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Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-(1-methylpropyl)-3 isoquinolinecarboxamide
Life Sciences, Vol. 32, pp. 1849-1856 Printed in the U.S.A.
Pergamon Press
PERIPHERAL BENZODIAZEPINE BINDING SITES: EFFECT OF PK 11195, I-(2-CHLOROPHENYL)-N-METHYL-(I-METHYLPROPYL)-3 ISOQUINOLINECARBOXAMIDE II.
IN VIVO STUDIES
G. Le Fur, F. Guilloux, P. Rufat, J. Benavides, A. Uzan, C. Renault, M.C. Dubroeucq and C. Gu~r~my PHARMUKA Laboratoires, 35, quai du Moulin de Cage, F 92231Gennevilliers, France (Received in final form January 25, 1983) SUMMARY Peripheral type of benzodiazepine binding sites wwre labelled in the kidney, the heart and the brain with [JH] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding : heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [ ~H] R95-4864 binding in vivo parallel relative affinities for [ ~HJRO5-4864 binding sites in isolated organs membranes : RO5-4864 > diazepam > clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [JH] RO5-4864 in vitro is also very effective (more than R05-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites. Earliest studies have demonstrated the feasibility of labelling benzodiazepine ~-eceptors "brain type" in in,tact animals after intravenous injection of i~H]flunitrazepam (1,2) or [JH]diazepam (3,4). A main advantage of estimating the in vivo binding of drugs is the possibility of directly measuring binding sites occupation in living animals, where the binding sites are in their physiological state, and to correlate the binding sites occupation with pharmaeolog%cal responses. L~gands for peripheral benzodiazepine binding sites, [~H]diazepam and [~H]RO5-4864, have been used in "in vitro" studies. The bin@ing of these ligands was always optimal at 0.4"C (5-9). For example when [~H]ROS-4864 is used as a ligand, its specific binding "in vitro" is 2 to 3 times less ~t 37"C than at 0.4"C. Such observations raise questions as to whether [JH]RO5-4864 binding occurs in vivo. We report here the successful in vivo labelling of heart, kidneys and b~'ain "peripheral type" benzodiazepine binding sites after I.V. injection of [~H]RO5-4864 to mice. We have also studied the effects of I.P. and oral administration of PK 11195, a new compound chemically unrelated to benzodiazepines, I- (2-chlorophenyl )-N-methyl-N~ (I-methylpropyl )- 3-isoquinolinecarboxamide, which is a potent inhibitor of [ H]R05-4864 binding in vitro (10). 0024-3205/83/161849-08503.00/0 Copyright (c) 1983 Perga~n Press Ltd.
1850
PK 11195 and Binding Sites: Part II
Vol. 32, No. 16, 1983
MATERIAL AND METHODS Restrazned male CDI mzce (23 - 2 g) Charles River~France strazn were I.V. injected into the tail vein with 200 ~Ci/kg of [~H]RO5-4864 (73.8 Ci/ m o l from NEN). After various intervals (mainly 2 min) mice were decapitated,their brains, hearts, kidneys rapidly removed, weighed and homogenized in 40 volumes of ice cold Tris HCI buffer (50 mM pH 7.7) using an Ultra turrax. Triplicate 500 u i aliquots were i~mediately filtered through GF/B Whatman glass fiber filters. The filters were washed twice with 5 ml of ice cold buffer and suspended in 10 ml Instagel (Packard). ~ Radioactivity was measured by liquid scintillation spectroscopy. Total [JH]R05-4864 in the organs was determined by counting 500 ul aliquots of the homogenates prior to filtration Specific binding is defined as ~he difference between particulate binding obtained after injection of [ H] R05-4864 alone and after co-injection with cold R05-4864 (3 mg/kg I.~.). This dose has been chosen because higher doses did not displace more [°H]RO5-4864. In order to quantify the relative amount of [3H]R05-4864 in organs radioagtivity the following set of experiments was carried out. 200 uCi/kg of [°H]R05-4864 was I.V. injected 2 min before sacrifice. The different organs were rapidly removed, homogenized and filtered. The radioactivity from the filters was extracted with ethanol under agitation during 15 min. Aliquots were evaporated to dryness under reduced pressure. The~residue was dissolved in 50 ul ethanol. For the identification of the ~ H ]R05-4864, 25 ul were placed on a thin layer chromatography plate (Silicagel F 254 Merck) and developed in two separate solvent systems : chloroform-methanol (~:I) and isopropanol-a~oniumhydroxide (28:1). It was found that unchanged [JH~O5-4864 accounted for at least 95 % of the total radioactivity. RESULTS After I.V.