Patent Description:
Myeloproliferative neoplasms (MPNs), or myeloproliferative diseases (MPDs), are a group of diseases of the bone marrow in which excess cells are produced. MPNs arise from precursors of the myeloid lineages in the bone marrow, and may encompass bone marrow fibrosis. Bone marrow (BM) fibrosis is characterized by continuous replacement of blood-forming cells in the bone marrow by excessive scar tissue. Primary myelofibrosis (PMF) is the prototypic example of progressive development of BM fibrosis. The hallmark feature of overt PMF is the excess deposition of extracellular matrix (ECM) which is accompanied by a progressive loss of hematopoiesis (cytopenia) causing transfusion dependency, splenomegaly due to extramedullary hematopoiesis and ultimately bone marrow failure. Currently the only available and potentially curative treatment of MPN with fibrosis is bone marrow transplantation. However, bone marrow transplantation is a complex treatment carrying a significant risk of serious complications. In addition, a majority of patients is not eligible for this challenging procedure e.g. due to age, disease stage, comorbidities and the lack of a suitable donor.

Although the molecular alterations in hematopoietic cells which drive the development of myeloproliferative neoplasms (MPN) have been largely defined (Vainchenker et al. , <NUM>; Rampal et al. , <NUM>), reactive cellular alterations in the non-hematopoietic compartment remain rather obscure and have not been studied at single cell level.

The bone marrow morphology in patients with PMF suggests that there is a stepwise evolution from an initial pre-fibrotic phase with absent to minimal fibrosis, to a fibrotic phase with marked reticulin or collagen fibrosis, and often accompanied by osteosclerosis. However, the initial changes and sequential events underlying the pre-fibrotic phase of the disease are not well characterized (Barbui et al.

The plethora of stromal cells in a normal, healthy HSC niche (Baryawno et al. , <NUM>; Tikhonova et al. , <NUM>) suggests that different stromal subtypes not only have distinct roles in normal hematopoiesis but also in bone marrow fibrosis, including PMF. In solid organ fibrosis, mesenchymal stromal cells (MSCs), which in this context are also often referred to as MSClike cells, are thought to be a major cellular source for fibrosis-driving cells (EI Agha et al. These findings are mainly derived from genetic fate tracing experiments which allow to study the cell fate in response to a pathological stimulus. The well accepted hypothesis in solid organ fibrosis is that in the presence of fibrosis-inducing insults, such as exposure to proinflammatory and pro-fibrotic cytokines, affects MSCs/MSC-like cells and leads to a fate switch towards myofibroblasts, which in turn drives fibrosis by depositing ECM.

One major open question in solid organ fibrosis is if MSCs are a distinct or heterogeneous cell population. In the bone marrow, it has been demonstrated that MSCs contribute to fibrosis-driving α-SMA+ myofibroblasts (Decker et al. , <NUM>; Schneider et al. , <NUM>) but it has remained unknown if they are the only source of fibrosis-driving cells in the bone marrow microenvironment or if other cell types of the non-hematopoietic bone marrow niche contribute to the fibrotic transformation.

<CIT> describes the use of an inhibitor of an S100 protein, such as S100A8 and S100A9, for the treatment of cancer, i. polycythemia vera and essential thrombocytosis, not however myelofibrosis.

Diklić M, et al. (Cell Biochem Funct. <NUM> Jun;<NUM>(<NUM>):<NUM>-<NUM>. doi: <NUM>/cbf. Epub <NUM> Dec <NUM>. PMID: <NUM>) and DiklićM, et al. (Haematologica, vol. <NUM>, June <NUM> (<NUM>-<NUM>), page <NUM>, XP009524513, & <NPL>) identify S100 proteins (particularly S100A8 and S100A9) as biomarkers and therapeutic targets for myeloproliferative neoplasms such as polycythemia vera, essential thrombocythemia and myelofibrosis.

<NPL>) mentions that tasquinimod, as inhibitor of an S100 protein, has been used with varying outcome in the treatment of some inflammatory diseases.

There is still a need in the art for an effective and/or earlier treatment of myeloproliferative neoplasms, in particular for the treatment of primary myelofibrosis.

The dependent claims depict other embodiments of the invention. Any embodiment not falling under the scope of the appended claims does not form part of the invention.

A first embodiment is an inhibitor of an S100 protein, as defined in the claims, for use in the prevention or treatment of a myeloproliferative neoplasm, wherein the myeloproliferative neoplasm is myelofibrosis.

In one embodiment, the S100 protein is at least one of S100A8 and S100A9.

The inhibitor for use according to any one of the preceding embodiments, is an inhibitor that inhibits S100 protein functional activity.

The inhibitor inhibiting S100 protein functional activity is a small molecule of formula (I)
<CHM>
or a pharmaceutically acceptable salt thereof,
wherein.

In some embodiments, R<NUM> is methyl or ethyl.

In some embodiments, R<NUM> is in para-position and is selected from the group consisting of methyl, methoxy, fluoro, chloro, trifluoromethyl, and trifluoromethoxy.

In some embodiments, the inhibitor is <NUM>-hydroxy-<NUM>-methoxy-N,I-dimethyl-<NUM>-oxo-N-[<NUM>-(trifluoromethyl)phenyl] -<NUM>,<NUM>-dihydroquino line-<NUM> -carboxamide (tasquinimod).

In some embodiments, the prevention or treatment is by administration of the inhibitor at a total daily dose of about <NUM> - <NUM>.

In some embodiments, the prevention or treatment is by oral administration of the inhibitor.

In some embodiments, the prevention or treatment further comprises at least one of radiation therapy, chemotherapy, and stem cell transplantation.

Various terms relating to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references:<NPL>; <NPL> and periodic updates; and the series <NPL>.

"A," "an," and "the": these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"About" and "approximately": these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±<NUM>% or ±<NUM>%, more preferably ±<NUM>%, even more preferably ±<NUM>%, and still more preferably ±<NUM>% from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about <NUM> to about <NUM> should be understood to include the explicitly recited limits of about <NUM> and about <NUM>, but also to include individual ratios such as about <NUM>, about <NUM>, and about <NUM>, and sub-ranges such as about <NUM> to about <NUM>, about <NUM> to about <NUM>, and so forth.

"And/or": The term "and/or" refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, with "at least" a particular value means that particular value or more. For example, "at least <NUM>" is understood to be the same as "<NUM> or more" i.e., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,.

"Comprising": this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

Exemplary: this term means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

As used herein "cancer" and "cancerous", refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also referred to as malignant neoplasm. A preferred cancer is a blood cancer, preferably the blood cancer is a myoloproliferative neoplasm.

As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with a further (second, third) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered.

As used herein "simultaneous" administration refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be provided orally whereas the other drug may be provided intravenously. Separate includes the administration of the drugs in separate form and/or at separate moments in time, but again, not necessarily via the same route of administration. Sequentially indicates that the administration of a first drug is followed, immediately or in time, by the administration of the second drug.

A used herein "compositions", "products" or "combinations" useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. The compositions, formulations, and products according to the disclosure invention normally comprise the drugs (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

As used in the context of the invention, the terms "prevent", "preventing", and "prevention" refers to the prevention or reduction of the recurrence, onset, development or progression of a disease, preferably a myeloproliferative neoplasm as defined herein, or the prevention or reduction of the severity and/or duration of the disease or one or more symptoms thereof.

