Eur J Clin Pharmacol (1991) 41:485-488 EuropeanJournalof

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1991

Dose-proportionality of eltoprazine Pharmacokinetics of single oral doses in healthy subjects M. H. de Vries 1, P. de Koning 2, H. L. Floor 1, A. Grahn6n 3, S. A . Eckerniis 3, M. Raghoebar 1, B. Dahlstriim 4, and L. E k m a n 4

Department of Drug Disposition and 2 Department of Clinical Research, Duphar BV, Weesp, The Netherlands and 3 PMC Drug Study Unit and 4 PMCAnalytical Services AB, Glunten, Uppsala, Sweden Received: November 19, 1990/Accepted in revised form: April 2, 1991

Summary. Eltoprazine. HC1 belongs to a new class of psychotropic drug, the serenics. The dose-proportionality and pharmacokinetics of eltoprazine HC1 has been investigated after single oral doses of 5, 10, 20 mg (18 subjects) and 30 mg (12 subjects) in a partly randomized, cross-over design. Eltoprazine was well tolerated and there were no relevant changes in safety parameters. All subjects showed irregular plasma-concentration-time profiles, some subjects demonstrating secondary peaks. The mean half-life was calculated to be about 6.5 h. The renal excretion of eltoprazine was characterized by net tubular secretion. A U C , peak plasma concentrations and the amount excreted unchanged in the urine were linearly related to the dose. Renal clearance and tu2 were independent of dose. Thus, eltoprazine HC1 was well tolerated orally and exhibited a linear pharmacokinetic profile. K e y words: Eltoprazine; pharmacokinetic, serenics, adverse effects

Eltoprazine HC1 ( D U 28853; Fig. 1) is a novel compound, which belongs to a new class of psychotropic drugs, the serenics [1]. It shows r e m a r k a b l e activity in animal models of aggression, in which it inhibits offensive aggression without causing sedation or m o t o r impairment. Defence mechanisms and social interactions are not compromised [2]. The effects on aggression in the animal experiments are considered to be related to its serotonergic (5-HT1A and 5-HT1B) activity [3, 4, 5, 6]. On the basis of its specific behavioural profile in animal models or agonistic behaviour [7] the c o m p o u n d is now being tested in several populations of patients who show destructive behaviour. Pharmacokinetic studies have indicated that eltopraZine. HC1 is very well [8] absorbed, with an absolute bioavailability of about 95%. The m a x i m u m plasma concentration of eltoprazine is attained within 1-4 h after administration, followed by a decrease in plasma concentration with a terminal half-life of 7-9 h. The cumulative

renal excretion of unchanged eltoprazine is about 40% [9]. The metabolic routes have yet to be elucidated. The present study was conducted to evaluate the dose proportionality of its pharmacokinetics after oral administration of four different single doses (5,10, 20 and 30 mg) to healthy volunteers. The tolerance and safety of eltoprazine in relation to dose was also evaluated.

Subjects and methods

Procedures The study followed a four-treatment, partly randomized, cross-over, single-dose design. The 5, 10 and 20 mg doses were given in blind, randomized, balanced order in the first 3 sessions. The 30 mg dose was administered in the fourth study session to subjects tolerating 20 mg, and so was not given in blind randomized order. The washout period between the study sessions was at least 10 days. Eighteen healthy male subjects (aged 20 to 32 y, with a weight range of 64 to 95 kg) volunteered for the study. The protocol was approved by the Uppsala University Institutional Review Board. Volunteers were screened for good general health by specific physical examination (including ECG) and blood and urine tests. Use of any drug (including over the counter drugs) within 2 weeks prior to the beginning of the study and at any time in the study was not allowed. Alcoholic beverages within 48 h prior to and during each study session were not permitted. The subjects were housed in the clinical unit the night before each session. Subjects received a light standard breakfast 1 h before dosing and were then kept fasting until lunch time. Eltoprazine was given as a capsule with 200 ml water. Blood samples were collected from a forearm vein before and at the following times after dosing: 0.5,1,1.5, 2, 2.5, 3, 4, 5, 6, 8,12,18, 24, 30, 36 h and 48 h (30 mg session only). Urine was collected over the intervals 0-1, 1-2, 2-4, 4-6, 6-8, 810, 10-12, 12-24, 24-48 and 48 72 h.

