Department of Laboratory Medicine and Pathology and
1 College of Pharmaceutics, School of Pharmacy, University of Minnesota, Minneapolis, MN 55455 and
2 National Cancer Institute, Bethesda, MD 20892, USA
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; GSD, geometric standard deviation; MMAD, mass median aerodynamic diameter; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
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Introduction |
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In subsequent work, it was found that budesonide administered by aerosol was much more potent in preventing pulmonary tumor formation than was dietary addition. Reductions in pulmonary adenoma formation by 80% or more were obtained (6). Although the dose levels were considerably lower than those used with dietary administration of glucocorticoids, further decreases are desirable to minimize possible side-effects. An important aim of this investigation was to obtain inhibition of pulmonary carcinogenesis with lower doses than employed previously. A target level is the dose range found to cause minimal side-effects in the human. Direct aerosol delivery to the lung can result in high concentrations of the protective agents reaching the target tissue compared with systemic distribution. A considerable amount of research has been directed towards identifying glucocorticoids with high topical potency in the lung and minimal systemic effects (913). Much of this research has been stimulated by the use of aerosol glucocorticoids for the treatment of bronchial asthma. Budesonide and beclomethasone dipropionate have favorable properties in this regard and, accordingly, were selected for studies of chemoprevention (911). In order for aerosols to be effective in preventing pulmonary neoplasia, the equipment employed should have two features: (i) production of particle sizes that can reach the periphery of the lung and (ii) delivery of concentrations of the test compounds sufficient for inhibitory efficacy under conditions of brief exposure. A nose-only aerosol delivery apparatus has been constructed which meets these requirements. It entails the use of a conventional jet nebulizer for aerosol generation. The glucocorticoid is dissolved in ethanol which is subsequently stripped away during passage along an aqueous trap. The budesonide particles produced have a mass median aerodynamic diameter (MMAD) of <1 µm. The amount of budesonide delivered was within the range necessary for efficacy. Using this apparatus, a high degree of inhibition of pulmonary tumor formation was obtained with dose levels of budesonide ranging from 23 to 126 µg/kg body wt delivered 6 days a week (6). In the present study, experiments using lower doses of budesonide have been carried out. Inhibition by a second glucocorticoid, beclomethasone dipropionate, with good topical efficacy, has also been investigated for comparison with budesonide.
In addition to administration of glucocorticoids as single agents, the effects of their combined administration with a second compound, myo-inositol, has also been investigated. myo-Inositol is a naturally occurring compound which has been previously shown to prevent pulmonary adenoma formation resulting from exposure to B[a]P or NNK in female A/J mice (1,2,4). Like the glucocorticoids, myo-inositol is effective when administered in the post-initiation period. It also has a small inhibitory effect when given throughout the pre-initiation and initiation periods. A maximum inhibition is produced when myo-inositol is fed during the entire duration of the experiment (4). In studies in which myo-inositol and dexamethasone were added to the diet, the inhibition obtained was greater with administration of the two agents than with either agent alone (4). An important feature of myo-inositol is its exceedingly low toxicity (14,15). Thus, it appeared useful to determine whether administration of myo-inositol would enhance the inhibitory effects of the aerosolized budesonide, thus reducing the amount of the glucocorticoid required for inhibition of pulmonary carcinogenesis.
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Materials and methods |
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Animal experiments
Female A/J mice obtained from the Jackson Laboratories (Bar Harbor, ME) were used in all experiments. The animals were fed a semi-purified diet consisting of 27% vitamin-free casein, 59% starch, 10% corn oil, 4% salt mix (USP XIV) and a complete mixture of vitamins (Teklad, Madison, WI). The mice were housed in a constant temperature facility with controlled lighting: lights on at 6 a.m. and off at 6 p.m. At 15 weeks of age, the mice were given the first of three administrations of 2 mg B[a]P in 0.2 ml cottonseed oil or cottonseed oil only (vehicle) by oral intubation. The time interval between the first and second doses was 4 days and between the second and third doses 3 days. Two experiments were performed. In the first experiment, dietary additions of myo-inositol were made to the designated groups 10 days prior to the first dose of B[a]P and these additions were continued for the duration of the protocol. In the second experiment, no such additions were employed. In both experiments, aerosol administrations were started 1 week following the last dose of B[a]P and were continued for the duration of the study. Mice were randomized by weight the day prior to the aerosol administrations and were re-weighed once a week. In both experiments, the aerosols were administered for 20 s, 3 days per week. The mice were exposed singly to the aerosol by placing their noses into the cone of the apparatus. Care was taken to handle the animals gently to minimize stress. The details of the aerosol apparatus and procedure are described below. In both experiments, the animals were killed at the termination of the protocol, which was 16 weeks after the last dose of B[a]P. The mice were autopsied and the lungs taken for pulmonary adenoma counts using the procedure of Shimkin as previously described (18,19).
