Imre Mezo

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.

Luteinizing hormone-releasing hormone analogs with increased anti-ovulatory activity

Abstract A series of LH-RH antagonist analogs has been developed in which inhibitory activities have been increased to a potentially clinically useful level. The new peptides, which are typified by [N-acetyl-D-p-Cl-Phe 1,2 , D-Trp 3 , D-Phe 6 ,D-Ala 10 ]-LH-RH and [N-acetyl-D-Trp 1,3 ,D-p-Cl-Phe 2 ,D-Phe 6 , D-Ala 10 ]-LH-RH, most importantly contain new modification to positions 1, 2 and 10, and induce full blockade of ovulation at single doses as low as 10 μg per rat (50 μg/kg). Various ring substituents on D-Trp or D-Phe in position 1 or other D-amino acid replacements in position 10 did not significantly improve anti-ovulatory activity. Incorporation of N-Me-Leu in position 7 was slightly detrimental to activity.

Effects of long-term administration of a superactive agonistic and an antagonistic GnRH analog on the pituitary-gonad system

A powerful GnRH antagonist: [Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]-GnRH (MI-1544) and a superactive GnRH agonist: [D-Phe6,desGly10]-GnRH(1-9)EA (OVURELIN) were used in long-term administration to compare their effects on the inhibition of ovulation, LH and progesterone (P) release, LH content of pituitaries as well as on the recovery period. Both analogs showed 100% inhibitory effects on ovulation in very low doses during the daily treatment for 21 days. The antagonist prevented LH release already after the first injection, decreased the serum P level to 40%, and increased the LH content of the pituitary up to 180%, inhibiting only the release but not the synthesis of LH. The agonist showed marked LH-releasing effects on the first day of the treatment, which were reduced to 12% on the 7th day. Serum P concentration was dropped to 68% by the end of the treatment. No change was found in the LH content of pituitaries in the group treated with the agonist. Ovaries showed polifollicular pictures in the antagonist-treated group, and persistent corpora lutea were seen in the ovaries from the agonist-treated group. Regular estrous cycles returned 13-15 days after ceasing the treatment with the antagonist and 3-5 days after ceasing the treatment with the agonist. No edema-inducing effect was observed after the injections of the antagonist in doses of 100 times higher than the single antiovulatory dose.

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-d-Trp1,3,d-Cpa2,d-Lys6,d-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-dl-Ala3.1)] (AcEAK) —a branched polypeptide having a polylysine backbone — resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%–35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 μM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%–15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%–50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and-insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.

Long-term inhibition of ovulation by a GnRH-antagonist at low dose level

Ac-D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10-GnRH has been prepared by solid phase synthesis. The peptide was found to completely inhibit ovulation when administered on proestrus day in a dose of 1.5 microgram/rat, s.c. The peptide completely inhibited ovulation for a period corresponding to three to four cycles when administered daily in a dose of 5 micrograms/rat, s.c. and caused 70% inhibition of ovulation in a dose of 3 micrograms/rat.

Antiovulatory doses of antagonists of LH-RH inhibit LH and progesterone but not FSH and estradiol release

The differential regulation of immunoactive FSH and LH secretion by endogenous LH‐RH was studied using LH‐RH antagonists (Ac‐D‐Trp1,2, D‐Cpa2, D‐Lys6, D‐Ala10LH‐RH (MI‐1544) and (Ac‐D‐Nal1, D‐Phe(pCI2), D‐Trp3, D‐Cit6, D‐Ala10LH‐RH (SB‐030) in ovariecto‐mized (OVX) and regularly cycling rats. Single injections of 10 μg and 100 μg doses and long‐term treatment with 10 μg doses of MI‐1544 were used in OVX animals. Serum and pituitary LH and FSH, as well as serum estradiol and progesterone was determined by RIA during and/or after the treatment. Single injections of MI‐1544 in OVX animals caused prompt (in 2 h) and long‐lasting (for more than 24 h) suppression of the serum LH, while no or late decrease (after more than 6 h) of the serum FSH. Long‐term treatment with the same analog decreased the serum LH (by 50%) and moderately increased the pituitary LH (by 21%) but did not change the serum and the pituitary FSH concentrations. In normal rats, long‐term treatment with both of our analogs also resulted in divergent alterations in the LH and FSH concentrations. Serum LH dropped to undetectable levels, while serum FSH did not change significantly. Pituitary LH increased (by 31 to 41%), while FSH decreased (by 27 to 38%). Marked depression was found in the serum progesterone (by 64%) but no significant change in the serum estradiol levels, after the long‐term treatment for 21 days. The ovarian cycles were interrupted, and no ovulation appeared during the treatment. Significant decrease was detectable in the weight of the ovaries (by 46%), whereas the weight of the uteri did not change or slightly elevated (by 22%), after the treatment with SB‐030 or MI‐1544, respectively. The clear antagonistic effect of the analogs on the LH‐RH‐stimulated LH and FSH release was tested in the in vitro superfused rat pituitary cell system. In this system our analogs provented both the LH‐and the FSH‐releasing effect of the LH‐RH. The inhibitory effect was longer‐lasting on the FSH than on the LH release. Our studies give evidences that the long‐term treatment with antiovulatory doses of LH‐RH antagonists suppress the LH and progesterone but not the FSH and estradiol secretion and indirectly support the earlier publications suggesting the presence of FSH‐releasing factor(s) in the CNS.

