KR101668462B1 - Pharmaceutical Composition for Preventing or Treating a Glaucoma Comprising Blood Components as Active Ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating glaucoma comprising a blood component as an active ingredient. According to the present invention, when monocyte extract or platelet extract is directly injected into the glaucoma-induced eyeball, the increase of glaucoma-induced glaucoma is mitigated, the retinal ganglion cell damage is protected, and the visual acuity is improved. Therefore, a composition comprising the blood component of the present invention as an active ingredient can be used for the development of a therapeutic agent for glaucoma.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating glaucoma,

The present invention relates to a pharmaceutical composition for preventing or treating glaucoma comprising a blood component as an active ingredient.

Glaucoma is a chronic optic nerve lesion, which is a severe refractory disease that results in gradual degeneration of the optic nerve, progressive loss of retinal ganglionic cells (RGC), and visual loss due to visual field defect. The number of domestic glaucoma patients is estimated to be about 480,000, and around 70 million people worldwide.

The risk factors for glaucoma include age, race, sex, hypertension, etc. However, intraocular pressure (IOP) elevation is known to be the most important cause of various glaucoma, especially open angle glaucoma (OAG) . In glaucoma, the extracellular matrix concentration of the trabecular meshwork (TM) is increased by the apoptosis of the eye, resulting in increased resistance to the release of aqueous humor (AH) (Alvarado et al., 1981; Grierson and Howes, 1987; Caballero et al., 2003; Tamm and Fuchshofer, 2007; Acott and Kelley, 2008; Baleriola et al., 2008; Tamm, 2009). In this study, we evaluated the effect of intraocular pressure (IOP) on the glaucoma severity in patients with glaucoma. In this study, .

For the management of glaucoma that should last a lifetime, there is a method of prescribing drugs that reduce the production of aquaria, irradiating a laser to temporarily control the intraocular pressure of the holding net, or creating a new ventilation drainage route surgically (Leske et al., 2007, 2008; McKinnon et al., 2008; Robert et al., 2013). However, there has been no attempt to fundamentally treat a holding net other than laser trabeculoplasty. Therefore, it is expected that the development of an effective net restoration method for restoring the intraocular pressure regulating function will be a breakthrough in the treatment of glaucoma.

In a study of various brain diseases, stem cells (SC) have been shown to improve irreversible brain damage through cell replacement, brain protection and inflammation inhibition. For example, neural stem cells (NSCs) and adipose tissue-derived stem cells (ASCs) inhibit inflammation to protect brain cells, replace damaged cells, (Park et al., 2012a, 2013a, 2013b), which are associated with brain diseases and aging of the brain. Growth Factors (GF) and neurotrophic factors (NF) have been reported to play a role in brain cell protection and regeneration (Park et al., 2013a, 2013b). Recently, transplantation of neural stem cells, bone marrow-derived stem cells (BMSC), and retinal progenitor cells (RPC) has been reported to be effective in protecting retinal cells (Park et al., 2012b; Hu Luo et al., 2013; Luo et al., 2014). It is believed that growth factors and neurotransmitters play an important role in this effect.

However, for the establishment of autologous stem cells and progenitor cells expressing functional growth factors or neurotrophin genes and its clinical application, it is difficult to secure the safety of various items as well as the efficacy in animals and the human body have.

Interestingly, recent studies have shown that monocytes infiltrate into the nets after selective laser-holding net plastic surgery, and that aqueous outflow is promoted by cytokines such as monocytes from cells (Alvarado et al., 2010). In addition, retinal ganglion cells were found to be protected when injected with monocyte protein 1 (Monocyte Chemoattractant Protein-1, MCP-1 / CCL2) in the eye (Chiu et al., 2010).

Therefore, the present inventors injected the blood component directly into the eyeball in consideration of the fact that the retinal ganglion cells in the eye are present in the isolated region where the blood supplying oxygen and growth / nutritional factors necessary for tissue regeneration can not reach. We found that the blood component improves glaucoma-induced elevation of IOP and decreased visual acuity.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to find a composition capable of protecting the glaucoma from elevated intraocular pressure, decreased visual acuity, and damage to retinal ganglion cells. As a result, the present inventors have completed the present invention by confirming that the intraocular injection of the monocyte extract or the platelet extract in human blood improves the intraocular pressure and the visual acuity increase due to glaucoma.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of glaucoma which comprises components of leukocytes or platelets as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating glaucoma, the composition comprising components of leukocytes or platelets in blood as an active ingredient.

