Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers W. Krause, Th. Duka & H. Matthes To cite this article: W. Krause, Th. Duka & H. Matthes (1991) Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers, Xenobiotica, 21:6, 763-774 To link to this article: http://dx.doi.org/10.3109/00498259109039516
Published online: 27 Aug 2009.
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Date: 07 November 2015, At: 00:51
XENOBIOTICA,1991,VOL.
21, NO. 6, 763-774
Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers W. KRAUSE, T H . D U K A and H . M A T T H E S Research Laboratories of Schering AG, Berlin, Germany
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Received 5 December 1989; accepted 23 November 1990
1. T h e pharmacokinetics of ahecarnil were studied in eight healthy male volunteers using I4C-abecarnil and an h.p.1.c. method for determination of unchanged drug. 2. Abecarnil was rapidly and nearly completely absorbed after an oral dose of 1Omg; bioavailahility was 40%. Plasma levels of the unchanged d r u g declined with a terminal halflife of 4 h . T h e total clearance of ahecarnil was 11 mlimin per kg. '4C-Ahecarnil was excreted rapidly and completely. T h e main route of elimination was in the faeces.
3. Ahecarnil was extensively metabolized resulting in ether cleavage at position 6 with subsequent glucuronidation or sulphation and, to a minor extent, in ester cleavage.
Introduction Anxiolytic, anticonvulsant and muscle-relaxant effects of benzodiazepines as well as their side-effects such as sedation, ataxia and amnesia are mediated by binding to the benzodiazepine-GABA-receptor complex. Recently, new chemical classes of compounds have been found to interact with the benzodiazepine receptor. Among these are the 8-carbolines (Braestrup et al. 1980)which have been shown to exhibit the full spectrum of benzodiazepine effects as full agonists (Moller et al. 1986),as partial agonists (Petersen and Jensen 1984,Dorow et al. 1987a,b) or as antagonists, effects which are opposite to those of benzodiazepines (Dorow et al. 1983,Jensen et al. 1984,Petersen 1983,Petersen and Jensen 1984. Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-~-carboline-3-carboxylate) is one of these 8-carboline derivatives which selectively bind to the benzodiazepine receptor. I n animal models of anxiety and epilepsy this new compound has been reported to exhibit potent anxiolytic and anticonvulsant activity (Turski et al. 1990). Abecarnil exhibited only weak ataxiogenic effects, whereas ataxia produced by benzodiazepines could be antagonized by the new substance. Additionally, abecarnil showed in comparison to benzodiazepines, decreased interaction with ethanol in animal studies (Stephens et al. 1990).T h e new substance therefore has been classified as a partial agonist at the benzodiazepine receptor. T h e pharmacokinetics of abecarnil have been studied extensively in a number of animal species including rat, rabbit, dog and monkey (Krause and Mengel 1990)and in man (Krause et al. 1990). T h e aim of the present study was to complete the interspecies comparison by adding data on the pharmacokinetics and biotransformation of abecarnil in healthy volunteers using radiolabelled drug.
Materials and methods Materials Abecarnil was '4C-lahelled at position 1 (figure 1) with a specific activity of 2.2GBq/mmol (59.4 mCi/mmol). Radiochemical purity was greater than 99%. T h e d r u g was diluted with unlabelled 0049-8254191 $3.00
0 1991 Taylor & Francis
Ltd.
W . Krause et al.
764
CH3 \
.O
CH3
0
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0
F
CH3
Figure 1. T h e structure of abecarnil *indicates position of I4C-label.
Table 1.
Characteristics of the subjects participating in the radiolabelled study.
Subject no.
Mean SD
Age (years)
Height ( 4
Weight (kg)
56 62 55 60 65 50 51 64
170 181 165 165 176 188 187 171
74 92.5 75 81.5 76 95 70.5 82.5
57.9 5.7
175.4 9.2
80.9 8.9
material for use in human experiments. T h e preparation constituents Intralipid, Myrj 53 and Klucel LI: were obtained from Pfrimmer Kahi (Erlangen, Germany), Atlas Chemie (Essen, Germany), and Hercules Inc. (Wilmington, MA, USA), respectively.
Experimental design Eight healthy male volunteers (age 58 & 6 years; weight 81 + Y kg; height 175 & 9 cm) participated in the study (table 1). They had given their written informed consent. According to German regulations, the administration of radioactive compounds to humans is only allowed if the subjects are 50 years o r older. T he investigation had been approved by an Institutional Review Board and by the German Authorities. T h e test subjects first received an i.v. injection of 1.88 m g (86pCi) and 2 weeks later an oral dose of 10.3 mg (IOlpCi). Treatments were performed o n an empty stomach, and standardized food was only allowed 1 h after drug administration. Abecdrnil was dissolved in Intralipid-10% at a concentration of 0.188mg/ml for i.v. injection and suspended in isotonic saline with 0.085% (w/v) Myrj 53/1% (w/v) Klucel LF (1.03 mg/ml) for oral use. Blood was collected from an arm vein contralateral to the injection site immediately before the administration o f ahecarnil and at 3, 10, 15, 3 0 ,4 5 ,6 0 m i n , 1.5, 2, 3 , 4 , 6 , 9 , 12, 15, 24, 3 6 , 4 8 , 7 2 , 9 6 and 120 h after injection. Following oral administration the time points for blood collection were: 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 15, 24, 36, 48, 72, 96 and 120 h. Blood and plasma (obtained by low-specd centrifugation of blood aliquots) were stored frozen until analysis. Urine was collected at 0-3, 3-6, 6-9,9-12, 12-24 h and then daily until day 8 after drug administration. All samples were kept frozen until analysed. Every faecal specimen passed after treatment was collected. At the 72-h time period, each subject ingested a capsule containing 250 m g of carmine red. All faecal specimens were collected for 8 days. By this time, if the carmine red stain had not appeared in th e stool, the collection was continued until the red stain was observed in the stool. Th is final specimen also was collected. All samples were stored frozen until analysis. Methods Measurement of radioactivity in plasma, urine and faeces was performed as described earlier (Bonelli et al. 1980). In short, 3 x 1 0 0 ~ fresh 1 blood was dried and combusted in an oxidizer (Packard, type 306).
