WO2015088094A1 - Pharmaceutical composition for preventing or treating scar from glaucoma filtering surgery - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing, as an active ingredient, a porcine chondrocyte-derived extracellular membrane (PCDECM), for preventing or treating a scar formed after a glaucoma filtering surgery, and more specifically, the porcine chondrocyte-derived extracellular membrane (PCDECM) prevents or treats a scar formed after a glaucoma filtering surgery by inhibiting fibrosis and angiogenesis of a wound site following a filtering surgery on a glaucoma patient, and thus problems of inflammation and loss of sight due to a failed filtering surgery, such as failure of filtering and controlling of ocular pressure due to a scar, can be resolved.

Description

Pharmaceutical composition for preventing or treating scar after glaucoma filtration

The present invention relates to a pharmaceutical composition containing a porcine chondrocyte derived extracellular membrane (PCDECM) derived from animal knee cartilage cells and provided for preventing or treating scar formation after filtration surgery in a glaucoma patient.

Filtration is the most effective treatment for reducing intraocular pressure (IOP) in glaucoma patients. However, postoperative conjunctival wounds and Tenon's capsules at sclerostomy sites cause filtration and failure of intraocular pressure control and consequent glaucoma optic papillary depression and vision loss. Evidence suggests a persistent inflammatory response around the blebs.

Failure of such glaucoma filtration is related to postoperative scar formation at the site of blebs. Recently, in order to reduce scar formation, substances such as 5-flururacil (5-FU) and mitomycin-C (MMC), which are antiproliferative agents that inhibit fibroblast replication and function, have been used as anti-scarring substances. Substances are toxic to corneal epithelial cells and cause serious side effects such as filter cloth leakage, eye infections, low eye pressure, and low eye cancer-related maculopathy, which requires the development of safer and more effective treatments to prevent scar formation. .

In wound healing, fibroblast proliferation and angiogenesis are important components, and especially angiogenesis is an important factor in the proliferation stage of wound healing, providing oxygen and nutrient supply for the rapid growth of cells that induce healing processes. It plays an important role, and such angiogenesis blockade can reduce the migration and proliferation of fibroblasts.

Recently, studies have been conducted on cell-derived ECM scaffolds composed of cartilage matrix molecules comprising glycosaminoglycan sulfates (GAGs) and collagen type II secreted from animal knee cartilage. It does not cause degradability and rejection to the living body, and shows the characteristic of porosity. It has also been reported that cartilage ECM molecules or fragments derived from ECM exhibit anti-angiogenic activity.

In another report, the porcine chondrocyte derived extracellular membrane (PCDECM) of animal knee cartilage cells showed low hypertrophy and vascular involvement in tissue-enhanced cartilage using mesenchymal stem cells on in situ.

Accordingly, the present inventors completed the present invention by confirming the effect of PCDECM inhibiting subangular epithelial neovascularization and fibrosis during the study of corneal neovascularization and visual acuity using PCDECM.

The present invention uses a pharmaceutical composition comprising a porcine chondrocyte derived extracellular membrane (PCDECM) from animal knee cartilage cells to prevent or treat scars formed on the wound site after filtration surgery for glaucoma patients. The present invention aims to provide a pharmaceutical composition for preventing or treating inflammation and visual acuity loss due to filtration and failure of IOP due to scarring.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating scar formation of the scar area after filtration surgery of a glaucoma patient by containing the chondrocyte-derived extracellular matrix as an active ingredient.

The chondrocyte-derived extracellular matrix may be chondrocyte-derived extracellular matrix formed by secretion from the animal's knee tube insulated bone cells.

In addition, the chondrocyte-derived extracellular matrix may inhibit scar formation by inhibiting fibrosis and neovascularization of the wound site after filtration surgery.

