allows estimation of receptor recovery in vivo. To assess benzodiazepine receptor recov- ery after benzodiazepine discontinuation, we treated mice with ...
Accelerated
benzodiazepine
receptor
recovery
after
lorazepam
discontinuation LAWRENCE Division New
G. MILLER,
of Clinical
England
Medical
to a behavioral
syndrome
LUMPKIN,
DAVID
discontinuation in animals
and
can lead
humans.
In
a
mouse model, this syndrome is associated with benzodiazepine receptor up-regulation. The protein-modifying reagent, (EEDQ),
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline has been used to irreversibly inactivate
ber of neurotransmitter
receptors thus allows
including estimation
a num-
benzodiazeof receptor
pine receptors, and recovery in vivo. To assess benzodiazepine receptor recovery after benzodiazepine discontinuation, we treated mice with lorazepam (LRZ), 2 mg. kg-1 day-1 for 1 wk. After 24 h, EEDQ (12.5 mg/kg) was administered, and benzodiazepine binding in the cortex and cerebellum was determined after 4-144 h. EEDQ treatment decreased receptor density in the cortex in both LRZ- and vehicletreated groups by approximately 50%, with no change in apparent affinity as previously reported. Binding in both groups returned to control values after 96 h. Kinetic analysis indicated a more rapid increase in binding in LRZcompared with vehicle-treated animals, with t1,2 for LRZ 19.1 h, and for vehicle, 30.8 h (P < 0.05). Receptor density was decreased in the cerebellum after EEDQ by approximately 40% in both treatment groups, with no change in apparent affinity. Receptor density returned to control values at 96 h, with no difference in kinetics in LRZ- compared with vehicle-treated mice. The decrease in receptor ti,2 associated with lorazepam discontinuation is consistent with the observed increase in benzodiazepine receptors in this setting. Miller, L. G.; Lumpkin, M.; Greenblatt, D. J.; Shader, R. I. Accelerated benzodiazepine receptor recovery after lorazepam discontinuation. FASEB J. 5: 93-97; 1991. Key
Words:
lorazepam
. discontinuation
benzodiaze-
BENZODIAZEPINE ADMINISTRATION is associated with the development of behavioral tolerance (1). Most neurochemical evidence indicates that chronic treatment is also associated with alterations in binding or function at the GABAA receptor (2). In particular, studies have indicated decreased benzodiazepine binding or decreased GABAdependent chloride uptake during administration of several benzodiazepines (3-6). Benzodiazepine discontinuation in humans and animals can produce a characteristic behavioral syndrome (7). Previous studies from our laboratory indicate that this syndrome is associated with increased binding and function at the GABAA receptor (8, 9). These data suggest a pattern of receptor down-regulation during treatment, followed by up-regulation after discontinuation. Such a pattern might be expected to result in altered rates of benzodiazepine synthesis. CHRONIC
1/0005-0093/$01
GREENBLATT,
AND
.50. © FASEB
RICHARD
I. SHADER
School of Medicine
and
The protein-modifying reagent, N-ethoxycarbonyl-2-ethoxy1,2-dihydroquinoline (EEDQ),’ has been used to irreversibly inactivatea number of neurotransmitter receptors,including cholinergic,dopaminergic, adrenergic, and serotonergic receptors (10-13). We have previously demonstrated that EEDQ inactivates approximately 50% of benzodiazepine receptors in cortical and cerebellar membranes both in vitro and in vivo (14). Use of this agent in vivo allows determination of receptor recovery and approximation of benzodiazepine receptor half-life. To assess the effects of chronic benzodiazepine administration on benzodiazepine receptor synthetic rate, we administered lorazepam (LRZ), 2 mg kg . day, to mice for 1 wk, followed by EEDQ (12.5 mg/kg). We evaluated benzodiazepine receptor binding in cortex and cerebellum at intervals from 4 to 144 h after EEDQ administration.
METHODS Materials Male CD1 mice, 6-8 wk of age, were purchased from Charles River (Wilmington, Mass.), maintained on a 12-h light/dark cycle, and given food and water ad libitum. [3H]Flunitrazepam (FNTZ, sp act 70 Ci/mmol) was purchased from New England Nuclear (Boston, Mass.). EEDQ was obtained from Aldrich (Milwaukee, Wis.). Flurazepam was a giftfrom Hoffmann-La Roche (Nutley, N.J.). Osmotic pumps were obtained from Alza (Palo Alto, Calif.). All other reagents were obtained from standard commercial sources.
