Phase I Study of Pazopanib and Ixabepilone in Patients With Solid Tumors.

OBJECTIVES: Pazopanib is a tyrosine kinase inhibitor predominantly acting on tumor endothelium, and ixabepilone is a semisynthetic analog of epothilone B that promotes microtubule stabilization inducing tumor and tumor endothelial cell apoptosis. The purpose of this study was to determine the optimal tolerated dose (OTD) of the combination of pazopanib and ixabepilone for the treatment of metastatic previously treated solid tumors. METHODS: Dose escalation started at 32 mg/m of ixabepilone and increased to 40 mg/m. Pazopanib was administered initially at 400 mg and escalated at 200 mg increments up to 800 mg. Pharmacokinetic analysis assessed effect of ixabepilone on pazopanib metabolism. Correlative studies evaluated changes in angiogenic cytokines. RESULTS: Thirty-one patients (20 male and 11 female; median age, 58 y) with ECOG PS of 0 or 1 were enrolled. Three patients had dose-limiting toxicities (fatigue and neutropenia) at dose level 2 (ixabepilone 40 mg/m and pazopanib 400 mg), and therefore the ixabepilone dose was decreased (32 mg/m) before escalating pazopanib to levels 3 and 4. One patient had a dose-limiting toxicity (thrombocytopenia) at dose level 4 (ixabepilone 32 mg/m and pazopanib 800 mg). Dose level 3 was determined to be the OTD (pazopanib 600 mg and ixabepilone 32 mg/m). The most common toxicities were cytopenias. A significant decrease in the level of sE-selectin was associated with improvement in progression free survival. CONCLUSIONS: The OTD for combination of pazopanib and ixabepilone was established. There was no impact of ixabepilone on pazopanib pharmacokinetics. The relationship between sE-selectin and progression free survival warrants further investigation.

Whole glucan particles as a vaccine against systemic coccidioidomycosis.

We reported previously that yeast-derived whole glucan particles (WGPs), with or without conjugation to BSA, used as a vaccine protected against systemic aspergillosis in mice. Here, we examined their utility as a potential vaccine against coccidioidomycosis. WGPs were prepared from Saccharomyces cerevisiae; conjugation with BSA (WGP-BSA) was done using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated conjugation. Heat-killed S. cerevisiae (HKY) was used as a positive-control vaccine. CD-1 mice were vaccinated with WGPs or WGP-BSA, HKY or PBS once weekly, beginning 21 days prior to infection. Mice were infected intravenously with arthroconidia of Coccidioides posadasii. In the low-mortality study, 50 % of PBS-treated controls died. Only WGP-BSA at 0.6 mg per dose induced significant protection compared with PBS treatment. All surviving mice were infected in all three organs examined. Those given WGP-BSA at 0.6 mg per dose had fewer c.f.u. in liver and lungs (P = 0.04), and those given WGPs at 6 mg per dose had fewer in lungs (P < 0.02), compared with PBS. In the high-mortality study, 90 % of PBS mice died. Vaccination with HKY, and WGPs or WGP-BSA at 6 or 12 mg per dose significantly prolonged survival (P ≤ 0.05). No surviving mice were free of infection. HKY and WGP-BSA at 12 mg per dose reduced c.f.u. in the liver and lungs (P < 0.05) and WGP-BSA at 6 mg per dose reduced c.f.u. in the lungs (P < 0.05); unconjugated WGPs did not reduce infection. WGPs or WGP-BSA acted as a vaccine that protected against mortality caused by coccidioidomycosis. Thus, WGP protection against coccidioidomycosis and aspergillosis provides the basis for development of a pan-fungal vaccine.

Whole glucan particles as a vaccine against murine aspergillosis.

Vaccination with heat-killed Saccharomyces cerevisiae (HKY) protects against experimental infection by pathogenic fungi of five genera. Here we tested whether purified Saccharomyces cell wall ß-glucan could induce protection against systemic aspergillosis. CD-1 mice were given three weekly vaccine doses subcutaneously prior to intravenous infection with Aspergillus fumigatus. Mice received PBS, 2.5 mg HKY, whole glucan particles (WGP), WGP conjugated to BSA (0.06 to 12 mg per dose), a soluble medium molecular mass (MMW) ß-glucan alone or MMW-BSA (≤24 mg per dose). Survival and c.f.u. were determined, and cytokine induction and anti-ß-glucan antibodies were assessed in vaccinated mice. Neither soluble MMW glucan, nor MMW-BSA was effective. HKY protected in two studies (survival and c.f.u. were reduced in brain and kidney organs, P<0.004). Six or 12 mg WGP or WGP-BSA prolonged survival (P≤0.004) and reduced c.f.u. in each organ (P≤0.015) in both experiments; 0.6 mg WGP or WGP-BSA prolonged survival (P≤0.015) and reduced c.f.u. (P≤0.015) in one experiment. Cytokine profiles in serum and bronchoalveolar lavage from uninfected vaccinated mice showed an innate and adaptive immune profile (i.e. upregulation of colony stimulating factors, interferons, TNF-α, chemokines such as MCP-1, MIP-1α, RANTES and KC, and Th17-activating cytokines such as IL-6, IL-1ß, IL-17). No anti-ß-glucan antibodies were in the sera, suggesting an adaptive T cell-mediated, not a B cell-mediated, protective response. Vaccination with WGP or WGP-BSA proved protective against systemic aspergillosis, equivalent to that of HKY, supporting the potential of particulate ß-glucans, alone or conjugated, as vaccines against aspergillosis.

