RU2352352C1 - Pharmaceutical composition for preventive maintenance of development and treatment of initial stage of age cataract (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns pharmaceutical compositions for preventive maintenance of development and treatment of an initial stage of an age cataract. The pharmaceutical composition (versions) contains the active beginning an admixture of two short-chain peptides and the suitable pharmaceutical carrier. In the first version - admixture of D-pantethine and N-acetylcarnosin at the following concentration of components: D-pantethine-not less than 0.001%, N-acetylcarnosin - not less than 0.001%, a normal saline solution - to 100%. In the second version - admixture D-pantethine and L-carnosin at the following concentration of components: D-pantethine - not less than 0.001%, L-carnosin - not less than 0.001%, a normal saline solution - to 100%.

EFFECT: high efficiency of the offered pharmaceutical composition is caused by that the components entering into composition; selectively protect different fibers of the lens of an eye.

2 cl, 7 dwg

Description

The invention relates to the field of medicine, veterinary medicine and pharmacology, specifically to a composition for the prevention of development and treatment of the initial stage of age-related cataract.

According to the WHO, cataracts account for 47.8% of all ocular pathology [World Health Organization Fact Sheet No. 282; WHO November 2004; available at www.who.int/mediacentre/factsheets/fs282/en/index.html]. Although surgical methods for treating this pathology are well developed, adverse effects also take place and are described in detail in the article [B. Malyugin. Cataract Surgery and Intraocular Aphakia Correction: Achievements, Problems, and Prospects for Development, Ophthalmology Bulletin, No. 1, p. 37- 41, 2006.]. In this regard, the search for conservative methods to prevent the development and treatment of cataracts, that is, the search for anticataract drugs, remains an urgent task.

The lens of the human eye and vertebrate animals is 90% composed of alpha, beta and gamma crystallins. The content of these proteins in the lens is approximately the same, however, they differ significantly in structure and in molecular weight. Alpha crystallins are chaperones and protect beta and gamma crystallins, which are most sensitive to damaging effects. It is believed that with age, the protective properties of alpha crystallin weaken and are associated with the development of senile cataracts.

Exposure to various denaturing factors, such as ultraviolet (UV) radiation or high temperatures, can cause crystalline aggregation. As a result of aggregation, large light-scattering conglomerates are formed, which lead to clouding of the lens, that is, to the development of cataracts.

Known tools based on short-chain peptides: either D-pantetin, or L-carnosine, or its derivative N - acetylcarnosine, for which patents were obtained as anticataract drugs.

So known aqueous solutions of N-acetylcarnosine, its derivatives or salts and the method of prevention and treatment of cataracts with their use (international application WO 2004/028536 A1, IPC ⁷ A61K 31/4164, publ. 08.04.2004).

Chemical retardation or reversal of cataract development by phase separation inhibitors is known from US Pat. No. 5,091,421, Chemical prevention or reversal of cataract by phase separation inhibitors, IPC ⁷ A61K 31/40, 31/66, publ. 02/25/1992).

The main disadvantage of the known pharmaceutical compositions for the treatment of the initial stage of age-related cataracts is that they do not have sufficient effectiveness.

The closest analogue to the proposed pharmaceutical composition are eye drops containing a short-chain peptide L-carnosine, hydrochloric acid and water, which delay the opacification of the lens of the eye and are used to treat the initial stage of age-related cataracts (patent RU 2071316 C1, IPC ⁷ A61K 9/00, 31/415 , published on January 10, 1997). This drug is used in the form of a 1-5% aqueous solution of carnosine. The preparation contains:

carnosine - 1.0-5.0 g,

concentrated hydrochloric acid - up to pH 6.5-7.2,

water - up to 100 ml.

The disadvantage of the closest analogue is the pharmaceutical composition for the prevention of development and treatment of the initial stage of age-related cataracts is its lack of effectiveness. In addition, it is known that an aqueous solution based on only one carnosine does not penetrate the lens sufficiently effectively. If such a solution is introduced in the form of instillations into the eye, then in aqueous humor on the way from the cornea to the lens, it will be destroyed by the enzyme carnosinase. In this case, histidine is formed, which easily turns into a histamine mediator of allergic reactions, which when administered from the outside, has the same effect as any allergen [Mark A. Babizhayev, Anatoly I. Deyev, Valentina N. Yermakova, Valerii V. Remenshchikov and Johan Bours , "Revival of the Lens Transparency with N-Acetylcarnosine", Current Drug Therapy, v.1, p.91-116, 2006].

The objective of the invention is the creation of more effective than known, drugs suitable for preventing or slowing down the process of clouding of the lens of the eye, that is, for the prevention of development and for the treatment of the initial stage of age-related cataract.

The problem is solved in that the proposed pharmaceutical composition for preventing the development and treatment of the initial stage of age-related cataract, as well as the closest analogue, contains at least one short-chain peptide.

