KR20150001693A - Water souble Heparin-DTX-TCA conjugates improved targeting and preparing method for the same - Google Patents

Abstract

The present invention relates to a target type water-soluble anticancer drug composed of a heparin-docetaxel-tourocholic acid conjugate and to a method for preparing the same and, more specifically, to a target type water-soluble anticancer drug in which a spherical nanoparticle is formed of a conjugate having aminized docetaxel and tourocholic acid linked to low-molecular weight heparin, and the docetaxel is introduced to a core of the nanoparticle and the tourocholic acid is introduced to a surface of the nanoparticle, and to a method for preparing the target type water-soluble anticancer drug, comprising the steps of removing a sodium salt from low-molecular heparin; introducing an amine group to docetaxel; introducing an amine group to tourcholic acid; mixing the sodium salt-removed heparin and the docetaxel with the introduced amine group and the tourocholic acid with the introduced amine group to self-aggregate the mixture; and purifying the self-aggregated heparin-docetaxel-tourocholic acid conjugate. According to the present invention, provided can be a target type water-soluble anticancer drug capable of overcoming the low solubility of docetaxel in water through the use of heparin and tourocholic acid, solving a problem in that heparin cannot be orally administered, improving the absorptivity in the ileum due to the introduction of bile acid, and expecting a synergy effect of simultaneously applying anticancer activity of docetaxel and anticancer activity of heparin.

Description

[0001] The present invention relates to a water-soluble anticancer agent comprising heparin-docetaxel-tocoloic acid conjugate,

The present invention relates to a water-soluble anticancer drug having improved hepatoprotective activity comprising heparin-docetaxel-tocolacic acid conjugate and a method for producing the same.

Recent cancer studies tend to be done through anti-angiogenic therapy. Tumor and cancer cells have been found to require excess nutrients because of their proliferation and abnormal growth. To supply a sufficient amount of nutrients for new cells, new blood vessels emerges. Anti-angiogenic agents can prevent the development of new blood vessels and inhibit the growth of cancer cells.

Low Molecular Weight Heparin (LMWH) is a well-known anticoagulant and anti-angiogenic agent. However, oral delivery of LMWH was restricted due to low water absorption. The reason for the low water absorption is due to the high molecular weight, the negatively charged structure and the hydrophilic structure in the natural state. Earlier researchers tried to solve this problem by conjugating it with deoxycholic acid (DOCA). DOCA is widely used as bile acid, and bile acid is used as a carrier to improve the absorption of heparin.

However, the major disadvantage of using DOCA is that DOCA is conjugated with LMWH to form micelles in aqueous solution and all DOCA is located inside micelles. As a result, notable maximum effect or absorption was not obtained. Water-soluble bile acids were required to be conjugated with LMWH to obtain maximum bioavailability of LMWH.

Docetaxole (DTX) is widely used as an anticancer agent and is known to be more effective than doxorubicin, paclitaxel and fluorouracil. Although DTX is an effective and widely used anti-cancer agent, its application is limited due to its route of administration.

Oral administration of DTX is required for better acceptance and convenience. Several formulations coated or loaded with a polymer similar to DTX have been proposed for oral administration. However, this type of formulation did not meet the desired bioavailability due to the lack of absorption enhancers.

In the present invention, it has been tried to prepare taurocholic acid (TCA), which is a kind of water-soluble bile acid, with LMWH in order to enhance the bioavailability of LMWH and finally to make micelles dispersible in aqueous solution by bonding DTX with heparin- Respectively.

As an example of achieving the above object, the water-soluble anticancer agent with improved targeting of the present invention comprises spherical nanoparticles as a conjugate in which docetaxel aminated with a low molecular weight heparin is bound to trocolic acid, and the core of the nanoparticles is doped with docetaxel Is introduced, and the surface is doped with tallowacetic acid.

At this time, it is preferable that the molecular weight of the low molecular weight heparin is in the range of 3000 to 5000 da.

As another example for achieving the above object, a method for producing a water-soluble anticancer drug having improved targeting of the present invention includes a process of removing a sodium salt of a low molecular weight heparin, a process of introducing an amine group into docetaxel, A step of mixing the sodium salt-eliminated heparin with the amine-introduced docetaxel and the tropolacol acid to cause self-aggregation, and a step of purifying the self-aggregated heparin-docetaxel-tocoloic acid conjugate. do.

The molar ratio of heparin to docetaxel is preferably in the range of 1: 1 to 10, and the molar ratio of the heparin and docetaxel to the heparin-docetaxel conjugate is preferably 1 to 10 molar equivalents.

