The metabolism and disposition of 3,4,4′-trichlorocarbanilide in the intact and bile duct-cannulated adult and in the newborn rhesus monkey (M. mulatta)

TOXICOLOGY

AND

APPLIED

46,593~608 (1978)

PHARMACOLOGY

The Metabolism and Disposition of 3,4,4’Trichlorocarbanilide in the Intact and Bile Duct-Cannulated Adult and in the Newborn Rhesus Monkey (M. mulatfa) R. A. HILES,’ The Procter

D. CAUDILL,

& Gamble Received

Company,

Miami

November

C. G. BIRCH, AND T. EICHHOLD Valley Laboratories,

24,1977;

accepted

May

Cincinnati,

Ohio 45247

22,1978

The Metabolism and Disposition of 3,4,4’-Trichlorocarbanilide in the Intact and Bile-DuctCannulated Adult and in the Newborn Rhesus Monkey (M. mulatta). HILES, R. A., CAUDILL, D.,

BIRCH,

C. G.,

AND

EICHHOLD,

T. (1978).

Toxicol.

Appl.

Pharmacol.

46, 593-608.

The

metabolism and disposition of intravenously infused radioactive 3,4,4’-trichloro[Wlcarbanilide (TCC) in the adult and newborn rhesus monkey have been evaluated. In adult animals the major metabolic reactions were N-glucuronide formation or ring hydroxylation followed by conjugation to glucuronic acid or sulfuric acid. Removal of 14Cfrom the plasma was biphasic; TCC and the N-glucuronides accounted for the fast phase, and the O-sulfate conjugates accounted for the slow phase. The major urinary metabolites were the N-glucuronides of TCC. The tissue residue of i4C was low in the monkeys and was limited primarily to tissues that are active in drug metabolism (liver, kidneys, and lungs). The bile was the major route of elimination with glucuronide conjugates as the major radioactive component. Enterohepatic circulation was extensive but did not affect the plasma concentrations or the elimination kinetics of TCC-derived material from the plasma. The newborn monkey also metabolized TCC by ring hydroxylation or N-glucuronidation. The plasma kinetics were similar to those observed in adults as was the tissue distribution. Unlike the.adult, there were only very low amounts of Oglucuronides and, instead, high amounts of O-sulfate conjugates. It is concluded that the infant monkey can readily metabolize and eliminate TCC.

The antibacterial agent 3,4,4’-trichlorocarbanilide (TCC) is used in toilet soaps and other cleaning agents. The primary biotransformation pathway for TCC in rats, adult monkeys, and humans is ring hydroxylation followed by conjugation to glucuronic acid (Jeffcoat ef al., 1977; Birch et al., 1978). Some O-sulfate conjugates were also found. In the rat, these metabolites are eliminated primarily in the bile (Hiles, 1977). The possible exposure of human infants to TCC has aroused concern because of known deficiencies in their capacity to metabolize drugs as well as their biliary functions (Anders et al., 1973; Vest, 1965; Nyhan, 1961; Done, 1964, 1966). The infant rhesus monkey would appear to be an appropriate model for evaluating drug metabolism in a system similar to the human infant. (Lucey et al., 1963; Dvorchik et al., 1974; Jacobson and Windle, 1960; Gartner and Lane, 1972). However, it must first be established that the adult rhesus monkey is a suitable model for studying TCC metabolism in humans. Birch et al. (1978) found similar biotransformation products of TCC in the urine and plasma of I Present address: Springborn Institute for Bioresearch, Spencerville, OH 45887. 0041-008X/78/0463~593%02.00/0 593 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

594

HILES

ET AL.

