JP2005097137A - Prophylactic or therapeutic agent for diabetic retinopathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicinal composition for prophylaxis or treatment of diabetic retinopathy. <P>SOLUTION: The medicinal composition comprises a CNTF (ciliary neurotrophic factor). The resultant composition as an eye drop can be used for the prophylaxis or treatment of diabetic retinopathy such as injury or degeneration of retinas, glaucoma, ophthalmic nerve disease or retinal nerve injury. The base sequence of human CNTF gene is made clear and the human CNTF is a protein of 200 amino acids and about 22 kD molecular weight. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition containing ciliary neurotrophic factor (CNTF) as an active ingredient, particularly to a prophylactic or therapeutic agent for diabetic retinopathy.

  Diabetic retinopathy is a disease that develops when hyperglycemia persists for a long period of time and damages or increases the permeability of retinal capillaries, and is the most common disease that currently causes blindness in humans. From its course, diabetic retinopathy is classified into three stages: simple retinopathy, preproliferative retinopathy and proliferative retinopathy. Proliferative retinopathy belongs to the most severe of the above stages and induces vitreous hemorrhage, traction retinal detachment or neovascular glaucoma due to angiogenesis in the retina and iris. As a result, it causes severe visual impairment. As a method for treating proliferative retinopathy, laser photocoagulation or vitrectomy is employed to remove abnormally proliferated blood vessels. However, these therapies are only symptomatic surgical procedures and not a fundamental cure for diabetic retinopathy.

  On the other hand, various neurotrophic factors and their receptors exist in retinal neurons and their processes (Non-patent Documents 1, 2, and 3). Among them, ciliary neurotrophic factor (Ciliary Neurotrophic Factor) : Hereinafter referred to as “CNTF”) is known to exist in large amounts in the retina (Non-patent Documents 4 and 5). In addition, there is a report example in which injection of a nutrition factor into the eyeball prevents retinal degeneration (Non-Patent Documents 6 and 7).

  CNTF is a protein isolated and identified as a factor that promotes the survival of ciliary ganglion parasympathetic neurons in chicken embryos. The nucleotide sequence of CNTF gene has been revealed in humans, mice, rats, rabbits, chickens, etc. It has become. For example, see GenBank Accession number X60542 for the human CNTF gene. And it is known that there is no homology with the gene family of nerve growth factor (NGF).

CNTF consists of 200 amino acids in humans and rats, and 199 amino acids in mice and rabbits, and has a molecular weight of about 22 kD. And, it has a function of promoting the survival or differentiation of various nerve cells and certain types of glial cells in vitro and promoting the survival maintenance of motor neurons in vivo (Non-patent Documents 8 and 9). CNTF also (i) keeps retinal neurons alive, (ii) suppresses retinal damage by light stimulation, or (iii) regulates degeneration delay and photoreceptor processes in mice with genetically altered retinal neurons. And plays an important role in the survival of the retina. On the other hand, the medical use of CNTF includes, for example, prevention of retinal damage and degeneration, treatment of glaucoma, treatment of optic nerve disease or brain disease, and treatment of retinal nerve damage. However, these are reports on the survival of the retina, but not on the suppression of retinal degeneration due to diabetes.
Gupta, SK et. Al. Biochem. Cell Biol. 75: 119-125, 1997 King, GL and Suzuma, KN Engl. J. Med. 342: 349-351, 2000 Beltran, WA et. Al. Invest. Ophthalmol. Vis. Sci. 44: 3642-3649, 2003 Kirsch, M. et. Al. J. Neurochem. 68: 979-990, 1997 Walsh, N. et. Al. Exp. Eye. Res. 72: 495-501, 2001 LaVail, M. et.al.Invest. Ophthalmol. 39: 592-602, 1998, Cayouette, M. et. Al. J. Neurosci. 18: 9282-9293, 1998 Ishiyama, T. et. Al. Exp. Neurol. 148: 247-255, 1997 Ikeda, K. et. Al. Ann. Neurol. 37: 47-54, 1995

  An object of the present invention is to provide a pharmaceutical composition containing CNTF.

  As a result of intensive studies to solve the above problems, the present inventor has found that diabetic retinopathy can be prevented or treated by instilling CNTF, and has completed the present invention.

  That is, the present invention is a pharmaceutical composition containing ciliary neurotrophic factor. The pharmaceutical composition of the present invention can be used for preventing or treating diabetic retinopathy. In this case, the dosage form of the pharmaceutical composition is preferably an eye drop from the viewpoint that it is less invasive to the patient and can be easily administered.

  The present invention provides a pharmaceutical composition containing CNTF. The pharmaceutical composition of the present invention is useful for preventing or treating diabetic retinopathy. In particular, it is useful as a prophylactic or therapeutic agent for suppressing retinal degeneration in diabetic retinopathy by instilling CNTF.

