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Absorption and excretion of 14C-labeled teroxalene hydrochloride in experimental animals and man
TOXICOLOGY
ANI)
APPLIED
PHARMACOLOGY
Absorption
and
Excretion
Hydrochloride JONATHAN
P.
Department
in
MILLER,
9, 455-462
EARL
of Pharmacology,
(1966)
of
‘“C-Labeled
Experimental V.
Animals
CARDINAL,
Abbott Received
AND
Laboratories, May
Teroxalene
16,
and
L. E. North
MICHAEL
Chicago,
Man CRAWFORU
Illinois
60064
1966
Teroxalene hydrochloride is a disubstituted piperazine effective orally in laboratory animals against Schistosoma mansoni (Bauman et al., 1964; Kimura et al., 1966). Previous studies on it were reported in this journal under the name of ABBOTT-16612 (Miller et al., 1966). The present data supplement the previous studies and include data obtained in schistosome-infected humans. This compound is l- (3-chloro-p-tolyl) -4- [ 6- (p-tert-pentylphenoxy) hexyl] piperazine hydrochloride, and the position of labeling with carbon-14 is shown in Fig. 1.
/ -x3FIG.
label
1. Structural in the sample
formula of teroxalene hydrochloride used in the present work.
For this report upon the recovery
\
Cl::H, \ CHs
-
showing
the
position
. HCI
of the
14C-labeled drug was used, and all the data reported of carbon-14 per se.
carbon-14
are based
METHODS
14C-Labeled teroxalene hydrochloride was administered orally to the experimental animals in a 0.3% tragacanth suspension and to humans as an encapsulated powder. Duplicate or triplicate radioassays were made on all samples by using a modified version of the Schijniger combustion procedure described by Oliverio et al. (1962). Blood, urine, and bile were assayed directly, and all tissues and feces were first homogenized. Homogenization was carried out in an instrument1 running at approximately 8000 rpm for 20 minutes (with cooling). Tissue and feces were homogenized in approximately 80% ethanol and aliquots of homogenates were pipetted into the combustion bags. Dog and monkey carcasses and skins were digested by refluxing in 6 N HCl followed by extraction of the supernatant fat with petroleum ether. Radioassay values on most of the fecal samples and many other samples were checked using other radioassay procedures, i.e., direct liquid scintillation counting and modification of the liquid combustion procedure described by Smith et al. (1964). 1 Sorvall
Omni-Mixer,
Ivan
Sorvall
Inc.,
Norwalk, 455
Connecticut.
456
JONATHAN
P.
MILLER
ET
AL.
RESULTS
Absorption
Studies
BZood ZeveEs.Groups of three female CFl mice were each administered tragacanth suspensionsof 14C-labeledteroxalene hydrochloride at dosagesof lo-50 mg/kg (2.4 PC). These groups were bled by heart puncture at 1 and 4 hours postadministration, and plasma levels of drug were determined. Considering the potential biologic variability, the relationship of dosageto plasma level (Table 1) is remarkably linear, and there is no doubt that the blood levels inEFFECT OF
DOSE
ON
TABLE 1 PLASMA RADIOACTIVITY AT 1 AND OF 14c-LABEL~0
TEROXALENE
4
HOURS AFTER
HYDROCHLORIDE
THE
ORAL ADMINISTRATION
T O MICE
Plasma concentrationa
(pg/ml)
Dosage (mg/kg)
1 hour
4 hours
10
1.94
1.24
20 30 40
2.64 3.24 5.68 5.30
1.88 3.24 4.24 5.80
50
0 Each value on plasma concentration was obtained from a pooled sample from three mice. Results are expressed as teroxalene hydrochloride calculated from measurement of carbon-14.
creaseas the dosageof drug is increased. Blood levels at 1 hour are rapidly changing so that these results are somewhat more erratic than the values obtained at 4 hours. Total absorption. Eight male Holtzman rats, weighing approximately 200 g each, were administered 1 mg/kg of teroxalene hydrochloride in tragacanth suspension.At hourly intervals one rat was sacrificed and the entire gastrointestinal tract and contents were removed. Radioactivity was then assayedin this sampleplus excreted feces. The remaining carcass plus excreted urine were also assayed as a measure of absorbed dose. The drug found in carcass and urine was defined as “absorbed.” Due to known high biliary excretion (Miller et al., 1966) with resultant excretion back into the gastrointestinal tract, this is a minimum value and actual absorption may be even somewhat greater. The results shown in Table 2 indicate that at least 20-25s of an oral dose of teroxalene hydrochloride is absorbed in rats. Excretion and Distribution Studies Monkey. A 2.73-kg male monkey received 10 mg/kg of teroxalene hydrochloride containing 222 uc of radioactivity. Total excreta were collected for 23 days; at the end of this time the animal was sacrificed and complete drug distribution studies were carried out. The results are shown in Tables 3 and 4. Dog. A 4.3-kg female dog received 9.74 mg/kg of teroxalene hydrochloride containing 122 uc of radioactivity. Total excreta were collected for 30 days at which time the animal was sacrificed and complete drug distribution studies were carried out. The results are shown in Tables 5 and 6.
