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Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat
'Brain Research, 290 (1984) 183-186 Elsevier
183
Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat RICHARD G. LISTER1,*, DAVID J. GREENBLATT2, DARRELL R. ABERNETHY2 and SANDRA E. FILE 1 1Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Sq., London WC1N ! A X (U. K.) 2Division of Clinical Pharmacology, Tufts-New England Medical Center, 171 Harrison Ave., Boston, MA 02111 (U.S.A.) (Accepted September 13th, 1983) Key words: Ro 15-1788- - benzodiazepine - - pharmacokinetics - - brain - - rat
Methods for determining Ro 15-1788in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788in the brain were reduced if chlordiazepoxide was coadministered.
In 1981, Hunkeler and coworkers reported that an imidazodiazepine (Ro 15-1788) with a high affinity for brain benzodiazepine binding sites, possessed no intrinsic pharmacological activity but reversed many of the behavioral actions of the benzodiazepines 9. Since that report, several groups of workers have demonstrated that this c o m p o u n d does in fact have behavioral actions of its own2,3,6-8,11,12, s o m e of which are potentiated by benzodiazepines7, 8. It is necessary to know of any pharmacokinetic changes resulting from coadministration of R o 15-1788 and a benzodiazepine before other explanations of these effects are discussed. The present study aimed at discovering whether such changes are important. In human subjects the plasma elimination half-life of Ro 15-1788 is less than 30 mint. It is perhaps surprising in view of its rapid elimination from plasma that Ro 15-1788 can reverse benzodiazepine effects for as long as 6 h after administration 4. One possible explanation is that R o 15-1788 is eliminated from the brain more slowly than from plasma. The present study compares the plasma and brain pharmacokinetics of Ro 15-1788 in the rat. The doses of Ro 15-1788 used were ones that had clear intrinsic behavioral actions6,7 and the dose of
chlordiazepoxide chosen was one that potentiated the effect of R o 15-17887 Ro 15-1788 was suspended in a water/Tween-20 vehicle to concentrations of 2 and 5 mg/ml. Chlordiazepoxide hydrochloride (Roche Products Ltd) was dissolved in water to a concentration of 2.5 mg/ml. Groups of male hooded-rats (Olac, Bicester), n = 7 per group, weighing approximately 350 g, received the following drug treatments: R o 15-1788 (4 or 10 mg/kg), chlordiazepoxide (5 mg/kg), or R o 151788 (4 or 10 mg/kg) plus chlordiazepoxide (5 mg/kg). Twenty minutes after receiving their i.p. injections, animals were killed by decapitation, trunk blood was collected and the brains were removed. In order to obtain information on the elimination of Ro 15-1788 further groups of animals were killed 40 and 80 min after an i.p. injection of R o 15-1788 (10 mg/kg). Plasma was separated by centrifugation and the concentrations of chlordiazepoxide and Ro 15-1788 were measured as described below. Abernethy and coworkers developed a method for determining R o 15-1788 in plasma using gas chromatography with nitrogen-phosphorus detection 1. We therefore modified this method to measure R o 151788 in brain tissue. Gas chromatography could not,
* Correspondence at present address: R. G. Lister, National Institute on Alcohol Abuse and Alcoholism, 10/ACRF Building, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
184 however, be used on samples that contained chlordiazepoxide, since this compound and its two major metabolites fragment on the column giving rise to a number of broad interfering peaks on the chromatographic trace 13. A second method was therefore developed for these samples, using high performance liquid chromatography (HPLC). We have previously used reverse phase HPLC to determine chlordiazepoxide and its metabolites, desmethylchlordiazepoxide (DMCDP) and demoxepam (DMX), in plasma and brain tissue and this technique was therefore used in the present study. The apparatus, chromatographic conditions and extraction procedure have already been described5,10. Standard tubes contained 10 ktg Ro 5-3027 (chlo-
J J ),
0
5
I0
15 20
0
5
10
15
20
Time (minutes)
Fig. 1. A chromatogram of a drug-free control brain extract (right) and a sample to which Ro 15-1788 (1.5 #g), chlordiazepoxide (CDP, 3/~g), desmethylchlordiazepoxide (DMCDP, 3/~g) and demoxepam (DMX, 3/~g), and the internal standard chlorodesmethyldiazepam (Ro 5-3027, 10#g) have been added (left).
