Pii: s0928-0987(97)00082-

European Journal of Pharmaceutical Sciences, 6 (1998) 219–223 Binding kinetics of budesonide to the human glucocorticoid receptor Institute of Pharmaceutical Chemistry, Westf ¨alische Wilhelms-U Munster, Hittorfstr. 58-62, 48149 Received 10 January 1997; accepted 10 July 1997 Abstract
Glucocorticoid receptor-ligand binding kinetics of budesonide, a glucocorticoid used for inhalation therapy, were determined and compared with dexamethasone and fluticasone propionate using glucocorticoid receptors from human lung tissue. From the association resulted the equilibrium dissociation constant KD of 1.32 nmol / l and a relative receptor affinity of 855 with dexamethasone as reference (100). The half-life of the budesonide-receptorcomplex was 4.6 h. Results agree with data derived from competition tests we performed earlier.
Keywords: Budesonide; Glucocorticoid receptor; Binding kinetics; Relative receptor affinity 1. Introduction
ternational plc (UK). [ H]Fluticasone propionate and [ H]budesonide as well as fluticasone propionate and The binding of glucocorticoids to the glucocorticoid budesonide were a generous gift from Glaxo GGR (Green- receptor and the formation of a receptor complex is a ford, UK). Radiochemical purity was demonstrated by prerequisite for their pharmacological and therapeutical thin-layer chromatography. Scintillation counting was per- formed with a scintillation beta counter, Rackbeta 1219 A more precise study of the interaction of glucocor- LKB (Germany) using Aquasafe 500 plus from Zinnser ticoids with their receptors is achieved with a kinetic analysis than with an equilibrium analysis, because the ethyl)-benzenesulfonyl fluoride hydrochloride) was pur- time to reach equilibrium in competition assays is in- chased from Boehringer Mannheim (Germany), DL-dithio- fluenced by (i) the concentration of the receptor, (ii) threitol (DTT) from Sigma Chemie (Deisenhofen, Ger- concentration of [ H]glucocorticoid, (iii) concentration of many), Norit A from Serva (Heidelberg, Germany). The the competitor, (iv) temperature and (v) by the differences other chemicals were obtained from E. Merck (Darmstadt, in affinities of the different glucocorticoids to the receptor.
Germany). Buffer A contained 10 mmol TRIS, 10 mM Therefore, in the present study the binding kinetics of NaMoO , 30 mM NaCl, 1 M glycerol, 4 mM DTT and 1 [ H]budesonide and [ H]dexamethasone were compared to determine the relative receptor affinities in addition tocompetition assays. Studies were performed on the glucocorticoid receptor in the cytosolic fraction fromhuman lung tissue. Binding kinetics of fluticasone propion- Peripheral lung tissue was obtained from a patient ate were included for comparison using the same lung undergoing lung resection due to lung cancer. The patient had no treatment with glucocorticoids at least 4 weeksbefore lung resection. The part of the lung lobe used forour investigations was normal lung tissue. The tissue was 2. Experimental
deep frozen immediately after resection and stored inliquid nitrogen; preparation of cytosol from this human [ H]Dexamethasone was obtained from Amersham In- lung tissue was performed as described previously ( ¨ Protein concentration of the cytosol was measured using Corresponding author. Fax: 149 251 8333310; e-mail: hogger@uni-  1998 Elsevier Science B.V. All rights reserved.
P I I : S 0 9 2 8 - 0 9 8 7 ( 9 7 ) 0 0 0 8 2 - 1 N. Esmailpour et al. / European Journal of Pharmaceutical Sciences 6 (1998) 219 –223 For the assay of non-specific binding, 10 parts of diluted cytosol, 1 part of [ H]glucocorticoid 1.2310 The affinity of a glucocorticoid to its receptor is 1 part of the respective non-labelled glucocorticoid 1.23 determined by the constants for the association step k and mol / l were mixed in polyethylene tubes and incu- bated at 208C. The assay for total binding was carried out in the same manner, but the part containing the non- labelled glucocorticoid was replaced with buffer A without were determined according to the method of For determination of the total [ H]glucocorticoid con- Baxter (Baxter and Tomkins, 1971). Knowing k and k centration [G], 20 m l of the above mentioned mixtures for the equilibrium dissociation constant K non-specific or total binding, respectively, were transferred into scintillation vials and counted.