Drug EEDQ
pine#{149} GABA
0892-6638/9
J.
Pharmacology and Departments of Pharmacology and Psychiatry, Tufts University Center, Boston, Massachusetts 02111, USA
Benzodiazepine
ABSTRACT
MONICA
administration
Lorazepam (2 mg. kg’ . day) was dissolved in PEG 400 and administered for 1 wk by subcutaneously implanted osmotic pumps as previously described (4). After 7 days, pumps were removed. After an additional 24 h to allow elimination of lorazepam, EEDQ (12.5 mg/kg) was administered i.p. This interval is required to prevent antagonism of EEDQ effects by residual lorazepam (14). Previous studies indicate that concentrations of lorazepam at 24 h after discontinuation using this protocol are negligible or undetectable (8). The dose ofEEDQwas chosen on the basis of previous studies indicating benzodiazepine receptor inactivation without mortality (14). EEDQ was dissolved in 50% ethanol and diluted in water to an ethanol concentration of 2.5%. Vehicle contained a similar ethanol concentration. At 4, 24, 48, 96, or 144 h after injection, the animals were killed and brains were rapidly
‘Abbreviations: quinoline; FNTZ,
EEDQ N-ethoxycarbonyl-2-ethoxy-1,2-dihydro[3H]flunitrazepam; LRZ, lorazepam.
93
removed (P2) were described
and dissected prepared from (4).
Benzodiazepine
on ice. Synaptosomal cortex and cerebellum
membranes as previously
binding
1.25
Benzodiazepine binding was performed as described previously (4). [3H]FNTZ, 0.1-20 nM in 50 mM Tris-HC1 (pH 7.4), was added to duplicate or triplicate samples. To an identical set of samples was added flurazepam, i0 M, to determine nonspecific binding. After incubation at 4#{176}C for 45 mm, samples were filtered using a Brandel M48R (Gaithersburg, Md.) onto Whatman GF/B filters. Filters were washed twice with cold buffer and counted by scintillation spectrometry. Data
1.50
0 0. C’
E 0
1.00
E 0.
-0--
S
E 0.75
analysis I
the EBDA programs. Data were analyzed by the Mann-Whitney test for two groups, analysis of variance for more than two groups, and linear regression for determination of rate constants. Receptor
analyses
were
performed
0
using
24
t’Eil!CLE
#{149}#{149}.-LORAZEPAM
-
48
A
72
TIME
96
120
144
(hours)
RESULTS Four hours after administration of EEDQ(12.5 mg/kg), benzodiazepine receptor binding was significantly decreased in the cortex of lorazepam- and vehicle-treated mice (Fig. 1). This decrement was caused in both groups by a decrease of approximately 50% in receptor density (Bm,,,;Fig. IA), as there was no change in apparent affinity at the receptor (Fig. 1B). Receptor density returned gradually to control values with both treatments by 96 h. Again, no changes were observed in apparent affinity during this period. However, density appeared to increase more rapidly in mice treated with lorazepam compared with vehicle-treated mice (Fig. 1A). To assess the kinetics of receptor turnover from these experiments, the procedure of Battaglia et al. (13) was used. Repopulation kinetics can be described briefly as:
1.50
1.00
t’EH!CLE
-0--
----#{149}-LOAZEPAM -I
ItII
0.50
[Rt]
=
r/k
x (l-e”)
where [Rt] = receptor concentration production rate constant, and K dation. Log transformation yields: In [Rss]/([Rss}
-
=
ERt])
at time t, r rate constant
=
kt
Vol. 5
January
1991
B
= receptor
48
72
TIME
96
120
144
(hours)
for degra-
Eq. 2
where (Rss] = receptor concentration at steady state. It should be added that this procedure assumes that receptor appearance occurs at a constant rate, and that the rate of receptor degradation is proportional to receptor concentration. These assumptions have been verified for several systems (13), and our previous studies of benzodiazepine receptors indicate that the assumptions appear valid for this system (14). A semi-logarithmic plot of the time course of benzodiazepine receptor recovery in cortex is presented in Fig. 2. Although [Rss] values are similar in lorazepamand vehicletreated mice ([Rss] = Bmax at day 6 minus Bmax at 4 h; lorazepam = 0.56 ± 0.08 pmol/mg prot; vehicle = 0.48 ± 0.09 pmol/mg protein), lorazepam-treated mice had rate constants approximately 40% greater than those in vehicletreated mice. Using the general relationship 11,2 = ln 2/k, this yields receptor half-lives (11/2) of 19.1 and 30.8 h for lorazepam and vehicle treatment, respectively (P < 0.05).