rodesmethyldiazepam, the internal standard) and 0.1, 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 #g chlordiazepoxide (CDP), DMX, D M C D P and Ro 15-1788. Blank brain homogenates were added to each tube. Standards were assayed with each set of unknowns. The retention times for Ro 15-1788, DMX, DMCDP, CDP and chlorodesmethyldiazepam were approximately 5.1, 6.9, 9.3, 12.2 and 14.6 min respectively (see Fig. 1). Plots of peak area or height ratio (relative to the internal standard) against concentration were linear for each compound up to concentrations of at least 10 #g/g brain tissue and gave correlation coefficients of greater than 0.99. Recoveries of 75-85% were obtained and concentrations as low as 0.1 #g/g brain tissue for CDP and its metabolites and as low as 50 ng/g could be determined for Ro 15-1788, starting from 0.5 g tissue. The apparatus and conditions for the gas chromatographic assay have been described previously1. Nmethylclonazepam served as the internal standard. A series of round-bottomed tubes was prepared containing 200 ng N-methylclonazepam. Each brain minus the cerebellum was bisected along the midline. Half-brains were weighed, homogenized in 3.5 ml of 0.4 M perchloric acid and the homogenate transferred to one of the tubes. The pH was adjusted to approximately 9 by the addition of 2 M sodium carbonate solution; 4 ml ethyl acetate were added, the tubes vortexed for 1 min and then centrifuged at 400 g for 10 min. The organic phase was transferred to a tube containing 1.5 ml of 4 M hydrochloric acid. The tubes were vortexed, centrifuged, and the organic phase discarded. After washing with ethyl acetate the pH of the aqueous phase was adjusted to approximately 9 by adding 2 M sodium carbonate solution; 3 ml ethyl acetate were added, the tubes were shaken and then centrifuged and the organic phase removed and evaporated to dryness at 40 °C under conditions of mildly reduced pressure. The residue was reconstituted with 50 #1 toluene (containing 15% isoamyl alcohol); 6 #1 were injected into the chromatograph. Calibration standards were prepared by adding 10, 25, 50, 100 and 200 ng of Ro 15-1788 to consecutive tubes containing 200 ng of N-methylclonazepam. Blank brain homogenates were added to each tube and taken through the assay with each set of unknown samples.
185
•
The H P L C m e t h o d was used to study the effects of C D P on the brain concentrations of R o 15-1788 and the gas c h r o m a t o g r a p h i c assay was used to investigate the elimination of R o 15-1788 from the brain. The concentrations of R o 15-1788, C D P and its metabolites in the d r u g - t r e a t e d animals are given in Table I. The administration of R o 15-1788 had no effect on the concentrations of chlordiazepoxide or its metabolites. This is consistent with a previous r e p o r t in human subjects showing that R o 15-1788 did not affect diazepam concentrations in plasma 4 and demonstrates that R o 15-1788 does not antagonize the effects of benzodiazepines by altering their bioavailability. H o w e v e r , the coadministration of chlordiaze-
/
poxide significantly r e d u c e d the concentrations of R o 15-1788 in rat brain following the 4 mg/kg dose of the imidazodiazepine (t = 4.0, P < 0.01). This reduction in brain concentration of R o 15-1788 clearly cannot explain chlordiazepoxide's e n h a n c e m e n t of the behavioral actions of the doses of R o 15-1788 studied 7. W e cannot rule out the possibility that R o 15-1788 has pharmacologically active metabolites. H o w e v e r , no additional peaks showed up on the chromatograms. The concentrations of R o 15-1788 in brain 20, 40 and 80 min after injection are shown in Fig. 3. It can be seen that R o 15-1788 is eliminated from the brain with an elimination half-life of a p p r o x i m a t e l y 16 min. Interestingly, the concentrations of R o 15-1788 in plasma were too low to be d e t e c t e d , and t h e r e f o r e less than 5 ng/ml at all time points. That R o 15-1788 was easily detectable in brain but not in p l a s m a at the time points studied suggests that the c o m p o u n d m a y be very rapidly m e t a b o l i z e d in the periphery. A l t e r -
\ t
t
t
0
5
10
[
0
5
i
10
T]ME(min) Fig. 2. A chromatogram of a drug-free control brain extract (left) and a sample to which Ro 15-1788 (75 ng) and methylclonazepam (200 ng) have been added (right). The retention times for R o 15-1788 and N-methylclonazepam were a p p r o x i m a t e l y 4.0 and 6.7 min respectively (see Fig. 2). Plots of p e a k height ratio (relative to the internal standard) against concentration were linear over the concentration range studied and gave correlation coefficients of greater than 0.99. Recoveries of 60-70% were obtained.
