml., respectively. In man, the administration of a single dose of 1 Gm. of DES resulted in a serum level of 6 mcg./ml. a t one and one-half hours. It was observed in three subjects that man excretes about ‘TO per cent of an oral dose of 500 mg., in biologically active form, within twenty-two hours. The development of a chemical procedure for the determination of DES (3) has made possible
EXPERIMENTAL
* Received March I . 1 W O . from the SterlitwWinthrtip Research Institute. Rensselaer, N. Y . 1 In the earlier literature dextrosulphenidol. or ~ d - t h r e o - 2 dichloracetamido - 1 - (4 - methylsulfonylphenyl) - 1.8 - propanediol, was referred t o as Thiocymetin. This trade name is now reserved for the dl-form (raceophenidol).
Albino rats weighing 170-230 Gm. were fasted for at least eight (but not more than fifteen) hours prior to medication and during the entire metabolic period, but they had access to water at all times. Xfter medication they were placed in metabolism cages, for the quantitative collection of the excreta. Generally speaking, no feces were eliminated in the brief period studied, and the urine volume was rather low. At various intervals postmedication the animals were sacrificed by decapitation, and representative tissues were removed for analysis. These included heart, lung, liver, kidney, spleen, and muscle; blood plasma was also obtained a t this time. The urine was analyzed for both free and total DES as previously described (3); the stomach plus content, and the intestines plus content were analyzed separately. Both oral and intravenous administration were studied in the rat. To improve solubility, the compound was dissolved in 50% propylene glycol; the dose of DES was 50 mg./Kg. and the dose of glycol was 2.500 mg./Kg. The results of these experimentsare presented in Table I. Similar data on the metabolism of DES in other animal species were obtained in one dog, one cat, and two rabbits. In order to obtain strictly comparable data on the several species, all of the animals were fasted for eight hours prior to medication, and during the sixteen-hour metabolic period but, again, they had access to water a t all times. The oral medications (50 mg./Kg.) were given in 50% propylene glycol, the total dose of the glycol again being 2,500 mg./Kg. Since these are the same experimental conditions as were used for one of the groups ol orally medicated rats, the data on the individual animals of this specific group are also given for comparative purposes. These data are presented in Table IT. One laboratory volunteer took a single 500-mg. dose ( t w o tablets) of DES and collected urine under toluene for six periods comprising the next fortyeight hours. These samples were analyzed both chemically and microbiologically (using P. septica as the test organism), with the results presented in Table 111.
762
5.0 f 12 f 15 f
0
8.4 f 49.2 f 12.7 f
Heart Lung Spleen Kidney Liver Muscle Plasma Stomach Intestine Urine Free Total Feces
Tissuesd G. I. tractC Urine
~
1.7 5.3 4.7
f 410 f 395
f f f f f
f f f f f
f f
0.7
1.9 1.5
f 185 f 56
6.7 f 39.4 f 21.5 f
0
1,765 1,830
16
1.5 1.0 26 8.2 2.5 2.6 0.6 0.8 0.4 f 19 f 2.3 f 1.4 f 1.1 f 0.02 f
f
0.5 f 0.5 1.6 f 0.5 19 f 16 12 f 2.9 4.9.f 1.1 0.9 f 0.2 0.6 f 0.01
Values recorded as mcg./Gm. (mean AS. E , ) .
8
1.9 0.9 13.1 1.7 1.3 0.3 0.1
6
ii
0.6 0.4 10
f 96 f 0.1 f 5 f 310
f
f f f
0
4,247 4,298
0.3 2.0 12 18 30 67 1.9 5.0 400
3.2 f 28.9 f 30.8 f
1.5 3.8 1.3
1.5 =k 16.5 f 53.8 f
0.5 2.7 1.4
f 85 f 115
0.3 i 0.6 f 9 f 4 f 6 f 20 f 0 i 2 f 230 f
Per Cent of Dose Present
2,41.0 f 47 2,510 f 109 0
~~
1.0 2.0 18 14 28 195 2.0 10 2,350
0.8 0.4 1.2 1.0 3.0 14 0.3 1.2 118
0.9 0.3 1.3 0.5 0.5 0.1 0.1
0.2 1.1 10.0
f 1,100
f 1,270
f f
f
f
f
f
f f
f
f f f f f f f
16 b
0.5 f 4.2 f 63.0 f
0
6,960 6,910
1.7 1.2 2.8 3.0 8.5 34 0.3 4.0 460
Total Dextrosulphenidol Present, mcg. (Mean f S. E.)
0.5 0.8 23 30 3.8 0.7 0.7
3.0 f 0.4 5.0 f 1.1 37 f 26 & ? f 55 ~69 f 30 360 f 46 11 f 3 30 f 10 3,320 f 120
4.0 3.9 41 48 12 4.7 2.8
4
Hours after Medication---
0.7 0.3 0.8 1.5 2.5 24 0.1 0.6 15
0.8 0.2 2.0 0.8 0.4 0.3 0.1
0.4 0.2 3.0
f 250
f 236
f f f f f f
f
f f
f
f
f
f f
f f
24
0.5 f 8.8 f 56.8 f
4,870 4,730 0
1.2 1.0 0.8 5.0 8.2 24 1.7 2.0 750
1.4 0.7 1.5 3.3 1.5 0.3 0.5
--___
f 0.8 f 0.4 f 6.9 f 2.8 f 0.9 f 0.5
.....