Oral administration of anticancer drugs is very convenient because of its ease of administration and patient satisfaction. However, a totally effective anticancer agent could be applied as IV because of the water-insolubility in aqueous solution.

Low Moelcular Weight Heparin (LMWH), Taurocholic acid (TCA) and Docetaxol (DTX) were conjugated to develop multifunctional delivery vehicles.

Both LMWH and DTX have been shown to have anticancer effects, so their synergistic effects can be predicted. TCA and DTX are conjugated with LMWH to form micelles in the aqueous phase due to the hydrophobic nature of DTX.

Bile acids, more specifically TCA, are located on the surface of micelles to enhance the absorption of LMWH through bile acid transporters.

In addition, bile acid is a component which is absorbed through the site of the ileum, and is applied to the present invention, thereby facilitating the absorption of the LMWH-TCA-DTX conjugate through the ileum. Due to such a feature, the LMWH-TCA-DTX conjugate of the present invention is easily absorbed through a site for absorbing succinic acid, so that it can be used for oral administration.

TCA and DTX molecules are conjugated with LMWH through amide bond formation. The bond between TCA and DTX with LMWH is confirmed by FT-IR and 1 H-NMR. The coupling ratio of TCA to DTX was also measured. The synthesized LMWH-DTX-TCA particles were found to have a diameter of 150 to 250 nm depending on the DTX junction ratio. These nanoparticles can be utilized as multifunctional carriers.

According to the present invention, it is possible to solve the problem that the hydrophobic segment possessed by docetaxel significantly restricts the use of docetaxel as an anticancer agent.

According to the present invention, a low-molecular-weight-heparin (LMWH) capable of improving compatibility with biocompatibility with blood, and a hydrophilic environment on the surface of a micelle structure by self-aggregating docetaxel and tororoic acid, Availability can be demonstrated.

In addition, according to the present invention, it is expected that an existing anticancer agent used only as an injectable agent can be used as an oral anticancer drug by natural products, by making micelles which are a conjugate of nanoparticle size by self-aggregating LMWH and docetaxel.

In addition, when the present invention is put to practical use, the present invention has the potential to be developed as an oral drug that can be more easily taken than existing drugs and can prevent cancer more easily.

In addition, according to the present invention, it is expected that the synergistic effect of LMWH and DTX is exhibited in cancer and angiogenesis therapy.

Fig. 1 shows the synthesis process (A) of the LMWH-TCA conjugate and the synthesis process (B) of the LMWH-DTX-TCA conjugate.
Figure 2 shows the chemical structure (A) of HDTA and the physical structure (B) of HDTA.
3 is a 1 H-NMR spectrum according to various molar ratios (LMWH: TCA, 1: 2 (A), 1: 4 (B) and 1: 7 (C)) of TCA bound to LMWH.
Figure 4 is a 1 H-NMR spectrum of HDTA according to various molar ratios (HTA: DTX, 1: 6 (A), 1: 8 (B) and 1:10 (C)) of TCA bound to HTA.

Hereinafter, the present invention will be described concretely with examples and the like, but the present invention is not limited by the following examples and the like.

Reference example. Component used

Low-molecular-weight heparin (LMWH, Fraxiparin1; average MW 4.5 kDa) was purchased from Mediplex Co., Ltd (Seoul, Korea). 4-NPC (4-nitrophenyl chloroformate), triethylamine (EDTA), tetrochloric acid sodium salt (TCA), docetaxel (N-hydroxysuccinimide), MMP (4-methylmorpholine), 1,4-dioxane, 2% ninhydrin reagent and trypsin- Lt; / RTI > (St. Louis, MO). DMF (N, N-dimethylformamide), ethylenediamine, formamide, and acetone were purchased from Sigma Chemical Co. (St. Louis, MO).

Example 1.

1) Preparation of LMWH-TCA (HTA) conjugate

Tocopherol (TCA) sodium salt was dissolved in DMF (4.6 mL) at 0 ° C, TEA and 4-NPC were added, reacted for 1 hour under the same conditions, and reacted at room temperature for 6 hours. The reaction solution was centrifuged and extracted with a separating funnel using ethanol and deionized water, and this was repeated three times. The extracted extract was concentrated by rotary evaporation to evaporate the organic solvent and lyophilized to obtain TCA-NPC powder. TCA-NPC powder was dissolved in DMF (5 mL), 4-MMP was added, and reacted at 50 ° C for 1 hour. After 1 hour, EDA was added dropwise to the solution, and the mixture was maintained at room temperature for 16 hours, and then an excessive amount of acetone was added thereto for crystallization. Finally, the crystallized portion was filtered and vacuum dried.