Leukocytes (leukocytes, white blood cells, WBC) are blood cells other than red blood cells, which are different from red blood cells. It is divided into granulocyte and agranulocyte depending on the presence of specific granules in cytoplasm. Granulocytic leukocytes have eosinophil, basophil and neutrophil, and granulocytic leukocytes have lymphocyte and monocyte. The proportion of neutrophils is 54-62%, followed by lymphocytes 25-45%, monocytes 4-8%, and eosinophils and basophils 5%. Leukocytes contain proteins such as various growth factors / nutrients and various cytokines.

The present invention is based on the active ingredient of a component of leukocyte, preferably a monocyte of leukocyte as an active ingredient, more preferably a soluble component obtained by disrupting monocyte of leukocyte. According to some embodiments of the present invention, when leukocytes are isolated from the blood of a human, and then the mononuclear cells are separated from the blood, and then the monocytes are disrupted, and the resulting soluble component is directly injected into the eye of the glaucoma-induced animal model, It shows the effect of alleviating.

Platelet (Platelet, Thrombocyte) is one of the solid components that plays an important role in blood clotting, and the cytoplasm of the megakaryocyte in the bone marrow has been torn into the blood. Thus, there is no nucleus, and one megakaryocyte produces about 400 to 8000 platelets. Platelets contain proteins such as various growth factors / nutrients and various cytokines.

The present invention comprises a platelet component as an active ingredient, preferably a soluble component obtained by disrupting platelets as an active ingredient. According to some embodiments of the present invention, when platelets are isolated from human blood, and the soluble component obtained by breaking the platelets is injected directly into the eye of the glaucoma-induced animal model, the effect of alleviating glaucoma symptoms is exhibited. The soluble component obtained by disruption of platelets seems to be more effective in relieving glaucoma symptoms than the soluble component obtained by disrupting monocytes, probably because monocytes and other growth / nutritional factors are present in the platelets.

The leukocyte or platelet component of the present invention is characterized by being injected directly into the eye of a glaucoma patient. Preferably, the glaucoma patient is intravitreal injected. Since normal cells do not have suspended cells in the vitreous body, it is much safer to use serum or cell extract than the cell itself, since artificially injected cells can cause adverse effects on the visual field Do. In the case of allogeneic stem cells or heterologous or xenogeneic stem cells, there is a possibility of inflammation and shock due to antibody production (Pigott et al., 2013), embryonic stem cells , ESC) are more likely to develop into tumors (Germain et al., 2012). Therefore, when the active ingredient is extracted from the self-derived blood or cells, the side effect can be excluded. According to some embodiments of the present invention, when a soluble component obtained by disrupting monocytes or platelets isolated from human blood is injected directly into the glaucoma-induced animal model, it exhibits an effect of remarkably reducing glaucoma symptoms.

The present invention relates to a pharmaceutical composition capable of treating glaucoma. The present invention relates to a pharmaceutical composition capable of treating glaucoma, and it is intended to provide a pharmaceutical composition capable of treating glaucoma by directly injecting a leukocyte or platelet derived component of blood, Thereby exhibiting an effect of improving visual acuity. Accordingly, the present invention suggests the possibility of developing a novel therapeutic agent capable of treating glaucoma without an effective therapeutic agent.

