JPH10273450A - Therapeutic agent for intraocular neovascular disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare the subject agent useful for treating especially retinopathy in immature babies by including a vascular permeability factor antagonist therein. SOLUTION: This therapeutic agent for intraocular neovascular diseases, e.g. a pharmaceutical preparation for injection is obtained by dissolving a refined vascular permeability factor [hereinafter referred to as a vascular endothelial growth factor (VEGF)/vascular permeability factor(VPF) antagonist (preferably an antibody against the VEGF/VPF, especially the one prepared by carrying out the chemical modification and reducing the antigenicity for administration to humans or a murine human chimeric antibody, a humanized antibody, etc.) in a solvent (a physiological saline, etc.), then adding an adsorption inhibitor [Tween 80 (R) or gelatin], etc., to the obtained solution and freeze-drying the resultant mixture with an excipient so as to dissolve and reconstitute the preparation before use. Thereby, the objective agent formulated for administration into a vitreous body is preferably obtained. The dose of the agent is within the range of 1-100 mg/kg for systemic administration and 0.01-10 mg/patient for local administration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a vascular permeability factor (V
PF) [also called vascular endothelial cell growth factor (VEGF),
In this specification, they are collectively referred to as "VEGF / VPF".] The present invention relates to a therapeutic agent for an intraocular neovascular disease, which comprises an antagonist as an active ingredient.

[0002]

2. Description of the Related Art Syndromes whose basic condition is abnormal proliferation of capillaries are collectively referred to as angiogenic diseases, and intraocular angiogenic diseases include diabetic retinopathy, neovascular glaucoma,
Senile discoid macular degeneration, retinopathy of prematurity, retinal vein occlusion, neovascular maculopathy, and Eales disease.

[0003] In the case of the eyeball, blood vessels do not exist in the cornea, eye chamber, lens, and vitreous through which light passes, and these are naturally transparent tissues, and angiogenesis occurs under physiological conditions in the eye. Absent. This is considered to be due to the fact that the angiogenesis inhibitory factor is superior in the balance between the angiogenesis promoting factor and the inhibitory factor in the tissue without blood vessels as described above.

However, when diabetic retinopathy causes retinal ischemia due to disruption of the blood-retinal barrier or occlusion of blood vessels, the balance between the above factors is disrupted, and the angiogenesis promoting factor becomes dominant, and the neovascularization by the proliferation of vascular endothelial cells occurs. Proliferation of blood vessels and other cells occurs, resulting in vitreous hemorrhage, traction retinal detachment, neovascular glaucoma, etc., resulting in significant visual impairment.

[0005] Retinal cell growth factor (RDGF), vascular endothelial cell growth factor (VEGF), lactic acid,
β-N-acetylglucosaminidase (β-N-acetyl
glucosaminidase) and hyaluronic acid degradation products are known. It has already been proposed as an idea to administer a VEGF antagonist for the treatment of diabetic retinopathy, neovascular glaucoma, and senile disk-shaped macular degeneration (Japanese Patent Application Laid-Open No. 8-502514 and International Publication WO96 / 96). No. 30046).

On the other hand, retinopathy of prematurity is a proliferative disease that occurs in developing retinal blood vessels. It is around the age of 40 weeks that the retinal blood vessels reach the outermost part of the fundus and complete the growth, and the retinal blood vessels of the premature infant born before this are often in the developing stage. As a result, a sudden change in the environment due to childbirth damages the capillary bed at the tip of the growth of retinal vessels where the most immature cells are present, and these vessels then regrow or proliferate in abnormal directions. Initially, it will be observed as fundovascular growth on fundus examination. Then, the number of the blood vessels increases more than normal, and a thick tissue is formed at the border between the vascular region and the avascular region, and if severe, progresses to retinal detachment.

