BIOPHARMACEUTICS & DRUG DISPOSITION, VOL. 12, 29-35 (1991)

A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE

DETERMINATION OF STOBADIN PHARMACOKINETICS IN SERUM

LADISLAV SOLTES,* ZOLTAN KALLAY, STEFAN BEZEK

Institute of Experimental Pharmacology, Slovak Academy of Sciences, CS-842 I6 Bratislava, Czrchoslo vukiu

AND VIERA FEDELESOVA Clinic of Pharmacotherapy. Institute of National Health, CS-833 08 Bratislava, Czechoslovakia

ABSTRACT

A high-performance liquid chromatographic method was developed to determine stobadin pharmacokinetics in dog and man. The relative bioavailability of stobadin dipalmitate compared with dihydrochloride was 46.4 per cent in dog. In man peak serum concentrations ranged from 12 to 289ngml-I after a single oral dose of stobadin dipalmitate (0.79 to 2.5mgkg-I).

KEY WORDS Pharmacokinetics Bioavailability Stobadin Gamma-carboline High-performance liquid chromatography

INTRODUCTION

Stobadin' (Fig. 1) is a gamma-carboline whose pharmacokinetics in experi- mental animal^^,^ and fate following liver microsomal incubation4 have been investigated in our Institute. Two preparations of stobadin, namely dihydro- chloride and dipalmitate salts, have been used. The dipalmitate form can be administered orally, while the dihydrochloride salt is preferred for intravenous or intraperitoneal administration due to the rather poor solubility of stobadin dipalmitate.

Several methods for stobadin determination in various biological matrices have been described, e.g. MarkoS used a spectrofluorometric analysis of extracts from serum and urine of dogs while Stefek and BeneS4 developed a gas-liquid chromatographic method to study the parent drug as well as two of its metab- olites formed during incubation with liver microsomes. A fluorometric assay of stobadin has been developed in our laboratory with a limit of detection in dog serum of 50ngml-1.6 Different approaches, described by SEasnar and Stefek7 and by Bittererova et ~ l . , ~ , ~ require the use of radioactively (3H) labeled

* Addressee for correspondence.

01 42-2782/9 11'0 10029-O7$05.00 0 1991 by John Wiley & Sons, Ltd.

Received 6 October 1989 Accepted 28 March 1990

30 L. SOLTES, z. KALLAY, S. BEZEK A N D v. FEDELESOVA

stobadin dihydrochloride to measure the unchanged drug by liquid scintillation counting.

This report describes a high-performance liquid chromatographic (HPLC) method developed for the determination of stobadin in serum. Application of the method is reported in which the time-course of stobadin is determined in serum after oral administration of the dihydrochloride and dipalmitate salts. Pharmacokinetic parameters including the relative bioavailability of stobadin dipalmitate are given.

Figure 1 . The structure of stobadin

METHODS

Material and chemicals Stobadin, both as dihydrochloride and dipalmitate salts, as well as the racemic

N-propyl analogue of stobadin, cis-( 5)-2,3,4,4a,5,9b-hexahydro-2-propyl-8- methyl- 1 H-pyrido[4,3-b]indole used as the internal standard, were synthesized at the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. Separcol SIC 18 disposable cartridges, containing 250mg of octadecylsilanized silica sorbent, were produced by the Centre for Chemical Research, Slovak Academy of Sciences, Bratislava, Czechoslovakia. HPLC grade acetonitrile and methanol were obtained from J. T. Baker Chemicals B.V., Holland. Dichloromethane puriss. was from Koch- Light Laboratories Ltd, England. n-Heptane from Loba Feinchemicals, Austria. Triethylamine from Lachema, Brno, Czechoslovakia was distilled and the fraction boiling at 89.4" was used. NaHCO, and K2C03 were purchased from Lachema, Brno, Czechoslovakia.

The carbonate buffer (pH 9.5) was a mixture of 1-3M NaHC03/1.3M K2CO3 = 312 (v/v).

