WO2026013116A1 - Lacto-n-fucopentaose i (lnfp-i) for use in lung cancer treatment - Google Patents

Abstract

The invention concerns LNFP-I for the prevention and/or treatment of cancer, preferably lung cancer, more preferably lung adenocarcinoma. The invention further concerns a composition comprising LNFP-I for the prevention and/or treatment of cancer, in particular lung cancer; and a pulmonary delivery device comprising the composition for the use.

Description

LACTO-N-FUCOPENTAOSE I (LNFP-I) FOR USE IN LUNG CANCER TREATMENT

FIELD OF THE INVENTION

The invention is in the field of cancer treatment with dietary substances and concerns the use of Lacto-N-fucopentaose I (LNFP-I) in the treatment of cancer, particularly lung cancer.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of mortality worldwide. Cancer is a disease in which abnormal, damaged, or mutated cells grow uncontrollably in the body. Cancerous cells can spread and invade nearby tissues as well as distant places in the body, a process called metastasis. The growth of cancer impairs the normal functioning of organs, eventually leading to organ failure and death. Carcinomas are the most common type of cancer. They are formed by epithelial cells, which are the cells covering the inside and outside surfaces of the body.

Among malignancies, lung cancer remains one of the leading causes of morbidity and mortality, leading to one quarter of cancer-related deaths worldwide. Non-small-cell lung cancer encompasses a variety of lung cancer types, including adenocarcinoma, squamous cell, and large-cell undifferentiated carcinoma. Non-small cell lung cancer accounts for about 80 per cent of all lung cancers, and about 75 per cent of patients are found in advanced and intermediate stages, with a low five-year survival rate.

Consistent research efforts have been invested worldwide into developing new therapeutic interventions to treat cancer. One of the most used interventions is chemotherapy. Chemotherapy involves the administration of one or more drugs that are cytotoxic to cancer cells, thus destroying them. The main drawback of this approach is that substances that are cytotoxic to cancer cells tend to be cytotoxic to healthy cells as well. This leads to sometimes severe side effects, such as nausea, vomiting, fatigue, hair loss, skin and mouth dryness. Chemotherapy may lead to patients being debilitated and frail. In recent years immunotherapy has shown great efficacy in clinical trials of a broad variety of malignancies, monoclonal antibodies directed against immune checkpoint PD-1/PD-L1 have shown beneficial results in treatment of non-small cell lung cancer patients, yet despite being generally well-tolerated there is a subset of patients wherein still some serious and even life-threatening adverse events related to treatment with immunotherapies occur. Another subset of non-small cell lung cancer is characterized by mutations in the Epidermal Growth Factor Receptor glycoprotein that responds to tyrosinase inhibitors. Nevertheless, while target therapy in NSCLC has provided disease control, the tumors inevitably develop drug resistance.

For these reasons, there is a growing interest in further exploring and investigating less-toxic naturally available substances, that are safe for human consumption, as chemotherapeutic agents.

Moreover, the mode of administration of chemotherapeutic agents plays a role in the development of side effect. Oral or injection administration exposes the whole body to toxicity. Thus, there is also a growing interest in localised drug delivery, targeting the area invaded by cancer. In the context of lung cancer, localised drug delivery may involve pulmonary delivery devices, such as inhalers or nebulizers. Such devices enable inhalation of small particles, solution, or suspensions containing an active substance, which gets directly delivered to the lungs (Al Khatib, A. O.; El-Tanani, M.; Al-Obaidi, H. “Inhaled Medicines for Targeting NonSmall Cell Lung Cancer” Pharmaceutics 2023, 15: 2777).

Against this background, Zhang et al. (Zhang, T.; Chen, Y; Ge, Y; Hu, Y; Lia, M.; Jin Y. “Inhalation treatment of primary lung cancer using liposomal curcumin dry powder inhalers” Acta Pharmaceutica Sinica B 2018; 8(3): 440-448) have developed liposomal curcumin dry powder inhalers for inhalation treatment of primary lung cancer via pulmonary delivery.

Human milk oligosaccharides (HMOs) have previously been investigated as adjuvant of chemotherapeutic agents. HMOs are complex carbohydrates as found in human milk. Numerous HMOs have been identified, made from the five basic monosaccharides glucose (Glc), galactose (Gal), N-Acetylglucosamine (GlcNAc), fucose (Fuc) and sialic acid (SA). Lactose, type 2 polylactosamine or type I Lacto-N-biose backbone structures can be sialylated in a2-3 and/or a2-6 linkages and/or fucosylated in al-2, al-3, and/or al-4 linkages. The structural complexity and abundance of these non-digestible oligosaccharides is unique for human milk, as in milk of other mammalian species the level and diversity of non-digestible oligosaccharides is much lower. HMOS are known to exert various biological functions, e.g., anti-infective (against bacteria, viruses, fungi, and parasites), signaling, anti- inflammatory/immunomodulatory, and prebiotic effects, with different HMOs having different specific effects. HMOs have received increasing attention in recent years due to these therapeutic properties and lack of toxicity (EFSA Panel on Nutrition Turck, D et al “Safety of lacto-N-fucopentaose I/2’-fucosyllactose (LNFP-E2’-FL) mixture as a novel food pursuant to Regulation (EU) 2015/2283” EFSA J. 2023; 21 (12): e8412). WO 2022/140324 teaches a composition comprising HMOs selected from 2’-FL, 3’-SL and 6’-SL in combination with an immune checkpoint inhibitor. The HMOs are used for enhancing the antitumor efficacy of the immune-oncology agent.

