Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8+ T-cell axis.

Activation of an anticancer innate immune response is highly desirable because of its inherent ability to generate an adaptive antitumor T-cell response. However, insufficient safety of innate immune modulators limits clinical use to topical applications. Toll-like receptor 5 (TLR5) agonists are favorably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in a number of animal models. Here, we decipher the molecular and cellular events underlying the metastasis suppressive activity of entolimod, a clinical stage TLR5 agonist that activates NF-κB-, AP-1-, and STAT3-driven immunomodulatory signaling pathways specifically within the liver. Used as a single agent in murine colon and mammary metastatic cancer models, entolimod rapidly induces CXCL9 and -10 that support homing of blood-borne CXCR3-expressing NK cells to the liver predominantly through an IFN-γ signaling independent mechanism. NK cell-dependent activation of dendritic cells is followed by stimulation of a CD8(+) T-cell response, which exert both antimetastatic effect of entolimod and establishment of tumor-specific and durable immune memory. These results define systemically administered TLR5 agonists as organ-specific immunoadjuvants, enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist.

Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.

Synthesis and Biological Evaluation of Novel 3'-Difluorovinyl Taxoids.

A series of 3'-difluorovinyl taxoids with C10 modifications, as well as those with C2 and C10 modifications, were strategically designed to block the metabolism by cytochrome P-450 3A4 enzyme and synthesized. These novel difluorovinyl taxoids were evaluated for their cytotoxicity against drug-sensitive human breast (MCF7), multidrug-resistant (MDR) human ovarian (NCI/ADR), human colon (HT-29) and human pancreatic (PANC-1) cancer cell lines. 3'-Difluorovinyl taxoids exhibit several to 16 times better activity against MCF7, HT-29 and PANC-1 cell lines and up to three orders of magnitude higher potency against NCI/ADR cell line as compared to paclitaxel. Structure-activity relationship study shows the critical importance of the C2 modifications on the activity against MDR cancer cell line, while the C10 modifications have a rather minor effect on the potency with some exceptions. The effect of the C2 modifications on potency against MCF7 cell line increases in the following order: H < F < Cl

Design and synthesis of de novo cytotoxic alkaloids by mimicking the bioactive conformation of paclitaxel.

Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics.

OT-82, a novel anticancer drug candidate that targets the strong dependence of hematological malignancies on NAD biosynthesis.

Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.

Use of an anti-vascular endothelial growth factor antibody in a pharmacokinetic strategy to increase the efficacy of intraperitoneal chemotherapy.

The efficacy of intraperitoneal chemotherapy for ovarian cancers is limited by poor penetration of drug into peritoneal tumors. Based on pharmacokinetic theory that suggests that penetration depth is primarily determined by the rate of drug removal via tumor capillaries, we have hypothesized that co-administration of antiangiogenic therapy will allow for decreased drug removal, increased drug concentrations in tumor, and increased efficacy of intraperitoneal chemotherapy. Pharmacokinetic modeling was conducted to simulate the effect of tumor blood flow on tumor concentrations of topotecan. Simulations predicted that tumor blood flow reductions, as potentially achieved by antiangiogenic therapy, would lead to substantial increases in tumor concentrations after intraperitoneal chemotherapy but would lead to a slight decrease after systemic chemotherapy. Pharmacokinetic studies performed using the A2780 xenograft tumor model showed that animals receiving combined intraperitoneal topotecan and an anti-vascular endothelial growth factor (VEGF) monoclonal antibody had approximately 6.5-fold higher (p = 0.0015) tumor topotecan concentrations compared with animals receiving intraperitoneal topotecan alone, whereas there was no significant (p = 0.16) difference for systemic topotecan. Therapeutic studies conducted with two different drugs, topotecan and cisplatin, showed that animals receiving combined intraperitoneal chemotherapy and anti-VEGF therapy displayed superior survival relative to animals treated with chemotherapy alone (i.e., cisplatin or topotecan), anti-VEGF alone, or intravenous chemotherapy with concomitant anti-VEGF therapy. Combined intraperitoneal topotecan and anti-VEGF resulted in the complete cure of four of 11 mice. The proposed combination of antiangiogenic therapy and intraperitoneal chemotherapy, which was predicted to be beneficial by pharmacokinetic simulations, may provide substantial benefit to patients with peritoneal malignancies.

Design, synthesis, and biological evaluation of new-generation taxoids.