  Hereinafter, the present invention will be described in detail.

  The present invention was completed for the purpose of developing a simple preventive or therapeutic method that prevents or fundamentally treats diabetic retinopathy and is less invasive, and contains CNTF as an active ingredient About.

Since pigment epithelial cells have a blood-retinal barrier, it has been conventionally said that a polymer therapeutic agent does not enter the retina under normal conditions. However, in diabetic patients, since serum proteins are deposited in the retina due to increased permeability of capillaries, it is considered that various factors having an activity of suppressing retinal degeneration enter the retina from within the blood vessel. Therefore, the present inventor paid attention to CNTF among the factors, and thought that CNTF, which is a polymer substance, also entered the retina from the blood. We examined whether retinopathy can be suppressed by instillation of CNTF. Specifically, pancreatic β-cells were destroyed by intravenous injection of streptozotocin into rats, and CNTF was administered by eye drop to the prepared diabetes model rats. As a result, it was found that degeneration of the retina was suppressed.
1. CNTF
As the CNTF contained in the pharmaceutical composition of the present invention, for example, a protein obtained by isolating the CNTF gene from an animal such as a rat, mouse, rabbit, chicken or human or expressed using an appropriate expression system may be used. it can. Since the pharmaceutical composition of the present invention requires a large amount of CNTF, it is preferable to use a recombinant protein obtained from an appropriate expression system, particularly a human-derived recombinant CNTF. CNTF may be directly isolated and purified from animals such as rats, mice, rabbits and chickens.

In order to express CNTF used in the present invention, for example, bacteria, yeast, insects or insect cells, or animals or animal cells can be used as a host. These hosts can be transformed with an expression vector having the CNTF gene used in the present invention to produce CNTF.
(1) Production of recombinant vector In the present invention, a recombinant vector can be obtained by linking (inserting) a CNTF gene into an appropriate vector. The vector for inserting the CNTF gene is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA.

  As plasmid DNA, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast and the like can be arbitrarily selected. Moreover, phage DNA, a viral vector, etc. can also be used.

The insertion of CNTF genes into vectors is well known in the art.
(2) Preparation of transformant A transformant is obtained by introducing the above recombinant vector into a host so that the CNTF gene can be expressed. Examples of the host include bacteria such as Escherichia and Bacillus, yeasts such as Saccharomyces and Schizosaccharomyces, insects or insect cells, or animals or animal cells.

  When a bacterium such as Escherichia coli is used as a host, it is preferable that the recombinant vector is capable of autonomous replication in the bacterium, and at the same time is composed of a promoter, a ribosome binding sequence, a CNTF gene, and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained.

  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used.

  The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used.

  As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.

  Silkworms are used when insects are used as hosts. As a method for introducing a recombinant vector into an insect, a calcium phosphate method, an electroporation method, a liposome method, or the like is used.

  When animal cells are used as hosts, COS cells, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used.

  Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

When animals are used as hosts, goats, mice, rats and the like are used. As a method for introducing a recombinant vector into an animal, a calcium phosphate method, an electroporation method, a liposome method, or the like is used.
(3) Purification of CNTF The CNTF can be obtained by culturing or raising the transformant and collecting it from the culture or the animal body.

  The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host. When CNTF is produced in cells or cells after culture, CNTF is extracted by disrupting the cells or cells.

  When CNTF is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. When insects or animals are used, for example, silkworms are collected from spouted raw silk, and goats are collected with milk.

  Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, Alternatively, CNTF can be isolated and purified from the collected material.

  When expressing CNTF using Escherichia coli as a host, it is possible to select whether (i) CNTF is expressed in a native form or (ii) expressed as a fusion protein with a tag. In the latter case, since it can be easily purified by affinity chromatography, it is desirable to express it as a fusion protein.

  Examples of tags to be fused to CNTF include glutathione-S-transferase (GST), maltose binding protein (MBP), histidine-tag (His-Tag) and the like. In the present invention, GST is preferable in that the length as a tag is short and the fusion protein can be easily purified.