WO 2017/103850 teaches a composition comprising a mix of HMOs, such as LNnT, LNT, 3’- SL, 6’-SL, 2’-FL and either DFL or 3-FL, and its use to increase the abundance of Bifidobacterium to increase the efficacy of anti-cancer agents.

Yin et al. (Yin H, Breastfeeding may reduce the effects of maternal smoking on lung cancer mortality in adult offspring: a prospective cohort study. Int J Surg. 2024 Aug l;110(8):4767- 4774) reports epidemiological findings suggesting that early-life breastfeeding reduces adult lung cancer mortality and mitigates the adverse impact of maternal smoking exposure.

WO20 18/215960 Al describes compositions comprising HMOs for reducing harmful proteolytic metabolites in the gastrointestinal tract. The HMOs are taught for use in managing colon cancer and liver damage.

These references advocate for HMOs as adjuvants of chemotherapeutic active agents. However, the efficacy of HMOs as therapeutic agents themselves has never been studied, let alone proven.

Therefore, there is an interest in investigating therapeutic effectiveness of HMOs in the context of cancer treatment.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that Lacto-N-fucopentaose I (LNFP-I) was cytotoxic to epithelial lung cancer cells. This effect was not observed for other oligosaccharides and HMOs, even when the concentration was doubled to 32 mg/ml. This is extremely surprising because HMOs to our knowledge have never before been found cytotoxic against cancer cells. Therefore, it is surprising that LNFP-I was found to be cytotoxic to cancer cells, while none of the other HMOs was. Even isomers of LNFP I which share the same (type I) backbone and just differ in the position of the Fucose at the backbone did not show any effect. In addition, LNFP- I was not cytotoxic to non-cancerous cells, specifically to Human Primary Peripheral Blood Mononuclear Cells (PBMCs). The findings indicate that LNFP-I can be used as therapeutic agent in the prevention and/or treatment of cancer, preferably in the treatment of cancer, in particular carcinoma, more in particular adenocarcinoma, such as adenocarcinoma of the lungs, breast, prostate, colon, rectum, pancreas, or stomach, most preferably adenocarcinoma of the lungs. In a particular embodiment it was found that LNFP-I is useful as therapeutic agent in the prevention and/or treatment of cancer, preferably lung cancer, most preferably non-small cell lung cancer.

Therefore, in a first aspect the invention concerns LNFP-I for use in the prevention and/or treatment of cancer. Worded differently, the invention concerns a method for the prevention and/or treatment of cancer comprising administration of LNFP-I to a subject suffering of cancer. Worded differently, the invention concerns the use of LNFP-I in the manufacture of a medicament for the prevention and/or treatment of cancer.

In a second aspect, the invention concerns a composition comprising LNFP-I for use in the prevention and/or treatment of cancer. Worded differently, the invention concerns a method for the prevention and/or treatment of cancer comprising administration of a composition comprising LNFP-I to a subject suffering of cancer. Worded differently, the invention concerns the use of a composition comprising LNFP-I in the manufacture of a medicament for the prevention and/or treatment of cancer. Preferably the composition comprising LNFP-I is selected from a nutritional composition, an intravenously injectable composition, or a pulmonary deliverable composition. In a preferred embodiment, the composition is a nutritional composition, preferably selected from a supplement or a complete nutritional composition. In yet another preferred embodiment, the composition is a pulmonary deliverable composition, preferably selected from finely ground or micronized particles, a suspension, a solution, or combinations thereof.

In a third aspect, the invention concerns a pulmonary delivery device comprising a pulmonary deliverable composition comprising LNFP-I.

In a fourth aspect, the invention concerns a method for the prevention and/or treatment of cancer comprising administering LNFP-I directly to the lungs of a subject suffering of cancer, by using a pulmonary delivery device comprising a pulmonary deliverable composition comprising LNFP-I.

In all aspects, preferably the cancer is lung cancer, more preferably lung adenocarcinoma, most preferably non-small-cell lung cancer.

LIST OF FIGURES Figure 1 shows the cytotoxicity (%) of different concentrations of oligosaccharides and HMOs on A549 ACE2-TMPRSS2 cells, assessed by CellTiter Gio (CTG) compared to a SLS cytotoxic control, in two biological duplicates (Fig. 1 A and IB).

Figure 2 shows the cytotoxicity (%) of different concentrations of oligosaccharides and HMOs on A549 ACE2-TMPRSS2 cells, assessed by lactate dehydrogenase (LDH) release, in two biological duplicates (Fig. 2A and 2B).

Figure 3 shows the cytotoxicity (%) of different concentrations of oligosaccharides and HMOs on PBMCs, assessed by lactate dehydrogenase (LDH) release in three biological replicates (Fig. 3 A to 3F)

LIST OF PREFERRED EMBODIMENTS

1. Lacto-N-fucopentaose I (LNFP-I) for use in the prevention and/or treatment of cancer, preferably lung cancer, more preferably non-small-cell lung cancer, most preferably lung adenocarcinoma.

2. LNFP-I for use according to embodiment 1, wherein LNFP-I is administered enterally, parenterally, by direct pulmonary delivery, or by combinations thereof, preferably by direct pulmonary delivery.

3. LNFP-I for use according to any one of the preceding embodiments, wherein the use comprises administering at least 3 mg/kg body weight/day, preferably 20 mg/kg/day - 1900 mg /kg body weight/day LNFP-I to a subject suffering from said cancer.

4. LNFP-I for use according to any one of the preceding embodiments, wherein LNFP-I is coadministered with immune-, chemo-, targeted- and/or radiotherapy.

5. LNFP-I for use according to any one of the preceding embodiments, wherein LNFP-I is a therapeutic agent, preferably wherein LNFP-I is not an adjuvant or an excipient.