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Fig. 1. Structure of eltoprazine HCI (DU 28853)

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M. H. de Vries et al.: Dose-proportionality of eltoprazine 70

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Fig.2. Mean (SEM) plasma concentration of eltoprazine HC1 after oral administration of 5, 10, 20 (n -- 18) and 30 (n = 12) mg to healthy volunteers. - O - 5 m g --~- 10mg -A- 20rag -@- 30rag

Blood samples were collected in heparinized tubes. Within 0.5 h after sampling, the blood was centrifuged cold. Plasma samples and aliquots of the urine were stored at - 20 0198C until analysis. At each treatment session, blood pressure and pulse rate were recorded before and 2, 4, 8 and 72 h after dosing. ECG, blood and urine analyses were done before and 24 h after dosing in each study session. Tolerance was assessed by means of general questioning.

Eltoprazine assay Unchanged eltoprazine in plasma and urine was measured by HPLC, After addition of D U 121334 (1-(1,3-benzodioxol-4-yl) piperazine hydrochloride) as the internal standard, biological samples and appropriate calibration standards were extracted on a preconditioned C-18 column (Baker 10 SPE no.7020-3, Deventer, The Netherlands). After washing with water, the column was eluted with 2 ml methanol containing 2% ammonia (25% w/w). The eluate was diluted with 2 ml water and extracted with 4 ml dichloromethane: acetone (80:20 v/v). After centrifugation, the organic layer was placed in a vial of suitable volume with a teflon-laminated closure, and was evaporated to dryness at 35 °C in a gentle stream of nitrogen. The residue was redissolved in 200 ~tl acetonitrile and 50 pl of a solution of the derivatization reagent dansylchloride (a solution of 10 mg 5-dimethylamino-l-naphthalene-sulphonylchloride in 5 ml acetonitrile). The derivatization reaction time was 10 min at room temperature. Water 500 gl and 50 gl potassium carbonate solution (60% w/v in water) were added and the reaction was continued for 5 min at 60°C. After further extraction with 5 ml diethylether: n-pentane (80 : 20 v/v), the organic layer was separated after rapid cooling in dryice. After evaporation, an aliquot of the residue was dissolved in 100 gl acetonitrile: H20 (65 : 35 v/v) and was injectedinto the HPLC system. This comprised a reversed phase (C-18) column and a fluorescence detector. The mobile phase was acetonitrile: H20 (65 : 35 % v/v) at a flow rate of 2 ml- min 1. The fluorescence detector was set at an excitation wavelength of 350 nm and at an emission wavelength of 515 nm. The method was validate d for line arity and reproducibility (CV = + 8% in plasma and urine) at concentrations up to 100 ng. ml- ain plasma and up tol gg-ml i in urine. The determination limit was set at 1 ng. ml- 1plasma and 0.05 gg. ml- ~urine.

minimum number of 5 data-points was employed. A U C vs time curves from 0 to 36 h were extrapolated to infinity (AUC) using individual Xz values. All other modeMndependent pharmacokinetic parameters were calculated using standard methods [10]. The pooled model dependent parameters were estimated from the Naive Pooled Data Approach according to Sheiner and Beal [11]. The disposition parameters for eltoprazine after oral administration were estimated from a one compartment model with first order absorption including lag time. The choice of the model was based on the best fit of the analysis of plasma concentrations vs time. Plasma concentration-time data were analysed by non-linear weighted (1/c 2) least-squares regression analysis (WLS). Goodness of fit of computed to observed data was based on on visual inspection, coefficient of correlation, residual plots, standard deviation of estimated parameters, correlation matrix and the Akaike information criterion. Data analysis was performed with the computer programme S I P H A R [12], which has been cross validated against SAS (release 5.16) and NONLIN. Statistical comparison of the parameters was performed with A N O V A for the randomized, balanced part of the study (5, 10 and 20 mg dose), The relationship between the pharmacokinetic parameters and dose was examined by linear regression analysis. Wilcoxon's signed rank test was used as a non-parametric test of differences in T .... P < 0.05 was considered to be significantly different. Laboratory safety data were compared to age-matched reference values. Particular attention was given to values deviating more than 2 SD from the population mean. Results