2.7 2.1 11.2 12.8 4.9 2.0
T/P Ratio<
-
b Intravenous medication. c Ratio of tissue concentration to plasma concentration determined concomitantly; mean fS. E. for 16 a The medication was oral except where otherwise indicated. animals. Two sixteen-hour i . v. animals with no detectable plasma level are excluded. d Total in the tissues analyzed. e In stomach, intestine, cecum:
1,070 1,070
68 522 30 160 000
60 .~ f
0.6 2.1 5.0 10 12 108 1.2 50 400
2.3 1.2 2.1 0.3
f
f f f
7.4
f
0.4
0.6
f
f
7.4 7.9 21 29 9.7 6.8 7.5
2
Heart Lung Spleen Kidney Liver Muscle Plasma
Material Analyzed
TABLEABSORPTION, TISSUE DISTRIBUTION, AND EXCRETION OF DEXTROSULPHENIDOL IN RATSRECEIVING DOSESOF 50 M G . / K G . ~ 0
ii
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JOURNAL
AMERICANPHARMACEUTICAL ASSOCIATION
Vol. 49, No. 12
TABLE 11.-COMPARATIVE METABOLISM OF DEXTROSULPHENIDOL I N SEVERAL ANIMAL SPECIES".b Material Analyzed
Rat
Heart Lung Spleen Kidney Liver Muscle Brain Pancreas Fat Bile Plasma
c
Rat
4.08 3.96 0 8.5 47.1
4.70 4.60 0 7.6 61.2
4.9 5.8 4.8 9.4 9.7 3.1 2.9
..
..
16
4.9 0.06 1s:0
200.0 235.0 10.8 350.0 67.3
52.5 52.3 0 107.0 49.0
Rabbit
Rabbit
0.2 0.2 0.6 4.0 0.4 0.3 1.2
1.4 0.3 2.0 2.0 2.4 0.7 1.2
i:o
0:s 52 0.5
74 0.5 0.54 0.93 6.45 43.5 43.8 0 137.5 31.9
0.41 0.48 7.50 44.4 42.5 0 133.0 31.9
All observations made sixteen hours after a single oral dose of 50 rng./Kg. Values recorded as mcg./Gm. Indicates material not analyzed.
AND
METABOLISM OF DEXTROSULPHENIDOL IN MANFOLLOWING A SINGLE ORAL ~ ~ O - M CDOSE .
-Interval of Urine Collection, hr.
0-4
4-8 8-12 12-24 24-32 32-48 Totals Means c
Cat
1.5 2.4 50.0 14.8 6.8 1.1
0 1.6 1.5 5.9 5.1 1.1
4.30 4.58 0 7.9 54.8
TABLE III.-EXCRETION
a b
Rat
2.1 0.4 0.8 2.7 0.8 1.3 . .c .. .. 0.7 .. 1.0 .. 0 30 0.6 0.7 0.6 0.7 Dextrosulphenidol Present, mg. 0.003 0.06 0.08 0.03 0.12 1:57 i.68 0.94 2.78
0 0.8 5.9 14.5 2.9 0.4
Stomach Small intestine Cecum-colon Urine Free DES Total DES Feces Dose, mg. Dose in urine, % o b
Species Dog
7 -
Volume output, ml .
Free DES, mcg./ml.
126 106 156 343 230 740
742 1,130 410 184 101 11
-
Chemical Assay Mg. Total Excreted DES.a in mcg./ml. interval b
732 1,130 420 175 110 13
-
Mg. Excreted per hr.
92.5 120.0 64.7 61.7 25.2 8.9
__
1,701
Microbiological Assayc Mg. Excreted in Mcg./ml. Interval
23 30 16 5 3 0.6
1200 400 200 100 20
7.8
234
800
373 222
101 127 62 69 23 15
397
22 1
Analysis involved preliminary drastic alkaline hydrolysis. Based on mean of free and total DES values. Determined by serial dilution techniques. using P. replica as the test organism.
DISCUSSION DES demonstrates in all of the animal species studied the typical metabolic pattern of a compound which is well absorbed, circulates freely in the extracellular fluids, and is excreted in both urine and bile. It has no outstanding tissue predilection except possibly for the spleen, and this exception has been observed in only one of the species (the rat). General comments on the results on the individual species studied follow. The Rut.-At the early intervals, the concentrations of DES in heart, lung, and muscle are essentially the same as those existing concurrently in the plasma. The concentrations in the kidney and liver are definitely higher than those existing concomitantly in the pkasina, as would be expected for the organs which are directly involved in the excretory processes. The tissue/plasma concentration ratios in the spleen are very erratic, varying from 2 to 80, even in two animals from the same group (the sixteen-hour, oral). A relatively high
splenic concentration is also characteristic of chloramphenicol metabolism (4). The biological half-life of DES in the rat, as estimated from the decreases in tissue and plasma levels with time, is about five hours; the similarly estimated value for chloramphenicol is one and one-half t o three hours (5). I t is evident from the data t h a t the r a t excretes DES in the urine entirely in unchanged form, since in no group of animals is there a significant difference between free and total DES. This eliminates the possibility that the rat excretes, via the urine, any measurable amount of DES as a glucuronide or as the free base.* Both of these derivatives have been detected in the urine following the administration of chloramphenicol (6). The urinary excretion o f DES increases with time. to a maximuni of 57y0 of the dose, in twenty-four hours. I t is of interest to note, however, that the sums of the amounts present -.ill the urine and the digestive tract remain nearly That panediol. f
is,
od-threo-l-(4-methylsulfonylpheny1)-1,3-pr~-
SCIENTIFIC EDITION
December 1960
i65
(b) It may be excreted in thc bilc as a glucuronide, a form which would not be extensively recycled and which would not be determined under the conditions used for DES analysis (3). This type of excretion has been demonstrated for chloramphenicol (7), but there is no entirely unambiguous procedure by which its occurrence can be demonstrated for DES, since no sample of its glucuronide is available for chemical or biological study. The point has been studied in the following way, however, based on the assumed properties of such a glucuronide : Finely powdered rat feces (obtained from the animals of Table IV) were suspended in 100 parts of methanol, the suspension was adjusted to pH 3.5 with hydrochloric acid, digested on the steam bath for fifteen minutes, and filtered. Two aliquot portions of this extract were evaporated separately to dryness and were analyzed for free and total DES in a manner analogous to that used for urine (3): one of the residues was partitioned between ethyl acetate and phosphate buffer of p H 6.2,and the amount of DES in the ethyl acetate was determined; the other residue was subjected t o a direct alkaline