As used herein, the terms "treat", "treating" and "treatment" refer to the reduction or amelioration of the progression, severity, and/or duration of a disease, preferably a myeloproliferative neoplasm as defined herein, and/or reduces or ameliorates one or more symptoms of the disease.

As used in the context of the invention, the terms "therapies" and "therapy" can refer to any protocol(s), method(s) and/or agent(s), preferably as specified herein below, that can be used in the prevention, treatment, management or amelioration of a disease, preferably a myeloproliferative neoplasm as defined herein below, or one or more symptoms thereof.

As used herein, the term "effective amount" refers to the amount of an agent, e.g., of an inhibitor as defined herein, which is sufficient to reduce the severity, and/or duration of a myeloproliferative neoplasm, ameliorate one or more symptoms thereof, prevent the advancement of the myeloproliferative neoplasm, or cause regression of the myeloproliferative neoplasm, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of the myeloproliferative neoplasm or one or more symptoms thereof. Alternatively or in addition, the effective amount of the inhibitor can be an amount that enhances or improves the prophylactic and/or therapeutic effect(s) of another therapy. Preferably, the effective amount of the inhibitor as defined herein prevents the onset, reduces and/or reverses the progression of primary myelofibrosis in a subject in need thereof. Preferably, the effective amount of the inhibitor as defined herein prevents the onset of primary myelofibrosis in a subject in need thereof. Preferably, the effective amount of the inhibitor as defined herein reduces the progression of primary myelofibrosis in a subject in need thereof. Preferably, the effective amount of the inhibitor as defined herein reverses the progression of primary myelofibrosis in a subject in need thereof.

Preferably, the effective amount of the inhibitor as defined herein prevents the onset, reduces and/or reverses the progression of primary myelofibrosis in a subject having a myeloproliferative neoplasm. Preferably, the effective amount of the inhibitor as defined herein prevents the onset of primary myelofibrosis in a subject having a myeloproliferative neoplasm. Preferably, the effective amount of the inhibitor as defined herein reduces the progression of primary myelofibrosis in a subject having a myeloproliferative neoplasm. Preferably, the effective amount of the inhibitor as defined herein reverses the progression of primary myelofibrosis in a subject having a myeloproliferative neoplasm.

The effective amount of the inhibitor used to practice the present invention for therapeutic treatment of a myeloproliferative neoplasm may vary depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. Thus, in connection with the administration of an inhibitor which, in the context of the current disclosure, is "effective against" a myeloproliferative neoplasm indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

The inhibitor as detailed herein is preferably an (active) agent. The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be a compound or a composition.

The medical use herein described is formulated as a compound as defined herein for use as a medicament for treatment of the stated disease(s) but could equally be formulated as a method of treatment of the stated disease(s) using a compound as defined herein, a compound as defined herein for use in the preparation of a medicament to treat the stated disease(s), and use of a compound as defined herein for the treatment of the stated disease(s) by administering an effective amount. Such medical uses are all envisaged by the present invention.

As used herein, the term "small molecule" can refer to compounds that are "natural product-like" but mostly will refer synthetic compounds. A small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than <NUM> Daltons (<NUM> kD), preferably less than <NUM> kD, still more preferably less than <NUM> kD, and most preferably less than <NUM> kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than <NUM> Daltons.

The term "protein" or "polypeptide" refers to a molecule consisting of a chain of amino acids, without reference to a specific mode of action, size, <NUM> dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred to as a "protein. " A protein as defined herein and as used in any method as defined herein may be an isolated protein. An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or animal host cell. Preferably, the protein comprises more than <NUM> amino acid residues.

Examples of pharmaceutically acceptable salts comprise salts with (as counter ion) an alkali metal ion, e.g. Li+, Na+ or K+, or with an alkaline earth ion, e.g. Mg++ or Ca++, or with any other pharmaceutically acceptable metal ion, e.g. Zn++ or Al<NUM>+; or pharmaceutically acceptable salts formed with organic bases, such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

The term "C1-C4 alkyl" refers to a branched or unbranched alkyl group having from <NUM>, <NUM>, <NUM> or <NUM> carbon atoms, i.e. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tertbutyl. The term methoxy refers to the moiety MeO-, or CH3O-. The term ethoxy refers to the moiety EtO-, or CH<NUM>CH<NUM>O-. The terms fluoro, chloro and bromo also may be represented by F, Cl and Br. The term trifluoromethyl refers to the moiety CF<NUM>-. The term trifluoromethoxy refers to the moiety CF<NUM>O-.

The inventors discovered that S100 proteins, and in particular the Alarmins S100A8 and S100A9, were upregulated in the blood cancer myeloproliferative neoplasms (MPN) in a murine model as well as in patient samples. Inhibiting the activity of S100A8 and S100A9 in an MPN mouse model strikingly improved disease associated symptoms and fibrosis.

The inventors discovered that inhibiting the activity of an S100 protein, in particular an S100A8/A9 protein, results in an effective and/or earlier treatment of myeloproliferative neoplasms, in particular the treatment of primary myelofibrosis. This novel treatment can prevent the progression of the myeloproliferative neoplasm, and/or reverses the disease. In addition, the treatment can be used in combination with another available treatment for a myeloproliferative neoplasm, in particular in combination with another treatment for primary myelofibrosis, preferably to target complementary pathways. The inventors further discovered that the S100 protein, preferably the S100A8/A9 protein, can function as a biomarker indicative of a myeloproliferative neoplasm, and may be used to stratify patients.

In a first aspect, the invention therefore pertains to an inhibitor of an S100 protein, as defined in the claims, for use in the prevention or treatment of a myeloproliferative neoplasm, which is myelofibrosis.

Preferably, the prevention or treatment as described herein is the prevention or treatment of a myeloproliferative neoplasm as defined herein in a mammalian subject, preferably a human subject.

A myeloproliferative neoplasm (MPN) is a type of blood cancer, characterized by the overproduction of at least one of red blood cells, white blood cells and platelets.

The MPN may be categorized by the presence or absence of the so-called Philadelphia chromosome, which is a genetic abnormality in chromosome <NUM>. More in particular, the (Philadelphia) chromosome <NUM> comprises a reciprocal translocation, t(<NUM>;<NUM>)(q34;q11), of genetic material between chromosome <NUM> and chromosome <NUM>, and contains the fusion gene BCR-ABL1. Preferably, the MPN is negative for the Philadelphia chromosome.

An MPN may be characterized by the presence, or development of, a fibrotic phase. Alternatively or in addition, the MPN is therefore preferably associated with a fibrotic phase. It is understood herein that the prevention or treatment as detailed herein can be before, during and/or after the onset of a fibrotic phase, preferably bone marrow fibrosis. The fibrotic phase is preferably characterized by at least one of collagenous fibrosis and reticulin fibrosis, preferably in the bone marrow. The MPN as defined herein preferably may result in, or is characterised by, an fibrotic phase, preferably a bone marrow fibrotic phase.

Alternatively or in addition, the MPN is preferably associated with a JAK2 mutation, preferably a JAK2 V617F mutation or a functional equivalent thereof, such as but not limited to, a JAK2 exon <NUM> mutation.

Essential thrombocythemia (ET) is a chronic blood cancer (myeloproliferative neoplasm) characterised by the overproduction of platelets (thrombocytes) by megakaryocytes in the bone marrow. Essential thrombocythemia may develop into myelofibrosis.

Polycythemia vera is a myeloproliferative neoplasm in which the bone marrow makes too many red blood cells. It may also result in the overproduction of white blood cells and platelets. Polycythemia vera may develop into myelofibrosis.

