KR20120120712A - Water souble Heparin-DTX-TCA conjugates improved targeting and preparing method for the same - Google Patents

Abstract

PURPOSE: A water soluble anticancer agent containing heparin-docetaxel-taurocholic acid conjugate and a method for preparing the same are provided to create hydrophilic environment on the surface of a micelle structure and to ensure bioavailability. CONSTITUTION: A targeting water soluble anticancer agent has a spherical nanoparticles comprising docetaxel in a core and taurocholic acid on the surface. The molecular weight of the heparin is 3000-5000 da. A method for manufacturing the anticancer agent comprises: a step of removing sodium salt of low molecular heparin; a step of introducing amine groups to the docetaxel; a step of introducing amine groups to taurocholic acid; a step of mixing the heparin, docetaxel, and taurocholic acid for self-aggregation; and a step of purifying the self-aggregated heparin-docetaxel-taurocholic acid assembly.

Description

Water souble Heparin-DTX-TCA conjugates improved targeting and preparing method for the same} consisting of heparin-docetaxel-torocholic acid conjugate

The present invention relates to a water-soluble anticancer agent with improved targeting consisting of a heparin-docetaxel-torocholic acid conjugate and a method for preparing the same.

Recent cancer research tends to be through anti-angiogenic therapy. Tumors 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 emerge. 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 has been limited in use due to its low absorption. The low absorbency is due to the high molecular weight, the negatively charged structure and the structure exhibiting hydrophilicity in nature. Early researchers tried to resolve this disorder by conjugating it with deoxycholic acid (DOCA). DOCA is widely used as a bile acid and attempted to improve the absorption of heparin using bile acid as a carrier.

However, the major disadvantage of using DOCA is that DOCA is conjugated with LMWH to form micelles in aqueous solution and all DOCAs are located inside the micelles. As a result, no noticeable maximum effect or absorption was obtained. In order to obtain the maximum bioavailability of LMWH, water-soluble bile acids were needed to be conjugated with LMWH.

Docetaxol (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 anticancer agent, its application is limited due to the route of administration.

For better acceptance and convenience, oral administration of DTX is required. Several formulations coated or loaded with polymers 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, toroic acid (taurocholic acid, TCA), which is a type of water-soluble bile acid, is conjugated with LMWH to enhance the bioavailability of LMWH. It was.

As an example for achieving the above object, the water-soluble anticancer agent of the present invention has improved spherical nanoparticles as a conjugate in which docetaxel and tocoholic acid, which are aminated in low molecular weight heparin, constitute a docetaxel in the core of the nanoparticles. Is introduced, and torocolic acid is introduced to the surface.

At this time, the low molecular weight heparin preferably has a molecular weight of 3000 to 5000 da range.

As another example for achieving the above object, a method for producing a highly water-soluble anticancer agent of the present invention includes a process for removing a sodium salt of low molecular weight heparin, introducing an amine group to docetaxel, and introducing an amine group to tocolic acid. And self-aggregating by mixing the docetaxel and tocoholic acid in which the heparin and the amine group into which the sodium salt has been removed are introduced, and purifying the self-aggregated heparin-docetaxel-tocoholic acid conjugate. do.

In this case, the heparin and docetaxel is preferably molar ratio of 1 to 1 to 10 range, the torocholic acid is preferably bound to 1 to 10 molar ratio range for 1 mole of heparin-docetaxel conjugate.

Oral administration of anticancer agents is very convenient because of the ease of administration and the satisfaction of patients. But totally effective anticancer agents were applicable to type IV because of their insolubility in aqueous solutions.

Low molecular weight heparin (LMWH), taroocholic acid (TCA) and docetaxol (Docetaxol, DTX) were conjugated to develop multifunctional transporters.

Since both LMWH and DTX have been observed to have anticancer effects, synergistic effects can be predicted. TCA and DTX are conjugated with LMWH to form micelles in the water phase due to the hydrophobic nature of DTX.

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

In addition, bile acid is a component that is absorbed through the ileum, it is applied to the present invention serves to facilitate the absorption of the LMWH-TCA-DTX conjugate through the ileum. Due to this feature, the LMWH-TCA-DTX conjugate of the present invention is easily absorbed through the ileum portion absorbing the bile acid, thereby adding a property that can be used for oral use.

TCA and DTX molecules are conjugated via LMWH and amide bond formation. Conjugation of TCA with DTX LMWH is confirmed by FT-IR and 1H-NMR. Coupling ratios of TCA and DTX were also measured together. The synthesized LMWH-DTX-TCA particles were found to have a diameter of 150 to 250 nm depending on the DTX conjugation ratio. Such nanoparticles can be utilized as multifunctional carriers.