94
24
Eq. 1
Figure 1. Benzodiazepine receptor recovery in cortex after treatment with EEDQ Mice were treated with lorazepam, 2 mg kg’ . day’, for I wk. At 24 h after discontinuation, animals were treated with EEDQ (12.5 mg/kg) and binding was determined at 4-144 h. Results are mean ± SEM, n = 3-5 at each point. A) Receptor density (Bm,,j. Box represents mean ± SEM for control Bm,, values. B) Apparent affinity (Kd).
In cerebellum, receptor density was reduced by approximately 40% at 4 h after EEDQ administration in both lorazepamand vehicle-treated groups (Fig. 3A). No change was observed in apparent affinity in cerebellum (Fig. 3B). Density returned to control values by 96 h in both treatment groups, with similar time courses. Apparent affinity was unchanged during this period. Analysis of rate constants (analysis described above, Fig. 4) yielded similar values for lorazepamand vehicle-treated mice: receptor half-life for lorazepam-treated mice was 35.0 h, and for vehicle-treated mice it was 42.3 h.
The FASEB Journal
MILLER ET AL.
CORTEX 5
VEHICLE
0
#{149} = LORAZEPAM
-
4
3
--
S
2
-
The current results with lorazepam suggest that alterations in benzodiazepine receptor half-life might contribute to the increase in benzodiazepine receptor binding afterbrazepam discontinuation. Receptor half-life was decreased by ap#{149}proximately 40% during the period beginning 24 h after lorazepam discontinuation. This time point was chosen for the administration of EEDQ as residual lorazepam before this point might interfere with EEDQ effects (14). Although EEDQ administration might affectthe time course for recepo (or alterations, the relatively rapid increase in binding at 24-48 h after EEDQ (48-72 h after drug discontinuation) corresponds in general to the period of benzodiazepine receptor overshoot noted previously (48-96 h). These results suggest that the decrease in half-life and the previously observed increase in binding may be related.
0
1.0
0.8
0 0
24
48
72
96
0 0.
0.6 Ci
TIME (hours) 2. Kinetics of benzodiazepine receptor recovery in cortex. Results are based on data shown in Fig. IA with analysis described in the text. The slopes of the regression lines are significantly different (P < 0.05). Half-life (t,,2) is calculated from Eq. 2 in the text as t,,2 = ln 2/k, where k is the slope of the regression line. Figure
E 0
E 0.
0.4
‘C
S
#{163}
-0-
VEH4E
---.--
WRAZEPAM
0.2
DISCUSSION 0
Our results are consistent with previous data indicating that EEDQ appears to inactivate approximately 50% of benzodiazepine receptors in the cortex, and a slightly smaller percentage in the cerebellum (14). The percentage of receptors inactivated was unchanged by administration of lorazepam or vehicle. In addition, duration of receptor recovery was also similar to that reported previously, approximately 96 h. Kinetic analysis indicates similar receptor 1/2 in cortex for vehicle-treated mice in the present study and untreated mice in our previous studies (30.8 and 25.3 h, respectively) (14). Half-life is also similar to calculated mean half-lifein cultured neurons (15). Thus, our results confirm the usefulness of EEDQ in estimating benzodiazepine receptor half-life in vivo. Several recent studies indicate that chronic benzodiazepine administration is associated with decreases in benzodiazepine receptor density in several brain regions (3, 4). However, other studies indicate little or no change in binding (16). Variations in results may be due to differences in drug, mode of administration, and receptor binding assays (2). Fewer reports have addressed effects of benzodiazepine discontinuation. In our previous studies, benzodiazepine receptor binding increased rapidly after termination of lorazepam administration and had returned to control values by 24 h after drug discontinuation (8). However, receptor binding increased further and was greater than control levels by 4 days after drug discontinuation, returning to control values after 7 days. Similar receptor alterations, with slight differences in time course, were observed for alprazolam (9) and clonazepam (W. R. Galpern, M. Lumpkin, D. J. Greenblatt, R. I. Shader, L. G. Miller, unpublished results).
