KR101732796B1 - A pharmaceutical composition for preventing or treating cancer comprising sugar chemical compound-taxane compound conjugate and a method for preparation thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a drug conjugate of a taxane compound having a carboxy group and a sugar compound having a hydroxy group, Reacting a taxane compound having a hydroxyl group with a sugar compound having a carboxyl group in the presence of 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide, , A method for producing a drug conjugate, and a carboxymethyl dextran-docetaxel polymer conjugate represented by Chemical Formula (1).
The drug conjugate of the present invention is capable of releasing a taxane compound by hydrolysis of the ester bond in the conjugate in a mildly acidic environment in a cancer microenvironment. Since no enzymes are required for the above process, it can be effectively used as a cancer therapeutic agent that minimizes adverse effects by releasing taxane compounds selectively at cancer lesion sites which are not normal tissues.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer including a sugar chemical compound-taxane compound conjugate, and a method for preparing the same. method for preparation thereof}

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a drug conjugate of a sugar compound and a taxane compound, a pharmaceutical composition comprising a taxane compound in combination with 4-dimethylaminopyridine and (N, N'-dicyclohexylcarbodiimide) in the presence of N, N'-dicyclohexylcarbodiimide, and a method for producing a drug conjugate comprising the step of reacting a carboxymethyl dextran ) - docetaxel polymer conjugate.

Taxane-based compounds, such as paclitaxel and docetaxel, are anticancer drugs approved by the FDA for use in clinical practice. They inhibit normal cell division by over-stabilizing the microtubules of the cells , And shows the effect. However, these anticancer agents are also injured in normal cells, particularly in normal cells, which are active in cell division as well as cancer cells. These drugs show adverse effects such as gastrointestinal mucosal damage, hair loss and abdominal pain, and the serious toxicity of the drug itself as well as its low solubility in aqueous solution, This has resulted in a halt. Therefore, it is essential to study the development of new cancer-targeting drug formulations that minimize the side effects of these drugs and maximize their effects in cancer cells.

Drug delivery systems are designed to exhibit physicochemical changes in response to stimuli such as pH, reduction, hypoxia, and reactive oxygen species. Of these potential cellular stimuli, pH has been extensively studied to develop drug delivery systems for cancer therapeutics, since the extracellular matrix of cancer tissues and intracellular compartments such as endosomes and lysosomes are weakly acidic. However, polymer conjugates that release docetaxel in a mildly acidic environment have not yet been studied as cancer drugs.

Recently, it has been proposed to improve the solubility of hydrophobic drugs, such as physical encapsulation of liposomes, polymeric micelles and polymeromas, as well as chemical conjugation to hydrophilic polymers, while at the same time improving the in vivo biodistribution Many strategies have been used to improve the drug conjugate. Polymer conjugates as anticancer drug delivery vehicles have high cancer targeting rates due to enhanced permeation and retention (EPR) effects. The conjugate system has the advantage that it can improve water solubility and change the pharmacokinetics and biodistribution of hydrophobic drugs.

In addition to these strategies, attempts have been made to produce conjugates using polymeric compounds that are biodegradable and biocompatible for effective delivery of drugs. However, the yield of the conventional drug conjugate was very low because it undergoes two or more chemical reactions. In addition, a polypeptide linker, which is mainly used for producing a complex, has a disadvantage in that it requires a complicated manufacturing process because it requires an enzyme specifically acting on the polypeptide.

Under these circumstances, the inventors of the present invention conducted a one-step esterification reaction using 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide to obtain hydroxy The present inventors prepared a drug delivery system in which docetaxel, a taxane compound having a carboxy group, was directly bound to carboxymethyldextran, a sugar compound having a carboxy group, To solve the problem of long-term toxicity in vivo, and to show a high therapeutic effect, thus completing the present invention.

One object of the present invention is to provide a method for preventing or treating cancer comprising as an active ingredient a drug conjugate of a taxane compound having a carboxy group and a sugar compound having a hydroxy group To provide a pharmaceutical composition.

It is another object of the present invention to provide a process for the preparation of a taxane compound having a hydroxyl group in the presence of 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide, Or a pharmaceutically acceptable salt thereof, with a compound.

