KR20030080735A - Pharmaceutical composition containing human integrin binding protein or peptide for treating ophthalmopathy - Google Patents

Abstract

PURPOSE: A therapeutic agent composition for treatment of ocular disease, containing saxatilin or human integrin binding protein having a similar sequence to the same as an effective ingredient and carriers is provided. The composition is effective in treatment of ocular disease such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, disease associated with angiogenesis such as angiogenesis-induced glaucoma, angiogenesis-induced corneal disease, etc. CONSTITUTION: The pharmaceutical composition for treatment of ocular disease associated with angiogenesis contains: saxatilin or human integrin binding protein having a similar sequence to the same; and furthermore carriers for suitable transmitting the drug. The carriers contain polyethylene glycol, dextran and a surfactant as a main component. The effective amount of the composition is in the range of 1microgram to 10mg per kg body weight in the case of vascular injection, 1ng to 1mg per eyeball in the case of ocular injection, and 1ng to 1mg per 1ml eye drop in the case of an eye drop.

Description

Pharmaceutical composition containing human integrin binding protein or peptide for treating ophthalmopathy}

The present invention relates to a composition for treating ocular disease, which is completed by mixing with a pharmaceutical agent that contains a recombinant disintegrin-like polypeptide as an active ingredient and can easily deliver drugs to intraocular retinal tissue. More specifically, it is a disintegrin-based protein containing the Arg-Gly-Asp (RGD) sequence, which is present in the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) factors. By blocking each of the integrin αvβ5 and αvβ3 is strongly inhibited angiogenesis, thereby strongly inhibiting abnormal angiogenesis to use as a therapeutic agent for the disease.

Angiogenesis refers to the process by which sprouts are formed from existing microvascular vessels, and these sprouts multiply to form new capillaries. This is a very important normal process in fetal growth, uterine saline, placental proliferation, corpus luteum formation and wound healing in the human body (Gunther G. et al ., Oncology 54: 177-184 (1997)). Solid cancer is typically a disease with many neovascularizations, but the neovascularization is not secondary to the etiology of cancer and is a secondary feature that is only produced for the supply of nutrients and oxygen to rapidly growing cancer tissues. In cancer, neovascularization can be suppressed to some extent, but the underlying treatment is impossible. On the contrary, there are diseases in which neovascularization itself is a etiology caused by abnormally increased neovascularization or failure to regulate normally. Representative diseases include neovascular diseases, rheumatoid arthritis, psoriasis, and pyogenic granulomas. These diseases can be treated fundamentally by inhibiting neovascularization.

In the eye, neovascularization must be turned off, but when the neovascularization is turned on due to a false signal, it causes serious eye disease, which is a major cause of acquired blindness (Lois EH et. al. , Nat Med. 5: 1390-1395 (1999). Representative neovascular diseases include diabetic retinopathy, blood vessels in the retina, retinopathy of prematurity, and age-related macular degeneration in which the blood vessels form in the choroid (Amal). AE et al. , Retina 11: 244-249 (1991); Constantin JP et al ., Ophthalmology 97: 1329-1333 (1990); Jin-Hong C. et al ., Current opinion in Ophthalmology 12: 242-249 ( Peter AC, J of Cellular Physiology 184: 301-310 (2000). Current treatments for these eye diseases include laser therapy, photocoagulation, cryocoagulation, and photodynamic therapy (Visudyne) (Edwin EB et al ., Ophthalmology 88: 101-107 (1981)). All of these treatments are surgical treatments, and medical treatments are still in development. Surgical treatment not only has a big limitation that it can not be applied to all patients, but also has a low success rate and a very high cost, which causes a great social and economic burden. Most patients who can't operate have blindness in the absence of special treatment. As human lifespan is prolonged, these diseases continue to increase, and the development of appropriate therapeutics is urgent. Therefore, research and development of angiogenesis inhibitors are currently being actively conducted, and most of the eye disease treatment agents under development include steroids, MMP inhibitors, and antibodies against angiogenic growth factors (Jeremy G. et al. , Am J Pathology 160: 1097-1103 (2002)). In the present invention, using a disintegrin that exhibits the effect of inhibiting neovascularization by acting as a different mechanism of action from the existing development of a therapeutic agent, a new ocular disease treatment agent for inhibiting angiogenesis of the retina and choroid was developed.

