Springer-Verlag 1988. Human studies on the benzodiazepine receptor antagonist fl-carboline ZK 93 426: antagonism of lormetazepam's psychotropic effects.
Psychopharmacology
Psychopharmacology (1988) 95:463~71
© Springer-Verlag 1988
Human studies on the benzodiazepine receptor antagonist fl-carboline ZK 93 426: antagonism of lormetazepam's psychotropic effects T. Duka, D. Goerke, R. Dorow, L. HOller, and K. Fichte Research Laboratories, Schering AG, Posffach 6503 tl, D-1000 Berlin 65 Abstract. The effects of lormetazepam (0.03 mg/kg IV) a benzodiazepine (BZ) derivative in combination with ZK 93 426 (0.04 mg/kg IV) a fl-carboline, benzodiazepine receptor antagonist were evaluated in humans. Independently, the effects of ZK 93 426 on its own were investigated. A psychometric test battery to evaluate sedation (visual analog scales (VAS), anxiolysis (state-trait-anxiety inventory scale (STAIG X1) and cognitive functions [logical reasoning test (LR), letter detection test (LD)] was applied before and several hours after initiation of treatment. Multiple sleep latency test (MSLT), which measures day time sleepiness, was also applied. Vigilosomnograms analysed from standard EEG recordings were evaluated shortly before and for 1 h after treatment. Treatment started with an intravenous injection of either lormetazepam (LMZ) or placebo (PLA), which was followed 30 rain later by administration of either ZK 93 426 or placebo; thus four treatment groups were created (PLA + PLA, L M Z + PLA, L M Z + ZK 93 426 and PLA + ZK 93 426). ZK 93 426 antagonized the sedative and hypnotic effect of LMZ as estimated by MSLT and vigilosomnograms, respectively. Impairment of cognitive functions (LR and LD) induced by LMZ was also antagonized by Z K 93 426. ZK 93 426 had no effect on the changes in the time estimation seen in the LMZ group. Furthermore, ZK 93 426 on its own increased vigilance (alertness) as measured by the vigilosomnogram. A competitive antagonism at the benzodiazepine binding site between ZK 93 426 and LMZ is suggested by their combination effects; the intrinsic activity of ZK 93 426 seems to be due to its weak partial inverse agonist component.
Key words: Lormetazepam - ZK 93 426 Multiple sleep latency test - Vigilosomnogram - Cognitive functions
The identification in the brain of high affinity binding sites for benzodiazepines and their relevance as regards the pharmacological effects of benzodiazepines have been well documented on the basis of in vivo and in vitro studies (Squires and Braestrup 1977; Mohler and Okada 1977; Duka et al. 1979). The recent development of ligands with high affinity but lack of efficacy at the benzodiazepine receptors offers a pharmacological tool for investigating the function of benzodiazepine receptors. One such compound, the imOffprint requests to .' T. Duka
idazo-benzodiazepine Ro 15-1788, which has been rather extensively investigated, selectively antagonizes most benzodiazepine effects in animals and in humans (Hunkeler et al. 1981 ; Darragh et al. 1981 ; Duka et al. 1986; Dorow et al. 1987), but it also has been shown to possess some intrinsic activity, behaving sometimes as a partial agonist and sometimes as an inverse agonist (for review see Pellow and File 1986). Recently, another BZ receptor antagonist, ZK 93 426 (ethyl-5-isopropyl-4-methyl-fl-carboline-3-carboxylate), a fl-carboline derivative, was synthesized. ZK 93 426 is a potent ligand at central benzodiazepine receptors whose interactions with the benzodiazepine/GABA receptor complex are mostly characteristic of a neutral benzodiazepine receptor antagonist with weak inverse agonist properties (Jensen et al. 1984; Stephens et al. 1986). Pharmacological studies revealed that ZK 93 426 reversed the effects of both agonists and inverse agonists (Jensen et al. 1984; Stephens et al. 1984) but that it also possessed weak intrinsic activity. Specifically, it exhibited proconffict activity in the lick suppression test (Jensen et al. 1984) and anxiogenic activity in the social interaction test (File et al. 1986), but it was found to be anticonvulsant against audiogenic seizures in DBA/2 mice (Jensen et al. 1984). Recently we reported that in humans ZK 93 426 possessed some weak intrinsic psychotropic effects ascribed to central activation (Duka et al. 1987). The aim of the current investigation is to examine whether ZK 93 426 antagonizes effects of benzodiazepines in humans. Specifically, we investigated the effects of ZK 93 426 on sedation, measured by the multiple sleep latency test or visual analog scales (see Methods), as well as on changes of the vigilosomnogram and cognitive functions induced by a benzodiazepine derivative, lormetazepam. Additionally, we studied the intrinsic activity of ZK 93 426 in the same parameters.