Another object of the present invention is to provide a carboxymethyl dextran-docetaxel polymer conjugate represented by the general formula (1).

In one aspect of the present invention, there is provided a pharmaceutical composition comprising a drug conjugate of a sugar compound having a carboxy group and a taxane compound having a hydroxy group as an active ingredient Or a pharmaceutically acceptable salt thereof.

The term "sugar compound having a carboxy group" of the present invention means a sugar compound having a functional group of -COOH, and a sugar compound means any compound having a sugar residue it means.

The term " taxane compound having a hydroxy group "of the present invention means a taxane compound having an '-OH' functional group, and the taxane compound is a substance which is widely used in chemotherapy Means a diterpen compound series.

The term "drug conjugate " of the present invention means a compound in which two or more compounds are linked to each other, and at least one compound among the above-mentioned linked compounds may be a pharmaceutically effective compound.

As a specific example, pharmaceutically effective compounds include, but are not limited to, paclitaxel, docetaxel, cabazitaxel, or protaxel, which are, for the purpose of the present invention, taxane compounds having a hydroxyl group.

Also, other compounds that are linked to a pharmaceutically active compound include, but are not limited to, carboxymethyl dextran, hyaluronic acid, chondroitin sulfate, heparin, ), Dermatan sulfate, alginate, and the like.

The term "cancer" of the present invention refers to a mass abnormally grown by autonomous overgrowth of the body tissue. Normally, abnormal cells to be killed are overproduced. In some cases, (Mass), and the state of destroying or deforming an existing structure can be defined as cancer.

The cancer may be, but is not limited to, lung cancer, oral cancer, esophageal cancer, bladder cancer, prostate cancer or cervical cancer.

The drug conjugate of a carboxy group-containing and a hydroxy group-containing taxane compound of the present invention may be prepared by combining 1 to 10 taxane compounds per 100 sugar residues of the sugar compound And specifically may be bonded with 2 to 9, and more specifically, with 3 to 8.

In one embodiment of the present invention, it was confirmed that docetaxel, which is a taxane compound having a hydroxyl group per 100 sugar residues of carboxymethyl dextran, which is a saccharide compound having a carboxyl group, is bound with 5 to 6 ).

The drug conjugate of the present invention is a pH-sensitive conjugate, and the release of the taxane compound may increase as the ester bond is decomposed by hydrolysis at the pH of the acidic condition. The pH of the acidic condition may be specifically from 3 to 6.9, more specifically from 4 to 6.8.

As a specific example, the release rate and cumulative release of the drug conjugate may increase as the pH decreases. More specifically, the conjugate of the present invention can be hydrolyzed at a pH of at least 60% to release the drug, and more particularly, the conjugate of the present invention is capable of hydrolyzing more than 80% at pH 5.0 to release the drug.

The term "prevention" of the present invention means any action to inhibit or delay the onset of cancer by administration of a pharmaceutical composition comprising the drug conjugate of a taxane compound having a carboxyl group and a saccharide compound having a carboxyl group of the present invention as an active ingredient .

The term "treatment" of the present invention means any suspicion of cancer by administration of the pharmaceutical composition, and any action in which the symptom of the affected subject is improved or changed.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants. In addition, the pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, oral formulations, external preparations, suppositories, . Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, ), Lactose, gelatin and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The compounds according to the present invention can be selectively hydrolyzed at cancer lesion sites having a lower pH than normal tissues to release the drug. Therefore, the compounds of the present invention can be formulated into injections and administered systemically.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administering" of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The term "individual" refers to all animals, including humans, mice, livestock, and the like, which have developed or are capable of developing cancer. As a specific example, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors.

Specifically, the composition of the present invention may be administered at a dose of 0.0001 to 100 mg / kg body weight per day, more specifically 0.001 to 100 mg / kg body weight, based on the solid content. The administration may be carried out once per day, or divided into several doses.

In another embodiment, the present invention relates to a process for preparing a taxane compound having a hydroxyl group in the presence of 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide in the presence of a carboxy group And reacting the compound with a compound of the present invention.