~injection of a tracer dose (200 uCi/kg corresponding to 0.86 ug/ kg) of [JH]RO5-4864 the highest concentration of total and particulate radioactivity was found in the kidney and the heart whereas much less was found into the brain. The particulate fraction contained respectively 70, 85 and 85 % of the brain, heart and kidney radioactivity. S~multaneous injection (I.V.) of 3 mg/kg of unlabelled RO5-4864 along with [JH]RO5-4864 (200 uCi/kg) decreased respectively by 65, 70 and 75 % of the radioactivity of the brain, heart and kidneys particulate fractions, whereas total radioactivity remained only weakly modified. As this displacement is not increased above 3 mg/kg, this dose was further used to determine the ~onspecific binding. The time course of the in vivo specific binding of [JH]R05-4864 (as defined in Material and Methods) is shown in Fig. I. The maxim~n was obtained at I or 2 min with the three organs. Thereafter levels of particulate radioactivity declined more rapidly in the heart and the kidneys than in the brain. For these r~asons the mice were sacrificed 2 minutes after the I.V. injection~of the [ ~ RO5-4864 for the displacement study. To determine whether [ °H ]RO5-4864 binding in these experiments might be an artifact of the homogenization procedure, kidneys, hearts or brains of untreated mice were homogenized together with [ JH]R05-4864 (at the concentrations found after ~.V. injection) and filtered in the same way as mice treated in vivo with [°H]R05-4864. Only 10 to 30 % of the tritium content of the different organs is particulate bound in contrast to 65 to 75 % particulate binding of radioactivity in mice receiving [ JH] RO5-4864 in vivo. Such a result suggests that the observed binding in vivo did not take place during tissue homogenization.
Vol. 32, No. 16, 1983
t
PK 11195 and Binding Sites: Part II
1851
•• BLAIN HEART • KIDNEY
25
FIG. I 18
o
Time ~ourse of the in vivo specific binding of [~H]R05-4864. Each value + s.e.m, represents the mean of at least 5 mice.
~
;
~
.:
TIME IN MINUTES
The most important criterion for determining whether bound [ 3HI R05-4864 labels "peripheral type" of benzodiazepine binding sites is to ascertain whether affinities of various drugs for binding sites in vivo parallel their affinities for this type of benzodiazepine binding sites in vitro. Mice received I.P. injections of various doses of be~odiazepines and PK 11195 30 rain prior to I.V. injections of 200 uCi/kg [~H]RO5-4864, were decapitated 2 rain later and their kidneys, hearts and brains assayed for bound and total radioactivity. The drugs tested had no or small effect on the total radioactivity, the displacement only occurred in the bound activity (Fig. 2). In all the organs, the following order of potency was found : PK 11195 > R054864 > diazepam > clonazepam (inactive at 10 mg/kg I.P.) (Fig. 2). The approximate I D ~ are shown in Table I. The ID5n of PK 11195 were relatively similar from o ~ organ to another although the dose response curves were different. R05-4864 appeared to be more effective in the heart than in the kidneys or in the brain whereas diazepam was more active in the brain than in the heart or in the kidney. After I.P. injectio9 of I mg/kg of PK 11195 the maximal inhibition of the in vivo binding of [~H]RO5-4864 was found at 30 rain (Fig. 3). Its effect still persists during 18 hours in the brain but only during 6 hours in the kidneys and the brain. PK 11195 was also active in the three organs after oral administration (Fig. 4) with IDgn around I mg/kg at 1 hour (Table I). The inhibition of the in vivo bindi~ of [~H]RO5-4864 by 10 mg/kg P.O. of PK 11195 is shown in Fig. 5. The maximal effect was found at I hour in the three organs. The effect was more rapid in the heart and kidneys than in the brain but more prolonged in the later organ (18 hours) than in the kidneys or heart (between 6 and 18 hours). Similar results have been found in the rat (data not shown).
