KR20070025135A - Composition for inhibiting stat3 activity comprising ceftriaxone - Google Patents

Abstract

A composition comprising ceftriaxone is provided to inhibit the activity of STAT3, thereby effectively treating or preventing cell proliferative disorders such as cancer. The composition for inhibiting STAT3 activity comprises ceftriaxone(C18H18N8O7S3:8-[2-(2-amino-1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino-4-[(2-methyl-5,6-dioxo-1H-1,2,4-triazin-3-yl)sulfanylmethyl]-7-oxo-2-thia-6-azabicyclo[4.2.0]oct-4-ene-carboxylic acid) represented by the formula(1), a salt thereof or a moist product thereof.

Description

Composition for inhibiting STAT3 activity comprising ceftriaxone including ceftriaxone

Figure 1 shows the results of examining the STAT3 inhibitory activity for each ceftriaxone concentration.

The present invention relates to a composition comprising ceftriaxone, a salt thereof, or a hydrate thereof having STAT3 inhibitory activity, and more particularly, to a composition having an anticancer effect by inducing apoptosis or inhibiting angiogenesis.

Cancer is the largest refractory disease, and the mechanism of cancer development and progression is closely related to diseases such as blood vessels, immunity, and rheumatism. Research has been conducted to develop various anticancer drugs. In particular, selective anticancer agents that act on specific molecular targets are attracting more attention as they can be applied to customized medicines and combination therapy as well as providing safe and effective treatments.

STATs are signaling and transcriptional regulator proteins. Activated by extracellular stimuli such as cytokines, hormones, growth factors, and the like, the tyrosine residues are phosphorylated, and the interactions of the SH2 domain form dimers that enter the nucleus and bind to specific promoters. The signaling system of these STAT proteins can be stopped by dephosphorylation and protein degradation.

STAT1, STAT3 and STAT5 activated forms are found in various carcinomas, especially STAT3, which is activated in hematological cancers such as leukemia, as well as in various solid cancers such as breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and prostate cancer. It acts as an important anticancer target (Hua Yu and Richard Jove, Nature Review Cancer (2004), 8 , 945).

STAT3 activity inhibits programmed cell death or apoptosis (Catlett-Falcone R. et al. , Immunity (1999), 10 , 105), induces angiogenesis (Niu, G. et al . , Oncogene (2002), 21 , 2000), inducing immune evasion (Wang T. et al ., Nature Medicine (2004), 10 , 48). Thus STAT3 acts as a target for cancer treatment (Bromberg, JF et al ., Cell (1999), 98 , 295; Niu, G., et al. , Cancer Research (1999), 59 , 5059), and the activity of STAT3 Inhibition is a complex anticancer mechanism to control the tumor, so anticancer effect can be expected higher than the conventional anticancer drugs. In addition, STAT3 protein is involved in not only tumors but also various intracellular functions, and thus, the discovery of inhibitors may also be used to develop immunosuppressants and diabetes treatments.

Ceftriaxone (C ₁₈ H ₁₈ N ₈ O ₇ S ₃ ; 8- [2- (2-amino-1,3-thiazol-4-yl) -2-methoxyimino-acetyl, a third-generation cephalosporin antibiotic) ] amino-4-[(2-methyl-5,6-dioxo-1H-1,2,4-triazin-3-yl) sulfanylmethyl] -7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-carboxylic acid) is an antibiotic with strong penetrating power, which is also used for infections of the brain and inner ear that are difficult to deliver. However, the anticancer efficacy of ceftriaxone is unknown.

Under these backgrounds, the present inventors confirmed that betalactam antibiotic ceftriaxone has excellent inhibitory activity on STAT3, and revealed that the ceftriaxone can be used as an anticancer agent and completed the present invention.

It is an object of the present invention to provide a composition having a STAT3 inhibitory activity comprising ceftriaxone, a salt thereof or a salt water salt thereof.

Another object of the present invention is to provide a method for preventing or treating a cell proliferative disorder by administering the composition to a patient.

In one embodiment, the present invention is to provide a composition having a STAT3 inhibitory activity comprising ceftriaxone, a salt thereof, or a water hydrate thereof represented by the formula (1).