6. A composition comprising LNFP-I for use in the prevention and/or treatment of cancer, preferably lung cancer, more preferably non-small-cell lung cancer, most preferably lung carcinoma.

7. The composition for use according to embodiment 6, wherein the use comprises administering an effective amount of LNFP-I to a subject suffering from cancer. 8. The composition for use according to embodiment 6 or 7, comprising at least 1 mg/ml LNFP- I, preferably 5-500 mg/ml, more preferably 8-300 mg/ml, even more preferably 8-40 mg/ml LNFP-I.

9. The composition for use according to any one of embodiments 6 to 8, wherein the composition is co-administered with immune-, chemo-, targeted- and/or radiotherapy.

10. The composition for use according to any one of embodiments 6 to 9, wherein LNFP-I is a therapeutic agent, preferably wherein LNFP-I is not an adjuvant or an excipient.

11. The composition for use according to any one of embodiments 6 to 10, wherein the composition is selected from an enteral composition, a parenteral composition, or a pulmonary deliverable (or inhalable) composition, more preferably selected from an oral composition, an intravenously injectable composition, a composition for intravenous infusion, or a pulmonary deliverable (or inhalable) composition.

12. The composition according to any one of embodiments 6 to 11, wherein the composition is a nutritional composition, preferably selected from a supplement or a complete nutritional composition.

13. The composition for use according to any one of embodiments 6 to 11, wherein the composition is a pulmonary deliverable (or inhalable) composition, preferably selected from finely-ground or micronized particles, a suspension, a solution, or combinations thereof.

14. A pulmonary delivery device comprising the pulmonary derivable (or inhalable) composition for use according to embodiment 13.

15. The pulmonary delivery device of claim 14, selected from an inhaler or a nebuliser.

DETAILED DESCRIPTION

Definitions

The term “nutritional composition” as used herein refers to a substance or formulation that satisfies at least a portion of a subject's nutrient requirements. The term “complete nutritional composition” as used herein refers to a nutritional composition which satisfies the daily nutritional requirements of a subject and can be used as main or sole source of nutrition. The term “supplement” as used herein refers to a nutritional composition which does not satisfy the daily nutritional requirements of a subject and is administered on top of the subject’s regular diet, preferably a well-balanced diet.

The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. It includes malignant tumors and benign tumors, metastatic tumors and non- metastatic tumors, particularly solid tumors. Unless stated otherwise, the term "cancer" also encompasses recurrent and non-recurrent cancers as well as cancer relapses, metastatic cancers, and multiple primary cancers.

The term "lung cancer" refers to a class of cancers originating in the tissues of the lung, typically in the cells lining the air passages.

Cancer is characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal lung tissue. It includes malignant tumors and benign tumors, metastatic tumors and non-metastatic tumors, particularly solid tumors. Unless stated otherwise, the term "lung cancer" also encompasses recurrent and non-recurrent lung cancers as well as lung cancer relapses, metastatic lung cancer, and multiple primary cancers with one of the primary cancers being lung cancer. “Metastatic lung cancer” refers to lung cancer that has spread from the lungs to distant organs or tissues, such as the brain, bones, liver, or adrenal glands. “Multiple primary cancers” refer to the presence of two or more distinct primary tumors arising independently in the same individual, which are not the result of metastasis or recurrence. These may occur synchronously (diagnosed at the same time) or metachronously (diagnosed at different times). Lung cancer may occur as one of such multiple primary cancers, either alongside primary tumors in other organs or as multiple primary lung cancers, where two or more distinct primary tumors arise independently within the lungs. The term “subject” as used herein refers to a "patient" that is either an animal, or preferably a human, which is afflicted with cancer as described herein.

The terms "non-malignant", "non-cancerous", or "non-cancer cells" refer to cells or tissues that do not exhibit the characteristics of cancer. These cells do not proliferate uncontrollably, do not invade surrounding tissues, and do not have the capacity to metastasize to distant sites. The terms may refer to normal, healthy cells or to benign cells that, although abnormal or proliferative, remain localized and lack malignant potential. Unless stated otherwise, these terms are used to distinguish such cells from malignant, cancerous, or transformed cells. The term “human milk oligosaccharide(s)” or “HMO(s)” as used herein refers to human milk oligosaccharide(s). HMOs are complex carbohydrates found in human breast milk (Urashima et al.: “Milk Oligosaccharides.” Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015); Thurl, S et al “Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography.” J. Chromatogr. B Biomed. Sci. Appl. 1991; 568 (2): 291-300). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes. HMOs can be composed of up to five different monosaccharides (glucose, galactose, N-acetylglucosamine, sialic acid (N-5-acetylneuraminic acid), and fucose). The diversity of HMOs is complex and based on allowed mammary gland metabolic pathways which result in specific sequences of monosaccharides, branching and decoration of HMO-backbone structures which can be decorated with fucose and/ or sialic acid residues.

HMOs can be divided into neutral HMOs which can either be fucosylated or non-fucosylated, and acidic HMOs that have at least one sialic acid residue in their structure but can also be fucosylated in addition. In the context of the present invention lactose is not regarded as an HMO species.

Examples of neutral non-fucosylated HMOs include lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para- lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).

A "fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. Examples of fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto- N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3 -FL), difucosyllactose (DFL), lacto-N- fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), lacto-N-difucohexaose-II (LNDFH II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para- lacto-N-hexaose l(FpLNH-I), fucosyl-paralacto-N-neohexaose II (F-pLNnH II) and fucosyl- lacto-N-neohexaose (FLNnH).