Novel second-generation taxoids with systematic modifications at the C2, C10, and C3'N positions were synthesized and their structure-activity relationships studied. A number of these taxoids exhibited exceptionally high potency against multidrug-resistant cell lines, and several taxoids exhibited virtually no difference in potency against the drug-sensitive and drug-resistant cell lines. These exceptionally potent taxoids were termed "third-generation taxoids". 19 (SB-T-1214), 14g (SB-T-121303), and 14i (SB-T-1213031) exhibited excellent activity against paclitaxel-resistant ovarian cancer cell lines with mutations in beta-tubulin as well, wherein the drug resistance is mediated by the beta-tubulin mutation. These taxoids were found to possess exceptional activity in promoting tubulin assembly, forming numerous very short microtubules similar to those formed by discodermolide. Taxoids 19 and 14g also showed excellent cytotoxicity against four pancreatic cancer cell lines, expressing three to four multidrug-resistant genes. Moreover, taxoid 19 exhibited excellent in vivo efficacy against highly drug-resistant CFPAC-1 pancreatic as well as DLD-1 human colon tumor xenografts in mice.

Design, synthesis, and biological evaluation of novel C14-C3'BzN-linked macrocyclic taxoids.

Novel macrocyclic paclitaxel congeners were designed to mimic the bioactive conformation of paclitaxel. Computational analysis of the "REDOR-Taxol" structure revealed that this structure could be rigidified by connecting the C14 position of the baccatin moiety and the ortho position of C3'N-benzoyl group (C3'BzN), which are ca. 7.5 A apart, with a short linker (4-6 atoms). 7-TES-14beta-allyloxybaccatin III and (3R,4S)-1-(2-alkenylbenzoyl)-beta-lactams were selected as key components, and the Ojima-Holton coupling afforded the corresponding paclitaxel-dienes. The Ru-catalyzed ring-closing metathesis (RCM) of paclitaxel-dienes gave the designed 15- and 16-membered macrocyclic taxoids. However, the RCM reaction to form the designed 14-membered macrocyclic taxoid did not proceed as planned. Instead, the attempted RCM reaction led to the occurrence of an unprecedented novel Ru-catalyzed diene-coupling process, giving the corresponding 15-membered macrocyclic taxoid (SB-T-2054). The biological activities of the novel macrocyclic taxoids were evaluated by tumor cell growth inhibition (i.e., cytotoxicity) and tubulin-polymerization assays. Those assays revealed high sensitivity of cytotoxicity to subtle conformational changes. Among the novel macrocyclic taxoids evaluated, SB-T-2054 is the most active compound, which possesses virtually the same potency as that of paclitaxel. The result may also indicate that SB-T-2054 structure is an excellent mimic of the bioactive conformation of paclitaxel. Computational analysis for the observed structure-activity relationships is also performed and discussed.

High-resolution magnetic resonance imaging of the efficacy of the cytosine analogue 1-[2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl]-N(4)-palmitoyl cytosine (CS-682) in a liver-metastasis athymic nude mouse model.

High-resolution magnetic resonance (MR) imaging techniques in a liver metastatic mouse model were used to assess CS-682, a novel 2'-deoxycytidine analogue of 1-[2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl]-N(4)-palmitoyl cytosine. The efficacy of CS-682 was visualized in real time by MR imaging of initial seeding and subsequent growth of liver metastases. The relative therapeutic efficacies of CS-682 and two agents used clinically, gemcitabine [2'-deoxy-2',2'-difluorocytidine monohydrochloride (DFDC)] and 5-fluorouracil (5-FU), were compared in this model. CS-682 was found to exhibit superior efficacy by delaying the onset and inhibiting the growth of liver metastasis compared with gemcitabine, 5-FU, and control. The overall occurrence of metastases was decreased 62% by CS-682, 18% by DFDC, and 35% by 5-FU. CS-682 increased the life span of the treated animals significantly, by 28 days above the 29-day median survival without treatment, compared with 11 days by DFDC and 14 days by 5-FU. The increased survival in CS-682-treated animals correlated with the antimetastatic activity of this compound. These preclinical findings support the potential clinical utility of CS-682 in the treatment of liver metastasis.

Toll-like receptor-5 agonist Entolimod broadens the therapeutic window of 5-fluorouracil by reducing its toxicity to normal tissues in mice.

Myelosuppression and gastrointestinal damage are common side effects of cancer treatment limiting efficacy of DNA-damaging chemotherapeutic drugs. The Toll-like receptor 5 (TLR5) agonist Entolimod has demonstrated efficacy in mitigating damage to hematopoietic and gastrointestinal tissues caused by radiation. Here, using 5-Fluorouracil (5-FU) treated mice as a model of chemotherapy-induced side effects, we demonstrated significant reduction in the severity of 5-FU-induced morbidity and increased survival accompanied by the improved integrity of intestinal tissue and stimulated the restoration of hematopoiesis. Entolimod-stimulated IL-6 production was essential for Entolimod's ability to rescue mice from death caused by doses of 5-FU associated with hematopoietic failure. In contrast, IL-6 induction was not necessary for protection and restoration of drug-damaged gastrointestinal tissue by Entolimod. In a syngeneic mouse CT26 colon adenocarcinoma model, Entolimod reduced the systemic toxicity of 5-FU, but did not reduce its antitumor efficacy indicating that the protective effect of Entolimod was selective for normal, non-tumor, tissues. These results suggest that Entolimod has clinical potential to broaden the therapeutic window of genotoxic anticancer drugs by reducing their associated hematopoietic and gastrointestinal toxicities.

