The present invention relates to a pharmaceutically compatible iron chelator or prodrug thereof for use in treating and/or preventing a disease caused or aggravated by STAT3 hyperactivity in effector cells in a subject; and to methods, kits, devices, uses, and combined preparations related thereto.

A common dogma of cancer physiology is that cancer cells exhibit higher intracellular iron levels than normal cells, which is believed to facilitate both initiation and growth of a tumor (Torti and Torti (2013), Nat Rev Cancer 13(5):342). On the one hand, via the Fenton reaction, intracellular iron induces the production of reactive oxygen species and therefore increased iron levels can facilitate the initiation of a tumor (Wans & Ahsan (2006), Journal of Carcinogenesis 5:14). On the other hand, cancer cells strongly depend on the activity of the iron dependent enzyme ribonucleotide reductase, which is necessary for DNA synthesis and thus proliferation (Sanvisens et al. (2013), Biomed J 36(2):51-8). Also, other iron-dependent enzymes have been implicated in the cancer-promoting effect of iron, e.g. Deoxyhypusine Hydroxylase (Clement et al. (2002), Int J. Cancer, 100:491), or Wnt-Signaling (Song et al. (2011), Cancer Res. 71:7628).

Ciclopirox (CPX) is clinically used as a topical antifungal agent to treat mycoses of the skin and nails. Although its exact mechanism of action is unclear, it is known to chelate intracellular iron (Shen & Huang (2016), Curr Pharm Des 22:4443) and anti-tumor properties have been reported (Zhou et al .(2010), Journal international du cancer 127(10):2467; Eberhard et al. (2009), Blood 114(14):3064). Recently, it was found that ciclopirox can induce senescence and apoptosis in human papillomavirus-positive cancer cells (Braun et al. (2019), Int J Cancer, doi: 10.1002/ijc.32709).

The STAT3 signaling cascade is considered pro-tumorigenic under most conditions and is hyperactivated in over 70% of all cancers. Many efforts have been undertaken to therapeutically block STAT3 for clinical cancer therapy, however, with only limited success. STAT3 is considered a therapeutic target also for cancers which are often accessible from the outside, such as squamous cell carcinomas of the skin or malignant melanoma. Moreover, STAT3 is also considered a therapeutic target for common non-malignant diseases, such as autoimmune disorders, asthma, cachexia, and fibrotic diseases. Moreover, STAT3 was reported to be a target for cancer prevention (Peyser et al (2016), Cancer Prev Res 9(8):657).

Thus, the development of novel strategies to interfere with STAT3 for tumor and immunological therapy is urgently required. It is therefore an objective of the present invention to provide means and methods to comply with the aforementioned needs, avoiding at least in part the disadvantages of the prior art. This problem is solved by compounds and the methods, kits and uses of the present invention. Embodiments, which might be realized in an isolated fashion or in any arbitrary combination, are listed in the dependent claims.

In accordance, the present invention relates to a pharmaceutically compatible iron chelator or prodrug thereof for use in treating and/or preventing a disease caused or aggravated by STAT3 hyperactivity in effector cells in a subject.

As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e. are equivalent to "comprising at least one".

Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

As used herein, the term "standard conditions", if not otherwise noted, relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. preferably, a temperature of 25°C and an absolute pressure of 100 kPa; also preferably, standard conditions include a pH of 7. Moreover, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1%, most preferably less than 0.1% by weight of non-specified component(s).

As used herein, the term "iron chelator" relates to a chemical compound forming a stable complex with iron ions, preferably Fe ²⁺ and/or Fe ³⁺ . Preferably, the iron chelator is a compound having a log stability constant for at least one of its iron complexes of at least 3, more preferably at least 5, more preferably 10, still more preferably at least 20, even more preferably at least 25, most preferably at least 30. Preferably, the log stability constant is the log of the equilibrium constant for the formation of the Fe/iron chelator complex in aqueous solution, preferably determined under standard conditions, preferably as specified elsewhere herein; preferably, the log stability constant of an iron chelator is determined in a solution consisting of water, iron ions, and iron chelator. Iron chelators are, in principle, known in the art and include compounds comprising at least one of a 2-pyridone structure, a hydroxamate structure, a (thio)semicarbazone structure, a bis(2-hydroxyphenyl)-lH-l,2,4-triazol structure, an alpha-hydroxyketone structure, an arylhydrazone structure, and a catechol structure.

The term "pharmaceutically compatible", as used herein, relates to a chemical compound which is pharmaceutically acceptable in the sense of being not deleterious to the recipient thereof and, preferably, being compatible with optional other ingredients of a formulation thereof. Preferably, a pharmaceutically compatible compound is a compound causing at most moderate adverse drug reactions, preferably causing at most mild adverse drug reactions. As used herein, the term "mild" adverse reactions relates to adverse reactions not requiring medical intervention, such as skin rashes, headaches, digestive disturbances, fatigue, and the like; "moderate" adverse reactions are adverse reactions requiring medical intervention, but not being potentially life threatening.

In accordance, a "pharmaceutically compatible iron chelator" is an iron chelator as specified herein above which is pharmaceutically compatible as specified above. Thus, preferably, the pharmaceutically compatible iron chelator is an iron chelator comprising a chemical compound in clinical use, preferably approved for clinical use by at least one of the Food and Drug Admininstration (FDA), the European Medicines Agency (EMEA), and the Bundesinstitut fiir Arzneimittel und Medizinprodukte (BfArM). More preferably, the pharmaceutically compatible iron chelator is an iron chelator comprising a chemical compound in clinical use as an iron chelator, preferably approved for clinical use by at least one of the aforesaid institutions. Thus, preferably, the iron chelator is ciclopirox (2(1H)- Pyridinone, 6-cyclohexyl-l-hydroxy-4-methylpyridin-2(lH)-one; CAS-No: 29342-05-0), Deferoxamine (DFO, CAS No. 70-51-9) or hydroxycarbamide (CAS No. 127-07-1, nitrofural (CAS No. 59-87-0), 3-aminopyridine-2-carboxaldehyde Thiosemicarbazone (Triapine, CAS No. 236392-56-6) or 5-Hydroxypyridine-2-carboxaldehyde Thiosemicarbazone (HPCT, CAS No. 19494-89-4), Deferasirox (CAS No. 201530-41-8), iv) Deferiprone (Cas No. 30652-11- 0), or N,N'N''-tris(2-pyridylmethyl)-cis,cis-l,3,5-triaminocyclohex ane (Tachpyr).

Preferably, the pharmaceutically compatible iron chelator is a 2-pyridone derivative and/or a prodrug thereof.

The term "prodrug" is understood by the skilled person to relate to a compound not having or having only to a reduced extent the relevant activity of being an iron chelator and being converted in the body of a subject to the actual active compound. Thus, preferably, a prodrug is an ether or preferably an ester of the iron chelator. More preferably, the prodrug is a glycosylate, a phosphate, a sulphate, or a macromolecule-conjugated, e.g. polyethyleneglycol (PEG) conjugated, derivative of the iron chelator.

The term "2-pyridone derivative", as used herein, relates to a compound comprising, at least in a tautomeric form, a Pyridin-2(lH)-one structure. More preferably, the 2-pyridone derivative is a chemical compound comprising a l-Hydroxy-Pyridin-2(lH)-one structure. More preferably, the 2-pyridone derivative is a compound comprising structure (I) with n being 0, 1, 2, 3, 4, 5, or 6;

R ¹ being H, an optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl;

R ² to R ⁵ being independently selected from -H, an optionally substituted organic side chain, and halogen.