Methods Subjects. Forty healthy male volunteers aged between 20
and 40 years (26_+ 5 years) and weighing between 60 and 85 kg (72_+ 7 kg), who had undergone a full physical examination including an extensive laboratory screen, participated in the study after informed consent. The subjects were free of centrally active drugs 4 weeks prior to treatment and were instructed to avoid alcohol the day before testing.
464 Table 1. Design and dose regimen
Injection of LMZ (0.03 mg/kg IV) or placebo Injection of ZK 93 426 (0.04 mg/kg IV) or placebo Tame
( - 30 rain) 0ram
EEG/ECG MSLT
X
30rain
X
60min
2h
4h
6h
8h
/Oh
X
X
X
X
X
-X
Vitalfunctions (Heart rate and blood pressure)
X
X
X
X
X
X
X
X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X X
X
X
x
X
x
X
X
Self rating VAS STAIG X1
Cognltive tests Letter detection (LD) Logical reasoning task (LR) Time estimation (TE)
Treatment schedule and experimental design. Subjects were randomly allocated to four independent treatment groups (ten/group); they received as first injection either lormetazepam or 25% propylenglycol solution as a placebo solution intravenously (4 ml/70 kg, injection time 1 min). Subjects received 30 rain later as second injection ZK 93 426 (0.04 mg/kg) or an Intralipid solution as placebo intravenously in the same volume (15 ml/70 kg, injection time 3 rain). All treatments were applied in a double-bfind fashion. Treatment groups can be divided as follows: 1. 2. 3. 4.
Placebo + placebo Placebo + ZK 93 426 Lormetazepam + placebo Lormetazepam + ZK 93 426
Subjects arrived at the ward at 9.00 p.m. on the night before drug testing. They were allowed to adapt to the test methods and questionnaires. EEG electrodes were then fixed according to a standardized configuration. A sleep EEG was documented by the Oxford Medilog System (which is analysed automatically) (HSller and Riemer 1986). Subjects were allowed to go to sleep at 11 p.m. (lights off). The same electrodes were used the next day for the EEG recordings. Subjects were woken at a fixed time (8 a.m.). The experimental procedure started 30 rain later when an indwelling catheter was inserted into the medial vein of each arm for the injection and the blood sampling. Thereafter subjects were instructed to complete the psychometric tests, and the first "multiple sleep latency test (MSLT)" evaluation took place. Then subjects were asked to rest in the supine position and keep their eyes closed for the EEG recordings. Lormetazepam (0.03 mg/kg) or placebo (propyleneglycol 25%) was injected in a volume of 4 ml/70 kg (time of injection 1 min). Thirty minutes later either ZK 93 426 (0.04 mg/kg) or placebo (volume 15 ml/70 kg, time of injection 3 rain) was injected through a catheter extension so that the subject would not be disturbed during the injection. Standardized
60 rain EEG recordings starting shortly before lormetazepam or placebo injection and continuing to 30 rain after ZK 93 426 or placebo injection, were evaluated as a vigilosomnogram according to the rules of Rechtschaffen and Kales. Drug administration was also followed by five 2hourly recordings of MSLT, measurements of vital functions as well as by performance in psychometric tests; vital functions and performance in psychometric tests were additionally measured 1 h after first injection (see Table 1). Standardized meals and beverages containing no alcohol or caffeine were offered throughout the day and subjects were free to do as they pleased between testing.
Physiological measures. EEG recordings were performed as described above; blood pressure and heart rate were evaluated immediately before and 1, 2, 4, 6, 8 and 10 h after the first injection (lormetazepam or placebo). ECG recordings were documented parallel to EEG measurements (Table 1).
Self rating. Bipolar visual analog scales (VAS) (0-100 mm range) were designed to assess activation (translated from the original German fresh worn out; wide awake sleepy). Subjects were instructed to consider the midpoint as their normal state and to put a mark spontaneously on the scale to indicate any deviation at the time of estimation. Measurements for both parameters took place before and 1, 2, 4, 6, 8 and 10 h after the first injection (LMZ or placebo) (Table 1). The state-trait-anxiety inventory scale (STAIG) (Spielberger et al. 1966) was used in its X1 version to estimate the response to a specific anxiety-provoking situation, in this case the treatment. It includes 20 questions which can be answered with either "not at all", "little", "moderately" or " a lot". STAIG X1 was applied before and 1, 2, 4, 6, 8 and 10 h after the first injection (LMZ or placebo) (Table 1).
