Aerosolized amphotericin B-liposomes for treatment of systemic Candida infections in mice.

Mice lethally infected with Candida albicans were exposed to small-particle aerosols containing amphotericin B-liposomes. The drug, when administered twice daily for 2 h (0.58 mg/kg of body weight per day) on days 1, 2, and 3 postinoculation, significantly reduced the numbers of Candida organisms in the kidneys. Aerosol treatment increased the survival time of mice given 2 2-h treatments once a week for 4 weeks. A twice-weekly, 2-h small-particle aerosol administration of amphotericin B-liposomes for 1, 2, or 3 weeks significantly increased both the mean time of survival and percent survival.

Characterization and administration of cyclosporine liposomes as a small-particle aerosol.

Systemically administered CsA has not consistently suppressed the pulmonary immunoreactivity that leads to rejection in lung transplant patients. Pulmonary T cells from patients given CsA systemically still retain their immunoreactivity, which can be suppressed with added CsA. Direct application of CsA by aerosol to the respiratory epithelium should achieve high lung concentrations with minimum systemic effects. In the present study, CsA was most efficiently incorporated into liposomes composed of egg yolk phosphatidylcholine at a molar ratio of CsA to egg yolk phosphatidylcholine of 1:20. These CsA liposomes retained their biological activity and were as effective as free CsA in the suppression of anti-CD3-stimulated [3H]thymidine incorporation by mouse spleen cells. The generation of a small-particle aerosol of CsA liposomes had no effect on this biological activity. CsA liposome aerosol particles have a mass median aerodynamic diameter of 2 microns, which allows for distribution of drug throughout the respiratory tract. Quantitation of CsA in the lungs and blood of mice exposed to CsA liposome aerosols for 4 days showed that as little as 15 min daily (0.11 mg/kg/day) was sufficient to achieve an estimated concentration of CsA in respiratory secretions of 6 micrograms/ml without detectable blood levels. Thus, CsA liposomes can be produced and aerosolized that achieve pulmonary concentrations with sufficient immunosuppressive activity to be effective in the treatment of lung diseases.

SP-303 small-particle aerosol treatment of influenza A virus infection in mice and respiratory syncytial virus infection in cotton rats.

A natural plant product, SP-303, was administered by small-particle aerosol to influenza A/HK virus-infected mice and RSV-infected cotton rats. Aqueous SP-303 at 2 mg/ml in the Collison nebulizer reservoir generated an aerosol with an output of 26 micrograms/l and a particle size distribution of 1.4 microns +/- 4.6 (MMAD +/- GSD). SP-303 at a dosage of 0.5-9.4 mg/kg per day administered for 3-4 days significantly increased both the rate and duration of survival of mice lethally infected with influenza A/HK virus. SP-303 was toxic to mice at 16 mg/kg per day as indicated by weight loss and a decrease in the duration of survival compared to control animals. From these data, a maximum therapeutic index (T.I.) of 12 was calculated. SP-303 given 3-4 days at dosages of 1.3-9.8 mg/kg per day was effective in reducing the pulmonary titer of RSV in infected cotton rats. However, at the 18.7 mg/kg per day dose a significant weight loss compared to control animals was observed; a T.I. of < or = 14 was estimated. These experiments demonstrate that aerosol administration of SP-303 was effective in the treatment of influenza A-infected mice and of RSV-infected cotton rats.

Aerosolized liposomal amphotericin B for treatment of pulmonary and systemic Cryptococcus neoformans infections in mice.