FIG. 1 is a photograph showing the effect of intraocular pressure drop of serum, monocyte extract (ME) and platelet extract (PE).
2 is a photograph showing the effect of improving the visual acuity of serum, monocyte extract (ME) and platelet extract (PE).
3 is a photograph showing an example of retinal ganglion cell protection by serum, monocyte extract (ME) and platelet extract (PE).
4 is a photograph showing the protective effect of retinal ganglion cells by serum, monocyte extract (ME) and platelet extract (PE).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Preparation of experimental animals

Female Sprague-Dawley rats (Rat, 10 wk, 240-260 g) were supplied from Koatech (Pyeongtaek, Korea) and used in the experiment after approximately one week of laboratory purification. The animals were housed in a cage for 2 rats, and the environment of the animal laboratory was maintained at a temperature of 23 ± 2 ° C., a relative humidity of 55 ± 10%, a ventilation frequency of 12 times / hour, a lighting cycle of 12 hours (07:00 to 19:00) , And the illumination was adjusted to 150-300 lux. Pelleted solid feed (Purina Rat Chow, BioPia, Korea) for laboratory animals was fed. Sterile purified water was allowed to drink freely. This study was carried out in accordance with the Standard Operation Procedures (SOP) of the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University.

Example 2: Preparation of test substance-monocyte extract, platelet extract

Blood from a 51-year-old male was collected from the vein, left at 4 ° C for 30 minutes, and centrifuged at 12,000 rpm to obtain serum. In the blood collection, some of the blood was diluted with 0.8% citric acid (Sigma-Aldrich, St. Louis, USA), 2.2% trisodium citrate (Sigma-Aldrich, St. Louis, USA) and 2% dextrose (dextrose, Sigma-Aldrich, St. Louis, USA), and then monocyte extract and platelet extract were obtained by the following method.

To obtain monocyte extracts, peripheral blood mononuclear cells (PBMCs) were first isolated by Percoll concentration gradient centrifugation (Waschbisch et al., 2014). The separated mononuclear cells were disrupted by ultrasonication for 1 minute and centrifuged in a capillary tube for 5 minutes to obtain a monocyte extract (ME).

Platelet-rich plasma (PRP) (Jang et al., 2013) was obtained by centrifuging the blood collected in an anticoagulant solution (citrate-dextrose) at 230 g for 10 minutes to obtain platelet extract. The PRP was further centrifuged at 800 g for 15 minutes to obtain platelets. The obtained platelets were washed with HEPES buffer (pH 7.4), disrupted by ultrasonication for 1 minute, and centrifuged in a capillary tube for 5 minutes to obtain a platelet extract (PE).

Example  3: Glaucoma induction and intraocular pressure measurement

Rats were anesthetized by intraperitoneal injection of ketamine-xylazine (Bayer, Toronto, Canada) and injected with 50 μl of 1.8 M hypertonic saline into episcleral veins in the left eye (He et al., 2013), which induced scarring in the nets by resistance of the anoxic water discharge pathway (He et al., 2013). The right eye was used as a control and the left eye and right eye were measured for intraocular pressure for 2 weeks at 2-day intervals before and after glaucoma induction using a Tonolab tonometer (Icare Finland Oy, Espoo, Finland) (Chiu et al. 2009, 2010). In order to minimize the time difference and the change of intraocular pressure by anesthesia, intraocular pressure was measured at 10 am and 15-30 minutes after anesthesia. To test the effect of the test substance, 4 days after glaucoma induction, the test substance (2 μl) was injected into the vitreous cavity and the intraocular pressure was measured at regular intervals.

All the test results were expressed as mean ± SEM. The difference between groups was analyzed using oneway analysis of variance (ANOVA, SPSS 12.0 version) and it was considered statistically significant when P value <0.05.

The intraocular pressure of the glaucoma-induced rats ranged from 50-70 mmHg in the normal range (40-80 mmHg) (Fig. 1). However, when glaucoma was induced by injection of saline solution (1.8 M) into the holding net, the intraocular pressure was increased to about 110 mmHg 4 days after injection, and reached to the maximum value of about 138 mmHg after 6 days Respectively.

This increase in intraocular pressure was alleviated when the test substance was injected. That is, when 2 μl of serum was injected into the vitreous body on the fourth day after glaucoma induction, the increase in the intraocular pressure tended to be somewhat relaxed. When the peripheral blood mononuclear cell extract (ME) was injected, the intraocular pressure slowly recovered to within the normal range on day 12 after 8 days. In particular, when intravenous injection of platelet extract (PE) was performed, the intraocular pressure rapidly dropped, and the intraocular pressure returned to normal level on the eighth day after the injection of platelet extract 4 days.