465 Table 2. Changes in heart rate (a) and blood pressure (systolic/diastolic) (b) following different treatment combinations Treatment
Time (h) after first injection 0
(a) Heart rate (l/min) (mean± SD) Lormetazepam+placebo 63± 6 Lormetazepam+ZK 93 426 56±10 Placebo+placebo 54± 8 Placebo + ZK 93 426 63± 5
1
2
4
6
8
t0
71±14 60± 6 64± 8 64± 9
71±11 64±11 64±10 65± 3
77± 9 67±12 64±15 66± 8
77± 5 74-t-10 65±10 71± 7
71± 8 71±11 65±14 68± 8
72± 9 72±11 66±10 70± 7
126±14/ 62± 7 126±10/ 72±11 122±10/ 67± 6 126±21/ 71±12
125±11/ 68± 7 125±11/ 65±11 120±14/ 64± 4 127±17/ 67± 7
126± 6/ 66± 5 132±11/ 68±13 121±14/ 63± 9 129±18/ 67±10
127± 5/ 63± 8 131±13/ 63±12 120±15/ 61± 7 127±15/ 65± 9
135± 9/ 68± 7 133±12/ 67±14 126+15/ 66± 6 t31±15/ 69± 8
(b) Blood pressure (ram Hg) systolic/diastolic (mean ± SD) Lormetazepam + placebo 122± 8/ 121±12/ 67± 9 62±12 Lormetazepam + ZK 93 426 119±13/ 124± 8/ 66±13 67±12 Placebo + placebo 116± 9/ 119±11/ 63± 8 69±10 Placebo + ZK 93 426 120±15/ 121 ±17/ 68±11 69± 9
Multiple sleep latency test ( M S L T ) . The multiple sleep latency test (Richardson et al. 1978) assesses daytime sleepiness, defined as the tendency to fall a sleep. For evaluation of MSLT, only subjects with a high proportion of occipital alpha frequencies were included in the study. Subjects were asked to lie in bed in a completely darkened, silent room and try to sleep. They were given 20 rain, during which EEG recordings are performed and analysed visually to register sleep latency, stages and their duration. MSLT measurements were performed before and 2, 4, 6, 8 and 10 h after drug administration (Table 1). Cognitive tests were used to assess an interaction of the two drugs (lormetazepam and ZK 93 426) on the subjects" ability to concentrate, and perform, as follows. Logical reasoning test (LR). The L R of Baddeley (1968) has been adapted for the German language in a form (Stephan and Dorow 1986) as follows: a series of statements in randomized order relating two letters, such as " A follows B", and a pair of letters are presented to the subjects. The subjects have to decide whether the statement describes the order of the letters correctly. In the present version, the constructions in the passive voice (e.g., " A is followed by B") are missing and the decision is somewhat complicated by the question whether the description is true: the subjects have to answer " y e s " or " n o " to continuously randomiTed questions (e.g., " A does not follow B", AB; true?). The test is computer based and a new question is presented immediately after a marked key is pressed. In the present version time is fixed (5 rain) and thus target variables are the total number of answers and the percentages of errors. The test in its cognitive complexity represents a measurement of working memory and involves high memory load (Baddeley 1968).
Letter detection (LD). In this test subjects have to decide whether there is an "E'" in a line of ten random letters. This version is computer based and does not require concellation of letters, thus minimizing the psychomotor component of the test (Stephan and Dorow 1986). Time is fixed, and the target variables are the number of answers and the percentage of errors.
All the cognitive tests were performed before and 1, 2, 4, 6, 8 and 10 h after drug administration (Table 1).