TABLE I Concentrations of Ro 15-1788, chlordiazepoxide (CDP), desmethylchlordiazepoxide (DMCDP) and of demoxepam (DMX) 20 min after i.p. injection of Ro 15-1788 (4 or 10 mg/kg) alone or in combination with CDP (5 rng/kg)
Scores are means + S.E.M. Drug treatment (mg/kg)
Brain concentration (ng/g) Ro 15-1788
CDP
DMCDP
DMX
CDP, 5 Ro 15-1788, 4 Ro 15-1788, 10 Ro 15-1788, 4 + CDP, 5 Ro 15-1788, 10 + CDP, 5
-98.8 + 7.4 200.0 +__36.7 63.6 + 5.7* 147.7 + 27.5
6060 _+90 --5980 ___250 5780 + 390
132 + 11 --112 + 11 130 + 26
218 + 29 --275 + 27 209 + 29
* Significantly different from Ro 15-1788, 4 mg/kg, P < 0.01 (t-test)
186 300
ma, allowing the drug to be measured in brain but not plasma.
c~
200 v
c
A study following the pharmacokinetics of this
u
oc t~
100
=
mm
.
c o m p o u n d in h u m a n plasma found the elimination half-life to be 28 min t , and yet it has been shown to reverse the effects of diazepam for at least 6 h 4. The
so
t tt3
o
.c_
results from the present study suggest that the com-
20 I
I
2O
40 Minutes
after
I
I
6O
8O
paratively long-lasting reversal of diazepam-induced sedation by Ro 15-1788 may result from the slower elimination of the imidazodiazepine from the brain.
Dose
Fig. 3. The concentration of Ro 15-1788 in rat brain 20, 40 and 80 min following i.p. administration of 10 mg/kg. The line was plotted using least squares regression analysis, indicating an apparent disappearance half-life of 16 min.
R . G . L . is supported by a School of Pharmacy postgraduate award. D . J . G . and D . R . A . are supported by Grants MH-34223 and AM-32050 from the U n i t e d States Public Health Service. S.E.F. is a Wellcome
natively, since Ro 15-1788 is a highly lipophilic compound like the benzodiazepines, brain concentrations are likely to considerably exceed those in plas-
Trust senior lecturer. The authors are grateful for the assistance of Dr. Richard I. Shader.
1 Abernethy, D. R., Arendt, R. M., Lauven, P. M. and Greenblatt, D. J., Determination of Ro 15-1788, a benzodiazepine antagonist, in human plasma by gas-liquid chromatography with nitrogen-phosphorus detection, Pharmacology, 26 (1983) 285-289. 2 Corda, M. G., Costa, E. and Guidotti, A., Specificproconvulsant action of an imidazobenzodiazepine (Ro 15-1788) on isoniazid convulsions, Neuropharmacology, 21 (1982) 91-94. 3 Dantzer, R. and Perio, A., Behavioural evidence for partial agonist properties of Ro 15-1788, a benzodiazepine receptor antagonist, Europ. J. Pharmacol., 81 (1982) 655-658. 4 Darragh, A., Lambe, R., Kenny, M., Brick, I., Taaffe, W. and O'Boyle, C., Ro 15-1788 antagonises the central effects of diazepam in man without altering diazepam bioavailability, Brit. J. clin. Pharmacol., 14 (1982) 677-682. 5 Divoll, M., Greenblatt, D. J. and Shader, R. I., Liquid chromatographic determination of chlordiazepoxide and metabolites in plasma, Pharmacology, 24 (1982) 261-266. 6 File, S. E., Lister, R. G. and Nutt, D. J., The anxiogenic action of benzodiazepine antagonists, Neuropharmacology, 21 (1982) 1033-1037. 7 File, S. E., Lister, R. G. and Nutt, D. J., Intrinsic actions of benzodiazepine antagonists, Neurosci. Lett., 32 (1982) 165-168.