.,r
i
0
Z Z
Z O J.
w .a
u.
v
u
m
Q
Z o
5(
I¢¢
•
LOG, OOS!
•
MG / K G
/
KIDNEY
MO/KG
BRAIN
LOG , DOSE
PK 11195
•• ,o-~ ... DIAZEPAM
PK 11198
•• DIAZEPAM , o - , 4,,,
• • •
LOG,
.
RO- 5 4864 DIAZEPAM PK 11195
.
.
.
MG/KG
FIG. 2
DOSE
HEART
Effect of PK 11%~5, R05-4864 and diazepam in vivo b~nding of [ JH]RO5-4864 in the mouse. Each value - s.e.m, represents the mean of 5 to 10 animals. The drugs were I.P. injected 30 min before sacrifice.
I
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Vol. 32, No. 16, 1983
PK 11195 and Binding Sites: Part II
1853
Table I DRUG EFFECTS ON BINDING OF [3H]R05-4864 IN VIVO
DRUGS
HEART
KIDNEYS
BRAIN
I.P.
0.07 (0.03 - 0.18)
0.25 (0.10 - 0.62)
0.25 (0.12 - 0.54)
P.O.
0.58 (0.25 - 1.35)
0.76 (0.40 - 1.44)
0.76 (0.49 - 1.16)
1.36 (0.98 - 1.90)
1.01 (0.53 - 1.94)
PK 11195
R05-4864
I.P
0.37 (0.13 - 1.05)
DIAZEPAM
I.P.
11.9 (3.9 - 36.1)
24.7 (x)
4.8 (3.9 -
5.9)
Results are expressed as approximate dose of drugs (~D~n) and 95 % confidence limits which half inhibits the specific binding of [ ~H~05-4864 (as defined in Material and Methods). They were calculated by log probit analysis of the displacement curves of figures 2 and 4. (x) by extrapolation 0 Z Q Z I
•
BRAIN
• ..,.,
'It
•
P K
11195
KIDNEY
qr 0 u
1OQ
m. a.
6(
Z 0 im. Z
TIME IN HOURS
FIG. 3 Time course of the inhibition of the in vivo binding of ~H]R05-4864 ~y PK 11195 after I.P. injection of I mg/kg in the mouse. Each value - s.e.m, represents the mean of 5 to 10 animals.
1854
PK 11195 and Binding Sites: Part II
• BRAIN • .,.., •
PK
Vol. 32, No. 16, 1983
11195
KIDNEY
z
v L
Z
_o
oi :
#
z
d.1
Z
0:3
t
:i
lb
LOG. DOS~E M G / KG
FIG. 4 Effect of oral administration of PK 11195 on the in vivo binding of [~H]R05-4864 in the mouse. Each value ~ s.e.m. represents the mean of 5 to 10 animals.
i
• BRAIN : :~2",,
PK
11195
1
? 0 ri
.~
o:st
:t
il
E
//-'--~ TIME IN HOURS
FIG. 5 T~me course of the inhibition of the in vivo binding of [ DH]RO5-4864 by PK 11195 after oral administration of 10 mg/kg in the mouse. Each value + s.e.m, represents the mean of 5 to 10 animals.
