Pulmonary Administration of Microparticulate Antisense Oligonucleotide (ASO) for the Treatment of Lung Inflammation.

Targeted delivery to the lung for controlling lung inflammation is an area that we have explored in this study. The purpose was to use microparticles containing an antisense oligonucleotide (ASO) to NF-κB to inhibit the production of proinflammatory cytokines. Microparticles were prepared using the B-290 Buchi Spray Dryer using albumin as the microparticle matrix. Physicochemical characterization of the microparticles showed the size ranged from 2 to 5 µm, the charge was - 38.4 mV, and they had a sustained release profile over 72 h. Uptake of FITC-labeled ASO-loaded microparticles versus FITC-labeled ASO solution by RAW264.7 murine macrophage cells was 5-10-fold higher. After pulmonary delivery of microparticles to Sprague-Dawley rats, the microparticles were uniformly distributed throughout the lung and were retained in the lungs until 48 h. Serum cytokine (TNF-α and IL-1ß) levels of rats after induction of lung inflammation by lipopolysaccharide were measured until 72 h. Animals receiving ASO-loaded microparticles were successful in significantly controlling lung inflammation during this period as compared to animals receiving no treatment. This study was successful in proving that microparticulate ASO therapy was capable of controlling lung inflammation.

Oral microparticulate vaccine for melanoma using M-cell targeting.

Cancer vaccines are limited in their use, because of their inability to mount a robust anti-tumor immune response. Thus, targeting M-cells in the small intestine, which are responsible for entry of many pathogens, will be an attractive way to elicit a strong immune response toward particulate antigens. Therefore, in the present investigation, we demonstrated that efficient oral vaccination against melanoma antigens could be accomplished by incorporating the antigens in an albumin-based microparticle with a ligand AAL (Aleuria aurantia lectin) targeted specifically to M-cells. The oral microparticulate vaccine effectively protected the mice from subcutaneous challenge with tumor cells in prophylactic settings. The animals were vaccinated with antigen microparticles having a size range of around 1-1.25 µm where one prime and four booster doses were administered every 14 days over 10 weeks of duration, followed by challenge with live tumor cells, which showed complete tumor protection after oral vaccination. With the inclusion of ligand in the microparticles, we observed significantly higher IgG titers (1565 µg/mL) as compared to the microparticle formulations without AAL (872 µg/mL). This data suggests that ligand loaded microparticles may have the potential to target antigens to M-cells for an efficient oral vaccination.

Potentiation of pro-inflammatory cytokine suppression and survival by microencapsulated dexamethasone in the treatment of experimental sepsis.

Cytokine inhibiting drugs are much more effective when delivered intracellularly to phagocytic cells in the microencapsulated form. Dexamethasone is a powerful inhibitor of TNF-α cytokine through inhibition of NF-κB which is a gene regulator of multiple pro-inflammatory cytokines. We have determined the effect of microencapsulated dexamethasone in pro-inflammatory cytokine release both in in vitro using whole blood model, and in vivo using peritonitis model of septic shock. Microspheres of 1-4 µm mean size were prepared by using albumin polymer matrix in a one-step spray drying method. Microencapsulated form of dexamethasone with concentration of 10(-1), 10(-2) and 10(-3) M was compared to an equivalent concentration of solution form of dexamethasone in the in vitro whole blood model. The results show microencapsulated dexamethasone inhibited tumor necrosis factor-alpha (TNF-α) and interleukin-beta (IL-1ß) significantly in comparison with the solution form of dexamethasone. The in vivo peritonitis model also demonstrated significant inhibition of TNF-α and IL-1ß cytokines in microencapsulated form in comparison with solution form of dexamethasone. In the in vivo study, the animal survival rate after 5 days was 90%, dexamethasone in solution with gentamicin was 40% and gentamicin alone was 30%. This study demonstrates significantly improved inhibition of TNF-α and IL-1ß both in vivo and in vitro when dexamethasone was used in microencapsulated form.

A novel microparticulate vaccine for melanoma cancer using transdermal delivery.

In this study, we formulated a microparticulate melanoma cancer vaccine via the transdermal route. The vaccine was delivered using microneedle-based Dermaroller® which is available for cosmetic purposes. Unlike subcutaneous injections, administration using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The vaccine microparticles were taken up by the antigen presenting cells which demonstrated a strong IgG titre level of 930 ug/mL in serum samples. The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size range of around 0.63-1.4 µm which acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 8 weeks duration, followed by challenge with live tumour cells which showed protection after transdermal vaccination.

Synergism in survival to endotoxic shock in rats given microencapsulated CNI-1493 and antisense oligomers to NF-kappaB.

