**4.1.2 Animal models for anticancer drug studies**

#### **4.1.2.1 Chemically induced lung cancer**

Several animal models of lung cancer have been developed and used to understand carcinogenesis and test anticancer agents. One of them is chemically-induced lung cancer. These animal models are usually used only to study carcinogenesis and cancer prevention because of the long time required for tumour development and the poor therapeutic response. Newborn A/J mice injected intraperitoneally with ethyl carbamate (urethane) begin to develop benign lung adenomas within few months, followed by degenerate adenocarcinomas similar to those of humans (Shimkin et al., 1975). Some of the many chemical agents that have been tested for their ability to induce lung tumours and study carcinogenesis are benzopyrene, metals, aflatoxin, and constituents of tobacco smoke such as polyaromatic hydrocarbons and nitrosamines (Liu et al., 2002). But chemical models have rarely been used in aerosol studies except for aerosolised chemopreventive treatment.

#### **4.1.2.2 Transgenic lung cancer**

Transgenic mouse models of lung adenocarcinoma expressing a mutant active K-ras transgene are available. Jonhson et al. (2001) created a mouse strain (K-ras LA1) carrying an oncogenic allele of K-ras that had an activating codon 12 mutation (gly to asp) on exon 1, that can be activated only on a spontaneous recombination event in the whole animal (Jonhson et al., 2001). This mutation, probably in combination with other somatically acquired mutations, leads to the development of multifocal lung adenocarcinomas in 100% of the mice, skin papillomas in 23%, and thymic lymphomas in 35%. Lung tumours can first be detected microscopically when the mice are 2 weeks old, and the number and size of tumours increase continuously until they essentially fill the thoracic cavity, causing the mouse to die of respiratory failure (mean survival 300 days). Extrathoracic metastases are rare and occur only at a very late stage of the disease, as in humans with adenocarcinoma (Wislez et al., 2003; Jonhson et al., 2001). The radiological appearance is also very similar to the human disease with multifocal lesions such as nodules with ground glass attenuation (Cody et al., 2005). We are presently using this model to analyze the administration of anticancer drugs *via* the airways.

#### **4.1.2.3 Human lung tumour xenografts**

Human lung tumour xenografts are widely used in cancer therapeutic studies. Nude mice or SCID immunosuppressed mice are injected intravenously or into the lungs with human tumour cells. Orthotopic models of lung cancer have also been evaluated for aerosol studies. Lung cancer cells, mainly H460 (large cell lung carcinoma) and A549 (bronchioloalveolar lung carcinoma) or disaggregated lung tumours are implanted endotracheally in immunocompromised mice. Orthotopic models are interesting because the tumours grow directly into the lungs, in an environment mimicking that of human lung neoplastic cells. Models of pulmonary metastases have also been used in aerosol studies. For example, LM7 and LM8 osteosarcoma cells were injected intravenously into immunosuppressed mice, leading to the development of lung metastases (Koshkina and Kleinerman, 2005). Lastly, tumours grow better in the lungs than in the subcutaneous compartment. The cell lines most frequently used in animal studies are H460 (large cell lung carcinoma) and A549 (lung carcinoma).

#### **4.2 Pharmacokinetics of anticancer agents delivered** *via* **the airways in animals 4.2.1 Lung deposition**

The concentrations in the lungs of anticancer drugs delivered by the pulmonary route are higher than the concentrations delivered by any other route. Cisplatin, one of the major drugs used to treat lung cancers, is administered systemically. Delivery of cisplatin *via* a catheter placed in right caudal lung lobe in dogs provided a concentration in the right caudal lobe that was 44 times higher than in other pulmonary lobes (Selting et al., 2008). The pulmonary delivery of other anticancer drugs that are clinically injected intravenously, but are unconventional for treating non small cell lung cancer, was also tested. The deposition of aerosolised liposomal camptothecin, a quinoline alkaloid, in the lungs was assessed in nude mice with colon, breast or lung tumour xenografts. The concentration of the encapsulated camptothecin in the lung was 100 times higher following airways administration than after intramuscular injection (Koshkina et al., 1999). Similarly, the concentration of aeorosolised 5- Fluorouracile (5-FU) in the lung tissue was 1000 times higher than in the serum of hamsters (Hitzman et al., 2006). High concentrations of 5-FU were detected in the trachea and bronchi of dogs after airways delivery, whereas a lower concentration was measured in the peripheral lung (Tatsumura et al., 1993). We have used near infrared imaging to analyze the distribution of cetuximab, an anti-EGFR antibody, in a xenograft model of lung tumour following systemic and pulmonary delivery, (Maillet et al., in press). The antibody accumulated rapidly and durably in the lungs (Figure 1), and the lung concentration was higher following airways delivery (not shown) (Maillet et al., in press).

