RGD(F/S/V)-Dex: towards the development of novel, effective, and safe glucocorticoids

Abstract

Dexamethasone (Dex) is an effective glucocorticoid in treating inflammation and preventing rejection reaction. However, the side effects limit its clinical application. To improve its druggable profile, the conjugates of RGD-peptide-modified Dex were presented and their enhanced anti-inflammation activity, minimized osteoporotic action, and nanoscaled assembly were explored. (RGD stands for Arg-Gly-Asp. Standard single letter biochemical abbreviations for amino acids have been used throughout this paper.) In respect of the rejection reaction, the survival time of the implanted myocardium of the mice treated with 1.43 µmol/kg/d of the conjugates for 15 consecutive days was significantly longer than that of the mice treated with 2.5 µmol/kg/d of Dex, and the conjugates, but not Dex, exhibited no toxic action. At a single dose of 14.3 µmol/kg (100 times minimal effective dose, 0.143 µmol/kg), the conjugates induced no liver, kidney, or systemic toxicity. At the dose of 1.43 µmol/kg, the conjugates, but not Dex, prolonged the bleeding time of the mice, and inhibited the thrombosis of the rats. In water and rat plasma, the conjugates formed nanoparticles of 14-250 and 101-166 nm in diameter, respectively. Since the nanoparticles of ~100 nm in size cannot be entrapped by macrophages in the circulation, RGDF-Dex would particularly be worthy of development, since its nanoparticle diameter is 101 nm.

Keywords: Dexamethasone; acute toxicity; coagulation; development; immunosuppression; nanomedicine; thrombus.

Figure 1

Structure of RGDV-Dex, RGDS-Dex, and RGDF-Dex. Abbreviations: RGDV, Arg-Gly-Asp-Val; RGDS, Arg-Gly-Asp-Ser; RGDF, Arg-Gly-Asp-Phe; Dex, dexamethasone.

Figure 2

TEM images showing the effects of concentration. Notes: (A) TEM images of 10⁻⁸, 10⁻¹⁰, and 10⁻¹² M of RGDF-Dex in ultrapure water (pH 7.0); (B) TEM images of 10⁻⁸, 10⁻¹⁰, and 10⁻¹² M of RGDS-Dex in ultrapure water (pH 7.0); (C) TEM images of 10⁻⁸, 10⁻¹⁰, and 10⁻¹² M of RGDV-Dex in ultrapure water (pH 7.0). Abbreviations: TEM, transmission electron microscope; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; RGDV, Arg-Gly-Asp-Val; Dex, dexamethasone.

Figure 3

AFM images. Notes: (A) AFM images of rat plasma alone; (B) AFM images of 10⁻⁸ M of RGDF-Dex in rat plasma; (C) AFM images of 10⁻⁸ M of RGDS-Dex in rat plasma; (D) AFM images of 10⁻⁸ M of RGDV-Dex in rat plasma; (E) AFM images of ultrapure water alone; (F) AFM images of 10⁻⁸ M of RGDF-Dex in ultrapure water; (G) AFM images of 10⁻⁸ M of RGDS-Dex in ultrapure water; (H) AFM images of 10⁻⁸ M of RGDV-Dex in ultrapure water. Inset in each figure shows magnified area identified by the red triangles. Abbreviations: AFM, atomic force microscopy; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; RGDV, Arg-Gly-Asp-Val; Dex, dexamethasone.

