A USE OF l'-CYANO-CYTARABINE FOR CANCER TREATMENT

FIELD OF THE INVENTION

The present invention relates to a cytidine deaminase (CDA)-resistant compound selected from the group consisting of Γ-cyano-cytarabine (l'-CN- Ara-C), Γ-cyano-gemcitabine, l'-cyano-5-azacytidine and l'-cyano-5-aza-2'- deoxycytidine, a prodrug or an acceptable salt thereof; a pharmaceutical composition comprising the same; and a method for preventing or treating a cancer using the compound.

BACKGROUND OF THE INVENTION Nucleoside analogs as a class of therapeutic agents are prevalent in the clinical treatment of cancer and viral disease (X. Liu, et al. , Expert Opin. Investig. Drugs, 2012, 21 : 541-555). Among them, anti-cancer nucleosides are grouped as antimetabolites, hypomethylating agents or others depending on their mechanism of action. They differ greatly in the means by which they cause cell death after they are incorporated into DNA. Especially, the key biochemical pathway responsible for the cytotoxic action of nucleoside analogs is mediated mainly by the incorporation of the drug into DNA which subsequently results in interfere with DNA replication or termination of DNA synthesis (A. J. Townsend, et al , Molecular Pharmacology, 1987, 32: 330-339; P. Huang et al., J. Bio. Chem., 1990, 265 : 16617- 16625)

Anti-cancer nucleosides are typically analogs of natural nucleosides of cytidine, adenosine, guanosine and thymidine (B. Ewald, et al. , Oncogene, 2008, 27: 6522-6537). The cytidine nucleoside analog 5-azacytidine (azacitidine), 5- aza-2'-deoxycytidine (decitabine), 1 -beta-D-arabinofuranosyl-5-azacytosine (fazarabine), 1-beta-D-arabinofuranosyl cytosine (cytarabine; Ara-C) and 2'- deoxy-2',2'-difluorocytidine (gemcitabine) have been used clinically in cancer treatment.

However, these cytidine anti-cancer nucleosides have clinical limits due to susceptibility to cytidine deaminase (CDA). CDA is an intracellular enzyme of the pyrimidine salvage pathways that catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively (S. Costanzi et al, Chem. Med. Chem., 2012, 6: 1452-1458). Importantly, human CDA recognizes as substrates a number of cytidine-based anti-cancer drugs such as Ara-C, gemcitabine, decitabine and azacytidine for the degradation of cytosine to uracil.

For example, Ara-C is a nucleoside cytotoxic anti-cancer agent useful for the chemotherapy of cancers, especially, hematological malignancies. It has been used as a standard treatment agent for acute myeloid leukemia since 1969. However, Ara-C rapidly undergoes deamination by CDA present in blood and tissues such as liver to become inactive metabolite, uracil- Ara-C. We excrete uracil-Ara-C through urine and Ara-C has no anti-cancer activity. Oral plasma exposure of Ara-C is very low, less than 20%, and clearance rate is high (O. Schiavon, et al, European Journal of Medicinal Chemistry, 2004, 39: 123-133). Therefore, it cannot be administered orally since it is rapidly deaminated once absorbed. In order to improve the efficacy of Ara-C, it is usually administerated in high doses by continuous-infusion. Accordingly, high dose to maximize a therapeutic effect results in association of unpleasant side effect for the patient such as colitis, intestinal mucostisis, myelosuppression, bone marrow suppression and thrombopenia (J. K. Christman, Oncogene 2002, 21 : 5483-5495). A small number of patients may have abnormal liver function, fever, rash and other side effects (J. M. Bennett, Leukemia Research, 2003, 27:761 ; and H. M. Kantarjian, Cancer, 2007, 109: 1007-1010). And also, Ara-C has no anti-cancer effect against solid tumors. As formation of uracil-Ara-C by CDA deamination is increased, active metabolite, triphosphate-Ara-C that plays a role in inhibiting DNA synthesis is diminished. Thus, very low plasma exposure of Ara-C brings out poor distribution into solid tumor tissues (J. P. Godefridus, Deoxynucleoside Analogs in Cancer Therapy, 2006, 139; W. A. Bleyer, Clinical Cancer Research, 1999, 5 : 3349-3351)