Fig. 1. Lung deposition of inhaled cetuximab at (A) 1h30 (B) 8h (C) 24h (D) 48h and (E) 72h.

#### **4.2.2 Blood passage**

58 Advances in Cancer Therapy

Several animal models of lung cancer have been developed and used to understand carcinogenesis and test anticancer agents. One of them is chemically-induced lung cancer. These animal models are usually used only to study carcinogenesis and cancer prevention because of the long time required for tumour development and the poor therapeutic response. Newborn A/J mice injected intraperitoneally with ethyl carbamate (urethane) begin to develop benign lung adenomas within few months, followed by degenerate adenocarcinomas similar to those of humans (Shimkin et al., 1975). Some of the many chemical agents that have been tested for their ability to induce lung tumours and study carcinogenesis are benzopyrene, metals, aflatoxin, and constituents of tobacco smoke such as polyaromatic hydrocarbons and nitrosamines (Liu et al., 2002). But chemical models have rarely been used in aerosol studies except for aerosolised chemopreventive treatment.

Transgenic mouse models of lung adenocarcinoma expressing a mutant active K-ras transgene are available. Jonhson et al. (2001) created a mouse strain (K-ras LA1) carrying an oncogenic allele of K-ras that had an activating codon 12 mutation (gly to asp) on exon 1, that can be activated only on a spontaneous recombination event in the whole animal (Jonhson et al., 2001). This mutation, probably in combination with other somatically acquired mutations, leads to the development of multifocal lung adenocarcinomas in 100% of the mice, skin papillomas in 23%, and thymic lymphomas in 35%. Lung tumours can first be detected microscopically when the mice are 2 weeks old, and the number and size of tumours increase continuously until they essentially fill the thoracic cavity, causing the mouse to die of respiratory failure (mean survival 300 days). Extrathoracic metastases are rare and occur only at a very late stage of the disease, as in humans with adenocarcinoma (Wislez et al., 2003; Jonhson et al., 2001). The radiological appearance is also very similar to the human disease with multifocal lesions such as nodules with ground glass attenuation (Cody et al., 2005). We are presently using this model to analyze the administration of

Human lung tumour xenografts are widely used in cancer therapeutic studies. Nude mice or SCID immunosuppressed mice are injected intravenously or into the lungs with human tumour cells. Orthotopic models of lung cancer have also been evaluated for aerosol studies. Lung cancer cells, mainly H460 (large cell lung carcinoma) and A549 (bronchioloalveolar lung carcinoma) or disaggregated lung tumours are implanted endotracheally in immunocompromised mice. Orthotopic models are interesting because the tumours grow directly into the lungs, in an environment mimicking that of human lung neoplastic cells. Models of pulmonary metastases have also been used in aerosol studies. For example, LM7 and LM8 osteosarcoma cells were injected intravenously into immunosuppressed mice, leading to the development of lung metastases (Koshkina and Kleinerman, 2005). Lastly, tumours grow better in the lungs than in the subcutaneous compartment. The cell lines most frequently used in animal studies are H460 (large cell lung carcinoma) and A549 (lung

**4.1.2 Animal models for anticancer drug studies** 

**4.1.2.1 Chemically induced lung cancer** 

**4.1.2.2 Transgenic lung cancer** 

anticancer drugs *via* the airways.

carcinoma).

