Field of the disclosure The present invention relates to use of ceritinib in disorder or a disease mediated by the activity of kinase. It also relates to pharmaceutical composition comprising ceritinib that is suitable for the treatment of such disorder or disease.

Background of the disclosure

FES (feline sarcoma) and FER belong to a distinct subfamily of the cytoplasmic non-receptor protein-tyrosine kinase family. Fes oncogene was first isolated from the tumour-causing feline retroviruses and once compared with cellular homologues from other species it was confirmed that it corresponds to the same gene. FER is closely related to FES (P Greer, Nature Reviews Molecular Cell Biology 3, 278-289, April 2002). In human, FES and FER are encoded by paralogous human FES and FER genes located at chromosome positions 15q26.1 and 5q21 , retrospectively (P Greer et al, Frontiers in Bioscience S4, 489-501 , 2012).

Beside the protein-tyrosine kinase domain, which is at carboxy-terminus, FES and FER proteins include also SRC-homology (SH2), coiled-coil (CC) and F-BAR (the Bin-Amphiphysin-Rvs) domains.

Ceritinib (also named LDK378) was disclosed as compound 66 in Example 7 of WO2008/073687. Chemical formula of ceritinib is 5-chloro-N2-(2-isopropoxy-5-methyl-4- (piperidin-4-yl)phenyl)-N4-[2-(propane-2-sulfonyl)-phenyl]-p yrimidine-2,4-diamine. Initially, it was developed as an anaplastic lymphoma kinase (ALK) inhibitor. ALK gene has been found to be rearranged, mutated, or amplified in a series of tumours, including non-small cell lung cancer.

Summary of the disclosure

It has been surprisingly identified that ceritinib interacts with FES and FER kinases in a dose dependent manner and inhibits them with the IC50 value being in the nM range (IC50 values determined for FES and FER inhibition were 10 nM and 8 nM, respectively). FES and FES- related kinase (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors for growth factors, cytokines and immunoglobulins, such as for example IL-6, FGF2, SCF and PDGF. The FES and FER kinases have been linked also to the regulation of cell-cell and cell-matrix interactions, as well as cell division, cell morphology, cell migration, and thus to tumorigenesis and development of metastasis in cancer. Therefore, it is postulated that inhibition of FES and FER kinases by ceritinib can be well employed in the treatment of a disorder or a disease mediated by the activity of FES and/or FER.

First aspect of the disclosure is ceritinib for use in the treatment of a disorder or a disease mediated by the activity of FES and/or FER. Similarly, the disclosure also provides use of ceritinib for the manufacture of a medicament for the treatment of a disorder or a disease in a subject mediated by the activity of FES and/or FER; or a method for the treatment of a disorder or a disease mediated by the activity of FES and/or FER comprising the step of administering to a subject a therapeutically effective amount of ceritinib.

Second aspect of the disclosure is a pharmaceutical composition comprising a ceritinib for use in the treatment of a disorder or a disease mediated by the activity of FES and/or FER.

Third aspect of the disclosure is a method of modulating FES and/or FER activity in a subject, comprising the step of administering to a subject a therapeutically effective amount of ceritinib.

Description of the drawings

Figure 1 Synthesis scheme of linker-Ceritinib

Figure 2 Effect of LDK378 on viability and apoptosis induction in confluent NRCs

Figure 3: siRNA-mediated knockdown of individual kinases

Figure 4: Effects of LDK378 on cell viability and apoptosis induction in LPS stimulated rat BM- derived macrophages after 5 h of treatment

Figure 5: Effects of Ceritinib on TNFa protein level in LPS-stimulated rat BM derived macrophages after 5 h of treatment

Detailed description of the disclosure

Functionality of ceritinib was assessed in a chemical proteomics experiment, where proteins that ceritinib binds to were identified. Surprisingly, the top hit of ceritinib chemical proteomics experiment, identified with the highest significance, was the non-receptor tyrosine kinase FER. Biochemical validation confirmed very potent inhibition of FER kinase by ceritinib (IC50 8 nM), as well as its related kinase FES (IC50 10nM). Based on the proven antitumor effect of ceritinib and the link between FES and FER kinases and certain tumor types, ceritinib can be utilized to treat a disorder or a disease mediated by the activity of FES and/or FER.

"FER" refers to tyrosine-protein kinase FER. Unless specifically stated otherwise, FER as used herein, refers to human gene and protein listed under accession numbers NM 005246.2 / NP_005237.2, respectively.

"FES" refers to tyrosine-protein kinase FES and all its isoforms 1 to 4. Unless specifically stated otherwise, FES as used herein, refers to human gene and protein listed under accession number NM_002005.3 / NP_001996.1 for tyrosine-protein kinase FES/Fps isoform 1 , NM_1 143783.1 / NP_001 137255.1 for tyrosine-protein kinase FES/Fps isoform 2, NM_001 143784.1 / NP_001 137256.1 for tyrosine-protein kinase FES/Fps isoform 3 and/or NM_001 143785.1 / NP_001 137257.1 for tyrosine-protein kinase FES/Fps isoform 4.

