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Human small-cell lung-cancer cells (SCLC) produce and secrete gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BN). There is some evidence to suggest that GRP is an autocrine regulator of SCLC cell growth. In the search for potent BN antagonists, several substance-P (SP) analogs were found to inhibit the growth of SCLC cells. We found that a known short-chain SP antagonist, pHOPA-DTrp-Phe-DTrp-Leu-Leu-NH2(NY3238), inhibits the binding of 125I-Tyr4-BN on Swiss 3T3 cell line expressing BN receptors, as well as the proliferation of NCI-H69 SCLC cells. In this study we tested several analogs of NY3238 and we found that NY3521 and NY3460 are more effective in inhibition of proliferation of SCLC cells but less potent in inhibition of binding of 125I-Tyr4-BN on Swiss 3T3 cells than NY3238. Furthermore, we detected specific binding of radiolabelled NY3238 even below 1 nM on NCI-H69 cells that could have been inhibited by SP and NY3460 rather than by BN. In addition to these in vitro studies, NY3460 proved to be effective in inhibiting the growth of NCI-H69 SCLC xenografts in nude mice in vivo. These analogs of NY3238 could be promising therapeutic agents in the treatment of SCLC.

The thymopoietin-type tripeptides TP3 (HArg-Lys-AspOH), TP(D-Asp)3(HArg-Lys-D-AspOH) and tetrapeptide TP4 (HArg-Lys-Asp-ValOH) were studied by one- and two-dimensional, 500 MHz 1H-NMR spectroscopy in H2O and D2O solutions at four different pH values. All proton resonances of the three oligopeptides were assigned by two-dimensional phase-sensitive TOCSY experiments at pH 12.2, 9.1, 5.9 and 3.6. At these pH-values well-defined stages of protonation and concomitant molecular charges exist, allowing different possibilities for intra-molecular and inter-residual orientations. Conformation-sensitive rotating frame nuclear Overhauser enhancement (ROESY) two-dimensional experiments were also performed at the above pH values. These experiments indicated no definite solution conformation of any of the molecules at any pH. Standard one-dimensional experiments were also carried out and three-bond coupling constants were measured for the NH--CH and the Asp CH--CH moieties. The coupling constants provided evidence that non-statistical orientations of the functional groups exist which are changed upon protonation of the basic sites.

Seventeen peptides, related to thymopoietin pentapeptide, L-arginyl-L-lysyl-L-aspartyl-L-valyl-L-tyrosine (thymopentin) were synthesized by the stepwise strategy in solution. Of these, L-arginyl-L-lysyl-L-aspartic acid and L-arginyl-L-lysyl-L-aspartyl-L-valine, shortened from the C-terminus of the pentapeptide, exhibit significant immuno-stimulating potencies, exceeding those of thymopentin, both in vitro and in vivo immunological tests. Studies on the structure-activity relationships suggest that the potencial active site of thymopoietins is very sensitive to the N- and C-terminal elongations of the peptide chain. For thymic hormones, an active site carrying cumulative chemical signals is proposed instead of well-defined active centers.

The immunostimulatory action of oligopeptides RGH-0205 (Arg-Lys-Asp), RGH-0206 (Arg-Lys-Asp-Val) and TP-5 (Arg-Lys-Asp-Val-Try) was measured using the B10LP to C57BL skin graft system and the determination of splenic T cell ratio. While thymectomy increased the period between skin grafting and rejection, each of the oligopeptides increased the number of splenic T cells and in some extent restored the rejection capacity of thymectomised C57Bl mice, presumably by restoration of thymic hormone function.

The immunomodulatory activities of new synthetic thymopoietin derivates TP4 (Arg-Lys-Asp-Val) and TP3 (Arg-Lys-Asp) have been compared to those of TP5 (Arg-Lys-Asp-Val-Tyr) which exhibits most of the biological activity of the native hormone and probably represents the active site. Both TP4 and TP3 are shown to exert similar immunomodulatory activities to TP5 affecting both humoral and cellular responses. Primary and secondary antibody responses of high responder mice were enhanced whilst the intensity of DTH reactivity was decreased. The effect on humoral immunity was particularly apparent following administration of TP4 or TP3 to mice undergoing primary antibody responses following immunization with sub-optimal doses of antigen or suppression by CY treatment. Administration of peptide(s) elicited DTH responses in mice previously shown to exhibit genetically determined unresponsiveness: in these animals antibody responses were not modulated. The data may be interpreted that the tetra- and tri-peptide representing the N-terminal sequence of TP5 possess immunomodulatory activity which is in many aspects similar to that of TP5. The experimental systems and protocols employed are shown to be appropriate for investigating the effect(s) of potential immunomodulators on humoral and cellular immunity.