Angiogenesis is achieved by vascular cell adhesions in smooth muscle and endothelial cells. Adhesion requires binding of matrix proteins to specific cell surface receptors, including the integrin family (Brian P. Eliceiri, Circ Res . 89: 1104-1110 (2001)). Angiogenesis has been reported to exist in two pathways by the vascular cell integrins αvβ3 and αvβ5 (Lin L. et al ., Exp. Eye Res . 70: 537-546 (2000)). These two integrins are expressed in angiogenic vascular cells and are known to play an important role in bFGF- and VEGF-induced angiogenesis (Guoquan Gao et al ., FEBS Lett. 489: 270-276). 2001); Joan W. Millers et al ., Ophthalmology 100: 9-14 (1993); Napoleone Ferrara, Curr Opin Biotech. 11: 617-624 (2000); Martin Friedlander et al ., Proc. Natl. Acad. Sci. 93: 9764-9769 (1996). In general, disintegrin is known as a potent antagonist of αvβ3 integrin expressed by bFGF, and the disintegrin used in the present invention is a polypeptide derived from snake venom (Sung-Yu H. et al ., Thrombosis Res) ., 105: 79-86 (2002)). Most disintegrins contain Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) sequences, which are structural motifs of fibrinogen. Reported studies have shown that disintegrins containing RGD sequences inhibit the formation of new blood vessels by preventing cells from adhering to the ECM (Victor IR and Michael SG Prostate 39: 108-118 (1999); Yohei M. et al ., J of Biological chemistry 276: 31959-31968 (2001).

It has been reported that angiogenesis pathways expressed by VEGF are more important in the eye, and we are the first to see whether disintegrins, which actually contain RGD sequences, can also inhibit intraocular neovascularization mainly involving VEGF but not bFGF. Clear results were obtained for.

In addition, considering the special structure of the eye, saxatrine, a polypeptide, can not be administered to the outside of the eye by itself, and the route of drug administration by direct injection is also impossible. Therefore, there has been a need for an eye-only pharmaceutical preparation capable of carrying and slowly releasing polypeptides that can exert an effective drug.

Therefore, the study was conducted to develop an ocular diseases caused by neovascularization using saxtrin, a disintegrin, and confirmed that saxatrin was effective in treating ocular diseases by inhibiting neovascularization, which is mainly caused by VEGF. As a result of pharmacological phenomena made by mixing Trin with eye drops in the form of eye drops and mixed with dextran, albumin, or biodegradable polymer gels and nanomolecules, it is effective and long-lasting. Pharmacological effect. Therefore, saxatrin strongly inhibits intraocular neovascularization, thereby confirming that it is effective in treating intraocular neovascularization such as diabetic retinopathy, prematurity retinopathy, and age-related macular degeneration.

The present invention has been made in view of the above problems, and the object of the present invention is to provide an ophthalmic disease therapeutic agent by devising an appropriate carrier capable of delivering a therapeutic agent and a drug using neovascular suppression.

1 is a surgical micrograph of saxatrin inhibiting corneal neovascularization of rabbits induced by bFGF.

Figure 2a is a fluorescence micrographs observed after treatment by concentration (0, 12.5, 25, 100ug / ml) to confirm the basic toxicity of saxatrin in primary cultured retinal cells.

Figure 2b is a graph of the lactate dehydrogenase enzyme released in the medium after treatment by concentration (0, 12.5, 25, 50, 100ug / ml) to determine the basic toxicity of saxatrin in primary cultured retinal cells.

Figure 3a is a photograph of the inhibition of saxatrin neovascularization in the animal model to lower the normal oxygen partial pressure after hyperbaric oxygen treatment (75%) to induce retinal neovascularization.

FIG. 3b is a photograph of observation of enhancing normal blood vessel production in a dose-dependent manner by saxatrin in an animal model in which retinal neovascularization is induced by lowering to normal oxygen partial pressure after hyperbaric oxygen treatment (75%).

Figure 4 shows the treatment of blood vessels after treatment with snake venom saxatrine, salmosin, and recombinant saxatrine at a concentration of 0.1 mg / ml using the HUVEC cell (primary cultured) proliferation test system, a research system for angiogenesis. It is a graph showing how slow growth is.