The chondrocyte-derived extracellular matrix may be contained in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

In embodiments, the pharmaceutical composition may be provided as eye drops or injections in the form of eye drops, or ophthalmic ointments or injections in the form of gels by mixing the chondrocyte-derived extracellular matrix with a pharmaceutically acceptable carrier.

Using a pharmaceutical composition comprising a porcine chondrocyte derived extracellular membrane (PCDECM) derived from animal knee cartilage cells according to the present invention, scars are suppressed by inhibiting fibrosis and neovascularization of the vesicle-forming site after filtration surgery in glaucoma patients. Preventing or treating the production can prevent or inhibit filtration surgery failure in glaucoma patients leading to vision loss.

1 is a result of staining the inflammatory cells in the edematous matrix of the PCDECM group and physiological saline group as a control group after surgery (A) hematoxyri and eosin staining of the PCDECM group, (B) mason trichrome of the PCDECM group Dyeing result.

(C) is the result of hematoxylin & eosin staining of physiological saline group after surgery, and (D) is the result of Mason Trichrome staining of physiological saline group and severe inflammatory infiltration in the scleral membrane of sclera membrane of physiological saline group as a control group. This was confirmed.

FIG. 2 shows the results of CD31 immunochemical staining analysis. (A) is a result of staining of the sclera membrane of the PCDECM group after surgery, and (B) is a result of staining of the sclera membrane of the control group. Confirmed.

The present invention provides a pharmaceutical composition for preventing or treating scar formation of a wound site of filtration surgery in a glaucoma patient by containing chondrocyte-derived extracellular matrix as an active ingredient.

The chondrocyte-derived extracellular matrix may be chondrocyte-derived extracellular matrix formed by secretion from the knee articular chondrocytes of an animal.

In addition, the chondrocyte-derived extracellular matrix may inhibit scar formation by inhibiting fibrosis and neovascularization of the wound site after filtration surgery.

More specifically, corneal wounds in experimental groups injected with porcine chondrocyte derived extracellular membrane (PCDECM) and control groups inoculated with saline after each rabbit's eye filtration. Inflammation, fibrosis and neovascularization of the subconjunctival area of the site were confirmed by microscopic analysis after tissue staining and immunochemical staining.

Referring to FIG. 1 and Table 1, the degree of fibrosis of the PCDECM group was 1.57 ± 0.53, and the control group was 3.00 ± 0.00, indicating that the fibrosis was suppressed in the PCDECM group as compared to the control group. Neovascularization was also confirmed that many neovascularizations were formed in the control group compared to the PCDECM group after filtration as shown in FIG.

From the above results, PCDECM was found to be able to prevent or treat scar formation by inhibiting fibrosis and neovascularization of the wound site.

In addition, the chondrocyte-derived extracellular matrix may be contained in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

The pharmaceutical composition may be provided as eye drops or injections in the form of eye drops, or ophthalmic ointments or injections in the form of gels by mixing the chondrocyte-derived extracellular matrix with a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating scars after glaucoma filtration surgery comprising the PCDECM as an active ingredient may be an eye drop, an injection, a granule, a powder, a tablet, a pill, a capsule, a suppository, Any formulation selected from the group consisting of gels, suspensions, emulsions, drops or solutions can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating scars after glaucoma filtration surgery comprising PCDECM as an active ingredient is a suitable carrier, excipient, disintegrant, sweetener, coating agent that is commonly used in the manufacture of pharmaceutical compositions. May further comprise one or more additives selected from the group consisting of swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatics, diluents, dispersants, surfactants, binders and lubricants.

Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.

The preferred dosage of the PCDECM may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

In order to help the understanding of the present invention will be described in detail with reference to Examples. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

The following reference examples are provided to provide a reference example applied to each embodiment according to the present invention.

Reference Example Statistical Analysis

Inflammation, fibrosis and neovascularization of the two groups were quantified and compared.

The comparison of the scores of inflammation, fibrosis and neovascularization between the surgery-PCDECM group and the surgery-physiological saline group was analyzed using the Wilcoxon signed rank test. The value of P <0.05 was considered significant and the results were ± mean. It is expressed as the standard deviation of.