The term "disorder or a disease mediated by FES and/or FER" denotes an ailment, symptom, condition, disorder or a disease caused by an increased differential expression of the nucleic acid or polypeptide, of gene number of FES and/or FER, or function or activity of FES and/or FER in a cell or tissue compared to a control. Disorder or a disease mediated by the activity of FES and/or FER is the ailment, symptom, condition, disorder or a disease can be alleviated, inhibited, prevented and/or ameliorated by inhibiting the increased differential expression of the nucleic acid or polypeptide, of gene number of FES and/or FER, or function or activity of FES and/or FER in a cell by a FES and/or FER inhibitor, such as Ceritinib, at least to the extent that the expression of the nucleic acid or polypeptide, of gene number of FES and/or FER, or function or activity of FES and/or FER in a cell or tissue is the same as in the control. An FES and/or FER inhibitor can also be an antibody.

Disorder or a disease mediated by FES and/or FER can be a proliferative disorder or disease. In one embodiment, the term means cancer. Cancer can mean presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone or may circulate in the blood stream as independent cells, such as leukemic cells. The cancer mediated by FES and/or FER can be hematological cancer or a solid tumor, particularly leukemia, lung, brain, colorectal or vascular (hemangioma) cancer. For example, Masanori Kawakami in Int J Clin Exp Pathol (2013, 6, 598-612) determined that FER overexpression is associated with poor postoperative prognosis and celcer-cell surivva in non-small cell lung cancer. Leukemia can be acute myeloid leukemia (AML) or chronic myelogenous leukemia. Ceritinib can be used to target FES and FER in leukemias, because the studies identified them to be potential downstream effectors of growth or survival signaling in leukemias (A Craig, Frontiers in Bioscience 17, 861 - 875, 2012). Further studies showed that the tyrosine kinase FES is an essential effector of KITD816V proliferation signal. Silencing of FES expression in KIT driven leukemias led to reduced cell growth (E Voisset et al, Blood 1 10, 2593-9, 2007). Further study in acute myeloid leukemia that was driven by constitutively activated internal tandem duplication (ITD) mutants of FLT3 receptor tyrosine kinase revealed that FES and FER were required for survival and growth, respectively (E Voisset et al, Leukemia, 24, 721 -8, 2010). The reference from Craig listed above reports of further studies where FES and FER were found to be involved in cancers like AML, mastocytosis and gastrointestinal stromal tumors, chronic myelogenous leukemia. Breitkopf et al identified FER as a potential target downstream of the BCR-ABL oncoprotein in chronic myelogenous leukemia (J Proteome Res, 2010). The two kinases carry the BAR domain. Proteins with the BAR domain have been found to play a major role in remodeling cellular membranes linked with organelle biogenesis, membrane trafficking, (Frost A, et all, Cell, 137(2):191 -6, 2009). The BAR domain superfamily of proteins is evolutionarily conserved with representative members present from yeast to man. The two kinases have also been implicated in cell interactions mediated by adherens junctions and focal adhesions (Condorelli et al, Curr Med Chem, 18, 2913-2920, 201 1 ). FER kinase is associated with cytoplasmic domain of N-cadherin (El Sayegh et al, Mol Biol Cell, 16, 5514-5527, 2005), as well as E-cadherin in adherence junction complexes of epithelial cells (Harder et al, Cell, 133, 1 1 18, 2008). By phosphorylating actin-interacting protein cortactin, FER kinase participates in the adherence junction interaction with cytoskeleton and regulating intercellular adhesion strength (Mege et al, Curr Opin Cell Biol, 18, 541 -548, 2006). FER kinase promotes breast cancer metastasis by regulating a6- and βΐ -integrin-dependent cell adhesion and anoikis resistance (Ivanova et al, Oncogene. 2013 Dec 1 2;32(50) :5582-92). FER kinase is elevated in NSCLC tumors and is important for cellular invasion and metastasis (Ahn et al, Mol Cancer Res. 2013 Aug;1 1 (8):952-63). In addition, FES absence attenuates tumor-associated angiogenesis and the metastasis-promoting functions of tumor-associated macrophages (Zhang et al., Cancer Res 201 1 ;71 :1465-1473). FES kinase promotes mast cell recruitment to mammary tumors via the Stem Cell Factor/KIT receptor signaling axis (Kwok et al, Mol Cancer Res. 201 2 Jul;1 0(7) :881 - 91 ). Therefore, it is expected that Ceritinib can reduce, delay or prevent metastasis, by reducing invasiveness of tumors or by the effect on the cells of the tumor niche.

A "control" refers to the expression of the nucleic acid or polypeptide, of gene number of FES and/or FER, or function or activity of FES and/or FER in a normal cell. For example, where cancer is driven by the activity of FES and/or FER, the expression, function or the activity of FES and/or FER can be compared between the cancer cell and a normal cell, such as for example noncancerous cell from a healthy tissue, or a healthy subject. Differential expression, function or activity means a measurable difference in expression, function or activity of FES and/or FER when compared to the control. The differential expression can mean upregulation of FES and/or FER compared to the control, meaning for example increased DNA copy number of the FES and/or FER, their genomic DNA, cDNA or RNA sequence copies; increased gene expression, protein expression, mRNA expression; functional effect of the protein; functional effect of the FES and/or FER gene, cDNA or mRNA; protein, cDNA, gene or mRNA activity, or mutation status like for example frame-shift mutation, deletion, translocation, insertion, duplication, inversion, functional mutation, or combinations thereof that translates into increased activity of FES and/or FER, compared to the control.