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Pharmacokinetics of abecarnil
765
14C-Activitywas measured in a liquid scintillation counter (Packard, type B 460 C). In plasma and urine I4C-activity was determined by liquid scintillation counting in 2 x 1OOpl and in 2 x 1 ml samples, respectively. Faeces were freeze dried, homogenized and 3 x c. 100mg combusted for '4C-measurement. The unchanged drug was determined by h.p.1.c. with fluorescence detection as described earlier (Krause et al. 1989). In short, after adding 30ng of the 5-benzyloxy analogue as internal standard, 0.1-1 ml plasma or 1 ml urine was extracted by rotating with 2.5 ml diethylether for 30min, followed by centrifugation (2000g, 10min) and freezing of the aqueous phase. The ethereal layer was taken to dryness by a slight stream of nitrogen and the residue was reconstituted in 20&250p1 mobile phase. 50-200 p1 was injected for h.p.1.c. analysis. In faeces, abecarnil was determined by rotating 1 g of freeze-dried and homogenized sample with lOml methanol for 30min, centrifuging for 10 min at 2000gand taking an aliquot of 1 ml of the extract to dryness by a slight stream of nitrogen. T h e residue was dissolved in 1 ml mobile phase and 10&250 p1 was injected for h.p.1.c. analysis. Calibration was performed by external standardization. Samples were automatically injected by a WISP ( T M ) (Waters) or a G I N A (Gynkotek) autosampler. The pumps used were Waters type 6000 A or type 510. T h e chromatographic column was Spherisorb ODS I1 (125 x 4 6 mm, 3 pm) and the mobile phase consisted of 0.01 M aqueous ammonium carbonate buffer-methanol (30: 70, v/v) at a flow rate of 1,5rnl/min. Excitation and emission wavelengths were 279 and 427nm, respectively. Evaluation of data was performed by calculating peak area ratios (drug/standard) and comparing with peak area ratios obtained in calibration runs. For quality control, approximately 10% of the samples analysed contained blank samples with known concentrations of abecarnil: 1 ng, l o n g and 50 ng per 0.5 ml plasma. The concentrations found were 1.4k0.4ng ( n = l l ) , 10.2+1.5ng (n=16) and 4 8 k 3 n g ( n = 3 ) per 0.5 ml plasma. The limits of detection were 0.2 ng/ml (plasma) and 0.5 ng/ml or ng/g (urine/faeces) respectively. The coefficient of variation was below 10% for plasma, urine and faeces. Metabolite patterns in urine and faeces were determined by radio-h.p.1.c. using a Raytest RamonaLS4 ( T M ) radioactivity monitor. Urines (24h) of the human subjects were pooled by combining 1% of the total urine volume of each fraction and volunteer. Approx. 9000c.p.m. was injected directly onto a Spherisorb ODS-I1 column (125 x 4,6mm, 3 pm) with an ammonium carbonate buffer-methanol gradient from 100 : 0 to 0 : 100 in 30 min. Metabolite patterns in faeces were determined by pooling 1% of total daily faecal excreta of volunteers 1-8 and work-up as described for unchanged drug analysis. Approximately 15-20000 c.p.m. was injected. For plasma, pools of 3min, 30min and 2 h after i.v. injection and 30min, 2 h and 4 h after oral treatment (1 100-4200 c.p.m.) were directly injected (1-1.5 ml) onto the h.p.1.c. column. Radioactivity was measured in 1-min fractions by liquid scintillation counting.
Pharmacokinetic calculations Pharmacokinetic parameters were obtained by fitting the experimental data with the computer program T O P F I T (Thomae, Biberach, Germany). Plasma levels of total radioactivity and of the unchanged drug were fitted by a two-compartment model. For urinary and faecal excretion data a onecompartment model was used.
Results Tolerability All treatments were well tolerated by the volunteers. T h e adverse reactions observed or reported (tiredness, headache, lack of concentration and unsteady gait) were mostly rated mild to moderate and were equally distributed among treatments.
Absorption T h e extent of drug absorption can be assessed either by comparing the areas under the plasma level time curves ( AUC) of I4C-labelled substances, by urinary excretion of 14C-activity or by the faecal excretion of unchanged drug after oral and i.v. administration and appropriate correction for dose. Comparison of the A U C values after i.v. and oral administration, which is only feasible when metabolites after both routes of treatment are identical both qualitatively and quantitatively, would result in 229%absorption (table 2). Urinary and faecel excretion data of radioactivity or unchanged drug gives 90% and 92% absorption, respectively (table 2 ) .
W . Krause et al.
766 Table 2.
Pharmacokinetic parameters and elimination of I4C-labe1ledcompounds in eight healthy male volunteers after i.v. injection and oral administration of I4C-abecarnil.
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Intravenous injection
Oral administration
Mean k SD
Range
Mean k SD
Range
Plasma (I4C) C',,, (ng equiv./ml) tm,, (h) AUC (hmg equiv./ml) CI,,, (mlimin per kg) t , / z (1) (h) t l , 2 (2) (h)
84k21 0.05 101 34 4.3 f 1.9 < 0.05 4.8t1.8
59-1 20 0.05 55-149 2.4 h 8.1
95k16 1.2f0.4 263 k478
62-1 12 1-2 392-1675
1005
01-0.8
2.2-7.2
0 4 f 0.2 22f11
Urine (14C) Excretion (% dose) t l / 2 (h)
24.6 k 5.9 10.4f4.3
17G35.3 4.7-1 8.2
22.2 k 4.5 1 5.6 5.0
16.9-31.3 96-23.8
58.9 k20.0 1 5.4 k 7.0 8 3 5 215
24.8-94.5 7.8-24.8 44.9-1 13.8
57.9 & 11.8 14.6 k 4.9 80.0 k 13.2
3 1.5-67.7 106-23'6 48.4-89.1
2.1 f 0.7
l.G-2.7
9.6 t 3.3
4+14.6
Faeces (14C) Excretion (% dose) (h) Recovery
tl/Z
(% dose)
Fueces (drug) Excretion (% dose) Data are mean values was 1.88mg.