At time intervals, 200 m l incubation mixture were mixed with 200 m l suspension of Norit A, incubated for 10 min on ice and centrifuged for 5 min at 0–48C. Two hundred ml of the supernatant were used for scintillation counting.
glucocorticoids (G) the relative receptor affinity of G maybe calculated: 2.4. Determination of the dissociation constant The constant for dissociation of the glucocorticoid-re- ceptor complex may be determined by measuring the 2.3. Determination of the association constant decrease of the concentration of the [ H]glucocorticoidbound to the receptor as a function of time, if no Following Rodbard (Rodbard et al., 1968) Eq. (4) reassociation of the complex occurs. Reassociation is enables the determination of k , if the dissociation may be prevented by adding a 500-fold surplus of non-labelled neglected because of a very slow dissociation rate constant glucocorticoid (Schaumburg, 1972). Under this condition Eq. (2) describes the first order reaction: Z 5 ]]]] 5 k ? t 1 ]]]] In a semi-logarithmic graph the concentration of re- Knowing the concentration of the free glucocorticoid at ceptor bound [ H]glucocorticoid is plotted against time, and the concentration of receptors without bound glucocorticoid [R ] as well as the concentration of Ten parts cytosol and 1 part [ H]glucocorticoid solution mol / l were incubated for 18 h at 0–48C (mixture of free receptor [R ] at time zero, a graph of Z vs. t 1). For the determination of non-specific binding, 10 parts enables us to calculate k from the slope of the resulting of cytosol, 1 part of [ H]glucocorticoid solution 6310 Cytosol from human lung tissue was incubated at 208C incubated for 18 h at 0–48C (mixture 2). Mixture 1 was with the [ H]glucocorticoid and at different times up to 1 mixed with one part of glucocorticoid 3310 h, total binding [B ] and non-specific binding [B at intervals 200 m l of the mixtures 1 and 2 were mixed determined. Knowing the used concentration of the with 200 m l Norit A suspension, incubated at 0–48C for 10 [ H]glucocorticoid [G] and the receptor concentration of min and thereafter centrifuged for 5 min at 48C. The the cytosol [R ] all parameters necessary for the calcula- supernatant was used for scintillation counting.
Experiments for dexamethasone and budesonide were performed in duplicate, the repetition of the kinetics offluticasone propionate binding was a single experiment.
For all experiments the same human lung tissue from one [R ] 5 [R ] 2 [B ] 1 [B Following the method of Toft and Gorski (1966) cytosol 3. Results
[ H]glucocorticoids alone and in the presence of an excess The time course of specific binding of budesonide N. Esmailpour et al. / European Journal of Pharmaceutical Sciences 6 (1998) 219 –223 Fig. 1. Association of glucocorticoids to the glucocorticoid receptor of the human lung at 208C. The data from the two experiments for budesonide (♦, m)and dexamethasone (j, ♦) have been combined in the figures. The data for fluticasone propionate (h) are from a single experiment.
indicated that the rate of association with the glucocor- plex of budesonide is more stable than the complex of the ticoid receptor was not as fast as for fluticasone propionate, but faster than that of dexamethasone (Fig. 1). However, Half-lives of the receptor complexes can be calculated the total amount bound after 1 h was not different between from the slopes of the linear graphs as t 5ln2 /k. The budesonide and fluticasone propionate, whereas specific half-life of the budesonide glucocorticoid receptor complex binding for dexamethasone was only approximately one (4.6 h) is 60% shorter than the half-life of the fluticasone third of the bound fraction of the two other glucocor- propionate-receptor complex (7.7 h). Again, the values of ticoids. The faster binding of fluticasone propionate and the half-life of the receptor complexes for dexamethasone budesonide is reflected in the association constants: k and fluticasone propionate agree well with our previous fluticasone propionate 27.4310 , k values for dexamethasone and fluticasone propionate agree 3.3. Equilibrium dissociation constant and relative well with our previous determinations (Table 1; The equilibrium dissociation constant K was calculated dissociation K for budesonide, fluticasone propionate and Dissociation of glucocorticoids from the receptor com- dexamethasone were 1.32, 0.55 and 11.25 nmol / l (Table plex follows first order kinetics (Fig. 2). Budesonide 1). The values for dexamethasone and fluticasone propion- ate are close to our previous calculations of 0.49 and 9.36 Hogger and Rohdewald, 1994). The relative , respectively), but the glucocorticoid receptor com- receptor affinity to dexamethasone was calculated accord- Table 1Kinetic constants of glucocorticoid-receptor complexes and relative receptor affinities N. Esmailpour et al. / European Journal of Pharmaceutical Sciences 6 (1998) 219 –223 Fig. 2. Dissociation of glucocorticoids from the glucocorticoid receptor of the human lung at 208C. The data from the two experiments with budesonide (d,m) have been combined in the figure. The data for fluticasone propionate (♦) and dexamethasone (j) are from a single experiment.
ing to Eq. (2). Results in Table 1 show that budesonide has In clinical practice, however, budesonide is not two half the relative receptor affinity of fluticasone propionate times less active than fluticasone propionate, but it is three and 8.5 times that of dexamethasone.
expected clinical activity could be caused by a greateruptake of fluticasone propionate in the human lung tissue 4. Discussion
compared to budesonide (Rohdewald et al., 1995) and / orby differences in the distribution between lung and sys- temic circulation of these glucocorticoids (Esmailpour et fluticasone propionate from our previous experiments Hogger and Rohdewald, 1994) with the data obtained in this study demonstrate the reproducibility of the kineticmeasurements (Table 1).
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