According to the present invention described above, the effect that can solve the problem that the hydrophobic segment of docetaxel has significantly limited the use of docetaxel as an anticancer agent can be expected.

According to the present invention, LMWH (Low-molecular-weight-heparin) and docetaxel and torocholic acid which self-aggregate to improve the compatibility of blood compatibility and biocompatibility, provide a hydrophilic environment on the surface of the micelle structure Demonstrate availability.

In addition, according to the present invention, the LMWH and docetaxel by the self-aggregation to make a micelle, a nano-particle sized conjugate can be expected to the effect that the existing anticancer agent used only as an injection can be used as an oral anticancer agent using natural products.

In addition, the present invention has the potential to be developed as an oral drug that can be used more easily than conventional drugs, as well as to prevent cancer when used in practice.

In addition, the present invention is expected to show synergistic effects of LMWH and DTX in cancer and angiogenesis.

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

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

Reference example. Ingredient

Low molecular weight heparin (Low-molecular-weight-heparin, LMWH, Fraxiparin1; average MW 4.5 kDa) was purchased from Mediplex Co., Ltd (Seoul, Korea). Taurorocic acid sodium salt (TCA)), docetaxel (Docetaxel, DTX), EDAC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimidehydrochloride), 4-NPC (4-nitrophenyl chloroformate), triethylamine (triethylamine), N-hydroxysuccinimide (HOSu), 4-methylmorpholine (MMP), 1,4-dioxane (1,4-dioxane), 2% ninhydrin reagent and trypsin-EDTA Sigma Chemical Co. (St. Louis, Mo.). DMF (N, N-dimethylformamide), ethylenediamine, formamide and acetone are listed in Sigma Chemical Co. (St. Louis, Mo.).

Example 1.

1) Preparation of LMWH-TCA (HTA) Conjugate

Toroic acid (TCA) sodium salt was dissolved in 0 ° C. DMF (4.6 mL), followed by addition of TEA and 4-NPC, and reaction for 1 hour under the same conditions, followed by reaction at room temperature for 6 hours. The reaction solution was centrifuged and extracted with separatory filter using ethanol and deionized water, which was repeated three times. The fractionated extract was concentrated under reduced pressure 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 kept at room temperature for 16 hours, followed by crystallization by addition of excess acetone. Finally the crystallized portion was filtered and vacuum dried.

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

The molar ratio of TCA-NH2 supplied was adjusted to obtain different coupling contents of TCA bound to LMWH. EDC was added to the LMWH solution, stirred for 5 minutes, NHS was added, followed by stirring for 30 minutes, TCA-NH ₂ and DTX solution were added thereto, and stirred at room temperature for 12 hours, followed by dialysis of the solution to unreacted material. Was removed 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 stirred at room temperature for 12 hours. Extraction of unreacted TEA and NPC with methanol and hexane solution was repeated three times.

MMP was added to active DTX containing methanol solution, stirred for 1 hour at room temperature, then EDA was added and maintained for at least 12 hours under the same conditions. Hexane was added to the solution to extract and remove unreacted MMP and EDA from the reaction solution, and concentrated under reduced pressure for 30 minutes to evaporate hexane from the mother liquor. Methanol solution containing N-docetaxol ethylenediamine was stored at 4 ° C. for further synthesis of the HDTA conjugate.

LMWH was dissolved in distilled water by weak heat and stirring. The molar ratio of N-docetaxol ethylenediamine fed was adjusted for different coupling contents of HTA and DTX. EDC was added to LMWH and stirred for 5 minutes, NHS was added and stirred for 30 minutes. Then, N-docetaxol ethylenediamine solution was added to the solution, stirred at room temperature for 12 hours, dialyzed with deionized water to remove unbound DTX, EDC, and NHS, and the entire solution was lyophilized to dry powder HDTA. The conjugate was obtained. Table 1 shows the feed molar ratio and coupling ratio of TCA and DTX and the particle size of HDTA to LMWH.

A synthesis process (A) of the LMWH-TCA conjugate and a synthesis process (B) of the LMWH-DTX-TCA conjugate are shown in FIG. 1.

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

HTA and HDTA conjugates were formed by amide bond formation between the carboxyl group of LMWH and the amine group of TCA and DTX, and were identified using FT-IR and 1H-NMR. HTA conjugates were scanned with solid powder for FT-IR. The coupling ratio of DTX and TCA of LMWH-DTX-TCA was determined by the sulfuric acid method, which was developed for the characteristics of polysaccharides modified with bile acids.

