KR101125737B1 - Apparatus for tranferring a drug using paclitaxel-loaded conjugated linoleic acid-coupled poloxamer hydrogels - Google Patents

Abstract

The present invention relates to a paclitaxel-loaded conjugated linoleic acid poloxamer hydrogel as a novel anti-tumor active topical drug delivery system loaded with paclitaxel with a conjugated linoleic acid poloxamer as a carrier. The hydrogel drug delivery system increases the cytotoxicity of PTX in intro and significantly reduces tumor volume in vivo by using CLA-binding poloxamer hydrogel as a system for topical delivery of PTX, resulting in synergistic antitumor activity. Has the effect of indicating.

Conjugated linoleic acid, paclitaxel, poloxamer, hydrogel

Description

Apparatus for tranferring a drug using paclitaxel-loaded conjugated linoleic acid-coupled poloxamer hydrogels}

The present invention relates to a paclitaxel loaded conjugated linoleic acid poloxamer hydrogel having antitumor activity, and more particularly, to a novel anti-tumor active topical drug delivery system in which paclitaxel is loaded with a conjugated linoleic acid poloxamer as a carrier. .

Paclitaxel (PTX) is one of a group of anti-tumor drugs that have been used for decades. It has been used to clinically treat a wide range of cancers such as breast cancer, ovarian cancer, skin cancer and lung cancer. However, paclitaxel has a low therapeutic index due to its high lipophilic properties and substantial water insoluble properties. Currently, adjuvant consisting of Cremophor EL (polyethoxylated castor oil) and anhydrous alcohol is used for the clinical administration of paclitaxel, which causes serious side effects including neurotoxicity, hypersensitivity reactions and nephrotoxicity. .

In order to overcome the problems caused by Cremophor EL and to enhance drug efficacy, recent research has focused on the development of new drug delivery systems. Various attempts have been studied including liposomes, polymer micelles, polymer biodegradable nanoparticles, water soluble prodrugs, albumin conjugates and hydrogels. A new alternative is the development of injectable prescription hydrogels.

In the present invention, a heat-sensitive conjugated linoleic acid-linked poloxamer hydrogel was developed as a carrier for topical delivery of PTX. The poloxamer consists of hydrophilic ethylene oxide (EO) and hydrophobic propylene oxide (PO) blocks arranged in the basic structure of EOaPObEOa. It has been widely developed as an in-situ -forming drug delivery carrier because of its unique sol-gel transition behavior in response to the temperature of aqueous solutions. These copolymers form spherical micelles in aqueous solution by hydrophobic interactions between intermediate PO compartments. At critical gelation temperatures and concentrations, the self-assembled micelles are tightly packed to form a physically crosslinked gel structure.

Conjugated linoleic acid (CLA) is a group of polyunsaturated fatty acids (PUFA) and exists as the position and stereoisomer of octadecadiic acid. Several studies have shown that PUFAs sensitize tumor cells for chemotherapy and radiotherapy in cell culture, tumor-bearing animals, and the human body.

We have reported that conventional CLA-binding poloxamer (F127) enhances anti-tumor activity compared to CLA (see Cancer letters 254 (2007) 244-254). In addition, CLA-binding poloxamer formed a more stable hydrogel than poloxamer itself at low concentrations due to the hydrophobic nature of CLA.

The inventors also found that CLA-binding poloxamers enhance the efficacy of the cancer chemotherapeutic PTX because the solubility of PTX is enhanced by CLA-binding poloxamer micelles and the anticancer CLA can be released from CLA-binding poloxamers as prodrugs. home would be able to, and, wherein the loading PTX- CLA- combination poloxamer-tumor activity was analyzed in breast xenografts and in in vitro against breast cancer cell lines in in vitro.

It is therefore an object of the present invention to provide a novel topical drug delivery system in which paclitaxel is loaded with conjugated linoleic acid poloxamer as a carrier.

An object of the present invention as described above is to prepare a PTX-loaded CLA-binding poloxamer by loading paclitaxel, an anti-tumor drug, using a poloxamer conjugated to conjugated linoleic acid as a carrier, and their anti-tumor activity in breast cancer in vitro This was achieved by analyzing for cell lines and for breast cancer xenografts in vitro .

