Ophthalmic drug and a method of treatment of diseases, pathological conditions and damage to the retina

 

(57) Abstract:

The invention relates to the field of medicine. The tool can be used to treat myopia, retinopathy, to relieve discomfort and eye fatigue, for the prevention and treatment of retinal detachment of different origin, including for prevention of retinal detachment with operating procedures. The tool contains an acidic glycoprotein isolated from the retina of mammals, calcium chloride and the media. The invention provides for the absence of adverse and allergic reactions and intolerance to the drug. 3 N. and 19 C.p. f-crystals, 1 table.

The invention relates to medicine, namely to ophthalmology, and can be used for the treatment of diseases and injuries of the retina, to prevent retinal detachment, surgical interventions on the retina in humans to improve the outcome of the operation, as well as to eliminate adverse effects when wearing contact lenses and to relieve discomfort and eye fatigue.

Among the diseases of the eye a special place on the severity and possible consequences of occupy pathology caused by dysfunction of the tissues of the retina. Neet to be caused by different reasons, including mechanical trauma, toxic lesions, hypertension stroke, and other retinal Detachment is a fairly common complication accompanying any abdominal surgical intervention.

Closest to the present invention is a tool that restores the function of the retina, which is a complex of low molecular weight polypeptides obtained from the retina of animals by extraction with 3% solution of acetic acid with addition of chloride of zinc and processing the supernatant acetone (EN 2073518, 20.02.97). The drug is called "Ritalin".

The technical objective of our invention is to improve the effectiveness of treatment and prevention of diseases, pathological conditions and damage the retina; improving the treatment and prevention of diseases, pathological conditions and injuries, accompanied by dysfunction of the retina; the way to prevent retinal detachment; with surgical interventions on the retina in humans a way to improve the outcome of the operation (prevention of retinal detachment, increased visual performance), as well as a way to relieve discomfort and weariness tool including the preparation of the retina and a pharmaceutically acceptable carrier, contains as drug retina acidic glycoprotein isolated from the retina of mammals, soluble in a saturated solution of ammonium sulfate having isoelectric point below 3.0 and the apparent molecular mass of 8 kDa to 200 kDa, and possibly calcium chloride, when following the preferred ratio of components, wt.%:

The above glycoprotein 110-16-110-8

Calcium chloride 0-0,01

Media else

As the pharmaceutically acceptable carrier ophthalmological tool contains, depending on the final pharmaceutical form solution for injection, drops, ointment, gel, such as, for example, saline, representing approximately 0,9% solution of chloride or sodium carboxymethylcellulose and water, where the concentration of carboxymethyl cellulose varies depending on the desired viscosity of the resulting gel; in the case of ointments as the carrier can be water, lanolin emulsion. The preferred content of calcium chloride in the composition of the funds committed funds is from 0.910-4to 0.01 wt.%.

Ophthalmological tool according to this invention derived from the glycoprotein isolated from the retina of mammals by the method lies in the extraction of the retina saline with Yes ammonium sulfate, separation otdelitelnogo supernatant fractions by the method of isoelectric focusing in the density gradient and further purification of acidic glycoprotein fraction by HPLC. Secreted acidic glycoprotein retinal soluble in a saturated solution of ammonium sulfate has an isoelectric point at pH below 3.0 and the apparent molecular mass of 8 kDa to 200 kDa. In the allocation process glycoprotein occurs dialysis in dialysis bags with limit bandwidth 8 kDa, resulting in the presence in selected fractions of low molecular weight peptides or glycopeptides is deleting them (unlike EN 2073518, 20.02.97, where the selected drug from the retina is a complex of low molecular weight polypeptides).

Cooking claimed ophthalmological preparations is the traditional volume-weight method. For cooking means using pyrogen-free deionized water. During cooking, if necessary, produce sterilization solutions.

Claimed in this invention ophthalmic drug can be used in the treatment of diseases of the eye prior to surgery, during the surgery the disgust of retinal detachment, the increase of view).

When applying this eye means no observed adverse and allergic reactions, there are no phenomena of intolerance.

The following examples illustrate but do not limit the invention.

Example 1

The selection of the glycoprotein from the retina of the eyes of cattle.

