Elsevier

Advanced Drug Delivery Reviews

Volume 133, August 2018, Pages 107-130
Advanced Drug Delivery Reviews

The potential to treat lung cancer via inhalation of repurposed drugs

https://doi.org/10.1016/j.addr.2018.08.012Get rights and content

Abstract

Lung cancer is a highly invasive and prevalent disease with ineffective first-line treatment and remains the leading cause of cancer death in men and women. Despite the improvements in diagnosis and therapy, the prognosis and outcome of lung cancer patients is still poor. This could be associated with the lack of effective first-line oncology drugs, formation of resistant tumors and non-optimal administration route. Therefore, the repurposing of existing drugs currently used for different indications and the introduction of a different method of drug administration could be investigated as an alternative to improve lung cancer therapy. This review describes the rationale and development of repositioning of drugs for lung cancer treatment with emphasis on inhalation. The review includes the current progress of repurposing non-cancer drugs, as well as current chemotherapeutics for lung malignancies via inhalation. Several potential non-cancer drugs such as statins, itraconazole and clarithromycin, that have demonstrated preclinical anti-cancer activity, are also presented. Furthermore, the potential challenges and limitations that might hamper the clinical translation of repurposed oncology drugs are described.

Introduction

Lung cancer, a highly invasive and prevalent disease, is one of the most deadly and frequent cancer-related deaths in the world, with more than 1.3 million cases reported annually [1]. Lung cancer is responsible for approximately 14% of new cancers worldwide, and 25% of cancer deaths [1,2]. In the United States alone, lung cancer causes more deaths than the next four leading cancer deaths combined (i.e., breast, pancreas, prostate and colon). Lung cancer is highly heterogeneous and can develop in different locations of the bronchial tree; therefore resulting in highly variable symptoms [3].

In general, lung cancer is classified into two major histopathology groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [3]. NSCLC comprises the majority of all lung cancer cases (85%) and can be expanded to include different lung cancer subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [2,4]. SCLS is a lethal, extremely aggressive subtype of lung cancer with high probability to metastasise to other types of cancer and accounts for 10–15% of lung cancer cases [4]. Furthermore, patients suffering from SCLS are often diagnosed at an advanced stage and have a short median survival (>1 year with chemotherapeutic intervention).

As established by the National Cancer Institute (2016), variable procedures for the detection and diagnosis of lung cancer stages are used in order to determine the appropriate treatments for each stage [5]. As a rule of thumb, treatment strategies vary according to the specific stage of lung cancer, patients’ susceptibility and health conditions, as well as the molecular profile of the disease. So far the treatment strategies include surgery, chemotherapy, external radiation therapy, radiofrequency ablation, stereotactic radiosurgery, targeted therapy, immunotherapy and palliative therapy [5]. Although surgery is the preferred option for early stage lung cancer, the low tolerance of some patients towards surgery does not allow this intervention. In addition, most lung cancer patients are diagnosed at an advanced stage and therefore chemotherapy alone, or in combination with other interventions, is the first choice of treatment (Table 1, Table 2).

Despite the advances in diagnostic techniques and discovery of novel chemotherapeutics, platinum-based chemotherapeutics such as cisplatin remain the most successful agents in the treatment of lung cancer and are often administered in combination with other chemotherapeutics [6]. Cisplatin and other platinum-core derivatives (i.e. carboplatin) are used as the first-line treatment for NSCLC. Other chemotherapeutics agents include paclitaxel (PTX), docetaxel (DTX), premetrexed and gemcitabine (GCB) [[7], [8], [9]]. In the case of lung cancer characterized by gene mutations, tyrosine kinase inhibitors (TKIs) are recommended as the first-line treatment instead [10]. Second-line treatments such as GCB, DTX and pemetrexed are often administered when patients fail to respond to first-line drugs [11] (Table 1, Table 2).

It is disheartening to acknowledge that despite the continued improvement in the understanding of cancer biology and the development of novel agents and cancer treatments, improvements in overall survival remain modest [12,13]. Furthermore, therapeutics for lung cancer are associated with a lengthy drug development process (from drug discovery to clinical use), exorbitant cost, high regulatory hurdles and staggering failure rates [13,14]. The average time for the transition of a novel drug from laboratory to bench side was 13 years and cost US$1.8 billion [15] (Fig. 1). Given the challenges involved drug development for lung cancer therapies, alternative strategies like the repurposing of ‘old’ drugs which have been approved for other indications are attractive. An added advantage of drug repurposing is the possibility that the drug can enter the clinical trial phase more rapidly than for a newly designed drug, since many of the existing drugs have already been thoroughly investigated for safety, dosage and toxicity.

Drug repurposing, also sometimes used interchangeably with drug repositioning, is the hypothesis that a new use can be identified for existing drugs outside of their originally approved indications [16]. This strategy also includes drugs under development for a specific target disease but that show promise to treat other diseases. Langedijk et al, linked drug repurposing to the treatment of a ‘new’ disease, which could involve new patient groups, dosage forms, or administration routes [17]. For instance, fentanyl was originally developed as a solution for infusion in the 1980s and was subsequently approved via other routes of administration using novel dosage forms such as nasal sprays, transdermal patches and buccal tablets [17] (Fig. 2). These new administration routes fall within the scope of drug repurposing as well. However, while these alternative formulation strategies are not novel, they allow the original active ingredient to realize additional therapeutic value.

In the case of cancer treatment, many first-line and second-line chemotherapeutics remain the preferred drug choice despite their accompanying side effects. Considering that these chemotherapeutic agents have multiple targets, they are continually being investigated in new therapeutic combinations (i.e., as theranostics, biomarkers and targeted agents), in new indications, via different routes of administration (i.e., inhalation, metronomic) and utilizing different dosage forms (i.e., nanoparticle, liposomes, micelles). These approaches to extend the functionality of existing oncology drugs to other cancer indications are often referred to as the ‘soft form’ of drug repurposing [18]. The repurposing of existing chemotherapeutics such as doxorubicin (DOX), paclitaxel (PTX) or cisplatin into a formulation for inhalation is therefore included and discussed in the latter part of the review.

In contrast, the ‘hard form’ of drug repurposing seeks to identify suitable non-cancer drugs with the clinical potential to achieve successful cancer treatment via a different mechanism from their original target indication [18]. These non-cancer drugs typically already possess a good long-term toxicology profile and if they achieve anti-cancer activities in both in vitro and in vivo models, then a putative mechanisms of action must be identified for their use in cancer therapy [18] (Fig. 3). The Repurposing Drugs in Oncology (ReDO) collaborative project has taken this approach and successfully elucidated the anti-cancer properties of the following drugs which were originally developed based on other clinical properties (in parenthesis): mebendazole (anti-helminthic), nitroglycerin (vasodilator), cimetidine (H2-receptor antagonist), clarithromycin (antibiotic), diclofenac (non-steroidal anti-inflammatory drug, NSAID), and itraconazole (anti-fungal) [18].

Table 3 summarizes some non-cancer drugs that have taken advantage of recent repurposing technologies and are currently undergoing clinical trials for oncology indications. In this table, we have included non-cancer drugs that have been re-purposed as inhalable formulations, showing promising preclinical data in lung cancer treatment. In addition, several putative and potential non-cancer drugs that have been demonstrated to be effective against lung cancer, but have yet to be investigated via pulmonary administration, are also included.

The inhalation route is becoming more popular due to the commercial success of inhalable drugs for treatment of chronic lung diseases such as asthma, chronic pulmonary obstructive disease (COPD) and cystic fibrosis. Pulmonary drug delivery to treat lung cancer is attractive because it bypasses limitations faced by other routes of administration [19]. The main justification for pulmonary delivery for oncology drugs is the same as the drugs repurposed to treat asthma or cystic fibrosis. Oral administration and intravenous (IV) injection of chemotherapeutic drugs typically reach the lung at extremely low concentrations [20]. Instead, the majority of the drug accumulates in the liver, kidney and spleen. In order to be therapeutically effective, higher doses or more frequent administrations have to be used which can be associated with severe side effects. Furthermore, systemic administration of chemotherapeutic drugs to treat lung cancer often fail to completely suppress tumor growth. Increasing the dose, dosing frequency, or the addition of multiple drugs not only has not enhanced their efficacy; it has resulted in the emergence of multidrug resistant tumors.

In contrast, local delivery of drugs via inhalation enhances accumulation of the drug in the lungs compared to IV or oral administration [[21], [22], [23], [24]]. Inhalation delivery limits the systemic bio-distribution of chemotherapeutics and therefore reduces systemic-associated toxicities. In addition, inhalation delivery typically improves the pharmacokinetics of the aerosolized agents. This route increases the retention of drugs in the lung [[21], [22], [23]]. In most cases, the area under the curve (AUC), half-life and maximum concentrations of drug in the lung were enhanced for drug delivered via inhalation compared to the oral or intravenous route [25]. Therefore, a lower inhaled dose can be administered to achieve therapeutic efficacy leading to a reduction in side effects.

Although repurposing strategies for inhalation should logically reduce the cost and time associated with the development of new cancer drugs, however, there are several points that must be taken into consideration. In order for any existing drug to be approved for inhalation, they have to undergo new formulation development, preclinical and clinical studies which include toxicity, pharmacokinetics, pharmacodynamics, tolerability, safety and efficacy studies. In addition, repurposed drug for inhalation commonly use different excipients in the formulation development and therefore the metabolism of both inhaled drugs and excipients have to be extensively investigated. In cases where the metabolism is significantly different from that for systemic administration, the repetitive aerosolization of these repurposed formulations could introduce adverse toxicological effects.

In addition, local delivery of high doses of chemotherapeutics could increase the risk of drug-induced toxicity and lung diseases. For instance, when a highly toxic anti-cancer drug delivered via inhalation is distributed equally via the lung parenchyma, both diseased and healthy lung cells would be in direct contact with the drug. In such case, healthy lung tissue is vulnerable to drug-associated toxicities leading to undesirable side-effects in the lung. Furthermore, at least 400 aerosolized drugs have been noted to potentially induce lung damage, accelerate the progression of lung diseases and enhance the severity of existing conditions [25,26]. It is common for lung cancer patients with or without a history of smoking to develop impaired pulmonary functions after undergoing chemotherapy treatment. In addition, lung metastasis is also associated with other lung diseases. The potential complications of inhalation of toxic anti-cancer drugs are relatively unknown and might exacerbate the severity of lung disease [25].

As mentioned earlier, inhalation delivery may enhance the pharmacokinetics compared to other administration routes (e.g., oral, IV). However, enhanced pharmacokinetic profile may not translate to increased therapeutic efficacy. One of the key factors is the ability to direct deposition of the drug onto the solid tumors in the lung which is influenced by several factors including: the tumor size, physicochemical properties of the anticancer drugs, drug-tumor interactions (i.e., penetration), the physiological condition of the patient, and whether the tumor is located in a ventilated airway, [27]. To be therapeutically effective, the concentration of the drug deposited in the proximity of the solid tumor needs to be sufficient to eradicate the cancerous tissue while being sufficiently safe locally. These outcomes depend also on the status of the patients, particularly the extent of their lung disease.

