JP7561433B2 - Drugs for preventing or treating diabetic retinopathy - Google Patents

Description

The present invention relates to a drug for the prevention or treatment of diabetic retinopathy.

Diabetic retinopathy is a microangiopathy that occurs in the retina due to diabetes, which not only reduces vision due to macular edema, but if it worsens, can lead to blindness due to fundus hemorrhage and retinal detachment. Preventing diabetic retinopathy requires controlling blood sugar levels, but if it does develop, laser photocoagulation, vitreous surgery, and intravitreal injection of steroids and anti-VEGF drugs are performed. However, the effects of these drug therapies are temporary, and more effective drug therapies are desired.

Meanwhile, the present inventors have investigated components secreted extracellularly under hypoxia-reoxygenation conditions, i.e., under oxidative stress conditions, and discovered secreted eIF5A (named ORAIP). The inventors have found that this secreted eIF5A is a protein in which the tyrosine residues of eIF5A are sulfated, and that it induces apoptosis in cells subjected to oxidative stress. Furthermore, the inventors have found that neutralizing antibodies against this secreted eIF5A inhibit apoptosis caused by oxidative stress and inhibit myocardial ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1).

International Publication No. 2009/144933

SCIENTFIC REPORTS 5, 13737(2015) JCI Insight 2, e90905(2017)

The objective of the present invention is to provide a new preventive or therapeutic drug for diabetic retinopathy, for which the development of an effective therapeutic drug is desired.

As a result of investigations conducted by the inventors to develop a therapeutic drug for the above-mentioned difficult-to-treat diseases, they discovered that neutralizing antibodies against secretory eIF5A exhibit excellent preventive and therapeutic effects against diabetic retinopathy, and thus completed the present invention.

That is, the present invention provides the following [1] to [12].

[1] A preventive or therapeutic agent for diabetic retinopathy, comprising as an active ingredient a neutralizing antibody against secretory eIF5A.
[2] The preventive or therapeutic agent according to [1], wherein the neutralizing antibody against secretory eIF5A is a monoclonal antibody.
[3] The preventive or therapeutic agent according to [1] or [2], wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
[4] Use of a neutralizing antibody against secretory eIF5A for the manufacture of a drug for the prevention or treatment of diabetic retinopathy.
[5] The use according to [4], wherein the neutralizing antibody against secretory eIF5A is a monoclonal antibody.
[6] The use of [4] or [5], wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
[7] A neutralizing antibody against secretory eIF5A for use in the prevention or treatment of diabetic retinopathy.
[8] The neutralizing antibody according to [7], wherein the neutralizing antibody against secretory eIF5A is a monoclonal antibody.
[9] A neutralizing antibody according to [7] or [8], which is a neutralizing antibody against secreted eIF5A having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
[10] A method for preventing or treating diabetic retinopathy, which comprises administering an effective amount of a neutralizing antibody against secretory eIF5A.
[11] The method described in [10], wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody.
[12] The method of [10] or [11], wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

By using the pharmaceutical or pharmaceutical composition of the present invention, a superior therapeutic effect for diabetic retinopathy can be obtained compared to conventionally used drugs, such as anti-VEGF drugs. This disease is one for which no excellent therapeutic drug exists, and the pharmaceutical of the present invention is extremely useful.

This shows edema and hemorrhage in a diabetic retinopathy model mouse in which pericytes were eliminated. 1 shows the edema/hemorrhage suppressing effect of a VEGF receptor decoy in a diabetic retinopathy model mouse. 1 shows the edema/hemorrhage-suppressing effect of the neutralizing antibody of the present invention (anti-ORAIP antibody) in a diabetic retinopathy model mouse. 1 shows the edema/hemorrhage-suppressing effect of the neutralizing antibody of the present invention (anti-ORAIP antibody) in a diabetic retinopathy model mouse. 1 shows the effects of an anti-VEGF drug and the neutralizing antibody of the present invention (anti-ORAIP antibody) on a pericyte-loss diabetic retinopathy model mouse. 1 shows the effects of an anti-VEGF drug and the neutralizing antibody of the present invention (anti-ORAIP antibody) on a pericyte-loss diabetic retinopathy model mouse. The amino acid sequence of the heavy chain variable region is shown. The amino acid sequence of the light chain variable region is shown.

The active ingredient of the pharmaceutical of the present invention is a neutralizing antibody against secreted eIF5A.

