JPH0789859A - Pharmaceutic composition containing pyrazine derivative and curing method for inhibiting neogenesis of new blood vessel - Google Patents

Abstract

PURPOSE: To provide a method and composition for preventing and/or treating ocular neovascularization (including neovascularization of cornea, iris, retina and choroid). CONSTITUTION: One kind of compound of pyrazine derivatives, e.g. amiloride or its derivative or homologue or one kind of their pharmaceutically permissible salts and/or solvated materials is administered to patients. The pyrazine derivative can topically be administered to ocular tissue with a proper carrier such as artificial tear carrier or petrolatum base ointment. The pyrazine derivative can be administered by oral administration, intravenous injection or intraocular implant.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the field of treatment of ocular tissue, and more particularly to a method of treating ocular tissue to prevent neovascularization. In particular, the present invention relates to corneal neovascularization,
Prevention of iris neovascularization, retinal neovascularization and choroidal neovascularization.

[0002]

BACKGROUND OF THE INVENTION Under certain pharmacological conditions of the eye, and as a result of certain eye treatments or as a result of traumatic injury to the eye, angiogenesis within the ocular tissue often occurs. Such angiogenesis is a neovascular phenomenon.
Known as ization), neovascularization is often an unwanted and unwanted phenomenon.

One example of unfavorable neovascularization of the eye is neovascularization of corneal tissue. Corneal neovascularization can occur after inflammatory or traumatic injury to the cornea. The main cause of this is corneal transplantation. However, corneal neovascularization also occurs after herpes keratitis, a fairly common corneal infection. In addition, corneal neovascularization occurs after corneal trauma, such as that which occurs after a car accident, exercise trauma, etc., which can lead to corneal fogging and reduced visual acuity.

Further, regarding corneal transplantation, graft rejection often occurs when corneal neovascularization occurs after corneal transplantation. Generally speaking, corneal grafts are generally considered to be less subject to patient rejection, as there is usually no blood supply to the corneal tissue. Therefore, ophthalmologists are in a unique position to not have to perform histocompatibility prior to performing corneal transplants. However, once corneal neovascularization occurs, histocompatibility becomes unavoidable to avoid tissue rejection, and the process of corneal transplantation becomes very complicated, especially if tissue rejection subsequently occurs. If the cornea is rejected, another graft is needed, increasing the risk of later rejection. This problematic situation is particularly important in the case of corneal transplants, as the recovery time after corneal transplantation to obtain useful vision is often as long as 6-8 months.

In the case of corneal neovascularization without the risk of graft rejection, ie after trauma or inflammation, corneal blood vessels induce local scarring and tissue edema, usually causing clouding of the clear cornea. This fog significantly reduces vision.

Although the data are not very accurate, it is estimated that approximately 36,000 people in the United States are at risk of developing corneal neovascularization each year, estimated by the inner circle. This number is predicted from the number of corneal transplants performed per year. In this way, about 36,000 people are at or at risk of loss of vision each year, and due to corneal neovascularization, they lack the ability to perform their work properly. Therefore, corneal neovascularization is considered both a serious medical problem and a serious economic problem.

Another case of unwanted neovascularization of the eye is neovascularization of retinal tissue. Neovascularization from the lining of the retina occurs after diabetic retinopathy, retinal branch vein occlusion, central retinal vein, sickle cell retinopathy, retinopathy of prematurity, and many other types of retinopathy. Retinal neovascularization has been a problem for quite some time. Since the discovery of insulin in the 1940s, the lifespan of diabetic patients has increased significantly and the number of diabetic retinopathy diagnoses has escalated as the incidence of diabetic retinopathy is directly proportional to the duration of diabetes.
Most diabetics have some form of diabetic retinopathy while they have been diabetics for 10-15 years, and almost all diabetics have impaired vision within 20 years. It suffers from significant diabetic retinopathy. There are approximately 50,000 new cases of diabetic retinopathy each year in the United States.

Retinal neovascularization is often accompanied by iris retinopathy, resulting in untreatable glaucoma.

On the contrary, neovascularization, which is generally called choroidal neovascularization, originating from the subretinal choroid occurs in various disease groups. These include age-dependent macular degeneration, histoplasmosis, various forms of eye trauma and many chorioretinal disorders. Chorioretinopathy results in varying degrees of visual loss and can also result in blindness.
Approximately 200,000 years of age-dependent macular degeneration in the United States
There are 0 new cases.

Furthermore, neovascularization from the subretinal choroid occurring during age-dependent macular degeneration and neovascularization from the retinal surface occurring during diabetic retinopathy are major causes of blindness in the workplace. Therefore, it is desirable to provide an ocular treatment that prevents neovascularization that occurs in retinal and choroidal tissue.

The only currently available therapies for the treatment of corneal neovascularization are anti-inflammatory agents, most commonly steroids, to reduce inflammatory factors, including neovascularization responses.
Is to treat the cornea.

As far as the treatment of retinal neovascularization is concerned, pan retinal photocoagulation
aggregation) is used. In this treatment, the laser creates a choroidal retinal scar in a scatter pattern around the retina. Scar tissue begins to block neovascularization. In particular, it has been found that the cells and substances within the scar tissue contain a myriad of pigment epithelium cells, and pigment epithelial cell-derived protease inhibitors (PEPI)
Is important for the inhibition of the potent protease urokinase-like plasminogen activator or uPA. This treatment appears to be effective in most cases, reducing the risk of blindness by about 1/2. However, there is a definite desire to provide treatments that prevent retinal neovascularization that are effective in more than half of the cases.