The compound "4-dimethylaminopyridine (DMAP)" used in the present invention is a pyridine derivative having the formula (CH ₃ ) ₂ NC ₅ H ₄ N, which is a colorless solid and has an anhydrides Esterification reaction, Baylis-Hillman reaction, hydrosilylation, tritylation, Steglich rearrangement, β-lactam star Staudinger synthesis of β-lactams and the like are usefully used as nucleophilic catalysts. It is also used as a catalyst for the Steglich esterification reaction.

The compound "N, N'-dicyclohexylcarbodiimide (DCC)" used in the present invention is an organic compound having the chemical formula of C ₁₃ H ₂₂ N _{2. In} the synthesis of an artificial peptide, It is mainly used for amino acid coupling. Under standard conditions, it exists as a white crystalline form with a dense stage. It is easy to handle because it has a low melting point and exists as a liquid. Very soluble in solvents such as dichloromethane, tetrahydrofuran, acetonitrile and dimethylformamide, but not soluble in water. It is used as a coupling agent in the stegyl esterification reaction.

As described above, 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide can be used together in the steglylisesterification reaction. The steric ester esterification reaction is a kind of esterification reaction using 4-dimethylaminopyridine as a coupling agent and N, N'-dicyclohexylcarbodiimide as a catalyst. The reaction can be generally carried out at room temperature. It is also preferred to carry out the reaction in an organic solvent. Non-limiting examples of the organic solvent include formamide, dimethylsulfoxide, and dichloromethane, and they may be mixed and used. The reaction conditions can be mild to obtain esters that can not be prepared by other methods, such as, for example, esters of sensitive 1,4-dihydroxybenzoic acid.

The molar ratio of 4-dimethylaminopyridine and N, N'-dicyclohexylcarbodiimide to the taxane compound having a hydroxyl group used in the above reaction may be specifically 1: 6 to 1: 12, but is not limited thereto .

In one embodiment of the present invention, 1 mM 4-dimethylaminopyridine and 1 mM N, N'-dicyclohexylcarbodiimide are added to 0.25 mM docetaxel, a taxane compound having a hydroxyl group, to form carboxymethyldext To-docetaxel drug conjugate was prepared (Fig. 1).

In another aspect, the present invention provides a carboxymethyl dextran-docetaxel polymer conjugate represented by the following general formula (1).

[Chemical Formula 1]

The term "conjugate " of the present invention means a compound in which two or more compounds are linked together. The linkage may be accomplished by non-covalent or covalent bonding, but specifically the conjugate of the present invention may be covalently linked by forming an esterified linkage through the carboxymethyldextran carboxyl group to the hydroxyl group of docetaxel.

Specifically, the polymer conjugate according to the present invention may have a weight average molecular weight (Mw) of 10,000 to 100,000.

In one experimental example of the present invention, docetaxel was chemically conjugated to the backbone of carboxymethyldextran through formation of a pH-sensitive ester bond so that docetaxel, a taxane compound, could easily be released from tumor tissues having weak acidic conditions (FIG. 1), and the chemical structure of the conjugate was confirmed using ¹ H NMR (FIG. 2).

In another experimental example of the present invention, it was confirmed that the release rate of docetaxel increased with decreasing pH (FIG. 3). In another experiment example of the present invention, free docetaxel and carboxymethyldextran released from the conjugate -Doxetaxel drug conjugate showed cancer cell toxicity in a concentration-dependent manner (FIG. 4), and the drug conjugate was absorbed in SCC7 cancer cells in a time-dependent manner (FIG. 5).

In another experimental example of the present invention, in order to demonstrate the cancer-target ability of the conjugate, the conjugate labeled with fluorescence was administered to a mouse having cancer. As a result, it was confirmed that a high level of fluorescence intensity exhibited by the conjugate was observed in cancer tissues (FIGS. 6 and 7), confirming that the drug conjugate exhibits excellent cancer targeting ability.