[0007] Regarding retinopathy of prematurity, there are many unclear points such as the mechanism of injury to immature blood vessels, but it is a general hypothesis that angiogenic factors are involved.
VEGF / VPF expression in the retina of a neonatal mouse with hyperoxia retinopathy that caused vascular proliferative lesions similar to retinopathy of human prematurity due to high oxygen load was examined by Northern blotting.The VEGF / VPF mRNA level of the retina was During the course of tissue ischemia and angiogenesis, a three-fold increase was observed.
As a result of in situ hybridization and immunohistochemical staining, it has been reported that VEGF / VPF was expressed in the inner nuclear layer (Pierce EA, et al. Proc Natl Acad Sci USA 92 (3)
905-909 (1995)). In addition, as a result of examining whether or not these newborn mice with hyperoxia retinopathy can be administered with a soluble VEGF / VPF receptor chimeric protein or an antisense oligonucleotide against VEGF / VPF to prevent the angiogenic lesion, It has been reported that body chimeric proteins and antisense oligonucleotides significantly reduced the number of neovascular cell nuclei invading the vitreous beyond the inner boundary layer by 47% and 31%, respectively (Aiello LP, et al.). Proc Natl Acad Sci USA. 92: 104
57-61 (1995), and Robinson GS, et al. Proc Natl A
cad Sci USA. 93: 4851-6 (1996)).

As an ophthalmic treatment using an anti-VEGF / VPF antibody, recently, administration of an anti-VEGF / VPF antibody to a retinal neovascularization model in which a monkey's retinal vein is occluded by photocoagulation significantly improves iris rubeosis. (Adamis AP, et al. Arch Ophthalmol. 114: 66
-71 (1996).).

[0009] However, administration of anti-VEGF / VPF antibodies for the treatment of intraocular neovascular diseases has not been studied yet, and there are no clinically satisfactory drugs. In particular, in ophthalmology, drugs must always be used with visual function in mind, suppressing angiogenesis, but affecting other eye functions, and consequently drugs that reduce visual function. I can not use it. Therefore, drugs that specifically suppress angiogenesis, such as eye drops and vitreous injection, are desired.

[0010]

SUMMARY OF THE INVENTION
The present inventors have conducted intensive studies for the purpose of providing a therapeutic agent useful for the treatment of intraocular neovascular diseases, particularly, retinopathy of prematurity and suitable for topical administration, and as a result, completed the present invention. It was.

[0011]

That is, the present invention relates to
The present invention relates to a therapeutic agent for an intraocular neovascular disease containing an EGF / VPF antagonist as an active ingredient.

In the present invention, VEGF / VPF antagonist refers to VEGF / V having a specific cell growth promoting activity on vascular endothelial cells or a vascular permeability promoting activity.
Refers to a substance having a function of inhibiting the function of the PF, and may have any form as long as it has the function.
Most commonly, an antibody that acts on VEGF / VPF, or a portion thereof, including, but not limited to, an inactive VEGF / VPF that inhibits the action of VEGF / VPF, or a portion thereof. , VEGF
/ VPF, which impairs the function of the receptor, for example, an antibody against a vascular permeability factor receptor, or a part thereof, and an agent that suppresses VEGF / VPF production itself.

VEGF / VPF contains 1 amino acid residue.
There are four subtypes, 21, 165, 189, or 206 (Masashi Shibuya, Clinical Immunity, 28 (6) 758).
-764 (1996)), and the antibody may be an antibody against any subtype. Examples of the antibody include a mouse antibody and the like, and it is preferable to use an antibody which has been treated to reduce side effects upon administration to humans. For example, a mouse monoclonal antibody that has been chemically modified with a substance such as polyethylene glycol to reduce antigenicity, a mouse / human chimeric antibody, a humanized antibody, or the like can be used. Furthermore, antibodies that are obtained by enzymatically cleaving them to reduce the molecular weight can also be used. As the chimeric antibody or humanized antibody,
IgG type or IgA type and the like.
Examples of G isotypes include IgG1, IgG2, and IgG.
3 or IgG4.

The therapeutic agent for an intraocular neovascular disease of the present invention is preferably administered parenterally, for example, systemically or locally by intravitreal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection or the like. Can be administered. In addition, it can take the form of a kit of pharmaceutical compositions together with at least one pharmaceutical carrier or diluent. The dose of the therapeutic agent for an intraocular angiogenic disease of the present invention to a human varies depending on the condition, 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, it can be administered in the range of 1 to 100 mg / kg,
For local administration, such as vitreous injection, it is administered as 0.01 to 10 mg / patient. However, the dosage of the therapeutic agent for an intraocular neovascular disease of the present invention is not limited to these dosages.