Fresh eyes of cattle (30 PCs) were isolated retina. Tissue was rinsed thoroughly in physiological solution (0.9% NaCl) and were extracted in an aqueous solution containing 9.0 g/l NaCl, 0.4 g/l KCl, 0.1 g/l l2and 0.35 g/l HEPES at a temperature of +4C for 2 hours. The extract obtained was centrifuged for 15 min at 3000 g, then decantation. To the obtained supernatant with vigorous stirring slowly added the dry ammonium sulphate to the formation of a saturated solution of salt (4C, pH 8.0 to 8.5). After keeping the mixture at 4C for 24 hours the precipitate of impurity proteins precipitated with centrifugation at 35000 g for 30 minutes the Supernatant was collected and the long-term were dialyzed against distilled water using cellulose film according to GOST 7730-89. During dialysis distilled water megacrania the excess solvent on a rotary evaporator, and repeated the procedure vysalivaniya in the same conditions. The obtained supernatant also were dialyzed against distilled water until complete removal of ammonium ions and then divided by the method of isoelectric focusing.

Isoelectrofocusing (IEF) was performed in the density gradient of sucrose, using a column LKB-440 (LKB, Sweden) and ampholytes (Serva, Germany) pH range of 3.5 to 10.0, at a temperature of 4C and the voltage 500-1500 In within 72 hours. The supernatant of the solution was injected into the heavy gradient solution in an amount of not more than 100 mg of total protein. After conducting IEF collected fraction volume of 10 ml, were detected them spectrophotometrically at 280 nm and pH was determined in each fraction. Combined fractions with pH<3.0 and spent dialysis combined acid fractions against distilled water containing 0.1 g/l CaCl2at 4C in dialysis bags with limit bandwidth 8 kDa ("Spectrapor, USA) to remove sucrose.

Final purification of the obtained peptide was carried out by HPLC on a column of Separon-NH2, eluent: 10 mm phosphate buffer, pH 3.0. Put 100 μl of the sample, the detection was performed at 280 nm, the sensitivity of 0.04 D, speed paper chart recorder 10 mm/min feed Rate eluent to 0.5 realizovannoy water at 4C in dialysis bags with limit bandwidth 8 kDa ("Spectrapor", USA) to remove salts.

The concentration of the glycoprotein in the resulting aqueous solution was determined spectrophotometrically (Darbre A. "Practical protein chemistry" // M.: Mir, 1989, S. 300-302).

Example 2

89 g of sodium chloride and 0.01 g of calcium chloride dissolved in 500 ml of pyrogen-free deionized water. To the resulting solution was added 10 ml of an aqueous solution containing 110-9g glycoprotein from the retina of cattle (solution with a concentration of 110-7g/l glycoprotein), obtained as described in example 1. The solution is stirred and brought its volume with stirring to 1 l of pyrogen-free deionized water. Then in a glass bowl put the resulting solution and add 9 liters of pyrogen-free deionized water. After stirring carrying cold sterilization solution by filtration under a pressure of 0.3 ATM through a filter with a pore size of 0.22 μm (firm "Pall", "Millipore", "Jeitz", "Vladipor" and others) and pour the preparation in a sterile vials of 5 ml and 1.5 ml tubes dropper polyethylene under high pressure. The vials are sealed with stoppers and aluminum caps, which then pass around. Filtering and bottling preparation is carried out in aseptic conditions article is made of themselves sterile colorless transparent solution without mechanical impurities, containing 110-10g/l glycoprotein from the retina of cattle, from 8.6 to 9.2 g/l sodium chloride solution (preferably 8,9 g/l) and from 0,0009 to 0.0011 g/l calcium chloride (preferably 0.001 g/l) and having a pH from 5.0 to 7.0.

Example 3

To 500 ml of pyrogen-free deionized water is added 10 ml of an aqueous solution containing 1 µg of glycoprotein from the retina of cattle (solution with a concentration of glycoprotein 0.1 mg/l), obtained as described in example 1. The solution is stirred and brought its volume with stirring to 1 l of pyrogen-free deionized water. Then in a glass bowl put the resulting solution and add 9 liters of pyrogen-free deionized water. After stirring carrying cold sterilization solution by filtration under a pressure of 0.3 ATM through a filter with a pore size of 0.22 μm (firm "Pall", "Millipore", "Jeitz", "Vladipor" and others) and pour the preparation in a sterile vials of 10 ml Vials sealed with stoppers and aluminum caps, which then pass around. Filtering and bottling preparation is carried out in aseptic conditions with sterile lids and laminar air flow. The obtained ophthalmic drug is sterilizing cattle and having a pH from 5.0 to 7.0.

Example 4

The influence of ophthalmic funds for the restoration of the function of the retina rats Campbell, genetically predisposed to retinal dystrophy.