Figure 5 is a graph showing whether the release of saxatrin (concentration 1mg / ml) loaded on the hydrogel using the HUVEC cell (primary culture cell) proliferation test system that has been developed as a research system for angiogenesis inhibits the growth of vascular cells to be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating angiogenesis-related ocular diseases comprising saxatrin or a human integrin binding protein having a similar sequence as an active ingredient.

Eye diseases that can be applied in the present invention are diabetic retinopathy, macular degeneration, prematurity retinopathy, neovascular glaucoma, neovascular corneal disease, neovascular iris disease and the like.

In addition, the composition of the present invention further comprises an ophthalmic carrier for drug penetration, which is mainly composed of polyethylene glycol, dextran and a surfactant, and an injectable drug release carrier which is a hydrogel or an injectable drug release carrier which is nano and microparticles. Include.

In addition, the effective amount of the composition of the present invention ranges from 1 ug to 10 mg per kg of body weight in the case of vascular injection, from 1 ng to 1 mg per human eye in the case of eye injection, and from 1 ng to 10 mg per 1 ml of eye drops. It is done.

In the range lower than the effective amount range, the drug efficacy is weak or hardly shown, and in the excess range, only the dose is increased in the state that does not show a large difference compared to the pharmacological effect appearing in the range below.

Hereinafter, the present invention will be described in detail.

By analyzing the effect of purely purified purified saxatrin on intraocular neovascularization under in vivo conditions, the following novel phenomena were discovered, and the composition characteristics of the appropriate pharmaceutical preparations for application to the eye were characterized by intraocular drug administration. At the time a new advantage was discovered. First, saxatrin strongly acts as an angiogenesis inhibitor by blocking αvβ5 integrin expressed by hypoxia in the eye. Second, the oxygen partial pressure animal model does not inhibit angiogenesis that saxatrin is normally generated at the developmental stage, but rather helps normal blood vessel formation. Third, biodegradable and water-soluble polymer gels (biodegrdable hydrogels) are non-toxic in the eye, effectively transport and stabilize saxatrin, and various drug release behavior conditions (day to year) depending on the polymer synthesis conditions. Fourth, the nanoparticle-type pharmaceutical preparation made of biodegradable polymers has the advantage that the particles can be administered to a special place in the eye through the light path because the particles do not cause light refraction and diffraction. Therefore, the use of the present invention is preferable for diseases in which abnormal neovascularization of eye diseases is induced, ie, diabetic retinopathy, senile macular degeneration, prematurity retinopathy, and the like.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1 Neovascular Inhibitory Effect of Saxatrin Induced by bFGF in Ocular Corneal Tissue.

Saxatrin is known to block angiogenesis by blocking the attachment of αvβ3, an integrin derived from vascular endothelial cells to extracellular matrix (ECM) by bFGF. However, no studies on saxatrin-induced vascular endothelial cell growth and angiogenesis inhibition have been reported. To investigate whether saxtrin affects intraocular neovascularization, a micropocket was made in the cornea of rabbit eye to induce a neovascularization by inserting a pellet containing 250ng of bFGF. At this time, in order to see the efficacy of saxatin, 1 mg of saxatrin was included in the pellet. Five days later, the eyes of the rabbits were examined for neovascularization using an anatomical microscope. As a result, it was confirmed that saxatrin effectively inhibits the growth of bFGF-induced ocular vascular endothelial cells. (See Fig. 1)

Inhibition of bFGF-induced angiogenesis in ocular avascular tissue by saxatrin can be explained by the fact that bFGF induced αvβ3 integrin and blocked saxatrin in blood vessels in the eye, like other blood vessels. have. On the other hand, no side effects such as corneal haze by saxatrin were observed in the rabbits used in the experiment.

Example 2 Basic Toxicity Test of Saxatrin in Primary Cultured Retinal Cells

The eye is part of the central nervous system. In particular, the retinal tissue is the optic nerve (optic nerve), various interneurons (interneuron), ganglion (retinal ganglion cell), and photoreceptor (photoreceptor) is the majority. These cells are so sensitive that apoptosis can occur even with small stimuli, and once killed, they do not regenerate. Therefore, it is very important to investigate whether saxatrin is toxic to these neurons. To verify these, primary cultures of the retina from the eyes of two-day-old ICR mice were treated with saxatrin concentrations (0, 12.5, 25, 50, 100 ug / ml), and cytotoxicity was determined by LDH assay and Hoechst-PI staining. Investigate. (See Figures 2A, 2B)

As a result, it was confirmed that the toxicity of saxatrin retinal cells in accordance with the concentration is almost no compared to the control group. Thus, when saxatrin is used for the treatment of eye diseases, at least there is no toxicity problem in the nervous system.