As described above, the carboxymethyldextran-docetaxel polymer conjugate, which is a drug conjugate of a taxane compound having a carboxy group and a hydroxy group according to the present invention, And can selectively release a large amount of drug selectively at a general pH of 6.5, which is a common pH of the cancerous microenvironment, which is lower than the physiological pH of 7.4. Therefore, even if systemic administration through intravenous injection is performed, cancer can be effectively treated while minimizing the side effects accumulated in other organs have.

The drug conjugate of the present invention is capable of releasing a taxane compound by hydrolysis of the ester bond in the conjugate in a mildly acidic environment in a cancer microenvironment. Since no enzymes are required for the above process, it can be effectively used as a cancer therapeutic agent that minimizes adverse effects by releasing taxane compounds selectively at cancer lesion sites which are not normal tissues.

Figure 1 a is a schematic of the synthesis of carboxymethyl dextran (CMD) -docetaxel (DTX) conjugates. Figure 1 (b) is a schematic diagram showing pH-responsive DTX release. DTX is released from the intracellular compartment of cancer cells because the ester linkage of the conjugate is easily degraded under mildly acidic conditions.
2 is a ¹ H NMR spectrum of a CMD-DTX conjugate contained in a D ₂ O / DMSO- d ₆ mixed solution (1: 1 volume ratio).
Figure 3 is a graph showing the behavior of DTX released from the CMD-DTX conjugate in vitro at 37 ° C as a function of time. The error bars in the graph represent standard curves (n = 3).
Figure 4 is a graph showing the in vitro cytotoxicity of free DTX and CMD-DTX conjugates under different pH conditions. The error bars in the graph represent the standard deviation (n = 5).
Figure 5 is a confocal micrograph of SCC7 cells cultured in CM5-DTX conjugate labeled with Cy5.5. a is 30 minutes, and b is 3 hours.
Figure 6a is an in vivo NIF fluorescence image showing the CM5-DTX conjugate labeled with Cy5.5 present in mice bearing cancer cells. 6 (b) and 6 (c) are graphs showing the intensity of fluorescence according to the function of time, b is the whole body, and c is the intensity of fluorescence present in the cancerous part of the living animal. The error bars in the graph represent the standard error for five animals per group.
Figure 7a is an ex vivo fluorescence image of the conjugate present in cancer and organs 24 hours after conjugate injection. b is a graph that quantifies the CMD-DTX conjugate present in cancer and organs. The error bars in the graph represent the standard error for five animals per group.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Carboxymethyldextran ( carboxymethyl dextran ; CMD ) - docetaxel ( docetaxel ; DTX ) Synthesis of conjugate

The esterification reaction was carried out in the presence of DCC (dicyclohexylcarbodiimide, Sigma Aldrich, St. Louis, MO, USA) and DMAP (dimethylaminopyridine, Sigma Aldrich, St. Louis, Mo., USA) Docetaxel (DTX, LC Laboratories, Woburn, MA, USA) was chemically conjugated to the backbone of carboxymethyl dextran (CMD, Sigma Aldrich, St. Louis, Mo., USA).

Briefly, 200 mg of carboxymethyldextran (0.84 mM) was dissolved in 30 mL of formamide, followed by addition of 25 mL of DMSO (dimethylsulfoxide), followed by stirring. 204 mg of docetaxel (0.25 mM) was dissolved in 5 ml of DMSO and then 122 mg of 4-dimethylaminopyridine (DMAP, 1 mM) and 206 mg of N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide; DCC, 1 mM) was added to the docetaxel solution. The docetaxel solution was slowly added to the carboxymethyldextran solution, and the mixture was stirred for 48 hours while blocking the light. The reaction solution was purified by centrifugation at 300 rpm for 5 minutes to remove dicyclohexylurea residues. Thereafter, dialysis was performed with distilled water adjusted to pH 7.4 for two days, followed by lyophilization. The prepared sample was dissolved in a mixed solution of D ₂ O and DMSO- d ₆ (1: 1 volume ratio), and the chemical structure of the conjugate was confirmed using 500 MHz ¹ H NMR (unity inova 500 MHz, VARIAN, USA).