From the formulation aspect, it is challenging to achieve selective drug targeting in specific areas of the lung followed by enhanced penetration into the tumor. Not many studies have determined that inhaled chemotherapy could achieve better tumor penetration compared to IV administration of drugs. The depth of tumor penetration is usually governed by the physicochemical properties of drugs (i.e., molecular weight, solubility, stability), tumor conditions (size, density) and tumor microenvironment [[27], [28], [29], [30]]. Hershey et al had reported that inhaled paclitaxel (PTX) or doxorubicin (DOX) were more effective against small nodules [31]. Therefore, a limited depth of penetration would reduce the drug concentration in tumor tissue and in turn decrease the therapeutic efficacy of inhaled anti-cancer drugs. In addition, the cellular internalization of inhaled chemotherapy to healthy cells and the corresponding toxicities have not been studied in detail. The risk of increased local toxicities to healthy cells in response to higher deposition of anti-cancer drugs via inhalation is uncertain. Therefore, opinions are quite divided regarding the safety of inhaled chemotherapy.

The aerosol deposition to targeted areas is also influenced by the degree of airway obstruction by the tumor; this changes as the disease progresses [32]. The presence of lung nodules could physically divert the airflow to other sections in the lung and thus reduce the deposition of inhaled chemotherapeutics to the site of the tumor [27]. Zhang et al demonstrated that increasing tumor size effectively decreased aerosol deposition via obstruction of the airway lumen [33]. Furthermore the optimal site of deposition varies for different lung cancer stages; i.e., primary lung cancer is located in the central airways while bronchioloalveolar carcinoma is located in the alveolar region [34].

Physiological bio-barriers also exist in the respiratory airways such as mucus, ciliated cells, transporters, enzymes and resident macrophages which can limit the localization, penetration, absorption and retention of drugs in the lung and must be taken in consideration during formulation of inhalable chemotherapeutics and devices [35,36]. For instance, one could consider adding a suppressor to the p-glycoprotein pump into the formulation to inhibit the cellular drug efflux transporter [25].

Non-steroidal anti-inflammatory drugs (NSAID) are used for the management of pain, fever and prevention of heart attack and stroke [37]. NSAIDs are inhibitors of cyclooxygenases (COX) enzymes and exist in three isoforms (COX-1, COX-2 and COX-3) [38,39]. COX enzymes are rate-limiting enzymes necessary for the conversion of arachidonic acid to prostaglandins and thromboxane A2 [37]. The COX-2/prostaglandin E2 signaling pathway was associated with the progression of lung cancer involving the evasion of apoptosis, uncontrolled cellular proliferation, angiogenesis, metastasis and suppression of immune response [40]. It is established that COX-2 is a highly inducible isoform and its expression is upregulated in many human tumors (i.e., colon, lung, breast, prostate and bladder) as a response to cytokines, growth factors and tumor promoters [[41], [42], [43]]. In many cases of lung cancer, overexpressed COX-2 proteins are often detected in lesions of human lung adenocarcinoma and are implicated with worse prognosis [44] (Fig. 4).

Celecoxib is the first specific inhibitor of COX-2 and received FDA approval for the treatment of osteoarthritis and rheumatoid arthritis pain. Celecoxib is also approved as an adjuvant therapy for patients with familial adenomatous polyposis (FAP) based on large clinical trials data [[45], [46], [47], [48]]. Celecoxib exerts its anti-cancer action through various intrinsic and extrinsic pathways associated with apoptosis, accompanied by downregulation of NF-kB, caspase-9, BAX and BCL-xL [[49], [50], [51], [52], [53]]. The inhibition of NF-kB, downstream of Akt, is associated with reduced COX-2 synthesis and other related events leading to cancer progression [54,55]. In addition, celecoxib binds to 3-phosphoinositide-dependent protein kinase-1 (PDK-1) to inhibit PDK1/Akt pathway via COX-independent mechanism [56,57]. The side effect profile of celecoxib is gastrointestinal toxicity, and similar to conventional NSAIDs, it is also associated with a higher risk of cardiovascular events.

Thus, there is rationale for an inhaled formulation of celecoxib to treat lung cancer. An aerosolized formulation of celecoxib demonstrated enhanced in vitro cytotoxic and apoptotic responses against human NSCLC (A549 and H460) [58]. This solution-based metered dose inhaler (MDI) formulation of celecoxib using HFA 134a as a propellant had a high respirable fraction (50.7%) with a medication delivery of 231.3 μg/shot. In vitro cytotoxicity studies of aerosolized celecoxib (2 shots) alone or solution-based docetaxel (DTX) alone against A549 cells provided a killing effect of approximately 50%. Synergistic effects were observed when aerosolized celecoxib was administered in conjunction with free DTX, whereby up to 80% of cancer cells were killed [58]. The enhanced cytotoxicity effect is accompanied with an increased induction of apoptosis as demonstrated by the increase in PPAR-γ and p53 expression. These findings also suggest the possible involvement of a COX-2 independent pathway that mediates the therapeutic activities of celecoxib against NSCLC. Both cPLA2 and 5-LOX expression in NSCLC cells were significantly reduced in the presence of aerosolized celecoxib. Under normal physiological conditions, cPLA2 and 5-LOX are involved in the regulation of arachidonic acid pathway, but both have been implicated in their role in promoting lung mouse tumorigenesis through the release of arachidonic acid from membrane lipids [58].

In another study, the in vitro cytotoxicity effect of aerosolized DTX and celecoxib against A549 cells was assessed using a 6-stage viable impactor [59]. A stable solution-based MDI formulation of DTX was formulated with 0.25% DTX, 15% ethanol and HFA 134a as propellant. The fine particle fraction (FPF), mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD) and median delivery of DTX-MDI formulation was 42.2%, 1.58 μm, 3.2 and 80 μg/shot, respectively. The administration of a one-shot combination of aerosolized DTX (80 μg) and celecoxib (10 μg/ml) onto A549 cells on a stage-5 Anderson Cascade Impactor (ACI) impactor was sufficient to achieve >90% cytotoxic effect [59]. The authors also found that combination administration of aerosolized DTX and celecoxib upregulated the expression of PPAR-γ, as evident from the significant higher apoptotic cell deaths [59].

The comparison of in vivo anti-tumor effect between aerosolized celecoxib in combination with DTX (i.v), and with oral celecoxib with DTX (i.v) was investigated using a human orthotopic NSCLC xenograft model [60]. A total deposited dose of 4.56 mg/kg/day of celecoxib (5 mg/ml) was aerosolized into A549 bearing nude mice via nose inhalation chamber using the PARI LC jet nebulizer for 30 min. Mice receiving a combination of celecoxib (aerosol/oral) and DTX (10 mg/kg) had lower lung weight and tumor volumes compared to the untreated groups. After 3 weeks of treatment, an approximately 60% reduction in tumor volume was observed for mice that underwent combination treatments compared to the untreated control [60]. The combinational approach enhanced anti-tumorigenesis through elevated apoptotic responses as mediated via the Fas pathway and inhibited angiogenesis through the reduction of Factor VIII, vascular endothelial growth factor (VEGF), PGE2 and IL-8 [60].

A novel inhalable nanoparticle formulation of celcoxib with particle size of 217 ± 20 nm was developed recently using solid triglycerides as the outer shell of the nanoparticle [61]. Formulation via the addition of liquid Migylol oil led to high encapsulation efficiency (95.6%), high physical stability (before and after aerosolization) and controlled release behavior of the nanoparticles. Celecoxib nanoparticles demonstrated time- and dose-dependent in vitro cytotoxicity against A549 cells, which is attributable to controlled release of celecoxib from the nanoparticle for a prolonged time (72 h for celecoxib nanoparticle vs 8 h for celecoxib in solution) and slower particle internalization [61]. In vitro aerosolization of celecoxib nanoparticle formulation using the Pari LC star jet nebulizer demonstrated an FPF, MMAD and GSD of 75.6 ± 4.6%, 1.6 ± 0.13 μm and 1.2 ± 0.21, respectively. In addition, the nebulization of celecoxib nanoparticle for 2 min on A549 cells directly inhibited 50% of the viable cells. Meanwhile, celecoxib nanoparticles showed a 4-fold higher AUCt/D in lung tissue and slower systemic clearance (0.93 l/h) compared to the solution formulation of celecoxib (20.03 l/h) [61].

In another study, metastatic A549 tumor model in Nu/Nu mice was used to evaluate in vivo response against the administration of aerosolized celecoxib nanoparticle in combination with DTX (i.v) [62]. Aerosol performance of these nanoparticles (217 ± 20 nm) indicated that more than 75% of the nebulized celecoxib nanoparticles were below 5 μm and could be deposited in the bronchioalveolar region of the lung. In vivo administration of the aerosolized celecoxib nanoparticle alone and DTX (i.v) alone resulted in 25 ± 4% and 37 ± 5% tumor volume reduction, respectively. Meanwhile, 68% suppression of tumor size was noted for the combination treatment groups [62]. Preclinical data suggests that activation of the apoptosis pathway, leading to increased expression of apoptotic proteins, such as caspase-9, caspase-3 and Bax as well as suppression of VEGF-associated downstream signaling pathways, is directly responsible for the significantly enhanced therapeutic action of the combination treatment [62].

An inhalable, stable biodegradable polymer nanoparticle co-encapsulating celecoxib and naringin (herbal compound), with an average particle size of 200 nm and a controlled release behavior, was successfully formulated [63]. The celecoxib/naringin loaded PLGA nanoparticle formulation displayed a good nebulization profile with a high deposition in the lower impinger and impactor stages. Biodistribution studies showed the highest accumulation in the lung tissues accompanied with systemic distribution to bones, brain and liver (common metastatic sites of lung cancer) after 30 min post-aerosolization. Interestingly, negligible histo-pathological changes in bronchioles, air alveoli and blood were detected for the inhaled formulation which suggested that PLGA nanoparticles exhibited low toxicity to rat lung tissue [63].

An aerosolized MDI of nimesulide with a similar aerodynamic characteristic to that of an approved HFA formulation of beclomethasone, was developed by Haynes et al. This formulation showed a dose, FPF and MMAD of 51.1 ± 3.3 μg/shot, 42.4 ± 4.2% and 1.1 ± 0.3 μm, respectively [64]. The presence of aerosolized nimesulide (40 shots) significant enhanced the doxorubicin (0.01 μg/ml) cytotoxicity activity against A549 cells [64]. In a recent work, the administration of high levels of phosphosulindac via inhalation was reported with minimal hydrolytic degradation to inactive metabolites [65]. Inhaled phosphosulindac (6.5 mg/kg) was sufficient to inhibit 75% lung tumorigenesis and increased survival of mice bearing A549 cells. The authors proposed that phosphosulindac acts as an anti-cancer agent via: (i) suppression of EGFR activation and consequent inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR cascades pathways; (ii) induction of oxidative stress leading to mitochondrial-related cell damages and, iii) activation of autophagy cell death [65].