The term "side chain" is understood by the skilled person and relates to an atom or chemical group attached covalently to the core part of a chemical compound as described herein, said core part also being referred to as "main chain" or "backbone". Preferably, the side chain is an organic side chain as described herein below. The term "substituted" side chain relates to a side chain substituted at one or more positions, preferably, at 1, 2, or 3 positions, wherein substituents may be attached at any available atom to produce a stable chemical compound. It is understood by the skilled person that the term "optionally substituted" side chain relates to an unsubstituted or to a substituted side chain.

The term "organic side chain", as used herein, relates to any, optionally substituted, side chain comprising at least one carbon atom. Preferably, the organic side chain is an, optionally substituted, alkyl, alkenyl, alkinyl, aryl, aralkyl, cycloalkyl, heterocycloalkyl, or heteroaryl side chain. Preferably, a substituted organic side chain is an organic side chain substituted with at least one substituent independently selected from -COO-, =0, -OH, -CN, halogen, - NH2, -NH(alkyl), -N(alkyl)2, -N(alkyl)3 ⁺ , -NH(aryl), N(aryl)2, -NO2, -O(alkyl), -0-(CH ₂ )n- OH, -0-(CH ₂ )n-0(alkyl), -O(aralkyl), -O(aryl), -0P0 ₃ ² , -PCri ² , -OSO3 ^· and -SO3-. Preferably, the alkyl, aryl, and aralkyl groups of the substituents are not further substituted by groups comprising alkyl, alkenyl, alkinyl, aryl, aralkyl, heterocycloalkyl, or heteroaryl groups. More preferably, the alkyl, aryl, and aralkyl groups of the substituents are not further substituted. Preferably, the organic side chain has of from 1 to 10 , more preferably of from 1 to 8, still more preferably of from 1 to 6, even more preferably of from 1 to 5, most preferably of from 1 to 4 C-atoms, preferably including any optional substituents.

The term "alkyl", as used herein, relates to a straight or branched chain, saturated hydrocarbon group, linked to the main chain by a covalent bond to at least one of its at least one carbon atoms. Preferred alkyl groups are straight chain alkyls, e.g., preferably, methyl, ethyl, propyl, butyl, pentyl, hexyl, or branched chain alkyl groups, e.g., preferably,

-CH(CH ₃ ) ₂ , -CH(CH ₂ CH ₃ )2, -C(CH ₃ ) ₃ , -C(CH ₂ CH ₃ ) ₃ , -CH(CH3)(CH ₂ CH ₃ ),

-CH ₂ CH(CH ₃ )2, -CH ₂ CH(CH3)(CH ₂ CH3), -CH ₂ CH(CH ₂ CH3)2, -CH ₂ C(CH ₃ )3, -CH ₂ C(CH ₂ CH3)3, -CH(CH3)CH(CH3)(CH ₂ CH3), -CH ₂ CH ₂ CH(CH3)2, -CH ₂ CH ₂ CH(CH3)(CH ₂ CH3), -CH ₂ CH ₂ CH(CH ₂ CH3)2, -CH ₂ CH ₂ C(CH3)3, -CH ₂ CH ₂ C(CH ₂ CH3)3, -CH(CH3)CH ₂ CH(CH3) ₂ , or -CH(CH3)CH(CH3)CH(CH )2. Accordingly, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. The term "cycloalkyl" relates to a circularly closed, hydrocarbon group, preferably with 5 to 8 carbon atoms. Preferred cycloalkyls are cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term "alkenyl" side chain relates to a side chain comprising at least one C=C double bond and linked to the main chain by a covalent bond to at least one of its at least two carbon atoms. Accordingly, the term "alkinyl" side chain relates to a side chain comprising at least one CºC triple bond linked to the main chain by a covalent bond to at least one of its at least two carbon atoms.

The term "cycloalkenyl" relates to a circularly closed hydrocarbon group, preferably with 5 to 12 carbon atoms, comprising at least one C=C double bond and linked to the main chain by a covalent bond to at least one of its at least two carbon atoms. The term "cycloalkinyl" relates to a circularly closed hydrocarbon group, preferably with 8 to 12 carbon atoms, comprising at least one CºC triple bond and linked to the main chain by a covalent bond to at least one of its at least two carbon atoms.

As used herein, the term "alkoxy" side chain relates to an -O-alkyl side chain, preferably having the indicated number of carbon atoms. Preferably, the alkoxy side chain is -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-sec-butyl, -O-tert-butyl, -O-pentyl, -O- isopentyl, -O-neopentyl, -O-hexyl, -O- isohexyl, or -O-neohexyl.

The term "aryl", as used herein, relates to an aromatic ring or ring system having 6 to 14 carbon atoms, preferably comprising one, two, or three aromatic rings. Preferred aryl side chains are phenyl, naphthyl, anthracenyl, and phenanthrenyl. The term "ring", in the context of the chemical compounds of the present invention, is understood by the skilled person; accordingly, the term "ring system" relates to a chemical structure comprising at least two rings sharing at least one covalent bond. Thus, preferably, "aryl" also includes aromatic ring systems fused with a cycloalkyl and/or a heterocycloalkyl ring.

As used herein, the tern "aralkyl" relates to an alkyl side chain, wherein at least one hydrogen is replaced by an aryl side chain. Preferably, aralkyl is benzyl or phenethyl.

The term "heterocycloalkyl", as used herein, relates to a saturated or partially unsaturated ring or ring system having 5 to 14 ring atoms, preferably 5 to 7 ring atoms, wherein at least one ring atom is a heteroatom selected from the group consisting of N, O, and S, said ring or ring system being linked to the main chain by a covalent bond to a C or N atom of said ring or ring system. Preferably, heterocycloalkyl is azepinyl, dihydrofuryl, dihydropyranyl, imidazolidinyl, imidazolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxazolidinyl, piperazinyl, piperidinyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuryl, tetrahydropyranyl, thiadiazolylidinyl, thiazolidinyl, or thiomorpholinyl.

As used herein, the term "heteroaryl" relates to an aromatic ring or ring system having 5 to 14 ring atoms, preferably 5 to 7 ring atoms, wherein at least one ring atom is a heteroatom selected from the group consisting of N, O, and S, said ring or ring system being linked to the main chain by a covalent bond to a C or N atom of said ring or ring system. Preferably, up to 4, more preferably up to 3, most preferably up to 2 ring atoms per ring are heteroatoms independently selected from the group of heteroatoms consisting of N, O, and S. Preferably, heteroaryl is pyridinyl, pyridazinyl, pyrazinyl, quinaoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, or indolyl.

More preferably, the pharmaceutically compatible iron chelator is a compound comprising structure (I), with n being 0, 1, 2, 3, 4, 5, or 6; R ¹ being H, an optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl; and R ² to R ⁵ being independently selected from -H, alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkoxy, aryl aralkyl, heterocycloalkyl, heteroaryl, and halogen.

Still more preferably, the pharmaceutically compatible iron chelator is a compound comprising structure (I), with n being 0, 1, 2, 3, 4, 5, or 6; R ¹ being H, optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl; R ² being selected from cyclohexyl, -H, C ₁ -C ₆ alkyl, Cs-Cx cycloalkyl, C ₁ -C ₆ alkoxy, and halogen; R ⁴ being selected from methyl, -H, C ₁ -C ₆ alkyl, Cs-Cx cycloalkyl, C ₁ -C ₆ alkoxy, and halogen; and R ³ and R ⁵ being independently selected from -H, C ₁ -C ₆ alkyl, Cs-Cx cycloalkyl, C ₁ -C ₆ alkoxy, and halogen.