8 Grecksch, G., Prado de Carvalho, L., Venault, P., Chapouthier, G. and Rossier, J., Convulsions induced by submaximal doses of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788, Life Sci., 32 (1983) 2579-2584. 9 Hunkeler, W., Moehler, H., Pieri, L., Polc, P., Bonetti, E. P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of benzodiazepines, Nature (Lond.), 290 (1981) 514-516. 10 Lister, R. G., Abernethy, D. R., Greenblatt, D. J. and File, S. E., Methods for the determination of lorazepam and chlordiazepoxide and metabolites in brain tissue: a comparison with plasma concentrations in the rat, J. Chrom., in press. 11 Lloyd, K. G., Bovier, P., Broekkamp, C. L. and Worms, P., Reversal of the antiaversive and anticonvulsant actions of diazepam, but not of progabide by a selective antagonist of benzodiazepine receptors, Europ. J. Pharmacol., 75 (1981) 77-78. 12 Nutt, D. J., Cowen, P. J. and Little, H. J., Unusual interactions of benzodiazepine antagonists, Nature (Lond.), 295 (1982) 436--438. 13 Sadee, W. and Van der Kleijn, E., Thermolysis of 1,4-benzodiazepines during gas chromatography and mass spectrometry, J. Pharm. Sci., 60 (1971) 135-137.
183
Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat RICHARD G. LISTER1,*, DAVID J. GREENBLATT2, DARRELL R. ABERNETHY2 and SANDRA E. FILE 1 1Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Sq., London WC1N ! A X (U. K.) 2Division of Clinical Pharmacology, Tufts-New England Medical Center, 171 Harrison Ave., Boston, MA 02111 (U.S.A.) (Accepted September 13th, 1983) Key words: Ro 15-1788- - benzodiazepine - - pharmacokinetics - - brain - - rat
Methods for determining Ro 15-1788in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788in the brain were reduced if chlordiazepoxide was coadministered.
In 1981, Hunkeler and coworkers reported that an imidazodiazepine (Ro 15-1788) with a high affinity for brain benzodiazepine binding sites, possessed no intrinsic pharmacological activity but reversed many of the behavioral actions of the benzodiazepines 9. Since that report, several groups of workers have demonstrated that this c o m p o u n d does in fact have behavioral actions of its own2,3,6-8,11,12, s o m e of which are potentiated by benzodiazepines7, 8. It is necessary to know of any pharmacokinetic changes resulting from coadministration of R o 15-1788 and a benzodiazepine before other explanations of these effects are discussed. The present study aimed at discovering whether such changes are important. In human subjects the plasma elimination half-life of Ro 15-1788 is less than 30 mint. It is perhaps surprising in view of its rapid elimination from plasma that Ro 15-1788 can reverse benzodiazepine effects for as long as 6 h after administration 4. One possible explanation is that R o 15-1788 is eliminated from the brain more slowly than from plasma. The present study compares the plasma and brain pharmacokinetics of Ro 15-1788 in the rat. The doses of Ro 15-1788 used were ones that had clear intrinsic behavioral actions6,7 and the dose of
chlordiazepoxide chosen was one that potentiated the effect of R o 15-17887 Ro 15-1788 was suspended in a water/Tween-20 vehicle to concentrations of 2 and 5 mg/ml. Chlordiazepoxide hydrochloride (Roche Products Ltd) was dissolved in water to a concentration of 2.5 mg/ml. Groups of male hooded-rats (Olac, Bicester), n = 7 per group, weighing approximately 350 g, received the following drug treatments: R o 15-1788 (4 or 10 mg/kg), chlordiazepoxide (5 mg/kg), or R o 151788 (4 or 10 mg/kg) plus chlordiazepoxide (5 mg/kg). Twenty minutes after receiving their i.p. injections, animals were killed by decapitation, trunk blood was collected and the brains were removed. In order to obtain information on the elimination of Ro 15-1788 further groups of animals were killed 40 and 80 min after an i.p. injection of R o 15-1788 (10 mg/kg). Plasma was separated by centrifugation and the concentrations of chlordiazepoxide and Ro 15-1788 were measured as described below. Abernethy and coworkers developed a method for determining R o 15-1788 in plasma using gas chromatography with nitrogen-phosphorus detection 1. We therefore modified this method to measure R o 151788 in brain tissue. Gas chromatography could not,
