Author's School

School of Engineering & Applied Science

Author's Department/Program

Energy, Environmental and Chemical Engineering

Language

English (en)

Date of Award

Spring 4-9-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Da-Ren Chen

Abstract

INTRODUCTION Chemoprevention is the use of natural or synthetic agents to inhibit either the initial development or the further progression of early lesions. Chemoprevention of lung cancer aims to decrease lung cancer morbidity and mortality, especially for former smokers. Many synthetic and natural compounds have been investigated for their potential chemopreventive efficacy. Conventional administration of these compounds: especially oral administration) is convenient, but may potentially result in adverse side effects. Aerosol delivery, on the other hand, offers many advantages over conventional routes of administration for diseases of the respiratory tract and the lungs. These advantages include the extensive pulmonary surface area available for drug deposition, the avoidance of first-pass metabolic degradation by the liver and/or intestines, the noninvasive nature of administration, and the localized effect of low doses of drugs, all of which lead to a rapid response with fewer systemic side effects. The efficacy of a given drug via aerosol administration depends on many factors such as particle size distribution, total particle mass concentration, and the physiochemical properties of drug particles. Genetically-engineered mice play an important role in drug screening and preclinical studies. However, because mice have much smaller lungs than those of human beings, the lung deposition data attained for humans cannot be applied to the mouse. Hence few studies have focused on particle deposition in the mouse lung. It is thus necessary to measure the mass deposition of particles in the mouse lung.

METHOD A spray-drying process was used to study the inhibitory effects of potential chemopreventive agents on carcinogen-induced tumors in the A/J mouse. The carcinogen in the studies was benzo[a]pyrene, unless otherwise specified. Synthetic and natural compounds were investigated individually or in combination. The compounds were aerosolized with a custom-built Collison atomizer. The resultant drug aerosols were delivered to the mice that were retained in a nose-only exposure chamber. Four small molecular inhibitors: gefitinib, erlotinib, lapatinib and wortmannin), and four natural agents: resveratrol, caffeine, anthocyanins, and protocatechuic acid), were considered as examples of single agents. Gefitinib and erlotinib were delivered in an aerosol form to reduce the cutaneous side effects. Lapatinib and wortmannin were each administered both via aerosol and oral gavage to compare the efficacy and toxicity as they were administered via different routes. Resveratrol was evaluated in two models with either vinyl carbamate or benzo[a]pyrene as the carcinogen. It was delivered via aerosol to avoid fast clearance in the blood before it reached the lung. Caffeine was delivered in aerosol to assess only its inhibitory effects and to avoid its negative effects on body weight. Anthocyanins were delivered via aerosol due to their poor bioavailability. Protocatechuic acid is a metabolite of anthocyanins and was also delivered via aerosol for comparison with anthocyanins. The combinations of aerosolized budesonide: a synthetic glucocorticoid) and dietary polyphenon E: a well-defined mixture of green tea extract) was discussed as one example of the combinational treatment.

In addition to the bioassays, drug deposition in the mouse lung was evaluated for both polydispersed and monodispersed drug particles for a better understanding of the delivery process and for future applications. Gefitinib was selected as the model agent. Polydispersed gefitinib particles were generated with the Collison atomizer used in the animal studies. Monodispersed particles were generated using the single-capillary electrospray technique. Lung and blood samples were harvested immediately after the aerosol treatment. The lung and plasma levels of gefitinib were measured with varied solution concentrations, exposure durations, and particle sizes. The aerial mass concentration in the chamber was also measured to estimate the doses.

