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 emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(<SCP>d</SCP>-Trp^1,3,<SCP>d</SCP>-Cpa²,<SCP>d</SCP>-Lys⁶,<SCP>d</SCP>-Ala¹⁰)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(X<INF>i</INF>-<SCP>dl</SCP>-Ala<INF>m</INF>)] (XAK) type polypeptides with poly(<SCP>l</SCP>-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free γ-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544−AcEAK conjugate with a 33% degree of substitution, (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 ¹¹¹In- and ¹²⁵I-labeled conjugates have demonstrated that 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 inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.