GnRH peptide analogs are widely used to treat diverse clinical conditions. However, they have poor oral activity and exhibit rapid metabolic clearance, thus requiring injection and depot formulation. Because
steroid hormones are bound to
plasma proteins, we explored the possibility of conjugating hydroxylated progesterones to
GnRH analogs to reduce metabolic clearance of the
peptides. Conjugation of [D-Lys6]
GnRH agonist to the alpha11-hydroxyl of alpha11-hydroxyl
progesterone via a hemi-
succinate bridge increased the plasma half-life after iv injection in rabbits by 3.6-fold while retaining high binding affinity, thus providing proof of concept. Five
GnRH antagonists were then synthesized with
21-hydroxyprogesterone conjugated via C21-hydroxyl to positions six (conjugates A and B) and position seven (conjugates C and D) of
GnRH antagonists. In the fifth compound the NH2 terminus of a
GnRH antagonist lacking the first two
amino acids was conjugated via the C21-hydroxyl to
21-hydroxyprogesterone (conjugate E). All five analogs bound to guinea pig
progesterone binding globulin with relatively high affinities (264-1020 nM). Moreover, all five conjugates retained high progestogenic activity in stimulating a
progesterone-response-element-driven
chloramphenicol acetyltransferase reporter gene in the T47D
breast cancer cell line. Conjugation via the epsilon-amino function of D-Lys6 (conjugates A and B) produced compounds with high binding affinity for the human
GnRH receptor (15 and 7 nM) comparable to that of the unconjugated
GnRH antagonists (4 and 26 nM). Conjugation via the epsilon-amino function of Lys7 (conjugates C and D) or the NH2 terminus of an N-terminally truncated antagonist (conjugate E) produced compounds of low binding affinity. Conjugates A and B also exhibited high functional antagonism of
GnRH stimulation of
inositol phosphate production in COS-7 cells expressing the human
GnRH receptor (2.6 and 16 nM) compared with the unconjugated antagonists (1.3 and 122 nM). In accordance with their poor receptor binding affinity, conjugates C, D, and E had poor functional antagonism. Preliminary dose-finding studies in female marmosets showed transitory
progesterone inhibition by 0.25 mg and prolonged suppression of 12 and 17 d by 0.5- and 1.0-mg doses. Injection of conjugate A in adult male marmosets (0.5 mg sc) rapidly suppressed plasma
testosterone levels, which remained suppressed for at least 3 d. In contrast, the unconjugated parent antagonist alone or with
progesterone suppressed
testosterone for only 8 h to 1 d. The findings demonstrate that conjugation of
progesterone to
GnRH antagonists conveys plasma binding and progestogenic properties and increases their efficacy and duration of action in vivo. These new
GnRH antagonists show promise as therapeutic agents for
hormone-dependent diseases and as
contraceptives.