Time estimation (TE). Subjects have to estimate a period of 15 s by pressing start and stop keys on a computer with an internal clock. This test is believed to identify changes in activation of behaviour (Elsass et al. 1979). Time estimation was performed before and 1, 2, 4, 6, 8 and 10 h after drug administration (Table 1). Statistics. Comparison of the two treatments (ZK 93 426 or placebo) in each main treatment part (lormetazepam or placebo) was systematically investigated. Hypothesis testing for target criteria was based on a) one-way multivariate analysis of variance, b) three-factorial analysis of variance, (factors: treatment, subject, time point and interaction treatment × time point) and c) U-test depending on different assumptions about correlations between measurement over time and the metric of the scales. The test of the hypothesis was based on the measuremerits after the second injection (ZK 93 426 or placebo) corrected for the basal values. In the group which received placebo instead of lormetazepam it was not possible to evaluate statistically the data obtained from performance in the letter detection test, logical reasoning test or anxiety scores from the STAIG X1, since basal values were not available for five of the subjects in these groups. We considered the data as significant when comparisons give P-values < 0.05 and as approaching significance when P-values < 0.1. All the data are presented as mean + SD. Results
Physiological measures Systolic and diastolic blood pressure as well as heart rate (Table 2) did not significantly differ between the two treatment groups (placebo versus ZK 93 426). A slight increase (n.s.) was observed in the group which received lormetazepam and then ZK 93 426 for both diastolic and systolic blood pressure when compared to the group which recdved lormetazepam and then placebo (Table2). Heart rate
466
Vlgllosomnogram
S~bj. No.
~,WJ~E
ST/k~E 1
14
1
8~IE 2 ~
3
~,td3,E 4
F~ ST~i~E 1
16
51TA~E 2 ~
3
~
4
t
t
8T~31EI I RE~ 8T/~[. 1
40
I
8T~3E 2
U
9~IE 3 ST,~61E 4
t
t ZK 93 426 0.04 mg/kg i.v.
LMZ 0.03 mg/kg i.v.
ab
cab [min.]
8T~
12
~Lr
Sm~ll: 2 ST~
3
t
8T~,E 0 REM
31
~
u
2
S'T~
3
t
F~ 8T~aE 1
32
~
m
8T~2
]
8 ~ 3
~'
t LMZ 0.03 rng/kg LV.
6
F~acebo
3o
slightly increased (n.s.) after lormetazepam injection but this increase was not altered by either consecutive treatment (ZK 93 426 or placebo) (Table 2). A similar increase was observed in the placebo + placebo group. As is shown in Fig. 1, after the administration of lormetazepam subjects reached sleep stages 2 to 3 within 10 rain. Thirty minutes later when the volunteers were still in slow wave sleep ZK 93 426 or placebo was injected. Whereas subjects who received ZK 93 426 woke up 10-20 rain after injection, subjects administered placebo remained in deep sleep stages up to 60 rain, at which time they were woken up (Fig. 1). Figure 2 shows the time spent by all ten subjects in each EEG stage. One-way multivariate analysis of variance for time spent in EEG stages 1 + 2 and 3 + 4 showed a treatment-time interaction effect (P=0.07) which was more related to the time spent in EEG stages 3 + 4 (P = 0.02) (lormetazepam + placebo versus lormetazepam + ZK 93
[min.]
Fig. 1. Individual vlgilosomnograms analysed visually for 60 rain after LMZ mje~tion followed by either placebo or ZK 93 426 (0.04 mg/kg)
426). Subjects who received placebo instead of lormetazepam reached sleep stages 1 2 within 20 rain, Thirty minutes later the injection itself (either ZK 93 426 or placebo) woke them up; 15-20 rain later subjects who were given placebo for the second time again reached sleep stages 1-2, whereas subjects who were injected with ZK 93 426 remained awake in the rest of EEG recordings (Fig. 3). In Fig. 4 is shown the time spent by all ten subjects in each EEG stage; oneway multivariate analysis of variance for time spent in EEG stages 1 + 2 showed a significant treatment-time interaction effect ( P < 0.01) (placebo + placebo versus placebo + ZK 93 426). S e l f ra tings
A minor sedative effect (n.s.) was indicated in the related VAS items (wide awake - sleepy and worn out - fresh)
467
m
Percefrtaoeof sleep 8tage~1+ 2
100-
1
Percec'Ca~eof ~eop stages3 + 4
Percentageof &~eopstages1+ 2
90-
[]
Be[ore2nd k'tJectlon(30 m~)
80-
•
Nte,r2nd t n ~
[] []
70-
Before2_IrlJeoUoe(30 mln) After 2_Inlec~ee(30 mln)
(30 mln)
6050403020100
LMZ/P'LA
LM7_/ZK93426
LMZJZK93426
PlJk/PLA Fig. 2. Percentages of different sleep stages of all subjects under different treatment combinations as evaluated from the EEG recordings (1 injection: LMZ; 2 injection placebo or ZK 93 426)
Subj.