HTA conjugate, LMWH was added to distilled water, heated to dissolve slightly, and 0.1 M HCl was added to maintain the pH within the range of 5.5 to 6 on average.

The molar ratio of TCA-NH2 fed was adjusted to obtain different coupling contents of TCA bound to LMWH. EDC was added to the LMWH solution, and the mixture was stirred for 5 minutes. NHS was added to the LMWH solution, and the mixture was stirred for 30 minutes. Then, TCA-NH ₂ and DTX solution were added and stirred at room temperature for 12 hours. And lyophilized to obtain HTA conjugate powder.

2) Preparation of LMWH-DTX-TCA (HDTA) conjugate

DTX was dissolved in DMSO and stirred to prepare a clear solution. Triethylamine and NPC (4-nitrophenyl chloroformate) were added, and the mixture was stirred at room temperature for 12 hours. Unreacted TEA and NPC were extracted with methanol and hexane solution and removed three times.

The MMP was added to the active DTX containing the methanol solution, stirred at room temperature for 1 hour, and then EDA was added and kept at the same condition for 12 hours or more. Hexane was added to the solution, unreacted MMP and EDA were extracted and removed from the reaction solution, and concentrated under reduced pressure for 30 minutes to evaporate hexane from the mother liquor. To further synthesize the HDTA conjugate, a methanol solution containing N-doxetaxol ethylenediamine was stored at 4 占 폚.

LMWH was dissolved in distilled water by mild heat and stirring. The molar ratio of N-docetaxol ethylenediamine supplied was adjusted for different coupling contents of HTA and DTX. EDC was added to LMWH, stirred for 5 minutes, NHS was added and stirred for 30 minutes. Then, an N-doxetaxol ethylenediamine solution was added to the solution, stirred at room temperature for 12 hours and then dialyzed with deionized water to remove unbound DTX, EDC and NHS, and the whole solution was lyophilized to obtain powder HDTA To obtain a conjugate. Table 1 below shows the molar ratios and coupling ratios of TCA and DTX to LMWH and the particle size of HDTA.

The process (A) for synthesizing the LMWH-TCA conjugate and the process (B) for synthesizing the LMWH-DTX-TCA conjugate are shown in FIG.

Sample Feed mole of LMWH: TCA Coupling mole of LMWH: TCA Feed mole ratio of HTA: DTX Coupling mole ration HTA: DTX Particle size in diameter (nm) HDTA 1 1: 2 1: 0.85 + 0.13 1: 6 1: 1.034 + - 0.45 242.0 ± 85.5 1: 8 1: 1.583 + - 0.135 251.3 ± 96.4 1:10 1: 2.387 + 0.651 220.7 ± 58.0 HDTA 2 1: 4 1: 1.35 + 0.39 1: 6 1: 0.98 + 0.162 217.3 ± 78.8 1: 8 1: 1.431 + 0.421 220.8 ± 50.5 1:10 1: 2.291 + 0.741 160.8 ± 47.5 HDTA 3 1: 7 1: 2.59 + - 0.46 1: 6 1: 0.765 + 0.264 177.7 + -53.9 1: 8 1: 1.128 + 0.368 164.9 ± 60.8 1:10 1: 1.794 + 0.642 181.1 + - 46.2

3) Characteristics of HTA and HDTA

The HTA and HDTA conjugates were formed by the formation of amide bonds between the carboxyl groups of LMWH and the amine groups of TCA and DTX, and were confirmed by FT-IR and 1 H-NMR. HTA conjugates were scanned as solid powder for FT-IR. The coupling ratio of DTX to TCA in LMWH-DTX-TCA was determined by the sulfuric acid method, which was developed for the characterization of bile acid-modified polysaccharides.

To summarize the experimental procedure, 140 μL of the solution in which the correct amount of LMWH was dissolved in distilled water was mixed with 360 μL of sulfuric acid at 80 ° C. for 3 minutes. The sample was cooled to room temperature, and the absorbance at 420 nm Were measured.

4) Size and morphology measurement

HTA and HDTA were dissolved in deionized water and mixed vigorously for several minutes to uniformly disperse. The size distribution of the two types of conjugates was measured using DLS (Otsuka, Japan). The morphology of the HDTA conjugate was measured using scanning electron microscopy (JEOL, Japan). SEM samples were prepared by dispersing 1 mg of the conjugate nanoparticles in 1 mL of water. 200 lL of dispersed nanoparticles were then pipetted into a bath, dehydrated, sputter coated with platinum, and then ramped to 25 kV.