Example 1 Preparation of PCDECM

ECM scaffolds by previously reported methods (Jin CZ, Park SR, Choi BH, Park KD, Min BH (2007) In vivo cartilage tissue engineering using a cell-derived extracellular matrix (ECM) scaffold.Artif Organs 31: 183-92) Was prepared from porcine chondrocytes. First, primary chondrocytes derived from pig knee articular cartilage were expanded by monolayer culture for 3 weeks, and further cultured for 3 weeks with 3D pellets. In order to remove cellular constituents of tissues such as three-dimensional cartilage, a porous sponge-like skeleton was prepared by lyophilization at -56 ° C. and 5 m Torr for 48 hours.

The membrane made by the above method was treated with 200 U / mL DNase I and washed three times with phosphate buffered saline (PBS; Gibco, Carlsbad, CA).

Example 2 Filtration of an Animal Model

Ketamine hydrochloride (30 mg / kg body weight, Huons, Jecheon, Korea) and xylazine chloride (2.5 mg / kg, Bayer Korea Ltd., Seoul, Korea) , Korea) was injected into the muscle of rabbits under general anesthesia and local anesthesia with alkane propparacaine eye drops (Alcon Inc., Seoul, Korea). Anesthetized rabbits underwent a modified trabeculectomy.

Form a 5 mm limbal-based conjunctival flap from the limbal, outline the limbal-based rectangular scleral flap with a triangular iron blade, and carefully scrub the partial thick sclera flap at 2 mm behind the limbal until the blade is visible on the anterior corneal matrix. Excised.

The conjunctival sections were closed with 8-0 polyglactin (Vicryl ^® , Ethicon, Somerville, NJ, USA) sutures in order to close and close the tenon sac and prevent water from entering.

In the above method, the right eye of all rabbits was filtered and randomly divided into 2 groups and 25 mg / mL immediately after surgery in another group of 6 eyes and the surgical-physiological saline group in which 0.9% NaCl was injected immediately into the 6 eyes as a control group. PCDECM was divided into surgical-PCDECM group injected with lysis in phosphorylated buffer (PBS; 10 mL).

Example 3 Pathological Analysis

3-1. Tissue staining

Pathological studies of rabbit eyes confirmed the inhibitory effects of PCDECM on wounds, fibrosis and neovascularization after surgery.

Six weeks after surgery, 250 mg of pentobarbital salt was rapidly perfused through the ear vein in rabbits, and 12 eyes of conjunctival and subconjunctival tissues were removed, and the eyes were fixed with formalin solution for 24 hours. It was then dried and embedded in paraffin wax.

The frontal section of the eyeball, including the cornea, sclera, conjunctiva and sclera plate, was vertically sectioned to 4 μm thick, dried and stained with hematoxylin-eosin on 4 μm thick tissue for pathological analysis of inflammation. The samples were also subjected to Mason Trichrome staining in order to display collagen fibers and to grade scar tissue formation, and were classified into grades 0-4 according to the reported method (Ozkan et al).

The inflammatory response consisted of lymphocytes, plasma cells, tissue cells and multinuclear leukocytes, and depending on the inflammatory grade, grade 0 was no inflammatory response; Grade 1 includes some lymphocytes and plasma cells below epithelial cells; 2 is not only densely packed under epithelial cells, but also with mild inflammation, lymphocytes, plasma cells and multinuclear leukocyte infiltration; Grade 3 is neutrophils in epithelial cells in addition to grade 2; Grade 4 was graded to the extent of ulcers and high concentrations of lymphocytes, plasma cells, multinuclear leukocytes and histocytes, as well as in the epithelium.