Most preferably, the pharmaceutically compatible iron chelator is a compound comprising structure (I), wherein n = 0, R ¹ is H, optionally substituted carbonyl, or optionally substituted phosphoryl; R ² is cyclohexyl; R ⁴ is methyl; and R ³ and R ⁵ are -H; ie. preferably, the pharmaceutically compatible iron chelator is ciclopirox (2(lH)-Pyridinone, 6-cyclohexyl- l-hydroxy-4-methylpyridin-2(lH)-one; CAS-No: 29342-05-0), preferably ciclopirox olamine.

The term "STAT3" is understood by the skilled person to relate to a member of the "signal transducer and activator of transcription" (STAT) family of proteins known under this designation, including its isoforms, such as splice variants. Preferably, STAT3 is mammalian STAT3, more preferably human STAT3. The gene and the polypeptide sequence of human STAT3 are known to the skilled person; e.g. human isoform 1 has the amino acid sequence as shown in Genbank Acc. No. NP_644805.1. Antibodies to STAT3 are commercially available, as also referred to herein in the Examples. Also, STAT3 target genes are known, e.g. from Carpenter & Lo (2014) Cancers (Basel) 6(2):897). Thus, the skilled person is well able to determine STAT3 expression, in particular its polypeptide levels, and STAT3 activity in a biological sample.

The term "STAT3 activity" is used herein in its usual meaning known to the skilled person to relate to the activity of STAT3 as a transcriptional regulator; as indicated above, STAT3 target genes are known in the art, thus the skilled person is able to determine STAT3 activity. In accordance, the term "STAT3 hyperactivity", as used herein, relates to an increased activity of STAT3 in an effector cell, preferably exceeding STAT3 activity in a corresponding normal cell, preferably by at least a factor 2, more preferably at least a factor of 3, still more preferably at least a factor of five, most preferably at least a factor of ten. In the above context, "corresponding normal cells" are cells of the same type from a subject or a group of subjects not suffering from a disease as specified herein; corresponding normal cells may, however, also be derived from the same subject but from a sample known not to be affected by STAT3 hyperactivity. Preferably, STAT3 hyperactivity is determined as STAT3 overexpression compared to a corresponding normal cell, preferably by at least a factor 2, more preferably at least a factor of 3, still more preferably at least a factor of five, most preferably at least a factor of ten, at the polypeptide level. More preferably, STAT3 hyperactivity is determined as increased amount of STAT3 phosphorylated at a position corresponding to Tyrosine 705 of the amino acid sequence of human STAT3 compared to a corresponding normal cell, preferably by at least a factor 2, more preferably at least a factor of 3, still more preferably at least a factor of five. Also preferably, STAT3 hyperactivity is determined as increased amount of STAT3 phosphorylated at a position corresponding to Serine 727 of the amino acid sequence of human STAT3 compared to a corresponding normal cell, preferably by at least a factor 2, more preferably at least a factor of 3, still more preferably at least a factor of five. Preferably, STAT3 hyperactivity and/or overexpression is determined in comparison to a reference; preferably, as detailed herein above, said reference provided from corresponding normal cells. Also preferably, the reference is a standard cultured cell, more preferably HeLa cells, still more preferably SiHa cells.

As used herein, the term "effector cell" relates to a cell causing or contributing to a disease as specified herein and showing hyperactivation of STAT3. Preferably, the effector cell is a cell causing or aggravating an immune hyperactivation, preferably causing and/or aggravating autoimmune disease and/or lymphoproliferative disease. Thus, preferably, the effector cell is an immune cell, preferred immune cells being T-cells, B-cells, NK cells, and dendritic cells. The effector cell may, however, also be any other cell contributing to a disease as specified, e.g. a skin cell in psoriasis. Also preferably, the effector cell is a cancer cell or a precancerous lesion cell. More preferably, the cancer cell is a papillomavirus (PV)-infected cell, in particular a human papillomavirus (HPV)-infected cell, preferably a PV- or HPV-infected cancer or precancerous lesion cell. Also preferably, the effector cells are cells of the growth and invasion rim of a tumor, preferably of an HPV positive tumor. In an embodiment, the effector cells is an HPV-negative cell, more preferably a PV-negative cell.

In accordance, the term "disease caused or aggravated by STAT3 hyperactivity in effector cells", as used herein, relates to any disease or disorder having the aforesaid cause. Corresponding diseases are known in the art. Preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is cancer, preferably cancer in which the cancer cells are suspected or known, preferably are known, to comprise STAT3 hyperactivity, preferably overexpress STAT3. Also preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is an immune hyperactivation, preferably an autoimmune disease and/or a lymphoproliferative disease; more preferably psoriasis, inflammatory bowel diseases (e. g. Crohn’s disease, ulcerative colitis) or rheumatoid arthritis. Also preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is asthma. Also preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is cachexia. Also preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is a fibrotic disease, such as lung fibrosis, liver fibrosis, or scleroderma. Symptoms and methods for the diagnosis of the aforesaid diseases caused or aggravated by STAT3 hyperactivity in effector cells are known to the skilled person and can be derived from standard medical textbooks. Also preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is STAT3 gain-of-function disease, a hereditary, autosomal dominant disease. Preferably, the disease caused or aggravated by STAT3 hyperactivity in effector cells is not an HPV-related disease, more preferably not a PV-related disease.

The term "cancer" is, in principle, understood by the skilled person and relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body. Preferably, also included by the term cancer is tumor recurrence, i.e. relapse. Thus, preferably, the cancer is a solid cancer, i.e. a cancer forming at least one detectable tumor, a metastasis, or a relapse thereof. Also preferably, the cancer is a non-solid cancer. Preferably, at least part of the cancer cells are effector cells as specified elsewhere herein, i.e., preferably, comprise an increased activity of STAT3 and/or overexpress STAT3. Preferably, the cancer is selected from the list consisting of aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and wilms tumor. More preferably, the cancer is cervix carcinoma.

The terms "treating" and “treatment” refer to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 10%, at least 20% at least 50% at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. Preferably, treating cancer is reducing tumor burden in a subject. As will be understood by the skilled person, effectiveness of treatment of e.g. cancer is dependent on a variety of factors including, e.g. cancer stage and cancer type. Preferably, treating causes effector cells to undergo senescence or apoptosis. Thus, preferably, treating has the effect of causing a tumor to stop growing, more preferably to cause regression of a tumor, more preferably of causing a tumor to resolve. As used herein, the above relates to treating a HPV-related lesion mutatis mutandis.

The term “preventing” refers to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the drug compound which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with the compound according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e. without preventive measures according to the present invention, would develop a disease or disorder as referred to herein. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools discussed elsewhere in this specification. As used herein, the above relates to preventing a HPV-related lesion mutatis mutandis.

Preferably, treating and/or preventing comprises topical application of the pharmaceutically compatible iron chelator, preferably to a mucosa, a skin, and/or a joint, preferably to a mucosa and/or a skin. Also preferably, treating and/or preventing cancer comprises inducing an irreversible proliferation arrest in a cancer cell, which may, preferably, proceed to cell death, in particular apoptosis, under prolonged treatment and/or increased dose of iron chelator. The term "irreversible proliferation arrest", as used herein, relates to all states of a cell in which no further cell division occurs. Preferably, the irreversible proliferation arrest is a cell death mechanism and/or senescence, more preferably is senescence, in a preferred embodiment is apoptosis. Cell death mechanisms are known to the skilled person and include in particular apoptosis, mitotic catastrophe, autophagy, and necrosis. As used herein, the above relates to treating and/or preventing a HPV-related lesion mutatis mutandis.