Therefore, cytidine-based nucleoside anti-cancer drugs do not meet the stability requirement against CDA deamination. In order to solve these problems and maximize anti-cancer efficacy, we have investigated the modified nucleoside structure not susceptible to CDA. Based on CDA resistance, this modified structure was aimed at achieving improved oral pharmacokinetic properties including high oral absorption via oral administration, high in vivo efficacy with a low dose and expanding indication area to solid tumors, as well as hematological malignancies. The synthesis of Γ-CN- Ara-C and its antiviral activity and in vitro anti-cancer activity in CCRF-HSB-2 and KB cell lines were reported in 1996 (Yoshimura et al , Nucleoside & Nucleotides, 1996, 15: 305- 324). Various compounds \ substituted at 2'-, 3'-, and 4'-position have been synthesized. Among them, the analog where a cyano group is attached at 2'- position with "up" configuration, l-(2-C-cyano-2-deoxy-l- -D- arabinosyl)cytosine (CNDAC) and its anticancer activities in vitro and in vivo were reported by Matsuda's group (Matsuda, A. et al , J. Med. Chem., 1993, 36, 4183-4189). The synthesis of thymidine analogs, 3'-C-cyano-3'-deoxythymidine (Matsuda, A et al , Tetrahedron 1988, 44: 625-636), and 4'-C-cyano-thymidine (Yang, et al , Tetrahedron letter 1992, 33 : 37-40) having cyano group instead of 3'-hydroxyl group and 4'-hyrogen, respectively, were reported (Prisbe, E. J. et al , Plenum Publishing Co. New York, 1993, 101- 1 13).

SUMMARY OF THE INVENTION

We surprisingly found that an addition of cyano (CN) group at the Γ- position of nucleoside in Ara-C represented by formula (I) maintained the intrinsic anti-cancer cell activity while selectively abolishing the degradation by CDA. This novel molecular mechanism of l'-CN- Ara-C represented by formula (II) having cyano group at l'-positon of nucleoside in Ara-C for CDA resistance was illustrated through molecular docking with human CDA enzyme (1MQ0) in Fig. 1 and CDA assay in Test Example 2 and Fig. 2. And also, this CDA resistance of -CN- Ara-C dramatically improved oral pharmacokinetic property and bioavailability compared to Ara-C in mouse, rat and dog in Fig. 3. Moreover, significantly enhanced efficacy in xenograft was achieved at the same dose compared to Ara-C (Fig. 4). Γ-CN- Ara-C was well distributed in HL-60 tumor tissues and exhibited high drug concentration in HL-60 tumor tissues implying that it will be effective against solid tumors (Fig. 5).

Accordingly, it is an object of the present invention to provide a cytidine deaminase (CDA)-resistant compound selected from the group consisting of Γ- cyano-cytarabine represented by formula (II), Γ-cyano-gemcitabine represented by formula (III), l'-cyano-5-azacytidine represented by formula (IV) and - cyano-5-aza-2'-deoxycytidine represented by formula (V), a prodrug or an acceptable salt thereof:

(H) (IV) X=OH

(V) X=H

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating a cancer, comprising the compound of the present invention as an active ingredient, and a pharmaceutically acceptable carrier or an excipient.

It is a further object of the present invention to provide a method for preventing or treating a cancer comprising administering a therapeutically effective amount of the compound of the present invention to the subject in need thereof.

