dioleoyl-phosphatidylethanolamine and (3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium

Patients with metastatic melanoma are immunosuppressed by the growing tumor. Allovectin-7 therapy is a form of active immunotherapy that aims at immunization of the host with substances designed to elicit an immune reaction that will eliminate or slow down the growth and spread of the cancer.. to describe the rationale for immunotherapy with Allovectin-7 and assess its safety profile and efficacy based on the results of completed melanoma clinical trials.. we reviewed both the published medical literature and the results of trials pending publication.. Allovectin-7 is a safe and active immunotherapeutic agent. It induces local and systemic durable responses in patients with metastatic melanoma.

Topics: Animals; beta 2-Microglobulin; Cancer Vaccines; Clinical Trials as Topic; DNA, Recombinant; Drug Screening Assays, Antitumor; Genetic Vectors; HLA-B7 Antigen; Humans; Immunotherapy, Active; Injections, Intralesional; Lipids; Lung Neoplasms; Macaca fascicularis; Melanoma; Mice; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Skin Neoplasms; Tumor Escape; Vaccines, DNA

Leuvectin (Vical Inc., San Diego, CA) is a gene transfer product in which a plasmid encoding the human interleukin-2 (IL-2) gene is complexed with the cationic lipid 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). In the current study, the authors investigated the safety and efficacy of in situ vaccination with Leuvectin in patients with metastatic renal cell carcinoma.. Thirty-one patients with metastatic renal cell carcinoma were treated with intratumorally administered Leuvectin at doses ranging from 0.75 to 4 mg. These patients subsequently were evaluated for response and for treatment-related toxicity.. Treatment was well tolerated: no Grade 3 or 4 toxicities were observed in association with the study agent. Documented side effects included Grade 1 pain at the injection site (20%); mild (i.e., Grade 1 or 2) constitutional symptoms, including malaise/myalgia, low-grade fever, and chills (74%); Grade 1 fatigue (19%); Grade 1 or 2 nausea (10%); and Grade 2 allergy (1 occurrence). Two patients experienced partial responses, which endured for 32 months and 6 years, respectively, and 1 patient currently is experiencing a pathologic complete response, which, to date, has persisted for 50 months; thus, the overall response rate was 10%. In addition, 7 patients (23%) experienced disease stabilization for a median of 8 months (range, 4-48 months). The median duration of survival from the start of Leuvectin treatment was 11 months (range, 2-72 months), with a 1-year survival rate of 48% and a 3-year survival rate of 19%. Laboratory analysis of tumor samples revealed the presence of IL-2 plasmid DNA in six of eight patients posttreatment, increased IL-2 expression in tumor cells in four of eight patients posttreatment, and increased tumor infiltration by CD8-positive lymphocytes in five of eight patients posttreatment.. Immunotherapy with intratumorally administered Leuvectin is safe and can lead to durable objective responses in patients with metastatic renal cell carcinoma.

Topics: Adult; Aged; Cancer Vaccines; Carcinoma, Renal Cell; CD8-Positive T-Lymphocytes; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Humans; Interleukin-2; Kidney Neoplasms; Lipids; Male; Middle Aged; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Survival Analysis

We synthesized new cationic lipids, analogue to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), in order to compare those containing a dodecyl chain with those having a relatively long chain with two or five double bonds, such as squalenyl and dihydrofarnesyl derivatives, or complex saturated structures, such as squalane derivatives. The fusogenic helper lipid dioleoylphosphatidylethanolamine (DOPE) was added to cationic lipids to form a stable complex. Liposomes composed of 50:50 w/w cationic lipid/DOPE were prepared and incubated with plasmidic DNA at various charge ratios and the diameter and zeta potential of the complexes were measured. The surface charge of the DNA/lipid complexes can be controlled by adjusting the cationic lipid/DNA ratio. Finally, we tested the in vitro transfection efficiency of the cationic lipid/DNA complexes using different cell lines. The transfection efficiency was highest for the dodecyloxy derivative containing a single hydroxyethyl group in the head, followed by the dodecyloxy and the farnesyloxy trimethylammonium derivatives. Instead the C27 squalenyl and C27 squalanyl derivatives resulted inactive.