Also preferably, in particular in case the disease caused or aggravated by STAT3 hyperactivity is an immune hyperactivation, the treatment further comprises administration of an immunomodulatory agent. Immunomodulatory agents are described elsewhere herein. Also preferably, in particular in case the disease caused or aggravated by STAT3 hyperactivity is cancer, the treatment further comprises administration of anticancer treatment as specified elsewhere herein, more preferably of chemotherapy and/or radiotherapy.

Preferably, in particular in case the effector cell is a PV positive cell, preferably a HPV positive cell, treating and/or preventing comprises preventing malignant transformation of a precancerous lesions. More preferably, the precancerous lesion is a histological cervical intraepithelial neoplasia (CIN) 1, CIN 2, or CIN 3, more preferably CIN 2 or CIN 3 lesion. In such case, the iron chelator or prodrug thereof may in particular be administered to a site of a precancerous lesion, preferably a mucosa, more preferably cervical mucosa or oropharyngeal mucosa. Also preferably, in particular in case the effector cell is a PV positive cell, preferably a HPV positive cell, treating and/or preventing comprises preventing tissue invasion. Since it was found in the work underlying the present invention that in HPV positive tumors STAT3 is in particular overexpressed in cells forming the outer shell of the tumor, in such case the cells forming this outer shell preferably are effector cells. Also preferably, in particular in case the effector cell is a PV positive cell, preferably a HPV positive cell, treating comprises avoiding concurrent apoptosis-inducing treatment and treatment with a pharmaceutically compatible iron chelator; in such case, it is preferred that treatment with an iron chelator is administered before or after apoptosis-inducing treatment. Preferably, the time span between apoptosis-inducing treatment and iron-chelator treatment in such case is at least 1 day, more preferably at least 3 days, more preferably at least one week, most preferably at least two weeks. The term "apoptosis-inducing treatment" is used herein in its usual meaning and preferably relates to any treatment causing at least a fraction of effector cells to undergo apoptosis, preferably at least 10%, more preferably at least 25%, still more preferably at least 50%, most preferably at least 75% of effector cells. Preferably, the apoptosis-inducing treatment is an anticancer treatment as specified elsewhere herein, more preferably is chemotherapy and/or radiotherapy.

The term "immunomodulatory agent" is used herein in a broad sense for all agents having the activity of causing the immune system of a subject to be modulated, i.e. to be repressed or activated, preferably repressed, preferably to a significant extent. Thus, the term includes all physical, chemical, and biological agents having the aforesaid effect. Thus, the immunomodulatory agent preferably is a physical agent, more preferably radiation, still more preferably ultraviolet (UV) radiation or infrared (IR) radiation, heat, or cold, most preferably is UV radiation. Also preferably, the immunomodulatory agent is a chemical agent, in particular a corticosteroid, a non-steroidal anti-inflammatory drug, vitamin D or an analogue thereof, a retinoid, a calcineurin inhibitor, or cyclosporine. Also preferably, the immunomodulatory agent is a biological agent, in particular etanercept, infliximab, adalimumab, ustekinumab, golimumab, apremilast, secukinumab, or ixekizumab; or an agent comprising immunomodulatory cells or mixtures thereof such as regulatory T-cells, a cytokine, or a checkpoint inhibitor, e.g. targeting CTLA4, PD-1, and/or PD-L1.

The term "anticancer treatment", as used herein, relates to measures administered to a subject to remove cancer cells from the subject, to kill cancer cells in the subject, to inhibit growth of cancer cells in the subject, and/or to cause the body of the subject to inhibit growth of or to kill cancer cells. Thus, anticancer treatment, preferably comprises surgery, radiotherapy, chemotherapy, anti-hormone therapy, targeted therapy, and/or cancer immunotherapy. Preferably, anticancer treatment comprises surgery; more preferably, anticancer treatment comprises surgery and radiotherapy; still more preferably, anticancer treatment comprises surgery, radiotherapy, and chemotherapy. Anticancer treatment may in principle be administered before, simultaneously to, and/or after preventing administration of an iron chelator or a prodrug thereof. The terms "radiation therapy" and "radiotherapy" are known to the skilled artisan. The terms relates to the use of ionizing radiation to treat or control cancer. The skilled person also knows the term "surgery", relating to invasive measures for treating cancer, in particular excision of tumor tissue. As used herein, the term "chemotherapy" relates to treatment of a subject with an antineoplastic drug. Preferably, chemotherapy is a treatment including alkylating agents (e.g. cyclophosphamide), platinum (e.g. carboplatin), anthracyclines (e.g. doxorubicin, epirubicin, idarubicin, or daunorubicin) and topoisomerase II inhibitors (e.g. etoposide, irinotecan, topotecan, camptothecin, or VP 16), anaplastic lymphoma kinase (ALK)-inhibitors (e.g. Crizotinib or AP26130), aurora kinase inhibitors (e.g. N-[4-[4-(4-Methylpiperazin-l-yl)-6-[(5-methyl-lH-pyrazol-3-y l)amino]pyrimidin-2- yl]sulfanylphenyl]cyclopropanecarboxamide (VX-680)), antiangiogenic agents (e.g. Bevacizumab), or Iodinel31-l-(3-iodobenzyl)guanidine (therapeutic metaiodobenzylguanidine), histone deacetylase (HDAC) inhibitors, alone or any suitable combination thereof. It is to be understood that chemotherapy, preferably, relates to a complete cycle of treatment, i.e. a series of several (e.g. four, six, or eight) doses of antineoplastic drug or drugs applied to a subject separated by several days or weeks without such application. The term "anti-hormone therapy" relates to cancer therapy by blocking hormone receptors, e.g. estrogen receptor or progesterone receptor, expressed on cancer cells, or by blocking the biosynthesis of a hormone the cancer is dependent on. Blocking of hormone receptors can preferably be achieved by administering compounds, e.g. tamoxifen, binding specifically and thereby blocking the activity of said hormone receptors. Blocking of hormone biosynthesis is preferably achieved by administration of inhibitors, e.g. in the case of estrogen, aromatase inhibitors like, e.g. anastrozole or letrozole may be used. It is known to the skilled artisan that anti-hormone therapy is preferably advisable in cases where tumor cells are expressing hormone receptors. The term "targeted therapy", as used herein, relates to application to a patient of a chemical substance known to block growth of cancer cells by interfering with specific molecules known to be necessary for tumorigenesis or cancer or cancer cell growth. Examples known to the skilled artisan are small molecules like, e.g. PARP-inhibitors (e.g. Iniparib), or monoclonal antibodies like, e.g., Trastuzumab. The term "cancer immunotherapy" as used herein relates to the treatment of cancer by modulation of the immune response of a subject. Said modulation may be inducing, enhancing, or suppressing said immune response. The term "cell based immunotherapy" relates to a cancer therapy comprising application of immune cells, e.g. T-cells, preferably tumor-specific NK cells, to a subject. As the skilled person understands, cancer immunotherapy, which aims at inhibiting or killing cancer cells, has a purpose different from treatment with an immunomodulatory agent as specified herein above, which aims at preventing a further immune overstimulation and the detrimental consequences thereof to a subject; nonetheless, the same or similar agents may be used in both treatments. The term "subject", as used herein, relates to a multicellular animal, preferably to a vertebrate, more preferably to a mammal. More preferably, the subject is a human, a cattle, a pig, a sheep, a goat, a horse, a cat, a dog, a guinea pig, a mouse, or a rat. Preferably, the subject is a laboratory animal, preferably a guinea pig, a mouse, or a rat. Also preferably, the subject is a livestock, preferably a cattle, a pig, a sheep, a goat, or a horse. Also preferably, the subject is a companion animal, preferably a cat, a dog, or a guinea pig. Most preferably, the subject is a human. Preferably, the subject is known or suspected to suffer from a disease caused or aggravated by STAT3 hyperactivity, preferably cancer, an immune hyperactivation disease, preferably auto immune disease, and/or STAT3 gain-of function disease. Also preferably, the subject was identified to profit from treatment with a pharmaceutically compatible iron chelator by the method according to the present invention.

