JP2838290B2 - Eyeball opacity prevention improver - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an eyeball opacity preventive improving agent,
More specifically, the present invention relates to an eyeball opacity preventing / improving agent containing a specific organic germanium compound or aminoguanidine as an active ingredient.

[Prior art] The lens in the eyeball contains about 65% of water and about 35% of protein as its main components, and contains protein in a higher ratio than other tissues. While undergoing various biological controls, it maintains transparency by forming and maintaining a hydrocolloid state with water in cells, but if for some reason this lens that should be transparent becomes cloudy, The amount of light that must reach the retina decreases, and, of course, the visual acuity decreases with the degree of turbidity.

The causes of the above-mentioned lens turbidity are extremely diverse,
Although not generally discussed, among the water-soluble proteins, membrane proteins, and insoluble proteins that constitute the protein,
Since SH (thiol) groups are abundant, these SH groups are converted into SS groups by in vivo reactions, which are said to lead to clouding of the lens. And sugar are converted to a substance called Amadori by the Maillard reaction, which is an irreversible non-enzymatic reaction,
It is said that this is one of the causes of lens turbidity,
Since it is difficult to culture the lens in a transparent state, research in this field has been delayed, and it can be naturally predicted that the lens is turbid due to a mechanism other than the above mechanism.

[Problems to be Solved by the Invention] The only thing that is clear is that at present, there is established a treatment method capable of preventing turbidity of the lens or improving turbidity when it occurs. It is not.

The present invention has been made with the background of the above-mentioned conventional technology as an object to provide a drug capable of preventing and improving opacity of an eyeball.

It is another object of the present invention to provide an eyeball opacity preventive / improving agent having no toxicity or side effects.

[Means for Solving the Problems] The configuration adopted by the present invention to achieve the above object is represented by the following formula: (Wherein, R _{1 to} R ₃ are a hydrogen atom or a lower alkyl group such as the same or different methyl group or ethyl group or a substituted or unsubstituted phenyl group, X is a hydroxyl group, an O-lower alkyl group,
An organic germanium compound represented by an amino group or O ^- Y ⁺ [Y represents a compound having a metal such as sodium or potassium, or a compound having a basic group such as lysozyme or a basic amino acid]. Alternatively, the present invention is characterized in that the compound represented by the formula (I) and aminoguanidine are used as active ingredients.

 Hereinafter, the present invention will be described in detail.

Since the eyeball turbidity preventive improver of the present invention contains a specific organogermanium compound represented by the above formula I as an active ingredient, the compound will be described first. This compound comprises three substituents R _{1 to} R ₃ and an oxygen functional group. The basic skeleton is germanylpropionic acid in which a propionic acid derivative having OX and a germanium atom are bonded, and germanium atoms and oxygen atoms in the basic skeleton are bonded at a ratio of 2: 3.

Here, the substituents R _{1 to} R ₃ are a hydrogen atom, a methyl group,
A so-called lower alkyl group such as an ethyl group, a propyl group or a butyl group, or a substituted or unsubstituted phenyl group; and the substituent X is a hydroxyl group, an O-lower alkyl group, an amino group or a carboxylic acid represented by O ^- Y ^+. Are shown respectively.

Y represents a metal such as sodium or potassium (but not limited to monovalent) or a compound having basicity represented by a basic amino acid such as lysozyme or lysine.

Further, the substituents R ₁ and R ₂ are bonded to the α-position of the germanium atom, and the substituent R ₃ is also bonded to the β-position.Therefore, examples of the organic germanium compound used in the present invention include the following. Can be.

Thus, the organogermanium compound having the above structure can be produced by various methods.

That is, the compound of the formula I wherein X = OH is, for example, a trihalogermil such as trichlorogermylpropionic acid (1) into which substituents R _{1 to} R ₃ have been previously introduced as shown in the following reaction formula. What is necessary is just to hydrolyze propionic acid.