An unfavorable side effect of retinal scar formation is the detrimental effect on the patient's peripheral and night vision. It would certainly be desirable to provide a treatment for retinal neovascularization that avoids the loss of peripheral and night vision in the patient.

With respect to choroidal neovascularization, this condition is treated with direct laser therapy, which erases new blood vessels based on the choroid. Unfortunately, this treatment is best practiced when the blood vessels are outside the avascular fossa area. 19
A study conducted in the 1980s found that only 20% of patients with choroidal neovascularization after age-dependent macular degeneration had lesions outside the avascular fossa area. Direct laser treatment is believed to result in reduced vision because treatment of the avascular fossa region destroys the cones within the fossa in this region. Therefore,
Only 20% of all patients were eligible for direct laser treatment. About 50% of these treatable patients are helped by laser treatment for the first 1-2 years. However, 60-70% of the initially treatable groups relapse with choroidal neovascularization, and recurrent symptoms usually do not cure with continued treatment. Therefore, it is clear that current treatments for choroidal neovascularization after age-dependent macular degeneration are terribly inadequate.

The scientific literature discloses that many substances show the ability to inhibit neovascularization in certain circumstances. Invests such as Berman . Offharmo
Le. Screw. Rhino. (Invest. Ophthalmo
l. Vis. Sci. ) 22, pp.191-9, 198
2, "Plasminogen activator (urokinase) causes corneal neovascularization (Plasminogen Ac
Tivator (Urokinase) Causes
Vascularization of the Cor
nea) "is chloromethyl ketone Phe-Al.
a-ArgCH ₂ Cl-2HCl inhibits rabbit uPA in rabbit cornea neovascularization be somewhat prevented is disclosed. Gold farb
b) Seminars in Thrombosis and
Mostasis (Seminars in Thrombo)
sis and Hemostasis) , vol. 12, 4
, 1986, "Plasminogen activator (urokinase) mediates neovascularization: a possible role in tumor angiogenesis (Plasminogen Activato).
rs (Urokinase) Media Neov
ascularization: Possible R
ole in Tumor Angiogenesi
In s) ", 3H diisopropylfluorophosphate ⁽³ H-DFP) inhibits rabbit corneas rabbit uPA, neovascularization is possible to some extent prevented is disclosed. Glaser, Jerdan
an) Invest such. Ophthalmole. Screw. Rhino.
(Invest. Ophthalmol. Vis. Sc
i. ) Vol. 29, 109 pp., 1988, urokinase inhibitor derived from "a human RPE cells prevents neovascularization in vivo (A Urokinase Inhibi
tor Derived from Human RP
ECells Inhibits Neovascul
“Arization in Vivo” ”discloses that a purified pigment epithelial protease inhibitor (PEPI) inhibits rabbit uPA in the rabbit cornea and to some extent inhibits neovascularization. Marbach
Business Week ,
January 22, 1990 edition, "This protein suppresses tumors (This Protein May Strang
le Tumors) ”, a natural protein called platelet factor 4 inhibits non-specific substances in non-specific animals,
Some inhibition of angiogenesis is disclosed. Science such as Maione ( Science )
e) 247, 77-9, 1990, "Inhibition of Angiogenesis by Recombinant Human Platelet Factor 4 and Related Peptides (Inhibition of Angiogene).
sis by Recombinant Human
Platelet Factor-4 and Rel
"Arated Peptides)" discloses that this factor blocks human vascular endothelial cell division in vitro. Folk man and other Saie
Nsu, 235, pp. 442-47, 1987, in the "angiogenic factor (AngiogenicFactors)", a mixture of heparin and cortisone discloses preventing neovascularization for tumor growth. Other substances that block neovascularization of tumorigenesis include copper and oxygen.

All of the above substances which have the property of inhibiting neovascularization have problems or other unfavorable characteristics.
For example, PEPI is a relatively large molecule and, as such, is relatively difficult to diffuse through the tissues of the eye. PEPI is a natural substance and is very expensive to isolate and synthesize. As a large molecule, PEPI can initiate an unwanted immune response.

Next, considering the DFP material, this is uPA.
It is a general serine protease inhibitor that is not selective for. DFP is routinely used to inhibit a wide range of enzymes, especially trypsin and chymotrypsin.
Moreover, DFP is extremely harmful and is not a very good inhibitor. This requires a long time for binding to the enzyme and is required at high concentration.

With respect to the uPA inhibitor disclosed in Bellman et al., High concentrations, long incubations and repeated use were required for this inhibitor to be effective. These inhibitors have little potential as therapeutic agents because they can be toxic due to their ability to alkylate various cellular nucleophiles.

[0019] Therefore, there is provided a substance capable of inhibiting neovascularization of the eye, which is generally available, non-toxic, specific, and whose pharmacological properties and side effects are well known. Is desirable.

The substances known as amiloride, particularly amiloride monohydrochloride dihydrate, are well known for their pharmacological properties and side effects, and are generally available substances. Amiloride monohydrochloride dihydrate is well known as an orally administered diuretic.

