Cyclosporin A aerosol improves the anticancer effect of paclitaxel aerosol in mice.

Paclitaxel (PTX) is a lipophilic agent with broad anticancer activity. In the present study we examined the antitumor effect and toxicity of co-administration of cyclosporine A (CsA) and PTX in liposomal aerosol using the Renca lung metastases mouse model. The untreated and PTX-only groups exhibited cancer growth while CsA aerosol plus PTX had more favorable effects on tumor growth. Weight loss was seen in mice treated with CsA/PTX+CsA by day 9 to 22. Histopathological examination showed no toxicity following treatment. The findings offer evidence that a combination of CsA and PTX may be suitable for aerosol treatment of lung cancer if it is possible to control toxicity of the therapy.

Camptothecins and lung cancer: improved delivery systems by aerosol.

Lung cancer is the largest and the most common cause of cancer-related deaths worldwide. The cure rate for lung cancer remains lowest among all malignancies. The discovery of new chemical agents with activity in first onset and recurrent disease is crucial for advancing treatment of patients with pulmonary tumors. Camptothecins are known as inhibitors of topoisomerase I, one of the key enzymes for DNA replication and subsequent cell proliferation. Preclinical and clinical studies had shown that the camptothecins are active against lung cancer and other solid malignancies. In this paper, we review the status of camptothecin and derivatives for treatment of pulmonary cancers, including the development of new formulations and, particularly, novel aerosol routes of drug administration, and their application in combination therapy.

Paclitaxel liposome aerosol treatment induces inhibition of pulmonary metastases in murine renal carcinoma model.

The present studies were undertaken to evaluate the pulmonary pharmacokinetics and therapeutic efficacy of paclitaxel (PTX) administered by aerosol. PTX was encapsulated into dilauroylphosphatidylcholine liposomal formulations (PTX-DLPC). The deposition and clearance of PTX-DLPC in the lungs administered by aerosol or i.v. at comparative doses was performed, and PTX was quantitatively determined in tissue extracts by high-performance liquid chromatography analysis. The murine renal carcinoma (Renca) pulmonary metastases model was used to determine the therapeutic effect of drug formulation administered by aerosol. PTX-DLPC aerosols were generated with the Aero-Mist jet nebulizer (cis-USA). The most effective schedule of treatment was when mice inhaled the drug for 30 min 3 days per week. There was a significant reduction of the lung weights and reduced number of visible tumor foci on the lung surfaces of mice treated with PTX aerosol (P < 0.004 and P < 0.01, respectively) compared with control groups. Inhalation of PTX-DLPC also led to prolonged survival in mice inoculated with Renca cells. The results of the present studies demonstrate the therapeutic potential of aerosol technology for lung cancer treatment.

Improved respiratory delivery of the anticancer drugs, camptothecin and paclitaxel, with 5% CO2-enriched air: pharmacokinetic studies.

PURPOSE: To increase pulmonary deposition of anticancer liposome aerosols in mice by modulation of respiratory physiology through the addition of 5% CO2 to the air source used to generate the aerosols. Breathing CO2-enriched aerosol increases pulmonary ventilation with concurrent increased deposition of inhaled particles. METHODS: Dilauroylphosphatidylcholine liposome formulations of two anticancer drugs, paclitaxel (PTX) and camptothecin (CPT), were investigated. The aerosol droplet size was measured using an Andersen cascade impactor. Drug concentrations in aerosol droplet fractions and tissues were determined by HPLC analysis. ICR mice were exposed to each liposome aerosol for 30 min. For each drug, one group of mice inhaled the drug-liposome aerosol generated with a mixture of 5% CO2 in air and another group inhaled the drug-liposome aerosols produced with normal air. Tissue distribution and pharmacokinetics were determined for both drug delivery systems. RESULTS: Significantly higher concentrations of PTX and CPT were found in organs of mice exposed to 5% CO2-air aerosols compared to organs of mice exposed to normal air aerosols. The highest concentrations of drug were detected in the lungs and were two- to fourfold higher with 5% CO2-air aerosols than with aerosols generated with normal air. Higher concentrations were also detected in liver, spleen, kidneys, blood, and brain. CONCLUSION: 5% CO2 enrichment of air increased respiratory tract deposition of inhaled aerosol particles containing PTX and CPT.

9-Nitrocamptothecin liposome aerosol treatment of melanoma and osteosarcoma lung metastases in mice.