On the other hand, a compound such as X ＝ O-lower alkyl group in the formula I is converted into the corresponding acid halide by, for example, reacting the above compound (1) with thionyl chloride or the like, and After reacting the alcohol corresponding to the alkyl group, it may be hydrolyzed,
Further, in the case of formula I where X = NH ₂ , for example, the acid halide may be hydrolyzed after allowing ammonia to act on the acid halide.

Further, in the formula (I), when X is represented by COO ^- Y ⁺ and Y is a metal, the compound (1) may be reacted with a corresponding metal hydroxide, and Y is a basic group. Compounds having the same may be in accordance with a known acid-base reaction.

The results of instrumental analysis such as nuclear magnetic resonance absorption (NMR) spectrum and infrared absorption (IR) spectrum of the organogermanium compound obtained as described above show that the compound is represented by the general formula I We support well.

Incidentally, the formula representing the organic germanium compounds corresponds to a state in which they are isolated as crystals, and in an aqueous solution, a germanium-oxygen bond undergoes hydrolysis. For example, in the compound (I1), It is known that it takes the following structure.

On the other hand, aminoguanidine used in the present invention has the formula Which is a compound known as a strong nucleophile.

Thus, the opacity preventive / improving agent for the eyeball of the present invention contains an organic germanium compound synthesized as described above or the above-mentioned aminoguanidine as an active ingredient, and is preferably boric acid, sodium chloride, sodium hydroxide or the like. Is formulated into an ophthalmic solution together with a known component such as benzalkonium chloride and administered.

The organic germanium compound which is the active ingredient of the present invention,
Since it is characterized by extremely low toxicity and almost no side effects, the amount of use can be determined relatively freely, but when instilled, for example, the range of 0.5 to 4% can be exemplified. When the organic germanium compound and aminoguanidine are the main components, the organic germanium compound is added to aminoguanidine, for example, 10%.
^{About -5 to} 10 ^-7 may be used.

Can be exemplified.

[Operation and Effect of the Invention] The effect of the present invention agent containing the above-described organogermanium compound as a main agent was examined using an aging-promoting mouse in which lens turbidity is likely to occur. The effect of the agent of the present invention by eye drops, such as bleeding and increase in the number of transparent eyes, is shown.

Also, α-crystallin derived from bovine lens (Crystallin
In an in vitro experimental system using e and glucose (Glucose), the agent of the present invention mainly composed of an organic germanium compound described above or the agent of the present invention mainly composed of an organic germanium compound and aminoguanidine shows the formation of Amadori. The effects of suppressing or improving the once formed Amadori to low molecular weight, thereby preventing or improving the opacity of the eyeball were shown.

EXAMPLES The present invention will be described below with reference to examples.

Example 1 (1) Method Eighteen 10-month-old aging-promoting mice (5 males and 13 females) were divided into two groups, and the present invention containing 4% of the organogermanium compound (I1) for a group of 10 mice The drug was instilled four times a day for 123 days at a rate of six days a week, and the remaining eight animals were used as a non-instilled control group, and both groups were bred under the same conditions. The lens of each individual was examined under a microscope under intraperitoneal injection anesthesia with 0.7 ml / Kg of Nembutal sodium (Sodium Nembutal) for a total of 4 times before, on the 39th, 78th and 123rd days after the start of the experiment.

(2) Results Survival of senescence-accelerated mice As shown in FIG. 1, the number of senescence-accelerated mice was 18 in both groups before the start of the experiment, but increased to 1 on day 39 and to 5 on day 78. However, by 12 months of age, there was a gradual decrease, and thereafter, by the 14th month of the 123rd day, there was a sharp decrease in the number of surviving animals to less than half.

Next, comparing the survival state on the 123rd day between the eye drop group and the control group, as shown in FIG. 2, the eye drop group died 3 out of 10 mice, whereas the control group 10 Six of them died, and it became clear that the instillation group had a longer life-span effect.