The rats Campbell, genetically predisposed to retinal dystrophy, was conducted series of experiments on the influence described ophthalmologic funds for the restoration of the function of the retina. As control were used Wistar rats without the introduction of drugs, and the rat line Campbell with the introduction of their physiological solution.

The development of the pathological process can be monitored using various methods, one of which is the method of determining the ratio of rhodopsin in the eye glass and, in fact, in the retinal tissue of the eye. In healthy animals the ratio is digital parameter whose value is about 1. This means that the protein mainly localized in the outer segments of the photoreceptors, and all the "waste" outer segments already absorbed by the cells of the pigment epithelium of the retina. In animals with hereditary dystrophy of the retina, the value of this parameter varies in the range of 0.5, which indicates the accumulation of oblack">

Ophthalmological tool, prepared as described in example 2, was administered to rats by intraperitoneal injection four times in 0.2 ml on the 10th, 20th, 30th and 40th days after birth. The results presented in the table. These data suggest that the introduction of the proposed framework improves the function of the retina in animals genetically unstable line (figure G/In characterizing the degree of destruction of the outer segments of photoreceptors, reached values corresponding to those in the normal functioning of the retina, i.e., in rats Wistar).

Example 5

The influence of eye means on the aggregation of cells in the retina of chicken embryos.

Experiments were performed on freshly prepared suspension culture cells of the retina of chicken embryo (breed Leghorn).

From 25-30 enucleated eyes chick embryo extract of the retina, which is then placed on 8-10 minutes at C in 1% trypsin solution (Sigma), prepared on the basis of physiological solution containing 10 mM calcium chloride. Then a solution of the enzyme was removed by pipette, the fabric is washed repeatedly with natural solution that contains trypsin inhibitor. To the washed fabric retinal add 2 ml of CPE is before the formation of a suspension of single cells. Determine the density of the suspension by counting cells in the cell Goryaeva. In each vial containing 1 ml of medium 199, 0.2 ml of bovine serum, add 0.1 ml of ophthalmological preparations, prepared as described in example 2 (in the control vials add 0.1 ml of physiologic saline) and a freshly prepared suspension of cells of the retina in the quantity necessary to produce the final density of the suspension 105-5105cells in the vial.

The vials are placed in the device for soft stirring of the liquid and leave it for 18-20 hours for the implementation of cellular aggregation. Then calculate a single cell.

In the experimental bottles (described ophthalmic remedy) number of single cells when the count is less than in the control (physical solution), at 2-3. In addition, in the experimental bottles are usually detected multicellular aggregates, which are absent in the control bottles.

Example 6

Patient K., 56 years. Diagnosis: Central chorioretinal dystrophy of the retina, the wet form. Visual acuity before treatment OD - 0,09, with correction equal to 0.4. Underwent treatment ophthalmic agent, prepared as described in example 2 - eye Institute of the physiological and peripheral indicators. After 3 weeks survey data and visual acuity is stable. Positive changes in the state of the retina.

Example 7

Patient M., aged 45. Diagnosis: Central chorioretinal dystrophy of the retina, dry form. Visual acuity before treatment OD is 0.8, not also corrected; OS - 0,7, not also corrected. Received medical treatment, ophthalmic agent, prepared as described in example 3. The drug was taken as follows: vial of 10 ml was dissolved in 1 l pranipatena chilled water and took the resulting solution to 100 ml before meals for 30 minutes 3 times a day for 1 month. After treatment, the visual acuity OD - 1,0; OS is 0.8. Improvement of visual functions is confirmed by the improvement of electrophysiological parameters and data of computer perimetric studies (increase foveolar sensitivity). The patient rarely uses the glasses, the disappearance of the sensation of flicker and "flies" in the eyes. When viewed through the month the data is stable.

Example 8

Patient L., 38 years. Diagnosis: retinal detachment of the left eye floater. Visual acuity before treatment OS is 0.2. Treatment: within 10 days received eye installation 2 drops 7 times a day ophthalmic vehicle, prepared to eye means; after surgery the patient received ocular instillation of 2 drops 7 times a day described ophthalmologic funds within 15 days. After treatment, OS - 0,6; the retina is adjoined. After 2 months OS - 0,7; survey data is stable.

As seen from the above examples, the stated vision tool on the basis of an acidic glycoprotein of the retina of mammals affect the adhesive interaction of cells of the retina, and restores the function of the retina in animals genetically predisposed to retinal dystrophy in humans.

In addition, it was found that the proposed means inhibits the activity of ATP-AZ retina, resulting in the accumulation of ATP, and this should have a positive impact on the core function of the retina (conducting visual signal), because ATP plays a key role in the biosynthesis of other necessary party photoreceptor act - GTP.