Budesonide, available as an inhaled solution, nasal spray and oral tablets, is approved for the treatment of asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis and ulcerative colitis. In addition to its anti-inflammatory activity, budesonide prevents the development of lung cancer in various mice models [[66], [67], [68], [69], [70]]. Its chemoprevention activity is postulated to be through growth arrest and apoptosis, via multiple signalling and associated downstream pathways such Mad2/3 and Bim/Blk pathways [66].

Wattenberg first explored the chemoprevention role of aerosolized budesonide to treat pulmonary carcinogenesis. Considering that extensive research has been devoted to develop aerosolized glucocorticoids with high local retention, therapeutic action and minimal systemic toxicity for chronic asthma, inhaled budesonide could be re-purposed as a possible candidate for lung neoplasia treatment [68]. Pulmonary administration of inhaled budesonide (23, 72 and 126 μg/kg) resulted in >80% inhibition of in vivo tumor formation compared to the aerosol control, thus demonstrating that low-dose local drug delivery is sufficient to prevent the occurrence of pulmonary carcinogenesis [68]. The same group further elucidated the chemopreventive effect of introducing a secondary dietary compound myo-inositol in conjunction with aerosolized budesonide or beclomethasone dipropionate (BDP), using female A/J mice model [67]. When inhaled budesonide/BDP was administered as the sole agent (10 μg/kg), an approximate 30% reduction in lung tumor formation was observed. The incorporation of 0.3% myo-inositol in the treatment regimen resulted in 2-fold higher tumor inhibition [67].

In another work, oral pioglitazone (anti-diabetic compound) was used in combination with inhaled budesonide to assess their synergistic potential on mouse lung cancer using female A/J mice with benzo(a)pyrene used as the carcinogen [71]. Co-administration of budesonide and pioglitazone achieved up to a 90% decrease in tumor load as opposed to a 60–78% reduction when a single agent was used. Inhaled budesonide was accumulated solely in the lung, whereas oral pioglitazone could be detected in the lung, blood, liver and spleen, thus confirming that the aerosol route is optimal for local delivery of therapeutics to the lung with minimal systemic drug circulation [71]. When oral pioglitazone was substituted with polyphenon E, similar results were also observed whereby both tumor multiplicity and tumor load were decreased by 65.4% and 87.3%, respectively. It should be noted that administration of dietary polyphenon E (7.5 mg/kg) as a sole agent did not exert any chemopreventive effect in this study, probably due to its low in vivo bioavailability [72]. In addition, both oral (0.25%) and aerosolized nicotinamide (15 mg/kg/day) were not effective to provide statistically significant relief in tumor load and multiplicity, compared to the untreated control [73]. Although nicotinamide failed to demonstrate anti-tumor activity, its presence enhanced the inhibitory effect of budesonide against lung adenoma formation. An increase by 20% in the tumor growth inhibition was observed at an early stage of intervention for co-administration treatment (aerosolized 25 μg/kg budesonide and 0.25% nicotinamide) compared to the sole agent (aerosolized budesonide) [73].

Chronic inflammation has been associated in the pathogenesis of lung cancer. In addition to suppressing airway and systemic inflammation, inhaled corticosteroids (ICS) could modulate the expression of prostaglandin E and inhibit pro-oncogene proteins in smokers [74,75]. The mechanisms of lung cancer development in COPD patients are not well established. However, there is a growing understanding that the repetitive oxidative stress in COPD patients exacerbates DNA damage, inhibits DNA repair mechanisms, stimulates continuous pro-inflammatory cytokine expression as well as promotes the uncontrolled growth of ‘cancerous’ cells. A long term epidemiological trial (1996–2001, n = 10474) on COPD showed a positive correlation between ICS and a lower risk of lung cancer [76]. Analysis showed that there is a dose-dependence in the decrease of lung cancer incidence in association with ICS (dose < 1200 μg/d: adjusted HR, 1.3; 95% confidence interval, 0.67–1.90; ≥ dose 1200 μg/d: adjusted HR, 0.39; 95% CI, 0.16–0.96). Even though the analysis has been adjusted for confounding factors, larger cohorts, especially for ICS users (n = 517), are required to provide definitive conclusions [76].

In another study, Kiri et al conducted a retrospective cohort study of COPD patients (n = 7,079) from 1989–2003 who had quit smoking and were under regular medications of ICS, ICS/LABA concomitantly, or short-acting bronchodilators (SABD) [77]. Out of the identified patients, 127 subsequently developed lung cancer and were matched with 1,470 individual controls. Only 6.0% of patients that were concomitant ICS/LABA users were diagnosed with lung cancer compared to 7.3% and 10.9% of ICS and SABD users, respectively. This observational trial suggests that ICS is chemopreventive against lung cancer and is stronger in combination with LABA (HR for ICS/LABA users is 0.50; 95% CI: 0.27–0.90 whereas HR for ICS users is 0.64; 95% CI: 0.42–0.98) [77].

A nested case control study based on the Korean national claims database was conducted to elucidate the association between ICS use and cancer diagnosis (lung and laryngeal) [78]. New adult users (n = 793,687) of inhaled medication between 2007 and 2010 were identified. Out of these eligible cohorts, 9177 individuals were diagnosed with lung cancer and were matched with 37,048 controls based on criteria such as age, sex, diagnosis of asthma/COPD, comorbidity index score and number of hospital visits. Of those, 408 individuals developed laryngeal cancer and were matched with 1,651 controls using the same selection criteria. After adjustment for multiple covariates differences, lung cancer incidence rate appears to be reduced with the use of regular ICS medication (adjusted odds ratio (aOR), 0.79; 95% CI, 0.69–0.90). However, no statistical difference was observed between ICS and non-ICS users for laryngeal cancer diagnosis [78].

Liu et al conducted a retrospective nationwide population-based cohort study to investigate the role of ICS in protecting female COPD patients against development of lung cancer [79]. A cohort of 13,686 female COPD patients (ICS users, n = 1,290, ICS non-users, n = 12,396) diagnosed from 1997 to 2009 was selected from Taiwan’s National Health Insurance database. Their analysis corroborated the other published findings in which the risk of lung cancer is associated with ICS in a dose-dependent manner. A therapeutic cumulative dose of >39.48 mg is significant to reduce lung cancer diagnosis after taking into consideration age, income, and comorbidities. In addition, non-ICS users were significantly more prone to be diagnosed with lung cancer compared to ICS users (10.75 vs. 9.68 years, p < 0.001) [79].

Lam et al conducted a phase IIb trial to determine the efficacy and safety of inhaled budesonide (Pulmicort Turbuhaler, 1,600 μg) in 112 heavy smokers (>30 pack-years) with bronchial dysplasia, which is considered the main precursor of squamous cell carcinoma [74,80]. After 6 months of intervention, both the response and disease progression rate for inhaled budesonide and placebo groups showed no statistical differences. Inhaled budesonide did not result in reduction of existing bronchial dysplasia or the prevention of new lesions [74].

Lazzeroni et al reported a first randomized, double blind placebo controlled trial focusing on persistent lung nodules (site where the majority of lung cancer arises) as target markers using low-dose computed tomography (LDCT) scan [81]. The efficacy of inhaled budesonide to individuals at high risk of lung cancer in the shrinkage of lung nodules was also nested among the screening study. Among the 4821 participants who underwent the second yearly LDCT screening in the European Institute of Oncology (EIO) CT screening trial, a total of 202 eligible participants were randomized in a 16-month period to receive either inhaled budesonide 800 μg or placebo twice daily for a year [82]. No significant differences in the nodule response between the treated and placebo arms were observed. The progressive disease rate as defined by existing nodules and appearance of new nodules for the budesonide and placebo arm was 7.7% and 10% (p = 0.41), respectively. In addition, the pre-existing lesions for participants receiving inhaled budesonide did not progress as opposed to approximately 5% growth of nodules in the placebo-treated arm [82]. As some of these nodules could be slow-growing adenocarcinoma precursors, therefore a long-term outcome (up to 5 years of follow-up) of target and non-target nodules in the inhaled budesonide and placebo arms was undertaken [83]. The mean size of non-solid nodules decreased significantly (p = 0.029) in the budesonide arm five years after baseline and treatment intervention. However, the reduction of solid nodules showed no differences between the placebo and budesonide arms (p = 0.252). Budesonide did not inhibit the appearance of new non-solid nodules or decrease the incidence of lung cancer. This is probably because 1 year of intervention followed by 5 years monitoring may not be long enough to detect the changes of premalignant lesions to carcinoma [83].

Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) agonist used commonly for the treatment of type II diabetes, with acceptable clinical toxicity. PPARγ, a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors, is important in the regulation of lipid metabolism and differentiation. The recruitment of PPARγ and retinoic X receptor heterodimers has been implicated in the differentiation events in several pre-neoplastic conditions. In addition, PPARγ appears to be overexpressed in NSCLC and is correlated with the histological type and grade of lung cancer.

Seabloom et al recently sought to repurpose pioglitazone for lung cancer treatment via the inhalation route [84]. The authors demonstrated that aerosolized pioglitazone was well tolerated and non-toxic to the lung parenchyma in A/J mice (up to 450 μg/kg bw/d); negligible evidence of inflammation events and hypersensitivity pneumonitis was observed. A week post-carcinogen challenge, early-stage intervention with aerosolized pioglitazone (50, 150 or 450 μg/kg bw/d) administered to mice for a period of 15 weeks clearly showed substantial effects on the reduction of adenomas (32% for 450 μg/kg bw/d) [84]. Zhang et al previously assessed the chemo preventive effect of oral pioglitazone in combination with aerosolized budesnonide or aerosolized 2-deoxy-D-glucose (target the glucose metabolism) on a lung carcinogenesis mouse model, but showed disappointing results in terms of inhibiting tumor load and multiplicity [72].

Angiotensin II receptor blockers are primarily used in the management of hypertension and have demonstrated a reduction in morbidity in the treatment of several cardiovascular indications such as stable coronary heart disease, acute myocardial infarction, and heart failure [[85], [86], [87]]. Telmisartan is an angiotensin II receptor blocker that is clinically indicated for hypertension therapy owing to its selective angiotensin II receptor type 2 (AT2) blocking activity. Telmisartan also exhibits anti-cancer activities through the activation of PPARγ which in turn suppresses tumor metastases.

