NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 3 267

Chronic Benzodiazepine Administration XI. Concurrent Administration of PKll195 Attenuates Lorazepam Discontinuation Effects John]. Byrnes, B.A., Lawrence G. Miller, M.D., Karen Perkins, M.D., DavidJ. Greenblatt, M.D., and Richard 1. Shader, M.D.

Btnzodiazepine discontinuation is associated with after lorazepam discontinuation; this effect was attenuated I1lerations in motor activity and gamma-aminobutyric by coadministration of PK11195 at 5 mglkg per day. rid-A receptor upregulation in a mouse model. Prior Lorazepam discontinuation effects were not altered by studies indicate that concurrent administration of the PK11195 at 1 mglkg per day, whereas the lO-mglkg dose compoundN-meth yl-N-(methyl-l-propyl)chloro-2-phenyl­ was not different from 5 mglkg per day. The competitive l-isoquinoline-3-carboxamide (PK1195), a "peripheral" ligand R05-4864 at 10 mglkg per day, blocked the effects site benzodiazepine antagonist, can attenuate the effects of of PKI1195 on lorazepam discontinuation. Benzodiazepine Iorazepamon tolerance and receptor alterations. To receptor binding in vivo was increased in the cortex and nwluate the effects of PK11195 administration on hippocampus at 4 days postlorazepam discontinuation. btnzodiazepine discontinuation, we administered This effect was attenuated in the hippocampus but not in Iorazepam (2 mglkg per day), PK 11195 (1 to 10 mglkg the cortex by concurrent administration of PK1195. ptrday) or the combination to mice for 7 days, and then These data indicate that concurrent administration of nwluated pentylenetetrazole-induced seizure threshold PK11195 may attenuate discontinuation effects of l11libenzodiazepine binding at days 1, 4, and 7 after lorazepam. [Neuropsychopharmacology 8:267-273, discontinuation. Seizure theshold was reduced at 4 days 1993J

KEY WORDS: Lorazepam; PK11195; Benzodiazepine; cases even seizures and mortality (Greenblatt et al. Gamma-aminobutyric acid 1990). We have previously described a mouse model of benzodiazepine discontinuation that demonstrates The abrupt discontinuation of benzodiazepine com­ both behavioral and neurochemical alterations (Miller pounds may evoke a variety of discontinuation syn­ et al. 1988b). In particular, hyperactivity and a reduced dromes,characterized by anxiety, insomnia, and in rare seizure threshold are associated with upregulation at the gamma-aminobutyric acid-A (GABAA) receptor complex. Generally similar results have been obtained

Fromthe Departments of Pharmacology and Experimental Thera­ with the benzodiazepines lorazepam, alprazolam, and peuticsand Psychiatry, Tufts University School of Medicine; and the clonazepam (Lopez et al. 1990; Galpern et al. 1990, Division of Clinical Pharmacology, New England Medical Center, 1991a). Boston, Massachusetts. Addresscorrespondence to: Dr.Lawrence G. Miller, Box 1007,New Several interventions have been proposed to pre­ England Medical Center, 750 Washington Street, Boston, Massa­ vent or limit the effectsof benzodiazepine discontinu­ chusetts 02111. ation, includingtapering doses, use of "partial agonist" ReceivedJune 18,1992;revised October 21,1992; accepted October 26. 1992. benzodiazepines, and treatment with anticonvulsants

C 1993American College of Neuropsychopharmacology Published by Elsevier Science Publishing Co., Inc. 655Avenue of the Americas, New York, NY 10010 0893-133XI93/$6.00 268 J.J. Byrnes et al. NEUROPSYCHOPHARMACOLOGY 1993- VOL. 8, NO.l