METABOLISM
ABSORPTION
0~ ORALLY
OF
14c
A~JMINI~TERED DOSAGE
TEROXALENE
TABLE 2 14C-LABELED OF 1 MG/KG
“Not-absorbed” (Digestive tract f feces)
457
HYDROCHLORIDE
TEROXALENE
HYDROCHLORIDE
AFTER A
TO RATS
“Absorbed” (carcass + urine)
Recovery
(5%)”
(%)a
(%I”
1
79.2
23.9
103.1
2
62.6
3
82.8 75.2
26.7 18.4
89.3 101.2
19.3
94.5
17.5 24.3
96.7 93.1
21.4
Time (hr)
4 5
79.2
6 7
68.8 76.6
8
69.5
23.7
98.0 93.2
74.2
21.9
96.1
Average a As percentage
EXCRETION
of the dose. TABLE 3 OF RADIOACTIVITW AFTER A SINGLE ORAL DOSE OF 10 MG/KG TEROXALENE HY~ROCHLOR~E TO A MONKEY
Time
Urine
(days)
(%)a
0.78
4.79
1.76 2.72
2.50 0.71
.3.70
0.32 0.14
4.81
OF r4C-LAn~L~o Feces (%)” 1.81 36.95 25.05 4.17 1.47
5.77
0.06
0.68
6.72 7.72
0.05 0.05
0.36 0.42
8.72
0.04
0.21
9.72 12.79
0.03
0.07
0.15 0.29
15.75
0.06
0.21
18.77
0.05
0.15
22.20 23.35
0.02 0.03
0.12 0.08
Total
8.92
72.12
a As percentage
of the dose.
Human. The patients studied were male in-patients who had a positive diagnosis of schistosomiasis by both rectal and liver biopsy as well as parasitologic examination. One patient (M.M.) weighed 56 kg and the other (H.) weighed 57 kg. Both patients were orally administered 10 mg/kg (40 ,uc) of encapsulated 14C-labeled teroxalene hydrochloride. Blood samples were then taken periodically and total excreta were collected for approximately 7 days. The results are shown in Tables 7 and 8 and in Fig. 2. DISCUSSION
Previous studies on the elimination of teroxalene hydrochloride (Miller et al., 1966) indicated a rather long-lived secondary drug elimination component. This caused
458
JONATHAN
P.
MILLER
TABLE DISTRIBUTION
OF RADIOACTIWTY OF 10 MC/KG
IN TISSUES
ET
4
OF A MONKEY
OF 14C-LABELED
AL.
23 Days
TEROXALENE Total
Organ
in organ C%)”
Brain Liver Kidneys Lungs Heart Spleen Gonads Gastrointestinal tract Skin Gall bladder with contents Bone marrow Whole blood Muscle (average of 6 sites) Adipose tissue (average of 6 sites) 1. Total attached t.o skin 2. Total in remaining carcass Remaining carcass Excreta
0.66 81.04
Total
86.18
recovery
DOSE
ORAL
Per
gram of tissue c%,n
-
O.lfO 0.021 0.016
0.009 0.003 0.003 0.496 0.160
-
0.018 -
0.009 0.013 0.00033 0.00045 o.cil42
1.87 1.55
-
of the dose. TABLE
EXCRETiON
A SINGLE
0.026
of dose
fl As percentage
AFTER
HYDROCHLOR~E
OF RADIOACTIVITY
AFTER TEROXALENE
Time (days)
A SINGLE
5 ORAL
HYDROCHLORIDE
Urine (%)a
Dose
OF 9.74
TO A DOG
MC/KG
OF l”C-LABELED
-___ Feces (%I”
1 .o 1.23 2 .o 3.0 4.0 5.0 6.0
6.3 1.15 0.20 0.09 0.07 0.04
74.46 2.96 6.63 0.78 0.21 -
7.0 9.0
0.08
0.69 -
10.0 12.0 15.0
0.05 0.04
17.0
0.015
20.0 23.0 26.0
0.023 0.021
0.26 0.12 0.13 0.076 0.089 0.087
0.018
0.045
29.0
0.015
30.0
0.010 8.352
0.070 0.019
Total a As percentage
of the dose.