A "sialylated oligosaccharide" is a charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having a sialic acid residue. It has an acidic nature. Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT). The HMOs as used herein are given their commonly used name that corresponds to the molecular name as provided in Table 1.

Table 1. Names of various HMO molecules The term “therapeutically effective amount” as used herein refers to an amount of HMO that is effective in preventing, reducing, eliminating, treating or controlling the symptoms of the herein-described oncologic diseases and conditions. As used herein, a "therapeutically effective amount" of a substance refers to an amount of said substance that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect. The term "controlling" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the oncologic diseases and conditions described herein, but does not necessarily indicate a total elimination of all disease and condition symptoms.

The terms “mixture (mix) of HMOs”, “HMOs mixture”, or “synthetic HMOs mixture (mix)” as used herein refer to HMO mixtures that are not chemically identical to mixtures naturally occurring in the specific ratios in mammalian milks and that can be obtained by chemical and/or biological processes and/or by blending of mammalian milk-derived purified or isolated HMOs.

HMOs may be isolated by chromatographic or filtration technology from a natural source such as animal milk and several may be produced by biotechnological means known in the art. Several HMOs such as but not limited to 2’-FL, 3-FL, 3 ’ -SI, 6’-SL, LNT, LNnT and DFL are commercially available and can be obtained from commercial sources such as Novonesis, BASF, DSM-Firmenich and others.

The terms “enteral administration” or “administered enterally” as used herein refer to the administration of a substance or composition via the gastrointestinal tract of a subject. Methods of enteral administration include oral, such as eating or tube feeding, sublingual, and rectal. Hence, an “enteral composition” refers to a composition which is suitable for enteral administration. Examples of an enteral composition include oral compositions such as a nutritional composition or a supplement, or a suppository.

The terms “parenteral administration” or “administered parenterally” as used herein refer to administration of a substance or composition by routes other than the digestive tract, typically by via injection or infusion. Examples of injection or infusion routes include intravenous, i.e. directly into a subject’s veins, intramuscular, i.e. into a subject's muscles, subcutaneous, i.e. beneath the skin, or intradermal, i.e. between the subject’s epidermis and hypodermis. Hence, an “injectable composition” refers to a composition suitable for parenteral administration. The term “intravenously injectable composition” as used herein refers to a composition suitable for intravenous injection. The term “composition for intravenous infusion” as used herein refers to a composition suitable for intravenous infusion. Examples of an intravenous injectable composition or a composition for intravenous infusion are saline or isotonic solutions, such as 0.9% sodium chloride solution, further containing one or more active substance(s). The term “(direct) pulmonary drug delivery” as used herein refers to a route of administration of a substance or composition in which a subject inhales the substance or composition, for example by using an inhaler or a nebulizer. Hence, a composition which is “(directly) pulmonary deliverable” is suitable to be inhaled and administered directly to the lungs of a subject via the respiratory system of said subject. The terms “(directly) pulmonary deliverable”, “(directly) deliverable to the lungs”, and “inhalable” are synonyms and used interchangeably throughout the application. Examples of a (directly) pulmonary deliverable composition are finely ground or micronized particles, a suspension, or a solution.

The term “chemotherapeutic agent” as used herein refers to a substance being cytotoxic to cancer cells. A chemotherapeutic agent is an active compound. The term “sole chemotherapeutic agent” as used herein refers to a substance being the only chemotherapeutic agent provided to a subject in need thereof.

The term “adjuvant” as used herein refers to a substance which increases the therapeutic or chemotherapeutic effect of another substance or composition but does not provide a therapeutic or chemotherapeutic effect when administered alone. An adjuvant is not an active principle or compound.

The term “excipient” as used herein refers to a substance which is formulated alongside (chemo)therapeutic agents and/or adjuvants in a pharmaceutical composition. Excipients may be used for example as bulking agents, fillers, diluents, binders, colours, and coatings. An excipient is not an active principle or compound.

The term “carcinoma” as used herein has the common meaning used in the art and refers to a malignancy that develops from epithelial cells. The term “adenocarcinoma” as used herein has the common meaning used in the art and refers to a neoplasia of epithelial tissue that has glandular origin, glandular characteristics, or both.

Herein, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. All percentages are by weight unless otherwise stated. The invention will now be described in further detail. It is noted that the various aspects, features, examples, and embodiments described in the present application may be compatible and/or combined.

LNFP-1

In a first aspect, the invention concerns LNFP-I for use in the prevention and/or treatment of cancer. In different jurisdictions, the first aspect of the invention may be worded as a method for the prevention and/or treatment of cancer comprising administration of LNFP-I to a subject suffering of cancer; or the use of LNFP-I in the manufacture of a medicament for the prevention and/or treatment of cancer. Preferably LNFP-I for use in the prevention and/or treatment of cancer concerns the use of LNFP-I as the sole active compound. LNFP-I as used herein is a therapeutic agent.

Preferably LNFP-I is administered to a subject suffering from cancer in a therapeutically effective amount. Preferably LNFP-I is administered to a subject suffering from cancer in an amount of at least 1 mg/ml, more preferably 5 - 500 mg/ml, even more preferably 8 - 300 mg/ml, most preferably 10-40 mg/ml. Per day, preferably LNFP-I is administered to a subject suffering from cancer in an amount of at least 3 mg/kg body weight/day, more preferably 10 - 2500 mg/kg body weight/day, most preferably 20 - 1900 mg/kg body weight/day.

Preferably LNFP-I is administered enterally, parenterally, by directly pulmonary delivery, or by a combination thereof, more preferably LNFP-I is administered orally, intravenously, by directly pulmonary delivery, or by a combination thereof, most preferably by directly pulmonary delivery, i.e. by inhalation.