As defined herein, the myeloproliferative neoplasm is myelofibrosis. Myelofibrosis (also called primary myelofibrosis, chronic idiopathic myelofibrosis, or agnogenic myeloid metaplasia) is a disorder in which normal bone marrow tissue is gradually replaced with a fibrous scar-like material. Over time, this may lead to progressive bone marrow failure. Myelofibrosis can the advanced stage of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Around one third of people with myelofibrosis have been previously diagnosed with polycythemia (post-polycythaemic myelofibrosis) or essential thrombocythaemia (post-ET myelofibrosis). In primary myelofibrosis, chemicals released by high numbers of platelets and abnormal megakaryocytes (platelet forming cells) may over-stimulate the fibroblasts. This can result in the overgrowth of thick coarse fibres in the bone marrow, which gradually replace normal bone marrow tissue. Over time this may destroy the normal bone marrow environment, preventing the production of adequate numbers of red cells, white cells and platelets, i.e. resulting in cytopenia. This can result in anaemia, low platelet counts (summarized as cytopenia) and the production of blood cells in areas outside the bone marrow for example in the spleen and liver, which become enlarged as a result.

Administration of the inhibitor of the S100 protein to prevent or treat an MPN as defined herein, may prevent, normalize or reduce at least one symptom associated with the MPN. Preferably, administration of the inhibitor of the S100 protein to prevent or treat an MPN as defined herein, may prevent, normalize or reduce at least one symptom associated with an MPN-related bone marrow fibrosis. Preferably administration of the inhibitor of the S100 protein to prevent or treat an MPN as defined herein, may prevent, normalize or reduce at least one of leucocytosis, splenomegaly, platelet counts, thrombocytosis and fibrosis grade. Preferably administration of the inhibitor of the S100 protein to prevent or treat an MPN as defined herein, may reduce the grade of myelofibrosis.

Currently, there is no therapy available to reduce or prevent myelofibrosis in patients suffering from an MPN. The inventors have now discovered that using an inhibitor of S100A8/A9, MPN disease progression can be halted, in particular progression into fibrotic phase is prevented and even reversed.

Hence an inhibitor of an S100 protein, as defined in the claims, preferably an inhibitor of at least one of S100A8/A9, can be used for the prevention and/or reduction of myelofibrosis in a subject in need thereof. In addition or alternatively, an inhibitor of an S100 protein, as defined in the claims, preferably an inhibitor of at least one of S100A8/A9, can be used to reduce grade <NUM> myelofibrosis to at least one of grade <NUM>, grade <NUM>, or grade <NUM> in a subject suffering from an MPN as defined herein. An inhibitor of an S100 protein, as defined in the claims, preferably an inhibitor of at least one of S100A8/A9, can be used to reduce grade <NUM> myelofibrosis to at least one of grade <NUM> or grade <NUM> in a subject suffering from an MPN as defined herein. An inhibitor of an S100 protein, as defined in the claims, preferably an inhibitor of at least one of S100A8/A9, can be used to reduce grade <NUM> myelofibrosis to grade <NUM> in a subject suffering from an MPN as defined herein.

The inventors discovered that the inhibition of an S100 protein can be used for the prevention or treatment of a myeloproliferative neoplasm as defined herein above. The S100 proteins are preferably a family of low-molecular-weight proteins characterized by two calcium-binding sites that have helix-loop-helix (ËF-hand type") conformation. There are at least <NUM> different S100 proteins and they are encoded by a family of genes whose symbols use the S100 prefix. They are considered Damage-associated molecular pattern molecules (DAMPs, also termed Alarmins). S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation.

The S100 protein to be inhibited in accordance with the invention preferably is a mammalian S100 protein, more preferably a human S100 protein. The S100 protein to be inhibited is preferably selected from the group consisting of S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7 (psoriasin), S100A8 (calgranulin A), S100A9 (calgranulin B), S100A10, S100A11, S100A12 (calgranulin C), S100A13, S100A14, S100A15 (koebnerisin), S100A16, S100B and S100P. More preferably the S100 protein to be inhibited is an S100A protein, of which at least one of S100A8 and S100A9 is preferred, of which S100A9 is most preferred. S100A8 and S100A9 form a heterodimer called calprotectin, which is secreted during inflammation. Other names for calprotectin include MRP8-MRP14, calgranulin A and B, cystic fibrosis antigen, L1, 60BB antigen, and 27E10 antigen. In an embodiment, the S100 protein to be inhibited in accordance with the invention is preferably the heterodimer calprotectin.

An inhibitor of an S100 protein, preferably an inhibitor of at least one of S100A8 and S100A9, is herein defined as any agent that reduces or eliminates the activity, preferably the specific activity, of said S100 protein in a cell. Preferably, the activity of the S100 protein is reduced or eliminated in the cell by reducing or inhibiting the functional activity of the S100 protein in the cell.

The inventors discovered that the inhibition of an S100 protein, in particular the inhibition of at least one of S100A8 or S100A9, can be used for the prevention or treatment of an MPN.

The inhibitor of the S100-protein, preferably at least one of S100A8 and S100A9, is an agent that inhibits S100 protein functional activity. The agent that inhibits S100 protein functional activity is a small moleculeas defined in the claims.

The skilled person may use any conventional means to determine whether the agent inhibits S100 protein functional activity as defined herein, preferably inhibits S100A8 or S100A9 protein functional activity. Such conventional means may include, but are not limited to, ELISA assays, e.g. to measure secretion or reduced inflammation, and western blotting (see e.g. <NPL>).

The inhibitor may inhibit at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of the functional activity of an S100 protein, preferably of an S100A8 or S100A9 protein, wherein <NUM>% indicates a complete absence of functional activity as compared to a control. The control is preferably the activity of the S100 protein, preferably the S100A8 or S100A9 protein, that is not exposed to the inhibitor.

The inhibitor is a quinoline carboxamide as defined in the claims. Processes for preparing therapeutically active quinoline carboxamides have been described in <CIT> and <CIT>. A deuterated form of a quinoline carboxamide is described in <CIT>. Pharmaceutical compositions containing a salt of a quinoline carboxamide having enhanced stability during long-term storage at room temperature, methods for the manufacture of such compositions, crystalline salts of quinoline carboxamides and methods for preparing crystalline salts of quinoline carboxamides are described in <CIT>.

The inhibitor is a small molecule of formula (I)
<CHM>
or a pharmaceutically acceptable salt thereof,
wherein R<NUM> preferably is selected from the group consisting of H, methyl, ethyl, n-propyl, iso-propyl, methoxy, ethoxy, fluoro, chloro, bromo, trifluoromethyl, and trifluoromethoxy. In some embodiments, R<NUM> is selected from methyl, ethyl, n-propyl, iso-propyl, methoxy, ethoxy, fluoro, chloro, bromo, trifluoromethyl, and trifluoromethoxy. In some other embodiments, R<NUM> is selected from ethyl, n-propyl, iso-propyl, methoxy, ethoxy, chloro, bromo, trifluoromethyl, and trifluoromethoxy. In still other embodiments, R<NUM> is selected from ethyl, methoxy, chloro, and trifluoromethyl. In some particular embodiments, R<NUM> is methoxy.

The moiety R<NUM> preferably is a C1-C4 alkyl radical, which radical may be branched or linear. In some embodiments, R<NUM> is a C1-C3 alkyl radical. In some embodiments, R<NUM> is methyl or ethyl. In some particular embodiments, R<NUM> is methyl.