Vol. 32, No. 16, 1983
PK 11195 and Binding Sites: Part II
1855
DISCUSSION The present investigation demonstrates the feasibility of the in ~ivo labelling "peripheral type" of benzodiazepine binding sites with [JH]RO5-4864 in the heart, the kidneys and the brain under physiological conditions. The experiments were performed ~ min after the injection of the ligand. At this time the metabolism of [~H]R05-4864 was not a confounding factor since greater than 95 % of the recovered activity has been shown to be unmetabolized R05-4864. The percentage of specific binding, as defined in Material and Methods, of this "in vivo" labelling (65 to 75 %) is smaller than the percentage found in the "in vitro" conditions (more than 90 %). A possible explanation might be that under "in vivo" conditions endogenous ligands or modulators could interfere with the exogenously applied benzodiazepine whereas under "in vitro" conditions these putative ligands or modulators might be washed out or be less effective because of the lower ~emperature (0-4"C). The tissue distribution of the in vivo binding of [JH]R05-4864 parallels the tissue distribution of the in vitro binding, the amounts of RO5-4864 in the kidneys and the heart being greater than in the brain. Moreover the pharmacological specificity of the "in vivo" labelling is the same as the pharmacological specificity of the in vitro binding : PK 11195> RO5-4864 > diazepam > clonazepam (inactive at 10 mg/kg I.P.).~ Thus, all these findings indicate that intravenous tracer doses of [~H]RO5-4864 appear to label the "peripheral type" of benzodiazepine binding sites "in vivo" allowing to evaluate the actions of drugs on such binding sites in vivo. T ~ s has been applied to PK 11195. This compound "in vivo" displaces [~H]RO5-4864 bound to the "peripheral type" of benzodiazepine binding sites in the kidneys, the heart and the brain both in the mice and the rats. PK 11195 was more potent than RO5-4864 when administered either intraperitoneally or per os. We have also studied its duration of action as displacing agent after I.P. injection or orale administration. The effect of PK 11195 on the "peripheral type" of benzodiazepine binding sites is selective since PK 11195, like RO5-4864, is inactive until 25 mg/kg I.P~ on the "brain type" of benzodiazepine binding sites in vivo labelled by [JH]flunitrazepam according to the method of Chang and Snyder (I) (data not shown). In conclusion this study demonstrates the presence of [3H]RO5-4864 binding sites in the heart, the kidneys and the brain "in vivo" and also emphasizes the feasibility of this technique to examine the effects of several pharmacological (PK 11195) and physiological manipulations of these binding sites in living animals. In our ease where the physiological relevance of these binding sites remains to be established, the fact that PK 11195 binds to these sites in vivo indicates that this compound might be used as a tool to investigate the possible physiological role of the "peripheral type" of benzodiazepine binding sites in intact animals. ACKNOWLEDGMENTS The authors wish to thank Hoffman-La Roche for their generous supply of R05-4864 and Mrs. E. Berthier for her excellent secretarial assistance. REFERENCES I. 2. 3.
R.S.L. CHAN~.and S.H. SNYDER, Europ. J. Pharmacol. 48 213-218 (1978) T. DUKA, V. HOLLT and A. HERZ, Brain Res. 179 147-156 (1979) M.J. WILLIAMSON, S.M. PAUL and P. SKOLNICK, Life Sci. 23 1935-1940 (1978)
1856
4.
PK 11195 and Binding Sites: Part II
Vol. 32, No. 16, 1983
J.F. TALLMAN, J.W. THOMAS and D.W. GALLAGER, Life Sci. 24 873-880 (1979) 5. C. BRAESTRUP and R.F. SQUIRES, Proc. Nat. Acad. Sci. USA 74 3805-3809 (1977) 6. J.K. WANG, T. TANIGUCHI and S. SPECTOR, Life Sci. 27 1881-1888 (1980) 7. T. TANIGUCHI, J.K. WANG and S. SPECTOR, Life Sci. 27 171-178 (1980) 8. T. TANIGUCHI, J.K. WANG and S. SPECTOR, Biochem. Pharmacol. 31 589-590 (1982) 9. H. SCHOEMAKER, M. BLISS and H.I. YAMAMURA, Europ. J. Pharmacol. 71 173-175 (1981) 10. G. LE FUR, M.L. PERRIER, N. VAUCHER, F. IMBAULT, A. FLAMIER, J. BENAVIDES, A. UZAN, C. RENAULT, M.C. DUBROEUCQ and C. GUEREMY. Preceding paper in this issue.
