KR101929199B1 - Anti-cancer compositions and formulations and preparation method thereof - Google Patents

Abstract

The present invention provides an anticancer composition and a formulation thereof that can overcome the disadvantages of conventional anticancer drugs and anticancer therapies, and includes anticancer activity pressure tamer and atelocollagen. The anticancer agent of the present invention can be used locally, has no side effects, and exhibits excellent anticancer effect.

Description

ANTI-CANCER COMPOSITIONS AND FORMULATIONS AND PREPARATION METHOD THEREOF FIELD OF THE INVENTION [0001]

More particularly, the present invention relates to an anticancer composition comprising atelocollagen and an aptamer having an anticancer activity, and a composition for topical application capable of topical application And a manufacturing method thereof.

Breast cancer is the most common cancer among women in advanced countries, including the United States and Europe, and is the leading cause of death among American women between the ages of 40 and 55. In one lifetime, one out of nine women develop breast cancer and the number of breast cancer patients also increases by about 15 percent annually. In Korea, breast cancer accounts for about 11.9% of female cancer patients in 1995, and it is the third most common cancer after cervical cancer and stomach cancer. It is the fifth most common cancer after gastric cancer, liver cancer, uterine cancer and lung cancer. Is increasing every year.

In the case of colorectal cancer, which is rapidly increasing in westernization of dietary habits, surgery or drug therapy is being tried as a method of treating colorectal cancer. The 5-fluorouracil (5-FU), an antineoplastic compound, is a major selective agent in the treatment of colorectal cancer and its use has only proven to be marginally effective. Although 5-FU can temporarily reduce colorectal cancer size, there is little evidence that the patient's lifespan has been substantially prolonged or "healed" (based on a 5 year reliever). Although chemotherapy with 5-FU has been used in patients with disease-to-liver metastases, only temporary improvement is observed in only 25% or less of such cases, and overall survival is not significantly affected.

In addition, excessive surgery is a problem in the case of thyroid cancer, which has recently been increasing rapidly, especially in Koreans.

6 = NapdU [5-(N-Napthylcarboxyamide)-2’-deoxyuridine]
A = 2’-deoxyAdenosine
G = 2’-deoxyGuanosine
C = 2’-deoxyCytidine
T = 2’-deoxyThymidine(Thymidine)
AP001-24(#11 short)의 서열번호는 1번에 해당함.Most of the drugs used for chemotherapy have serious side effects or toxicity. In recent years, they have developed a platamer that specifically binds to cancer-related receptors and has been used to treat breast cancer alone or in combination with drugs And a variety of other cancers. The present inventors have already developed aptamer that specifically binds to the Her-2 receptor and have been published in International Patent Publication Nos. WO2012176952 and Korean Patent Registration No. 10-1279584. The sequence of the ERBB2 plasmid (ERBB2 plasmid = NapdU-modified DNA aptamer, 6 = NapdU) is shown in the following table.

6 = NapdU [5- (N-Napthylcarboxyamide) -2'-deoxyuridine]
A = 2'-deoxyadenosine
G = 2'-deoxyGuanosine
C = 2'-deoxyCytidine
T = 2'-deoxyThymidine (Thymidine)
The sequence number of AP001-24 (# 11 short) corresponds to No. 1.

In addition, it is not easy to completely remove micro cancer or residual cancer deeply surrounding tissue after removal of primary tumor when performing conventional cancer resection. After the primary cancer surgery, the patients are not given chemotherapy or radiotherapy for 1 ~ 2 weeks until their physical strength is restored.

At this time, active treatment methods of residual cancer or micro cancer have not been conducted until now. However, using the biomedical device product developed through the present invention, the first method of active treatment in this period is proposed, Maximizing the survival rate of patients and reducing the risk of recurrence can be expected.

International Patent Publication No. WO2012176952

It is an object of the present invention to provide an anticancer composition and a formulation thereof which can overcome the disadvantages of conventional anticancer drugs and anticancer therapies.

The inventors of the present invention have found that when such an aptamer related to cancer is mixed with atelocollagen and used as an anticancer agent, the effect can be maximized, and the present invention has been completed.

The anticancer agent of the present invention can be used locally, has no side effects, and exhibits excellent anticancer effect.