"PTX" in the present invention is to refer to paclitaxel (anti-tumor drug).

In the present invention, "CLA" refers to a group of polyunsaturated fatty acids, conjugated linoleic acid.

In the present invention, CLA-binding poloxamer refers to a compound in which CLA is bound to poloxamer.

In the present invention, "PTX-loaded CLA-binding poloxamer" refers to the loading of PTX with CLA-binding poloxamer as a carrier.

The present invention provides a drug delivery system in which paclitaxel (PTX) is loaded onto a conjugated linoleic acid-binding poloxamer hydrogel.

The present invention also provides an anti-tumor active pharmaceutical composition containing the PTX-loaded CLA-binding poloxamer hydrogel drug delivery material as an active ingredient.

The antitumor activity is characterized in that the breast cancer inhibiting activity.

Local delivery of anti-tumor drugs is expected to provide high local concentrations to reduce the incidence of side effects commonly observed in systemic therapies. The hydrogel system can be used as a delivery system for direct administration to mouse body or tumor tissue. In the present invention, a thermosensitive CLA-binding poloxamer hydrogel containing PTX, a chemotherapeutic agent, was prepared for local delivery of PTX. In the present invention, it was anticipated that CLA-bound poloxamer hydrogel could be an effective thermosensitive injection delivery system for topical delivery of anticancer agents without serious structural side effects. Furthermore, the present invention assumed that CLA-binding poloxamer hydrogels would exhibit synergistic anti-tumor activity with PTX because they have enhanced anti-tumor activity as prodrugs.

As shown in Figure 3, there was no significant difference in tumor volume after treatment when comparing the control, poloxamer hydrogel treatment and CLA-linked poloxamer hydrogel treatment. On the other hand, in the other two treatments, including the PTX treatment, a significant difference was found in comparison with the above three treatments. Moreover, PTX-incorporated CLA-binding poloxamer hydrogels showed significant inhibitory activity compared to PTX-incorporated poloxamer hydrogels. The results of the present invention show that the excellent anti-tumor activity of the PTX-incorporated CLA-binding poloxamer hydrogel delivery system has been shown to enhance the solubility of PTX promoted by CLA-binding poloxamer and the anti-tumor activity of CLA-binding poloxamer as prodrug. It is closely related.

PTX confers its effects through stabilization of microtubules, induction of cell death arrest in G2-M, and proapoptotic signal activation. According to conventional studies, PTX treatment results in the induction of apoptosis pathways, cyclin-dependent kinases and phosphorylation of Bcl-2. Moreover, CLA-binding poloxamers induce apoptosis in MCF-7 cancer cells by changing the expression levels of Bcl-2 and other proteins involved in apoptosis and cell cycle. These findings are consistent with the results of the present invention that PTX-incorporated hydrogels reduced the expression levels of Bcl-2 and altered the expression levels of cell cycle stimulation and apoptosis related proteins (see FIGS. 4 and 5). The results of the present invention indicate that PTX-incorporated CLA-binding poloxamer hydrogels induce apoptosis and cell cycle stimulation in tumor tissues. The present invention contributes to the enhancement of antitumor activity of the PTX-incorporated CLA-binding poloxamer hydrogel delivery system as compared to the CLA-binding poloxamer.

In our previous studies, it has been reported that administration of CLA-binding poloxamer as a prodrug enhances anticancer efficacy by inducing apoptosis compared to administration of CLA alone. PUFA is an important structural component of cell membranes. Plasma membranes enriched in PUFAs are associated with lower order parameters or greater fluidity and greater permeability. Feeding the tumor with PUFA results in an increase in the fraction of tumor phospholipids by PUFA, leading to an increase in membrane fluidity, an increase in unsaturated index and an increase in transport potential. This accumulates selective anticancer drugs and increases the activity of the selected drug, activating enzymes and altering signaling pathways important for cancer progression. In addition, a number of transporter and receptor proteins are located in the membrane and across the lipid bilayer, and changes in membrane composition can affect the properties of these proteins to change the cellular uptake of the drug. In the present invention, CLA-binding poloxamer increased the cytotoxicity of paclitaxel in both MCF-7 and MDA-MB-231 cells in vitro . Moreover, PTX-incorporated CLA-binding poloxamer hydrogels significantly reduced tumor volume in vivo by causing cell cycle stimulation and apoptosis.