Example  2: CMD - DTX  Junction DTX  Emission Behavior Evaluation Experiment

In vitro, a water-soluble buffer solution (phosphate buffer solution of pH 7.4, and acetate solution of pH 6.5 and pH 5.0) having different pH values as the medium was used to evaluate the drug release behavior of the conjugate according to pH Respectively.

Briefly, CMD-DTX was dispersed in a buffer solution containing 0.1% Tween 80 surfactant and transferred to a cellulose membrane tube (MWCO = 3500 Da). After placing the dialysis tube in the release medium, it was stirred at 100 rpm in a constant temperature water bath at 37 캜. The release medium was changed at regular intervals and the amount of docetaxel released was detected using high performance liquid chromatography (HPLC, Agilent 1200 series, Agilent Technologies, USA). The CMD-DTX was dissolved in acetonitrile / distilled water (51:49 v / v, 1 mg / ml) and then subjected to HPLC analysis using a symmetry column (Waters Corporation, MA, USA). The mobile phase consisted of acetonitrile / distilled water (51:49 v / v) at a flow rate of 1.0 ml / min. The diode array detector was set at 227 nm and all samples were analyzed based on the standard curve of free DTX.

Example  3: CMD - DTX  Toxicity evaluation experiment of conjugate

Squamous cell carcinoma (Squamous cell carcinoma; SCC7) cells (American Type Culture Collection, Rockville, MD, USA) to 10% in a 5% CO ₂ incubator for 37 ℃ (v / v) FBS and 1% (w / v) And cultured in RPMI1640 medium containing penicillin-streptomycin.

The cytotoxicity of CMD-DTX was evaluated by MTT assay.

Simply described, 1 × 10 ⁴ cells in 96-well plates were dispensed at a concentration of cells / well. After 24 hours, the cells were rinsed twice with DPBS and cultured for one day in media containing various concentrations of free DTX and conjugate. In this experiment, the pH of the medium was adjusted to 7.4, 6.5 or 5.0. Then, 25 μl of MTT solution (0.5 mg / ml in RPMI 1640) was added to each well and the cells were further incubated for 4 hours. Then, the medium was removed and 200 쨉 l of DMSO was added to the cells. Finally, cytotoxicity of the conjugate was assessed by measuring absorbance at 570 nm using a microplate reader (BioTek, Seoul, Korea).

Example  4: CMD - DTX  Cancer cell absorption behavior of conjugate

CMD-DTX conjugate was uptake to cancer cell SCC7.

SCC7 cells (1x10 ⁵ cells / well) were dispensed in 6-well plates onto gelatin-coated coverslips and cultured for 24 hours. The cells were then rinsed twice with DPBS and fixed with 4% formaldehyde solution. For nuclear staining, cells were incubated with 4,6-diamino-2-phenylinodol (DAPI) for 10 minutes and rinsed with DPBS. The intracellular location of CMD-DTX conjugates labeled with Cy5.5 (Amersham Biosciences, Piscataway, JN, USA) using a LSM 510 META NLO confocal laser scanning microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) Respectively.

Example  5: Using disease animal models CMD - DTX In vivo (in vivo ) Distribution and x-bibo vivo ) Distribution by organ Behavior evaluation experiment

All animal experiments were carried out in accordance with the relevant laws and institutional guidelines of SungKyunKwan University and conducted with the approval of the relevant institutional committee.

In order to evaluate the in vivo distribution behavior of the CMD-DTX conjugate, firstly, 1 x 10 ⁶ SCC7 (1: 1) dispersed in 100 [mu] l of medium in the left flank of 5.5-week-old male nude mice (20-25 g) Cells were injected subcutaneously to create a tumor animal model. After 14 days, when the size of the tumor reached 200 to 250 mm ³ , the conjugate labeled with Cy5.5 was intravenously injected via the tail vein. The in vivo distribution of the conjugate was evaluated as a function of time using an eXplore Optix system (ART Advanced Research Technologies Inc., Montreal, Canada). The cancer-targeting characteristics of the conjugate were evaluated by measuring the fluorescence intensity of the NIR marker (NIR dye, Amersham Biosciences, Piscataway, JN, USA) at the tumor site (n = 5). The results were analyzed using the region of interest (ROI) of the Analysis Workstation software (ART Advanced Research Technologies Inc., Montreal, Canada) and the values were expressed as mean ± SD in groups of five animals.

