Potential of Z-100, extracted from Mycobacterium tuberculosis strain Aoyama B, as a hot tumor inducer.

The tumor microenvironment is one of the most important factors determining the efficacy of cancer immunotherapy. In particular, variability in efficacy has been linked to whether tumors are hot or cold, with hot tumors exhibiting greater T cell infiltration and responding better to immunotherapy. Z-100 extracted from Mycobacterium tuberculosis Aoyama B strain has been reported to increase cytokine production from immune cells. In this study, we examined its effect on the tumor microenvironment and its potential as a hot tumor inducer. The antitumor effect of Z-100 was confirmed in a mouse oral squamous cell carcinoma (Sq-1979) tumor model by starting administration before tumor injection. Treated tumors were collected to identify infiltrating CD8+ T cells. The antitumor effects of Z-100 were additionally examined in mice treated with anti-CD8 antibody and in IL-12p40 knockout (KO) mice. We found that Z-100 had strong antitumor effects and increased the proportion of CD8+ T cells in tumors. Moreover, the CD8+ T cells infiltrating tumors were identified as effector memory CD8+ T cells. Furthermore, the antitumor effects of Z-100 were abolished in mice treated with an anti-CD8 antibody and in IL-12p40 KO mice. Thus, Z-100 induces its antitumor effects by increasing tumor-infiltrating CD8+ T cells, suggesting that Z-100 may be a useful cancer therapy by acting as a hot tumor inducer.

Peptidoglycan-Like Components in Z-100, Extracted from Mycobacterium tuberculosis Strain Aoyama B, Increase IL-12p40 via NOD2.

Background: Z-100 is a hot-water extract of the human-type Mycobacterium tuberculosis strain Aoyama B. While Z-100's macrophage-mediated immunomodulatory effects have been reported, the mechanistic details have not been fully clarified. Here, we studied the immunomodulatory effects of Z-100 on mouse bone marrow-derived cells, human CD14+ cells, and skin. Methods: Mouse bone marrow-derived cells and CD14+ cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor, differentiated into macrophage-like cells, and then stimulated with Z-100. Furthermore, since Z-100 is subcutaneously administered clinically, we injected Z-100 into mice and measured gene expression in the skin. Results: While Z-100 stimulation increased the production of interleukin- (IL-) 12p40 and IL-1ß in mouse bone marrow-derived macrophages, levels of IL-1ß were low. In contrast, TNF-α production did not increase. Meanwhile, stimulation of human CD14+ cells with Z-100 increased production of IL-12p40, TNF-α, and IL-1ß. Because Z-100 appeared to have the most stable effect on IL-12p40, we examined the components of Z-100 that induce IL-12p40 production. We found that Z-100 contained peptidoglycan-like components. In addition, an siRNA study showed that Z-100 increased the production of IL-12p40 via nucleotide-binding oligomerization domain 2 (NOD2). Further, subcutaneous administration of Z-100 to mice significantly elevated expression of IL-12p40 and IL-1ß and showed a trend towards increasing TNF-α in the skin. Conclusion: Z-100 induced the production of immunomodulatory cytokines from various types of macrophages and specifically increased IL-12p40 production through peptidoglycan-like components via NOD2.

Z-100, extracted from Mycobacterium tuberculosis strain Aoyama B, promotes TNF-α production via nucleotide-binding oligomerization domain containing 2 (Nod2)-dependent NF-κB activation in RAW264.7 cells.