Preferably, the iron chelator or prodrug thereof is comprised in a composition. The term “composition”, as used herein, relates to a mixture of compounds comprising at least a compound as specified herein and, preferably, at least one carrier. The compounds, preferably the iron chelator or prodrug thereof, comprised in the composition are described herein above. The composition may have any consistency deemed appropriate by the skilled person. Preferably, the composition is a solid composition, e.g. a tablet or a powder, a semisolid composition, e.g. a gel, or, more preferably, a liquid, e.g. a solution or an emulsion.

The composition preferably comprises a carrier. The carrier(s) preferably is/are acceptable in the sense of being compatible with the other ingredients of the composition and being not deleterious to a potential recipient thereof. The carrier(s) preferably is/are selected so as not to affect the biological activity of the composition. Preferably, the composition is sterile, more preferably a sterile solution, most preferably a sterile solution for injection. The carrier is selected by the skilled person such as to achieve the consistency intended and may be, for example, a gel or, preferably, a liquid, more preferably an aqueous liquid. Examples of such carriers are distilled water, physiological saline, Ringer's solutions, dextrose solution, phosphate-buffered saline solution, and Hank's solution. The carrier may include one or more solvents or other ingredients increasing solubility of the compounds comprised in the composition. Further examples of liquid carriers are syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Suitable carriers comprise those mentioned above and others well known in the art. As is understood by the skilled person, the composition, in particular the pharmaceutical composition, may comprise one or more further compounds; preferably, such additional compounds are selected so as to not affect the biological activity of the composition, in particular of the active compounds, such as the iron chelator, and/or is acceptable in the sense of being compatible with the other ingredients of the composition and being not deleterious to a potential recipient thereof.

Preferably, the composition according to the present specification is a pharmaceutical composition; thus, preferably, the carrier is a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, stabilizers and/or other compounds deemed appropriate by the skilled person, e.g. for galenic purposes. As referred to herein, the compound as specified herein above, in particular the iron chelator or prodrug thereof as specified, is the "active compound" of the preparation, although "further active compounds", which are referred to under this term, may be present. Preferably, the active compound and the further active compound, i.e. preferably the active compounds, are pharmaceutically active compounds. Specific pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound referred to herein above, preferably in admixture or otherwise associated with at least one pharmaceutically acceptable carrier or diluent. For making those specific pharmaceutical compositions, the active compound(s) will usually be mixed with a carrier or the diluent. The resulting formulations are to be adapted to the mode of administration, i.e. in the forms of tablets, capsules, suppositories, solutions, suspensions or the like. Dosage recommendations shall be indicated in the prescriber's or user's instructions in order to anticipate dose adjustments depending on the considered recipient.

The pharmaceutical composition is, preferably, administered systemically or, more preferably, topically. Suitable routes of administration conventionally used for drug administration are topical, intravenous, or parenteral administration as well as inhalation. Preferably, administration is systemic, more preferably intravenous. However, depending on the nature and mode of action of the specific compound(s) administered and on the clinical situation, the pharmaceutical composition may be administered by other routes as well. Also, the pharmaceutical composition may be administered topically, e.g. as a tablet, in particular as a time-delay preparation, which is preferably implanted during surgery removing a tumor, or e.g. as a gel administered to a mucosa. Other modes of administration are specified elsewhere herein. Moreover, the pharmaceutical composition can be administered in combination with other further active compounds either in a common pharmaceutical composition or as separated pharmaceutical compositions wherein said separated pharmaceutical compositions may be provided in form of a kit of parts.

The pharmaceutical composition is, preferably, administered in conventional dosage forms prepared by combining the active compound with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to obtain the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Similarly, the carrier or diluent may include time delay material well known in the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. A therapeutically effective dose refers to an amount of the active compound to be used in a pharmaceutical composition of the present invention which provides the effect referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depend upon many factors, which may include the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 pg to 1000 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 100 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 1 mg units per kilogram of body weight per minute, respectively. Preferably, the pharmaceutical composition is administered once to the subject, i.e., preferably, is used as a one-time treatment. Depending on the subject and the mode of administration, the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 100 mg per kg body mass. The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from two to 50 times, more preferably from five to 50 times. Preferably, administration is adjusted to maintain an effective concentration in the body of a subject for the time period intended. Also, as indicated above, the pharmaceutical preparation may be administered topically at the site of a tumor as a depot; in such case, the depot preferably is adjusted to maintain an effective dose until at least after additional cancer treatment, e.g. chemotherapy, was administered or, preferably, until such additional cancer treatment has been completed. Progress can be monitored by periodic assessment.

Advantageously, it was found in the work underlying the present invention that iron chelators are effective in inducing STAT3 degradation in living cells and in ameliorating or preventing the effects of STAT3 hyperactivation.

The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

The present invention further relates to a method for identifying a subject profiting from treatment with an iron chelator, comprising

(a) determining STAT3 activity and/or expression in a sample of said subject;

(b) identifying a subject profiting from treatment with an iron chelator in case STAT3 overexpression is determined.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to pre-processing a sample for step a), e.g. by fixing and embedding, or comparing the STAT3 activity and/or expression determined in step a) to a reference in step b). Moreover, one or more of said steps may be performed by automated equipment.

The term "profiting from treatment with an iron chelator", as used herein, relates to an improvement of health and/or of survival probability by iron chelator treatment. It is to be understood that profiting from treatment with an iron chelator may not be effective in all subjects treated. However, the term preferably requires that improvement of health and/or survival is observed for a statistically significant portion of subjects of a cohort or population. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e. without iron chelator treatment, would develop the adverse health-related event, would develop more severe adverse health-related events, and/or would show increased mortality. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, preferably as specified elsewhere herein.

Methods for determining STAT3 activity and/or expression are known in the art and are described elsewhere herein. STAT3 activity is preferably determined e.g. in a reporter gene assay, preferably using one of the known STAT3 regulated genes; STAT3 expression is preferably determined as STAT3, more preferably P-STAT3, polypeptide levels, which are preferably determined by immunological methods, preferably using STAT3- or P-STAT3- specific antibodies, but may also be determined by other methods, e.g. by mass spectrometry. Preferably, the STAT3 activity and/or amount of STAT3 gene product determined is compared to a reference; suitable references have been described herein above. Thus, STAT3 hyperactivity and/or overexpression is determined in case STAT3 activity and/or the amount of STAT3 gene product is found increased compared to a reference. As the skilled person understands in view of the Examples provided herein, STAT3 expression levels can be used as a surrogate marker of STAT3 activity; thus, it is preferably not always required to determine STAT3 activity as such.

The term "sample", as used herein, refers to any sample suspected or known to comprise effector cells; thus, the sample preferably is a biological sample. Preferably, the sample is a sample of a body fluid, a sample of separated cells, a sample from a tissue or an organ, or a sample of wash/rinse fluid obtained from an outer or inner body surface of a subject. Preferably, the sample is a body fluid like blood, plasma, serum, urine, saliva, or lacrimal fluid. More preferably, the sample is a tissue sample, more preferably a tumor tissue or inflammation tissue sample, known or suspected to comprise effector cells. Samples can be obtained by well-known techniques which include, preferably, scrapes, swabs or biopsies. Such samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or by surgical instrumentation. Preferably, the sample is a biopsy, a tumor, or part thereof. Separated cells and/or cell-free liquids may be obtained from cell culture supernatants, body fluids, or the tissues or organs by separating techniques such as filtration, centrifugation, or cell sorting. It is to be understood that the sample may be further processed in order to carry out a method of the present invention.

