WO1999013909A1

WO1999013909A1 - Corneal neovascularization inhibitors

Info

Publication number: WO1999013909A1
Application number: PCT/JP1998/004114
Authority: WO
Inventors: Shigeru Yatoh; Yasushi Kawakami; Yukichi Okuda; Tadahiko Kozawa; Toshiaki Segawa
Original assignee: Toagosei Co., Ltd.
Priority date: 1997-09-12
Filing date: 1998-09-11
Publication date: 1999-03-25
Also published as: JPH1180024A

Abstract

Practically useful corneal neovascularization inhibitors which are capable of not only inhibiting neovascularization in corneitis or corneal grafting but also preventing corneal clouding or edematization accompanying the same. These inhibitors contain as the active ingredient VEGF/VPF antagonists. The VEGF/VPF antagonists are preferably exemplified by an antibody against VEGF/VPF. These inhibitors are also usable as corneal clouding/edematization inhibitors and promote the take of grafts.

Description

Specification

Corneal angiogenesis inhibitor Technical field

The present invention relates to vascular permeability factor (VPF) [also referred to as vascular endothelial cell growth factor (VEGF), and collectively referred to as “VEGF / VPF” in the present specification]. The present invention relates to an inhibitor, particularly to an agent that suppresses vascularization and corneal opacity and / or edema during corneal transplantation. Background art

Since the eye is said to be the hardest tissue in the body to recognize foreign bodies, human keratomoplasty has a high success rate even without intensive immunosuppressive drugs like kidney transplantation. It is 70%. On the other hand, 80% of the remaining 30% rejected is thought to be able to survive by suppressing rejection using appropriate anti-inflammatory and immunosuppressive agents.

In fact, steroid and non-steroid anti-inflammatory drugs are used to prevent rejection during corneal transplantation. However, both steroid and non-steroid anti-inflammatory drugs have significant problems. In other words, steroid eye drops have a strong anti-inflammatory effect, but have serious side effects. These side effects include exacerbation of infections, induction of infections, delayed wound healing, cataracts, glaucoma, and suppression of adrenal gland system function. When medication is stopped, rebound phenomena may cause exacerbation of inflammation. Non-steroid anti-inflammatory drugs do not have as severe side effects as steroids, but have the disadvantage of being less effective. There are only a limited number of immunosuppressive drugs that can be used to prevent rejection during corneal transplantation, and at present, corneal transplants have weak efficacy even for drugs that can be used for other organ transplants. The use of immunosuppressants that specifically suppress T lymphocyte function, such as cyclosporin A and FK506, may also be effective, but it is difficult to formulate for ophthalmic use because it is poorly soluble in water. There are many issues in development, such as maintaining pharmacokinetics and confirming clinical efficacy.

On the other hand, it is known that allograft rejection during corneal transplantation involves corneal neovascularization (Jpn. J. Ophthalmol., 38 (3), 311-316 (1994)). This suggests that inhibition of angiogenesis after corneal transplantation can suppress graft rejection. Examples of such angiogenesis inhibitors include, for example, prominin (Taylor, S. et al., Nature, 297, 307, 1982), and a combination drug of heparin and cortisone (Folkman, J. et al.). al "Science, 221, 71, 1983), prednisolone 'acetate (Robin, B., Arch. Opthalmol., 103, 284, 1985), sulfated polysaccharide (JP-A-63-119500), and harpimycin A ( JP-A-63-295509), fumagillin (JP-A-1-79828), interferon /? (International Publication WO96 / 29092) and the like are known.

Regarding VEGFATF, it has been suggested that an anti-VEGF VPF antibody may prevent excessive angiogenesis in transplantation of cornea and other tissues (Japanese Patent Application Laid-Open No. Hei 8-520254). However, its practicality is still unknown. Especially in the ophthalmology area, drugs must always be used with the visual function in mind, and it is necessary to confirm that not only vascularization during corneal transplantation is suppressed, but also opacity and edema of the transplanted cornea do not occur. Without it, usefulness or practicality cannot be recognized. Accordingly, the present inventors have developed a practical corneal neovascularization inhibitor that not only suppresses keratitis and vascularization during corneal transplantation but also does not cause corneal opacity or edema. As a result of intensive research for the purpose of providing the present invention, the present invention was completed. Disclosure of the invention

That is, the present invention relates to a corneal angiogenesis inhibitor comprising VEGF / VPF angulinist as an active ingredient. The present inventors conducted a rat corneal transplantation experiment (Invest. Ophthalmol. Visual Sci., 26, 23 (1985)) using an anti-VEGFVPF antibody capable of neutralizing the activity of VEGFVPF, and As a result of finding that not only suppression of angiogenesis but also significant improvement in blood vessel size, edema, clarity, etc. as compared with the control group, the present invention was completed.