Evaluation of combined bevacizumab and intraperitoneal carboplatin or paclitaxel therapy in a mouse model of ovarian cancer.

PURPOSE: To evaluate the pharmacokinetics of bevacizumab following IP and IV administration, and to investigate combined bevacizumab therapy (IP or IV) with IP paclitaxel or carboplatin in a mouse model of ovarian cancer. METHODS: Bevacizumab pharmacokinetics were investigated following IV or IP dosing, and mice bearing peritoneal A2780 xenografts were treated with vehicle, IV or IP bevacizumab, IP paclitaxel, IP paclitaxel with co-administration of IV or IP bevacizumab, IP carboplatin, and IP carboplatin with co-administration of IV or IP bevacizumab. Survival time was defined as the time to death or the time to reach 120% of baseline body weight. RESULTS: Following IP administration, bevacizumab was rapidly absorbed and bioavailability was 92.8%. Median survival time, which was 33 days for control mice, was increased by 24% with IP paclitaxel. IP carboplatin failed to increase survival time when administered alone. IV and IP bevacizumab increased survival time by 42 and 33%. Combined bevacizumab and IP paclitaxel was superior to paclitaxel alone (P = 0.01 for IV and P = 0.04 for IP bevacizumab), and combined bevacizumab and IP carboplatin was superior to carboplatin alone (P = 0.002 for IV and P = 0.02 for IP bevacizumab). There were no significant differences in survival between groups receiving bevacizumab IV or IP, either alone (P = 0.586), in combination with paclitaxel (P = 0.467), or in combination with carboplatin (P = 0.149). CONCLUSIONS: Following IP administration to mice, bevacizumab demonstrates rapid and near complete absorption. Bevacizumab therapy, initiated prior to IP carboplatin or paclitaxel administration, increased survival time significantly in mice, and results were not dependent on the route of bevacizumab administration (IV vs. IP).

Increased tumor uptake of chemotherapeutics and improved chemoresponse by novel non-anticoagulant low molecular weight heparin.

BACKGROUND: Recent prospective clinical trials of low molecular weight heparins (LMWHs) have demonstrated that these agents may provide significant advantages in terms of progression-free and overall survival in certain subgroups of cancer patients. The mechanisms of improved survival associated with LMWHs are not known, and may involve direct and/or indirect effects on tumor growth. The purpose of this study was to investigate the effects of LMWH and a sulfated non-anticoagulant LMWH (S-NACH) on tumor chemotherapeutic uptake and chemoresponse. MATERIALS AND METHODS: LMWH and S-NACH were tested for their ability to reduce tumor growth and tumor-associated angiogenesis using three different in vivo models. Biodistribution studies were undertaken to determine the effect of these agents on uptake of paclitaxel (PACL) and doxorubicin (Dox) by breast cancer tumor xenografts. RESULTS: LMWH and S-NACH (10 mg/kg s.c. daily) effectively limited tumor growth of human A549 lung adenocarcinoma xenografts in the nude mouse. In an MDA453/LCC6 breast tumor xenograft model, PACL plus S-NACH showed significant (p < 0.01) tumor growth suppression and improved survival when compared to PACL alone. LMWH increased [(124-)I]-PACL uptake into MDA453/LCC6 tumors, with tumor:muscle ratios several fold greater than that of [(124-)I]-PACL alone 24 h post-injection. Similarly, LMWH and S-NACH significantly (p < 0.01) increased the uptake of Dox by 1.5-2 fold in MCF7 Dox-resistant tumor xenografts. CONCLUSION: Protocols utilizing adjuvant or neo-adjuvant therapy with LMWH or S-NACH could lead to increased tumor chemo responsiveness, potentially overcoming tumor chemoresistance.

Curaxins: anticancer compounds that simultaneously suppress NF-κB and activate p53 by targeting FACT.

Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from "chromatin trapping" of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser(392) by casein kinase 2 and inhibition of NF-κB-dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

Syntheses and evaluation of novel fatty acid-second-generation taxoid conjugates as promising anticancer agents.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA), linolenic acid, and linoleic acid were linked to the C-2' position of the second-generation taxoids that could overcome MDR caused by overexpressed ABC transporters. The new conjugates, tested in vivo, exhibited strong activity against drug-resistant colon cancer and drug-sensitive ovarian cancer xenografts in mice. Two of the new conjugates, DHA-SB-T-1214 and DHA-SB-T-1213, were found to achieve the total regression of drug-resistant and drug-sensitive tumors, respectively, in the animal models with substantially reduced systemic toxicity.