Animal study

Stobadin (dipalmitate or dihydrochloride) was administered orally to a mon- grel dog (lokg) in a dose of 5mg of the drug base per 1 kg of body weight. Blood was withdrawn from the cephalic vein at different time intervals following administration.

HPLC OF STOBADIN IN SERUM 31

Human volunteers

Nine healthy volunteers of both sexes aged 2 3 4 0 years, weighing 50-96 kg participated in the study. All subjects gave their informed consent. Capsules containing stobadin dipalmitate (25 mg of the base) were ingested after over- night fasting along with 200ml of water. The dose administered (cf. Table 2) was contained in 1 to 5 capsules. Blood was withdrawn from the antecubital vein at appropriate time intervals.

Sample treatment

Separcol SIC 18 disposable cartridges were conditioned before use by wash- ing with 2ml of methanol followed by 2ml of water. The sample applied to the cartridge consisted of 4ml of serum to which 2.5pg of the internal standard (dissolved in 25 pl of methanol) and 400 pl of carbonate buffer had been added. After the sample had passed through the cartridge, the octadecyl- silanized silica support was washed with 2ml of water followed by lml of acetonitrile. The acetonitrile was blown off and discarded. The retained mater- ial was eluted with 2.5ml of methanol which was then evaporated under a stream of N2 at ambient temperature. The residue was redissolved in 25pl of CH2C12 and, after centrifugation, a sample ( 1 0 ~ 1 ) was injected onto the HPLC column.

Chromatography

Chromatographic separation was performed in a compact glass cartridge column (3-3 X 150mm) packed with Separon SGX sorbent of 5pm silica par- ticles (Laboratory Works, Prague, Czechoslovakia). The mobile phase was a quarternary mixture of n-heptane/dichloromethane/methanol/triethylamine = 76:19.95:4:0.05 (v/v). The composition of the eluent was achieved by using a Spectra Physics SP 8000 high-performance liquid chromatograph pump sup- plied from three reservoirs of helium purged solvents, i.e. n-heptane, methanol, and 0.25 per cent solution (v/v) or triethylamine in dichloromethane. The elution rate was maintained constant at 0*5mlrnin-* and the column jacket was thermo- stated at 25". Detection was by spectrophotometry (SP 770) at 302nm for high drug concentration or 247nm for the lowest.

Pharmacokinetic analysis

The time-courses of stobadin in the dog were approximated by a function representing a one-compartment open linear pharmacokinetic model with first- order absorption and a time delay between administration and the onset of the drug absorption.

32 L. SOLTES, z. K ~ L L A Y , 5 . BEZEK AND v. FEDELESOVA:

c ( t ) = Pl[e-P2('-7'1agI - e-PXt-L,) 1 The estimation of parameters of the model equation was carried out using an extended least squares nonlinear regression program8 with reciprocal weighting of the data. The pharmacokinetic parameters were calculated using standard methods9 and their standard deviations were obtained using the asymptotic estimates and covariances of the parameters of the applied exponential func- tion.'O

i L

4 t

CI

i Human (blank) Dog

Figure 2. HPLC chromatograms of blank dog serum, blank human serum, and dog serum after administration of stobadin dipalmitate

RESULTS AND DISCUSSION

HPLC analytical method

Stobadin as well as its N-propyl analogue, the internal standard used, were quantitatively removed from serum on octadecylsilanized silica. No peaks at the HPLC elution times of either of these substances were observed in the acetonitrile eluate from Separcol SIC 18; the methanolic eluate contained 100 per cent of both compounds studied. The chromatographic profile of samples prepared with an acetonitrile wash was much smoother than that without aceto- nitrile." Similar behaviour has been observed with other, mainly strong bases on elution from C18-silanized ~ i l i ca .~*- '~ In our opinion, this is due to the effects of the residual silanol groups of the support which alter partition mechanisms by polar as well as cation-exchange processes. Therefore we now prefer HPLC

HPLC OF STOBADIN IN SERUM 33

1001 I I I I 1 1 I 1 I I i 0 120 240 360 48Q mo

lime (min)

