European Journal of Clinical Pharmacology © by Springer-Verlag 1979

Eur. J. Clin. Pharmacol. 16, 141-147 (1979)

Pharmacokinetics of Prazepam in Man M. T. Smith 1, L. E. J. Evans 2, M. J. Eadie 1, and J. H. Tyrer 1 ~Departmentof Medicineand 2Departmentof Psychiatry,Universityof Queensland,ClinicalSciencesBuilding,RoyalBrisbaneHospital, Brisbane, Australia Summary. An original electron-capture gas chromatographic assay was developed for simultaneous measurement of plasma levels of the benzodiazepine derivative prazepam and of its principal unconjugated metabolite, N-desmethyldiazepam. The assay was used to study the pharmacokinetics of the drug and its comparative bioavailability from tablets and from a specially prepared solution. Nine healthy adult volunteers were studied. Each volunteer on one occasion took 30 mg of the drug in tablet form, and on another occasion 30 mg of the drug in solution. In all subjects, N-desmethyldiazepam appeared in plasma shortly after prazepam appeared and reached a peak within four hours of prazepam ingestion. Thereafter plasma N-desmethyldiazepam levels were much higher than plasma prazepam levels throughout. Prazepam became undetectable within six hours of intake, whereas its metabolite could still be measured in plasma fourteen days after dosage. Thus much of the pharmacological action of prazepam may be mediated through its metabolite, N-desmethyldiazepam. In five of the nine subjects, areas under the plasma level curves for the metabolite were not markedly different for the tablet and solution formulations studied. In the other four subjects the area under the curve for the tablets was 50% to 80% of the area under the curve for the solution. The time to reach peak plasma level for the metabolite was shorter after the solution formulation (mean 2.0 + SD 1.2 h) than after the tablet formulation (mean 4.2 _+ SD 1.7 h).

Key words: prazepam, N-desmethyldiazepam; bioavailability, pharmokinetics, electron-capture gasliquid chromatography

Prazepam is a member of the 1,4-benzodiazepine group of drugs. It has an N-l-cyclopropylmethyl group in place of the N-l-methyl group of diazepam. It was suggested by earlier workers (di Carlo et al., 1970; Vesell et al., 1972) that the presence of the cyclopropytmethyl group on the molecule at position 1 hindered the metabolism of the molecule at this point. Prazepam and diazepam form a common metabolite (N-desmethyldiazepam) by N-dealkylation at the N-1 position (Fig. 1). N-desmethytdiazepam is the major metabotite which occurs unconjugated in plasma (di Carlo et al., 1970). Two pharmacokinetic studies on prazepam in man have been published (di Carlo et al., 1970; Vessel et al., 1972). In both studies prazepam was assayed by total radioactivity counting in conjunction with thin-layer chromatography. Since these previous studies on prazepam employed a potentially nonspecific method, it was decided to carry out further pharmacokinetic investigations on the drug after developing a more specific gas chromatographic assay. Since the completion of the present pharmacokinetic investigations, Greenblatt and Shader (1978) have published the outline of a study in which prazepam was measured by gas-liquid chromatography. Materials and Methods

Analytical Method A gas-liquid Chromatographic (GLC) assay for prazepam was developed. The assay employed flunitrazepam as the internal standard, since this substance was chromatographically resolved from diazepam, prazepam and N-desmethyldiazepam in a reasonable time. 0031-6970/79/0016/0141/$01.40

142

M.T. Smith et al.: Pharmacokinetics of Prazepam

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HYDROXYLATION C! "

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N-DEALKYLATION

H

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N- DESMETHYLDIAZEPAM

OXAZEPAM

Fig. 1. Biotransformation of prazepam as proposed by di Carlo, Viau, Epps and Haynes (1970)