The moiety R<NUM> preferably is selected from methyl, methoxy, hydrogen, fluoro, chloro, bromo, trifluoromethyl, and trifluoromethoxy. In some embodiments, R<NUM> is selected from methyl, methoxy, fluoro, chloro, trifluoromethyl, and trifluoromethoxy. In some embodiments, R<NUM> is hydrogen or trifluoromethyl. In some embodiments, R<NUM> is trifluoromethyl.

R<NUM> is preferably selected from hydrogen, fluoro and chloro, with the proviso that R<NUM> is selected from fluoro and chloro only when R<NUM> is selected from fluoro and chloro. In some embodiments, R<NUM> is hydrogen or fluoro. In some particular embodiments, R<NUM> is hydrogen.

In some particular embodiments, in a compound of formula (I),.

In some other particular embodiments, in a compound of formula (I),.

In some embodiments, R<NUM> is in para-position, i.e. the compound for use as defined herein may be represented by formula (la)
<CHM>
wherein R<NUM>, R2, R<NUM> and R<NUM> preferably are as defined herein above.

For example, in some embodiments of a compound of formula (la),.

In some other particular embodiments, in a compound of formula (la),.

As noted herein above, in some embodiments, R<NUM> is hydrogen. In those embodiments, the compound of formula (I) may be represented by formula (Ib)
<CHM>
wherein R<NUM>, R<NUM> and R<NUM> preferably are as defined herein above.

For example, in some embodiments of a compound of formula (Ib),.

In some particular embodiments of a compound of formula (I), R<NUM> is in para-position and R<NUM> is H, and the compound for use as defined herein may then be represented by formula (Ic)
<CHM>
wherein R<NUM>, R<NUM> and R<NUM> are as defined herein above.

In some particular embodiments of a compound of formula (Ic),.

For the purpose of the present invention, any reference to a compound of formula (I) also should be understood as a reference to a compound of any one of the formulas (Ia), (Ib) and (Ic), unless otherwise specified or apparent from the context.

In one embodiment, the compound of formula (I) is <NUM>-hydroxy-<NUM>-methoxy-N,<NUM>-dimethyl-<NUM>-oxo-N-[<NUM>-(trifluoromethyl)phenyl]-<NUM>,<NUM>-dihydroquinoline-<NUM>-carboxamide (tasquinimod), of the structural formula:
<CHM>.

A preferred inhibitor is tasquinimod or a functional equivalent thereof. A preferred inhibitor is tasquinimod or analogues thereof, for instance a small molecule of formula (I) as defined herein. Preferably, the inhibitor is tasquinimod. Tasquinimod (ABR-<NUM>) is a known inhibitor of S100A9 (see e.g. <NPL>). Compounds of formula (I), pharmaceutically acceptable salts thereof, deuterated forms thereof, crystalline salts thereof, and pharmaceutical compositions containing the compounds and their salts, as well as methods for preparing such compounds, their salts, deuterated forms and pharmaceutical compositions containing the compounds and their salts have been described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

In some embodiments, any reference to a compound of formula (I) also encompasses the deuterated form of thereof. As mentioned herein above, a deuterated form of tasquinimod is described in <CIT>. The person of ordinary skill in the art will be capable of preparing analogously deuterated compounds of formula (I) by following the description of in said WO pamphlet. In some embodiments, thus, the compound of formula (I) has a deuterium enrichment in the moiety R<NUM> of formula (I) of at least <NUM>%, more preferably at least <NUM>%. For example, in some embodiments, R<NUM> is methyl having a deuterium enrichment of at least <NUM>%,more preferably at least <NUM>%. In some particular embodiments, the compound of formula (I) is tasquinimod having a deuterium enrichment in the amide-N methyl group of at least <NUM>%, more preferably at least <NUM>%. In some other embodiments, the compound of formula (I) is non-deuterated, having a deuterium content corresponding to the natural abundance of deuterium.

In an embodiment, the compound is the quinolone-<NUM>-carboxamide paquinimod (ABR-<NUM>), having the structural formula:
<CHM>
Paquinimod is a known inhibitor of S100A9 (see e.g. <NPL>. Hence in some embodiments, the inhibitor for use according to the invention has structural formula I),wherein R<NUM> and R<NUM> are ethyl and wherein R<NUM> and R<NUM> are hydrogen.

In an embodiment, the inhibitor of an S100 protein, as defined herein above is for the prevention or treatment of an MPN as defined herein above, wherein the inhibitor is administered at a total daily dose of about <NUM> - <NUM>.

It is understood that the terms "daily dose" and "total daily dose" are used interchangeably herein. In particular, the total daily dose can be administrated over one or several units (doses) per day as detailed herein below.

Preferably, the total daily dose is therapeutically effective and preferably also well tolerated, e.g. does not cause a side effect. A side effect is herein defined as an effect, whether therapeutic or adverse, that is secondary to the one intended. The side effect is preferably an adverse effect. Side effects can be graded as mild (grade <NUM>), moderate (grade <NUM>), severe (grade <NUM>) or potentially life threatening (grade <NUM>). Preferably, the administered total daily dose is well-tolerated, e.g. does not cause any side effects that are grade <NUM>, <NUM>, <NUM> and/or <NUM>, preferably the administered total daily dose does not cause any side effects that are grade <NUM>, <NUM> and/or grade <NUM> and more preferably the administered total daily dose does not cause any side effects that are grade <NUM> or <NUM>.

Preferably, the administered total daily dose does not cause a very common, common, uncommon, rare or very rare side effect. More preferably the administered dose does not cause a very common, common, uncommon or rare side effect, even more preferably the administered total daily dose does not cause a very common, common or uncommon side effect and even more preferably the administered total daily dose does not cause a very common or common side effect. Most preferably the administered total daily dose does not cause a very common side effect.

A very common side effect is herein defined as the probability (chance) of experiencing the side effect is >=<NUM>/<NUM>, a common (frequent) side effect is herein defined as the probability of experiencing the side effect is >=<NUM>/<NUM> and <<NUM>/<NUM>, an uncommon (infrequent) side effect is herein defined as the probability of experiencing the side effect is >=<NUM>/<NUM> and <<NUM>/<NUM>, a rare side effect is herein defined as the probability of experiencing the side effect is >=<NUM>/<NUM> and <<NUM>/<NUM> and a very rare side effect is herein defined as the probability of experiencing the side effect is <<NUM>/<NUM>.

The inhibitor can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the inhibitor as defined herein may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with any further active agents, the active agents can be formulated as separate compositions that are given at the same time or different times, or the active agents can be given as a single composition.

The total daily dose of the S100 protein inhibitor is preferably in the range of <NUM> - <NUM>. Preferably, the total daily dose of a small molecule S100 protein inhibitor as defined herein is in the range of <NUM> - <NUM>. Preferably, the total daily dose of tasquinimod is in the range of <NUM> - <NUM>.

Preferably the total daily dose is in the range of about <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM>, <NUM> - <NUM> or <NUM> - <NUM>. Preferably, the total daily dose is about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or about <NUM>. Preferably, the total daily dose is about <NUM>, <NUM> or <NUM>. Preferably, the total daily dose is about <NUM>.