* Correspondence at present address: R. G. Lister, National Institute on Alcohol Abuse and Alcoholism, 10/ACRF Building, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
184 however, be used on samples that contained chlordiazepoxide, since this compound and its two major metabolites fragment on the column giving rise to a number of broad interfering peaks on the chromatographic trace 13. A second method was therefore developed for these samples, using high performance liquid chromatography (HPLC). We have previously used reverse phase HPLC to determine chlordiazepoxide and its metabolites, desmethylchlordiazepoxide (DMCDP) and demoxepam (DMX), in plasma and brain tissue and this technique was therefore used in the present study. The apparatus, chromatographic conditions and extraction procedure have already been described5,10. Standard tubes contained 10 ktg Ro 5-3027 (chlo-
J J ),
0
5
I0
15 20
0
5
10
15
20
Time (minutes)
Fig. 1. A chromatogram of a drug-free control brain extract (right) and a sample to which Ro 15-1788 (1.5 #g), chlordiazepoxide (CDP, 3/~g), desmethylchlordiazepoxide (DMCDP, 3/~g) and demoxepam (DMX, 3/~g), and the internal standard chlorodesmethyldiazepam (Ro 5-3027, 10#g) have been added (left).
rodesmethyldiazepam, the internal standard) and 0.1, 0.25, 0.5, 1.0, 2.0, 5.0 and 10.0 #g chlordiazepoxide (CDP), DMX, D M C D P and Ro 15-1788. Blank brain homogenates were added to each tube. Standards were assayed with each set of unknowns. The retention times for Ro 15-1788, DMX, DMCDP, CDP and chlorodesmethyldiazepam were approximately 5.1, 6.9, 9.3, 12.2 and 14.6 min respectively (see Fig. 1). Plots of peak area or height ratio (relative to the internal standard) against concentration were linear for each compound up to concentrations of at least 10 #g/g brain tissue and gave correlation coefficients of greater than 0.99. Recoveries of 75-85% were obtained and concentrations as low as 0.1 #g/g brain tissue for CDP and its metabolites and as low as 50 ng/g could be determined for Ro 15-1788, starting from 0.5 g tissue. The apparatus and conditions for the gas chromatographic assay have been described previously1. Nmethylclonazepam served as the internal standard. A series of round-bottomed tubes was prepared containing 200 ng N-methylclonazepam. Each brain minus the cerebellum was bisected along the midline. Half-brains were weighed, homogenized in 3.5 ml of 0.4 M perchloric acid and the homogenate transferred to one of the tubes. The pH was adjusted to approximately 9 by the addition of 2 M sodium carbonate solution; 4 ml ethyl acetate were added, the tubes vortexed for 1 min and then centrifuged at 400 g for 10 min. The organic phase was transferred to a tube containing 1.5 ml of 4 M hydrochloric acid. The tubes were vortexed, centrifuged, and the organic phase discarded. After washing with ethyl acetate the pH of the aqueous phase was adjusted to approximately 9 by adding 2 M sodium carbonate solution; 3 ml ethyl acetate were added, the tubes were shaken and then centrifuged and the organic phase removed and evaporated to dryness at 40 °C under conditions of mildly reduced pressure. The residue was reconstituted with 50 #1 toluene (containing 15% isoamyl alcohol); 6 #1 were injected into the chromatograph. Calibration standards were prepared by adding 10, 25, 50, 100 and 200 ng of Ro 15-1788 to consecutive tubes containing 200 ng of N-methylclonazepam. Blank brain homogenates were added to each tube and taken through the assay with each set of unknown samples.