PLA/ZK93426
Fig. 4. Percentages of different sleep stages of all subjects under different treatment combinations as evaluated from the EEG recordings (1 injection: placebo; 2 injection: placebo or ZK 93 426)
Vigilosomnogmm
No.
s ~
I
IF U
8T~
27
-
-
L_
j
8T~13& 1 ST~CE 2 8TP~3E 3
--1__~
AW~KE
1
STaGe Q
21
STY1 S~tE 2 gr~ 3 ~ 4
t Racebo
3b
cob [mln.]
AW,'L~E ~AGIE J 8~G4_ 1 ~ 2 ST~C~E 3
t
3T/~3 E 4
22
AWANE b~fM21EO RE~ BT~I ST~!31z 2 ~ 3
t
ST~ R~ ST~3E 1
26
S]~ 2 ST~3E 3
t
~T~
Placebo
Z_K 93 426 0.04 mg/kg i.v.
3:o
6b [mJn.]
Fig. 3. Individual vigilosomnograms analysed visually for 60 min after placebo injection followed by either placebo or ZK 93 426 (0.04 mg/kg)
468 Table 3. Changes in VAS performance after different treatment combinataons Treatment
Time (h) after t'u'st injection 0
1
2
4
6
8
10
1. VAS ("worn out - fresh") 0-100 mm mean+ SD Lormetazepam + placebo 50 ± 21 46 ± 28 Lormetazepam + ZK 93 426 47±17 39±20 Placebo +placebo 48 + 4 59 ± 17 Placebo + ZK 93 426 52 ± 20 52 ± 27
45 ± 19 52±20 58± 18 50 + 21
51 + 23 50±26 52± 19 50 ± 26
52 ± 19 58±15 48 ± 16 49 ± 23
52 ± 27 53±19 49± 17 50 + 18
52 ± 29 48±21 53 ± 19 53 ± 20
2. VAS ("wide awake - sleepy") I~100 mm mean± SD Lormetazepam + placebo 49 ± 25 60 ± 27 Lormetazepam+ZK 93 426 54±22 56±24 Placebo + placebo 52 ± 6 42 ± 14 Placebo + ZK 93 426 55±27 44-/-21
49 + 25 54±23 43 ± 16 44±20
43 ± 26 49±27 50 ± 21 46+25
42 ± 23 45±22 51 ± 18 50±22
48 ± 27 40±24 50 ± 17 44±13
38 ± 22 34±18 43 ± 20 44±12
Table 4. Changes in STAIG X~ scores following different treatment combinations state anxiety scores (mean ± SD)
Treatment
Lormetazepam + placebo Lormetazepam + ZK 93 426 Placebo+placebo Placebo + Z K 93 426
Time Oa) after first injection 0
t
2
4
6
8
10
39±4 43±4 44±6 40+5
40±6 43-r4 44±4 40±3
39±4 41+3 44±5 41 ±3
38+6 41±4 45±5 40±5
37±5 425:3 44±5 40±6
37±5 41±3 43±5 39-t-5
38±4 43+3 43±6 40±4
1 and 2 h after l o r m e t a z e p a m injection (Table 3). Z K 93 426 slightly altered this marginal l o r m e t a z e p a m effect (Table 3). N o effects o f either substance could be detected in the STAIG-X1 rating (Table 4).
Multiple sleep latency test (MSLT)
Later~
to sloop stage 1 (e13ochee)
......
L M Z - Plac.el:~ L M Z - Z.K 9 3 4 2 6
%%%N
L o r m e t a z e p a m reduced the latency to sleep stage 1 measured 2 h after injection. This effect was detected up to 6 h, and values were reinstated 8 h after injection (Fig. 5). Z K 93 426 antagonized this effect o f lormetazepam only up to 90 min after administration (three-factorial analysis of variance: P = 0.02). Subjects who received placebo instead o f l o r m e t a z e p a m also showed a reduced latency to sleep stage 1 (Fig. 5) but not until 4 h after injection, the time which coincided with the observed afternoon sleepiness. It is noteworthy that the group which received as second injection Z K 93 426 instead o f placebo did not reach this low p o i n t o f m i d d a y tiredness (Fig. 5).
.I
- ......
P't.ace,b,o - Ptacebo Place,13o - Z K 9.3426
fS
Logical reasoning test ( LR ) In the logical reasoning test an impairment of performance as indicated by the total number o f answers and the percentage of errors was found in the group which received lormetazepam followed 30 rain later by placebo but n o t in the group which received Z K 93 426 as the second injection (three-factorial analysis o f variance: P = 0 . 0 7 lormetazep a m + placebo versus l o r m e t a z e p a m + Z K 93 426) (Fig. 6). These differences were observed between the two groups only in the first 3 h after l o r m e t a z e p a m injection. N o differences were found between placebo + placebo and placebo + Z K 93 426 groups (data not shown).
