KR20020010230A - Use of 5-[(2-chlorophenyl) methyl]-4,5,6,7-tetrahydrothieno [3,2-c] pyridine or hydrochloride thereof, ticlopidine, as an inhibitor of angiogenesis - Google Patents

Abstract

PURPOSE: A method for treatment diseases related to angiogenesis by use of angiogenesis inhibitors containing ticlopidine as an effective component is provided. Whereby, the composition can be used as a treating agent for diseases caused by angiogenesis such as hemangioma, hemangiofibroma, arthritis, diabetic retinopathy, retinopathy of a premature baby, glaucoma caused by angiogenesis, corneal disease caused by angiogenesis or the like. CONSTITUTION: The pharmaceutical composition for inhibition of angiogenesis contains 5-£(2-chlorophenyl)methyl|-4,5,6,7-tetrahydrothieno£3,2-c|pyridine or hydrochlorate thereof as an effective component and pharmaceutically acceptable salts. The composition can be used as an anticancer agent or a metastasis inhibitor and is orally administered at a daily dose of 5 to 800mg.

Description

Use of 5-[(2-chlorophenyl) methyl] -4,5,6,7-tetrahydrothieno [3,2-C] pyridine or its hydrochloride ticlopidine as an angiogenesis inhibitor {USE OF 5-[( 2-CHLOROPHENYL) METHYL] -4,5,6,7-TETRAHYDROTHIENO [3,2-C] PYRIDINE OR HYDROCHLORIDE THEREOF, TICLOPIDINE, AS AN INHIBITOR OF ANGIOGENESIS}

5-[(2-chlorophenyl) methyl] -4,5,6,7-tetrahydrothieno [3,2-C] pyridine, which is used as an anticoagulant by inhibiting aggregation of platelets Or it relates to the use of the hydrochloride hydrochloride Ticlopidine (Ticlopidine) a new effect of inhibiting angiogenesis, as a therapeutic agent for diseases related to angiogenesis

Angiogenesis is a process in which new capillaries are formed from existing microvascular vessels. Normally, angiogenesis occurs when embryonic development occurs, tissue regeneration and wound treatment, and periodic female genital system Changes occur only when the corpus luteum develops and is strictly controlled (Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int Rev Exp Pathol 16, 207-248 (1976)).

The formation of new blood vessels is strictly divided into vasculogenesis and angiogenesis. Angiogenesis is defined as angiogenesis of endothelial cells from differentiated blood levels in early mesodermal mesoderm. Embryo vasculature begins when simple diffusion does not meet the needs of nutrition to rapidly improve cell proliferation for complex forms. Angiogenesis is a process of growing out of endothelial cells from existing blood vessels, and usually forms terminal veins and posterior capillaries of microvascular vessels.

In adults, vascular endothelial cells do not divide relatively well, and the rate of endothelial division is typically months to years. However, as a result of hormonal signal responses, the release of pro-angiogenesis cytokines in inflammatory cells, the activity of hydrolytic enzymes releasing angiogenic mediators isolated from extracellular matrix, or stimulation of angiogenesis factors Angiogenesis will occur. Angiogenesis is generally caused by the formation of lumen due to the breakdown of the basal membrane, proliferation, proliferation and differentiation of vascular endothelial cells due to proteases, resulting in new capillaries.

However, there are diseases caused by continuous pathological growth when angiogenesis is not controlled autonomously. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as arteriosclerosis, angiogenesis, Ophthalmologic diseases caused by edema and angiogenesis include corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, neovascularized corneal disease, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granuloconjunctivitis Chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, acne, and dermatological diseases such as acne. Cancer growth and metastasis depend on angiogenesis (Ophthalmol 102, 1261-1262, 1995). J Am Acad Derm 34 (3): 486-497, 1996; Circulation 93 (4): 672-682, 1996; Cell 86: 353-364, 1996).

Diseases caused by abnormal angiogenesis are deeply involved in the development or progression of the various diseases, and studies are actively underway to find substances that inhibit angiogenesis for the prevention or treatment of these diseases.

Especially in cancer, neovascularization of cancer cells plays an important role in the growth and metastasis of cancer cells. If cancer cells fail to angiate or enter and exit blood vessels, most cancers will not grow to more than 1 to 2 mm in diameter and remain localized in the primary location (Folkman and Tyler, Cancer Invasion and metastasis, Biologic). mechanisms and Therapy (SB Day ed.) Raven press, New York, 94-103 (1977).

Angiogenesis plays two important roles in tumors; first, it provides the nutrition and oxygen needed for the growth and proliferation of primary and metastasized secondary tumors; It gives you a chance to enter the circulatory system, causing cancer cells to spread throughout your body and become metastasis.