The scientific literature also discloses the use of amiloride in several other areas. Bassari
Lili) and Belin's Phebs Letters
(FEBS Letters) , Volume 214, 187-1
On page 91, amiloride is discussed in relation to blocking human uPA-catalyzed hydrolysis of thiobenzyl benzyloxycarbonyl-L-lysinate substrates in vitro. In addition, the article by Bassari and Berrin showed that amiloride competitively tested uPA for plasminogen in vitro.
It inhibits the catalytic activity of uPA and the catalytic activity of uPA against the serpin family of macromolecular antiproteases and does not reduce the activity of tissue-type plasminogen activator (tPA) or plasmin. However, the authors suggest and conclude further work involving amiloride homologues, and amiloride itself is anti-u in vivo.
It is suggested that it is not specific enough to be useful as a PA agent. Anti-Cancer such as Kelen
Research
8: 1373-1376 (1988), "Anti-metastatic effect of amiloride in an animal tumor model (Anti.
metastaticEffect of Amilo
Ride in an Animal Tumor
Model) ”discloses how amiloride administered orally to rats inhibited the metastasis of circulating cancer cells to the rat lung. The authors suggest that amiloride blocks migration of tumor cells through the extracellular matrix. In vitro amiloride competitively inhibited the catalytic activity of uPA towards plasminogen.

It is noted that some of the above references relate to experiments performed in vitro and some relate to experiments performed in vivo. Fes such as Kristensen
Bus Letters , 168, 33-37, 1984,
“Human Endothelial Cells Contain One Plasminogen Activator (Human Endothelial Cells
Content One Type of Plas
"Minogen Activator") mentions a contradiction between these two types of experiments, and the authors attribute this discrepancy to the fact that cultured cells do not necessarily represent intact cells in vivo for plasminogen activator synthesis. Suggests.

From the above, it is clear that amiloride or a pharmaceutically acceptable salt or solvate thereof is not known as a therapeutic agent for ocular neovascularization, particularly corneal, iris, retina and choroidal neovascularization. Is.

[0024]

Accordingly, it is an object of the present invention to provide a commonly available substance which inhibits neovascularization of the eye, the pharmacological properties and side effects of which are well known.

Another object of the present invention is to provide a substance which inhibits neovascularization of the eye, which can be used directly in the eye of a patient or by means of an implantable device, by oral administration or by intravenous administration. .

Another object of the present invention is to provide a substance which inhibits retinal neovascularization, which can be used directly in the eye of a patient, or by oral administration or by intravenous administration.

Another object of the present invention is to provide a substance which inhibits corneal neovascularization, which can be used directly in the eye of a patient, or by oral administration or by intravenous administration. Yet another object of the present invention is to provide a substance that inhibits neovascularization of the eye without being rejected by the body's immune system. Yet another object of the present invention is to provide a method for treating the eye that suppresses neovascularization that occurs during age-dependent macular degeneration and diabetic retinopathy.

Another object of the present invention is to provide a method for treating the eye which suppresses neovascularization occurring in the retina and choroid tissue.

Yet another object of the present invention is to provide a method of treating retinal neovascularization which avoids the loss of peripheral and night vision of the patient.

[0030]

Some of the other objects, advantages and novel features of the present invention are set forth in the following description, some of which will become apparent to those skilled in the art upon reviewing the following description. Or it may be learned by practice of the invention.
The objects and advantages of the invention will be realized and attained by means of the instruments and combinations particularly pointed out in the appended claims.

In order to achieve the above and other objectives, in accordance with the objects of the present invention described herein, an improved method of treating ocular tissue is directed to the ocular tissue directly or systemically to prevent or prevent neovascularization of the eye. , An amiloride or related pyrazine derivative or a pharmaceutically acceptable salt and / or solvate thereof is administered. In particular, pyrazine derivatives block neovascularization of the cornea, iris, retina and choroid.

Other objects of the invention will be readily apparent to one skilled in the art from the following description. Detailed Description of the Preferred Embodiments Amiloride is administered as an eye drop comprising amiloride or a pharmaceutically acceptable salt or solvate thereof and a carrier, such as an artificial tear solution. The artificial tear carrier solution is a buffered isotonic aqueous solution.

For the purposes of the present invention, the term amiloride-related compound as used herein is the free base form of the amiloride-related compound and / or a pharmaceutically acceptable salt and / or
Or understood to include solvates. In particular, when a ready-made amiloride-related compound or salt and / or solvate is dissolved in an aqueous solution in the presence of a pharmaceutically acceptable buffer, it contains a free base form, a protonated cation form, and an anion form. A complex equilibrium mixture is obtained. Such a mixture is administered to the patient.

Some formulations of the pharmaceutical composition of the present invention are described herein. Such formulations and the like can be used to prevent neovascularization of the cornea, iris, retina and choroid. Amiloride has a molecular weight of 229.65, and amiloride monohydrochloride dihydrate (or amiloride-HCl · 2H ₂
It is noted that the molecular weight of O) is 303.4.

[0035]

Example 1 About 3.03 g of amiloride-HCl.2H ₂ O was dissolved in an artificial tear solution to make a volume of 1 liter. This formulation forms a 10 millimolar solution of amiloride-HCl in an artificial tear carrier for administration to a patient by eye dropper.
This solution is approximately 0.29% by weight amiloride-HCl.