Lens findings of aging-promoting mice Survival status When the lens of a 10-month-old aging-promoting mouse was examined under a microscope, individual differences were observed in the onset of turbidity. We were able to. That is, in the transparent lens, there is no abnormal finding in the lens itself, retinal blood vessels can be seen through, and in the lens determined to be nuclear sclerosis, nuclear sclerosis extends to the cortical intermediate layer,
After that, a ripple-like pattern was formed on the pole side surface.Histologically, there was a boundary layer with different staining properties between the perinuclear zone and the cortical surface layer. Admitted.

On the other hand, a lens determined to be opaque is a state in which axle-shaped opacity is usually generated in the cortex, and no serious one is observed until 78 days after the start of the experiment.

Eye Drop Effect of the Agent of the Present Invention The lens findings of the administration group and the control group before, at 39 days, 78 days and 123 days after the start of the experiment are shown in Table 1 below.

Fig. 3 shows the result of conversion to a percentage, and in the eye drop group, 20% of the cloudy eyes were before the eye drop.
The ophthalmic instillation of the present invention showed that the clear eyes decreased to 11% or 15% before administration, but once disappeared on day 39 after instillation, but increased to 38% on day 78. The effect is shown.

On the other hand, in the control group, the deterioration of symptoms is remarkable, for example, the number of transparent eyes decreases with age and the number of opaque ones increases.

4 and 5 show the effect of the agent of the present invention in the same eye. In the lens shown in FIG. 4, slight opacity is seen, but FIG. 5 shows one month after instillation. It can be seen that the transparency is considerably improved.

Others, such as those in which the number of prints seen multiple times in the lens determined to be sclerosing decreased after instillation and those in which the age-related change in the equatorial bow construction decreased after instillation Some findings showed that the symptoms were alleviated.

Example 2 (1) Method Method using bovine α-Crystalline 20 mg of α-Crystalline (manufactured by Sigma) derived from bovine lens
/ ml and 200 mM D-Glucose were mixed in equal amounts and 100 μl was dispensed on a 96-well microtiter plate. Aminoguanidine hydrochloride at a concentration of 5-fold dilution from 200 mM and the organogermanium compound (I1) at a concentration of 5-fold dilution from 20 mM.
x Titration method, collected over time at 7μ / well, diluted with 50mM phosphate buffer 133μ, TSK-G3000S
By high performance liquid chromatography equipped with W column, 350
A fluorescence detector having a wavelength of 440 nm and an ultraviolet detector having a wavelength of 280 nm were connected at the same time, and the measurement was performed simultaneously.

As described above, the Amadori formed by α-Crystalline and D-Glucose was collected, washed, resuspended in 0.5 mol phosphate buffer, redistributed on a 96-well microtiter plate, and the organogermanium was collected. The effects of compound (I1) and aminoguanidine were examined.

Method using bovine serum albumin An equal amount of bovine serum albumin (200 mg / ml) and 400 mM D-Glucose solution in 0.5 M phosphate buffer (PH7.4) were mixed,
₃ mM NaN ₃ was added. The organic germanium (I1) and aminoguanidine were adjusted to various concentrations by the box titration method and added to this system, and the influence on the Amadori formation was examined using the fluorescence concentration as an index. On the other hand, a mixture of bovine serum albumin and D-Glucose was heated at 37 ° C. for a long time to form Amadori. Then, the effect was assayed by high performance liquid chromatography.

(2) Results Method Using Bovine α-Crystalline It was found that administration of aminoguanidine alone promoted Amadori formation at a low concentration, but the present invention agent using the above-mentioned organogermanium compound (I1) was considerably low. From the concentration to the high concentration, it is effective in suppressing Amadori formation. In addition, in the case of the present invention using the organogermanium compound (I1) and aminoguanidine, the Amadori formation caused by the single administration of aminoguanidine is suppressed. Acceleration was suppressed and lower values than the positive control were obtained over the entire concentration range.

In addition, the effect on Amadori formed from α-Crystalline was 40 and 20% when aminoguanidine was administered alone.
Although the reversible solubilization was observed at 0 mM, the inventive agent based on the organic germanium compound (I1) was found to be 20 mM, and the inventive agent based on the organic germanium compound (I1) and aminoguanidine, Germanium compound: 800μ
M, aminoguanidine: It was found to be effective even at a low concentration of 320 μM.