Effects of continuous and repetitive administration of a potent analog of GH-RH(1-30)NH2 on the GH release in rats

We examined the desensitization and/or sensitization phenomenon in the pituitary GH responsiveness induced by continuous infusion and multiple pulses at different frequencies of a potent GH-RH analog [D-Ala2, Leu15, Nle27, GABA30-GH-RH(1-30)amide]. Further, we investigated the correlation between doses and GH responses, as well as between pulse frequency and GH responses in male rats in vivo and in vitro. Long-term, continuous administration was attained by osmotic minipumps releasing low and high doses of the analog for 14 days. The effects of repetitive administration of the GH-RH analog on the pituitary GH release was investigated by injecting 4-6 pulses of the analog at different doses and pulse frequencies. The in vitro experiments were performed in the superfused rat anterior pituitary cell system. Pituitary cells were challenged with continuous, repetitive and simultaneous continuous and repetitive perfusion of the analog. Continuous infusion with low doses of the GH-RH analog in vivo induced sensitization of the pituitary GH-secretory responsiveness and resulted in moderately increased GH releases (129% of the control) to additional bolus injections of the same analog, whereas continuous stimulation of the pituitary with high doses of the GH-RH analog evoked desensitization and resulted in blunted GH responses (29% of the control). Despite the desensitization of the pituitary GH-secretory responsiveness, high doses of the analog elevated the serum GH concentration to 310% and induced acceleration of body weight gain (160% of the control). Repetitive pulsatile administration of the GH-RH analog evoked both sensitization and desensitization of the pituitary GH-secretory responsiveness, depending on the dose and pulse frequency administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin‐releasing hormone (GnRH) analogues

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF‐7 and Ishikawa human cancer cell lines was studied. GnRH‐III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI‐1544, MI‐1892, and GnRH‐III with poly(N‐vinylpyrrolidone‐co‐maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P‐X‐GnRH‐III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Potent GnRH agonists containing L-amino acid derivatives in the six position

New agonists related to gonadotropin-releasing hormone (GnRH) have been synthesized that are comparable in potency to the GnRH and its superagonists for release of LH and estrus suppression without substitutions with D- or unnatural amino acids in position 6. We now report a series of L-beta-aspartyl-6 GnRH analogs containing only naturally occurring L-amino acids in the whole sequence, exhibiting considerable in vivo biological activity. Dose and time dependent LH release capability of the different analogs in adult male mice, estrus suppression comparisons and blockade of ovulation in female rats are given. The incorporation of L-Asp-OMe and L-Asp-OBzl in position 6 of GnRH resulted in the most potent GnRH agonists (to 12-20xGnRH potency) in this series inducing a biphasic biological response similar to the D-amino acid-6 substituted superactive GnRH analogs. A correlation between the LH releasing potencies of the analogs and their HPLC retention times was also investigated. Peptide synthesis were achieved using either solid phase or solution phase methodology.

Electrochemical stimulation of the median eminence evokes FSH but not LH release after LHRH antagonist treatment in vivo and in vitro.

Experimental data suggest that a follicle stimulating hormone‐releasing factor (FSH‐RF) distinct from luteinizing hormone‐releasing hormone (LHRH) exists. In the present study, we investigated, in short‐term ovariectomized (OVX) rats, whether FSH‐RF(s) can be released from nerve terminals by electrochemical stimulation (ECS) of the median eminence. To prevent the effect of LHRH liberated by ECS, 100 μg of a potent LHRH antagonist (MI‐1544) was administered to one group of OVX rats 60 min before ECS. Two groups of OVX rats were used as controls. One group was treated with the solvent of the LHRH antagonist 60 min before the ECS; the other group received sham‐ECS only. In‐vitro experiments using a hypothalamus‐pituitary coperifusion system were also performed to investigate the direct effect of ECS of the median eminence on LH and FSH release from pituitary cells. ECS in vivo induced 4.6‐fold (P<0.01) and 10.2‐fold (P<0.01) elevation of serum LH concentration, measured by RIA at 10 min and 60 min after ECS, respectively. Serum FSH concentrations increased 1.35‐fold at 10 min (P<0.01) and 1.50‐fold at 60 min (P<0.01) after ECS, compared with sham‐stimulated controls. Administration of LHRH antagonist attenuated the ECS‐induced release of LH by 44% at 10 min and prevented it entirely at 60 min after ECS. However, the ECS‐induced release of FSH was not modified by the antagonist at 10 min and was diminished by only 17% at 60 min after ECS, compared with solvent‐treated and stimulated controls. Immunohistological examination of the hypothalami showed that LHRH‐immunoreactivity was depleted in the region of ECS. In the study in vitro, substances released from the fragments of mediobasal hypothalami bearing ECS in the median eminence induced significant release of both LH and FSH, and the induced release of LH, but not FSH, was prevented by the LHRH antagonist. The present study suggests that FSH‐releasing factor(s) different from LHRH can be released from the median eminence and that a significant portion of FSH secretion is independent of the control of LHRH.