Therapeutic treatment of lung metastases

Abstract

The present invention relates to a therapeutic treatment of lung metastases. In particular, a nebulized BCG composition is administered directly to the lungs to increase uptake therein.

Description

[0001] Therapeutic treatment of lung metastases

[0002] Technical field of the invention

[0003] The present invention relates to a therapeutic treatment of lung metastases. In particular, a nebulized BCG composition is administered directly to the lungs to increase uptake therein.

[0004] Background of the invention

[0005] Pulmonary metastases represent a significant challenge in cancer treatment, profoundly impacting societal health. Metastasis, the spread of cancer cells from the primary tumor to distant organs, is responsible for the majority of cancer-related deaths. Lung metastases, in particular, are associated with poor prognosis and high mortality rates. This condition not only reduces patients' quality of life and life expectancy but also imposes a substantial economic burden on healthcare systems due to the need for complex and prolonged treatments.

[0006] Treating lung metastases is particularly challenging due to several factors. The metastatic tumors often exhibit resistance to conventional therapies, including chemotherapy and radiation therapy, making them difficult to eradicate. Additionally, the presence of multiple metastatic sites complicates surgical interventions. The heterogeneity of cancer cells within metastases further complicates treatment, as different cells may respond differently to the same therapy. These challenges necessitate the development of more effective and targeted treatment strategies.

[0007] Aggressive types of cancer, such as melanoma or sarcomas, are frequently the cause of lung metastases and therefore present a particular concerning group of cancers that are difficult to treat. The metastatic cells typically resemble the cells of the primary tumours, and when cancer spreads to a new location in the body, the metastatic cancer cells retain the characteristics of the original cancer cells. While this may assist a clinician with identifying the origin of the cancer, it also implies that different lung metastases may respond differently to the same treatment.

[0008] One approach that has been explored is the use of Bacillus Calmette-Guerin (BCG) therapy, which has shown promise in treating certain types of cancer, such as bladder cancer. BCG works by stimulating the immune system to attack cancer cells. However, its application in treating lung metastases has been limited by several shortcomings. The effectiveness of BCG in lung cancer, or lung metastases per se, is not well- established, and the treatment can cause significant side effects, including severe

[0009] P96599PC inflammation and infection. Additionally, the response to BCG therapy can be highly variable among patients, making it an unreliable option for widespread use.

[0010] As such, no reliable treatment for lung metastases originating from melanoma or sarcomas is currently available to patients. Accordingly, there is a great need to provide an effective method for treatment of these lung metastases.

[0011] In particular, it would be advantageous to provide a treatment for lung metastases originating from melanoma or sarcomas that efficiently remove, inhibit or ameliorate the lung metastases with minimal systemic exposure and adverse effects.

[0012] Summary of the invention

[0013] The present invention addresses the challenges of treating lung metastases by introducing a novel approach that utilizes nebulized Bacillus Calmette-Guerin (BCG) therapy. The invention has been demonstrated to be efficient in the treatment of lung metastases derived from melanoma or sarcomas. By delivering BCG directly to the lungs in a nebulized form, the treatment stimulates the immune system to target and eliminate cancer cells while ensuring minimal exposure to unaffected tissues. The treatment therefore offers a promising therapeutic strategy against lung metastases originating from melanoma or sarcomas, ultimately improving patient outcomes and reducing the overall burden of cancer on healthcare systems.

[0014] Thus, an object of the present invention relates to a treatment of lung metastases derived from melanoma or sarcomas.

[0015] Thus, an aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a sarcoma.

[0016] Another aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a melanoma.

[0017] P96599PC An embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs.

[0018] Brief description of the figures

[0019] Figure 1 shows the metastatic burden in the lungs of each mouse at experimental endpoint, either displayed as tumour area (A) or as tumour foci count (B). Untreated control mice are shown as white circles and the BCG-Nebulized mice are shown as black circles. (C) Photo of harvested lungs.

[0020] The present invention will in the following be described in more detail.