Figure 4

Nanoproperties of 10⁻⁸ M solution of RGDF-Dex, RGDS-Dex, and RGDV-Dex in water. Notes: (A) Faraday–Tyndall effect of RGDF-Dex: (i) in ultrapure water without laser beam irradiation, (ii) in pH 7.0 ultrapure water with 650 nm laser beam irradiation, (iii) in pH 2.0 ultrapure water with 650 nm laser beam irradiation; (B) Faraday–Tyndall effect of RGDS-Dex: (i) in ultrapure water without laser beam irradiation, (ii) in pH 7.0 ultrapure water with 650 nm laser beam irradiation, (iii) in pH 2.0 ultrapure water with 650 nm laser beam irradiation; (C) Faraday–Tyndall effect of RGDV-Dex: (i) in ultrapure water without laser beam irradiation, (ii) in pH 7.0 ultrapure water with 650 nm laser beam irradiation, (iii) in pH 2.0 ultrapure water with 650 nm laser beam irradiation; (D) Faraday–Tyndall effect of ultrapure water: (i) in pH 7.0 ultrapure water without laser beam irradiation, (ii) in pH 7.0 ultrapure water with 650 nm laser beam irradiation, (iii) in pH 2.0 ultrapure water with 650 nm laser beam irradiation; (E) size distribution of RGDF-Dex: (left) in pH 2.0 ultrapure water, (right) in pH 7.0 ultrapure water; (F) size distribution of RGDS-Dex: (left) in pH 2.0 ultrapure water, (right) in pH 7.0 ultrapure water; (G) size distribution of RGDV-Dex: (left) in pH 2.0 ultrapure water, (right) in pH 7.0 ultrapure water; (H) ζ-potential of RGDF-Dex in pH 7.0 ultrapure water; (I) ζ-potential of RGDS-Dex in pH 7.0 ultrapure water; (J) ζ-potential of RGDV-Dex in pH 7.0 ultrapure water. Abbreviations: St dev, standard deviation; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; RGDV, Arg-Gly-Asp-Val; Dex, dexamethasone.

Figure 5

The effect of RGDV-Dex, RGDS-Dex, and RGDF-Dex on ConA-induced proliferation of the spleen lymphocytes of BALB/C mouse, n=3. Abbreviations: Dex, dexamethasone; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; RGDV, Arg-Gly-Asp-Val; ConA, concanavalin; IC₅₀, half maximal inhibitory concentration; SD, standard deviation.

Figure 6

The survival time of the implanted myocardium in the “pocket” near the distal edge of the ear of BALB/C mice, n=12. Abbreviations: Dex, dexamethasone; CMCNa, carmellose sodium; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; CsA, cyclosporine A; SD, standard deviation.

Figure 7

Tail bleeding time of RGDV-Dex-, RGDS-Dex-, and RGDF-Dex-treated mice, n=12. Abbreviations: CMCNa, carmellose sodium; Dex, dexamethasone; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; SD, standard deviation.

Figure 8

Anti-thrombotic activities of Dex, RGDV-Dex, RGDS-Dex, and RGDF-Dex, n=12. Abbreviations: CMCNa, carmellose sodium; Dex, dexamethasone; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; SD, standard deviation.

Figure 9

Body weight of the treated BALB/C mice, n=12. Abbreviations: Dex, dexamethasone; CMCNa, carmellose sodium; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; CsA, cyclosporine A; SD, standard deviation.

Figure 10

Spleen index of the treated BALB/C mice, n=12. Abbreviations: Dex, dexamethasone; CMCNa, carmellose sodium; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; CsA, cyclosporine A; SD, standard deviation.

Figure 11

Liver index of the treated BALB/C mice, n=12. Abbreviations: Dex, dexamethasone; CMCNa, carmellose sodium; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; CsA, cyclosporine A; SD, standard deviation.

Figure 12

Serum ALT levels of the mice on which acute toxicity assay was performed, n=12. Abbreviations: ALT, alanine transaminase; CMCNa, carmellose sodium; Dex, dexamethasone; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; SD, standard deviation.

Figure 13

AST levels of the mice on which acute toxicity assay was performed, n=12. Abbreviations: AST, aspartate transaminase; CMCNa, carmellose sodium; Dex, dexamethasone; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; SD, standard deviation.

Figure 14

Serum Cr levels of the mice on which acute toxicity assay was performed, n=12. Abbreviations: Cr, creatinine; CMCNa, carmellose sodium; Dex, dexamethasone; RGDV, Arg-Gly-Asp-Val; RGDF, Arg-Gly-Asp-Phe; RGDS, Arg-Gly-Asp-Ser; SD, standard deviation.