or benzodiazepine antagonists (Miller et al. 1992). In and 7 after lorazepam discontinuation. As previously the mouse model, treatment with the anticonvulsant reported, concurrent administration of PKl1195 does carbamazepine attenuates the effects of alprazolam not alter lorazepam concentrations in brain (Miller et discontinuation (Galpern et al. 1991b). We have re­ al. 1992). Also as previously reported (Miller et aI. cently reported that administration of the "peripheral" 1988a), lorazepam isundetectable in cortex at day 1 post· benzodiazepine antagonist N-methyl-N-(methyl-1- discontinuation and subsequently thereafter. propyl)chloro-2-phenyl-1-isoquinoline-3-carboxamide (PK11195) limits the development of tolerance to loraze­ Pentylenetetrazole-Induced Seizures pam (Miller et al. 1992), as was previously reported in a model of diazepam tolerance in the rat (Massotti et As previously described (Schatzki et al. 1989), unre­ al. 1990). strained mice were infused intravenously with a solu· In view of the postulated relationship between tion of penytlenetetrazole, 7.5 mg/ml (5.43 mmol/mI), tolerance and withdrawal, the present study was de­ at 0.30 ml/min. Infusion was terminated at the onset signed to evaluate the effectsof chronic administration of a tonic-clonic seizure as determined by two ob­ of PKl1195 on benzodiazepine discontinuation in the servers. mouse model. Mice received chronic lorazepam, with or without PK11195 or the competitive ligand R05-4864, Benzodiazepine Binding for 1 week, and then induced seizure thresholds and benzodiazepine binding were evaluated. Benzodiazepine binding in vivo was performed as pre­ viously described (Miller et al. 1988a). Briefly, micewere injected intravenously with 3 /.lCi PH]Ro15-1788. Af· ter 20 minutes, animals were sacrifIcedand brains rap­ METHODS idly removed and dissected on ice. After weighing, Materials brain regions were dissolved in Solvable (40°C for 24 hours) and then counted by scintillation spectrometry. Male CD1 mice,6 to 8 weeks of age (Charles River, Wil­ For nonspecifIcbinding, mice were treated with clonaze­ mington, MA)were maintainedon a 12-hour light/dark pam, 5 mg/kg (16.13 mmol/kg) intraperitoneally 30 cycle and given food and water ad libitum. Osmotic minutes prior to radioligand injection and samples were pumps were obtained from Alza (Palo Alto, CA). PEG processed as above. 400 was obtained from J.T. Baker (St. Louis, MO). For benzodiazepine binding in vitro, synaptosomal pH]flunitrazepam (specifIc activity 70 Ci/mmol), membranes from mouse cerebral cortex were prepared [3H]Ro15-1788 (flumazenil; specifIc activity 81 Ci/ and binding was performed as previously described mmol), and Solvable were purchased from New En­ (Miller et al. 1988a). Briefly, PHjFNTZ at 0.1 to 10 gland Nuclear (Boston, MA). Flunitrazepam and nmollL was added to duplicate or triplicate samples. clonazepam were gifts from Hoffmann-LaRoche (Nut­ Flunitrazepam at 10-5 mollL was added to an identi· ley, NJ). Lorazepam was a gift from Wyeth (Radnor, cal set of samples. After incubation at 4°C for 45 PA). PKl1195 and R05-4864 were obtained from Re­ minutes, samples were fIltered using a Brandel M48R search Biochemicals (Natick, MA). All other reagents (Gaithersburg, MD) onto Whatman GF/B fIlters.Filters were obtained from standard commercial sources. were washed three times with cold bufferand counted by scintillation spectrometry.

Drug Administration Lorazepam (2 mg/kg per day; 6.23 mmollkg per day), Data Analysis PK11195 (1 to 10 mg/kg per day; 2.79 to 27.9 mmollkg Binding data were analyzed using the EBDA programs per day) and R05-4864 (10 mg/kg per day; 32.8 mmollkg (McPherson 1983). Data were compared using analy­ per day) were administered by subcutaneously im­ sis of variance with Dunnett's test or Student's t-test. planted osmotic pumps as previously reported (Miller et al. 1988a). The doses of lorazepam and PK11195 were based on prior studies (Miller et al. 1992). The study RESULTS groups were lorazepam alone, PK11195 alone, R05-4864 Pentylenetetrazole-Induced Seizures alone, lorazepam plus PKl1195, lorazepam plus R05- 4864, and all three compounds. For lorazepam and At 1 day postdiscontinuation, there was no difference PK11195 and for all three compounds, drugs were ad­ between mice treated with lorazepam or the combina­ ministered via the same pump. In allcases, pumps were tion of lorazepam and PKl1195 (Fig. 1). However, at removed after 7 days of administration. All drugs were day 4 postdiscontinuation, seizure threshold was dissolved in PEG 400. Mice were studied at days 1, 4, markedly decreased in mice treated with lorazepam NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 3 Discontinuation after PKI1195/Lorazepam 269