For example, when applied to a gene, it can refer to the differential mutation status of a gene compared to a normal, wildtype gene in a normal cell. A differentially mutated gene can be mutated as compared to the wildtype gene, which causes activation and increased function of the mutated gene. It can also refer to the differential production of the mRNA transcribed and/or translated from the gene or the protein product encoded by the gene. A differentially expressed gene may be overexpressed (or upregulated) or underexpressed (or downregulated) as compared to the expression level of a normal or control cell. However, as used herein, increased differential expression or upregulation is an increase in gene expression and generally is at least 1 .25 fold or, alternatively, at least 1 .5 fold or, alternatively, at least 2 fold, or alternatively, at least 3 fold or alternatively, at least 4 fold expression over that detected in a normal or control counterpart cell or tissue. The term "differentially expressed" also refers to where expression in a cancer cell or cancerous tissue is different compared to the expression in a control (for example control cell or normal tissue, e.g. non-cancerous cell or tissue) . Similar comparison can be applied when measuring if FES and/or FER are "activated" compared to the control. Activated FES and/or FER would mean that the activity of the proteins is more pronounced in treated cell compared to control, for example phosphorylation level of a substrate is at least 1 .5 fold or, alternatively, at least 2 fold, or alternatively, at least 3 fold or alternatively, at least 4 fold higher in cancerous or treated cell compared to control. Methods used in measuring the expression, function or activity of FES and/or FER are generally known in the art, and in any event would be applied the same to the cell and the control. As an example, expression of the nucleic acid can be assessed by measuring levels of messenger RNA using a RT-PCR. Levels of protein and phosphorylated protein can be monitored by using FES and/or FER specific antibodies. Level of FES and/ or FER activity can be monitored by phosphorylation level of its direct protein substrates.

The disorder or a disease mediated by the activity of FES and/or FER is also an ailment, symptom, condition, disorder or a disease in a subject that can be alleviated, inhibited, prevented and/or ameliorated by administering Ceritinib at the dose that causes blood concentration to reach C i ₀ o _d of 10 μΜ or less, preferably at 5 μΜ, preferably C i ₀ o _d of 2 μΜ or less. Therefore, a method of modulating FES and/or FER activity in a subject is also provided herein, the method comprising the step of administering to a subject a therapeutically effective amount of ceritinib. The term "a therapeutically effective amount" of a compound of the present disclosure refers to an amount of ceritinib that will elicit the biological or medical response of a subject. For example, the effect can be reduction or inhibition of FES and/or FER, or the protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression etc. In one non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound of the present disclosure that, when administered to a subject, is effective to (1 ) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by FES and/or FER and/or mediated by FES and/or FER activity, or (ii) characterized by activity (normal or abnormal) of FES and/or FER; or (2) reduce or inhibit the activity of FES and/or FER; or (3) reduce or inhibit the expression of FES and/or FER. The therapeutically effective amount of Ceritinib would be in the range of 1 mg - 1500 mg, preferably from 100 mg to 1000 mg, more preferably between 150 mg and 750 mg. The dose can be administered daily in a single or in divided doses.

The compound to be used in the treatment of the disorder or disease is to be administered to a subject, which refers to an animal. Typically the animal is a mammal. A subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In more specific embodiment, the subject is a human. Ceritinib can be formulated in a pharmaceutical composition in order to be used for the purposes described above, particularly for use in the treatment of a disorder or a disease mediated by FES and/or FER. The composition can contain further pharmaceutical excipients. The pharmaceutically acceptable excipients used can be for example filler, disintegrant, glidant, and/or lubricant, or mixture thereof. Further excipients like antioxidants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof can also be added. The composition can comprise one or more fillers, for example microcrystalline cellulose, silicified microcrystalline cellulose, lactose (anhydrous, monohydrate), mannitol, sorbitol, calcium phosphate (dibasic anhydrous, dibasic hydrate, tribasic), isomalt, sucrose, Hydroxypropyl Cellulose Low-substituted; one or more disintegrants, for example Croscarmellose Sodium, Crospovidone, Sodium starch glycolate; further binders, such as for example starch,

Hydroxypropyl Cellulose Low-substituted, Hypromellose (Hydroxypropylmethyl cellulose), Copovidone (Copolyvidone), gelatine, polymethacrylates; one or more glidants, for example Silica, colloidal silica, talc, hydrophobic colloidal silica, magnesium silicate; or one or more lubricants, for example Magnesium stearate, calcium stearate, zinc stearate, glyceryl mono fatty acid, glyceryl monostearate, glyceryl dibehenate, glyceyryl palmito stearic ester, or

polyoxyethylen glycol; or mixtures thereof. They can be mixed in the composition in a powder form, as separately granulated granules, or dissolved in a fluid, which can then be substantially removed by drying throughout the process, or added after going through the process of briquetting or slugging, and optionally sieving. The pharmaceutical composition can be formulated for example in a tablet or a capsule by tableting or filling the composition in a capsule, respectively.