Eukaryotic translation initiation factor (eIF) 5A is a substance that has been identified as a translation initiation factor, as its name suggests. eIF5A is expressed in the cytoplasm, deoxyhypusinated (deoxyhypusine eIF5A) by deoxyhypusine synthase (DHS), and then hypusinated (hypusinated eIF5A) by deoxyhypusine hydroxylase (DOHH), and this hypusinated eIF5A is known to exhibit cell proliferation activity. In addition, eIF5A is secreted outside the cell and changes to secreted eIF5A in which tyrosine residues are sulfated. This secreted eIF5A induces apoptosis in cells subjected to oxidative stress. And, a neutralizing antibody against this secreted eIF5A strongly suppresses apoptosis caused by oxidative stress and suppresses myocardial and cerebral ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1). However, it is not known what effect this neutralizing antibody has on diabetic retinopathy.

The neutralizing antibody used in the present invention may be of any origin, type (monoclonal, polyclonal) or shape as long as it binds to the secreted eIF5A protein. Specifically, known antibodies such as mouse antibodies, rat antibodies, chicken antibodies, human antibodies, chimeric antibodies and humanized antibodies can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. The monoclonal antibody is preferably a monoclonal antibody produced by a hybridoma deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under NITE P-02955.

A preferred example of a neutralizing antibody used in the present invention is a neutralizing antibody having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:6.
A further preferred example of a neutralizing antibody is a neutralizing antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8. Here, the above-mentioned "comprising an amino acid sequence" includes cases in which one to several amino acids have been deleted, substituted or added in the amino acid sequence.

The neutralizing antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies using known means. As the neutralizing antibodies used in the present invention, mammalian or avian monoclonal antibodies are preferred. In particular, mammalian monoclonal antibodies are preferred. Mammalian monoclonal antibodies include those produced by hybridomas and those produced in hosts transformed by genetic engineering techniques with an expression vector containing an antibody gene.

Monoclonal antibody-producing hybridomas can basically be prepared using known techniques as follows: That is, eIF5A protein in which tyrosine residues are sulfated, eIF5A protein, hypusinated eIF5A protein in which tyrosine residues are sulfated, hypusinated eIF5A protein, partial peptides of these proteins, or the like are used as sensitizing antigens, which are then immunized according to a conventional immunization method, the resulting immune cells are fused with known parent cells by a conventional cell fusion method, and monoclonal antibody-producing cells are screened by a conventional screening method to prepare the monoclonal antibody-producing hybridomas.
Specifically, monoclonal antibodies can be prepared as follows.

Purified secreted eIF5A protein, eIFA protein, partial peptides of eIF5A protein containing tyrosine residues that can be sulfated, etc. can be used as sensitizing antigens. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human eIF5A protein, or by incorporating a part of the eIF5A gene into an expression vector, or by decomposing natural human eIF5A protein with a protease. The part and size of the human eIF5A protein used as the partial peptide are not limited.

There are no particular limitations on the mammal to be immunized with the sensitizing antigen, but it is preferable to select one taking into consideration its compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, and hamsters, or birds, rabbits, monkeys, etc. are used.

Immunization of animals with sensitizing antigens is carried out according to known methods. For example, a common method is to inject the sensitizing antigen intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is appropriately diluted and suspended in PBS (Phosphate-Buffered Saline) or physiological saline, etc., and mixed with an appropriate amount of a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it is desirable to bind it to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

After immunizing a mammal in this manner and confirming that the desired antibody level has increased in the serum, immune cells are collected from the mammal and subjected to cell fusion, with preferred immune cells being spleen cells in particular.

Mammalian myeloma cells are used as the other parent cell to be fused with the immune cell. These myeloma cells may be any of various known cell lines, such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), M PC-11 (Margulies. D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al. al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S.J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used.

The cell fusion between the immune cells and myeloma cells can basically be carried out according to known methods, such as the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

More specifically, the cell fusion is carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an auxiliary agent such as dimethyl sulfoxide can be added to increase the fusion efficiency.

The ratio of immune cells to myeloma cells can be set as desired. For example, it is preferable to use 1 to 10 times the immune cells relative to the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI 1640 culture medium, MEM culture medium, or other normal culture medium used for this type of cell culture, which are suitable for the growth of the myeloma cell line, can be used, and serum supplements such as fetal calf serum (FCS) can also be used in combination.

Cell fusion is performed by thoroughly mixing a predetermined amount of the immune cells and myeloma cells in the culture medium, adding a PEG solution (for example, average molecular weight of about 1000 to 6000) pre-warmed to about 37°C, usually at a concentration of 30 to 60% (w/v), and mixing to form the desired fused cells (hybridomas). Next, an appropriate culture medium is successively added, and the process of centrifuging and removing the supernatant is repeated to remove cell fusion agents and other substances that are unfavorable to the growth of the hybridomas.