In the present invention, the VEGF / VPF antagonist is a substance having a function of inhibiting the function of VEGF / VPF having a specific cell growth promoting activity or a vascular permeability promoting activity on vascular endothelial cells, It may be in any form as long as it has the function, most commonly, an antibody acting on VEGF / VPF, or a part thereof, but is not limited thereto, for example, VEGF Inactive VEGF / VPF that inhibits the action of VGF / VPF, or a part thereof, impairs the function of VEGF / VPF receptor, such as vascular endothelial cell growth factor / vascular permeability factor receptor (eg, FLT or KDR And a part thereof, and an agent that suppresses VEGF / VPF production itself.

VEGF / VPF contains 121, 165, 189 or 206 amino acid residues There are four subtypes (see Masashi Shibuya, Clinical Immunity, 28 (6) 758-764 (1996)), but the antibody may be an antibody against any subgroup. Examples of the antibody include a mouse antibody and the like. However, it is desirable to use an antibody which has been treated to reduce side effects in administration to humans. For example, a mouse monoclonal antibody which has been chemically modified with a substance such as polyethylene glycol to reduce its antigenicity, a mouse-human chimeric antibody or a humanized antibody can be used. Furthermore, antibodies that have been degraded by enzymatic cleavage can also be used. Examples of the chimeric antibody or humanized antibody include IgG type and IgA type, and examples of the IgG isotype include IgGl, IgG2, IgG3 and IgG4. The chimeric antibody or humanized antibody is, for example, an anti-VEGFVPF mouse monoclonal antibody MV833 (neutralizing antibody) that neutralizes the proliferation of human umbilical cord vascular endothelial cells (HUVEC) by VEGFAHPF with the same neutralizing activity as an anti-VEGFVPF ゥ sagi polyclonal antibody. A known conversion technique from mouse monoclonal antibodies to quinula antibodies and humanized antibodies (see, for example, a report on the production of chimeric antibodies: Cancer Res., 47,999 (1987), extra edition of Experimental Medicine). , 6 (10), 965-971 (1988), Production Examples of Humanized Antibodies: see JP-A-62-296890, JP-A-04-502408, etc.).

Note that anti-VEGF / VPF monoclonal antibodies are commercially available from various companies, and any of them can be used in the present invention. Examples of commercially available products include R & D Systems, INC. And Austral Biologicals (USA). Mouse monoclonal antibody sold on the market.

The corneal angiogenesis inhibitor of the present invention is preferably parenterally, for example, instillation, It can be administered systemically or locally by vitreous injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc. Furthermore, the pharmaceutical composition can be in the form of a kit together with at least one pharmaceutical carrier or diluent. The dose of the corneal angiogenesis inhibitor of the present invention to humans varies depending on the disease state, age and administration method of the patient, but it is necessary to select an appropriate amount as appropriate. For example, in the case of systemic administration, 1 ~: L 0 can be administered in the range of mg / kg, per body weight, administered as lg~lm _g / patient in the topical administration, such as eye drops. However, the corneal angiogenesis inhibitor of the present invention is not limited to these doses.

The corneal angiogenesis inhibitor of the present invention can be formulated according to a conventional method. For example, injectable preparations are prepared by dissolving purified VEGF / VPF antagonists in a solvent such as physiological saline or buffer, and adding an anti-adsorption agent such as Tween80, gelatin, or human serum albumin (HSA). ) Or lyophilized for reconstitution before use. As an excipient for freeze-drying, for example, sugar alcohols such as mannitol and glucose and sugars can be used. Similarly, it can be used in the form of an ophthalmic solution, an ointment ointment, an emulsifier, or the like, or can be used as a lipo-microsphere to prolong the retention.