86.626
METABOLISM
OF
14c
TEROXALENE
TABLE DISTRIBUTION
OF RADIOACTIVITY 9.74
M&KG
IN TISSUES OF 1W-LABELED
6
OF A Doe
30 DAYS
TEROXALENE
AFTER
0.461 94.978
Total
96.12
recovery
of dose
ORAL
DOSE
OP
in organ (70)”
Brain Liver Kidneys Lungs Heart Spleen Gonads Gastrointestinal tract Skin Bile Bone marrow \Vhole blood Muscle (average of 7 sites) Adipose tissue (average of 6 sites) 1. Total in remaining carcass Remaining carcass Excreta
‘& ils percentage
A SINGLE
HYDROCHLORIDE
Total Organ
459
HYDROCHLORIDE
0.005
0.092 0.008 C.006 0.011 0.002
0.0004 0.167
-
O.OOOJ 0.00004
0.006 0.00005 0.00024 0.0012
of the dose.
concern, and it was further investigated. Our final results indicate that there is no such long-lived component and our previous results arose from radioassay difficulties with fecal samples. These difficulties generally fall into two categories: (1) problems associated with representative sampling of fecal homogenates, and (2) problems associated with Schiiniger combustion of fecal samples. The first was completely eliminated by more vigorous homogenization in the presence of a mixture of water and alcohol. The latter was largely solved by decreasing the amount of total dry solids to be combusted to approximately 30-60 mg. The Schijniger combustion procedure is generally capable of handling larger samples, but evidently fecal samples of teroxalene hydrochloride, or its metabolites, can be difficult to combust because of either physical or chemical properties. This problem was also investigated using other radioassay techniques such as direct liquid scintillation counting of homogenates and “wetcombustion” procedures (Smith et al., 1964). These results were entirely compatible with those obtained using the Schiiniger technique. Using the above radioassay techniques, teroxalene hydrochloride, or its metabolites, was found to be nearly completely eliminated in the human, as well as in experimental animals, within a relatively short time. The approximate biologic half-life in the human is 10 hours (Fig. 2), and there appears to be much less than 0.1% of the dose remaining in the body within 3-4 days after a single oral dose of the drug. As might be expected from such a nonpolar compound, there seems to be a general tendency for this drug to gradually migrate to adipose tissue. This tendency is reflected in the relatively high concentrations of radioactivity found in adipose tissue, as compared to other tissues, after several weeks (Tables 4 and 6). In spite of the
460
JONATHAN
P.
MILLER
TABLE EXCRETION
OF RADIOACTIVITY
AFTER
TEROXALENE
Urinea (%b)b
Time 0-x) 22.25
DOSE OF 10 MG/KG
ORAL
TO Two
HYDROCHLORIDE
Patient
AL.
7
A SINGLE
OF I%-LABELED
PATIENTS
M.M.
Patient
2.25
23.25 47.00 48.75
ET
o.so
53.75
H.
Feces (%)h
Urinea (%b)b
Feces (%b)b
40.09 -
2.08
-
-
6.87 71.52
43.87
0.67
-
10.32 -
71.25 75.00
0.35
3.16
119.25
-
0.66 -
119.75 142.50
0.30
0.21
93.25
0.35
16.39 -
-
3.48
143 .oo 155.20
-
0.45 -
0.30 -
0.10
-
0.20
Total
3 20
98.76
3.60
a Urine values b As percentage
represent accumulated of the dose.
totals
during
TABLE BLOOD
LEVELS
OP RADIOACTIVITY
14C-LABELED
TIZROXALENE
Patient Time (hr)
AFTER
cited
time
3.70 0.65 102.61
intervals.
8 A SINGLE
HYDROCHLORIDE
ORAL
DOSE
TO Two
OF 10 MG/I(G
Patient Packed cells (%P
Serum (%I”
OF
PATIENTS
M.M.
Packed cells (k)”
-
H.
Serum (%)”
2
0.0024
0.0073
0.0047
0.016
4 8
0.033 0.011
0.093 0.057
0.0136
0.056
12 24
0.010 0.0059
0.048 0.030
0.0088 0.0120 0.0065
0.038 0.033 0.023
48 72
0.0038 0.0034
0.017 0.0096
0.0070 0.0061
0.019 0.014
96 120
0.0031
0.0049 0.005 I
0.0066 0.0058
0.010 0.007
144 168
-
0.0029 0.0014
0.0047 0.0060
0.006 0.005
a As percentage
of the dose per 100
ml
of packed
red cells or serum.
drug being so nonpolar and water insoluble, there seems to be a significant amount, about 20%, of radioactivity absorbed within even 1 hour post-administration (Table 2). From biliary concentrations it is evident that a significant amount of drug undervalues cited in Table 2 are goes enterohepatic circulation so that the “absorbed” probably low. Studies in mice (Table 1) indicate that the plasma concentration of drug increases with dose, reaching a value of 5-6 pg/ml at a dosage of 50 mg/kg. As shown in previous animal studies (Miller EL al., 1966), serum levels peak at about 4 hours post-administration and reach levels in the human of 0.06-0.097, of
METABOLISM
OF
14c
TEROXALENE
361
HYDROCHLORIDE
the dose per 100 ml (Table 8). This would equal a serum drug level of approximately 4 mg/lOO ml in a SO-kg man at a dosage of 10 mg/kg. This serum drug level does not show any great decrease for almost 24 hours, after which time the concentration drups rather rapidly. It would appear that serum radioactivity levels remain relatively high during the transit time of teroxalene hydrochloride through the gut, i.e., the biological half-life of drug radioactivity is about 10 hours. It is interesting that significant amounts of radioactivity seem to be associated with the packed red blood ESTIMATED
BIOLOGICAL
HALF-LIFE
-
PATIENT
M.M.