The main cause of death of cancer patients is metastasis, which is a phenomenon in which cancer cells are isolated from primary tumor blood and grow on anatomically distant tissues or organs (Sugarbaker, Weingard and Rosoman, Cancer Invasion and Metastasis (LA Liotta and IR Hart ed.) Boston, Nijhoff, 427-465 (1982). Currently, the clinic uses chemotherapy or immunotherapy to improve the therapeutic performance of cancer. Although these treatments have some effects in some tumors, they do not contribute to improving the survival rate of cancer patients. Because of the transition.

Tumors remain dormant for a period of time if angiogenesis does not occur and can no longer grow. However, when the ends of the expanding tumor approach proximal capillaries, diffuse angiogenic factors are released from the tumor, stimulating endothelial cells to grow and move and form capillary networks. This transition from angiogenesis to angiogenesis involves an exponential increase in tumors. Tumor cells circulate new blood vessels. It produces diffuse angiogenic factors that grow into developing tumors that directly activate endothelial cells. They produce cytokines and produce factors that further increase angiogenesis by attracting and activating macrophages, mast cells and neutrophils. It is also possible to stimulate angiogenesis by generating enzymes that release isolated angiogenic factors in organs of cells. Angiogenesis is essential for cancer growth and metastasis because angiogenesis occurs along the blood vessels that have penetrated the cancer tissue (PJ Polverini, Critical Reviews in Oral Biology, Vol. 6, No. 3, 230-247 (1995). )).

Arthritis, a representative disease of inflammatory diseases, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis, thereby destroying cartilage without blood vessels. With the help of cytokines that induce such inflammation, synovial cells and vascular endothelial cells proliferate in the synovial cavity, and as angiogenesis progresses, cartilage, which acts as a cushion, destroys cartilage, which acts as a cushion, forming joint layers of connective tissue. (Kocb AE, Polverini PJ and Lcibovich SJ, Arth Rheum 29, 471-479 (1986); Stupack DG, Storgard CM and Cheresh DA, Braz J Med Biol Rcs 32, 578-581 (1999), Koch AE, Atrhritis Rheum 41 , 951-962 (1998).

Many ocular diseases, which cause millions of blindness worldwide each year, are also caused by angiogenesis (Jeffrey MI and Takayuki A, J Clin Invest 103, 1231-1236 (1999)).

Diseases such as macular degeneration, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma and corneal diseases caused by neovascularization are diseases that cause angiogenesis in the elderly (Adamin AP, Aiello LP). and D'Amato RA, Angiogenesis 3, 9-14 (1999)). Among them, diabetic retinopathy is a complication of diabetes, in which capillaries in the retina invade the vitreous body and eventually become blind.

It is believed that these diseases are caused by the release of growth factors from the ischemic retina, and our bodies are trained to respond to these hypoxia by supplying new blood. The eye is the most bloodless tissue, and the growth of blood vessels means blindness. Ocular disease caused by angiogenesis does not have a suitable treatment, and if steroid or antibiotic is used, further progression may result in cautery or photocoagulation of blood vessels, but the effect is temporary and prevents blood vessel proliferation. Therefore, to prevent the angiogenesis can be a fundamental treatment.

Psoriasis, which is characterized by red spots and skin, is also a chronic proliferative disease of the skin. It does not heal and involves pain and malformations. In normal cases, keratinocytes proliferate once a month, whereas psoriasis patients proliferate at least once a week. This rapid proliferation requires a large supply of blood and angiogenesis is bound to occur actively (Folkman J, J Invest Dermatol 59, 40-48 (1972)). Angiogenesis inhibitors can be applied as novel therapeutics for such psoriasis.

In addition to the diseases caused by angiogenesis so far, various diseases caused by angiogenesis are currently difficult to treat unless there is a therapeutic agent and inhibit angiogenesis. Therefore, efforts to treat the diseases by inhibiting angiogenesis have been made. It is true. However, even a compound that is excellent in inhibiting angiogenesis can be used as a medicine only when safety, such as toxicity, has been demonstrated. Therefore, the inventor of the present invention completed the present invention by revealing that the toxicology test is completed and ticlopidine, which is currently used as a thrombolytic agent, has an angiogenesis inhibitory effect.

The present invention provides a blood vessel comprising 5-clo ((2-chlorophenyl) methyl] -4,5,6,7-tetrahydrothieno [3,2-C] pyridine or hydrochloride hydrochloride Ticlopidine as an active ingredient. It is an object of the present invention to provide a method of treating angiogenesis-related diseases using an angiogenesis inhibitor.