hydrolysis, followed by a determination of the free DES bases in the hydrolysate. I n all of the extracts so studied the “total” DES exceeded the “free” (i. e., chemically unaltered) by 253570. When the values for total fecal DES are calculated on this basis (Table IV, column 4) it is found that the total recovery becomes 85-9970 of the amount administered. S i c e the procedure used to extract the supposed DES glucuronide from the feces is incapable of direct experimental verification, it cannot be assumed that such a procedure would necessarily extract the supposed glucuronide quantitatively and it is not necessary, therefore, to assume the existence of still another metabolic product to account for the remainder of the dose (in groups B and C Table IV). (c) It may be converted to the free base by loss of the dichloroacetyl group (6)and the base may be further degraded by oxidative processes. This point was studied by administering the bases intravenously (35 mg./Kg.) to two groups of three rats each. Excreta were collected as described above and the base was determined in the urine as for total DES (3), except that the alkaline hydrolysis was omitted. The finely ground feces were extracted with methanol, followed by alkaline hydrolysis of the residue so extracted. The results are presented in Table V: they demonstrate that no substantial oxidation of the propanediol side chain occurs, and that the excretion of the base is largely renal. The results described in sections ( a ) and (c) TABLEIV.-URINARY AND FECALEXCRETION OF of this discussion may be interpreted to mean that DEXTROSULPHENIDOL I N THE RATFOLLOWING AN either DES base or DES glucuronide is excreted in ORALDOSEOF 50 MG./KG.~ the bile and feces of rats. Of these possibilities, the latter is much more likely, in view of the data of In Urine, In Feces Total DES Table V. The data of Table I fit readily into a 0-48 hr. Total logical scheme if it is assumed that about 65% of the ReI324 24-48 Free Total administered DES is eventually excreted in the Group) hr. hr. DES DES covered urine of (fasting) rats, and 35Y0 is excreted in the 98.8 37.8 54.4 A 38.7 5.7 36.6 49.5 86.0 B 31.9 4.6 hile as a glucuronide. The latter would not be
constant during tlic twenty-four-hour period, all of these sums being between 59 and 70% of the dose. While the animal is effecting this transfer of DES from intestine to urine, the amouttts in the tissues decrease steadily, from 8.5y0 of the dose a t two hours, to o.5yOat twenty-four hours. A considerable part of the dose (about 30%) is not accounted for and must be present in the tissues or the digestive tract in a form which is not extractable and, therefore, not determinable by the analytical method used. Extensive metabolic degradation is clearly ruled out as a possibility. The results on the intravenously medicated animals demonstrate that excretion into the intes tine definitely occurs. Residues in the intestine of the orally medicated animals, therefore, do not represent, exclusively, unabsorbed material. Tissue levels in the i. v. animals, sixteen hours after medication, are as low as those observed in the twenty-four-hour orally medicated animals, indicating a more rapid physiological disposition of the compound when it is administered parenterally. The higher level of urinary excretion in the intravenously medicated animals, as compared t o the orally medicated animals sacrificed at the same time interval, points to the same conclusions. Some possibilities for the fate of the unaccountedfor remainder (about 30%) of the orally or intravenously administered DES are as follows: (a) It may be excreted in the urine, but at a later time. This seems improbable in view of the fact that only 0.5% of the dose is found in the tissues analyzed a t twenty-four hours postmedication; nevertheless, the point was studied by administering 50 mg./Kg. of DES orally in 50y0 propylene glycol to three pairs of rats (200-250 Gm. each) and collecting excreta for forty-cight hours thereafter. These rats had been fasted for fifteen hours prior to medication, but food was provided during the experimental period. The results of this experiment are presented in Table IV, and they demonstrate that delayed urinary excretion does not account for a very large proportion of the dose. However, it is also clear that the results in Table I V are not entirely comparable to those presented in Table 1. The presence of food in the intestine a t any time evidently alters the absorption and excretion of DES very materially, probably by stimulating the flow of bile. The result is that much more of the drug is excreted in the feces and much less in the urine of the fed animals than is the case when the animals receive no food whatsoever, after eight hours premedication.
.
C
Mean
41.1 37.2
4.6 5.0
29.8 34.7
40.0 48.0
85.7 90.2
(i All values in the table are given as % ’ of dose. 8 TWO rats per group.
Kindly supplied by Mr. S . Schalit of this Institute. The product used was the crystalline dl-form, m. p. 123-121’. % N = 5.82. J
766
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Vol. 49, No. 12
TABLEV.-URINARV A N D FECAL EXCRETION" OF ( 6 ) . The total urinary excretion of DES in fortyDEXTROSULPHENIDOL BASE"I N THE RATFOLLOWINGcight hours (in this one subjcct) is 7.570 of the dos;.. A N INTRAVENOUS DOSEOF 35 MG./KG. which contrasts markedly with the 5-6y0 of chloramphenicol so excreted (8-11).* Such excretion 0; -In Urine -. Total (unchanged) chloramphenicol as does occur, further0-24 24-48 In Feces, ReGroupc hr. hr. 0-48 hr. covered more, is mostly in the first six hours after administraA 80.0 7.5 5.7 93.2 tion. 5.8 81.5 B 72.0 6.7 SUMMARY a All values in the table are as % ' of dose. b See text footnote 3.
c
Three rats per group.
