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.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

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 microg 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.

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(pCl2), D- Trp3, D-Cit6, D-Ala10LH-RH (SB-030) in ovariectomized (OVX) and regularly cycling rats. Single injections of 10 micrograms and 100 micrograms doses and long-term treatment with 10 micrograms 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.

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

To assess the efficacy of a potent GH-releasing hormone (GH-RH) analog (D-Ala2,Nle27,Gaba30-GH-RH-(1-30)-amide) in the treatment of GH deficiency, we investigated the effects of chronic administration of this analog (A-495) on growth responses in monosodium glutamate (MSG)-lesioned rats. Basal serum GH concentrations, GH responses to bolus injections of GH-RH, as well as acceleration of body gain and linear growth were compared after long-term continuous and repetitive administration of A-495. The effects of continuous and repetitive administration of the analog on GH responses in vitro were also compared using the superfused pituitary cell system method. Treatment with MSG reduced the body weight and linear growth of the animals (-22% and -11%, respectively), the basal serum GH concentration (-66%), and the GH-RH-induced absolute GH responses (-61%) but did not alter the relative GH responses (to basal GH concentrations). Repetitive administration of 10 micrograms daily doses of A-495 at 24 h intervals for 2 weeks highly increased the GH responsiveness to GH-RH and induced catch-up growth, by which MSG-treated animals achieved the growth rate of normal controls. However, basal serum GH concentrations were only modestly enhanced. Continuous infusion of A-495 at the same daily dose resulted in slight increases in the GH-RH-induced GH rises, moderate acceleration of body gain, and no change in linear growth. Basal serum GH concentrations were not significantly influenced by this treatment. These results demonstrate that exogenous GH-RH pulses administered at lower frequency than the frequency of the physiological GH secretion are able to fully restore the normal growth rate of the GH deficient rats. The effectivity of the treatment is rather dependent on the magnitude of GH rises than the basal GH level. Although continuous administrations of the GH-RH is also have some effect on the body gain, repetitive administration is more effective at the same daily dose. Our results from in vitro experiments show that, in addition to the low magnitude of the GH-RH-stimulated GH rises, desensitization of the GH secretory response might also be accounted for the low effectivity of the continuously administered GH-RH. Present results demonstrate the therapeutic usefulness of our new GH-RH analog and are the first to evidence that GH-RH need not be administered as frequently as the appearance of the endogenous GH pulses to restore the normal growth of the GH deficient rats.

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 microM. 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.

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.

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C 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.

The role of N-acyl groups in the inhibitory activity of LH-RH analogues.

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp1, acetyl-D-Trp1, valeryl-D-Trp1, tartaryl-D-Trp1, diacetyl-tartaryl-D-Trp1, acetyl-Gly1, and acetyl-Sar1 successively replacing the position one in the analogue [D-Trp1, D-p-Cl-Phe2, D-Trp3, D-Phe6, D-Ala10]-LH-RH. The formyl-D-Trp1 and acetyl-D-Trp1 analogues yielded 100% blockade of ovulation at the 10 micrograms dose; the others were less potent and inhibited ovulation at the 50 micrograms dose. The inhibitory potency seems to correlate with the polarity of the acyl group.

LH-RH antagonists: further analogs with ring-substituted aromatic residues.

Although the systematic substitution of benzene and other aromatic ring systems with various atoms and groups has been a standard approach in conventional pharmaceutical research, it has only recently received the attention it deserves in peptide research. The observation that D-p-Cl-Phe inserted in position 2 of certain LH-RH (luteinizing hormone-releasing hormone) analogs results in large improvements in antagonist activity led us to examine the effect of this and other substituents on position 1 and 2 D-phenylalanyl analog side chains. Analogs containing two D-p-Cl-phenylalanines were found to be particularly powerful competitive inhibitors when assayed in cycling rat for blockade of ovulation. Analogs with non-aromatic amino acids in the first position exhibited much lower activities.

Diverse effects of a potent LH-RH antagonist on the LH and FSH release.

A potent LH-RH antagonist (Ac-D-Trp1,2, D-Cpa2, D-Lys6, D-Ala10 LH-RH (Antag) was used to study the differential regulation of FSH and LH secretion by endogenous LH-RH in ovariectomized (OVX) and regularly cycling rats. The endogenous LH-RH was suppressed by single injections of Antag in OVX animals and by long-term treatment with Antag in normal and OVX rats. Serum and pituitary LH and FSH, as well as serum estradiol (E2) and progesterone (P) was determined by RIA during and/or after the treatment. The direct effect of the antagonistic analog on the ovarian P release was tested in vitro using the isolated luteal cell system. Single injections of the Antag in OVX animals caused prompt and marked suppression of the serum LH (-80%), while no decrease of the serum FSH. Long-term treatment with the same analog decreased the serum LH by 50% but did not modify the serum FSH. In normal rats, serum LH dropped to undetectable levels, while serum FSH did not change significantly. Long-term treatment with the antagonist also resulted in divergent alterations in the pituitary gonadotropin concentrations. In OVX animals, the pituitary LH content moderately elevated (+21%), however, the FSH did not change. In normal rats, ovarian cycles were interrupted, and no ovulation appeared during the treatment. The pituitary LH concentration increased by 46%, while the FSH decreased by 43%. Marked depression was found in the serum P (-60%) but no significant change in the serum E2 levels. Incubation of isolated luteal cells with the Antag did not influence the HCG-induced P secretion in vitro, demonstrating that the in vivo inhibitory effect of the antagonistic LH-RH analog on the P secretion asserts not directly on the ovarian LH-RH receptors, but through inhibition of the endogenous LH-RH. Our studies give evidence that the long-term treatment with LH-RH antagonist suppress the LH and P but not the FSH and E2 secretion, and provide new data suggesting the presence of a FSH-releasing factor in the CNS.

