~ @ S ~ e F S Receptor Analysis and Drug Monitoring

B 12

Radioreceptor Assay of Brotizolam in Human Plasma

W. D. Bechtel

Abt. Biochemie, Boehfinger Ingelheim KG, D-6507 Ingelheim, Federal Republic of Germany

Radioreceptorassay f'fir Brotizolam in Humanplasma

Introduction. Brotizolam (We 941, Lendormin <~) is a newly de- veloped highly effective hypnotic [1]. Giving therapeutic doses of 0.25 mg maximum plasma levels of 4 - 6 ng/ml of the unchanged drug were reached [2]. Up to now brotizolam plasma levels in humans were determined using specific methods like radio- activity measurement [2], radioimmunoassay (Bechtel, unpub- lished), and gas liquid chromatography [3-5] . Because broti- zolam shows extremely high affinity to the benzodiazepine (BZD) receptor binding sites a radioreceptorassay (RRA) was developed.

In humans brotizolam is almost completely metabolized to hydroxylated compounds which were excreted in the urine as conjugates only [2]. The main metabolites in humans which occur in a 4:1 relation are hydroxylated at the methyl group of brotizolam (We 964) or at the 3-position of the diazepine ring (We 1061). Both compounds also show a high affinity to BZD binding sites and are pharmacologically active. Thus, not only brotizolam as given drug but also its main metabolites in a considerable portion are determined using the RRA.

Methods. Human plasma (0.5 ml) at pH 8.0 was extracted with ether. The extracts were evaporated to dryness and dissolved again in Krebs-Tris buffer, pH7.4 at 0~ The RRA followed the usual BZD receptor binding procedure [6] using [3H]flunitrazepam as radioactive ligand. A standard assay con- tained 20gl of 12nmole/1 [3H]flunitrazepam corresponding to about 50,000 dpm, 100 gl Krebs-Tris buffer, pH 7.4 at 0~ 920 gl extract of plasma or of brotizolam standards in the same buffer and 960 gl freshly prepared membrane suspension corresponding to 10mg tissue wet weight. Incubations (30min, 0~ were terminated by rapid filtration and washing with ice-cold buffer under vacuum through Whatman GF/B filters. Specific [3H]flunitrazepam binding was defined as total binding minus blank which was obtained in the presence of 10-5 M diazepam. Usual liquid scintillation counting was applied.

Results. The reproducibility of the standard curves during a series of plasma level studies can be seen from Fig. 1. All the curves were established including the extraction of brotizolam from an aqueous buffered solution making unnecessary a factor calcu- lation for plasma level determinations.

The excellent reproducibility can also be seen from the [3H]flunitrazepam binding parameters. The total binding of the tfitiated benzodiazepine was constantly 25.3 +2 .1% of the radioactivity applied to each test tube (mean _+ SD, n : 20). Blanks were always less than 1% of the total binding. Accuracy and precision of the radioreceptor assay can be taken from 1 ng and 10 ng standards running in parallel during all the plasma analyses performed. As a mean of 20 assays over a period of time of 3 months 1.03 + 0.46 ng and 9.57 + 1.52 ng, resp., was found (mean + SD). The intra-assay variation of both, I ng and 10 ng brotizolam, was 11% at the most, the majority was found to be in the range of 2 - 5 % (CV).

The high affinity binding ofbrotizolam and its metabolites to the BZD receptor binding sites is described by these IC 50 values: Brotizolam (We 941) 1.0 nmole/1, Methyl-OH brotizolam (We 964) 1.6 nmole/l, Diazepine-OH brotizolam (We 1061) 4.3 nmole/1.

714

~a ~

100

50

0.1 0.3 1 3 10 30 100 ng 8rotizolam

Fig. I. Mean of 5 brotizolam radioreceptor assay standard curves _+ SD, plotted as B/Bo versus brotizolam concentration. Blank ( = 9 8 d p m ) subtracted, maximum specific binding equalled 11,000 dpm

ng eq./mt Brotizo[em

2O

10

5

2

1

0.5

0'.5 i i �88 6 8 1'0 I I 24 Time after edministretion (h}

Fig. 2. Plasma level of brotizolam and its main metabolites after giving a single oral dose of 0.5 mg. Mean values of 24 volunteers • SD

From the IC 50 values the calculated cross-reactivities are 25 % and 65 %, resp., for the We 1061 and the We 964 metabolite.

A set of samples with a constant amount of 3ng of brotizolam plus 6 different amounts of the main metabolite We 964 (0.3-100 ng) was prepared and analyzed following the procedure described. Theoretical results based on the 65 ~o cross- reactivity of the metabolite We 964 were calculated and com- pared to the measured ng equivalents of brotizolam. The differences between the theoretical and measured results were in the range of 0 -10 ,2 %.

After giving tablets of 0.5 mg brotizolam to 24 healthy male subjects the numerous plasma samples collected were analyzed by RRA. A mean plasma level curve shown in Fig. 2 was obtained. The basic pharmacokinetic parameters determined from the measured data are tma x = 0.5 h (median), Cm~ = 9.5 + 3.8 ng eq./ml and the elimination half-life of 5.7 _+ 2.0 h (mean + SD each).

