Vernon Knight

Clinical evaluation of the delivery and safety of aerosolized liposomal 9-nitro-20(S)-camptothecin in patients with advanced pulmonary malignancies

Purpose: The purpose is to evaluate the feasibility and safety of aerosol administration of the topoisomerase I inhibitor, 9-nitrocamptothecin, in a liposome formulation, and to recommend a dosage for a Phase II trial for an 8-week daily treatment schedule. Experimental Design: Patients with primary or metastatic lung cancer received aerosolized liposomal 9-nitrocamptothecin for 5 consecutive days/week for 1, 2, 4, or 6 weeks followed by 2 weeks of rest to determine feasibility. For the Phase I part, the dose was increased stepwise from 6.7 up to 26.6 μg/kg/day Monday to Friday for 8 weeks followed by 2 weeks of rest. Results: Twenty-five patients received treatment. The mean baseline forced expiratory volume in 1 second for all patients was 85% of predicted. A dose-limiting toxicity was chemical pharyngitis seen after 1 week in 2 of 2 patients at 26.6 μg/kg/day. At 20.0 μg/kg/day, grade 2 and 3 fatigue prompting a dose reduction was seen after 4 weeks in 2 of 4 patients. Grade 2 toxic effects included nausea/vomiting (9 patients), cough and bronchial irritation (6 patients), fatigue (5 patients), anemia (4 patients), neutropenia (2 patients), anorexia (1 patient), and skin rash around the face mask (1 patient). 9-Nitro-20(S)-camptothecin (9NC) was absorbed systemically. Partial remissions were observed in 2 patients with uterine cancer, and stabilization occurred in 3 patients with primary lung cancer. Conclusions: Aerosol administration of liposomal 9NC was found to be feasible and safe. 9NC delivered as an aerosol was detected in patient’s plasma shortly after the start of treatment. The recommended dose for Phase II studies is 13.3 μg/kg/day (equivalent to 0.5 mg/m2/day), which constitutes two consecutive 30-min nebulizations/day from a nebulizer reservoir with 4 mg of 9NC in 10 ml of sterile water, Monday to Friday for 8 weeks every 10 weeks.

Mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis.

Ribavirin, a guanosine analogue, is a broad spectrum antiviral agent which is effective in the treatment of influenza. In this study, the effect of ribavirin on influenza virus ribonucleoprotein (RNP) synthesis and nucleotide pool sizes was simultaneously measured. Ribavirin (100 microM) reduced viral RNP synthesis 94% as measured by UTP incorporation. Intracellular GTP pools, measured by high performance liquid chromatography, were reduced approximately 45% in ribavirin treated cells, while other nucleotides remained near control values. Attempts to reverse ribavirins inhibitory effects on viral RNP synthesis by addition of exogenous guanosine (50 microM) resulted in only a partial restoration of viral RNP synthesis, despite full restoration of the GTP pool. Dose-response experiments indicated that the GTP pool was significantly reduced (65% of control) at 25 microM ribavirin, and increasing concentrations of the drug caused only a small further reduction in the GTP pool (5-10% at 100 microM). In contrast, RNP synthesis was inhibited by 50% at 25 microM ribavirin and was further decreased to 5% of control at 100 microM ribavirin. Thus, ribavirins antiviral activity may result from a reduction of the GTP pool size combined with direct effects on viral replicative enzymes.

Anticancer exffect 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 μm ± 2.0 mass median diameter to deliver doses of usually less than 200 μg/kg daily, 5 days per week. 9-NC liposome aerosols were generated with a Aerotech II nebulizer (CIS-USA) flowing at 10 l/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.

Amantadine and ribavirin aerosol treatment of influenza A and B infection in mice.

Ribavirin, amantadine, and the two drugs in combination given in small-particle aerosol were highly effective in the treatment of influenza A infection in mice. Treatment was started 72, 96, and 120 h after inoculation and was given continuously for 4 days. With increasing delay in start of treatment, there was a pronounced reduction in effectiveness of ribavirin but not in that of amantadine. The combination treatment reflected the loss of ribavirin activity. Leukocyte infiltration and virus titers in the lungs were inversely related to the effectiveness of treatment. Influenza B infection treated 72 h after inoculation responded only to ribavirin, as indicated by the criteria described for influenza A. Intraperitoneal administration of drug begun 72 h after inoculation in regimens equivalent to aerosol afforded less protection than aerosol treatment.

A study of 99mtechnetium-labelled beclomethasone dipropionate dilauroylphosphatidylcholine liposome aerosol in normal volunteers

Abstract 99mTechnetium (99mTc) was bound to preformed beclomethasone dipropionate (Bec) dilauroylphosphatidylcholine (DLPC) liposomes (Bec-DLPC) in the presence of the reducing agent, stannous chloride. Labelling efficiency was 96–99% as determined by micropartition and chromatographic analysis. Andersen cascade impactor analysis showed close correlation of the distribution of 99mTc, DLPC, and Bec over the full range of particle sizes sampled. In mouse biodistribution studies, approximately one-half of 99mTc activity delivered to the lungs was retained at 24 h. 99mTc clearance was almost exclusively via the gastrointestinal tract. In contrast, free 99mTc (administered unbound to Bec-DLPC) liposomes was cleared from mouse lungs within a few minutes. Six normal volunteers inhaled 20 breaths of the labelled Bec-DLPC liposome aerosol from each of two nebulizers (Aerotech II, MMAD 1.5μ, GSD 2.4) (Spira, MMAD 3.6μ, GSD 2.5). Immediately following inhalation, the gamma camera analysis showed 17% pulmonary deposition of inhaled 99mTc-Bec-DLPC with the Aerotech II and 14% with the Spira nebulizer. At 3 h after Aerotech II exposure, 93% of deposited activity was retained in the lung and 82% was retained from the Spira nebulizer. These findings suggest that there is a stable association of 99mTc with the Bec-DLPC liposomes and that inhaled liposomes were cleared slowly from the lungs of normal volunteers. The smaller particles produced by the Aerotech II nebulizer are presumably responsible for the somewhat larger deposition and longer persistence of pulmonary activity. These findings encourage further study of this modality of treatment for asthma and related conditions.