2.5 1.2 �• LRZ/PK 1.0 2.0 i a ! g 0.8 • Ilol .. ... 0 1.5 0 'N N C C It II: 0.6 � to- • Ilol � to- • 1.0 Ilol Z Z • Ilol .. ... 0.4 ,. > � to- Z Z • Ilol � 0.5 11- 0.2

0.0 0.0 4 7

PK11195 DOSE + LRZ DAYS AFTER DISCONTINUATION

Figure1. Pentylenetetrazole-induced seizures after loraze­ Figure 2. Dose effects of PK1195 (PK) in combination with pam(LRZ) and PK11195 (PK). Mice treatedwith LRZ (2 mg/kg lorazepam (LRZ). Mice treated with LRZ (2 mg/kg per day) per day)or LRZplus (5 mg/kg per day) for 7 days were evalu­ or LRZ plus PK (1 to 10 mg/kg per day) for 7 days were evalu­ liedat days 1, 4, and 7 postdiscontinuation. Umestrained ated at day 4 postdiscontinuation. Umestrained mice were mi:ewere injected intravenously with pentylenetetrazole, 7.5 injected intravenously with pentylenetetrazole, 7.5 mg/ml at mg/mlat 0.30ml/min. Infusion was discontinued at the on­ 0.30 ml/min. Infusion was discontinued at the onset of a setof a tonic-clonic seizure. Pentylenetetrazole = quantity tonic-clonic seizure. Results are mean ± SEM, n = 6 to 13. requiredto induce a seizure. Results are mean ± SEM, n = * P < .05 for PK 5 and 10 mg/kg compared to LRZ. 6109.·p< .05 compared to LRZ at days 1 and 7; ** P < .05 comparedto LRZ/PK at days 1 and 7, and to LRZ at day 4.

concurrently with Ro5-4864, which competes with aloneas previously reported (Schatzki et al. 1989). Mice PK11195 for similar sites, and evaluated at 4 days post­ treated with both lorazepam and PKl1195 had a discontinuation (Fig. 3). Administration of Ro5-4864, signibcantly higher seizure threshold compared to 10 mg/kg per day, in combination with lorazepam was Iorazepam alone, although the threshold for combined not signifIcantly different from lorazepam alone when treatmentremained reduced compared to day 1. Results seizure threshold was evaluated at 4 days after lor­ atday 7 postdiscontinuation were similar to day 1 and azepam discontinuation. Similarly, when Ro5-4864 was showed no differences between the two treatment coadministered with PKl1195 and lorazepam, thresh­ groups.Seizure threshold in mice treated with PK11195 olds were unchanged from lorazepam alone, and sig­ alonewas similar to vehicle at days 1, 4, and 7 postdis­ nifIcantly different from the combination of PKl1195, continuation (data not shown). 5 mg/kg per day, and lorazepam. To evaluate the dose-response effect of PK11195, two additional doses, 1 and 10 mg/kg per day, were Benzodiazepine Binding In Vivo administered in combination with lorazepam, and sei­ zure thresholds were determined at 4 days postdis­ As previously reported, benzodiazepine binding in the continuation (Fig. 2). Seizure threshold was not cortex and hippocampus was similar at 1 and 7 days significantlyaltered by administration of PK11195 at 1 after lorazepam discontinuation, but was increased mglkgper day compared to lorazepam. As noted above, signifIcantly at 4 days compared to 1 and 7 days after PK11195 at 5 mg/kg per day signifIcantly reversed the drug discontinuation (Figure 4; Milleret al. 1988b). Simi­ effects of lorazepam discontinuation. Administration lar results were observed in the cortex in animals treated ofPK11195 at 10 mg/kg per day demonstrated a fur­ with the combination of lorazepam and PK11195 at 5 thersmall, nonsignifIcantincrease beyond the 5-mg/kg mg/kg per day. In the hippocampus, however, concur­ perday dose, and also differed signifIcantlyfrom loraze­ rent administration of lorazepam and PK11195 led to pamalone. Pentylenetetrazole-induced seizure thresh­ signifIcantly reduced binding at day 4 postdiscontinu­ olds were not altered by PK11195 alone at 1 and 10 ation compared to lorazepam alone. There were no mglkg per day. signifIcant changes in binding in any group in hypo­ To evaluate the specifIcity of PKl1195 in altering thalamus, cerebellum, or pons-medulla (data not Iorazepam discontinuation effects, mice were treated shown). In mice treated with PKl1195 alone, binding 270 J.J. Byrnes et al. NEUROPSYCHOPHARMACOLOGY 1993- VOL. 8, NO. J