Cryptococcus infections of the lung and central nervous system have become major problems in immuno-compromised patients, leading to the need for additional treatment protocols. We have utilized a Cryptococcus-mouse model that mimics human cryptococcal disease to evaluate the efficacy of amphotericin B-liposomes (AmpB-Lip) when delivered by small-particle aerosol (SPA). In the model, initial intranasal inoculation leads to a pulmonary infection that spreads after 2 to 3 weeks to distant organs, including the brain. Aerosols of AmpB-Lip that were generated by a Collison nebulizer had mass median aerodynamic diameters of 1.8 microns and contained 10.3 micrograms of AmpB per liter. When AmpB-Lip SPA was begun at 24 h postinoculation, a single 2-h treatment (0.3 mg of AmpB per kg of body weight) was effective in reducing pulmonary Cryptococcus infection. This regimen was more effective than intravenous administration of AmpB-Lip given for 3 continuous days. This single 2-h exposure to AmpB-Lip also was effective in reducing pulmonary Cryptococcus infection when treatment was delayed for 7 or 14 days. At day 21, when organisms had spread to the brain in all animals, the single 2-h aerosol treatment reduced the number of cryptococci in the brain as well as in the lungs. AmpB-Lip SPA administered once for 2 h on days 7, 14, and 21 also was effective in increasing the duration of survival of infected animals. These results demonstrate that aerosolized AmpB-Lip can be effective in treating both local, pulmonary Cryptococcus disease and systemic disease.

Further studies with short duration ribavirin aerosol for the treatment of influenza virus infection in mice and respiratory syncytial virus infection in cotton rats.

Ribavirin aerosol administration has been shown to be effective in the treatment of respiratory syncytial virus (RSV) infections in infants and in influenza A and B virus infections in young adults. Long treatment schedules and potential for environmental contamination have stimulated the search for alternative dosing schedules. Thus, we attempted to determine the length of time of ribavirin aerosol necessary for effective treatment of influenza and RSV. In RSV-infected cotton rats, aerosolization for just 30 min with high-dose ribavirin (HDR:60 mg ribavirin/ml in reservoir), 3 times daily, reduced viral lung titers/gm of tissue by 1.1 log10. In influenza virus-infected mice, 15 min of aerosolized HDR, 3 times daily, was effective in reducing both mortality and pulmonary virus titers (1.1 log10 reduction). When the intervals between aerosol administration each day were equally divided (i.e., q.8 h), the treatments were most effective. Treatment for 45 min, once daily, was not as effective as divided doses. Calculations of ribavirin concentrations in respiratory secretions following 15 min treatment in mice with HDR indicated that drug levels dropped below the ED50 for influenza viruses after about 9 h. A daily dosage of ribavirin, estimated to be 8-15 mg/kg, was effective for the treatment of influenza and RSV infections.

Aerosol and intraperitoneal administration of ribavirin and ribavirin triacetate: pharmacokinetics and protection of mice against intracerebral infection with influenza A/WSN virus.

Ribavirin is active in vitro but not in vivo against a number of viruses capable of causing encephalitis. Ribavirin triacetate (RTA), a lipophilic derivative, has been reported to be more effective than ribavirin in protecting animals from encephalitis. By using an influenza A/WSN virus encephalitis model, we demonstrated that RTA administered by small-particle aerosol was able to decrease the death rate and increase the time of survival. To determine if this beneficial effect was due to increased delivery of drug, the pharmacokinetic properties of ribavirin and RTA when administered as an aerosol or by intraperitoneal injection were examined. Aerosol administration of ribavirin or RTA gave significantly higher concentrations of ribavirin in the lungs and serum of mice than did intraperitoneal injection. There was no difference, however, in ribavirin levels when either ribavirin or RTA was administered by small-particle aerosol. In brain tissue, ribavirin concentrations increased with time and did not appear to decrease as rapidly as in lungs and serum. Mean peak ribavirin concentrations in the brain were higher following aerosol administration of ribavirin than RTA, and both were higher than that following intraperitoneal injection of either drug. Administration of ribavirin or RTA by intraperitoneal injection failed to protect mice from a lethal intracerebral inoculation of influenza A/WSN virus, while aerosolized RTA did protect mice. The pharmacokinetics of ribavirin in brain tissue following aerosol administration of either drug did not explain the advantage of RTA over ribavirin in protecting mice from intracerebral infection with influenza A/WSN virus.

Safety and pharmacokinetics of rimantadine small-particle aerosol.