[0021] Detailed description of the invention

[0022] Definitions

[0023] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0024] Primary solid tumour

[0025] In the present context, the term "primary solid tumour" refers to an abnormal mass of tissue that grows uncontrollably. The primary solid tumour is the original, or first, in the body. The primary solid tumour may spread to other parts of the body and form secondary tumours called metastases. These metastases are the same type of cancer as the primary solid tumour.

[0026] The primary solid tumour may be either benign or malignant. For the treatment disclosed herein, the primary solid tumour is typically malignant as these tumours are more likely to metastasize than benign tumours.

[0027] The primary solid tumour is to be distinguished from liquid tumours that circulate around the body through the bloodstream.

[0028] Lung metastases

[0029] In the present context, the term "lung metastases" refers to secondary tumours located in the lungs which have spread from a primary solid tumour. Typically, lung metastases are located in the lung parenchyma or pleura.

[0030] The terms "lung metastases" and "pulmonary metastases" may be used interchangeably.

[0031] P96599PC Sarcoma

[0032] In the present context, the term "sarcoma" refers to a malignant tumour of transformed cells of mesenchymal origin. The affected mesenchymal cells may be present in different types of connective tissue, such as bone, cartilage, fat, or vascular tissue.

[0033] Sarcomas are primary solid tumours that may give rise to metastases elsewhere in the body, such as the lungs.

[0034] Melanoma

[0035] In the present context, the term "melanoma" refers to a cancer which develops in melanin-producing cells called melanocytes. Melanocytes are a type of mesenchymal cells which are derived from neural crest cells. Accordingly, melanoma is to be considered a sarcoma.

[0036] Carcinoma

[0037] In the present context, the term "carcinoma" refers to a malignant tumour of transformed cells of epithelial origin. Carcinomas forms in a tissue that lines the inner or outer surfaces of the body and include, but is not limited to, breast cancer, prostate cancer, lung cancer and colorectal cancer.

[0038] Carcinomas may be categorised according to their histological types as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma.

[0039] In the context of the present treatment, carcinomas are considered primary solid tumours that may give rise to metastases elsewhere in the body, such as the lungs.

[0040] Bacillus Calmette-Guerin (BCG) composition

[0041] In the present context, the term "BCG composition" refers to any composition comprising live attenuated Mycobacterium bovis BCG as active ingredient. The BCG composition may comprise one or more pharmaceutically acceptable carriers, excipients and / or diluents so that it is suitable for administration to a subject, such as a human patient. Administration of the BCG composition can be done in nebulized form.

[0042] The BCG composition may be a commercially available BCG vaccine.

[0043] Tumour burden

[0044] P96599PC In the present context, the term "tumour burden" refers to the total amount of cancer present in a subject's body. The total amount of cancer may be represented as the volume of tumours and metastases.

[0045] Tumour burden may be measured using imaging techniques such as CT, MRI or PET scanning.

[0046] The terms "tumour burden" and "tumour load" may be used interchangeably.

[0047] Pharmaceutically acceptable

[0048] In the present context, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are suitable for use with human patients without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0049] Nebulization and nebulized

[0050] In the present context, the term "nebulization" refers to the process of converting a liquid composition to a fine spray, aerosols or mist. Thus, a nebulized composition is in the form of a fine spray, aerosols or mist.

[0051] Nebulization may be achieved using a nebulizer, such as a jet nebulizer, ultrasonic nebulizer, or mesh nebulizer.

[0052] Mechanical ventilation

[0053] In the present context, the term "mechanical ventilation" refers to the use of a ventilator to fully or partially provide artificial ventilation.

[0054] The terms "mechanical ventilation", "assisted ventilation" and "intermittent mandatory ventilation" may be used interchangeably.

[0055] About

[0056] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, concentrations, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about

[0057] P96599PC 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0058] Treatment of lung metastases

[0059] Lung metastases present a significant clinical challenge, particularly when originating from aggressive cancers such as sarcoma or melanoma. These metastases are often resistant to conventional therapies, including chemotherapy and radiation therapy, due to the complex microenvironment of the lungs. Sarcomas and melanomas are known for their high metastatic potential and ability to evade the immune system, making them particularly difficult to treat. Consequently, there is a critical need for innovative treatments that can effectively target and eradicate lung metastases from these malignancies.

