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I. Kinetics and metabolism of theobromine in male rats
Toxicology, 30 (1984) 327--341 Elsevier Scientific Publishers Ireland Ltd.
I. KINETICS AND METABOLISM OF THEOBROMINE IN MALE RATS
M. BONATIa, R. LATINI a'*, B. SADURSKATM, E. RIVA a, F. GALLETTIa, J.F. BORZELLECA b , S.M. TARKAc , M.J. ARNAUD d and S. GARATTINIa
aIstituto di Ricerche Faramcologiche "Mario Negri" Via Eritrea 62, 20157Milan (Italy), bDepartment of Pharmacology, Medical College of Virginia, Richmond, VA (U.S.A.) CLife Sciences Division, Hershey Foods Technical Center, Hershey, PA., 17033 (U.S.A.) and dNestld Products Technical Assistance Co., Ltd., Research Department, CH-1814 La Tour-de-Peilz (Switzerland) (Received May 4th, 1983) (Accepted November 9th, 1983)
SUMMARY
On the basis of general pharmacological information (blood cells/plasma partition, plasma protein binding) and using HPLC as the principal analytical m e t h o d , we investigated the kinetics and metabolism of theobromine (a caffeine metabolite) in male rats after a single dose and after a 2 week chronic application. Doses in both conditions varied between 1 and 100 mg/ kg. In in vitro and in vivo the fraction of theobromine u n b o u n d to plasma proteins averaged 0.90 over a wide range of concentrations. No significant difference was f o u n d in the pharmacokinetic profile of the drug after acute or chronic treatment at different doses except for a reduction in the absorption rate constant as the dose increased. AUC values increased in proportion to the dose. The 2 treatment schedules were also similar as regards metabolism, at least 50% of the administered dose o f theobromine being excreted unchanged, and 25% as 6-amino-5-[N-methyl-formylamino]l-methyluracil. Only at the highest doses was there a tendency for theobromine to accumulate at the expense o f its major metabolite (a uracil compound).
Key words: Theobromine; Pharmacokinetics; Metabolism; Rats
*Present address: Cardiology Division, Stanford University Medical Center, Stanford, CA, U.S.A.
**Visiting scientist: Instytut Biofarmacji, Zaklad Biochemii, UL. Banacha 1, 02-097 Warszawa, Poland. All correspondence should be addressed to: Dr. Maurizio Bonati, Department of Clinical Pharmacology, Mario Negri Institute, Via Eritrea, 62, 20157 Milan, Italy. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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INTRODUCTION Theobromine (3,7-dimethylxanthine; 3,7-DMX) is a naturally occurring purine derivative present in cocoa, chocolate products and tea. Pharmacologically, 3,7-DMX has a moderate diuretic effect on the kidneys, causes mild cardiac stimulation and is virtually inactive as a central nervous system stimulant [ 1]. Recent concern about the safety of caffeine has raised questions regarding all methylxanthines [2 ]. There is a relative paucity of data on the toxicology of theobromine in laboratory animals and man [1]. Continuous short-term administration of high doses o f theobromine to rodents is known to result in thymic and testicular atrophy and growth depression [3,4]. In dogs, right atrial cardiomyopathy has been described [5]. Accurate information on the disposition of 3,7-DMX is essential for interpretation of toxicological data in laboratory animals. This paper reports the kinetics and metabolism of 3,7-DMX in male rats given oral doses ranging from I to 100 mg/kg/day on an acute and chronic basis. METHODS
Chemicals [8-14C]theobromine (3,7-DMX) (7.8 mCi/mmol), unlabelled 3,7-DMX, 3-methylxanthine (3-MX), 3-methyluric acid (3-MU), 7-methylxanthine (7-MX}, 7-methyluric acid (7-MU) and 3,7-dimethyluric acid (3,7-DMU) were generously supplied b y CMA (Chocolate Manufacturers Association of the United States}; 6-amino-5-{N-methyl-formylamino) 1-methyluracil(6AM-1-MU) was kindly supplied by Dr. Philipposian (Nestle, La Tour de Peilz, Switzerland). Reagents (LiChrosolv, Merck, Darmstadt, G.F.R.) were U.V. grade. Animals, housing and dosing CD-COBS mature male rats (Charles River, Italy} weighing 250--300 g were acclimatized to the research facility for 1 week before the study, receiving standard laboratory chow and water ad libitum. Animal rooms had controlled temperature (22°C) and light cycle (12/12 h). 3, 7-DMX kinetics after single oral doses Animals fasted for 16 h received 3,7-DMX by gavage in the free form suspended in a 1% water solution of Emulphor-EL-620, 24 h after carotid artery cannulation according to Popovic and Popovic [6]. Doses of 1, 5, 10, 50, 100 mg/kg b o d y weight were administered to groups of 6 animals. Blood samples from 0.05 to 0.20 ml were taken at various times (0, 15, 30, 60, 90, 120, 180, 240, 480 min and up to 24 h for the highest doses). Hematocrit was checked every 3 h. Fresh blood transfusions of 1--2 ml from a donor rat were given only for the dose of 1 mg/kg at 360 min where larger volumes had to be sampled. Blood samples were immediately diluted in 1.0 ml of 0.5% acetic acid in water and kept at 4°C until analysis {within 24 h).
328
Multiple dose study 3,7-DMX doses of 1, 10 and 100 mg/kg were administered by gavage every morning for 14 days to 3 groups of 6 animals, kept in the same conditions as in the acute experiment. Body weight was recorded daily. On the 7th day blood samples were taken from the tail vein of some individual animals, in order to establish a trough blood level profile during treatment. On day 12 (before the 12th 3,7-DMX dose) the carotid artery was cannulated. After the last dose (day 14) blood samples were drawn. On days 1, 7 and 13, instead of the unlabeUed c o m p o u n d the same dose of [8J4C]3,7-DMX (2 X 106 dpm/ rat) was administered to 3 animals (always the same ones) and feces and urine were collected separately for 24 h in metabolic cages. After measurement of the volume, urine was centrifuged at 15 000 rpm, and the supernatant was kept at - 2 0 ° C with other specimens until analysis.
Analy tical procedures Blood. One millilitre o f acetic acid (0.5%) and 50 ul of internal standard (10 pg/ml theophylline) were added to blood samples which were then processed according to a previously described caffeine assay [7] slightly modified. The samples were shaken for 30 min with 7 ml of chloroform/isopropyl alcohol (90 : 10), then the organic phase was transferred and evaporated in a water bath at 60°C under a gentle nitrogen stream. The residue was dissolved in 100 ~l of the chromatographic mobile phase and transferred to a water bath at 37°C for 10 min, so as to more easily dissolve 3,7-DMX, especially at higher doses. A b o u t 30 ul of sample were injected into the liquid chromatograph (Perkin-Elmer, Norwalk, C.T., U.S.A. -- series 2/2) equipped with an LC 15 s p e c t r o p h o t o m e t e r (280 nm) and a reversed phase column (Hibar RP-8, 7 ~m, 250 mm X 4 mm, Merck, Darmstadt, G.F.R.). A mobile phase of acetonitrile/acetic acid 0.5% ( 9 : 9 1 ) was used for elution at a flow rate of 1.3 ml/min. Urine. Urine (0.1 ml} was poured into a vial for total radioactivity counting, and 0.1 ml was injected into the high-pressure system together with an unlabelled standard mixture of 3,7-DMX and its metabolites (100 ug/ml). The chromatograph was equipped with a reversed phase column (Hibar RP-18, 7 ~m, 250 mm X 4 mm, Merck, Darmstadt, G.F.R.) and elutions were carried o u t with a linear gradient from 0% to 5% acetonitrile in 0.5% acetic acid (v/v) in water within 40 min, at a flow rate of 1.3 ml/min. The column eluent was collected in fractions directly into scintillation vials (by hand) using the UV absorption tracing on the recorder as reference (Fig. 1). After addition of 15 ml of scintillation mixture (Biofluor, NEN, England}, the vials were placed in a liquid scintillation counter (Beckman, Mod. B 2450}. The dpm of the various fractions were assigned to the different metabolites according to routine laboratory m e t h o d o l o g y [8] using the retention time on a UV tracing. Feces. Feces were collected on aluminium foil, weighed and kept a t - 2 0 ° C until analysis. They were homogenized in acetonitrile ( 1 : 1 0 , w : v ) , centrifuged at 15 000 rev./min, and 1 ml of supernatant was counted for radio-
329
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:E
w w
:E
2
cr
O {:Ex w
-rr~
O n
b- c~
L C~
i
I
0
2a
TIME
(min)
Fig. 1. Representative high-pressure liquid chromatogram o f 3,7-DMX and its metabolites. Corresponding fractions collected to measure radioactivity are marked (o).