Example  4: Measurement of vision

The animal's visual acuity was assessed using the Morris water-maze system (Smart v2.5; Panlab Technology, Barcelona, Spain). That is, a water tank of 1.8 m in diameter was filled with water of 22 ± 2 ° C., and the water tank was divided into four zones, and a platform having a height of 5 cm above the water surface (a diameter of 10 cm) was installed in one zone. On the 14th day after glaucoma induction at the end of the test, the animals were placed in the opposite side of the platform toward the wall of the tank, and the latency time until the platform was found was measured. (Hu et al., 2013; Park et al., 2013a).

Approximately 11 seconds were required to visit the visible platform through swimming in normal animals, but in the animal 14 days after glaucoma induction, the platform required about 44 seconds to reach the platform (Fig. 2). The time to reach the platform was shortened when the test substance was injected. In other words, when serum was injected into glaucoma 4 days after glaucoma induction, it took about 35 seconds, and the time to reach the platform was remarkably reduced to about 23 seconds in the monocyte extract administration group and about 18 seconds in the platelet extract administration group. These results indicate a possible improvement in visual acuity by blood components.

Example  5: Number of retinal ganglion cells  Measure

Retrograde labeling was performed by applying fluorogold (Fluorochrome, Denver, CO, USA) to the superior colliculus (SC) surface 4 days before the autopsy of the animal to determine retinal ganglion cell count al., 2008, 2009, 2010). That is, the scalp of the rat was cut at the midline, and a dental drill was applied to the left and right parietal bone at a distance of 0.5 mm from the sagittal suture and the transverse suture to form a hole having a diameter of 2 mm "He said. After removal of the cerebral cortex, the four marginal margins were identified through a surgical microscope and a thin gelatin sponge (UpJohn, Kalamazoo, MI, USA) moistened with 6% fluorogold was placed on top of the top of the cortex, (Chu et al., 2008; Hu et al., 2010). The skull skin was sutured and bupreorphine (100 mg / kg) was orally administered for 5 days.

Eyes were removed on the day of the autopsy, fixed in formalin solution for 60 minutes, and cut horizontally in the upper eyecups. Paraffin sections were made by fixing the upper optic having the complete optic nerve. The retina was separated from the underlying sclera in the lower compartment, flattened with the vitreous side up, and attached to the slides using a fluorescent mounting medium (Dako, Carpentaria, CA, USA) (Chiu et al. 2009, 2010). Fluorogold-labeled retinal ganglion cells (cytoplasmic fluorogold granule confirmation) were observed using a fluorescence microscope equipped with a UV-385 filter (Nikon, Kawasaki, Japan).

In the retinal ganglion cell layer of normal animals, retinal cells exhibiting a strong fluorogold-positive response were clearly observed, but fluorescence responses were significantly reduced in glaucoma-induced animals, indicating that much of the retinal cells were lost (Fig. 3). Such cell loss was somewhat restored when the serum of the test material was injected. When the monocyte extract or the platelet extract was injected, it was confirmed that retinal cells were remarkably protected.

The change in the number of cells in the retinal ganglion was quantitatively analyzed (Fig. 4). In the case of glaucoma-induced ganglion cells, approximately 51% of the cells were normal, indicating that nearly 50% of the cells were lost. When serum was injected, the number of cells was normalized to about 61% when the number of cells was normal, indicating that retina cells were protected to some degree compared with the case where only glaucoma was induced. When the monocyte extract was injected, the number of cells was about 81% when the number of cells was normal. When the platelet extract was injected, the number of cells was about 89% when the number of cells was normal. These results show that the protective effect of retinal cells on monocyte extract or platelet extract is remarkable.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

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Claims (6)

A pharmaceutical composition for preventing or treating glaucoma comprising, as an active ingredient, soluble components of platelets obtained by breaking platelets.
delete delete 2. The composition of claim 1, wherein the composition is injected directly into the eye of a glaucoma patient. 5. The composition of claim 4, wherein said composition is intravitreal injection of intravitreal injection of eye of a glaucoma patient. 2. The composition of claim 1, wherein the composition is characterized by decreased intraocular pressure and protection of retinal ganglion cells.

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