Anal. Chem. 1995, 67,2655-2658
Nonisotopic Receptor Assay for Benzodiazepine Drugs Using TimelResolved Fluorometry Toshifumi Takeuchi,* Takashi Nishikawa,t Ritsuko Matsukawa, and Jun Matsui Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, 151-5Ozuka, Numata-cho, Asaminami-ku, Hiroshima 731-31, Japan
A nonisotopic receptor assay for benzodiazepine drugs using a time-resolvedfluorometric technique is described.
A novel europium chelate-labeledbenzodiazepine (Eu10124) was prepared as a probe ligand for the binding assay based on the competitive inhibition of Eu-1012-S binding by benzodiazepine drugs to their receptors. In this system, the degree of the inhibition was proportional to the aflinity of drugs for the receptor. Background fluorescencedid not interfere with the measurement, due to the long-lifetime fluorescence of Eu-1012-S. The proposed nonisotopic assay may provide an alternative to radioreceptorassays of benzodiazepines in biological systems and could also be used to screen new benzodiazepine-likecompounds drom nature. Receptors are generally classified as membrane-localized proteins with high sensitivity and specificity for their target molecules such as hormones, neurotransmitters, and drugs. These receptors play an important role in triggering or modulating cellular events. The binding of ligands to the receptor induces conformationalchanges of the receptor, resulting in the activation or deactivation of, for example, receptor-coupled enzymes or ionchannels. In many cases, the release of secondary messengers will act as a chemical signal for subsequent signaling systems. The final biological response is achieved by many different mechanisms, such as the induction of translatory activity. In the fields of pharmacology, physiology, neurobiology, and clinical analysis, receptors have been used as analytical tools for bioactive compounds.' Although the receptors used in these tests were isolated and the assays were carried out in vitro, without the elements of the secondary signaling system and the final metabolic control elements of the in vivo system, good correlation has been reported between these in vitro receptor affinity studies and the evaluation of pharmacological activity of drugs by animal tests? Benzodiazepine drugs have been primarily used for the treatment of anxiety and insomnia? and it has been widely accepted that these pharmacological effects are mediated through specific receptors in the central nervous system! In vitro receptor-ligand interactions have been used to investigate the mechanism of the pharmacological effects of benzodiazepines and to screen new drugs with activity similar to benzodiazepine. These ~~
' Department of Clinical Pathology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan. (1) Yamamura, H. I., Enna, S. J., Kuhar, M. J., Eds. Neurotransmitter Receptor Binding, 2nd ed.; Raven Press: New York, 1985. (2) (a) Braestrup, C.; Albrechtsen, R.; Squires, R. F. Nature 1977,269, 7024. (b) Mahlar, H.; Okada, T. Life Sci. 1978,22,985-96. (3) Freeman, H., Rue, Y., Eds. The Benzodiazepines in Current Clinical Practice; Royal Society of Medicine Services: London, 1987. 0003-2700/95/0367-2655$9.00/0 0 1995 American Chemical Society
assays are based upon competitive binding of target compounds with labeled ligands to their receptors. At present, radioisotopelabeled ligands are being routinely used for the receptor-based assays as many radioligands are commercially a~ailable.~ Radioligands have the advantage of simple measurement and, as radiolabeling generally does not change the ligand molecular structure, these ligands have the same binding properties as the parent compounds. However, there are some disadvantages associated with the use of radioisotopes such as limited shelf life and special safety measures for their handling and disposal. Nevertheless, such radioligand receptor assays are frequently performed in laboratories as no feasible nonisotopic assays using drug receptors have been reported. For the development of nonisotopic receptor assays, two factors have to be carefully considered: the label may not significantly reduce the ligand af6nity to the receptor, and at the same time, a highly sensitive measurement method for the labeled ligand must be available, so sensitivity comparable to radioisotopic methods can be achieved. Under consideration of the two factors, we demonstrate a new approach for the development of nonisotopic receptor assays for benzodiazepine receptor binding drugs, in order to improve upon the limitationsassociated with radioisotopic methods. In the present study, we employed time-resolved fluorometry using an europium chelate-labeled ligand. As europium chelates emit long-lifetime fluorescence after excitation with pulsed light, time-resolved fluorometry can be performed without interference from short-lifetime fluorescence of common fluorophores. This technique improves the sensitivityof this type of assays, which has been successfully used for immunological assays and DNA hybridization assays: In this paper we describe the preparation of the novel europium-labeled ligand Eu-1012-S, the evaluation of the Eu-10124 binding properties, and the procedure for the time-resolved fluorometric receptor assay for the determination of benzodiazepine receptor binding drugs. EXPERIMENTAL SECTION