[0060] Sarcomas are a rare group of cancers that originate in the connective tissues of the body, such as bones, muscles, fat, blood vessels, nerves, and tendons. Despite their diversity, sarcomas share several key characteristics. They can develop in almost any part of the body and often present as a painless lump or swelling. All types of sarcomas arise from mesenchymal cells, which are the cells that form connective tissues. This common origin means that, regardless of the specific type, sarcomas tend to exhibit similar patterns of growth and spread, often invading nearby tissues and potentially metastasizing to distant parts of the body.

[0061] Treating sarcomas is particularly challenging due to several factors. Firstly, their rarity means that there is less clinical experience and fewer research studies compared to more common cancers. Sarcomas often grow near critical structures like nerves and blood vessels, making surgical removal difficult. Furthermore, they are frequently diagnosed at a later stage, reducing the effectiveness of treatments. These complexities necessitate a highly specialized and individualized approach to treatment, often involving a combination of surgery, radiation, and chemotherapy.

[0062] Herein it is found that administration of a BCG composition can reduce the tumour burden of lung metastases originating from sarcomas. This is surprising and encouraging because the mechanism of action of BCG in the treatment of cancer is broad and not fully elucidated. In particular, while BCG has been trialed as a cancer treatment against a broad range of cancer types, its success has been very limited. The most notable examples are the use of BCG in the treatment of non-muscle invasive bladder cancer (NMIBC).

[0063] P96599PC The inventors of the present invention have devised a simple method for efficiently treating lung metastases derived from melanoma or sarcoma by delivering a BCG composition to a subject. In particular, it was surprisingly found that administration of the BCG composition directly to the lungs of the subject caused a substantial reduction of metastatic burden and foci.

[0064] In the present context treatment may be construed as the elimination or reduction of already established lung metastases, i.e. with the goal of curing the disease. Amelioration is covering slowing down disease progression, e.g. delaying how fast disease is spreading and new symptoms manifest themselves. Inhibition can be construed as the stopping of disease progression.

[0065] Thus, an aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a sarcoma.

[0066] An embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

[0067] Melanoma is a type of cancer that originates in the melanocytes, the cells responsible for producing melanin, the pigment that gives skin its colour. Melanomas often present as unusual moles or skin lesions that change in size, shape, or colour. Diagnosing melanoma typically involves several steps to ensure accurate detection and staging. Initially, a healthcare professional will conduct a thorough physical examination, focusing on any suspicious moles or skin lesions. They will look for signs such as asymmetry, irregular borders, multiple colours, large diameter, and any evolving changes in the mole, often referred to as the ABCDEs of melanoma.

[0068] If a suspicious lesion is found, a biopsy is performed to remove a sample of tissue for laboratory analysis. The biopsy helps determine whether the cells are cancerous and, if so, the thickness and depth of the melanoma, which are crucial for staging the

[0069] P96599PC cancer. Once melanoma is confirmed, additional tests may be conducted to determine if the cancer has spread to other parts of the body. This can include imaging tests such as X-rays, CT scans, MRI, and PET scans.

[0070] If lung metastases have formed, conventional treatment options include chemotherapy, immunotherapy and radiation therapy. All of these conventional treatments are unfortunately liable to severe adverse effects caused by the damaging of healthy cells as well as cancer cells and autoimmune reactions. Accordingly, patients experience hair loss, mouth sores, loss of appetite, weight loss, nausea, vomiting, diarrhea, constipation, fatigue and inflammation of lungs.

[0071] Herein it is demonstrated that tumour burden of lung metastases derived from melanoma can be significantly reduced by administration of a nebulised BCG composition. This treatment is not only effective but also gentle to the patient with minimal adverse effects compared to conventional treatments.

[0072] Accordingly, an aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a melanoma.