The safety and pharmacokinetics of rimantadine administered by small-particle aerosol were assessed in healthy adults and adults with acute influenza virus infection. Aerosolized rimantadine delivered at a concentration of 40 micrograms/liter of air was associated with nasal burning and irritation in normal volunteers. A concentration of 20 micrograms/liter of air was well tolerated for up to 12 h by normal volunteers and was not associated with any changes in pulmonary function, as measured by routine spirometry, plethysmography, or diffusion capacity of carbon monoxide. Mean peak levels of drug in serum were approximately 10-fold lower after 12 h of aerosol administration than they were after oral administration of 200 mg (29.7 versus 255 ng/ml, respectively), while mean nasal wash levels were approximately 100-fold higher (6,650 versus 66.6 ng/ml, respectively). Elimination half-lives were similar after both aerosol and oral administration (24.1 and 25.2 h, respectively), and rimantadine urinary excretion was less than 1% per 24 h in both groups. Twenty micrograms of aerosolized rimantadine per liter of air given 12 h daily for 3 days to nine adults with acute influenza virus infection was well tolerated. Levels in plasma after 12 h of aerosol inhalation were similar to those in normal volunteers, but were higher at the end of the third treatment than they were at the end of the first treatment (88.3 versus 47.9 ng/ml, respectively). Thus, rimantadine delivered via small-particle aerosol at a dose of 20 micrograms/liter of air was well tolerated in normal volunteers and in those with acute influenza and was associated with high local concentrations.

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

Nine children (aged 6 weeks to 7 years) with suspected respiratory syncytial virus infection received aerosal treatment with ribavirin, 60 mg/ml for 2-hour periods three times daily for up to 5 days. Five children received treatment via an endotracheal tube and four via an oxygen hood. Blood samples (3 to 17 per patient) and respiratory secretions (4 to 23 per patient) were assayed for ribavirin with reverse-phase high-performance liquid chromatography. Ribavirin triphosphate in erythrocytes was determined by ion-exchange high-performance liquid chromatography. The mean (+/- SD) peak ribavirin level after the first dose was 1725 +/- 2179 mumol/L in secretions and 3.8 +/- 2.6 mumol/L in plasma. Ribavirin in the secretions was rapidly cleared, with a mean (+/- SD), half-life of 1.9 +/- 0.8 hours. Plasma ribavirin increased with treatments to reach a steady state of 5 to 10 mumol/L. Mean peak ribavirin triphosphate levels were 15- to 300-fold higher than plasma ribavirin levels by the end of therapy. More than 98% reduction of viral load without the emergence of resistant virus was noted on day 3 of therapy. High-dose treatment was compatible with the aerosol equipment routinely used (small-particle aerosol generator, model 2-6000) for ribavirin administration and with ventilators. High-dose, short-duration ribavirin therapy was well tolerated by all patients, permitted easier accessibility for patient care, and may result in less environmental exposure of health care workers.

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.

Activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes.

Enviroxime has been shown to inhibit the replication of rhinoviruses and other enteroviruses in concentrations as low as nanograms per milliliter in in vitro assays but is markedly less effective in clinical trials. The marked hydrophobicity and water insolubility of this compound may be a factor for this disparity. To overcome this handicap, we incorporated enviroxime into liposomes and then tested the antirhinovirus activity and toxicity of the liposome-incorporated enviroxime (LE) in cell culture and studied its administration by small-particle aerosol. Free enviroxime and LE were found to have equivalent efficacies against rhinovirus strains 1A and 13 in in vitro assays; however, preparations of LE were 10- to greater than or equal to 50-fold less toxic to tissue culture cells than was free enviroxime. In contrast to free enviroxime, which could not be delivered by small-particle aerosol because of its water insolubility, LE (4 mg/ml) was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tracts of mice; after just 20 min, significant levels of enviroxime were detected in the lungs and noses of exposed mice. Moreover, mice exposed to aerosols of liposomes containing both enviroxime and fluorescein isothiophosphatidylethanolamine showed accumulations of the fluorescent marker in the lungs, particularly in or around the tall columnar epithelial cells lining the bronchi and bronchioles.

Efficacy of high dose-short duration ribavirin aerosol in the treatment of respiratory syncytial virus infected cotton rats and influenza B virus infected mice.