humans and the rhesus monkey. Hiles and Birch (1978) have studied TCC metabolism in humans. The experiments in this communication were designed to further compare TCC metabolism in the adult rhesus monkey (Mucuca muluttu) and man. In addition, knowledge concerning TCC metabolism was extended using techniques such as iv infusion and bile duct cannulation in the monkey which were inappropriate in our human studies. METHODS Chemicals. TCC and [r4ClTCC (4’-chlorophenyl ring labeled; specific activity 40 ,uCi/mg) were obtained from Monsanto Chemical CO.~ They were judged to be >95% chemically pure and >99% radiochemically pure by high-pressure liquid chromatography (hplc) analyses (Jeffcoat et al., 1977). Acetone3 was reagent grade. Animals. Adult male rhesus monkeys (M. muluttu) were housed in individual cages until the study began, and were fed a diet of biscuits4 and ad libitum water. When the study began they were placed in restraining chairs5 and were then fed biscuits4 supplemented with fresh fruit, cookies,‘j or cereal.’ Water containing Oralyte-A8 electrolytes was available ad libitum. Monkeys born 12 to 19 hr before the initiation of drug dosing and weighing 405 to 455 g were used in the infant studies.9 The infants were full term and were delivered by the colony-reared mothers without human assistance. They remained with their mothers at least 4 hr before being moved to incubators at 30°C. A cloth surrogate was placed in the incubators. Infants were fed as follows: 10% dextrose the first day, a 50/50 mixture of 10% dextrose and Similaclo the second day, and Similac only thereafter. Infants were removed from the incubators for weighing, for blood sampling, and for the surgical implantation of venous catheters. Surgical preparations. Some adult animals were surgically prepared with a bile duct cannula and a duodenal cannula as previously described (Cloyd et al., 1977). These monkeys had completely recovered from surgery as judged by “normal” bile flow (Cloyd et al., 1977) before being used for metabolism studies. For iv infusions of drug, a section of PE/90 tubing” was inserted into a femoral .vein or a feline catheterI (20 ga, 2”) was placed in a saphenous vein under Sernylan13 (phencyclidine . HCl) anesthesia. Preparation of dosing solutions. Human plasma (<21 days old) was filtered through an 8-m filter.14 A known amount of TCC and [i4ClTCC dissolved in N 1 ml of acetone was added dropwise with stirring to 140 ml of the filtered plasma for adult doses. An additional milliliter of acetone was used to rinse the TCC into the plasma. Stirring was r Monsanto Chemical Co., St. Louis, MO. 3 MCB Manufacturing Chemists, Norwood, OH. 4 Purina Monkey Biscuits, Ralston Purina Co., St. Louis, MO. 5 Plas-Labs, Plastics Manufacturing & Supply, Inc., Lansing, MI. 6 Oreo, Nabisco, E. Hanover, NJ. ’ Freakies, Ralston Purina Co., St. Louis, MO. * Elanco Products Division of Eli Lilly Co., Indianapolis, IN. 9 Wisconsin Regional Primate Research Center, Madison, WI. lo Ross Laboratories, Columbus, OH. ii Intramedic polyethylene tubing, Clay-Adams, New York, NY. I2 Sherwood Medical Division of Brunswick Co., St. Louis, MO. i3 Bio-centic Laboratories, Inc., St. Joseph, MO. I4 Type SC, Millipore, Bedford, MA.

595

DISPOSITION OF TCC IN THE RHESUS

continued for 30 min while a stream of nitrogen was directed at the surface to remove the acetone. A sample was then removed for 14C analysis, and the remaining plasma was filtered again to remove any undissolved TCC. A second sample was removed for r4C analysis, and the TCC-plasma solution was used for IV infusion. Proportionately smaller amounts of dosing solutions were prepared for infants using undiluted [14CITCC. Dosing. Intravenous infusion was metered with a Harvard” pump through a section of polyethylene tubing surgically implanted in a saphenous or femoral vein. The exact dosage was determined by weighing the amount of solution administered. A summary of the dosings of adult monkeys is presented in Table 1. Infants received TCC over a 5TABLE

1

SUMMARY OF EXPERIMENTALCONDITIONS FOR ~,~,~'-TRICHLORO[~~C]CARBANILIDE ADULTRHESUS MONKEYS

Experiment AH-46 AH-6 1 AH-76 AH-80 AH-95 AH-97 AH- 106 AH- 107 AH-108 AH-109

Monkey number

Animal weight 0%)

198

4.5

251 (1)” 257(2) 257(3)

4.8 4.8 4.8 4.6 7.2 4.1 6.6 7.2. 6.6

259 339 (1) 264 340(l) 339(2) 340(2)

Infusate concentration Infusion time (Ctmol Biliary system TCC/ml) (hr) Intact Intact Intact Intact 5% diversion Intact Intact 5% diversion Intact 100% diversion

0.29 0.09 0.02 0.23

0.21 0.22 0.28

0.21 0.26 0.21

24 24 24 24 24 24 12 12 12 12

Infusion rate (nmol TCC/kg/hr) 350 104 24 244 252 172 732 352 405 347

INDOSING

Total dose (,umol TCC) 37.8 12.0 2.8 28.1 27.8 29.7 36.0 27.9 35.0 27.5

a Numbers in parenthesesindicate the monkeywas used more than once.

to 6-hr period at a rate of 1.08 ml/hr. The doses for the three infants (AH-98, AH-104, and AH-105) were 1.3, 0.9, and 1.0 pol/kg/hr, respectively. During these infusions, infants were restrained (Caudill, 1977) but afterwards were allowed to roam in the incubators. Sample collections. Urine was collected from adults in a stainless-steel pan while feces were collected on a screen suspended above the pan. Feces were not collected for 14C analysis from infants as they were watery and were generally distributed over the surface of the animal, the surrogate, and the incubator. Much effort was made to keep the environment clean and to prevent accidental ingestion of feces. Urine samples were collected by gently pressing the bladder. Blood samples from all animals were drawn from a saphenous vein or a femoral vein with heparinized equipment from the limb not used for infusion and the cells and plasma were separated by centrifugation. Animals were sacrificed by an overdose of sodium pentobarbital and exsanguination. I5 Harvard Apparatus Co., Millis, MA.

596

HILES ET AL.