determinable in feces or intestinal content, except as described in (b). The Cal.-So far as it is justifiable to draw conclusions from one animal, it may be said that the metabolism of DES in the cat differs significantly from that in the dog, rat, and rabbit. For example, the tissue levels in the cat sixteen hours postmedication are higher than those in any of the other species studied. However, the plasma level is correspondingly high, with the result that the tissue/ plasma ratios remain nearly 1/1, except for the two organs directly involved in the excretory processes. It is of interest to note that the ratio of biliary to plasma concentration in the cat is only 3/1, as compared to 100/1 for the rabbit and 40/1 for the dog. The relatively low level of biliary excretion in the cat may correlate with the high tissue levels in this species, since its sixteen-hour urinary excretion is a t least average for the animals studied. The Dog.-Only in the dog is there a definite difference between free and total urinary DES. The sixteen-hour renal excretion is the highest of any of the species studied, and the amount remaining in the intestine of the dog is correspondingly low. The tissue levels in the dog are comparable to those of the twenty-four-hour rats (Table I, column 6 ) and of the sixteen-hour rabbits. The only DES found in the feces of the dog, however, is in a n (analytically)unchanged form. The Rabbit.-The results on this species differ most noticeably in the presence of considerable drug residues in the stomach, and in the low urinary excretion. These appear to be interrelated observations since, in spite of the fact that the rabbits had been fasted for eight hours prior to medication, very bulky food residues remained in all levels of theii digestive tracts. It has been noted above that the presence of such residues affects the absorption and excretion rates of DES appreciably. Biliary excretion is evidently a very important process in the rabbit. Man.-The only determinable form of DES excreted in the urine of man is the unchanged drug, as is shown by the fact that the chemically-determined values for free and total drug exactly coincide (see Table 111). These observations are further confirmed by the fact that the microbiological assay of each sample is in very close agreement with the chemical analysis for total DES. N o significant amount of glucuronide (determinable by difference), therefore, can be present, although this is the principal pathway of chloramphenicol metabolism
T h e metabolism of dextrosulphenidol has been studied i n t h e rat, dog, cat, rabbit, and man. In all species b u t one (the dog) t h e only urinary excretory product is the unchanged drug; in this one species about 15 per cent of the dose is evidently excreted in a conjugated form. Absorption and excretion of dextrosulphenidol appear t o be delayed b y t h e presence of food residues i n the digestive tract; in their absence t h e urinary excretion in sixteen hours, regardless of t h e species, is about 60 per cent of t h e dose. Biliary excretion is also an important process, with the result that t h e drug continues to be found i n the intestine as long as it is present in the tissues. Tissue :plasma concentration ratios are generally about 1 : 1 except in the liver and kidney, and possibly the spleen. There is some evidence t h a t t h e drug is present in the intestine of t h e r a t other t h a n as unchanged dextrosulphenidol. This form could b e either the free base or a glucuronide conjugate, but is more likely the latter. In man 75 per cent of a n oral dose is excreted in unchanged form within forty-eight hours.
REFERENCES (1) Shaffer, M . F., Milner, K. C . . Clemmer. D . I., Potash, L., and Shaffer, L. S . , Anfibiofics & Ckemofkcrapy, 4. 992 ( 1954). (2) Deitz. W. H., unpublished data, Sterling-Winthrop Research Institute. (3) McChesney. E. W., Shekosky. J. M., Eckert, H . W., 49,28(1960). and Koss R . F. THISJOURNAL (4) Lhvine, 'J.. and Fishbach, H . , Anfibiofics & Chcmotherapy. 1, 59(1951). (5) Orr. W. W.. Preisser. W. G.. Ross.. S . . Burke. F. G . . and'Ricei~E:. C. ibjd. 1 63(1951). ' (6) Glazko, 'A. J . . ' D h , W. A , , and Rebstock, M. C., J . B i d . Ckem. 183 679(1950). (7) Glaiko. k. J., Dill, W. A,. and Wolf. L. M., J . Pkarmacol. ExPf1. Thcrafi. 104,452(1952). (8) Glazko. A. J.. Woli, L. M., Dill, W. A,,and Bratton, A. C.. ibid., 96, 445(1949). (9) Ley, H. L.. Jr., Smadel. J. E.. and Crocker, T. T., Proc. Soc. E x p f l . Bad. Med.. 68, 9(1948). (10) Shuck 0. Cholmsky K. Smahel 0. and GraRneterova. J . . Anli6iofic Mad. & Cl&. Tkerody, 6: 98(1959). ( 1 1 ) Trivellato. E., and Vettori, G., Minerua med.. 48,
___~ --.,_ "
d i t . i ( t 4x7)
(12) Knnin. C. M.,and Finland, M., Proc. Soc. E x p f l . Biol. Mcd.. 103. 246(1960). 4 Recently Kunin and Finland (12) have studied comparatively the absorption and excretion of chloramphenicol and dextrosulphenidol in man. They found that an oral dose of 500 mu. of the latter gave a peak blood level of 4-6 mcg./ml. at two hours postmedication. and a cumulative forty-eighthour urinary excretion (in six subjects) of 336 f 42 mg.. when assayed against S. lulea.
a more detailed study of its metabolism. This procedure consists essentially of ethyl acetate extraction, alkaline hydrolysis, oxidation with periodate a t pH i . 5 , and final estimation of @methylsulfonylbenzaldehyde as an alkali salt of its p-nitrophenylhydrazone. Data on the absorption, tissue distribution, and excretion of DES in rat, dog, cat, rabbit, and man are presented in this paper.