RESULTS Aerosolized erlotinib: 5 mg/ml) did not inhibit tumor multiplicity but reduced tumor load by 63.8%: P < 0.05). Aerosolized gefitinib in three separate doses: 5, 10, and 15 mg/ml) inhibited tumor multiplicity by ~30% for all three doses when the tumors were induced by one dose of benzo[a]pyrene: 100 mg/kg body weight), but the results were not statistically significant. Aerosolized gefitinib showed consistent inhibitory effects on tumor load, and the inhibition rate increased as the dose increased. The tumor load was reduced by 39.0%, 46.2%, and 56.4%: P < 0.05) for 5, 10, and 15 mg/ml gefitinib solutions, respectively. The highest dose: 15 mg/ml) of gefitinib was repeated in mice whose tumors were induced by two doses of benzo[a]pyerene: 100 mg/kg body weight, one week apart) and it inhibited both tumor multiplicity: by 49.8%, P < 0.001) and tumor load: by 57.0%, P < 0.001). No visible skin alteration was observed in mice treated with aerosolized gefitinib or erlotinib. Both aerosolized lapatinib: 50 mg/ml) and orally-administered lapatinib: 100 mg/kg body weight) showed inhibitory effects. Aerosolized lapatinib reduced tumor multiplicity by 39.6%: P < 0.05) and tumor load by 41.7%: P < 0.05). Orally-dosed lapatinib reduced tumor multiplicity by 37.6%: not significant) and tumor load by 42.4%: P < 0.05). At the current doses of lapatinib, no adverse side effect was observed in either the aerosol group or the orally-dosed group. Wortmannin showed striking inhibitory effects via aerosol inhalation and per os. Oral wortmannin: 1.0 mg/kg body weight) inhibited tumor multiplicity by 85.5%: P < 0.001) and tumor load by 77.9%: P < 0.05). In the same model, aerosolized wortmannin: 2.0 mg/ml) inhibited tumor multiplicity by 50.8%: P < 0.05) and tumor load by 79.7%: P < 0.05). Despite the efficacy of oral wortmannin, the accompanying systemic adverse effects were not negligible. Reduced body weight and death were observed in the orally-dosed mice, but not in the aerosol treated mice. Thus, aerosolized wortmannin was evaluated a second time in the bioassay with two doses of benzo[a]pyrene, and it was found to reduce tumor multiplicity and tumor load by 66.7%: P < 0.001) and 80.4%: P < 0.0001), respectively, with a slight decrease in body weight. Resveratrol inhibited the proliferation of cells in the human lung cancer cell lines A549 and H1129, which indicates that resveratrol could possibly be an effective inhibitor of human lung cancer. Aerosolized resveratrol was shown to inhibit the tumor load in both vinyl carbamate- and benzo[a]pyrene-induced models. The decrease in tumor load was 26.3%: P < 0.05) and 36.0%: P < 0.01) for 7.5 and 15 mg/ml solutions, respectively, in the vinyl carbamate-induced model. In the benzo[a]pyrene-induced model, aerosolized resveratrol: 15 mg/ml) significantly reduced tumor multiplicity by 37.1%: P < 0.05) and tumor load by 72.0%: P < 0.01). Pharmacokinetic studies showed that more resveratrol was delivered to the lung by aerosol inhalation than by oral gavage. Aerosolized caffeine: 10 mg/ml) inhibited tumor multiplicity by 31.9%: P < 0.05) and tumor load by 44.3%: P < 0.05) without causing a reduction in the body weight gain, in contrast to the orally-administered caffeine, which did cause body weight loss. Aerosolized protocatechuic acid: 12 mg/ml) reduced tumor multiplicity by 47.8%: P < 0.05) and tumor load by 44.9%: P < 0.05). However, the inhibitory effects of anthocyanins: 5 mg/ml, extracted from black raspberries) were marginal: 14.5% on the tumor multiplicity and 30.4% on the tumor load, not significant).

The particle deposition in the mouse lung was estimated using gefitinib as the model compound. For the Collison atomizer, the aerosol mass concentration in the exposure chamber increased linearly from 12.3 to 179.8 μg/L as the solution concentration increased from 1 to 50 mg/ml. The lung and plasma levels of gefitinib increased monotonically with increased solution concentration and exposure time, and the concentration in the lung was much higher than that in the plasma. The deposition efficiency is defined as the ratio of the mass deposited in the lung to the dose, and it is a function of particle size. In general, monodispersed particles have a higher delivery efficiency than polydispersed particles. For polydispersed particles, the 2.5 mg/ml solution: with a mass mean aerodynamic diameter, MMAD, at 120 nm) had the highest efficiency. For monodispersed particles, 100 nm particles showed the highest deposition efficiency.

CONCLUSIONS Aerosol delivery is a promising approach for the chemoprevention of lung cancer. Many natural and synthetic compounds showed inhibitory effects on benzo[a]pyrene-induced lung tumorigenesis in A/J when they are delivered via aerosol inhalation. In contrast to oral administration, aerosol delivery of the agents mitigated systemic toxicities with comparable inhibitory effects and improved the efficacy of some agents by increasing their bioavailability in the lung. The current aerosol delivery system was characterized and the mass deposition in the mouse lung was positively correlated with both the solution concentration and the exposure time. Aerosols with an MMAD around 100 nm may have the highest delivery efficiency, for both polydispersed and monodispersed distributions.

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Permanent URL: http://dx.doi.org/10.7936/K7GF0RJB

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