The synthesis of TNF may be inhibited at the transcriptional level by antisense to either TNF or NF-kappaB or at the post-transcriptional level by CNI-1493, a guanylhydrazone compound which inhibits p38 MAP kinase activity. Previous studies have demonstrated that targeting macrophages and other phagocytic cells by intracellular drug delivery using albumin microcapsules containing either antisense oligomers to NF-kappaB or CNI-1493 greatly enhances intracellular drug concentration and survival in both endotoxic shock and sepsis models. It is the purpose of this study to determine if microencapsulated drugs acting at different stages in the synthesis of TNF are synergistic. Four groups of 10 rats each were given 15 mg kg(-1) of E.coli endotoxin and treated with (1) CNI-1493 1 mg kg(-1), (2) antisense oligomers to NF-kappaB at 100 mcg, (3) CNI-1493 1 mg kg(-1) plus antisense kappa to NF- at 100 mg kg(-1) and (4) CNI-1493 200 mg kg(-1) plus antisense oligomer to NF-kappaB at 200 mg kg(-1). TNF and IL1 were measured by ELISA at 4, 8, 24 and 48 h. The rats were observed for 5 days. The combination of CNI-1493 and antisense oligomers to NF-kappaB inhibited TNF 41% greater that CNI alone and 51% greater than antisense oligomers to NF-kappaB alone at 4 h after endotoxin administration. Survival at 5 days with CNI alone was 0%, 20% with antisense oligomers to NF-kappaB and 60% with the combination. In conclusion, synergism in survival occurs using microencapsulated drugs acting at different points in the synthesis of TNF was demonstrated using an in-vivo model of endotoxic shock. Both the amount of TNF inhibition and the mortality were significantly improved with combination therapy. Multiple drugs acting at different sites in the synthesis of TNF may be useful in the treatment of disease states characterized by pro-inflammatory cytokine release.

The effect of intracellular delivery of catalase and antisense oligonucleotides to NF-kappaB using albumin microcapsules in the endotoxic shock model.

UNLABELLED: Microencapsulated (MC) catalase has been shown to inhibit H(2)O(2) and tumor necrosis factor (TNF) in vitro after endotoxin stimulation. It is the purpose of this study to determine whether MC catalase improves pro-inflammatory cytokine inhibition and mortality in an endotoxic shock model in vivo. We also examined whether MC catalase and antisense oligonucleotides (ASO) to nuclear factor kappaB (NF-kappaB) together improved survival by inhibiting pro-inflammatory cytokines using different mechanisms. METHODS: Albumin microcapsules containing catalase and ASO to NF-kappaB were prepared 2-7 microm in size by using a Büchi spray dryer. Progressively increasing doses of MC catalase, MC ASO to NF-kappaB, and the combination were given to rats before the administration of Escherichia coli endotoxin. Results demonstrated 60% survival in rats given 15 mg/kg MC catalase, 70% survival with 20 mg/kg MC ASO NF-kappaB, and 80% survival with the combination. TNF was inhibited by 53% in the MC catalase group 4 h after endotoxin administration, 43% in the ASO NF-kappaB group, and 78% in the combination group compared to controls. In conclusion, this study demonstrates the effectiveness of MC intracellular delivery of the naturally occurring antioxidant catalase in improving animal survival. The addition of ASO to NF-kappaB improved both cytokine inhibition and animal survival in endotoxic shock.

A methodology for quantitation and characterization of oligonucleotides in albumin microspheres.

A fluorescence assay was developed to quantify oligonucleotides (ODNs) encapsulated in bovine serum albumin (BSA) microspheres using antisense to Nuclear Factor-kappaB (NF-kappaB) as a model ODN and employing Oligreen as the fluorescent dye. Methodologies were optimized for the suspension of the microspheres as well as release of the encapsulated ODN using protease digestion. This was followed by the detection and quantitation of the ODN using the Oligreen dye. The Oligreen fluorescence assay gave a concentration-dependent fluorescent interaction with the ODN. Further characterization of the ODN with respect to their structural integrity in non-irradiated and gamma-irradiated antisense encapsulated in BSA microspheres was performed using HPLC, infrared spectroscopy and polyacrylamide gel electrophoresis. Results showed no structural modification of antisense in the BSA microspheres as determined by HPLC retention times for the pure antisense and microsphere-encapsulated ODN. The migration pattern of the antisense in polyacrylamide gels confirmed the absence of significant alterations as a result of the encapsulation process or due to gamma-irradiation. The infrared spectra of non-irradiated and gamma-irradiated antisense to NF-kappaB microspheres also displayed peaks characteristic of the functional groups. The fluorescence assay could also detect NF-kappaB antisense in the serum of rats administered with encapsulated antisense by oral and intravenous routes. This methodology should be valuable for the analysis of BSA-encapsulated antisense ODN and for pharmacokinetic studies during antisense therapy.

Reversal of LPS induced endothelial cell TNF synthesis and increased permeability with microencapsulated antisense oligomers to NF-kappaB.