NVESTIGATION of the metabolism of dextroIsulphenidol (DES) has thus far been limited by the lack of adequate chemical assay methods, the data which are presently available having been obtained by microbiological assay (tube dilution techniques). Using these methods, Shaffer, et al. (l), have shown DES to be well absorbed by chicks when administered orally; nevertheless, concentrations which were inhibitory to the test organism remained in the intestinal tract for several hours after a dose of 100 mg./Kg. With doses of 400 mg./Kg., serum levels of 31-62 mcg./ml. were maintained for as long as three to six hours, and levels of 4-8 mcg./ml. were detected as late as twelve hours postmedication. Drug levels in the bile were higher and more prolonged than those in the plasma. Using similar methods, one of us (W. H. D.) has studied the absorption of DES in dogs and man (2). Single doses of 55 and 150 mg./Kg. have been given to mongrel dogs: the former dose produced peak serum levels of 50 mcg./ml. a t two and four hours, and by twelve hours postmedication this level had fallen to 3 mcg./ml. The latter dose gave a four-hour level of 100 mcg./ml. and a twelve-hour level of 7 mcg./ml. Dogs also received and tolerated well doses of 50, 40, or 80 mg./Kg. twice daily for twenty-one days The initial doses resulted in two-hour serum levels of 6,10,and 22 mcg.,/ml., respectively. In man, the administration of a single dose of 1 Gm. of DES resulted in a serum level of 6 mcg./ml. a t one and one-half hours. It was observed in three subjects that man excretes about ‘TO per cent of an oral dose of 500 mg., in biologically active form, within twenty-two hours. The development of a chemical procedure for the determination of DES (3) has made possible
EXPERIMENTAL
* Received March I . 1 W O . from the SterlitwWinthrtip Research Institute. Rensselaer, N. Y . 1 In the earlier literature dextrosulphenidol. or ~ d - t h r e o - 2 dichloracetamido - 1 - (4 - methylsulfonylphenyl) - 1.8 - propanediol, was referred t o as Thiocymetin. This trade name is now reserved for the dl-form (raceophenidol).
Albino rats weighing 170-230 Gm. were fasted for at least eight (but not more than fifteen) hours prior to medication and during the entire metabolic period, but they had access to water at all times. Xfter medication they were placed in metabolism cages, for the quantitative collection of the excreta. Generally speaking, no feces were eliminated in the brief period studied, and the urine volume was rather low. At various intervals postmedication the animals were sacrificed by decapitation, and representative tissues were removed for analysis. These included heart, lung, liver, kidney, spleen, and muscle; blood plasma was also obtained a t this time. The urine was analyzed for both free and total DES as previously described (3); the stomach plus content, and the intestines plus content were analyzed separately. Both oral and intravenous administration were studied in the rat. To improve solubility, the compound was dissolved in 50% propylene glycol; the dose of DES was 50 mg./Kg. and the dose of glycol was 2.500 mg./Kg. The results of these experimentsare presented in Table I. Similar data on the metabolism of DES in other animal species were obtained in one dog, one cat, and two rabbits. In order to obtain strictly comparable data on the several species, all of the animals were fasted for eight hours prior to medication, and during the sixteen-hour metabolic period but, again, they had access to water a t all times. The oral medications (50 mg./Kg.) were given in 50% propylene glycol, the total dose of the glycol again being 2,500 mg./Kg. Since these are the same experimental conditions as were used for one of the groups ol orally medicated rats, the data on the individual animals of this specific group are also given for comparative purposes. These data are presented in Table IT. One laboratory volunteer took a single 500-mg. dose ( t w o tablets) of DES and collected urine under toluene for six periods comprising the next fortyeight hours. These samples were analyzed both chemically and microbiologically (using P. septica as the test organism), with the results presented in Table 111.
762
5.0 f 12 f 15 f
0
8.4 f 49.2 f 12.7 f
Heart Lung Spleen Kidney Liver Muscle Plasma Stomach Intestine Urine Free Total Feces
Tissuesd G. I. tractC Urine
~
1.7 5.3 4.7
f 410 f 395
f f f f f
f f f f f
f f
0.7
1.9 1.5
f 185 f 56
6.7 f 39.4 f 21.5 f
0
1,765 1,830
16
1.5 1.0 26 8.2 2.5 2.6 0.6 0.8 0.4 f 19 f 2.3 f 1.4 f 1.1 f 0.02 f
f
0.5 f 0.5 1.6 f 0.5 19 f 16 12 f 2.9 4.9.f 1.1 0.9 f 0.2 0.6 f 0.01
Values recorded as mcg./Gm. (mean AS. E , ) .
8
1.9 0.9 13.1 1.7 1.3 0.3 0.1
6
ii
0.6 0.4 10
f 96 f 0.1 f 5 f 310
f
f f f
0
4,247 4,298
0.3 2.0 12 18 30 67 1.9 5.0 400
3.2 f 28.9 f 30.8 f
1.5 3.8 1.3
1.5 =k 16.5 f 53.8 f
0.5 2.7 1.4
f 85 f 115
0.3 i 0.6 f 9 f 4 f 6 f 20 f 0 i 2 f 230 f
Per Cent of Dose Present
2,41.0 f 47 2,510 f 109 0
~~
1.0 2.0 18 14 28 195 2.0 10 2,350
0.8 0.4 1.2 1.0 3.0 14 0.3 1.2 118
0.9 0.3 1.3 0.5 0.5 0.1 0.1
0.2 1.1 10.0
f 1,100
f 1,270
f f
f
f
f
f
f f
f
f f f f f f f
16 b
0.5 f 4.2 f 63.0 f
0
6,960 6,910
1.7 1.2 2.8 3.0 8.5 34 0.3 4.0 460
Total Dextrosulphenidol Present, mcg. (Mean f S. E.)
0.5 0.8 23 30 3.8 0.7 0.7
3.0 f 0.4 5.0 f 1.1 37 f 26 & ? f 55 ~69 f 30 360 f 46 11 f 3 30 f 10 3,320 f 120
4.0 3.9 41 48 12 4.7 2.8
4
Hours after Medication---
0.7 0.3 0.8 1.5 2.5 24 0.1 0.6 15
0.8 0.2 2.0 0.8 0.4 0.3 0.1
0.4 0.2 3.0
f 250
f 236
f f f f f f
f
f f
f
f
f
f f
f f
24
0.5 f 8.8 f 56.8 f
4,870 4,730 0
1.2 1.0 0.8 5.0 8.2 24 1.7 2.0 750
1.4 0.7 1.5 3.3 1.5 0.3 0.5
--___
f 0.8 f 0.4 f 6.9 f 2.8 f 0.9 f 0.5
.....
