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The effect of phenobarbital on chlorphentermine-induced alveolar foam cells in rats
171-173(1977)
TOXICOLOGYANDAPPLIEDPHARMACOLOGY‘~&
SHORT The
COMMUNICATION
Effect of Phenobarbital Induced Alveolar
on ChlorphentermineFoam Cells in Rats
The Effect of Phenobarbital on Chlorphentermine-Induced Alveolar Foam Cells in Rats. SVENDSEN, 0. (1977). Toxicol. Appl. Phurmacol. 40, 171-173. Treatment of rats 5 days/week for 4 weekswith chlorphentermine (50 mg/kg) produced pulmonary intra-alveolar accumulations of typical foam cells. Alveolar foam cells did not develop in the lungs from rats treated simultaneously with chlorphentermine and phenobarbital (30 mg/kg). The findings suggest that treatment of rats with phenobarbital increases the metabolic degradation of chlorphentermine and that this is the mechanism for the observed protection afforded by phenobarbital. The anorexigenic drug chlorphentermine is known to produce pulmonary intra-alveolar accumulations of foam cells in rats (Franken et al., 1970; Magnusson and Magnusson, 1972; Hruban et al., 1973; Parwaresch et al., 1973; Karabelnik et al., 1974). Chlorphentermine is highly concentrated in the lungs of rats (Dubnick et al., 1968), especially after chronic treatment (Liillmann et al., 1973b), and it is slowly excreted. The plasma halflife in rats is 3.9 hr from the a-slope of the disposition curve, but the concentration in plasma does not change much from 9 to 24 hr after chlorphentermine administration (Eichelbaum et al., 1970). The half-life in lungs is about 50 hr (Eichelbaum et al., 1970). The present investigation was initiated in order to elucidate the influence of phenobarbital on chlorphentermine-induced alveolar foam cells. METHODS Fifteen Wistar/Af/Han/Mol (Han 67) male rats (SPF), weighing 195-222 g, were used for the experiment. The rats were divided into three groups, each consisting of five animals. One group served as a control group and was given 0.9 ‘A NaCl. The second group received chlorphentermine, 50 mg/kg/day po (1 ml/100 g), in the morning 5 days/week, while the third group received phenobarbital (30 mg/kg/day, po) in the afternoon 5 days/week in addition to chlorpentermine. Chlorphentermine and phenobarbital were dissolved in 0.9% NaCl. The rats were weighed before and every day throughout treatment, which lasted for 4 weeks. All rats were anesthetized by iv injection of Narcodorm (enibomal-Na) and killed by exsanguination in the morning the day after the final dosing. The trachea was ligated in order to avoid collapse of the lungs during excision. The lungs were weighed and fixed in phosphate-buffered 10 % formalin. Longitudinal slices of both lungs were embedded in paraffin, sectioned 6 pm thick, and stained with hematoxylin and eosin. Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
171 ISSNOO41-008X
172
SHORT
RESULTS
COMMUNICATION
AND
DISCUSSION
Diffuse accumulations of typical foam cells were found intra-alveolarly in lungs from the animals treated with chlorphentermine only. The lungs from rats receiving both chlorphentermine and phenobarbital were histologically normal and no foam cells could be observed except from occasional small subpleural foci. Similar foci were also found in the lungs from the control rats and it is a well-known spontaneous entity in the lungs of the rat (Flodh et al., 1974). The relative weight of the lungs from each group is shown in Table 1. The mean relative weight of the lungs from the group receiving chlorphentermine only was significantly higher (p < 0.05, Student’s t test) than that of the control group. The mean relative weight of the lungs from the group receiving both chlorphentermine and phenobarbital was not changed by the treatment. TABLE
1
RELATIVE WEIGHT OF THE LUNGS OF RATS TREATED WITH CHLORPHENTERMINE
Treatment Animal number 1 2 3 4 5
Mean rt SD
Chlorphentermine 0.9 % NaCl
(50 mg/W
0.51” (306)b 0.41 (325) 0.45 (298) 0.46 (290) 0.52 (295) 0.47 f 0.044
0.70 0.52 0.56 0.64 0.52 0.59
(245) (297) (275) (272) (259) + 0.080’
Chlorphentermine + Phenobarbital (30 m&d 0.46 0.54 0.47 0.44 0.46 0.48
(290) (250) (299) (300) (261) k 0.034
a Values are in grams per 100 grams body weight. * The final body weights of the animals are shown in parentheses. c Statistically different (p < 0.05) from the control group.