1.2 Table 1. Benzodiazepine Receptor Density in Cortex In Vitro

1.0 Days after Discontinuation Iii g 1 4 7 0.' w ... 0 N Lorazepam 0.97 ± 0.09 1.33 ± 0.21 1.05 ± 0.12 C a:: PK11195 1.18 ± 0.23 1.24 ± 0.19 1.14 ± 0.21 � 0.8 W PK/LRZ 1.06 ± 0.11 1.37 ± 0.20 1.15 ± 0.17 � W Z w Binding was performed in mice treated with lorazepam (LRZ;2 ... 0.4 > mg/kg per day), PKl1195 (PK, 5 mg/kg per day), or the combina­ � z tion (PK/LRZ) for 1, 4 and 7 days. Bindin was performed in corti­ W � IL cal synaptosomal membranes (P2) using [ H]flunitrazepam. Results 0.2 are mean ± SEM in pmollmg protein, n = 3 to 4 membranes at each point. Comparisons were performed using analysis of variance for each treatment group across the discontinuation period. There an 0.0 no signiflcant differences.

PK11195 DOSE + LRZ

Figure 3. Effectsof R05-4864(R05) and PK11195 (PK) in com­ bination with lorazepam (LRZ). Mice treated with LRZ (2 the combination of lorazepam and PKI1195 (Table 1). mg/kg per day), LRX plus PK (5 mg/kg per day) or R05 (10 Receptor density was unchanged at I, 4, and 7 days mg/kg per day), or all three drugs for 7 days were evaluated after PK11195 alone. Apparent affinity at the receptor at day 4 postdiscontinuation. Unrestrainedmice were injected site was unaffected in any group evaluated (data not intravenously with pentylenetetrazole, 7.5 mg/ml at 0.30 shown). ml/min. Infusion was discontinued at the onset of a tonic-clonic seizure. Results are mean ± SEM, n = 7 to 13. * P < .05 for PK/LRZ compared to LRZ. DISCUSSION

These results indicate that concurrent administration was unchanged at I, 4, and 7 days after lorazepam dis­ of PK11195 and lorazepam markedly attenuated, but continuation (data not shown). did not totally eliminate, the effects of lorazepam dis­ continuation both on seizure threshold and receptor up­ regulation. SpecifIcally, pentylenetetrazole-induced Benzodiazepine Binding In Vitro seizure threshold was decreased after lorazepam dis­ In cortical synaptosomal membranes, benzodiazepine continuation, as previously reported (Schatzki et aI. receptor density was increased, but not signifIcantly, 1989), but this effect was attenuated after concurrent at day 4 compared to days 1 and 7 after lorazepam or PKI1195 treatment compared to lorazepam alone. This

CORTEX HIPPOCAMPUS

* * * 2000 :g; � 1600 0 '0 � 1600 � 1200- CJ CJ z z 1200 C C z z 800 iii iii 800 U u ii: 400 ii: LRZ U U PKlLRZ 400 W W I� Q. A. (IJ (IJ 0 �-r����-rT-�-r�+0 0 1 2 3 4 5 6 7 2345678 DAYS AFTER DISCONTINUATION DAYS AFTER DISCONTINUATION

