Brian E. Gilbert

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.

Augmented Lung Inflammation Protects against Influenza A Pneumonia

Background Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia. Methods and Findings To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001). Conclusions Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia.

Essential Role for Autophagy in the Maintenance of Immunological Memory Against Influenza Infection

Vaccination has been the most widely used strategy to protect against viral infections for centuries. However, the molecular mechanisms governing the long-term persistence of immunological memory in response to vaccines remain unclear. Here we show that autophagy has a critical role in the maintenance of memory B cells that protect against influenza virus infection. Memory B cells displayed elevated levels of basal autophagy with increased expression of genes that regulate autophagy initiation or autophagosome maturation. Mice with B cell–specific deletion of Atg7 (B/Atg7−/− mice) showed normal primary antibody responses after immunization against influenza but failed to generate protective secondary antibody responses when challenged with influenza viruses, resulting in high viral loads, widespread lung destruction and increased fatality. Our results suggest that autophagy is essential for the survival of virus-specific memory B cells in mice and the maintenance of protective antibody responses required to combat infections.

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.

High-dose, short-duration ribavirin aerosol therapy compared with standard ribavirin therapy in children with suspected respiratory syncytial virus infection

Children with suspected respiratory syncytial virus infection were examined prospectively in a randomized evaluation of standard ribavirin aerosol therapy (6 gm/300 ml water for 18 hours daily) compared with high-dose, short-duration ribavirin aerosol therapy (6 gm/100 ml water given for a period of 2 hours three times a day) by means of an oxygen hood (n = 20) or a ventilator (n = 12). Viral shedding was quantitated daily; clinical observations were recorded daily by 2 physicians aware and one unaware of treatment assignments. Study characteristics evaluated at entry were not significantly different in the high-dose and the standard-dose groups. Viral titers and clinical scores decreased similarly in both groups during the study; pulmonary function test results were also similar at discharge in children not receiving mechanical ventilation. Potential complications related to aerosol therapy were noted in three patients (one hood patient who was receiving standard therapy; two patients with an endotracheal tube in place who were receiving high-dose therapy); substantial crystallization was noted in the tubing of the patients undergoing intubation and receiving high-dose therapy. Environmental sampling revealed that ribavirin was nearly undetectable near patients supported by mechanical ventilation who were receiving either form of therapy, and was significantly decreased on a daily basis in patients without an endotracheal tube who were receiving high-dose therapy compared with those receiving standard therapy. The effects of high-dose, short-duration aerosol ribavirin therapy were similar to those of standard-dose therapy in our study patients and resulted in a decreased release of ribavirin into the room of patients receiving therapy by means of an oxygen hood.

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.

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

The objective of this study was to assess the effect of cyclosporin A liposome aerosol on the anticancer activity of paclitaxel (PTX) liposome aerosol against renal cell carcinoma (Renca) pulmonary metastases in mice. Cyclosporin A (CsA) was administered as a liposome aerosol for one-half hour before starting one-half hour treatment with PTX liposome aerosol (CsA/PTX), and in a second groups of animals cyclosporin A liposome aerosol was given before PTX for one-half hour and also later by mixing a second dose of cyclosporin A aerosol with PTX aerosol and extending the treatment period to one hour (CsA/PTX + CsA). In one experiment, PTX and CsA/PTX aerosols were significantly more effective compared to untreated controls against renal cell cancer as measured by lung weights and tumor surface areas. CsA/PTX was significantly better that PTX alone as measured by lung weights and tumor area. In a second experiment, tumor areas of PTX and CsA/PTX treated mice were significantly reduced compared to untreated controls and CsA/PTX treated mice had significantly smaller tumor areas than PTX treated mice. In contrast, tumor numbers were not significantly fewer than controls in either therapeutic group. In a third experiment, tumor numbers and tumor areas were significantly fewer in mice treated with CsA/PTX and CsA/PTX + CsA compared to untreated controls. Mice treated with CsA/PTX + CsA had significantly fewer tumors and less tumor area than mice receiving CsA/PTX. While PTX treated mice were not different than untreated controls with respect to tumor numbers or tumor volumes, PTX treated mice had significantly greater tumor numbers and tumor areas than CsA/PTX and CsA/PTX + CsA treated mice. Co-administration of CsA with PTX demonstrated significant dose dependent anticancer effects against renal cell pulmonary metastases in mice. Toxicity manifested by weight loss was associated with the highest dose of CsA.

Treatment of influenza A (H1N1) virus infection with ribavirin aerosol.

In a randomized, controlled study of ribavirin aerosol treatment of influenza A(H1N1) virus infection among college students, treated patients had a significantly shorter duration of fever than control patients. There was a trend of more rapid recovery in treated patients. Virus shedding was similar in treated and control patients, declining gradually from a 50% tissue culture infective dose of 3.5 log10 per ml at admission to 1.8 log10 per ml at 53 h after admission. There was no local or systemic intolerance and no hematological or biochemical abnormalities associated with ribavirin treatment.