Major organs and tumors were obtained 24 hours after intravenous injection of the Cy5.5-labeled conjugate into the SCC7 tumor mouse model. NIR fluorescence intensities of extracted organs and tumors were observed using the eXplore Optix system.

The contents confirmed through the above examples are summarized in the following experimental examples.

Experimental Example  One. CMD - DTX  Fabrication of the bonded body

The CMD-DTX conjugate was simply prepared by chemically bonding DTX to the backbone of CMD through formation of a pH-sensitive ester bond (Fig. 1 (a)).

Polymer conjugates or nanoparticles have been shown to accumulate in cancer tissues due to improved permeation and preservation effects following systemic administration, but nanoparticles that can selectively release drugs that recognize the intracellular and extracellular microenvironment of tumor tissue are produced Many studies have not been conducted. Thus, the present inventors have found that DTX with biological activity is preferentially released from the conjugate in weak acidic conditions such as extracellular matrix and intracellular compartments (e. G., Endosomes and lysosomes) of tumor tissue because ester linkages are unstable in acid (Fig. 1 (b)).

The 2'- and 7-hydroxy groups of paclitaxel (PTX) structurally similar to DTX are capable of structural modification and conjugation. However, this reaction preferentially occurs in the 2'-hydroxy group because the steric hindrance reduces the reactivity of the 7-hydroxy group. Thus, as shown in Figure 1, the conjugate was composed of hydrophobic DTX twigs and a hydrophilic CMD backbone. ¹ was confirmed using the chemical structure of the H NMR and CMD, DTX, and CMD-DTX, exhibited the spectrum of the ¹ H NMR in Figure 2.

As a result, the conjugate showed characteristic peaks of CMD and DTX. The number of DTX per 100 sugar residues of CMD was found to be about 5.6, and the results were obtained from the peak integral ratio of the carboxymethyl group (4.10 ppm) of CMD to the t-butyl group (1.34 ppm) of DTX Respectively. Because of the hydrophilicity of CMD, the conjugate was water-soluble in water.

Experimental Example  2. CMD - DTX  Junction In vitro < RTI ID = 0.0 > DTX  Emission evaluation

The release behavior of DTX from the conjugate was evaluated under various pH conditions as a function of time and the experimental results of the present invention demonstrated that the release rate of DTX was highly dependent on the pH of the buffer solution.

The DTX release rate increased with decreasing pH, and over the entire test period, DTX release at pH 7.4 and pH 5.0 were 44.3 2.50% and 81.5 3.59%, respectively (FIG. 3).

The results indicate that rapid release of DTX is possible by ester linkage under acidic conditions simulating intracellular compartments of cancer cells such as endosomes and lysosomes.

Experimental Example  3. CMD - DTX  Junction In vitro < RTI ID = 0.0 >  Cytotoxicity Assessment

In SCC-7 cancer cells, cytotoxicity of CMD-DTX was assessed via MTT assay. Various concentrations of DTX and the conjugate were exposed to cells for days at different pH conditions.

As shown in Fig. 4, both free DTX and CMD-DTX showed concentration-dependent cytotoxicity. The cytotoxicity of free DTX was not dependent on the pH of the medium. Conjugates, on the other hand, showed higher toxicity with decreasing pH. At pH 7.4, the ester bond was not readily degraded, and the biologically active DTX was slowly released from the conjugate and showed lower toxicity than free DTX. Notably, at pH 5.0, the cytotoxicity of the conjugate was comparable to the cytotoxicity of free DTX and could be expected due to the rapid release of DTX under mildly acidic conditions. The results were consistent with the release behavior of DTX as shown in Fig.

Experimental Example  4. CMD - DTX  Junction In vitro < RTI ID = 0.0 >  Evaluation of cell absorption behavior

To investigate cellular uptake behavior, SCC7 cancer cells were incubated with Cy5.5-labeled conjugates and monitored by confocal microscopy at different time intervals.