Previous studies have reported that linoleic acid or seal oil may improve the cytotoxicity exhibited by PTX and may make tumor cells more apoptotic in both MCF-7 and MDA-MB-231 cells. The results of the present invention correspond to these results, indicating the potential for increasing the efficacy of chemotherapeutic drugs because CLA-binding poloxamers have an inhibitory effect on the growth of cancer cells. In conclusion, CLA-bound poloxamer hydrogels were used as a system for topical delivery of PTX, which showed synergistic anti-tumor activity.

The use of thermosensitive CLA-binding poloxamer hydrogels as carriers in the formulation of chemotherapeutic drugs will be beneficial for the treatment of various cancers.

The PTX-loaded CLA-binding poloxamer hydrogel drug delivery system according to the present invention increases the cytotoxicity of PTX in introtro and increases tumor volume in vivo by using CLA-binding poloxamer hydrogel as a system for topical delivery of PTX. Significantly decreases the effect of showing synergistic antitumor activity.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example

CLA used in the present invention was provided by HK Biotech (Seoul, Korea) and contained 45.98% cis-9, trans-11 and 49.88% trans-10, cis-12 CLA. Pluronic F127 (Mw: 12,600) was provided by BASF Korea Inc. (Seoul, Korea) and used without further purification. Paclitaxel was purchased from Samyang Genex Corporation (Daejion, South Korea).

MDA-MB-231 and MCF-7 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC), Rockville, MD, USA. MDA-MB-231 and MCF-7 human breast cancer cells were cultured in DMEM supplemented with 10% heat-inactive FBS and 1% antibiotics (100 penicillin and 0.1 streptomycin). Cells were maintained at 37 ° C. in a wet incubator containing 5% CO ₂ .

For data analysis, quantification of Western blots was performed using Multi Gauge version 2.02 software (Fujifilm). All results are expressed as mean ± SE. Results were analyzed by Student's t-test (GraphPad Software, San Diego, Calif.). P <0.05 was considered significant and P <0.01 was considered significant compared to the control.

Example 1 Synthesis of CLA-binding Poloxamer

CLA was simply bound to poloxamer through ester bonds between the carboxyl group of CLA and the hydroxyl group of poloxamer in the molten state without the need for a catalyst or solvent. The synthesis process is as follows:

5 g (17.8 mmol) of CLA and 5 g (0.4 mmol) of poloxamer were combined in a 25 mL round bottom flask and the mixture was heated with continuous stirring to give a well mixed useful phase and reacted at 150 ° C. for 5 hours. The resulting solution was dropped in ethyl ester to remove unreacted CLA to give CLA-bound poloxamer. Finally, the precipitated CLA-bound poloxamer was dried at room temperature under reduced pressure for 1 day. The composition of the CLA-bound poloxamer conjugate was measured by ¹ H-NMR spectroscopy (Avance ™ 500, Brker, Germany).

As shown in FIG. 1A, CLA-linked poloxamers were successfully synthesized via ester bonds between the carboxyl groups of CLA and the hydroxyl groups of poloxamers. The composition of CLA in the synthesized CLA-binding poloxamer was 49.2 mol% as determined by ¹ H-NMR spectroscopy. The gelling behavior of the CLA-bound poloxamer formulation is shown in FIG. 2, which was sol (A) at 4 ° C. and gelled at 37 ° C. (B).

Example 2 Loading PTX into CLA-Binding Poloxamers

A “cooling method” was applied to load PTX into CLA-bound poloxamers as described in the previous report. The amount of CLA-binding poloxamer and PTX required for each formulation was carefully quantified and mixed in a flat bottom vial. After the required amount of distilled water was added, the mixture was stirred at 4 ° C. until the CLA-bound poloxamer and PTX were well suspended.

Example 3 in vitro cytotoxic effect of PTX-loaded CLA-binding poloxamer

Cell viability was measured using an MTT proliferation kit (Sigma) as described by the supplier.