Endothelial cells form the barrier between the circulation and interstitial space. Changes in permeability of endothelial cells allow penetration of inflammatory cells such as polymorphonuclear cells and macrophages to respond to infections and other inflammatory stimuli. Endothelial cells have also been shown to be phagocytic and produce pro-inflammatory cytokines such as TNF. It is the purpose of this study to evaluate endothelial cell phagocytosis of albumin microspheres containing antisense oligonucluetide to NF-kappaB (MASO), the effect of MASO on TNF synthesis after LPS stimulation and the effect of TNF inhibition on the permeability of endothelial cells in vitro. Results were (1) endothelial cells avidly phagocytozed albumin miocrospheres 1.0 and 1.7 microm in size, (2) phagocytosis of microspheres was potentiated by LPS, (3) TNF is synthesized by endothelial cells in cell culture with the peak concentrations occurring 4 h after stimulation with LPS, (4) MASO results in high intracellular concentration of oligomer, (5) MASO inhibits TNF synthesis to a greater extent than equivalent amounts of NF-kappaB antisense in solution and (6) the inhibition of TNF by MASO significantly decreases the permeability of albumin through endothelial cells in vitro.

Pro-inflammatory cytokine inhibition in the primate using microencapsulated antisense oligomers to NF-kappaB.

PRIMARY OBJECTIVE: Antisense oligomers to NF-kappaB (ASO) were incorporated into albumin microspheres to determine if microcapsules containing ASO inhibit pro-inflammatory cytokines to a greater extent than comparable doses of ASO in solution. Phagocytosis of microcapsules and intracellular release of ASO in macrophages was evaluated. RESEARCH DESIGN: Comparable doses of microencapsulated ASO and ASO in solution were evaluated in non-human primates. METHODS: Blood was sampled and stimulated with Escherichia coli endotoxin ex vivo. TNF, IL-1 and IL-6 concentrations were compared for 72 hrs. The intracellular concentration of ASO was measured in macrophages in vitro to evaluate the difference in intracellular penetration of microencapsulated ASO. RESULTS: Microencapsulated ASO produced significantly greater cytokine inhibition at all time points compared to ASO in solution. There were no side effects to ASO in the baboons. Intracellular ASO concentration was 10 fold greater in macrophages using microencapsulation. CONCLUSIONS: Microencapsulated ASO to NF-kappaB is more effective than ASO in solution in pro-inflammatory cytokine inhibition in non-human primates.

Treatment of experimental septic shock with microencapsulated antisense oligomers to NF-kappaB.

NF-kappaB is an ideal target for inhibition of proinflammatory cytokines. The purpose of this study was to determine if microencapsulated antisense oligomer to NF-kappaB can inhibit proinflammatory cytokine release in response to Escherichia coli endotoxin and bacteria. Microencapsulation takes advantage of the phagocytic function of the macrophage to deliver the oligomer intracellularly and enhance the effect. Albumin microcapsules 1 microm in size were prepared by a nebulization method containing antisense oligomers to NF-kappaB. E. coli endotoxin was incubated in 1 ml aliquots of whole blood. Microencapsulated antisense to NF-kappaB was given, and the inhibition of tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and IL-8 was compared with similar amounts of oligomer in solution. Endotoxic shock was produced in rats using E. coli endotoxin (15 mg/kg). Peritonitis was induced by injecting 10(10) CFU E. coli. Cytokines were measured after simultaneous and delayed (4 h) administration of antisense to NF-kappaB in microcapsules and solution form. TNF was suppressed by 81% in whole blood, 56% in the endotoxic shock model, 89% in the peritonitis model (simultaneous treatment), and 56% in the delayed treatment group. Survival was 70% in the endotoxic shock group, 80% in the simultaneous peritonitis group, and 70% in the delayed treatment group. Microcapsule treatment using antisense to NF-kappaB suppressed TNF and IL-1 levels and mortality significantly better than all solution treatment groups in the whole blood model, endotoxic shock model, and peritonitis model.

Microencapsulation of tumor necrosis factor oligomers: a new approach to proinflammatory cytokine inhibition.

Antisense oligonucleotides offer great therapeutic potential provided adequate intracellular penetration can be achieved. In this study, we evaluated the effectiveness of microencapsulating antisense oligonucleotides to tumor necrosis factor (TNF) in suppressing TNF release in vitro and in vivo. Microencapsulation of TNF oligomers was performed using albumin to produce microcapsules 0.6-1.0 mum in size that target phagocytic cells. Albumin microcapsules containing fluoresceinated TNF oligomers were incubated with U-937 cells to observe uptake. Microcapsules were added to whole blood and stimulated with Escherichia coli endotoxin. Endotoxin was given intravenously (i.v.) to rats along with 100 mug microencapsulated TNF oligomers to determine TNF inhibition and animal survival. E. coli was given intraperitoneally (i.p.) along with gentamicin and microencapsulated TNF oligomers to assess TNF inhibition and animal survival. The duration of microencapsulated antisense TNF oligomers was also determined in vivo. The results demonstrated rapid uptake of the microcapsules by macrophages after 2 h and 4 h incubation. There was improvement in TNF inhibition in vitro and improved animal survival by microencapsulated antisense in both endotoxin (100% survival) and peritonitis models (70% survival) compared with free antisense oligomers in solution. Microencapsulation extended the duration of action of the oligomers to 72 h. Intracellular targeting of macrophages with antisense oligomers to TNF by microencapsules as a delivery system improves TNF inhibition using the models of whole blood endotoxin stimulation and endotoxic shock and peritonitis in rats.