Bile sampling. When the bile duct-cannulated animals were not being used for experiments, 5% of the total bile output was collected over dry ice and 95% was pumped into the duodenal cannula and thus returned to the animal. This fractionation was accomplished with a device designed by Berger (1974).16 Such diversion does not affect bile production or composition (Dowling et al., 1970). In experiments labeled “5% bile diversion,” the bile was fractionated as above. In one experiment where total bile was collected, bile which contained no 14C and which had been stored frozen was infused into the duodenal cannula at a rate equal to the average daily output of the individual animal. This infusion of control bile assured that the animal maintained normal functional biliary capacity (Dowling et al., 1970). Sample analyses. i4C levels in plasma, tissues, feces, blood cells, and dosing solutions were determined by burning them in oxygen, trapping the CO,, and counting it by liquid scintillation methods. Aliquots of urine and bile were placed directly in scintillation fluid. Quenching was corrected by external standardization. The concentrations of the TCC in the dosing solutions were determined from the levels of the i4C in the plasma before and after filtration, and the known quantity of TCC added originally to the plasma. By comparing the count rates of the various samples to that in the dosing solution standards, the amounts of TCC-derived materials (expressed as equivalents of TCC) were calculated. The metabolite composition of selected bile, urine, and plasma samples was determined by hplc analyses as previously described (Jeffcoat et al., 1977; Birch et al., 1978). Data analyses. Kinetic parameters and constants were estimated from semilogarithmic graphs of the data on the initial assumption of first-order processes. Using these estimates, the best mathematical expressions were calculated using a linear or nonlinear digital computer program. Half-lives were calculated from the rate constants. RESULTS

Adult Monkey A summary of the experiments in adult monkeys is presented in Table 1. hplc analysis showed that TCC was not metabolized when it was mixed with the human plasma dosing medium. The data in Fig. 1 depict the time course of the 14C in the plasma and blood cells of an intact adult monkey infused for 24 hr with [14ClTCC (expt AH-61, Table 1). The plasma 14C concentration increased rapidly up to N 12 hr and appeared to be approaching steady state as it increased only very slowly between 12 and 24 hr. When infusion was stopped, the 14C was removed from the plasma in two distinct phases. This pattern of total plasma 14C was observed in all experiments. An approximately linear relationship was found to exist between the infusion rate of [i4ClTCC in intact monkeys and the plasma levels of TCC-derived materials after 12 hr (Fig. 2, inset). Initial assumptions of first-order metabolism were further verified by demonstrating that the normalized plasma 14C elimination data obtained at the various dosing levels (24 to 732 nmol/kg/hr) were superimposable (Wagner, 1975) and fit the I6 Suburban Electronics,Peabody, MA.

DISPOSITION

: -

591

OF TCC IN THE RHESUS

SbP YiO" S.' i

0.001

1 0

n ' c a c " c 1 / " .20 40 60 80 100 120

I ""I 140 160 180

" I ' 200 220

Time (hrs.)

FIG. 1. The plasma and blood cell concentrations of TCC-derived materials in an intact adult monkey intravenously infused with 3,4,4’-trichloro[‘4Clcarbanilide. The data are from Expt AH-61 in Table 1. The elimination of r4C from the plasma, as determined by the method of least squares, fits the equation: concentration = 0.29 e-“.r9* + 0.053 e- 0.‘3’3961 n = Plasma, A = blood cells. 200

1.0

0

20

I

I

I

I

40

60

80

100

I

120

I

140

I

160

I

180

I

200

I

220 240

Time (hr.) FIG. 2. The relationship between the steady-state plasma levels of 3,4,4’-trichloro[14Clcarbanilide derived material after 12 hr of infusion and the dose rate of drug and the rate of the elimination of r4C from the plasma of adult monkeys. The 12-hr infusion plasma levels (inset) were determined using iv infusion on intact monkeys (Table 1). Each point represents a different experiment. In calculating the least squares fit, 0 = 0 was designated as an absolute point. The normalized elimination data were pooled from the same seven experiments used for the inset.

598

HILES

ETAL.

expression: percentage of t = 0 plasma level remaining = 75 e-o.‘46’ + 19 e-O*OOSt (Fig. 2). If it is assumed that the two phases of elimination represent two independent processes, then the half-lives for the fast and slow phases were 4.9 and 84 hr, respectively. The excretion pattern of 14C after iv infusion of [14ClTCC was determined in intact and bile duct-cannulated animals (Table 2). The urine contained -20% of the 14C, and most of the remaining radioactivity in the intact animals was in the feces. In one experiment (AH-109) the total bile output was collected and was found to contain 82% of the dosed 14C. In this animal, feces contained only 1% of the dosed radioactivity. TABLE 2 DISPOSITION

OF RADIOACTIVITY FROM

~,~,~'-TRIcHLORO~~~~~CARBANILIDE MONKEYS

IN ADULT

RHESUS

Percentage of dose radioactivity Experiment

Urine

Feces“

AH-46 AH-6 1 AH-76 AH-SO’ AH-95d AH-97

30 20 21 1.5 24 22 28 18 8 15

55 65 71 61 55 63 61 51 48 1

AH-106e AH- 107d

AH-108C AH- 109f

Bile 5c 12

Tissuesb 11

-

1 9 2c 82

30

-

Recovery 85 85 93 92 91 85 90 78 88 98

n Feces include gastrointestinal wash and tract and cage wash for sacrificed animals. b Tissues are the same as in Table 3. c Necropsy at 12 to 24 hr after starting infusion when the plasma 14C would be at a maximum. Bile was taken from gallbladder. d 5% bile diversion. e Necropsy at 228 hr after dosing. / 100% bile diversion.