Example 3 Inhibition of Saxtrin's Angiogenesis in Mouse Model Inducing Retinal Angiogenesis Using Oxygen Partial Pressure Change

Artificial retinal neovascularization caused by oxygen partial pressure difference is similar to that of prematurity and diabetic retinopathy in humans. When the mice exposed to high oxygen environment (75%) at the beginning of birth were returned to normal oxygen partial pressure (20%), they were experimented on the principle of spontaneously generating abnormal neovascularization. To this end, after 7 days of birth in a device capable of adjusting the oxygen partial pressure, it is left for 5 days in a high-pressure oxygen environment that maintains a 75% oxygen partial pressure and then left for 5 days again at a normal oxygen partial pressure of 20% oxygen. At this time, saxatrin was administered once daily for 5 days at 0.001, 0.01, 0.1, and 1 mg / kg of mice to observe the retinal neovascular suppression of saxatrin. In order to observe the blood vessels, a solution of 50 mg of FITC-dextran having a molecular weight of 2 × 10 ^{6 in} 1 ml of saline was injected through the left ventricle. Immediately after injection, the mice's eyes were removed. The extracted eye was washed with physiological saline, fixed with 4% paraformaldehyde for 4 to 24 hours, the lens was removed from the eye, the retina was flattened on a glass slider, and then sutured with glycerin-gelatin and observed using a tube microscope. . (See FIG. 3A)

As a result, it was found that neovascularization was severely formed in the periphery of the retina of the mouse treated only with saline after hyperbaric oxygen treatment, but the neovascularization was effectively inhibited in the group treated with saxatrin of 0.01 mg / Kg or more. The neovascular suppression pattern was dose-dependent. These results confirm that saxatrin acts as an antagonist of integrin αvβ5 expressed by VEGF from the existing results of VEGF-induced and neovascularization in the retina in this oxygen partial pressure animal model.

On the other hand, young mice that were left in hyperbaric oxygen did not develop vascular tissues of the retina more properly than mice that grew properly. However, in the group treated with saxatrin, dose-dependently occurring blood vessels were observed to be properly formed (see FIG. 3B). This is very interesting in that saxatrin helps the growth of normal blood vessels beyond the advantage of having no effect on normal blood vessels. In addition to angiogenesis inhibitory effects, the effects of improving the formation of these normal blood vessels were the first to be found. It is inferred that soluble polypeptides, such as saxatrin, are attached to integrins and carry signaling associated with cell growth. These saxatrin effects help normal blood vessel development in addition to neovascularization in retinopathy in premature infants. There is a big advantage to playing a role.

Example 4 Comparison of Salmonine with Saxatrine, Another Disintegrin in Inhibiting Angiogenesis

The following experiment was conducted to determine whether other proteins having similar sequences to saxatrine perform similar functions to saxattrin. Like saxatrine, the salmonine extracted from snake venom has a sequence similar to saxatrine. The effects of salmonine, saxatrine, and recombinant saxatrine purified from snake venom on blood vessel formation were investigated. HUVEC cell (primary cultured cell) proliferation test system, which has been developed as an angiogenesis research system, was used to observe how salmosin, saxatrine and recombinant saxatrin inhibit the growth of vascular cells. Salmonine, saxatrine, and recombinant saxatrin were treated with HUVEC cells at a concentration of 0.1 mg / ml, followed by incubation for 48 hours. Cultured cells were monocellularized with trypsin and cell number was measured. As a result, all three disintegrins, such as salmosin, saxatrine, and recombinant saxatrin, inhibited the growth of blood vessels in similar proportions (see Figure 4). To suppress it.

