Eur J ClinPharmacol(1991) 40:181-185 0031697091000520

esMecel;e © Springer-Verlag 1991

Single and multiple dose pharmacokinetics of etizolam in healthy subjects C. Fracasso, S. Confalonieri, S. Garattini, and S. Caccia Istituto di RicercheFarmacologiche"MarioNegri",Milan,Italy Received: March 22, 1990/Acceptedin revised form:May 2,1990

Summary. The pharmacokinetics of etizolam, a new thienodiazepine derivative, has been examined after single and multiple (0.5 mg tablet) (0.5 mg b.d for 1 week) oral therapeutic doses in healthy volunteers. The singledose kinetic profile of etizolam suggested that absorption after oral dosage was reasonably rapid, the maximum plasma concentration (Cmax)being attained within 0.5-2 h in all subjects. The mean elimination half-life (tu2) averaged 3.4 h. Consistent with this, steady-state concentration were rapidly achieved and accumulation was extremely limited. Predicted average plasma concentrations (Cp) did not differ significantly from those actually measured at steady-state, suggesting that the kinetics of etizolam was linear, at least at therapeutic doses. The mean wash-out tu2 was comparable to the elimination tl/2 of the single dose, which means that the drug probably has no effect on hepatic microsomal enzymes and other kinetic variables after repeated dosing. At steady state plasma concentrations of the main metabolite, c~-hydroxyetizolam, were higher and disappeared more slowly (mean tl/2 8.2h) than those of the parent compound. Taken with the fact that in animals the metabolite shows almost the same potency of pharmacological action as etizolam, this suggests that it may contribute significantly to the clinical effects of the parent compound. Based on the kinetic characteristics of the parent drug and its metabolite, etizolam can be regarded as a short-acting benzodiazepine, with elimination kinetics between those of shortintermediate derivatives and ultra-rapidly eliminated benzodiazepines.

marked anticonvulsive effect, attenuates conflict behaviour, inhibits aggessive behaviour and has a muscle relexant effect in laboratory animals [3]. Quantitative EEG studies indicate that its mode of action is similar to the benzodiazepine hypnotic-anxiolytics [4]. It acts as an agonist on the GABA-benzodiazepine receptor complex (Yoshitomi Pharmaceutical Industries, unpublished data). It has been recently approved for use in the treatment of anxiety at the dosage of 0.5 mg b.d [5]. Previous kinetic and metabolic studies in human volunteers given 2 mg etizolam orally (Yoshitomi Pharmaceutical Industries, unpublished data) indicated that the drug was well absorbed and rapidly eliminated by oxidative metabolic pathways primarily involving hydroxylation at various positions of the molecule, followed by conjugation with glucuronic acid [6]. Elimination was rapidly in healthy subjects, with a mean elimination tl/a of about 5 h, comparable to that of well-known ultra-short acting benzodiazepine derivatives. To characterize further the pattern of absorption and elimination of etizolam after a single oral dose and to determine the pattern of accumulation during chronic therapy, the present study was undertaken in healthy volunteers given a therapeutic dose. The study included a preliminary assessement of the kinetic properties of a-hydroxyetizolam, 6-(o-chlorophenyl)-8-(1-hydroxyethyl)-l-methyl4H-s-triazolo[3,4-c]thieno[2,3-e][1,4]diazepine, the main metabolite of etizolam in humans, which has been found to have almost the same pharmacological potency and high affinityfor benzodiazepine receptors as etizolam (Yoshit omi Pharmaceutical Industries, unpublished).

Key words: Etizolam, ~-hydroxyetizolam, healthy subjects, kinetics Materials and methods Etizolam (Fig. 1) is a thienodiazepine derivative originating from the research of Tahara et al., 1978 [1] on 6-aryl4H-s-triazolo[3,4-c]thieno[2,3-e][1,4]diazepines, a series of benzodiazepine derivatives which have a thieno moiety in place of the benzo moiety of the benzodiazepine ring. Like thienodiazepines in general [2], etizolam shows a

Study design Six healthy males, aged 20 to 32 y (mean 26 y), weight 66 to 78 kg (mean 72 kg), with normal clinical and biochemical profiles took part in the study.Each subject was screened for suitabilityfor entry and was accepted only if the medical history was satisfactoryand

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C. Fracasso et al,: Etizolam kinetics

CH ' - ' ~ N N N s3 \

//

CH3CH2~N---~ Ct

Fig. 1. Chemical structure of etizolam, 6-(0-chlorophenyl) -8-ethyl-l-methyl4H-s-triazolo[3,4-c]thieno [2,3-e][1,4]diazepine

there was no evidence of diabetes, cardiovascular, hepatic, renal or gastrointestinal disease, psychotic disorders or depressive illness. Each subject was given a full explanation of the purpose of the study and signed on informed consent form approved by the local Ethical Committee. All subjects were free from medication for at least i week before the study. After an overnight fast, each subj ect took one tablet of 0.5 mg etizolam with water (100 ml). Food and drink, apart from water, were withheld for 4 h after dosing, then each subject was free to follow his normal eating and drinking routine. Blood samples were collected by direct venepuncture immediately pre-dose and nominally 0.25, 0.5, 1, 2, 3, 4.5, 6, 9, 12 and 24 h after dosing. Plasma was separated and was stored frozen until analysed. Urine was collected for 30 h after dosing and stored at - 20°C. During the multiple-dose schedule the same subjects received etizolam 0.5 mg tablets at 12 h intervals (9.00 and 21.00 h), each dose with 100 ml of water, daily for 1 week. Venous blood was sampled before each morning dose an Days 2, 5 and 8, Blood samples were also collected 0.25, 0.5, 1, 2, 3, 4.5, 6, 9,12 and 24 h after the final dose (morning of Day 8).