The total daily dose can be divided over several doses per day. These separate doses may differ in amount. For example for each total daily dose, the first dose may have a larger amount of the compound than the second dose or vice versa. However preferably, the compound is administered in similar or equal doses. The inhibitor may be administered <NUM>, <NUM>, <NUM>, <NUM>, <NUM> times per day, or more often. Preferably, the inhibitor is administered once a day. The inhibitor may also be administered once every <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more days.

The inhibitor, or a composition comprising the inhibitor, as defined herein may be administered enterally, orally, parenterally, sublingually, by inhalation (e. as mists or sprays), rectally, or topically, preferably in dosage unit formulations containing conventional nontoxic pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles as desired. Preferably the inhibitor, or a composition comprising the inhibitor, is administered orally.

For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e. via nasal mucosa), subdural, rectal, gastrointestinal, and the like, and directly to a specific or affected organ or tissue, e.g. a cancerous tissue. For delivery to the central nervous system, spinal and epidural administration, or administration to cerebral ventricles, can be used. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

The inhibitor of an S100 protein as defined herein can be mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for the desired route of administration. The inhibitor as defined herein may be administered by supplementation via gastric or percutaneous tubes.

In a preferred embodiment the invention pertains to an inhibitor as defined herein above, for use in treating, preventing, or suppressing symptoms associated with the MPN by administration of an effective total daily dose for a specified period of time.

The dosage form for oral administration can be a solid oral dosage form. The class of solid oral dosage forms consists primarily of tablets and capsules, although other forms are known in the art and can be equally suitable. When used as a solid oral dosage form, an inhibitor or a composition comprising an inhibitor as defined herein, may e.g. be administered in the form of an immediate release tablet (or a capsule and the like) or a sustained release tablet (or a capsule and the like). Any suitable immediate release or sustained release solid dosage forms can be used in the context of the invention as will be evident for the skilled person.

A composition as described herein, for a use as described herein, can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, and in other suitable forms. The composition can also be administered in liposome formulations. The composition comprising the S100 protein inhibitor can be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.

Preferably a composition comprising a compound as defined herein, preferably a sulphonamide as defined herein, is administered orally or parenterally.

In an aspect, the invention pertains to a composition comprising an inhibitor of an S100 protein as defined herein. The composition may be suitable for use in cell culture, preferably animal cell culture, more preferably mammalian cell culture. The composition preferably is a pharmaceutical composition. The composition is preferably for a use in the prevention or treatment of a myeloproliferative neoplasm (MPN), preferably an MPN as defined herein above.

A composition may comprise one type of compound as defined herein or a combination of compounds as defined herein, e.g. a combination of S100 protein inhibitors. A composition may comprise at least <NUM>, <NUM>, <NUM> or more different types of S100 protein inhibitors.

The composition may comprise an S100 protein inhibitor together with a physiologically acceptable carrier. In particular, the composition can be formulated as pharmaceutical composition by formulation with additives such as pharmaceutically or physiologically acceptable excipients, carriers, and vehicles.

Suitable pharmaceutically or physiologically acceptable excipients, carriers and vehicles can include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "<NPL>), and "<NPL>).

Pharmaceutical compositions as described herein for use according to the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. In a preferred embodiment, a composition as defined herein is administered in a solid form or in a liquid form.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms the composition, preferably a composition comprising a S100 protein inhibitor as described herein, may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Preferably, the composition is in the form of a capsule.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or saline. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers / liquid dosage forms contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof.

A composition as described herein can be admixed with an aqueous solution prior to administration. The aqueous solution should be suitable for administration and such aqueous solutions are well known in the art. It is further known in the art that the suitability of an aqueous solution for administration may be dependent on the route of administration. The aqueous solution is an isotonic aqueous solution. The isotonic aqueous solution preferably is almost (or completely) isotonic to blood plasma. In an even more preferred embodiment, the isotonic aqueous solution is saline.

The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, flavorants and the like. Preferred flavorants are sweeteners, such as monosaccharides and / or disaccharides. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the composition as defined herein can be prepared by mixing with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the compound and/or immune checkpoint blocking agent.

For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions for use in the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

Time-release, sustained release or controlled release delivery systems may be used for administration of one or more of the compositions as described herein, such as a diffusion controlled matrix system or an erodible system, as described for example in: <NPL> and <NPL>, in "<NPL>. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g. , by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes).

A composition as defined herein can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound, immune checkpoint blocking agent or a combination of a compound and an immune checkpoint blocking agent as defined herein, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, <NPL>).

A pharmaceutical composition as defined herein, i.e. comprising an S100 protein inhibitor, can comprise a unit dose formulation, wherein the unit dose is a dose sufficient to have a therapeutic or preventive effect of a myeloproliferative neoplasm as defined herein, and/or an amount effective to reduce, or knock out, the expression of a S100 protein in a cell, preferably a mesenchymal stromal cell, preferably a mesenchymal stromal cell located in the bone marrow.

The unit dose may be sufficient as a single dose to have a preventive and/or therapeutic effect on an MPN as defined herein. Alternatively, the unit dose may be a dose administered periodically in a course of treatment of an MPN as defined herein. During the course of the treatment, the concentration of the subject compositions may be monitored to insure that the desired level is maintained.

While the compound for use as described herein can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents known in the art to be used for the treatment or prevention of an MPN.

The current goal for the treatment of an MPN as defined herein is to reduce the number of increased blood cells and to help control any complications as bleeding or blood clotting.

Venesection (or phlebotomy) is a procedure in which a controlled amount of blood is removed from the bloodstream. For many people, particularly younger patients and those with mild disease, regular venesection (every few months) may be all that is needed to control their disease for many years. Chemotherapy is a further option, mostly myelosuppressive agents as hydroxyurea (oral capsule chemotherapy). Interferon is sometimes prescribed for younger patients to help control the production of blood cells. Interferon can be given as an injection under the skin (subcutaneous injection). Many people are prescribed daily doses of aspirin, which have been shown to reduce the risk of thrombosis. Anagrelide hydrochloride (Agrylin) is a drug used to reduce high platelet counts. Anagrelide can be taken in capsule form by mouth.

Myelofibrosis is typically associated with enlargement of the spleen (splenomegaly). Chemotherapy such as hydroxyurea, or thalidomide may be used to reduce an enlarged spleen. In some cases, the surgical removal of the spleen (splenectomy) may be considered. Small doses of radiation to the spleen can also be given to reduce its size. This usually provides temporary relief for about three to six months. Myelofibrosis is generally regarded as incurable. Some younger patients who have a suitably matched donor may be offered an allogeneic (donor) stem cell transplant. Stem cell transplants carry significant risks and are only suitable for a small minority of younger patients (usually under <NUM> years). A common other treatment in myelofibrosis are JAK2 inhibitors which work by blocking the activity of the JAK2 protein, which may lead to a reduction in splenomegaly and decreased symptoms. They also work in patients with myelofibrosis without the JAK2 mutation. A number of JAK2 inhibitors are in clinical trials. The problem is that some patients never respond to JAK2 inhibition or become resistant over time. Moreover JAK2 inhibitors are known to cause cytopenia (a reduction in the number of mature blood cells), rendering JAK2 inhibitors unsuitable for use in the treatment of patients having myelofibrosis in combination with cytopenia.

Prior to the present invention, there was no treatment available for a subject suffering from myelofibrosis in combination with cytopenia. The S100 protein inhibitors as described herein, in particular the S100A8/A9 protein inhibitors described herein, may thus be particularly useful for the treatment of a subject suffering from myelofibrosis in combination with cytopenia.