Pergamon Press
PERIPHERAL BENZODIAZEPINE BINDING SITES: EFFECT OF PK 11195, I-(2-CHLOROPHENYL)-N-METHYL-(I-METHYLPROPYL)-3 ISOQUINOLINECARBOXAMIDE II.
IN VIVO STUDIES
G. Le Fur, F. Guilloux, P. Rufat, J. Benavides, A. Uzan, C. Renault, M.C. Dubroeucq and C. Gu~r~my PHARMUKA Laboratoires, 35, quai du Moulin de Cage, F 92231Gennevilliers, France (Received in final form January 25, 1983) SUMMARY Peripheral type of benzodiazepine binding sites wwre labelled in the kidney, the heart and the brain with [JH] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding : heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [ ~H] R95-4864 binding in vivo parallel relative affinities for [ ~HJRO5-4864 binding sites in isolated organs membranes : RO5-4864 > diazepam > clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [JH] RO5-4864 in vitro is also very effective (more than R05-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites. Earliest studies have demonstrated the feasibility of labelling benzodiazepine ~-eceptors "brain type" in in,tact animals after intravenous injection of i~H]flunitrazepam (1,2) or [JH]diazepam (3,4). A main advantage of estimating the in vivo binding of drugs is the possibility of directly measuring binding sites occupation in living animals, where the binding sites are in their physiological state, and to correlate the binding sites occupation with pharmaeolog%cal responses. L~gands for peripheral benzodiazepine binding sites, [~H]diazepam and [~H]RO5-4864, have been used in "in vitro" studies. The bin@ing of these ligands was always optimal at 0.4"C (5-9). For example when [~H]ROS-4864 is used as a ligand, its specific binding "in vitro" is 2 to 3 times less ~t 37"C than at 0.4"C. Such observations raise questions as to whether [JH]RO5-4864 binding occurs in vivo. We report here the successful in vivo labelling of heart, kidneys and b~'ain "peripheral type" benzodiazepine binding sites after I.V. injection of [~H]RO5-4864 to mice. We have also studied the effects of I.P. and oral administration of PK 11195, a new compound chemically unrelated to benzodiazepines, I- (2-chlorophenyl )-N-methyl-N~ (I-methylpropyl )- 3-isoquinolinecarboxamide, which is a potent inhibitor of [ H]R05-4864 binding in vitro (10). 0024-3205/83/161849-08503.00/0 Copyright (c) 1983 Perga~n Press Ltd.
1850
PK 11195 and Binding Sites: Part II
Vol. 32, No. 16, 1983
MATERIAL AND METHODS Restrazned male CDI mzce (23 - 2 g) Charles River~France strazn were I.V. injected into the tail vein with 200 ~Ci/kg of [~H]RO5-4864 (73.8 Ci/ m o l from NEN). After various intervals (mainly 2 min) mice were decapitated,their brains, hearts, kidneys rapidly removed, weighed and homogenized in 40 volumes of ice cold Tris HCI buffer (50 mM pH 7.7) using an Ultra turrax. Triplicate 500 u i aliquots were i~mediately filtered through GF/B Whatman glass fiber filters. The filters were washed twice with 5 ml of ice cold buffer and suspended in 10 ml Instagel (Packard). ~ Radioactivity was measured by liquid scintillation spectroscopy. Total [JH]R05-4864 in the organs was determined by counting 500 ul aliquots of the homogenates prior to filtration Specific binding is defined as ~he difference between particulate binding obtained after injection of [ H] R05-4864 alone and after co-injection with cold R05-4864 (3 mg/kg I.~.). This dose has been chosen because higher doses did not displace more [°H]RO5-4864. In order to quantify the relative amount of [3H]R05-4864 in organs radioagtivity the following set of experiments was carried out. 200 uCi/kg of [°H]R05-4864 was I.V. injected 2 min before sacrifice. The different organs were rapidly removed, homogenized and filtered. The radioactivity from the filters was extracted with ethanol under agitation during 15 min. Aliquots were evaporated to dryness under reduced pressure. The~residue was dissolved in 50 ul ethanol. For the identification of the ~ H ]R05-4864, 25 ul were placed on a thin layer chromatography plate (Silicagel F 254 Merck) and developed in two separate solvent systems : chloroform-methanol (~:I) and isopropanol-a~oniumhydroxide (28:1). It was found that unchanged [JH~O5-4864 accounted for at least 95 % of the total radioactivity. RESULTS After I.V.