SYNTHESIS, CONFORMATION, BIODISTRIBUTION, AND HORMONE-RELATED IN-VITRO ANTITUMOR-ACTIVITY OF A GONADOTROPIN-RELEASING-HORMONE ANTAGONIST-BRANCHED POLYPEPTIDE CONJUGATE

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much empha sis was put on the development of retarded-release devices (e.g. micro capsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the de sign of bioconjugates acting as (a) prodrugs producing prolonged relea se or (b) macromolecular therapeutics. In order to evaluate the feasib ility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp(1,3),D-Cpa(2),D-Lys(6),D-Ala(10))-GnR H (MI-1544). As a carrier, a representative of a new generation of syn thetic, biodegradable branched poly[Lys(X(i)-DL-Ala(m))] (XAK) type po lypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic poly peptide with free gamma-carboxyl groups was conjugated to MI-1544, whi ch has only a single amino group at position 6. In this paper, we desc ribe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substi tution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone -related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with In-111- and I-125-labeled conjugates have demonstrated t hat in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibiti on of ovulation even after single subcutaneous injection. These data s uggest that it is feasible to use a biodegradable polymeric polypeptid e for development of a macromolecular therapeutic with GnRH antagonist s.