In the search for selective and long-acting analogs of somatostatin, nearly 200 compounds were synthesized by solid-phase methods, purified, and tested biologically. Among these octapeptides, some contained N-terminal D-Phe, Ac-D-Phe, or AcPhe followed by hexapeptide showed a prolonged duration of action and were able to inhibit growth hormone release for at least 3 hr. Analogs of both Phe-3/Thr-6 and Tyr-3/Val-6 classes also suppressed the release of insulin and glucagon in rats and pentagastrin-induced secretion of gastric acid in dogs, but their potencies in these tests were much smaller than the growth-hormone-release inhibitory activity. Some of these analogs possessed antitutnor activities as shown by the inhibition of growth of animal models of prostate, mammary, and ductal pancreatic tumors.The tetradecapeptide somatostatin (also called somatostatin -14) is of little therapeutic value since it has a broad spectrum of biological actions and a short half-life (1). Conformational analyses and structure-function studies on somatostatin analogs indicate that the sequence required for biological activity consists of the B-turn fragment Phe-Trp-Lys-Thr corresponding to the residues 7-10 of somatostatin (2, 3). Many somatostatin analogs with smaller and more rigid rings have been designed and synthesized in the search for compounds with selective, enhanced, and prolonged activity (2-6). Veber and co-workers (3, 4) reported that cyclic hexapeptide analogs cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) and cyclo(N-MeAla-Phe-D-Trp-Lys-Thr-Phe) were highly active in tests on the inhibition of growth hormone (GH), insulin, and glucagon release. More recently, the same group synthesized a new hexapeptide, cyclo(N-MeAla-Tyr-D-Trp-LysVal-Phe), containing tyrosine and valine in positions corresponding to the residues 7 and 10, respectively, of somatostatin, and found it to be 50-100 times more potent than the parent molecule (5). Bauer et al. (6) synthesized another series of highly potent octapeptide analogs of somatostatin. They retained the sequence of residues 7-10 of somatostatin, Phe-Trp-Lys-Thr, and incorporated this sequence with the tryptophan residue in the D configuration into a series of cystine-bridged analogs of which D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol) (code no. SMS 201-995) containing a C-terminal amino alcohol was the most active (6). This analog has been subjected to careful clinical evaluation (7,8).Recently, nearly 200 octapeptide amide analogs of somatostatin related to compound SMS 201-995 made by Bauer et al. (6) have been synthesized by solid-phase methods in our laboratory (9, 10). This paper reports the synthesis and the evaluation of biological activities of some of these analogs. MATERIALS AND METHODSSynthesis. The analogs were synthesized in a Beckman model 990 automatic peptide synthesizer using standard solid-phase procedures (11). Benzhydrylamine resin (0.50 mmol/g) was used as starting material. Amino acids were coupled as their NV-tert-butoxycarbonyl (Boc) derivatives, and reactive side ch...

To create cytotoxic hybrid analogs of somatostatin (

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1–29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. “Agmatine analogs”, MR-356 (N-Me-Tyr1-JI-38), MR-361(N-Me-Tyr1, D-Ala2-JI-38) and MR-367(N-Me-Tyr1, D-Ala2, Asn8-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr1, showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr1 and Arg29-NHCH3 as in MR-403 (N-Me-Tyr1, D-Ala2, Arg29 -NHCH3 -JI-38), MR-406 (N-Me-Tyr1, Arg29 -NHCH3 -JI-38) and MR-409 (N-Me-Tyr1, D-Ala2, Asn8, Arg29-NHCH3 -JI-38), and MR-410 (N-Me-Tyr1, D-Ala2, Thr8, Arg29-NHCH3 -JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa30-NH2 such as MR-326 (N-Me-Tyr1, D-Ala2, Arg29, Apa30-NH2 -JI-38), and with Gab30 -NH2, as MR-502 (D-Ala2, 5F-Phe6, Ser28, Arg29, Gab30 -NH2 -JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