Fibrosis was also classified into grades 0-4 based on the amount of collagen formation, grade 0 being no fibrosis; Grade 1 is a mild peripheral fibrosis reaction (a thin band right next to the muscle that can only be found through collagen staining); Grade 2 is a thick band that is easily identified; Grade 3 is a well developed and dense collagen band; Grade 4 was classified as severe fibrosis, replacing most areas.

3-2. Immunohistochemical Analysis

In addition, immunohistochemical analysis was performed using 4 μm thick tissue.

First, tissues were fixed with 3.5% paraformaldehyde, permeated with 0.1% Triton X-100, and then inactivated with 2% bovine serum albumin (BSA; all from Sigma, St. Louis, MO) to anti- primary antibody. CD31 (1: 1,000; Bioss Antibodis, Woburn, Mass.) Was treated and incubated overnight at 4 ° C.

3-3. New blood vessel formation confirmation

Angiogenesis was assessed by microvascular counts (MVC) performed at high power magnification (× 200), with mean microvascular counts of the five largest vascular regions selected through MVD, representing the absolute number of microvessels per 0.74 mm ² . Got.

3-4. result

All rabbits were spared for sacrifice for pathological analysis.

Microscopic examination of the surgical site of rabbits revealed that the subconjunctival soft tissue of the surgical-physiological saline group was almost completely replaced by fibrosis, whereas the surgical-PCDECM group, which was injected with PCDECM in the subconjunctival space, It was confirmed that it was preserved, and the whole test showed no inflammatory reactions at the site of surgery of all rabbits.

As a result of the microscopic analysis of the two groups, the average values of inflammation, fibrosis and microvascular number are shown in Table 1.

Pathological findings of scleral flap and subconjunctival connective tissue. Referring to Table 1 and FIG. 1, after 6 weeks, the surgery-PCDECM group showed a significant difference in the control-physiological saline group and the inflammation score (P = 0.19). However, as shown in Table 1, FIG. 1 and FIG. 2, the surgery-PCDECM group showed less fiber formation (P = 0.002) and neovascularization (P = 0.036) than the control group.

Table 1 Inflammation versus fibrosis by pathological evaluation

	Surgery-PCDECM Group	Surgery-physiological saline group (control)	P value
Inflammation	2.00 ± 1.00	2.25 ± 0.50	0.19 ＊
Fibrosis	1.57 ± 0.53	3.00 ± 0.00	0.002 ＊
blood vessel	18.67 ± 5.24	31.33 ± 4.93	0.002 †

(* wilcoxon signed rank test. Significance P <0.05, † independent t-test. Significance P <0.05)

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The pharmaceutical composition containing the chondrocyte derived extracellular matrix (CDECM) derived from animal knee cartilage cells according to the present invention prevents scar formation by inhibiting fibrosis and neovascularization of the vesicle-forming site after filtration surgery in glaucoma patients. Or showed an excellent effect on treatment. Therefore, the pharmaceutical composition can prevent or suppress failure of filtration due to scar formation after filtration of glaucoma patients leading to visual loss. In addition, it is very useful because it can be used as a therapeutic composition of various forms.

Claims (5)

1. A pharmaceutical composition for preventing or treating scar formation of a wound site after filtration surgery of a glaucoma patient by containing a matrix of chondrocyte-derived cells as an active ingredient.
2. The pharmaceutical composition according to claim 1, wherein the chondrocyte-derived extracellular matrix is a chondrocyte-derived extracellular matrix formed by secretion from an animal's knee joint chondrocytes.
3. The pharmaceutical composition according to claim 1, wherein the chondrocyte-derived extracellular matrix inhibits scar formation by inhibiting fibrosis and neovascularization of the wound site after filtration surgery.
4. The pharmaceutical composition according to claim 1, wherein the chondrocyte-derived extracellular matrix is contained in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total pharmaceutical composition.
5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is provided as eye drops or injections in the form of eye drops or ophthalmic ointments or injections in the form of gels by mixing the chondrocyte-derived extracellular matrix with a pharmaceutically acceptable carrier.

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