In a preferred embodiment, the composition comprising LNFP-I that is administered enterally, more preferably orally, to a subject suffering from cancer has a concentration of at least 1 mg/ml, preferably 5 - 500 mg/ml, more preferably 8 - 300 mg/ml, most preferably 10 -40 mg/ml. Preferably LNFP-I is administered enterally, more preferably orally, to a subject suffering from cancer in an amount of at least 30 mg/kg body weight/day, more preferably 100- 2500 mg/kg body weight/day, most preferably 200 -1900 mg/kg body weight/day.

In another preferred embodiment, the composition comprising LNFP-I that is administered parenterally, more preferably intravenously, to a subject suffering from cancer has a concentration of at least 1 mg/ml, preferably 5 to 500 mg/ml, more preferably 8 to 300 mg/ml, even more preferable 8-40 mg/ml. Preferably LNFP-I is administered parenterally, more preferably intravenously, to a subject suffering from cancer in an amount of at least 3 mg/kg body weight/day, more preferably 10 -250 mg/kg body weight/day, most preferably 20 - 190 mg/kg body weight/day.

In yet another preferred embodiment, the composition comprising LNFP-I that is administered by direct pulmonary delivery, preferably by inhalation, to a subject suffering from cancer, particularly lung cancer, has a concentration of at least 1 mg/ml, preferably 5 - 500 mg/ml, more preferably 8 - 300 mg/ml. Preferably LNFP-I is administered by direct pulmonary delivery, i.e. by inhalation, to a subject suffering from cancer in an amount of at least 3 mg/kg body weight/day, more preferably 10-2500 mg/kg body weight/day, most preferably 30 -1900 mg/kg body weight/day.

In yet another preferred embodiment, LNFP-I is administered by a combination of at least two of enteral administration, parenteral administration, and direct pulmonary delivery, in the amounts reported above. More preferably, LNFP-I is administered by a combination of at least two of oral administration, intravenous administration, and direct pulmonary delivery, in the amounts reported above. Most preferably, LNFP-I is administered by a combination of direct pulmonary delivery and at least one, most preferably both, of oral administration and intravenous administration.

LNFP-I may be co-administered together with other cancer treatments. Preferably LNFP-I is co-administered with immune-therapy, chemotherapy, targeted therapy, radiotherapy, or combination thereof. In a preferred aspect LNFP-I is not co-administered with immune checkpoint inhibitors.

LNFP-I for use according to the invention is a therapeutic agent. LNFP-I for use according to the invention is not an adjuvant or excipient. In one embodiment, LNFP-I is not administered in combination with an immune checkpoint inhibitor.

Preferably LNFP-I is not administered in combination with other HMOs.

LNFP-I is commercially available (CAS No. 7578-25-8), for example from Biosynth (Product Code OL05676) or from Dextra UK (Product Code: L502). LNFP-I can also be synthesized as known in the art, for example as reported by Zhang et al. “Strategies for synthesizing human milk lacto-N-fucopentaoses oligosaccharides” Journal of Agriculture and Food Research 2023; 14: 100724. Composition

In a second aspect, the invention concerns a composition comprising LNFP-I for use in the prevention and/or treatment of cancer. In different jurisdictions, the second aspect of the invention may be worded as a method for the prevention and/or treatment of cancer comprising administration of a composition comprising LNFP-I to a subject suffering of cancer; or the use of a composition comprising LNFP-I in the manufacture of a medicament for the prevention and/or treatment of cancer. LNFP-I in the composition as used herein is a therapeutic agent.

Preferably LNFP-I present in the composition in a therapeutically effective amount. Worded differently preferably the use of the composition or method comprises administering to a subject suffering from cancer LNFP-I in a therapeutically effective amount. Preferably the composition comprises LNFP-I in an amount of at least 1 mg/ml, more preferably 5 - 500 mg/ml, most preferably 8 - 300 mg/ml. Preferably the composition provides an amount of LNFP-I at least 3 mg/kg body weight/day, more preferably 10 - 2500 mg/kg body weight/day, most preferably 20 - 1900 mg/kg body weight/day.

Preferably the composition is an enteral composition, a parenteral composition, or a pulmonary deliverable (or inhalable) composition. More preferably the composition is an oral composition, intravenously injectable composition, or a pulmonary deliverable (or inhalable) composition.

In a preferred embodiment, the composition is a nutritional composition, preferably selected from a supplement or a complete nutritional composition. The nutritional composition preferably comprises LNFP-I in an amount of at least 1 mg/ml, preferably 5 - 500 mg/ml, more preferably 8 - 300 mg/ml. Preferably the nutritional composition provides LNFP-I in an amount of at least 30 mg/kg body weight/day, more preferably 100-2500 mg/kg body weight/day, most preferably 200-1900 mg/kg body weight/day.

Preferably the nutritional composition has an energy content of 50 - 300 kcal, more preferably 100 - 200 kcal per 100 ml. Preferably the nutritional composition comprises one or more of protein, carbohydrates, lipid, minerals, and vitamins.

Preferably the nutritional composition comprises protein, more preferably whey protein. A high protein content ensures the maintenance of adequate nutrition and contributes to prevent and treat cachexia in subjects suffering from cancer. Preferably the nutritional composition comprises protein is in an amount of 5 - 20 g, more preferably 8 - 15 g per 100 ml. Preferably the protein provides 10 - 50%, more preferably 20 - 40% of the total energy of the composition. Preferably the nutritional composition comprises digestible carbohydrates. Preferably the digestible carbohydrates are selected from monosaccharides, more preferably glucose and/or fructose, disaccharides, more preferably lactose, maltose and/or sucrose, polysaccharides, and combinations thereof. Preferably the nutritional composition comprises carbohydrates in an amount of 5 - 30 g, more preferably 10 - 20 g per 100 ml. Preferably the digestible carbohydrates provide 20 - 60%, more preferably 30 - 50% of the total energy of the composition.