Example 2 About 3.33 g of amiloride-HCl.2H ₂ O is dissolved in an artificial tear solution. Then, about 10 g of methylcellulose is also added to this solution. The solution is made up to 1 liter with isotonic sodium chloride artificial tear solution. This formulation forms an 11 millimolar solution of amiloride-HCl in an artificial tear carrier containing about 1% methylcellulose for administration to a patient by eyedropper. This solution is about 0.31 wt% amiloride-HCl. Example 3 About 3.64 g of amiloride-HCl · 2H ₂ O was dissolved in an artificial tear solution together with 14 g of polyvinyl alcohol,
Make up to 1 liter with artificial tear solution. This formulation forms a 12 millimolar solution of amiloride-HCl in an artificial tear carrier containing about 1.4% polyvinyl alcohol for administration to a patient by eye dropper. This solution is about 0.34 wt% amiloride-HCl.

Example 4 About 3.94 g of amiloride-HCl.2H ₂ O was dissolved in an artificial tear solution to make a volume of 1 liter. This formulation forms a 13 millimolar solution of amiloride-HCl in an artificial tear carrier for administration to patients by eye dropper.
This solution is about 0.37 wt% amiloride-HCl.

Example 5 About 4.24 g of amiloride-HCl · 2H ₂ O was dissolved in an artificial tear solution to make a volume of 1 liter. This formulation forms a 14 millimolar solution of amiloride-HCl in an artificial tear carrier for administration to a patient by eye dropper.
This solution is about 0.40 wt% amiloride-HCl.

Example 6 The following components: Amiloride-HCl.2H ₂ O 4.55
Another ophthalmic formulation is prepared by mixing a fixed amount of g and an isotonic sodium chloride artificial tear solution with stirring to form 1 liter of amiloride-HCl solution. This forms a 15 millimolar solution of amiloride. This solution is about 0.43% by weight amiloride-HC
1 solution.

Generally speaking, eyedrop formulations are 10-15
It is a millimolar concentration. About 1-5 amiloride in one drop
% Is believed to penetrate the cornea. It is believed that about 0.1% of the amiloride in a drop penetrates the retina. The presence of a surfactant, such as benzalkonium chloride, included in Examples 9-14 as an antibacterial agent facilitates penetration into internal ocular tissues.

Ointments may also be formulated for amiloride administration (in the examples below). EXAMPLE 7 3.03 g amiloride-HCl.2H ₂ O was thoroughly mixed with 997.0 g petrolactam vehicle (ointment base) to obtain about 0.29% by weight amiloride-HCl in petrolactam for ocular tissues. The following ointments can be made by forming an ointment suitable for administration.

Example 8 4.55 g amiloride-HCl.2H ₂ O was thoroughly mixed with 995.4 g petrolatum vehicle (ointment base) to give ocular tissue containing about 0.43% by weight amiloride-HCl in petrolatum. The following ointment can be produced by forming an ointment suitable for administration.

Additional eye drop formulations are described below.

Example 9 About 3.03 g of amiloride monohydrochloride dihydrate was added to
0329% (w / v) monosodium phosphate monohydrate,
Add to an aqueous solution containing 0.0613% (w / v) disodium phosphate dihydrate, 0.0040% (w / v) benzalkonium chloride and 2.43% (w / v) glycerin. The pH of the solution is adjusted to pH 6.8 with 1N sodium hydroxide or 1N hydrochloric acid, if necessary, and the solution is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 10 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.29% by weight amiloride-hydrochloride.

Example 10 Amiloride monohydrochloride dihydrate About 3.33 g is dissolved in an aqueous solution containing the components mentioned in Example 9. The pH is adjusted as above and the volume is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 11 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.31
% Amiloride-hydrochloride.

Example 11 Amiloride monohydrochloride dihydrate About 3.64 g is dissolved in an aqueous solution containing the components mentioned in Example 9. The pH is adjusted as above and the volume is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 12 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.34
% Amiloride-hydrochloride.

Example 12 Amiloride monohydrochloride dihydrate About 3.94 g is dissolved in an aqueous solution containing the components mentioned in Example 9. The pH is adjusted as above and the volume is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 13 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.37
% Amiloride-hydrochloride.

Example 13 Amiloride monohydrochloride dihydrate About 4.24 g is dissolved in an aqueous solution containing the components mentioned in Example 9. The pH is adjusted as above and the volume is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 14 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.40
% Amiloride-hydrochloride.

Example 14 Amiloride monohydrochloride dihydrate About 4.55 g is dissolved in an aqueous solution containing the components mentioned in Example 9. The pH is adjusted as above and the volume is brought to the final volume of 1 liter by addition of water. This formulation forms an approximately 15 millimolar solution of amiloride monohydrochloride in a buffered isotonic solution for administration to a patient by eye dropper. This solution is about 0.43
% Amiloride-hydrochloride.

Instead of the petrolactam-based vehicle,
Water-soluble gels can also be used as carriers for amiloride.

Other eye drop formulations may include ethylenediaminetetraacetic acid which acts as a stabilizer. Hyaluronic acid can also be added to the eye drop formulation for prolonged contact of the amiloride-HCl with the eye, or the amiloride can be administered in a collagen or hyaluronic acid gel. As described above, in order to promote the transfer of the amiloride compound from the outside of the eyeball to which the eye drop is administered to the internal tissues of the eye such as the choroid or the retina, a surfactant such as benzalkonium chloride is used as an eye drop. Can be added to.

Another mode of administration of amiloride is contact lenses soaked in amiloride-HCl solution.
Contact lenses soaked in amiloride-HCl solution have the following properties:
It can be composed of collagen. Amiloride
It can also be administered by polymeric microspheres containing amiloride-HCl.