The present invention also relates to a method for treating a disease caused or aggravated by STAT3 overexpression in effector cells in a subject suffering therefrom, comprising

(A) administering an iron chelator or a prodrug thereof to said subject, and

(B) thereby treating said disease caused or aggravated by STAT3 overexpression.

The method for treating of the present invention, preferably, is an in vivo method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to diagnosing the disease caused or aggravated by STAT3 overexpression and/or identifying the subject as profiting from treatment with an iron chelator, preferably according to the method specified above, before step a); thus, preferably, the method comprises the further steps of (i) determining or having determined STAT3 activity and/or expression in a sample of said subject; and (ii) identifying or having identified a subject profiting from treatment with an iron chelator in case STAT3 hyperactivity and/or overexpression is determined. Also, a further step may comprise co-treating the subject as specified herein above in step b). Moreover, one or more of said steps may be performed by automated equipment.

The present invention also relates to a kit comprising an iron chelator or a prodrug thereof and a means of determining STAT3 expression.

The term “kit”, as used herein, refers to a collection of the aforementioned components. Preferably, said components are combined with additional components, preferably within an outer container. Examples for such components of the kit as well as methods for their use have been given elsewhere in this specification. Suitable means for determining STAT3 expression are known to the skilled person and include in particular a STAT3 -specific antibody, a hybridization probe specific for a STAT3 encoding sequence or part thereof, PCR primers specific for a STAT3 encoding sequence or part thereof, and the like. The kit, preferably, contains the aforementioned components in a ready-to-use formulation. The kit preferably comprises further components, preferably a cancer therapeutic agent as specified herein above, a diluent, and/or a means of administration, in particular a syringe and/or a needle, and/or an IV infusion equipment. Preferably, the kit may additionally comprise instructions, e.g., a user’s manual for carrying out a method of the present invention. Details are to be found elsewhere in this specification. Additionally, such user’s manual may provide instructions about correctly using the components of the kit. A user’s manual may be provided in paper or electronic form, e.g., stored on CD or CD ROM. The present invention also relates to the use of said kit in any of the methods according to the present invention.

The present invention further relates to a device comprising (i) an analysis unit comprising a means for determining STAT3 activity and/or expression in a sample, and, operatively connected thereto (ii) an evaluation means comprising tangibly embedded executable instructions for performing a method according to the present invention.

The term “device”, as used herein, relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the determination. Typical means for determining STAT3 activity and/or expression, and means for carrying out the determination are disclosed herein above. As is understood by the skilled person, means for determining STAT3 activity and/or expression include means capable for determining an amount of STAT3 polypeptide, such as an ELISA reader, means for determining an amount of STAT3 mRNA, such as a quantitative PCR device, or means for determining STAT3 activity, such as an optical unit detecting a signal of a reporter gene assay. An evaluation means is any means capable of providing the analysis as specified; preferably, the evaluation means is a data processing means, such as a microprocessor, a handheld device such as a mobile phone, or a computer. How to link the means in an operating manner will depend on the type of means included into the device. In an embodiment, the means are comprised by a single device. Said device may accordingly include (i) an analyzing unit for the measurement of the amount and/or activity of STAT3 in a sample and a (ii) computer unit for processing the resulting data for the evaluation. Typical means for detection are disclosed herein above. Preferably, the instructions and interpretations are comprised in an executable program code comprised in the device, such that, as a result of determination a classification of the subject as profiting or not profiting from iron chelator treatment may be output to a user. Typical devices are those which can be applied without the particular knowledge of a specialized technician, e.g., test stripes or electronic devices which merely require loading with a sample. The results may be given as output of raw data which need interpretation by a technician. In an embodiment, the output of the device is, however, processed, i.e. evaluated, raw data, the interpretation of which does not require a technician. Further typical devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the peptide, Plasmon surface resonance devices, NMR spectrometers, mass- spectrometers etc.) or evaluation units/devices referred to above in accordance with the method of the invention. Preferably, the the device further comprises a memory unit, preferably comprising a database comprising at least one reference value for a STAT3 activity and or expression.

The present invention also relates to a use of an iron chelator or prodrug thereof for increasing apoptosis sensitivity in a STAT3 -overexpressing cell, preferably a STAT3 overexpressing cancer cell.

As the skilled person will understand, the term "increasing apoptosis sensitivity" is used herein relative to an otherwise identical STAT3-overexpressing cells, which was not treated with an iron chelator. Preferably, said use is an in vitro use.

The present invention also relates to a combined preparation comprising (i) a pharmaceutically compatible iron chelator or prodrug thereof and (ii) an anticancer agent and/or an immunomodulatory agent for use in treating and/or preventing a disease caused or aggravated by STAT3 overexpression in effector cells in a subject.

The term “combined preparation”, as referred to in this description, relates to a preparation comprising the pharmaceutically active compounds of the present invention in one preparation. Preferably, the combined preparation is comprised in a container, i.e. preferably, said container comprises all pharmaceutically active compounds of the present invention. Preferably, said container comprises the pharmaceutically active compounds of the present invention as separate formulations, i.e. preferably, one formulation of the active compound as specified herein and one formulation of a cancer therapeutic agent and/or an immunomodulatory agent. As will be understood by the skilled person, the term "formulation" relates to a, preferably pharmaceutically acceptable, mixture of compounds, comprising or consisting of at least one pharmaceutically active compound. Preferably, the combined preparation comprises the active compound and the further active compound in a single solid pharmaceutical form, e.g. a tablet or, preferably, a solution; more preferably, the active compounds of the present invention are comprised in two separate, preferably liquid, formulations; said separate liquid formulations, preferably are for injection, preferably at different parts of the body of a subject.

Preferably, the combined preparation is for separate or for combined administration. "Separate administration", as used herein, relates to an administration wherein at least two active compounds are administered via different routes and/or at different parts of the body of a subject. E.g. one compound may be administered by enteral administration (e.g. orally), whereas a second compound is administered by parenteral administration (e.g. intravenously). Preferably, the combined preparation for separate administration comprises at least two physically separated preparations for separate administration, wherein each preparation contains at least one active or further active compound; said alternative is preferred e.g. in cases where the pharmaceutically active compounds of the combined preparation have to be administered by different routes, e.g. parenterally and orally, due to their chemical or physiological properties. Conversely, "combined administration" relates to an administration wherein the active compounds are administered via the same route, e.g. orally or, preferably, intravenously.

Also preferably, the combined preparation is for simultaneous or for sequential administration. "Simultaneous administration", as used herein, relates to an administration wherein the active compounds are administered at the same time, i.e., preferably, administration of the pharmaceutically active compounds starts within a time interval of less than 15 minutes, more preferably, within a time interval of less than 5 minutes. Most preferably, administration of the pharmaceutically active compounds starts at the same time, e.g. by swallowing a tablet comprising the pharmaceutically active compounds, or by swallowing a tablet comprising one of the pharmaceutically active compounds and simultaneous injection of the second compound, or by administering an intravenous injection of a solution comprising an active compound and injecting a further active compound in a different part of the body, or by administering an intravenous injection of a solution comprising the active compound and the further active compound. Conversely, "sequential administration", as used herein, relates to an administration causing plasma concentrations of the active compounds in a subject enabling the synergistic effect of the present invention, but which, preferably, is not a simultaneous administration as specified herein above. Preferably, sequential administration is an administration wherein administration of the active compounds, preferably all active compounds, starts within a time interval of 1 or 2 days, more preferably within a time interval of 12 hours, still more preferably within a time interval of 4 hours, even more preferably within a time interval of one hour, most preferably within a time interval of 5 minutes.