ACCELERATED
BENZODIAZEPI
NE RECEPTOR RECOVERY
24
72
48
A
TIME
96
120
144
120
144
(hours)
3.00
2.00 C
1.00
-0-
VENE
----#{149}-LOFAZEPAM
0
B
24
48
72
TIME
96
(hours)
Figure 3. Benzodiazepine receptor recovery in cerebellum after treatment with EEDQ. Mice were treated with lorazepam, 2 mg kg’ . day’, for I wk. At 24 h after discontinuation, animals were treated with EEDQ (12.5 mg/kg) and binding was determined at 4-144 h. Results are mean ± SEM, n = 3-5 at each point. A) Receptor density (Bm,,). Box represents mean ± SEM for control Bm1,, values. B) Apparent affinity (Ku).
95
recent study indicates that chronic borazepam administration might have different regional effects based on subtype distribution (19). Studies of GABAA receptor subunit mRNA may elucidate this issue. In conclusion, our results indicate the usefulness of the protein-modifying reagent EEDQ in assessing receptor kinetics in vivo. These data indicate that benzodiazepine receptor half-life is decreased after lorazepam discontinuation, consistentwith the increase in receptor binding that occurs in this situation.
CEREBELLUM 4
o
= VEHICLE = LORAZEPAM
--
3
The
authors
Shim
2
for
thank
Wendy
assistance.
and AG-01006
MH-34223,
L. G. M. is the recipient cal Pharmacology ation
Galpern,
Supported
Shelley
in
part
Development
the Pharmaceutical
and
grants
from the U.S. Public
of a Faculty
from
Chesley,
by
Young
DA-05258,
Health
Service.
Award
in Clini-
Manufacturers
Associ-
Foundation.
REFERENCES
0
0
48
24
TIME
96
72
(hours)
Figure 4. Kinetics of benzodiazepine receptor recovery in lum. Results are based on data shown in Fig. IA, with described in the text. The slopes of the regression lines significantly different. Half-life (t112) is calculated from Eq. text as 11,2 = In 2/k, where k is the slope of the regression
These receptor ate tor
results do not necessarily synthesis. Analysis using
alterations degradation,
in synthesis increased
cerebel-
analysis are not 2 in the line.
indicate an alteration in EEDQ cannot differenti-
from potential turnover of
changes receptors,
in recepor even
cellular pool (13, 17). Finally, it is possible that chronic drug treatment alters receptors response to EEDQ itself. Thus, although it is tempting to speculate that our results represent increases in receptor synthesis, the data cannot directly confirm this hypothesis. In addition, EEDQ affects other neurotransmitter receptors as noted above; in several systems studies, EEDQ reduces receptor density by 70% or more (10-13). It is possible that effects at these sites might contribute indirectly to the observed alterations at the benzodiazepine site. The differential effects observed in this study of cortex compared with cerebellum are consistent with previous studies involving EEDQ. This compound affects slightly fewer benzodiazepine receptors in cerebellum than in cortex (14), perhaps because of differential abundance of receptor subtypes in the two regions (18). However, rate of receptor recovery appears to be similar in the two regions, as previously reported (14). With regard to effects of lorazepam discontinuation, in a previous study we found a small but significant increase in benzodiazepine binding in cerebellum (8). This increment was substantially less than that which occurred in the cortex. The lack of change in receptor 1112 in cerebellum despite the increase in binding may result from insufficient sensitivity of the EEDQ/receptor binding methodology, especially in light of the lower level of binding in the cerebellum compared with that in the cortex. Alternatively, receptor recovery might be less affected in cerebellum than in cortex, perhaps because of receptor subtype differences. A mobilization
96
Vol. 5
of receptors
January
1991
from
another
1. Greenblatt, D. J., and Shader, R. I. (1978) Dependence, tolerance and addiction to benzodiazepines: clinical and pharmacokinetic considerations. Drug Metab. Rev. 8, 13-28 2. Miller, L. G., Greenblatt, D. J., Lopez, F., Schatzki, A., Heller, j,, Lumpkin, M., and Shader, R. I. (1990) Chronic benzodiazepine administration: effects in vivo and in vitro. In GABAA Receptor Subtypes (Biggio, G., and Costa, E., eds) Raven, New York pp. 167-176 3. Tietz, E. I., Rosenberg, H. C., and Chiu, T. H. (1986) Autoradiographic localization of benzodiazepine receptor lation. j Pharmacol. Exp. T/zer 236, 284-291
downregu-
4. Miller, L. G., Greenblatt, D. J., Barnhill, J. G., and Shader, R. I. (1988) Chronic benzodiazepine administration. I. Tolerance is associated with benzodiazepine receptor downregulation and decreased y-aminobutyric acidA receptor function. j Pharmacol. Exp. Ther 246, 170-176 5. Miller, L. G., Woolverton, S., Greenblatt, D. J., Lopez, F, and Shader, R. I. (1989) IV. Rapid development
Chronic benzodiazepine administration. of tolerance and receptor downregulawith alprazolam administration. Biochem. Phar-
tion associated macol. 38, 3773-3777 6. Marley, R. J., and Gallager, treatment stimulated 7. Petursson,
dence. 8. Miller,
D. W. (1989) Chronic
diazepam
produces regionally specific changes in GABAchloride influx. Eur. J. Phar,nacol. 159, 217-223 H., and Lader, M. H. (1981) Benzodiazepine depenBr. j Addict. 76, 133-145
L. G., Greenblatt,
D.