---
FUTIENT
Ii.
= IO Hrr.
\ \ \ \
\ \ \ \ \
2
\ \ I 24
I 48
HOURS FIG. 2. Rate of excretion single oral dose of 10 mg/kg tion shown in Table 7.
of 1%labeled (40 UC). These
I” 72
I 96
(01 120
POST-ADMINISTRATION
teroxalene hydrochloride in humans following a results are based on the determination of total excre-
cells since these values are approximately one-third of the serum values at the 4-hour peak. The excretory pattern of radioactivity after a single oral dose of 14C-labeled teroxalene hydrochloride is similar in the dog and monkey (Tables 3 and 5). Within 2 days post-administration, approximately 60-8070 of the dose is excreted in the feces and 6-7s in the urine. The amount of radioactivity excreted in human urine was approximately one-half that found in the dog and monkey, and this may reflect lower absorption due to a difference in the dosage form (encapsulated powder VS. tragacanth suspension).
462
JONATHAN
P.
MILLER
ET
AL.
As would be expected, tissue concentrations of radioactivity are very low in experimental animals 23-30 days after administration (Tables 4 and 6). The gastrointestinal tract and contents contained many times as much radioactivity as other tissues,and this may be related to prolonged excretion via the biliary route. SUMMARY The absorption of I%-labe!ed teroxalene hydrochloride was studied in mice and rats, and the distribution and excretion of the drug was studied in one dog, one monkey, and two schistosomeinfected humans. All subjects received a single oral dose. Serum drug levels of radioactivity are linearly related to dose up to 50mg/‘kg, and at least 20% of an orally administered dose appears to be absorbed as evidenced by studies in mice and rats. Peak blood levels occur at approximately 4 hours after oral administration of the drug. There is some indication, as evidenced by urinary excretion, that total absorption can be influenced by the dosage form used for oral administration. The radioactive drug, or its metabolites, appears to be nearly completely eliminated, in a linear fashion, with almost all of the dose appearing in the feces and 4-8s excreted in the urine. The approximate biological half-life of the drug is 10 hours in the human. Tissue distribution studies in the dog and monkey show very low drug concentrations 23-30 days after administration with a tendency for migration of the radioactive teroxalene, or its metabolites, to adipose tissue. At the time of sacrifice, the gastrointestinal tract and its contents contained several orders of magnitude more radioactivity than any of the other tissues except adipose tissue. ACKNOWLEDGMENTS We gratefully acknowledge the valuable assistance and contributions by Dr. R. L. Herting and Miss Grace Dillon of Cook County Hospital, Chicago, Illinois, during the human studies. We also acknowledge the valuable technical assistance of Messrs. Carleton Pierce and Jerry Netwal as well as that of Miss Ann Dalziel. We are especially indebted to Mr. .4rthur Alter, radiochemist, for the actual synthesis and labeling of this compound. REFERENCES BAUMAN, P. M., GEISZLER, A. O., and OTTO, G. F. (1964). Antischistosome activity of N-(3chloro-4-methylphenyl) -N’- (4’+amylphenoxyhexamethylene) piperazine and analogs against laboratory infections of Schistosoma mansoni. Presented at Thirteenth Annual Meeting, The American Society of Tropical Medicine and Hygiene, November 4-7, New York City. KIMURA, E. T., RICHARDS, R. K., EBERT, D. M., YOUNG, P. R., and BAUMAN, P. M. (1966). Species differences in the toxicity of the schistosomicide .4BBOTT-16612. Tosicol. Appt. Pharmacol. 8, 57-63. MILLER, j. P., ALTER, A., CARDINAL. E. V., DALZIEL, A., and BAUMAN, P. M. (1966). Distribution and excretion of C14-labeled ABBOTT-16612 in experimental animals. Toxicol. .4ppZ. Pharmacol. 8, 295-305. OLIVERIO, V. T., DENHAM, C., and DAVIDSON, J. D. (1962). Oxygen flask combustion in determination of Cl” and Ha in biological materials. Anal. Biochem. 4, 188189. SMITH, G. N., LUDWIG, P. D., WRIGHT, K. C., and BAURIEDEL, W. R. (1964). Simple apparatus pesticides for residue analysis. J. Agr. Food for combustion of samples containin, rr Cl*-iabeled Chem. 12. 172-175.