activity as described above. No chromatographic analysis was performed because of the small amount of radioactivity found. Quality control of 3,7-DMX blood assay. According to our previously described procedure [9], 1 and 20 ug/ml of 3,7-DMX were added to 2 pooled samples of rat blood. Each pool was divided into 1-ml aliquots and stored at - 2 0 ° C until analysis. Analysis was done in triplicate for both concentrations (1 and 20) during routine assays over 1 year. The percentage coefficient of variation (CV%) was calculated as the standard deviation divided by the mean times 100. Blood cells/plasma partition. The blood cells/plasma partition of 3,7-DMX was calculated from whole blood and plasma levels of the compound 180 min after administration of 5, 10 and 50 mg/kg body weight to rats. Blood cell concentration was calculated from the following formula: BC=
Cb-Cp(1-Ht)
Ht where: BC = blood cell concentration Cb = whole blood concentration Cp = plasma concentration H t - - h e m a t o c r i t {measured in 20-pl heparinized glass capillary tubes}.
330
Plasma protein binding. 3,7-DMX plasma protein binding was studied: (i) in vitro: the unlabelled c o m p o u n d was added to rat plasma at 37°C under magnetic stirring to give the required concentrations (1, 10, 50 and 100 ug/ ml), and (ii) in vivo: blood samples were drawn from animals at different times after various 3,7-DMX doses, according to the data obtained in kinetic studies. Plasma protein binding was measured by equilibrium dialysis using a Dianorm apparatus [10]. The drug was assayed by HPLC in the 2 sides of dialytic cells (plasma and phosphate buffer (pH 7.4)). Data analysis Blood curves of 3,7-DMX concentrations vs. time were analysed following a one,compartment open model system after oral administration. Experimental points were fitted by a non-linear regression iterative program (Carl Peck, Uniformed Services University, Bethesda, MD, U.S.A.) on a HP-85 desk c o m p u t e r (Hewlett-Packard, U.S.A.). Areas under the blood concentrations vs. time curves (AUC) were calculated by the trapezoidal rule and extrapolated to infinity. For the calculation of apparent volume of distribution (aVd = f • D/(AUC • kel) and blood clearance (C1b = aVd. kel), the bioavailable fraction (f) of the administered 3,7-DMX dose (D) was considered equal to 1. Statistical analysis was b y ANOVA using a completely randomized block design [ 11 ].
RESULTS Analy tical procedures The reliability of blood 3,7-DMX assay was confirmed by quality control. The average "within d a y " coefficients of variation for i and 20 ~g/ml were 7.1% and 5.1%, and the "day-to-day" coefficients were 10.1% and 9.3%, respectively. The m e t h o d developed for analysis of 3,7-DMX and its metabolites in urine (Fig. 1) is definitely an improvement over existing ones, it specifically permits the quantitation of 3-MU and o f an u n k n o w n (polar metabolite(s)) peak, clearly separated from interfering peaks. Blood cells/plasma partition Blood cells/plasma ratios (180 min after dose) were slightly higher (0.86) after low doses of 3,7-DMX (5 ug/ml) than after high ones (0.70), the difference reaching statistical significance (P < 0.05). Blood/plasma ratios ranged from 0.93 to 0.85 (see Table I) enabling us to measure 3,7-DMX reliably in whole blood and to calculate plasma concentrations from the hematocrit values. Plasma protein binding. No significant difference in plasma protein-binding was detected when 3,7-DMX was incubated in vitro (1--100 ug/ml) with male rat plasma first
331
¢.o
1.19 _+0.08 a 4.21 +_ 0.58 3 9 . 3 9 +_ 1.87
5 10 50
a Mean +_ S.E. (n ~- 5).
Whole b l o o d ( u g / m l ) (Cb)
Dose (mg/kg) 1.27 +_ 0.80 4.84 _+0.63 4 6 . 7 4 + 2.16
Plasma ( u g / m l ) (Cp) 0.93 _+0.01 0.87 +_0.01 0 . 8 5 _+0.04
Blood/plasma ratio 0.47 + 0 . 0 0 4 0.46 + 0 . 0 0 5 0.49 _+0 . 0 0 8
Hematocrit (Ht)
1.10 _+0.09 3.48 + 0.53 3 2 . 0 4 +_ 3.20
B l o o d cells (BC) (/~g/ml)
0.86 _+0.03 0.71 +_0.02 0 . 6 9 _+0.07
BC/plasma ratio
T H E O B R O M I N E P A R T I T I O N IN B L O O D , P L A S M A A N D B L O O D C E L L S O F M A L E R A T S 1 8 0 m i n A F T E R D I F F E R E N T O R A L DOSES (5, 10 a n d 50 m g / k g )
TABLE I
for a range of intervals (1--6 h), though the 4-h incubation was selected for further experiments. The values of the u n b o u n d fraction ranged from a minimum of 0.91 + 0.02 to a m a x i m u m of 0.99 + 0.07 (mean 0.95). Similar results were obtained after in vivo administration of 3,7-DMX (1--100 mg/ kg orally), taking blood samples at various intervals (0.5--3 h). The u n b o u n d fraction ranged from 0.83 + 0.05 to 0.92 + 0.02 (mean 0.88).
Bioavailability The same rats received [8-14C] 3,7-DMX intravenously (1 mg/kg) and orally (1, 10 and 100 mg/kg) at different times. Recovery of radioactivity in urine was 80% as calculated from the i.v. experiment. Very little radioactivity (less than 2.5% after the i.v. dose) was present in feces. Bioavaflability, corrected for the recovery, was about 100% for the oral doses of 1 and 10 mg/kg and 95% for 100 mg/kg of 3,7-DMX. Kinetics after a single oral dose Five doses o f 3,7-DMX (1, 5, 10, 50 and 100 mg/kg) were given orally to groups of 6 rats. The average blood levels at different intervals are reported in Fig. 2 and kinetic parameters are summarized in Table II. No major changes occurred in the kel, aVd and C1b as a result of the administered dose.
100-
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1440 TIME
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Fig. 2. Average b l o o d c o n c e n t r a t i o n vs. t i m e curves f o r 3 , 7 - D M X in male rats a f t e r differ e n t oral d o s e s (6 a n i m a l s f o r e a c h ) : 1 m g / k g (o), 5 m g / k g (A), 10 m g / k g (=), 50 m g / k g ( : ) a n d 1 0 0 m g / k g (o).
