Effect of Z-360, a novel orally active CCK-2/gastrin receptor antagonist on tumor growth in human pancreatic adenocarcinoma cell lines in vivo and mode of action determinations in vitro.

PURPOSE: Gastrin is known to enhance the growth of pancreatic carcinoma via the cholecystokinin (CCK)-2/gastrin receptor. We investigated the anti-tumor effect of Z-360 (calcium bis [(R)-(-)-3-[3-{5-cyclohexyl-1-(3,3-dimethyl-2-oxo-butyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl}ureido]benzoate]), a novel orally active CCK-2 receptor antagonist alone or combined with the chemotherapeutic agent, gemcitabine in human pancreatic adenocarcinoma cell lines. RESULTS: Z-360 potently inhibited specific binding of [3H]CCK-8 to the human CCK-2 receptor, with a Ki value of 0.47 nmol/l, and showed antagonistic activity for this receptor. The anti-tumor effect of Z-360 alone or combined with gemcitabine was assessed using subcutaneous xenografts of MiaPaCa2 and PANC-1 and an orthotopic xenograft model (PANC-1). Oral administration of Z-360 significantly inhibited the growth of MiaPaCa2 (41.7% inhibition at 100 mg/kg, P<0.01). Combined administration of Z-360 and gemcitabine significantly inhibited subcutaneous PANC-1 tumor growth compared with either agent alone (27.1% inhibition compared to effect with gemcitabine, P<0.05), and significantly prolonged survival compared with the vehicle control (median survival of 49 days in vehicle compared to 57 days in the combination group, P<0.05). In vitro studies showed that Z-360 significantly inhibited gastrin-induced proliferation of human CCK-2 receptor-expressing cells, and also significantly reduced gastrin-induced PKB/Akt phosphorylation to the level of untreated controls. CONCLUSION: In the present study, we have shown that Z-360 combined with gemcitabine can inhibit pancreatic tumor growth and prolong survival in a pancreatic carcinoma xenograft model, on a possible mode of action being the inhibition of gastrin-induced PKB/Akt phosphorylation through blockade of the CCK-2 receptor. Our results suggest that Z-360 may be a useful adjunct to gemcitabine for the treatment of pancreatic carcinoma and a therapeutic option for patients with advanced pancreatic cancer.

Effect of polaprezinc on taste disorders in zinc-deficient rats.

The effect of polaprezinc, a chelate compound consisting of zinc ion and L-carnosine, on abnormalities of taste sensation induced by feeding a zinc-deficient diet to rats was examined by using the two-bottle preference test (quinine hydrochloride as a bitter taste and sodium chloride as a salty taste). Rats were fed either a zinc-deficient or a zinc-sufficient diet. The zinc-deficient diet increased the preference for both taste solutions, while polaprezinc (at doses of 3 and 10 mg/kg) restored the altered taste preferences. We also evaluated the proliferation of taste bud cells using 5-bromo-2'-deoxyuridine (BrdU). The BrdU incorporation into taste bud cells was significantly reduced in rats fed a zinc-deficient diet compared with rats fed a zinc-sufficient diet (from 50.8% to 45.0%, p<0.05) and this reduction was reversed by polaprezinc at doses of 1, 3, and 10 mg/kg, increasing to 50.2%, 53.5%, and 52.5%, respectively. These findings indicate that zinc deficiency induces the delayed of proliferation of taste bud cells, while polaprezinc improves cell proliferation. In conclusion, polaprezinc had a therapeutic effect in a rat model of abnormal taste sensation. Its mechanism of action was suggested to involve improvement of the decrease in taste bud cell proliferation caused by zinc deficiency.

Inhibition of human immunodeficiency virus type 1 replication by Z-100, an immunomodulator extracted from human-type tubercle bacilli, in macrophages.

Z-100 is an arabinomannan extracted from Mycobacterium tuberculosis that has various immunomodulatory activities, such as the induction of interleukin 12, interferon gamma (IFN-gamma) and beta-chemokines. The effects of Z-100 on human immunodeficiency virus type 1 (HIV-1) replication in human monocyte-derived macrophages (MDMs) are investigated in this paper. In MDMs, Z-100 markedly suppressed the replication of not only macrophage-tropic (M-tropic) HIV-1 strain (HIV-1JR-CSF), but also HIV-1 pseudotypes that possessed amphotropic Moloney murine leukemia virus or vesicular stomatitis virus G envelopes. Z-100 was found to inhibit HIV-1 expression, even when added 24 h after infection. In addition, it substantially inhibited the expression of the pNL43lucDeltaenv vector (in which the env gene is defective and the nef gene is replaced with the firefly luciferase gene) when this vector was transfected directly into MDMs. These findings suggest that Z-100 inhibits virus replication, mainly at HIV-1 transcription. However, Z-100 also downregulated expression of the cell surface receptors CD4 and CCR5 in MDMs, suggesting some inhibitory effect on HIV-1 entry. Further experiments revealed that Z-100 induced IFN-beta production in these cells, resulting in induction of the 16-kDa CCAAT/enhancer binding protein (C/EBP) beta transcription factor that represses HIV-1 long terminal repeat transcription. These effects were alleviated by SB 203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), indicating that the p38 MAPK signalling pathway was involved in Z-100-induced repression of HIV-1 replication in MDMs. These findings suggest that Z-100 might be a useful immunomodulator for control of HIV-1 infection.

Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, augments anti-tumor activities of X-ray irradiation against B16 melanoma in association with the improvement of type 1T cell responses.