Another method that can be used to administer the amiloride compound according to the present invention is to use a sustained release implant in the stroma, in the vitreous cavity, or an implant attached to the intraocular lens. Such implants can be inserted during eye surgery and joined by surgical methods. The vitreous implant can be biodegradable, so that the implant disappears when the amiloride is delivered.

Yet another mode of administration of amiloride is with an implant in the subconjunctival fornix.

Yet another mode of administration of amiloride is by oral administration. Intravenous administration can also be used.

Another mode of administration is to use intrascleral implants, especially under the macular region of the retina.

A series of experiments were performed using the rabbit model. A sustained release pellet containing a stimulator of neovascularization [eg prostaglandin E ₁ (PGE ₁ )] was injected into a small slit in each cornea of 21 rabbits. The implants were pellets made from ethylene-vinyl acetate copolymer. Within 3 days, neovascularization was clearly detected in the cornea adjacent to the infused implant.
At this time, the amiloride-HCl · 2H ₂ O-containing agarose pellet was injected into the second slit on one side of each PGE ₁ pellet slit at a predetermined distance from the area of active neovascularization. A blank control agarose pellet was similarly injected as a control into the third slit opposite the PGE ₁ pellet at the same predetermined distance from the area of active neovascularization. Amiloride dose-dependently blocked neovascularization. See Table 1 below.

[0058] Table 1 Effect dose of amiloride corneal vessels to growth N E / C E / C E / C 2 Day 4 Day 6 Day 12μg 4 0.80 (± 0.15) 0.95 (± 0.52) 1.21 (± 0.44) 25 μg 4 0.86 (± 0.19) 0.88 (± 0.24) 1.21 (± 0.35) 50 μg 4 0.65 (± 0.21) 0.42 (± 0.20) 0.54 (± 0.2 1) 100 μg 5 0.36 (± 0.13) 0.16 (± 0.05) 0.54 (± 0.10) 400 μg 3 0.63 (± 0.13) 0.28 (± 0.20) 0.29 (± 0.13) 500 μg 1 0.71 (-) 0.20 (-) 0.21 (-) Numerical values are mean ± SEM.

E / C = experimental blood vessel length / control blood vessel length ratio Amiloride, besides blocking or inhibiting further neovascularization, even reverses some of the neovascularization already generated. Has been observed. Therefore, amiloride can be used to treat existing cases of neovascularization for reversal of neovascularization.

In addition to preventing further neovascularization from occurring in patients who have some degree of neovascularization, as well as reversing existing neovascularization, ocular neovascularization It can also be administered prophylactically to patients who are predisposed to develop.

So far, only amiloride has been disclosed as suitable for preventing neovascularization of the eye. Amiloride is also known by the following name: 3,5-
Diamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide; 3,5-diamino-N- (aminoiminomethyl) -6-chloropyrazinecarboxamide;
N-amidino 3,5-diamino-6-chloropyrazinecarboxamide; N-amidino 3,5-diamino-6
-Chloropyrazinamide; 1- (3,5-diamino-6
-Chloropyrazinecarboxyl) guanidine; 1-
(3,5-diamino-6-chloropyrazinoyl) guanidine; guanamprazine; amiprazine; amipramizide; MK-870; amical; amylar; collectril; midamol; and modamide. Amiloride is usually used as amiloride monohydrochloride dihydrate.

Amiloride has been disclosed as a suitable therapeutic agent for ocular tissues to prevent retinal neovascularization,
There are many other amiloride-related pyrazine derivatives, analogs or congeners that are suitable for preventing neovascularization. Table II below lists amiloride and 16 derivatives. table
II includes a code combining letters and numbers for an amiloride derivative, analog or homolog; a chemical structure; and a generic name for the derivative.

Table II

[0064]

Table II (continued)

[0066]

Table II (continued)

[0068]

In particular, the method of preventing neovascularization of the eye involves 3,5-diamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide, which is also known as amiloride.
3-Amino-5-dimethylamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide, also known as-(N, N-dimethyl) amiloride or DMA, 5- (N, N-hexamethylene) amiloride or HMA Known 3-amino-5- (1-homopiperidinyl) -6-chloro-N- (diaminomethylene)
Pyrazinecarboxamide, 5- (N-ethyl-N-isopropyl) amiloride or 3-amino-5- (N-ethyl-N-isopropylamino) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, also known as EIPA, 5- ( N-methyl-N-isobutyl)
3-Amino-5- (N-methyl-N-isobutylamino) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, also known as amiloride or MIBA, 3,5-diamino-6-, also known as phenamyl
Chloro-N- (phenylaminoaminomethylene) pyrazinecarboxamide, 5- (N-4-chlorobenzyl)-
3-Amino-5- (N-4-chlorobenzylamino) -6-chloro-N-[(2,4-dimethylbenzyl) aminoaminomethylene] pyrazinecarboxamide, also known as 2 ', 4'-dimethylbenzamyl or CBDMB , 5-Chloroamiloride or 3-Amino-5,6-dichloro-N- (diaminomethylene) pyrazinecarboxamide, also known as 5-CIA, 3,5-diamino-6-fluoro, also known as 6-fluoroamiloride or 6-FA N- (diaminomethylene) pyrazinecarboxamide, 3,6-amino-N- (diaminomethylene) pyrazinecarboxamide, also known as 5,6-dihydroamiloride or 5,6-HA, also known as 6-iodoamiloride or 6-IA , 5-Diamino-6-iodo N- Diaminomethylene) pyrazinecarboxamide, 6- hydrochloride amiloride or 6-H
3,5-diamino-N- (diaminomethylene) pyrazinecarboxamide, also known as A, 6-iodo5-
3-Amino-5- (N, N-dimethylamino) -6-iodo N- (diaminomethylene) pyrazinecarboxamide, 6-bromo-5-hydroamiloride or 6-also known as (N, N-dimethyl) amiloride or IDMA br-5-HA as well known 3-amino-6- bromo-N- (diaminomethylene) pyrazinecarboxamide, also known as MK-875 3,5-diamino 6-chloro-N ² - (diaminomethylene) pyrazine hydrazide, 3,5-Diamino-6-bromo-N- (diaminomethylene) pyrazinecarboxamide, also known as 6-bromoamiloride or 6-BrA, 3,5-diamino-6-, also known as benzamyl
Chloro-N- (benzylaminoaminomethylene) pyrazinecarboxamide, and 5- [N-methyl-N- (guanidinocarbonylmethyl)] amiloride or MG
3-Amino-6-chloro-5-also known as CMA
Amiloride selected from the group consisting of [N-methyl-N- (guanidinocarbonylmethylamino)]-N- (diaminomethylene) pyrazinecarboxamide in free base form or a pharmaceutically acceptable salt and / or solvate Administration of a pyrazine derivative compound is included.