333
5.24 5.20 1.83 1.32 0.71
1 5 10 50 100
-+ 2 . 1 1 d +-1.64 +_0.42 e +-0.20 e _+0.15 e
b aVd
FROM
MALE
0.20 + 0.02 0.28 -+0.03 f 0.15-+0.01 0 . 1 7 -+ 0 . 0 1 0.17 -+0.02
kel ( h -~ )
PARAMETERS
a C a l c u l a t e d b y t h e t r a p e z o i d a l rule. = f " D / ( A U C • kel ). c C1 b = a V d . k e l . a M e a n _+ S.E. (n - 6). Dunnet's test: e p < 0.05. f P < 0.01.
kab s ( h -~ )
Dose (mg/kg)
PHARMACOKINETIC THEOBROMINE
T A B L E II AFTER
3.78 2.57 4.87 4.23 4.18
-+ 0 . 4 5 -+0.27 f -+0.52 -+0.35 "+0.50
ORAL
2 0 3 -+ 26 832-+ 122 3096-+ 162 13 3 5 1 - + 1 1 5 7 2 8 6 8 9 _+3649
Auca._. ~ ( m g • 1-~ • r a i n )
DIFFERENT
Mean elimination half-life ( h )
RATS
(1/kg)
aVd b
+- 0 . 2 0 "+0.09 -+0.13 -+0.13 -+0.13
(1, 5, 50 a n d
1.68 1.38 ,1.36 1.40 1.31
DOSES
5.34 6.54 3.27 3.90 3.74
-+ 0 . 6 9 +0.75 f +-0.15 e _+0.41 e +-0.41 e
Clb c (ml/min/kg)
100 mg/kg) OF
Only for the dose of 5 mg/kg was there a significantly higher (P < 0.01) elimination rate constant (and consequent differences in related parameters). Because of the wide variability in estimated values, the Clb after the 1 mg/kg dose was at the limit of statistical significance (P ~ 0.05). The absorption rate constant (kabs) decreased (P < 0.05) as the dose increased. There was a proportional increase in the area under the curve (AUC) as the dose increased (Fig. 3). To reduce the individual variability between animals in the elimination rate constant and related parameters, in order to relate AUC to dose, the AUC values were multiplied by the estimated kel, as suggested by Wagner [12]. Regression for the 5 different 3,7-DMX doses (mg/kg) vs. AUC0-.oo • kel reached statistical significance at the P < 0.009 level (r = 0.99, F = 1464). In separate experiments (not shown here in detail) rats were injected i.v. with 3,7-DMX (1 and 5 mg/kg). The calculated kinetic parameters fell within the range o f those obtained in rats given the same dose of 3,7-DMX orally.
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3,7-DMX DOSE ( m g l k g ) Fig. 3. R e l a t i o n s h i p b e t w e e n oral 3,7-DMX d o s e a n d AUC 0 -~o • kel in male rats (6 animats f o r each point).
335
Average values after 1 and 5 mg/kg were 0.27 and 0.24 h -1 for kel, 1.05 and 0.94 1/kg for aVd and 299 and 1330 m g • 1-' • rain for AUC.
Kinetics after multiple doses 3,7-DMX was given orally at doses of 1, 10 or 100 mg/kg/day for 14 days. Average concentrations on days 3, 7, 14 and 15 (24 h after the last dose) are reported in Table III. At the lowest dose 3,7-DMX blood levels were not measurable (<0.05 ~g/ml). Despite the broad inter-individual variability, average concentrations after 10 mg/kg were relatively constant, while with the 100 mg/kg dose there was a tendency to accumulation. On days 14 and 15 blood levels rose by the same factor as the dose (10 times) (Table III). The kinetic parameters after 14 days of treatment with 3 doses are reported in Table IV. Although not statistically different, kel, aVd and Clb were slightly lower (obviously the elimination half-life was longer) after repeated than after single 3,7-DMX doses (see Table II). Only AUC values tended to be higher after chronic treatment but the difference was not statistically significant. Metabolic studies The 24 h urine collected after a single dose of 3,7-DMX (1, 10 and 100 mg/ kg) was analyzed for the presence of 3,7-DMX and a number of metabolites (Table V). The amount of identified products in relation to the total radioactivity present in urine was approximately 100% for each dose. 3,7-DMX was excreted, as such, in relatively large amounts, a b o u t 51--74% of the total identified radioactivity. The major metabolite was 6-amino-5-(N-methylformylamino) 1-methyluracil (6-AM-1-MU). The other metabolites identified, namely 3,7-dimethyluric acid (3,7-DMU), 3-methylxanthine (3-MX), 7-methylxanthine (7-MX), 7-methyluric acid (7-MU) and 3-methyluric acid (3-MU), only amounted to a small percentage of the dose. For 3 of the 6 male rats given 1 mg/kg {oral) urine was collected for 48 h. During the 24--48 h period average recovery of administered radioactivity was very low (4.3%) compared
T A B L E III T H E O B R O M I N E C O N C E N T R A T I O N S (ug/ml) IN B L O O D O F M A L E R A T S D U R I N G C H R O N I C T R E A T M E N T I M M E D I A T E L Y B E F O R E THE N E X T DOSE Dose (mg/kg)
10 100
Days o f t r e a t m e n t 3
7
14 a
15 b
0.30 _+0.07 c 0.96 -+0.39
0.14 _+0.06 0.67 + 0 . 1 5
0.48 _+0.17 4.27 -+0.25
0.41 +_0.21 5.52 _+ 1.72
a Last dose. b 24 h after t h e last dose. c Mean _+ S.E. (n = 6).
336
5.46 +_ 1.06 d 2.48 +_0.81 e 0.51 +_0.09 e
1 10 100
d M e a n + S.E. ( n = 6). e Dunnet's test: P ~ 0.01.
a Calculated b y t h e t r a p e z o i d a l rule. b a V d ~ f o D / ( A U C - kel ). c CI b ~_ a V d ° kel.
kab s (h-')
Dose (mg/kg) 0.14 _+0.01 0 . 1 6 +_0.02 0.17 _+0.02
kel (h-') 5.13 _+0.59 4.37 +_0 . 6 0 4 . 2 5 +_ 0.46
Elimination half-life ( h )
3 1 5 +_ 4 8 5313 _+ 874 37 3 0 7 _+6 7 3 7
Auc0a--,~ (mg 1-' r a i n )
1.44 +_0.19 0.83 +_0.16 1.25 +_0.35
(l/kg)
aVd b
3.52 +_ 0 . 7 4 2.16 +_0.36 3.72 +_ 1.27
Clb c (ml/min/kg)
P H A R M A C O K I N E T I C P A R A M E T E R S F R O M M A L E R A T S A F T E R C H R O N I C D A I L Y T R E A T M E N T (14 D A Y S ) WITH D I F F E R E N T O R A L D O S E S (1, 10 a n d 100 m g / k g ) O F T H E O B R O M I N E
T A B L E IV
TABLE V
(% U R I N A R Y
URINARY METABOLIC PATTERN BROMINE IN M A L E RATS AFTER 100 mg/kg) a 6-AM-1-MU
Days o f treatment
REPEATED
ACTIVITY) OF [8J4C]THEO D A I L Y O R A L D O S E S (1, 10 and
7-MU
7-MX
3-MX
3,7-DMU
3,7-DMX
2.3+-0.4 2.0+_0.6
4.2+-0.7 4.7+_0.7
6.0+_0.8 6.7+_0.9
5.0+_0.7 1.7+_0.9
56.2+_5.4 51.0+_4.9
2.0+-0.0 1.3+0.3
3.3+-0.7 2.7+_0.3
6.0+-1.0 3.7+_0.3
5.0+_0.6 4.0+_0.6
58.3+_5.4 72.3+_3.3
1.7+_0.3 2.0+_0.0
2.0 +- 0.0 2.3+_0.3
3.7+_0.3 3.0+_1.0
4.0+_0.0 4.0+_1.1
73.7+_1.2 72.3+3.4
Dose 1 mg/kg 1b 13
25.8+3.2 c 28.0+_2.6
DoselOm~kg 1 13
25.0+-3.2 16.3+_1.7
Dose 100 mg/kg 1 13
15.3+_0.3 16.0+_1.1
a Polar m e t a b o l i t e ( s ) and (3-MU) were <1 in all fractions. bn=6. c Mean +_ S.E. (n = 3).
POLAR METABOLITE($) (0 7 %) o
o
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Fig. 4. Urinary m e t a b o l i c p a t h w a y (0--48 h) o f [8-~4C]3,7-DMX in male rats a f t e r oral a d m i n i s t r a t i o n (1 m$/kg).