ANI)
APPLIED
PHARMACOLOGY
Absorption
and
Excretion
Hydrochloride JONATHAN
P.
Department
in
MILLER,
9, 455-462
EARL
of Pharmacology,
(1966)
of
‘“C-Labeled
Experimental V.
Animals
CARDINAL,
Abbott Received
AND
Laboratories, May
Teroxalene
16,
and
L. E. North
MICHAEL
Chicago,
Man CRAWFORU
Illinois
60064
1966
Teroxalene hydrochloride is a disubstituted piperazine effective orally in laboratory animals against Schistosoma mansoni (Bauman et al., 1964; Kimura et al., 1966). Previous studies on it were reported in this journal under the name of ABBOTT-16612 (Miller et al., 1966). The present data supplement the previous studies and include data obtained in schistosome-infected humans. This compound is l- (3-chloro-p-tolyl) -4- [ 6- (p-tert-pentylphenoxy) hexyl] piperazine hydrochloride, and the position of labeling with carbon-14 is shown in Fig. 1.
/ -x3FIG.
label
1. Structural in the sample
formula of teroxalene hydrochloride used in the present work.
For this report upon the recovery
\
Cl::H, \ CHs
-
showing
the
position
. HCI
of the
14C-labeled drug was used, and all the data reported of carbon-14 per se.
carbon-14
are based
METHODS
14C-Labeled teroxalene hydrochloride was administered orally to the experimental animals in a 0.3% tragacanth suspension and to humans as an encapsulated powder. Duplicate or triplicate radioassays were made on all samples by using a modified version of the Schijniger combustion procedure described by Oliverio et al. (1962). Blood, urine, and bile were assayed directly, and all tissues and feces were first homogenized. Homogenization was carried out in an instrument1 running at approximately 8000 rpm for 20 minutes (with cooling). Tissue and feces were homogenized in approximately 80% ethanol and aliquots of homogenates were pipetted into the combustion bags. Dog and monkey carcasses and skins were digested by refluxing in 6 N HCl followed by extraction of the supernatant fat with petroleum ether. Radioassay values on most of the fecal samples and many other samples were checked using other radioassay procedures, i.e., direct liquid scintillation counting and modification of the liquid combustion procedure described by Smith et al. (1964). 1 Sorvall
Omni-Mixer,
Ivan
Sorvall
Inc.,
Norwalk, 455
Connecticut.
456
JONATHAN
P.
MILLER
ET
AL.
RESULTS
Absorption
Studies
BZood ZeveEs.Groups of three female CFl mice were each administered tragacanth suspensionsof 14C-labeledteroxalene hydrochloride at dosagesof lo-50 mg/kg (2.4 PC). These groups were bled by heart puncture at 1 and 4 hours postadministration, and plasma levels of drug were determined. Considering the potential biologic variability, the relationship of dosageto plasma level (Table 1) is remarkably linear, and there is no doubt that the blood levels inEFFECT OF
DOSE
ON
TABLE 1 PLASMA RADIOACTIVITY AT 1 AND OF 14c-LABEL~0
TEROXALENE
4
HOURS AFTER
HYDROCHLORIDE
THE
ORAL ADMINISTRATION
T O MICE
Plasma concentrationa
(pg/ml)
Dosage (mg/kg)
1 hour
4 hours
10
1.94
1.24
20 30 40
2.64 3.24 5.68 5.30
1.88 3.24 4.24 5.80
50
0 Each value on plasma concentration was obtained from a pooled sample from three mice. Results are expressed as teroxalene hydrochloride calculated from measurement of carbon-14.
creaseas the dosageof drug is increased. Blood levels at 1 hour are rapidly changing so that these results are somewhat more erratic than the values obtained at 4 hours. Total absorption. Eight male Holtzman rats, weighing approximately 200 g each, were administered 1 mg/kg of teroxalene hydrochloride in tragacanth suspension.At hourly intervals one rat was sacrificed and the entire gastrointestinal tract and contents were removed. Radioactivity was then assayedin this sampleplus excreted feces. The remaining carcass plus excreted urine were also assayed as a measure of absorbed dose. The drug found in carcass and urine was defined as “absorbed.” Due to known high biliary excretion (Miller et al., 1966) with resultant excretion back into the gastrointestinal tract, this is a minimum value and actual absorption may be even somewhat greater. The results shown in Table 2 indicate that at least 20-25s of an oral dose of teroxalene hydrochloride is absorbed in rats. Excretion and Distribution Studies Monkey. A 2.73-kg male monkey received 10 mg/kg of teroxalene hydrochloride containing 222 uc of radioactivity. Total excreta were collected for 23 days; at the end of this time the animal was sacrificed and complete drug distribution studies were carried out. The results are shown in Tables 3 and 4. Dog. A 4.3-kg female dog received 9.74 mg/kg of teroxalene hydrochloride containing 122 uc of radioactivity. Total excreta were collected for 30 days at which time the animal was sacrificed and complete drug distribution studies were carried out. The results are shown in Tables 5 and 6.