Fifteen to 20 mg/ml ribavirin administered as a small particle aerosol for 10-18 h per day is currently the regimen generally used to treat experimental or naturally-occurring respiratory syncytial (RS) or influenza virus infections in humans. To determine if such prolonged treatment schedules could be reduced, cotton rats and mice were inoculated with RS or influenza B virus, respectively, and then treated with different concentrations of ribavirin small particle aerosols. Aerosols generated from reservoirs containing 60 mg/ml ribavirin given 2 h twice daily, protected cotton rats from RS virus and mice from influenza B virus as well as aerosols generated from reservoirs containing 20 mg/ml ribavirin given 11 h daily. Aerosols generated from reservoirs containing 40 or 20 mg/ml given 2 h daily were less efficacious. There was no evidence of intolerance or pulmonary histopathology in infected or uninfected animals exposed to any of the doses of ribavirin tested. These studies indicate that use of aerosols containing higher concentrations of ribavirin than generally used to treat respiratory virus diseases may permit significantly shorter treatment schedules without loss of efficacy or increase in toxicity.

Immunoglobulin administration and ribavirin therapy: efficacy in respiratory syncytial virus infection of the cotton rat.

We studied the effects of combined administration of human immunoglobulin (IVIG) and ribavirin aerosol on respiratory syncytial virus (RSV) infection in cotton rats (Sigmodon hispidus). Cotton rats assigned to receive combined therapy were administered Gamimune, a preparation of purified IVIG with a high titer of anti-RSV neutralizing activity, intraperitoneally 24 h prior to intranasal RSV challenge and then treated with ribavirin aerosol 3 days after challenge. Lung viral titers from these cotton rats (geometric mean titers [GMT] log10 = 0.15 +/- 0.5) were lower than titers from untreated animals (GMT, log10 = 3.7 +/- 0.6) and animals treated with either IVIG alone (GMT, log10 = 1.8 +/- 0.9) or ribavirin alone (GMT, log10 = 1.9 +/- 1.1). Only one of 12 cotton rats treated with both IVIG and ribavirin had a demonstrable titer of virus after RSV challenge. When IVIG administration was delayed until day 3 after virus challenge, lung viral titers were still lowest in animals receiving both IVIG and ribavirin. In comparison, there was no additive antiviral effect between IVIG and ribavirin against RSV infections of HEp-2 cells in vitro. Pathologic changes on histologic examination of pulmonary tissues from animals challenged with RSV were least prominent in animals treated with both IVIG and ribavirin. Despite the apparent absence of in vitro additive antiviral effect, combined use of IVIG and ribavirin was more efficacious against RSV infection in the cotton rat than use of either agent alone.

Protection of mice from lethal influenza virus infection with high dose-short duration ribavirin aerosol.

An aerosol generated from a reservoir containing 60 mg of ribavirin per ml given for 2 h twice daily for 4 days afforded the same high level of protection against lethal influenza virus infection of mice as a longer, conventional treatment schedule (20 mg/ml given for 11 h daily for 4 days). Incremental decreases in ribavirin concentration made while maintaining the 2-h intermittent schedule provided progressively less protection of mice. Mice exposed to the 60-mg/ml doses had significantly increased pulmonary and serum drug levels when compared with mice given 20 mg of drug per ml, these increases were transient, and no evidence of pulmonary intolerance was detected. These studies suggest that protective effects of ribavirin against influenza virus infection can be achieved without untoward effects if higher doses and shorter periods of administration are used.

Limited efficacy of aerosolized recombinant alpha interferon against virulent influenza A/HK infection in mice.

Hybrid recombinant human alpha interferon A/D (rIFN A/D) is unusual in that it has equivalent antiviral activity in in vitro assays using mouse or human tissue cells. This interferon was delivered to outbred Swiss mice in small particle aerosols before and/or after these mice were inoculated with virulent influenza A/HK/68 virus. Although rIFN A/D was effective in inhibiting influenza virus replication in vitro in primary mouse embryo cells, it had only a limited degree of effectiveness in the in vivo tests; only animals exposed to rIFN A/D for 4 hr and inoculated with influenza virus 4 hr later exhibited a significant decrease in mortality (approximately 25% reduction in deaths; P less than 0.025) compared with that of the untreated control animals. The limited effectiveness of rIFN A/D seen in these studies indicated that the use of this or similar recombinant alpha interferons alone to prevent or treat influenza virus infection in humans is not promising.

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.

Duration of effect of interferon aerosol prophylaxis of vesicular stomatitis virus infection in mice.