338
with the 0--24 h period (85.3%). No difference was found in the urinary metabolic pathway for the 2 collection periods. The 48 h urinary metabolic pathway of 3,7-DMX is schematically represented in Fig. 4. Metabolic studies in rats treated chronically (14 days) with 1, 10 or 100 mg/kg of 3,7-DMX orally yielded a pattern of urinary excretion similar to that after a single dose (Table V and Fig. 4). DISCUSSION 3,7-DMX is almost equally distributed in plasma and in blood cells and binds insignificantly to plasma proteins. Theobromine is well absorbed by the oral route, with absolute bioavailability close to unity; this is in good agreement with Shively and Tarka [13] who recovered 94--106% of administered labelled 3,7-DMX in urine. However the absorption rate constant markedly decreases as the dose of 3,7-DMX increases. Consequently, the peak blood level tends to appear later with larger doses of 3,7-DMX. It is noteworthy that over a wide range of doses, the elimination half-life and other pharmacokinetic parameters do not change significantly while the AUC and the steady-state blood levels after 10 and 100 mg/kg increase proportionally to the dose (Table III). In this respect, 3,7-DMX is quite different from caffeine which shows markedly dose-dependent kinetics in rats [14]. The t½ was variable in rats, the lowest value being 1.9 h and the highest 6.4 h. The average tl/~ was similar to that reported for man (6.1 + 0.7 h) by Drouillard et al. [15]. The metabolism of 3,7-DMX agrees closely with the data reported in the literature. Theobromine is excreted unchanged in the urine amounting to 50% of the administered dose. Thus, urinary excretion is one of the major processes of disposal of 3,7-DMX. Arnaud and Welsch [16] and more recently, Cornish et al. [17] found that the uracil metabolite (6-AM-1-MU) is by far the major metabolite of 3,7-DMX in rats. The uracil metabolite resulting from the metabolism of caffeine has been shown to have mild cytotoxic activity [18--20] but the role of other uracil metabolites in the toxicology of methylxanthines has not yet been established. The other N-demethylated metabolites (3-MX and 7-MX) and the metabolite (3,7-DMU) resulting from the oxidation of 3,7-DMX accounted for less than 10% of the administered dose while N-demethylated and oxidized products (7-MU and 3-MU) were relatively minor metabolites. The metabolic pathway of 3,7-DMX in rats is qualitatively similar to that reported for man [16,21]. Repeated administration, does not substantially affect the kinetics or metabolism of 3,7-DMX. Only at the highest dose used (100 mg/kg) was there a tendency to accumulation of 3,7-DMX to the detriment of its uracil metabolite (towards saturation of 3,7-DMX metabolism?). Although toxicological studies with 3,7-DMX have usually been performed at daffy doses exceeding 200 mg/kg [3,4], such a high dose was not included in the kinetic study since it would not be employed in long-term toxicological
339
experiments. The effects introduced into the distribution and possibly the toxicological profile of 3,7-DMX by such accumulation should be taken into account when extrapolating from animal toxicological data to the action of lower doses in animals and man. ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e C h o c o l a t e M a n u facturers' Association of the United States, McLean, VA. REFERENCES
1 S.M. Tarka Jr., The toxicology of cocoa and methylxanthines: A review of the literature. CRC Crit. Rev. Toxicol., 9 (1982) 275. 2 SGOGS-89, Evaluation of health aspects of caffeine as a food ingredient, prepared for Bureau of Foods, F o o d and Drug Administration, Department of Health, Education and Welfare, Washington, D.C. Contract No. FDA 223-75-2004 (1978). 3 S.M. Tarka Jr., B.L. Zoumas and J.H. Gans, Short-term effects of graded levels of theobromine in laboratory rodents. Toxicol. Appl. Pharmacol., 49 (1979) 127. 4 S.M. Tarka Jr., B.L. Zoumas and J.H. Gans, Effects of continuous administration of dietary theobromine on rat testicular weight and morphology. Toxicol. Appl. Pharmacol., 58 (1981) 76. 5 J.H. Gans, R. Korson, M.R. Cater and C.C. Ackerly, Effects of short-term and longterm theobromine administration to male dogs. Toxicol. Appl. Pharmacol., 53 (1980) 481. 6 V. Popovic and P. Popovic, Permanent cannulation o f aorta and vena cava in rats and ground squirrels. J. Appl. Physiol., 15 (1960) 727. 7 M. Bonati, R. Latini, F. Galletti, J.F. Young, G. Tognoni and S. Garattini, Caffeine disposition after oral doses. Clin. Pharmacol. Ther., 32 (1982) 98. 8 M. Bonati, R. Latini, E. Marzi, R. Cantoni and G. Belvedere, [2-14C]Caffeine metabolism in control and 3-methylcholanthrene induced rat liver microsomes b y high pressure liquid chromatography. Toxicol. Left., 7 (1980) 1. 9 M. Bonati, D. Castelli, R. Latini and S. Garattini, Comparison of gas-liquid chromatography with nitrogen-phosphorus selective detection and high-performance liquid chromatography methods for caffeine determination in plasma and tissues. J. Chromatogr., 164 {1979) 109. 10 H.J. Weder and M.H. Bickel, Interactions of drugs with proteins II: Experimental methods, treatment o f experimental data, and thermodynamics of binding reactions of thymoleptic drugs and model dyes. J. Pharm. Sci., 59 (1970) 1563. 11 M. Rocchetti and M. Recchia, SPBS: Statistical programs for biological sciences - Minicomputer software for applying routine biostatistical methods. Comput. Programs Biomed., 14 (1982) 7. 12 J.F. Wagner, A method of estimating relative absorption of a drug in a series of clinical studies in which blood levels are measured after single/or multiple doses. J. Pharm. Sci., 56 (1967) 652. 13 C.A. Shively and S.M. Tarka, Jr. Theobromine metabolism and pharmacokinetics in pregnant and non-pregnant female rats. Toxicol. Appl. Pharmacol., 67 (1983) 376. 14 R. Latini, M. Bonati, D. Castelli and S. Garattini, Dose-dependent kinetics of caffeine in rats. Toxicol. Lett., 2 (1978) 267. 15 D.D. Drouillard, E.S. Vesell and B.H. Dvorchik, Studies on theobromine disposition in normal subjects. Alterations induced b y dietary abstention from or exposure to methylxanthines. CI.in. Pharmacol. Ther., 23 (1978) 296.
340
16 M.J. Arnaud and C. Welsch, Metabolic pathway of theobromine in the rat and identification of two new metabolites in h u m a n urine. J. Agric. Food Chem., 27 (1979) 524. 17 H. Cornish, M. Brabec, S. Tarka, G. Miller, L. Radulovic and R. DeWit, A comparative study of the metabolism of theobromine in mammalian species. Toxicologist, 1
(1981) (1). 18 R. Latini, M. Bonati, E. Marzi and S. Garattini, Urinary excretion of an uracilic metabolite from caffeine by rat, monkey and man. Toxicol. Lett., 7 (1981) 267. 19 S. Garattini, E. Erba, L. Morasca, G. Peri, A. Mantovani, S. Filippeschi, F. Spreafico and M. Arnaud, In vitro and in vivo cytotoxicity of 6 amino-5-formylmethylamino1,3-desmethyl uracil, a uracilic metabolite of caffeine. Toxicol. Lett., 10 (1982) 313. 20 G. Peri, N. Polentarutti, S. Filippeschi, C. Malfiore, G. Amato, S. Pepe, M. Bonati, S. Garattini and A. Mantovani, In vitro and in vivo cytotoxicity of possible uracil metabolites of methylxanthines. Toxicol. Lett., 18 (1983) 133. 21 H.H. Cornish and A.A. Christman, A study of the metabolism of theobromine, theophylline, and caffeine in man. J. Biol. Chem., 228 (1957) 315.