1 "- D I A Z E P A M 2 =

PRAZEPAM

3 = N-DESMETHYLDIAZEPAM 4 =

FLUNITRAZEPAM

3 2

preparation." A sample of plasma (0.1 ml-2 ml, containing the drugs to be assayed) was added to a screw-cap tube. To this was added 1 ml of borate buffer (pH = 9). The contents were then mixed by vortexing for approximately ten seconds. Ten ml of diethyl ether were added and the tube shaken for thirty minutes. The ether layer was then transferred to another tube by pasteur pipette, and evaporated to dryness under a stream of nitrogen. The dry residue was reconstituted in 0.1 ml of hexane acetone (80: 20) mixture and 5 gl of this final solution was injected into the gas chromatograph. The gas chromatograph used was a Varian 2100 model equipped with a Scandium 3H electron-capture detector. The operating temperatures were: column (250°C); injector (285°C) and detector (300 ° C). The nitrogen flow rate was 35 ml min-1. The column was packed with 3% OV-225 on gaschrom Q (A. W. 80-100 mesh). The four drugs were eluted from this column within 12 min (Fig. 2). The electron-capture detector response was linear over a limited range of drug concentration only. Calibration of the assay was performed using peak height ratios i. e. Height of Drug Peak RH

Height of Internal Standard Peak

These ratios were plotted against drug concentration. The calibration curves were linear over the concentration range 0-100 ng" m1-1 for both prazepam and N-desmethyldiazepam. The minimum detectable concentration of prazepam and of N-desmethytdiazepam was 2.5 ng" m1-1, using a i ml plasma sample. The calibrations were performed using triplicate samples of blank plasma to which prazepam and Ndesmethyldiazepam had been added in six concentrations. The coefficient of determination for the line of best fit through the N-desmethyldiazepam data was 0.990 and that for the prazepam data was 0.988. A set of three standards was run on the gas chromatograph every day and all plasma samples were assayed in duplicate. The analysis of six plasma samples to which 100 ng. m1-1 of N-desmethyldiazepam and of prazepam were added gave recoveries of 101.6 _+ 6.5 ng. m1-1 for N-desmethyldiazepam and 101.9 _+ 5.1 ng" m1-1 for prazepam. Similarly the recoveries of N-desmethyldiazepam and prazepam from six plasma samples to which 10 ng- m1-1 of these drugs had been added were 10.6 + 1.2 ng • m1-1 and 9.6 _+ 1.2 ng. m1-1 respectively.

Pharmacokinetic Studies 12 m i n

Fig.2. Gas chromatogramillustratingretention times of various benzodiazepines

After a 12 h fast, nine healthy, young adult volunteers, whose informed consent to the study had been

143

M.T. Smith et al.: Pharmacokinetics of Prazepam

obtained in writing, were each given 3 0 m g of prazepam in a specially prepared solution (Table 1). None of the subjects had hepatic, cardiac or renal abnormalities. Personal details of the subjects are shown in Table 2. Venepunctures were performed at 0.5, 1.0, 1.5, 2.0, 3, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 192, 240, 288 and 336 h after dosage administration. The plasma was immediately separated and frozen until the time of assay. One month later, the same nine volunteers were each given a 30 mg dose of prazepam (3 × 10 mg tablets)L Venepunctures were performed as on the previous occasion. As this study was a subsidiary part of another investigation, it was not possible to divide the subjects into two groups such that half the subjects took each preparation at each time of study.

Method of Data Analysis In two subjects the elimination of prazepam appeared possibly to be biexponential. In the remaining seven of the nine subjects, the distributive phase appeared so rapid that the decline in prazepam levels was adequately described by a one-compartment model. For the one-compartment model

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where A U C is the area under the plasma level-time curve.

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Table 2. Personal details of the subjects studied Subject

Sex

Age (y)

Weight (kg)

Concurrent Drugs

J.P. A.D. M.D.

M M F

22 30 28

70 83 63

J.A.