Reagents. Lyophilized benzodiazepine receptor preparations from cow brain were purchased from Research Biochemicals (Natick, MA). A membrane fraction of rat brain homogenate was prepared in the same manner as the previous work? [3H](4) (a) Olsen, R W., Venter, J. C., Eds. Benzodiazepine/GABA Receptors and Chloride Channels; Alan R. Liss: New York, 1986. (b) Muller, W. E. The Benzodiazepine Receptor; Cambridge University Press: Cambridge, U.K, 1987. (c) Squires, R F., Ed. GABA and Benzodiazepine Recepton; CRC Press: Boca Raton, FL, 1988. (5) Bruhwyler, J.; Hassoun, A.J. Anal. Toxicol. 1992,16, 244-52. (6) (a) Diamandis, E. P.; Christopoulos, T. K Anal. Chem. 1990,62, 1149A57A. 01) Hemmil, I.; Haju, R. Bioanalytical Applications of Labelling Technologies; Wallac: Turku, Finland, 1994; Chapter 5.
Analytical Chemistry, Vol. 67, No. 15,August 1, 1995 2655
0 I1
i
CNH(CH,LNHCNH
EU3'
r c o oFigure I. Structure of Eu-10124.
Flunitrazepam was obtained from NEN Research Products (Boston, MA). A ligand for the benzodiazepine receptor, 1012-S,was kindly provided by Shionogi & Co., Ltd. (Osaka, Japan). A europium labeling kit, including a europium labeling reagent, N-(p isothiocyanatobenql)diethylenetriamineN,N,",M'-te!macetic acid, was purchased from Wallac (Turku, Finland). Microtiter plates were obtained from Sumitomo Bakelite (Tokyo, Japan). Other chemicals of analytical grade were purchased from commercial sources and used without further purification. Preparation of Eu-1012-S. Eu-1012-S (Figure 1) was prepared by mixing 0.2 mg of the europium labeling reagent with 0.7 mg of 1012-S in 500 pL of 50 mM carbonate buffer @H 9.0). After incubation overnight at room temperature, the product was purified by an HPLC system including a Hitachi pump L6200 (Tokyo, Japan), a Rheodyne sample injector Model 7125 with 1 mL sample loop (Cotati, CA), a Tosoh column of Phenyl-5PW (75 mm x 7.5 mm id., Tokyo, Japan) with an eluent of 50 mM carbonate buffer @H 9.0,l.Z mL/min), and a Hitachi UV detector L3000. Alkaline conditions were employed during the labeling procedure and the HPLC purification to prevent the dissociation of Eu3+ions from Eu-1012-S. Purified Eu-1012-S was stored in the dark at 4 "C. Concentrations of Eu- 1012-S were estimated spectrophotometrically by comparison with an absorption spectrum of unmodified 1012-S. Calculations were based on an apparent molar extinction coefficient of 1012-S at 270 nm of 1.1 x lo4 M-' cm-'. Binding of Eu-1012-S to the Benzodiazepine Receptor. Mixtures of the receptor suspension (400 pL) prepared by mixing 400 mg of the lyophilized receptor preparation with 8 mL of 25 mM phosphate buffer at pH 7.4 (PB), 50pL of Eu-1012-S (0.94120 nM), and 50pL of PB were incubated in 1.5 mL polypropylene microcentrifuge tubes for 1 h at 4 "C. After centrifugation at 20600g for 15 min at 4 "C (Sakuma Model M 150-W, Tokyo, Japan), the supernatants (2OpL) were pipeted into microtiter plate wells, to each of which Enhance Solution (200 pL, Wallac Oy, Turku, Finland) was then added in order to enhance and stabilize the fluorescence. After shaking for 5 min on a microtiter plate shaker (Model 1296002, Wallac Oy) , time-resolved fluorescence were measured with the Delfia fluorometer (Model 1232, Wallac Oy). To estimate nonspecific binding of Eu-1012-S to the receptor, a large excess of unlabeled ligand 1012-S (50pL, 10p M in PB) was added to the mixture of the receptor suspension and Eu-10124 instead of 50 pL of PB. The specific binding was estimated by the difference in signals corresponding to the incubation mixtures with (the nonspecific binding) and without (7) Nishikawa, T.; Nishida, A; Ohtani, H.; Sunaoshi, W.; Miura, H.; Sudo, Y.: Kubo, H. T h e y . DrugMonit. 1989,1 2 , 483-6. 2656
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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Initial Eu-1012Sconc., nM Figure 2. Binding properties of Eu-1012-S to the benzodiazepine receptors. (@) specific binding; (0)nonspecific binding. Mixtures of the receptor suspension in 25 mM phosphate buffer at pH 7.4 (400 pL, 50 mg/mL), Eu-10124 (50 pL, 0.94-120 nM), and either the phosphate buffer or 10 pM 10126 in the phosphate buffer (50 pL) were incubated for 1 h at 4 "C.After centrifugation, the supernatant (20 pL) was taken, and the time-resolved fluorometric measurement was performed as described in the Experimental Section.