Figure 1 compares the serum half-life of the ERBB2 platemaker and the atelocollagen-ERBB2 platamater complex.
Figures 2a-2e compare the retention and half-life of the localized area after implanting the aptamer and atelocollagen complexes in mice.
Figure 3 shows the results of MTT assay for B-474 cell line of ERBB2 platelets and atelocollagen-ERBB2 platelets complex.
FIG. 4 shows FDG PET images obtained 30 days after treatment with ATELO collagen-ERBB2 platemater and ERBB2 platemaker.
Figure 5 compares the size of tumors measured after ERBB2-platemater and atelocollagen-ERBB2 platemater complex treatment in a BT-474 tumor model.
Figure 6 compares the size of tumors extracted at 30 days after treatment with Atelocollagen-ERBB2 platemater and ERBB2 platemater in a tumor model.

The anticancer agent of the present invention comprises atelocollagen and an aptamer. A dispersion of atelocollagen is prepared, and then an extruder is added and homogenized. These compositions are currently formulated into ointments and directly applied or cross-linked matrices are applied in patch form to the skin near the site of breast cancer, colon cancer, or thyroid cancer, and are generally injected into the body for injection or oral administration The anticancer agent can act more directly on the cancer cells than the anticancer agent, so that the effect is excellent and the side effect is remarkably reduced. Atelocollagen dissolves slowly, so that it acts directly on the cancer cells in the form of slowing the platamater. By adding hyaluronic acid to atelocollagen during matrix preparation, it is possible to reinforce weak porous membranes and to have the function of increasing cell migration upon binding with collagen fibers.

For example, (a) a method in which a collagen dispersion is homogenized by adding an aptamer to a dispersion of a predetermined thickness to form a uniform film having a predetermined thickness, and then lyophilized to form a collagen porous layer (B) after the step (a), separately dispersing the collagen dispersion to a predetermined thickness, applying pressure to the porous adsorption plate to leach moisture, and bringing the collagen particles into close contact with each other to form a dense collagen hard layer (C) drying the collagen hard layer formed in step (b) at room temperature for about 10 to 20 minutes, laminating the collagen porous layer prepared in step (a) and blowing to form the collagen hard layer (D) combining the collagen hard layer primarily bound through the step (c) and the collagen hard layer with the collagen hard layer By crosslinking using crosslinking means of the layer comprises the step of combining the collagen, the hard layer and the porous collagen layer secondarily.

The amount of platemaker contained in atelocollagen may vary depending on the type of platemaker, the weight of the patient, the size of the tumor to be treated, the distance from the skin, and the like.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1. Preparation of the composition

First, atelocollagen corresponding to 2 wt% was added to distilled water, and the mixture was stirred at 40 DEG C for 40 hours to obtain a collagen dispersion. To this dispersion was added 0.5N NaOH to adjust the pH to 7.4, To obtain a collagen dispersion (A). Further, a 0.1 mM solution of ERBB2 platemer prepared by the method described in International Patent Publication No. WO2012176952 was added to the obtained atelocollagen dispersion (A) to obtain a homogenized dispersion (B).

4% by weight of atelocollagen was added to distilled water, and the mixture was stirred at 30 ° C for 30 hours to obtain a collagen dispersion. To this dispersion was added 0.5N NaOH to adjust the pH to 7.4. To obtain a collagen dispersion (E).

A collagen-based matrix was prepared as follows using the prepared atelocollagen and platemar dispersion (B) and the hard film-forming atelocollagen dispersion (E) as follows:

(1) a uniform membrane of 0.5 mm was formed by diffusing Atelocollagen and platamer dispersion (B) on a plate of releasing property, and then lyophilized in a freeze dryer maintained at -70 ° C for 30 hours to prepare a porous membrane;

(2) Atelocollagen dispersion (E) for hard film formation was diffused on the surface of a flat plate of a releasing property and a pressure of 10 psi was applied to prepare a hard film of 0.5 mm;

(3) The hard membrane obtained in the above step (2) was dried at room temperature for 15 minutes, lightly laminated the porous membrane obtained in the above step (1) and air-dried at room temperature for 30 hours to obtain a double membrane by combining the hard membrane and the porous membrane.