Chlorphentermine-induced accumulations of pulmonary foam cells in rats are thus prevented by simultaneous treatment with phenobarbital. Phenobarbital has wellknown multipotent inducing properties of drug metabolizing enzyme systems (Conney, 1967). Chlorphentermine has proved to be very stable to enzyme degradation in vitro (Dubnick et al., 1968). Studies by Opitz and Weischer (1966) and Dubnick et al. (1968) suggest that chlorphentermine is mainly excreted unchanged by rats, and no evidence of microsomal biotransformation has been found (Dubnick et al., 1968). However, a small portion is biotransformed in rats (Dubnick et al., 1968; Ryrfeldt, 1970; Caldwell et al., 1975). Based on the finding of a more polar and hydrolyzable metabolite in bile (Ltillmann et al., 19736) and in urine of rats (Ryrfeldt, 1970; Caldwell et al., 1975), it is concluded that the metabolite may be a conjugate between some endogenous substance and the amino group of the chlorphentermine molecule. The findings in the present study suggest that treatment with phenobarbital increased the metabolic degradation of chlorphentermine. A phenobarbital-induced increase in
SHORT
173
COMMUNICATION
metabolic degradation of chlorphentermine or metabolites might be elicited through a preexisting metabolic pathway or by induction of a new one. Phospholipid-drug complexes are accumulated in the alveolar foam cells (Liillmann et al., 1973a). Therefore, the possibility could also be considered that phenobarbital stimulated enzymes not involved in the metabolic biotransformation of chlorphentermine and otherwise related to the catabolism of phospholipid-drug complexes. ACKNOWLEDGMENTS
The author wishesto thank ProfessorTage Moller for the evaluation of the histological slides. REFERENCES J., KBSTER, U., SMITH, R. L., AND WILLIAMS, R. T. (1975).Speciesvariations in the N-oxidation of chlorphentermine.Biochem. Pharmacol. 24,2225-2232. CONNEY, A. H. (1967). Pharmacologicalimplications of microsomal enzyme induction. CALDWELL,
Pharmacol.
Rev. 19,317-366.
C. A., HARTIGAN, J. M., AND PHILLIPS, G. E. (1968).Distribution and metabolismof chlorphentermine-Cl4in rats and mice. Biochem. Pharmacol. 17, 1243-1250. EICHELBAUM, M., HENGSTMANN, J. H., AND DENGLER, H. J. (1970).Das Verteilungsmusterdes Chlorphenterminsbei Ratte, Kaninchenund Schwein.Arch. Pharmacol. 267, 446456. FLODH, H., MAGNUSSON, G., AND MAGNUSSON, 0. (1974).Pulmonary foam cells in rats of different age.2. Versuchstierk. 16,299-312. FRANKEN, G., LULLMANN, H., AND SIEGFRIEDT, A. (1970).The occurrenceof hugecellsin pulmonary alveoli of rats treated by an anorexicdrug. Arzneim.-Forsch. 20, 417-419. HRUBAN, Z., SLESERS, A., AND ASCHENBRENNER, I. (1973).Pulmonary intra-alveolarhistiocytosisinducedby drugs. Toxicol. Appl. Pharmacol. 26,72-85. KARABELNIK, D., ZBINDEN, G., AND BAUMGARTNER, E. (1974).Drug-induced foam cell reactions in rats. I. Histopathologicand cytochemicalobservationsafter treatment with chlorphentermine,RMI 10.393,and Ro 4-4318.Toxicol. Appl. Pharmacol. 27,395-407. LOLLMANN, H., L~LLMANN-RAUCH, R., AND WASSERMANN, 0. (1973a).Drug-induced phospholipidosis.Ger. Med. Month. 3, 128-135. L~LLMANN, H., ROSSEN, E., AND SEILER, K. -U. (1973b).The pharmacokinetics of phentermine and chlorphenterminein chronically treated rats. J. Pharm. Pharmacol. 25,239-243. MAGNUSSON, G., AND MAGNUSSON, 0. (1972).Cloforex-inducedpulmonary changesin rats. DUBNICK,
B., TOWNE,
Beitr. Path. Anat. 146, 79-88. K., AND WEISCHER, M. -L. (1966).Die AusscheidunganorexigenerPhenylalkylamine mit demHarn. Arzneim.-Forsch. 16, 1311-1313. PARWARESCH, M. R., REIL, G. -H., AND SEILER, K. -U. (1973).Uber die Tier- und Organspezifitat morphologischerVeranderungennach chronischerChlorphentermingabe.Res. Exp. OPITZ,
Med. 161,272-288.
AA. (1970).The distribution, in the mouseand rat. Acta Pharmacol.
