European Journal o f Pharmacology, 55 (1979) 319--322 © Elsevier/North-Holland Biomedical Press
319
Short communication BRAIN DISTRIBUTION OF PROPRANOLOL IN THE RAT JEAN-LUC ELGHOZI, GABRIO BIANCHETTI *, PAOLO L. MORSELLI * and PHILIPPE MEYER Unitd de Recherches de Physiologie et Pharmacologie Vasculaire et Rdnale, INSERUM U7, HSpital Necker 161 rue de S~vres, 75015 Paris, and * L.E.R.S.-Synthdlabo, Ddpartement de Recherche Clinique, Groupe de Pharmacocindtique, 58 rue de la Glaci~re, 75013 Paris, France Received 19 February 1979, accepted 23 February 1979
J.-L. ELGHOZI, G. BIANCHETTI, P.L. MORSELLI and P. MEYER, Brain distribution ofpropranolol in the rat, European J. Pharmacol. 55 (1979) 319--322. The distribution and kinetics of D,L-propranolol in rat brain were examined after an intravenous injection of the drug. Measurements in brain areas and blood were performed by means of a sensitive and specific gas liquid chromatographic method. The disappearance rate in cortical areas paralleled that in blood. However D,L-propranolol decreased at a slower rate in hypothalamic and medullary nuclei. Since propranolol is believed to have central hypotensive effects, its retention by certain central nuclei involved in blood pressure regulation is of interest. Brain
Pharmacokinetics
D,L-Propranolol
1. Introduction The mode of action of the antihypertensive drug propranolol remains unclear (Prichard, 1978). The hypothesis of a central action of the drug is supported by several observations: (i) a blood pressure decrease results from the intracerebroventricular administration of propranolol to conscious animals, an effect which is specific for the L-isomer (Day and Roach, 1974; Reid et al., 1974), (ii) the hypotensive phase following an intravenous injection of D,L-propranolol to the conscious rabbit is accompanied by a reduction in the frequency of sympathetic nerve discharges (Lewis and Haeusler, 1975), (iii) propranolol has been detected in the brain after peripheral administration (Garvey and Ram, 1975; Myers et al., 1975; Saelens et al., 1977; Schneck et al., 1977). However, these findings cannot be considered as entirely conclusive. Large doses were used for the cerebroventricular injections and a leakage of propranolol from cerebrospinal fluid to plasma may have accounted
Rat
for a peripherally mediated effect of the drug. The reduction of sympathetic nervous activity observed during drug-induced hypotension may simply indicate an increased sensitivity of arterial baroreceptors. The purpose of the present study was to reinvestigate the distribution of intravenously administered D,L-propranolol in discrete brain areas of conscious rats. 2. Materials and methods
2.1. Animals and procedure Male Wistar rats weighing between 190 g and 210 g were used. D,L-propranolol (Avlocardyl®, I.C.I.) was administered intravenously (saphenous vein) to conscious rats in a dose of 5 mg/kg. Animals were killed by decapitation at 30, 60, 120, 180, 240, 300 and 360 min. Blood was collected into heparinized tubes and stored a t - - 2 0 ° C . Brains were quickly removed, frozen in liquid nitrogen and kept at --20°C until dissection. The areas punched
320
o u t were anterior neocortex, amygdala nuclei, hippocampus, anterior and posterior nuclei of the hypothalamus (together with a part of the periventricular nucleus). Two areas were punched o u t in the medulla. Firstly t h e nucleus reticularis lateralis, which includes the A1 cell group and the C1 cell group, designated here as C1 for the sake of simplicity. The letter A corresponds to the topographic description of aminergic cell groups and nerve terminals which had been recognized b y a histofluorescence technique. The letter C corresponds to the topographic description of nerve cell groups in the medulla oblongata which contain the enzyme PNMT, obtained with an immunohistochemical technique. Secondly we have punched o u t a group of cells which extend along the long axis of the dorsal medulla. This area includes the nucleus tractus solitarii, the caudal part o f which contains the A2 cell group, nucleus intercalatus, nucleus originis dorsalis vagi and nucleus originis nerve hypoglossi. This second area which includes the C2 group is designated C2 in this study. The punched samples were dessicated and weighed. The minimal dry weight for propranolol measurement was 0.5 mg. Each nucleus could therefore be studied separately. In the second part of the study D,L-alprenolol (Gubernal®, Geigy) which also penetrates into the brain was administered intravenously, 10 mg/kg, 30 min before sacrifice and the brain distribution o f propranolol was followed as indicated above. This experiment was performed in order t o see if an excess of alprenolol could d i s p l a c e propranolol from binding sites with limited capacity.