It is still further object of the present invention to provide a use of the compound of the present invention for the manufacture of a medicament for preventing or treating a cancer. BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Fig. 1 : View of the complex between human CDA (PDB ID: 1MQ0) and

Ara-C (a) as resulting from molecular docking; -CN-Ara-C (b);

Fig. 2: Relative susceptibility of cytidine, Ara-C and -CN- Ara-C to CDA of Test Example 2; Fig. 3: Pharmacokinetics of the Ara-C (mouse) and -CN-Ara-C (mouse, rat and dog) of Test Example 4 through IV and PO administration routes;

Fig. 4: The tumor volume of IP and PO Ara-C and Γ-CN- Ara-C administered mice in HL-60 human leukemia xenograft of Test Example 5; and Fig. 5: Γ-CN- Ara-C concentration in plasma and tumor tissues in HL-60 human leukemia xenograft of Test Example 6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a cytidine deaminase (CDA)-resistant compound selected from the group consisting of -cyano-cytarabine represented by formula (II), Γ-cyano-gemcitabine represented by formula (III), l'-cyano-5- azacytidine represented by formula (IV) and l'-cyano-5-aza-2'-deoxy cytidine represented by formula (V), a prodrug or an acceptable salt thereof:

(ii) (lit) (IV) X-OH

(V) X=H

The compound of the present invention, a prodrug or an acceptable salt thereof is highly resistant to deamination by CDA and highly stable against CDA.

The high level of CDA resistance of the compound of the present invention affords animals such as mouse, rat and dog excellent pharmacokinetic properties and in vivo efficacy by PO or IV administration. Further, the present invention provides a pharmaceutical composition for preventing or treating a cancer, comprising the compound of the present invention as an active ingredient, and a pharmaceutically acceptable carrier or an excipient.

The cancer may be selected from the group consisting of hematological malignancies, epithelial tumors, melanoma, leukemia, acute promyelocyte leukemia, lymphoma, osteogenic sarcoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, lung cancer and a combination thereof.

In one embodiment of the present invention, the compound may be formulated in a pharmaceutical composition, comprising one or more of the compound and a pharmaceutically acceptable carrier or an excipient.

The compound can be mixed with a pharmaceutically acceptable carrier or an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the compound. Thus, the pharmaceutical compositions can be in the form of tablets, pills, powers, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, and other orally ingestible formulations.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, magnesium carbonate, water, ethanol, propylene glycol, syrup, and methyl cellulose.

The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl-hydroxybenzoates, sweetening agents; and flavoring agents. The compositions of the present invention can also be formulated so as to provide quick, sustained or delayed release of the novel compound after administration to the patient by employing procedures known in the art.

As used herein, the term "pharmaceutically acceptable carrier" refers to those components in the particular dosage form employed which are considered inert and are typically employed in the pharmaceutical fields to formulate a dosage form containing a particular active compound. This may include without limitation solids, liquids and gases, used to formulate the particular pharmaceutical product. Examples of carriers include diluents, flavoring agents, solubilizers, suspending agents, binders or tablet disintegrating agents, encapsulating materials, penetration enhancers, solvents, emollients, thickeners, and dispersants, sustained release forms, such as matrices, transdermal delivery components, buffers, stabilizers, and the like.

The pharmaceutical composition of the present invention may be formulated in the absence or presence of carriers or excipients that provide a sustained released effect.

The pharmaceutical composition of the present invention may be prepared according to methods known in the art. It is contemplated that administration of such compositions may be via oral, injectable and/or parenteral routes depending upon the needs of the subject. The pharmaceutical composition of the present invention can also be administered by nasal or oral inhalation, oral ingestion, intramuscular injection, intravenous injection, intraperitoneal injection, transdermally, or other forms of administration.

Aerosol formulations for use in this invention typically include propellants, such as a fluorinated alkane, surfactants and co-solvents and may be filled into aluminum or other conventional aerosol containers which are then closed by a suitable metering valve and pressurized with propellant, producing a metered dose inhaler. Aerosol preparations are typically suitable for nasal or oral inhalation, and may be in powder or solution form, in combination with a compressed gas, typically compressed air. Additionally, aerosols may be useful topically. Topical preparations useful herein include creams, ointments, solutions, suspensions and the like. These may be formulated to enable one to apply the appropriate dosage topically to the affected area once daily, up to 3-4 times daily as appropriate. Topical sprays may be included herein as well.