Kinetics The time course of eltoprazine concentrations after each d o s e is s h o w n in F i g . 2 . T h e c o r r e s p o n d i n g p h a r m a c o kinetic parameters derived from plasma and urine data a r e g i v e n in T a b l e I. A N O V A o f t h e d o s e - a d j u s t e d A U C , Cmax a n d A e d a t a s h o w e d n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e 5, 10 a n d 20 m g t r e a t m e n t s . F o r all t r e a t m e n t s , A U C , C . . . . A e w e r e l i n e a r l y r e l a t e d to t h e e l t o p r a z i n e d o s e ( P < 0 . 0 0 0 1 ;

Table 1. Mean (SD) pharmacokinetic parameters derived from

plasma and urine after oral administration of 5, 10, 20 and 30 mg eltoprazine HC1 to healthy volunteers Parameter

Dose 5 mg

10 mg

20 mg

30 mg

from plasma data AUC(0_t)[ng. ml-~, h] AUC [ng. ml-1. hi Residual AUC [%] Cm~x[ng/ml] tma×[h] )vz[l/h] tl/2, z [h] MRT [h]

125 (44.7) 136 (44.5) 9.2 (5.9) 12.9 (3.6) 2.5 (1.1) 0.108 (0.03) 6.7 (1.3) 11.2 (1.9)

243 (66.0) 254 (66.6) 4.6 (2.5) 22.8 (5.3) 2.8 (1.3) 0.116 (0.02) 6.2 (1.0) 10.8 (1.2)

491 (151) 506 (158) 2.7 (1.5) 42.9 (9.6) 3.9 (1.0) 0.118 (0.02) 5.9 (0.9) 10.6 (1.4)

735 (225) 746 (227) 1.5 (0.7) 62.9 (13.8) 3.5 (1.8) 0.120(0.02) 5.9 (0.9) 10.5 (1.2)

from urine data Ae [mg] fe [%] CLR [ml. min-~] tl/2 [h]

2.06 (0.43) 41.2 (8.7) 271 (80) 6.3 (1.6)

4.18(0.98) 8.47(1.81) 41.8 (9.8) 42.3 (9.0) 291 (91) 293 (75) 6.7 (2.5) 5.9 (1.3)

12.5(1.9) 41.6 (6.3) 299 (82) 6.0 (1.4)

6.9 0.0 170 11.4

6.8 0.4 284 11.5

6.7 0.1 869 11.5

Model independent

Data analysis

Model dependent

Phamacokinetic parameters were first calculated utilizing a compartment-independent approach. The elimination rate constant (Xz) was estimated individually by regression analysis of the terminal part of the log plasma-concentration-time curves (8-10 to 36-48 h). A

t1,,2,z [h] hag [hi AUC [ng.ml- t.h] MRT [hi

6.7 0.3 583 11.9

487

M. H. de Vries et al.: Dose-proportionality of eltoprazine 1200

no deviation which could be ascribed to drug administration. There was a clear relation between the dose of eltoprazine and the number and duration of reported signs and symptoms (Table II). The recorded sedation in some patients referred to reports of difficulty in concentrating on a book. At higher dose levels the sedation was of longer duration rather than of higher severity. This side effect was not considered bothersome and, according to the physician present at the sessions, it was qualitatively different from the sedation following intake of neuroleptics. None of the reported adverse effects were considered serious, although the nausea at higher dose levels was not easily tolerated. Hiccup was reported by one subject at 20 mg and by 3 subjects at 30 rag. There were no drop-outs. None of the subjects volunteering to participate in the 30 mg session had shown a safety or tolerance problem which would have precluded their participation at this, the highest dose level.

