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British Journal of Haematology Nov 2008Oblimersen sodium plus rituximab was evaluated in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) patients. Oblimersen was administered as a continuous intravenous...
Oblimersen sodium plus rituximab was evaluated in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) patients. Oblimersen was administered as a continuous intravenous infusion at a daily dose of 3 mg/kg/d for 7 d on alternate weeks for 3 weeks. Rituximab was given at a weekly dose of 375 mg/m(2) for six doses. Patients with stable disease or objective response were allowed to receive a second course of treatment. The overall response rate (ORR) was 42% with 10 complete responses (CR) and eight partial responses (PR). Twelve (28%) patients achieved a minimal response or stable disease. Among the 20 patients with follicular lymphoma the ORR was 60% (eight CR, four PR). Three of the responders were refractory to prior treatment with rituximab, and two of the responses occurred in patients who had failed an autologous stem cell transplant. Median duration of response was 12 months. Most toxicities were low grade and reversible. In conclusion, oblimersen sodium can be safely combined with rituximab. The combination appears to be most beneficial in patients with indolent NHL and warrants further investigation in a large randomized trial.
Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Drug Administration Schedule; Female; Humans; Lymphoma, B-Cell; Male; Middle Aged; Oligonucleotides, Antisense; Recurrence; Rituximab; Survival Analysis; Thionucleotides; Treatment Outcome
PubMed: 18764869
DOI: 10.1111/j.1365-2141.2008.07353.x -
Journal of the American Chemical Society Feb 2022The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites...
The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the β-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of β strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded β-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.
Topics: Binding Sites; Cholesterol; Humans; Ion Channel Gating; Ligands; Lipid Bilayers; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Thionucleotides; Voltage-Dependent Anion Channel 1
PubMed: 35164499
DOI: 10.1021/jacs.1c09848 -
British Journal of Haematology Dec 2004New strategies have evolved in the treatment of patients with non-Hodgkin's lymphoma (NHL). Anti-sense oligonucleotides (ASO) and monoclonal antibody (mAb) therapy,...
New strategies have evolved in the treatment of patients with non-Hodgkin's lymphoma (NHL). Anti-sense oligonucleotides (ASO) and monoclonal antibody (mAb) therapy, though proven to be safe and effective, have not demonstrated to be curative when used as single agents. We tested an innovative combination strategy involving various mAbs and ASO against Bcl-2 (G3139) in aggressive preclinical models. G3139, under optimal transfection conditions, decreased the proliferation rate of lymphoma cells by 60-75% when compared with controls. In addition, apoptosis was demonstrated in Raji (25%) and DHL-4 cells (30%) treated with Genasense following downregulation of Bcl-2 protein. Downregulation of Bcl-2 by G3139 was associated with a higher degree of rituximab-associated, complement-mediated cytotoxicity and antibody dependent cellular cytotoxicity when compared with rituximab alone-treated controls. In vivo studies in severe combined immunodeficiency (SCID) mice clearly demonstrated synergistic activity between G3139 and rituximab. Treatment of lymphoma-bearing SCID mice with G3139 for two consecutive days prior to each rituximab dose resulted in better disease control and survival than treatment with either agent alone or controls. Our findings suggest that Bcl-2 downregulation by G3139, followed by the administration of rituximab is an efficient anti-tumour strategy associated with improved survival in lymphoma-bearing SCID mice.
Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Combined Modality Therapy; Gene Expression Regulation; Genes, bcl-2; Genetic Therapy; Humans; Immunotherapy, Active; Lymphoma, Non-Hodgkin; Mice; Mice, SCID; Oligonucleotides, Antisense; Rituximab; Thionucleotides; Transfection; Transplantation, Heterologous
PubMed: 15566355
DOI: 10.1111/j.1365-2141.2004.05239.x -
Molecular Pharmaceutics 2009G3139, an antisense oligodeoxyribonucleotide (ODN) against Bcl-2, contains two CpG dinucleotides and has shown immunostimulatory activities in preclinical studies. It...