MDA-MB-231 and MCF-7 cells were treated with PTX alone and in combination with 25 μM CLA-binding poloxamer. After 24 hours cell viability was measured by MTT assay. As shown in FIG. 3, cell viability was significantly reduced in all cells when PTX was added to CLA-bound poloxamer compared to PTZ alone treatment.

Example 4 of PTX-loaded CLA-binding poloxamer In vivo  Anti-tumor activity measurement

Human breast cancer cell line MDA-MB-231 was inoculated into 6 week old (1920 female BALB / C nu / nu athymic (nude) mice (Orient Bio Inc., South Korea). Cancer cells were 0.2 mL at 4 to 5 × 10 ⁶ . The cells were suspended in culture medium and subcutaneously inoculated into the left flank of mice using 1.0 syringe Animals were housed in a specific pathogen free (SPF) facility and fed free of diet and drink.

When tumor volume reached 30 mm ³ after 2 weeks of inoculation, poloxamer, CLA-bound poloxamer, PTX loaded poloxamer or PTX loaded CLA-bound poloxamer (concentrations of poloxamer and PTX were 200 mg / ml and 2 mg, respectively) 0.2 ml) was injected subcutaneously next to the tumor using a 1.0 syringe. Conventional saline was injected as a control. Tumor volume of nude mice was measured at two values over two days up to 30 days using veneer calipers.

Tumor volume measurements were performed using Formula 0.5 × (a × b ² ), where a means maximum diameter and b means minimum diameter. Tumor volume doubling time; the (doubling time DT) equation _{DT = (T F Ti) ×} log2 / logV F logVi ( wherein, V _F is the final tumor volume, V _i is the initial tumor volume, T is V _F and V _i is the number of days between measurements). Animals were killed at the end of the experiment and tumor masses were incubated for further analysis and imaging.

PTX promotes the polymerization of tubulin. Microtubules formed in the presence of PTX are extremely stable, but cause dysfunction, destroying normal tubulin vitality required for mitotic cell division, resulting in cell death. In particular, PTX promotes aggregation and stabilization of microtubules to block cells on G2 / M. In order to investigate the anti-tumor efficacy of PTX-incorporated CLA-binding poloxamer hydrogels in vivo , a human tumor xenograft model was established by transplanting human breast cancer MDA-MB-231 cell lines into nude mice. Mice bearing the MDA-MB-231 species were treated with 20 mg / kg PTX by subcutaneous injection. As shown in FIG. 4, administration of PTX-incorporated CLA-binding poloxamer hydrogels inhibited tumor growth compared to controls and other treatments, suggesting that chemotherapy efficiency was enhanced in vivo . Mean MDA-MB-231 tumor volume at baseline was similar in all groups. The average tumor volume on seeds 30 days after treatment initiation, as shown in Figure 4A, control was 970.3 mm ^3, poloxamer hydrogel treatment is 940.7 mm ^3, CLA- combination poloxamer hydrogel treatment is 834.6 mm ^3, PTX- incorporated The poloxamer hydrogel treatment was 313.9 mm ³ and the PTX-incorporated CLA-linked poloxamer hydrogel treatment was 122.5 mm ³ . PTX alone inhibited tumor growth, but PTX-incorporated hydrogel administration inhibited tumor growth more strongly than PTX alone. The effect of PTX-incorporated CLA-binding poloxamer hydrogel was significantly greater than that of other treatments. Images of tumor mass were taken at killing time and shown in FIG. 4B.

Pharmacokinetics of tumor growth corresponding to therapy were measured by measuring tumor volume doubling time (DT). As shown in Table 1, tumor volume doubling times in control, poloxamer and CLA-bound poloxamer hydrogels were 3.8 ± 0.2, 3.9 ± 0.3, and 4.3 ± 2.0 days, respectively.

TABLE 1

process Tumor volume doubling time (days) Control 3.8 ± 0.2 Poloxamer Hydrogel 3.9 ± 0.3 CLA-linked poloxamer hydrogel 4.3 ± 2.0 PTX / P Hydrogel 6.6 ± 1.4 ^a PTX / CLA-c-P Hydrogel 5.9 ± 2.8 ^{b, c}

^a significant difference (p <0.05) between control and PTX-incorporated poloxamer hydrogels.