*
-
-
8-3 8
SD of n = 8 and range. Oral dose of I4C-abecarnil was 10.3 mg and i . v . dose
Plasma levels of abecarnil After i.v. injection plasma levels of the unchanged drug declined from 84 ng/ml at 3 min post administration (figure 2) in two phases with half-lives of < 3 min and 3-7h (table 3). Mean transit time was 4.2 h. T h e total clearance of unchanged abecarnil was calculated as 11 ml/min per kg. The volume of distribution V,, was 2.6 l/kg. After oral administration maximum concentrations of 14ng/ml were observed at 0.8 h. T h e half-life of absorption was 26 min. Disposition was biphasic with half-lives of 0-3h and 6.6 h. Mean transit time was calculated as 8.2 h. Bioavailability, not corrected for the unabsorbed proportion of dose, was 39%. Total radioactivity in the plasma Following intravenous injection of 14C-abecarnil (figure 3), plasma levels of total ''C declined in two phases with half-lives of < 0.05 h and 4.8 h (table 2). Mean transit time was 6.5 h. T h e total clearance of ''C-activity was calculated as 4 3 ml/min per kg after oral administration, maximum concentrations of 95 ng equiv./ml were observed at 1.2 h. T h e half-life of absorption was 0 8 h. Disposition again was biphasic with half-lives of 0.4 h and 22 h. Mean transit time was 28 h. Total radioactivity in the blood After i.v. injection a mean blood/plasma ratio of 14C-activity of 2.0 was obtained by measuring both blood and plasma levels of labelled compounds during the total observation period and calculated on the basis of dose/total volume. Oral administration resulted in a mean ratio of 1.5 indicating partitioning of the unchanged drug into blood cells, but no partitioning of metabolites.
767
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Pharmacokinetics of abecarnil
Time after dosing ( h )
(*I Figure 2.
Plasma levels of abecarnil in eight healthy male volunteers after i.v. injection (a) and oral administration (b).
Mean concentrationsf SD are given. Oral dose was 103mg, i.v. dose was 1.88mg.
Table 3 .
Pharmacokinetic parameters of unchanged abecarnil in eight healthy male volunteers after i.v. injection and oral administration of I4C-abecarnil. Intravenous injection Range
Mean f SD
Range
84+23 0.05
51-120 0.05
*+
.rl
4 4
lxm 2 1
mir
21.0
16.0
85.0
Ret en t io n t i m e (min)
3
(6) Figure 6.
Metabolite patterns obtained by radicz-h.p.1.c. of plasma of eight healthy male volunteers after i.v. injection (a) or oral administration (b) of 14C-abecarnil.
Oral dose was 10.3mg, i.v. dose 1.88mg. Patterns were obtained from native pool plasma of n = 8. Without extraction peaks represent 6-hydroxy glucuronide (l), 6-hydroxy-sulphate (2). 6hydroxy analogue (3), two unknowns (4,4')and unchanged drug (5). Concentrations (y-axis) are given in relative units.
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W . Krause et al.
Discussion In the present study the pharmacokinetics and biotransformation of the anxiolytic b-carboline derivative abecarnil have been investigated using the 14C-labelleddrug, doses of 1.88 mg i.v. and 10.3 mg given orally, in sequence to eight elderly healthy male volunteers. Elderly subjects were chosen because according to German regulations radioactive drugs may not be administered to persons below the age of SO. Following i.v.injection, plasma levels declined in two phases with a terminal halflife of approximately 4 h (table 3, figure 2). T h e total clearance of abecarnil-which was practically identical to the metabolic clearance as the drug was nearly totally metabolized with only 2% of dose excreted unchanged (table 2)-- was calculated as 11 ml/min per kg with a range from 6 to 20ml/min per kg (table 3), indicating interindividual differences in the metabolic handling of the drug. In an earlier study with unlabelled drug (Krause et al. 1990) almost identical data have been obtained with young healthy male volunteers (mean age: 2 7 + 5 years). T h e terminal disposition half-life was 3.4 h and the total clearance of abecarnil was evaluated as 13 ml/min per kg. After oral administration of a microcrystalline suspension, peak plasma levels were rapidly reached (GO.8 h, table 3, figure 2). I n five of eight subjects the first sampling point (0.5 h) was identical with C,,,. Absorption, as evaluated from faecal excretion of the unchanged drug, was practically complete (92%, table 2). Comparing urinary elimination of labelled compounds after oral and i.v. administration (table 2), the extent of absorption was calculated as 90 & 20%. Due to the relatively high first-pass effect, metabolite patterns in the plasma were quantitatively different after i.v. and oral treatment (figure 6), so that absorption cannot be calculated from an A UC comparison of I4C-activity in the plasma. In the above-mentioned former study oral dosing had been performed with encapsulated 2.5 mg tablets (total dose: 10 mg). Accordingly, t,,, was reached considerably later (1.7 vs. 0.8 h). However, in spite of nearly complete absorption in the present investigation, A U C was greater by 50% in young volunteers (125 h.ng/ml) compared to elderly test subjects (80h.ng/ml, table 3). As a consequence of identical clearance values, bioavailability also differed by 50% (65 vs. 39%). With the low number of volunteers tested (n= 6 or 8), the significance of this finding remains unclear as yet. 14C-Labelled compounds exhibited a much longer disposition half-life in the plasma compared to the unchanged drug (22 vs. 7 h, tables 2,3), especially after oral treatment. Multiple oral dosing using a t.i.d. regimen should therefore lead to accumulation of metabolites. This issue will be addressed in a future study. Comparing the terminal disposition half-lives of 14C-labelled compounds in the plasma after i.v. and oral administration reveals great differences (4.8 vs. 22 h, table 2). Possible explanations for these observations might either be different pharmacokinetic behaviour of individual metabolites depending on the route of administration (quantitative aspects) or the overlap of a third disposition phase after oral treatment which is not seen after i.v. injection. From the blood/plasma ratio of labelled substances after i.v. and oral treatment, the partitioning of the unchanged drug into corpuscular blood constituents was concluded. Metabolites, on the other hand-as can also be deduced from their markedly increased polarity in h.p.1.c.-were not able to cross the membranes. This has been confirmed by animal studies in which abecarnil was able to pass the
Pharmacokinetics of abecarnil
773
Table 4. Interspecies comparison of pharmacokinetic parameters of abecarnil. Man
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Absorption (% dose) Rioavailability (% absorbed dose) Cl,,, (ml/min per kg) tl/Zlermi"al(h) Elimination of radioactivity (urineifaeces)
92 42 11 3.7 1 : 2.4
Cynomolgus monkey
80 32 38 1.7 1 : 1.4
Rat
Rabbit
Mouse
100 32 25 1.7 1 :3.5
100 23 76 0.75 1 : 2.0
ND 20 15 1.0
ND
Data represent mean values. Doses were 0.1 mg/kg i.v. (mouse and monkey), 1 mg/kg (rat and rabbit) or 1.88mg (man) and 10mg/kg oral (animal species) or 10.3mg (man). Data on pharmacokinetic parameters of abecarnil in animal species are cited from Krause and Mengel (1 990).