The effective dosage of the compound to treat the disorder or disease mediated by FES and/or FER may vary depending on a pharmaceutical composition employed, the mode of administration, the condition being treated, and the severity of the condition being treated. Thus, a dosage regimen for the treatment would be selected in accordance with a variety of factors including the route of administration and the renal and hepatic function of the patient. A clinician or physician of ordinary skill can determine and prescribe the effective amount of the single therapeutic agents required to alleviate, counter or arrest the progress of the condition.

Examples

The following Examples illustrate the disclosure described above; they are not, however, intended to limit the scope of the disclosure in any way. The beneficial effects of the ceritinib for use in the treatment according to the present disclosure, or methods as disclosed herein can also be determined by other test models known as such to the person skilled in the pertinent art.

Example 1 : Chemical proteomics experiment

Synthesis of linker-Ceritinib (Fig 1)

In short, the linker was synthesized from the commercially available 4-chlorobutan-1 -ol after treatment with sodium azide in a solution of DMSO at 85°C. In order to prepare the iodo- derivative thereof, the hydroxyl moiety was first transformed into its corresponding mesylate under standard conditions. The iodine was inserted after nucleophilic substitution, using sodium iodide in acetone at room temperature. The coupling of the linker with Ceritinib was successfully achieved in presence of potassium carbonate in DMF, after 24h of trituration. Final reduction of the azide into the desired amine-derivative was realized under Staudinger conditions, using triphenylphosphine in wet tetrahydrofuran (94 %). The expected linker-Ceritinib was obtained as a pure compound and further utilized for competition experiment. Preparation of 4-Azidobutan-1-ol, compound 2:

To a solution of 4-chlorobutan-1 -ol (500 mg, 4.61 mmol, 1 eq.) in DMSO (5 mL) was added sodium azide (599 mg, 9.21 mmol, 2 eq.) in one portion at room temperature. The reaction mixture was then heated at 85°C for 16 h and monitored by LCMS until completion. After full conversion of 4-chlorobutan-1 -ol into the corresponding azide, the reaction mixture was poured into water and extracted with EtOAc (2x 100 mL). The organic layers were combined, washed successively with water (1 x 100 mL) and saturated NaCI (1 x 100 mL), dried over MgS0 ₄ , filtered and evaporated under reduced pressure. Compound 1. was recovered as a colorless oil (385 mg, 72% yield). The analytic data were consistent with the structure of the expected product. Rf 0.35 (EtOAc/hexane, 2:1 ); ¹ HNMR (400 MHz, CDCI ₃ ) δ 3.70 (t, J = 5 Hz, 2H), 3.35 (t, J = 6.5 Hz, 2H), 1 .72-1 .67 (m, 4H), 1 .54 (s, 1 H); ¹³ C NMR (75 MHz, CDCI ₃ ) δ 62.2, 51 .3, 29.8, 25.4.

Preparation of 4-lodobutan-1-ol, compound 3:

To a solution of 4-azido-1 -butanol 2 (380 mg, 3.30 mmol, 1 eq.) in dry DCM (10 mL), at

0°C, was added dropwise methanesulfonyl chloride (0.257 mL, 3.30 mmol, 1 eq.), followed by DMAP (403 mg, 3.30 mmol, 1 eq.) and Et ₃ N (0.460 ml, 3.30 mmol, 1 eq.). After 1 h at 0°C, the reaction mixture was poured into ice/water (50 mL) and extracted with DCM (3x 50 mL). The combined organic layers were washed with HCI (1 M, 2x 25 mL) and saturated NaCI (3x 50 mL), dried over MgS0 ₄ , filtered and evaporated. Flash chromatography, using DCM/hexane (2:1 ) as eluent, gave the corresponding 4-azidobutyl methanesulfonate intermediate (594 mg, 93%) as a colorless oil. Rf 0.54 (EtOAc/hexane, 1 :2); ¹ H NMR (400 MHz, CDCI ₃ ) δ 4.32 (t, J = 6.1 Hz, 2H), 3.42 (t, J = 5.9 Hz, 2H), 3.07 (s, 3H), 1 .94 (m, 2H), 1 .80 (m, 2H); ¹³ C NMR (75 MHz, CDCI ₃ ) δ 69.1 , 50.7, 37.4, 26.4, 25.0.

To a solution of the 4-azidobutyl methanesulfonate (590 mg, 3.05 mmol, 1 eq.) in dry acetone (10 mL) was added sodium iodide (915 mg, 6.1 1 mmol, 2 eq.) and the reaction mixture was stirred at room temperature for 16 h. Reaction mixture was diluted with a portion of ether (30 mL) and a precipitate was formed. After filtration of the reaction mixture, the mother liquor was recovered and further concentrated under reduced pressure. The residue was dissolved in Et ₂ 0, washed with a solution of sat. Na ₂ S ₂ 0 ₃ (1 x 30 mL) followed by an ice-cold 1 N HCI (1 x 30 mL), and a sat. NaHC0 ₃ (1 x 30 mL) and dried over MgS0 ₄ . The volatiles were evaporated and the desired 4-iodobutan-1 -ol dried under vacuum to afford a colorless oil (642 mg, 93 %). ¹ H NMR (400 MHz, CDCI ₃ ) δ 3.40 (t, J = 6.0 Hz, 2H), 3.28 (t, J = 6.6 Hz, 2H), 2.01 (m, 2H), 1 .81 (m, 2H); ¹³ C NMR (75 MHz, CDCI ₃ ) δ 69.1 , 50.7, 37.4, 26.4, 25.0.