In addition or alternatively, an S100 protein inhibitor for use in the prevention or treatment of an MPN as defined herein can be used in combination with at least one of radiation therapy, chemotherapy, and stem cell transplantation. The stem cell transplantation is preferably a bone marrow stem cell transplantation.

An S100 protein inhibitor for use in the prevention or treatment of an MPN as defined herein can be used in combination with a myelosuppressive agent. An S100 protein inhibitor for use in the prevention or treatment of an MPN as defined herein can be used in combination with an agent selected from the group consisting of hydroxyurea, thalidomide, interferon, aspirin, anagrelide hydrochloride and an JAK2 inhibitor, or any combination thereof. The S100 protein inhibitor for use in the prevention or treatment of an MPN as defined herein can be used in combination with a JAK2 inhibitor. A preferred JAK2 inhibitor may be ruxolitinib or fedratinib. The inventors discovered that a combined inhibition of the inflammatory axis through S100A8/S100A9 in combination with Ruxolitinib will lead to more efficient inhibition of MPN-associated inflammation (data not shown).

In addition or alternatively, the S100 protein inhibitor for use in the prevention or treatment of an MPN as defined herein can be used in combination with at least one of venesection, splenectomy and a stem cell transplantation.

Further representative agents useful in combination with the inhibitor, for the treatment of an MPN include, but are not limited to, Coenzyme Q, vitamin E, idebenone, MitoQ, EPI-<NUM>, vitamin K and analogues thereof, naphtoquinones and derivatives thereof, other vitamins, and antioxidant compounds.

When further active agents are used in combination with an inhibitor as defined herein, the further active agents may generally be employed in therapeutic amounts as indicated in the <NPL>), or such therapeutically useful amounts as would be known to one of ordinary skill in the art.

A composition as described herein, can be prepared as a medicinal or cosmetic preparation or in various other media, such as foods for humans or animals, including medical foods and dietary supplements.

A "medical food" is a product that is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements exist. By way of example, but not limitation, medical foods may include vitamin and mineral formulations fed through a feeding tube (referred to as enteral administration).

A "dietary supplement" shall mean a product that is intended to supplement the human diet and is typically provided in the form of a pill, capsule, and tablet or like formulation. By way of example, but not limitation, a dietary supplement may include one or more of the following ingredients: vitamins, minerals, herbs, botanicals; amino acids, dietary substances intended to supplement the diet by increasing total dietary intake, and concentrates, metabolites, constituents, extracts or combinations of any of the foregoing. Dietary supplements may also be incorporated into food, including, but not limited to, food bars, beverages, powders, cereals, cooked foods, food additives and candies.

The subject composition thus may be compounded with other physiologically acceptable materials which can be ingested including, but not limited to, foods. In addition or alternatively, the compositions for use as described herein may be administered orally in combination with (the separate) administration of food.

One or more of the pharmaceutical compositions as defined herein may be comprised in a kit of parts. In an aspect, the invention relates to a kit of parts comprising one or more compositions, preferably pharmaceutical compositions, as defined herein above. Preferably, a kit of parts as defined herein is for a use in the treatment of an MPN as defined herein.

Optionally, the kit of parts further comprises a leaflet. The leaflet may comprise instructions for use. In addition or alternatively, the leaflet may be at least one of a patient information leaflet and a Summary of Product Characteristics (an SmPC). Preferably the instructions specify the use of the inhibitor of an S100 protein as defined herein, or the composition comprising the inhibitor, for the prevention or treatment of an MPN as defined herein.

For retroviral and lentiviral transduction, c-kit+-enriched cells from <NUM>-<NUM>-week-old WT mice were isolated by crushing compact bone and cells were lineage depleted by magnetic separation (Miltenyi Biotec). c-kit+ BM cells were pre-stimulated for <NUM> hours in CellGro media (Corning) supplemented by murine stem-cell factor (m-Scf, 50ng/ml, Peprotech) and murine thrombopoietin (m-Tpo, 50ng/ml, Peprotech). Oncoretroviral vectors were pseudotyped with ecotropic envelope and produced using standard protocols. Retroviral transduction was performed on retroNectin (Takara Bio)-coated cell culture dishes loaded with unconcentrated virus. Cells were resuspended in virus containing medium in the presence of 4µg/ml polybrene at <NUM> for a minimum of <NUM> hours. Lentiviral particles were produced by transient transfection with lentiviral plasmid together with pSPAX and VSVG packaging plasmids using Fugene. Lentivirus and retrovirus particles were concentrated by ultracentrifugation at <NUM>.

After sacrifice bones (femurs, hip, spine) were crushed in PBS/<NUM>% FCS on ice. The cells were dissociated by filtering through a <NUM> nylon mesh. For magnetic activated cell sorting cells were incubated with biotinylated antibodies directed against lineages (CD11b, GR1, NK1. <NUM>, TER119, CD4, CD8 and B220), CD45, CD71 and CD41 (biolegend; 6µg/ml/1e7 cells) for <NUM> minutes. After centrifugation for <NUM> minutes at <NUM> × g and <NUM>°, cells were resuspended in 80µl/1e7 cells PBS/<NUM>% FCS, mixed with 20µl/1e7 cells magnetic anti-biotin beads (Biolegend) and incubated for <NUM> minutes at <NUM> with gentle agitation. For magnetic depletion, cells were resuspended in <NUM> PBS/<NUM> % FCS and placed into a cell separation magnet (BD) for <NUM> minutes at <NUM>. For additional purification, the negative fraction was transferred to a new FACS-tube and placed into the cell separation magnet for another <NUM> minutes. The supernatant was centrifuged for <NUM> minutes at <NUM> × g at <NUM>.

Cells were resuspended in 300µl PBS/<NUM>% FCS and stained at <NUM> for <NUM> minutes. Washing was performed by adding <NUM> PBS/<NUM>% FCS and centrifuging for <NUM> minutes at <NUM> × g, <NUM>. After resuspension and addition of Hoechst (<NUM>:<NUM>), lineage/CD45/Ter119 negative cells were sorted into 50µl DMEM/<NUM>% FCS (BD Aria III). Unstained cells were used as negative controls to define gating. All antibodies were acquired from Biolegend. The following fluorochrome conjugated antibodies were used: CD41-APC-Cy7, CD3-APC-Cy7, CD11b-APC-Cy7, Gr1-APC-Cy7, Ter119-APC-Cy7, B220-APC-Cy7, CD45. <NUM>-APC-Cy7, CD45. <NUM>-APC-Cy7, Sca1-PerCP, CD31-APC, CD51-PE.

The libraries were prepared using the Chromium Single Cell <NUM>' Reagent Kits (v2): Single Cell <NUM>' Library & Gel Bead Kit v2 (PN-<NUM>), Chromium Single Cell A Chip Kit v2 (PN-<NUM>) and i7 Multiplex Kit (PN-<NUM>) (10x Genomics), and following the Single Cell <NUM>' Reagent Kits (v2) User Guide (manual part no. CG00052, Rev D). Finalized libraries were sequenced on a Novaseq6000 platform (Illumina), aiming for a minimum of <NUM> reads/cell using the 10x Genomics recommended number of cycles (<NUM>-<NUM>-<NUM>-<NUM> cycles).