Anti-proliferative effects of telmisartan against renal cell carcinoma, colon cancer, lung cancer, bladder cancer, prostate cancer and cervical cancer lines were recently discovered [[88], [89], [90], [91], [92], [93]]. From the context of inhalation therapy, a recent attempt was undertaken to assess the intratumoral distribution and therapeutic action of losartan and telmisartan nanoparticles [93]. Losartan and telmisartan nanoparticles were found to be highly effective against lung cancer (A549 and H1650) and well tolerated by human normal fibroblast (WI-38) cells in in vitro settings. Based on the aerodynamic size distribution from ACI measurements, both losartan and telmisartan possess satisfactory aerosol performance thus indicating that these drugs could be delivered as inhalation aerosols for lung cancer treatment. Both telmisartan (~1.12 mg/kg) and losartan (~4.5 mg/kg) were administered to tumor bearing animals using a nebulizer at alternate days for 4 weeks. Although in vitro aerosol performance for both drugs was comparable, intratumoral distribution of telmisartan was approximately 2.7-fold higher than losartan, thus exhibiting a significantly higher anticancer and antifibrotic effect in orthotopic and metastatic lung tumor models [93].

Wortmannin is a steroid metabolite originally isolated from the fungus Penicillium wortamanni. It is a potent inhibitor of phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related kinases (PIKKs) including the DNA-dependent protein kinase (DNA-PK). The P13K signalling pathway is critical for the regulation of cell growth, proliferation, survival, cell motility, activation of integrins and angiogenesis. Therefore, abnormalities in P13k/AKY pathways are often associated with cancer progression, invasion and metastasis and subsequently leading to therapy resistance. In addition, both p85 and p110 subunits of PP13K are overexpressed in primary lung carcinoma while these are not seen in healthy lung tissues. Wortmannin has thus demonstrated superior efficacy to inhibit human NSCLC in vitro and in vivo [94,95]. In addition, it also acts as an excellent radio-sensitizer to increase the susceptibility of NSCLC cells to ionizing radiation [96]. However, in reality wortmannin is an example of an abandoned drug that encountered several toxicities, pharmacological and drug delivery challenges, resulting in an unsuccessful translation in the clinic. To revive the clinical translational potential of wortmannin, several strategies have been proposed, mainly focusing on reducing the hepatoxicity and enhancing the solubility of the wortammin formulation. These include the modification of the molecular structure of wortmannin to produce structural analogues such as PX-866 and DJM-166 [97,98]. In addition, another approach used is the synthesis of conjugates through the binding of wortmannin to water-soluble compounds [[99], [100], [101]]. In other studies, a polymeric nanoparticle formulation of wortmannin seems to have resolved the solubility, stability and toxicity issues of the drugs in vivo [102,103].

Since wortmannin delivered via the oral route caused severe liver toxicity, an alternative administration approach was used to reduce the accumulation of drug in the liver and gastrointestinal tract. In a study by Zhang et al, wortmannin was formulated into nanoparticles via spray drying and directly aerosolized into the lung via inhalation to reduce systemic uptake and reduce systemic toxicity. The inhibitory effects of wortmannin on lung tumorigenesis were compared between oral and inhalation delivery using carcinogen-induced tumors in the A/J mouse model [72]. Aerosolized wortmannin (2.0 mg/ml) inhibited tumor multiplicity and load by 50.8% and 79.7%, respectively, after receiving scheduled treatment five days/week for 20 consecutive weeks. Reduction in body weights and deaths were not observed with this treatment group. Although oral wortmannin (1.0 mg/kg body weight, tumor multiplicity 85.5% and tumor load 77.9%) was more effective than aerosol wortmannin to inhibit lung tumorigenesis, it is accompanied with systemic toxicities that are not acceptable for long-term chemoprevention treatment.

Noscapine, a natural non-opiate alkaloid, possesses antitussive, antimalarial and analgesic properties [104]. Recent preclinical studies have elicited the anti-cancer properties for noscapine [[104], [105], [106], [107]]. Some of the mechanisms involved include inhibitions of microtubule assembly, suppression of anti-apoptotic proteins such as Bcl-2, activation of c-Jun NH2 terminal kinase, downregulation of survivin and enhanced expression of pro-apoptotic factors (p53 and p21) [[108], [109], [110]]. In a recent work, 9-bromo-noscapine (9BM) was formulated into inhalable lipid-based nanoparticles (9BM-NP) and rapid release inhalable lipid based nanoparticles (9BM-RR-NP) to achieve deep lung deposition and enhanced cytotoxic activity against lung cancer [111]. To generate inhalable particles with rapid release properties, the nanoparticles (<100nm) were spray dried with lactose in conjunction with effervescent particles. The aerodynamic size for both spray-dried formulations ranged between 2.3 to 3.1 μm. It was observed that the 9BM-RR-NP (IC50=5.1μM) formulation was superior at inducing apoptosis and cytotoxic killing against A549, followed by 9-BM-NP (IC50 = 6.8 μM) and free 9-BM solution (IC50 > 64 μM), which may be attributable to its rapid internalization via endocytosis. In addition, pharmacokinetic and distribution analyses following nose-only inhalation showed that 9-BM-RR-NPs displayed an enhanced residence time and lowered the elimination/clearance rate compared to 9-BM-NPs and free 9-BM. In addition, the half-life of 9-BM-RR-NPs was 1.12 and 1.75 fold higher than 9-BM-NPs and free 9-BM, respectively [111].

Methotrexate is a folic acid analog that inhibits dihydrofolate reductase, an enzyme needed for DNA synthesis, repair and cellular replication. Shaik et al reported the feasibility of delivering cytotoxic concentrations of MTX to the lower respiratory tract for lung cancer treatment using in vitro viable impaction [112]. An MDI formulation of cryo-milled MTX was formulated using HFA 134a containing 0.67% MTZ and 10% ethyl alcohol. Cryo-milled MTX in the presence of pluronic F77 was not successful to obtain the desired size for respiration, as noted in the low FPF varying between 14.2% and 17.1%. However, exposure of HL60 cells placed in the third, fourth, fifth and sixth stage of the ACI to aerosolized MTX resulted in >50% cell death, thus indicating that a sufficient therapeutic dose could potentially be delivered to the lower respiratory tract [112].

Cyclosporine is an immunosuppressant medication administered using the oral or injection route to prevent organ rejection after a kidney, heart, or liver transplant. It is also used to treat rheumatoid arthritis, psoriasis and Crohn’s disease. In a study by Knight et al, co-delivery of aerosol cyclosporine A and PTX liposome demonstrated effective tumor suppression using a Renca lung metastases mouse model [113,114]. The effect was exemplified when cyclosporine A was administered 1 h prior to actual treatment with cyclosporine + PTX liposome to sensitize resistant cells towards chemotherapeutics. Some of the mechanisms of action include: (i) competitive binding to multi-drug resistant efflux pumps (i.e., p-glycoprotein, as cyclosporine A exhibits high binding affinity towards p-gp, its presence therefore competes with other chemotherapeutics for binding with p-gp, which in turn prevents the PTX elimination from cells by efflux pumps) and, (ii) inhibition of cytochrome P450 mediated pathways (first pass metabolism of PTX) by cyclosporine A [113,114].

The use of isotretinoin as cancer preventive agents is lacking, despite promising preliminary clinical trials data owing to the side effects associated with the doses used [115]. From the therapeutic efficacy point of view, administration of >300 mg/kg weekly failed to prevent lung cancer formation in the A/J mouse model [116]. Therefore, direct and localized delivery of isotretinoin to the lung via the inhalation route could be a better approach to overcome these drawbacks [117,118]. In a study by Dahl et al, aerosolized isotretinoin was administered to carcinogens-exposed male A/J mice (tobacco smoke or lung carcinogens) using the Pari LC-plus nebulizer for 45 min daily, to achieve a total weekly deposited dose of 0.24 mg/kg (low), 1.6 mg/kg (mid) and 24.9 mg/kg (high), of which 16% was estimated to be deposited in the lungs. The MMADs for the low, mid and high doses were 1.00 μm, 1.33 μm and 1.64 μm, respectively. Meanwhile the GSD for these three doses were 2.08, 1.76 and 2.61, respectively [117]. The highest dose was associated with lethal toxicity whereas the lowest dose failed to achieve an optimal therapeutic effect for lung cancer chemoprevention. In contrast, carcinogen-exposed mice treated with the mid dose of aerosolized isotretinoin had tumor nodule multiplicity reduced by 66–88%. Although isotretinoin delivered via inhalation was only 0.5% of an oral dose used in previous studies, the potent anti-cancer efficacy coupled with negligible toxicity of this inhaled formulation suggested localized drug delivery via inhalation could be a better approach compared to the oral or intravenous route for lung cancer prevention [117].

In this section, we highlight some potential non-cancer drugs that could be repurposed as inhalable formulations for the treatment of lung cancer such as itraconazole, clarithromycin and statins (Table 4). These potential agents have demonstrated in vitro and in vivo therapeutic efficacy towards various cancers, using either the oral or injection route. Although the original intention of the existing inhalable formulations of these drugs has not been directed towards lung cancer treatment, they will be discussed in this section to highlight the future possibility of employing this route as an alternative therapy.

Itraconazole was one of the first compounds in the ReDO projects for which an interest was shown to generate clinical evidence in oncology indications [126]. The original indications of itraconazole include the prevention and systemic treatment of a broad range of fungal infections such as aspergillosis, blastomycosis, candidiasis and histoplasmosis [126]. Early pre-clinical evidence demonstrated that at clinically relevant doses, itraconazole is a potent anti-cancer agent including the reversal of multi-drug resistance (MDR) mediated by p-glycoprotein, inhibition of the Hedgehog signaling pathway, inhibition of the mechanistic target of rapamycin, suppression of angiogenesis and lymphangiogenesis, induction of autophagic cell death and possibly interference with cancer-stromal cell interactions [[127], [128], [129], [130], [131], [132]] (Fig. 5). The anti-angiogenic activity of itraconazole with respect to endothelial cell functions was investigated using HUVEC cells and panels of NSCLC cell lines [133]. Itraconazole consistently demonstrated specific and dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to angiogenic growth factor stimulations. Using primary xenograft models of NSCLC, oral itraconazole, either administered alone or in combination with cisplatin, inhibited tumor growth and reduced tumor vascular area compared to the control [133]. In multiple xenograft models of NSCLC and in a clinical study, the presence of itraconazole enhanced the cytotoxic efficacy of chemotherapeutic agents [133,134]. Its potential was clearly evident in a phase II clinical trial in which 67% of patients receiving a combination of itraconazole and pemetrexed were tumor progression-free after 3 months of initiation, compared to only 29% in the control group receiving pemetrexed alone [133].

