Saffie, Roghieh; (2001) Preparation, characterisation and in vivo evaluation of dhea-protein conjugates. Doctoral thesis (Ph.D.), University College London.

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Abstract

Dehydroepiandrosterone (DHEA) is the major secretory steroid product of the adrenal gland, and it has been found that its endogenous level declines with ageing. Exogenous administration has been suggested for cardiovascular diseases, cancer, rheumatoid arthritis, weight loss, diabetes etc. DHEA used here as a model lipophilic drug is highly insoluble in water making its administration parenterally difficult to achieve. In attempt to improve the water solubility of DHEA, it was covalently linked to the s-amino groups of the lysine residues of natural macromolecular carriers such as immunoglobulin G (IgG) and bovine serum albumin (BSA) through a succinyl spacer which provides an opportunity to formulate soluble DHEA without toxic solubilisers. The 3'-hydroxy group of DHEA was esterified with succinic anhydride. The evidence for 3'-succinyl-DHEA was obtained by HPLC, UV-visible spectroscopy and mass spectrometry. After the reaction of 3'-succinyl-DHEA with N-hydroxy-3-sulfo-succinimide in the presence of carbodiimide, the carboxylate function on the succinyl spacer produced an active ester which is highly reactive to the lysyl amino groups of the proteins. Methods for the characterisation of the IgG-succinyl-DHEA and BSA-succinyl- DHEA included gel filtration chromatography, UV-visible spectroscopy , the TNBS method, radiolabelling and SDS-PAGE electrophoresis. It was found that the degree of derivatisation (number of drug molecules per molecule of protein) was related to the concentration of 3'-sulfoNHS-succinyl-DHEA in DMF and the ratio of the protein; 3'- sulfoNHS-succinyl-DHEA. Double radiolabelling was used to follow the two moieties of the conjugates (DHEA and protein) simultaneously for in vitro and in vivo studies. The conjugates were stable in mouse plasma after incubation for 24h, After intravenous administration, the clearance of the conjugates was evaluated and compared to that of the free drug. The clearance of the conjugates was found to be much slower than that of the free drug in a two compartment model. The pharmacokinetic parameters showed that the clearance of the conjugates was not dependent on the degree of derivatisation. Judging from radioactivity measurements, the tissue distribution study of the conjugates revealed that the conjugates altered the disposition of DHEA and prolonged the retention of DHEA in the tissues as compared with the free drug. The results suggested that IgG and BSA could be used as drug carriers to improve the solubility of insoluble drugs, prolong their circulation in the blood and alter their biodistribution in the tissue.

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