G3139, an antisense oligodeoxyribonucleotide (ODN) against Bcl-2, contains two CpG dinucleotides and has shown immunostimulatory activities in preclinical studies. It has been suggested that immunoactivation, rather than antisense activity, is primarily responsible for the therapeutic efficacy of G3139. Nanoparticle formulations naturally target phagocytic antigen presenting cells and therefore might enhance the immunological effects of G3139. In this study, a novel formulation of lipid nanoparticles (LNPs) encapsulating G3139 was synthesized and evaluated in mice bearing L1210 subcutaneous tumors. Intravenous injection of G3139-LNPs into mice led to increased serum levels of IL-6 and IFN-gamma, promoted proliferation of natural killer (NK) cells and dendritic cells (DCs), and triggered a strong antitumor immune response in mice. The observed effects were much greater than those induced by free G3139. Correspondingly, the G3139-LNPs more effectively inhibited tumor growth and induced complete tumor regression in some mice. In contrast, free G3139 was ineffective in tumor growth inhibition and did not prolong survival of the tumor-bearing mice. These results suggest that G3139-LNPs are a potential immunomodulatory agent and may have applications in cancer therapy.
Topics: Adaptation, Biological; Animals; Antineoplastic Agents; Cell Line, Tumor; Down-Regulation; Female; Humans; Immunity, Innate; Lipids; Mice; Mice, Inbred DBA; Nanoparticles; Neoplasm Transplantation; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Thionucleotides
PubMed: 19072654
DOI: 10.1021/mp800146j -
Anticancer Research Mar 2011Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid...
BACKGROUND
Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid nanoparticles (LNPs) in a reproducible manner on a laboratory scale.
MATERIALS AND METHODS
A 3-inlet microfluidic (MF) chip-based device was used to synthesize LNPs at the lipid/ON ratio of 10/1 (w/w) and at flow rates ranging from 50 to 1100 μl/min. A series of LNPs containing either antisense oligodeoxyribonucleotide (AS-ODN) or small-interfering RNA (siRNA) were synthesized. Bulk mixing was used as control.
RESULTS
The MF method was shown to be particularly useful for synthesis of LNPs loaded with AS-ODN. The optimal range of flow rates for AS-ODN LNPs was found to be 100 to 200 μl/min. MF synthesis produced LNPs with lower polydispersity values. However, the MF was less effective in preparing LNPs loaded with siRNA, which may have been due to greater rigidity of double-stranded siRNA comparing to single-stranded AS-ODN.
CONCLUSION
MF technology is a simple, affordable and reproducible method for production of ON-LNPs.
Topics: Cell Line; Gene Silencing; Humans; Lipids; Luciferases; Microfluidics; Nanoparticles; Oligonucleotides; RNA, Small Interfering; Rheology; Thionucleotides
PubMed: 21498694
DOI: No ID Found -
Biophysical Journal Aug 2007Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and...
Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of -3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (approximately 0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.
Topics: Animals; Ion Channel Gating; Kinetics; Membranes, Artificial; Mitochondria, Liver; Oligonucleotides, Antisense; Organothiophosphorus Compounds; Poly T; Protein Conformation; Rats; Thionucleotides; Voltage-Dependent Anion Channels
PubMed: 17483171
DOI: 10.1529/biophysj.107.105379 -
British Journal of Haematology Jun 2008Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target...
Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target modulation were characterized in cell lines with t(4;11) and BCL-2 expression was examined in MLL and non-MLL infant/paediatric leukaemia cases by Western blot analysis and/or real-time polymerase chain reaction. Cytotoxicity of Genasensetrade mark (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single-agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4-11 cells to select anti-leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S-phase progression. The abundant BCL-2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4-11 cells, and these effects occur through apoptosis.
Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 4; Dose-Response Relationship, Drug; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; Translocation, Genetic; Tumor Cells, Cultured
PubMed: 18422996
DOI: 10.1111/j.1365-2141.2008.07100.x -
The Prostate Dec 2005Bcl-2 is anti-apoptotic and overexpression is associated with prostate tumor aggressiveness. We hypothesized that Bcl-2 has a role in prostate cancer radiation (RT)...
BACKGROUND
Bcl-2 is anti-apoptotic and overexpression is associated with prostate tumor aggressiveness. We hypothesized that Bcl-2 has a role in prostate cancer radiation (RT) response. The relationship of Bcl-2 expression in four prostate cancer cell lines, and the effect of modulating expression with a Bcl-2 antisense oligonucleotide (G3139, Genasense, oblimersen sodium, Genta Incorporated), to RT was examined.