In other words, the present invention has been proven to be safe, such as toxicity, Ticlopidine currently used as a thrombolytic agent to inhibit vascular endothelial cell formation using human vascular endothelial cells and to quantitatively measure the fetal ureteric assay and angiogenesis, which are in vivo experiments Experimental mouse matrigel model revealed that the antiangiogenic activity, the composition containing ticlopidine as well as arthritis treatment, diabetic retinopathy, prematurity retinopathy, neovascular glaucoma, degeneration, spot degeneration, pterygium, retina Dermatological diseases such as hemangiomas, angiofibromas, psoriasis, capillary dilatation, purulent granulomatosis, seborrheic dermatitis, acne, for the treatment of ocular diseases caused by angiogenesis such as degeneration, posterior capsular fibrosis, granuloconjunctivitis and neovascular corneal diseases It is used as a therapeutic agent for various diseases caused by angiogenesis such as anticancer agent and metastasis inhibitor. And that for the purpose.

1 is an experiment observing the effect on the tube formation of HUVE cells (Human Umbilical Vein Endothelial cells) by thyclopidine at a concentration of 1 mM.

Figure 2 is an experiment showing that 2 mg of ticlopidine inhibits angiogenesis in a mouse Matrigel model.

Figure 3 is a photograph showing the angiogenesis inhibitory effect of ticlopidine through the fetal calculus ureteral assay.

The present invention relates to angiogenesis inhibitory effect, a new use of ticlopidine, which is used as a thrombolytic agent, and is characterized by the first disclosure that it is possible to treat angiogenesis diseases by preventing angiogenesis by using angiogenesis inhibitory effect of ticlopidine. There is this. In other words, the present invention is a drug for the treatment of diseases caused by angiogenesis, for example, hemangioma, hemangiofioma, arthritis, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma, corneal disease caused by neovascularization, degeneration of plaque, degeneration , Pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, acne and the like, and can be used as anticancer agents or metastatic inhibitors.

In the present invention, in order to measure the angiogenesis inhibitory effect of ticlopidine, the effect on the formation of capillary-like structures by human vascular endothelial cells was observed. After gelling the Matrigel and culturing vascular endothelial cells on the Matrigel, tube formation can be thought of as a process of angiogenesis. At this time, when ticlopidine was treated at 1 mM concentration, the tube was strongly inhibited. The cells formed do not form well. In addition, the mouse matrigel assay, an in vivo experiment to measure angiogenesis, revealed that ticlopidine has the effect of inhibiting angiogenesis.

The pharmaceutical composition formulations of the present invention comprising ticlopidine may comprise a suitable pharmaceutical diluent known to be useful in pharmaceutical compositions. Such diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should be suitable for the mode of administration.

The ticlopidine compound of the present invention may be administered in one or more pharmaceutical compositions with a pharmaceutically acceptable excipient. When administered to a human patient, it is apparent that the total daily usage of the pharmaceutical composition of the present invention can be determined by the practitioner within the scope of good medical judgment. The specific therapeutically effective amount for a particular patient may include the type and degree of response to be achieved; Specific compositions, including whether other agents are optionally used; The age, body weight, general health, sex and diet of the patient; Time of administration, route of administration, and rate of secretion of the composition; Duration of treatment; It will depend on a variety of factors, including drugs used in conjunction with or concurrently with the specific composition (eg, chemotherapeutic agents) and similar factors well known in the medical arts. Suitable formulations known in the art are described in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Therefore, the "effective amount" of ticlopidine suitable for the purposes of the present invention is determined in consideration of the foregoing.

When ticlopidine is used as an angiogenesis inhibitor, the dosage and administration method vary depending on the age and weight of the patient as well as the type and extent of the disease caused by angiogenesis. Typically, when ticlopidine is used as an angiogenesis inhibitor, the dose is 5-800 mg once, and preferably 50-600 mg once orally 1-4 times daily.

The pharmaceutical compositions of the invention can be administered in a conventional manner via the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes. As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion.

For parenteral administration, the ticlopidine compounds of the present invention are pharmaceutically acceptable carriers, i.e., nontoxic to the receptor at the concentrations and dosages employed and compatible with other formulation components and in unit dosage forms, under the desired purity. It is common to prepare by mixing in (solution, suspension or emulsion). For example, the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to the human body.

In general, formulations are prepared by uniformly and sufficiently contacting the ticlopidine compound with a liquid carrier or particulate solid carrier or both. If necessary, the product is then shaped into the desired formulation. Examples of such carrier excipients are water, saline, Ringer's solution and dextrose solution. Suitable formulations known in the art are disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1 Effect of Ticlopidine on the Tubular Formation of HUVE Cells in Matrigel