 Table 2 below shows some of the results.

The numerical values in Table 2 represent the integrated value of the fluorescence intensity measured by the fluorescence detector having the excitation wavelength of 350 nm and the emission wavelength of 440 nm.

The value of α-Crystalline alone is 137, and *
Represents a numerical value when D-Glucose is added to α-Crystalline.

Method Using Bovine Serum Albumin The formation of Amadori corresponding to a fluorescence intensity exceeding 4 × 10 ⁴ in 50 days was confirmed with bovine serum albumin and D-Glucose. Observation of this system by high performance liquid chromatography reveals that serum albumin dimers and trimers are significantly increased.

Then, when the agent of the present invention containing 4 mM of the organic germanium compound (I1) and 200 mM of aminoguanidine was added, significant suppression of amadori formation was observed, and the synergistic action of the organic germanium compound (I1) and aminoguanidine was confirmed. The result is shown in FIG.

Observation of this system by high performance liquid chromatography shows that the peaks corresponding to serum albumin dimers and trimers are low, while peaks with lower molecular weight are observed.

Further, bovine serum albumin and D-Glucose were added at 37 ° C for 9 hours.
After heating for a week to sufficiently form Amadori, to which the present invention was added using 20 mM of an organic germanium compound (I1) and 200 mM of aminoguanidine, significant suppression of Amadori formation was observed as shown in FIG. The synergistic action of the organogermanium compound (I1) and aminoguanidine was also confirmed in this system.

Observation of this system by high-performance liquid chromatography showed that the peak corresponding to the trimer of serum albumin became the main component, while the peak of lower molecular weight showed strong UV absorption and reduced fluorescence. The low molecular weight of the constituent proteins is suggested together with the impaired formation.

As described above, the present invention is excellent as an eyeball opacity preventive improving agent.

[Brief description of the drawings]

FIG. 1 is a diagram showing the survival status of senescence-accelerated mice, FIG. 2 is a diagram comparing the survival status between an eye drop group and a control group, FIG. 3 is a diagram obtained by converting the number of eyes of lens findings into percentages, FIG. 4 is a photograph of a morphology of an organism showing an eye determined to be cloudy before instillation, and FIG.
The figure is a photograph of the morphology of the organism showing one month after instillation of the same eye as the eye in FIG. 4, and FIG. 6 is a photograph of bovine serum albumin and D-Gl.
FIG. 7 shows the formation of Amadori by ucose and the effect of the agent of the present invention on the formation of Amadori by D-Glucose. FIG. 7 shows the relationship between bovine serum albumin and D-Glucose.
It is a figure which shows the effect of this invention agent with respect to Amadori formed by -Glucose.

Continuation of the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) A61K 31/28 A61K 31/155 A61K 37/54

Claims (2)

(57) [Claims] (1) Expression (Wherein, R _{1 to} R ₃ are a hydrogen atom or a lower alkyl group such as the same or different methyl group or ethyl group or a substituted or unsubstituted phenyl group, X is a hydroxyl group, an O-lower alkyl group,
An organic germanium compound represented by an amino group or O ^— Y ⁺ [Y represents a metal having sodium or potassium or a compound having a basic group such as lysozyme or a basic amino acid] is used as an active ingredient. An eyeball opacity prevention and improvement agent. (2) (Wherein, R _{1 to} R ₃ are a hydrogen atom or a lower alkyl group such as the same or different methyl group or ethyl group or a substituted or unsubstituted phenyl group, X is a hydroxyl group, an O-lower alkyl group,
An organic germanium compound represented by an amino group or O ^- Y ⁺ [Y represents a compound having a basic group such as a metal such as sodium or potassium or lysozyme or a basic amino acid], and aminoguanidine. An opacity preventing / improving agent for an eyeball, characterized by comprising as a component.