[0073] The nebulized BCG composition may advantageously be administered to the lungs. This may be accomplished by inhalation, e.g. through mouth and / or nose via a nebulizer. Some nebulizers are designed to deliver medication through both the nose and mouth, which can be beneficial for treating conditions affecting the upper respiratory tract. This administration can deliver the BCG composition to the lungs, but herein it is preferred to administer the BCG composition specifically through the mouth to ensure that the medication reaches the lungs and are not upheld in the mucous membranes of the nasal cavity. Administration exclusively through the mouth may be facilitated by inhalation of the nebulized BCG composition through a mouthpiece or mask.

[0074] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs.

[0075] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation.

[0076] P96599PC A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation through the mouth and / or nose.

[0077] A still further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation through the mouth.

[0078] To further guide the BCG composition directly to the target site in the lungs, the administration may entirely circumvent the upper respiratory tract and be performed by intubation. By delivery of the BCG composition directly to the lungs via intubation, the mucosa of the upper airways (nose, throat, etc.) are bypassed, ensuring that none of the active ingredient is retained in the mucosa of the nasal and oral cavity. Intubation of the patient may be executed via any conventional means.

[0079] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs via a tracheal tube.

[0080] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the tracheal tube is an endotracheal tube.

[0081] Yet another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the route of administration is orotracheal or nasotracheal, preferably orotracheal.

[0082] As a further means for distributing the BCG composition directly to the lungs, the administration may be performed while the patient is under conditions of mechanical ventilation. Mechanical ventilation refers to the use of a ventilator to fully or partially provide artificial ventilation. Non-invasive mechanical ventilation may be accomplished for patients that are conscious and can use a face or nasal mask. Invasive mechanical ventilation relates to the situation wherein placement of an instrument to create an airway that is placed inside the trachea, such as an endotracheal tube or nasotracheal tube, is utilised.

[0083] Administering aerosolized medications to a patient under mechanical ventilation may enhance distribution to the lungs, i.e. to achieve higher concentration at the target

[0084] P96599PC site. Furthermore, mechanical ventilation can improve patient comfort in that it allows co-administration of sedatives and analgesics to keep patients that are otherwise uncomfortable with the situation calm and relaxed.

[0085] The two main types of mechanical ventilations are positive pressure ventilation in which air is pushed into the lungs through the airways, and negative pressure ventilation in which air is pulled into the lungs. It is contemplated herein that positive pressure ventilation is particularly advantageous for delivery of the BCG composition to pulmonary metastases.

[0086] Positive pressure ventilation may be enacted by increasing the patient's airway pressure through an endotracheal tube. The positive pressure allows air to flow into the airway until the ventilator stream is stopped. Then, the airway pressure drops to zero, and the elastic recoil of the chest wall and lungs push the breath out through passive exhalation.

[0087] Without being bound by theory, it is contemplated herein that positive pressure ventilation is favourable because the distal areas of the lungs are better inflated and therefore the BCG composition is more completely distributed in the lungs.

[0088] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered under conditions of mechanical ventilation of the subject.

[0089] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered under conditions of positive pressure ventilation.

[0090] The active ingredients intended to assert their effect at a target site in the lungs of patients are delivered in the form of a nebulized composition, such as an aerosol. The nebulized composition may be prepared by any conventional means.

[0091] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is nebulized by pneumatic, mechanical or electrical means.

[0092] The BCG composition described herein is best administered as soon as lung metastases have been identified to maximize its therapeutic effect. Thus, once metastases are

[0093] P96599PC detected, it is diligent to administer the BCG composition promptly. In some situations, it may even be preferred to administer the BCG composition as soon as a primary solid tumour is identified. This is particularly the case when the primary tumour is a sarcoma or melanoma, both of which are known to aggressively metastasize, and there is a fair expectation that lung metastases have already developed.

[0094] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered after diagnosis of a primary solid tumour.

[0095] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered after diagnosis of a lung metastasis.