~injection of a tracer dose (200 uCi/kg corresponding to 0.86 ug/ kg) of [JH]RO5-4864 the highest concentration of total and particulate radioactivity was found in the kidney and the heart whereas much less was found into the brain. The particulate fraction contained respectively 70, 85 and 85 % of the brain, heart and kidney radioactivity. S~multaneous injection (I.V.) of 3 mg/kg of unlabelled RO5-4864 along with [JH]RO5-4864 (200 uCi/kg) decreased respectively by 65, 70 and 75 % of the radioactivity of the brain, heart and kidneys particulate fractions, whereas total radioactivity remained only weakly modified. As this displacement is not increased above 3 mg/kg, this dose was further used to determine the ~onspecific binding. The time course of the in vivo specific binding of [JH]R05-4864 (as defined in Material and Methods) is shown in Fig. I. The maxim~n was obtained at I or 2 min with the three organs. Thereafter levels of particulate radioactivity declined more rapidly in the heart and the kidneys than in the brain. For these r~asons the mice were sacrificed 2 minutes after the I.V. injection~of the [ ~ RO5-4864 for the displacement study. To determine whether [ °H ]RO5-4864 binding in these experiments might be an artifact of the homogenization procedure, kidneys, hearts or brains of untreated mice were homogenized together with [ JH]R05-4864 (at the concentrations found after ~.V. injection) and filtered in the same way as mice treated in vivo with [°H]R05-4864. Only 10 to 30 % of the tritium content of the different organs is particulate bound in contrast to 65 to 75 % particulate binding of radioactivity in mice receiving [ JH] RO5-4864 in vivo. Such a result suggests that the observed binding in vivo did not take place during tissue homogenization.
Vol. 32, No. 16, 1983
t
PK 11195 and Binding Sites: Part II
1851
•• BLAIN HEART • KIDNEY
25
FIG. I 18
o
Time ~ourse of the in vivo specific binding of [~H]R05-4864. Each value + s.e.m, represents the mean of at least 5 mice.
~
;
~
.:
TIME IN MINUTES
The most important criterion for determining whether bound [ 3HI R05-4864 labels "peripheral type" of benzodiazepine binding sites is to ascertain whether affinities of various drugs for binding sites in vivo parallel their affinities for this type of benzodiazepine binding sites in vitro. Mice received I.P. injections of various doses of be~odiazepines and PK 11195 30 rain prior to I.V. injections of 200 uCi/kg [~H]RO5-4864, were decapitated 2 rain later and their kidneys, hearts and brains assayed for bound and total radioactivity. The drugs tested had no or small effect on the total radioactivity, the displacement only occurred in the bound activity (Fig. 2). In all the organs, the following order of potency was found : PK 11195 > R054864 > diazepam > clonazepam (inactive at 10 mg/kg I.P.) (Fig. 2). The approximate I D ~ are shown in Table I. The ID5n of PK 11195 were relatively similar from o ~ organ to another although the dose response curves were different. R05-4864 appeared to be more effective in the heart than in the kidneys or in the brain whereas diazepam was more active in the brain than in the heart or in the kidney. After I.P. injectio9 of I mg/kg of PK 11195 the maximal inhibition of the in vivo binding of [~H]RO5-4864 was found at 30 rain (Fig. 3). Its effect still persists during 18 hours in the brain but only during 6 hours in the kidneys and the brain. PK 11195 was also active in the three organs after oral administration (Fig. 4) with IDgn around I mg/kg at 1 hour (Table I). The inhibition of the in vivo bindi~ of [~H]RO5-4864 by 10 mg/kg P.O. of PK 11195 is shown in Fig. 5. The maximal effect was found at I hour in the three organs. The effect was more rapid in the heart and kidneys than in the brain but more prolonged in the later organ (18 hours) than in the kidneys or heart (between 6 and 18 hours). Similar results have been found in the rat (data not shown).