Preferably the nutritional composition comprises non-digestible oligosaccharides (fibres). Preferably the nutritional composition comprises non-digestible oligosaccharides in an amount of 0.1 - 5 g, more preferably 0.5 - 3 g per 100 ml.

Preferably the nutritional composition comprises lipid, wherein more preferably the lipid comprises unsaturated fatty acids, even more preferably monosaturated fatty acids, polysaturated fatty acids, or combinations thereof. Most preferably the lipid comprises linoleic acid (LA) and alfa-linoleic acid (ALA). Preferably the nutritional composition comprises lipid in an amount of 1 - 15 g, more preferably 2 - 10 g per 100 ml. Preferably the lipid provides 1- - 50 %, more preferably 20 - 40% of the total energy of the composition.

Preferably the nutritional composition comprises choline. Choline improves cell membrane integrity and promotes the synthesis of vital neurotransmitters. Preferably the nutritional composition comprises 20 - 60 mg, more preferably 30 - 50 mg choline per 100 ml.

Preferably the nutritional composition comprises vitamins, more preferably selected from vitamin A, vitamin D, vitamin E, vitamin K, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6, vitamin B9 (folic acid), vitamin Bl 2, vitamin B7 (biotin), vitamin C, and combinations thereof, most preferably all of vitamin A, vitamin D, vitamin E, vitamin K, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6, vitamin B9 (folic acid), vitamin B12, vitamin B7 (biotin), vitamin C. Vitamin D has several biological activities that slow or prevent the development of cancer, including promoting cellular differentiation, decreasing cancer cell growth, stimulating cell death (apoptosis), reducing tumor blood vessel formation (angiogenesis), and decreasing tumor progression and metastasis. Preferably the nutritional composition comprises vitamin D in an amount of 1 - 15 pg, more preferably 2 - 10 pg per 100 ml. Preferably the nutritional composition comprises minerals, more preferably selected from sodium, potassium, calcium, phosphorous, magnesium, iron, zinc, copper, manganese, fluoride, molybdenum, selenium, chromium, iodine, and combinations thereof.

In another preferred embodiment, the composition is an intravenously injectable composition or a composition for intravenous infusion. The intravenously injectable composition or a composition for intravenous infusion preferably comprises LNFP-I in an amount of at least 1 mg/ml, preferably 5-500 mg/ml, more preferably 8 to 300mg/ml. Preferably the intravenously injectable composition provides LNFP-I in an amount of at least 3 mg mg/kg body weight/day, more preferably 10-250 mg/kg body weight/day, most preferably 20 -190 mg/kg/body weight per day/day.

In yet another preferred embodiment, the composition is a pulmonary deliverable composition, or inhalable composition, more preferably selected from finely ground or micronized particles, a suspension, a solution, or combinations thereof. The pulmonary deliverable, or inhalable, composition preferably comprises LNFP-I in an amount of at least 1 mg/ml, preferably 5 - 500 mg/ml, more preferably 8 to 300 mg/ml, most preferably 8 - 40 mg/ml. Alternatively worded, when the deliverable, or inhalable, composition is in powder form said powder comprises LNFP-I in an amount of at least 1 mg/g, preferably 5 - 500 mg/g, more preferably 8 to 300 mg/g, most preferably 8 - 40 mg/g powder. Preferably the pulmonary derivable, or inhalable, composition provides LNFP-I in an amount of at least 3 mg/kg body weight/day, more preferably 10 to 25 mg/kg body weight/day, most preferably 20-190 mg/kg body weight/day.

A subject suffering from cancer may receive one or more of the compositions of the invention, such as one or more of the nutritional compositions, intravenously injectable composition or composition for intravenous infusion, and pulmonary deliverable composition. Preferably, the use or method according to the invention comprise administering to a subject suffering from cancer at least two of the enteral composition comprising LNFP-I, the parenteral composition comprising LNFP-I, and the pulmonary deliverable composition comprising LNFP-I. More preferably, the use or method according to the invention comprise administering to a subject suffering from cancer at least two of the oral composition comprising LNFP-I, the intravenously injectable composition comprising LNFP-I, the composition for intravenous infusion comprising LNFP-I, and the pulmonary deliverable composition comprising LNFP-I. Even more preferably, the use or method according to the invention comprise administering to a subject suffering from cancer at least two of the nutritional composition comprising LNFP-I, the intravenously injectable composition comprising LNFP-I, the composition for intravenous infusion comprising LNFP-I, and the pulmonary deliverable composition comprising LNFP-I. Most preferably, the use or method according to the invention comprise administering to a subject suffering from cancer the pulmonary deliverable composition comprising LNFP-I and at least one, most preferably two, of the nutritional composition comprising LNFP-I, the intravenously injectable composition comprising LNFP-I, and the composition for intravenous infusion comprising LNFP-I.

The composition of the invention may be co-administered together with other cancer treatments. Preferably the composition is co-administered with immune-therapy, chemotherapy, targeted therapy, radiotherapy, or combination thereof.

In the composition for use according to the invention, LNFP-I is a therapeutic agent. In the composition for use according to the invention, LNFP-I is not an adjuvant or excipient. In one embodiment, LNFP-I is the sole therapeutic agent in the composition.

