Evaluation of the in vitro and in vivo activity of the l‐, d,l‐ and d‐Cit6 forms of the LH‐RH antagonist Cetrorelix (SB‐75)

J. PINSKI, A. V. SCHALLY, T. YANO, K. GROOT, G. SRKALOVIC, P. SERFOZO, T. REISSMANN, M. BERND, W. DEGER, B. KUTSCHER, J. ENGEL

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Abstract

The objective of this study was to examine the in vivo and in vitro gonadotropin-inhibiting potencies, edematogenic activities and the receptor binding affinities of the D-Cit6, D,L-Cit6 and L-Cit6 forms of the LH-RH antagonist Cetrorelix (SB-75) [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH. In order to demonstrate the suppressive effects of two different diastereomers of SB-75 and their racemic mixture on LH and FSH release, [D-Cit6] SB-75 was injected subcutaneously in doses of 2.5 and 10 μg/rat, [D,L-Cit6]-SB-75 in doses of 5 and 20 μg/rat and [L-Cit6] SB-75 in doses of 12.5 and 50 μg/rat to castrated male rats. Two hours after administration, there was no difference in LH levels between rats injected with the L-form and control animals, indicating a low activity and/or a rapid enzymatic degradation of this peptide. The (1:1) diastereomeric mixture was only about half as potent in suppression of LH release compared to [D-Cit6] SB-75, Serum FSH levels were suppressed significantly (p < 0.01) for more than 48 h after the administration of 10 μg [D-Cit6] SB-75 and 20 μg of [D,L-Cit6] SB-75, respectively. CD-Cit6] SB-75 administered at a dose of 2 μg/rat induced 100% inhibition of ovulation, while 4 μg/rat of the D,L-Cit6 peptide were necessary to produce the same effect. [L-Cit6] SB-75 given at a high dose of 40 μg/rat produced only 14% inhibition of ovulation. The D-Cit6 form of SB-75 produced skin lesions with a much smaller diameter than the L-isomer, and was about 34 times less edematogenic. [D-Cit6] SB-75 was bound more powerfully to high-affinity pituitary LH-RH receptors than either D,L-Cit6 or L-Cit6 analogues. In vitro assays based on the superfusion of dispersed rat pituitary cells on a column, followed by radioimmunoassay for LH, also demonstrated a lower inhibitory activity for the L-Cit6 analogue, but the differences between D-, D,L- and L-citrulline analogues were smaller than in vivo. Our results indicate that the LH-RH antagonist [D-Cit6] SB-75 is more effective in suppression of gonadotropin release in vivo and in vitro, less edematogenic and possesses higher binding affinity to pituitary LH-RH receptors than the D,L- and L-citrulline decapeptide analogues.

Original languageEnglish (US)
Pages (from-to)410-417
Number of pages8
JournalInternational journal of peptide and protein research
Volume45
Issue number5
DOIs
StatePublished - May 1995

Keywords

  • Edematogenic activity
  • LH-RH antagonist
  • Pituitary-gonadal axis

ASJC Scopus subject areas

  • Biochemistry

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