Discussion and Conclusions. The RRA of brotizolam presented is linear within the region of interest (about 1 to 10 ng per assay tube) and, moreover, up to 100 rig. The detection limit of the assay is about 0.3 ng. Accuracy and precision of standards and of

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standard curves even during a longer period of time are very good. The complete extraction of the main metabolite could be demonstrated. According to their degree of affinity to the BZD receptor binding sites the metabolites of brotizolam are included into the measurement. Thus, the sum of all pharmacologically active substances is determined.

Plasma Cm, x values determined with the RRA were found to be nearly double the plasma concentration determined by specific analytical methods ( 5 - 7 ng/ml). In contrast to this result the half-life is very similar to those determined by specific procedures ( 4 - 7 h). We conclude from this finding that the main meta- bolites were excreted with half-lives which were not very different from that of the parent compound.

Other benzodiazepines, of course, if present in the plasma do interfere with the radioreceptor assay according to their affinity

t o the BZD receptor. We thank Karin Menk and Reinhard Beckhaus for their excellent technical assistance.

References

I. Nicholson AN, Stone BM, Pascoe PA (1980) Brit J Clin Pharmacol 10:75

2. Bechtel WD (1983) Brit J Clin Pharmacol 16:279S 3. Jochemsen R, Wesselman JGJ, Hermans J, van Boxtel CJ,

Breimer DD (1983) Brit J Clin Pharmacol 16:285S 4. Greenbtatt DJ, Locniskar A, Shader RJ (1983) Sleep 6:72 5. Bun H, Ba BB, Coassolo P, Aubert C, Cano JP (1983) Anal

Lett 16:327 6. M6hler H, Okada T (1977) Life Sci 20:2101 7. Gray EG, Whittaker VP (1962) J Anat 96:79

Fresenius Z Anal Chem (1984) 317:714-715 �9 Springer-Verlag 1984

B 13 Analytical Performance of a Mechanized Substrate-Labelled Fluorescent Immunoassay System Evaluated by Therapeutic Drug Monitoring

U. Klotz

Dr. Margarete Fischer-Bosch-Institut ftir Klinische Pharmakologie, AuerbachstraBe 112, D-7000 Stuttgart 50, Federal Republic of Germany

Vergleichende Messungen von Arzneimittelkonzentrationen zur Therapiekontrolle mit Hilfe eines mechanisierten Substrat-markierten Fluorescenz-Immuno-Assays

Introduction

Plasma level monitoring of drugs with a narrow therapeutic index is increasingly performed by many laboratories. In the past mainly GLC, HPLC and different immunoassays have been applied. However, substrate-labelled fluorescence immunoassays (SLFIA) have focused recent interest [1]. Therefore, we tested a new automated SLFIA-system (Optimate/TDA I) and compared its analytical performance to established reference assays for 6 different drugs [2].

Material and Methods

HPLC (theophylline, carbamazepine), GLC (phenytoin, pheno- barbital, valproic acid) and fluometry (quinidine) were used as reference methods. The Optimate-system with its TDA reagents in kit form were kindly provided by Miles/Sparte Ames (Mfinchen, FRG). Quality control samples in the lower thera- peutic and toxic range were purchased from Fisher Diagnostics (Mfinchen, FRG). Therapeutic quality controls (Meditrol) were also obtained from Dr. Jacobsen (Inst. Toxicol. Med. Chem., Singen, FRG). Routine plasma specimen of in- and outpatients on multidrug therapy were used for comparative measurements to test the analytical performance of Optimate/TDA by applying linear regression analysis.

Results

In the lower therapeutic range the within- and between-run precision (coefficient of variation; n = 10) of the reference methods used were for valproic acid 6.5 and 6.6 %, for pheno- barbital 11.4/14.7%, respectively, for phenytoin 1.9/6.2%, for quinidine 3.0/4.2%, for theophylline 7.2/12.5% and for carbamazepine 4.3/8.2%. The corresponding figures for the new Optimate/TDA-system were comparable-phenobarbital: 2.4/2.9 %, phenytoin: 4.5/7.2 %, valproic acid: 2.8/4.0 %, qui- nidine 3.0/22.8%, theophylline: 3.2/4.6%, carbamazepine 4.8/4.7 %. With the Optimate/TDA differently spiked specimens

Table 1. Linear regression analysis for evaluation of analytical performance

Drug Concentration Sample Slope Intercept Correlation Mean range (n) (b) (a), ~tg/ml coefficient of x, ~tg/ml (gg/ml) (reference

assays)

Mean of y, ~tg/ml (Optimate)

Phenobarbital 0 - 43.5 45 0.99 0.75 0.98 19.8 20.3 Phenytoin 0 - 42.8 50 0.97 -0 .35 0.99 8.8 8.2 Quinidine 0 - 4.1 32 0.94 0.11 0.96 2.0 2.0 Theophylline 0 - 37.3 44 1.0 0.43 0.99 9.0 9.4 Carbamazepine 1 .3 - 9.6 44 0.85 a 0.49 0.90 5.5 5.2 Valproic acid 0 - 142 42 1.05 1.04 0.97 54.6 58.5

a Slope significantly (P = 0.05) different from unity; all other values for a and b correspond to the hypothesis a = 0 and b = 1

1 Ames Division Miles, Miinchen, FRG

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