Figure 3. Time-course of serum concentrations of stobadin in dog. The dose was 5 mg kg-' of stobadin base administered as dipalmitate (O), or dihydrochloride (0)

Table 1. Pharmacokinetic parameters of stobadin in the d o g

Parameter Stobadin dihydrochloride dipalmitate

t 1 /2 abs (min) elim (min)

lag-time (min) t,,, cm,, (ng m1-7 AUC; (ng min m1-I) CI,,, (ml min-I) Fr,, ("4

24.2 f 2.5 32.0 f 4.6 196.2 f 20.6

2.3 f 5.2 14.7 f 1.2 96.1 f 5.3 100.0 f 7.1

823.2 f 27.2 331827 f 18015

6.99 f 0.37 15.07 f 0.82 46.4

3 12.2 f 28.8

1281.7 f 44.0 715526 f 38254

iliZ abs: absorption half-life; r , / ? elim: elimination half-life; t,,,: time to reach maximal plasma concentration; C,,,: maximal plasma concentration; AUC,": area under the plasma stobadin con- centration-time curve; Cll0,: total body clearance; Frel: ( AUCdipalmi,at~AUCd,hydrochloride) 100.

separation on bare, rather than bonded, silica. Under the experimental con- ditions used the peaks of stobadin and internal standard eluted at 7.2 and 4.6min and both were sharp and symmetrical (cf. Fig. 2). Although the alkaline elution conditions provided relative advantages, detection was less sensitive. Photometric detection at 302nm was satisfactory for relatively high concen- trations of stobadin measured in dog serum; however, the lower wavelength

34 L. SOLTES, z. KALLAY, 5 . BEZEK AND v. FEDELESOVL

Table 2. Concentration of stobadin in human volunteers after administration of different doses

Dose Drug concentration Time interval to Number of (mg kg-l) a t peak (ng m1-I) reach peak subjects

2.23-2.5 45-289 90 min 2 1 '62- 1 '79 82-1 14 50-1 50 min 3 0.79-0.83 12-38 90-1 20 min 3

1 0.27 not detected -

of 247nm improved the limit of detection when stobadin was injected as the pure substance. In samples from human volunteers there was a minor interfering endogenous peak which eluted immediately after stobadin.

At a drug concentration of 500ngml-l the relative standard deviation was 4.4 per cent while at the concentration of 50ngml-' it was 8.1 per cent in dog serum. The lowest detectable drug concentration in human volunteer serum was IOngrnl-'.

Pharmacokinetic analysis

Figure 3 shows the stobadin concentration-time profiles in dog serum after oral administration of dihydrochloride and dipalmitate salts as single doses of 5mgkg-I. A rapid increase in drug concentration after administration of the dihydrochloride salt was followed by a monoexponential decline. Compared with the dihydrochloride, stobadin dipalmitate administration resulted in a lower peak concentration and a lag-time was observed. Table 1 shows the calculated pharmacokinetic parameters for both stobadin salts used. The di- hydrochloride salt is readily absorbed with an absorption half-life of 24 min and peak serum concentration of 1282ngml-I. No significant lag time could be obtained from the data. Following stobadin dipalmitate administration slower absorption was observed with a half-life of 32min and a peak concen- tration of 823ngml-'. The lag-time of 14.7min is most probably related to the poor solubility of the dipalmitate salt in the acidic gastric milieu, absorption starting presumably only after the drug has reached the intestine. The difference in total body clearance for the two salts studied (C1= Dose/AUC) is a conse- quence of the difference in AUCs. The relative bioavailability of stobadin dipal- mitate is 46.4 per cent.

Table 2 shows the peak concentrations of drug in volunteers receiving dif- ferent drug doses. The time to reach the peak ranged from 50 to 150min. The peak drug concentrations reached were dependent on the dose administered (cf. Table 2). The detection limit of the HPLC method, i.e. lOngml-', allowed a complete pharmacokinetic evaluation for only the highest dose used. For lower dose studies an analytical method is needed which will permit sub-nano- gram per millilitre concentrations to be determined.

HPLC OF STOBADIN IN SERUM 35

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