[0096] Identifying lung metastases typically involves one or more imaging techniques, including, but not limited to, X-ray, CT scanning, MRI and PET scanning. Chest X- rays are often the first step, providing a basic view of the lungs to detect any obvious abnormalities. Upon identification of at least one lung metastasis, a formal diagnosis of lung metastasis may be made. However, small metastases can be missed on X-rays, so more detailed imaging is usually required. Computed Tomography (CT) scanning is more precise and can reveal the size, shape, and position of lung metastases. This method involves taking multiple X-ray images from different angles and combining them to create a detailed cross-sectional view of the lungs. Positron Emission Tomography (PET) scanning is also commonly used and involves injecting a small amount of radioactive sugar into the bloodstream. Cancer cells, which consume more sugar than normal cells, will appear as bright spots on the scan. PET scanning is often combined with CT scanning (PET / CT) to provide both metabolic and anatomical information.

[0097] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein identification of lung metastases is performed by one or more techniques selected from the group consisting of X-ray, CT scanning, MRI, PET scanning, and intraoperative visualization.

[0098] Cancer diseases are notoriously known to be difficult to prognosticate, and the extent of treatment regimens varies from patient to patient. Pulmonary metastases are no different, the required amount of treatment is difficult to predict. However, the vast majority of patients require a repeated administration of effective doses to obtain a

[0099] P96599PC positive clinical outcome. The BCG composition herein may be administered repeatedly, if necessary, without any severe adverse effects.

[0100] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered as a single effective dose or as multiple effective doses.

[0101] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the number of effective doses of the BCG composition administered is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 10, such as at least 15, such as at least 20, such as at least 30, such as at least 40, such as at least 50.

[0102] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered daily, every other day, three times a week, weekly, bi-weekly or monthly.

[0103] Without being bound by theory, it is contemplated that the therapeutic effect of the BCG composition against lung metastases may be caused by its ability to act as an immune stimulating component that efficiently drive maturation of immune cells, such as dendritic cells and macrophages, and mobilise them at the target site to fight cancerous tissue. Importantly, the BCG composition may induce a downstream production of pro-inflammatory cytokines that stimulate a tumour-targeted cytotoxic T-cell and NK cell responses, which results in the elimination of the cancer cells. Overall, the administration of BCG in nebulized form may remove the immunosuppressive environment created by the cancer tissue and induce a pro- inflammatory state that leads to elimination of the lung metastases.

[0104] The BCG composition may comprise any Mycobacterium bovis strain that induces maturation of the immune cells and facilitates a strengthened immune response against the lung metastases, i.e. the present BCG composition is not limited to one particular bacterial strain.

[0105] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

[0106] P96599PC Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein BCG composition comprises a bacterial strain or substrain selected from the group consisting of Pasteur 1173 P2, Danish 1331, Glaxo 1077, Tokyo 172-1, Russian BCG-I, and Moreau RDJ strains.

[0107] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331.

[0108] The BCG composition may function with conventional pharmaceutically acceptable carriers, excipients and diluents and is not limited to any particular one. However, some conventional carriers, excipients and diluents are known to specifically work well with BCG, including, but not limited to, those comprised in BCG vaccines. Thus, the BCG composition may comprise or consist of a commercially available BCG vaccine. Such vaccines are known to be safe and without serious adverse effects.

[0109] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition comprises one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0110] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the one or more pharmaceutically acceptable carriers, excipients and / or diluents are selected from the group consisting of sodium glutamate, magnesium sulphate heptahydrate, dipotassium phosphate, citric acid monohydrate, L-asparagine monohydrate, ferric ammonium citrate, and glycerol.

[0111] Other carrier solutions include, but are not limited to, buffered saline solutions, such as Hank's balanced salt solution, Tris buffered saline, HEPES buffered solutions, and others.

[0112] A preferred embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is a BCG vaccine.

[0113] Herein it is demonstrated that administration of nebulized BCG reduces the tumour burden of subject with lung metastases significantly. Tumour burden refers to the total amount of cancer present in a patient's body. This can be measured in several ways, including the number of cancer cells, the size of a tumour, or the overall volume of

[0114] P96599PC cancerous tissue. It is an important factor in determining the stage of cancer, assessing the effectiveness of treatment, and predicting patient outcomes.