**4.1.2.3 Human lung tumour xenografts** 

Most studies have shown that the concentration of an anticancer drug in the bloodstream is lower after airways delivery than after systemic injection. For example, the concentration of cisplatin in the serum was 15.6 times lower after pulmonary administration than after intravenous (i.v.) injection (Selting et al., 2008). Gemcitabine was given to 3 baboons *via* the airways and its concentration in the blood was 25 times lower than after its systemic delivery (Gagnadoux et al., 2006). Dogs with spontaneous pulmonary metastases were given aerosolized paclitaxel and doxorubicin and the drug concentrations in the bloodstream were measured 1 minute later (Hershey et al., 1999). The serum concentrations were lower than when the drugs were delivered intravenously. However a pharmacokinetic analysis is

The Airways: A Promising Route for the Pulmonary Delivery of Anticancer Agents 61

al., 2001). There were significantly fewer tumours in the treated mice than in the mice given liposome alone. Survival was improved, with 40% of the mice treated with liposomal paclitaxel being alive after 45 days, whereas all the mice treated with liposome alone were dead. A recent study found that adding vitamin E to the aerosolised liposomal-encapsulated paclitaxel enhanced the anticancer response to paclitaxel in murine model of lung metastases from primary breast cancer (Latimer et al. 2009). Lastly, the feasibility of aerosolising paclitaxel encapsulated in lipid nanoparticles was tested *in vitro*. Preclinical studies in animal models of lung cancer are expected to evaluate whether encapsulation

Gagnadoux et al. evaluated the dose-limiting toxicity of gemcitabine delivered endotracheally to Wistar rats with a sprayer (Gagnadoux et al., 2005). The maximum tolerated dose was 4 mg/kg. They detected no toxicity, except for decreases in platelet and red blood cell counts, when gemcitabine had been inhaled once a week for 9 weeks. They also evaluated the antitumor efficacy of aerosolised gemcitabine in nude mice implanted intrabronchially with H460 cells (Gagnadoux et al., 2005). Of the 13 mice given aerosolised gemcitabine, 4 had no visible tumour at the end of the experiment. And those tumours remaining in the treated mice were smaller (2.05 mm) than those in the control group (5 mm). Aerosolised gemcitabine also significantly reduced tumour numbers in a pulmonary metastases murine model of osteosarcoma (Koshkina et al., 2005). Also, dogs with spontaneous lung metastases of osteosarcoma were treated in a preclinical study with aerosolised gemcitabine combined with a standard treatment regimen (Rodriguez et al., 2010). Almost half (46%) of the treated dogs showed over 50% tumour necrosis, but there

Hershey et al. treated 18 dogs with spontaneous primary or secondary lung tumours with aerosolised doxorubicine using a jet nebuliser (Hershey et al., 1999). There were 4 partial responses and treatment was well tolerated except for some coughing by half of the subjects, cardiotoxicity in two animals, and mild to moderate pneumonitis in almost all the dogs.

Aerosolised liposomal 9-nitro-20(S)-camptothecin (L9-NC) was first assessed in mice with xenografts of breast, colon and lung tumours injected subcutaneously (Knight et al., 1999). Treatment started 1 to 4 weeks after tumour implantation and was given 5 days a week. The growth of subcutaneous tumours was 7- fold lower in treated animals with breast cancer than in the controls and 7 to 10-fold lower in the mice with colon cancers. The lung tumour xenografts were also smaller in the mice treated with aerosolised L9-NC than in the controls. The antitumor effect of L9-NC was better when it was delivered through the airways than orally in all the systems. L9-NC was also evaluated in nude mice with lung metastases produced by the i.v. injection of melanoma (B16) or osteosarcoma (LM6) cells (Koshkina et al., 2000). The mice were given aerosolized L9-NC 5 days a week for three weeks. The treated mice with lung metastases of melanoma had statistically smaller (mean diameter 32 mm) tumour foci than the control group (85 mm). Similarly, 10 of the 11 control mice with LM6 lung metastase, had visible tumours, whereas none of 11 treated mice had tumours,

leads to the controlled release of the drug (Hureaux et al., 2009).

**4.3.3 Gemcitabine** 

**4.3.4 Doxorubicine** 

**4.3.5 Camptothecin** 

was no necrosis in the untreated animals.

required to support this conclusion. We have recently determined the pharmacokinetics of cetuximab delivered systemically and *via* the lungs. Little cetuximab was found in the bloodstream after airways delivery (only 11% of the fraction delivered to the lungs) and its passage was slow with a peak around 48 hours (Maillet et al., in press).

Fig. 2. Serum concentrations of cetuximab delivered systemically or *via* the pulmonary route.