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Fig.3 a-e. Relationship between the AUC, Cmax and Ae of eltoprazine. HCI and the oral dose

Fig.3). Dose-adjusted (10mg) AUC-ratios were 0.96 (0.17), 0.95 (0.22), 0.97 (0.22) for the 10, 20 and 30 mg doses in comparison with 5 mg. A N O V A analysis of the half-life data revealed that tl/2 was significantly higher for the lowest dose compared to the higher doses. Utilising the population kinetic approach, as suggested by Sheiner and Beal [11], the so called Naive P o o l e d D a t a A p p r o a c h , a similar trendwas detected,but no significant differences in tm values was observed, tmax showed a slight trend to increase with increasing dose up to 20 mg. MRT, re, CLm tmaxand the tl/2 calculated from the urine data was independent of dose. A N O V A analysis revealed a significant period effect in CLa.

Safety and tolerance The most prominent finding in the laboratory safety data was a low haemoglobin and related values due to the blood sampling. ECG, blood pressure and pulse showed

The current study has shown that the pharmacokinetics of eltoprazine, HC1 is linear in the dose-range 5 to 30 mg following a single oral dose administration. The major pharmacokinetic processes thus seem to be of the first order type. The plasma concentrations of eltoprazine increased in a linear, dose-dependent manner, as assessed both by the A U C and C . . . . Plasma eltoprazine levels peaked at 1.5-5 h. tmax showed a trend to increase with increasing dose up to 20 mg. From dissolution tests of the 5, 10 and 20 mg capsules (Duphar; data on file) it was concluded that the initial dissolution rate decreased with increasing eltoprazine content. There appeared to be some correlation between the rate of in vitro dissolution and t .... The disposition parameters reflecting elimination (eg CLR and tl/2) were independent of dose. The fraction of dose excreted unchanged in urine (re) and MRT reflected both absorption and elimination and was independent of the dose. Utilizing the Naive Pooled Data Approach, no significant difference in half-life or M R T between doses was observed; the mean half-life was about 6.5 h and the mean MRT was about 12 h. The half-life estimated from plasma data was in close agreement with that estimated from urine data and was somewhat shorter than has previously been reported [9]. All subjects showed irregular plasma-concentrationtime profiles, some subjects demonstrating second peaks after oral administration. The irregularity and double peak phenomena were not dose-dependent. The mechaTable 2. Signs and symptoms after oral eltoprazine Session numberof sub- totalnumber predominantcomplaints jectsin session of complaints 5 mg 18 5 sedation 10mg 18 17 sedation, GI discomfort 20 mg 18 26 sedation,GI discomfort,dizziness 30raga 12 20 sedation,GI discomfort,hiccup Subjects in this session were not blind to the dose administered

488 nism behind the appearance of the multiple peaks is not dear. Double plasma peaks after oral administration (as well as i. v.) have been demonstrated for m a n y drugs, especially for weak bases, such as cimetidine and ranitidine. It has been suggested that gastric secretion and subsequent entrapment in the gut mucosa followed by reabsorption may account for the p h e n o m e n o n [13, 14]. The renal excretion of eltoprazine was characterized by net tubular secretion, as the renal clearance of eltoprazine was about twice the glomerular filtration rate, a finding similar to that made in previous studies of eltoprazine [9]. A b o u t 40% of a given dose was recovered in urine as unchanged eltoprazine. The urinary excretion rate was directly proportional to the plasma concentrations in the concentration range covered here (0-95 ng/ml). Thus, the renal elimination followed a first order process. It should be emphasized that the renal clearance of eltoprazine was subject to time-dependent variability (e. g., CLR varied from one day to another). The variability was also shown not to be uniformly distributed, as statistical analysisrevealed asignificantperiod effect. Suchvariabilityhas been reported for m a n y drugs with a high renal clearance [15]. It is probably due to competing tubular secretion processes between the drug and other (endogenous) substances. The variability must be kept in mind when undertaking bioavailability studies of eltoprazine, due to the fact that if clearance (renal clearance is 40% of total) is not constant over time, the laws of corresponding areas and corresponding urinary excretions are not valid. In fact, R a g h o e b a r et al. [9] reported that variability in the absolute bioavailability of eltoprazine was reduced after correction of the bioavailability ratio (p. o./i. v.) for differences in CLR. Adjusting the data for variability in CLR, the absolute bioavailability of eltoprazine was about 95% in most subjects. The reported signs and symptoms and the absence of relevant changes in safety parameters are consistent with previous findings following administration of single doses of eltoprazine. Thus, eltoprazine was well tolerated orally and exhibited a linear pharmacokinetic profile.