Tissue distribution of 14C was determined (Table 3) in two animals sacrificed when the plasma 14C was at a maximum (Expts AH-80 and AH-108, Table 2) and in one animal sacrificed 228 hr after dosing (Expt AH-106, Table 2). Liver and kidneys had the greatest potentials to concentrate 14C. By 228 hr after dosing, all of the tissue concentrations were less than the plasma except these two tissues. During infusion, concentrations of 14C were greater in both urine and bile than in plasma. By 228 hr, only bile maintained a concentration above plasma. Lipid levels of 14Cwere low as were those of brain. The bile was the major route of 14C elimination (Table 2). The importance of enterohepatic circulation can be estimated in bile duct-cannulated animals where only 5% of the bile was removed. Simple normalization of the percentage of dosed 14Cin this bile to 100% collection projects that 180 to 240% of the dosed 14C passed through the bile. Two experiments (AH-107 and AH-109, Table 1) were run on the same animal;

DISPOSITION

599

OF TCC IN THE RHESUS

one with 5% bile diversion and one with 100% bile diversion. The plasma 14C curves in the two experiments were almost identical (Fig. 3). A semiquantitative analysis of plasma, urine, and bile was done as a function of time in several experiments. Examples of the chromatographic results from Expt AH-107 are shown in Figs. 4, 5, and 6. The letters are those that were assigned to the various peaks by Birch et al. (1978) as follows; A and B = unknown, C and D = TCC N- and N’glucuronide, F = 3’-OH-TCC glucuronide, H = ortho-OH-TCC glucuronide (2’-OH or TABLE 3 DISTRIBUTION

AT NECROPSY

OF RADIOACTIVITY AND IN INFANT

FROM RHESUS

IN ADULT

Concentration of TX-derived

~,~,~‘-T~~~HLoRo[‘~CI~ARBANILIDE MONKEYS

material (equiv nmol of TCC/g)

Adult monkey experiments

Sample”

Plasma Blood cells Liver

Kidneys Brain Fat Lungs Gallbladder

AH-80 necropsy during

AH- 108

AH-106

necropsy

during

necropsy at

infusion

infusion

240 hr

0.29 0.29 2.63

3.91 0.36

1.97 0.11 -

Large intestines

Smallintestines 424 BileC UrineC 19.4

2.3 1.5 16.6

9.7 0.6 1 5.21 2.6

-

236 21.6

o.054b 0.012 0.68 0.41 <0.003 0.05 0.04

-

Infant monkey experiments AH-98 necropsy

AH- 104 necropsy

AH-105 necropsy

at

at

102hr

240 hr

240 hr

2.2

0.2

0.1

-

0.1 0.7

0.02 0.4 0.4

2.5 1.7 0.04

0.3

0.1


2.3 1.3

0.2 2.3

1.1

0.1

0.99

3.6 134

0.03

-

0.2 0.2

-

at

0.01 0.05 0.2

1.1 0.04 0.07 33

-

OOther samples taken but which were found to have concentration: of 14C near to or below those of plasma included lymph nodes, pancreas, spleen, gonads, adrenals, heart, esophagus, thymus, thyroid, skin, skeletal muscle, bone, bone marrow, and remaining carcass. h Steady-state plasma level = 2.77 equiv mnol/g.

cTakenat necropsy.

6-OH), J = 3’-OH-TCC sulfate, K = ortho-OH-TCC sulfate, and L = TCC. In addition, M = 3’-OH-TCC and N = ortho-OH-TCC. During TCC infusion (Fig. 4, 12-hr sample), the major plasma components were Nglucuronides, O-sulfate conjugates, TCC, and some unknowns. Hydroxylated but unconjugated TCC compounds (which would be eluted between K and L) were not observed. TCC and the N-glucuronides were more rapidly removed than the other components, leaving a plasma composed mainly of unknown A and the sulfate conjugates of hydroxylated TCC (Fig. 4; 14 and 18 hr) after the fast phase elimination was essentially completed (Fig. 2). The major urinary components during drug infusion were the N- and N’-glucuronides of TCC (Fig. 5). At later times (> 18 hr), the 14C in the urine was divided among

HILES ETAL.

600

numerous compounds (not including the N-glucuronides) which were not identified. Neither the O-sulfate conjugates, the 0-glucuronide conjugates, nor TCC were detected in urine. Figure 6 is a chromatographic profile of the biliary 14C at two different times. The major components at 10 to 11 and 118 to 125 hr were 0-glucuronide conjugates of hydroxylated TCC. Only very small amounts of N-glucuronides of TCC were observed in the 10 to 11 hr sample and none was found in the 118 to 125 hr bile sample. Sulfate conjugates of hydroxylated TCC were only minor components of the bile. In addition, some hydroxylated but unconjugated derivatives of TCC were found.

.

..

. .

1

I

0

20

60

I

I

I

I

I

100 Time

I

I1

L

140

I

. .

I

I

180

I

. .

I

I

. . I

81

220

(hr.)