blood/brain barrier achieving concentrations in rat brain similar to those in the plasma, whereas metabolites could not be detected in the brain (Krause and Mengel 1990). 14C-Activity was excreted mainly by the faecal route (approx. 60% of dose, table 2 ) , whereas in the urine c. 25% of the administered dose was recovered. Total 14C-balance was SO% (oral) and 84% (i.v.), respectively. Elimination was considered complete. T h e remaining c . 20% of dose was probably lost either due to technical problems or because of incomplete collection b y the volunteers. I n particular subject no. 4 had extremely low recovery data (45 and 48% of dose, respectively). An interspecies comparison of pharmacokinetic parameters of abecarnil is given in table 4. In all the species investigated the drug was more or less completely absorbed (80-100%) after oral administration, Bioavailability varied in a very low range from 20% in mouse to 42% in man. Terminal half-life of the unchanged drug ranged from 0.75 h in rabbit to 3.7 h in man. Only with total clearance a broader spectrum of values was found from 11 ml/min per kg in humans to 76 ml/min per kg in rabbit. Rabbit, however, was the only species in which total clearance was not identical to metabolic clearance since in this species unchanged drug was found in the faeces after i.v. injection of abecarnil (Krause and Mengel 1990). Elimination of labelled compounds proceeded in all the species mainly via the faeces with urine/faeces ratios of I4C-activity ranging from 0.3 to 0-7. T h e preliminary metabolite patterns of abecarnil in plasma, urine and faeces indicated phase I biotransformation at the 6-hydroxy group followed by conjugation both with glucuronic acid and with sulphate. Using cochromatography with previously isolated or synthesized compounds, practically all metabolites of abecarnil could tentatively be identified. At present, tests on pharmacological and toxicological activity of these metabolites are in progress.
References BONEILI,J., HITZENBERCER, G., KRAUSE, W., WENDT,I I . , and SPECK,U., 1980,Pharmacokinetics and pharmacodynamics of mepindolol sulphate. International Journal of Clinical Pharmacology Therapeutics and Toxicology, 18, 169-1 76. BRAESTRUP, C.,NIELSEN, M., and OLSEN,C. E., 1980,Urinary and brain ~-carboline-3-carboxylates as potent inhibitors of brain benzodiazepine receptors. Proceedings of the National Academy of Sciences of the United States of America, 77, 2288-2292. DOROW, R., HOROWSKI, R., PASCHELKE, G., AMIN,M., and BRAESTRUP, C., 1983,Severe anxiety induced by FG 7142,a j-carboline ligand for benzodiazepine receptors Lancet, ii, 98. DOROW, R., DUKA, T.,IIOLLER,L., and SAUERBREY, N., 1987a.Clinical perspectives of 8-carbolines from first studies in humans. Brain Research, 19, 319-326.
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DOROW,R., DUKA,T.. SAUERBREY, N., and HOLLER,L., 1987 b, B-Carbolines: new insights into the clinical pharmacology of benzodiazepine receptor ligands. In Clinical Pharmacology in Psychiatry ; selectivity in psychotropic drug action-promises or problems, edited by S. Dahl, I,. F. Gram, S. M. Paul and W. Z. Potter. Psychopharmacology (Berlin) Suppl., 3, 37-51. JBNSEN,L. H., PETERSEN, E. N., BRAESTRUP, C., HONORE,T . , KEHR, W., STEPHENS,D. N., SCHNEIDER, H. H., SEIDELMANN, D., and SCHMIECHEN, R., 1984, Evaluation of the /I-carboline ZK 93 426 as a benzodiazepine receptor antagonist. Psychopharmacology (Berlin), 83, 349-356. KRAUSE, W., and MENGEL, H., 1990, Pharmacokinetics of the anxiolytic 8-carholine derivative, abecarnil, in the mouse, the rat, the rabbit, the dog, the cynomolgus monkey and the baboon. Studies on species differences. Drug Research, 40, 522-529. KRAUSE, W., MENGEL, H., and NORDHOLM, L., 1989, Determination of j-carboline derivatives in biological samples by high-performance liquid chromatography with fluorescence detection. Journal of Pharmaceutical Sciences, 78, 622-626. KRAUSE, W., SCHUTT,B., and DUKA, T., 1990, Pharmacokinetics and acute toleration of the b-carboline derivative, abecarnil (ZK 112 119), in man. Drug Research, 40, 529-532. MOLLER, A., BLATT-LYON, B., SKRUMSAGER, B. K., and DAM,M., 1986, Antiepileptic effect of Bcarbolines. In Golden Jubilee Conference and Northern European Epilepsy Meeting, York, p. 85. PETERSEN, E. N.,1983, DMCM: a potent convulsive benzodiazepine receptor ligand. European Journal of Pharmacology, 94, 117-1 24. PETERSEN, E. N., and JENSEN, L. I%, 1984, Proconflict effect of benzodiazepine receptor inverse agonists and other inhibitors of GABA function. European Journal of Pharmacology, 103, 91-97. STEPHENS, D. N., SCHNEIDER, H. H., KEHR, W., ANDREWS, J. S., RETTIG, K.-J.,TURSKI, L., SCHMIECHEN, R., TURNER, J. D., JENSEN, L., PETERSEN, E. N., HONORE,T., and BONDOHANSEN, J., 1990, Abecarnil, a metabolically stable anxioselective B-carboline acting at benzodiazepine receptors. Journal of Pharmacology and Experimental Therapeutics, 253, 334-343. TIJRSKI, L., STEPHENS, D. N., JENSEN,L. H., PETERSEN, E. N., MELDRUM, B. S., PATEL,S., BONDO HANSEN, J., LOSCHER, W., SCHNEIDER, H. H., and SCHMIECHEN, R., 1990, Anticonvulsant action of the B-carboline abecarnil: studies in rodents and baboon, Papiopapio. Journal of Pharmacology and Experimental Therapeutics, 253, 34+352.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers W. Krause, Th. Duka & H. Matthes To cite this article: W. Krause, Th. Duka & H. Matthes (1991) Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers, Xenobiotica, 21:6, 763-774 To link to this article: http://dx.doi.org/10.3109/00498259109039516
Published online: 27 Aug 2009.