In this study, the effects of combination therapy consisting of X-ray irradiation and Z-100 on the survival time of C57BL/6 mice inoculated with B16F10 melanoma were investigated. Survival time was significantly prolonged in B16F10 melanoma-bearing mice treated with the X-ray irradiation (5 Gy) and Z-100 (10 mg/kg s.c.) combination therapy compared with mice irradiated with X-rays alone. The weight of primary tumors and number of metastatic colonies were also significantly suppressed by the combination therapy compared with that in the X-ray irradiation group. These results indicated that Z-100 could enhance the anti-tumor effects of radiotherapy against B16F10 melanoma. On the other hand, the survival time of CD4 knockout mice bearing the same tumors was not prolonged by the combination therapy compared with mice irradiated with X-rays alone, suggesting that CD4+ cells are partly involved in augmentation of the anti-tumor effect of radiotherapy by Z-100. In addition, type 1 cytokine (IL-2, IFN-gamma) production was significantly increased and type 2 cytokine (IL-4, IL-10) production was significantly suppressed in the tumor-bearing mice treated with the combination therapy compared with the X-ray irradiation group. Moreover, interleukin-12 production by CD11c+ cells was also significantly increased in mice treated with the combination therapy compared with the X-ray irradiation group. These results indicate that Z-100 augmented the anti-tumor effects of X-ray irradiation. Moreover, we demonstrated that the effects of Z-100 were expressed at least in part, by the improvement of the T cell responses from type 2-dominant to type 1-dominant via up-regulation of IL-12 production.

Antimetastatic effect of an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, Z-100, through the production of interleukin-12.

In this study, the role of interleukin (IL)-12 on the antimetastatic effect of Z-100 was investigated using wild-type C57BL/6 mice or IL-12p40 knockout (IL-12p40 KO) mice inoculated with highly metastatic B16F10 melanoma. When C57BL/6 mice were inoculated with B16F10 melanoma (2x10(5) cells/mouse i.v.), Z-100 (10 mg/kg i.p.) significantly suppressed the pulmonary metastasis of B16F10 melanoma 14 d after tumor inoculation. On the other hand, the antimetastatic effect of Z-100 was not observed in IL-12p40 KO mice inoculated with B16F10 melanoma. These results indicate that IL-12 is essentially required for the appearance of the antimetastatic effect of Z-100. Since helper T (Th) 2 cell responses have been reported to have a role in tumor metastasis, the regulatory effect of Z-100 on the immune balance of Th1/Th2 cell responses was investigated. In both C57BL/6 mice and IL-12p40 KO mice bearing B16F10 melanoma, Th1 cytokine production (IL-2, interferon-gamma) was significantly suppressed as compared with those in normal mice. On the other hand, Th2 cytokine production (IL-4, IL-10) in these mice was increased. The administration of Z-100 (10 mg/kg i.p.) in C57BL/6 mice bearing B16F10 melanoma improved the balance of Th1/Th2 cell responses from the Th2-dominant state to the normal state. However, the improvement of Th1/Th2 cell responses by Z-100 was not observed in IL-12p40 KO mice bearing the same tumors. In addition, Z-100 significantly increased IL-12 production by macrophages in a concentration-dependent manner, while Z-100 significantly decreased IL-10 production by these cells in vitro. These results suggested that up-regulation of IL-12 production and down-regulation of IL-10 production by Z-100 are related to the improvement of Th1/Th2 cell responses from the Th2-dominant state to the normal state, which resulted in suppression of tumor metastasis.

Anti-tumor mechanism of Z-100, an immunomodulatory Arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, on pulmonary metastases of B16F10 melanoma: restoration of helper T cell responses via suppression of glucocorticoid-genesis.

In the present study, the anti-tumor mechanism of Z-100 was investigated with the use of pulmonary metastasis of B16F10 melanoma. In B16F10 mice, Th1 cytokine production (IL-2, IFN-gamma) was suppressed in comparison with normal mice. On the other hand, Th2 cytokine production (IL-4, IL-10) was increased in the B16F10 mice. The administration of Z-100 to B16F10 mice restored the balance of Th1/Th2 cell responses from the Th2 dominant state to the normal state. Z-100 significantly suppressed the pulmonary metastasis of B16F10 melanoma in a dose-dependent manner. These results suggest that Z-100 restored the breakdown of Th1 cell responses, resulting in the suppression of pulmonary metastasis of B16F10 melanoma. Moreover, Z-100 decreased the corticosterone levels, which is known to suppress the Th1 cell responses, in both serum specimens and splenic tissue, and the steroidogenic CYP11A1 mRNA expression in CD4+ T cells. These results suggest that a suppression of pulmonary metastasis and restoration of Thl/Th2 cell responses by Z-100 may be due to the decrease in the corticosterone levels and the steroidogenic CYP11A1 mRNA expression of CD4+ T cells in B16F10 mice. Further, the role of Th1 cytokine, IFN-gamma, on these activities of Z-100 was examined. The suppressive effects of Z-100 on pulmonary metastasis and restoration of Th1/Th2 cell responses were eliminated by the administration of anti-IFN-gamma mAb. Moreover, the suppressive effects of Z-100 on glucocorticoid-genesis were eliminated by the administration of anti-IFN-gamma-mAb. These results suggest that Z-100 restores the balance of Th1/Th2 cell responses via the suppression of glucocorticoid-genesis by Z-100-induced IFN-gamma. IFN-gamma acts as a key cytokine in anti-tumor activities of Z-100.