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.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

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.

Direct anticancer activity of gonadotropin-releasing hormone-III.

In previous studies GnRH-III, a variant of the hypothalamic neurohormone GnRH, was isolated from the brain of the sea lamprey and structurally characterized. GnRH-III is a hypothalamic neurohormone in both female and male sea lampreys. In the present work biological activities of GnRH-III in mammalian systems were examined. In superfused rat pituitary cells, GnRH-III at 1 nM to 100 nM neither induced LH-secretion nor inhibited the LH-secretion elicited by native GnRH and elicited LH release only at 1 microM. At high dose (500 microg/day) in vivo, GnRH-III behaved as a GnRH agonist, though, it was 1000-fold less active than ovurelin. The in vitro and in vivo results were in good agreement in showing that GnRH-III is only a weak agonist of the endocrine activity of GnRH. GnRH-III specifically bound to receptors on cancer cells and recognized not only the high-, but also the low-affinity binding sites. GnRH-III significantly suppressed growth of human cancer cells which have GnRH receptors. The inhibitory effect of GnRH-III on growth of cancer cells was specific and direct since the peptide did not have endocrine activity in the concentration range found to be effective in anticancer assays. GnRH-III inhibited equally the growth of ER-positive and -negative breast and TeR-positive and negative prostate cells.

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.

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Comparative analysis of somatostatin analog peptides by capillary electrophoresis and micellar elektrokinetic chromatography.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25-11.0, containing sodium dodecyl sulfate (SDS) (0-100 mM) for analysis of somatostatin-analog peptides were developed. The method presented here was compared with the reversed-phase high performance liquid chromatographic (RP-HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na-borate buffer containing 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP-HPLC, with highly hydrophobic peptides-migrating slower than peptides without hydrophobic moieties.

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.

Effect of gonadotropin-releasing hormone analogs and their conjugates on gonadotropin-releasing hormone receptor--positive human cancer cell lines.

The purpose of the present investigation was to develop new gonadotropin-releasing hormone (GnRH) antagonists and to increase their stability and antitumor effect by conjugation with carrier macromolecules. Antitumor effect was evaluated using clonogenic assay, cell counting for antiproliferation, and sulforhodamine B method. The presence of GnRH-binding sites in human cancer cell lines (MCF-7, MDA-MB-231, Ishikawa, LNCaP) was proved. The direct growth inhibition of tumor cell lines is achieved with relatively high analog concentrations (10(-10)- 10(-5) M). We have developed new GnRH analogs of human and chicken origin. MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)GnRH and the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3, D-Cpa2, Lys5, [beta-Asp(DEA)]6, Gln8, D-Ala10)-GnRH have stronger direct antitumor properties than the agonists. The antagonists inhibited proliferation of GnRH receptor-positive human cancer cell lines by 28 to 38%. GnRH peptide analogs were coupled with macromolecules through biodegradable groups, to enhance their antitumor effects. The antagonists reduced survival of MCF-7 and MDA-MB-231 cells by 38 to 48% and 20 to 41%, respectively. They showed less activity against human endometrial and prostate cancer cells (10-20%). The copolymer (P) as polyanionic carrier molecule reached only 15 to 20% survival reduction in all cell lines. However, the copolymer GnRH antagonist conjugates P-X-1892 and P-X-1544 killed 95 to 98% of cells at doses corresponding to the GnRH analog concentration. These compounds having antitumor activity could be tried for the treatment of prostate, breast, and endometrium cancer.

In vivo studies of the new gonadotropin-releasing hormone antagonist--copolymer conjugates having antitumor activity.

The aim of the study was to test in vivo the gonadotropin-releasing hormone (GnRH) antagonists and their conjugates showing antitumor activities in vitro. The in vivo experiments with the human GnRH antagonist MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH, the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3,D-Cpa2,Lys5,/beta-Asp(DEA)/6,Gln8,D-Al a10)-GnRH, and their copolymer conjugates were carried out on MCF-7 and MDA-MB-231 human breast tumors xenografted in immunosuppressed CBA/Ca HRIJ-T6 female mice and on MXT mouse mammary tumors in BDF1 mice. The P-X-1544 and P-X-1892 conjugates were prepared by coupling the GnRH antagonists to macromolecule copolymer through biodegradable spacers. MI-1544 and its conjugate had strong, whereas MI-1892 and its conjugate had slight, castration effect in rats. All of them showed selective antitumor activity. The conjugates, given daily, inhibited both types of xenografts by 42 to 49%. Their activity was stronger in MXT mammary tumor (72 to 61%). The in vivo effect of GnRH antagonists was largely increased by coupling them to nonbiodegradable macromolecule carriers of polyanionic character. P-X-1544 and P-X-1892 GnRH antagonist-macromolecule conjugates might become important therapeutic agents for the treatment of breast cancer.

Synthesis of GnRH analogs having direct antitumor and low LH-releasing activity.

New chicken I GnRH agonists and antagonists have been synthesized and tested for their biological activities. The common feature of these analogs was that the molecules had a beta-L-aspartyl residue inserted in position 6. The agonist bound to the pituitary still had low endocrinological activity. On the other hand, it exhibited direct antitumor effect in in vitro assays. The endocrinological activity of the antagonist was low; however, it showed potent, direct antitumor activity. These observations might lead to the development of new GnRH analogs with selective antitumor effect.

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.

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)

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.

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.

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.

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.

New Gaba-containing analogues of human growth hormone-releasing hormone (1-30)-amide: I. Synthesis and in vitro biological activity.

Analogues of human growth hormone-releasing hormone (1-30)-amide have been developed. All analogues have been modified in position 27 with Nle and with Gaba in position 30. Additional D-amino-acids have been inserted in the GHRH(1-30)-NH2 sequence: A-1741: Nle27,Gaba30-GH-RH(1-30)-NH2 A-495: D-Ala2,Nle27,Gaba30-GH-RH(1-30)-NH2 A-515: D Ala2,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2 A-527: D-Ala2,D-Arg11,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2. Our analogues were synthesized by solid phase peptide synthesis and were tested is two different in vitro systems and in rat pituitary cell cultures. A-495 and A-1741 were found to be the most active in releasing GH, however they showed different activities in the two different test systems. A-495 exhibited higher potency in the superfusion system (1.63 fold potency of the GHRH (1-29)-amide), while A-1741 evoked higher GH release from cultured pituitary cells (1.5-2.5 times higher than the GH-RH(1-44)-amide). The other analogues (A-515 and A-527) were found to be equipotent to the standard molecule. We can conclude that Nle27 and Gaba30 substitutions appeared to be a good modification in in vitro test systems, and Gaba30 substitution served as a good spacer during the synthesis, since it made the coupling of the C-terminal amino acids easier and produced quantitative coupling. In addition to the advantageous properties in the synthesis these modifications with or without D-Ala at the N-terminus increased the in vitro biological activity to 1.5-2.5 fold of the GHRH molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

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)