Preparation of A/,A -(4-(1-(4-azidobutyl)piperidin-4-yl)-2-isopropoxy-5- methylphenyl)-5-chloro- Ν,Ν,Ν,Ν -(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine, compound 4: To a white suspension of Certinib® (200 mg, 0.305 mmol, 1 eq.) in dry DMF (3 mL) were added, compound 3 (103 mg, 0.457 mmol, 1 .5 eq.) followed by potassium carbonate (126 mg, 0.914 mmol, 3 eq.) at 0°C.The reaction mixture was then allowed to stir further for 16 h until completion and monitored by LCMS. After completion of the reaction, the crude mixture was washed with a 1 :1 mixture of EtOAc (50 mL) and water (50 mL). The 2 layers were separated and the organic layers were extracted and subsequently washed with water (1 x 50 mL), saturated NaCI (2x 50 mL), dried over MgS0 ₄ , filtered and evaporated under reduced pressure. The crude residue was purified by flash chromatography, poured onto a column of silica (25 g) and eluted with a gradient of heptane/EtOAc. Fractions containing the desired product were combined and concentrated under reduced pressure. Compound 4 was obtained as a white solid (133 mg, 66 %). The analytical data supported the structural assignment expected for compound 4. LCMS for C32H43CIN ₈ 0 ₃ S; retention time =1.28 mins, MH ⁺ = 655.0. ¹ H NMR (400 MHz, DMSO) δ 9.37 (br.s, 1 H), 8.37 (d, J = 1 1 Hz, 1 H), 8.16 (s, 1 H), 7.97 (s, 1 H), 7.74 (d, J = 1 1 Hz, 1 H), 7.53 (m, 1 H), 7.41 (br.s, 1 H), 7.26 (m, 1 H), 6.75 (s, 1 H), 4.48 (p, J = 7.4 Hz, 1 H), 3.98 (dt, J = 12; 4 Hz, 1 H), 3.40 (m, 1 H), 2.91 (br.d, J = 10 Hz, 2H), 2.60 (m, 1 H), 2.25 (m, 2H), 2.04 (s, 3H), 1 .92 (m, 3H), 1 .58 (m, 4H), 1 .49 (m, 4H), 1 .13 (d, J = 6.3 Hz, 6H), 1 .09 (d, J = 6.5Hz, 6H).

Preparation of A/,A -(4-(1-(4-aminobutyl)piperidin-4-yl)-2-isopropoxy-5- methylphenyl)-5-chloro- Ν,Ν,Ν,Ν -(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine, compound 5: To a solution of compound 4 (100 mg, 0.153 mmol, 1 eq.) in THF/water (10:1 ), was added in one portion PPh ₃ (60.0 mg, 0.229 mmol, 1 .5 eq.) at room temperature. The reaction mixture was stirred at room temperature for 16 h until completion. The crude mixture was diluted with EtOAc (20 mL) and the organic phase was washed with 1 M HCI (2x 20 mL). The 2 layers were separated and a 1 M solution of NaOH was added to the aqueous phase until the pH was basic (pH ~ 10). The aqueous layer was extracted with EtOAc (3x 20 mL) and the organic phases were combined and washed successively with water (2x 20 mL), saturated NaCI (2x 20 mL), and dried over MgS0 ₄ . After filtration and evaporation under reduced pressure, the crude residue was quickly purified through a pad of celite to afford the desired compound 5 (90.6 mg, 94%) as a white solid and with high purity (>95%). LCMS for C ₃ 2H45CIN ₆ 0 ₃ S; retention time =1.04 mins, MH ⁺ =629.3. ¹ H NMR (400 MHz, CDCI) δ 9.42 (br.s, 1 H), 8.51 (d, J = 8 Hz, 1 H), 8.01 (m, 1 H), 7.92 (s, 1 H), 7.85 (d, J = 8 Hz, 1 H), 7.56 (m, 1 H), 7.47 (br.s, 1 H), 7.19 (m, 1 H), 6.77 (s, 1 H), 4.51 (p, J = 7.4 Hz, 1 H), 4.05 (dt, = 12; 4 Hz, 1 H), 3.19 (m, 1 H), 3.04 (br.d, J = 10 Hz, 2H), 2.69 (t, J = 6.7 Hz, 2H), 2.59 (m, 1 H), 2.35 (t, J = 6.7 Hz, 2H), 2.07 (s, 3H), 2.01 (m, 3H), 1 .69 (m, 4H), 1 .53 (m, 2H), 1 .46 (m, 2H), 1 .28 (d, J = 6.3 Hz, 6H), 1 .25 (d, = 6.5Hz, 6H).

This compound was further subjected to our kinase panel to reveal a clean activity. Data is summarized below in the table 1 .