Preferably the composition comprises further ingredients, more preferably excipients.

Preferably the composition does not comprise HMOs other than LNFP-I.

Pulmonary delivery device

In a third aspect, the invention concerns a pulmonary delivery device comprising the pulmonary deliverable, or inhalable, composition for use according to the invention.

The pulmonary delivery device may be any device known in the art and suitable for direct delivery of the composition of the invention directly to the lungs of a subject suffering from cancer, particularly lung cancer. Preferably the pulmonary delivery device is selected from a pressurised inhaler or a nebuliser.

In a preferred embodiment, the pulmonary delivery device is an inhaler, selected from a pressurised metered-dose inhaler, a breath-actuated pressurised metered-dose inhaler, a dry powder inhaler, or a soft mist inhaler.

In another preferred embodiment, the pulmonary device is a nebuliser selected from a jet nebuliser, an ultrasonic nebuliser, or a mesh nebuliser.

The pulmonary deliverable, or inhalable, composition comprised in the pulmonary delivery device of the invention is a pulmonary deliverable, or inhalable, composition according to the invention. Preferably the pulmonary deliverable, or inhalable, composition comprised in the pulmonary delivery device comprises finely ground or micronized particles of LNFP-I, is a suspension of LNFP-I, is a solution of LNFP-I or is combinations thereof.

The pulmonary delivery device of the invention can be used by a subject suffering from cancer, particularly lung cancer, to inhale a composition comprising LNFP-I.

Thus, in a fourth aspect, the invention concerns a method for the prevention and/or treatment of cancer comprising administering LNFP-I directly to the lungs of a subject suffering of cancer, particularly lung cancer, by using a pulmonary delivery device comprising a pulmonary deliverable composition comprising LNFP-I.

Application

The LNFP-I, composition, or pulmonary delivery device of the invention are for use in the prevention and/or treatment of cancer. Preferably the cancer is a carcinoma, more preferably selected from basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ, invasive ductal carcinoma, or adenocarcinoma, most preferably adenocarcinoma.

Preferably the cancer is adenocarcinoma selected from adenocarcinoma of the lungs, breast, prostate, colon, rectum, pancreas, or stomach, more preferably adenocarcinoma of the lungs.

In a preferred embodiment, the cancer is lung cancer, more preferably non-small-cell lung cancer, most preferably lung carcinoma. Preferably the lung cancer is selected from lung adenocarcinoma, squamous cell carcinoma, or large-cell undifferentiated carcinoma, more preferably lung adenocarcinoma.

EXAMPLES

Example 1 - sample preparation

In this example mixtures of oligosaccharides and/or human milk oligosaccharides (HMOs) were prepared. Solubility and pH were tested.

Methods

The oligosaccharides and HMOs tested are reported in Table 2.

Table 2. Samples of example 1. oligosaccharide.

DNLST was obtained from Creative Biolabs (NY, USA). All other individual HMOS were obtained from Chemily Glycoscience (GA USA). The HMO Mix5 (sample 2) was a mixture of 48.5% 2'-fucosyllactose, 11.6% 3-fucosyllactose, 26.0% lacto-N-tetraose, 4.5% 3’-sialyllactose, and 5.2% 6'-sialyllactose and was obtained from Chr. Hansen HMO GmbH, Rheinbreitbach, Germany.

The HMO Mix5 + scGOS + IcFOS (sample 3) was a mixture of HMO Mix5 + scGOS/lcFOS with an overall ratio of short chain (sc) to long chain (1c) oligosaccharides of 9: 1. scGOS/lcFOS (sample 4) was a mixture at a ratio of 9:1 of short chain GOS derived from Vivinal GOS and long chain FOS derived from RaftilineHP. pAOS was obtained from Suedzucker. The Total HMOs (sample 5) represents all HMOs which can be found in mature human milk in the respective weight ratios as observed in mature human breast milk and ranging in DP between 3 and 28 or higher. This HMO fraction was prepared by depleting the Lactose and mineral content from a total carbohydrate mineral fraction extracted from mature pooled human milk (Chia, L.W.; Mank, M.; et al. “Cross-feeding between Bifidobacterium infantis and Anaerostipes caccae on lactose and human milk oligosaccharides.” Benef Microbes. 2021; 12 (1): 69-83).

Stock solution with a concentration of 32mg/ml were prepared in cell culture medium. The solubility was confirmed by visual observation and the pH was tested by pH paper. The 32 mg/ml stock solutions of samples 1-16 were serially diluted to obtain solutions of 1, 2, 4, 8, 16 mg/ml.

Results

The results are reported in Table 2. All the samples were soluble at 32 mg/ml. The pH of all samples was similar to that of the medium. Hence, no pH adjustments were carried out.

Example 2 - cytotoxicity

The cytotoxicity of the samples of Example 1 to a lung carcinoma cell line was tested.

Methods

The carcinoma cell line used was A549-hACE2-TMPRSS2, which are lung epithelial carcinoma cells expressing human ACE2 and human TMPRSS2.

A549-hACE2/TMPRSS2 cells were seeded in 96 well plates (Costar, 3610) in culture medium (DMEM Glutamax, 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin-streptomycin, 0.5pg/mL Puromycine, 300pg/mL hygromycine B, lOOmM sodium pyruvate) and incubated at 37 °C and 5% CO2 overnight.

