-
Molecular Pharmaceutics 2009Human serum albumin (HSA)-coated liposomal formulations were synthesized and evaluated for the delivery of antisense oligodeoxyribonucleotide (ODN) G3139 in KB human...
Human serum albumin (HSA)-coated liposomal formulations were synthesized and evaluated for the delivery of antisense oligodeoxyribonucleotide (ODN) G3139 in KB human oral carcinoma cells. Liposomes composed of dimethyldioctadecyl ammonium bromide/egg phosphatidylcholine/alpha-tocopheryl polyethylene glycol 1000 succinate (58:40:2 molar ratio) complexed with G3139 and coated with HSA were investigated for Bcl-2 downregulating activity. Cellular uptake of HSA-coated liposome-ODN complexes was more efficient than the uncoated liposome-ODN complexes. Treatment of the cells with HSA-coated liposome-ODN complexes resulted in efficient Bcl-2 mRNA downregulation that was approximately 3-fold greater than with uncoated liposomes (p < 0.05) and 6-fold greater than with free ODN. The transfection efficiency of liposome-ODN complexes coated with HSA was dependent on the concentration of HSA used and on the contents of alpha-helix and beta-strand in HSA. HSA-coated liposomes are effective delivery vehicles for antisense ODN.
Topics: Blotting, Western; Cell Line, Tumor; Circular Dichroism; Electrophoresis, Agar Gel; Humans; Liposomes; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Serum Albumin; Thionucleotides
PubMed: 19725564
DOI: 10.1021/mp900150g -
American Journal of Physiology. Cell... Apr 2007G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the...
G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.
Topics: Adenosine Diphosphate; Animals; Cell Line, Tumor; Cytochromes c; Humans; In Vitro Techniques; Ion Channel Gating; Liposomes; Mitochondria, Liver; Mitochondrial Membranes; Mitochondrial Swelling; Oligonucleotides, Antisense; Permeability; Phospholipids; Proto-Oncogene Proteins c-bcl-2; Rats; Thionucleotides; Voltage-Dependent Anion Channels
PubMed: 17135295
DOI: 10.1152/ajpcell.00490.2006 -
Journal of Clinical Oncology : Official... Nov 2009A randomized trial of oblimersen plus fludarabine/cyclophosphamide (OBL-FC; n = 120) versus FC (n = 121) was conducted in patients with relapsed/refractory chronic... (Randomized Controlled Trial)
Randomized Controlled Trial
5-year survival in patients with relapsed or refractory chronic lymphocytic leukemia in a randomized, phase III trial of fludarabine plus cyclophosphamide with or without oblimersen.
PURPOSE
A randomized trial of oblimersen plus fludarabine/cyclophosphamide (OBL-FC; n = 120) versus FC (n = 121) was conducted in patients with relapsed/refractory chronic lymphocytic leukemia (CLL). The primary end point was met: the complete response (CR) rate, defined as complete or nodular partial response, was significantly greater with OBL-FC than with FC (17% v 7%; P = .025). Among patients with CR, response duration was significantly longer with OBL-FC than with FC (median not reached; > 36 months v 22 months; P = .03). Maximum benefit with OBL-FC, including a four-fold increase in CR rate and a survival benefit with 3 years of follow-up (hazard ratio, 0.53; P = .05), was observed in patients with fludarabine-sensitive disease. We evaluated long-term survival and poststudy CLL therapy among all randomly assigned patients.
METHODS
Poststudy CLL treatment information was collected. Patients were observed for survival for up to 5 years from the date of random assignment.
RESULTS
Poststudy CLL treatment was balanced between arms. Intent-to-treat analysis of 5-year survival showed no significant between-treatment difference (hazard ratio, 0.87; P = .34). Among the greater than 40% of patients with complete or partial remission, a significant 5-year survival benefit was observed with OBL-FC (hazard ratio, 0.60; P = .038). Among patients with fludarabine-sensitive disease who had previously demonstrated maximum benefit with OBL-FC, the previously observed survival benefit improved: a 50% reduction in the risk of death was observed (P = .004).
CONCLUSION
In relapsed/refractory CLL, OBL combined with FC offers patients who achieve complete or partial remission, as well as those who have fludarabine-sensitive disease, a significant survival benefit.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Female; Humans; Kaplan-Meier Estimate; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Recurrence, Local; Thionucleotides; Vidarabine
PubMed: 19738118
DOI: 10.1200/JCO.2009.22.5748 -
Anticancer Research 2006Folate receptors (FRs) are cellular surface markers for numerous solid tumors and myeloid leukemias. The aim of this study was to develop an antisense...
BACKGROUND
Folate receptors (FRs) are cellular surface markers for numerous solid tumors and myeloid leukemias. The aim of this study was to develop an antisense oligodeoxyribonucleotide (ODN) carrier targeting FR-overexpressing cancer cells using folate (FA) as the targeting moiety. G3139, a phosphorothioate antisense ODN against human bcl2 mRNA, was evaluated in this study.