It will be appreciated that the pyrazine derivatives of Table II have substituents at the 2-, 3-, 5- and 6-positions of the pyrazine ring. In addition, Table II DMA, HMA, EIP
A, MIBA, Phenamyl, CBDMB, 6-HA, 6
-FA, 6-IA, IDMA, 6-BrA, benzamyl and MGCMA pyrazine derivatives have a carboxamide substituent at the 2-position and the nitrogen atom of the carboxamide moiety is-(aminoiminomethyl),-(arylaminoiminomethyl), It is also recognized that they have a-(aralkylaminoiminomethyl) or [(substituted aralkyl) aminoiminomethyl] group. Further, the pyrazine derivative DM of Table II
A, HMA, EIPA, MIBA, Phenamyl, CBD
MB, 6-FA, 6-HA, 6-IA, IDMA, 6-
It is recognized that BrA, benzamil and MGCMA have an amino substituent at the 3 position, an amino or substituted amino group at the 5 position and a halogeno substituent or hydrogen at the 6 position.

Table III lists the amiloride and 16 derivatives listed in Table II. Table III shows the relative inhibition of uPA at 100 micromolar dose.
ibit) and uPA relative inhibition at 20 micromolar dose are also listed.

Table III Inhibition of uPA activity% Compound 20 mM Inhibitor 100 mM Inhibitor Amiloride 20.5 46.6 DMA 41.1 48.3 HMA 51.1 77.0 EIPA 28.3 56.0 MIBA 18.7 57.3 Phenamyl 1.7 5.5 CBDMB 41.7 63.4 5-CIA 5.0 20.7 6-FA 7.1 27.9 5, 6-HA 2.1 11.1 6-IA 64 .4 83.9 6-HA 15.3 38.2 IDMA 40.3 73.1 6-Br-5-HA 1.0 7.7 MK-875 1.9 7.3 6-BrA 65.6 69. .9 Benzamil 1.2 7.7 MGCMA 29.1 43.0 The relative inhibition values included in Table III are obtained by the following method.

In vitro assays include synthetic, commercially available synthetic chromogenic substrates.
A commercially available human uPA is used with glutaryl glycyl-arginyl-p-nitroanilide. uPA catalyzes the hydrolysis between the arginyl moiety and the p-nitroanilide moiety. The p-nitroaniline released upon hydrolysis of the substrate absorbs at a wavelength of 405 nm. Enzyme activity is expressed as a function of increasing absorbance of the solution at 405 nm as measured by a spectrophotometer.

For the data in Table III, the numbers in the table are u
Percentage inhibition of p-nitroaniline release from the substrate observed upon inclusion of each compound at the indicated concentrations in the PA assay.
Is. The higher the number in the table, the greater the ability of the amiloride-related pyrazine derivative compound to inhibit uPA activity. To obtain the data, a stock solution of amiloride-related pyrazine derivative compound was included in the assay,
P with change in absorbance of the reaction mixture at 405 nm
-Nitroaniline release was compared to a control compound without amiloride-related pyrazine derivative compound.

Although the phenomenon caused by treatment of the eye with the amiloride-related pyrazine derivatives of the invention to prevent retinal neovascularization is not fully understood, a theoretical interpretation is provided to further understand the effect of the invention. Is described here. Briefly, the already formed main body of the capillaries, from the inside to the outside, the luminal surface,
Vascular endothelial cells, ablumina surface
and the basement membrane. Capillaries have unicellular tips that penetrate into the extracellular matrix as the capillaries grow. Capillaries in fact grow longitudinally from behind the tip during cell division, and the tip penetrates the surrounding matrix and tissues. At the tip of newly growing capillaries, urokinase-type plasminogen activator (uPA) is present. uPA can activate enzymes that degrade extracellular matrix components, thus allowing cells to permeate surrounding tissues. Once the new blood vessels penetrate the surrounding tissue, they grow into established blood vessels. This growth process involves the formation of cell indirect points, the formation of luminal and abluminal endothelial cell surfaces, and the remodeling of the surrounding specialized extracellular matrix (basement membrane).