341
I. KINETICS AND METABOLISM OF THEOBROMINE IN MALE RATS
M. BONATIa, R. LATINI a'*, B. SADURSKATM, E. RIVA a, F. GALLETTIa, J.F. BORZELLECA b , S.M. TARKAc , M.J. ARNAUD d and S. GARATTINIa
aIstituto di Ricerche Faramcologiche "Mario Negri" Via Eritrea 62, 20157Milan (Italy), bDepartment of Pharmacology, Medical College of Virginia, Richmond, VA (U.S.A.) CLife Sciences Division, Hershey Foods Technical Center, Hershey, PA., 17033 (U.S.A.) and dNestld Products Technical Assistance Co., Ltd., Research Department, CH-1814 La Tour-de-Peilz (Switzerland) (Received May 4th, 1983) (Accepted November 9th, 1983)
SUMMARY
On the basis of general pharmacological information (blood cells/plasma partition, plasma protein binding) and using HPLC as the principal analytical m e t h o d , we investigated the kinetics and metabolism of theobromine (a caffeine metabolite) in male rats after a single dose and after a 2 week chronic application. Doses in both conditions varied between 1 and 100 mg/ kg. In in vitro and in vivo the fraction of theobromine u n b o u n d to plasma proteins averaged 0.90 over a wide range of concentrations. No significant difference was f o u n d in the pharmacokinetic profile of the drug after acute or chronic treatment at different doses except for a reduction in the absorption rate constant as the dose increased. AUC values increased in proportion to the dose. The 2 treatment schedules were also similar as regards metabolism, at least 50% of the administered dose o f theobromine being excreted unchanged, and 25% as 6-amino-5-[N-methyl-formylamino]l-methyluracil. Only at the highest doses was there a tendency for theobromine to accumulate at the expense o f its major metabolite (a uracil compound).
Key words: Theobromine; Pharmacokinetics; Metabolism; Rats
*Present address: Cardiology Division, Stanford University Medical Center, Stanford, CA, U.S.A.
**Visiting scientist: Instytut Biofarmacji, Zaklad Biochemii, UL. Banacha 1, 02-097 Warszawa, Poland. All correspondence should be addressed to: Dr. Maurizio Bonati, Department of Clinical Pharmacology, Mario Negri Institute, Via Eritrea, 62, 20157 Milan, Italy. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
327
INTRODUCTION Theobromine (3,7-dimethylxanthine; 3,7-DMX) is a naturally occurring purine derivative present in cocoa, chocolate products and tea. Pharmacologically, 3,7-DMX has a moderate diuretic effect on the kidneys, causes mild cardiac stimulation and is virtually inactive as a central nervous system stimulant [ 1]. Recent concern about the safety of caffeine has raised questions regarding all methylxanthines [2 ]. There is a relative paucity of data on the toxicology of theobromine in laboratory animals and man [1]. Continuous short-term administration of high doses o f theobromine to rodents is known to result in thymic and testicular atrophy and growth depression [3,4]. In dogs, right atrial cardiomyopathy has been described [5]. Accurate information on the disposition of 3,7-DMX is essential for interpretation of toxicological data in laboratory animals. This paper reports the kinetics and metabolism of 3,7-DMX in male rats given oral doses ranging from I to 100 mg/kg/day on an acute and chronic basis. METHODS
Chemicals [8-14C]theobromine (3,7-DMX) (7.8 mCi/mmol), unlabelled 3,7-DMX, 3-methylxanthine (3-MX), 3-methyluric acid (3-MU), 7-methylxanthine (7-MX}, 7-methyluric acid (7-MU) and 3,7-dimethyluric acid (3,7-DMU) were generously supplied b y CMA (Chocolate Manufacturers Association of the United States}; 6-amino-5-{N-methyl-formylamino) 1-methyluracil(6AM-1-MU) was kindly supplied by Dr. Philipposian (Nestle, La Tour de Peilz, Switzerland). Reagents (LiChrosolv, Merck, Darmstadt, G.F.R.) were U.V. grade. Animals, housing and dosing CD-COBS mature male rats (Charles River, Italy} weighing 250--300 g were acclimatized to the research facility for 1 week before the study, receiving standard laboratory chow and water ad libitum. Animal rooms had controlled temperature (22°C) and light cycle (12/12 h). 3, 7-DMX kinetics after single oral doses Animals fasted for 16 h received 3,7-DMX by gavage in the free form suspended in a 1% water solution of Emulphor-EL-620, 24 h after carotid artery cannulation according to Popovic and Popovic [6]. Doses of 1, 5, 10, 50, 100 mg/kg b o d y weight were administered to groups of 6 animals. Blood samples from 0.05 to 0.20 ml were taken at various times (0, 15, 30, 60, 90, 120, 180, 240, 480 min and up to 24 h for the highest doses). Hematocrit was checked every 3 h. Fresh blood transfusions of 1--2 ml from a donor rat were given only for the dose of 1 mg/kg at 360 min where larger volumes had to be sampled. Blood samples were immediately diluted in 1.0 ml of 0.5% acetic acid in water and kept at 4°C until analysis {within 24 h).
328
Multiple dose study 3,7-DMX doses of 1, 10 and 100 mg/kg were administered by gavage every morning for 14 days to 3 groups of 6 animals, kept in the same conditions as in the acute experiment. Body weight was recorded daily. On the 7th day blood samples were taken from the tail vein of some individual animals, in order to establish a trough blood level profile during treatment. On day 12 (before the 12th 3,7-DMX dose) the carotid artery was cannulated. After the last dose (day 14) blood samples were drawn. On days 1, 7 and 13, instead of the unlabeUed c o m p o u n d the same dose of [8J4C]3,7-DMX (2 X 106 dpm/ rat) was administered to 3 animals (always the same ones) and feces and urine were collected separately for 24 h in metabolic cages. After measurement of the volume, urine was centrifuged at 15 000 rpm, and the supernatant was kept at - 2 0 ° C with other specimens until analysis.
Analy tical procedures Blood. One millilitre o f acetic acid (0.5%) and 50 ul of internal standard (10 pg/ml theophylline) were added to blood samples which were then processed according to a previously described caffeine assay [7] slightly modified. The samples were shaken for 30 min with 7 ml of chloroform/isopropyl alcohol (90 : 10), then the organic phase was transferred and evaporated in a water bath at 60°C under a gentle nitrogen stream. The residue was dissolved in 100 ~l of the chromatographic mobile phase and transferred to a water bath at 37°C for 10 min, so as to more easily dissolve 3,7-DMX, especially at higher doses. A b o u t 30 ul of sample were injected into the liquid chromatograph (Perkin-Elmer, Norwalk, C.T., U.S.A. -- series 2/2) equipped with an LC 15 s p e c t r o p h o t o m e t e r (280 nm) and a reversed phase column (Hibar RP-8, 7 ~m, 250 mm X 4 mm, Merck, Darmstadt, G.F.R.). A mobile phase of acetonitrile/acetic acid 0.5% ( 9 : 9 1 ) was used for elution at a flow rate of 1.3 ml/min. Urine. Urine (0.1 ml} was poured into a vial for total radioactivity counting, and 0.1 ml was injected into the high-pressure system together with an unlabelled standard mixture of 3,7-DMX and its metabolites (100 ug/ml). The chromatograph was equipped with a reversed phase column (Hibar RP-18, 7 ~m, 250 mm X 4 mm, Merck, Darmstadt, G.F.R.) and elutions were carried o u t with a linear gradient from 0% to 5% acetonitrile in 0.5% acetic acid (v/v) in water within 40 min, at a flow rate of 1.3 ml/min. The column eluent was collected in fractions directly into scintillation vials (by hand) using the UV absorption tracing on the recorder as reference (Fig. 1). After addition of 15 ml of scintillation mixture (Biofluor, NEN, England}, the vials were placed in a liquid scintillation counter (Beckman, Mod. B 2450}. The dpm of the various fractions were assigned to the different metabolites according to routine laboratory m e t h o d o l o g y [8] using the retention time on a UV tracing. Feces. Feces were collected on aluminium foil, weighed and kept a t - 2 0 ° C until analysis. They were homogenized in acetonitrile ( 1 : 1 0 , w : v ) , centrifuged at 15 000 rev./min, and 1 ml of supernatant was counted for radio-
329
uJ Z C)
C) a]
hJ 2:
:E
w w
:E
2
cr
O {:Ex w
-rr~
O n
b- c~
L C~
i
I
0
2a
TIME
(min)
Fig. 1. Representative high-pressure liquid chromatogram o f 3,7-DMX and its metabolites. Corresponding fractions collected to measure radioactivity are marked (o).
activity as described above. No chromatographic analysis was performed because of the small amount of radioactivity found. Quality control of 3,7-DMX blood assay. According to our previously described procedure [9], 1 and 20 ug/ml of 3,7-DMX were added to 2 pooled samples of rat blood. Each pool was divided into 1-ml aliquots and stored at - 2 0 ° C until analysis. Analysis was done in triplicate for both concentrations (1 and 20) during routine assays over 1 year. The percentage coefficient of variation (CV%) was calculated as the standard deviation divided by the mean times 100. Blood cells/plasma partition. The blood cells/plasma partition of 3,7-DMX was calculated from whole blood and plasma levels of the compound 180 min after administration of 5, 10 and 50 mg/kg body weight to rats. Blood cell concentration was calculated from the following formula: BC=
Cb-Cp(1-Ht)
Ht where: BC = blood cell concentration Cb = whole blood concentration Cp = plasma concentration H t - - h e m a t o c r i t {measured in 20-pl heparinized glass capillary tubes}.