[0015] The therapeutic agent for an intraocular neovascular disease of the present invention can be formulated according to a conventional method. For example, an injectable formulation may be obtained by dissolving a purified VEGF / VPF antagonist in a solvent, for example, saline, a buffer, or the like,
It may also contain an adsorption inhibitor, such as Tween 80, gelatin, human serum albumin (HSA), or may be lyophilized for reconstitution before use. As an excipient for lyophilization, for example, sugar alcohols such as mannitol and glucose and sugars can be used.

According to the present invention, VEGF / VPF induces angiogenesis by the action of a VEGF / VPF antagonist.
By suppressing the action of VPF and suppressing intraocular neovascularization, it is considered that the action of treating intraocular neovascular diseases is exhibited.

[0017]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 (Preparation of GST-VEGF / VPF and Preparation of Anti-VEGF / VPF Rabbit Polyclonal Antibody) Human cervical cancer cell HeLa / v5 (deposited with the Research Institute of Microorganisms: Deposit No. No. 13185 ”), cDNA of VEGF / VPF isolated from Escherichia coli was expressed as a fusion protein with glutathione S-transferase (GST), and the obtained protein was used as an antigen to immunize rabbits in a conventional manner. IgG was fractionated from the serum with the increased antibody titer by DEAE chromatography.

In order to confirm that the obtained antibody reacts with active VEGF / VPF, a protein was taken out from a medium in which HeLa / v5 was cultured by salting out with ammonium sulfate, and developed by 12% SDS-PAGE, followed by gelation. Was transferred to a nylon membrane, and the antibody obtained by the above immunization was reacted and subjected to Western blot. VEGF / V
Specific cross-reaction was observed at a position corresponding to the molecular weight of PF, confirming that the prepared antibody recognized active VEGF / VPF molecules.

Example 1 (Evaluation of Anti-VEGF / VPF Antibody Administration Effect on Intraocular Neovascular Disease Model)

(1) Preparation of Newborn Mouse with Hyperoxia Retinopathy Hyperoxia retinopathy of neonatal rats / mouse is obtained by rearing newborn rats / mouse under 75-100% oxygen high oxygen condition. After occlusion of the polar retinal vessels, the rat /
A vascular proliferative lesion with angiogenesis similar to retinopathy of prematurity induced by hypoxia in the retinal non-perfused area due to the vascular occlusion by rapidly returning the mouse to the atmosphere of normal oxygen concentration. Widely used as a model for endovascular diseases (Ricci B, et al. Pediatrics. 82: 1
93-8 (1988), Reynaud X, et al. Invest Ophthalmol Vis
Sci. 35: 3169-77 (1994), and Smith LE, et al. Inv.
est Ophthalmol Vis Sci. 35: 101-11 (1994).).

The method of Smith et al. (Smith et al. (1994) supra)
Invest Ophthalmol Vis Sci. 35: 101-11.)
Neonatal mice were bred under high oxygen conditions to induce angiogenic retinal lesions.

First, the pregnancy C57B1 / whose plug was confirmed on the same day
Purchase 6 mice (Charles River Japan, Yokohama) 6 belly,
They were bred for one week before parturition and acclimated to the environment. After childbirth, the pups of each parent mouse are exchanged with each other, and each parent is pre-adjusted so that the proportions of breeding parents of the pups to be bred are equal,
In addition, all parental mice were alternately rotated once every two days so that the lactation conditions of all offspring mice were as equal as possible. On day 7 after birth, 30 randomly selected pups among the pups were arbitrarily divided into three groups, each group comprising 10 mice, which were designated as group A, group B and group C. Group A was raised as a control group under atmospheric pressure and normal air throughout the course of the experiment. For groups B and C, retinal neovascular lesions were induced by breeding under 75% oxygen for 5 days from the 7th day after birth. That is, the inside of a 50 cm square tempered glass sealed case equipped with a carbon dioxide absorption device and connected to an oxygen cylinder was maintained at 1 atm by an oxygen concentration detector (OV-621, RIKEN KEIKI, Tokyo) to maintain the oxygen concentration at 75 atm. Adjust to ± 0.7vol.%,
The pups of the groups B and C were placed together with three parent mice. In consideration of the burden on the respiratory system of the parent mouse caused by inhalation of high oxygen, the three parent mice were changed every three hours with the three mice kept under air. Rearing room temperature is 22 ° C, free feeding,
It was drinking water and was turned on and off every 12 hours.