In accordance with the present invention, an amiloride-related pyrazine derivative material is used in uPA to inhibit ocular neovascularization.
It turned out to appear to inhibit the action. In the presence of amiloride, plasmin generation in the eye tissue, cleavage of peptide substrate, and interaction of uPA with specific macromolecular proteinase substrate are all blocked. On the contrary,
Bassari and Verin report that amiloride does not inhibit tissue-type plasminogen activator (tPA), plasmin, plasma kallikrein or thrombin in vitro. UPA exceeds tPA inhibition of amiloride
Specificity for inhibition has important therapeutic implications. u
PA is involved in extracellular matrix degradation during cell migration,
tPA is involved in the degradation of fibrin clots, a process that is associated with the biological hemostatic balance.
lance) is essential. Inappropriate inhibition of tPA can result in dangerous side effects.

In summary, the numerous advantages resulting from the application of the principles of the invention have been described. According to the present invention, there is provided an amiloride composition that inhibits neovascularization of the eye.
Amiloride-HCl · 2H ₂ O has been extensively studied for its pharmacological properties as a diuretic, the side effects of its use as a diuretic are well known and are readily available substances. According to the present invention, there is provided an amiloride-HCl containing composition that can be used directly on the eye of a patient. Amiloride-HCl can also be administered orally. Amiloride-related compositions are useful in preventing neovascularization of the cornea, iris, retina and choroid.

Furthermore, the present invention provides an ocular therapeutic method that can be used for preventing neovascularization that occurs in age-dependent macular degeneration and diabetic retinopathy.

According to the application of the present invention, there is provided a local inhibitor of ocular neovascularization, which can improve the postoperative treatment of corneal transplant. The present invention provides an eye treatment method for preventing neovascularization that occurs in retinal tissue and choroidal tissue. According to the application of the present invention, there is provided a method for treating retinal neovascularization that avoids a reduction in peripheral vision and night vision of a patient.

The above description of the invention is for purposes of illustration and explanation. It is not intended to be exhaustive, that is, to limit the invention in the precise form disclosed. Obvious modifications or variations are possible in light of the above disclosure. The embodiments best illustrate and explain the principles of the invention and its practical application, and those skilled in the art will appreciate that in various embodiments, various modifications will be made to suit the particular application envisioned. Are selected and described so that they can be optimally used. The scope of the invention is intended to be defined by the appended claims.

Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C07D 241/28 (72) Inventor Hugh Verner, Maryland 21030, Cockeysville, Lingold Valley・ Circle 318

Claims (15)