Given the evidence presented, the rationale exists for repurposing itraconazole as an inhalable product for localized lung cancer therapy. One possible example that has been described is a chitosan-based nanoparticle formulation for pulmonary delivery [136]. The co-spray drying of chitosan-based itraconazole with both leucine and mannitol and lactose improved the aerosol performance of the dried powder. The addition of leucine and mannitol altered the roughness of the microparticles, thus resulting in an increase in the FPF from 16.3% to 42.9% [136]. In a recent study, a solventless fine solid-crystal suspension consisting of mannitol and itraconazole was developed using a combination of hot melt extrusion and jet milling [137]. The resulting itraconazole-mannitol DPI exhibited enhanced dissolution, saturation solubility and good aerosolization performance (FPF of 50.6 ± 0.7%) [137]. The relatively high dissolution rate, coupled with a high respirable fraction and saturation solubility should provide a sufficiently high in situ drug concentration to diffuse and become internalized into target lesions in the lung.

Another example of an itraconazole DPI formulation with enhanced properties was one that was manufactured via co-spraying of itraconazole with polymeric surfactant and mannitol [138]. The incorporation of polymeric surfactant (i.e., TPGS) was beneficial to increase the dissolution and saturation solubility of the itraconazole, but negatively impacted its aerodynamic behavior. The authors revealed that the optimal formulation consisted of 10% intraconazole content in the spray-dried particles while maintaining the ratio of itraconazole: TPGS at 10% [138].

In another study, self-assembled lipid-based nanotransfersomes were used as vesicles for pulmonary delivery of itraconazole [139]. The optimal itraconazole-loaded nanotransfersome had a mean diameter of 518 nm and was formulated with lecithin: Span® 60 in the ratio of 9:1. In addition, co-spray dried DPI formulations of aggregated itraconazole-loaded nanotransfersomes and mannitol at 2:1 resulted in the highest aerosolization efficiency (FPF = 37%) [139].

Clarithromycin, a macrolide antibiotic, has demonstrated its potential to treat various tumors with the highest efficacy towards lung cancer, multiple myeloma, lymphoma and chronic myeloid leukemia [140]. Putative mechanisms of action underlying its anti-cancer activity are associated with autophagy inhibition, anti-angiogenesis, suppression of NF-kB pathway as well as immune modulating and anti-inflammatory actions [[140], [141], [142], [143], [144], [145]]. Hamade et al reported that treatment of clarithromycin alone (10 mg/kg/day) retarded the growth of Lewis lung carcinoma and effectively reduced the number of tumor nodules in C57BL/6 mice [146]. Post-administration of clarithromycin a week after treatment with either cisplatin (6 mg/kg) or vindesine sulfate (7 mg/kg) displayed significant anti-tumor effect in Lewis lung cancer bearing C57BL/6 mice. Clarithromycin as an adjuvant therapy acted as an immune-stimulant to increase the expansion of helper T cell subsets, interferon-4 producing T cells and interferon-γ producing T cells as well as to enhance cytotoxicities of natural killer cell and CD8+ cells [146]. In addition, suppression of pulmonary metastasis was also observed in B16BL6 melanoma mice model receiving 50 mg/kg/day of clarithromycin [147].

Similar to itraconazole, the development of clarithromycin for inhalation is intended for lung cancer treatment [148,149]. Clarithromycin liposomal dry powder formulations created using ultrasonic spray freeze drying possessed improved aerosolization and stability [150]. This liposomal formulation remained unchanged in terms of physicochemical and aerodynamic characteristics after 3 months storage at 60% RH and 25 °C (FPF 43–50%). In addition, the incorporation of co-lyoprotectants (mannitol and sucrose) allowed for successful reconstitution of intact liposomes with a narrow size distribution and high entrapment efficiency [150].

Recently, an inhalable clarithromycin formulation prepared as a pMDI solution containing 10% ethanol as co-solvent in HFA134a demonstrated physical stability for a month between 4 and 37 °C and a relatively high fine particle fraction (47%) as assessed using the Andersen Cascade impactor [151]. The authors also showed that the clarithromycin pMDI formulation retained its immunomodulatory properties through the regulation of inflammatory cytokine (IL-8) expression [152]. Development of a nanoparticle-in-microparticle dry powder formulation containing clarithromycin was attempted in which the antibiotic was encapsulated in PLGA polymeric nanoparticles prior to spray drying to yield micron-sized particles suitable for inhalation [153]. Addition of low concentrations of leucine during spray drying resulted in stabilization of the microparticles through a reduction of surface cohesiveness. As a consequence, the emitted dose, FPF and yield process were significantly enhanced [153].

Statins are competitive inhibitors of hydroxymethylglutaryl-CoA reductase enzyme (HMG-CoA), inhibiting the mevalonate pathway that initiates cholesterol biosynthesis. Statins are conventionally used for hypercholesterolemia treatment and are effective to reduce morbidity and mortality in patients with coronary heart diseases. In recent years, the mevalonate pathway has been implicated in tumorigenesis in which the ectopic expression of HMG-CoA reductase in cooperation with the Ras oncogene contributed to breast cancer progression, thus implying the role of HMG-CoA as ‘metabolic oncogene’ [154,155]. Other enzymes in the mevalonate pathway such as farnesyl diphosphate synthetase (FDPS) and geranylgeranyl pyrophosphate (GGPP), critical for prenylation of proteins, are also involved in both tumor progression and resistance to chemotherapy [156] (Fig. 6). These data suggest that inhibition of HMG-CoA using statins may disrupt cell cycle progression and proliferation and thus impose anti-cancer effects [157,158]. Simvastatin, one of the groups of drugs in the statin family, exerts its anti-proliferative action against lung cancer through the inhibition of NF-κB activation. This may lead to a disruption in the cell cycle, enhanced apoptosis, and down-regulation of cyclin-D1, cyclin-dependent kinases and MM9 expression [159]. Other findings show that simvastatin is able to regulate tumor necrosis factor-β receptor II in A549 cells, as well as IL-8 levels in human lung adenocarcinoma cell line GLC-82 [160,161].

A micronized simvastatin DPI formulation was successfully fabricated with an appropriate particle size distribution for delivery to the lung following inhalation [162]. The DPI formulation was chemically stable for long periods of time and maintained its aerosol performance. No chemical degradation of simvastatin into its metabolites was noted for up to 9 months. As for the aerosol stability, the FPF values for day 0 (initial preparation), 180 and 270 corresponded to 44.6 ± 5.8%, 39.6 ± 3.8 and 41.2 ± 1.4%, respectively, without statistical differences [162]. In addition to the DPI formulation, simvastatin showed versatility to be developed into a pMDI solution using ethanol as a co-solvent for treatment of airway-associated inflammations [163]. In vitro aerosol performance assessed using ACI demonstrated a FPF, MMAD and GSD of 31.9%, 1.58 μm and 2.12, respectively [163]. In addition, the inhibitory effects of simvastatin on the inflammatory mediators (IL6, IL8, TNF-α) and oxidative stress potentiated the role of simvastatin in the chemoprevention of lung cancer [164].

Cisplatin, or cis-diamminedichloroplatinum (II) [Pt(NH3)Cl2], is a platinum based chemotherapeutic agent with broad-spectrum activity against various cancers, including lung cancer. The modes of action of cisplatin include binding to DNA and disturbing cell repair mechanisms. However, IV administration of cisplatin is associated with severe renal toxicity, anemia and chronic neurotoxicity [165]. Therefore, repurposing of cisplatin as an inhalable formulation could overcome the above-mentioned limitations by reducing its toxic side effects and enhancing its drug bioavailability in lung tissue (Table 5).

For this reason, a comprehensive experiment was conducted to evaluate the efficacy of delivering anti-cancer drugs including cisplatin using different jet nebulizers (Maxineb®, Sunmist® and Invacare®) and different residual cups with different drug loadings [166]. Six residual cups were used where four cups had a capacity of up to 6 ml (B, C, F, and G) and two cups had a capacity of up to 10 ml (A and D). The efficacy was evaluated based on the fraction of droplets <5 μm. For all anti-cancer drugs tested, all combinations of jet nebulizers and residual cups successfully generated droplets <5 μm, suitable for lung delivery. For cisplatin and carboplatin, the optimal combination was the Maxineb® nebulizer with residual cup “D” design and 8 mL loading. To further minimize the toxic side effects of inhaled cisplatin towards healthy tissue, controlled-release inhalable formulation may be a better option to release the drug in a slow manner upon deposition [166].

A controlled-release dry powder formulation of cisplatin for lung cancer treatment was optimized recently by incorporating solubilized tristearin and/or a PEGylated component during spray drying [167]. These cisplatin microcrystalline formulations embedded in solid lipid microparticles exhibited high in vitro lung deposition with the FPF ranging from 37.3% to 51.5% [167]. In addition, the cisplatin in these formulations was covered with thick layers of lipids and exhibited a controlled-release behavior with a limited burst.

The controlled release behavior of different cisplatin DPI formulations (immediate and controlled release; with or without PEGylated excipients) administered using the pulmonary route was documented in a subsequent in vivo pharmacokinetic and biodistribution study [168]. The DPI formulations with PEGylated excipients provided a retention of platinum for up to 48 h, irrespective of the drug release behavior. However, owing to the rapid uptake and clearance by alveolar macrophages, the residence time of the non-PEGylated formulations was relatively short and independent of their drug release kinetics [168].

Singh et al attempted to improve the local concentration of cisplatin in the lung via aerosol delivery of chitosan based DPI formulations using lactose as a carrier [169]. The importance of lactose is reflected in the higher FPF of chitosan/lactose-based cisplatin DPIs (33.4%) compared to non-lactose DPIs. The formulation was stable for over six months at different storage conditions (40 °C/75% relative humidity (RH) or 25 °C/75% RH) in terms of physical (i.e., FPF and moisture content) and chemical properties (i.e., drug content). The cytotoxicity of chitosan/lactose-based cisplatin was lower than cisplatin solution, which may be due to slower release of cisplatin from the microspheres. It took >700 min for the release of cisplatin from the chitosan/lactose-based DPIs to reach a plateau as compared to <100 min for the cisplatin solution [169].

Inhalation delivery of hyaluronan (HA)-cisplatin conjugates directly to the lung is another approach for effective lung cancer and metastasis treatment by reducing the systemic toxicity and enhancing cisplatin retention in lung tissue and the surrounding mediastinal lymph [165]. In a study by Xie et al, the chemical conjugation of cisplatin and HA was found to effectively preserve the in vitro anti-tumor action of cisplatin. In vivo pharmacokinetics was conducted using Sprague–Dawley rats randomized into four groups to receive: (i) IV. cisplatin (3.5 mg/kg), (ii) IV HA-cisplatin conjugate (3.5 mg/kg equivalent cisplatin), (iii) lung instillation cisplatin and, (iv) lung instillation HA-cisplatin conjugate [165]. The ratio of cisplatin in lung tissue to plasma was higher for the lung instillation group compared to the IV administered groups. For instance, the total platinum level was 5.7-fold and 1.2-fold higher at 24 h and 96 h, respectively for rats receiving the formulation via pulmonary delivery. Drug delivered via lung instillation was highly localized in lung tissue whereby low levels of cisplatin were found accumulated in the brain and kidney and non-detectable in the spleen and liver. In addition, the occurrence of inflammation by cisplatin conjugated to hyaluronan was significantly reduced after instillation [165]. In vitro cytotoxicity test of the novel nano-HA cisplatin conjugate formulation showed effective attenuation of cell growth in 2D and 3D spheroid cultures corresponding to an IC50 of 2.62 and 5.36 μM, respectively.