STAT3 protein is a transcription factor (txn factor), apoptosis inhibitors (eg Bcl-xL, Mcl-1 and survivin), cell-cycle regulators (eg cyclin D1 and c-Myc), neovascularization It is known to function as an important mediator of oncogenic signaling through the transcriptional activity of genes encoding formation inducers (eg, vascular endothelial growth factor). . As the direct link between STAT3 and the carcinogenic process was revealed, STAT3 was recognized as a new target for chemotherapy. Indeed, using STAT3-specific siRNA to inhibit STAT3 expression inhibits cell growth and induces apoptosis (Gao LF et al., Acta Pharmacol Sin (2005), 6 , 377), Inhibiting STAT3 expression using STAT3-specific antisense oligonucleotides inhibits cell growth by inhibiting G1 to S cell cycle transition. (Ma X et al., Beijing Da Xue Xue Bao (2003), 18 , 50).

In the present invention, the term "signal transducers and activators of transcription 3 (STAT3) inhibition" means that STAT3 is activated to act as an electronic regulatory protein to reduce or block the activity of STAT3 in the carcinogenic process. Inhibition of STAT3 induces apoptosis and inhibits neovascular formation, thereby preventing or treating cell proliferative diseases.

The term “cell proliferative disease” refers to a disease in which malignant and nonmalignant hyperproliferative cell populations are formed that look morphologically different from surrounding tissue, and a representative example of such cell proliferative disease is cancer.

Ceftriaxone is a substance having a molecular formula of C ₁₈ H ₁₈ N ₈ O ₇ S ₃ and is widely used as an antibiotic. The present inventors found that cetriaxone exhibits STAT3 inhibitory activity. The beta-lactam compound of Formula 1 showed very strong STAT3 activity with IC ₅₀ value of 1.6 μM as a result of STAT3 inhibitory activity measurement. The results demonstrate that cetriaxone has STAT3 inhibitory activity and thus has therapeutic activity against cytostatic diseases such as cancer. Cancers that can be prevented or treated with ceftriaxone of the present invention include all cancers in which STAT3 is overexpressed. STAT3 is widely expressed in almost all cancers, and specific examples include colorectal cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, prostate cancer, head and neck cancer, and leukemia.

As used herein, the term "prevention" means any action that inhibits or delays the onset of a disease caused by STAT3 overexpression by administration of a composition comprising ceftriaxone, a salt or a salt water salt thereof, and "treatment" means By administration of a composition comprising ceftriaxone, a salt thereof, or a salt of a salt thereof, it is meant any action in which the symptoms of a disease already caused by STAT3 overexpression improve or benefit.

Ceftriaxone is already used as a broad-spectrum antibiotic and has been tested and approved as a drug that can be used at high concentrations depending on the symptoms of infection. In addition, Ceftriaxone exhibits strong permeability through the brain-blood barrier, so when Ceftriaxone is used as an anticancer agent, it can be effectively used for carcinoma of organs deep in the body such as pancreatic cancer, which is difficult to penetrate the drug. Can be. In this case, access to early primary cancers, where cancer neovascularization has not yet formed, is easy to prevent and treat cancer, and in particular, due to such good permeability, actual use can be reduced, and drug side effects and resistance may be prevented.

Ceftriaxone that can be used in the composition having the STAT3 inhibitory activity of the present invention includes carboxylic acid forms (C ₁₈ H ₁₈ N ₈ O ₇ S ₃ ) as well as salts or hydrates thereof. Preferably, the sodium salt _{(C 18 H 16 N 8 O} 7 S 3 Na 2) or a function of water _{(C 18 H1 6 N 8 Na} 2 O 7 S 3 · 3.5H 2 O).

The composition may be formulated into a human or veterinary preparation for oral or parenteral use, including a pharmaceutically acceptable carrier.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate ( Calcium carbonate, sucrose or lactose, and gelatin are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used. Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

In another aspect, the present invention relates to a method of preventing or treating a cell proliferative disease by administering the composition to a patient.