(2) Treatment with Anti-VEGF / VPF Antibody From the end of the high oxygen exposure (ie, 12 days after birth), all the pups were returned to normal air and bred for another 5 days (ie, up to 17 days after birth). Continued. For the mice of group B, the anti-human VEGF / VPF121 rabbit polyclonal antibody obtained in Reference Example 1 was used once a day for 100 days at a time for 5 days (that is, from day 13 to day 17 after birth).
(Diluted in saline to adjust the volume to 0.1 ml)
Per mouse / subcutaneous injection. For the pups of group C, the same anti-VEGF / VPF was injected into the right eye vitreous only once two days after the end of the high oxygen exposure (ie, 14 days after birth).
1 μl of the antibody (20 μg / μl) was administered. For intravitreal administration, pups should be anesthetized with ether and
The eyelid was slightly incised with the blade of a No. 1 scalpel and injected using a 31-gauge needle (custom-made) and a syringe (1 μl) for HPLC sample injection (both from Hamilton, UK). Liquid, Daiichi Pharmaceutical, Tokyo). The dose of anti-VEGF / VPF antibody in this experiment was determined by a previous study (Kondo, et al. Biochem Bioph.) Which showed that the antibody suppressed the growth of tumors transplanted into rats.
ys Res Commun 194 (3) 1234-1241 (1993)), and was determined based on the dose that exerted the tumor growth inhibitory effect.

As for the control of these administration methods, the relationship between the number of mice that can be accommodated in the glass case of the high oxygen load device at one time and the Association for Research, which prohibits simultaneous treatment on both eyes of experimental animals, was prohibited. in
Vision and Ophthalmology Statement on the Use of A
Another preliminary experiment was performed due to limitations of nimals in Ophthalmic and Vision Research. That is, physiological saline (0.
1ml) or normal rabbit IgG (DAKO X903, USA) 100μ
g / mouse (diluted with physiological saline to 0.1 ml) was injected subcutaneously according to the same schedule as above, and 1 μl of physiological saline or normal rabbit IgG (20
1 μl (μg / μl) was also administered intravitreally on the same schedule as above. It was previously confirmed that these treatments did not affect the formation of retinal neovascular lesions in this model.

(3) Treatment of eye specimen and evaluation of angiogenesis On day 17 after birth, all offspring mice in groups A, B and C were
After anesthesia by pentobarbital intraperitoneal overdose, both eyes were excised. The excised eyes were fixed with 4% paraformaldehyde 3% saccharose 0.1M PBS (pH 7.4) at 4 ° C. for 24 hours.

The preparation of the specimen and the method of evaluating the new blood vessels are described in Smit.
h et al. (Smith et al. (1994) Invest Ophthalmol Vis, supra)
Sci. 35: 101-11.). That is, after dehydration and paraffin embedding, a continuous section of 6 μm in thickness was obtained from the entire eye along the sagittal section from the ear pole to the nasal pole. Of these, each eye has a distance of 60 μm, centered on the optic disc.
Eight intact sections at m intervals were evaluated (ie,
Evaluated over a width of 468 μm).

This was stained with PAS-hematoxylin and examined microscopically, and the total number of neovascular cell nuclei invading the vitreous body beyond the inner boundary layer was counted and averaged for eight sections.
The number of nuclei per section of each eye was shown, and the measured values of each group are shown in FIG. The measured values are shown as the mean ± standard deviation. For statistical analysis, Mann-Whitney U test and one-way ANOVA, Fis
P <0.05 was considered significant using cher's PLSD.

As a result, no neovascularization was observed in the retinas of Group A pups bred under normal oxygen concentration, and the number of neovascular cell nuclei crossing the inner limiting membrane was 4.3 ± 0.6 as shown in FIG. There were very few pieces / section. On the other hand, of the pups of the B and C groups exposed to hyperoxia, the left eye of the pups of the C group left untreated showed marked retinal neovascularization, and the vitreous body crossed the inner limiting membrane. As shown in FIG. 1, the number of neovascular cell nuclei that had invaded into the cells was 86.7 ± 17.3 / section.