Depending upon the compound, transdermal delivery may be an option, which can provide a relatively steady state delivery of the medication. Transdermal delivery typically involves the use of a compound in solution, with an alcoholic vehicle, optionally a penetration enhancer, such as a surfactant and other optional ingredients. Transdermal delivery systems of matrix and reservoir type are examples of suitable transdermal systems. Transdermal delivery differs from conventional topical treatment in that the dosage form delivers a systemic dose of medication to the patient.

The present invention includes a method for inhibiting the growth of a cell using a compound of the present invention. In particular, the present invention provides a method for inhibiting the growth of a cancer cell in a subject, which comprises administering a therapeutically effective amount of the compound to the subject in need thereof.

Also, the present invention provides a method for preventing or treating a cancer in a subject, which comprises administering a therapeutically effective amount of the compound to the subject in need thereof.

The compound of the present invention, a prodrug or an acceptable salt thereof may be administered orally or intravenously. Combination therapy includes combining the method of preventing or treating cancer as described in the invention and one or more cancer therapeutic methods. The cancer therapeutic methods include surgical therapy, radiation therapy, administering an anticancer agent (including, for example, antineoplastics, novantrone, bicalutamide, esterified estrogens, goserelin, histrelin, leuprolide, nilandron, triptorelin pamoate, docetaxel, taxotere, carboplatin, and cisplatin angiogenesis inhibitors), immunotherapy, antineoplastons, investigational drugs, vaccines, and less conventional therapies (sometimes referred to as novel or innovative therapies, which include, for example, chemoembolization, hormone therapy, local hyperthermia, photodynamic therapy, radiofrequency ablation, stem cell transplantation, and gene therapy).

In embodiments of the method of preventing or treating cancer, the compound may be administered in combination with other active agents. Generally, the compound used in the methods of prevention or treatment is administered in an amount which effectively achieves the desired therapeutic result in a subject. The compound may be administered as a pharmaceutical composition, or in the absence of a carrier or diluent. Naturally, the dosages of the compound will vary somewhat depending upon the components of the compound, the rate of in vivo hydrolysis, etc. Those skilled in the art can determine the optimal dose of the compound based on clinical experience and the treatment indication.

Preferably, the amount of compound administered to a subject is about 0.1 to about 100 mg/kg of body weight, more preferably, about 5 to about 40 mg/kg. Other preferred dosages include about 0.1 to about 10 mg/kg of body weight, about 1 to about 100 mg/kg of body weight, about 1 to about 60 mg/kg of body weight, about 1 to about 10 mg/kg of body weight, about 10 to about 100 mg/kg of body weight, about 10 to about 60 mg/kg of body weight, about 20 to about 60 mg/kg of body weight and about 30 to about 50 mg/kg of body weight.

The compound can also be converted into a pharmaceutically acceptable salt or pharmaceutically acceptable solvate or other physical forms (e.g., polymorphs) by methods known in the art field.

The subjects that may be treated using the compounds of the present invention may be mammals including humans. The present invention also includes a use of the compound of the present invention for the manufacture of a medicament for preventing or treating a cancer.

The present invention also includes a kit comprising one or more of the compound of the present invention and instructions for its use.

The following Examples are intended to further illustrate the present invention without limiting its scope. TEST EXAMPLE 1: Molecular docking with CDA structure

The coordinates of the X-ray structures of the human CDA in complex with cytidine (PDB ID: 1MQ0) were retrieved from the protein data bank. The structure was then subjected to the Protein Preparation Wizard workflow implemented in the Schrodinger package. The molecules were docked in the Gold program with Goldscore. After the scoring, the pose of ligand was selected with the best Goldscore. In the co-crystal structure of human CDA (1MQ0) with l-bata-ribofuranosyl-l,3-diazepinone (reference), goldscore showed 52.6. For clarity, only the side chains of the residues are shown in Fig. 1. All the residues shown in Fig. 1 belong to human CDA monomer Al .

As shown in Fig.1 (a), Ara-C favorably interacts with the E67 through the H-bonds and with the hydrophobic V38 and C65 with preserved goldscore 51.9 compared to reference goldscore 52.6. However, this interaction is disrupted by the presence of CN in 1 '-position of Ara-C. As shown in Fig.l (b), Γ-CN group causes crash in binding pocket with reduced goldscore 45.8.