Submit your article to this journal
Article views: 7
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [NUS National University of Singapore]
Date: 07 November 2015, At: 00:51
XENOBIOTICA,1991,VOL.
21, NO. 6, 763-774
Pharmacokinetics and biotransformation of the anxiolytic abecarnil in healthy volunteers W. KRAUSE, T H . D U K A and H . M A T T H E S Research Laboratories of Schering AG, Berlin, Germany
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Received 5 December 1989; accepted 23 November 1990
1. T h e pharmacokinetics of ahecarnil were studied in eight healthy male volunteers using I4C-abecarnil and an h.p.1.c. method for determination of unchanged drug. 2. Abecarnil was rapidly and nearly completely absorbed after an oral dose of 1Omg; bioavailahility was 40%. Plasma levels of the unchanged d r u g declined with a terminal halflife of 4 h . T h e total clearance of ahecarnil was 11 mlimin per kg. '4C-Ahecarnil was excreted rapidly and completely. T h e main route of elimination was in the faeces.
3. Ahecarnil was extensively metabolized resulting in ether cleavage at position 6 with subsequent glucuronidation or sulphation and, to a minor extent, in ester cleavage.
Introduction Anxiolytic, anticonvulsant and muscle-relaxant effects of benzodiazepines as well as their side-effects such as sedation, ataxia and amnesia are mediated by binding to the benzodiazepine-GABA-receptor complex. Recently, new chemical classes of compounds have been found to interact with the benzodiazepine receptor. Among these are the 8-carbolines (Braestrup et al. 1980)which have been shown to exhibit the full spectrum of benzodiazepine effects as full agonists (Moller et al. 1986),as partial agonists (Petersen and Jensen 1984,Dorow et al. 1987a,b) or as antagonists, effects which are opposite to those of benzodiazepines (Dorow et al. 1983,Jensen et al. 1984,Petersen 1983,Petersen and Jensen 1984. Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-~-carboline-3-carboxylate) is one of these 8-carboline derivatives which selectively bind to the benzodiazepine receptor. I n animal models of anxiety and epilepsy this new compound has been reported to exhibit potent anxiolytic and anticonvulsant activity (Turski et al. 1990). Abecarnil exhibited only weak ataxiogenic effects, whereas ataxia produced by benzodiazepines could be antagonized by the new substance. Additionally, abecarnil showed in comparison to benzodiazepines, decreased interaction with ethanol in animal studies (Stephens et al. 1990).T h e new substance therefore has been classified as a partial agonist at the benzodiazepine receptor. T h e pharmacokinetics of abecarnil have been studied extensively in a number of animal species including rat, rabbit, dog and monkey (Krause and Mengel 1990)and in man (Krause et al. 1990). T h e aim of the present study was to complete the interspecies comparison by adding data on the pharmacokinetics and biotransformation of abecarnil in healthy volunteers using radiolabelled drug.
Materials and methods Materials Abecarnil was '4C-lahelled at position 1 (figure 1) with a specific activity of 2.2GBq/mmol (59.4 mCi/mmol). Radiochemical purity was greater than 99%. T h e d r u g was diluted with unlabelled 0049-8254191 $3.00
0 1991 Taylor & Francis
Ltd.
W . Krause et al.
764
CH3 \
.O
CH3
0
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0
F
CH3
Figure 1. T h e structure of abecarnil *indicates position of I4C-label.
Table 1.
Characteristics of the subjects participating in the radiolabelled study.
Subject no.
Mean SD
Age (years)
Height ( 4
Weight (kg)
56 62 55 60 65 50 51 64
170 181 165 165 176 188 187 171
74 92.5 75 81.5 76 95 70.5 82.5
57.9 5.7
175.4 9.2
80.9 8.9
material for use in human experiments. T h e preparation constituents Intralipid, Myrj 53 and Klucel LI: were obtained from Pfrimmer Kahi (Erlangen, Germany), Atlas Chemie (Essen, Germany), and Hercules Inc. (Wilmington, MA, USA), respectively.
Experimental design Eight healthy male volunteers (age 58 & 6 years; weight 81 + Y kg; height 175 & 9 cm) participated in the study (table 1). They had given their written informed consent. According to German regulations, the administration of radioactive compounds to humans is only allowed if the subjects are 50 years o r older. T he investigation had been approved by an Institutional Review Board and by the German Authorities. T h e test subjects first received an i.v. injection of 1.88 m g (86pCi) and 2 weeks later an oral dose of 10.3 mg (IOlpCi). Treatments were performed o n an empty stomach, and standardized food was only allowed 1 h after drug administration. Abecdrnil was dissolved in Intralipid-10% at a concentration of 0.188mg/ml for i.v. injection and suspended in isotonic saline with 0.085% (w/v) Myrj 53/1% (w/v) Klucel LF (1.03 mg/ml) for oral use. Blood was collected from an arm vein contralateral to the injection site immediately before the administration o f ahecarnil and at 3, 10, 15, 3 0 ,4 5 ,6 0 m i n , 1.5, 2, 3 , 4 , 6 , 9 , 12, 15, 24, 3 6 , 4 8 , 7 2 , 9 6 and 120 h after injection. Following oral administration the time points for blood collection were: 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 15, 24, 36, 48, 72, 96 and 120 h. Blood and plasma (obtained by low-specd centrifugation of blood aliquots) were stored frozen until analysis. Urine was collected at 0-3, 3-6, 6-9,9-12, 12-24 h and then daily until day 8 after drug administration. All samples were kept frozen until analysed. Every faecal specimen passed after treatment was collected. At the 72-h time period, each subject ingested a capsule containing 250 m g of carmine red. All faecal specimens were collected for 8 days. By this time, if the carmine red stain had not appeared in th e stool, the collection was continued until the red stain was observed in the stool. Th is final specimen also was collected. All samples were stored frozen until analysis. Methods Measurement of radioactivity in plasma, urine and faeces was performed as described earlier (Bonelli et al. 1980). In short, 3 x 1 0 0 ~ fresh 1 blood was dried and combusted in an oxidizer (Packard, type 306).