The response rates of relapsed osteosarcoma and melanoma pulmonary metastases to traditional i.v. chemotherapeutic regimens have been disappointing. Direct drug delivery of chemotherapy to the lungs could increase the drug concentration in the tumor area and may offer a new therapeutic approach for these patients. Previous studies demonstrated that drugs delivered to the respiratory tract in liposomal formulation resulted in high pulmonary drug concentration, reduced systemic toxicity, and reduced dosage requirements compared with parenteral and oral administration. To determine whether this approach has utility against pulmonary metastases, the efficacy of aerosol therapy with liposome-encapsulated 9-nitrocamptothecin (L-9NC) was determined using two different experimental lung metastasis models. C57BL/6 mice were treated the day after the i.v. injection of B16 melanoma cells with aerosol L-9NC for 1 h (153 microg 9-nitrocamptothecin/kg) for 5 days per week for up to 3 weeks. Aerosol L-9NC treatment resulted in a reduction in lung weights (P = 0.005) and number of tumor foci (P < 0.001). Visible tumor nodules were fewer and smaller in the 9-nitrocamptothecin-treated group than in untreated control mice (P < 0.001). Using a newly developed human osteosarcoma experimental metastasis model in nude mice, we demonstrated that aerosol L-9NC was also effective against established lung metastases. Aerosol therapy initiated on the ninth week after i.v. tumor injection and continued for 8 or 10 weeks produced highly significant reductions in the number of animals with both visible and microscopic disease (P < 0.02), the total number of tumor foci in the lungs (P < 0.005), and the size of the individual tumor nodules (P < 0.02). These data suggest that L-9NC aerosol therapy may offer significant advantage over existing methods in the treatment of melanoma and osteosarcoma pulmonary metastases.

9-Nitrocamptothecin liposome aerosol treatment of human cancer subcutaneous xenografts and pulmonary cancer metastases in mice.

The purpose of this study was to test the anticancer properties of the water-insoluble derivative of camptothecin, 9-nitrocamptothecin (9NC), administered in a liposome formulation (L-9NC) in aerosol to mice with subcutaneous xenografts of three human cancers and in mice with murine melanoma and human osteosarcoma pulmonary metastases. The drug was formulated with dilauroylphosphatidylcholine and nebulized in particle sizes of 1.2-1.6 microns mass median aerodynamic diameter and a geometric standard deviation of 2.0. The aerosol was generated with the nebulizer flowing at 10 l/min and delivered to mice in sealed plastic cages or in a nose-only exposure chamber. Aerosol was administered for 15 min to 2 hr daily, delivering deposited doses in the respiratory tract of 8.1-306.7 micrograms of 9NC/kg. With subcutaneous tumors, growth was greatly inhibited or tumors were undetectable after several weeks of treatment. We also showed that oral dosage with L-9NC had no detectable effect on cancer growth, and thus the benefit from aerosol treatment was due to pulmonary deposition and not the larger fraction of drug deposited in the nose of mice during aerosol treatment which is promptly swallowed. Intramuscular L-9NC in slightly larger doses than given in the aerosol had detectable anticancer activity, but it was significantly less than in mice receiving the drug by aerosol. With metastatic pulmonary cancers, treated animals showed highly significantly less cancer growth than control animals. L-9NC aerosol showed a major therapeutic benefit in the treatment of subcutaneous human cancer xenografts in nude mice, suggesting that cancers at systemic sites might be responsive to this treatment. In addition, the strong anticancer effect of L-9NC aerosol on pulmonary metastases offers a therapeutic approach for treatment of pulmonary cancers. Thus, L-9NC aerosol may have applicability in the treatment of cancers throughout the body.

Anticancer effect of 9-nitrocamptothecin liposome aerosol on human cancer xenografts in nude mice.

PURPOSE: To test the anticancer properties of the water-insoluble derivative of camptothecin, 9-nitrocamptothecin (9-NC) against human breast, colon and lung cancer xenografts in nude mice when administered in liposome aerosol. METHODS: The drug was formulated with dilauroylphosphatidylcholine and nebulized in a particle size of 1.6 microm +/- 2.0 mass median diameter to deliver doses of usually less than 200 microg/kg daily, 5 days per week. 9-NC liposome aerosols were generated with a Aerotech II nebulizer (CIS-USA) flowing at 101/min from a compressed air source and delivered to mice in sealed plastic cages or in a nose-only exposure chamber. RESULTS: Tumor growth was greatly reduced or tumors were undetectable after several weeks of treatment. Colon tumor was least responsive. 9-NC was better than the parent compound, camptothecin, also water-insoluble, tested by aerosol in a similar liposomal preparation. Equivalent doses of 9-NC liposome preparations administered by mouth were substantially without effect while there was some effect, but limited, of the liposome preparation given intramuscularly. CONCLUSIONS: 9-NC liposome aerosol was strikingly effective in the treatment of three human cancer xenografts growing subcutaneously over the thorax in nude mice at doses much smaller than those traditionally used in mice administered by other routes.