MATERIALS AND METHODS
G3139-containing liposomes were prepared using an ethanol dilution method. For the targeted formulation, 0.5 mol% of folate-PEG-DSPE was incorporated as a targeting ligand into cationic liposomes composed of DC-Chol/egg PC/PEG-DSPE at 25:65:10 mol/mol. Particle size and surface charge were measured and cellular uptake was assessed by fluorescence microscopy and flow cytometry. The ODN-containing formulations were evaluated in FR+ KB cells for Bcl2 down-regulation measured by Western blot. The cytotoxicity of the formulations was determined by MTT assay.
RESULTS
The G3139-containing liposomes had an average diameter of 80-90 nm with high ODN entrapment efficiency (70-80%). Incorporation of the folate ligand did not significantly alter the particle size and entrapment efficiency. The formulation exhibited colloidal stability in a serum-containing environment. In uptake studies, the folate-targeted formulation showed ligand concentration-dependent uptake that was up to 6-fold more efficient than that of the non-targeted formulation (p < 0.05). The uptake could be blocked by an excess amount of free folate, thus indicating an FR-dependent mechanism.
CONCLUSION
FR-targeted G3139-containing liposomes showed promising transfection activity in KB cells. FR-targeted formulations were capable of specific targeting to FR-overexpressing cell lines and optimizing the amount of folate ligand in the liposomal formulation can result in more efficient antisense delivery.
Topics: Antibiotics, Antineoplastic; Carrier Proteins; Daunorubicin; Down-Regulation; Folate Receptors, GPI-Anchored; Folic Acid; Humans; KB Cells; Liposomes; Microscopy, Fluorescence; Particle Size; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Receptors, Cell Surface; Thionucleotides
PubMed: 16619505
DOI: No ID Found -
The Journal of Investigative Dermatology Apr 2000Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is...
Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is thought to derive from the neuroendocrine (Merkel) cells of the skin, although in contrast to fetal and especially adult Merkel cells, Merkel cell carcinomas express high levels of the Bcl-2 oncoprotein. Bcl-2 is capable of blocking programmed cell death and has been shown to play an important role in normal cell turnover, tumor biology, and chemoresistance. High Bcl-2 expression leading to prolonged survival of cells may therefore be of importance in the biological and clinical characteristics of Merkel cell carcinoma. In a SCID mouse xenotransplantation model for human Merkel cell carcinoma, we investigated the influence of the bcl-2 antisense oligonucleotide G3139 (Genta) on tumor growth in comparison with control oligonucleotides or cisplatin. Bcl-2 antisense treatment, targeting the first six codons of the bcl-2 mRNA, resulted in either a dramatic reduction of tumor growth or complete remission, whereas reverse sequence and two-base mismatch control oligonucleotides or cisplatin had no significant antitumor effects compared with saline-treated controls. Apoptosis was enhanced 2.4-fold in the bcl-2 antisense treated tumors compared with the saline-treated group, and no other treatment showed a comparable increase in apoptosis. Our findings suggest that bcl-2 antisense treatment may be a novel approach to improve treatment outcome of human Merkel cell carcinoma.
Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Merkel Cell; Cell Division; Combined Modality Therapy; Female; Humans; Mice; Mice, SCID; Models, Biological; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; Transplantation, Heterologous
PubMed: 10733680
DOI: 10.1046/j.1523-1747.2000.00937.x -
International Journal of Cancer Jul 2006Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV), and the EBV oncoprotein, latent membrane protein 1 (LMP-1), is expressed in the majority...
Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV), and the EBV oncoprotein, latent membrane protein 1 (LMP-1), is expressed in the majority of NPCs. LMP-1 upregulates antiapoptotic genes, including bcl-2, and Bcl-2 protein is overexpressed in NPC. Given the antiapoptotic and chemoprotective effects of Bcl-2, it represents a rational therapeutic target in NPC. We have investigated the antitumor and chemosensitizing effects of the Bcl-2 antisense oligodeoxynucleotide G3139 (oblimersen, Genasense) in NPC. For these studies, we used the C666-1 line, a stably infected NPC-derived line that co-expresses LMP-1 and Bcl-2. We have shown that G3139 treatment of C666-1 in vitro caused sequence-dependent suppression of Bcl-2 protein, inhibition of cell growth and enhanced sensitivity to cisplatin (CDDP), as measured by increased antiproliferative and apoptotic effects. In vivo, G3139 treatment (25 mg/kg every 3 days x 5 doses) delayed engraftment and significantly inhibited growth of established C666-1 xenografts in SCID mice compared to control oligo-treated animals. However, G3139 alone did not prevent engraftment or cure established tumors in any animals. In contrast, G3139 treatment (25 mg/kg every 3 days x 5 starting on day 7) in combination with CDDP (8 mg/kg on day 14) completely abrogated tumor engraftment in 80% of animals compared to CDDP (0%) or CDDP + control oligo (0%). When treatment was delayed until tumor was established, G3139 in combination with CDDP significantly inhibited tumor growth compared to CDDP or CDDP + control oligo, and cured 69% animals with established tumors. No animals treated with G3139, CDDP or CDDP + control oligo were cured. Tumor burden and response to treatment correlated with EBV DNA load in serum, measured by real-time PCR. Western blots of tumor extracts obtained during oligo treatment showed that Bcl-2 levels were significantly decreased in G3139-treated animals. Our studies have demonstrated that the Bcl-2 antisense oligodeoxynucleotide, G3139, has proapoptotic effects in C666-1, and in combination with CDDP, is curative in C666-1 NPC xenograft tumors in vivo. The sequence-dependency of these effects is consistent with an antisense mechanism. These studies suggest that Bcl-2 may represent a biologically relevant target for the development of novel combinatorial therapies for NPC.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Carcinoma; Cisplatin; DNA, Viral; Herpesvirus 4, Human; Mice; Mice, SCID; Nasopharyngeal Neoplasms; Thionucleotides; Transplantation, Heterologous; Tumor Burden; Viral Load; Viral Matrix Proteins
PubMed: 16477627
DOI: 10.1002/ijc.21804 -
Blood Jan 2013Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral...
Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif-mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)-conjugated lipopolyplex nanoparticle (RIT-INP)- and Bcl-2-targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell-targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP-G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed.
Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal, Murine-Derived; Antimetabolites, Antineoplastic; Cell Line, Tumor; CpG Islands; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Gene Silencing; Genes, bcl-2; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Liposomes; Mice; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Nanoparticles; Neoplasm Proteins; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Rituximab; Thionucleotides; Toll-Like Receptor 9; Vidarabine; Xenograft Model Antitumor Assays
PubMed: 23165478
DOI: 10.1182/blood-2012-01-407742 -
American Journal of Clinical Oncology Apr 2009Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine malignancy of the skin. Preclinical studies have identified up-regulation of the critical antiapoptosis...
OBJECTIVES
Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine malignancy of the skin. Preclinical studies have identified up-regulation of the critical antiapoptosis gene bcl-2 in MCC. We conducted a multicenter phase II trial of the novel bcl-2 antisense agent (G3139, Genasense) in patients with advanced MCC.
METHODS
Twelve patients (9 men, 3 women) with histologically confirmed metastatic or regionally recurrent MCC were enrolled. Ten patients (83%) had received prior chemotherapy. Eight patients (67%) had Karnofsky performance status of 90 to 100. Patients received continuous IV infusion of G3139 (7 mg/kg/d) via central venous access in an outpatient setting for 14 days, followed by a 7-day rest period. Response was assessed at 6-week intervals. Patients were allowed to continue therapy until unacceptable toxicity or disease progression.
RESULTS
No objective responses were observed. The best response was stable disease in 3 patients and progressive disease in 9 patients. A median of 4 doses per patient (total 46 doses) was administered. Dose delays and/or reductions were required in 6 patients. One patient developed grade 4 lymphopenia. One patient developed grade 3 renal failure characterized by grade 3-elevated creatinine and grade 4 hyperkalemia. Other grade 3 events included cytopenia (n = 5), aspartate aminotransferase/alanine aminotranferease elevation (n = 3), hypophosphatemia (n = 2), and pain (n = 1). The most frequent grade 1 to 2 toxicities were elevated creatinine, ALT elevation, hypokalemia, lymphopenia, and fatigue.
CONCLUSIONS
Bcl-2 antisense therapy (G3139) was well tolerated among patients with advanced MCC. Although probable antitumor activity was documented in 1 patient, no objective responses per Response Evaluation Criteria in Solid Tumors criteria were observed.
Topics: Aged; Carcinoma, Merkel Cell; Female; Humans; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Oligonucleotides, Antisense; Prognosis; Skin Neoplasms; Survival Rate; Thionucleotides
PubMed: 19307957
DOI: 10.1097/COC.0b013e31817eebf8 -
Clinical Cancer Research : An Official... May 2008This phase I trial assessed the safety and tolerability of G3139 when given in combination with carboplatin and paclitaxel chemotherapy. The effect of G3139 treatment on...
A phase I pharmacokinetic and pharmacodynamic correlative study of the antisense Bcl-2 oligonucleotide g3139, in combination with carboplatin and paclitaxel, in patients with advanced solid tumors.