M. H. de Vries et al.: Dose-proportionality of eltoprazine 3. Olivier B, Mos J, Van der Heyden H, Schipper J, Tulp M, Berkelmans B, Bevan P (1987) Serotonergic modulation of agonistic behaviour. In: Olivier B, Mos J, Brain PF (eds) Ethopharmacology of agonistic behaviour in animals and humans. Nijhoff, Dordrecht, pp 162-186 4. Olivier B, Mos J, ]Salp M, Schipper J, Den Daas S, Van Oortmerssen G (1990) Serotonergic involvement in aggressive behaviour in animals. In: Van Praag HM, Plutchik R, Apter A (eds) Violence and suicidality: perspectives in clinical and psychobiological research. Brunner/Mazel, New York, pp79-137 5. Bevan R Olivier B, Schipper J, Mos J (1990) Serotonergic function and aggression in animals. In: Serotonin: actions, receptors, pathophysiology. Mylecharane MA, Angus JA, de la Lande IS, Humphry PPA (eds) Macmillan, England, pp 101 108 6. Schipper J, Tulp M, Sijbesma H (1990) Neurochemical profile of eltoprazine. Drug Metabol Drug Interact 8:85-114 7. Olivier B, Mos J, Rasmussen D (1990) Behavioural pharmacology of the serenic, eltoprazine. Drug Metabol Drug Interact 8: 31-83 8. Van Harten J, Mathlener IS, Raghoebar M (1990) Pharmacokinetics of eltoprazine in healthy subjects. Drug Metabol Drug Interact 8:149-158 9. Raghoebar M, Mak M, Cournot A, Pistorius MCM, van Harten J, Roseboom H (1990) Pharmacokinetics of eltoprazine hydrochloride in healthy male subjects after single dose oral and intravenous administration. Br J Clin Pharmaco130:879-883 10. Gibaldi M, Perrier D (1975) Pharmacokinetics. Dekker, New York ll. Sheiner L, Beal S (1980) Evaluation of methods for estimating population pharmacokinetic parameters. I.Michaelis-Menten Model: Routine clinical pharmacokinetic data. J Pharmacokinet Biopharm 8:553-571 12. Gomeni R (1984) Pharm. An interactive graphic program for individual and population pharmacokinetic parameter estimation. Comput Biol Med 14:24-34 13. Grahn6n A, von Bahr C, Lindstr6m B, Rosdn A (1979) Bioavailability and pharmacokinetics of cimetidine. Eur J Clin Pharmacol 16:335-340 14. Grahn6n A (1984) Cimetidine bioavailability and variable renal clearance. Eur J Clin Pharmaco127:623-624 15. Grahn6n A (1985) The impact of time dependent phenomena on bioequivalence studies. In: Breimer DD, Speiser P (eds) Topics in pharmaceutical sciences 1985, Elsevier, Amsterdam, pp 179190

Acknowledgements. We thank Ms. M. van Vliet, M.Stoker and M. Pistorius for their excellent technical assistance.

References 1. Olivier B, Van Dalen D, Hartog J (1986) A new class of psychoactive drugs: serenics. Drugs Future 11:473-494 2. Olivier B, Mos J (1986) Serenics and aggression. Stress Med 2: 197-209

Dr. M. H. de Vries Drug Disposition Department Duphar B. V. R O. Box 900 NL-1380 DA Weesp The Netherlands