The fusion protein with GST can be purified using a glutathione-Sepharose column. Further, the obtained fusion protein was treated with thrombin or factor X to cleave and separate the GST part and CNTF used in the present invention, and then passed through the affinity column again without passing through the column. The fraction is collected as CNTF. Further, by passing the collected fraction through a benzamide sepharose 6B column, thrombin and the like can be removed, and pure CNTF can be obtained.
2. Pharmaceutical composition CNTF obtained as described above can be used as a prophylactic or therapeutic agent for diabetic retinopathy, as will be apparent from the examples described later, in order to suppress retinal degeneration in diabetic model rats. it can. If CNTF can form a pharmaceutically acceptable salt, such a salt may be formed. As the salt, salts with physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.) are used, and physiologically acceptable acid addition salts are particularly preferred. . Examples of the salt include a salt with an inorganic acid (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or an organic acid (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Salts with tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

  The CNTF (including salts thereof) can be used parenterally as a mixture with water or other pharmaceutically acceptable solution, or a mixture with a suspending agent. For example, the above CNTF is mixed with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in unit dosage forms required for the practice of accepted formulations. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

  It is desirable that the pharmaceutical composition of the present invention be administered in the form of eye drops.

  In addition to the above active ingredients, the pharmaceutical composition of the present invention may further contain known excipients, other active ingredients, and the like. Examples of these include sodium bicarbonate, sodium carbonate, sodium L-aspartate, aminoethylsulfonic acid, sodium chondroitin sulfate, naphazoline hydrochloride, retinol and the like.

  Additives usually used in eye drops, for example, refreshing agents such as camphor and borneol, pH regulators such as sodium hydroxide and hydrochloric acid, isotonic agents such as glycerin and propylene glycol, and viscous agents such as hydroxyethyl cellulose Containing thickeners, ethanol, polysorbate 80, solubilizing agents such as polyethylene glycol, stabilizers such as sodium hydrogen sulfite, preservatives such as chlorobutanol and benzalkonium chloride, and sterilized water in the amounts normally used. it can.

  The pharmaceutical composition of this invention can be manufactured using a normal manufacturing method. For example, it can be obtained by dissolving the active ingredients and additives in sterilized water, sterilizing and filtering with a membrane filter, and filling a sterilized container.

  When the above pharmaceutical composition is used in humans to treat diabetic retinopathy, the content of CNTF contained in one drop (approximately 50 μl) of eye drops is approximately 10 μg, preferably 15 μg, more preferably 20 μg. . The administration period is considered to be necessary for 3 months or more, preferably about 6 months. The effective dose per day is approximately 100 μl per eye, preferably 200 μl, more preferably 300 μl, and the number of daily doses is preferably 3 times (number of drops per drop: 2 drops, CNTF 20 μg). is there.

  Hereinafter, the present invention will be specifically described by showing examples. However, the present invention is not limited to the examples.

Preparation of CNTF cDNA of human CNTF was prepared by extracting mRNA from the collected sciatic nerve, preparing First DNA with reverse transcriptase, and amplifying it by PCR using the DNA as a template. This cDNA is incorporated into an E. coli expression vector (pGEX-2T: Pharmacia), then introduced into E. coli (JM109: TOYOBO), and induced with IPTG (isopropyl-beta-D-thiogalactopyraanoside) to produce human CNTF. Protein [fusion protein with glutathione S-transferase (GST)] was expressed. This fusion protein was purified using a glutathione-sepharose column (Amersham Biosciences), and then the resulting fusion protein was treated with thrombin to cleave and separate the GST part and the human CNTF part, and then again the glutathione-sepharose column. The fraction that passed through without being bound to the column was collected. Subsequently, the fraction was passed through a benzamide sepharose 6B column to remove thrombin and obtain pure human CNTF. SDS-PAGE confirmed human CNTF as a single band (FIG. 1).

Production of diabetes model rats and instillation of human CNTF
Streptozotocin (STZ) (50 mg / Kg body weight) was administered into the tail vein of 8-week-old male male SD rats, and the rats whose blood glucose level reached 300 mg / ml or more after 24 hours were regarded as diabetic model rats. Confirmation that the diabetic model rat developed retinopathy was performed by confirming a decrease in the amplitude of the a wave and the b wave of the retinal potential. Statistical processing was performed by analysis of variance. Human CNTF (2 μg / 10 μl physiological saline) was instilled twice daily in the morning and evening for one month continuously in both eyes of diabetic model rats (n = 20) 24 hours after STZ administration. As a control group, diabetic model rats (n = 20) and healthy rats (n = 20) were instilled with 10 μl of physiological saline. Table 1 shows the body weight and blood glucose level after 1 month for each group of rats.

Measurement of retinal potential Rats were raised in the dark for 24 hours, then anesthetized with ketamine (32 mg / kg) and xylazine (20 mg / kg) under a red lamp, and mydriatic with 1% atropine sulfate and 1% phenylephrine hydrochloride. Retinal potential (PE-2000, TOMEY) was measured.

  The results are shown in FIG. The a wave is the photoreceptor in the process of the photoreceptor cell, and the b wave is the Mueller cell. In the diabetic rat group, both a and b waves decreased compared to the healthy rat group (*: p <0.05). On the other hand, the b wave in the human CNTF ophthalmic diabetic rat group showed significantly higher amplitude than the diabetic rat group (*: p <0.05).