TEST EXAMPLE 2: Cytidine deaminase (CDA) assay

Activities of human cytidine deaminase on cytidine, Ara-C and Γ-CN- Ara-C were compared. His ₆ -tagged, E. co/ -purified recombinant human cytidine deaminase (CD A) was incubated with 0.01 mM of each of the cytidine, Ara-C and l '-CN- Ara-C, respectively, in a 1 mL reaction, containing 20 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 mM EDTA, 100 mM NaCl and 40% glycerol at 25°C. Changes in observance at OD ₂ 82 were recorded at every 10 seconds for 3 minutes using the continuous spectrophotometric method (A. Amici, et al., Br. J. Haematol , 1989, 73 : 392-395).

[Table 1]

As shown in Table 1 , and Fig. 2, -CN- Ara-C was not a substrate of CD A, and completely resistant to deamination by CDA in vitro.

TEST EXAMPLE 3: Anti-proliferative activity test

Anti-proliferative activities in IC ₅₀ (uM) of Ara-C and l '-CN- Ara-C against hematological cancer (ML-2, MOLT-4, MV-4- 1 1, and HL-60), colon cancer (HCT- 1 16) and liver cancer (Hep G2) cell lines were compared.

CellTiter-Glo ^® Luminescent Cell Viability Assay (Promega #G7572) was used to measure the 50% inhibition concentration (IC ₅₀ ) of the Ara-C and l '-CN- Ara-C against ML-2 (human acute myeloma leukemia), MOLT-4 (human acute lymphoblastic leukemia), MV-4- 1 1 (human biphenotypic B myelomonocytic leukemia), HL-60 (human acute promyelocyte leukemia), Hep G2 (human hepatocellular carcinoma), HCT 1 16 (human colorectal carcinoma) cell lines. Six hundred cells in 90 μΐ media were plated per well in duplicates in a 96-well plate. Cells were incubated in a humidified incubator at 37°C with 5% CO ₂ during the assay. DMSO was added to the test compounds with the final DMSO concentration being 0.1% [v/v] of the culture. 100 μΐ of CellTiter-GkT was added to the equal volume of cultured cells to read luminescence in En Vision Multi Label Reader. The results are shown in Table 2 below. [Table 2]

As shown in Table 2, l '-CN- Ara-C exhibited the proliferative activities on hematological cancer, liver cancer and colon cancer cells.

TEST EXAMPLE 4: Animal pharmacokinetics

Pharmacokinetics of Ara-C and l'-CN- Ara-C were evaluated in ICR mice following intravenous (IV) administration at 10 mg/kg and per oral (PO) administration at 10 mg/kg dose level, respectively.

For serum compound analysis, blood samples were collected by cardiac puncture over 24 hr time course. 20 μΐ of spiked plasma was added into 96 well plate, adding ten volumes of internal standard (IS) in acetonitrile (ACN) to precipitate proteins, mixed fully, centrifuged for 10 min at 4,000 rpm. 150 μΐ of supernatant was transferred to another pre-labeled 96 well plate, mixed with 150 μΐ of water, then injected 5 μΐ or 10 μΐ into the liquid chromatography-mass spectrometry (LC-MS) system under the following conditions:

Column: API-4000 + Waters UPLC (TCLM08);

Mobile phase: water : MeOH = 100:0, 30:70, 5 :95 and 100:0 (v/v %);

Flow rate: 0.45 mL/min. Lower limit of quantification (LLOQ) was at 1 ng/mL. Pharmacokinetic parameters of half-life (T _1/2 ), clearance (CL), maximum plasma concentration (C _max ), time when the maximal serum concentration of compound is achieved (T _max ) and bioavailability (F%), exposure (AUC) and volume of distribution (V _ss ) of the compound were calculated using Phoenix WinNonlin 6.3 (non- compartmental model). The results are shown in Table 3, and Fig. 3.