The efficacy was comparable to unconjugated cisplatin in Lewis lung carcinoma cells [170]. However, the in vivo therapeutic efficacy of the nanoformulation was 2-fold stronger compared to the unconjugated cisplatin at 7.5 mg/kg (the highest tolerated dose of free cisplatin). This was confirmed in the TUNEL assay which revealed that apoptotic populations were higher for mice treated with the nanoformulation compared to unconjugated cisplatin. A single dose of nanoformulation (7.5 mg/kg) delivered via intratracheal aerosol spray inhibited 90% of tumor nodule numbers and growth of lung allografts in mouse lungs. The administration of a higher dose of nanoformulation (15 mg/kg), which was above the LD50, did not cause acute toxicity or animal death and suppressed 94% of tumor nodules [170].

An epidermal growth factor receptor (EGFR) targeted gelatin nanoparticle (GP) based drug delivery system exploiting active targeting to the over-expressed EGFR in cancerous cells was explored as an alternative therapy for lung cancer [171]. The aerosol size revealed that 99% of the nebulized biotinylated EGF-modified GP had a MMAD with suitable range for lower airway deposition. The absence of acute lung inflammation in nude mice following inhalation of nanoparticles suggests this formulation is well tolerated. In addition, a 3-fold increase in accumulation of aerosolized biotinylated EGF-modified GP in lung tumors compared to healthy cells confirms specific targeting to EGFR-overexpressing cells. The accumulation of biotinylated EGF-modified GP in the organs of tumor mice after 24 h of inhalation was highest in the lung and lowest in the liver. In contrast, for normal mice, the distribution of this formulation was the lowest in the lung [171]. Following this, the authors reported the successful delivery of cisplatin loaded EGF-modified GP via nebulization with improved therapeutic efficacy towards lung cancer [172]. The IC50 of cisplatin loaded EGF-modified GP against A549 cells was 1.2 μg/ml while free cisplatin and cisplatin-GP was 2.5 μg/ml and 9.3 μg/ml, respectively. In addition, in vivo tumor reduction assay showed that the formulation harboring a specific targeting moiety could reduce approximately 70% of tumor burden after 17 days of treatment. In contrast, only 20% reduction was observed for free cisplatin [172].

An open-label phase Ib/IIa trial was performed primarily to assess the efficacy and safety of inhaled cisplatin to recurrent osteosarcoma patients with pulmonary metastases [173]. Inhaled cisplatin administered via nebulizer every 2 weeks (24 mg/m2) was well tolerated and had low systemic drug exposure. The toxicity associated with inhaled cisplatin treatment was localized (i.e., cough), transient and reversible. Meanwhile, the serum concentration of cisplatin was much lower than that reported for IV cisplatin, at doses of 100–120 mg/m2 [173,174]. Pre-treatment with human ATP-binding cassette (ABC) [ABCA10 gene] protein prior to aerosolization of cisplatin was conducted in an attempt to improve absorption of the drug into lung tissue [175]. In this work, a high intracellular concentration of cisplatin within the lung tumor tissue was coupled with minimal damage to the lung parenchyma [175].

A phase I dose escalating study of aerosolized Sustained Release Lipid Inhalation Targeting (SLIT) cisplatin was conducted to evaluate the dose limiting toxicity, safety profile and pharmacokinetics in seventeen patients with primary or metastatic lung carcinoma [176]. The aerosolized cisplatin treatment did not show dose-limiting toxicity at the maximum delivered dose. The concentrations of platinum in plasma were very low even with the longest repetitive inhalation. Side effects commonly associated with cisplatin treatment such as hematologic toxicity, nephrotoxicity, ototoxicity, or neurotoxicity was also non-existent, with the most common adverse events being nausea, vomiting, dyspnea, fatigue and hoarseness [176].

In another trial, an open-label phase Ib/IIa study was conducted to investigate the safety and effectiveness of inhaled lipid cisplatin (ILC) in recurrent osteosarcoma patients who only had pulmonary metastases (n=19) [173]. The patients were split into two dose groups (seven patients with a 24 mg/m2 dose and twelve patients with a 36 mg/m2 dose) who received ILC via nebulizer every 2 weeks (= 1 cycle). ILC was well tolerated in these osteosarcoma patients with absence of the typical side effects associated with IV delivery of cisplatin such as hematologic toxicity, nephrotoxicity or ototoxicity. In addition, the systemic cisplatin exposure was minimal using the inhalation route. In general, the patients responded well to the treatment; i.e., two patients had stable disease after 2 cycles and could then undergo metastasectomy and remained free from pulmonary recurrence. Furthermore, one patient showed sustained partial response while the lesions in eight patients were less than 2 cm [173].

Paclitaxel (PTX) is considered a first-line drug in lung cancer therapy. The commercial IV formulation of PTX, Taxol®, contains Cremophor E and dehydrated ethanol and is associated with adverse reactions and toxicities such as muscle pain, neurologic and cardiac toxicities and hypersensitivity. Various approaches have been used to minimize the adverse reactions while achieving a high therapeutic index. These repositioning strategies include the engineering of PTX into new dosage forms with the exclusion of Cremophor E (i.e., liposomes, micelles, or nanoparticles) and a new administration route (aerosol inhalation) (Table 5).

One example is PTX loaded microporous poly(lactic-co-glycolic acid) [PLGA] microparticles formulated via an emulsion technology. They demonstrated high drug encapsulation, controlled release kinetics, comparable aerosol performance and superior in vitro cytotoxicity against lung cancer cells (A549 and Calu-6) [177]. Plasma concentrations of PTX in Sprague-Dawley rats receiving this formulation via the endotracheal route were in the therapeutic range and lasted 4-times longer than those administered by the IV route. In addition, lung-targeting behavior for PTX loaded PLGA microparticles was also approximately 12-fold higher for the inhalation route compared to IV. injection [177]. In contrast, a dry powder formulation of PTX loaded alginate microparticles for inhalation showed poor aerosol performance with an MMAD, FPF and GSD of 5.9 ± 0.33 μm, 13.9 ± 0.6% and 1.8, respectively. The PTX/alginate microparticles showed only comparable in vitro anti-proliferative effect against A549 and Calu-6 to free PTX [178]. Another way to improve the efficiency of delivery of anti-cancer drugs via inhalation is through the modulation of respiration parameters [179]. The utilization of 5% CO2-enriched air during nebulization increased both pulmonary ventilation and deposition of inhaled PTX liposomes. In this study, PTX was encapsulated into dilauroylphosphatidylcholine (DLPC) multilamellar liposomes with a mean diameter of 12.49 ± 8.06 μm prior to nebulization. The size of the PTX liposomes was reduced following nebulization to between 0.12 and 0.23 μm [179]. More than a 4-fold increase in the deposition of PTX in mice lungs was observed for aerosols generated with CO2-enriched air (23.1 ± 4.3 μg/g) compared to normal air (5.5 ± 0.2 μg/g) [179]. The authors next compared the in vivo tumor activity and pharmacokinetics of PTX liposome administered intravenously and as an aerosol generated in CO2-enriched air [180]. Based on the lung area under the curve (AUC) after administration of a single dose of PTX liposomes (5 mg/kg), the concentration of PTX delivered via the inhalation route (33.4 mg-h/g) was 26-fold higher compared to IV injection (1.3 mg-h/g). In addition, the clearance of inhaled PTX from the lungs was much slower as evident from the half-life (t1/2) of 0.02 h for IV and 0.71 h for inhalation [180]. To investigate the therapeutic effectiveness of PTX liposomes, mice bearing murine renal carcinoma (Renca) pulmonary metastases received aerosolized drug for 30 min three times weekly for 2 weeks (equivalent dose 5 mg/kg PTX). Compared to the untreated group, the numbers of tumors (and their size) were reduced from 478 (0.88 mm) to 323 (0.59 mm). This was accompanied with prolonged survival of the Renca cell bearing mice following inhalation therapy [180].

Luo et al recently explored the use of 6 kDa and 20 kDa PEG-PTX conjugates as a strategy to increase the tolerability of mice towards PTX, prolong its residence time within the lungs and thereby increase its efficacy [181]. PEG-PTX conjugates with varying molecular weight of linear PEG were prepared through conjugation of PTX and PEG at both hydroxyl ends using azide linker and ester bonds. Interestingly, PEG-PTX conjugates drastically increased the maximum tolerated dose (MTD) by 100-fold following intratracheal delivery to healthy mice. The MTD for PEG-PTX 6 kDa, PEG-PTX 20 kDa and Taxol® was 50 mg/kg, 20 mg/kg and 0.5 mg/kg, respectively [181]. Intratracheal delivery of Taxol® (0.5 mg/kg) is associated with local toxicity as evidenced by an increase in total protein, lactate dehydrogenase (LDH) and neutrophils. In contrast, using the same dose and delivery route, PEG-PTX conjugates did not contribute to any changes in these biochemical markers, thus demonstrating the ability of PEGylation to attenuate the local toxicity of PTX. Furthermore, both PEG-PTX conjugate formulations demonstrated superior anti-tumor activity in Lewis lung carcinoma bearing mice. As a comparison, Taxol® failed to exhibit significant lung tumor reduction irrespective of administration route [181].

In another similar study, PTX loaded PEG-DSPE micelles were also studied for their distribution and pharmacokinetics in Sprague-Dawley rats using two different administration methods (intratracheal and IV) [182]. Intratracheal administration resulted in enhanced AUC of PTX in the lung compared to IV injection. Similarly, the incorporation of PEG into a micelle formulation resulted in the accumulation of PTX in the lung for a prolonged period of time. After 12 h of intratracheal administration, PTX concentrations for micelle formulation and Taxol® were >200 ng/ml and <50 ng/ml, respectively [182]. Meenach et al recently reported the engineering of high performing DPI formulations comprising co-spray dried PTX, lung surfactant and lipopolymers with varying PEG chain lengths [183]. Although the encapsulation efficiency and drug loading was not statistically different, the variation in the PEG chain length affected the aerosol performance significantly. The use of PEG with higher molecular weight caused an increase in the MMAD and GSD while reducing the FPF and respirable fraction.

