Recombinant interleukin-2 significantly augments activity of rituximab in human tumor xenograft models of B-cell non-Hodgkin lymphoma.

Recombinant interleukin-2 (rIL-2) is a pleiotropic cytokine that activates select immune effector cell responses associated with antitumor activity, including antibody-dependent cellular cytotoxicity (ADCC). Rituximab is an anti-CD20 monoclonal antibody that activates ADCC in non-Hodgkin lymphoma (NHL). The ability of rIL-2 to augment rituximab-dependent tumor responses was investigated. The efficacy of rIL-2 in combination with rituximab was evaluated in 2 NHL tumor xenograft models: the CD20hi, rituximab-sensitive, low-grade Daudi model and the CD20lo, aggressive, rituximab-resistant Namalwa model. Combination of rIL-2 plus rituximab was synergistic in a rituximab-sensitive Daudi tumor model, as evidenced by significant tumor regressions and increased time to tumor progression, compared with rIL-2 and rituximab single agents. In contrast, rituximab-resistant Namalwa tumors were responsive to single-agent rIL-2 and showed an increased response when combined with rituximab. Using in vitro killing assays, rIL-2 was shown to enhance activity of rituximab by activating ADCC and lymphokine-activated killer activity. Additionally, the activity of rIL-2 plus rituximab F(ab')2 was similar to that of rIL-2 alone, indicating a critical role for immunoglobulin G1 Fc-FcgammaR-effector responses in mediating ADCC. Antiproliferative and apoptotic tumor responses, along with an influx of immune effector cells, were observed by immunohistochemistry. Collectively, the data suggest that rIL-2 mediates potent tumoricidal activity against NHL tumors, in part, through activation and trafficking of monocytes and natural killer cells to tumors. These data support the mechanistic and therapeutic rationale for combination of rIL-2 with rituximab in NHL clinical trials and for single-agent rIL-2 in rituximab-resistant NHL patients.

CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo.

PURPOSE: Chk1 kinase is a critical regulator of both S and G(2)-M phase cell cycle checkpoints in response to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumor effects of a novel Chk1 inhibitor, CHIR124. EXPERIMENTAL DESIGN: CHIR-124 was evaluated for its ability to abrogate cell cycle checkpoints, to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling induced by topoisomerase I poisons in human tumor cell line and xenograft models. RESULTS: CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC(50) = 0.0003 micromol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determined by isobologram or response surface analysis. CHIR-124 abrogates the SN-38-induced S and G(2)-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G(2)-M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53. We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1 for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G(2)-M checkpoint and increasing tumor apoptosis. CONCLUSIONS: CHIR-124 is a novel and potent Chk1 inhibitor with promising antitumor activities when used in combination with topoisomerase I poisons.

CHIR-258: a potent inhibitor of FLT3 kinase in experimental tumor xenograft models of human acute myelogenous leukemia.

PURPOSE: Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. Given the relevance of FLT3 mutations in AML, we investigated the activity of CHIR-258, an orally active, multitargeted small molecule, with potent activity against FLT3 kinase and class III, IV, and V RTKs involved in endothelial and tumor cell proliferation in AML models. EXPERIMENTAL DESIGN: CHIR-258 was tested on two human leukemic cell lines in vitro and in vivo with differing FLT3 mutational status [MV4;11 cells express FLT3 internal tandem duplications (ITD) versus RS4;11 cells with wild-type (WT) FLT3]. RESULTS: Antiproliferative activity of CHIR-258 against MV4;11 was approximately 24-fold greater compared with RS4;11, indicating more potent inhibition against cells with constitutively activated FLT3 ITD. Dose-dependent down modulation of receptor phosphorylation and downstream signaling [signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase] in MV4;11 cells with CHIR-258 confirmed the molecular mechanism of action. Target modulation of phospho-FLT3, phospho-STAT5, and phospho-ERK in MV4;11 tumors was achieved at biologically active doses of CHIR-258. Tumor regressions and eradication of AML cells from the bone marrow were shown in s.c. and bone marrow engraftment leukemic xenograft models. Tumor responses were characterized by decreased cellular proliferation and positive immunohistochemical staining for active caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting cell death was mediated in part via apoptosis. CONCLUSIONS: Our data indicate that CHIR-258 may be an effective therapy in FLT3-associated AML and warrants clinical trials.

Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice.

We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139). In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells. PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively. The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines. In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent. PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments. The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.

Pharmacokinetics of Bcl-2 antisense oligonucleotide (G3139) combined with doxorubicin in SCID mice bearing human breast cancer solid tumor xenografts.

PURPOSE: To evaluate the pharmacokinetic (PK) properties of Bcl-2 antisense oligodeoxynucleotide G3139 when combined with the anthracycline anticancer drug doxorubicin (DOX) in a model of MDA435/LCC6 human breast cancer in severely compromised immunodeficient (SCID) mice. METHODS: An orthotopic model of MDA435/LCC6 solid breast tumors was developed by bilateral implantation of passaged cells in female SCID-RAG2 mice. The G3139 plasma profile was compared for two common routes of administration (i.v. or i.p.) in single and multiple dose treatment regimens of 5 mg/kg G3139 alone or with simultaneous DOX (5 mg/kg) administration. At selected times, plasma and major organs were assayed for [3H]G3139 using scintillation counting and DOX determined using HPLC. The molecular integrity of G3139 was analyzed using SDS-PAGE. The PKs of G3139 and DOX were evaluated using a two-compartment model. RESULTS: G3139 administered i.v. at 5 mg/kg revealed a biexponential plasma concentration-time curve with a Cmax of 99.9 microg/ml and elimination half-lives of 0.03 h and 9.8 h, respectively, which resulted in an area under the concentration-time curve (AUC) of 15.9 microg x h/ml. G3139 administered i.p. showed a plasma absorption, distribution and elimination profile typical of this route of administration, characterized by half-lives of 0.03 h, 0.2 h and 8.9 h, respectively and a Cmax of 8.6 microg/ml. Based on AUC comparisons, the bioavailability of G3139 injected i.p. was 84% compared to i.v. administration. Subtle changes were observed in G3139 PKs after three prior i.p. doses of G3139. Specifically, a six-fold slower absorption rate, lower Cmax (6.9 microg/ml), increased Tmax (0.2 h), and an AUC of 17.4 microg x h/ml were observed, consistent with concentrations approaching saturation levels in tissue sites to which G3139 distributes. Coadministration of DOX had significant effects on the PK properties of G3139, manifested by an increased Cmax (11.2 microg/ml), higher AUC (19.7 microg x h/ml), and ninefold lower plasma clearance for single-dose G3139 administration. G3139 in plasma remained largely intact (< 17% degraded in plasma over 4 h), and increased plasma protein association occurred as a function of time. G3139 was detected in both healthy and tumor tissue after i.v. and i.p. administration. The highest tissue levels of G3139 were observed in the kidneys (40 microg/g), and low levels (< 2 microg/g) were detected in lung, heart and muscle. The rate of accumulation of G3139 in organs was dependent upon G3139 levels in plasma and the presence of coadministered DOX. Significant accumulation of G3139 was observed in solid tumors, with peak levels of approximately 5 microg G3139/g tumor, and approximately a two-to threefold tumor/muscle AUC ratio. The kinetics of G3139 accumulation in tumor tissue increased with increasing circulating G3139 concentration. The tissue distribution properties of DOX were also altered in the presence of coadministered G3139: in the presence of G3139, tumor exposure to DOX increased two-to threefold without alteration in plasma DOX PKs. CONCLUSIONS: These findings indicate that drug-drug interactions between G3139 and DOX are modest and favorable in that elevated tumor DOX levels are achieved without compromising G3139 tumor uptake or significantly altering plasma drug concentrations.