[0115] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein administration of the BCG composition causes reduction of tumour burden.

[0116] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein administration of the BCG composition causes reduction of tumour burden stemming from lung metastases.

[0117] As for many other cancer treatments, the present administration of the BCG composition may be combined with administration of another immunotherapeutic composition. The immunotherapeutic composition may be an already commercially available composition or product. The combination treatment may be administered concomitantly or separated in time.

[0118] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, further comprising administration of an immunotherapeutic composition.

[0119] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the immunotherapeutic composition is administered before, simultaneously, or after administration of the BCG composition.

[0120] Alternatively, the BCG composition is combined with treatment directed at the primary tumour. Such treatments may be any conventional treatment, including surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, stem cell treatment. The BCG composition may be combined with personalized treatments, such as advanced therapy medicinal products (ATMPs), hereunder gene therapy, cell therapy, and tissue engineering.

[0121] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein said BCG composition is administered as part of a combination therapy that comprises administering a treatment targeting the primary tumour.

[0122] P96599PC Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein said treatment targeting the primary tumour is selected from the group consisting of surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, gene therapy, cell therapy, such as CAR-T cell therapy or stem cell therapy, or combinations thereof.

[0123] An aspect of the present invention relates to a method of treatment, inhibition, or amelioration of lung metastases in a subject, the method comprising administration of a BCG composition in nebulized form.

[0124] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0125] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the medical use of the BCG composition, which embodiments may readily be part of the method of treatment described herein. Embodiments and features of the present invention are also outlined in the following items.

[0126] Items

[0127] XI. A Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a sarcoma.

[0128] X2. The BCG composition for use according to item XI, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

[0129] X3. The BCG composition for use according to any one of items XI or X2, wherein the primary solid tumour is a melanoma.

[0130] P96599PC X4. A Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a melanoma.

[0131] X5. The BCG composition for use according to any one of items X1-X4, wherein the use is for treatment of lung metastases in a subject.

[0132] X6. The BCG composition for use according to any one of items X1-X4, wherein the use is for amelioration of lung metastases in a subject.

[0133] X7. The BCG composition for use according to any one of items X1-X4, wherein the use is for inhibition of lung metastases in a subject.

[0134] X8. The BCG composition for use according to any one of the preceding items, wherein the subject is a human.

[0135] X9. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered to the lungs.

[0136] X10. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation.

[0137] XI 1. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation through the mouth and / or nose.

[0138] X12. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation through the mouth.

[0139] X13. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered to the lungs via a tracheal tube.

[0140] X14. The BCG composition for use according to item X13, wherein the tracheal tube is an endotracheal tube.

[0141] P96599PC X15. The BCG composition for use according to any one of the preceding items, wherein the route of administration is orotracheal or nasotracheal, preferably orotracheal.

[0142] X16. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered under conditions of mechanical ventilation of the subject.

[0143] X17. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered under conditions of positive pressure ventilation.

[0144] X18. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is nebulized by pneumatic, mechanical or electrical means.

[0145] X19. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered after diagnosis of a primary solid tumour.

[0146] X20. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered after diagnosis of a lung metastasis.

[0147] X21. The BCG composition for use according to any one of the preceding items, wherein identification of lung metastases is performed by one or more techniques selected from the group consisting of X-ray, CT scanning, MRI, PET scanning, and intraoperative visualization.

[0148] X22. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered as a single effective dose or as multiple effective doses.

[0149] X23. The BCG composition for use according to any one of the preceding items, wherein the number of effective doses of the BCG composition administered is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 10, such as at least 15, such as at least 20, such as at least 30, such as at least 40, such as at least 50.

[0150] X24. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered daily, every other day, three times a week, weekly, bi-weekly or monthly.

[0151] P96599PC X25. The BCG composition for use according to any one of the preceding items, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

[0152] X26. The BCG composition for use according to any one of the preceding items, wherein BCG composition comprises a bacterial strain or sub-strain selected from the group consisting of Pasteur 1173 P2, Danish 1331, Glaxo 1077, Tokyo 172-1, Russian BCG- I, and Moreau RDJ strains.