330
Plasma protein binding. 3,7-DMX plasma protein binding was studied: (i) in vitro: the unlabelled c o m p o u n d was added to rat plasma at 37°C under magnetic stirring to give the required concentrations (1, 10, 50 and 100 ug/ ml), and (ii) in vivo: blood samples were drawn from animals at different times after various 3,7-DMX doses, according to the data obtained in kinetic studies. Plasma protein binding was measured by equilibrium dialysis using a Dianorm apparatus [10]. The drug was assayed by HPLC in the 2 sides of dialytic cells (plasma and phosphate buffer (pH 7.4)). Data analysis Blood curves of 3,7-DMX concentrations vs. time were analysed following a one,compartment open model system after oral administration. Experimental points were fitted by a non-linear regression iterative program (Carl Peck, Uniformed Services University, Bethesda, MD, U.S.A.) on a HP-85 desk c o m p u t e r (Hewlett-Packard, U.S.A.). Areas under the blood concentrations vs. time curves (AUC) were calculated by the trapezoidal rule and extrapolated to infinity. For the calculation of apparent volume of distribution (aVd = f • D/(AUC • kel) and blood clearance (C1b = aVd. kel), the bioavailable fraction (f) of the administered 3,7-DMX dose (D) was considered equal to 1. Statistical analysis was b y ANOVA using a completely randomized block design [ 11 ].
RESULTS Analy tical procedures The reliability of blood 3,7-DMX assay was confirmed by quality control. The average "within d a y " coefficients of variation for i and 20 ~g/ml were 7.1% and 5.1%, and the "day-to-day" coefficients were 10.1% and 9.3%, respectively. The m e t h o d developed for analysis of 3,7-DMX and its metabolites in urine (Fig. 1) is definitely an improvement over existing ones, it specifically permits the quantitation of 3-MU and o f an u n k n o w n (polar metabolite(s)) peak, clearly separated from interfering peaks. Blood cells/plasma partition Blood cells/plasma ratios (180 min after dose) were slightly higher (0.86) after low doses of 3,7-DMX (5 ug/ml) than after high ones (0.70), the difference reaching statistical significance (P < 0.05). Blood/plasma ratios ranged from 0.93 to 0.85 (see Table I) enabling us to measure 3,7-DMX reliably in whole blood and to calculate plasma concentrations from the hematocrit values. Plasma protein binding. No significant difference in plasma protein-binding was detected when 3,7-DMX was incubated in vitro (1--100 ug/ml) with male rat plasma first
331
¢.o
1.19 _+0.08 a 4.21 +_ 0.58 3 9 . 3 9 +_ 1.87
5 10 50
a Mean +_ S.E. (n ~- 5).
Whole b l o o d ( u g / m l ) (Cb)
Dose (mg/kg) 1.27 +_ 0.80 4.84 _+0.63 4 6 . 7 4 + 2.16
Plasma ( u g / m l ) (Cp) 0.93 _+0.01 0.87 +_0.01 0 . 8 5 _+0.04
Blood/plasma ratio 0.47 + 0 . 0 0 4 0.46 + 0 . 0 0 5 0.49 _+0 . 0 0 8
Hematocrit (Ht)
1.10 _+0.09 3.48 + 0.53 3 2 . 0 4 +_ 3.20
B l o o d cells (BC) (/~g/ml)
0.86 _+0.03 0.71 +_0.02 0 . 6 9 _+0.07
BC/plasma ratio
T H E O B R O M I N E P A R T I T I O N IN B L O O D , P L A S M A A N D B L O O D C E L L S O F M A L E R A T S 1 8 0 m i n A F T E R D I F F E R E N T O R A L DOSES (5, 10 a n d 50 m g / k g )
TABLE I
for a range of intervals (1--6 h), though the 4-h incubation was selected for further experiments. The values of the u n b o u n d fraction ranged from a minimum of 0.91 + 0.02 to a m a x i m u m of 0.99 + 0.07 (mean 0.95). Similar results were obtained after in vivo administration of 3,7-DMX (1--100 mg/ kg orally), taking blood samples at various intervals (0.5--3 h). The u n b o u n d fraction ranged from 0.83 + 0.05 to 0.92 + 0.02 (mean 0.88).
Bioavailability The same rats received [8-14C] 3,7-DMX intravenously (1 mg/kg) and orally (1, 10 and 100 mg/kg) at different times. Recovery of radioactivity in urine was 80% as calculated from the i.v. experiment. Very little radioactivity (less than 2.5% after the i.v. dose) was present in feces. Bioavaflability, corrected for the recovery, was about 100% for the oral doses of 1 and 10 mg/kg and 95% for 100 mg/kg of 3,7-DMX. Kinetics after a single oral dose Five doses o f 3,7-DMX (1, 5, 10, 50 and 100 mg/kg) were given orally to groups of 6 rats. The average blood levels at different intervals are reported in Fig. 2 and kinetic parameters are summarized in Table II. No major changes occurred in the kel, aVd and C1b as a result of the administered dose.
100-
= ~
= =
I----I
E
~
/"~:1C~,~
~
ID---~C]
10
lmglkg 5mg/kg lOmglkg 50mg/kg
.0-~ -~--o lOOmg/kg
v Z
_o z ,,,
1,0
z 12) 123 0 o._1
0.1
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i
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i
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i
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1440 TIME
(min)
Fig. 2. Average b l o o d c o n c e n t r a t i o n vs. t i m e curves f o r 3 , 7 - D M X in male rats a f t e r differ e n t oral d o s e s (6 a n i m a l s f o r e a c h ) : 1 m g / k g (o), 5 m g / k g (A), 10 m g / k g (=), 50 m g / k g ( : ) a n d 1 0 0 m g / k g (o).
333
5.24 5.20 1.83 1.32 0.71
1 5 10 50 100
-+ 2 . 1 1 d +-1.64 +_0.42 e +-0.20 e _+0.15 e
b aVd
FROM
MALE
0.20 + 0.02 0.28 -+0.03 f 0.15-+0.01 0 . 1 7 -+ 0 . 0 1 0.17 -+0.02
kel ( h -~ )
PARAMETERS
a C a l c u l a t e d b y t h e t r a p e z o i d a l rule. = f " D / ( A U C • kel ). c C1 b = a V d . k e l . a M e a n _+ S.E. (n - 6). Dunnet's test: e p < 0.05. f P < 0.01.