FIG. 3. The effect of enterohepatic circulation on the plasma levels and kinetics of elimination of 3,4,4’trichloro[Wlcarbanilide derived materials in the adult monkey. In the first experiment (AH-107) (@), 5% of the bile was collected and 95% was circulated into the duodenum. In the second experiment (AH-109) (A), 100% of the bile was diverted away from the animal and collected.

Infant Mmkey Tissue and fluid distribution of 14C were determined in one animal which was sacrificed when the plasma level was relatively high (2.2 equiv nmol of TCC at 102 hr), and in two which were sacrificed at 240 hr when the plasma level was less than onetenth this value (Table 3). Material balance could not be determined because of the problems of excretion collections (see Methods). Very little of the 14C was found in fat or in the central nervous system. The organs of highest accumulation were the liver, kidneys, and lung. High levels of i4C were generally seen in the bile, gallbladder, and intestinal tract. All of the tissues and fluids contained much less i4C at 240 hr than at 102 hr. A similar distribution pattern was found in adult monkeys as well as in rats (Hiles, 1977). The time courses of the plasma i4C in Expts AH-104 and AH-105 are depicted in Fig. 7. Although the infusion of TCC was stopped at 5.5 hr, the plasma concentrations of i4C continued to increase, reaching a maximum value at 10 to 20 hr. Assuming first-

DISPOSITION

OF TCC IN THE RHESUS

601

order elimination, the 14C appeared to be removed in two phases: a short fast phase which afforded too little data for estimation of its t+ and a slow phase with an estimated t+ = 36 to 60 hr (Expts AH-104 and AH-105). The blood cells contained slightly less 14C than the plasmas and followed the same pattern of 14C concentration with time. Similar patterns of r4C elimination were observed in adult monkeys and humans (Hiles and Birch, 1978) except that the early rapid phase was more easily observed in the adult monkey since the plasma 14C decreased as soon as infusion was terminated. 12 hours

Relative

Retention

Time

FIG. 4. A representative chromatographic “profile” of the 14C derived from 3,4,4’trichloro[14Clcarbanilide in adult monkey plasma. The three plasma samples are from Expt AH-107 (12.hr infusion) (Fig. 3) and were taken at the times indicated. The definitions of the letters denoting different compounds are A and B = unknown, C and D = N-glucuronides of TCC, J = 3’-OH-TCC sulfate, K = o&o-OH-TCC sulfates, and L = TCC.

The composition of several plasma samples was determined, and the results are depicted in Fig. 7. The components can be divided into three categories: (1) TCC and the Nglucuronides of TCC which, upon cessation of infusion, quickly disappeared from the plasma, (2) 3’-OH-TCC sulfate and 0-glucuronide conjugates of TCC which, upon cessation of infusion, continued to increase in concentration before being eliminated at a relatively rapid rate and (3) the ortho-OH-TCC sulfates (2’-OH- or 6-OH-) which accounted for most of the increase in plasma r4C after the infusion was stopped and which were removed from the plasma slowly (t+ = 36 to 60 hr). Thus, the continued

HILES

602

ET AL.

Relative

Retention

Time

5. A representative chromatographic “profile” of the r4C derived from 3,4,4’trichloro[14Clcarbanilide in adult monkey urine (AH- 107). The definitions of the letters denoting different compounds are C = TCC IV-glucuronide and D = TCC N’-glucuronide. FIG.

lo-l

1 hours J

z f! Q .-f

H 118-

125

hours

3 d

F B I

Relative

Retention

Time

6. A representative chromatographic “profile” of the r4C derived from 3,4,4’trichloro[14Clcarbanilide in adult monkey bile. The samples are from Expt AH-107 (Fig. 3) where 5% of the total bile production was diverted from the animal. The samples were taken at the times noted. The definition of the letters are B = unknown, C and D = the IV-glucuronides of TCC, F = 3’-OH-TCC glucuronide, H = orrho-OH-TCC glucuronides, J = 3’-OH-TCC sulfate, K = o&o-OH-TCC sulfates, M = 3’-OH-TCC, and N = or&o-OH-TCC. FIG.

DISPOSITION

OF TCC

IN THE RHESUS

603

increase in plasma 14C after ceasing infusion was due primarily to the ortho-OH-TCC sulfates. The metabolic tissues (liver?) must remove TCC from the plasma and metabolize it to these sulfate conjugates more rapidly than they can be eliminated. These metabolites are then infused into the plasma from the tissue depot until the rate of

Time (hs)

FIG. 7. The time course of plasma and blood cell r4C of iv infused radioactive 3,4,4’-trichlorocarbanilide (TCC) and the plasma metabolites in an infant rhesus monkey. 0 = plasma total‘KC &rived mate&& n = TCC, v = N- or N’-glucuronides of TCC, 0 = ortho (2’- or 6-)-hydroxy-TCC glucuronide, v = 3’hydroxy-TCC sulfate, and A = ortho (2’- or 6->hydroxy-TCC sulfate. The arrow indicates where infusion of drug ceased. The dashed lines project to the point dh the graph where the detection limit was for samples which contained too low a level of metabolite or drug to be detected.

elimination becomes greater than the rate of synthesis. This phenomenon was not observed in adult monkeys where O-glucuronides are the major metabolites and are readily excreted in the bile. The metabolite composition of the bile samples taken at 102 and 240 hr are shown as chromatographic profiles of 14C in Fig. 8. Most of the 14C in the 102-hr sample was in the form of o&o-OH-TCC sulfates (69%) mixed with smaller amounts of 3-OH-TCC sulfate (4%), the ortho-OH-TCC glucuronides (5%), and the N-glucuronides of TCC

604

HILES

ET

AL.