Also, pharmacokinetics of Ara-C and -CN-Ara-C were evaluated in Sprague Dawley (SD) rats following IV administration at 3 mg/kg dose level, and PO administration at 3 mg/kg dose level, respectively. The analytical procedures were as described in the above and the results of rat pharmacokinetics are shown in Table 3, and Fig. 3. Further, pharmacokinetics of Ara-C and l'-CN- Ara-C were evaluated in beagle dogs following IV administration at 3 mg/kg and PO administration at 3 mg/kg dose level, respectively. The analytical procedures were as described in the above and the results are shown in Table 3, and Fig. 3. [Table 3]

Dose T C CI RT , v _z

Compounds animal route F%

(mg/kg) (hr) (u hr) (uM) (mL/min/kg) (hr) (L/Kg)

IV 10 0.8 33.3 20.5 1.4

Ara-C mouse

PO 10 0.7 8.6 5 26

IV 10 4.4 25.0 24.9 1.1 9.5 l '-CN-Ara-C mouse

PO 10 4.4 17.2 5 2.3 69

0.5

IV 3 4.4 25.1 7.5 1.0

l '-CN- Ara-C rat (V _ss )

PO 3 3.0 7.1 2.0 2.8 28

0.8

IV 3 3.9 47.4 3.9 3.1

l '-CN-Ara-C dog (V _ss )

PO 3 3.7 40.8 7.4 4.5 87 As shown in Table 3, -CN- Ara-C showed significantly improved oral bioavailability, longer half-life and increased clearance in dog.

TEST EXAMPLE 5: Tumor growth inhibition efficacy of l'-CN-Ara- C in HL-60 human leukemia xenograft

Tumor growth inhibition (TGI) activities of IP-administered Ara-C, and IP/PO-administered l'-CN- Ara-C were compared in HL-60 human leukemia xenografts.

Tumor growth inhibitions were observed in in vivo subcutaneous mouse xenograft model (HL-60 cell line, Corning #3603). NOD/SCID female mice of 6-8 week age and weighing approximately 18-22 g, were used for tumor inoculation. Each mouse was inoculated subcutaneously at the right flank with HL-60 cancer cells (1 x 10 ⁷ cells) in mixture of 100 μΐ of PBS and 100 μΐ of matrigel™ for tumor development. Eight mice were used for each group (n=8 / group). The amounts of treated compounds are represented in milligram (mg) of compound per kilogram (kg) of animal body weight (mg/kg, mpk). Administration route of the test compound was either intraperitoneal (IP) injection or peros (PO, oral) injection. Three groups, i.e., vehicle group (Control group), Ara-C group (treatment at 40 mpk of cytarabine through IP injection; Comparative group) and Γ-CN- Ara-C group (treatment at 40 mpk through IP and 60 mpk through PO injection) of mice were used. The overall administration schedule was 2 cycles of treatment for 5 days (5d ON), followed by 2 days of non-treatment (2d OFF). Tumor sizes were measured using a caliper, once every 4 days, and the tumor volume was expressed in mm using Equation 1 :

[Equation 1]

V = 0.5 a x b ²

Wherein, a and b are the long and short diameters of the tumor, respectively. The tumor volume (mm ) and body weight (g) results of the three groups are shown in Fig. 4. Also, tumor growth inhibition (% TGI) and body weight change (% BW change) compared to that of Ara-C were shown in Table 4.

[Table 4]

As shown in Table 4 and Fig. 4, oral (PO) dosing of Γ-CN- Ara-C shows a good potency.

Further, IP dosing of Γ-CN- Ara-C exhibited superior efficacy (73% TGI) to IP dosing of Ara-C at the same dose (40 mpk).

TEST EXAMPLE 6: Drug concentration in plasma/tumor tissue

The concentration of Ara-C and -CN- Ara-C in plasma/tumor tissue were evaluated in Test Example 5 in HL-60 human leukemia xenografts.

As shown in Fig. 5, Γ-CN- Ara-C retained high intracellular drug concentration in plasma/tumor tissue from leukemia HL-60 cell derived xenograft model.