PTX loaded solid lipid nanoparticles (SLN) exhibited 20 times higher efficacy compared to free PTX to inhibit the proliferation of MXT-B2 cells [184]. Prolonged inhalation of PTX-SLN (1 mg/kg/dose) to MXT-B2 harboring B6D2F1 female mice was sufficient to completely inhibit the spread of lung metastases, invasion and tumor growth, with negligible lung injury. Complete lung regression coupled with widespread metastases was observed for the mice receiving IV free PTX (2.4 mg/kg/dose) [184]. Although the lipid-based nanoparticle formulation exhibited superior anti-lung cancer activity, large-scale production of nanoparticle-based chemotherapeutic agents without compromising the physicochemical and aerodynamic properties is challenging from the industrial point of view. Therefore, Hureaux et al investigated the feasibility of scaling-up the production of PTX encapsulated lipid nanoparticles for aerosol delivery [185]. Various parameters were considered including the particle size, osmolarity, pH, sterility, pyrogenicity, aerosol performance and preservation of therapeutic activity after storage. Both PTX-lipid nanoparticles and Taxol® formulations which underwent freeze and thawing process were able to preserve the anti-proliferative activity compared to fresh Taxol®. Out of the three different types of nebulizers tested (jet, ultrasonic and mesh), only the mesh nebulizers were able to create aerosols without compromising their lipid structures [185].

It is hypothesized that nanoparticles engineered using endogenous pulmonary surfactant mimetic material that offers a pH responsive release behavior could maintain normal airway potency, reduce pulmonary toxicity and simultaneously achieve targeted aerosol delivery to solid tumors [186]. To mimic the pulmonary surfactant microenvironment, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the most abundant phospholipid in lung surfactant and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were used to form lipid nanovesicles (LN) with a size distribution of 100–200 nm for aerosol delivery of PTX to metastatic lung cancer [186]. Owing to pH responsive characteristics of PTX-LN, selective targeting to the acidic microenvironment of cancer cells resulted in specific killing in B26F10 melanoma cells without exerting any toxicity to normal lung fibroblasts. More than 70% of metastasis inhibition was noted for the B16F10 bearing mice exposed to aerosolized PTX-LN (0.5 mg PTX/ml, 30 min exposure, five times weekly for a duration of four weeks). This was significantly higher compared to IV Taxol® (10 mg/kg) and IV Abraxane® (10 mg/kg). Furthermore, the numbers of metastatic nodules were also significantly lower compared to conventional chemotherapeutic treatments [186].

Doxorubicin (DOX) is one of the leading chemotherapeutic agents used for oncology treatment, including lung cancer. This anthracycline based drug holds great promise to control cancer growth and progression via intercalation into the DNA double helix which suppresses the progression of topoisomerase II enzyme (responsible for DNA replication). However, DOX causes severe cardiac toxicity when delivered via the IV route and thus repositioning into an inhalable formulation may minimize side effects without compromising its therapeutic efficacy (Table 5).

For this purpose, a comparison of administration routes for DOX was conducted using a lung metastatic mouse model [187]. Intrapulmonary administration resulted in high local concentrations of DOX in the lung in conjunction with low systemic circulation (plasma level). Interestingly, DOX was found widely localized in tumor regions with p-gp expression following intrapulmonary delivery. This was accompanied with more effective anti-tumor activities. The binding saturation of active efflux pumps decreased the removal rate of DOX from cells, which in turn contributed to high local intracellular drug concentrations and increased therapeutic action. In contrast, IV injection of DOX resulted in undetectable drug levels in the tumor region and poor therapeutic efficacy [187]. Mainelis et al also concluded that simultaneously bypassing both pump and non-pump resistances could substantially enhance the chemotherapy effect [188]. This was influenced by the selection of a suitable administration route and the presence of pump inhibitors, such as antisense oligonucleotides (ASO) or small interfering RNA (siRNA). Inhalation therapy reduced the tumor burden in mice by more than 90%, versus 40% for the IV group. Another advantage of the inhalation route was the successful delivery of ASO and siRNA to targeted tissue as evident from the suppression of MRP1 and BCL2 proteins [188]. ASO and siRNA delivered intravenously encountered significant inactivation in the bloodstream and often failed to penetrate cancer cells [189]. Similarly, in another work, multifunctional mesoporous silica nanoparticles (MSN) incorporating chemotherapeutic drugs (cisplatin and DOX), a specific tumor targeted moiety (LHRH peptides) and two different siRNAs were developed for inhalation treatment of lung cancer [190]. Antisense oligonucleotides MRP1 and BCL2 mRNA were used for targeted suppression of pump and nonpump cellular resistance in lung carcinoma respectively. Their findings showed that inhalation delivery is a suitable administration route to preserve the specific activity of the chemotherapeutics and siRNA. Microscopic evaluation also confirmed that the MSN carrier localized DOX in both the cytoplasm and the nuclei of A549 cells, while siRNA was mainly located in the perinuclear region of the cytoplasm. Simultaneous delivery of DOX and suppressor proteins via inhalation to A549-bearing NCR nude mice significantly enhanced its cytotoxic effect and resulted in higher accumulation of DOX in the lung compared to IV administration. The expression of BCL2 and MRP1 genes were reduced by 58% and 56%, respectively, which in turn confirmed that siRNA was released in the cytoplasm [190]. In their next work, a luteinizing hormone-releasing hormone (LHRH) was additionally incorporated into their existing formulations as a targeting moiety to receptors overexpressed in the plasma membrane of lung cancer with the goal to produce highly tumor-specific and effective therapeutic action [191]. Superior chemotherapeutic effect of this multifunctional formulation following pulmonary delivery was observed as demonstrated in the complete regression of tumors in vivo. Comparatively, this target-specific formulation was 100-fold more effective than PTX or DOX [191]. Other studies showed that the functionalization of inhalable liposomes with transferrin or phospholipase A2 produced encouraging results by targeting transferrin receptors and inflamed lung tissues, respectively [192,193]. In addition to transferrin, functionalization of inhalable nanoparticles with other active ligands such as epidermal growth factor (EGF), tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and haloperidol were also proposed for pulmonary delivery to treat drug resistant lung cancer and lung metastasis [[194], [195], [196]]. EGF-modified gelatin nanoparticles released DOX in a sustained manner intracellularly and were effectively taken-up by EGFR, overexpressing A549 and H26 cancer through receptor-mediated endocytosis. As a consequence, these nanoparticles were retained in the lungs in vivo at high therapeutic concentrations for more than 24 h and achieved approximately 90% tumor inhibition [196]. TRAIL (apoptosis-inducing protein) conjugated DOX loaded albumin nanoparticles were deposited evenly in the mouse lung via aerosolization and demonstrated a prolonged drug release behavior. As expected, active-targeted nanoparticles coupled with DOX were superior compared to single DOX nanoparticles and TRAIL alone in vivo against H226 cell-induced metastatic tumors [195]. Overall, ligand-mediated nanoformulations were robust and remained active after inhalation, specifically-targeted lung tumors and reduced the side effects associated with IV DOX [[194], [195], [196]].

DOX loaded PLGA microparticles with high residency and cytotoxic properties were manufactured to treat lung metastasis [197]. The PLGA-based formulation was retained in the lung for up to 2 weeks after inhalation and the cytotoxicity effect towards B16F10 cells was effective within 24 h. In addition, tumor number and mass as well as lung weight were reduced compared to the untreated control [197]. The same group took a step further to co-deliver DOX and PTX for in situ lung cancer treatment using PLGA microspheres as a carrier [198]. Interestingly, the combination therapy showed synergism towards B16F10 cells when DOX was the major component while antagonistic effects were observed when PTX was the main compound. DOX/PTX at a ratio of 5:1 demonstrated the highest synergistic therapeutic effect with fewer lesions in the lung [198].

In another study, a dendrimer (polyamidoamine)-based DOX conjugate improved the bioavailability and pharmacokinetic profile of DOX in lung cancer tissue [199]. DOX was conjugated to polyamidoamine via a pH-sensitive linker to provide stimuli-responsive release in the acidic tumor microenvironment. PEG was introduced to enhance the aqueous solubility of the DOX conjugate to allow high DOX payloads and confer stealth-like characteristic to avoid drug clearance. To achieve high local delivery, a novel PEGylated DOX-dendrimer pseudosolution MDI formulation with enhanced aerosol characteristics was developed and optimized. Interestingly, the presence of PEG in the formulation was beneficial to achieve exceptional aerosol performance with an FPF of 78% [199]. DOX-dendrimer demonstrated delayed in vitro cytotoxicity effect against A549 cells, whereas free DOX solution demonstrated instant toxicity and was time-independent. The differences in the therapeutic action are associated with the makeup of the formulations. Solubilized free DOX may be rapidly internalized into the cells. Meanwhile, PEGylated DOX was retained in the conjugate form at extracellular physiological conditions and the DOX was released in a sustained manner within the nucleus [199]. Mice bearing metastatic cells had a higher survival rate and a decreased tumor burden (fewer and smaller tumor nodules) following inhalation of the MDI-based DOX formulation [200]. In addition, a significant reduction in cardiac accumulation was noted upon inhalation delivery of this formulation [200].

Garbuzenko et al recently initiated an in-depth evaluation of the effect of the physico-chemical characteristics of the nanocarrier (i.e., size, shape and type) and the administration route on drug accumulation, residency and efficacy in the lung [23]. Based on their findings, the PEGylated liposomes, PEG-DSPE micelles, and PEG polymer resulted in higher accumulation and were retained relatively longer in the lung compared to MSN, dendrimer and quantum dots. Regarding the administration route, DOX loaded liposomes delivered via IV were less effective than inhalation to induce apoptosis and overall solid tumor treatment [23]. In addition, other polymer-based nanoparticles proven to be efficacious for lung cancer treatment include poly(butylcyanoacrylate), Janus, chitosan and PLGA nanoparticles [[201], [202], [203]].

Gemcitabine (GCB), 2,2-difluorodeoxycytidine is a prodrug belonging to the nucleoside analog family and must be phosphorylated in the nucleus by deoxycitidine kinase to convert it to its active form. This drug is effective for NSCLC treatment but not against highly metastatic tumors, such as osteosarcoma, owing to inefficient or inappropriate delivery methods. For instance, intraperitoneal administration of GCB failed to prevent the occurrence of lung metastasis due to limited GCB bioavailability in the lung [223,224].

GCB has shown remarkable potential as the Fas upregulating agent in both in vitro and in vivo studies [223,225,226]. Dysregulation of the Fas pathway is associated with tumor growth and progression. Several studies had evaluated the role of Fas in the development of osteosarcoma pulmonary metastases since osteosarcoma is known to metastasize exclusively to the lung [223,225,226]. Upregulated expression of FasL is inversely correlated to the metastatic tendency of osteosarcoma in the lung. Highly metastatic tumors are often found in lung tissues with minimal cell surface Fas expression.