Distribution of camptothecin after delivery as a liposome aerosol or following intramuscular injection in mice.

PURPOSE: The plant alkaloid camptothecin (CPT) has shown significant antitumor activity against a wide variety of human tumors xenografted in nude mice. In previous studies we have found that administration of dilauroylphosphatidylcholine (DLPC) liposome aerosols containing 9-nitrocamptothecin (9-NC) inhibits the growth of human breast, colon and lung cancer xenografts. The purpose of this study was to analyze the pharmacokinetics and tissue distribution of inhaled CPT formulated in DLPC liposomes. METHODS: C57BL/6 mice with subcutaneous Lewis lung carcinoma, Swiss nu/nu mice with human lung carcinoma xenografts and BALB/c mice without tumors were used for pharmacokinetic studies of CPT administered as a liposome aerosol and BALB/c mice were given CPT intramuscularly. RESULTS: After 30 min inhalation of CPT liposome aerosol, drug was deposited in the lungs (310 ng/g) and was followed promptly by the appearance of high concentrations in the liver (192 ng/g) and with lesser amounts appearing in other organs. Drug concentration in the brain was 61 ng/g. After intramuscular injection of CPT dissolved in DMSO, drug was released from the site of injection very slowly and accumulated mainly in the liver (136 ng/g). Only trace amounts appeared in the lungs (2-4 ng/g). These results demonstrate a prompt pulmonary and later systemic distribution of CPT following liposome aerosol administration. CONCLUSIONS: The substantial concentrations of CPT in lungs and other organs following inhalation of liposome aerosol suggest the possible benefit of it and of its more active derivative, 9-NC, in the treatment of lung, liver, kidney and brain cancer in humans.

[The immunological activity of Neisseria meningitidis lipo-oligosaccharide incorporated into liposomes]. / Immunologicheskaia aktivnost' lipooligosakharida Neisseria meningitidis, vkliuchennogo v liposomy.

Immune response to lipooligosaccharide (LOS), isolated from group B N. meningitidis cell wall, was studied after its incorporation into liposomes. The adjuvant effect produced by liposomal LOS, more pronounced in comparison with that produced by native LOS, was observed after the injection of the preparation by different routes (intravenous, intraperitoneal and subcutaneous injections), but the highest level of response was achieved after the intravenous and intraperitoneal injections of the preparation. Experiments aimed at the study of the dynamics of plaque-forming cells and the level of antibodies after intraperitoneal immunization of CBA mice with LOS revealed that immune response to liposomal LOS was more intense and prolonged than that to the free antigen. Liposomal LOS induced genetically nonrestricted and T-independent immune response. This fact was confirmed in experiments on mice having different haplotypes, such as CBA (k), C57BL/6 (b), BALB/c (d) and in nude mice with congenital thymic aplasia. In addition, liposomal LOS induced mainly the accumulation of IgM and IgG and did not produce the effect of immunological memory.

Non-specific modulation of the immune response with liposomal meningococcal lipopolysaccharide: role of different cells and cytokines.

The immunomodulating action of Neisseria meningitidis lipopolysaccharide (LPS) incorporated into liposomes and the activation of different populations of immunocompetent cells or the secretion of cytokines were studied. LPS stimulated an anti-sheep red blood cell (SRBC) plaque-forming cell response in the spleen of mice after simultaneous injection of LPS and SRBC but if LPS was administered 3 days before the immunization with SRBC the response to SRBC was strongly suppressed. After the incorporation of LPS into liposomes the stimulation index was increased from 6 to 19 and the liposomal LPS did not suppress the immune response to SRBC. The incorporation of LPS into liposomes leads to enhancement of B-mitogenic properties of LPS, as liposomal LPS stimulated the proliferation of splenocytes in mice better than free LPS and has no influence on the thymocytes. The liposomal LPS induced more prolonged and significant accumulation of IgM-secreting cells in the spleen of mice in comparison with the free LPS. Liposomal LPS also induced more active accumulation of IFN-gamma in human peripheral blood mononuclear cells and less active accumulation of monokines, contributing to the realization of the toxic properties of endotoxin (IL-1 alpha, TNF-alpha, IL-6 and GM-CSF). These results demonstrated that the incorporation of N. meningitidis LPS into liposomes dramatically changed its immunomodulating activity. The data obtained are important for the construction of an adjuvant formulation for synthetic immunogens capable of inducing genetically unrestricted immune responses.

Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.

FITC-labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies.

FITC-labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC-LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxicity decrease in actinomycin D-sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC-LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co-sonication of lipid suspension and FITC-LPS. Following FITC-LPS fluorescence polarization it was shown that the substance seems to be clusterized in the liposomal membrane starting from FITC-LPS/lipid molar ratio 1:800.