Figure 4. Benzodiazepine binding in vivo after lorazepam (LRZ) and PK11195 (PK). Mice treated with LRZ (2 mg/kg per day) alone or in combination with PK (5 mg/kg per day) for 7 days were evaluated at days 1, 4, and 7 postdiscontinuation. Benzodiazepine binding was determined by specifIcuptake of [3H]flumazenil. Results are mean ± SEM, n = 5 to 9. • p< .05 for both LRZ and PK/LRZ compared to days 1 and 7. ** P < .05 for LRZ compared to days 1 and 7 and to PK/LRZ day 4. NEUROPSYCHOPHARMACOLOGY 1993-VOL.8, NO.3 Discontinuation after PK11195/Lorazepam 271

effect was incomplete, since seizure thresholds re­ region-specifIceffects ofPKl1195; that is, effects on both mainedre duced compared to day 1 after coadministra­ receptor downregulation during chronic exposure and tionofPK11195. Also as previously reported (Miller et receptor upregulation following discontinuation were ai.1988b), benzodiazepine receptor binding increased observed in the hippocampus, but not signifIcantly in in the cortex and hippocampus at day 4 following the cortex. Similar relative specifIcityhas been observed Iorazepamdiscontinuation. Concurrent treatment with based on choice of benzodiazepine compounds; loraze­ PK11195showed similar results in the cortex, but in hip­ pam affects both the cortex and hippocampus (Miller pocampus, there was a small, nonsignifIcant increase et aI. 1988a,b), whereas alprazolam affects only the cor­ inbinding at day 4. tex (Miller et aI. 1989; Lopez et aI. 1990; Galpern et a1. In addition, there appeared to be a dose-response 1990). The mechanism for this regional specifIcity may effect,since a lower dose of PKl1195, 1 mg/kg per day, be related to receptor subtype differences in these didn ot alter Iorazepam-induced threshold reductions regions (Olsen and Tobin 1990). Evidence based on au­ at day 4. Conversely, a higher dose of PK11195, 10 toradiography and in situ hybridization studies sug­ mg/kg per day, exerted a small, nonsignificant incre­ gests differential distribution of receptor subtypes and ment compared to the 5 mg/kg per day, suggesting that subunit messenger ribonucleic acids, although the func­ the maximal effect of PK11195 had been reached at 5 tional consequences of these data are unknown. It mg/kg per day. Finally, the effectof PK11195 appeared should be pointed out that these changes in binding tobe site speci fic. The compound Ro5-4864, which com­ were observed based in in vivo binding techniques. Use petes withPK11195 at the peripheral benzodiazepine of in vitro methods in the cortex revealed qualitatively site (Basile et aI. 1989) and the putative chloride chan­ similar changes but the differences were not signifIcant. nel site (Gee 1987), blocked the effects of PKl1195 at This incomplete correspondence between in vivo and a dose twice that of PKl1195. This compound in com­ in vitro binding has been reported previously in studies bination with lorazepam did not exert a signifIcant of benzodiazepine tolerance and dependence (Miller effect, arguing in favor of pharmacologic specifIcity at et a1. 1988a,b, Miller et aI. 1989; Lopez et a1. 1990; GaI­ these sites. pern et aI. 1991a), and may reflect effectsof tissue prep­ Data with regard to seizure thresholds in the pres­ aration and assay conditions (Miller et aI. 1987). entstudy corroborate prior data that indicate an associ­ The mechanism for the effects of PKll195 on ben­ ation between lorazepam discontinuation, decreased zodiazepine tolerance and dependence remains uncer­ seizurethres hold, and GABAA receptor upregulation tain. This compound has been shown to bind with high (Schatzkiet aI. 1989). Intuitively, it might be expected affinityat the "peripheral-type" benzodiazepine recep­ thatreceptor upregulation would be associated with an tor, which appears to be present on nonneuronal cells increasein seizure threshold. However, in view of the in brain and at low density on neurons (Olson et a1. unknownnature of the networks involved in convul­ 1988; Backus et aI. 1988; Basile et a1. 1989). In several sant effects and the potential subtype-specifIc effects situations, including oxygen consumption (Hirsch et ofGABAA receptors, it is plausible that receptor up­ aI. 1989; Larcher et a1. 1989) and cell multiplication and regulation may be linked to convulsant sensitivity. It differentiation (Wang et aI. 1984; Bisserbe et aI. 1986), should also be pointed out that changes in receptor PKl1195 functions as an antagonist at this site. How­ binding may not always reflect changes in receptor func­ ever, the effects of PK11195 at this site in the central tion (Miller 1991). nervous system are unknown. It has been hypothesized Priorreports have addressed the effectsof PKll195 that PK11195 might modulate glial production of on benzodiazepine-induced tolerance. In a behavioral steroids, which in turn can exert effectson the neuronal study in rats, Massotti et aI. (1990) reported that con­ GABAA receptor (Krueger 1991). Alternatively, current administration of PKl1195 prevented the de­ PKl1195 also binds to an incompletely characterized site velopment oftolerance. In a mouse model, we have pre­ at the chloride channel of the GABAA receptor (Gee viouslydemonstrated that tolerance and accompanying 1987; Gee et a1. 1988). The effects of PKl1195 on toler­ receptordownregulation are attenuated, but not com­ ance and dependence could be mediated at this site. pletelyprevented, by PK11195 administration (Miller As noted above, in both cases PK11195 competes et aI. 1992). Taken together with the current results, with Ro5-4864 for binding and in most functional as­ thesedata suggest thatPK11195 administration can at­ says. For example, in some behavioral paradigms Ro5- tenuate both tolerance and discontinuation phenom­ 4864 exhibits a benzodiazepine inverse agonist-like ena. In addition, these results support a relationship effect, reversed by PKl1195 (File and Pellow 1983). In between tolerance and discontinuation, as has been neurochemical slice studies, Ro5-4864 also had effects postulated in a number of neurotransmitter receptor similar to inverse agonists, whereas PK11195 exerted systems (Miller 1991). the opposite effects (Simmonds 1985). Thus, it is likely The neurochemical data in this study and in a prior that PK11195 and Ro5-4864 are competing ligands, but studyof tolerance (Miller et aI. 1992) support relatively the blockade ofPKl1195 effectsby Ro5-4864 in the pres- 272 J.J. Byrnes et al. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.3