kab s ( h -~ )
Dose (mg/kg)
PHARMACOKINETIC THEOBROMINE
T A B L E II AFTER
3.78 2.57 4.87 4.23 4.18
-+ 0 . 4 5 -+0.27 f -+0.52 -+0.35 "+0.50
ORAL
2 0 3 -+ 26 832-+ 122 3096-+ 162 13 3 5 1 - + 1 1 5 7 2 8 6 8 9 _+3649
Auca._. ~ ( m g • 1-~ • r a i n )
DIFFERENT
Mean elimination half-life ( h )
RATS
(1/kg)
aVd b
+- 0 . 2 0 "+0.09 -+0.13 -+0.13 -+0.13
(1, 5, 50 a n d
1.68 1.38 ,1.36 1.40 1.31
DOSES
5.34 6.54 3.27 3.90 3.74
-+ 0 . 6 9 +0.75 f +-0.15 e _+0.41 e +-0.41 e
Clb c (ml/min/kg)
100 mg/kg) OF
Only for the dose of 5 mg/kg was there a significantly higher (P < 0.01) elimination rate constant (and consequent differences in related parameters). Because of the wide variability in estimated values, the Clb after the 1 mg/kg dose was at the limit of statistical significance (P ~ 0.05). The absorption rate constant (kabs) decreased (P < 0.05) as the dose increased. There was a proportional increase in the area under the curve (AUC) as the dose increased (Fig. 3). To reduce the individual variability between animals in the elimination rate constant and related parameters, in order to relate AUC to dose, the AUC values were multiplied by the estimated kel, as suggested by Wagner [12]. Regression for the 5 different 3,7-DMX doses (mg/kg) vs. AUC0-.oo • kel reached statistical significance at the P < 0.009 level (r = 0.99, F = 1464). In separate experiments (not shown here in detail) rats were injected i.v. with 3,7-DMX (1 and 5 mg/kg). The calculated kinetic parameters fell within the range o f those obtained in rats given the same dose of 3,7-DMX orally.
100-
g
t
u
50
o
'
'
'
'
' 5O
'
'
'
'
1(~0
3,7-DMX DOSE ( m g l k g ) Fig. 3. R e l a t i o n s h i p b e t w e e n oral 3,7-DMX d o s e a n d AUC 0 -~o • kel in male rats (6 animats f o r each point).
335
Average values after 1 and 5 mg/kg were 0.27 and 0.24 h -1 for kel, 1.05 and 0.94 1/kg for aVd and 299 and 1330 m g • 1-' • rain for AUC.
Kinetics after multiple doses 3,7-DMX was given orally at doses of 1, 10 or 100 mg/kg/day for 14 days. Average concentrations on days 3, 7, 14 and 15 (24 h after the last dose) are reported in Table III. At the lowest dose 3,7-DMX blood levels were not measurable (<0.05 ~g/ml). Despite the broad inter-individual variability, average concentrations after 10 mg/kg were relatively constant, while with the 100 mg/kg dose there was a tendency to accumulation. On days 14 and 15 blood levels rose by the same factor as the dose (10 times) (Table III). The kinetic parameters after 14 days of treatment with 3 doses are reported in Table IV. Although not statistically different, kel, aVd and Clb were slightly lower (obviously the elimination half-life was longer) after repeated than after single 3,7-DMX doses (see Table II). Only AUC values tended to be higher after chronic treatment but the difference was not statistically significant. Metabolic studies The 24 h urine collected after a single dose of 3,7-DMX (1, 10 and 100 mg/ kg) was analyzed for the presence of 3,7-DMX and a number of metabolites (Table V). The amount of identified products in relation to the total radioactivity present in urine was approximately 100% for each dose. 3,7-DMX was excreted, as such, in relatively large amounts, a b o u t 51--74% of the total identified radioactivity. The major metabolite was 6-amino-5-(N-methylformylamino) 1-methyluracil (6-AM-1-MU). The other metabolites identified, namely 3,7-dimethyluric acid (3,7-DMU), 3-methylxanthine (3-MX), 7-methylxanthine (7-MX), 7-methyluric acid (7-MU) and 3-methyluric acid (3-MU), only amounted to a small percentage of the dose. For 3 of the 6 male rats given 1 mg/kg {oral) urine was collected for 48 h. During the 24--48 h period average recovery of administered radioactivity was very low (4.3%) compared
T A B L E III T H E O B R O M I N E C O N C E N T R A T I O N S (ug/ml) IN B L O O D O F M A L E R A T S D U R I N G C H R O N I C T R E A T M E N T I M M E D I A T E L Y B E F O R E THE N E X T DOSE Dose (mg/kg)
10 100
Days o f t r e a t m e n t 3
7
14 a
15 b
0.30 _+0.07 c 0.96 -+0.39
0.14 _+0.06 0.67 + 0 . 1 5
0.48 _+0.17 4.27 -+0.25
0.41 +_0.21 5.52 _+ 1.72
a Last dose. b 24 h after t h e last dose. c Mean _+ S.E. (n = 6).
336
5.46 +_ 1.06 d 2.48 +_0.81 e 0.51 +_0.09 e
1 10 100
d M e a n + S.E. ( n = 6). e Dunnet's test: P ~ 0.01.
a Calculated b y t h e t r a p e z o i d a l rule. b a V d ~ f o D / ( A U C - kel ). c CI b ~_ a V d ° kel.
kab s (h-')
Dose (mg/kg) 0.14 _+0.01 0 . 1 6 +_0.02 0.17 _+0.02
kel (h-') 5.13 _+0.59 4.37 +_0 . 6 0 4 . 2 5 +_ 0.46
Elimination half-life ( h )
3 1 5 +_ 4 8 5313 _+ 874 37 3 0 7 _+6 7 3 7
Auc0a--,~ (mg 1-' r a i n )
1.44 +_0.19 0.83 +_0.16 1.25 +_0.35
(l/kg)
aVd b
3.52 +_ 0 . 7 4 2.16 +_0.36 3.72 +_ 1.27
Clb c (ml/min/kg)
P H A R M A C O K I N E T I C P A R A M E T E R S F R O M M A L E R A T S A F T E R C H R O N I C D A I L Y T R E A T M E N T (14 D A Y S ) WITH D I F F E R E N T O R A L D O S E S (1, 10 a n d 100 m g / k g ) O F T H E O B R O M I N E
T A B L E IV
TABLE V
(% U R I N A R Y
URINARY METABOLIC PATTERN BROMINE IN M A L E RATS AFTER 100 mg/kg) a 6-AM-1-MU
Days o f treatment
REPEATED
ACTIVITY) OF [8J4C]THEO D A I L Y O R A L D O S E S (1, 10 and
7-MU
7-MX
3-MX
3,7-DMU
3,7-DMX
2.3+-0.4 2.0+_0.6
4.2+-0.7 4.7+_0.7
6.0+_0.8 6.7+_0.9
5.0+_0.7 1.7+_0.9
56.2+_5.4 51.0+_4.9
2.0+-0.0 1.3+0.3
3.3+-0.7 2.7+_0.3
6.0+-1.0 3.7+_0.3
5.0+_0.6 4.0+_0.6
58.3+_5.4 72.3+_3.3
1.7+_0.3 2.0+_0.0
2.0 +- 0.0 2.3+_0.3
3.7+_0.3 3.0+_1.0
4.0+_0.0 4.0+_1.1
73.7+_1.2 72.3+3.4
Dose 1 mg/kg 1b 13
25.8+3.2 c 28.0+_2.6
DoselOm~kg 1 13
25.0+-3.2 16.3+_1.7
Dose 100 mg/kg 1 13
15.3+_0.3 16.0+_1.1
a Polar m e t a b o l i t e ( s ) and (3-MU) were <1 in all fractions. bn=6. c Mean +_ S.E. (n = 3).
POLAR METABOLITE($) (0 7 %) o
o
H-N I ' ~ , F - ~ N - C H I
3
H- N ~ I - I
I
t
O H ~ ' ~
N_
/
CH3 THEOBROMINE (/.,81 Ol,) "-~
o DIME THYL-ALLANTOIN
o
H_N I ~ - . - . - - N , - C H 3 0 . ~ N I ~ N 7~.,j
H- N i ~ " ~ - - ~ - N - H o ~ N ]J~ N ~_.J
H
1
N- METHYLUREA
O-~ ~NI ~ ~-NH 2 CH 3 3?-DAU (29.6°1o)
I
CH3 H 3.7-DMU (5 0%)
CH3
?-MX (5.1%)
'
/CH3 N~cHO
3-MX(?.4°/o)
'
~
0
oJ","Jl'", A° H H 7-MU ( 29%)
0
._N
I
CH3 H 3-MU (~1°1o)
Fig. 4. Urinary m e t a b o l i c p a t h w a y (0--48 h) o f [8-~4C]3,7-DMX in male rats a f t e r oral a d m i n i s t r a t i o n (1 m$/kg).