.,r
i
0
Z Z
Z O J.
w .a
u.
v
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m
Q
Z o
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I¢¢
•
LOG, OOS!
•
MG / K G
/
KIDNEY
MO/KG
BRAIN
LOG , DOSE
PK 11195
•• ,o-~ ... DIAZEPAM
PK 11198
•• DIAZEPAM , o - , 4,,,
• • •
LOG,
.
RO- 5 4864 DIAZEPAM PK 11195
.
.
.
MG/KG
FIG. 2
DOSE
HEART
Effect of PK 11%~5, R05-4864 and diazepam in vivo b~nding of [ JH]RO5-4864 in the mouse. Each value - s.e.m, represents the mean of 5 to 10 animals. The drugs were I.P. injected 30 min before sacrifice.
I
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Vol. 32, No. 16, 1983
PK 11195 and Binding Sites: Part II
1853
Table I DRUG EFFECTS ON BINDING OF [3H]R05-4864 IN VIVO
DRUGS
HEART
KIDNEYS
BRAIN
I.P.
0.07 (0.03 - 0.18)
0.25 (0.10 - 0.62)
0.25 (0.12 - 0.54)
P.O.
0.58 (0.25 - 1.35)
0.76 (0.40 - 1.44)
0.76 (0.49 - 1.16)
1.36 (0.98 - 1.90)
1.01 (0.53 - 1.94)
PK 11195
R05-4864
I.P
0.37 (0.13 - 1.05)
DIAZEPAM
I.P.
11.9 (3.9 - 36.1)
24.7 (x)
4.8 (3.9 -
5.9)
Results are expressed as approximate dose of drugs (~D~n) and 95 % confidence limits which half inhibits the specific binding of [ ~H~05-4864 (as defined in Material and Methods). They were calculated by log probit analysis of the displacement curves of figures 2 and 4. (x) by extrapolation 0 Z Q Z I
•
BRAIN
• ..,.,
'It
•
P K
11195
KIDNEY
qr 0 u
1OQ
m. a.
6(
Z 0 im. Z
TIME IN HOURS
FIG. 3 Time course of the inhibition of the in vivo binding of ~H]R05-4864 ~y PK 11195 after I.P. injection of I mg/kg in the mouse. Each value - s.e.m, represents the mean of 5 to 10 animals.
1854
PK 11195 and Binding Sites: Part II
• BRAIN • .,.., •
PK
Vol. 32, No. 16, 1983
11195
KIDNEY
z
v L
Z
_o
oi :
#
z
d.1
Z
0:3
t
:i
lb
LOG. DOS~E M G / KG
FIG. 4 Effect of oral administration of PK 11195 on the in vivo binding of [~H]R05-4864 in the mouse. Each value ~ s.e.m. represents the mean of 5 to 10 animals.
i
• BRAIN : :~2",,
PK
11195
1
? 0 ri
.~
o:st
:t
il
E
//-'--~ TIME IN HOURS
FIG. 5 T~me course of the inhibition of the in vivo binding of [ DH]RO5-4864 by PK 11195 after oral administration of 10 mg/kg in the mouse. Each value + s.e.m, represents the mean of 5 to 10 animals.