Experimental example.

1) Synthesis of HTA

In the synthesis of HTA, the amine group of N-taurocholylethylenediamine bound to the carboxyl group of LMWH in the presence of EDAC (Fig. A). The precision of the synthesis was confirmed by FT-IR and 1 H-NMR. In the < 1 > H-NMR spectrum, the amide peak at 8 ppm indicates the presence of amide bonds in all HTAs and the peak in the 0.65-2.1 ppm region indicates that the taurocholate (TCA) (Fig. 3). To obtain the anticoagulant activity of various HTAs, various molar ratios of reactants were provided and their coupling ratios were measured.

Figure 2 shows the chemical structure (A) of HDTA and the physical structure (B) of HDTA. LDTA nanoparticles, TCA appeared on the surface of the micelle and DTX was located on the core.

FIG. 3 is a 1 H-NMR spectrum according to various molar ratios of TCA bound to LMWH (LMWH: TCA, 1: 2 (A), 1: 4 (B) and 1: 1 H-NMR spectrum of HDTA according to various molar ratios (HTA: DTX, 1: 6 (A), 1: 8 (B) and 1:10 (C)) of TCA bound. The red circle represents the amide bond between LMWH and TCA and DTX. Deuterium water (D2O) was used as the solvent for all analyzes.

2) Synthesis of HDTA

In the synthesis of HDTA, N-docetaxol ethylenediamine binds LTH. The hydroxyl group of DTX is activated by NPC and replaced with EDA to form an amine group (Fig. 1 (b)). DTX causes LMWH to bond with TCA to form amide bonds and react with the remaining carboxyl groups. The amide bond formed between HTA and DTX was confirmed by FT-IR and NMR. Peaks of TCA and DTX were observed in both the FT-IR and the 1 H-NMR spectra, confirming that all of these residues were included in the LMWH (FIG. 3). DTX and TCA are conjugated to LMWH to form micelles in aqueous solution, DTX is located in the core of micelles, and TCA is located on the surface of micelles (FIG. 2).

3) Characteristics of HTA and HDTA conjugates

The coupling ratio, expressed as the amount of TCA and DTX bound in the HTA, is determined by the sulfuric acid method. The coupling ratios of HTA range from 1 to 2, with a coupling ratio of about 2 for fully saturated forms. These results indicate that two moles of TCA bind to one mole of HTA. The properties of HTA and the molar ratios of each reactant are shown in Table 1. The coupling ratio of DTX ranges from 1 to 2, indicating that up to 2 moles of DTX is bound to 1 mole of HTA.

4) Measurement of size and morphology

While particle size analysis was performed by DLS and SEM, particle formation was not detected in HTA conjugates due to nature-like conditions and high hydrophilic LMWH. However, SEM and DLS data show that about 200 nm diameter particles are formed in HDTA micelles, which is due to micelle formation. SEM data indicate that nanoscale spherical particles are uniformly distributed without agglomeration.

As described above, TCA and DTX are conjugated with LMWH molecules to predict an effective oral anticancer agent. TCA helps strengthen the absorptivity through the bile acid transporter of the small intestine. DTX as well as LMWH exhibit anti-cancer effects through anti-angiogenic processes. The coupling ratio between TCA and DTX can be increased or decreased by controlling the amount of feed.

In Figures 3 and 4, the red circle is able to identify the amide bond formed between TCA, DTX and LMWH.

Claims (5)

A water-soluble anticancer agent which is spherical nanoparticles composed of docetaxel conjugated with low molecular weight heparin and tocoloic acid, wherein the core of the nanoparticles is doped with docetaxel,
The method according to claim 1,
Wherein the low molecular weight heparin has a molecular weight in the range of 3000 to 5000 Da.
The process of removing the sodium salt of low molecular weight heparin,
The process of introducing an amine group into docetaxel,
A step of introducing an amine group into the tororic acid,
A step of mixing heparin from which the sodium salt has been removed with docetaxel and an amino group-introduced docetaxel, followed by self-aggregation,
The process of purifying the self-aggregated heparin-docetaxel-tocoloic acid conjugate
Wherein the water-soluble anticancer agent is a water-soluble anticancer agent.
The method of claim 3,
Wherein the heparin and the docetaxel are combined in a molar ratio of 1: 1 to 10.
The method of claim 3,
Wherein the tocolic acid is bound to the heparin-docetaxel conjugate in a molar ratio of 1 to 10 moles per mole of the heparin-docetaxel conjugate.

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