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22

60

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60 56 86 95 57

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the administered drug dose ka = absorption rate constant (first-order) D 1 -----CIV I = amount of drug in the central compartment concentration of drug in the central compartment C1 = V1 --= the volume of the central compartment c2 = concentration of drug in the peripheral compartment D 2 = C2V 2 = the amount of drug in the peripheral compartment the apparent first-order intercompartmental disk21 and k12 tribution rate constants, and klo is the apparent first-order elimination rate constant from the central compartment a a n d fl = complex hybrid rate constants, each influenced by all of the rate constants of the system. a = 1/2 {(k12 + k2t + kio) + (kt2 + k21 + klo) 2 - 4k12klo} /3 = i/, {(kl 2 + k21 + k10) _ (k12 + k21 + k10)2 _ 4kt2klo} Now,

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where A = IzD = quantity of dose absorbed (where F is the bioavailability and D is the dose administered) ka = absorption rate constant Ab = amount of drug in the body Vd = apparent volume of distribution k = elimination rate constant (metabolism, excretion) Ae = amount of drug eliminated Since In% = lnCp° - kt, a plot of InCp versus time is linear with slope = - k and a y intercept of lnCp°. The elimination half-life (tv2) can be calculated from the relation: tv2 = 0.693/k. The apparent volume of distribution, Vd, may be determined from the calculated values for the area under the plasma level - time curve and from the elimination rate constant.

vd= - -

Table 1. Formulation of the specially prepared prazepam solution

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+

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+

144

M.T. Smithet al.: Pharrnacokineticsof Prazepam

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320

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Fig. 3. Semilogarithmicplots of prazepam and n-desmethyldiazepamplasmalevels versus time 60

(a)

ORAL

A t s o m e t i m e f o l l o w i n g a d m i n i s t r a t i o n , t h e t e r m s , e -k~t a n d e -at a p p r o a c h z e r o a n d s i n c e D I = C1V1 t h e n C 1 = D 1 / V l Thus

SOLUTION

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F i g . 4. T i m e c o u r s e o f m e a n u n c h a n g e d p r a z e p a m p l a s m a levels a f t e r 3 0 m g p r a z e p a m d o s e s , g i v e n as t a b l e t s a n d a s a s o l u t i o n

It would appear that the absorption rate constant of prazepam was sufficiently close to a in seven of the nine subjects studied for the distributive phase not to be observed following first-order input. Thus in these seven subjects, the two-comparlment kinetics may be reduced to one-compartment kinetics as described above (Wagner, 1971). Area under the plasma level - time curves was determined by trapezoidal rule integration to t hours, and AUC? by dividing Ct by k or fl (Gibaldi and Perrier, 1975). The elimination rate constant, k, or the hybrid constant 13, were estimated by performing a least squares linear regression on the data from the terminal linear portion of the semilogarithmic plasma level - time curve plot. Other pharmacokinetic parameters were calculated as above. The terminal half-life (tl/2) of N-desmethyldiazepam was calculated from the slope of the terminal log Cp versus time plot. The terminal phase was usually reached within approximately 24 h from dosing./3, the disposition rate constant of N-desmethyldiazepam, is equal to the quotient of 0.693 and the tl/2 of N-desmethyldiazepam. If one assumed 100% bioavailability of prazepam and total conversion of this prazepam to Ndesmethyldiazepam, and assumes that no metabolite is forming during the period over which/3 is calculated, several pharmacokinetic parameters for the metabolite may be calculated, The total plasma clear-

M. T. Smith et at.: Pharmacokinetics of Prazepam

145

Table 3. Pharmacokinetic and Bioavailability Parameters for Prazepam and derived N - d e s m e t h y l d i a z e p a m Parameter

Tablet Measured Cpr~x ( n g . rnl q ) tmax (h) fl or kdim (h -1) tv: (h) V d (1 k g -1) C1 (1 k g ~ h -~) A U C ratios

N-Desmethyldiazepam

Prazepam

6.6