Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol

L.W. Wattenberg3, T.S. Wiedmann1,3, R.D. Estensen, C.L. Zimmerman1, A.R. Galbraith, V.E. Steele2 and G.J. Kelloff2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This investigation is part of an effort to develop chemoprevention for carcinogenesis of the lung. It focuses on the efficacy of low doses of synthetic glucocorticoids administered either as single agents or in combination with a second compound, myo-inositol. Glucocorticoids are potent inhibitors of carcinogenesis. The use of low doses is important to avoid potential side-effects. The synthetic glucocorticoid budesonide, administered by aerosol for 20 s three times a week, was studied to determine its effects on benzo[a]pyrene-induced pulmonary adenoma formation in female A/J mice. Two dose levels were employed, 10 and 25 µg/kg body wt. The lower dose produced a 34% reduction in lung tumor formation and the higher dose level a 60% reduction in lung tumors. In additional groups of mice, the effects of 0.3% myo-inositol added to the diet was found to reduce pulmonary tumor formation by 53%. The two agents given in combination resulted in a greater inhibition of lung tumor formation than either by itself. Budesonide at 10 µg/kg body wt plus 0.3% myo-inositol reduced the number of tumors by 60% and budesonide at 25 µg/kg body wt plus 0.3% myo-inositol reduced lung tumor formation by 79%. To determine whether a glucocorticoid other than budesonide would have inhibitory effects in this experimental model, beclomethasone dipropionate administered by aerosol for 20 s three times a week was studied as a single agent and showed almost identical inhibitory properties to budesonide. The doses of the glucocorticoids calculated on a daily basis are within the range of those used widely for control of chronic allergic respiratory diseases in the human. The capacity of low doses of inhaled glucocorticoids to prevent pulmonary neoplasia and the enhancement of this preventive effect by myo-inositol, an essentially non-toxic compound, are findings that should encourage further work to evaluate the applicability of these agents to the prevention of neoplasia of the lung in the human.

Abbreviations: B[a]P, benzo[a]pyrene; GSD, geometric standard deviation; MMAD, mass median aerodynamic diameter; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This investigation is part of a continuing effort to develop effective chemoprevention for carcinogenesis of the lung (16). In the present study, low doses of budesonide administered by aerosol as a single agent have been studied as well as the effects of its combination with myo-inositol fed in the diet. The efficacy of a second glucocorticoid, beclomethasone dipropionate, in inhibiting pulmonary neoplasia has also been investigated. The aim of this work is to develop optimal use of these agents in the prevention of pulmonary neoplasia. Glucocorticoids are effective chemopreventive agents (48). However, they can have side-effects which may compromise their use (9). The occurrence of these side-effects is governed by factors such as the route of administration and dose (9). For effective chemoprevention, it is very important to minimize any adverse reactions. In initial experiments with glucocorticoids as inhibitors of pulmonary neoplasia, the synthetic compounds dexamethasone and budesonide were evaluated. Both compounds reduced pulmonary adenoma formation in female A/J mice. In these experiments, the glucocorticoids were added to the diet beginning 1 week subsequent to administration of either benzo[a]pyrene (B[a]P) or the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). However, high doses were required (4,5).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The chemicals used were budesonide (>99% purity), beclomethasone dipropionate (>98% purity) and myo-inositol (>99% purity) (Sigma Chemical Co., St Louis, MO), and B[a]P (>98% purity) (Aldrich Chemical Co., Milwaukee, WI).

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 3/4 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 3/4 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, Kruskal–Wallis 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the first experiment are shown in Table IGo. All groups of mice were administered B[a]P. The designations used for experimental groups in Table IGo contain two components. The first refers to the exposure to aerosol (C, none; SC, exposure to solvent only; Bud 0.56, exposure to budesonide at 0.56 mg/ml, etc.). The second designation refers to the dietary addition of myo-inositol (C, none; Inos 0.1, inositol at 0.1% of the diet, etc.). Two controls were employed along with the groups receiving the test compounds. The first, designated C-C, was not exposed to aerosol treatment of any type. The second, designated SC-C, was exposed to the solvent (ethanol) used to dissolve the glucocorticoids. As discussed previously, nebulization of ethanol in the equipment employed does not result in any significant amount of ethanol reaching the nose cone (i.e. <3 µl/l). However, these animals were subject to the same handling as was used for the various treatment groups. The two control groups developed similar numbers of pulmonary adenomas, with the SC-C group showing slightly fewer tumors. The SC-C group was used to evaluate the effects of test agents on the occurrence of pulmonary adenomas. The two doses of aerosolized budesonide produced significant inhibition of pulmonary adenoma formation, as did myo-inositol added to the diet. When the two agents were combined, the inhibitory effect obtained was greater than for either agent administered alone. The final body weights of all groups were similar. The spleen weights were reduce in the mice receiving budesonide, which is an indication of systemic exposure to glucocorticoids (19,20). The results of the second experiment are shown in Table IIGo. All groups of mice received B[a]P. The designations for the experimental groups in Table IIGo are: C, no aerosol; SC, exposure to solvent only; Bud, exposure to budesonide; Bec 0.26, exposure to beclomethasone dipropionate at 0.26 mg/ml, etc. Beclomethasone dipropionate was found to inhibit pulmonary adenoma formation. A direct comparison in this experiment of the chemopreventive effects of beclomethasone dipropionate and budesonide given at the same dose level, i.e. 0.023 µmol/kg body wt (budesonide 10 µg/kg body wt and beclomethasone dipropionate 12 µg/kg body wt) showed that they were similar.