The hybridomas thus obtained are selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture in the HAT culture medium is continued for a period of time (usually several days to several weeks) sufficient for cells other than the desired hybridoma (non-fused cells) to die. Then, a conventional limiting dilution method is performed to screen and single-clone hybridomas that produce the desired antibody.

Screening and single cloning of the desired antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, an antigen may be bound to a carrier such as polystyrene beads or a commercially available 96-well microtiter plate, reacted with the hybridoma culture supernatant, and after washing the carrier, reacted with an enzyme-labeled secondary antibody or the like, to determine whether the culture supernatant contains the desired antibody that reacts with the sensitizing antigen. Hybridomas producing the desired antibody may be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used as the antigen.

The hybridomas producing the monoclonal antibodies produced in this manner can be subcultured in normal culture medium and can be stored for long periods in liquid nitrogen.

To obtain monoclonal antibodies from the hybridoma, the hybridoma is cultured according to a conventional method and the culture supernatant is obtained, or the hybridoma is administered to a compatible mammal to grow and the ascites is obtained. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

The neutralizing antibody used in the present invention is not limited to the entire antibody molecule, but may be an antibody fragment or a modified antibody, as long as it binds to and neutralizes the secreted eIF5A protein, and includes both bivalent and monovalent antibodies. For example, antibody fragments include Fab, F(ab')2, Fv, Fab/c having one Fab and a complete Fc, or single chain Fv (scFv) in which the Fv of the H chain or L chain is linked with an appropriate linker. Specifically, antibodies are treated with enzymes such as papain and pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, for example, Co, M.S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A.H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.). Press, Inc. , Lamoyi, E. , Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al. , Methods in Enzymology (1989) 121, 663-669, Bird, R. E. et al. , TIBTECH (1991) 9, 132-137).

An scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described herein. As the peptide linker linking the V regions, for example, any single-chain peptide consisting of 12 to 19 amino acid residues can be used.

DNA encoding the scFv can be obtained by using the DNA encoding the H chain or H chain V region of the antibody, and the DNA encoding the L chain or L chain V region, as a template, and amplifying the entire sequence or the DNA portion encoding the desired amino acid sequence by PCR using a primer pair that specifies both ends, and then further amplifying it in combination with DNA encoding a peptide linker portion and a primer pair that specifies that both ends are linked to the H chain and L chain, respectively.

In addition, once DNA encoding scFv has been prepared, an expression vector containing it and a host transformed with the expression vector can be obtained in a conventional manner, and by using the host, scFv can be obtained in a conventional manner.

These antibody fragments can be produced by a host by obtaining and expressing the genes in the same manner as described above. In the present invention, the "antibody" also includes these antibody fragments.

Furthermore, the neutralizing antibody used in the present invention may be a bispecific antibody. The bispecific antibody may have antigen-binding sites that recognize different epitopes on the molecule, or one antigen-binding site may recognize the secreted IF5A protein and the other antigen-binding site may recognize a labeling substance or the like. A bispecific antibody can be produced by combining the HL pairs of two types of antibodies, or it can be obtained by fusing hybridomas that produce different monoclonal antibodies to produce bispecific antibody-producing fusion cells. Furthermore, it is also possible to produce a bispecific antibody by genetic engineering techniques.

As shown in the Examples below, a neutralizing antibody against secretory eIF5A has an excellent preventive and therapeutic effect against diabetic retinopathy, and the effect is superior to that of an anti-VEGF drug.
Regarding diabetic retinopathy, the neutralizing antibody of the present invention specifically exhibits the effects of suppressing retinal edema and suppressing bleeding in retinal capillaries.
Furthermore, when the neutralizing antibody of the present invention is used in combination with an anti-VEGF drug, the effects of suppressing edema and hemorrhage in diabetic retinopathy are synergistically enhanced. Therefore, the combination therapy of the neutralizing antibody of the present invention with an anti-VEGF drug is useful for treating diabetic retinopathy.
Anti-VEGF drugs include aflibercept, bevacizumab, ranibizumab, pegaptanib sodium, and the like.

The pharmaceutical of the present invention can be formulated by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, or the like, the neutralizing antibody together with a pharma- ceutically acceptable carrier well known in the art, and used in the form of a pharmaceutical composition.