We use cellranger (version <NUM>. <NUM>) to align reads to mouse genome mm10 and for detection of cells with default parameters. Next, we used Seurat (v2. <NUM>) for high level analysis of the scRNA-seq (<NPL>). We filtered cells only keeping ones with low amount of mitochondrial genes (< <NUM>), low amount of ribosomal genes (< <NUM>). We removed cells with more than <NUM> UMIs as they represent potential duplets. Finally, we filtered genes detected in less than <NUM> cells. Next, we regressed out cell cycle, the proportion of mitochondrial, ribosomal and UMI counts and performed a log-normalization of read counts using Seurat. Data of ThPO (or Jak2) experiments were then integrated with a canonical correlation analysis (CCA) based on the first <NUM> CCs (Butler et al, supra). Next, we performed a clustering with a SNN of k = <NUM> and produced a t-SNE representation with perplexity set as <NUM>. Differential gene expression analysis was performed with Seurat to find cluster specific genes or genes changing between distinct phenotypes.

GO and pathway enrichment analysis were based on clusterProfiler (Version <NUM>. <NUM>; <NPL>) and CAMERA from edgR (Version <NUM>. <NUM>; <NPL>). We have also proposed gene sets describing "hematopoiesis-support", "MSC progenitor phenotype", "non-collagenous ECM" and "collagenous ECM". Pre-ranked GSEA analysis was performed as described (Version <NUM>; <NPL>). All P-values were corrected by Benjamini-Hochberg correction.

Patient samples were collected and supplied by the University Clinic Hamburg-Eppendorf (UKE); University Hospital RWTH Aachen and University. Samples were deidentified at the time of inclusion. All patients provided informed consent and the data collection was performed in accordance with the Declaration of Helsinki. Control plasma was obtained from the Institute for Clinical Chemistry at Erasmus Medical Center in Rotterdam. Surplus material was collected from dermatological and cardiological patients after diagnostics according to the ethical vote MEC-<NUM>-<NUM> and processed on the day of acquisition. All subjects had no history or indication of any hematological or non-hematological malignancy. Plasma was isolated from whole blood anticoagulated with EDTA by centrifugation (<NUM>×g, <NUM> minutes) and stored at -<NUM>. Samples were thawed gently on ice and centrifuged for <NUM> minutes at <NUM>×g before further processing. Samples were diluted <NUM>:<NUM> and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-<NUM>) according to the manufacturer's instructions.

Murine organs were fixed in <NUM>% paraformaldehyde for <NUM> hours and transferred to <NUM>% ethanol. Femurs were decalcified in <NUM>% EDTA/Tris-HCI (pH <NUM>) solution for <NUM> hours, dehydrated and paraffin embedded. H&E and reticulin staining were performed on <NUM> sections according to established routine protocols.

Human bone marrow biopsies from the patients whose plasma was used for S100A8 quantification in plasma were chosen for histological examination from archived patient samples of paraffin-embedded tissue from the Biobank of Dr. Büsche at the Department of Pathology, Hannover Medical School, Hannover, Germany. Biopsies were primarily taken during earlier hospitalization. Bone marrow biopsies were fixed for <NUM> hours using the Hannover Solution (<NUM> % buffered formaldehyde plus <NUM> % methanol), decalcified (EDTA), dehydrated and embedded in paraffin. Control bone marrow slides were acquired from the Department of Pathology at Erasmus Medical Center. All reference patients showed normal bone marrow characteristics and had evidence for or history of hematological malignancies.

For immunohistochemical analysis of S100A8, antigen retrieval was performed using citrate buffer in a conventional lab microwave (Vector, antigen unmasking solution). Sections were treated with <NUM>% H<NUM>O<NUM> and blocked with Avidin/Biotin blocking kit (Vector), and subsequently incubated with primary antibody (rabbit-anti-S100A8: ab92231 (Abcam), <NUM>:<NUM>; rabbit-anti-CD56: RBK050 (Zytomed Systems), <NUM>:<NUM>) for one hour at room temperature. Biotinylated monoclonal goat anti-rabbit-antibody (Vector) was used as a secondary antibody for <NUM> minutes at room temperature. Slides were incubated with AB complex for <NUM> minutes at room temperature, washed and incubated for a further <NUM> minutes with DAB substrate. Slides were stained with hematoxylin and mounted with glass coverslip using DPX mountant (Sigma).

Statistical analysis - excluding that for single cell RNAseq Data - was conducted using either GraphPad Prism Version or R Version <NUM>. Data are presented as mean ± SEM. Two independent groups with (approximately) normally distributed samples were compared with an unpaired t-test. When normality could not be assumed, Wilcoxon Rank Sum test was used. For ≥ <NUM> groups, ANOVA was employed with post-hoc paired t-test and Bonferroni correction for multiple hypothesis testing.

For JAK2V617F studies, WT BM cells were transduced with JAK2(V617F) (JAK2) retrovirus or control pMIG retrovirus (control: JAK2 empty vector) and transplanted into <NUM> Gy-irradiated <NUM>-<NUM>-week-old female B6. SJL recipients. Mice were randomly assigned to transplant groups. Mice from the early ThPO cohort were sacrificed <NUM> weeks post-transplant, whilst mice in the late ThPO cohort were sacrificed <NUM> weeks post-transplant. Mice used in the JAK2 experiment were sacrificed at <NUM> weeks post-BM transplant, when hemoglobin levels decreased, owing to the varying phenotypic severity of JAK2V617F.

Blood was periodically collected from mice via submandibular bleeds into Microtainer tubes coated with K<NUM>EDTA (Becton Dickinson, NJ, USA) and complete blood counts were performed on a Horiba Scil Vet abc Plus hematology system.

For tasquinimod studies, WT recipient mice (n=<NUM>-<NUM>/group) were lethally irradiated (<NUM>. 5Gy) and received ckit+ cells transduced with JAK2(V611F)- or JAK2(WT) retroviral virus. Tasquinimod treatment (ABR-<NUM>, Biorbyt) was administered via drinking water at <NUM>/kg/day dissolved in <NUM>% sucrose in autoclaved water. Tasquinimod was first dissolved in DMSO and mixed with <NUM>% sucrose, <NUM>% PEG300 (Sigma) in water. Vehicle-treated groups received DMSO-, PEG300-treated water with <NUM>% sucrose. Drinking bottles were renewed twice a week. Treatment was started at <NUM> weeks post-transplant until <NUM> post-transplant, and resumed at <NUM> weeks post-transplant until sacrifice at <NUM> weeks post-transplant. Mice were randomly assigned to transplant and treatment groups.

