Preclinical toxicity and toxicokinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase R2.

PURPOSE: GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRNA sequence of human ribonucleotide reductase R2. GTI-2040 has been shown to inhibit human cancer cell proliferation by downregulation of R2 expression in vitro and to significantly inhibit tumor growth in xenograft models of human cancer in mice. As part of the safety evaluation for human clinical trials, the toxicity and toxicokinetics of GTI-2040 were determined in Sprague-Dawley rats and rhesus monkeys. METHODS: GTI-2040 was administered to rats at 2, 10, and 50 mg/kg/day by bolus intravenous injection every second day for 21 days with a 21-day recovery. In monkeys, an acute study was performed with single, escalating doses of GTI-2040 ranging from 10 to 80 mg/kg given as a 24-h continuous intravenous infusion. As well, a 21-day, continuous intravenous infusion study with GTI-2040 was conducted in monkeys at 2, 10, and 50 mg/kg/day, with a 3-week recovery. Blood sampling was done to measure GTI-2040 plasma concentrations, metabolites, and pharmacokinetic parameters, and tissues were collected to assess the distribution of GTI-2040 and/or metabolites. RESULTS: The toxicities of GTI-2040 in both rats and monkeys were typical for the phosphorothioate oligonucleotide class of compounds. In monkeys, there was a dose-related increase in GTI-2040 plasma levels with concomitant increase in complement activation and prolongation of activated partial thromboplastin time. In both rats and monkeys, the tissues having the highest concentrations of GTI-2040 (kidney, liver, spleen) had the largest dose-related toxic effects. Adverse effects were diminished or absent in the recovery animals. CONCLUSIONS: GTI-2040 was well tolerated when infused over 24 h at doses up to 80 mg/kg in monkeys. In rats and monkeys, GTI-2040 was reasonably well tolerated and showed reversible toxicities when administered at doses up to 50 mg/kg/day for 21 days. The no observed adverse effect dose level for GTI-2040 in both animal species was 2 mg/kg/day. There were no apparent sequence-specific effects related to the interaction of GTI-2040 with the R2 component of the mRNA expressing ribonucleotide reductase.

IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo.

Interleukin-17E (IL-17E) belongs to a novel family of cytokines that possess significant homology to IL-17. IL-17E has potent inflammatory effects in vitro and in vivo. Overexpression of IL-17E in mice results in a T helper-2 (Th2)-type immune response, which includes the expansion of eosinophils through the production of IL-5, and elevated gene expression of IL-4 and IL-13 in multiple tissues. In this study, we show that IL-17E has antitumor activity in vivo, a previously unrecognized function of IL-17E. Antitumor efficacy of IL-17E was examined in a variety of human tumor xenograft models, including melanoma, breast, lung, colon, and pancreatic cancers. Injection of recombinant IL-17E every other day resulted in significant antitumor activity in these tumor models. In addition, the combination of IL-17E with chemotherapy or immunotherapy agents showed an enhanced antitumor efficacy in human tumor xenograft models in mice as compared to either agent alone. Antitumor activity was demonstrated using different routes of administration, including intraperitoneal, intravenous, and subcutaneous injection. Anticancer activity was shown for both mouse and human forms of IL-17E, which have a high degree of sequence identity. Tumor-bearing mice treated with IL-17E showed a significant increase in serum levels of IL-5 and increased numbers of eosinophils in peripheral blood compared to the control group. Spleens isolated from IL-17E-treated mice showed a significant increase in eosinophils that correlated with antitumor activity of IL-17E in a dose-response manner. Finally, we demonstrate that B cells are necessary for IL-17E-mediated antitumor activity and that IL-17E was found to activate signaling pathways in B cells in vitro. Taken together, these data demonstrate that IL-17E has antitumor activity in vivo, and support further investigation of the potential clinical use of IL-17E as an anticancer agent.

11-Phenyl-[b,e]-dibenzazepine compounds: novel antitumor agents.

A series of 11-phenyl-[b,e]-dibenzazepine compounds were synthesized and shown to be inhibitors of tumor cell proliferation with IC(50) values ranging from submicromolar to micromolar concentrations. Flow cytometric analyses of several active compounds demonstrated inhibition of cell cycle progression at the G(0)-G(1) phase transition resulting in G(0)-G(1) arrest.

Virulizin induces production of IL-17E to enhance antitumor activity by recruitment of eosinophils into tumors.

Virulizin has demonstrated strong antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Our previous studies have demonstrated that macrophages, NK cells, and cytokines are important in the antitumor mechanism of Virulizin. Virulizin treatment of tumor bearing mice results in the expansion as well as increased activity of monocytes/macrophages and production of cytokines IL-12 and TNFalpha and activation of NK cells. In this study we show that the inflammatory cytokine IL-17E (IL-25) is induced by Virulizin treatment and is part of its antitumor mechanism. IL-17E is a proinflammatory cytokine, which induces a T(H)2 type immune response, associated with eosinophil expansion and infiltration into mucosal tissues. IL-17E was increased in sera of Virulizin-treated mice bearing human melanoma xenografts, compared to saline-treated controls, as shown by 2D gel electrophoresis and ELISA. Treatment of splenocytes in vitro with Virulizin resulted in increased IL-17E mRNA expression, which peaked between 24 and 32 h post-stimulation. Both in vitro and in vivo experiments demonstrated that B cells produced IL-17E in response to Virulizin treatment. Furthermore, Virulizin treatment in vivo resulted in increased blood eosinophilia and eosinophil infiltration into tumors. Finally, injection of recombinant IL-17E showed antitumor activity towards xenografted tumors, which correlated with increased eosinophilia in blood and tumors. Taken together, these results support another antitumor mechanism mediated by Virulizin, through induction of IL-17E by B cells, leading to recruitment of eosinophils into tumors, which may function in parallel with macrophages and NK cells in mediating tumor destruction.

Virulizin, a novel immunotherapy agent, stimulates TNFalpha expression in monocytes/macrophages in vitro and in vivo.

Virulizin, a novel biological response modifier, has demonstrated broad antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Previous studies have demonstrated a significant role of macrophages and NK cells in the antitumor mechanism of Virulizin. Increased activity and expansion of macrophages and NK cells has been observed in mice treated with Virulizin. In the present study, the effects of Virulizin on TNFalpha expression were investigated in vitro and in vivo. CD-1 nude mice were treated with Virulizin daily for 5 days. Quantitative RT-PCR demonstrated that the level of TNFalpha mRNA increased in peritoneal macrophages isolated from Virulizin-treated mice as compared to the control group. An increase in TNFalpha protein expression was also observed, as assessed by flow cytometric analysis. Increased levels of TNFalpha mRNA were seen in human tumor xenografts following treatment of tumor-bearing mice with Virulizin. In the presence of LPS, Virulizin also stimulated TNFalpha protein secretion and mRNA expression in human monocytic U937 cells and mouse macrophage RAW264.7 cells in vitro in a time- and dose-dependent manner. U937 cells treated with Virulizin showed a significantly enhanced cytotoxicity that was eliminated upon neutralization of TNFalpha. Virulizin also induced the phosphorylation of IkappaB, suggesting that induction of TNFalpha expression by Virulizin is mediated by activation of NFkappaB. The results indicate that Virulizin-induced TNFalpha expression contributes to modulation of immune responses and antitumor activities.

Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome.

The Pseudomonas aeruginosa chromosome was fractionated with the enzymes SpeI and DpnI, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.