T - - T - - T - - ' [ - - - [ O 1 2 3 4
[ 5
r 6
T 7
T - - " I ' ~ 8 9
hc,ur8
Fig. 5. Multiple sleep latency test (MSLT) measurements (time required to reach sleep stage 1) after different combination treatments. Test duration was 20 rain = 60 epochs
469
Letter d e t e c l k m test - N u m b e r of a n s w e r s
Loglcal reasoning test - N u m b e ~ of a n s w e r s
- ......
- ......
LMZ - P l a c e b o [k,tZ - ZK 93426
LMZ - P ~ c e b o LM,Z - Z K 9 3 4 2 6
±
Loglc,al ~ n g
test - Percentage of erro¢~
Letter deteclJon test - Percentage of errors
j
3
4
5
6
7
8
9
hours
/'1
...........
-r
r
r
T
1"
]
1
]"
r
- r - -
0
1
2
3
4
5
6
7
8
9
hours
Fig. 6. Subjects' performance in logical reasoning test evaluated by the number of answers and the percentage of errors, after different treatment combinations
Fig. 7. Subjects' performance on letter detection test, evaluated by the number of answers and the percentage of errors after different treatment combinations
Letter detection
mal studies have already shown that ZK 93 426 can antagonize several benzodiazepine effects, such as its anxiolytic, anticonvulsant and ataxic actions (Jensen et al. 1984; Stephens et al. 1986). In this study the hypnotic effect of lormetazepam (as measured by the vigilosomnogram) and the sedative effect evaluated through the daytime sleepiness were both antagonized by ZK 93 426. This later effect of lormetazepam was only short lasting; it was first measured 2 h after lormetazepam injection and did not reach values higher than those seen in the placebo group during midday sleepiness. Although only a minor lormetazepam sedative effects was seen in the subjective ratings (VAS), this effect remained mostly unchanged when ZK 93 426 was given in combination. No anxiolytic effect of lormetazepam could be detected, since our volunteers were not anxious as estimated in the STAIG X1 scores. The present study also further supports clinical data showing that be~zodiazepines cause an impairment of cognitive performance (Ghoneim et al. 1984; Lister 1985; Brosan et al. 1986). Lormetazepam decreased the number of answers and increased the incidence of errors in a reasoning test which involves high memory load and higher mental processes (Baddeley 1968). Lack of concentration and impairment of attentional processes could count for such an effect. Impairment of memory processes induced by lormetazepam (Dorow et al. 1987) could also be involved. In another test, the "Letter Detection" in which concentration and activation of behaviour is
Performance in the letter detection test did not differ significantly between the two groups (LMZ + placebo and lormetazepam + ZK 93 426) (Fig. 7). An increase in the percentage of errors found 1 h after lormetazepam injection only in the group which received placebo instead of ZK 93 426 was not considered to be significant. No differences were found between placebo +placebo and placebo + ZK 93 426 groups (data not shown).
Time estimation ( TE) Time estimation showed a marginal increase 1 h only after lormetazepam injection (n.s.) which then returned to near normal values and no differences were found between the two groups (lormetazepam + placebo, lormetazepam + ZK 93 426) (Fig. 8). In the group which received placebo instead of lormetazepam minor diurnal changes could be observed which were similar for the two additional treatments (placebo or ZK 93 426) (Fig. 8). Discussion
The present study demonstrates that ZK 93 426, a fl-earboline derivative, antagonizes several effects of the benzodiazepine lormetazepam in a dose which shows only marginal intrinsic activity (Duka et al. 1987 and in this study). Ani-
470
"nme o*tlmatkm (~,c)
- -
LMZ - R a c e b o I._MZ- Z K 93426
......
%%%%" /
T l m e e ~ r r m l t o n (sec)
- ......