Topics: Animals; Cations; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; DNA; Humans; Lipids; Liposomes; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection

We have evaluated the apoptotic and DNA damaging activity of Idarubicin (IDA) on K-562 cells alone and following the uptake of modified antisense-oligodeoxynucleotides (AS-ODNs) targeting b3a2 mRNA of bcr/abl hybrid gene, after treatment with AS-ODNs/DCChol-DOPE (liposomes) complexes. The uptake of FITC-labeled oligonucleotide-liposomes complexes (FITC-ODNs/DCChol-DOPE) was analyzed by flow cytometry and fluorescence microscopy. Both techniques indicated cytoplasmic accumulation of labeled liposome complexes following 24h of exposure. In absence of liposomes, AS-ODNs uptake was minimal. Pre-treatment of cells with AS-ODNs/DCChol-DOPE increased the capability of IDA to induce apoptosis as determined by morphology and the comet assay. In contrast, the use of a non-sense oligodeoxynucleotide conjugated with liposomes, in the presence of IDA, did not increase K-562 cell apoptosis. Nevertheless, DNA damage in IDA treated cells was not related to ODNs/liposomes pre-treatment, as determined by the comet assay. Our data suggests that DCChol-DOPE increases the uptake of ODNs in K-562 cells, and these modified AS-ODNs increase IDA induced apoptosis by decreasing p210(bcr/abl) levels in K-562 cells.

Topics: Apoptosis; Biological Transport; Cations; Cholesterol; Comet Assay; DNA Damage; DNA, Neoplasm; Drug Synergism; Flow Cytometry; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Idarubicin; K562 Cells; Lipids; Liposomes; Microscopy, Fluorescence; Neoplasm Proteins; Oligodeoxyribonucleotides, Antisense; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Messenger; RNA, Neoplasm

The overproduction of the cytokine TNF-alpha is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-alpha production with ribozymes directed against TNF-alpha mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-alpha ribozymes were more effective inhibitors of TNF-alpha secretion than catalytically inactive ribozyme controls. Inhibition of TNF-alpha secretion was proportional to the concentration of ribozyme administered, with an IC50 of approximately 10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated approximately 6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-alpha secretion by approximately 50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1alpha. Ribozyme-specific TNF-alpha mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.

Topics: Animals; Ascitic Fluid; Base Sequence; Cations; Cells, Cultured; Female; Hydrolysis; Injections, Intraperitoneal; Kinetics; Lipids; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Peritoneum; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Catalytic; RNA, Messenger; Spermine; Tumor Necrosis Factor-alpha

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2, 3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i. p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-gamma and GM-CSF. Taken together, these findings indicate that i. p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.

Topics: Animals; Antineoplastic Agents; Ascites; Cytokines; DNA, Bacterial; Dose-Response Relationship, Immunologic; Female; Growth Inhibitors; Injections, Intraperitoneal; Interleukin-2; Lipids; Mice; Mice, Inbred C3H; Mice, Nude; Ovarian Neoplasms; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Teratocarcinoma

Aerosol delivery of gene therapy for treatment of lung diseases allows topical treatment of the airways with DNA concentrations not obtainable by systemic administration. We have investigated delivery of cationic liposomes complexed to plasmid DNA in a small particle aerosol. Plasmid cDNA-DMRIE/DOPE complexes were nebulized using either an Aerotech II or Puritan-Bennett 1600 (PB1600) nebulizer. Reservoir sampling showed that DNA-DMRIE/DOPE complexes were damaged to a significant degree during nebulization, such that activity of transfected gene was diminished. Of the nebulizers analyzed, DNA-DMRIE/DOPE complexes were more stable in the PB1600. The loss of effective transfection by DNA-DMRIE/DOPE, as detected by decreased reporter gene activity in A549 lung cells, was consistent with denaturation of the DMRIE/DOPE. In contrast, nebulized DNA-DOSPA/DOPE complexes retained complete ability to transfect. Adjustments to flow rate and reservoir volume of the PB1600 allowed a longer period of delivery of active DNA-DMRIE/DOPE particles. DNA-DMRIE/DOPE was radiolabeled with Technetium-99m (99mTc), nebulized, and the output captured in either an Andersen Sampler (AS) (Andersen, 1958) cascade impactor particle size analyzer or an all glass impinger. cDNA-cationic lipid complexes were detected in size ranges of 0.4-10 microns, with most particles found between 1-2 microns. Aerosol output was consistent from 0 to 5 min. These results show the feasibility of aerosol delivery of DNA-cationic lipids for the purposes of gene therapy to the lung.

Topics: Aerosols; Cations; DNA; Gene Transfer Techniques; Humans; Lipids; Liposomes; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Tumor Cells, Cultured

The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cations; DNA; Epithelial Cells; Epithelium; Gene Transfer Techniques; HeLa Cells; Humans; Lipids; Luciferases; Myristic Acids; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Trachea; Transfection