As shown in Figure 5, weak fluorescence signals were detected from the cytoplasm after incubation with the conjugate for 30 minutes. However, strong fluorescence signals were observed not only in the cytoplasm but also in the nucleus after 3 hours.

These results imply that SCC7 cells absorb the conjugate through time-dependent, nonspecific endocytosis.

Experimental Example  5. CMD - DTX of Invivo (in vivo ) Distribution behavior and XVibo (ex vivo ) Long-term  Distribution behavior evaluation experiment

To demonstrate the cancer-target ability of the conjugates, the in vivo distribution behavior experiments of the conjugates were evaluated using NIR fluorescence imaging techniques, and then mice bearing SCC-7 cancer cells were injected with Cy5.5-labeled CMD -DTX was performed (Fig. 6 (a)).

NIR fluorescence was gradually reduced in the entire body for 12 hours due to circulation of the conjugate in the bloodstream (Fig. 6 (b)). Notably, during the entire time, strong fluorescence was observed at the tumor site and there was a clear difference between the subcutaneous tumor and normal tissue. As shown in FIG. 6 (c), the fluorescence intensity at the tumor site reached a maximum at 9 hours, and then gradually decreased.

The CM5-DTX conjugate labeled with Cy5.5 was intravenously administered in a tumor animal model. After 24 hours, mice were killed and the organ was excised and the Cy5.5 type light amount in each organ was measured (Fig. 7a).

As a result, high fluorescence intensity was observed in the kidney of the tumor animal model, and the result is considered to be due to the excretion effect of the conjugate. Except for kidney, weak intensity was observed in the remaining organs including the liver, spleen and heart. Quantitative analysis confirmed that high levels of fluorescence intensity were observed in other organs except the kidney, especially in cancer tissues (FIG. 7b).

These results are evidence that the conjugate exhibits excellent cancer targeting ability and imply that it is due to increased circulation and enhanced penetration and retention.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (14)

A carboxymethyldextran-docetaxel conjugate represented by the following Formula 1:
[Chemical Formula 1]

The method according to claim 1,
Wherein the conjugate has a weight average molecular weight (Mw) of 10,000 to 100,000.
The method according to claim 1,
Wherein said conjugate is conjugated to 5-6 of docetaxel per 100 of said carboxymethyldextran sugar moieties.
The method according to claim 1,
Wherein said conjugate has increased release of docetaxel at a pH of from 3 to 6.9.
The method according to claim 1,
Wherein said conjugate has increased release of docetaxel at a pH of between 4 and 6.8.
The method according to claim 1,
Wherein said conjugate releases more than 60% docetaxel by hydrolysis at pH 6.5.
The method according to claim 1,
Wherein said conjugate releases more than 80% docetaxel by hydrolysis at pH 5.0.
A pharmaceutical composition for preventing or treating cancer comprising the conjugate of any one of claims 1 to 7 as an active ingredient.
9. The method of claim 8,
Wherein said cancer is at least one selected from the group consisting of lung cancer, oral cancer, esophageal cancer, bladder cancer, prostate cancer and cervical cancer.
A method for preparing a sugar chemical compound-taxane compound conjugate,
A taxane compound having a hydroxyl group is reacted with a carboxyl group-bearing sugar in the presence of a 4-dimethylaminopyridine (DMAP) catalyst and N, N'-dicyclohexylcarbodiimide (DCC) Lt; / RTI > with a compound.
11. The method of claim 10,
Wherein the molar ratio of the 4-dimethylaminopyridine catalyst to the taxane compound having the hydroxyl group and the N, N'-dicyclohexylcarbodiimide coupling agent is from 1: 6 to 1:12.
11. The method of claim 10,
The saccharide compound having a carboxyl group is preferably a group consisting of carboxymethyl dextran, hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, and alginate. ≪ / RTI >
11. The method of claim 10,
Wherein the taxane compound having a hydroxyl group is selected from the group consisting of paclitaxel, docetaxel, cabazitaxel, and protaxel.
11. The method of claim 10,
Wherein the conjugate is a compound of Formula 1 prepared by reacting docetaxel of Formula 2 and carboxymethyldextran of Formula 3:
[Chemical Formula 1]

(2)

(3)

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