For oral administration, the neutralizing antibodies can be formulated together with pharma- ceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. into dosage forms such as tablets, pills, sugar-coated tablets, soft capsules, hard capsules, solutions, suspensions, emulsions, gels, syrups, slurries, etc.

For parenteral administration, the neutralizing antibody can be formulated with pharma- ceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., into injection solutions, suspensions, emulsions, creams, ointments, inhalants, suppositories, etc. In the formulation for injection, the neutralizing antibody can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. Furthermore, the composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the pharmaceutical composition can be prepared in the form of a powder, and an aqueous solution or suspension can be prepared using sterile water, etc., before use. For administration by inhalation, the neutralizing antibody can be powdered and made into a powder mixture with a suitable base such as lactose or starch. Suppository formulations can be prepared by mixing the neutralizing antibody with a conventional suppository base such as cocoa butter. Furthermore, the pharmaceutical of the present invention can be encapsulated in a polymer matrix, etc., and formulated as a sustained release preparation.

The dosage and frequency of administration will vary depending on the dosage form and route of administration, as well as the patient's symptoms, age, and body weight, but in general, neutralizing antibodies can be administered once or several times a day to achieve a daily dose in the range of about 0.001 mg to 1000 mg per kg of body weight, preferably about 0.01 mg to 10 mg per kg of body weight.

The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto in any way.

Example 1
(Preparation of neutralizing antibodies against secreted eIF5A)
The anti-ORAIP antibody (clone YSP5-45-36) was prepared by binding a peptide consisting of amino acid residues (44-72) of human eIF5A protein (including the 50th lysine residue to be hypusinated and the 69th tyrosine residue to be sulfated) to keyhole limpet hemocyanin (KLH) as an antigen. A hybridoma was prepared by cell fusion of spleen cells of a mouse immunized with this antigen with mouse myeloma cells. From the monoclonal antibodies produced by the hybridoma, an antibody that specifically reacts with the amino acid residues (44-72) of the human eIF5A protein described above was selected by ELISA, and a monoclonal antibody (clone YSP5-45-36) was established. The obtained hybridoma was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under the name NITE P-02955.

Example 2
(Analysis of CDR regions of monoclonal antibodies)
Total cytoplasmic RNA was collected from the hybridoma clone YSP5-45-36.
cDNA was synthesized by RT reaction using primers specific to mouse IgG (H chain, L chain) sequences.
Using the synthesized cDNA as a template, 5' RACE analysis was carried out using SMARTer ^™ RACE 5'/3' Kit (TaKaRa Code Z4859N).
The resulting consensus sequence was analyzed for CDR regions using the analysis tool IMGT ^™ , the international ImMunoGeneTics information system ^™ http://www.imgt.org.
Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and CDR3 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. The heavy chain variable region is shown in SEQ ID NO: 7 and Figure 7, and the light chain variable region is shown in SEQ ID NO: 8 and Figure 8.

Example 3
(Effects on diabetic retinopathy)
In diabetic retinopathy, the disappearance of pericytes that cover the capillary walls is believed to cause a series of vascular abnormalities. In fact, when an inhibitory antibody against platelet-derived growth factor receptor β (PDGFRβ) is administered once intraperitoneally to a one-day-old mouse to eliminate pericytes in the retinal vascular walls, edema and bleeding similar to those in diabetic retinopathy can be reproduced after the eighth day of age (FIG. 1, Non-Patent Document 2). By using this diabetic retinopathy model mouse, the retinal edema and bleeding suppression effect of various drugs, including anti-VEGF drugs, can be verified (FIG. 2). In the present invention, a single anti-PDGFRβ monoclonal antibody was administered intraperitoneally to a one-day-old mouse, and then the neutralizing antibody of the present invention (anti-ORAIP antibody) was administered intraperitoneally every day from the seventh day of age, and it was confirmed that retinal edema and bleeding on the eleventh day of age were significantly suppressed (FIGS. 3 and 4).