METABOLISM
ABSORPTION
0~ ORALLY
OF
14c
A~JMINI~TERED DOSAGE
TEROXALENE
TABLE 2 14C-LABELED OF 1 MG/KG
“Not-absorbed” (Digestive tract f feces)
457
HYDROCHLORIDE
TEROXALENE
HYDROCHLORIDE
AFTER A
TO RATS
“Absorbed” (carcass + urine)
Recovery
(5%)”
(%)a
(%I”
1
79.2
23.9
103.1
2
62.6
3
82.8 75.2
26.7 18.4
89.3 101.2
19.3
94.5
17.5 24.3
96.7 93.1
21.4
Time (hr)
4 5
79.2
6 7
68.8 76.6
8
69.5
23.7
98.0 93.2
74.2
21.9
96.1
Average a As percentage
EXCRETION
of the dose. TABLE 3 OF RADIOACTIVITW AFTER A SINGLE ORAL DOSE OF 10 MG/KG TEROXALENE HY~ROCHLOR~E TO A MONKEY
Time
Urine
(days)
(%)a
0.78
4.79
1.76 2.72
2.50 0.71
.3.70
0.32 0.14
4.81
OF r4C-LAn~L~o Feces (%)” 1.81 36.95 25.05 4.17 1.47
5.77
0.06
0.68
6.72 7.72
0.05 0.05
0.36 0.42
8.72
0.04
0.21
9.72 12.79
0.03
0.07
0.15 0.29
15.75
0.06
0.21
18.77
0.05
0.15
22.20 23.35
0.02 0.03
0.12 0.08
Total
8.92
72.12
a As percentage
of the dose.
Human. The patients studied were male in-patients who had a positive diagnosis of schistosomiasis by both rectal and liver biopsy as well as parasitologic examination. One patient (M.M.) weighed 56 kg and the other (H.) weighed 57 kg. Both patients were orally administered 10 mg/kg (40 ,uc) of encapsulated 14C-labeled teroxalene hydrochloride. Blood samples were then taken periodically and total excreta were collected for approximately 7 days. The results are shown in Tables 7 and 8 and in Fig. 2. DISCUSSION
Previous studies on the elimination of teroxalene hydrochloride (Miller et al., 1966) indicated a rather long-lived secondary drug elimination component. This caused
458
JONATHAN
P.
MILLER
TABLE DISTRIBUTION
OF RADIOACTIWTY OF 10 MC/KG
IN TISSUES
ET
4
OF A MONKEY
OF 14C-LABELED
AL.
23 Days
TEROXALENE Total
Organ
in organ C%)”
Brain Liver Kidneys Lungs Heart Spleen Gonads Gastrointestinal tract Skin Gall bladder with contents Bone marrow Whole blood Muscle (average of 6 sites) Adipose tissue (average of 6 sites) 1. Total attached t.o skin 2. Total in remaining carcass Remaining carcass Excreta
0.66 81.04
Total
86.18
recovery
DOSE
ORAL
Per
gram of tissue c%,n
-
O.lfO 0.021 0.016
0.009 0.003 0.003 0.496 0.160
-
0.018 -
0.009 0.013 0.00033 0.00045 o.cil42
1.87 1.55
-
of the dose. TABLE
EXCRETiON
A SINGLE
0.026
of dose
fl As percentage
AFTER
HYDROCHLOR~E
OF RADIOACTIVITY
AFTER TEROXALENE
Time (days)
A SINGLE
5 ORAL
HYDROCHLORIDE
Urine (%)a
Dose
OF 9.74
TO A DOG
MC/KG
OF l”C-LABELED
-___ Feces (%I”
1 .o 1.23 2 .o 3.0 4.0 5.0 6.0
6.3 1.15 0.20 0.09 0.07 0.04
74.46 2.96 6.63 0.78 0.21 -
7.0 9.0
0.08
0.69 -
10.0 12.0 15.0
0.05 0.04
17.0
0.015
20.0 23.0 26.0
0.023 0.021
0.26 0.12 0.13 0.076 0.089 0.087
0.018
0.045
29.0
0.015
30.0
0.010 8.352
0.070 0.019
Total a As percentage
of the dose.