2.7 2.1 11.2 12.8 4.9 2.0
T/P Ratio<
-
b Intravenous medication. c Ratio of tissue concentration to plasma concentration determined concomitantly; mean fS. E. for 16 a The medication was oral except where otherwise indicated. animals. Two sixteen-hour i . v. animals with no detectable plasma level are excluded. d Total in the tissues analyzed. e In stomach, intestine, cecum:
1,070 1,070
68 522 30 160 000
60 .~ f
0.6 2.1 5.0 10 12 108 1.2 50 400
2.3 1.2 2.1 0.3
f
f f f
7.4
f
0.4
0.6
f
f
7.4 7.9 21 29 9.7 6.8 7.5
2
Heart Lung Spleen Kidney Liver Muscle Plasma
Material Analyzed
TABLEABSORPTION, TISSUE DISTRIBUTION, AND EXCRETION OF DEXTROSULPHENIDOL IN RATSRECEIVING DOSESOF 50 M G . / K G . ~ 0
ii
764
OF THE
JOURNAL
AMERICANPHARMACEUTICAL ASSOCIATION
Vol. 49, No. 12
TABLE 11.-COMPARATIVE METABOLISM OF DEXTROSULPHENIDOL I N SEVERAL ANIMAL SPECIES".b Material Analyzed
Rat
Heart Lung Spleen Kidney Liver Muscle Brain Pancreas Fat Bile Plasma
c
Rat
4.08 3.96 0 8.5 47.1
4.70 4.60 0 7.6 61.2
4.9 5.8 4.8 9.4 9.7 3.1 2.9
..
..
16
4.9 0.06 1s:0
200.0 235.0 10.8 350.0 67.3
52.5 52.3 0 107.0 49.0
Rabbit
Rabbit
0.2 0.2 0.6 4.0 0.4 0.3 1.2
1.4 0.3 2.0 2.0 2.4 0.7 1.2
i:o
0:s 52 0.5
74 0.5 0.54 0.93 6.45 43.5 43.8 0 137.5 31.9
0.41 0.48 7.50 44.4 42.5 0 133.0 31.9
All observations made sixteen hours after a single oral dose of 50 rng./Kg. Values recorded as mcg./Gm. Indicates material not analyzed.
AND
METABOLISM OF DEXTROSULPHENIDOL IN MANFOLLOWING A SINGLE ORAL ~ ~ O - M CDOSE .
-Interval of Urine Collection, hr.
0-4
4-8 8-12 12-24 24-32 32-48 Totals Means c
Cat
1.5 2.4 50.0 14.8 6.8 1.1
0 1.6 1.5 5.9 5.1 1.1
4.30 4.58 0 7.9 54.8
TABLE III.-EXCRETION
a b
Rat
2.1 0.4 0.8 2.7 0.8 1.3 . .c .. .. 0.7 .. 1.0 .. 0 30 0.6 0.7 0.6 0.7 Dextrosulphenidol Present, mg. 0.003 0.06 0.08 0.03 0.12 1:57 i.68 0.94 2.78
0 0.8 5.9 14.5 2.9 0.4
Stomach Small intestine Cecum-colon Urine Free DES Total DES Feces Dose, mg. Dose in urine, % o b
Species Dog
7 -
Volume output, ml .
Free DES, mcg./ml.
126 106 156 343 230 740
742 1,130 410 184 101 11
-
Chemical Assay Mg. Total Excreted DES.a in mcg./ml. interval b
732 1,130 420 175 110 13
-
Mg. Excreted per hr.
92.5 120.0 64.7 61.7 25.2 8.9
__
1,701
Microbiological Assayc Mg. Excreted in Mcg./ml. Interval
23 30 16 5 3 0.6
1200 400 200 100 20
7.8
234
800
373 222
101 127 62 69 23 15
397
22 1
Analysis involved preliminary drastic alkaline hydrolysis. Based on mean of free and total DES values. Determined by serial dilution techniques. using P. replica as the test organism.