According to the present invention, by suppressing the action of VEGF / VPF, which induces angiogenesis by the action of VEGF / VPF antagonist, the angiogenesis accompanying corneal transplantation is suppressed, and the occurrence of opacity and edema of the transplanted cornea is suppressed. It is considered to suppress the activity and promote engraftment without causing rejection of the transplanted cornea, maintain transparency, and contribute to the healing of the transplanted cornea. In addition, since the animal experiment model in the following examples is considered to be a keratitis model, the VEGF / VPF angel It is contemplated that it could also be used to treat angiogenesis due to meningitis. Diseases that cause such keratitis include Stevens-Johnson syndrome, diseases related to Stevens-Johnson syndrome, pemphigus ophthalmopathy, diseases related to ocular pemphigus, corneal erosion (alkali, acid, surfactant, These include various solvents, volatile gases, and various other cytotoxic agents), long-term contact lens wear, keratitis due to viral, bacterial, or fungal infections, keratitis due to foreign substances, keratitis due to allergies, etc. No. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the clarity of the graft when an anti-VEGF / VPF antibody was instilled into a corneal transplant model rat. FIG. 2 is a graph showing a range of new blood vessels when an anti-VEGF / VPF antibody was applied to a corneal transplant model rat. FIG. 3 is a graph showing the size of new blood vessels when an anti-VEGF / VPF antibody was instilled into a corneal transplant model rat. FIG. 4 is a graph showing the occurrence of edema in a graft when an anti-VEGF / VPF antibody was instilled into a corneal transplant model mouse. FIG. 5 is a graph showing the determination of the range of neovascularization by an ophthalmologist on day 14 after instilling an anti-VEGF / VPF antibody into a corneal transplant model mouse. FIG. 6 is a graph showing the determination of neovascular size by an ophthalmologist on day 14 after instilling anti-VEGF / VPF antibody into a horn transplant model rat. FIG. 7 is a graph showing the determination of the occurrence of edema by an ophthalmologist on the 14th day after instilling an anti-VEGF / VPF antibody into a corneal transplant model rat. FIG. 8 is a photomicrograph showing the state of the transplanted cornea on day 19 after instilling the egret immunoglobulin fraction into a corneal transplant model rat. FIG. 9 is a microscopic photograph showing the condition of the transplanted cornea on day 19 when an anti-VEGF VPF antibody was applied to a corneal transplant model rat. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described with reference to Reference Examples and Examples, but the present invention is not limited to these. Reference Example 1 (Preparation of GST-VEGF / VPF and preparation of anti-VEGF / VPF ゥ Sagi polyclonal antibody) VEGF / VPF cDNA isolated from cultured cells of human origin was purified by glutathione-S-transferase (GST). Was integrated into Escherichia coli, expressed as a fusion protein (GST-VEGF / VPF), and the resulting protein was used as an antigen to immunize egrets in a conventional manner. The IgG fraction was fractionated from the serum with increased antibody titer by DEAE chromatography. Since the resulting antibody to confirm that react with active VEGF / VPF incorporate VEGF _I21 gene, the human uterine cancer cells HeLa / v5 (Fermentation Research Institute began to VEGF is highly expressed Deposit: Deposit the protein from the medium in which the accession number “Denken No. 13185 ·” has been cultured by ammonium sulfate precipitation, develop it by 12% SDS-PAGE, transfer the protein from the gel to a nylon membrane, The antibody obtained by the immunization was reacted and a Western blot was performed. Specific cross-reactivity was observed at a position corresponding to the molecular weight of VEGF / VPF, confirming that the prepared antibody recognized active VEGF / VPF molecules. Example 1 (Evaluation of anti-VEGF / VPF antibody administration effect on corneal transplanted rats)

(1) Preparation of corneal transplant model rat

Lewis rats (male, 8 weeks old, Charles River Japan) were removed under Nembutal anesthesia, the cornea of the left eye was removed with a trepan of 3 mm in diameter, and Fisher rats (male, 8 weeks old, Charles River Japan) were removed. The cornea extracted from 1) with a trepan having a diameter of 3 mm was implanted by suturing it at four places with a 10-0 lip.

(2) Prevention of rejection reaction by anti-VEGF / VPF antibody

The corneal transplanted rats were divided into three groups A to C by 10 rats. In group C, the anti-VEGF / VPF ゥ sagi polyclonal antibody prepared using the VEGF 121-GST (glutathione-S-transferase) fusion protein as the antigen in Reference Example 1 was used 5 times a day (5, 9, 13, and 17). , 22 o'clock) I instilled 10 1 for 10 days. This antibody was used after diluting the immunoglobulin (IgG) fraction obtained in Reference Example 1 with a protein concentration of 14 mg / ml to 10-fold with physiological saline. Group B was prepared by diluting the egret immunoglobulin fraction (DAKO Japan, 20 mg / ml) 10-fold with physiological saline, and then, as with the anti-VEGF VPF antibody, 10〃1 / time, 5 times a day, It was instilled for 10 days. Group A was performed using physiological saline in the same manner as the anti-VEGF / VPF antibody. '