185
•
The H P L C m e t h o d was used to study the effects of C D P on the brain concentrations of R o 15-1788 and the gas c h r o m a t o g r a p h i c assay was used to investigate the elimination of R o 15-1788 from the brain. The concentrations of R o 15-1788, C D P and its metabolites in the d r u g - t r e a t e d animals are given in Table I. The administration of R o 15-1788 had no effect on the concentrations of chlordiazepoxide or its metabolites. This is consistent with a previous r e p o r t in human subjects showing that R o 15-1788 did not affect diazepam concentrations in plasma 4 and demonstrates that R o 15-1788 does not antagonize the effects of benzodiazepines by altering their bioavailability. H o w e v e r , the coadministration of chlordiaze-
/
poxide significantly r e d u c e d the concentrations of R o 15-1788 in rat brain following the 4 mg/kg dose of the imidazodiazepine (t = 4.0, P < 0.01). This reduction in brain concentration of R o 15-1788 clearly cannot explain chlordiazepoxide's e n h a n c e m e n t of the behavioral actions of the doses of R o 15-1788 studied 7. W e cannot rule out the possibility that R o 15-1788 has pharmacologically active metabolites. H o w e v e r , no additional peaks showed up on the chromatograms. The concentrations of R o 15-1788 in brain 20, 40 and 80 min after injection are shown in Fig. 3. It can be seen that R o 15-1788 is eliminated from the brain with an elimination half-life of a p p r o x i m a t e l y 16 min. Interestingly, the concentrations of R o 15-1788 in plasma were too low to be d e t e c t e d , and t h e r e f o r e less than 5 ng/ml at all time points. That R o 15-1788 was easily detectable in brain but not in p l a s m a at the time points studied suggests that the c o m p o u n d m a y be very rapidly m e t a b o l i z e d in the periphery. A l t e r -
\ t
t
t
0
5
10
[
0
5
i
10
T]ME(min) Fig. 2. A chromatogram of a drug-free control brain extract (left) and a sample to which Ro 15-1788 (75 ng) and methylclonazepam (200 ng) have been added (right). The retention times for R o 15-1788 and N-methylclonazepam were a p p r o x i m a t e l y 4.0 and 6.7 min respectively (see Fig. 2). Plots of p e a k height ratio (relative to the internal standard) against concentration were linear over the concentration range studied and gave correlation coefficients of greater than 0.99. Recoveries of 60-70% were obtained.
TABLE I Concentrations of Ro 15-1788, chlordiazepoxide (CDP), desmethylchlordiazepoxide (DMCDP) and of demoxepam (DMX) 20 min after i.p. injection of Ro 15-1788 (4 or 10 mg/kg) alone or in combination with CDP (5 rng/kg)
Scores are means + S.E.M. Drug treatment (mg/kg)
Brain concentration (ng/g) Ro 15-1788
CDP
DMCDP
DMX
CDP, 5 Ro 15-1788, 4 Ro 15-1788, 10 Ro 15-1788, 4 + CDP, 5 Ro 15-1788, 10 + CDP, 5
-98.8 + 7.4 200.0 +__36.7 63.6 + 5.7* 147.7 + 27.5
6060 _+90 --5980 ___250 5780 + 390
132 + 11 --112 + 11 130 + 26
218 + 29 --275 + 27 209 + 29
* Significantly different from Ro 15-1788, 4 mg/kg, P < 0.01 (t-test)
186 300
ma, allowing the drug to be measured in brain but not plasma.
c~
200 v
c
A study following the pharmacokinetics of this
u
oc t~
100
=
mm
.
c o m p o u n d in h u m a n plasma found the elimination half-life to be 28 min t , and yet it has been shown to reverse the effects of diazepam for at least 6 h 4. The
so
t tt3
o
.c_
results from the present study suggest that the com-
20 I
I
2O
40 Minutes
after
I
I
6O
8O
paratively long-lasting reversal of diazepam-induced sedation by Ro 15-1788 may result from the slower elimination of the imidazodiazepine from the brain.