J. ELGHOZI
2.3. Pharmacokinetic and statistical analysis The pharmacokinetic analysis of the data was carried o u t using the IGPHARM programme (Gomeni and Gomeni, 1978) on a Tektronix 4051 graphic system. The pharmacokinetic parameters were evaluated b y the method of residuals according to a two-compartment open model. The statistical analysis of the kinetic parameters thus obtained was performed using a two-way analysis of variance.
3. Results
The concentrations of D,L-propranolol in blood and discrete brain areas a r e shown in fig. 1. Pharmacokinetic parameters are detailed in table 1. The brain concentration of D,Lpropranolol appeared to be consistently higher than that of blood. At 30 min the highest concentrations of the drug were found in the cortical areas (cortex frontalis, hippocampus, amygdala). The elimination rate of D,L-propranolol from these structures paralleled that in blood and whole brain and could be described b y a single ~compartment model, with an apparent T1/2 ranging between 60 and 70 min.
4
4
2.2. Chemical analysis Propranolol measurements in blood a n d brain were performed b y means of a sensitive and specific GLC method as previously described (Di Salle et al., 1973). T h e dry discrete brain areas were homogenized in a micro Potter homogenizer using 1 ml of HC1 0.1 N. The whole homogenate was used for analysis.
: ~ " ' ,, 3060 120 I1~) 240 300 360
03()60 120 180 2~103()0 360
Fig. 1. D,L-propranolo] concentrations in blood and discrete brain areas of the rat after i.v. injection of 5 mg/kg (mean of 3 determinations). Ordinate: log propranolol concentration (ng/ml or ng/g); abscissa: time (rain).
B R A I N D I S T R I B U T I O N O F P R O P R A N O L O L IN T H E R A T
321
TABLE 1 D , L - P r o p r a n o l o l p h a r m a c o k i n e t i c s in b l o o d a n d d i s c r e t e brain areas o f t h e rat: ~ and ~ r e f e r t o d i s t r i b u t i o n and e l i m i n a t i o n phases respectively; T 1 / 2 c o r r e s p o n d s t o half-life. Tissue
0~ (rain -1 )
T 1 / 2 0l (min)
~ (rain -1 )
T1/2 (rnin)
Blood Whole brain Anterior neocortex Hippocampus A m y g d a l a (nuclei) H y p o t h a l a m u s (N. ant.) H y p o t h a l a m u s (N. post. ) Medulla (C1) Medulla (C2)
-----0.0261 0.0261 0.0266 0.0198
-----26.4 26.4 26.8 34.8
0.0133 0.0115 0.0102 0.0097 0.0116 0.0046 0.0039 0.0015 0.0017
52 60 67 71 59 149 174 440 385
The disappearance rate in hypothalamic and medullar structures was quite different. At first D,L-propranolol decreased rapidly with a T1/2 of about 30 min. Later the disappearance rate slowed with a T1/2 of about 150 min for hypothalamus and 400 min for medulla, indicating a marked retention of the drug within these structures. The T1/2 of D,L-propranolol in hypothalamus and medulla was significantly longer than in the other structures (P < 0.01). D,L-Alprenolol did not modify the distribution pattern of D,L-propranolol in the rat brain and its rate of disappearance.