Effect of ribavirin triphosphate on primer generation and elongation during influenza virus transcription in vitro

These studies examine the effect of ribavirin triphosphate (RTP) on two replicative functions associated with influenza virus nucleocapsids, primer generation and its subsequent elongation. To study primer generation influenza virus cores were added to beta-globin mRNA in the presence of only [32P]GTP. To examine elongation, ATP and CTP were added to the reaction mixture to permit limited elongation, and products from both reactions were separated on polyacrylamide gels and quantified. Under these conditions, the 50% inhibitory concentration of RTP for primer generation was 3.0 mM, and the 50% inhibitory concentration for elongation was 0.6 mM. RNA polymerase activity associated with cores isolated from clinical strains of influenza A and B viruses reacted as did the laboratory strain of influenza virus and was equally susceptible to inhibition by RTP.

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.

Growth suppression of established human osteosarcoma lung metastases in mice by aerosol gene therapy with PEI-p53 complexes

Lung metastases are a frequent complication of osteosarcoma and a treatment that would reduce the severity of this complication would be of great benefit to patients. We have used a formulation consisting of polyethyleneimine (PEI) and a p53 gene administered in aerosol to treat established lung micrometastases as a model of human osteosarcoma in nude mice. The SAOS-LM6 cell line, a metastatic derivative of the p53 null SAOS-2 line, expresses high levels of p53 protein after in vitro transfection with PEI– p53 complexes as determined by ELISA, and transfection with both p53 wt and the p53 variant, p53 -CD(1–366) in vitro , results in a marked inhibition of SAOS-LM6 cell proliferation. Aerosol delivery of plasmid DNA containing either the p53 gene or a p53-CD(1–366) variant gene formulated with PEI to mice resulted in highly significant reductions in the numbers and size of tumors ( P <.001), the total number of tumor foci in the lungs ( P <.001) and the size of individual tumor nodules in treated animals compared to untreated, PEI only–treated and PEI–CAT–treated control animals. The different tissues examined did not reveal any signs of toxicity or inflammation after repeated exposure to PEI–DNA. The aerosol delivery of PEI-based formulations of p53 or synthetic p53 variant genes represents a promising new strategy for the treatment of established human osteosarcoma lung metastases. The noninvasive nature of aerosol delivery coupled with low toxicity also make this therapeutic approach potentially appropriate for combination therapy with either radio- or chemotherapy. Cancer Gene Therapy (2001) 8, 619–627.

Ribavirin small-particle aerosol treatment of infections caused by influenza virus strains A/Victoria/7/83 (H1N1) and B/Texas/1/84.

In a double-blind study of influenza in a population of college students in 1984, ribavirin small-particle aerosol treatment of 38 patients (18 treated, 20 control) infected with a new antigenic variant, influenza virus strain A/Victoria/7/83 (H1N1), was associated with statistically significant reductions in the height and duration of fever, systemic symptoms, and virus shedding. Patients received a total of 2.4 g of ribavirin over 42 h during 68 h of hospitalization without any side effects. In addition, in a study of patients infected with influenza virus strain B/Texas/1/84 (seven treated, eight control) treated with ribavirin aerosol showed a trend of more rapid recovery than control patients.

Small particle aerosols of enviroxime-containing liposomes

Enviroxime inhibits the replication of all rhinoviruses tested in vitro at very low concentrations (10-100 ng/ml), but evaluations in humans have not consistently shown efficacy. Lack of an appropriate method for administering this water-insoluble drug may have contributed to the latter result. The present report describes the characteristics and utilization of small particle aerosols to continuously deliver enviroxime-containing liposomes (LE) throughout the respiratory tract. The enviroxime content of liposomes and biological fluids of exposed individuals was quantified by high performance liquid chromatography using C18 resin, a mobile phase of 60:40 acetonitrile:water, and monitoring at 215 nm. Small particle aerosols of LE generated by Puritan-Bennett nebulizers had mass median diameters ranging from 2.4 to 3.1 microns. The concentration of enviroxime in aerosol particles was proportional to the reservoir concentration; during the first hour of operation, the mean concentration was 20 micrograms of enviroxime/l of aerosol. Liposome particles in the reservoir, although initially heterogeneous in size (less than 0.1 to greater than 1 micron), were processed by passage through the nebulizer to smaller, more homogeneous particles; the majority were less than 0.2 micron. In a preliminary study to evaluate short term tolerance and toxicity, five volunteers were exposed to small particle aerosol of LE for 1 h. At 1 h post-treatment, large amounts of enviroxime were still present in the nasal wash as determined both by HPLC and biological assay. Enviroxime was not detected in any urine sample and was detected in only 1 of 5 serum samples. No side effects were noted. This data suggest that liposome aerosols offer a method for the delivery of hydrophobic compounds for the treatment of respiratory diseases.