Organizational analysis
(1) Tissue treatment and slices Rats were perfused from the heart with cacodyl buffer containing 2.5% glutaraldehyde and 1% paraformaldehyde and fixed. Thereafter, the ocular tissue was taken out and placed in the same fixing solution overnight, further fixed with 1% osmium, and embedded with Epon 812. The embedded tissue was sliced into 1 μm and stained with toluidine blue. A photograph of the retina image of each rat is shown in FIG. In diabetic rats, a large number of vacuoles (pigment epithelial cell foot processes (= the retina-blood barrier)) are engulfed in the retinal pigment epithelial cells. Observations) were observed (arrows in FIG. 3). On the other hand, in human CNTF ophthalmic diabetic rats, the number and size of vacuoles were not significantly different from controls.
(2) Measurement of thickness of each layer of retina The thickness of each layer of the retina was measured using an ocular micrometer. The division of each layer of the retina is shown in FIG. The thickness of the inner plexiform layer (IPL) is from the center of the optic ganglion cell layer to the innermost side of the inner granular layer, and the thickness of the inner granular layer (INL) is from the innermost side to the outer side of the inner granular layer. (OPL) thickness is from the outermost side of the INL to the innermost side of the outer granular layer (ONL), the outer granular layer (ONL) is from the outermost side to the inner side of the ONL, and the photoreceptor projection layer (PSL) is outside From the outermost side of the granular layer (ONL) to the apical surface of the pigment epithelial cell layer, the retinal layer (RL) extends from the center of the optic ganglion cell layer to the apical surface of the pigment epithelial cell layer. The region of each layer is shown in FIG. 4, and the thickness comparison is shown in FIG. When statistical analysis was performed by analysis of variance, the thickness of IPL was significantly reduced in the diabetic rat group (FIG. 5, IPL: p <0.05).

However, in the human CNTF ophthalmic diabetic rat group, the IPL thickness was significantly thicker than that in the diabetic rat group, and was not different from the control (p <0.05).
(3) Electron microscopic (electron microscopic) findings of the retina The retinas of healthy rats, diabetic rats, and CNTF ophthalmic diabetic rats were observed with an electron microscope and the findings were analyzed. The most prominent findings in diabetic rats were observed in retinal pigment epithelial cells. In diabetic rats, a large number of vacuoles (cytopathic findings) formed by the depressions of the basal infoldings of the pigment epithelial cells being engulfed into the cells (stars in FIG. 6) were observed. In contrast, in the CNTF ophthalmic diabetic rat group, the number of vacuoles was not different from that of normal controls (FIGS. 6 and 7).
(4) Examination of the number of vacuoles observed in the retinal pigment epithelial cells The number observed per 250 μm length of epithelium was statistically processed with Kruskal-Wallis H-test. As a result, a large number of vacuoles were formed in the retinal pigment epithelium of diabetic rats, which were completely suppressed by CNTF instillation (p <0.05) (Fig. 7).

The photograph which shows the result of SDS-polyacrylamide gel electrophoresis of CNTF. The figure which showed the retinal potential a wave and b wave of the healthy rat group, the diabetic rat group, and the human CNTF ophthalmic diabetic rat group. On the other hand, the b wave in the human CNTF ophthalmic diabetic rat group showed significantly higher amplitude than the diabetic rat group. *: P <0.05. The photograph which showed the result of having dye | stained and analyzed the retinal section | slice of a healthy rat, a diabetic rat, and a human CNTF ophthalmic diabetic rat. In the human CNTF administration group, there is no decrease in the thickness of the retina (atrophy) and the pigment epithelial cell cavity (arrow) seen in the diabetic group. Bar: 40μm A photograph showing the measurement range of each layer of the retina. Bar: 40μm The figure which shows the thickness of each retina of a healthy rat group, a diabetic rat group, and a human CNTF ophthalmic diabetic rat group. In the human CNTF administration group, there is no decrease in IPL (inner granule layer) thickness (atrophy). *: P <0.05. Electron micrograph of retinal pigment epithelial cells. In diabetic rats, many vacuoles are seen, but in CNTF ophthalmic diabetic rats it is completely suppressed. The figure which shows the number of vacuoles of a retinal pigment epithelial cell. The large number of vacuoles found in diabetic rats are completely suppressed in CNTF ophthalmic diabetic rats.

Claims (3)

  A pharmaceutical composition comprising a ciliary neurotrophic factor.   The pharmaceutical composition according to claim 1 for preventing or treating diabetic retinopathy. The pharmaceutical composition according to claim 1 or 2, which is an eye drop.

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