DISCUSSION DES demonstrates in all of the animal species studied the typical metabolic pattern of a compound which is well absorbed, circulates freely in the extracellular fluids, and is excreted in both urine and bile. It has no outstanding tissue predilection except possibly for the spleen, and this exception has been observed in only one of the species (the rat). General comments on the results on the individual species studied follow. The Rut.-At the early intervals, the concentrations of DES in heart, lung, and muscle are essentially the same as those existing concurrently in the plasma. The concentrations in the kidney and liver are definitely higher than those existing concomitantly in the pkasina, as would be expected for the organs which are directly involved in the excretory processes. The tissue/plasma concentration ratios in the spleen are very erratic, varying from 2 to 80, even in two animals from the same group (the sixteen-hour, oral). A relatively high
splenic concentration is also characteristic of chloramphenicol metabolism (4). The biological half-life of DES in the rat, as estimated from the decreases in tissue and plasma levels with time, is about five hours; the similarly estimated value for chloramphenicol is one and one-half t o three hours (5). I t is evident from the data t h a t the r a t excretes DES in the urine entirely in unchanged form, since in no group of animals is there a significant difference between free and total DES. This eliminates the possibility that the rat excretes, via the urine, any measurable amount of DES as a glucuronide or as the free base.* Both of these derivatives have been detected in the urine following the administration of chloramphenicol (6). The urinary excretion o f DES increases with time. to a maximuni of 57y0 of the dose, in twenty-four hours. I t is of interest to note, however, that the sums of the amounts present -.ill the urine and the digestive tract remain nearly That panediol. f
is,
od-threo-l-(4-methylsulfonylpheny1)-1,3-pr~-
SCIENTIFIC EDITION
December 1960
i65
(b) It may be excreted in thc bilc as a glucuronide, a form which would not be extensively recycled and which would not be determined under the conditions used for DES analysis (3). This type of excretion has been demonstrated for chloramphenicol (7), but there is no entirely unambiguous procedure by which its occurrence can be demonstrated for DES, since no sample of its glucuronide is available for chemical or biological study. The point has been studied in the following way, however, based on the assumed properties of such a glucuronide : Finely powdered rat feces (obtained from the animals of Table IV) were suspended in 100 parts of methanol, the suspension was adjusted to pH 3.5 with hydrochloric acid, digested on the steam bath for fifteen minutes, and filtered. Two aliquot portions of this extract were evaporated separately to dryness and were analyzed for free and total DES in a manner analogous to that used for urine (3): one of the residues was partitioned between ethyl acetate and phosphate buffer of p H 6.2,and the amount of DES in the ethyl acetate was determined; the other residue was subjected t o a direct alkaline hydrolysis, followed by a determination of the free DES bases in the hydrolysate. I n all of the extracts so studied the “total” DES exceeded the “free” (i. e., chemically unaltered) by 253570. When the values for total fecal DES are calculated on this basis (Table IV, column 4) it is found that the total recovery becomes 85-9970 of the amount administered. S i c e the procedure used to extract the supposed DES glucuronide from the feces is incapable of direct experimental verification, it cannot be assumed that such a procedure would necessarily extract the supposed glucuronide quantitatively and it is not necessary, therefore, to assume the existence of still another metabolic product to account for the remainder of the dose (in groups B and C Table IV). (c) It may be converted to the free base by loss of the dichloroacetyl group (6)and the base may be further degraded by oxidative processes. This point was studied by administering the bases intravenously (35 mg./Kg.) to two groups of three rats each. Excreta were collected as described above and the base was determined in the urine as for total DES (3), except that the alkaline hydrolysis was omitted. The finely ground feces were extracted with methanol, followed by alkaline hydrolysis of the residue so extracted. The results are presented in Table V: they demonstrate that no substantial oxidation of the propanediol side chain occurs, and that the excretion of the base is largely renal. The results described in sections ( a ) and (c) TABLEIV.-URINARY AND FECALEXCRETION OF of this discussion may be interpreted to mean that DEXTROSULPHENIDOL I N THE RATFOLLOWING AN either DES base or DES glucuronide is excreted in ORALDOSEOF 50 MG./KG.~ the bile and feces of rats. Of these possibilities, the latter is much more likely, in view of the data of In Urine, In Feces Total DES Table V. The data of Table I fit readily into a 0-48 hr. Total logical scheme if it is assumed that about 65% of the ReI324 24-48 Free Total administered DES is eventually excreted in the Group) hr. hr. DES DES covered urine of (fasting) rats, and 35Y0 is excreted in the 98.8 37.8 54.4 A 38.7 5.7 36.6 49.5 86.0 B 31.9 4.6 hile as a glucuronide. The latter would not be
constant during tlic twenty-four-hour period, all of these sums being between 59 and 70% of the dose. While the animal is effecting this transfer of DES from intestine to urine, the amouttts in the tissues decrease steadily, from 8.5y0 of the dose a t two hours, to o.5yOat twenty-four hours. A considerable part of the dose (about 30%) is not accounted for and must be present in the tissues or the digestive tract in a form which is not extractable and, therefore, not determinable by the analytical method used. Extensive metabolic degradation is clearly ruled out as a possibility. The results on the intravenously medicated animals demonstrate that excretion into the intes tine definitely occurs. Residues in the intestine of the orally medicated animals, therefore, do not represent, exclusively, unabsorbed material. Tissue levels in the i. v. animals, sixteen hours after medication, are as low as those observed in the twenty-four-hour orally medicated animals, indicating a more rapid physiological disposition of the compound when it is administered parenterally. The higher level of urinary excretion in the intravenously medicated animals, as compared t o the orally medicated animals sacrificed at the same time interval, points to the same conclusions. Some possibilities for the fate of the unaccountedfor remainder (about 30%) of the orally or intravenously administered DES are as follows: (a) It may be excreted in the urine, but at a later time. This seems improbable in view of the fact that only 0.5% of the dose is found in the tissues analyzed a t twenty-four hours postmedication; nevertheless, the point was studied by administering 50 mg./Kg. of DES orally in 50y0 propylene glycol to three pairs of rats (200-250 Gm. each) and collecting excreta for forty-cight hours thereafter. These rats had been fasted for fifteen hours prior to medication, but food was provided during the experimental period. The results of this experiment are presented in Table IV, and they demonstrate that delayed urinary excretion does not account for a very large proportion of the dose. However, it is also clear that the results in Table I V are not entirely comparable to those presented in Table 1. The presence of food in the intestine a t any time evidently alters the absorption and excretion of DES very materially, probably by stimulating the flow of bile. The result is that much more of the drug is excreted in the feces and much less in the urine of the fed animals than is the case when the animals receive no food whatsoever, after eight hours premedication.
.
C
Mean
41.1 37.2
4.6 5.0
29.8 34.7
40.0 48.0
85.7 90.2
(i All values in the table are given as % ’ of dose. 8 TWO rats per group.