Table 1 Kinase panel

EPK CE

EPK CE _{Ερκ ΓΕ} EPK CE EPK CE EPK CE EPK CE FGFR3 EPK CE EPK CE EPK CE EPK CE EPK CE EPK CE EGFR

LCK (1- 7 , K7DR ,( _™ 80-7, EPK CE EPK CE

- " " MET (956- EPHA4 EPHB4 (411- JAK2

PDPK1 CDK2A PRKACA BTK PRKCA (668- 1 ³⁹ °) (574-986) (566-987) K650E- 113

2.4 > io > 10 > 10 > 10 0.45 > 10 0.96 1.1 > 10 > 10 > 10 > 10 8.5 > 10

^■ 66.4127 -1.74419 -36.3583 -0.12981 -37.1353 -93.9775 0 ^■ 99.8548 -90.0993 -80.6568 -45.6281 -47.9021 -6.43275 -30.6808 -53.2158 -1.30719 iilll

■0.84399 -1.74419 -0.01359 52.26096 0 -0.07102 0 ^■ 1.82564 -0.02269 -0.2459 -0.20375 -0.28825 -6.43275 -0.08327 -0.01193 -1.30719

S iSa&* 0.48358 0.740627 4.534833 0.887339 0.933589 0.940794 0.659805 0.534232 0.511468 0.556975 0.813278

EPK CE EPK CE

EPK CE EPK CE

EPK CE EPK CE EPK CE EPK CE EPK CE PDGFRa ABL1 EPK CE EPK CE EPK CE

ALK TYK2 EPK CE EPK CE EPK CE

KIT (544- INSR (871 JAK3 (811 JAK1 (86e MAP3K8 (551- (229- RET (658- ROCK2 AXL (515- (1066- PRKCQ PKN1 CDK4D1

976) 1343) 1124) 1154) (30-404) V561D- T315I- 1072) (6-553) 885)

1459) 1187)

1089) 500)

isjftSiitiSj HI > io ;;;¾¾|6.;;;! > 10 > 10 2.1 > 10 > 10 6.9 1.4 0.24 0.89

84.9506 99.9373 2.217293 -99.9974 -0.54945 1.953129 -2.55591 -51.8879 -81.7987 -31.001 -2.61628 -59.1823 -75.3176 -95.5588 -91.0007 i!fi!

0.03043 1.95151 2.217293 -44.6641 -0.54945 1.953129 -2.55591 -0.1618 -0.00775 200.2641 -2.61628 -0.00184 -0.46691 -0.1966 -0.02088 il iiif

iSlillS i 0.872355 1.002239 0 0.95776 0 0 0 0.577 0.973487 0 1.000959 0.574978 0.825361 0.957515

Affinity enrichment on immobilized linker-ceritinib and quantitative Mass Spectrometry

Rat biliopancreatic duct tissue was quickly removed during necropsy, washed in PBS, and immediately flash frozen in liquid N ₂ to minimize degradation processes. Several ducts were pooled. Subsequently rat biliopancreatic duct tissue was thawed and homogenized in lysis buffer (50 mM Tris-HCI , pH 7.4, 100 mM NaCI, 2 mM MgCI ₂ , 5 % glycerol, 0.8 % NP-40 and protease inhibitors) at a volume to weight ratio of 8. After clearing by centrifugation, the lysate was split in 4 equal samples, each containing approximately 10 mg of protein. Lysate samples were then incubated with either DMSO or 0.3, 3, or 30 μΜ of ceritinib for 30 min at 4°C.

Subsequently, 100 μΙ of Sepharose-bound linker-ceritinib was added and incubation was continued for another 2 h at 4°C. Sepharose beads were then separated from the unbound proteins by centrifugation, transferred to a spin column and washed with 10 ml of lysis buffer. Finally, bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized with colloidal Coomassie Blue for excision. Bands were excised from each lane at an equidistant space. Disulfide bonds from the protein were reduced using dithiotreitol and the free thiols that were generated alkylated with iodoacetamide. Proteins were subsequently digested in-gel by trypsin. Peptides were eluted from the gel pieces and labeled with iTRAQ reagent.

Table 2 Labeling of SDS-PAGE derived peptides with ITRAQ reagents

Peptides from bands of the same Mw across the gel were pooled and analyzed by LC-MS. Peptides were identified by querying against humanJPI database using the Mascot search algorithm and proteins were quantified by combining iTRAQ reporter ions across all identified peptides using an in-house analysis pipeline. Specific binders to Ceritinib were identified based on their depletion from the bound protein fraction in the samples containing the competitor compound relative to the DMSO control (Table 1 ). Kinase inhibitory potency of identified kinases were measured at Nanosys.

Competition experiments with Ceritinib identified competing proteins at 30 μΜ ceritinib suggesting binding to these proteins. Among the proteins FER kinase showed clearest dose- dependent effects. Follow-up kinase inhibition experiments confirmed that FER (and its related kinase FES) were inhibited by Ceritinib in the low nanomolar range (8 nM and 10 nM, respectively), strengthening the significance of the identified proteins. mRNA extraction / expression analysis in target tissues

Tissue samples

Animals: Na ^' i ^' ve male Han: rats, 3-6 months old.