In view of the above, the following embodiments are particularly envisaged:

Embodiment 1. A pharmaceutically compatible iron chelator or prodrug thereof for use in treating and/or preventing a disease caused or aggravated by STAT3 hyperactivity in effector cells in a subject.

Embodiment 2. The pharmaceutically compatible iron chelator or prodrug thereof for use of embodiment 1, wherein said pharmaceutically compatible iron chelator comprises at least one of a 2-pyridone structure, a hydroxamate structure, a (thio)semicarbazone structure, a bis(2-hydroxyphenyl)-lH-l,2,4-triazol structure, an alpha-hydroxyketone structure, an arylhydrazone structure, and a catechol structure.

Embodiment s. The pharmaceutically compatible iron chelator or prodrug thereof for use of embodiment 1 or 2, wherein said pharmaceutically compatible iron chelator is a 2- pyridone derivative and/or a prodrug thereof.

Embodiment 4. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 3, wherein said 2-pyridone derivative and/or prodrug comprises structure with n being 0, 1, 2, 3, 4, 5, or 6;

R ¹ being H, optionally substituted carbonyl, optionally substituted phosphoryl, or optionally substituted sulfonyl;

R ² being selected from cyclohexyl, -H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen;

R ⁴ being selected from methyl, -H, C1-C6 alkyl, C5-C8 cycloalkyl, C1-C6 alkoxy, and halogen; and

R ³ and R ⁵ being independently selected from -H, C1-C6 alkyl, C5-C8 cycloalkyl, Cl- C6 alkoxy, and halogen.

Embodiment 5. The pharmaceutically compatible iron chelator for use of any one of embodiments 1 to 4, wherein n = 0,

R ¹ is H, optionally substituted carbonyl, or optionally substituted phosphoryl;

R ² is cyclohexyl;

R ⁴ is methyl; and R ³ and R ⁵ are -H.

Embodiment 6. The pharmaceutically compatible iron chelator for use of any one of embodiments 1 to 5, wherein said pharmaceutically compatible iron chelator is ciclopirox (2(lH)-Pyridinone, 6-cyclohexyl- l-hydroxy-4-methylpyridin-2(lH)-one; CAS-No: 29342-05- 0), preferably ciclopirox olamine.

Embodiment 7. The pharmaceutically compatible iron chelator for use of embodiment 1 or 2, wherein said pharmaceutically compatible iron chelator is (i) a hydroxamate iron chelator, more preferably Deferoxamine (DFO, CAS No. 70-51-9) or hydroxycarbamide (CAS No. 127-07-1, (ii) a (thio)semicarbazone iron chelator, preferably nitrofural (CAS No. 59-87-0), 3-aminopyridine-2-carboxaldehyde Thiosemicarbazone (Triapine, CAS No. 236392-56-6) or 5-Hydroxypyridine-2-carboxaldehyde Thiosemicarbazone (HPCT, CAS No. 19494-89-4), (iii) a bis(2-hydroxyphenyl)-lH-l,2,4-triazol iron chelator, preferably Deferasirox (CAS No. 201530-41-8), iv) an alpha-hydroxyketone iron chelator, preferably Deferiprone (Cas No. 30652-11-0), or (v) N,N'N"-tris(2-pyridylmethyl)-cis,cis-l,3,5- triaminocyclohexane (Tachpyr). Embodiment 8. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 7, wherein said disease caused or aggravated by STAT3 hyperactivity in effector cells is (i) cancer, (ii) an autoimmune disease, preferably psoriasis, rheumatoid arthritis, or an inflammatory bowel disease, in particular Crohn’s disease or ulcerative colitis; (iii) asthma, (iv) cachexia, or (v) a fibrotic disease, in particular lung fibrosis, liver fibrosis, or scleroderma..

Embodiment 9. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 8, wherein said effector cells are HPV-positive cells.

10. The pharmaceutically compatible iron chelator or prodrug thereof for use of embodiment 9, wherein said treating and/or preventing comprises preventing malignant transformation and/or preventing tissue invasion.

Embodiment 11. The pharmaceutically compatible iron chelator or prodrug thereof for use of embodiment 9 or 10, wherein said treating comprises avoiding concurrent apoptosis- inducing treatment and treatment with a pharmaceutically compatible iron chelator.

Embodiment 12. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 11, wherein said treating and preventing comprises topical administration of said pharmaceutically compatible iron chelator.

Embodiment 13. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 12, wherein said treating and/or preventing further comprises administration of apoptosis-inducing treatment, preferably chemotherapy and/or radiotherapy.

Embodiment 14. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 13, wherein said effector cells are cells causing or aggravating an immune hyperactivation.

Embodiment 15. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 14, wherein said effector cells are cells causing and/or aggravating autoimmune disease and/or lymphoproliferative disease. Embodiment 16. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 15, wherein said disease is STAT3 gain-of- function disease.

Embodiment 17. The pharmaceutically compatible iron chelator or prodrug thereof for use of any one of embodiments 1 to 16, wherein said subject was identified to profit from treatment with a pharmaceutically compatible iron chelator by the method according to any one of embodiments 18 to 20.

Embodiment 18. A method for identifying a subject profiting from treatment with an iron chelator, comprising

(a) determining STAT3 activity and/or expression in a sample of said subject;

(b) identifying a subject profiting from treatment with an iron chelator in case STAT3 hyperactivity and/or overexpression is determined.

Embodiment 19. The method of embodiment 18, wherein said sample is known or suspected to comprise effector cells.

Embodiment 20. The method of embodiment 18 or 19, wherein said method comprises the further step of comparing the STAT3 activity and/or expression determined in step (a) to a reference; and wherein a subject profiting from treatment with an iron chelator is identified in case STAT3 activity and/or expression is higher than in the reference is determined.

Embodiment 21. A method for treating a disease caused or aggravated by STAT3 overexpression in effector cells in a subject suffering therefrom, comprising

(A) administering an iron chelator or a prodrug thereof to said subject, and

(B) thereby treating said disease caused or aggravated by STAT3 overexpression.

Embodiment 22. The method of embodiment 21, wherein said method further comprises the steps of

(i) determining or having determined STAT3 activity and/or expression in a sample of said subject; and

(ii) identifying or having identified a subject profiting from treatment with an iron chelator in case STAT3 hyperactivity and/or overexpression is determined, and wherein step (a) is administering an iron chelator to said subject identified to profit from treatment with an iron chelator.

Embodiment 23. A kit comprising an iron chelator or a prodrug thereof and a means of determining STAT3 expression.

Embodiment 24. A device comprising (i) an analysis unit comprising a means for determining STAT3 activity and/or expression in a sample, and, operatively connected thereto (ii) an evaluation means comprising tangibly embedded executable instructions for performing a method according to any one of embodiments 18 to 21.

Embodiment 25. Else of an iron chelator or prodrug thereof for increasing apoptosis sensitivity in a STAT3 -overexpressing cell, preferably a STAT3 overexpressing cancer cell.

Embodiment 26. A combined preparation comprising (i) a pharmaceutically compatible iron chelator or prodrug thereof and (ii) an anticancer agent and/or an immunomodulatory agent for use in treating and/or preventing a disease caused or aggravated by STAT3 overexpression in effector cells in a subject.