4. Discussion The present study, in agreement with previous investigations (Garvey and Ram, 1975; Myers et al., 1975; Saelens et al., 1977; Schneck et al., 1977) shows that D,L-propranolol rapidly enters the brain after peripheral administration. The brain concentration appeared to be higher than the blood concentration. The high concentrations of D,L-propranolol observed in the cortical areas during the first 30 min may be related to the rich blood supply to these areas. The most important finding of the present study was the low disappearance rate in hypothalamic and medullary nuclei. Myers et al.
(1975) have also observed a retention of D,L-propranolol in hypothalamus and medulla-pons after intravenous infusion of D,Lpropranolol. This accumulation is particularly interesting in view of the fact that these various nuclei are involved in blood pressure regulation. The hypothalamus, and also the amygdala and hippocampus, appear to control the activity of the nuclei located in the medulla oblongata and belonging to the baroreflex loop. The C2 area including nucleus tractus solitarii and nucleus originis dorsalis vagi therefore corresponds to the first synapse of the afferent barosensitive nerves and to the origin of the vagal efferent to the heart in rats. The C1 area corresponds to the origin of descending fibres which have their synapse in the spinal cord with preganglionic sympathetic neurones. In order to determine if the drug measured in the brain was bound to sites with a limited capacity, s u c h a s ~-adrenoceptor sites, an experiment was performed with D,L-alprenolol in concentrations 2-fold higher than those of D,L-propranolol. No inhibitory effect of D,L-alprenolol on D,L-propranolol distribution was observed. However, the dose of the competitor may have been too small for inhibiting D,L-propranolol concentration. Higher doses of D,L-alprenolol appeared to be lethal andthus no conclusions could be drawn. Since propranolol is believed to have central
322
hypotensive effects its retention by certain central nuclei involved in blood pressure regulation is of interest. Other peripheral mechanisms can o f course play an important role.
References Day, M.D. and A.G. Roach, 1974, Cardiovascular effects of /3-adrenoceptor blocking drugs after intracerebroventricular administration to conscious normotensive cats, Clin. Exptl. Pharmacol. Physiol. 1,347. Di Salle, E., K.M. Baker, S.L. Bareggi, W.D. Watkins, C.A. Chidsey, A. Frigerio and P.L. Morselli, 1973, A sensitive gas chromatographic method for the determination of propranolol in the human plasma, J. Chromatography 8 4 , 3 4 7 . Garvey, H.L. and N. Ram, 1975, Centrally induced hypotensive effects of beta-adrenergic blocking drugs, European J. Pharmacol. 33, 283. Gomeni, C. and R. Gomeni, 1978, IGPHARM: Inter-
J. ELGHOZI active graphic package for pharmacokinetic analysis, Comp. Biomed. Res. 11,345. Lewis, P.J. and G. Haeusler, 1975, Reduction in sympathetic nervous activity as a mechanism for hypotensive effect of propranolol, Nature 256,440. Myers, M.G., P.J. Lewis, J.L. Reid and C.T. DoUery, 1975, Brain concentration or propranolol in relation to hypotensive effect in the rabbit with observations on brain propranolol levels in man, J. Pharmacol. Exptl. Therap. 192,327. Prichard, B.N.C. 1978, fl-Adrenergic receptor blockade in hypertension, past, present and future, Brit. J. Clin. Pharmacol. 5, 379. Reid, J.L., P.J. Lewis, M.G. Myers and C.T. Dollery, 1974, Cardiovascular effects of intracerebroventricular d-, 1- and d,l-propranolol in the conscious rabbit, J. Pharmacol. Exptl. Therap. 188,394. Saelens, D.A., T. Walle, T.E. Gaffney and P.J. Privitera, 1977, Studies on the contribution of active metabolites to the anticonvulsant effects of propranolol, European J. Pharmacol. 42, 39. Schneck, D.W., J.F. Prichard and A.H. Hayes, 1977, Studies on the uptake and binding of propranolol by rat tissues, J, Pharmacol. Exptl. Therap. 203, 621.