Animals were sacrificed by C02 inhalation, followed by organ extraction (liver, pancreas, duodenum, biliopancreatic duct) by using In Situ Ductal Perfusion method. RNA isolation was performed immediately after. RNA was isolated using Rneasy plus mini kit (Qiagen; cat n°74134), according to the manufacturer's instruction. cDNA synthesis and quantitative PCR Total RNA was reverse transcribed to cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, cat n° 4368813) according to the manufacturer instructions, using the DNA Engine Dyad PTC-220 (BioRad). cDNA was synthesized starting from 300 ng of total RNA and adding a mix preparation to a final volume of 100 μΙ_. Real-time PCR was performed using ABI prism™ 7900HT (Applied Biosystems) in combination with TaqMan® Fast Universal PCR Master Mix (Applied Biosystems, cat n°4352042). The PCR conditions were as follow: one cycle at 95°C for 20 sec, followed by 40 cycles of amplification: a denaturation step at 95°C for 1 s and annealing/elongation step at 60°C for 20 sec. Human probe/primer sets was also used on rat RNA, purchased commercially from Applied Biosystems. 18S (Hs99999901_s1 ) was used as internal standard for real time PCR analysis. The relative quantification of gene expression changes was performed using the standard curve method. The standard curve is generated from 8 dilution series (10 fold dilution) constructed from a "reference" sample. "Reference" sample was a commercial mRNA from rat liver (Ambion cat n° 09508186B). The standard curve was used to generate expression data expressed in molecule number.

Table 3 TaqMan probes list

mRNA expression of kinases Fert2 and Fes was higher in epithelial tissues such as duodenum as compared to liver and pancreas, and correlated well with Keratin 19 expression level. On the other hand, the expression of Alk kinase, primary Ceritinib target, was virtually absent from all the tissues analyzed.

Experiment 2: Normal Rat Cholangiocyte (NRC-1) cell model

Cell maintenance and treatment

Normal rat cholangiocyte (NRC-1 ) are primary derived bile duct epithelial cells (Bogert and LaRusso 2007). NRC-1 cells were cultivated in NRC-1 medium. Cells were maintained up to passage 36 into T75 collagen I coated flask (Biocoat). For splitting cells were washed with 2:1 PBS:Trypsin-EDTA (Gibco), and then incubated at 37°C/5 % C02 for approximately 10-15 min with 1 ml of trypsin-EDTA. Once all cells were detached, 9 ml of NRC-1 medium were added and cells were counted. Then 1 .10E6 cells were seeded into a T75 collagen I flask for 48 hours maintenance. Viability and apoptosis testing

For testing the effect of compounds on cell viability and determining non-cytotoxic range, NRC-1 were seeded at 8000 or 16000 cells/well in BD BioCoat™ Collagen I, 96-well plates (BD, #354650). Cells were grown for 2 days at 37°C, 5 % C02, at which point 8000 cells/well reached 70 % confluence, while 16000 cells/well reached 100 % confluence. Medium was then replaced with fresh NRC-1 medium containing compound dilutions (1 .3 ηΜ-100μΜ) for 3 h or 24 hours. NRC-1 medium containing 1 % DMSO was used as control. Cell viability after treatment was determined by measuring ATP content using CellTiter-Glo® Luminescent Cell Viability Assay (Promega), according to manufacturer's instruction. For determining the effect of Ceritinib on confluent, barrier-forming cells, cells were seeded at 20000 cells/well in BD BioCoat™ Collagen I, 96-well plates (BD, #354650). Cells were grown for 4 days at 37°C, 5 % C0 ₂ , at which point they were fully confluent. Cell seeding density corresponded to seeding for barrier function analysis using ECIS technology. Medium was then replaced with fresh NRC-1 medium containing Ceritinib dilutions (1 μΜ-32μΜ) for 24 hours. NRC-1 medium containing 1 % DMSO was used as a control. Treatment with 20 μΜ taxol and 1 μΜ staurosporine was used as a positive control for apoptosis induction. Cell viability after treatment was determined by using CellTiter-Blue® Cell Viability Assay (Promega), according to manufacturer's instruction.

Apoptosis induction was quantified by using Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega), according to manufacturer's instruction. For statistical analysis, one-way ANOVA was used, assuming Gaussian distribution, with Dunnett correction for multiple comparisons. Values were considered significant: ^* = p< 0.05, ^** = p< 0.01 and ^*** = p< 0.001 . (Fig. 2)

Compound treatment

On the day of measurement, NRC-1 medium was removed from each well and replaced by 300 μΙ of fresh NRC-1 medium. Four to six hours baseline measurement at 37°C, 5 % C0 ₂ was performed in order to allow barrier stabilization. Cells were stimulated with 33 μΙ of 10x compounds solution pre-diluted into DMEM-F12 (Gibco). During compounds injection, measurement was paused for few seconds.

Example 3: siRNA transfection.

Cells were transfected by electroporation with Amaxa 96-well plate system (Lonza), with P1 primary cell 96-well Nucleofector Kit (Lonza cat # V4SP-1096). Cells were grown up to 80 % confluence in T75 collagen I flask and trypsinized in order to prepare a cell suspension. From the cell suspension, after 5 minutes centrifugation at 800 rpm, a solution of 3.5x10E6 cells/ml was prepared in P1 complemented nucleofector solution, in order to have 0.7x10E5 cells/20 μΙ in each well. Each condition was prepared in Eppendorf tubes with 300 nM of the corresponding siRNA. From these cells/siRNA mixes, 20μΙ replicates were dispensed into each well.