RYRFELDT,
elimination,
and biotransformation
of 14C-Cloforex
Toxicol. 28,391X)5. OVE SVENDSEN
Department of Pharmacology and Toxicology, H. Lundbeck & Co. A/S, DK 2500 Copenhagen- Valby, Denmark Received October 25, 1976; accepted December 19, 1976
TOXICOLOGYANDAPPLIEDPHARMACOLOGY‘~&
SHORT The
COMMUNICATION
Effect of Phenobarbital Induced Alveolar
on ChlorphentermineFoam Cells in Rats
The Effect of Phenobarbital on Chlorphentermine-Induced Alveolar Foam Cells in Rats. SVENDSEN, 0. (1977). Toxicol. Appl. Phurmacol. 40, 171-173. Treatment of rats 5 days/week for 4 weekswith chlorphentermine (50 mg/kg) produced pulmonary intra-alveolar accumulations of typical foam cells. Alveolar foam cells did not develop in the lungs from rats treated simultaneously with chlorphentermine and phenobarbital (30 mg/kg). The findings suggest that treatment of rats with phenobarbital increases the metabolic degradation of chlorphentermine and that this is the mechanism for the observed protection afforded by phenobarbital. The anorexigenic drug chlorphentermine is known to produce pulmonary intra-alveolar accumulations of foam cells in rats (Franken et al., 1970; Magnusson and Magnusson, 1972; Hruban et al., 1973; Parwaresch et al., 1973; Karabelnik et al., 1974). Chlorphentermine is highly concentrated in the lungs of rats (Dubnick et al., 1968), especially after chronic treatment (Liillmann et al., 1973b), and it is slowly excreted. The plasma halflife in rats is 3.9 hr from the a-slope of the disposition curve, but the concentration in plasma does not change much from 9 to 24 hr after chlorphentermine administration (Eichelbaum et al., 1970). The half-life in lungs is about 50 hr (Eichelbaum et al., 1970). The present investigation was initiated in order to elucidate the influence of phenobarbital on chlorphentermine-induced alveolar foam cells. METHODS Fifteen Wistar/Af/Han/Mol (Han 67) male rats (SPF), weighing 195-222 g, were used for the experiment. The rats were divided into three groups, each consisting of five animals. One group served as a control group and was given 0.9 ‘A NaCl. The second group received chlorphentermine, 50 mg/kg/day po (1 ml/100 g), in the morning 5 days/week, while the third group received phenobarbital (30 mg/kg/day, po) in the afternoon 5 days/week in addition to chlorpentermine. Chlorphentermine and phenobarbital were dissolved in 0.9% NaCl. The rats were weighed before and every day throughout treatment, which lasted for 4 weeks. All rats were anesthetized by iv injection of Narcodorm (enibomal-Na) and killed by exsanguination in the morning the day after the final dosing. The trachea was ligated in order to avoid collapse of the lungs during excision. The lungs were weighed and fixed in phosphate-buffered 10 % formalin. Longitudinal slices of both lungs were embedded in paraffin, sectioned 6 pm thick, and stained with hematoxylin and eosin. Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
171 ISSNOO41-008X
172
SHORT
RESULTS
COMMUNICATION
AND
DISCUSSION
Diffuse accumulations of typical foam cells were found intra-alveolarly in lungs from the animals treated with chlorphentermine only. The lungs from rats receiving both chlorphentermine and phenobarbital were histologically normal and no foam cells could be observed except from occasional small subpleural foci. Similar foci were also found in the lungs from the control rats and it is a well-known spontaneous entity in the lungs of the rat (Flodh et al., 1974). The relative weight of the lungs from each group is shown in Table 1. The mean relative weight of the lungs from the group receiving chlorphentermine only was significantly higher (p < 0.05, Student’s t test) than that of the control group. The mean relative weight of the lungs from the group receiving both chlorphentermine and phenobarbital was not changed by the treatment. TABLE
1
RELATIVE WEIGHT OF THE LUNGS OF RATS TREATED WITH CHLORPHENTERMINE
Treatment Animal number 1 2 3 4 5
Mean rt SD
Chlorphentermine 0.9 % NaCl
(50 mg/W
0.51” (306)b 0.41 (325) 0.45 (298) 0.46 (290) 0.52 (295) 0.47 f 0.044
0.70 0.52 0.56 0.64 0.52 0.59
(245) (297) (275) (272) (259) + 0.080’
Chlorphentermine + Phenobarbital (30 m&d 0.46 0.54 0.47 0.44 0.46 0.48
(290) (250) (299) (300) (261) k 0.034
a Values are in grams per 100 grams body weight. * The final body weights of the animals are shown in parentheses. c Statistically different (p < 0.05) from the control group.