86.626
METABOLISM
OF
14c
TEROXALENE
TABLE DISTRIBUTION
OF RADIOACTIVITY 9.74
M&KG
IN TISSUES OF 1W-LABELED
6
OF A Doe
30 DAYS
TEROXALENE
AFTER
0.461 94.978
Total
96.12
recovery
of dose
ORAL
DOSE
OP
in organ (70)”
Brain Liver Kidneys Lungs Heart Spleen Gonads Gastrointestinal tract Skin Bile Bone marrow \Vhole blood Muscle (average of 7 sites) Adipose tissue (average of 6 sites) 1. Total in remaining carcass Remaining carcass Excreta
‘& ils percentage
A SINGLE
HYDROCHLORIDE
Total Organ
459
HYDROCHLORIDE
0.005
0.092 0.008 C.006 0.011 0.002
0.0004 0.167
-
O.OOOJ 0.00004
0.006 0.00005 0.00024 0.0012
of the dose.
concern, and it was further investigated. Our final results indicate that there is no such long-lived component and our previous results arose from radioassay difficulties with fecal samples. These difficulties generally fall into two categories: (1) problems associated with representative sampling of fecal homogenates, and (2) problems associated with Schiiniger combustion of fecal samples. The first was completely eliminated by more vigorous homogenization in the presence of a mixture of water and alcohol. The latter was largely solved by decreasing the amount of total dry solids to be combusted to approximately 30-60 mg. The Schijniger combustion procedure is generally capable of handling larger samples, but evidently fecal samples of teroxalene hydrochloride, or its metabolites, can be difficult to combust because of either physical or chemical properties. This problem was also investigated using other radioassay techniques such as direct liquid scintillation counting of homogenates and “wetcombustion” procedures (Smith et al., 1964). These results were entirely compatible with those obtained using the Schiiniger technique. Using the above radioassay techniques, teroxalene hydrochloride, or its metabolites, was found to be nearly completely eliminated in the human, as well as in experimental animals, within a relatively short time. The approximate biologic half-life in the human is 10 hours (Fig. 2), and there appears to be much less than 0.1% of the dose remaining in the body within 3-4 days after a single oral dose of the drug. As might be expected from such a nonpolar compound, there seems to be a general tendency for this drug to gradually migrate to adipose tissue. This tendency is reflected in the relatively high concentrations of radioactivity found in adipose tissue, as compared to other tissues, after several weeks (Tables 4 and 6). In spite of the
460
JONATHAN
P.
MILLER
TABLE EXCRETION
OF RADIOACTIVITY
AFTER
TEROXALENE
Urinea (%b)b
Time 0-x) 22.25
DOSE OF 10 MG/KG
ORAL
TO Two
HYDROCHLORIDE
Patient
AL.
7
A SINGLE
OF I%-LABELED
PATIENTS
M.M.
Patient
2.25
23.25 47.00 48.75
ET
o.so
53.75
H.
Feces (%)h
Urinea (%b)b
Feces (%b)b
40.09 -
2.08
-
-
6.87 71.52
43.87
0.67
-
10.32 -
71.25 75.00
0.35
3.16
119.25
-
0.66 -
119.75 142.50
0.30
0.21
93.25
0.35
16.39 -
-
3.48
143 .oo 155.20
-
0.45 -
0.30 -
0.10
-
0.20
Total
3 20
98.76
3.60
a Urine values b As percentage
represent accumulated of the dose.
totals
during
TABLE BLOOD
LEVELS
OP RADIOACTIVITY
14C-LABELED
TIZROXALENE
Patient Time (hr)
AFTER
cited
time
3.70 0.65 102.61
intervals.
8 A SINGLE
HYDROCHLORIDE
ORAL
DOSE
TO Two
OF 10 MG/I(G
Patient Packed cells (%P
Serum (%I”
OF
PATIENTS
M.M.
Packed cells (k)”
-
H.
Serum (%)”
2
0.0024
0.0073
0.0047
0.016
4 8
0.033 0.011
0.093 0.057
0.0136
0.056
12 24
0.010 0.0059
0.048 0.030
0.0088 0.0120 0.0065
0.038 0.033 0.023
48 72
0.0038 0.0034
0.017 0.0096
0.0070 0.0061
0.019 0.014
96 120
0.0031
0.0049 0.005 I
0.0066 0.0058
0.010 0.007
144 168
-
0.0029 0.0014
0.0047 0.0060
0.006 0.005
a As percentage
of the dose per 100
ml
of packed
red cells or serum.
drug being so nonpolar and water insoluble, there seems to be a significant amount, about 20%, of radioactivity absorbed within even 1 hour post-administration (Table 2). From biliary concentrations it is evident that a significant amount of drug undervalues cited in Table 2 are goes enterohepatic circulation so that the “absorbed” probably low. Studies in mice (Table 1) indicate that the plasma concentration of drug increases with dose, reaching a value of 5-6 pg/ml at a dosage of 50 mg/kg. As shown in previous animal studies (Miller EL al., 1966), serum levels peak at about 4 hours post-administration and reach levels in the human of 0.06-0.097, of
METABOLISM
OF
14c
TEROXALENE
361
HYDROCHLORIDE
the dose per 100 ml (Table 8). This would equal a serum drug level of approximately 4 mg/lOO ml in a SO-kg man at a dosage of 10 mg/kg. This serum drug level does not show any great decrease for almost 24 hours, after which time the concentration drups rather rapidly. It would appear that serum radioactivity levels remain relatively high during the transit time of teroxalene hydrochloride through the gut, i.e., the biological half-life of drug radioactivity is about 10 hours. It is interesting that significant amounts of radioactivity seem to be associated with the packed red blood ESTIMATED
BIOLOGICAL
HALF-LIFE
-
PATIENT
M.M.
