Binding kinetics of budesonide to the human glucocorticoid receptor

https://doi.org/10.1016/S0928-0987(97)00082-1Get rights and content

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 constant of 18.9×105 l mol−1 min−1 and the dissociation constant of 25.0×10−4 min−1 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-receptor complex was 4.6 h. Results agree with data derived from competition tests we performed earlier.

Introduction

The binding of glucocorticoids to the glucocorticoid receptor and the formation of a receptor complex is a prerequisite for their pharmacological and therapeutical action.

A more precise study of the interaction of glucocorticoids with their receptors is achieved with a kinetic analysis than with an equilibrium analysis, because the time to reach equilibrium in competition assays is influenced by (i) the concentration of the receptor, (ii) concentration of [3H]glucocorticoid, (iii) concentration of the competitor, (iv) temperature and (v) by the differences in affinities of the different glucocorticoids to the receptor.

Therefore, in the present study the binding kinetics of [3H]budesonide and [3H]dexamethasone were compared to determine the relative receptor affinities in addition to competition assays. Studies were performed on the glucocorticoid receptor in the cytosolic fraction from human lung tissue. Binding kinetics of fluticasone propionate were included for comparison using the same lung tissue.

Section snippets

Experimental

[3H]Dexamethasone was obtained from Amersham International plc (UK). [3H]Fluticasone propionate and [3H]budesonide as well as fluticasone propionate and budesonide were a generous gift from Glaxo GGR (Greenford, UK). Radiochemical purity was demonstrated by thin-layer chromatography. Scintillation counting was performed with a scintillation beta counter, Rackbeta 1219 LKB (Germany) using Aquasafe 500 plus from Zinnser (Frankfurt/Main, Germany). Pefabloc SC™ (4-(2-aminoethyl)-benzenesulfonyl

Association constant

The time course of specific binding of budesonide indicated that the rate of association with the glucocorticoid receptor was not as fast as for fluticasone propionate, but faster than that of dexamethasone (Fig. 1). However, the total amount bound after 1 h was not different between budesonide and fluticasone propionate, whereas specific binding for dexamethasone was only approximately one third of the bound fraction of the two other glucocorticoids. The faster binding of fluticasone

Discussion

Comparison of k1 and k−1 for dexamethasone and fluticasone propionate from our previous experiments (Högger and Rohdewald, 1994) with the data obtained in this study demonstrate the reproducibility of the kinetic measurements (Table 1).

The association of glucocorticoids with the glucocorticoid receptor is almost complete within 1 h in vitro. However, the association rate differs considerably between the glucocorticoids tested. Budesonide binds to the glucocorticoid receptor twice as fast as

References (11)

There are more references available in the full text version of this article.

Cited by (44)

  • Glucocorticoid cell priming enhances transfection outcomes in adult human mesenchymal stem cells

    2016, Molecular Therapy
    Citation Excerpt :

    This variation is hypothesized to be dependent on physiological GC receptor binding affinity. Physiological Kd values are between 1–50 nmol/l for DEX, depending on donor age, disease status, tissue type, and stage of the cell cycle.39,40,41 Due to the increase in DEX concentration required to produce enhanced transfection outcomes beyond physiological Kd, it is hypothesized that the optimal DEX dose (90–360 nmol/l) is the concentration at which receptors become saturated with GC rather than 50% bound.

  • Structural classification of steroid-binding sites on proteins by coarse-grained atomic environment and its correlation with their biological function

    2015, Steroids
    Citation Excerpt :

    The similarity in the binding site was evidently reflected to the binding constant of the steroid molecule to the proteins. The dissociation constant of 17β-estradiol from oestrogen receptor, for instance, is approximately 0.9 nM [18], that of progestin is 0.3 nM [19], and that of budesonide, a glucocorticoid for inhalation therapy, is 4–1.32 nM [20]. This tight-binding property was also found in non-receptor proteins in G2, such as human sex hormone-binding globulin (SHBG) [21] and the N-terminal domain of membrane-bound Niemann-Pick C1 (NPC1) [22].

  • Budesonide loaded nanoparticles with pH-sensitive coating for improved mucosal targeting in mouse models of inflammatory bowel diseases

    2014, Journal of Controlled Release
    Citation Excerpt :

    The glucocorticoid budesonide served as a model drug and was entrapped in PLGA core which was enteric coated with a methacrylate copolymer (Eudragit® S100). Budesonide has a strong affinity for glucocorticoid receptor [17] but due to extensive metabolism by cytochrome P-450 enzyme, a very low amount of the drug reaches the systemic circulation [18]. Thus a carrier system for local delivery of budesonide was designed.

View all citing articles on Scopus
View full text