319
Short communication BRAIN DISTRIBUTION OF PROPRANOLOL IN THE RAT JEAN-LUC ELGHOZI, GABRIO BIANCHETTI *, PAOLO L. MORSELLI * and PHILIPPE MEYER Unitd de Recherches de Physiologie et Pharmacologie Vasculaire et Rdnale, INSERUM U7, HSpital Necker 161 rue de S~vres, 75015 Paris, and * L.E.R.S.-Synthdlabo, Ddpartement de Recherche Clinique, Groupe de Pharmacocindtique, 58 rue de la Glaci~re, 75013 Paris, France Received 19 February 1979, accepted 23 February 1979
J.-L. ELGHOZI, G. BIANCHETTI, P.L. MORSELLI and P. MEYER, Brain distribution ofpropranolol in the rat, European J. Pharmacol. 55 (1979) 319--322. The distribution and kinetics of D,L-propranolol in rat brain were examined after an intravenous injection of the drug. Measurements in brain areas and blood were performed by means of a sensitive and specific gas liquid chromatographic method. The disappearance rate in cortical areas paralleled that in blood. However D,L-propranolol decreased at a slower rate in hypothalamic and medullary nuclei. Since propranolol is believed to have central hypotensive effects, its retention by certain central nuclei involved in blood pressure regulation is of interest. Brain
Pharmacokinetics
D,L-Propranolol
1. Introduction The mode of action of the antihypertensive drug propranolol remains unclear (Prichard, 1978). The hypothesis of a central action of the drug is supported by several observations: (i) a blood pressure decrease results from the intracerebroventricular administration of propranolol to conscious animals, an effect which is specific for the L-isomer (Day and Roach, 1974; Reid et al., 1974), (ii) the hypotensive phase following an intravenous injection of D,L-propranolol to the conscious rabbit is accompanied by a reduction in the frequency of sympathetic nerve discharges (Lewis and Haeusler, 1975), (iii) propranolol has been detected in the brain after peripheral administration (Garvey and Ram, 1975; Myers et al., 1975; Saelens et al., 1977; Schneck et al., 1977). However, these findings cannot be considered as entirely conclusive. Large doses were used for the cerebroventricular injections and a leakage of propranolol from cerebrospinal fluid to plasma may have accounted
Rat
for a peripherally mediated effect of the drug. The reduction of sympathetic nervous activity observed during drug-induced hypotension may simply indicate an increased sensitivity of arterial baroreceptors. The purpose of the present study was to reinvestigate the distribution of intravenously administered D,L-propranolol in discrete brain areas of conscious rats. 2. Materials and methods
2.1. Animals and procedure Male Wistar rats weighing between 190 g and 210 g were used. D,L-propranolol (Avlocardyl®, I.C.I.) was administered intravenously (saphenous vein) to conscious rats in a dose of 5 mg/kg. Animals were killed by decapitation at 30, 60, 120, 180, 240, 300 and 360 min. Blood was collected into heparinized tubes and stored a t - - 2 0 ° C . Brains were quickly removed, frozen in liquid nitrogen and kept at --20°C until dissection. The areas punched
320
o u t were anterior neocortex, amygdala nuclei, hippocampus, anterior and posterior nuclei of the hypothalamus (together with a part of the periventricular nucleus). Two areas were punched o u t in the medulla. Firstly t h e nucleus reticularis lateralis, which includes the A1 cell group and the C1 cell group, designated here as C1 for the sake of simplicity. The letter A corresponds to the topographic description of aminergic cell groups and nerve terminals which had been recognized b y a histofluorescence technique. The letter C corresponds to the topographic description of nerve cell groups in the medulla oblongata which contain the enzyme PNMT, obtained with an immunohistochemical technique. Secondly we have punched o u t a group of cells which extend along the long axis of the dorsal medulla. This area includes the nucleus tractus solitarii, the caudal part o f which contains the A2 cell group, nucleus intercalatus, nucleus originis dorsalis vagi and nucleus originis nerve hypoglossi. This second area which includes the C2 group is designated C2 in this study. The punched samples were dessicated and weighed. The minimal dry weight for propranolol measurement was 0.5 mg. Each nucleus could therefore be studied separately. In the second part of the study D,L-alprenolol (Gubernal®, Geigy) which also penetrates into the brain was administered intravenously, 10 mg/kg, 30 min before sacrifice and the brain distribution o f propranolol was followed as indicated above. This experiment was performed in order t o see if an excess of alprenolol could d i s p l a c e propranolol from binding sites with limited capacity.