New in the proposed pharmaceutical composition is that it contains as an active principle a mixture of two short chain peptides and a suitable pharmaceutical carrier. In the first embodiment, a mixture of D-pantetin and N-acetylcarnosine with the following concentration of components:

D-pantetin - not less than 0.001%,

N-acetylcarnosine - not less than 0.001%,

saline solution - up to 100%.

In the second embodiment, a mixture of D-pantetin and L-carnosine with the following concentration of components:

D-pantetin - not less than 0.001%,

L-carnosine - not less than 0.001%,

saline solution - up to 100%.

The content of the active principle in the composition is from 0.002% to 10%, and the content of each of the short chain peptides is from 0.001% to 5%. The carrier may be a liquid material, acting as a carrier, excipient, or medium for the active component. An example of a suitable carrier is saline.

The increase in the effectiveness of the anti-cataract properties of the two proposed variants of the pharmaceutical composition in comparison with the known preparations is based on the new mechanism of action of the active principle of the created pharmaceutical composition revealed by the authors.

The preferred dosage form for this composition may be drops, and the proposed method for their use is installation in the eye. Another possible way to use variants of the composition may be intraperitoneal injection.

The authors tested the proposed variants of the composition in vitro in vitro and in experimental animals in vivo at various concentrations of the active principle. The lowest concentration is determined by the boundary of the manifestation of the positive effect, and the maximum concentration is determined by the potential toxicity of the preparations and the high viscosity of the solution.

The following are the components that make up the proposed pharmaceutical compositions:

1) in the first embodiment, a mixture of short-chain peptides:

N-acetylcarnosine of the general formula:

and D-pantethine of the general formula:

2) in the second embodiment, a mixture of short-chain peptides of L-carnosine of the General formula

and D-pantetin, which are pharmaceutical compositions for the prevention of development and treatment of the initial stage of age-related cataract.

Obtained during testing of the proposed compositions in vitro and in vivo experimental dependencies are presented in figures 1-7.

Figure 1 shows the dose dependences of the power of the probe radiation scattered in the beta-crystallin solution (λ = 633 nm) from the ultraviolet (UV) dose when various short-chain peptides are added to the solution.

Figure 2 presents the dose dependences of the power of the test radiation scattered in a mixture of alpha and beta crystallins from the UV dose when various short-chain peptides and the proposed composition are added to the solution.

Figure 3 shows the dependence of the cataract development rate (points) in the lens in different groups of experimental animals - rats (control group; UV-irradiated; UV-irradiated, which received the composition in the form of eye drops) on the time of observation (evaluation number).

Figure 4 presents the dependence of the cataract development rate (points) in the lens in different groups of experimental animals - rats (control group; UV-irradiated; UV-irradiated, which received the composition in the form of intraperitoneal injections at a concentration of 150 mg / kg) from the time of observation (evaluation number).

Figure 5 presents the dependence of the cataract development rate (points) in the lens in different groups of experimental animals - rats (control group; UV-irradiated; UV-irradiated, which received the composition in the form of intraperitoneal injections at a concentration of 25 mg / kg) from the time of observation (evaluation number).

Figure 6 shows the dependences of the cataract development rate (points) in the anterior cortical lens layers of different groups of experimental animals - rats (control group; UV-irradiated; UV-irradiated, which received the composition in the form of eye drops; UV-irradiated, which received composition in the form of intraperitoneal injections at a concentration of 25 mg / kg or 150 mg / kg) from the time of observation (days).

Figure 7 presents the dependences of the cataract development rate (scores) in the middle nuclear layers of the lens in different groups of experimental animals - rats (control group; UV-irradiated; UV-irradiated, which received the composition in the form of eye drops; UV-irradiated, which received composition in the form of intraperitoneal injections at a concentration of 25 mg / kg or 150 mg / kg) from the time of observation (days).

The high effectiveness of the proposed pharmaceutical compositions (options) for the prevention of development and treatment of the initial stage of age-related cataracts is due to the fact that their components, as established by the authors, selectively protect different lens proteins of the eye.

The authors found that L-carnosine and N-acetylcarnosine significantly slow down the rate of aggregation of beta-crystallin, while the effect of D-pantetin on aggregation of beta-crystallin is practically absent (see figure 1). However, D-pantethine slows the photoaggregation of a mixture of beta and alpha crystallins and a mixture of beta, alpha and gamma crystallins by maintaining the chaperone-like (protective) properties of alpha crystallin (see figure 2). Based on these studies, the authors selected short-chain peptide compositions with chaperone-like activity.