J.,
Summer,
R. I. (1988) Chronic benzodiazepine continuation syndrome is associated aminobutyric acidA receptor binding macoL Exp. Ther. 246, 177-182
W.
R.,
and
administration: with upregulation and function.
D. J., Chesley, administration
9. Lopez, F., Miller, L. G., Greenblatt, A., and Shader, R. I. (1990) Chronic
Shader,
II. j
Disof y-
Phar-
S., Schatzki,
of benzodi-
azepine: V. Rapid onset of behavioral and neurochemical alterations after discontinuation of alprazolam. Neuropharmacology
29,
237-241
10. Norman,
A. B., Wachendorf,
T.
J.,
and
Sanberg,
P. R. (1989)
Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [‘H]SCH23390 and [‘H]forskolin binding in rat striatum. Life Sci. 44, 831-836 11. SaIler, C. F., Kreamer, L. D., Adamovage, F., and Salama, A. I. (1989) Dopamine receptor occupancy in vivo: measurement using N-ethoxycarbonyl-l-ethoxy-1,2-dihydroquinoline (EEDOJ. Life Sci. 45, 917-929 12. Pile,A., Vetulani, J., Nomura, S., and Enna, S. J. (1989) An in vivo procedure for the selective inactivation of ratbraincerebral
cortical
es-adrenoceptors
using N-ethoxycarbonyl-2-ethoxy-1,2Brain Res. 493, 8-13 13. Battaglia, G., Norman, A. B., and Creese, I. (1987) Differential serotonin2 receptor recovery in mature and senescent rat brain after irreversible receptor modification: effect of chronic reserdihydroquinoline
pine
The FASEB Journal
treatment.
(EEDQ).
j
Pharinacol.
Exp.
They. 243,
69-75
MILLER ET AL.
14. Miller, L. G., and Shader, binding and droquinoline In press
Lumpkin, M., Galpern, W. R., Greenblatt, D. J., R. I. (1990) Modification of GABAA receptor function by N-ethoxycarbonyl-2-ethoxy-1,2-dihyin vitro and in vivo: effects of aging. J. Neurochem.
L. A., Czajkowski, C., Chan, C. Y., and Farb, D. H. (1984) Benzodiazepine receptor synthesis and degradation by neurons in culture. Science 226, 857-860 16. Gallager, D. D., Lakoski, J. M., Gonsales, S. F, and Fauch, S. L. (1984) Chronic benzodiazepine treatmentdecreasespostsynaptic GABA sensitivity. Nature (London) 308, 74-77 17. Hess, E. J., Norman, A. B., and Creese, I. (1988) Chronic treat15. Borden,
ACCELERATED
BENZODIAZEPI
NE RECEPTOR RECOVERY
ment with dopamine receptor antagonist: behavioral and pharmacological effects on D1 and D2 dopamine receptors. j Neurosci. 8, 2361-2370 18. Sieghart, W. (1989) Multiplicityof GABAA-benzodiazepine receptors. TIPS 10, 407-411 19. Galpern, W. R., Miller, L. G., Greenblatt, D. J., and Shader, R. I. (1990) Differential effectsof chronic borazepam and alprazolam on benzodiazepine binding and GABAA receptor function. Br. j P/zarrnacoL In press Received for publication July Accepted for publication September
23,
1990.
17, 1990.
97