-
-
1
~
2
3
4
5
T 6
Placebo - Place,bo ~ - Z K 93426
r
T
1
7
8
9
hour8
Fig. 8. Time estimation (approximation of 10 s) calculated by the subjects after different treatment combinations
thought to be involved lormetazepam had a marginal effect: it increased only the percentage of errors, but did not influence the number of answers given by the subjects. This test, which is possibly sensitive only to high degree of sedation, was impaired by lormetazepam 1 h after injection and returned to normal values 2 h after injection. Both lormetazepam effects were completely antagonized when ZK 93 426 was given in combination, demonstrating an additional effect of ZK 93 426, i.e. to antagonize BZ behavioural changes on cognitive performance. Thus, not only sedation but also impairment of a complex cognitive function measured in logical reasoning test appears to be BZ-receptor mediated. The lormetazepam effect on time estimation, which shows that the internal clock becomes slower for the first 2 h after administration, is in agreement with previous studies with other psychotropic drugs (Elsass et al. 1979; Stephan and Dorow 1986). Interestingly, this effect was not even marginally antagonized by ZK 93 426. This may suggest either that other mechanisms unrelated to BZ receptors may be involved in this effect or that ZK 93 426 possesses a weak intrinsic activity which may contribute to this effect of lormetazepam. For instance, in a previous study subjective mood ratings indicated a "relaxing" effect of ZK 93 426 (Duka et al. 1987). Changes induced by lormetazepam in physiological responses, specifically an increase in heart rate, were not antagonized by ZK 93 426. Since this lormetazepam effect has been antagonized by the benzodiazepine
receptor antagonist Ro 15-1788 (Duka et al. 1986), it is suggested that the absence of a ZK 93 426 effect is probably related to time measurements and pharmacokinetics of the drug (haft-life for both is estimated between 50 and 60 rain: measurements were performed 20 rain after injection in the case of Ro 15-1788 and 2 h in the case of ZK 93 426). The present results also demonstrate that ZK 93 426 is pharmacologically active by itself, and are thus in agreement with data obtained from animal studies (Jensen et al. 1984; File et al. 1986; Stephens et al. 1986) and previous human studies (Duka et al. 1987). This intrinsic activity of ZK 93 426 is shown in animals as a proconflict (Jensen et al. 1984), anxiogenic (File et al. 1986) and in some cases anticonvulsant action (Jensen et al. 1984), and in humans it is ascribed to central activation (Duka et al. 1987). EEG recordings for 1 h, during which treatments were taken, clearly demonstrated that volunteers who were given ZK 93 426 remained awake compared with those given placebo, who reached sleep stage 2; another effect of ZK 93 426 indicative of a central stimulating effect was shown in the daytime sleepiness measured in the multiple sleep latency test, which is believed to be a valid and accurate reflection of normal variations in alertness and sleepiness (Richardson et al. 1978). Thus, subjects administered ZK 93 426 did not reach a sleepiness stage throughout the day, in contrast to subjects who received placebo. Similar changes of EEG profiles indieative of a central stimulation have been also shown for the BZ antagonist Ro 15-1788 (Sch6pf et al. 1984; Ziegler et al. 1986). The absence of an effect of ZK 93 426 on its own on logical reasoning test (data not shown) - a finding that is not in agreement with data obtained in a previous study (Duka et al. 1987) - may be due a) to the different version of the test (see methods) and b) to the small number of subjects (n = 5) from whom basal values were obtained. In summary, the present study demonstrates that the fl-carboline ZK 93 426 can antagonize several of the effects of a benzodiazepine agonist without producing marked effects of its own. However, a presumptive activating effect of ZK 93 426 noted especially in EEG recordings may indieate some slight inverse agonist properties of the compound. The existence of high affinity binding sites for benzodiazepines in the brain (M6hler and Okada 1977; Squires and Braestrup 1977) makes it easier to understand a competitive antagonism in this ease of two substances with similar affinity for the receptor, but one with efficacy (LMZ) and one with no or with minor efficacy (ZK 93 426). What obviously remains limited at this time is an understanding of the intrinsic action of this compound. Further experimental work will be necessary to better define the psychotropic effects of ZK 93 426.