338
with the 0--24 h period (85.3%). No difference was found in the urinary metabolic pathway for the 2 collection periods. The 48 h urinary metabolic pathway of 3,7-DMX is schematically represented in Fig. 4. Metabolic studies in rats treated chronically (14 days) with 1, 10 or 100 mg/kg of 3,7-DMX orally yielded a pattern of urinary excretion similar to that after a single dose (Table V and Fig. 4). DISCUSSION 3,7-DMX is almost equally distributed in plasma and in blood cells and binds insignificantly to plasma proteins. Theobromine is well absorbed by the oral route, with absolute bioavailability close to unity; this is in good agreement with Shively and Tarka [13] who recovered 94--106% of administered labelled 3,7-DMX in urine. However the absorption rate constant markedly decreases as the dose of 3,7-DMX increases. Consequently, the peak blood level tends to appear later with larger doses of 3,7-DMX. It is noteworthy that over a wide range of doses, the elimination half-life and other pharmacokinetic parameters do not change significantly while the AUC and the steady-state blood levels after 10 and 100 mg/kg increase proportionally to the dose (Table III). In this respect, 3,7-DMX is quite different from caffeine which shows markedly dose-dependent kinetics in rats [14]. The t½ was variable in rats, the lowest value being 1.9 h and the highest 6.4 h. The average tl/~ was similar to that reported for man (6.1 + 0.7 h) by Drouillard et al. [15]. The metabolism of 3,7-DMX agrees closely with the data reported in the literature. Theobromine is excreted unchanged in the urine amounting to 50% of the administered dose. Thus, urinary excretion is one of the major processes of disposal of 3,7-DMX. Arnaud and Welsch [16] and more recently, Cornish et al. [17] found that the uracil metabolite (6-AM-1-MU) is by far the major metabolite of 3,7-DMX in rats. The uracil metabolite resulting from the metabolism of caffeine has been shown to have mild cytotoxic activity [18--20] but the role of other uracil metabolites in the toxicology of methylxanthines has not yet been established. The other N-demethylated metabolites (3-MX and 7-MX) and the metabolite (3,7-DMU) resulting from the oxidation of 3,7-DMX accounted for less than 10% of the administered dose while N-demethylated and oxidized products (7-MU and 3-MU) were relatively minor metabolites. The metabolic pathway of 3,7-DMX in rats is qualitatively similar to that reported for man [16,21]. Repeated administration, does not substantially affect the kinetics or metabolism of 3,7-DMX. Only at the highest dose used (100 mg/kg) was there a tendency to accumulation of 3,7-DMX to the detriment of its uracil metabolite (towards saturation of 3,7-DMX metabolism?). Although toxicological studies with 3,7-DMX have usually been performed at daffy doses exceeding 200 mg/kg [3,4], such a high dose was not included in the kinetic study since it would not be employed in long-term toxicological
339
experiments. The effects introduced into the distribution and possibly the toxicological profile of 3,7-DMX by such accumulation should be taken into account when extrapolating from animal toxicological data to the action of lower doses in animals and man. ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e C h o c o l a t e M a n u facturers' Association of the United States, McLean, VA. REFERENCES
1 S.M. Tarka Jr., The toxicology of cocoa and methylxanthines: A review of the literature. CRC Crit. Rev. Toxicol., 9 (1982) 275. 2 SGOGS-89, Evaluation of health aspects of caffeine as a food ingredient, prepared for Bureau of Foods, F o o d and Drug Administration, Department of Health, Education and Welfare, Washington, D.C. Contract No. FDA 223-75-2004 (1978). 3 S.M. Tarka Jr., B.L. Zoumas and J.H. Gans, Short-term effects of graded levels of theobromine in laboratory rodents. Toxicol. Appl. Pharmacol., 49 (1979) 127. 4 S.M. Tarka Jr., B.L. Zoumas and J.H. Gans, Effects of continuous administration of dietary theobromine on rat testicular weight and morphology. Toxicol. Appl. Pharmacol., 58 (1981) 76. 5 J.H. Gans, R. Korson, M.R. Cater and C.C. Ackerly, Effects of short-term and longterm theobromine administration to male dogs. Toxicol. Appl. Pharmacol., 53 (1980) 481. 6 V. Popovic and P. Popovic, Permanent cannulation o f aorta and vena cava in rats and ground squirrels. J. Appl. Physiol., 15 (1960) 727. 7 M. Bonati, R. Latini, F. Galletti, J.F. Young, G. Tognoni and S. Garattini, Caffeine disposition after oral doses. Clin. Pharmacol. Ther., 32 (1982) 98. 8 M. Bonati, R. Latini, E. Marzi, R. Cantoni and G. Belvedere, [2-14C]Caffeine metabolism in control and 3-methylcholanthrene induced rat liver microsomes b y high pressure liquid chromatography. Toxicol. Left., 7 (1980) 1. 9 M. Bonati, D. Castelli, R. Latini and S. Garattini, Comparison of gas-liquid chromatography with nitrogen-phosphorus selective detection and high-performance liquid chromatography methods for caffeine determination in plasma and tissues. J. Chromatogr., 164 {1979) 109. 10 H.J. Weder and M.H. Bickel, Interactions of drugs with proteins II: Experimental methods, treatment o f experimental data, and thermodynamics of binding reactions of thymoleptic drugs and model dyes. J. Pharm. Sci., 59 (1970) 1563. 11 M. Rocchetti and M. Recchia, SPBS: Statistical programs for biological sciences - Minicomputer software for applying routine biostatistical methods. Comput. Programs Biomed., 14 (1982) 7. 12 J.F. Wagner, A method of estimating relative absorption of a drug in a series of clinical studies in which blood levels are measured after single/or multiple doses. J. Pharm. Sci., 56 (1967) 652. 13 C.A. Shively and S.M. Tarka, Jr. Theobromine metabolism and pharmacokinetics in pregnant and non-pregnant female rats. Toxicol. Appl. Pharmacol., 67 (1983) 376. 14 R. Latini, M. Bonati, D. Castelli and S. Garattini, Dose-dependent kinetics of caffeine in rats. Toxicol. Lett., 2 (1978) 267. 15 D.D. Drouillard, E.S. Vesell and B.H. Dvorchik, Studies on theobromine disposition in normal subjects. Alterations induced b y dietary abstention from or exposure to methylxanthines. CI.in. Pharmacol. Ther., 23 (1978) 296.
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16 M.J. Arnaud and C. Welsch, Metabolic pathway of theobromine in the rat and identification of two new metabolites in h u m a n urine. J. Agric. Food Chem., 27 (1979) 524. 17 H. Cornish, M. Brabec, S. Tarka, G. Miller, L. Radulovic and R. DeWit, A comparative study of the metabolism of theobromine in mammalian species. Toxicologist, 1
(1981) (1). 18 R. Latini, M. Bonati, E. Marzi and S. Garattini, Urinary excretion of an uracilic metabolite from caffeine by rat, monkey and man. Toxicol. Lett., 7 (1981) 267. 19 S. Garattini, E. Erba, L. Morasca, G. Peri, A. Mantovani, S. Filippeschi, F. Spreafico and M. Arnaud, In vitro and in vivo cytotoxicity of 6 amino-5-formylmethylamino1,3-desmethyl uracil, a uracilic metabolite of caffeine. Toxicol. Lett., 10 (1982) 313. 20 G. Peri, N. Polentarutti, S. Filippeschi, C. Malfiore, G. Amato, S. Pepe, M. Bonati, S. Garattini and A. Mantovani, In vitro and in vivo cytotoxicity of possible uracil metabolites of methylxanthines. Toxicol. Lett., 18 (1983) 133. 21 H.H. Cornish and A.A. Christman, A study of the metabolism of theobromine, theophylline, and caffeine in man. J. Biol. Chem., 228 (1957) 315.
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