Example 5 Inhibition of Angiogenesis of Saxatrin by Drug-Release Carrier for the Treatment of Eye Disease

Saxatrin is a polypeptide and has a relatively high molecular weight, so a carrier suitable for eye tissue characteristics is essential for use in eye diseases. It is difficult to reach the target position that exists deeply inside the eye, such as the retina, through the eye drops. And systemic injections are likely to cause a lot of cost and side effects. Therefore, there is a need for a vehicle of direct intraocular injection. The eye also needed a carrier that was characterized by a long release in a single dose because repeated injections were not possible due to the risk of infection. And as mentioned above, because of neurotoxicity, the carrier must not be neurotoxic. Water-soluble biodegradable polymer gel was most suitable as a carrier capable of satisfying these conditions. Polymer gels can synthesize gels having different graft rates according to reaction conditions and thus have different release behavior rates, which is a great advantage. Saxatrin was loaded on hydrogel, a biodegradable polymer gel, to analyze the effect on angiogenesis under laboratory conditions. Using the HUVEC cell (primary cultured cell) proliferation test system, which has been developed as a research system for angiogenesis, it was observed whether the saxatrin loaded on the hydrogel was released to inhibit the growth of vascular cells. 1 mg of saxatrin was mixed with 1 ml of hydrogel, which could be released within 48 hours, and 40ul was added to 400ul of cell culture solution (24 well tissue culture vessel). The cells were then treated with bFGF lysed at a concentration of 1 ng / ml, further incubated for 72 hours, and then cells were monocellularized with trypsin and cell number was measured. As a result, the control group increased more than two-fold cell proliferation by bFGF, proliferation was inhibited in the hydrogel mixed with saxatrin (see Fig. 5). Thus, these results show that the controlled release of saxatrin is well occurring in hydrogels. Depending on the synthesis conditions of the gel to make a gel having a different release rate can be used to control the saxatrin release according to the application. In addition, it is possible to configure the gel form in the form of nanoparticles to solve the problems of light scattering and permeation, etc. can have the advantage as an eye-only carrier.

The present invention proposes a new treatment method through a therapeutic drug in addition to the neovascular disease-related ophthalmic disease treatment method that was only dependent on the existing surgical operation. Surgical operation is expensive and has limitations that cannot be applied to all patients, but the present invention is a breakthrough in the treatment of neovascular diseases related to ocular diseases, and prevents blindness. Intraocular neovascular suppression characteristics of saxatrin have no effect on the formation of existing normal blood vessels and normal neovascularization of the newly developing stage. Rather, it helps to produce normal blood vessels in the developmental stage, which is a great advantage for patients in developmental stages such as prematurity retinopathy. If you suppress all neovascularization, it cannot be applied to prematurity retinopathy. Therefore, the value of saxatrin as a therapeutic agent for retinopathy of prematurity is very high. In addition, diabetic retinopathy or senile macular degeneration show chronic characteristics and require long-term treatment. Therefore, the free control of the drug behavior time by the pharmaceutical preparation of the present invention is expected to meet the needs of doctors and patients in the treatment and to maximize the ease of use, and most of all minimize the risk of infection during treatment. In addition, the formulation according to the present invention is most suitable for ophthalmic treatments and works for a long time, so it is excellent in efficiency.

Claims (7)

A pharmaceutical composition for treating angiogenesis-related ocular disease, comprising as saxtrin or a human integrin binding protein having a similar sequence as an active ingredient. The method of claim 1, The ocular disease is diabetic retinopathy, age-related macular degeneration, prematurity retinopathy, neovascular glaucoma, neovascular corneal disease, neovascular iris disease, characterized in that the pharmaceutical composition for treating ocular disease. The method of claim 1, The composition is a pharmaceutical composition for treating ocular disease, characterized in that it further comprises an eyedrop carrier for drug penetration, the main component of the polyethylene glycol, dextran and surfactant. The method of claim 1, The composition of the pharmaceutical composition for treating ocular disease, characterized in that it further comprises a drug injection carrier for injection or a drug release drug for nano and micro-particles that is a hydrogel. The method according to any one of claims 1 to 4, When the effective amount of the composition of the vascular injection range of 1ug ~ 10mg per kg body weight of the pharmaceutical composition for the treatment of eye diseases. The method according to any one of claims 1 to 4, When the effective amount of the composition of the eye injection is in the range of 1ng ~ 1mg per human eye, the pharmaceutical composition for the treatment of eye diseases. The method according to any one of claims 1 to 4, When the effective amount of the composition is an eye drop, the range is 1ng ~ 10mg per 1ml of eye drops, characterized in that the pharmaceutical composition for treating eye diseases.