#### **4.3 Efficacy and safety of anticancer agents delivered** *via* **the airways in animals 4.3.1 Cisplatin**

Selting and co-workers aerosolised cisplatin directly into the right caudal lobes of healthy dogs (Selting et al., 2008). They found no haematological toxicity and this delivery of aerosolised cisplatin was generally very well tolerated. Radiological and histological analyses indicated that all the dogs developed mild to moderate pneumonitis in the right caudal lobe where the drug was delivered. Chemotherapy with both cisplatin and gemcitabine was also delivered through the pulmonary route in healthy dogs using the same method (Selting et al., 2011). As previously reported, the only adverse event observed was focal pneumonitis in the right caudal lung lobe. Overall, the local-regional delivery of cisplatin either alone or in combination was well tolerated, suggesting that aerosolised platin-based chemotherapy may be used safely to treat humans. However, this method may be difficult to administer to patients with chronic respiratory failure.

#### **4.3.2 Paclitaxel**

Dogs with spontaneous primary or secondary lung tumours were given aerosolised paclitaxel using a jet nebuliser (Hershey et al., 1999). One of the 15 dogs had a partial response and one had a complete response with long term survival. The thirteen remaining had stabilised or progressive disease. In another study, mice with pulmonary metastases were given paclitaxel encapsulated in liposomes using a pneumatic nebuliser (Koshkina et al., 2001). There were significantly fewer tumours in the treated mice than in the mice given liposome alone. Survival was improved, with 40% of the mice treated with liposomal paclitaxel being alive after 45 days, whereas all the mice treated with liposome alone were dead. A recent study found that adding vitamin E to the aerosolised liposomal-encapsulated paclitaxel enhanced the anticancer response to paclitaxel in murine model of lung metastases from primary breast cancer (Latimer et al. 2009). Lastly, the feasibility of aerosolising paclitaxel encapsulated in lipid nanoparticles was tested *in vitro*. Preclinical studies in animal models of lung cancer are expected to evaluate whether encapsulation leads to the controlled release of the drug (Hureaux et al., 2009).

#### **4.3.3 Gemcitabine**

60 Advances in Cancer Therapy

required to support this conclusion. We have recently determined the pharmacokinetics of cetuximab delivered systemically and *via* the lungs. Little cetuximab was found in the bloodstream after airways delivery (only 11% of the fraction delivered to the lungs) and its

Aerosolized cetuximab

Intravenous cetuximab

Fig. 2. Serum concentrations of cetuximab delivered systemically or *via* the pulmonary

0 2 8 24 48 72 168 336 Time (hours)

**4.3 Efficacy and safety of anticancer agents delivered** *via* **the airways in animals** 

be difficult to administer to patients with chronic respiratory failure.

Selting and co-workers aerosolised cisplatin directly into the right caudal lobes of healthy dogs (Selting et al., 2008). They found no haematological toxicity and this delivery of aerosolised cisplatin was generally very well tolerated. Radiological and histological analyses indicated that all the dogs developed mild to moderate pneumonitis in the right caudal lobe where the drug was delivered. Chemotherapy with both cisplatin and gemcitabine was also delivered through the pulmonary route in healthy dogs using the same method (Selting et al., 2011). As previously reported, the only adverse event observed was focal pneumonitis in the right caudal lung lobe. Overall, the local-regional delivery of cisplatin either alone or in combination was well tolerated, suggesting that aerosolised platin-based chemotherapy may be used safely to treat humans. However, this method may

Dogs with spontaneous primary or secondary lung tumours were given aerosolised paclitaxel using a jet nebuliser (Hershey et al., 1999). One of the 15 dogs had a partial response and one had a complete response with long term survival. The thirteen remaining had stabilised or progressive disease. In another study, mice with pulmonary metastases were given paclitaxel encapsulated in liposomes using a pneumatic nebuliser (Koshkina et

route.

Serum concentratio

n of cetuximab (µ

g/ml)

**4.3.1 Cisplatin** 

0,00

2,00

4,00

6,00

8,00

10,00

12,00

**4.3.2 Paclitaxel** 

passage was slow with a peak around 48 hours (Maillet et al., in press).