Investigation of the chemical stability of a new growth hormone-releasing hormone (GHRH) analogue by HPLC.

The stability of a new active growth hormone-releasing hormone analogue (D-Ala2,Nle27,(gamma-amino-butyric acid)30-GHRH(1-30)-NH2) was investigated during storage at different temperatures in aqueous solution. Samples stored for various periods of time were analysed by HPLC. It is concluded that in aqueous solution D-Ala2, Nle27,(gamma-amino-butyric acid)30-growth hormone-releasing hormone (1-30)-NH2 is stable: at least for 36 days at 4 degrees C; for 28 days at 25 degrees C; and for 10 days at 37 degrees C.

New Gaba-containing analogues of human growth hormone releasing hormone (1-30)-amide: II. Detailed in vivo biological examinations.

Analogues of human growth hormone-releasing hormone-(1-30)-amide [GH-RH(1-30)-amide] were tested for their ability to stimulate GH release in vivo by injecting the peptides intravenously (iv), subcutaneously (sc), and intramuscularly (im). The analogues involved derivatization with Nle27 and Gaba substituents at the C-terminus with or without D-amino acid(s) in the peptide chain. The potency of the analogues was compared to that of GH-RH(1-29)-amid testing their ability to release GH at 5, 15 and 30 min after the administration. In iv test the potency of the analogues was 1.2-2 times higher than that of the GH-RH(1-29)-amide, and no significant differences were detected between the potencies of the analogues with or without D-amino acid. In the sc test the analogue with D-Ala2, Nle27, and Gaba30 substitutions expressed 8.0-51.7 times higher potency than the GH-RH(1-29)-amide, however, the analogue with similar modifications but with L-Ala2 showed the same low potency (1.2-2.1) as in the iv test. Results from the im experiments were similar to those of SC test. The most potent analogues were those which had D-Ala2, Nle27, and Gaba30 substitutions with Gly15 or Leu15. Circular dichroism (CD) spectra of the analogues showed that Leu in position 15 increased the stability of the predominant alpha-helix conformation, which improved the absorption of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational study of a series of somatostatin analogues with antitumor and/or GH inhibitory activity.

A series of somatostatin analogues with varying activities have been studied by 1H NMR in CD3OH at low temperature in order to find a possible structural explanation for the differentiation of biological activities. In somatostatin analogues with GH release inhibitory activity a beta-turn/beta-sheet backbone conformation is present, which is shown to be characteristic of somatostatin-derived peptides exhibiting this biological activity. On the other hand, among the analogues with antitumor activity, a deviation from these typical structural features is clearly observed, but not general conformational model can be proposed.

Comparison of high-performance liquid chromatography and capillary electrophoresis in the analysis of somatostatin analogue peptides.

HPLC and CE methods were developed for analysis of somatostatin analogue (S-analogue) peptides utilizing triethylammonium phosphate-organic solvent modifier solvents as the CE buffer and HPLC eluent. Acetonitrile, methanol, ethanol and 2-propanol were applied as organic modifiers. The applicability of HPLC and CE systems was evaluated and compared. Optimum conditions for the separation were determined for both methods. Retention (migration) time, elution order and selectivity can be influenced by modifying the composition of the eluent (buffer) with organic solvents not only in HPLC but also in CE. Although the HPLC system reacted to changes in the organic solvent concentration in a much more sensitive way than the CE system did (from the point of view of retention time), CE proved to be a more suitable method for separating the peptides investigated. Baseline separation could be achieved within 6-9 min by CE, a result which was impossible to achieve with HPLC working in the isocratic mode. In CE the effect of the alcohols on migration times proved to be opposite to that of acetonitrile. Whereas ACN decreased, the alcohols increased the migration times in a concentration-dependent way. The results suggest that CE can be applied very advantageously in peptide analysis. Its performance regarding selectivity, resolution, theoretical plate number, duration and cost is comparable or sometimes superior to that of HPLC.

Octapeptide analogs of somatostatin exhibiting greatly enhanced in vivo and in vitro inhibition of growth hormone secretion in the rat.

Analogs of a superactive somatostatin (SRIF) octapeptide (code named SMS 201-995 (1)) were synthesized using solid-phase synthetic methodology and assayed for their ability to inhibit growth hormone release from cultured rat anterior pituitary cells and in sodium pentobarbital-anesthetized rats. One analog: (Formula: see text) exhibited greatly enhanced in vitro inhibitory activity (greater than 1,000x) relative to both the parent octapeptide molecule and to the 14 amino acid SRIF molecule. This analog which was also very potent in vivo contains a tyrosine residue and, given its high in vitro activity, may be of investigative importance as a radioiodinated ligand in receptor assays. An octapeptide retro-inverso analog also exhibited significant SRIF-like activity. Several very low activity octapeptide analogs were synthesized and were found to be devoid of SRIF-antagonist activity. A dodecapeptide analog previously shown to be superactive in vivo also demonstrated high in vitro activity.

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.