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765
14C-Activitywas measured in a liquid scintillation counter (Packard, type B 460 C). In plasma and urine I4C-activity was determined by liquid scintillation counting in 2 x 1OOpl and in 2 x 1 ml samples, respectively. Faeces were freeze dried, homogenized and 3 x c. 100mg combusted for '4C-measurement. The unchanged drug was determined by h.p.1.c. with fluorescence detection as described earlier (Krause et al. 1989). In short, after adding 30ng of the 5-benzyloxy analogue as internal standard, 0.1-1 ml plasma or 1 ml urine was extracted by rotating with 2.5 ml diethylether for 30min, followed by centrifugation (2000g, 10min) and freezing of the aqueous phase. The ethereal layer was taken to dryness by a slight stream of nitrogen and the residue was reconstituted in 20&250p1 mobile phase. 50-200 p1 was injected for h.p.1.c. analysis. In faeces, abecarnil was determined by rotating 1 g of freeze-dried and homogenized sample with lOml methanol for 30min, centrifuging for 10 min at 2000gand taking an aliquot of 1 ml of the extract to dryness by a slight stream of nitrogen. T h e residue was dissolved in 1 ml mobile phase and 10&250 p1 was injected for h.p.1.c. analysis. Calibration was performed by external standardization. Samples were automatically injected by a WISP ( T M ) (Waters) or a G I N A (Gynkotek) autosampler. The pumps used were Waters type 6000 A or type 510. T h e chromatographic column was Spherisorb ODS I1 (125 x 4 6 mm, 3 pm) and the mobile phase consisted of 0.01 M aqueous ammonium carbonate buffer-methanol (30: 70, v/v) at a flow rate of 1,5rnl/min. Excitation and emission wavelengths were 279 and 427nm, respectively. Evaluation of data was performed by calculating peak area ratios (drug/standard) and comparing with peak area ratios obtained in calibration runs. For quality control, approximately 10% of the samples analysed contained blank samples with known concentrations of abecarnil: 1 ng, l o n g and 50 ng per 0.5 ml plasma. The concentrations found were 1.4k0.4ng ( n = l l ) , 10.2+1.5ng (n=16) and 4 8 k 3 n g ( n = 3 ) per 0.5 ml plasma. The limits of detection were 0.2 ng/ml (plasma) and 0.5 ng/ml or ng/g (urine/faeces) respectively. The coefficient of variation was below 10% for plasma, urine and faeces. Metabolite patterns in urine and faeces were determined by radio-h.p.1.c. using a Raytest RamonaLS4 ( T M ) radioactivity monitor. Urines (24h) of the human subjects were pooled by combining 1% of the total urine volume of each fraction and volunteer. Approx. 9000c.p.m. was injected directly onto a Spherisorb ODS-I1 column (125 x 4,6mm, 3 pm) with an ammonium carbonate buffer-methanol gradient from 100 : 0 to 0 : 100 in 30 min. Metabolite patterns in faeces were determined by pooling 1% of total daily faecal excreta of volunteers 1-8 and work-up as described for unchanged drug analysis. Approximately 15-20000 c.p.m. was injected. For plasma, pools of 3min, 30min and 2 h after i.v. injection and 30min, 2 h and 4 h after oral treatment (1 100-4200 c.p.m.) were directly injected (1-1.5 ml) onto the h.p.1.c. column. Radioactivity was measured in 1-min fractions by liquid scintillation counting.
Pharmacokinetic calculations Pharmacokinetic parameters were obtained by fitting the experimental data with the computer program T O P F I T (Thomae, Biberach, Germany). Plasma levels of total radioactivity and of the unchanged drug were fitted by a two-compartment model. For urinary and faecal excretion data a onecompartment model was used.
Results Tolerability All treatments were well tolerated by the volunteers. T h e adverse reactions observed or reported (tiredness, headache, lack of concentration and unsteady gait) were mostly rated mild to moderate and were equally distributed among treatments.
Absorption T h e extent of drug absorption can be assessed either by comparing the areas under the plasma level time curves ( AUC) of I4C-labelled substances, by urinary excretion of 14C-activity or by the faecal excretion of unchanged drug after oral and i.v. administration and appropriate correction for dose. Comparison of the A U C values after i.v. and oral administration, which is only feasible when metabolites after both routes of treatment are identical both qualitatively and quantitatively, would result in 229%absorption (table 2). Urinary and faecel excretion data of radioactivity or unchanged drug gives 90% and 92% absorption, respectively (table 2 ) .
W . Krause et al.
766 Table 2.
Pharmacokinetic parameters and elimination of I4C-labe1ledcompounds in eight healthy male volunteers after i.v. injection and oral administration of I4C-abecarnil.
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Intravenous injection
Oral administration
Mean k SD
Range
Mean k SD
Range
Plasma (I4C) C',,, (ng equiv./ml) tm,, (h) AUC (hmg equiv./ml) CI,,, (mlimin per kg) t , / z (1) (h) t l , 2 (2) (h)
84k21 0.05 101 34 4.3 f 1.9 < 0.05 4.8t1.8
59-1 20 0.05 55-149 2.4 h 8.1
95k16 1.2f0.4 263 k478
62-1 12 1-2 392-1675
1005
01-0.8
2.2-7.2
0 4 f 0.2 22f11
Urine (14C) Excretion (% dose) t l / 2 (h)
24.6 k 5.9 10.4f4.3
17G35.3 4.7-1 8.2
22.2 k 4.5 1 5.6 5.0
16.9-31.3 96-23.8
58.9 k20.0 1 5.4 k 7.0 8 3 5 215
24.8-94.5 7.8-24.8 44.9-1 13.8
57.9 & 11.8 14.6 k 4.9 80.0 k 13.2
3 1.5-67.7 106-23'6 48.4-89.1
2.1 f 0.7
l.G-2.7
9.6 t 3.3
4+14.6
Faeces (14C) Excretion (% dose) (h) Recovery
tl/Z
(% dose)
Fueces (drug) Excretion (% dose) Data are mean values was 1.88mg.