^b Significant difference (p <0.01) between control and PTX-incorporated CLA-binding poloxamer hydrogels.

^c Significant difference (p <0.05) between PTX-incorporated poloxamer and PTX-incorporated CLA-binding poloxamer hydrogels.

When PTX was injected into poloxamer hydrogels, tumor volume doubling time increased to 6.6 ± 1.4 days. Tumor volume doubling time was most impressive at 15.9 ± 2.8 days when PTX was loaded onto CLA-bound poloxamer hydrogels.

Example 5 Regulation of Cell Cycle

After measuring the protein concentration of the homogenized eluate using the Bradford kit (Bioford, Bio-Rad, Hercules, CA, USA), an equivalent amount (40 μg) of the protein was separated on the SDS-PAGE, and the nitrocellulose membrane was Transferred. The membrane was blocked for 1 hour in Tris-buffered saline + Tween 20 (TBST) containing 5% skim milk. Membranes were incubated overnight at 4 ° C. with their corresponding primary antibodies for immunodripping. Cyclin A, Cyclin B, Primary Antibody of Cyclin D3, Cyclin Dependent Kinase 2 (CDK2), Bcl-2, Bcl-xL, and Actin are purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) It was.

Caspase-3 antibodies were obtained from Cell Signaling (Beverly, Mass., USA). After washing in TBST, the membrane was incubated with horseradish peroxidase (HRP) -labeled secondary antibody and the band of interest was detected using a luminescence image analyzer LAS-3000 (Fujifilm, Japan). The results obtained were quantified using the multi gauge version 2.02 program of the LAS-300.

Western blot analysis as described above analyzed the effect of PTX-incorporated CLA-binding poloxamer hydrogels on cell cycle-regulating proteins in tumor tissues of mice. The results showed that local delivery of PTX inhibited proteins important for cell cycle regulation compared to the control. As shown in FIG. 5A, expression of cyclin A, cyclin B, cyclin D ₃ , and CDK2 in the treated group was significantly down-regulated compared to the control.

Example 6 Apoptosis Detection

Formalin-fixed, paraffin-embedded tumor tissue slides were deparaffinized in xylene and rehydrated via alcohol gradient. The slides were washed with PBS and the nicked DNA ends were labeled with the TUNEL method using an in situ cell death detection kit (ROCHE, Basel, Switzerland) following the manufacturer's protocol. As a final step, tissue sections were counterstained with methyl green (Trevigen, Gaithersburg, MD, USA).

Apoptosis is the active response of cell death that occurs in response to various agents, including anticancer drugs. As shown in FIG. 6, the expression level of anti-apoptotic Bcl-2 and Bcl-xL in the treated group was significantly reduced compared to the control group. In contrast, the expression level of caspase-3 increased significantly. In order to confirm the result of the western blot analysis, TUNEL analysis was performed. The results showed that a specific apoptosis process occurred in the treated group as confirmed by the presence of fractionated DNA, a direct evidence of apoptosis cell death in the treated group compared to the control.

1 is a diagram showing the ¹ H-NMR spectrum of CLA-binding poloxamer in CDCL ₃ .

FIG. 2 shows sol (A) -gel (B) variation of CLA-binding poloxamer formulations.

3A and 3B show the effect of PTX alone and combination with CLA-binding poloxamer on cell viability in MCF-7 and MDA-MB-231 breast cancer cells after 24 h incubation.

4A and 4B show the effect of PTX-incorporated CLA-binding poloxamer hydrogels on tumor growth.

5a and 5b show the results of Western blot analysis of cell cycle signal proteins.

6A to 6C are diagrams showing the results of Western blot analysis and TUNEL analysis of apoptosis signal protein expression.

Claims (3)

Anti-tumor activity PTX characterized in that the conjugated linoleic acid (CLA) and poloxamer was ester-bonded to synthesize CLA-linked poloxamer, mixed with paclitaxel (PTX), stirred, and stirred at 4 ° C. to load PTX. Load conjugated linoleic acid (CLA) -binding poloxamer hydrogels. A pharmaceutical composition having breast cancer inhibitory activity, comprising the PTX-loaded CLA-binding poloxamer hydrogel according to claim 1 as an active ingredient. delete

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