Macrophages are a major component of the innate immune system, and the cytokines they secrete are involved in antitumor responses. Z-100 is obtained from hot-water extract of human-type Mycobacterium tuberculosis strain Aoyama B and activates the innate immune response. However, while Z-100 is known to modulate macrophage activity, the mechanism behind this modulation is not fully understood. We evaluated the effects of Z-100 on the murine macrophage cell line RAW264.7. Tumor necrosis factor-alpha (TNF-α) production from RAW264.7 cells was strongly induced by Z-100 and interferon-gamma (IFN-γ) stimulation but only weakly induced by Z-100 alone. Quantitative gene expression analysis showed that nucleotide-binding oligomerization domain containing 2 (Nod2) expression was up-regulated by IFN-γ treatment in RAW264.7 cells while Z-100-induced TNF-α production was attenuated by Nod2 gene silencing. Further, componential analysis demonstrated that muramic acid and amino acids distinctive of muramyl dipeptide (MDP) were contained within Z-100 and Z-100Fr I, the low-molecular-weight fraction containing components <3 kDa in size. In addition, Z-100Fr I enhanced TNF-α production in RAW264.7 cells and promoted NOD2-dependent nuclear factor-kappa B (NF-κB) activation in murine NOD2-expressing SEAP reporter HEK293 (HEK-Blue-mNOD2) cells. Taken together, these results suggest that Z-100 contains MDP-like molecules and augments NF-κB signaling via the direct activation of Nod2 in macrophages, which might be one mechanism driving the innate immune responses induced by Z-100 in cancer immunotherapy.

Z-100, an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B, prevents spontaneous lymphatic metastasis of B16-BL6 melanoma.

Lymphatic metastasis is common in advanced-stage carcinoma and is associated with a poor prognosis. However, few effective treatments to inhibit it are available. Z-100 is an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B that contains polysaccharides such as arabinomannan and mannan. Here, we investigated the inhibitory effect of Z-100 on spontaneous lymphatic metastasis. C57BL/6N mice injected subcutaneously with B16-BL6 melanoma cells in the right hind footpad were administered Z-100 subcutaneously in the right inguinal region on a daily basis. On day twenty-one after the injection, the right inguinal lymph nodes were excised, and the extent of metastasis, the number of immune cells, and the amount of granzyme B protein in the lymph nodes were examined. We also investigated the combined effect of Z-100 and irradiation in this model. Results showed that Z-100 reduced number of animals with metastasis, with respective metastasis rates of 85.7%, 42.9%, 7.1% and 0.0% in saline, 0.1 mg/kg Z-100, 1 mg/kg Z-100 and 10 mg/kg Z-100 group. Further, mice that had been given Z-100 were found to have more immune cells and granzyme B protein in the lymph nodes than control mice. The combination of low dose Z-100 and irradiation also inhibited spontaneous lymph node metastases. These findings suggest that Z-100 may be beneficial in preventing lymphatic metastasis by enhancing the immune response.

Acotiamide hydrochloride (Z-338), a new selective acetylcholinesterase inhibitor, enhances gastric motility without prolonging QT interval in dogs: comparison with cisapride, itopride, and mosapride.

Acotiamide hydrochloride (acotiamide; N-[2-[bis(1-methylethyl) amino]ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl) amino] thiazole-4-carboxamide monohydrochloride trihydrate, Z-338) has been reported to improve meal-related symptoms of functional dyspepsia in clinical studies. Here, we examined the gastroprokinetic effects of acotiamide and its antiacetylcholinesterase activity as a possible mechanism of action in conscious dogs. Acotiamide increased postprandial gastric motor activity in conscious dogs with chronically implanted force transducers and, like itopride, mosapride, and cisapride, exhibited gastroprokinetic activity in these dogs. Furthermore, acotiamide improved clonidine-induced hypomotility and delayed gastric emptying. Acotiamide-enhanced postprandial gastroduodenal motility was suppressed completely by pretreatment with atropine, a muscarinic receptor antagonist. In in vitro studies, acotiamide enhanced acetylcholine- but not carbachol-induced contractile responses of guinea pig gastric antrum strips. Moreover, like itopride and neostigmine, acotiamide inhibited recombinant human and canine stomach-derived acetylcholinesterase (AChE) activity in vitro. The mode of the AChE inhibitory action of acotiamide was selective and reversible. Unlike itopride or mosapride, acotiamide showed no affinity for dopamine D(2) or serotonin 5-HT(4) receptors. With regard to cardiovascular side effects, unlike cisapride, acotiamide did not affect myocardial monophasic action potential duration, QT interval, or corrected QT interval in anesthetized dogs. These results suggest that acotiamide stimulates gastric motility in vivo by inhibiting AChE activity without affecting QT interval. Acotiamide thus represents a beneficial new drug for the treatment of functional dyspepsia involving gastric motility dysfunction, with differences from other prokinetic agents.