Analytical procedures Plasma and urine concentrations of etizolam were quantified by a modification of an HPLC procedure provided by Yoshitomi Pharmaceutical Industries Ltd. Briefly, 15 gl alprazolam in methanol (1 gg/ml) and 2 N sodium hydroxide 0.1 ml were added to 2 ml plasma or urine. Samples were gently mixed with a Vortex, extracted with 6 ml benzene, shaken for 15 rain and centrifuged. Approximately 5 ml organic phase was transferred to a tube containing 1 N hydrochloric acid 2,5 ml and the tube was shaken for 15 min and centrifuged. The acidic aqueous phase was transferred to a clean tube, made alkaline with 5 N sodium hydroxide 0.4 ml and re-extracted with 6 ml benzene. After centrifugation, 5 ml benzene phase was collected, 2 ml McIlvaine buffer solution (pH 3) was added and the samples were shaken for 15 rain and centrifuged. Approximately 4.5 ml organic phase was evaporated to dryness and the residue was reconstituted in 100gl of the mobile phase. Samples of 90 ~1 were injected into the chromatograph. Analysis was performed in a Beckman system (Beckman Instruments, Inc, Berkeley, CA) equipped with a Zorbax SIL column ( 1 5 c m x 4 . 6 m m I.D.), maintained at room temperature. The mobile phase was methylene chloride-methanol-32% aqueous ammonia solution (959:40:1 v/v) at a flow rate of 1 ml. min-1. Approximate retention times were 6 rain for etizolam and 7 min for alprazolam. Column eluate was monitored at 254 nm. Standard curves ranging in concentration from I (approximately the limit of sensitivity) to 10 ng. ml- ~were linear (r > 0, 99) and were used in the analysis of unknown plasma and urine samples. Intraassay precision was assessed on control plasma and urines spiked with different amounts of etizolam (1-10 ng-ml- t). The precision of the analysis was good at all concentrations and the coefficient of variation (C. V) was less than 3% in all cases. Inter-assay precision was evaluated by analyzing quality control plasma and urine samples containing i and 10 ng-ml-1 etizolam throughout the kinetic study.

The results indicated that the method had good reproducibility, in all cases the C. V being less than 5% at both concentrations. The metabolite c~-hydroxyetizolam could not be measured by the same procedure, because its extraction characteristics and chromatographic behaviour differed. A separate analytical procedure for its determination in human plasma was developed and, when possible, biological samples of the subjects who had taken part in the etizolam kinetic study were reanalysed. After addition of 4-hydroxyestazolain as internal standard, plasma samples were alkalinized with 2 N sodium hydroxide 0.1 ml and then extracted twice with 8 ml benzene-cloroform (80:20). The combined extracts were evaporated to dryness and redissolved in 100 gl of the mobile phase. Samples of 90 gl were injected into the chromatograph. The chromatographic system and the mobile phase was as for etizolam. The flow rate was 1.2 ml. rain- 1. Retention times for the metabolite (z-hydroxyetizolam and the internal standard were 17 and 15 rain, respectively. Standard curves from 2.5 to 10 ng. ml- ~were linear (r > 0.99) and were used in the analysis of unknown plasma samples. Like etizolam, intra-assay C. V was assessed on control plasma spiked with different amounts of the compound and was less than 10% in all cases. The inter-assay C,V of the method was between 10 and 16%. The method has a sensitivity limit of approximately 2.5 ng.ml -I, using 2.0 ml of plasma.

Pharmacokinetic analysis The area under the plasma concentration-time curve from zero to the last plasma concentration, A U C (0-12) was determined by the trapezoidal rule, and extrapolated to infinity, AUC, by a conventional extrapolation method. The terminal slope was determined by a nonlinear least-squares regression program [7], utilizing the data points of the terminal log linear region of the plasma concentrationtime curves. A weighting factor of l/y 2 was employed. The elimination tv2 was determinded from the terminal slope by the usual equation [8]. The observed maximum plasma concentrations (C~x) and the time when they were reached (G~) were taken directly flom the

10

8 o

0.1

0

'

I

3

i

I

6 Time (h)

9

I

I

12

Fig.2. Plasma concentration-time curves of etizolam in healthy subjects after single (0.5 mg) and repeated oral doses (0.5 mg b.d). Results of the first ( • ) and 15th ( [] ) doses are expressed as mean (SD; n=6)

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C. Fracasso et al.: Etizolam kinetics Table 1. Single-dose oral kinetics of etizolam Parameters

Mean (SD) 0.9 (0.7) 8.3 (1.7) 0.9 (0.2)

Time to maximum concentration (h) Maximum concentration (ng-ml ~) Apparent volume of distribution (1)~ Oral clearance (1. h-l)a Elimination half-life (h) Urinary excretion (% of dose)

13.8 (3.5) 3.4 (0.3)

Single and multiple dose pharmacokinetics of etizolam in healthy subjects.

The pharmacokinetics of etizolam, a new thienodiazepine derivative, has been examined after single and multiple (0.5 mg tablet) (0.5 mg b.d for 1 week...
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