Vol. 32, No. 16, 1983
PK 11195 and Binding Sites: Part II
1855
DISCUSSION The present investigation demonstrates the feasibility of the in ~ivo labelling "peripheral type" of benzodiazepine binding sites with [JH]RO5-4864 in the heart, the kidneys and the brain under physiological conditions. The experiments were performed ~ min after the injection of the ligand. At this time the metabolism of [~H]R05-4864 was not a confounding factor since greater than 95 % of the recovered activity has been shown to be unmetabolized R05-4864. The percentage of specific binding, as defined in Material and Methods, of this "in vivo" labelling (65 to 75 %) is smaller than the percentage found in the "in vitro" conditions (more than 90 %). A possible explanation might be that under "in vivo" conditions endogenous ligands or modulators could interfere with the exogenously applied benzodiazepine whereas under "in vitro" conditions these putative ligands or modulators might be washed out or be less effective because of the lower ~emperature (0-4"C). The tissue distribution of the in vivo binding of [JH]R05-4864 parallels the tissue distribution of the in vitro binding, the amounts of RO5-4864 in the kidneys and the heart being greater than in the brain. Moreover the pharmacological specificity of the "in vivo" labelling is the same as the pharmacological specificity of the in vitro binding : PK 11195> RO5-4864 > diazepam > clonazepam (inactive at 10 mg/kg I.P.).~ Thus, all these findings indicate that intravenous tracer doses of [~H]RO5-4864 appear to label the "peripheral type" of benzodiazepine binding sites "in vivo" allowing to evaluate the actions of drugs on such binding sites in vivo. T ~ s has been applied to PK 11195. This compound "in vivo" displaces [~H]RO5-4864 bound to the "peripheral type" of benzodiazepine binding sites in the kidneys, the heart and the brain both in the mice and the rats. PK 11195 was more potent than RO5-4864 when administered either intraperitoneally or per os. We have also studied its duration of action as displacing agent after I.P. injection or orale administration. The effect of PK 11195 on the "peripheral type" of benzodiazepine binding sites is selective since PK 11195, like RO5-4864, is inactive until 25 mg/kg I.P~ on the "brain type" of benzodiazepine binding sites in vivo labelled by [JH]flunitrazepam according to the method of Chang and Snyder (I) (data not shown). In conclusion this study demonstrates the presence of [3H]RO5-4864 binding sites in the heart, the kidneys and the brain "in vivo" and also emphasizes the feasibility of this technique to examine the effects of several pharmacological (PK 11195) and physiological manipulations of these binding sites in living animals. In our ease where the physiological relevance of these binding sites remains to be established, the fact that PK 11195 binds to these sites in vivo indicates that this compound might be used as a tool to investigate the possible physiological role of the "peripheral type" of benzodiazepine binding sites in intact animals. ACKNOWLEDGMENTS The authors wish to thank Hoffman-La Roche for their generous supply of R05-4864 and Mrs. E. Berthier for her excellent secretarial assistance. REFERENCES I. 2. 3.
R.S.L. CHAN~.and S.H. SNYDER, Europ. J. Pharmacol. 48 213-218 (1978) T. DUKA, V. HOLLT and A. HERZ, Brain Res. 179 147-156 (1979) M.J. WILLIAMSON, S.M. PAUL and P. SKOLNICK, Life Sci. 23 1935-1940 (1978)
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Vol. 32, No. 16, 1983
J.F. TALLMAN, J.W. THOMAS and D.W. GALLAGER, Life Sci. 24 873-880 (1979) 5. C. BRAESTRUP and R.F. SQUIRES, Proc. Nat. Acad. Sci. USA 74 3805-3809 (1977) 6. J.K. WANG, T. TANIGUCHI and S. SPECTOR, Life Sci. 27 1881-1888 (1980) 7. T. TANIGUCHI, J.K. WANG and S. SPECTOR, Life Sci. 27 171-178 (1980) 8. T. TANIGUCHI, J.K. WANG and S. SPECTOR, Biochem. Pharmacol. 31 589-590 (1982) 9. H. SCHOEMAKER, M. BLISS and H.I. YAMAMURA, Europ. J. Pharmacol. 71 173-175 (1981) 10. G. LE FUR, M.L. PERRIER, N. VAUCHER, F. IMBAULT, A. FLAMIER, J. BENAVIDES, A. UZAN, C. RENAULT, M.C. DUBROEUCQ and C. GUEREMY. Preceding paper in this issue.