Pharmacological evaluation of analgesic effects of the cholecystokinin2 receptor antagonist Z-360 in mouse models of formalin- and cancer-induced pain.

Z-360, a novel cholecystokinin(2) (CCK(2)) receptor antagonist, has been developed as a therapeutic drug for pancreatic cancer and showed pain relief action in phase Ib/IIa clinical trial. This study was attempted to elucidate the analgesic efficacy of Z-360 in mice. Oral administration of Z-360 (30-300 mg/kg) showed a dose-dependent inhibitory effect on the late phase of nociceptive responses to formalin. YF476, another CCK(2) receptor antagonist, was without effects at 1 and 10 mg/kg. In contrast, the CCK(1) receptor antagonist devazepide inhibited the nociceptive responses to formalin. In a mouse model of cancer pain, significant anti-allodynic effect of Z-360 was observed after single and repeated oral administration of 100 and 300 mg/kg doses. Anti-allodynic effect was also observed after repeated administration of devazepide. Combined single treatment with morphine and Z-360 caused an increase inhibition of pain-related responses in the pain models produced by formalin and cancer. Although Z-360 has lower affinity for CCK(1) receptor than for CCK(2) receptor, Z-360 exhibited an inhibitory effect on sulfated CCK-8-induced gallbladder emptying, a CCK(1) receptor-mediated effect, at a dose of 100 mg/kg. These results suggest that Z-360 inhibits inflammatory and cancer pain probably through the blockade of CCK(1) receptors. Z-360 is expected to become a useful drug for the pancreatic cancer with analgesic effects as well as the prolongation of survival.

Polaprezinc, a zinc compound, is distributed to the lingual epithelium and increases its zinc concentration in zinc-deficient rats.

AIMS: To clarify the mechanism underlying the effect of polaprezinc on hypogeusia, we investigated the uptake of polaprezinc by the tongue in rats. MAIN METHODS: Rats were fed a zinc-sufficient (Zn(+)) or zinc-deficient (Zn(-)) diet. After 4weeks on the Zn(-) diet, polaprezinc (1, 3, or 10mg/kg) or [(65)Zn] polaprezinc (10mg/kg) was administered orally once a day. The zinc concentration or the (65)Zn radioactivity of the tongue was measured by inductively-coupled plasma mass spectrometry or gamma counting, respectively. In addition, the distribution of (65)Zn in the tongue was analyzed by microautoradiography and the proliferative activity of taste bud cells was measured from the uptake of 5-bromo-2'-deoxyuridine. KEY FINDINGS: The zinc concentration of the lingual epithelium, but not the whole tongue, was markedly decreased in Zn(-) rats compared with Zn(+) rats. After administration of polaprezinc to Zn(-) rats at doses of 1, 3, and 10mg/kg, the zinc concentration in the lingual epithelium increased significantly from 85+/-4 to 105+/-7 (p<0.05), 120+/-3 (p<0.001), and 124+/-3 (p<0.001) microg/g, respectively. After administration of [(65)Zn] polaprezinc, the (65)Zn radioactivity of the tongue and serum were higher in Zn(-) rats than in Zn(+) rats. (65)Zn was mainly detected in the epithelium on microautoradiograms of the tongue in Zn(-) rats. In addition, polaprezinc (3 and 10mg/kg) improved the reduced proliferation of taste bud cells due to zinc deficiency. SIGNIFICANCE: Polaprezinc is distributed to the lingual epithelium and restores its zinc concentration in Zn(-) rats resulting in improvement of cellular functions, especially proliferation.

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.