*
-
-
8-3 8
SD of n = 8 and range. Oral dose of I4C-abecarnil was 10.3 mg and i . v . dose
Plasma levels of abecarnil After i.v. injection plasma levels of the unchanged drug declined from 84 ng/ml at 3 min post administration (figure 2) in two phases with half-lives of < 3 min and 3-7h (table 3). Mean transit time was 4.2 h. T h e total clearance of unchanged abecarnil was calculated as 11 ml/min per kg. The volume of distribution V,, was 2.6 l/kg. After oral administration maximum concentrations of 14ng/ml were observed at 0.8 h. T h e half-life of absorption was 26 min. Disposition was biphasic with half-lives of 0-3h and 6.6 h. Mean transit time was calculated as 8.2 h. Bioavailability, not corrected for the unabsorbed proportion of dose, was 39%. Total radioactivity in the plasma Following intravenous injection of 14C-abecarnil (figure 3), plasma levels of total ''C declined in two phases with half-lives of < 0.05 h and 4.8 h (table 2). Mean transit time was 6.5 h. T h e total clearance of ''C-activity was calculated as 4 3 ml/min per kg after oral administration, maximum concentrations of 95 ng equiv./ml were observed at 1.2 h. T h e half-life of absorption was 0 8 h. Disposition again was biphasic with half-lives of 0.4 h and 22 h. Mean transit time was 28 h. Total radioactivity in the blood After i.v. injection a mean blood/plasma ratio of 14C-activity of 2.0 was obtained by measuring both blood and plasma levels of labelled compounds during the total observation period and calculated on the basis of dose/total volume. Oral administration resulted in a mean ratio of 1.5 indicating partitioning of the unchanged drug into blood cells, but no partitioning of metabolites.
767
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Pharmacokinetics of abecarnil
Time after dosing ( h )
(*I Figure 2.
Plasma levels of abecarnil in eight healthy male volunteers after i.v. injection (a) and oral administration (b).
Mean concentrationsf SD are given. Oral dose was 103mg, i.v. dose was 1.88mg.
Table 3 .
Pharmacokinetic parameters of unchanged abecarnil in eight healthy male volunteers after i.v. injection and oral administration of I4C-abecarnil. Intravenous injection Range
Mean f SD
Range
84+23 0.05
51-120 0.05
*+
.rl
4 4
lxm 2 1
mir
21.0
16.0
85.0
Ret en t io n t i m e (min)
3
(6) Figure 6.
Metabolite patterns obtained by radicz-h.p.1.c. of plasma of eight healthy male volunteers after i.v. injection (a) or oral administration (b) of 14C-abecarnil.
Oral dose was 10.3mg, i.v. dose 1.88mg. Patterns were obtained from native pool plasma of n = 8. Without extraction peaks represent 6-hydroxy glucuronide (l), 6-hydroxy-sulphate (2). 6hydroxy analogue (3), two unknowns (4,4')and unchanged drug (5). Concentrations (y-axis) are given in relative units.
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W . Krause et al.
Discussion In the present study the pharmacokinetics and biotransformation of the anxiolytic b-carboline derivative abecarnil have been investigated using the 14C-labelleddrug, doses of 1.88 mg i.v. and 10.3 mg given orally, in sequence to eight elderly healthy male volunteers. Elderly subjects were chosen because according to German regulations radioactive drugs may not be administered to persons below the age of SO. Following i.v.injection, plasma levels declined in two phases with a terminal halflife of approximately 4 h (table 3, figure 2). T h e total clearance of abecarnil-which was practically identical to the metabolic clearance as the drug was nearly totally metabolized with only 2% of dose excreted unchanged (table 2)-- was calculated as 11 ml/min per kg with a range from 6 to 20ml/min per kg (table 3), indicating interindividual differences in the metabolic handling of the drug. In an earlier study with unlabelled drug (Krause et al. 1990) almost identical data have been obtained with young healthy male volunteers (mean age: 2 7 + 5 years). T h e terminal disposition half-life was 3.4 h and the total clearance of abecarnil was evaluated as 13 ml/min per kg. After oral administration of a microcrystalline suspension, peak plasma levels were rapidly reached (GO.8 h, table 3, figure 2). I n five of eight subjects the first sampling point (0.5 h) was identical with C,,,. Absorption, as evaluated from faecal excretion of the unchanged drug, was practically complete (92%, table 2). Comparing urinary elimination of labelled compounds after oral and i.v. administration (table 2), the extent of absorption was calculated as 90 & 20%. Due to the relatively high first-pass effect, metabolite patterns in the plasma were quantitatively different after i.v. and oral treatment (figure 6), so that absorption cannot be calculated from an A UC comparison of I4C-activity in the plasma. In the above-mentioned former study oral dosing had been performed with encapsulated 2.5 mg tablets (total dose: 10 mg). Accordingly, t,,, was reached considerably later (1.7 vs. 0.8 h). However, in spite of nearly complete absorption in the present investigation, A U C was greater by 50% in young volunteers (125 h.ng/ml) compared to elderly test subjects (80h.ng/ml, table 3). As a consequence of identical clearance values, bioavailability also differed by 50% (65 vs. 39%). With the low number of volunteers tested (n= 6 or 8), the significance of this finding remains unclear as yet. 14C-Labelled compounds exhibited a much longer disposition half-life in the plasma compared to the unchanged drug (22 vs. 7 h, tables 2,3), especially after oral treatment. Multiple oral dosing using a t.i.d. regimen should therefore lead to accumulation of metabolites. This issue will be addressed in a future study. Comparing the terminal disposition half-lives of 14C-labelled compounds in the plasma after i.v. and oral administration reveals great differences (4.8 vs. 22 h, table 2). Possible explanations for these observations might either be different pharmacokinetic behaviour of individual metabolites depending on the route of administration (quantitative aspects) or the overlap of a third disposition phase after oral treatment which is not seen after i.v. injection. From the blood/plasma ratio of labelled substances after i.v. and oral treatment, the partitioning of the unchanged drug into corpuscular blood constituents was concluded. Metabolites, on the other hand-as can also be deduced from their markedly increased polarity in h.p.1.c.-were not able to cross the membranes. This has been confirmed by animal studies in which abecarnil was able to pass the
Pharmacokinetics of abecarnil
773
Table 4. Interspecies comparison of pharmacokinetic parameters of abecarnil. Man
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Absorption (% dose) Rioavailability (% absorbed dose) Cl,,, (ml/min per kg) tl/Zlermi"al(h) Elimination of radioactivity (urineifaeces)
92 42 11 3.7 1 : 2.4
Cynomolgus monkey
80 32 38 1.7 1 : 1.4
Rat
Rabbit
Mouse
100 32 25 1.7 1 :3.5
100 23 76 0.75 1 : 2.0
ND 20 15 1.0
ND
Data represent mean values. Doses were 0.1 mg/kg i.v. (mouse and monkey), 1 mg/kg (rat and rabbit) or 1.88mg (man) and 10mg/kg oral (animal species) or 10.3mg (man). Data on pharmacokinetic parameters of abecarnil in animal species are cited from Krause and Mengel (1 990).