(4) EDC was added to a 95 wt% ethanol solution at a concentration of 50 mM, and the mixture was stirred at 4 ° C for 15 minutes to obtain a mixed solution;

(5) To the mixed solution obtained in the step (4), the double membrane obtained in the step (3) was immersed so as to be submerged, and then the porous membrane and the hard membrane were crosslinked at 4 DEG C for 40 hours;

(6) In the step (5), the EDC was washed 5 times with distilled water for 15 minutes to remove the EDC from the crosslinked bilayer membrane;

⑦ The washed double membrane was again lyophilized at -70 ℃ for 30 hours to obtain a formal double membrane;

(8) The double membrane obtained in the above step (7) was cut to a size of 10 mm x 15 mm x 15 mm at a maximum size of 200 mu m x 200 mu m x 200 mu m to prepare a matrix.

Example 2. Serum Stability Experiment

100 uL of ERBB2 platamater and atelocollagen-ERBB2 platamater complex were prepared at a specific concentration (0.5 nmole). Add 40 uL (4 nmoles) of each platemaker solution to 200 uL of Human Serum (Human male AB plasma / Sigma H4522), vortex and dispense 20 μL into each of the ten E-tubes and place in a 37 ° C incubator. Each sample was stored in a cryocooler in the order of 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 21 h, 24 h, 30 h, 48 h and 72 h and 20 uL of distilled water was added thereto before analysis. The serum stability of ERBB2-platemater and atelocollagen-ERBB2 platamater complexes was determined by analyzing the results of quantitative analysis and the residual amounts in the serum. Serum stability of erbB2 erythromycin was confirmed to be stable for more than 72 hours (t1 / 2 in serum> 72 hr).

Compared with 3 hours (t1 / 2 = 3 hr) in the serum half-life of ERBB2 platemer, the atelocollagen-ERBB2 platamateral complex has a half-life of 70 hours (t1 / 2 in serum = 70 hr) (Fig. 1).

Example 3: Stability experiment through animal experiment

Cythermal labeled tympanum was synthesized at the end of tympanum. Samples were prepared by compositing 100 ul of Atelocollagen 0%, 0.3%, 2.0% and 10 uM of platamer. Subcutaneous injection was performed in a mouse using a 1 ml syringe, and the amount of residual remnants in the mouse was measured using a fluorescence imaging device. Compared with the uniquely injected sample, the control group, aptamer, was able to confirm that the aptamer combined with the 2.0% collagen solution continued for up to 5 weeks. Therefore, it has been confirmed that the combination of atelocollagen and platamer is maintained in a stable state for a long time at a local site in an animal body. (Figs. 2A to 2E)

Example 4. In vitro efficacy of the composition

MTT assay for BT-474 cell line: The cell inhibition effect of ERBB2-platemater and atelocollagen-ERBB2 platamater complex in ERBB2-overexpressed breast cancer cell line BT-474 cell was confirmed in vitro by MTT assay. We compared the cell viability and proliferation of cells through the MTB assay of the ATELO collagen-ERBB2 platamater complex and ERBB2 platemer, confirming that the atelocollagen-ERBB2 platamater complex is superior to the ERBB2 platemer.

Experimental method: Breast cancer cell line BT-474 (ATCC, IMDM + 10% FBS) was seeded on a 96-well plate with 2.5 ~ 5X105 cells / well determined by cell test to determine the optimal cell concentration. ERBB2 aptamers were heated at 95 ° C for 5 minutes, cooled slowly at room temperature, and then directly treated in each well. The BT-474 cells were incubated for 72 h in a 5% CO 2 incubator and then incubated with 20 μl each of the MTT assay reagent solution (Cell Proliferation kit II, Roche) for 10 min, 30 min and 1 hr. (Fig. 3).

Example 5. In vivo efficacy of the composition

Experimental Method

Production of animal models: 6-week-old Balb / c nude female mice were used to model tumors in vivo. The average weight of mice used in the experiment was 20g on average. An estradiol pellet was implanted in the subcutaneous tissue of the mice. Human breast cancer cell line BT-474 cells were purchased from ATCC and propagated. Pellet Injection After this, BT-474 cells proliferated were injected subcutaneously into the thigh of a mouse in an amount of 5X107 cells to form a tumor. The tumor was observed daily for 2-3 weeks after the injection and the tumor size was measured using a caliper.