As an in vitro experiment to indirectly understand the effects on angiogenesis, the effect on the formation of capillary-like structures by human vascular endothelial cells was observed. In order to perform the tube formation (tube formation) experiments were first performed HUVE cells (Human Umbilical Vein Endothelial cells). In other words, after the caesarean section, the endothelial cells of the vein were isolated from the umbilical cord of the fresh human, and the cells obtained by culturing the vascular endothelial cells were successfully isolated by quantitative cytochemical staining using antibodies of the factor 후. Vascular endothelial cells were cultured on Matrigel (commercially available from Collaborative Biomedical Products). After gelling the Matrigel and culturing the endothelial cells on the Matrigel, two-dimensional tubular formation occurs. The reticular tubular structure observed after 16-18 hours of incubation at 37 ° C can be considered as a process of angiogenesis. At this time, the effect on angiogenesis was indirectly confirmed by observing the results obtained by treating ticlopidine at a concentration of 1 mM. When ticlopidine was treated at a concentration of 1 mM, tube formation was inhibited by the inhibition of tube formation, and when the area of the formed tube was analyzed by Image-Pro Plus (Media Cybernetics), only 70% of the tube was formed. (Fig. 1).

Example 2 Animal Experiments Measuring Angiogenesis (Mouse Matrigel Model)

The angiogenesis inhibitory effect of ticlopidine was measured using a mouse matrigel model, an animal experiment that can quantitatively measure the inhibition of angiogenesis. Subcutaneous injection of 0.4 ml of Matrigel, 1-100 ng / ml of basic Fibroblast Growth Factor (FGF) and 20-100 unit / ml of heparin in C57BL / 6 mice of 6-8 weeks showed strong angiogenesis. . After 3-5 days, the epidermis was removed and the matrigel was carefully separated to recover the gel, and then the amount of hemoglobin was obtained using a Drabkin reagent (commercially available from Sigma), a reagent that measures the total amount of hemoglobin in the blood. Measured. As a result of quantitatively measuring the hemoglobin content when 2 mg of ticlopidine was mixed with the Matrigel, as shown in FIG. 2, angiogenesis was suppressed by 95% compared to the control group.

Hemoglobin content (g / dL) Control 66.4 ± 27.6 Ticlopidine 2.4 ± 2.0

Example 3 A Fetal Villi Urinary Assay to Measure Angiogenesis

(CAM assay; Chorioallantoic membrane assay)

Incubate the fertilized egg for 3 days in a thermo-hygrostat with a humidity of 70% or higher, extract 2-3 ml of albumin with a 26 gauge syringe, seal it with a transparent tape, and drill a 1 × 1 cm window in the center of the fertilized egg. ) Dissolve 60 ug of ticlopidine in 15 ul of saline, drop onto a Thermanox disc (available from Miles Scientific), dry, place it on a Chorioallantoic membrane exposed through a window, and use transparent tape. It was sealed and incubated for 3 days in a 37 degreeC thermostat. In the control group, 15ul of saline instead of ticlopidine was dropped on a disc, dried, and the same method was performed to compare vascular changes. In order to distinguish between the blood vessels distributed in the ureter cavity, a 26-gauge syringe was used to inject fat into the ureter cavity to cover the blood vessels distributed in the ureter cavity. In the fertilized egg treated with ticlopidine, it was clearly shown that capillary formation of the fetal villi of the fetus was suppressed and all the fertilized eggs used (n = 15) inhibited 100% angiogenesis (FIG. 3).

Compositions containing 5-[(2-chlorophenyl) methyl] -4,5,6,7-tetrahydrothieno [3,2-C] pyridine or its hydrochloric acid, ticlopidine, are only used to treat arthritis by angiogenesis. However, ocular diseases such as diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma, degeneration, spot degeneration, pterygium, retinal degeneration, posterior lens fibrosis, granuloconjunctivitis and corneal disease caused by neovascularization It is effective in the treatment of various diseases caused by angiogenesis such as hemangiomas, hemangiofibromas, psoriasis, capillary dilatation, purulent granulomatosis, seborrheic dermatitis, acne and anticancer agents, metastatic inhibitors.

Claims (5)

For inhibiting angiogenesis comprising 5-[(2-chlorophenyl) methyl] -4,5,6,7-tetrahydrothieno [3,2-C] pyridine or its hydrochloride Ticlopidine as an active ingredient Pharmaceutical composition. The method of claim 1, Hemangioma, hemangiofibroma, arthritis, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma, corneal disease caused by neovascularization, degenerative plaque, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, psoriasis, capillary A pharmaceutical composition for inhibiting angiogenesis, which is used as a therapeutic agent for diseases caused by angiogenesis such as bloat, purulent granulomas, seborrheic dermatitis and acne. The method of claim 1, Pharmaceutical composition for inhibiting angiogenesis used as an anticancer agent or metastasis inhibitor. The method of claim 1, Pharmaceutical composition for inhibiting angiogenesis, further comprising a pharmaceutically acceptable carrier. The method according to any one of claims 1 to 4, Pharmaceutical composition, characterized in that a single dose of ticlopidine is 5-800 mg