((1%). The 240-hr sample contained only the ortho-OH-TCC sulfates. No TCC or aconjugates of hydroxylate TCC was observed. In adult monkeys, the major biliary metabolites of TCC were the 0-glucuronides. The following observations indicate that the bile is the major excretory route for TCC metabolites in the infant monkey as it was in the adult: (1) the high ratio of bile-14C to plasma-14C, (2) the high level of 14C in the gallbladder and intestines, and (3) the major plasma metabolite was also the major biliary metabolites.

120 hr.

GD

.-I% ‘, b = ~

240

H J h

hr.

II Relative

Retention

Time

FIG. 8. The metabolites of iv infused radioactive 3,4,4’-trichlorocarbanilide (TCC) found in the bile of infant rhesus monkeys. The bile was obtained at necropsy at 120 hr (Expt AH-98, Table 1) or 240 hr (Expt AH-105, Table 1) after initiating the dosing. The metabolites were identified (Birch et al., 1978) as follows: C + D = N- or N’-glucuronide of TCC, H = ortho (2’- or 6-)-hydroxy-TCC glucuronide, J = 3’-hydroxyTCC sulfate, and K = ortho (2’. or 6-)-hydroxy-TCC sulfate.

Several urine samples were analyzed and were found to contain all the detectable 14C divided approximately equally between N- and N’-TCC glucuronide. DISCUSSION

In adult rhesus monkeys, using iv infusion as a model for dermal absorption, we determined that the steady-state plasma level of TCC-derived materials was approached after -12 hr of infusion and that the elimination kinetics were first order and thus independent of dose up to a dose rate of -800 nmol of TCC/kg/hr. It is appropriate to assume that within this dose range biotransformation, distribution, and excretion are also independent of dose. The elimin&on of the 14C from plasma occurred in two distinct phases with tt -5 and 80 hr (Figs. 1 and 2). Humans eliminate 14C from an oral dose of TCC in a similar fashion but with t+ N 2 and 20 hr (Hiles and Birch, 1978). The rapid elimination phase in humans was due to the removal of TCC and the N-glucuronides of TCC, while the

DISPOSITION

OF TCC

IN THE

RHESUS

605

slower phase resulted from elimination of the sulfate conjugates of hydroxylated TCC. A similar pattern was observed in rhesus monkeys. A biphasic elimination pattern for 14C was also observed in human urine with a fast phase due to the excretion of the N-glucuronides and a slow phase resulting from the removal of numerous compounds which did not cochromatograph with any known substances (Hiles and Birch, 1978). We observed a similar pattern in the adult rhesus monkey. Thus, on the basis of the results reported here and reported by Hiles and Birch (1978) and Birch et al. (1978), it is concluded that the adult rhesus monkey is a reasonable model for humans for studying TCC metabolism and disposition. The bile was found to be the major route (>75%) of elimination of TCC metabolites from the rat (Hiles, 1977). However, from orally dosed drug, one could not confirm this in humans. Using bile duct-cannulated monkeys we found that >80% of the i4C from iv dosed TCC is eliminated in the bile. It seems reasonable that the bile plays an important role in the metabolism of TCC in humans (Hiles and Birch, 1978). An analysis of bile obtained from bile duct-cannulated monkeys shows glucuronide conjugates of hydroxylated TCC to be the major metabolites. The bile collected in our study differed from the bile that was collected at necropsy from the bladder and frozen immediately (Birch et al., 1978), in that it contained aconjugates of hydroxylated TCC. We believe this to be an artifact of the collection method since hydrolysis could have occurred during the time (as much as 6 hr) that the bile took to travel through the tubing and fractionator from the liver to the collection vessel. The data of Birch et al. (1978) indicate that most of the aconjugates we observed were originally glucuronides. Enterohepatic circulation of these biliary metabolites was extensive, yet this recirculation had no detectable effect on the plasma 14C pharmacokinetics. The liver is thus highly efficient in forming and eliminating glucuronide conjugates of hydroxylated TCC. Only small amounts of the major urinary metabolites (N- and N’- glucuronide of TCC) were found in the bile. Why this metabolite with its close similarity in polarity and molecular weight to the glucuronides of hydroxylated TCC should be almost excluded from the bile (Smith, 1973) and excreted in the urine is unknown. No general pattern of N-glucuronide excretion from other N-conjugates has been established (Bridges et al., 1968; Boyland et al., 1957; Axelrod et al., 1958). Some N-giucuronides are formed without the assistance of enzymes while others require enzymes (Bridges et al., 1968; Bridges and Williams, 1962). The observation that rats do not form them from TCC while they are important metabolites in both man (12-23%) and monkey (Birch et al., 1978; Hiles and Birch, 1978) lead us to think that the N-glucuronides of TCC are formed by an enzymatic reaction. The metabolite analyses in the bile, plasma, and urine produced an unexpected result: the sulfate conjugates of hydroxylated TCC, which are the major plasma metabolites and which are slowly eliminated (tt N 80 hr), are not detectable components of the urine and are only minor components of the bile. Detailed pharmacokinetic studies using glucuronide and sulfate conjugates would be required to properly understand the relative roles of the two conjugates in hydroxylated TCC removal. However, of broad metabolic and toxicological significance is the fact that one cannot properly evaluate the biotransformation of this drug by analyzing only the urine, the bile, or the plasma.