Chlorphentermine-induced accumulations of pulmonary foam cells in rats are thus prevented by simultaneous treatment with phenobarbital. Phenobarbital has wellknown multipotent inducing properties of drug metabolizing enzyme systems (Conney, 1967). Chlorphentermine has proved to be very stable to enzyme degradation in vitro (Dubnick et al., 1968). Studies by Opitz and Weischer (1966) and Dubnick et al. (1968) suggest that chlorphentermine is mainly excreted unchanged by rats, and no evidence of microsomal biotransformation has been found (Dubnick et al., 1968). However, a small portion is biotransformed in rats (Dubnick et al., 1968; Ryrfeldt, 1970; Caldwell et al., 1975). Based on the finding of a more polar and hydrolyzable metabolite in bile (Ltillmann et al., 19736) and in urine of rats (Ryrfeldt, 1970; Caldwell et al., 1975), it is concluded that the metabolite may be a conjugate between some endogenous substance and the amino group of the chlorphentermine molecule. The findings in the present study suggest that treatment with phenobarbital increased the metabolic degradation of chlorphentermine. A phenobarbital-induced increase in
SHORT
173
COMMUNICATION
metabolic degradation of chlorphentermine or metabolites might be elicited through a preexisting metabolic pathway or by induction of a new one. Phospholipid-drug complexes are accumulated in the alveolar foam cells (Liillmann et al., 1973a). Therefore, the possibility could also be considered that phenobarbital stimulated enzymes not involved in the metabolic biotransformation of chlorphentermine and otherwise related to the catabolism of phospholipid-drug complexes. ACKNOWLEDGMENTS
The author wishesto thank ProfessorTage Moller for the evaluation of the histological slides. REFERENCES J., KBSTER, U., SMITH, R. L., AND WILLIAMS, R. T. (1975).Speciesvariations in the N-oxidation of chlorphentermine.Biochem. Pharmacol. 24,2225-2232. CONNEY, A. H. (1967). Pharmacologicalimplications of microsomal enzyme induction. CALDWELL,
Pharmacol.
Rev. 19,317-366.
C. A., HARTIGAN, J. M., AND PHILLIPS, G. E. (1968).Distribution and metabolismof chlorphentermine-Cl4in rats and mice. Biochem. Pharmacol. 17, 1243-1250. EICHELBAUM, M., HENGSTMANN, J. H., AND DENGLER, H. J. (1970).Das Verteilungsmusterdes Chlorphenterminsbei Ratte, Kaninchenund Schwein.Arch. Pharmacol. 267, 446456. FLODH, H., MAGNUSSON, G., AND MAGNUSSON, 0. (1974).Pulmonary foam cells in rats of different age.2. Versuchstierk. 16,299-312. FRANKEN, G., LULLMANN, H., AND SIEGFRIEDT, A. (1970).The occurrenceof hugecellsin pulmonary alveoli of rats treated by an anorexicdrug. Arzneim.-Forsch. 20, 417-419. HRUBAN, Z., SLESERS, A., AND ASCHENBRENNER, I. (1973).Pulmonary intra-alveolarhistiocytosisinducedby drugs. Toxicol. Appl. Pharmacol. 26,72-85. KARABELNIK, D., ZBINDEN, G., AND BAUMGARTNER, E. (1974).Drug-induced foam cell reactions in rats. I. Histopathologicand cytochemicalobservationsafter treatment with chlorphentermine,RMI 10.393,and Ro 4-4318.Toxicol. Appl. Pharmacol. 27,395-407. LOLLMANN, H., L~LLMANN-RAUCH, R., AND WASSERMANN, 0. (1973a).Drug-induced phospholipidosis.Ger. Med. Month. 3, 128-135. L~LLMANN, H., ROSSEN, E., AND SEILER, K. -U. (1973b).The pharmacokinetics of phentermine and chlorphenterminein chronically treated rats. J. Pharm. Pharmacol. 25,239-243. MAGNUSSON, G., AND MAGNUSSON, 0. (1972).Cloforex-inducedpulmonary changesin rats. DUBNICK,
B., TOWNE,
Beitr. Path. Anat. 146, 79-88. K., AND WEISCHER, M. -L. (1966).Die AusscheidunganorexigenerPhenylalkylamine mit demHarn. Arzneim.-Forsch. 16, 1311-1313. PARWARESCH, M. R., REIL, G. -H., AND SEILER, K. -U. (1973).Uber die Tier- und Organspezifitat morphologischerVeranderungennach chronischerChlorphentermingabe.Res. Exp. OPITZ,
Med. 161,272-288.
AA. (1970).The distribution, in the mouseand rat. Acta Pharmacol.
RYRFELDT,
elimination,
and biotransformation
of 14C-Cloforex
Toxicol. 28,391X)5. OVE SVENDSEN
Department of Pharmacology and Toxicology, H. Lundbeck & Co. A/S, DK 2500 Copenhagen- Valby, Denmark Received October 25, 1976; accepted December 19, 1976