Acknowledgement. We would like to thank H. Riemer, M. KnabeRfihl, R. Obeng-Gyan and C. Jacob for their valuable assistance in carrying out the study. K. Stephan for his valuable help with the cognitive tests and B. Getflhar for his excellent assistance in evaluating the data. We also want to thank J. Horkulak for his help in preparing the manuscript and A. Dahrmann for typing It. References
Baddeley AD (1968) A 3 min reasoning test based on grammatical transformation. Psychon Sci 10:341-342 Brosan L, Broadbent D, Nutt D, Broadbent M (1986) Performance
471 effects of diazepam during and after prolonged administrataon. Psychol Med 16:510-522 Darragh A, Lambe R, Scully M, Brick I, O'Boyle C, Downie WW (1981) Investigation in man of the efficacy of a benzodiazepine antagonist, Ro 1%1788 Lancet II: 8-10 Dorow R, Berenberg D, Duka T, Sauerbrey N (1987) Amnesic effects of lormetazepam and their reversal by the benzodiazepine antagonist Ro 1%1788. Psychopharmacology 93:507514 Duka T, H6Ut V, Herz A (1979) In VlVOreceptor occupation by benzodiazepines and correlation with the pharmacological effect. Brain Res 179:147-156 Duka T, Ackenheil M, Noderer J, Doenicke A, Dorow R (1986) Changes in noradrenaline plasma levels and behaviourgl responses induced by benzodiazepine agonists with the benzodiazeplne antagonist Ro 15-1788. Psychopharmacology 90:351 357 Duka T, Stephens DN, Krause W, Dorow R (1987) Human studies on the benzodiazepine receptor antagonist fl-carboline ZK 93 426: Preliminary observations on psychotropic activity. Psychopharmacology 93:421~427 Elsass P, Mellerup ET, Rafaelsen OJ, Theilgaard A (1979) Lithium effects on ttme estimation and mood on manic-melancholic patients. A study of diurnal variations. Acta Psychiatr Scand 60:263-271 File SE, Pellow S, Jensen LH (1986) Actions of the fl-carboline ZK 93 426 in animal test of anxiety and the holeboard: interactions with Ro 15-1788. J Neural Transm 65:103-114 Ghoneim MM, Mewaldt SP, Hinrichs JV (1984) Dose-response analysis of the behavioral effects of diazepam : II. Psychomotor performance, cognition and mood. Psychopharmacology 82 : 296-300 H611er L, Riemer H (1986) Comparison of visual analysis and automatic sleep stage scoring (Oxford Medilog 9000 System). Eur Neurol [Suppl] 2:36-45 Hunkeler W, Mohler H, Pieri L, Polc P, Bonetti EP, Cumin R, Schaffner R, Haefely W (1981) Selective antagonists of benzodiazepines. Nature 290:514-516 Jensen LH, Petersen EN, Braestrup C, Honor6 T, Kehr W, Stephens DN, Schneider H, Seidelmann D, Schmiechen R (1984)
EvaluaUon of the fl-carboline ZK 93 426 as a benzodiazepine receptor antagonist. Psychopharmacology 83 : 249-256 Lister RG (1985) The amnesic action of benzodiazepines in man. Neurosci Biobehav Rev 9:87-93 Mohler H, Okada T (1977) Benzodiazepine receptors: demonstration in the CNS. Science 198:849-851 Pellow S, File SE (1986) Intrinsic actions of the benzodiazepine receptor antagonist Ro 15-1788. Psychopharmacology 88 : 1-1 l Richardson G, Carskadon MA, Flagg W, van den Hoed J, Dement WC, Mitler MM (1978) Excessive daytime sleepiness in man, multiple sleep latency measurement in narcoleptic and control subjects. Electroencephalogr Clin Neurophysiol 45 : 621~527 SchSpf J, Laurian S, Le PK, Gaillard JM (1984) Intrinsic activity of the benzodiazepine antagonist Ro I%1788 in man: an electrophysiological investigation. Pharmacopsychiatry 17:79~3 Spielberger CD, Gorsuch RL, Lushene RE (1966) Manual for the State Trait Anxiety Inventory. Consulting Psychologist Press, Palo Alto, California Squires RF, Braestrnp C (1977) Benzodiazepine receptors in rat brain. Nature 266:732-734 Stephan K, Dorow R (1986) The influence of the new antidepressant rolipram on alertness, performance and mood in healthy volunteers varied with time of day and diurnal type of subject. In: Labrecque G (ed) Biological rhythms and medications. Annual review of chronophaxmacology, vol 3. Pergamon Press, Oxford New York, pp 437-440 Stephens DN, Shearman GT, Kehr W (1984) Discriminative stimulus properties of fl-carbolines characterized as agonists and inverse agonists at central benzodiazepine receptors. Psychopharmacology 83 : 233-239 Stephens DN, Kehr W, Duka T (1986) Anxiolytic and anxiogenic fl-carbolines: tools for the study of anxiety mechanisms. In: Baggio G, Costa E (eds) Gabaergic transmission and anmety. Raven Press, New York Ziegler G, Ludwig L, Fritz G (1986) Effects of the specific benzodiazepine antagonist Ro 15-1788 on sleep. Pharmacopsychiatry 19 : 200-201 Received March 11, 1987 / Final version March 14, 1988