The advantages of the drug created by the authors and the results of its tests, which showed a positive effect in slowing down UV-induced cataracts in rats, are described in the works:

1) Soustov L.V., Chelnokov E.V., Sapogova N.V., Bityurin N.M., Nemov V.V., Karpova O.E., Sheremet N.L., Polunin G.S., Avetisov S.E., Ostrovsky M.A. Investigation of the effect of chaperone-like (protective) activity of short-chain peptides on the rate of beta-crystallin aggregation of UV-induced excimer laser // Bulletin of Ophthalmology, (accepted).

2) Avetisov S.E., Polunin G.S., Sheremet N.L., Makarov I.A., Fedorov A.A., Karpova O.E., Muranov K.O., Timofeeva A.K., Soustov L.V., Chelnokov E.V., Bityurin N.M., Sapogova N.V., Nemov V.V., Boldyrev A.A., Ostrovsky M.A. Studying the effects of a mixture of di- and tetra-peptides on a “prolonged” model of ultraviolet-induced cataract in rats // Bulletin of Ophthalmology, (accepted)

The proposed compositions more effectively slow down the aggregation of a mixture of crystallins forming the lens of the eye than L-carnosine, N-acetylcarnosine or D-pantetin separately (Fig.1,2). Based on each of these substances, anti-cataract preparations have previously been proposed [see analogues of the above patents: RU 2071316; US 5091421; WO 2004/028536], however, compared with L-carnosine or N-acetylcarnosine, the effectiveness of the proposed compositions is 1.5 times higher, and 2.5 times higher than that of D-pantetine.

Preclinical trials of the proposed compositional options in rats showed the absence of acute and chronic toxicity, allergenic, irritating, mutagenic and teratogenic effects, as well as good tolerance to high doses of drugs. According to the available classification, these compositions belong to harmless substances.

The invention is as follows.

The following examples, carried out both in vitro and in vitro in experimental animals, testify to the influence of the developed pharmaceutical composition on slowing down the aggregation of crystallins forming the lens of the eye.

Example 1

Proteins of the lens of the eye of a bull alpha, beta and gamma crystallin, as well as D-pantetin purchased in the company "Sigma", (USA). L-carnosine and N-acetylcarnosine were purchased from Hamari Chemicals Ltd., (Japan).

Proteins in an amount of 0.5 mg / ml were dissolved in phosphate buffer (0.1 M NaCl, 10 mm Na ₂ NRA ₄ , 3 mm K ₂ NRA ₄ , pH 7.2) and passed through membrane filters with pore size 0 , 45 microns and 0.22 microns (firm "Sartorius", Germany). To eliminate insoluble impurities, the test tube with the solution was centrifuged for 15 minutes at room temperature with an acceleration of 5000 g. In the study of a mixture of crystallins, beta-L and alpha-crystallin were used in a 1: 1 ratio.

An excimer pulsed XeCl laser LPX-200 (Lambda Physik, Germany), the radiation wavelength of which was λ = 308 nm, was used as a source of UV radiation.

The laser irradiation mode was used, which is closest to the conditions of illumination of the lens with natural sunlight. The experiments were carried out at a temperature of (22 ± 1) ° C, and the heating of the solution during irradiation did not exceed 2 ° C. The degree of protein aggregation was estimated by the value of the probe power of the HeNe laser scattered in the solution (λ = 633 nm). The height of the quartz cuvette was 10 mm; the length along the HeNe and XeC1 laser beams was 10 mm and 5 mm, respectively. To study the dynamics of protein aggregation, the dependences P (D), the power P of the probe radiation scattered in the protein solution with λ = 633 nm, were measured as a function of the UV exposure D (i.e., the dose of UV irradiation in J / cm ² ). The parameter characterizing the aggregation rate is the exposure value of UV radiation (dose), with which the light scattering of the probe radiation in the solution begins to increase. We denote this parameter as D *.

The results of exposure to beta-crystallin UV-susceptibility of the same concentrations of D-pantetin, L-carnosine or N-acetylcarnosine individually are shown in FIG. It can be seen from the experimental dependences that L-carnosine and N-acetylcarnosine significantly slow down the beta-crystallin photoaggregation, and the degree of deceleration of aggregation by these peptides is the same up to a measurement error, and the addition of D-panthenin practically does not lead to a decrease in D *, i.e. on the rate of aggregation of beta-crystallin.

Example 2

Figure 2 shows the change in the photoaggregation rate of a mixture of alpha and beta crystallins with the addition of the same concentrations of D-pantetin, or L-carnosine, or N-acetylcarnosine, as well as only one of the proposed compositions, namely, a mixture of D-pantetin with N- acetylcarnosine, since the effect of another composition is the same with an accuracy of measurement errors. From a comparison of the exposures at which scattering of the probe beam in a mixture of alpha and beta crystallins begins and when peptides or their compositions are added to the solution, the better protective effect of the proposed compositions is clearly seen in comparison with the action of L-carnosine, N-acetylcarnosine or D- pantetin separately.