In experiments conducted in vitro systems, it has been shown antioxidant property of the proposed tools, i.e. its ability to inhibit the processes of lipid peroxidation, which can be regarded as a universal mechanism of damage of membrane also the aging process.

Due to the fact that the stated means of restoring the function of the retina, it can be argued that the use of funds is indicated for the prevention and treatment of diseases, injuries and pathological conditions involving abnormal function of the retina, as well as for prevention and treatment of diseases, injuries and pathological conditions of the retina. Shows use specified funds for the treatment of myopia and retinopathy of various etiologies.

The application stated ophthalmological preparations recommended to all those who suffer from myopia people as a means of ensuring the removal of the voltage, regulating the curvature of the lens of the eye muscles and because of this removal of the associated discomfort and eye fatigue.

Especially important is the use of specified funds for the prevention and treatment of retinal detachment, because retinal detachment occurs when the violation or weakening of the adhesive interactions between the retina and the pigment epithelium, and acidic glycoprotein that is part of the declared funds, enhances these interactions. Otsloenie retina is the most rasprostranenie, including mechanical trauma, toxic lesions, hypertension stroke, and other retinal Detachment is a very frequent complication arising as a consequence of surgical intervention in the eye cavity. The use of the above agents in the preoperative period during surgery or in the postoperative period is a good protector, warning retinal detachment.

1. Ophthalmic drug, including the preparation of the retina and a pharmaceutically acceptable carrier, characterized in that it contains as drug retina glycoprotein isolated from the retina of mammals, soluble in a saturated solution of ammonium sulfate having isoelectric point below 3.0 and the apparent molecular weight of from 8 to 200 kDa, and possibly calcium chloride.

2. Means under item 1, characterized in that it contains components in the following ratio, g/l:

Glycoprotein 110-15-110-7

Media Else

3. Means under item 1, characterized in that it contains components in the following ratio, g/l:

Glycoprotein 110-15-110-7

Calcium chloride 0,0009-0,1

5. Means under item 4, wherein the glycoprotein contains from 3 to 5 residues of phosphoric acid.

6. Tool for PP.1-3, characterized in that the sulfated glycoprotein.

7. Tool for PP.1-3, characterized in that the glycoprotein contains residues of mannose, galactose and N-acetylglucosamine.

8. Means under item 7, wherein the glycoprotein contains residues of mannose, galactose and N-acetylglucosamine in the following ratio: 5-6 mannose residues have 2-3 galactose residue 2 residue N-acetylglucosamine.

9. Tool for PP.1-8, characterized in that as the pharmaceutical carrier contains saline.

10. Means under item 9, characterized in that it contains components in the following ratio, g/l:

Glycoprotein 110-10

Calcium chloride 0,0009-0,0011

Sodium chloride is 8.6 to 9.2

Water Up to 1 l

11. Tool for PP.1, 2, 4-8, characterized in that it contains components in the following ratio, g/l:

Glycoprotein 110-7

Water Up to 1 l

12. Method for the treatment and prevention of diseases, pathological conditions and injuries of the retina, characterized in that the pathological conditions and injuries, accompanied by dysfunction of the retina, characterized in that the use of pharmacological tool for PP.1-11.

14. The method of treatment and prophylaxis for PCP.12 and 13, characterized in that the means for PP.1-11 parenteral use, inside retrobulbarno parabulbarno, subkonyunktivalno, locally, as well as ocular instillation, skin application and in physiotherapeutic effects in the composition of any of the dosage form or composition, as the only means of medicatie or in combination with other medicines.

15. The method of treatment according to PP.12-14, characterized in that used vehicle for PP.1-11 for the treatment of myopia.

16. The method of treatment according to PP.12-14, characterized in that used vehicle for PP.1-11 as a protector, to prevent retinal detachment.

17. The method of treatment according to PP.12-14, characterized in that used vehicle for PP.1-11 for the treatment of retinopathy of various etiologies.

18. The method of treatment according to PP.12-14, characterized in that used vehicle for PP.1-11 for the treatment of maculopathy.

19. The method of treatment according to PP.12-14, characterized in that used vehicle for PP.1-11 to relieve discomfort and Ustina period to prevent retinal detachment and higher vision.

21. The method of treatment according to p. 16, characterized in that the means for PP.1-11 are used during surgery to prevent retinal detachment and higher vision.

The method of treatment according to p. 16, characterized in that the means for PP.1-11 are used in the postoperative period to prevent retinal detachment and higher vision.

 

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