Electroporation was performed in the 96-well plate using the Amaxa electroporation machine

(96-EA-104 electroporation program). Once electroporation was performed, cells were left for 10 minutes to recover, and 80 μΙ of 37°C pre-warmed medium was added to each well. With these electroporated cells different follow up experiments were performed. RNA extraction and gene expression analysis

For RNA extraction, 50μΙ of electroporated cells were seeded per well, in duplicates, into collagen I coated 96 well plates containing 150 μΙ/well of pre-warmed NRC-1 medium. Cells were grown for 72 hours at 37°C/5 % C0 ₂ in order to reach confluence and form barrier, then medium was washed-out and 150 μΙ of RLT lysis buffer (Qiagen RNeasy 96 Kit #74181 ) were added into each well. RNA was extracted according to manufacturer's instruction.

Table 4 siRNA and Taqman probes catalog numbers.

As shown on the figure 3, only parital knockdown of Fert2 mRNa expression was achieved, and Fert2 protein level remained stable.

Experiment 4: Macrophage model - Bone marrow (BM)-derived macrophage

differentiation and stimulation

As Ceritinib potently inhibited both FER and FES kinase in a biochemical assay, activity of Ceritinib in LPS-stimulated rat bone marrow (BM)-derived macrophages has been tested. Fes kinase was shown to negatively regulate TLR-triggered cytokine production in macrophages (Xu et al, Nature Immunology Vol 13, No 6, June 2012). The purpose was to demonstrate Ceritinib potency against FES kinase in a cellular context, as well as to verify Ceritinib effects on viability and cytokine production in bone marrow-derived macrophages.

Femurs were taken from rats. BM cells were isolated by flushing the femurs with HBSS

(Invitrogen) using a cell strainer (Falcon), counted, adjusted to 1 x106 cells/mL and differentiated to BM-derived macrophages in complete RPMI-1640 medium (RPMI-1640, 1 x Sodium- Pyruvate, 1 x Penicillin/Streptomycin, 1 x Glutamax, 1 x MEM non-essential amino acids, 50 μΜ 2- betamercaptoethanol and 10 % FCS, all Invitrogen) supplemented with 40 ng/mL

recombinant rat M-CSF (Peprotech). Cell suspensions were put then into 175 cm2 flasks (Falcon). After one day of culture, non-adherent cells and medium were harvested, centrifuged (1400 rpm, 5 minutes, room temperature) and only the cell-free supernatants were put back into the respective flasks for further six days. After differentiation to BM-derived macrophages, medium was removed and adherent cells were detached by cold PBS/2 mM EDTA

(Gibco/Promega) and scratching. After two washing steps (1400 rpm, 10 minutes, room temperature), cells were counted and adjusted to 1 x106 cells/mL in complete RPMI-1640 medium. All conditions in the experiments were performed in triplicates. In pre-experiments, a sub-optimal LPS concentration was determined. Briefly, 100 μί of cell suspension was transferred to 96-well flat bottom plates (Falcon) and incubated with different concentrations of LPS (0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL or 100 ng/mL; Sigma) for 4 hours and cytokine levels (TNFa, IL-6) in the supernatants were measured. After determination of the sub optimal LPS concentration, 100 μΙ_ of cell suspension were transferred to 96-well flat bottom plates (black with clear bottom, Costar) and pre-incubated either with vehicle, different concentrations of Ceritinib (0.04 μΜ, 0.2 μΜ, 1 μΜ, 5 μΜ or 25 μΜ) or staurosporin (1 μΜ) for 1 hour or 2 hours. LPS (0.1 ng/mL) was added on top of the cells for additional 4 hours. All compound dilutions were prepared freshly in complete RPMI-1640 medium. After the incubation time, supernatants were transferred to 96-well round bottom plates (Falcon) for ELISA measurements and cell pellets were re-suspended in 100 μί complete RPMI 1640 medium and further analysed. From every experiment and every rat, 2 mL of non-treated cell suspensions were used for standard curve adjustments.

Viability and apoptosis testing in macrophages

Viability of stimulated BM-derived macrophages after treatment was determined by using CellTiter-Blue® Cell Viability Assay (Promega), according to manufacturer's instruction.

Apoptosis induction was quantified by using Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega), according to manufacturer's instruction. A broad kinase inhibitor staurosporin was used as positive control for apoptosis induction.

Ceritinib treatment induced dose-dependent reduction in cell viability in both LPS-stimulated and control cells, with no evident apoptosis induction (Fig 4).

Cytokine production measurements by ELISA

Supernatants from stimulated BM-derived macrophages were analyzed using rat TNFa

(eBioscience) and rat IL-6 (R&D) ELISA kits according to the manufacturer's instructions. IL-6 was under detection limit in this experiment. Ceritinib in different concentrations (without LPS addition) had no effects on TNFa release. In contrast, LPS induced a strong TNFa protein release compared to control and Ceritinib showed a dose-dependent decrease in LPS-induced TNFa levels after 5 h of treatment (Fig 5). Identical results were obtained after 6 hours of treatment.

Experiment 5: Effect of ceritinib on BaF3 cell line

Ceritinib was tested in BaF3 cells driven by FES or FER kinase. The measured IC50 was 3.65 μΜ and 1 .65 μΜ in BaF3/FES and BaF3/FER line, respectively.