Mice were exposed for 8 h to continuous small-particle aerosols containing natural mouse alpha interferon (estimated dosage 100 U per mouse) or one of two concentrations of hybrid recombinant alpha interferon A/D bgl (estimated dosages of 100 and 10,000 U per mouse, respectively). On days 1, 3, 5, 7, and 9 after exposure to these interferons, three mice from each group were inoculated intranasally with 100 PFU of vesicular stomatitis virus. Control mice were exposed to aerosols of saline or inoculated intraperitoneally with either natural mouse alpha interferon (350 U) or one of two doses of hybrid recombinant alpha interferon A/D bgl (350 or 35,000 U) and challenged similarly. Of mice injected intraperitoneally, only those given 35,000 U of hybrid recombinant alpha interferon A/D bgl 24 h before virus challenge were protected from pulmonary infection, compared with the saline-treated control mice. Of mice given 100 U of either interferon by small-particle aerosol, only those exposed 24 h before inoculation of vesicular stomatitis virus had reduced pulmonary titers of the virus. However, of mice given ca. 10,000 U of hybrid recombinant alpha interferon A/D bgl by small-particle aerosol, all groups except those exposed 9 days before virus inoculation had significantly reduced lung virus titers.

Interferon aerosol suppression of vesicular stomatitis virus replication in the lungs of infected mice.

Mice were inoculated intranasally with vesicular stomatitis virus 16 to 22 h after being exposed to small-particle aerosols of saline, natural mouse alpha interferon, recombinant human alpha interferon A, or hybrid recombinant human alpha interferon A/D bgl for 2, 4, or 8 h. Compared with comparably inoculated, untreated mice, significantly reduced levels of vesicular stomatitis virus were observed in the lungs of animals treated with any interferon preparation for 8 h and in groups treated with mouse alpha interferon or hybrid recombinant human alpha interferon A/D bgl for 4 h. No significant reductions in lung virus titers were observed in any group treated with interferon for 2 h or in any of the groups treated with saline.

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.

Pulmonary deposition and clearance of aerosolized interferon.

Small particle aerosols of a hybrid DNA recombinant human alpha interferon, A/D bgl, and a related DNA recombinant leukocyte interferon, A, were generated and delivered to mice for 23.5 h a day for 4 consecutive days. The antiviral activity of these interferons in delivery reservoirs, in the aerosols generated, and in the lungs of test mice was monitored during and after aerosol administration in cytopathic effect inhibition assays, using vesicular stomatitis virus as the indicator virus. In addition, the activity of these interferons in primary mouse embryo cells against influenza A/HK/68 (H3N2) virus was determined. The results obtained indicated that the interferon particles generated in the continuous aerosol therapy system used in these studies remained biologically active and could be readily detected in both aerosol mists and lungs of test mice; levels of exogenous interferon in the lungs equalled or exceeded levels of interferons produced endogenously during experimentally induced influenza virus infection. Titers of the exogenously administered interferons decreased gradually and disappeared from the lungs between 24 and 48 h after cessation of aerosolization. Recombinant human alpha interferon A/D, but not recombinant leukocyte alpha interferon A, significantly inhibited replication of A/HK/68 virus in primary mouse embryo cells in the in vitro studies.

Efficacy of aerosolized recombinant interferons against vesicular stomatitis virus-induced lung infection in cotton rats.

Cotton rats (Sigmodon hispidus) were inoculated intranasally with vesicular stomatitis virus (VSV) and exposed to different regimens of small particle aerosols of either recombinant human alpha interferon A (rIFN-alpha A) or hybrid recombinant human alpha interferon A/D (rIFN-alpha A/D). Preliminary in vitro tests indicated that both recombinant IFNs were effective in protecting primary cotton rat pulmonary cells against VSV replication. However, rIFN-alpha A/D was 20-fold more active than rIFN-alpha A in these tests. In the in vivo tests, in contrast to control animals inoculated with VSV, but not treated, or treated only with aerosols of saline, animals exposed to either rIFN-alpha A or -alpha A/D for 8 h per day before and/or following VSV inoculation had no detectable virus titers in their lungs. In experiments in which groups of animals were treated for shorter periods, rIFN-alpha A was less effective than rIFN-alpha A/D in suppressing replication of VSV in lungs of these animals.