PURPOSE
This phase I trial assessed the safety and tolerability of G3139 when given in combination with carboplatin and paclitaxel chemotherapy. The effect of G3139 treatment on Bcl-2 expression in peripheral blood mononuclear cells (PBMC) and paired tumor biopsies was also determined.
EXPERIMENTAL DESIGN
Patients with advanced solid malignancies received various doses of G3139 (continuous i.v. infusion days 1-7), carboplatin (day 4), and paclitaxel (day 4), repeated in 3-week cycles, in a standard cohort-of-three dose-escalation schema. Changes in Bcl-2/Bax transcription/expression were assessed at baseline and day 4 (prechemotherapy) in both PBMCs and paired tumor biopsies. The pharmacokinetic interactions between G3139 and carboplatin/paclitaxel were measured.
RESULTS
Forty-two patients were evaluable for safety analysis. Primary toxicities were hematologic (myelosuppression and thrombocytopenia). Dose escalation was stopped with G3139 at 7 mg/kg/d, carboplatin at area under the curve of 6, and paclitaxel at 175 mg/m(2) due to significant neutropenia seen in cycle 1 and safety concerns in further escalating chemotherapy in this phase I population. With G3139 at 7 mg/kg/d, 13 patients underwent planned tumor biopsies, of which 12 matched pairs were obtained. Quantitative increases in intratumoral G3139 with decreases in intratumoral Bcl-2 gene expression were seen. This paralleled a decrease in Bcl-2 protein expression observed in PBMCs.
CONCLUSIONS
Although the maximal tolerated dose was not reached, the observed toxicities were consistent with what one would expect from carboplatin and paclitaxel alone. In addition, we show that achievable intratumoral G3139 concentrations can result in Bcl-2 down-regulation in solid tumors and PBMCs.
Topics: Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Combined Modality Therapy; Down-Regulation; Female; Gene Expression; Genes, bcl-2; Humans; Male; Neoplasms; Oligonucleotides, Antisense; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; bcl-2-Associated X Protein
PubMed: 18451239
DOI: 10.1158/1078-0432.CCR-07-1490 -
Annals of Oncology : Official Journal... Aug 2004Expression of the Bcl-2 protein confers resistance to various apoptotic signals. G3139 [oblimersen sodium (Genasense)] is a phosphorothioate antisense... (Clinical Trial)
Clinical Trial
PURPOSE
Expression of the Bcl-2 protein confers resistance to various apoptotic signals. G3139 [oblimersen sodium (Genasense)] is a phosphorothioate antisense oligodeoxynucleotide that targets Bcl-2 mRNA, downregulates Bcl-2 protein translation, and enhances the antitumor effects of subtherapeutic doses of docetaxel (Taxotere).
PATIENTS AND METHODS
We performed a phase I trial to determine the maximum tolerated dose (MTD) and safety profile of combined therapy with G3139 and weekly docetaxel in patients with advanced Bcl-2-positive solid tumors. Cohorts of three to six patients were enrolled to escalating doses of G3139 and a fixed dose of weekly docetaxel using either of two schedules. In part I, G3139 was administered by continuous infusion for 21 days (D1-22), and docetaxel (35 mg/m2) was given weekly on days 8, 15 and 22. In part II, G3139 was given by continuous infusion for 5 days before the first weekly dose of docetaxel, and for 48 h before the second and third weekly docetaxel doses. For both schedules, cycles were repeated every 4 weeks.
RESULTS
Twenty-two patients were enrolled. Thirteen patients were treated on the part I schedule with doses of G3139 escalated from 1 to 4 mg/kg/day. Nine patients were on the part II schedule of shorter G3139 infusion at G3139 doses of 5-9 mg/kg/day. Hematologic toxicities were mild, except for one case of persistent grade 3 thrombocytopenia in part I. The most common adverse events were cumulative fatigue and transaminase elevation, which prevented further dose escalation beyond 4 mg/kg/day for 21 days with the part I schedule. In part II of the study, using the abbreviated G3139 schedule, even the highest daily doses were tolerated without dose-limiting toxicity or the need for dose modification. Objective tumor response was observed in two patients with breast cancer, including one whose cancer previously progressed on trastuzumab plus paclitaxel. Four patients had stable disease. Pharmacokinetic results for G3139 were similar to those of other trials.
CONCLUSIONS
G3139 in combination with standard-dose weekly docetaxel was well tolerated. The shortened and intermittent G3139 infusion had less cumulative toxicities and still allowed similar total G3139 delivery as the longer infusion. Further studies should examine the molecular effect of the regimen, as well as clinical activities in malignancies for which taxanes are indicated.
Topics: Adult; Aged; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Docetaxel; Drug Administration Schedule; Female; Humans; Infusions, Intravenous; Maximum Tolerated Dose; Middle Aged; Neoplasms; Taxoids; Thionucleotides; Treatment Outcome
PubMed: 15277270
DOI: 10.1093/annonc/mdh317