Culture medium was removed from A549-hACE2/TMPRSS2 cell plates and replaced with the oligosaccharides/HMO samples of Example 1. Plates were incubated at 37 °C and 5% CO2 for Ih. Culture medium was then removed from plates; all wells were washed once with IX Phosphate-Buffered Saline (PBS) then replenished with 100 pl of fresh medium. Plates were incubated for 48 h at 37 °C, 5% CO2. The oligosaccharide and HMO samples were tested at six concentrations, i.e. 1, 2, 4, 8, 16, and 32 mg/ml, in technical triplicates and biological duplicates. The cytotoxicity of each sample was assessed using a CellTiter-Glo® Luminescent Cell Viability Assay (CTG) according to the manufacturers protocol (Promega, Cat Nb G8461 CellTiter-Glo^R One Solution Assay) and compared to cells exposed to sodium lauryl sulphate (SLS) (Fig. 1) and by assessing lactate dehydrogenase (LDH) release using an LDH release assay according to the manufacturer’s protocol (Thermofi scher scientific, Cat nb V20301. CyQUANT™LDH Cytotoxicity Assay Kit) (Fig. 2). Experiments were repeated with HepG2 and HT29 wherein the cell lines were exposed to increasing concentrations of LNFP-I

Results

The results are shown in Figures 1 and 2. Both tests showed that LNFP-I was cytotoxic to the lung cancer cells at a concentration as low as 16 mg/ml. All the other oligosaccharides and HMOs samples did not show any cytotoxicity, even up to the concentration of 32 mg/ml. In contrast LNFP-I was found to be not cytotoxic to HepG2 or HT29 cells (data not shown).

Example 3- Evaluation of cytotoxicity in non-malignant cells.

The cytotoxicity of oligosaccharides and HMO to non-malignant cells was tested. In particular the cytotoxicity of 2’ -FL, HMO Mix 5, LNDFH-I, LNFP-I, LNFP-II and LNFP-III was tested in Human Primary Peripheral Blood Mononuclear Cells (PBMCs).

Methods

Human PBMCs from three different donors were thawed and rested overnight prior to experimentation. Cells were plated at a density of l / IO per well in 96-well plates (Costar, 3610) containing 100 pl of X-VIVO® 15 medium without serum (X-VIVO® 15, with L- Glutamine, gentamicin, recombinant transferrin, and phenol red, xenofree). 100 pl of HMO solution was added per well. Three concentrations of HMOs were tested of 4 mg/ml, 8 mg/ml, or 16 mg/ml. Incubation was carried out at 37 °C and 5% CO2 for 48 hours.

The cytotoxicity of each sample was measured by assessing lactate dehydrogenase (LDH) release using an LDH release assay according to the manufacturer’s protocol (Thermofi scher scientific, Cat nb V20301. CyQUANT™LDH Cytotoxicity Assay Kit) (Fig. 3). Results The results are shown in Figures 3. LNFP-I was not cytotoxic to PBMCs cells in any concentration up to 16 mg/ml, while another oligosaccharide, LNDFH-I was toxic to PBMCs in a concentration of 16 mg/ml.

Conclusions These results show that the HMO LNFP-I is cytotoxic to lung cancer cells, while it is not toxic to non-malignant, healthy cells. Therefore, it can be used as therapeutic agent to prevent and/or treat lung cancer.

Claims

1. Lacto-N-FucoPentaose I (LNFP-I) for use in the prevention and/or treatment of lung cancer, more preferably lung adenocarcinoma, most preferably non-small-cell lung cancer.

2. LNFP-I for use according to claim 1, wherein LNFP-I is administered enterally, parenterally, by direct pulmonary delivery, or by combinations thereof, preferably by direct pulmonary delivery.

3. LNFP-I for use according to claim 1 or 2, wherein the use comprises administering at least 3 mg/kg body weight/ day, preferably 10-2500 mg/kg body weight/day LNFP-I more preferably 20 - 1900 mg/kg body weight/day to a subject suffering from said cancer.

4. LNFP-I for use according to any one of the preceding claims, wherein LNFP-I is coadministered with immune-, chemo-, targeted- and/or radiotherapy.

5. LNFP-I for use according to any one of the preceding claims, wherein LNFP-I is the therapeutic agent, preferably wherein LNFP-I is not an adjuvant or an excipient.

6. A composition comprising LNFP-I for use in the prevention and/or treatment of lung cancer, more preferably non-small-cell lung cancer, most preferably lung adenocarcinoma.

7. The composition for use according to claim 6, wherein the use comprises administering an effective amount of LNFP-I to a subject suffering from said cancer.

8. The composition for use according to claim 6 or 7, comprising at least 1 mg/ml LNFP-I, preferably 5-500 mg/ml LNFP-I.

9. The composition for use according to any one of claims 6 to 8, wherein the composition is co-administered with immune-, chemo-, targeted- and/or radiotherapy.

10. The composition for use according to any one of claims 6 to 9, wherein LNFP-I is the therapeutic agent, preferably wherein LNFP-I is not an adjuvant or an excipient.

11. The composition for use according to any one of claims 6 to 10, wherein the composition is selected from an enteral composition, a parenteral composition, or a pulmonary deliverable (or inhalable) composition, more preferably selected from an oral composition, an intravenously injectable composition, or a pulmonary deliverable (or inhalable) composition.

12. The composition according to any one of claims 6 to 11, wherein the composition is a nutritional composition, preferably selected from a supplement or a complete nutritional composition.

13. The composition for use according to any one of claims 6 to 11, wherein the composition is a pulmonary deliverable (or inhalable) composition, preferably selected from finely ground or micronized particles, a suspension, a solution, or combinations thereof.

14. A pulmonary delivery device comprising the pulmonary derivable (or inhalable) composition for use according to claim 13.

15. The pulmonary delivery device of claim 14, selected from an inhaler or a nebuliser.