Aerosol procedure
The aerosol apparatus consisted of a MiniHeart Nebulizer (Central Medical Services, Naperville, IL) held vertically in an ice bath and connected directly to a 300 mm glass Leibig2 condenser. This was connected to a second identical condenser by a 75° glass connector and a 6 inch section of flexible inch diameter TygonTM tubing. Both condensers were heated to 60°C with a heated circulating water bath (VWR model 1130A; VWR Scientific Products, McGaw Park, IL). The solvent/drug aerosol passed up the first condenser and down through the second to ensure complete evaporation of the droplets. The aerosol then exited the condenser through a three-joint 75° glass connector and into the first of two 600 mm horizontal glass tubes, each with an inner diameter of 1 inch. Water was pumped counter currently to the aerosol flow through the glass tubes by an electric circulator pump (Little Giant; VWR Scientific Products). The solvent vapor was removed by dissolution in the stream of water passing through each of the horizontal tubes only to the midpoint. Because the jet nebulizer was operated with nitrogen to prevent any oxidation of the drug during nebulization, it was necessary to add sufficient oxygen to the dried aerosol cloud at this point to give a final gas composition of 80% nitrogen and 20% oxygen. The aerosol cloud then passed through a
inch TygonTM tube to a nose cone for administration to the test animal. The nose cone was fabricated from a 6 ml disposable syringe casing (Monoject), from which 5 mm of the nose end was removed. Fourteen holes of ~2 mm in diameter were cut in the nose cone to allow exit of the aerosol after passing by the animal's nose.
For nebulization, budesonide or beclomethasone dipropionate was dissolved in ethanol and placed in the jet nebulizer. In these studies, the starting volume was 12 ml and the nebulizer was chilled to 0°C in an ice bath 5 min prior to starting each run. The ethanol concentration was determined by gas chromatography at the apex of the nose cone, i.e. the position the nose of the mouse would occupy in the cone of the apparatus. The concentration was <3 µl ethanol/l air. The nebulizer had an output flow rate of 1.75 l/min of nitrogen when operated at an input pressure of 30 p.s.i. The volume of oxygen added after drying of the aerosol stream was 0.44 l/min. With these conditions, the nebulizer had an output rate of 323 µl solution/min.
Budesonide aerosol concentration
The concentration of glucocorticoids in the aerosol was determined by replacing the nose cone with a collection filter assembly with Whatman glass fiber filters. The aerosol was captured for a fixed time and then budesonide was extracted from the filter. The concentration was determined spectrophotometrically using appropriate standards. The aerosol concentration was calculated as the mass collected in 1 min divided by the air flow rate.
Aerosol particle sizing
A (low flow rate) cascade impactor (InTox, Alburqueque, NM) was used to determine the size distribution of the aerosol budesonide particles. The MMAD and geometric standard deviation (GSD) were obtained from the cumulative undersized mass collected given as a function of the logarithm of the cut-off diameter. For a budesonide solution of 1.4 mg/ml, the MMAD was 0.91 µm with a GSD of 2.1. At a solution concentration of 0.56 mg/ml, the MMAD was 0.78 µm with a GSD of 2.0.
Monitoring dose delivery
The dose to the animal was estimated from the aerosol concentration (µg budesonide/l air) as follows:
dose = (aerosol concentrationxrespiratory minute volumex
exposure time)/body weight(1)
where the respiratory minute volume was estimated with Guyton's formula (18), the exposure time was 1 min and the body weight was taken to be 0.025 kg. The inherent assumption of this approach is that the inspired aerosol is completely deposited in the lung.
Statistical analyses
Non-parametric tests were used to compare the different treatments, KruskalWallis analysis to test the null hypothesis of no difference among the groups. Since this null hypothesis was rejected, the Wilcoxon rank sum test for pairwise comparisons was used. No further adjustment for multiple comparisons were applied because the comparisons of interest had been determined a priori. Statistical analysis was carried out by means of a statistical software package (SAS).
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Results |
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Discussion |
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In addition to their chemoprevention potency, glucocorticoids have another positive feature which is of considerable pragmatic importance. They have widespread use as medicinals. Chemoprevention of carcinogenesis of the lung in the human is likely to require long-term administration. Thus, the availability of information pertaining to chronic administration of a potential chemopreventive agent in the human, such as side-effects, dosage regimes and potential problem areas, is useful. In the case of the glucocorticoids, a very large amount of data of this nature comes from their chronic administration by aerosol to asthmatics (9). The dose ranges used for treating asthmatics and for inhibition of carcinogenesis of the lung in the animal experiments reported are similar. The doses used in asthmatics are related to the severity of the disease. For budesonide, the dose required to treat patients with moderately severe asthma is 400600 µg/day (5.78.6 µg/kg body wt/day for a 70 kg individual). At this dose level, adverse effects from chronic administration are minimal (9). In the animal studies reported in this investigation, inhibition of pulmonary carcinogenesis occurs at doses of 10 and 25 µg/kg body wt using an administration schedule in which the aerosols were given 3 times a week. This schedule provides average daily doses of budesonide of 4.3 and 10.8 µg/kg body wt. These data indicate that the doses of aerosol glucocorticoids employed in the experimental model might be acceptable for use for chemoprevention of pulmonary neoplasia in the human. The additional inhibition provided by combined administration of myo-inositol with glucocorticoids offers a further option. The data obtained pertaining to chemoprevention of pulmonary carcinogenesis by glucocorticoids and myo-inositol in the mouse are provocative. Whether or not it will prove applicable to the human remains to be determined. Clinical trials utilizing aerosolized budesonide as a means of preventing pulmonary carcinogenesis are under consideration.
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Acknowledgments |
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Notes |
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References |
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