Summarizing the experimental procedure, 140 μL of a solution of the correct amount of LMWH in distilled water was mixed with 360 μL of sulfuric acid for 3 minutes at 80 ° C., cooled to room temperature, and then absorbed at 420 nm using a sample of the same composition as a control. Was measured.

4) Size and Morphology Measurement

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

Experimental example.

1) Whether HTA is synthesized

In the synthesis of HTA, the amine group of N-taurocholylethylenediamine was combined with the carboxyl group of LMWH in the presence of EDAC (Fig. (A)). The precision of the synthesis was confirmed by FT-IR and 1H-NMR. In the 1H-NMR spectrum, the amide peak at 8 ppm indicates the presence of amide bonds in all types of HTA, and the peak in the 0.65-2.1 ppm region indicates the successful introduction of tarocholate (TCA) residues. [FIG. 3]. In order to acquire the anticoagulant activity of various HTA, various molar ratios of reactants were provided and their coupling ratios were measured.

2 shows the chemical structure (A) of HDTA and the physical structure (B) of HDTA. LDTA nanoparticles are shown, TCA appears on the surface of the micelles and DTX is located in 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: 7 (C)), FIG. 1 H-NMR spectra of HDTA according to various molar ratios of TCA bound (HTA: DTX, 1: 6 (A), 1: 8 (B) and 1:10 (C)). Red circle represents the amide bond between LMWH and TCA and DTX. Deuterium water (D2O) was used as solvent for all analyses.

2) Synthesis of HDTA

In the synthesis of HDTA, N-docetaxol ethylenediamine binds to LTH. The hydroxyl group of DTX is activated by NPC and replaced with EDA to form an amine group (FIG. 1B). DTX reacts with the remaining carboxyl groups after LMWH binds to TCA to form an amide bond. The amide bond formed between HTA and DTX was confirmed by FT-IR and NMR. Peaks of TCA and DTX appeared in both the FT-IR and 1H-NMR spectra, indicating 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 at the core of the micelles and TCA is located on the surface of the micelles [Fig. 2].

3) Characteristics of HTA and HDTA Conjugates

Coupling ratios, expressed as the content of TCA and DTX bound in HTA, are determined by the sulfuric acid method. Coupling ratios of HTA range from 1 to 2, and they are about 2 in the fully saturated form. This result indicates that 2 moles of TCA are combined with 1 mole of HTA. The properties of HTA and the molar ratios supplied for each reactant are shown in Table 1. The coupling ratio of DTX ranges from 1 to 2, indicating that at most 2 moles of DTX are combined with 1 mole of HTA [Table 1].

4) Measurement of size and morphology

While particle size analysis was performed by DLS and SEM, the formation of particles was not detected in the case of HTA conjugates due to the nature-like state and high hydrophilic LMWH. However, SEM and DLS data indicate that about 200 nm diameter particles were formed in HDTA micelles due to micelle formation. SEM data indicate that nanosized spherical particles are uniformly distributed without aggregation.

As noted above, TCA and DTX are conjugated with LMWH molecules to predict effective oral anticancer agents. TCA helps enhance absorption through the bile acid transporter in the small intestine. DTX, as well as LMWH, has anticancer effects through antiangiogenic procedures. The coupling ratio of TCA and DTX can be increased or decreased by adjusting the supply amount.

3 and 4, the red circle identifies the amide bond formed between TCA and DTX and LMWH.

Claims (5)

A target-type water-soluble anticancer agent comprising spherical nanoparticles as a conjugate in which docetaxel and torocolic acid, which are aminated in low molecular weight heparin, are introduced to the core of the nanoparticles, and torocolic acid is introduced to the surface thereof.
The method according to claim 1,
The low molecular weight heparin is a targeted water-soluble anticancer agent, characterized in that the molecular weight ranges from 3000 to 5000 da.
The process of removing the sodium salt of low molecular weight heparin,
Introducing an amine group into docetaxel,
Introducing an amine group into torocolic acid,
A process of self-aggregation by mixing docetaxel and tocoholic acid in which the heparin from which the sodium salt is removed and the amine group are introduced, and,
Purifying the self-aggregated heparin-docetaxel-torocholic acid conjugate
Method for producing a targeted water-soluble anticancer agent comprising a.
The method according to claim 3,
The heparin and docetaxel is a molar ratio of 1: 1 to 10 characterized in that the manufacturing method of the targeted water-soluble anticancer agent.
The method according to claim 3,
The torocholic acid is a method for producing a targeted water-soluble anticancer agent, characterized in that bound in the range of 1 to 10 molar ratio with respect to 1 mole of heparin-docetaxel conjugate.

Images