[0153] X27. The BCG composition for use according to any one of items X25 or X26, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331.

[0154] X28. The BCG composition for use according to any one of the preceding items, wherein the BCG composition comprises one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0155] X29. The BCG composition for use according to item X28, wherein the one or more pharmaceutically acceptable carriers, excipients and / or diluents are selected from the group consisting of sodium glutamate, magnesium sulphate heptahydrate, dipotassium phosphate, citric acid monohydrate, L-asparagine monohydrate, ferric ammonium citrate, and glycerol.

[0156] X30. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is a BCG vaccine.

[0157] X31. The BCG composition for use according to any one of the preceding items, further comprising administration of an immunotherapeutic composition.

[0158] X32. The BCG composition for use according to item X31, wherein the immunotherapeutic composition is administered before, simultaneously, or after administration of the BCG composition.

[0159] X33. The BCG composition for use according to any one of the proceeding items, wherein administration of the BCG composition causes reduction of tumour burden.

[0160] X34. The BCG composition for use according to any one of the proceeding items, wherein administration of the BCG composition causes reduction of tumour burden stemming from lung metastases.

[0161] P96599PC X35. The BCG composition for use according to any one of items X19-X34, wherein said BCG composition is administered as part of a combination therapy that comprises administering a treatment targeting the primary tumour.

[0162] X36. The BCG composition for use according to item X35, wherein said treatment targeting the primary tumour is selected from the group consisting of surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, gene therapy, cell therapy, such as CAR-T cell therapy or stem cell therapy, or combinations thereof.

[0163] Yl. A method of treatment, inhibition, or amelioration of lung metastases in a subject, the method comprising administration of a BCG composition in nebulized form.

[0164] Examples

[0165] Example 1: Induction of lung metastasis in a melanoma mouse model and treatment scheme

[0166] This example describes establishment of a tumour model for determining the effect of therapeutic treatment of lung metastases. Tumour cells were injected in a mouse recipient to simulate a primary tumour metastasizing. After allowing time for lung metastases to establish, treatment was initiated to evaluate the therapeutic effect.

[0167] Method

[0168] Tumour cells

[0169] B16-F10 Iuc2 (CRL-6475 LUC2) cells were cultured in DMEM +10% FBS (foetal bovine serum, heat inactivated), Penicillin-streptomycin and Blasticidin (10 ug / mL). For tumour cell inoculation, the cells were harvested, washed in DPBS and counted. The cells were resuspended in DPBS to a final concentration of 5xl06cells / mL. Tumour cells were transported to the animal facility on ice.

[0170] Tumour cell inoculation

[0171] Mice were gently warmed under a heat lamp for 5-10 minutes to make the tail vein dilated. The tumour cells were carefully resuspended by using a ImL syringe (without needle). The mice were inoculated with 100 pL tumour cell suspension (2xl05cells / mouse) by intravenous injection in the lateral tail vein using a 27g needle. Control mice did not receive any injection.

[0172] Anaesthesia

[0173] P96599PC Mice in the treatment group were anesthetized with ZRF cocktail (3.3 mg zolazepam, 3.3 mg tiletamine, 0.5 mg xylazine and 2.6 pg fentanyl per 1 mL 0.9% NaCI). ZRF was administered by intraperitoneal (i.p.) injection (max 100 pL / lOg). The mice were kept on a heating mat until awake.

[0174] Preparation of BCG for administration

[0175] BCG was resuspended in DPBS so that one vial of BCG gives 10 doses (one vial Mycobacterium Bovis BCG has 2-8xl06CFU attenuated BCG (Bacillus Calmette-Guerin, Danish strain 1331). One dose BCG has 2-8xl05CFU BCG. For the nebulization of BCG, one vial was resuspended in 2000 pL to give 10 doses of 200 pL each.