J. ELGHOZI
2.3. Pharmacokinetic and statistical analysis The pharmacokinetic analysis of the data was carried o u t using the IGPHARM programme (Gomeni and Gomeni, 1978) on a Tektronix 4051 graphic system. The pharmacokinetic parameters were evaluated b y the method of residuals according to a two-compartment open model. The statistical analysis of the kinetic parameters thus obtained was performed using a two-way analysis of variance.
3. Results
The concentrations of D,L-propranolol in blood and discrete brain areas a r e shown in fig. 1. Pharmacokinetic parameters are detailed in table 1. The brain concentration of D,Lpropranolol appeared to be consistently higher than that of blood. At 30 min the highest concentrations of the drug were found in the cortical areas (cortex frontalis, hippocampus, amygdala). The elimination rate of D,L-propranolol from these structures paralleled that in blood and whole brain and could be described b y a single ~compartment model, with an apparent T1/2 ranging between 60 and 70 min.
4
4
2.2. Chemical analysis Propranolol measurements in blood a n d brain were performed b y means of a sensitive and specific GLC method as previously described (Di Salle et al., 1973). T h e dry discrete brain areas were homogenized in a micro Potter homogenizer using 1 ml of HC1 0.1 N. The whole homogenate was used for analysis.
: ~ " ' ,, 3060 120 I1~) 240 300 360
03()60 120 180 2~103()0 360
Fig. 1. D,L-propranolo] concentrations in blood and discrete brain areas of the rat after i.v. injection of 5 mg/kg (mean of 3 determinations). Ordinate: log propranolol concentration (ng/ml or ng/g); abscissa: time (rain).
B R A I N D I S T R I B U T I O N O F P R O P R A N O L O L IN T H E R A T
321
TABLE 1 D , L - P r o p r a n o l o l p h a r m a c o k i n e t i c s in b l o o d a n d d i s c r e t e brain areas o f t h e rat: ~ and ~ r e f e r t o d i s t r i b u t i o n and e l i m i n a t i o n phases respectively; T 1 / 2 c o r r e s p o n d s t o half-life. Tissue
0~ (rain -1 )
T 1 / 2 0l (min)
~ (rain -1 )
T1/2 (rnin)
Blood Whole brain Anterior neocortex Hippocampus A m y g d a l a (nuclei) H y p o t h a l a m u s (N. ant.) H y p o t h a l a m u s (N. post. ) Medulla (C1) Medulla (C2)
-----0.0261 0.0261 0.0266 0.0198
-----26.4 26.4 26.8 34.8
0.0133 0.0115 0.0102 0.0097 0.0116 0.0046 0.0039 0.0015 0.0017
52 60 67 71 59 149 174 440 385
The disappearance rate in hypothalamic and medullar structures was quite different. At first D,L-propranolol decreased rapidly with a T1/2 of about 30 min. Later the disappearance rate slowed with a T1/2 of about 150 min for hypothalamus and 400 min for medulla, indicating a marked retention of the drug within these structures. The T1/2 of D,L-propranolol in hypothalamus and medulla was significantly longer than in the other structures (P < 0.01). D,L-Alprenolol did not modify the distribution pattern of D,L-propranolol in the rat brain and its rate of disappearance.
