A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer.

PURPOSE: Bcl-2 is a negative prognostic indicator in prostate cancer, implicated in the development of androgen independence and treatment resistance, and is overexpressed in hormone-refractory prostate cancer (HRPC). Genasense is a phosphorothioate antisense oligonucleotide complementary to the bcl-2 mRNA open reading frame that in preclinical studies has shown significant activity in inhibiting expression of Bcl-2, delaying androgen independence, and improving chemosensitivity in prostate and other cancer models. In this dose escalation study, we evaluated the combination of Genasense and mitoxantrone, a standard chemotherapy for patients with HRPC. DESIGN: Twenty-six patients with HRPC were treated at seven dose levels receiving Genasense at a dose ranging from 0.6 to 5.0 mg/kg/day and mitoxantrone from 4 mg/m(2) to 12 mg/m(2). Genasense was administered as a 14-day i.v. continuous infusion every 28 days with mitoxantrone given as an i.v. bolus on day 8. RESULTS: No dose-limiting toxicities were observed. Hematological toxicities were transient and included neutropenia, thrombocytopenia, and lymphopenia. Nonhematological toxicities included fatigue, fever, nausea, arthralgias, myalgias, and transient elevations in serum creatinine, none of which were severe. Two patients had >50% reductions in prostate-specific antigen. One patient, who received six cycles of Genasense at 1.2 mg/kg/day and a low dose (4 mg/m(2)) of mitoxantrone, also had symptomatic improvement in bone pain. Peripheral blood lymphocyte Bcl-2 protein expression decreased in five of five patients given Genasense at 5mg/kg/day (mean change from baseline, -12.8%; SD, 16.4%) as assessed by flow cytometry. Serum concentrations of Genasense given at doses of 3 mg/kg/day and greater, exceeded 1 microg/ml. CONCLUSIONS: Genasense and mitoxantrone are well tolerated in combination, and mitoxantrone can be delivered at a standard dose with biologically active doses of Genasense without significant additional toxicity. This observation allays concerns about trials that combine Genasense with full doses of other cytotoxic agents seeking greater evidence of activity.

Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis.

BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.

Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense oligonucleotide G3139 combined with free and liposomal doxorubicin.

Bcl-2 is a key apoptosis-regulating protein that has been implicated in mechanisms of chemoresistance for a variety of malignancies by blocking programmed cell death. This study investigated the activity of the Bcl-2 antisense oligodeoxynucleotide (AS ODN) G3139 combined with free doxorubicin (F-DOX) or sterically stabilized liposomal doxorubicin (SL-DOX) to determine the role that drug pharmacodistribution properties may have on antitumor activity using a Bcl-2-expressing human breast solid tumor xenograft model. Administration of G3139 was able to delay the growth of MDA435/LCC6 cells compared with control ODN-treated animals; however, in all of the cases, tumors reestablished after AS ODN treatment. Western blot analyses of Bcl-2 levels of solid tumors showed a sequence-specific down-regulation of the Bcl-2 protein after four daily doses of G3139, which correlated with histological evidence of tumor cell death. Interestingly, the expression of Bcl-2 returned to pretreatment levels during the course of subsequent ODN administration, which suggested the development of resistance to continued Bcl-2 ODN treatment. The antitumor activity of ODN given in conjunction with either F-DOX or SL-DOX was also examined. The combination of G3139 and F-DOX was able to suppress the growth of MDA435/LCC6 cells beyond that obtained with either of the treatments given alone, indicative of synergistic action. Examination of the pharmacokinetics of F-DOX with systemic G3139 administration revealed that elevated tumor drug DOX levels were obtained compared with DOX treatment in the absence of G3139. This effect was sequence-specific and plasma DOX levels were unaffected by G3139 treatment, which indicated possible positive ODN-drug interactions at the tumor site. Combining G3139 with SL-DOX further increased the degree of antitumor activity. The improved efficacy of this combination was attributed to increased tumor drug levels that arise from the ability of SL-DOX to passively accumulate in solid tumors. These results suggest that additional benefits of Bcl-2 antisense ODN may be obtained when it is combined with liposomal formulations of anticancer drugs such as DOX.

Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo.

Circulating malignant CD19(+) B cells have been implicated in the pathogenesis and relapse of multiple myeloma (MM). This study investigated the therapeutic applicability of using long-circulating liposome-encapsulated doxorubicin (DXR) targeted against the internalizing CD19 antigens present on human MM cells. In vitro binding studies using the CD19(+) MM cell line ARH77 demonstrated that CD19-directed immunoliposomes (SIL[anti-CD19]) specifically attached to these cells. Formulations of immunoliposomal doxorubicin (DXR-SIL[anti-CD19]) showed a higher association with, and higher cytotoxicity against, ARH77 cells than did non-targeted liposomal doxorubicin (DXR-SL) or isotype-matched controls (DXR-NSIL[IgG2a]). By using the pH-sensitive fluorophore, 1-hydroxypyrene-3,6, 8-trisulfonic acid, binding of SIL[anti-CD19] to CD19 antigens was shown to trigger receptor-mediated internalization of the antibody-antigen complexes into endosomes. Targeting of SIL[anti-CD19] to CD19(+) B cells was also demonstrated in a heterogeneous mixture of peripheral blood mononuclear cells (PBMC) from MM patients. A decrease in cellular DNA (which is an indicator of apoptosis) caused by the cytotoxicity of DXR-SIL[anti-CD19] to myeloma PBMC was determined by using flow cytometry. While PBMC treatment with free DXR resulted in non-specific cytotoxicity to both B and T cells, DXR-SL were only minimally cytotoxic to either. In contrast, DXR-SIL[anti-CD19] were selectively cytotoxic for B cells in PBMC, indicating that this treatment may be effective in eliminating circulating malignant B cells in MM patients.

In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma.

The ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the therapeutic effectiveness of the liposomal preparations as well as reduce adverse side effects associated with chemotherapy. Long-circulating formulations of liposomes containing lipid derivatives of poly(ethyleneglycol) [sterically stabilized liposomes (SLs)] have been described previously, and new techniques have recently been developed for coupling monoclonal antibodies (Abs) at the poly(ethyleneglycol) terminus of these liposomes. Ab-targeted SLs [immunoliposomes (SILs)] containing entrapped anticancer drugs are predicted to be useful in the treatment of hematological malignancies such as B-cell lymphomas or multiple myeloma, in which the target cells are present in the vasculature. The specific binding, in vitro cytotoxicity, and in vivo antineoplastic activity of doxorubicin (DXR) encapsulated in SILs coupled to monoclonal Ab anti-CD19 (SIL[anti-CD19]) were investigated against malignant B cells expressing CD19 surface antigens. Binding experiments with SIL[anti-CD19] resulted in a 3-fold higher association of the SILs with a human CD19+ B lymphoma cell line (Namalwa) in comparison with nontargeted SLs. Using flow cytometry, fluorescently labeled SIL[anti-CD19] bound to B cells with no recognition of T cells in a mixture of B cells and T cells in culture. Nontargeted SLs demonstrated significantly lower recognition of either B cells or T cells. Targeted DXR-SIL[anti-CD19] displayed a higher cytotoxicity to B cells relative to DXR entrapped in nontargeted SLs. Therapeutic experiments in severe combined immunodeficient mice implanted with Namalwa cells by the i.v. or i.p. routes resulted in significantly increased effectiveness of DXR-SIL[anti-CD19] compared to similar amounts of free DXR, DXR-SL (no Ab), or isotype-matched nonspecific Abs attached to DXR-SL. Single doses (3 mg/kg) of DXR-SIL[anti-CD19] administered i.v. resulted in a significantly improved therapeutic benefit, including some long-term survivors. From our results, we infer that targeted anti-CD19 liposomes containing the anticancer drug DXR may be selectively cytotoxic for B cells and may be useful in the selective elimination of circulating malignant B cells in vivo.