The JAK2V617F mutation is found in the majority (<NUM>-<NUM>%) of patients with PMF. The retroviral expression of the JAK2V617F mutation in transplanted HSPCs leads to an MPN phenotype including splenomegaly due to extramedullary hematopoiesis and the development of myelofibrosis after a short latency with a high degree of penetrance (<NPL>; <NPL>). The inventors thus either transplanted c-kit+ HSCPs expressing the JAK2V617F mutation or the control empty vector (EV) into lethally irradiated mice. Around <NUM> weeks after transplantation, bone marrow histology demonstrated severe fibrosis, characterized by thick, intercrossing reticulin-positive fibers and also severe osteosclerosis, indicative of grade <NUM> myelofibrosis (<FIG>). Unsupervised clustering of <NUM> high quality non-hematopoietic niche cells lead to the identification of <NUM> distinct clusters/populations (<FIG>): <NUM>-<NUM>) clusters of mesenchymal stromal cells (MSC-<NUM>: adipogenic, MSC-<NUM>: osteogenic, MSC-<NUM>: transition, MSC-<NUM>: interferon high), <NUM>) and <NUM>) clusters of Schwann cell precursors (SCPs; <NUM>: non-myelinating SCPs, nmSCPs; <NUM>: myelinating SCPs, mSCPs), <NUM>) osteoblastic lineage cells (OLCs) and <NUM>) arterial cells (ACs). MSC-<NUM> and MSC-<NUM> showed the highest up-regulation of extracellular matrix (ECM) proteins, in particular "core matrisome" and collagens, confirming that these two MSC populations are the cellular drivers of the fibrotic progression and acquire myofibroblast characteristics in fibrosis (<FIG>). The most significantly de-regulated genes in MSCs could be grouped into <NUM>) down-regulated genes and <NUM>) up-regulated genes with onset of the MPN phenotype (<FIG>). The down-regulated genes comprised <NUM>) MSC markers (Vcam, Lepr, Adipoq) and <NUM>) hematopoiesis-support (Kitl, II7, Igf1, Cxcl2, Csf1, Bmp4). In particular MSC-<NUM> showed a highly significant down-regulation of MSC markers and genes involved in the regulation of hematopoietic stem cells. A significant down-regulation of Vcam1 occured in MSC-<NUM>, MSC-<NUM> and -<NUM>, further indicating a spatial activation and migration of MSC as VCAM1 plays a central role in the firm adhesion of MSC to the ECM. The up-regulated genes were defined through four major biological processes: <NUM>) secreted factors (S100a8, S100a9, Pdgfa), <NUM>) osteogenesis (Mgp, Fndc1), <NUM>) neoangiogenesis (Vegfa, C3) and <NUM>) ECM synthesis (Col8a1, Col1a1). Interestingly, among the secreted factors the alarmins S100a8/S100a9, danger-associated molecular patterns (DAMPs) - known contributors to inflammatory responses - were significantly up-regulated in their expression in MSC-<NUM> and -<NUM>, the subpopulations showing the most significant changes.

The inventors aimed to validate the findings of transcriptional changes in the bone marrow microenvironment in patient bone marrow biopsies. Single cells of the bone marrow microenvironment were isolated from a PMF patient with grade <NUM> (advanced) myelofibrosis from a small piece of a bone marrow punch biopsy and from two age-matched control patients with peripheral lymphoma without contribution to the bone marrow. The HE analysis of the bone marrow showed a hypercellular marrow in the PMF patient with dysplastic megakaryocytes and thick coarse black fibers in the Reticulin staining (<FIG>). In contrast, control patients showed a normocellular bone marrow without any fibers present. Unsupervised clustering of high-quality non-hematopoietic niche cells lead to the identification of <NUM> distinct clusters/populations (<FIG>): <NUM>) mesenchymal stromal cells (MSCs), <NUM>) megakaryocytes (Meg), <NUM>) fibroblasts (Fib), <NUM>) myeloid/granulocyte (myel) and <NUM>) Schwann Cells (SCs). Exactly as seen in the murine model of PMF/MPN MSCs were functionally reprogrammed, lost their hematopoiesis support (support of normal blood formation) and MSC signature and were the fibrosis-driving cells, significantly up-regulating Collagens (<FIG>). Interestingly, and again exactly as seen in the murine model, MSC, that usually do not express S100A8/S100A9 at all, showed strong up-regulation of S100A8/S100A9 (<FIG>). This indicates that the up-regulation of S100A8/S100A9 is a disease-specific/MPN-specific mechanisms that can be both a disease marker and a therapeutic target. Looking into the interaction of S100A8 with down-stream receptors, it became obvious, that mainly the interaction with RAC1/RAC2 increased (<FIG>).

The inventors asked if S100A8 might serve as a marker for progression of (human) bone marrow fibrosis as they also observed increased S100a8/S100a9 expression in murine models in MSCs already in pre-fibrosis (compared to control) which then significantly increased in fibrotic stages in almost all niche cells, suggesting S100A8 as a sensitive marker for disease/fibrosis progression.

The inventors therefore quantified S100A8 in plasma samples (peripheral blood) from MPN patients (n = <NUM>) and age-matched patients without a primary hematological disease (n = <NUM>) by ELISA. S100A8 was significantly increased in MPN patients (<FIG>) and clearly demarcated the transition from MF0 to MF1 and higher MF grades in general, indicating S100A8 as a sensitive biomarker for fibrosis onset or progression in PMF (when follow-up samples are taken; <FIG>). In previous work, the inventors linked increased S100A8 expression in the stroma (in MDS) to decreased hematopoiesis-support (<NPL>), underlining that S100A8 is a good marker for the reprogramming of the stroma from hematopoiesis-support towards fibrotic transformation, in particular if followed up in patients over time.

The inventors further performed immunohistochemistry for S100A8 in bone marrow biopsies from patients and controls (<FIG>). In control bone marrow biopsies (MF0), S100A8 specifically marks neutrophils (S100A8 grade <NUM>). Already in low grades of fibrosis, S100A8 is not only expressed in hematopoietic cells but stains positive in the interstitium (grade <NUM>-<NUM>), while in progressed fibrosis (MF3) stromal cells are strongly positively stained (grade <NUM>). The quantification of the S100A8 score showed that it correlates with disease severity reflected by the MF grade (<FIG>).

To further validate the role of S100A8/S100A9 in the fibrotic initiation and progression, the inventors sought to use a S100A9-inhibitor known as tasquinimod (ABR-<NUM>) in mice developing JAK2(V617F)-mediated myelofibrosis. Tasquinimod, a quinoline compound with anti-angiogenic and anti-tumor activity under investigation for solid tumors (Isaacs et al. , <NUM>, supra), binds to S100A9 protein and impedes its interaction with TLR4 and RAGE receptors. Tasquinimod treatment was administered via drinking water starting at <NUM> weeks post-transplant, until sacrifice at <NUM> weeks post-transplant with a treatment break at <NUM> weeks post-transplant to minimize deleterious effects on normal granulocyte function (<FIG>).

Crucially, JAK2(V617F) mice receiving tasquinimod did not develop MPN-specific leukocytosis, which was observed in the JAK2(V611F)-vehicle-treated mice (<FIG>). Severe splenomegaly, which is a major clinical manifestation in PMF patients, was observed in vehicle-treated JAK2(V617F) mice but completely normalized with tasquinimod treatment (<FIG>).

The inventors observed a progressive increase in peripheral platelet counts until <NUM> weeks post-transplant in JAK2(V617F) mice, followed by a sharp decrease in platelets associated with the progressive replacement of hematopoietic cells in the bone marrow (<FIG>).

Claim 1:
An inhibitor of an S100 protein for use in the prevention or treatment of a myeloproliferative neoplasm, wherein the myeloproliferative neoplasm is myelofibrosis, and wherein the inhibitor is a compound of formula (I)
<CHM>
or a pharmaceutically acceptable salt thereof,
wherein
R<NUM> is selected from the group consisting of H, methyl, ethyl, n-propyl, iso-propyl, methoxy, ethoxy, fluoro, chloro, bromo, trifluoromethyl, and trifluoromethoxy;
R<NUM> is C1-C4 alkyl;
R<NUM> is selected from the group consisting of methyl, methoxy, hydrogen, fluoro, chloro, bromo, trifluoromethyl, and trifluoromethoxy; and
R<NUM> is selected from the group consisting of hydrogen, fluoro and chloro, with the proviso that R<NUM> is selected from fluoro and chloro only when R<NUM> is selected from fluoro and chloro.