ent study does not differentiate between activity at the tration. IX. Attenuation of alprazolam discontinuation peripheral site or the apparent chloride channel site. effects by carbamazepine. Biochem Pharmacol 42:S99- 5104 It should also be pointed out that initial evidence based on transient complementary dioxyribonucleic acid ex­ Gee KW (1987): Phenylquinolines PK8165 and PK9084 al­ pression methods indicates that in some cases,PKl1195 losterically modulate [35S]t-butylbicyc1ophosphorothio­ nate binding to a chloride ionophore in rat brain via a may not antagonize Ro5-4864 (P uia et al. 1989). These novel R05 4864 binding site. J Pharmacol Exp Ther data argue in favor of additional sites of action for Ro5- 240:747-753 4864; such sites could be involved in the effectsobserved Gee KW, Bolger MB, Brinton RE, Coirini H, McEwen BS in this study. (1988): Steroid modulation of chloride ionophore in rat In view of the problematic nature of benzodiaze­ brain. J Pharmacol Exp Ther 246:803-812 pine tolerance and dependence in clinical use, concur­ Greenblatt OJ, Miller LG, Shader RI (1990): Benzodiazepine rent administration of PKl1195 or similar compounds discontinuation syndromes. J Psychiatric Res 24 (Suppl may have a role in drug discontinuation after chronic 2):73-79 treatment with benzodiazepines. Additional studies of Hirsch JD, Beyer CF, Malkowitz L, Beer B, Blume AL (1989): similar compounds may elucidate the mechanism of Mitochondrial benzodiazepine receptors mediate inhi­ bition of mitochondrial respiratory control. Mol Phar­ PK11195 effects. macoI 35:157-163 Krueger KK (1991): Peripheral-type benzodiazepine recep­ tors: A second site of action for benzodiazepines. Neu­ ACKNOWLEDGMENTS ropsychopharmacology 4:237-244 Larcher J-p, Vayssiere J-L, Le Marquer FJ, Cordeau LR, Keane The authors thank Young Shim for assistance. This work PE, Bachy A, Gros F, Croizat BP (1989): Effectsof periph­ was supported in part by Grants OA-05258, MH-47598, and eral benzodiazepines upon the oxygen consumption of H-34223 from the U.S. Public Health Service. Dr. Miller is neuroblastoma cells. Eur J Pharmacol 171:197-202 the recipient of a Faculty Development Award in Clinical Phar­ macology from the Pharmaceutical Manufacturers Associa­ Lopez F, Miller LG, Greenblatt DJ, Chesley 5, Schatzki A, tion Foundation. Dr. Perkins was the recipient of the 1991 Shader RI (1990): Chronic administration of benzodiaze­ ACNP-Upjohn Minority Summer Student Research Fellow­ pines: V. Rapid onset of behavioral and neurochemical ship Award. alterations after discontinuation of alprazolam. Neu­ ropharmacology 29:237-241 Massotti M, Mele L, DeLuca C (1990): Involvement of the "pe­ REFERENCES ripheral" benzodiazepine receptor type omega 3 in the tolerance to the electroencephalographic effectsof ben­ Backus KH, Kettenmann H, Schachner M (1988): Effectof ben­ zodiazepines in rats. Pharmacol Biochem Behav 35: zodiazepines and pentobarbital on the GABA-induced 933-936 depolarization in cultured astrocytes. Glia 1:132-140 McPherson GA (1983): A practical computer-based approach Basile AS, Bolger GT, Lueddens HW, Skolnick P (1989): Elec­ to the analysis of radioligand binding experiments. Com­ trophysiological actions of R05-4864 on cerebellar Pur­ put Prog Biomed 17:107-114 kinje neurons: Evidence for "peripheral" benzodiazepine Miller LG (1991): Chronic benzodiazepine administration: receptor-mediated depression. J Pharmacol Exp Ther From the patient to the gene. J Clin PharmacoI31:492-495 248:463-469 Miller LG, Greenblatt DJ, Barnhill JG, Shader RI (1988a): Bisserbe JC, Patel J, Eskay RL (1986): Evidence that the Chronic benzodiazepine administration. I. Tolerance is peripheral-type benzodiazepinereceptor ligand R05-4864 associated with benzodiazepine receptor downregulation inhibits beta-endorphin release from AtT-20 cells by and decreased y-aminobutyric acidA receptor function. blockade of voltage-dependent calcium channels. J Neu­ J Pharmacol Exp Ther 246:170-176 rochem 47:1419-1424 Miller LG, Greenblatt DJ, Summer WR, Shader RI (1988b): File SE, Pellow 5 (1983): R05-4864, a ligand for benzodiaze­ Chronic benzodiazepine administration: II. Discontin­ pine micromolar and peripheral binding sites: An­ uation syndrome is associated with upregulation of tagonism and enhancement of behavioural effects.Psy­ y-aminobutyric acidA receptor complex binding and chopharmacology 80:166-170 function. J Pharmacol Exp Ther 246:177-182