(the total binding) a large excess of the unlabeled ligand. The equilibrium dissociation constant (KD)was estimated by Scatchard analysis. Two independent receptor binding experiments were performed, and each sample was analyzed in duplicate. In the measurement procedure, excitation light (340 nm) was flashed at 1 ms intervals, and fluorescence detected at 615 nm was integrated over the period beginning at 400 ps and ending at 800 ps after the excitation flash was extinguished. This process was repeated lo00 times, and the summation of all measurements was recorded. CompetitiveBinding of Eu- 10124 andTritium-Labeled Flunitrazepam. Binding experiments of Eu-1012-S and tritiumlabeled flunitrazepam ( [3H]flunitrazepam) to the receptors were performed. Eu-1012-S (31 nM-5 mM, 50 pL) was added to the suspension of the membrane fraction of rat brain in 67 mM phosphate buffer at pH 7.4 (400 pL) and then [3Hlflunitrazepam (40 nM, 50 pL) was added to the mixture. After a 2 h incubation at room temperature, the suspension was filtered through a GF/C glass fiber membrane (Whatman International Ltd., Maidstone, England). The membrane was washed with the phosphate buffer and then [3Hlflunitrazepam on the filter was counted by a liquid scintillation counter LS2800 (Beckman Instruments, Fullerton, CA). To estimate the nonspecific binding, a large excess of unlabeled ligand was added. Two independent competitive binding experiments were performed, and each sample was analyzed in duplicate. Time-ResolvedFluorometric Receptor Assay for Benzodiazepines. Mixtures of the receptor suspension (50 mg/mL in PB, 400 pL), Eu-1012-S (30 nM in PB, 50 pL) and benzodiazepine drugs (10-5-10-12 M in PB, 50 pL) were incubated in 1.5 mL microcentrifuge tubes for 1 h at 4 "C. The measurement procedure was in the same manner as described before. Two to four independent receptor assays were performed, and each sample was analyzed in duplicate. RESULTS AND DISCUSSION
Binding Characteristicsof Eu-1012-Sto the Benzodiazepine Receptor. A typical binding profile of Eu-1012-S to the benzodiazepine receptor is shown in Figure 2. The specific binding of Eu-10126, calculated by subtracting nonspecific binding from total binding, showed a saturable profile, while the
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1
" 0
i
1 0 2 0 3 0 4 0 5 0
Eu-1012sbound, pmol g-' Figure 3. Scatchard analysis of the Eu-10124 binding. According to the Scatchard equation, B/F = S,,, - WKD, the dissociation constant (KD)was estimated to be 1.2 nM by using the data in Figure 2, where B is the amount of specific binding of Eu-10124, F i s the concentration of unbound Eu-10124, and B,,, is the apparent maximum number of binding sites.