Potentiation of the anti-tumor effects of imidazoquinoline immune response modifiers by cyclophosphamide.

The aim of the current study was to evaluate the influence of chemotherapeutic drugs on immunotherapy with Imidazoquinoline Toll-like Receptor (TLR) agonists in cancer. First, the previously described antitumor efficacy of TLR agonists [i.e. a TLR7 agonist (852A) and a dual TLR7/8 agonist (3M-011)] was confirmed in additional cancer models, and second the therapeutic potential of TLR agonists in combination with cyclophosphamide was investigated. The antitumor potential was evaluated against a panel of syngeneic tumor models; namely B16-F10 melanoma, M3 melanoma and MC-26 colon carcinoma. Systemic administration of either 3M-011 or 852A in these various syngeneic models induced significant antitumor activity as evidenced by delays in tumor growth curves. Combination of cyclophosphamide with either 3M-011 or 852A demonstrated that cyclophosphamide does not negatively interfere with the TLR agonist's antitumor effects, but may, depending on the dosing schedule, to actually potentiate the effect. These findings suggest that the immunomodulatory TLR agonists may be used in combination with cytotoxic agents in the treatment of cancer.

Effect of 3M-003, an imidazoquinoline, on phagocyte candidacidal activity directly and via induction of peripheral blood mononuclear cell cytokines.

3M-003, like related imidazoquinoline immunomodulators, interacts with Toll-like receptor-7 (TLR-7) and TLR-8. TLRs are important in the defense against fungal pathogens. The effect of 3M-003 on killing of Candida was evaluated on mouse (BALB/c) effector cell lineages: monocytes, neutrophils, and macrophages. After direct application, 3M-003 (1-80 microg mL(-1)) enhanced (P<0.05-0.01) macrophage killing, comparable to killing by interferon-gamma-activated macrophages. 3M-003 did not directly enhance the candidacidal activity of monocytes or neutrophils. To test an effect mediated by leukocytes, BALB/c peripheral blood mononuclear cells (PBMC) were stimulated in vitro with 3M-003 to generate cytokine-containing supernatants. 3M-003 at 1 or 3 microM was optimal for the stimulation of PBMC to produce tumor necrosis factor-alpha and interleukin-12p40 in 24 h. For indirect tests, monolayers were treated with supernatants for 18 h, the supernatants were removed, and effector cells were tested; the supernatants enhanced (P<0.05-0.01) killing, in 2-4-h assays, by neutrophils from 42% to 73%, macrophages from 0% to 23%, and monocytes from 0% to 20%. 3M-003, presumably through TLRs, acts directly on macrophages to enhance fungal killing and stimulates PBMC to produce soluble factors that enhance killing by neutrophils, macrophages, and monocytes. 3M-003 could be a candidate for antifungal immunotherapy.

NK1.1+ cells mediate the antitumor effects of a dual Toll-like receptor 7/8 agonist in the disseminated B16-F10 melanoma model.

Innate immune stimulation with Toll-like receptor (TLR) agonists is a proposed modality for immunotherapy of melanoma. Here, a TLR7/8 agonist, 3M-011, was used effectively as a single systemic agent against disseminated mouse B16-F10 melanoma. The investigation of the mechanism of antitumor action revealed that the agonist had no direct cytotoxic effects on tumor cells tested in vitro. In addition, 3M-011 retained its effectiveness in scid/B6 mice and scid/NOD mice, eliminating the requirement for T and B cells, but lost its activity in beige (bg/bg) and NK1.1-immunodepleted mice, suggesting a critical role for natural killer (NK) cells in the antitumor response. NK cytotoxicity was enhanced in vivo by the TLR7/8 agonist; this activation was long lasting, as determined by sustained expression of the activation marker CD69. Also, in human in vitro studies, 3M-011 potentiated NK cytotoxicity. TLR7/8-mediated NK-dependent antitumor activity was retained in IFN-alpha/beta receptor-deficient as well as perforin-deficient mice, while depletion of IFN-gamma significantly decreased the ability of 3M-011 to delay tumor growth. Thus, IFN-gamma-dependent functions of NK cell populations appear essential for cancer immunotherapy with TLR7/8 agonists.