In comparison, in the right eye of group C mice to which the anti-VEGF / VPF antibody was administered intravitreally, the number of neovascular cell nuclei was remarkably 41.9 ± 22.3 / section as shown in FIG. Significant decrease was observed (P <0.01). In the mice of group B to which the anti-VEGF / VPF antibody was subcutaneously administered, the number of the neovascular cells nuclei crossing the inner limiting membrane was 71.1 ± 10.5 / section, and the control C
Although a significant (P <0.05) decrease was observed compared to the left eye of the group (normal IgG administration side), the decrease was significantly greater in the right eye of Group C administered intravitreally.

When the left and right eyes of each individual in Group C were compared, in most individuals, the right eye to which the anti-VEGF / VPF antibody was administered intravitreally had a higher intraretinal content than the control left eye. A similar degree of neovascular growth was observed, but the number of neovascular cell nuclei invading the vitreous beyond the inner limiting membrane was:
As is clear from FIG. 2, the number was considerably small.

From the above, when the anti-VEGF / VPF antibody administration side was compared with the untreated control side, the same degree of neovascular growth was observed on both sides in the retina.
Antibody administration was clearly shown to prevent neovascular invasion into the vitreous. This indicates that the anti-VEGF / VPF antibody injected into the vitreous can prevent the invasion of new blood vessels in the retina into the vitreous cavity.
Fig. 4 shows the efficacy of treatment with an PF antibody for intraocular neovascular diseases such as retinopathy of prematurity.

The subcutaneous systemic administration did not reach the effect of a single intravitreal administration despite the daily administration of a high dose of anti-VEGF / VPF antibody to which an antitumor effect could be expected. This is because 1) the antibody concentration in the local area of the eye was not sufficiently increased by systemic administration, and 2) the antibody was metabolized and decomposed by subcutaneous administration, whereas it remained in the vitreous without being decomposed for a relatively long time. 3) The presence of the retinal blood barrier may make it difficult for antibodies present in blood to migrate to intraocular tissues. In addition to the above reasons, VEGF / VPF is likely to play an important role in early growth,
In order to avoid the possibility of side effects due to systemic administration of anti-VEGF / VPF antibodies, local administration is preferable for clinical application to retinopathy of prematurity.

In this example, a heterologous antibody was used for the mouse, which is a rabbit antibody against human VEGF / VPF.
Since the species difference of GF / VPF was extremely small, angiogenesis could be suppressed by suppressing the activity of VEGF / VPF in mice. When such a heterologous antibody is administered to an animal to be treated,
Antibodies to foreign body reactions and administered heterologous antibodies are produced,
Problems such as an anaphylactic reaction and a diminished effect of the administered antibody are also expected during repeated administration. In this regard, those skilled in the art will be able to chemically modify the antibody,
It can be easily improved by reconstituting a mouse / human chimeric antibody or a humanized antibody having reduced immunogenicity of the antibody itself without changing the specificity or binding ability to the antigen, and administering the antibody. Would. Alternatively, a mouse / human chimeric antibody or a humanized antibody may be cleaved with an enzyme or the like to reduce the molecular weight, for example, F (ab) ₂ or F (ab).

The above administration route of vitreous puncture injection is considered to be advantageous as a combination therapy during vitreous surgery.

[0036]

According to the present invention, administration of a VEGF / VPF antagonist suppresses intraocular neovascularization, and in particular, prevents retinopathy of prematurity by inhibiting the invasion of neovessels formed in the retina into the vitreous cavity. And other intraocular neovascular diseases.

[Brief description of the drawings]

FIG. 1. Anti-VEGF against an intraocular neovascular disease model mouse
Graph showing the inhibitory effect of the number of neovascular cell nuclei invading the vitreous body when the / VPF antibody is administered subcutaneously or intravitreally (N
= 10, mean ± standard deviation).

FIG. 2 is a graph showing the effect of intravitreal administration of an anti-VEGF / VPF antibody on retinal neovascular lesions for each mouse model of intraocular neovascular disease (N = 10).

Claims (4)

[Claims] 1. A therapeutic agent for an intraocular neovascular disease comprising a VEGF / VPF antagonist as an active ingredient. 2. The therapeutic agent according to claim 1, wherein the intraocular neovascular disease is retinopathy of prematurity. 3. The method of claim 1, wherein the VEGF / VPF antagonist is VE.
3. The therapeutic agent according to claim 1, which is an antibody against GF / VPF. 4. The method of claim 1, wherein the composition is for intravitreal administration.
Or the therapeutic agent of claim 2.