nonspecific binding increased linearly with the initial concentration. This suggests that the binding sites for Eu-10124 were limited and that, up to the initial concentration of 6 nM, Eu-1012-S bound to the specific binding sites rather than to the nonspecific binding sites. The nonspecific binding was -27% of the total binding at the initial Eu-10124 concentration of 0.75 nM. The degree of the nonspecific binding may be changed, depending upon the purity of the receptor preparation used. The equilibrium dissociation constant (KD)of the Eu-1012S-receptor complex was estimated by Scatchard analysis (Figure 3). From the slope, a KDvalue was calculated to be 1.2 nM under the given experimental conditions. This figure is -7 times less than that of the previously reported fluorophore-labeled ligand, N-(7-amino-4-methylcoumarin-2-acetyl) Ro7-1986/1 (8.6 nM),&and comparable with [3Hlflunitrazepam (1.1 nM) .g The reasonably high aflinity of Eu-10123 to the benzodiazepine receptor indicates that Eu-10123 is well suited to fulfill the requirements for a competitive receptor assay. Competitive binding experiments of Eu-1012-S with t3H1flunitrazepam were performed in order to determine the binding sites on the receptors. The binding of [3Hlflunitrazepam was competitively inhibited by the addition of Eu-1012-S in the range of 10-9-10-6 M (Figure 4). Although the pharmacological properties of Eu-1012-S have not been examined, these results suggests that Eu-10124 binds to the same pharmacologically relevant sites as flunitrazepam. The parent ligand, 10124, offers a number of advantages in the development of the time-resolved fluorescence receptor assay, because 1012-Swas reported to have a high displacement potency for the binding of [3Hlflunitrazepamto solubilized benzodiazepine receptors, with an IC50 value of 60 pM in the presence of 1 nM [3Hlflunitrazepam,10 meeting the requirementfor the development of a nonisotopic assay. Moreover, 1012-S has a primary amino group which can be reacted easily with a phenylisothiocyanate group of the commercially available europium labeling reagent, facilitating preparation of the europium chelate-labeled ligand. (8) (a) Takeuchi, T.; Rechnitz, G. A Anal. Biochem. 1991,194,250-5. (b)
Velazquez, J. L.; Thompson, C. L.; Barnes, E. M., Jr.; Angelides, K. J. J. Neurosci. 1989,9, 2163-9. (c) McCabe, R. T.; De Costa, B. R; Miller, R. L;Havunjian, R H.; Rice, K. C.; Skolnick, P.FASEE]. 1990,4,2934-40. (d) Havunjian, R. H.; De Costa, B. R.: Rice, K C.; Skolnick, P.J. Biol. Chem. 1990,265, 22181-6. (9)Braestrup, C.; Nielsen, M. In Handbook of Psychophamacology; Iversen, L. L., Iversen, S. D., Snyder, S. H., Eds.; Plenum: New York, 1983;Vol. 17, pp 285-384. (10) Taguchi, J.; Kuriyama, K. Brain Res. 1984,323,219-26.
Log[Eu-l012S,MI Figure 4. Competitive binding of Eu-10124 with [3H]flunitrazepam to the receptor. [3H]Flunitrazepam (40 nM, 50 pL) and Eu-10124 (31 nM-5 mM, 50 pL) were added to the receptor prepared from rat brain homogenate in the phosphate buffer (400 pL). After the incubation, the suspension was filtered and the [3H]flunitrazepamon the filter was counted.
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Log [Benzodiazepine, MI Figure 5. Inhibition of the Eu-10124 binding by benzodiazepine drugs: (1) triazolam; (2) midazolam; (3) flunitrazepam; (4) diazepam; (5) estazolam; (6) nitrazepam. Mixtures of the receptor suspension (50 mg/mL, 400pL), Eu-10124 (30 nM, 50pL), and benzodiazepine drugs (10-5-10-12 M, 50 pL) were incubated for 1 h at 4 "C. The measurement procedure was described in the Experimental Section. Percent inhibition is calculated as 1OO(x - yj/(z - yj. The value x represents the relative fluorescence intensity obtained. The value y represents the signal obtained from the reaction mixture of the receptor preparation containing 3 nM Eu-1012-S. The value z represents the signal obtained from the reaction mixture of the receptor preparation containing 3 nM Eu-10124 and 1 pM 1012-S. Consequently, values calculated by (x - yj express specific binding of Eu-10124 decreased by the addition of the competitive binders, and a value calculated by (z - yj expresses a maximum displaceable binding of Eu-1012-S.
