Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified, and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH-I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recLH displayed a 5-10-fold higher affinity than rat pitLH, but about 100-fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.

1994-10-01·Journal of the Society for Gynecologic Investigation

In Vivo Effects of a Potent GnRH Antagonist ORG 30850

Article

作者: Richard T. Scott ; Helenius J. Kloosterboer ; Keith Gordon ; Hubert J. J. Loozen ; Gary D. Hodgen ; Douglas R. Danforth ; Robert F. Williams

OBJECTIVE:

Our purpose was to determine the pituitary responsiveness to exogenous GnRH in GnRH antagonist-suppressed ovariectomized monkeys.

METHODS:

This was a prospective experimental non-human primate study performed at the research laboratories of The Jones Institute for Reproductive Medicine. Seventeen long-term ovariectomized cynomolgus monkeys were studied.

INTERVENTIONS:

The GnRH antagonist ORG 30850 was administered to long-term ovariectomized monkeys assigned to one of six groups: single subcutaneous injections in group A (n = 4), 0.3 mg/kg; group B (n = 4), 1.0 mg/kg; and group C (n = 3), 3.0 mg/kg; and six consecutive daily subcutaneous injections in group D (n = 2), 0.3 mg/kg; group E (n = 2), 1.0 mg/kg; and group F (n = 2), 3.0 mg/kg. Blood samples were collected daily from 10 days before treatment until 22 days after treatment, then weekly for 6 additional weeks. Intravenous GnRH stimulation tests (10 micrograms/kg) were performed on the day after vehicle injection (control) and the day after completion of treatment(s), and then at weekly intervals. The main outcome measures were serum levels of LH, FSH, and ORG 30850.

RESULTS:

Administration of ORG 30850 resulted in suppression (P < .05) of LH and FSH in all treatment groups. Long-term suppression (greater than 2 weeks) was evident in all primates receiving a cumulative dose of at least 1 mg/kg. Paradoxically, the responsiveness of the pituitary to exogenous GnRH was accentuated during the time of maximal tonic LH/FSH suppression.

CONCLUSIONS:

ORG 30850 is a potent long-acting GnRH antagonist. Furthermore, the present in vivo demonstration of heightened pituitary responsiveness to exogenous GnRH emphasizes the divergent mechanisms of action of GnRH antagonists and GnRH agonists.

1993-04-01·Journal of medicinal chemistry1区 · 医学

The effect of NMeTyr5 substitution in luteinizing hormone-releasing hormone antagonists

1区 · 医学

Article

作者: Holst, Mark R. ; Haviv, Fortuna ; Swenson, Rolf E. ; Fitzpatrick, Timothy D. ; Nichols, Charles J. ; Bush, Eugene N. ; Nguyen, A. T. ; Mort, Nicholas A. ; Diaz, Gilbert

Three LH-RH antagonists, N-Ac-D-4ClPhe-D-4ClPhe-D-3Bal-Ser-N-MeTyr-D-Lys-Leu-Arg-Pro-D-AlaNH₂ (I), N-Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-N-MeTyr-D-Cit-Leu-Arg-Pro-D-AlaNH₂ (II), and N-Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-N-MeTyr-D-Lys(Nic)-Leu-Lys(Isp)-Pro-D-AlaNH₂ (A 75998) and their des-N-Me analogs, the known antagonists: Org 30850, SB 75, and antide, were synthesized and their biol. activities compared [3Bal = 3-(3-benzothienyl)alanine; Lys(Isp) = lysine(N-ε-isopropyl); Lys (Nic) = lysine(N-ε-nicotinyl); 2Nal = 3-(2-naphthyl)alanine; 3Pal = 3-(3-pyridyl)alanine; 4ClPhe = 3-(4-chlorophenyl)alanine].The compounds were tested in vitro for LH-RH receptor binding, suppression of LH release, and histamine release.LH suppression was also measured in vivo in castrate male rats following the s.c. administration of a 30μg/kg dose.While I had in vitro activity similar to the parent compound, its LH suppression in vivo showed a shorter duration.In the SB 75 series, the N-methylation improved both receptor binding and in vitro LH inhibition; in vivo, the suppression was again a little shorter lived than the parent.Antagonists I,II,Org 30850, and SB 75 were similar in histamine release with ED₅₀ of ∼1 μg/mL.A 75998, however, showed increase in activity over antide in inhibition of LH release in vitro.In the castrate rat the LH suppression of A 75998 was longer lived than that of antide, but shorter than that of NalGlu.While the histamine release ED₅₀ of A 75998 was 26-fold lower than antide, it was >10-fold higher than the standard antagonist NalGlu.All the N-methylated compounds exhibited over 12-fold increase in water solubilityThe N-MeTyr⁵ substitution appears to produce 2 major effects: (1) it introduces a backbone constraint that maintains or increases intrinsic potency which implies that it favors the bioactive conformation: and (2) it improves water solubility by enhancing solute-solvent interactions.

100 项与 Org-30850 相关的药物交易

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