View this table:
[in this window]
[in a new window]
 
Table I. Effects of aerosolized budesonide and dietary myo-inositol administered alone or in combination on B[a]P-induced pulmonary adenoma formation in female A/J micea
 

View this table:
[in this window]
[in a new window]
 
Table II. Effects of budesonide and beclomethasone dipropionate administered by aerosol on B[a]P-induced pulmonary adenoma formation in female A/J micea
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemoprevention of carcinogenesis of the lung has proved to be very difficult in both humans and experimental animals. The most commonly employed animal model is pulmonary carcinogenesis in the A strain mouse (17). In this model, a number of compounds show efficacy when administered in the initiation period or during both the initiation and post-initiation periods. Inhibitory effects occurring in the initiation period are frequently the result of carcinogen detoxification rather than impinging on basic mechanisms of the cellular events occurring during carcinogenesis. Very few compounds inhibit when administered solely during the post-initiation period. However, one group of compounds, the glucocorticoids, are potent inhibitors of pulmonary carcinogenesis in the A strain mouse when given only during the post-initiation period.

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 400–600 µg/day (5.7–8.6 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.


    Acknowledgments
 
This work was supported by the National Cancer Institute, contract no. N01-CN-75114.


    Notes
 
3 These authors were equal contributors to whom correspondence should be addressed: L.W.Wattenberg (animal prevention studies); T.S.Wiedmann (aerosol technology) Email: watte002{at}maroon.tc.umn.edu

Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Estensen,R.D. and Wattenberg,L.W. (1993) Studies of chemopreventive effects of myo-inositol on benzo[{alpha}]-pyrene-induced neoplasia of the lung and forestomach of female A/J mice. Carcinogenesis, 14, 1975–1977.[Abstract]
  2. Wattenberg,L.W. (1995) Chalcones, myo-inositol and other novel inhibitors of pulmonary carcinogenesis. J. Cell. Biochem., 22 (suppl.), 1162–1168.
  3. Wattenberg,L.W. (1993) Prevention, therapy, basic science and the resolution of the cancer problem. Cancer Res., 53, 5890–5896.[ISI][Medline]
  4. Wattenberg,L.W. and Estensen,R.D. (1996) Chemopreventive effects of myo-inositol and dexamethasone on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary carcinogenesis in female A/J mice. Cancer Res., 56, 5132–5135.[Abstract]
  5. Wattenberg,L.W. and Estensen,R.D. (1997) Studies of chemopreventive effects of budesonide on benzo[a]pyrene-induced neoplasia of the lung and forestomach of female A/J mice. Carcinogenesis, 18, 2015–2017.[Abstract]
  6. Wattenberg,L.W., Wiedmann,T.S., Estensen,R.D., Zimmerman,C.L., Steele,V.E. and Kelloff,G.K. (1997) Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice. Cancer Res., 57, 5489–5492.[Abstract]
  7. Belman,S. and Troll,W. (1972) The inhibition of croton oil-promoted mouse skin tumorigenesis by steroid hormones. Cancer Res., 32, 450–454.[ISI][Medline]
  8. Verma,A.K. Garcia,C.T., Ashendel,C.L. and Boutwell,R.K. (1983) Inhibition of 7-bromomethylbenz[a]anthracene-promoted mouse skin tumor formation by retinoic acid and dexamethasone. Cancer Res., 43, 3045–3049.[Abstract]
  9. National Heart, Lung and Blood Institute (1997) Guidelines for the Diagnosis and Management of Asthma: Expert Panel Report. National Heart, Lung and Blood Institute, Bethesda, MD, pp. 57–79.
  10. Barnes,P.J. (1997) Inhaled glucocorticoids for asthma. N. Engl. J. Med., 332, 868–875.[Free Full Text]
  11. Barnes,P.J. (1996) Inhaled glucocorticoids: new developments relevant to updating asthma management guidelines. Resp. Med., 90, 379–384.[ISI][Medline]
  12. Ryrfelt,Å., Andersson,P., Edsbacker,S., Tonnesson,M., Davies,D. and Pauwels,R. (1982) Pharmacokinetics and metabolism of budesonide, a selective glucocorticoid. Eur. J. Resp. Dis., 63 (suppl. 122), 86–95.
  13. Pederson,S. and Hansen,O.R. (1994) Budesonide treatment of moderate and severe asthma in children: a dose-response study. J. Allergy Clin. Immunol., 95, 29–33.[ISI]
  14. Gregersen,G. (1987) myo-Inositol supplementation. In Dyck,P.J., Thomas,P.K., Asbury,A.K., Winegrad,A.J. and Porte,D. (eds) Diabetic Neuropathy. W.B.Saunders, Philadelphia, PA, pp. 188–189.
  15. Levine,J., Barak,Y., Gonzales,M., Szor,H., Elizur,A., Kofman,O. and Belmaker,R.H. (1995) Double-blind, controlled trial of inositol treatment of depression. Am. J. Psychiat., 152, 792–794.[Abstract]
  16. Shimkin,M.B. (1940) Induced pulmonary tumors in mice. II. Reactions of lungs in strain A mice to carcinogenic hydrocarbons. Arch. Pathol., 29, 235–255.
  17. Shimkin,M.B. (1955) Pulmonary tumors in experimental animals. Adv. Cancer Res., 3, 223–267.[ISI]
  18. Guyton,A.C. (1947) Measurement of the respiratory volumes of laboratory animals. Am. J. Physiol., 150, 70–77.[ISI]
  19. Dougherty,T.F. (1952) Effects of hormones on lymphatic tissue. Physiol. Rev., 32, 379–401.[Free Full Text]
  20. Claman,H.N. (1972) Corticosteroids and lymphoid cells. N. Engl. J. Med., 287, 388–397.[ISI][Medline]
Received June 23, 1999; revised September 23, 1999; accepted October 7, 1999.