FDG PET imaging: FDG PET imaging was performed to confirm tumor formation. Fasting was performed in addition to water for 24 hours before the test. The mice were injected intraperitoneally with F-18 FDG (F-18 Fluorodioxyglucose, Cytologic Centers for Severe Inflammation and Cyclotron Center) 220-230, and images were obtained 1 hour later using Siemens Inveon animal PET (Positron emission tomography). FDG PET images were obtained again using the same protocol for efficacy after treatment.

Treatment: The patches prepared in Example 1 and the patches made only with platamer without using atelocollagen were attached and fixed to the skin on the area confirmed by the image, and the size of the tumor was caliper at 5-day intervals. And FDG PET images were obtained 30 days later. The results are shown in Fig.

Example 6. Ex-vivo validation

The ATELO collagen-ERBB2 platamater complex is superior to the ERBB2 platemer alone in preventing cancer. It was confirmed that 80% of the cancer inhibition was suppressed compared to administration of ERBB2 platemaker alone (Fig. 5)

Methods: Tumor size was measured on the horizontal and vertical axes at 5-day intervals from the start of treatment and tumors were removed from each group after 30 days of treatment. The tumor size of the ATELO collagen-ERBB2 platamater complex and the ERBB2 platemer were compared (Fig. 6)

result

Tumor Size Change: A significant tumor size change when attached with the matrix of the present invention. The tumor size inhibition effect persisted for more than 2 weeks after the treatment was terminated.

In addition, the degree of glucose metabolism in the tumor was confirmed using FDG PET, and a significant decrease in the glucose metabolism level was observed after the treatment with the ATLAC-ERBB2 complex. The tumor size and glucose metabolism were evaluated to determine the anticancer effect of BT-474 breast cancer cell line of the ATELO collagen-ERBB2 platemater combination.

The atelocollagen-abtamma combination preparation of the present invention is not limited to the atelocollagen-ERBB2-platemulsion combination preparation but may be used as the atelocollagen-abtamer combination preparation by using various compressors. When the complex formulation is used The stability of the platemer and the anticancer effect can be expected to be increased. The combined preparation of the present invention is useful for the treatment of various cancers including breast cancer, thyroid cancer and colon cancer depending on the type of abdominal cancer. For example, in removing cancer cells after cancer surgery, It can be useful.

<110> Interoligo <120> ANTI-CANCER COMPOSITIONS AND FORMULATIONS AND PREPARATION METHOD          THEREOF <130> 10-2016-0082330 <150> KR 10-2015-0094110 <151> 2015-07-01 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> AP001-24 ERBB2 aptamer <220> <221> modified_base <222> (1) (40) <223> n is NapdU; [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine] <400> 1 angnnagagn nngccngagn gccncgcaag ggcgnaacaa 40

Claims (4)

And a complex of AP001-24 ERBB2 platemer of SEQ ID NO: 1 as an active ingredient and the complex is a porous membrane made of a dispersion liquid homogenized by adding the plaster to the dispersion of atelocollagen An anticancer composition for treating breast cancer.
A topical anti-cancer agent for breast cancer comprising the composition of claim 1.
3. The preparation according to claim 2, wherein the topical anticancer agent has a patch form.
(a) homogenizing AP001-24 ERBB2 plateler represented by SEQ. ID. NO: 1 to a collagen dispersion to form a homogeneous membrane having a predetermined thickness by diffusing the mixture to a predetermined thickness and lyophilizing it to prepare a collagen porous layer; (b) diffusing the collagen dispersion to a predetermined thickness separately after the step (a), applying pressure to the porous adsorption plate to leach water, and bringing the collagen particles into close contact with each other to form a dense collagen hard layer; c) drying the collagen hard layer formed in the step (b) at room temperature for about 10 to 20 minutes, laminating the collagen porous layer prepared in the step (a), and air drying to form the collagen hard layer and the collagen porous layer (D) crosslinking the collagen hard layer and the collagen porous layer, which are primarily bound through the step (c) By cross-linking by the method of producing a patch for the treatment of breast cancer, comprising the step of combining the collagen, the hard layer and the porous collagen layer secondarily used.

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