Galpern WR, Miller LG, Greenblatt DJ, Shader RI (1990): Miller LG, GalpernWR, Byrnes JJ, Greenblatt OJ, Shader Rl Differentialeffects of chronic lorazepam and alprazolam (1992): Chronic benzodiazepineadministration. X. Co n­ on banzodiazepine binding and GABAA receptor func­ current administration of the peripheral ligand PK11195 tion. Br J Pharmacol 101:839-842 attenuates chronic effectsof lorazepam. J Pharmacol Exp Galpern WR, Lumpkin M, Greenblatt DJ, Shader RI, Miller Ther 261:285-289 LG (1991a): Chronic benzodiazepineadministration. VII. Olsen RW, Tobin AJ (1990): Molecular biology of GABAA Behavioral tolerance and withdrawal and receptor alter­ receptors. FASEB J 4:1469-1480 ations associated with clonazepam administration. Psy­ Olson JMM, Cilix BJ, Mancini WR, Young AB (1988): Pres­ chopharmacology 104:225-230 ence of peripheral-type benzodiazepine binding sites on Galpern WR, Miller LG, Greenblatt DJ, Szabo GK, Browne human erythrocyte membranes. Eur J Pharmacol 152: TR, Shader RI (1991b):Chronic benozdiazepineadminis- 47-53 NtUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 3 Discontinuation after PK11195/Lorazepam 273

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