(3) Evaluation of rejection prevention effect

The evaluation was based on the clarity of the transplanted cornea, the extent of angiogenesis and the size of the blood vessel, and the degree of edema.On the day of the transplant operation, 1, 2, 4, 6, 8, 11, 1, 12, 14, and 19 The eyes were photographed with a stereomicroscope and examined. Furthermore, on the 14th day of surgery, without informing the treatment group, the ophthalmologist was asked to randomly evaluate the extent of neovascularization, blood vessel size, and the degree of edema under a stereoscopic microscope. The evaluation criteria for each evaluation are as follows: • Graft clarity

0: completely transparent

1: Faint haze, but iris and pupil are easily visible 2: opaque but iris and pupil are visible

3: Opaque and iris and pupil cannot be easily seen

4: Completely opaque and no pupil visible

• Range of neovascularization by ophthalmologist

The area of neovessels on the cornea under a microscope was evaluated by percentage.

0: No blood vessel at the edge of the graft

1: There is a blood vessel at the edge of the graft

2: 25% of the distance from the edge to the center of the graft has blood vessels

3: Vessel 50% of distance from graft edge to center

4: There is a blood vessel from the edge to the center of the graft

• Neovascular size

0: No blood vessels

1: There are blood vessels that can be seen under a microscope

2: There are blood vessels that can be easily observed under a microscope

3: There are blood vessels that can be easily seen without a microscope • Degree of edema of the graft

0: No edema

1: There is edema but light

2: Severe edema protruding at the edge From Figure 1, the clarity of group C (anti-VEGFVPF antibody-administered group) is more significant than that of group B (ゥ egan IgG-administered group) from day 4 to day 14. 4 to 12, it was shown to be significantly better than Group A (saline administration group). FIG. 2 shows that the range of angiogenesis in group C was significantly smaller than groups B and A on days 6-19.

From FIG. 3, it was shown that the thickness of the new blood vessels in the group C was significantly smaller than that in the groups B and A, according to the statement from 6 to 19.

FIG. 4 shows that the edema of the group C was significantly suppressed on the 6th to 19th days compared to the groups B and A.

In addition, Fig. 5 shows that the range of neovascularization in group C was 14 days, and that the range of neovascularization in group C was significantly narrower in group B and group A, as determined by the ophthalmologist. . From FIG. 6, the ophthalmologist determined the size of the new blood vessel, and as a result, the thickness of the new blood vessel in the group C was significantly smaller than that in the groups および and で in 14 words. As shown in FIG. 7, the ophthalmologist determined that edema was present. The edema in group C was significantly more suppressed on day 14 than in groups B and A.

Further, on the 19th day after the transplantation, the rat was sacrificed, and the cornea-transplanted eyes were removed. After fixing the extracted eyeball, a section was prepared and subjected to pass staining. The results are shown in the photographs of FIGS. The photograph in Fig. 8 shows the Egret immunoglobulin fraction. The instillation and the photograph in Fig. 9 show the instillation of the anti-VEGF ゥ sagi polyclonal antibody. It can be seen in Fig. 9 that the swelling of the transplanted cornea is clearly suppressed. From the above, the anti-VEGF / VPF antibody has a function of neutralizing the action of VEGF / VPF, and during administration, it significantly suppresses angiogenesis occurring in the transplanted corneal slices, and at the same time, It was shown that corneal transplant rejection was suppressed by keeping the clarity of the sera good and preventing the occurrence of edema. Industrial applicability

According to the present invention, the administration of VEGF / VPF antagonists suppresses angiogenesis of a transplanted corneal piece, maintains the clarity of the graft well, and prevents the occurrence of edema. It can prevent corn transplantation rejection, and can also be used as a therapeutic agent for keratitis, and as a turbidity preventer or edema preventer for transplants. The agent of the present invention can be used in combination with an immunosuppressant, an anti-inflammatory agent, an anti-cytokine agent and the like in order to further improve the angiogenesis inhibitory effect.

Claims

The scope of the claims

1. A corneal angiogenesis inhibitor containing VEGF / VPF angiogonist as an active ingredient.

2. The corneal neovascularization inhibitor according to claim 1, which is used as a corneal transplant rejection inhibitor.

3. The corneal neovascularization inhibitor according to claim 1, which is used as a therapeutic agent for keratitis.

4. The corneal angiogenesis inhibitor according to claim 1, wherein the VEGF / VPF antagonist is an antibody against VEGF / VPF.