As used herein, the term "patient" is intended to include both humans and livestock such as cows, horses, rabbits, cats, dogs, sheep, and the like with cell proliferative diseases caused by STAT3 overexpression. By administering to a patient a composition of the present invention comprising ceftriaxone, a salt thereof, or a salt function thereof, cell proliferative diseases caused by the above-mentioned STAT3 overexpression, specifically colon cancer, lung cancer, breast cancer, ovarian cancer, Prostate cancer, pancreatic cancer, prostate cancer, head and neck cancer, leukemia, etc. can be effectively prevented or treated. The composition of the present invention can be administered in parallel with existing anticancer agents and the like.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as the target tissue can be reached. May be administered. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

Compositions of the present invention comprising ceftriaxone, salts thereof, or a salt water salt thereof may be administered alone or co-administered with adjuvants, cytokines and the like known in the art.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the sex, age, severity, activity of the drug of the patient. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The effective dose of the composition of the present invention is preferably 1 to 10 mg / kg once per adult.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  1: due to double reporter gene Stat 3  Protein activity measurement

Inhibition of the activity of STAT3 by ceftriaxone was measured by a luciferase assay.

STAT3 regulates the expression of genes by recognizing and binding to specific sequences present in the promoter region of genes that are regulated by STAT3. At this time, if luciferase is inserted into a specific gene, the amount of STAT3 is proportional to the degree of luciferase expression. Luciferase binds to the substrate and exhibits luminescence by luminescence reaction, and the amount of light at this time can be measured to determine the degree of activity of STAT3. In the present invention, a "dual luciferase assay" was devised and applied to the experiment. It simultaneously expresses fireflies luciferase and Renilla luciferase in one treatment, and then treats two separate substrates (Beetle luciferin and coelenterazine) specific to the two enzymes in sequence. It is a method of measuring the activity of STAT3 by measuring activity. Renilla luciferase corrects for nonspecific cytotoxicity and internal deviations that can occur in each experiment, so that accurate values expressed only by STAT3 can be derived. The introduction of the luciferase reporter gene into the cell was performed by transfection method. When introducing the reporter gene into the cell, the plasmid expressing Renilla luciferase was simultaneously infected regardless of the activity of STAT3. , Interference with luminescence reactions due to cytotoxicity. The luminescence reaction was measured using a wallacluminometer (Luminometer, Wallac 1420). The cell line used was gastric cancer cell line KatoIII with high transfection yield, and the plasmid was 21-pSTAT3 recombined with 21 copies of the STAT3 binding sequence (SEQ ID NO: 1) at the promoter region of pSTAT3-TA-Luc. -TA-Luc (firefly luciferase), pTA-Luc and pRL-TK ( renilla luciferase) were used. DNA primers containing a cis-acting element (TGCTTCCCGAACGT: SEQ ID NO: 1) sequence to which STAT3 binds were purchased from Genotech. 21-pSTAT3-TA-Luc was prepared by amplifying the purchased primers by PCR method and cloned into luciferase expression vector pSTAT3-TA-Luc (CLONTECH Laboratories, Inc.) having four STAT3 enhancer elements. At this time, XhoI site of expression vector was used for cloning.

(RLU: relative luminescence unit)

Blank: [21-pSTAT3-TA-Luc] + [pRL-TK] + [DMSO]

Control: [pTA-Luc] + [pRL-TK]

Sample: [21-pSTAT3-TA-Luc] + [pRL-TK] + [Compound]

By dissolving ceftriaxone in dimethylsulfoxide (DMSO) by the above method, the inhibition of STAT3 activity was observed at concentrations ranging from 0 to 50 ㎍ / ml. It can be seen that (Fig. 1). As a result of measuring a STAT3 inhibitory activity IC ₅₀ are given in Table 1.

compound IC ₅₀ (μM) Ceftriaxon 1.6

As described above, the compositions of the present invention comprising ceftriaxone, salts or salts thereof, inhibit the activity of STAT3, effectively treating or preventing cell proliferative diseases, more specifically cancer.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Composition for inhibiting STAT3 activity comprising ceftriaxone <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 14 <212> DNA <213> Artificial Sequence <220> 223 STAT3 binding sequence <400> 1 tgcttcccga acgt 14

Claims (2)

A composition having a STAT3 inhibitory activity comprising ceftriaxone, a salt thereof, or a water hydrate thereof according to Formula 1. Formula 1

The composition of claim 2 having an anticancer effect.

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