blood/brain barrier achieving concentrations in rat brain similar to those in the plasma, whereas metabolites could not be detected in the brain (Krause and Mengel 1990). 14C-Activity was excreted mainly by the faecal route (approx. 60% of dose, table 2 ) , whereas in the urine c. 25% of the administered dose was recovered. Total 14C-balance was SO% (oral) and 84% (i.v.), respectively. Elimination was considered complete. T h e remaining c . 20% of dose was probably lost either due to technical problems or because of incomplete collection b y the volunteers. I n particular subject no. 4 had extremely low recovery data (45 and 48% of dose, respectively). An interspecies comparison of pharmacokinetic parameters of abecarnil is given in table 4. In all the species investigated the drug was more or less completely absorbed (80-100%) after oral administration, Bioavailability varied in a very low range from 20% in mouse to 42% in man. Terminal half-life of the unchanged drug ranged from 0.75 h in rabbit to 3.7 h in man. Only with total clearance a broader spectrum of values was found from 11 ml/min per kg in humans to 76 ml/min per kg in rabbit. Rabbit, however, was the only species in which total clearance was not identical to metabolic clearance since in this species unchanged drug was found in the faeces after i.v. injection of abecarnil (Krause and Mengel 1990). Elimination of labelled compounds proceeded in all the species mainly via the faeces with urine/faeces ratios of I4C-activity ranging from 0.3 to 0-7. T h e preliminary metabolite patterns of abecarnil in plasma, urine and faeces indicated phase I biotransformation at the 6-hydroxy group followed by conjugation both with glucuronic acid and with sulphate. Using cochromatography with previously isolated or synthesized compounds, practically all metabolites of abecarnil could tentatively be identified. At present, tests on pharmacological and toxicological activity of these metabolites are in progress.
References BONEILI,J., HITZENBERCER, G., KRAUSE, W., WENDT,I I . , and SPECK,U., 1980,Pharmacokinetics and pharmacodynamics of mepindolol sulphate. International Journal of Clinical Pharmacology Therapeutics and Toxicology, 18, 169-1 76. BRAESTRUP, C.,NIELSEN, M., and OLSEN,C. E., 1980,Urinary and brain ~-carboline-3-carboxylates as potent inhibitors of brain benzodiazepine receptors. Proceedings of the National Academy of Sciences of the United States of America, 77, 2288-2292. DOROW, R., HOROWSKI, R., PASCHELKE, G., AMIN,M., and BRAESTRUP, C., 1983,Severe anxiety induced by FG 7142,a j-carboline ligand for benzodiazepine receptors Lancet, ii, 98. DOROW, R., DUKA, T.,IIOLLER,L., and SAUERBREY, N., 1987a.Clinical perspectives of 8-carbolines from first studies in humans. Brain Research, 19, 319-326.
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DOROW,R., DUKA,T.. SAUERBREY, N., and HOLLER,L., 1987 b, B-Carbolines: new insights into the clinical pharmacology of benzodiazepine receptor ligands. In Clinical Pharmacology in Psychiatry ; selectivity in psychotropic drug action-promises or problems, edited by S. Dahl, I,. F. Gram, S. M. Paul and W. Z. Potter. Psychopharmacology (Berlin) Suppl., 3, 37-51. JBNSEN,L. H., PETERSEN, E. N., BRAESTRUP, C., HONORE,T . , KEHR, W., STEPHENS,D. N., SCHNEIDER, H. H., SEIDELMANN, D., and SCHMIECHEN, R., 1984, Evaluation of the /I-carboline ZK 93 426 as a benzodiazepine receptor antagonist. Psychopharmacology (Berlin), 83, 349-356. KRAUSE, W., and MENGEL, H., 1990, Pharmacokinetics of the anxiolytic 8-carholine derivative, abecarnil, in the mouse, the rat, the rabbit, the dog, the cynomolgus monkey and the baboon. Studies on species differences. Drug Research, 40, 522-529. KRAUSE, W., MENGEL, H., and NORDHOLM, L., 1989, Determination of j-carboline derivatives in biological samples by high-performance liquid chromatography with fluorescence detection. Journal of Pharmaceutical Sciences, 78, 622-626. KRAUSE, W., SCHUTT,B., and DUKA, T., 1990, Pharmacokinetics and acute toleration of the b-carboline derivative, abecarnil (ZK 112 119), in man. Drug Research, 40, 529-532. MOLLER, A., BLATT-LYON, B., SKRUMSAGER, B. K., and DAM,M., 1986, Antiepileptic effect of Bcarbolines. In Golden Jubilee Conference and Northern European Epilepsy Meeting, York, p. 85. PETERSEN, E. N.,1983, DMCM: a potent convulsive benzodiazepine receptor ligand. European Journal of Pharmacology, 94, 117-1 24. PETERSEN, E. N., and JENSEN, L. I%, 1984, Proconflict effect of benzodiazepine receptor inverse agonists and other inhibitors of GABA function. European Journal of Pharmacology, 103, 91-97. STEPHENS, D. N., SCHNEIDER, H. H., KEHR, W., ANDREWS, J. S., RETTIG, K.-J.,TURSKI, L., SCHMIECHEN, R., TURNER, J. D., JENSEN, L., PETERSEN, E. N., HONORE,T., and BONDOHANSEN, J., 1990, Abecarnil, a metabolically stable anxioselective B-carboline acting at benzodiazepine receptors. Journal of Pharmacology and Experimental Therapeutics, 253, 334-343. TIJRSKI, L., STEPHENS, D. N., JENSEN,L. H., PETERSEN, E. N., MELDRUM, B. S., PATEL,S., BONDO HANSEN, J., LOSCHER, W., SCHNEIDER, H. H., and SCHMIECHEN, R., 1990, Anticonvulsant action of the B-carboline abecarnil: studies in rodents and baboon, Papiopapio. Journal of Pharmacology and Experimental Therapeutics, 253, 34+352.