METHODS
The four cell lines studied were LNCaP (wild type-p53), PC3 (p53 null), Bcl-2 stably transfected LNCaP (LNCaP-BST), and Bcl-2 stably transfected PC3 (PC3-BST) cells. Cells were treated with antisense (AS) Bcl-2 alone or with RT (2-6 Gy). Following RT, cells were processed at 3-6 hr for Western blots, 18 hr for Annexin V staining and flow cytometric analysis, 24 hr for caspases 3+7 quantification by fluorometric assay, and immediately for clonogenic survival.
RESULTS
AS caused a significant reduction in Bcl-2 expression in all cell lines. P53 expression was elevated following RT treatment in LNCaP and LNCaP-BST cells. P21 was increased by RT treatment in all cell lines. AS caused a significant increase in caspase 3+7 activity over the mismatch (MM) controls in all cell lines. When AS was combined with RT, caspase 3+7 activity was further increased significantly over all other groups in all cell lines. Moreover, AS+RT resulted in significantly reduced clonogenic survival over MM+RT, which was dampened in the Bcl-2 overexpressing lines.
CONCLUSIONS
To our knowledge, these data demonstrate for the first time that a Bcl-2 specific AS oligonucleotide sensitizes prostate cancer cells to RT. p53 is not required for this effect.
Topics: Apoptosis; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Combined Modality Therapy; Cyclin-Dependent Kinase Inhibitor p21; DNA, Antisense; Flow Cytometry; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transfection; Tumor Suppressor Protein p53
PubMed: 16015611
DOI: 10.1002/pros.20303 -
Archives of Biochemistry and Biophysics Dec 2015Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and...
Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 μM PPIX and PK11195 at concentrations of 50 μM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening.
Topics: Animals; Brain; Calcium; Carrier Proteins; Cations, Divalent; Isoquinolines; Ligands; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Permeability; Rats; Receptors, GABA-A; Thionucleotides; Voltage-Dependent Anion Channels
PubMed: 26498031
DOI: 10.1016/j.abb.2015.10.012 -
Medical Hypotheses 2007The antisense drug G3139 (oblimersen sodium, Genta, Inc.) is a phosphorothioate oligodeoxynucleotide (ODN) containing unmethylated CpG units, which is targeted to...
The antisense drug G3139 (oblimersen sodium, Genta, Inc.) is a phosphorothioate oligodeoxynucleotide (ODN) containing unmethylated CpG units, which is targeted to suppress Bcl-2. To date, its effectiveness in cancer clinical trials has been minimal. Some suggestions are provided for that disappointment and recent citations are provided that support the idea that G3139 may be effective at clearing viral infections, specifically HIV. At the time G3139 was conceived as an anti-cancer drug candidate, it was viewed optimistically because Bcl-2 was widely believed to be the most important protein blocking p53-dependent apoptosis caused by internal stress. Since that time, we have learnt that Bcl-2 is not the only protein that inhibits apoptosis and that p53 itself is frequently malfunctioning in tumors. Thus, the anti-cancer utility of suppressing Bcl-2 in cancer cells is limited. Moreover, Bcl-2 has a role in halting the cell cycle (though p27), which may slow down tumor growth; and Bcl-2 even has pro-apoptotic roles in the execution of apoptosis initiated by external death signals (via Fas/CD95 and caspase 3). Overall, in the clinical setting, G3139 usually has statistically significant but medically unimportant benefit. These results have greatly diminished the enthusiasm for the drug especially when the side effects are considered. Specifically, the unmethylated CpG ODN (and/or the phosphorothioate group) activates the immune system, but this potentially important anti-cancer effect is lost when the immune cells undergo premature apoptosis apparently because their Bcl-2 levels have been lowered by the antisense effect of G3139. While this effect on immune cells is usually undesirable, it is exactly what would be useful for activating immune cells, initiating provirus transcription in retrovirus-infected cells, and facilitating selective apoptosis of these infected cells. In general, G3139 might have benefit in clearing chronic infections by intracellular parasites including viruses (HIV, SIV, HTLV, HBV, coronavirus, etc.). Indeed, G3139 has been shown to cause apoptosis in EBV-infected cells leading to clearance of the virus.
Topics: Animals; Anti-HIV Agents; Apoptosis; Clinical Trials as Topic; Genes, p53; HIV Infections; Humans; Immune System; Models, Biological; Mutation; Neoplasms; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Thionucleotides; Treatment Outcome
PubMed: 17363184
DOI: 10.1016/j.mehy.2007.01.044