[Claims] 1. A fixed amount of a compound of pyrazine derivatives, and / or a pharmaceutically acceptable salt and / or solvate thereof, which is effective in inhibiting ocular neovascularization in an animal or human patient; and A pharmaceutical composition comprising a carrier for the pyrazine derivative, and / or a pharmaceutically acceptable salt thereof, and / or a solvate, which is suitable for topical use on ocular tissues. 2. The pyrazine derivative has a substituent at the 3-position, 5-position and 6-position of the pyrazine ring; has a carboxamide substituent at the 2-position, and the nitrogen atom of the carboxamide moiety is-(aminoiminomethyl), -(Arylaminoiminomethyl),-(aralkylaminoiminomethyl),
Or having a [(substituted aralkyl) aminoiminomethyl] group; having an amino substituent at the 3-position; having an amino substituent or a substituted amino substituent at the 5-position; having a halogeno substituent or hydrogen at the 6-position The pharmaceutical composition according to claim 1, which is a pyrazine derivative and a pharmaceutically acceptable salt and / or solvate. 3. The pharmaceutical composition according to claim 1, wherein the pyrazine derivative is amiloride, and / or a pharmaceutically acceptable salt and / or solvate thereof. 4. The 3,5-diamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N, N), wherein the pyrazine derivative is also known as amiloride.
3-Amino-5-dimethylamino-6-chloro-N-, also known as -dimethyl) amiloride or DMA
(Diaminomethylene) pyrazinecarboxamide, 5-
(N, N-hexamethylene) amiloride or HMA
Also known as 3-amino-5- (1-homopiperidinyl) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N-ethyl-N-isopropyl) amiloride or 3-amino-5- (EIPA) also known as. N-ethyl-N-isopropylamino) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N-methyl-N-isobutyl) amiloride or 3-amino-5-also known as MIBA.
(N-methyl-N-isobutylamino) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 3,5-diamino-6-chloro-N- (phenylaminoaminomethylene) pyrazinecarboxamide, also known as phenamyl, 5- (N -4-chlorobenzyl) -2 ',
3-amino-5- (N-4-chlorobenzylamino) -6-chloro-N-[(2,4-dimethylbenzyl) aminoaminomethylene] pyrazinecarboxamide, also known as 4'-dimethylbenzamyl or CBDMB,
3,5-diamino-6-fluoro-N- (diaminomethylene) pyrazinecarboxamide, also known as 6-fluoroamilolide or 6-FA, 3,5-diamino-6-iodo, also known as 6-iodoamiloride or 6-IA 3,5-diamino-N- (diaminomethylene) pyrazinecarboxamide, also known as N- (diaminomethylene) pyrazinecarboxamide, 6-hydroamiloride or 6-HA, 6-iodo 5- (N,
3-amino-5- (N, N-dimethylamino) -6, also known as N-dimethyl) amiloride or IDMA
3,5-diamino-6-bromo-N- (diaminomethylene) pyrazinecarboxamide, also known as iodo N- (diaminomethylene) pyrazinecarboxamide, 6-bromoamiloride or 6-BrA, 3,5-diamino-also known as benzamyl 6-chloro-N- (benzylaminoaminomethylene) pyrazinecarboxamide, and 5- [N-methyl-N- (guanidinocarbonylmethyl)] amiloride or 3-amino-6-chloro-5- [N-methyl-N, also known as MGCMA.
2. The pharmacology of claim 1 selected from the group consisting of the free base form of-(guanidinocarbonylmethylamino)]-N- (diaminomethylene) pyrazinecarboxamide or a pharmaceutically acceptable salt and / or solvate. Composition. 5. The composition wherein the carrier is a buffered isotonic composition,
Ointment base, water-soluble gel, and artificial tear composition (arti
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has a shape selected from the group consisting of physical tear composition). 6. The artificial tear composition is methyl cellulose,
The pharmaceutical composition according to claim 5, comprising a cellulose ether selected from the group consisting of hydroxypropylmethyl cellulose and hydroxyethyl cellulose. 7. The pharmaceutical composition according to claim 5, wherein the artificial tear composition comprises polyvinyl alcohol. 8. The group wherein the pyrazine derivative, and / or a pharmaceutically acceptable salt and / or solvate, comprises retinal neovascularization, corneal neovascularization, choroidal neovascularization, and iris neovascularization. The pharmaceutical composition according to claim 1, which is present in an amount effective to inhibit neovascularization of the eye of the type selected from: 9. A method for treating a human for inhibiting, reducing or preventing ocular neovascularization, which comprises a compound of pyrazine derivatives and / or a pharmaceutically acceptable salt thereof.
And / or a solvate comprising administering to a human an effective amount of the agent for inhibiting, reducing or preventing ocular neovascularization. 10. A compound of pyrazine derivatives, and / or
10. The method of claim 9, wherein the pharmaceutically acceptable salt and / or solvate thereof is administered by direct use to ocular tissues. 11. A compound of pyrazine derivatives, and / or
The method according to claim 9, wherein the pharmaceutically acceptable salt, and / or solvate thereof is orally administered. 12. A compound of pyrazine derivatives, and / or
The method according to claim 9, wherein the pharmaceutically acceptable salt and / or solvate thereof is administered by intraocular implant. 13. A compound of pyrazine derivatives, and / or
3,5-diamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N, N-dimethyl) amiloride, or a pharmaceutically acceptable salt and / or solvate thereof which is also known as amiloride, 3-Amino-5-dimethylamino-6-chloro-N- (diaminomethylene) pyrazinecarboxamide, also known as DMA, 5- (N, N-hexamethylene) amiloride or 3-amino-5, also known as HMA.
(1-Homopiperidinyl) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N-ethyl-N-isopropyl) amiloride or 3-amino-5- (N-ethyl-N-isopropylamino) -also known as EIPA. 6-chloro-N- (diaminomethylene) pyrazinecarboxamide, 5- (N-methyl-N-)
3-Amino-5- (N-methyl-N-isobutylamino) -6-chloro-N- (diaminomethylene) pyrazinecarboxamide, also known as isobutyl) amiloride or MIBA, 3,5-diamino-6-chloro-N, also known as phenamyl -(Phenylaminoaminomethylene) pyrazinecarboxamide, 5- (N-4-chlorobenzyl) -2 ', 4'-dimethylbenzamyl or C
Also known as BDMB, 3-amino-5- (N-4-chlorobenzylamino) -6-chloro-N-[(2,4-
Dimethylbenzyl) aminoaminomethylene] pyrazinecarboxamide, 6-fluoroamiloride or 6-F
3,5-diamino-6-fluoro-N, also known as A
-(Diaminomethylene) pyrazinecarboxamide, 6
3,5-diamino-6-iodo N- (diaminomethylene) pyrazinecarboxamide, also known as iodoamilolide or 6-IA, 3,5-diamino-N-, also known as 6-hydroamiloride or 6-HA
(Diaminomethylene) pyrazinecarboxamide, 6-iodo 5- (N, N-dimethyl) amiloride or I
3-amino-5- (N, N-dimethylamino) -6-iodo N- (diaminomethylene), also known as DMA
3,5-Diamino-6-bromo-N- (diaminomethylene) pyrazinecarboxamide, also known as pyrazinecarboxamide, 6-bromoamiloride or 6-BrA, 3,5-diamino-6-, also known as benzamil
Chloro-N- (benzylaminoaminomethylene) pyrazinecarboxamide, and 5- [N-methyl-N- (guanidinocarbonylmethyl)] amiloride or MG
3-Amino-6-chloro-5-also known as CMA
A free base form or a pharmaceutically acceptable salt and / or solvate of [N-methyl-N- (guanidinocarbonylmethylamino)]-N- (diaminomethylene) pyrazinecarboxamide. 9. The method described in 9. 14. The group wherein the pyrazine derivative and / or a pharmaceutically acceptable salt and / or solvate comprises retinal neovascularization, corneal neovascularization, choroidal neovascularization, and iris neovascularization. 10. The method of claim 9, which is present in an amount effective to prevent, reduce or prevent ocular neovascularization of the type selected from. 15. A method of treating humans for the inhibition, reduction and prevention of ocular neovascularization, which comprises inhibiting a compound that inhibits a urokinase-like plasminogen activator (uPA) from ocular neovascularization. A method comprising oral administration in an effective amount for alleviation and prevention.