Gagnadoux et al. evaluated the dose-limiting toxicity of gemcitabine delivered endotracheally to Wistar rats with a sprayer (Gagnadoux et al., 2005). The maximum tolerated dose was 4 mg/kg. They detected no toxicity, except for decreases in platelet and red blood cell counts, when gemcitabine had been inhaled once a week for 9 weeks. They also evaluated the antitumor efficacy of aerosolised gemcitabine in nude mice implanted intrabronchially with H460 cells (Gagnadoux et al., 2005). Of the 13 mice given aerosolised gemcitabine, 4 had no visible tumour at the end of the experiment. And those tumours remaining in the treated mice were smaller (2.05 mm) than those in the control group (5 mm). Aerosolised gemcitabine also significantly reduced tumour numbers in a pulmonary metastases murine model of osteosarcoma (Koshkina et al., 2005). Also, dogs with spontaneous lung metastases of osteosarcoma were treated in a preclinical study with aerosolised gemcitabine combined with a standard treatment regimen (Rodriguez et al., 2010). Almost half (46%) of the treated dogs showed over 50% tumour necrosis, but there was no necrosis in the untreated animals.

#### **4.3.4 Doxorubicine**

Hershey et al. treated 18 dogs with spontaneous primary or secondary lung tumours with aerosolised doxorubicine using a jet nebuliser (Hershey et al., 1999). There were 4 partial responses and treatment was well tolerated except for some coughing by half of the subjects, cardiotoxicity in two animals, and mild to moderate pneumonitis in almost all the dogs.

#### **4.3.5 Camptothecin**

Aerosolised liposomal 9-nitro-20(S)-camptothecin (L9-NC) was first assessed in mice with xenografts of breast, colon and lung tumours injected subcutaneously (Knight et al., 1999). Treatment started 1 to 4 weeks after tumour implantation and was given 5 days a week. The growth of subcutaneous tumours was 7- fold lower in treated animals with breast cancer than in the controls and 7 to 10-fold lower in the mice with colon cancers. The lung tumour xenografts were also smaller in the mice treated with aerosolised L9-NC than in the controls. The antitumor effect of L9-NC was better when it was delivered through the airways than orally in all the systems. L9-NC was also evaluated in nude mice with lung metastases produced by the i.v. injection of melanoma (B16) or osteosarcoma (LM6) cells (Koshkina et al., 2000). The mice were given aerosolized L9-NC 5 days a week for three weeks. The treated mice with lung metastases of melanoma had statistically smaller (mean diameter 32 mm) tumour foci than the control group (85 mm). Similarly, 10 of the 11 control mice with LM6 lung metastase, had visible tumours, whereas none of 11 treated mice had tumours,

The Airways: A Promising Route for the Pulmonary Delivery of Anticancer Agents 63

We have recently evaluated the antitumor activity of aerosolised cetuximab in a xenograft model of lung tumours. Aerosolised cetuximab limited the growth of the lung tumours (Maillet et al., in press). This proof-of-concept study demonstrates that the airways can be suitable for delivering aerosolised monoclonal antibodies to treat lung cancer, but further studies are required to determine whether the pulmonary route really does increase the

Cisplatin is the standard drug for treating non small cell lung cancers. It is given combined with another chemotherapeutic agent. Aerosolised cisplatin was assessed in a phase I study on 17 patients with lung cancer, including 16 with non-small-cell-lung cancer and one with small cell lung cancer (Wittgen et al., 2007). The cancers were progressing despite all previous treatment. Increasing doses of cisplatin encapsulated in liposomes were given. Each aerosol treatment was for 20 minutes with a jet nebuliser. The particles produced had a MMAD of about 3 µm. Each patient was given cisplatin on 1 to 4 consecutive days over a period of 21 days. The initial dose was 24 mg/m², which was doubled to 48 mg/m² without toxicity. The cycle was then shortened from 3 to 2 weeks, and finally to 1 week. Nebulised cisplatin was administered 3 times a day instead of twice. No dose-limiting toxicity was observed and the maximum tolerated dose was not reached. Dyspnea was reported in 11 patients and a productive and irritating cough in 5. Eosinophilia was observed in 4 of these 11 (36.4%) patients. Cisplatin was detected in the blood of only 4 patients. The disease of 12 of the 17 patients became stable and it progressed in five of them, but the study was not constructed to assess the response to treatment. The dose-limiting toxicity was not reached,