Two osteosarcoma lung metastasis models with different invasive activities were utilized to evaluate the effectiveness and tolerability of aerosolized GCB to mice bearing metastatic tumors [223]. Human osteosarcoma lung metastatic cells (LM7) develop lung metastases in mice directly following IV injection, while metastatic murine subline (LM8) cells grew subcutaneously in mice followed by metastatic progression to the lung [223]. Complete physical regression in lung metastasis (tumor diameter, number metastases and tumor area) was observed following GCB aerosol treatment at 1.0 mg/kg for twice a week for 6.5 weeks [223]. In another study, mice were free from any signs of toxicity after receiving a high dose of GCB (5 mg/ml) twice a week for 5 weeks via pulmonary delivery [226]. The authors also made important findings using a K7M2 murine osteosarcoma mice model: (i) inhibition of Fas pathway prolonged the retention of K7M3 cells in the lungs and, (ii) FasL was needed to induce the cytotoxicity of GCB for lung metastasis treatment [226]. Gordon and Kleinerman further showed that the induction of Fas expression in K7M3 and DLM8 mouse models was also dependent on the administration route. GCB delivered via the intraperitoneal route failed to inhibit the metastatic progression to lung. Additionally, IV injection of GCB exhibited a fairly low rate of prevention of lung metastasis compared to the inhalation route (8-fold difference) [225]. Using a larger animal model such as the baboon, aerosol GCB was deposited in the peripheral lung compartment without pulmonary and systemic toxicity [208]. Relatively larger dog populations (n = 30) responded well to inhalation of GCB twice weekly in terms of body weight maintenance without signs of gastrointestinal toxicity [227]. Regarding the therapeutic potential, all aerosol treated dogs displayed intratumoral necrosis and approximately 85% of metastases exhibited >25% necrosis [227].

Safety of GCB administration to rats was compared between the oral and pulmonary route. The maximum tolerated dose of healthy Wistar rats to both aerosol and oral GCB was the same at 4 mg/kg, but a higher death rate was observed for dosages exceeding 6 mg/kg using oral delivery [228]. Gagnadoux et al evaluated the effect of high GCB doses on the growth and infiltration of tumors using an orthotopic model of large cell carcinoma via inhalation [229]. About 31% of mice had no visible tumor and 69% of mice showed more than a 50% reduction in tumor diameter (5 ± 0.3 mm to 2.05 ± 0.7) when the treatment regimen was scheduled at a single dose of 8 mg/kg weekly for five consecutive weeks. Even though higher doses (12 mg/kg) were significantly superior to achieve total inhibition of tumor growth, however this concentration was associated with fatal pulmonary oedema [229]. In another work, Selting et al investigated the tolerance and safety of intrapulmonary delivery of combination chemotherapeutics (GCB and cisplatin) to mechanically ventilated healthy dogs using the AeroProbe® Intracorporeal Nebulizing Catheter (INC) [230]. Sequential escalating doses of GCB (1, 2, 3 or 6 mg/kg of a 40 mg/ml solution) with a fixed dose of cisplatin were administered and changes in interstitial, bronchiolar and alveolar radiology were monitored. It was reported that radiographic changes worsened with sequential treatment of combination drugs and eventually progressed to severe grade after four treatments over a period of 8 weeks. All treated dogs developed severe pneumonitis and peribronchial fibrosis in the adjacent lobe. Owing to the absence of excessive clinical toxicity (i.e., cough, vomiting, diarrhea), it was concluded that the treated dogs were unaffected clinically and these histological changes could be acceptable considering the potential chemotherapeutic benefits of this combinational treatment [230].

In light of encouraging results from preclinical experiments, a proof-of-concept Phase I clinical trial was undertaken in eleven patients with primary NSCLC to establish the pharmacokinetics, distribution and feasibility of dose escalation of nebulized GCB [231]. Patients were treated with doses of between 1 mg/kg and 4 mg/kg using the Aeroneb Pro with the Idehaler Chamber. Approximately 42% of the total dose of GCB was delivered to the lung with no hematologic toxicity, nephrotoxicity or neurotoxicity reported [231].

Camptothecin (CPT) and its derivative 9-nitrocamptothecin (9-NC) possesses anti-cancer effect towards multiple types of cancerous cells including human ovarian, breast, prostate, colon, lung and malignant melanoma [[232], [233], [234], [235]]. Despite that, both CPT and 9-NC share low aqueous solubility and a cytotoxicity effect towards healthy cells; thus, encapsulating these drugs into carriers could overcome these limitations. For this reason, Knight et al tested a liposomal encapsulated 9-NC formulation against a wide range of xenograft models including breast, colon and lung cancer via aerosolization [236]. In the treatment of xenografts of human breast cancer, the increase in tumor size after receiving 9-NC liposome (8.1 μg/kg per day) for 5 days per week for 31 days, was seven times lower (3.8% per day) compared to the non-treatment group (27.2% per day). Complete regression of xenografts bearing colon cancer was achieved with treatment using a higher dose of 9-NC (38.3 μg/kg) for 5 days per week. Treatment using oral delivery did not translate into successful tumor size reduction as evidenced by the continual increase in tumor size for all xenograft models comparable to the untreated control [236].

Healthy BALB/c mice, D57BL/6 mice with subcutaneous Lewis lung carcinoma and Swiss nu/nu mice with human lung carcinoma xenografts, were used to measure the distribution and pharmacokinetics of an aerosolized liposomal CPT formulation [237]. Irrespective of the animal models, drug delivered via inhalation showed the highest deposition in the lung, followed by the liver with lesser amounts in other organs (i.e., blood, brain and kidney). In addition, the biodistribution of CPT in the organs was dependent to the administration method. For example, the highest concentration of CPT was found in lung (310 ng/g of tissue) with aerosol; however, both the IV and oral routes resulted in the highest deposition in the kidney at 243 ng/g and 109 ng/g tissue, respectively [237]. Encouraged by the promising distribution profile of drug in the lung following aerosol delivery, the efficacy of liposomal 9-NC formulations was evaluated in melanoma and osteosarcoma lung metastases mice models [238]. In their study, C57BL/6 mice harboring B16 melanoma cells were given aerosolized 9-NC for 1 h (153 mg 9-nitrocamptothecin/kg) for 5 days/week for three consecutive weeks. At the end of treatment, the number and size of tumors and lung weight were visibly reduced compared to untreated groups. In addition, tumor nodules and tumor foci in osteosarcoma lung metastases nude mice were smaller and fewer for the liposomal 9-NC-treated groups [238]. In another study, the slower release of 9-NC from the liposomal formulation increased the mean residence time (3.4-fold) and AUC (2.2-fold) of 9-NC in the lungs compared to the 9-NC solution [239]. This liposomal formulation increased the targeting efficiency coupled with the ability to reduce pulmonary damage. According to the histological images, severe lung edema and congestion were noticeable in rats after receiving intratracheal instillation of 9-NC solution for 5 consecutive days. In contrast, this was not observed for liposomal 9-NC formulations using the same treatment conditions [239]. In a phase I study which evaluated the feasibility and toxicity of liposomal 9-NC formulations delivered via inhalation, side effects higher than grade 2 were not observed in any cohorts [240]. While 9-NC was observed systemically, there was a rapid reduction of 9-NC from 36.7 ng/ml to 4.9 ng/ml after 24 h [240].

Temozolomide (TMZ) is used clinically for the treatment of aggressive cancer with poor prognosis, such as melanoma and glioblastoma. TMZ exhibits well-accepted safety features which seldom results in the discontinuation of therapy. Dry powder formulations of TMZ with or without excipients were recently prepared using spray drying [241]. The presence of cholesterol in the formulation provided lipid-coating properties and improved the FPF to 51%. However, the higher amount of low melting point phospholipids caused the particles to agglomerate, thus reducing the aerosol performance (FPF of 26%) [241]. Self-assembled folate-grafted PEGylated TMZ nanomicelles for inhalation as a DPI formulation with improved drug solubility were recently manufactured [242]. TMZ-based nanomicelles were more toxic against A549 and M109-HiFR cells compared to raw TMZ alone [242]. A phospholipid stabilizing the TMZ microcrystals in suspension was prepared to treat B16F10 melanoma pseudo-metastatic lung model via endotracheal administration [243]. Irrespective of the incubation time and treatment dosage, IC50 values for TMZ microcrystals against A549, B16F10 and T98G cells were relatively identical, thus indicating that this formulation has broad-spectrum activity towards cancer cells.

Both endotracheal and IV administration of TMZ (160 mg/kg) were relatively safe in the pseudo-metastatic lung model with no deaths or significant reduction of weight observed within the experimental period. When the frequency of dosage was increased from once weekly to thrice weekly, it significantly affected the survival of mice, but was therapeutically effective to achieve complete tumor eradication using this treatment regimen [243]. Antitumor activities of TMZ in different types of formulations were evaluated with the urethane induced lung cancer BALB/c mouse model through intratracheal inhalation [244]. The lactate dehydrogenase (LDH), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) activities in mice were much lower in all tested formulations [gold nanoparticles alone (GNP), liposome-embedded gold nanoparticles (LGNP), TMZ loaded gold nanoparticle (TGNP) and liposome embedded TCNP (LTGNP)], compared to the urethane control group. Similar observations were also found on the expression of lung oxidative stress proteins [lipid peroxidation (MDA), reduced glutathione (GSH)], inflammatory markers [tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β)] when treated with the same formulations above [244]. Among the four formulations, the effect of LTGNP was the most pronounced towards the change in biological activities in vivo [244].

Hitzman et al prepared a respirable nanoformulation of 5-fluorouracil (5-FU) with a thick lipid shell that was readily aerosolized by ultrasonic nebulization without impacting release kinetics [245]. Preliminary in vivo release characteristics of 5-FU nanoparticles following aerosol and intratracheal administration demonstrated successful deposition of 5-FU into the lower airways of male Syrian Golden hamsters. The concentrations of 5-FU in the lung and serum remained detectable even after 24 h of aerosol administration [245]. In their next study, detailed pharmacokinetics and distribution of 5-FU in the hamster following inhalation therapy was investigated. A dose of 30 mg nanoparticles/kg body weight (equivalent to 1.5 mg/kg 5-FU) was nebulized over an 8 mins interval. Targeted delivery of aerosolized 5-FU (MMAD = 0.95 μm, GSD = 1.57) to the respiratory tract was successfully achieved, with the deposition following the decreasing order: larynx (13.24 ± 0.77%) > esophagus (7.53 ± 0.85%) > trachea (2.51 ± 0.36%) > lung (1.38 ± 0.05%). The concentrations of 5-FU were extremely low initially in the gastrointestinal tract and stomach, thus suggesting effective local targeting of 5-FU as an adjuvant therapy for lung cancer. However, 5-FU was rapidly cleared from the lung whereby more than 99% was eliminated after 24 of administration [246].

Section snippets

Concluding remarks

Lung cancer remains one the most life-threatening malignant diseases that is influenced by a multitude of factors including both internal (genetic) and external (environmental). Owing to the complexity as well as the rapid and aggressive development of drug resistance in lung cancer, it signals the urgency for the discovery of wonder drugs which can ideally completely kill, eliminate and inhibit tumor progression without development of drug resistance. Therefore, we have seen a boom in

Acknowledgements

WH Lee is the recipient of Early Career Fellowship from Cancer Institute New South Wales (CINSW).

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