Dose
Fig. 3. The concentration of Ro 15-1788 in rat brain 20, 40 and 80 min following i.p. administration of 10 mg/kg. The line was plotted using least squares regression analysis, indicating an apparent disappearance half-life of 16 min.
R . G . L . is supported by a School of Pharmacy postgraduate award. D . J . G . and D . R . A . are supported by Grants MH-34223 and AM-32050 from the U n i t e d States Public Health Service. S.E.F. is a Wellcome
natively, since Ro 15-1788 is a highly lipophilic compound like the benzodiazepines, brain concentrations are likely to considerably exceed those in plas-
Trust senior lecturer. The authors are grateful for the assistance of Dr. Richard I. Shader.
1 Abernethy, D. R., Arendt, R. M., Lauven, P. M. and Greenblatt, D. J., Determination of Ro 15-1788, a benzodiazepine antagonist, in human plasma by gas-liquid chromatography with nitrogen-phosphorus detection, Pharmacology, 26 (1983) 285-289. 2 Corda, M. G., Costa, E. and Guidotti, A., Specificproconvulsant action of an imidazobenzodiazepine (Ro 15-1788) on isoniazid convulsions, Neuropharmacology, 21 (1982) 91-94. 3 Dantzer, R. and Perio, A., Behavioural evidence for partial agonist properties of Ro 15-1788, a benzodiazepine receptor antagonist, Europ. J. Pharmacol., 81 (1982) 655-658. 4 Darragh, A., Lambe, R., Kenny, M., Brick, I., Taaffe, W. and O'Boyle, C., Ro 15-1788 antagonises the central effects of diazepam in man without altering diazepam bioavailability, Brit. J. clin. Pharmacol., 14 (1982) 677-682. 5 Divoll, M., Greenblatt, D. J. and Shader, R. I., Liquid chromatographic determination of chlordiazepoxide and metabolites in plasma, Pharmacology, 24 (1982) 261-266. 6 File, S. E., Lister, R. G. and Nutt, D. J., The anxiogenic action of benzodiazepine antagonists, Neuropharmacology, 21 (1982) 1033-1037. 7 File, S. E., Lister, R. G. and Nutt, D. J., Intrinsic actions of benzodiazepine antagonists, Neurosci. Lett., 32 (1982) 165-168.
8 Grecksch, G., Prado de Carvalho, L., Venault, P., Chapouthier, G. and Rossier, J., Convulsions induced by submaximal doses of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788, Life Sci., 32 (1983) 2579-2584. 9 Hunkeler, W., Moehler, H., Pieri, L., Polc, P., Bonetti, E. P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of benzodiazepines, Nature (Lond.), 290 (1981) 514-516. 10 Lister, R. G., Abernethy, D. R., Greenblatt, D. J. and File, S. E., Methods for the determination of lorazepam and chlordiazepoxide and metabolites in brain tissue: a comparison with plasma concentrations in the rat, J. Chrom., in press. 11 Lloyd, K. G., Bovier, P., Broekkamp, C. L. and Worms, P., Reversal of the antiaversive and anticonvulsant actions of diazepam, but not of progabide by a selective antagonist of benzodiazepine receptors, Europ. J. Pharmacol., 75 (1981) 77-78. 12 Nutt, D. J., Cowen, P. J. and Little, H. J., Unusual interactions of benzodiazepine antagonists, Nature (Lond.), 295 (1982) 436--438. 13 Sadee, W. and Van der Kleijn, E., Thermolysis of 1,4-benzodiazepines during gas chromatography and mass spectrometry, J. Pharm. Sci., 60 (1971) 135-137.