Anthracycline is usually administered intravenously to treat various cancers, including small cell lung cancer. The most severe adverse response is cardiac toxicity. Delivering the drug by inhalation is an interesting alternative route which may limit the cardiac toxicity. A phase I study was conducted by Otterson et al. to assess the safety of inhaled doxorubicin (Otterson et al., 2007). The 53 patients taking part included 16 patients with a primary lung tumour without histology details. The remaining 37 had pulmonary metastases of sarcoma (n=19), osteosarcoma (n=6), colorectal (n=4), thyroid (n=3) and other primary tumours (n=5). They were given doxorubicin with a pneumatic nebuliser fitted with a device that captured any fugitive aerosol to protect health worker. The starting dose was 0.4 mg/m². Inhalation was given every 3 weeks. The majority of adverse events were pulmonary with cough (n=27), dyspnea (n=9), chest pain (n=5), wheezing (n=4), hoarseness (n=3), hemoptysis (n=1), and bronchospasm (n = 1). While 5 patients suffered grade 3-4 pulmonary toxicity such as hypoxemia or decreased lung function tests, it was difficult to differentiate the adverse effects of treatment and the consequences of the pulmonary disease. Two patients suffered severe toxicity in response to a dose of 9.4 mg/m², with respiratory distress in 1 patient and bilateral ground glass infiltrates in the other. The lung function tests

therapeutic benefit of monoclonal antibodies to patients with lung cancer.

**5. Airways delivery of anticancer agents in clinical studies 5.1 Aerosolisation of anticancer drugs in primary lung cancer** 

**4.3.9 Monoclonal antibodies** 

**5.1.1 Cisplatin** 

precluding a phase II study.

**5.1.2.1 Safety and pharmacokinetic data** 

**5.1.2 Doxorubicin** 

suggesting that these tumors were very sensitive to aerosolised L9-NC. Gilbert et al. evaluated the safety of aerosolised L9-NC in healthy dogs. They found no toxicity after treatment with this drug for 5 days a week for 8 weeks (Gilbert et al., 2002).

#### **4.3.6 Temozolomide**

Wauthoz and co-workers evaluated aerosolised temozolomide, a recent alkylating agent, in mice with pulmonary metastases obtained by injecting B16 melanoma cells i.v. (Wauthoz et al., 2010). The treatment seemed to be very well tolerated because the maximum tolerated dose was not reached. The antitumor activity of temozolomide was similar whether it was delivered through the airways or intravenously and both routes gave significantly better results than the control group. And three of the mice were long-term survivors. These encouraging results might lead to clinical trials.

#### **4.3.7 Interleukin-2 (IL-2)**

IL-2 is a cytokine that induces an antitumor immune response. Anderson et al. evaluated immunotherapy with aerosolised IL-2 using mice with pulmonary metastases of sarcoma (Anderson et al., 1990). The antitumor activity of IL-2 delivered *via* the airways appeared to be better than *via* the other routes tested. Aerosolisation of IL-2 encapsulated in liposomes gave better survival than free IL-2 or liposome alone delivered *via* the same route. Tumour size was reduced by 50% to 85% by aerosolised liposomal IL-2, which was better than the results with free IL-2. This suggested that liposomal IL-2 was better than free IL-2.

The efficacy of airways delivery of free IL-2 and liposomal IL-2 were also compared in healthy dogs (Khanna et al. 1996). There were increases in white cells and effector cells in the broncho-alveolar lavage fluid of dogs treated with liposomal IL-2 but no haematological or clinical toxicity. A preclinical study in which dogs were treated with aerosols of liposomal IL-2 gave spectacular results (Khanna et al., 1997). Aerosolised liposomal IL-2 was used to treat 9 dogs, 7 with pulmonary metastases of a primary osteosarcoma and 2 with primary lung carcinomas. The pulmonary nodules in 2 of the 4 dogs with metastatic pulmonary osteosarcomas were in complete regression. No relapse was observed over 12 and 20 months, respectively. The disease of one of the two dogs with a primary lung carcinoma was stabilized for up to 8 months. Aerosolised IL-2 was widely evaluated in clinical trials to treat pulmonary metastases of renal cell carcinoma but there are few published preclinical studies in animals with IL-2 delivered *via* the pulmonary route. Clinical trials were probably proposed based on results obtained with IL-2 administered i.v. (Rosenberg et al., 1994)

#### **4.3.8 Celecoxib**

Celecoxib is a non-steroidal anti-inflammatory drug that inhibits cyclooxygenase 2. Treatment with aerosolised celecoxib was assessed in association with intravenous docetaxel in an orthotopic lung tumour model (A549) (Fulzele et al., 2006). Animals were given aerosolised celecoxib plus i.v docetaxel or oral celecoxib plus i.v. docetaxel, each of the drugs alone or a placebo. The aerosol was produced with a pneumatic nebuliser. Aerosolised celecoxib plus i.v. docetaxel reduced lung tumour volume by 61% compared to the placebo group while oral celecoxib plus i.v. docetaxel reduced it by 54%. Moreover, the combined treatments gave better results than the drugs alone.