4. Discussion The present study, in agreement with previous investigations (Garvey and Ram, 1975; Myers et al., 1975; Saelens et al., 1977; Schneck et al., 1977) shows that D,L-propranolol rapidly enters the brain after peripheral administration. The brain concentration appeared to be higher than the blood concentration. The high concentrations of D,L-propranolol observed in the cortical areas during the first 30 min may be related to the rich blood supply to these areas. The most important finding of the present study was the low disappearance rate in hypothalamic and medullary nuclei. Myers et al.
(1975) have also observed a retention of D,L-propranolol in hypothalamus and medulla-pons after intravenous infusion of D,Lpropranolol. This accumulation is particularly interesting in view of the fact that these various nuclei are involved in blood pressure regulation. The hypothalamus, and also the amygdala and hippocampus, appear to control the activity of the nuclei located in the medulla oblongata and belonging to the baroreflex loop. The C2 area including nucleus tractus solitarii and nucleus originis dorsalis vagi therefore corresponds to the first synapse of the afferent barosensitive nerves and to the origin of the vagal efferent to the heart in rats. The C1 area corresponds to the origin of descending fibres which have their synapse in the spinal cord with preganglionic sympathetic neurones. In order to determine if the drug measured in the brain was bound to sites with a limited capacity, s u c h a s ~-adrenoceptor sites, an experiment was performed with D,L-alprenolol in concentrations 2-fold higher than those of D,L-propranolol. No inhibitory effect of D,L-alprenolol on D,L-propranolol distribution was observed. However, the dose of the competitor may have been too small for inhibiting D,L-propranolol concentration. Higher doses of D,L-alprenolol appeared to be lethal andthus no conclusions could be drawn. Since propranolol is believed to have central
322
hypotensive effects its retention by certain central nuclei involved in blood pressure regulation is of interest. Other peripheral mechanisms can o f course play an important role.
References Day, M.D. and A.G. Roach, 1974, Cardiovascular effects of /3-adrenoceptor blocking drugs after intracerebroventricular administration to conscious normotensive cats, Clin. Exptl. Pharmacol. Physiol. 1,347. Di Salle, E., K.M. Baker, S.L. Bareggi, W.D. Watkins, C.A. Chidsey, A. Frigerio and P.L. Morselli, 1973, A sensitive gas chromatographic method for the determination of propranolol in the human plasma, J. Chromatography 8 4 , 3 4 7 . Garvey, H.L. and N. Ram, 1975, Centrally induced hypotensive effects of beta-adrenergic blocking drugs, European J. Pharmacol. 33, 283. Gomeni, C. and R. Gomeni, 1978, IGPHARM: Inter-
J. ELGHOZI active graphic package for pharmacokinetic analysis, Comp. Biomed. Res. 11,345. Lewis, P.J. and G. Haeusler, 1975, Reduction in sympathetic nervous activity as a mechanism for hypotensive effect of propranolol, Nature 256,440. Myers, M.G., P.J. Lewis, J.L. Reid and C.T. DoUery, 1975, Brain concentration or propranolol in relation to hypotensive effect in the rabbit with observations on brain propranolol levels in man, J. Pharmacol. Exptl. Therap. 192,327. Prichard, B.N.C. 1978, fl-Adrenergic receptor blockade in hypertension, past, present and future, Brit. J. Clin. Pharmacol. 5, 379. Reid, J.L., P.J. Lewis, M.G. Myers and C.T. Dollery, 1974, Cardiovascular effects of intracerebroventricular d-, 1- and d,l-propranolol in the conscious rabbit, J. Pharmacol. Exptl. Therap. 188,394. Saelens, D.A., T. Walle, T.E. Gaffney and P.J. Privitera, 1977, Studies on the contribution of active metabolites to the anticonvulsant effects of propranolol, European J. Pharmacol. 42, 39. Schneck, D.W., J.F. Prichard and A.H. Hayes, 1977, Studies on the uptake and binding of propranolol by rat tissues, J, Pharmacol. Exptl. Therap. 203, 621.