HILES

606

ET AL.

Like the adult, the infant rhesus could readily metabolize and eliminate TCC. Because the drug metabolism and developmental patterns of rhesus and human are similar (Jacobson and Windle, 1960; Rane and Ackerman, 1972; Dvorchik et al., 1974; Pelkonen and Klrki, 1973) it is reasonable to assume that the infant rhesus would handle TCC in a manner similar to the human infant. The studies of TCC metabolism in monkeys have added additional knowledge about the relationship between infant and adult monkeys and their importance as a model for human drug metabolism. Unlike many laboratory animals, primate fetuses (Dvorchik et al., 1974) and human fetuses (Rane et al., 1973; Rane and Ackerman, 1972) and neonates (Vest, 1959) can readily oxidize aromatic drugs. Our studies establish that the neonatal rhesus monkey is similar to humans in this respect. It is well established that human fetuses and neonates are deficient in their ability to form 0-glucuronide conjugates (Dutton, 1959; Yu and Aldrich, 1960; Vest, 1965; Vest, 1959; Rane et al., 1973); the deficiency is expressed as physiologic jaundice. The newborn simian is the only animal that exhibits a similar jaundice condition (Gartner and Lane, 1972; Lucey et al., 1963). In the rhesus, the plasma bilirubin levels return to normal within -72 hr after birth. In our studies with TCC, all animals were dosed within -24 hr after birth. As expected, the level of 0-glucuronide metabolites, which are the major TCC metabolites in adult rhesus monkeys, were very low. Levy et al. (1975) and Miller et al. (1976) demonstrated that in human neonates deficient 0-glucuronide conjugation of drugs could be compensated for by increased Osulfate conjugation. Our studies demonstrate a similar compensatory mechanism in the rhesus neonate. The O-sulfate conjugates of TCC are the major plasma and biliary metabolite in the neonate, whereas, in the adults, the major biliary metabolites are the 0-glucuronide conjugates. The N-glucuronides of TCC were the major urinary metabolites in infants and in adult monkeys and also in humans (Hiles and Birch, 1978) but were not observed in rats (Birch et al., 1978). If, as previously discussed, the N-glucuronides of TCC are formed enzymatically, then our data show that, as hypothesized (Arius, 1961; Isselbacher, 1961), the N-transferase and 0-transferase involved in glucuronide conjugations are different enzymes. Whether the N-glucuronide formation is enzymatic or chemical, we have established that the formation of 0-glucuronides in the primate neonate is a function of transferase deficiency and not the lack of glucuronic acid substrate. ACKNOWLEDGMENTS

The assistance of H. Lampe, R. Schneider, and R. Bruce was appreciated. A special note of gratitude is due to Drs. R. Goy and D. Houser of the Wisconsin Regional Primate Research Center for their assistancein procuring the infant monkeys used in this study. REFERENCES ANDERS,

R., SJGQUIST,

F., AND ORRENIUS,

S. (1973). Drugs and fetal metabolism. Gin. Pharm.

Therap. 14,666-672.

ARIUS, I. M. (1961). Ethereal and N-linked glucuronide formation by normal and Gunn rats in vitro and in vivo. Biochem.Biophys.Res.Commun.6,81-&l.

DISPOSITION OF TCC IN THE RHESUS

607

J., INSCOE, J. K., AND TOMKINS, G. M. (1958). Enzymatic synthesisof Nglucosyluronicacidconjugates.J. Biol. Chem. 232, 835-841. BERGER, J. (1974). Fraction collector using ultrasonic technique, IEEE Transactionson BiomedicalEngineering,May, 241-243. BIRCH, C. G., HILES, R. A., EICHHOLD, T. H., JEFFCOAT, A. R., HANDY, R. W., HILL, J. M., WILLIS, S. L., HESS,T. R., ANDWALL, M.E. (1978).Biotransformationproductsof 3,4,4’trichlorocarbanilide.Drug Metab. Dispos. 6, 169-176. BOYLAND,E., MANSON,D., ANDOaa, S. F. D. (1957).The biochemistryof aromaticamines: The conversionof arylaminesinto arylsulphamicacid and arylamine-N-glucosiduronic acids. Biochem. J. 65,4 17-423. BRIDGES, J. W., KIBBY, M. R., WALKER, S. R., AND WILLIAMS, R. T. (1968).Species differences in the metabolismof sulphadimethoxine. Biochem. J. 109, 85 l-856. BRIDGES, J. W., AND WILLIAMS, R. T. (1962). N-Glucuronideformation in vivo and in vitro. AXELROD,

Biochem. J. 83,27.