Figure 1 shows a diabetic retinopathy model mouse used in the present invention. When a single dose of anti-PDGFRβ monoclonal antibody is administered intraperitoneally to wild-type C57BL/6 mice one day after birth, pericytes in the retinal vascular walls disappear, causing retinal edema and hemorrhage to develop after eight days after birth. In Figure 1, retinal edema and hemorrhage on day 10 after birth are evaluated on a four-point scale.
Grade 1: No retinal hemorrhage or edema Grade 2: Localized hemorrhage or edema in the retina Grade 3: Retinal edema of less than half the circumference Grade 4: Retinal tissue destruction Figure 2 shows the edema and hemorrhage suppression effect of VEGF receptor decoy in diabetic retinopathy model mice. A single dose of anti-PDGFRβ antibody (40 μg) was administered intraperitoneally to 1-day-old wild-type C57BL/6 mice (5 mice), and human immunoglobulin G (IgG) Fc was administered to one eye and VEGF receptor decoy (aflibercept, 5 μg) was administered to the other eye on the 8th day after birth, and analysis was performed on the 11th day after birth.
3 and 4 show the effect of suppressing edema and bleeding in a mouse model of diabetic retinopathy. A single dose of anti-PDGFRβ antibody (40 μg) was administered intraperitoneally to 1-day-old C57BL/6 wild-type mice (11 mice), and from 7 days after birth, mouse IgG was administered intraperitoneally to 5 mice and the neutralizing antibody of the present invention (anti-ORAIP antibody) (100 μg) was administered intraperitoneally to 6 mice every day. Both eyes of each mouse were analyzed on the 11th day after birth.

Example 4
Figure 5 shows the effects of monotherapy with an anti-VEGF drug (Aflibercept) and combination therapy with an anti-VEGF drug (Aflibercept) and an anti-ORAIP antibody on retinal edema and hemorrhage using a diabetic retinopathy model mouse in which pericytes had been lost.

The experimental method is as follows.
(Experimental method using a diabetic retinopathy model mouse in which pericytes were eliminated)
(1) On day 1 after birth, 50 μg of anti-PDGFRβ antibody (clone APB5) was administered intraperitoneally. (2) On day 8 after birth, the mice were divided into three groups and the following solutions (1 μL) were administered intraocularly to each group.
1) PBS
2) Anti-VEGF drug (Aflibercept) 2mg/mL in PBS
3) Anti-VEGF drug (Aflibercept) 2mg/mL
+ Anti-ORAIP antibody (clone YSP5-45-36) 9.614mg/mL in PBS
(3) Determine the grade of retinal edema and hemorrhage on the 13th day after birth.

Figure 5A shows photographs of the retina of each eye in the three groups. Figure 5B shows the grade of retinal hemorrhage and edema in each group. Compared to the PBS-administered group, the proportion of retinas showing lower grades increased in the group administered anti-VEGF drug (Aflibercept) alone. The proportion of retinas showing lower grades further increased in the group administered anti-VEGF drug (Aflibercept) and anti-ORAIP antibody (YSP5-45-36) in combination, compared to the group administered anti-VEGF drug (Aflibercept) alone.
These findings strongly suggest that the combination of anti-ORAIP antibody with anti-VEGF drugs has an additive effect on suppressing edema and bleeding.

Therefore, the severity of Grades 1 to 4 was conveniently scored from 0 to 3 (Grading score), and the effects of monotherapy and combination therapy on PBS were compared, as shown in Figure 7. The grading scores for the PBS, monotherapy, and combination therapy groups were (2.86 ± 0.38 [mean ± SE], n=7), (2.33 ± 0.33, n=9), and (1.89 ± 0.31, n=9), respectively, indicating a tendency for the combination therapy to improve severity compared to the monotherapy.
Furthermore, when the grading scores were statistically compared, no significant decrease in the grading scores was observed between the PBS-administered group and the monotherapy group (*P=0.1864, not significant [NS]), whereas a significant decrease was observed between the PBS-administered group and the combination therapy group (**p=0.0296; Dunnett's multiple comparison test) (Figure 7).
These findings suggest that the combination of anti-ORAIP antibody with anti-VEGF drugs has an additive effect on suppressing retinal edema and hemorrhage.

By using the pharmaceutical or pharmaceutical composition of the present invention, the therapeutic effect of conventional drugs such as anti-VEGF drugs on diabetic macular edema can be enhanced. This disease has a problem of frequent recurrence even when anti-VEGF drugs can be used to treat it, and many patients are refractory to anti-VEGF drugs, so the pharmaceutical of the present invention is extremely useful.

Claims (4)

A preventive or therapeutic drug for diabetic retinopathy, comprising a neutralizing antibody against secretory eIF5A as an active ingredient,
A prophylactic or therapeutic drug, wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. The preventive or therapeutic drug according to claim 1, wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody. The preventive or therapeutic drug according to claim 1 or 2, which is used in combination with an anti-VEGF drug. The preventive or therapeutic drug according to claim 3, wherein the anti-VEGF drug is one or a combination of two or more selected from the group consisting of aflibercet, bevacizumab, ranibizumab, and pegaptanib sodium.