[0176] Administration of BCG by nebulization

[0177] Mice were under anaesthesia during the whole procedure. Endotracheal intubation was performed before BCG administration. The intubated mice were connected to the ventilator with settings: 120 bpm priority press, 15 cm H2O pressure, 0,145 mL tidal volume, 17 mL / min minute volume (SomnoSuite® Low-Flow Anaesthesia System with the RoVent® Automatic Ventilator Module, Kent scientific). The BCG dose (200 pL) was added to the Nebulizer (Aeroneb Lung Instillation Nebulizer Delivery System) and the mice received the nebulized BCG directly into the lungs via the endotracheal tube. The mice were disconnected from the ventilator system and the endotracheal tube removed when the nebulization chamber was empty (indication that the whole dose was administered).

[0178] Untreated controls

[0179] Untreated controls received no treatment.

[0180] Treatment regimen

[0181] All mice were intravenously injected with tumour cells on Day 0. The mice received a total of two treatments (of 2-8xl05CFU). The BCG was administered to the lung via intubation / nebulization at Day 14 and Day 18 post tumour cell inoculation. The mice were sacrificed on Day 22 post tumour cell inoculation and lung tissue was harvested for further examination.

[0182] Example 2: Quantitative assessment of lung metastases by macroscopic analysis.

[0183] This example describes the analysis of data acquired from the experiments of example 1.

[0184] P96599PC Method

[0185] Euthanasia, lung harvest and photography of the lung tissue

[0186] Mice were euthanised by cervical dislocation. The lungs were then removed from the mice, rinsed in DPBS and placed on a white plastic board for photography of the lungs. The lungs were carefully spread out on the board to get the largest surface area. A paper note below each lung was used to indicate the mouse number. One side of the lung was photographed.

[0187] Measurement of tumour area in the lungs

[0188] The tumour area in the lung was measured by ImageJ software (area in % of lung surface). A threshold was applied to the images to select areas of dark pigmentation (tumour tissues). The images were then converted to black-and-white images (8-bit). The selected area corresponding to tumour tissues was then quantified using the area measurement tool (ImageJ) and reported as a percentage of the total lung area.

[0189] Counting of lung metastases (metastatic foci)

[0190] The number of lung metastases (metastatic foci) was counted using ImageJ software. The number of foci was counted manually using the point tool to ensure all foci were counted and to avoid duplicated counts.

[0191] Results

[0192] Mice tolerated the handling well and the model consistently resulted in mice developing lung metastases (figure 1C, control). The presence of lung metastases was quantified as the metastatic burden (figure 1A) and count of metastatic foci (figure IB). Upon therapeutic treatment with BCG nebulization there was clear reduction in prevalence and size of lung metastases compared to untreated mice.

[0193] Conclusion

[0194] The Example demonstrates the advantage of utilizing BCG nebulization as a therapeutic treatment of lung metastases from a sarcoma, here exemplified by melanoma.

[0195] P96599PC

Claims

22Claims1. A Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, or amelioration of lung metastases in a subject, wherein the BCG composition is administered in nebulized form, and wherein the primary solid tumour from which the lung metastases are derived is a sarcoma.

2. The BCG composition for use according to claim 1, wherein the subject is a human.

3. The BCG composition for use according to any one of claims 1 or 2, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

4. The BCG composition for use according to any one of the preceding claims, wherein the primary solid tumour is a melanoma.

5. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered to the lungs.

6. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered via inhalation.

7. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered via inhalation through the mouth.

8. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered to the lungs via a tracheal tube.

9. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered after diagnosis of a primary solid tumour.

10. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered after diagnosis of a lung metastasis.P96599PC11. The BCG composition for use according to any one of the preceding claims, wherein identification of lung metastases is performed by one or more techniques selected from the group consisting of X-ray, CT scanning, MRI, PET scanning, and intraoperative visualization.

12. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered as a single effective dose or as multiple effective doses.

13. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

14. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is a BCG vaccine.

15. The BCG composition for use according to any one of the proceeding items, wherein administration of the BCG composition causes reduction of tumour burden.P96599PC

Images