Gastric cancer (GC) ranks as the fourth most frequent in incidence and second in mortality among all cancers worldwide. The development of effective treatment approaches is an urgent requirement. Growth hormone-releasing hormone (GHRH) and GHRH receptor (GHRH-R) have been found to be present in a variety of tumoral tissues and cell lines. Therefore the inhibition of GHRH-R was proposed as a promising approach for the treatment of these cancers. However, little is known about GHRH-R and the relevant therapy in human GC. By survival analyses of multiple cohorts of GC patients, we identified that increased GHRH-R in tumor specimens correlates with poor survival and is an independent predictor of patient prognosis. We next showed that MIA-602, a highly potent GHRH-R antagonist, effectively inhibited GC growth in cultured cells. Further, this inhibitory effect was verified in multiple models of human GC cell lines xenografted into nude mice. Mechanistically, GHRH-R antagonists target GHRH-R and down-regulate the p21-activated kinase 1 (PAK1)-mediated signal transducer and activator of transcription 3 (STAT3)/nuclear factor-κB (NF-κB) inflammatory pathway. Overall, our studies establish GHRH-R as a potential molecular target in human GC and suggest treatment with GHRH-R antagonist as a promising therapeutic intervention for this cancer.GHRH receptor | GHRH-R antagonist | PAK1 | stomach cancer | prognostic predictor

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We investigated the effect of bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095 and other analogs on the growth of Hs746T human gastric cancer cells implanted in nude mice or cultured in vitro and on the binding of bombesin to its receptors. Nude mice bearing xenografts of the Hs746T cell line received s.c. injections of RC-3095 (10 micrograms twice daily) or the vehicle (control) for 21 days. Administration of antagonist RC-3095 inhibited the growth of Hs746T tumors. Treatment with RC-3095 produced a significant decrease in tumor volume, prolonged the tumor volume doubling time from 3.6 days to 5.1 days, and decreased the tumor growth rate by 76.9%. The tumor growth delay time in mice treated with RC-3095 was 2.8 days. Treatment with RC-3095 also decreased the final tumor weight by 88.3% and reduced DNA and protein contents in tumors by 91.5% and 89.5%, respectively, as compared to controls. The presence of specific receptors for bombesin/GRP was investigated on the crude membranes of implanted tumors of Hs746T cells. Saturation binding assays showed that the binding of [125I-Tyr4]bombesin to the membranes was saturable and reversible. Scatchard analysis indicated the presence of a single class of binding sites with a high affinity (Kd = 0.24 +/- 0.07 nM) and a low binding capacity (Bmax = 57.0 +/- 0.9 fmol/mg protein). In displacement studies, the binding of [125I-Tyr4]bombesin was inhibited in a dose-dependent manner by unlabelled bombesin(1-14), [Tyr4]-bombesin and GRP (14-27), but not by structurally unrelated peptides. Synthetic bombesin/GRP antagonists RC-3095, RC-3110, and RC-3950-II were all able to inhibit effectively the binding of [125I-Tyr4]bombesin to the membranes of Hs746T cells. RC-3950-II showed a higher binding affinity for bombesin receptors than RC-3095 or RC-3110. Addition of the non-hydrolyzable guanine-nucleotide analog GTP [S] to the binding buffer caused a significant reduction in the amount of [125I-Tyr4]bombesin bound to the cells, indicating that the bombesin receptor is coupled to a G-protein. In cell cultures, bombesin significantly stimulated the growth of Hs746T cells in vitro as shown by an increase in the uptake of [3H]thymidine. Bombesin antagonist RC-3095 could effectively inhibit the bombesin-stimulated growth of Hs746T cells in cultures. These observations suggest that bombesin/GRP may act as growth factors through specific receptors present on the membranes of Hs746T cells. Bombesin/GRP antagonists appear to nullify the effects of bombesin/GRP and may be useful for the treatment of gastric cancers.

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Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 μg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.