Embodiment 27. The combined preparation of embodiment 27, wherein said effector cells are HPV-free.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends

Figure 1: Repression of STAT3 protein expression by CPX; immunoblot analysis of STAT3 levels in HPV16-positive SiHa and CaSki cells, as well as in HPV18-positive SW756 and HeLa cervix-carcinoma cells after treatment with 10 mM CPX for the indicated time frames. EtOH: solvent control; Vinculin: loading control. Figure 2: Reduction of phospho(Tyr705)-STAT3 (P-STAT3) levels and of Survivin expression by CPX; immunoblot analysis of SiHa and SW756 cells after treatment with 10 mM CPX for the indicated timeframes; EtOH: solvent control; Vinculin, beta-actin: loading controls.

Figure 3: Repression of STAT3 expression is caused by iron depletion: (a) SiHa (upper panel) and HeLa (lower panel) cells were treated with 100 pM DFO for the indicated timeframes and STAT3 expression was determined by immunoblotting; EtOH: solvent control; beta-actin: load control; (B) SiHa cells were treated with 10 pM CPX for 48 hours, with or without addition of a 3fold molar excess of iron. (FAC: ferric ammonium citrate; FeS0 ₄ ) or zinc (ZnCh; ZnS0 ₄ ); immunoblot analysis of STAT3 and the phospho(Tyr705) STAT3 (P- STAT3) levels; ¾0: solvent control; ferritin heavy chain (HC): marker for bioavailable iron; beta-actin: loading control.

Figure 4: CPX treatment and STAT3 transcript levels: SiHa and SW756 cells were treated with 10 pM CPX for the indicated time frames, followed by determining STAT3 mRNA levels via qRT-PCR; mRNA levels are indicated as relative mRNA levels compared to a solvent control (EtOH), which was set to a value of 1.0; similar results were also obtained for CaSki and HeLa cells.

Figure 5: Stability measurements on the STAT3 protein: (a) SW756 cells were preincubated for 8 hours with 10 pM CPX or solvent control (EtOH) and treated from time 0 additionally with 10 pg/ml CHX for the indicated timeframes; additionally, a part of the cells was treated additionally with 10 pM MG- 132 or the corresponding solvent control (DMSO); (B) densitometric measurements (normalized to beta-actin) showing a STAT3 halflife in control SW756 cells of approximately 39 hours and in CPX-treated cells of approximately 8.5 hours. A strong reduction of STAT3 expression by CPX was also in all other tumor cells evaluated, including HPV-negative tumor cells.

Figure 6: Canonical and non-canonical STAT3 transcriptional regulation are inhibited by CPX treatment: SiHa and SW756 cells were treated with 10 pM CPX or solvent control for 8, 24 or 48 hours followed by total protein extraction (A) or nuclear-cytoplasmic fractionation (B, C) and analyzation of STAT3 or P-STAT3 levels by immunoblotting. beta-actin was analyzed as loading control. Vinculin was analyzed as cytoplasmic loading and contamination control and lamin A/C as nuclear control.

Figure 7: Downregulation of total STAT3 levels in head & neck cancer cells, melanoma cells and colon cancer cells. FaDu head & neck cancer cells, MeWo and SK-Mel28 melanoma cells, and HCT116 colon cancer cells were treated with 10 mM CPX or solvent control (EtOH) for 24 h or 48 h. Total STAT3 protein levels were measured by immunoblot analysis. beta-actin served as loading control..

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1 : iron chelators repress STAT3 protein expression

HPV 16 positive SiHa cervix carcinoma cells were treated with CPX or not, and it was found that STAT3 is one of the most highly regulated proteins after 24 hours of treatment, being reduced by approximately 65% (p = 0.009).

These observations were confirmed by immunoblot analysis, by which method a clear repression of STAT3 expression could be verified after CPX treatment in HPV16-positive SiHa and CaSki cells, and in HPV18-positive HeLa and SW756 cells (Fig. 1).

To evaluate whether the reduction of STAT3 levels correlate with the reduction of P-STAT3, immunoblot analysis with P-STAT3 specific antibodies was performed, which detect phospho(Tyr705)-STAT3 (Fig. 2). These experiments show a clear reduction of P-STAT3 amounts under CPX treatment, as well as a reduction of the anti-apoptotic Survivin protein, expression of which is activated by P-STAT3 (Aoki et al. (2003), Blood 101:1535).

The iron chelating activity of CPX has been known for some time (reviewed by Shen & Huang (2016), Curr Pharm Des 22:4443). To confirm that CPX activity is mediated via this iron chelator activity, the further, structurally non-related, iron chelator Deferoxamine (DFO), was tested and it was found that treatment of tumor cells with DFO also leads to a strong reduction of STAT3 protein levels (Fig. 3 A). Moreover, it was evaluated whether the CPX induced STAT3 repression can be reverted by increasing iron concentration. As shown in Fig. 3B, an excess of iron, but not of zinc, reverts the CPX mediated repression of STAT3 and P-STAT3, showing that the decrease in STAT3 expression by CPX is mediated by its iron chelating activity.

Example 2: CPX induces a destabilisation of the STAT3 protein

The effect of CPX on STAT3 mRNA levels was evaluated. Under the same conditions which lead to strong reduction in STAT3 protein levels (cf. Fig. 1), qRT-PCR analysis showed that the amounts of STAT3 mRNA do not change significantly (Fig. 4).

The STAT3 polypeptide was reported to have a halflife of 24 hours or more (Nie et al. (2015), Drug Des Devel Ther 9:5611). Since STAT3 mRNA levels did not show significant changes upon CPX treatment, it was evaluated whether the treatment destabilizes the STAT3 protein. To this end, HPV-positive cells were treated with CPX and cycloheximide (CHX), a protein biosynthesis inhibitor, and STAT3 protein levels were analyzed over time. It was found that CPX strongly reduces the halflife of the STAT3 protein (Fig. 5). Since this destabilization of STAT3 can be reverted by the protesome inhibitor MG- 132 (Fig. 5), it can be concluded that CPX induces degradation of STAT3 probably via the proteasome pathway.

Example 3: Canonical and non-canonical STAT3 transcriptional function are inhibited by CPX

The levels of phosphorylated STAT3 (P-STAT3) were analyzed using Western blotting after 8, 24 or 48 hours of 10 mM CPX treatment or solvent control. It was found that levels of P- STAT3 were reduced in both SiHa and SW756 starting after 24 hours (Figure 6A). At 8 hours no reduction of P-STAT3 could be observed (Figure 6A), correlating with the levels of total STAT3.

To evaluate the amounts of STAT3 in the nucleus of cells, nuclear fractionation was performed when harvesting the protein of SiHa and SW756 cells treated with 10 pM CPX or EtOH as a solvent control for 8, 24 and 48 hours to see whether total nuclear STAT3 protein, i.e. the phosphorylated and unphosphorylated form together, is reduced as well. Analysis of the nuclear and the cytoplasmic protein fractions by western blot shows that total STAT3 is indeed also downregulated in the nucleus after CPX treatment (Figure 6B) similar to the total cellular STAT3 levels. In SW756 a reduction could be even observed at the 8-hour time-point already. Moreover, it was found that STAT3 is mainly present in the cytoplasm both with and without treatment with CPX. The levels of lamin A/C and vinculin demonstrate only low levels of cross-contamination between nuclear and cytoplasmic proteins or vice versa , respectively (Figure 6B).

CPX can also strongly diminish total STAT3 protein amounts in cells other than cervical cancer cells. Figure 7 depicts examples of STAT3 repression upon CPX treatment in head & neck cancer cells, in melanoma cells, and in colon cancer cells. Aoki et al. (2003), Blood 101:1535

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