Kindly supplied by Mr. S . Schalit of this Institute. The product used was the crystalline dl-form, m. p. 123-121’. % N = 5.82. J
766
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Vol. 49, No. 12
TABLEV.-URINARV A N D FECAL EXCRETION" OF ( 6 ) . The total urinary excretion of DES in fortyDEXTROSULPHENIDOL BASE"I N THE RATFOLLOWINGcight hours (in this one subjcct) is 7.570 of the dos;.. A N INTRAVENOUS DOSEOF 35 MG./KG. which contrasts markedly with the 5-6y0 of chloramphenicol so excreted (8-11).* Such excretion 0; -In Urine -. Total (unchanged) chloramphenicol as does occur, further0-24 24-48 In Feces, ReGroupc hr. hr. 0-48 hr. covered more, is mostly in the first six hours after administraA 80.0 7.5 5.7 93.2 tion. 5.8 81.5 B 72.0 6.7 SUMMARY a All values in the table are as % ' of dose. b See text footnote 3.
c
Three rats per group.
determinable in feces or intestinal content, except as described in (b). The Cal.-So far as it is justifiable to draw conclusions from one animal, it may be said that the metabolism of DES in the cat differs significantly from that in the dog, rat, and rabbit. For example, the tissue levels in the cat sixteen hours postmedication are higher than those in any of the other species studied. However, the plasma level is correspondingly high, with the result that the tissue/ plasma ratios remain nearly 1/1, except for the two organs directly involved in the excretory processes. It is of interest to note that the ratio of biliary to plasma concentration in the cat is only 3/1, as compared to 100/1 for the rabbit and 40/1 for the dog. The relatively low level of biliary excretion in the cat may correlate with the high tissue levels in this species, since its sixteen-hour urinary excretion is a t least average for the animals studied. The Dog.-Only in the dog is there a definite difference between free and total urinary DES. The sixteen-hour renal excretion is the highest of any of the species studied, and the amount remaining in the intestine of the dog is correspondingly low. The tissue levels in the dog are comparable to those of the twenty-four-hour rats (Table I, column 6 ) and of the sixteen-hour rabbits. The only DES found in the feces of the dog, however, is in a n (analytically)unchanged form. The Rabbit.-The results on this species differ most noticeably in the presence of considerable drug residues in the stomach, and in the low urinary excretion. These appear to be interrelated observations since, in spite of the fact that the rabbits had been fasted for eight hours prior to medication, very bulky food residues remained in all levels of theii digestive tracts. It has been noted above that the presence of such residues affects the absorption and excretion rates of DES appreciably. Biliary excretion is evidently a very important process in the rabbit. Man.-The only determinable form of DES excreted in the urine of man is the unchanged drug, as is shown by the fact that the chemically-determined values for free and total drug exactly coincide (see Table 111). These observations are further confirmed by the fact that the microbiological assay of each sample is in very close agreement with the chemical analysis for total DES. N o significant amount of glucuronide (determinable by difference), therefore, can be present, although this is the principal pathway of chloramphenicol metabolism
T h e metabolism of dextrosulphenidol has been studied i n t h e rat, dog, cat, rabbit, and man. In all species b u t one (the dog) t h e only urinary excretory product is the unchanged drug; in this one species about 15 per cent of the dose is evidently excreted in a conjugated form. Absorption and excretion of dextrosulphenidol appear t o be delayed b y t h e presence of food residues i n the digestive tract; in their absence t h e urinary excretion in sixteen hours, regardless of t h e species, is about 60 per cent of t h e dose. Biliary excretion is also an important process, with the result that t h e drug continues to be found i n the intestine as long as it is present in the tissues. Tissue :plasma concentration ratios are generally about 1 : 1 except in the liver and kidney, and possibly the spleen. There is some evidence t h a t t h e drug is present in the intestine of t h e r a t other t h a n as unchanged dextrosulphenidol. This form could b e either the free base or a glucuronide conjugate, but is more likely the latter. In man 75 per cent of a n oral dose is excreted in unchanged form within forty-eight hours.
REFERENCES (1) Shaffer, M . F., Milner, K. C . . Clemmer. D . I., Potash, L., and Shaffer, L. S . , Anfibiofics & Ckemofkcrapy, 4. 992 ( 1954). (2) Deitz. W. H., unpublished data, Sterling-Winthrop Research Institute. (3) McChesney. E. W., Shekosky. J. M., Eckert, H . W., 49,28(1960). and Koss R . F. THISJOURNAL (4) Lhvine, 'J.. and Fishbach, H . , Anfibiofics & Chcmotherapy. 1, 59(1951). (5) Orr. W. W.. Preisser. W. G.. Ross.. S . . Burke. F. G . . and'Ricei~E:. C. ibjd. 1 63(1951). ' (6) Glazko, 'A. J . . ' D h , W. A , , and Rebstock, M. C., J . B i d . Ckem. 183 679(1950). (7) Glaiko. k. J., Dill, W. A,. and Wolf. L. M., J . Pkarmacol. ExPf1. Thcrafi. 104,452(1952). (8) Glazko. A. J.. Woli, L. M., Dill, W. A,,and Bratton, A. C.. ibid., 96, 445(1949). (9) Ley, H. L.. Jr., Smadel. J. E.. and Crocker, T. T., Proc. Soc. E x p f l . Bad. Med.. 68, 9(1948). (10) Shuck 0. Cholmsky K. Smahel 0. and GraRneterova. J . . Anli6iofic Mad. & Cl&. Tkerody, 6: 98(1959). ( 1 1 ) Trivellato. E., and Vettori, G., Minerua med.. 48,
___~ --.,_ "
d i t . i ( t 4x7)
(12) Knnin. C. M.,and Finland, M., Proc. Soc. E x p f l . Biol. Mcd.. 103. 246(1960). 4 Recently Kunin and Finland (12) have studied comparatively the absorption and excretion of chloramphenicol and dextrosulphenidol in man. They found that an oral dose of 500 mu. of the latter gave a peak blood level of 4-6 mcg./ml. at two hours postmedication. and a cumulative forty-eighthour urinary excretion (in six subjects) of 336 f 42 mg.. when assayed against S. lulea.