CAUDILL,D. (1977).A restrainingdevicefor the infant monkey.Lab. Anim. Sci. 27,406. CLOYD, G. G., HILES, R. A., AND CAUDILL, D. (1977).Chronicbile duct cannulationof rhesus macaques(Macaca mulatta) without causingbiliary fistulas.Amer. J. Vet. Med. Res. 38, 1607-1610. DONE, A. K. (1964).Developmentalpharmacology.Clin. Pharmacol. Ther. 5,423-479. DONE,A. K. (1966).Parenteralpharmacology.Annu. Rev. Pharmacol. 6,189-208. DOWLING, R. H., MACK, E., AND SMALL, D. M. (1970).Effects of controlledinterruptionof the enterohepaticcirculation of bile salts by biliary diversion and by ileal resectionon bile secretion,synthesis,andpoolsizein the rhesusmonkey.J. Clin. Invest. 49,232-242. Du-I-~oN,G. J. (1959). Glucuronidesynthesisin foetal liver and other tissues.Biochem. J. 71, 141-148. DVORCHIK,B. H., STENGER, V. G., AND QUALTROPANI,S. L. (1974). Fetal hepatic drug metabolismin nonhumanprimate,Macaca arctoides. Drug Metab. Dispos. 2, 539-544. GARTNER,L. M., ANDLANE,D. L. (1972).The physiologyof physiologichyperbilirubinemiaof the newborn.Medical Primatology. Proc. 3rd Conf. Exp. Med. Surg. Primates,Lyon 1972, Part I, pp. 237-247. HILES, R. A. (1977). Metabolism and toxicity of halogenatedcarbanilides:absorption, distribution andexcretionof radioactivity from 3,4,4’-trichloro[14Clcarbanilide (TCC) and 3trifluoromethyl-4,4’-dichloro-[14Clcarbanilide (TFC) in rats. Food Cosmet. Toxicol. 15, 205211. HILES,R. A., ANDBIRCH,C. G. (1978).The metabolism of 3,4,4’-trichlorocarbanilide(TCC) in humans.Drug Metab. Dispos. 6, 177-183. ISSELBACHER, J. J. (1961). Solubilizationand purificationof glucuronyl transferase from rabbit liver microsomes. Biochem. Biophys. Res. Commun. 5,243. JACOBSON, H. N., AND WINDLE, W. F. (1960). Observationson mating, gestation,birth, and postnataldevelopmentof Macaca mulatta. Biol. Neonate 2, 105-120. JEFFCOAT, A. R., HANDY, R. W., FRANCIS, M. T., WILLIS, S., WALL, M. E., BIRCH, C. G., AND HILES, R. A. (1977). The metabolismand toxicity of halogenatedcarbanilides:biliary metabolitesof 3,4,4’-trichlorocarbanilideand 3’-trifluoromethyl-3,4’-dichlorocarbanilide in the rat. Drug Metab. Dispos. 5, 157-166. LEVY, G., KHANNA, N. N., SODA, D., TSUZUKI, O., AND STERN, L. (1975).Pharmacokinetics of acetaminophen in the humanneonate:Formationof acetaminophen glucuronideand sulfate in relation to plasmabilirubin concentrationand D-gluconicacid excretion. Pediatrics 55, 818-825. LUCEY, J. F., BURLINGTON, J. T., BEHRMAN, R. E., AND WARSHAW, A. L. (1963).Physiologic jaundicein newborn rhesusmonkey.Amer. J. Dis. Child. 106,350-35.5. MILLER, R. P., ROBERTS, R. J., AND FISCHER, L. F. (1976).Acetaminopheneliminationkinetics in neonates,childrenandadults.Clin. Pharmacol. Ther. 19,284-294. NYHAN, W. I. (1961).Toxicity of drugsin the neonatalperiod.J. Pediatr. 59, l-20. PELKONEN, O., AND KARKI, N. T. (1973).Drug metabolism in humanfetal tissues.Life Sci. 13, 1163-1180.

608

HILES

ET AL.

A., AND ACKERMAN, E. (1972). Metabolism of ethylmorphine and aniline in human fetal liver. Clin. Pharmacol. Ther. 13,663-670. RANE, A., SJ~QLJIST, F., AND ORRENIUS, S. (1973). Drugs and fetal metabolism.Clin.

RANE,

Pharmacol. Ther. 14,666-672. SMITH, R. L. (1973).The Excretory Fnxction aSBile,pp. 3-99. Chapmanand Hall, London.

M. (1959).Studieson the developmentof the ability of the liver to form glucuronidein the newborn-the behavior of aminophenolglucuronide in the blood after administrating acetanilide.Schweiz. Med. Wochensch. 4, 102-105. VEST, M. (1965). The developmentof conjugation mechanismsand drug toxicity in the newborn.Biol. Neonate 8,258-266. WAGNER, J. G. (1975). Fundamentals of Clinical PharmaEokinetics, pp. 247-284. Drug IntelligencePublications,Hamilton,IL. Yu, W. L., AND ALDRICH, R. A. (1960). The glucuronyl transferasesystemin the newborn infant. Pediatr. Clin. N. Amer. I, 381-396. VEST,