#### **4.3.9 Monoclonal antibodies**

62 Advances in Cancer Therapy

suggesting that these tumors were very sensitive to aerosolised L9-NC. Gilbert et al. evaluated the safety of aerosolised L9-NC in healthy dogs. They found no toxicity after

Wauthoz and co-workers evaluated aerosolised temozolomide, a recent alkylating agent, in mice with pulmonary metastases obtained by injecting B16 melanoma cells i.v. (Wauthoz et al., 2010). The treatment seemed to be very well tolerated because the maximum tolerated dose was not reached. The antitumor activity of temozolomide was similar whether it was delivered through the airways or intravenously and both routes gave significantly better results than the control group. And three of the mice were long-term survivors. These

IL-2 is a cytokine that induces an antitumor immune response. Anderson et al. evaluated immunotherapy with aerosolised IL-2 using mice with pulmonary metastases of sarcoma (Anderson et al., 1990). The antitumor activity of IL-2 delivered *via* the airways appeared to be better than *via* the other routes tested. Aerosolisation of IL-2 encapsulated in liposomes gave better survival than free IL-2 or liposome alone delivered *via* the same route. Tumour size was reduced by 50% to 85% by aerosolised liposomal IL-2, which was better than the

The efficacy of airways delivery of free IL-2 and liposomal IL-2 were also compared in healthy dogs (Khanna et al. 1996). There were increases in white cells and effector cells in the broncho-alveolar lavage fluid of dogs treated with liposomal IL-2 but no haematological or clinical toxicity. A preclinical study in which dogs were treated with aerosols of liposomal IL-2 gave spectacular results (Khanna et al., 1997). Aerosolised liposomal IL-2 was used to treat 9 dogs, 7 with pulmonary metastases of a primary osteosarcoma and 2 with primary lung carcinomas. The pulmonary nodules in 2 of the 4 dogs with metastatic pulmonary osteosarcomas were in complete regression. No relapse was observed over 12 and 20 months, respectively. The disease of one of the two dogs with a primary lung carcinoma was stabilized for up to 8 months. Aerosolised IL-2 was widely evaluated in clinical trials to treat pulmonary metastases of renal cell carcinoma but there are few published preclinical studies in animals with IL-2 delivered *via* the pulmonary route. Clinical trials were probably proposed based on results obtained with IL-2 administered i.v.

Celecoxib is a non-steroidal anti-inflammatory drug that inhibits cyclooxygenase 2. Treatment with aerosolised celecoxib was assessed in association with intravenous docetaxel in an orthotopic lung tumour model (A549) (Fulzele et al., 2006). Animals were given aerosolised celecoxib plus i.v docetaxel or oral celecoxib plus i.v. docetaxel, each of the drugs alone or a placebo. The aerosol was produced with a pneumatic nebuliser. Aerosolised celecoxib plus i.v. docetaxel reduced lung tumour volume by 61% compared to the placebo group while oral celecoxib plus i.v. docetaxel reduced it by 54%. Moreover, the

combined treatments gave better results than the drugs alone.

results with free IL-2. This suggested that liposomal IL-2 was better than free IL-2.

treatment with this drug for 5 days a week for 8 weeks (Gilbert et al., 2002).

**4.3.6 Temozolomide** 

**4.3.7 Interleukin-2 (IL-2)** 

(Rosenberg et al., 1994)

**4.3.8 Celecoxib** 

encouraging results might lead to clinical trials.

We have recently evaluated the antitumor activity of aerosolised cetuximab in a xenograft model of lung tumours. Aerosolised cetuximab limited the growth of the lung tumours (Maillet et al., in press). This proof-of-concept study demonstrates that the airways can be suitable for delivering aerosolised monoclonal antibodies to treat lung cancer, but further studies are required to determine whether the pulmonary route really does increase the therapeutic benefit of monoclonal antibodies to patients with lung cancer.