Time-ResolvedFluorometricAssay for Benzodiazepines. The binding properties of Eu-10124 were examined in the presence of benzodiazepine drugs that are known to be receptor binders. The binding of Eu-10124 was inhibited by benzodiw epines tested with an order of potencies proportional to their pharmacological response (Figure 5). The results showed the same trend as those obtained by using [3Hlflunitrazepam.11 Regarding the reproducibility, repetitive assays were performed for 10124. A typical value of coefficient of variation was calculated to be 5% (6 nM, n = 4). These competitive binding phenomena could be applied as a nonisotopic binding assay for the analytical evaluation of benzodiazepine receptor binding drugs. The major advantage of receptor assays is to detect any substances with benzodiazepine(11) (a) Lund, J. Scand. /. Clin. Lab. Inoesf., 1981,41,275-80. @) Dorow, R G.; Seidler, J.; Schneider, H. H. Br. J Clin. Phamacol. 1982,13,561-5. (c) Nishikawa, T.; Sumki, S.; Ohtani, H.; Eizawa, N. W.; Sugiyama, T.; Kawaguchi, T.; Miura, S. Am. J. Clin. Puthol. 1994,102,605-10. Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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like activity, due to the recognition of all therapeutically active benzodiazepines and their metabolites by the receptor. The presented europium chelate label assay could substitute radioisotope assays for the determination of benzodiazepines and the discovery of new compounds with biological activities for GABAbenzodiazepine receptor complexes. Additionally, the assay system is simpler and safer than conventional radioreceptor assays. Because the commercially available benzodiazepine receptor preparations are only partially purified, their supernatants exhibit strong background fluorescence and may interfere with the measurement of fluorophore-labeled ligands. Therefore, in this study, we did not employ common fluorophores, but a europium chelate was chosen as the label. Europium chelates have unique fluorophore properties such as (1)larger Stokes shifts preventing scattering interference, (2) a narrow emission spectrum at 615 nm with no overlap of most other naturally occurring fluorophores, and (3) long fluorescence lifetime enabling the application of microsecond time-resolved fluorometry. These properties are suitable for the determination of europium chelates in complicated matrixes with background fluorescence which is always problematic in fluorometry. In practice, time-resolved fluorometry seems well suited for the measurement of Eu-1012-S in the supernatant of receptor preparations, due to the low level of background fluorescence, which was approximately double the intensity of empty microtiter wells.12
Europium chelate labeling has been a successful improvement of our previously reported nonisotopic benzodiazepine receptor assays in which either fluorophore-labeledE or biotin-labeled ligands was used.I3 In the fluorophore-labeled ligand system, the endogenous fluorophores in the receptor preparation interfered with the measurement, making pretreatments necessary to eliminate the interferences88 In the biotin-labeled ligand system, although the affinity of the ligand (0.3 nM) was higher than that of Eu- 1012-S,the measurement procedure was time-consuming and tedious due to the use of an enzymatic method and the avidin-biotin interaction.13b The combination of longlifetime fluorescence of Eu-1012-S and the time-resolved measurement as presented in this paper allows both sensitive measurement and establishment of a simple assay procedure: single-step incubation, centrifugation, and subsequent time-resolved fluorescence measurement.
(12) Takeuchi, T.;Yoshida, M.; Kabasawa, Y.; Matsukawa, R; Tamiya, E.; Karube, I. Anal. Lett. 1 9 9 3 , 26, 1535-45. (13) (a) Takeuchi, T.; Tanaka. S.; Rechnitz, G. A. Anal. Lett., 1991,24,211121. (b) Takeuchi. T.; Tanaka, S.;Rechnitz, G. A. Anal. Biochem. 1992,203, 158-62. (c) Tanaka, S.; Takeuchi, T.; Rechnitz. G. A.J. Chromatogr. 1 9 9 2 . 597,443-8.
Received for review March 7, 1995. Accepted May 22,
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ACKNOWLEWMENT
The authors gratefully acknowledge Shionogi & Co., Ltd. (Osaka, Japan) for providing 1012-S. We also thank Dr. Otto Doblhoff-Dier, a visiting scientist from the Institute for Applied Microbiology at the University of Agriculture (Vienna, Austria) for helpful discussion. The present work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “New Development of Rare Earth Complexes” (06241219) from the Ministry of Education, Science and Culture.
1995.B AC9502312 Abstract published in Advance ACS Abstracts, July 1, 1995.