---
FUTIENT
Ii.
= IO Hrr.
\ \ \ \
\ \ \ \ \
2
\ \ I 24
I 48
HOURS FIG. 2. Rate of excretion single oral dose of 10 mg/kg tion shown in Table 7.
of 1%labeled (40 UC). These
I” 72
I 96
(01 120
POST-ADMINISTRATION
teroxalene hydrochloride in humans following a results are based on the determination of total excre-
cells since these values are approximately one-third of the serum values at the 4-hour peak. The excretory pattern of radioactivity after a single oral dose of 14C-labeled teroxalene hydrochloride is similar in the dog and monkey (Tables 3 and 5). Within 2 days post-administration, approximately 60-8070 of the dose is excreted in the feces and 6-7s in the urine. The amount of radioactivity excreted in human urine was approximately one-half that found in the dog and monkey, and this may reflect lower absorption due to a difference in the dosage form (encapsulated powder VS. tragacanth suspension).
462
JONATHAN
P.
MILLER
ET
AL.
As would be expected, tissue concentrations of radioactivity are very low in experimental animals 23-30 days after administration (Tables 4 and 6). The gastrointestinal tract and contents contained many times as much radioactivity as other tissues,and this may be related to prolonged excretion via the biliary route. SUMMARY The absorption of I%-labe!ed teroxalene hydrochloride was studied in mice and rats, and the distribution and excretion of the drug was studied in one dog, one monkey, and two schistosomeinfected humans. All subjects received a single oral dose. Serum drug levels of radioactivity are linearly related to dose up to 50mg/‘kg, and at least 20% of an orally administered dose appears to be absorbed as evidenced by studies in mice and rats. Peak blood levels occur at approximately 4 hours after oral administration of the drug. There is some indication, as evidenced by urinary excretion, that total absorption can be influenced by the dosage form used for oral administration. The radioactive drug, or its metabolites, appears to be nearly completely eliminated, in a linear fashion, with almost all of the dose appearing in the feces and 4-8s excreted in the urine. The approximate biological half-life of the drug is 10 hours in the human. Tissue distribution studies in the dog and monkey show very low drug concentrations 23-30 days after administration with a tendency for migration of the radioactive teroxalene, or its metabolites, to adipose tissue. At the time of sacrifice, the gastrointestinal tract and its contents contained several orders of magnitude more radioactivity than any of the other tissues except adipose tissue. ACKNOWLEDGMENTS We gratefully acknowledge the valuable assistance and contributions by Dr. R. L. Herting and Miss Grace Dillon of Cook County Hospital, Chicago, Illinois, during the human studies. We also acknowledge the valuable technical assistance of Messrs. Carleton Pierce and Jerry Netwal as well as that of Miss Ann Dalziel. We are especially indebted to Mr. .4rthur Alter, radiochemist, for the actual synthesis and labeling of this compound. REFERENCES BAUMAN, P. M., GEISZLER, A. O., and OTTO, G. F. (1964). Antischistosome activity of N-(3chloro-4-methylphenyl) -N’- (4’+amylphenoxyhexamethylene) piperazine and analogs against laboratory infections of Schistosoma mansoni. Presented at Thirteenth Annual Meeting, The American Society of Tropical Medicine and Hygiene, November 4-7, New York City. KIMURA, E. T., RICHARDS, R. K., EBERT, D. M., YOUNG, P. R., and BAUMAN, P. M. (1966). Species differences in the toxicity of the schistosomicide .4BBOTT-16612. Tosicol. Appt. Pharmacol. 8, 57-63. MILLER, j. P., ALTER, A., CARDINAL. E. V., DALZIEL, A., and BAUMAN, P. M. (1966). Distribution and excretion of C14-labeled ABBOTT-16612 in experimental animals. Toxicol. .4ppZ. Pharmacol. 8, 295-305. OLIVERIO, V. T., DENHAM, C., and DAVIDSON, J. D. (1962). Oxygen flask combustion in determination of Cl” and Ha in biological materials. Anal. Biochem. 4, 188189. SMITH, G. N., LUDWIG, P. D., WRIGHT, K. C., and BAURIEDEL, W. R. (1964). Simple apparatus pesticides for residue analysis. J. Agr. Food for combustion of samples containin, rr Cl*-iabeled Chem. 12. 172-175.