Examples 1 and 2 show the selective effect of short chain peptides on the rate of crystalline photoaggregation. If L-carnosine or N-acetylcarnosine directly slows down the aggregation of beta-crystallin, then D-pantetin slows down the aggregation of a mixture of beta-and alpha-crystallins, that is, it supports the chaperone (protective) activity of alpha-crystallin. Based on these results, the proposed compositions were created: a mixture of D-pantetin with N-acetylcarnosine or a mixture of D-pantetin with L-carnosine, which slow down the photoaggregation of crystalline lens of the eye more than L-carnosine, or N-acetylcarnosine, or D- pantetin separately.

Example 3

Studies of the anticataract effect of the proposed compositions in an in vivo experiment on the development of UV-induced cataracts in rats were carried out. The experiment was conducted on 33 rats of male Wistar species (66 eyes) aged 20 to 23 days with a body weight of 39-41 g. The observation period was 10 months (43 weeks). All animals were randomly divided into 5 groups.

The first group consisted of 8 animals, which initiated the formation of UV cataract, but did not introduce the proposed composition (preparations).

The second (control) group consisted of 7 rats that were not exposed to UV radiation and did not receive drugs.

In the 3rd group (6 animals, 12 eyes), the irradiated animals received preparations in the form of installations in the eye of a 5% solution of each peptide from the proposed combinations.

In the 4th group (6 animals, 12 eyes), the irradiated animals received preparations in the form of intraperitoneal injections at a dose of 25 mg / kg of each peptide from the proposed combinations.

In the 5th group (6 animals, 12 eyes), the irradiated animals received a peptide composition in the form of intraperitoneal injections at a dose of 150 mg / kg of each peptide from the proposed combinations.

Installations and intraperitoneal injections were performed once every day during the entire observation period (10 months).

The development of UV cataract was initiated using two ultraviolet quartz irradiators OUF-K-01 "Sun" (λ = 280-400 nm) located at a distance of 0.3 m above the cage with animals. Sessions of exposure lasting 16 minutes were carried out every other day. For wavelengths of 313 and 366 nm, the radiation intensity of the irradiators according to Parker and Hutchard was 23.7 ± 4.4 ¹ and 21.5 ± 4.0 J / (s · m ² ), respectively. ( ¹ Data are presented in the form M ± σ, where M is the average value of intensity, σ is the standard deviation.)

A subjective assessment of the dynamics of cataract development was carried out using the method of expert estimates on digital photographs of the lens. The study was performed by three independent experts. When analyzing photographs, we used our own rating scale.

A biomicroscopic study of the lenses (expert assessment method) revealed the dynamics of the formation of lens opacities in the animals of the studied groups during the experiment. So, already after the first assessment, carried out a month after the start of irradiation, statistically significant differences between the 1st (irradiation) and 2nd (control) group were revealed. The significance of differences was maintained throughout the observation period. By the end of the experiment, the formation of a moderate severity of a uniform cloud-like cloudiness of the lens, more intense in the nuclear layers (cataract 5 points), was observed in the irradiated group, while in the control group the lens retained its transparency to a much greater extent (cataract 2-3 points).

In the main groups receiving the proposed composition on the background of UV radiation, the dynamics of the increase in opacities in the lenses were not the same (see Figs. 3, 4, 5). The earliest statistically significant differences compared with the irradiated (1st) group occurred in groups 4 and 5 (irradiation and administration of 25 mg / kg and 150 mg / kg of the drug, respectively, intraperitoneally) already at the 2nd month of the experiment. However, by the end of the experiment, on day 313, a more pronounced anti-cataract effect of the proposed compositions was obtained in group 3 (irradiation and drug installations). At the last observation, cataract in this group corresponded to 2-3 points. At the end of the experiment, group 4 was rated 3 points in group 4, and 3-4 points in group 5.

Example 4

An objective assessment of the dynamics of the development of lens opacities was carried out by microdensitometry of biomicroscopic sections of the lens. Biomicroscopy was performed on a slit lamp SL-75 (Opton, Germany) with a tilt angle of the illuminator 45 ° and the opening of the diaphragm 0.1 mm. Studies were performed monthly. In the course of the study, a degree of severity of opacities in the anterior cortical layers, in the anterior, middle, and posterior third of the nucleus and posterior cortical layers of the lens was quantified.

After 10 months, the animals were removed from the experiment by an overdose of chloroform anesthesia according to the "Statement for the Use of Animals in Ophthalmic and Visual Research" [7]. The lenses were fixed and semi-thin sections were made according to a standard technique. Light-optical and morphometric studies of the anti-cataract effect of the preparations were carried out on a Photomicroscope III (Opton, Germany) using the hardware-software complex of automatic morphodensitometry of the DiaMorf company (DiaMorf Objective).

Microdensitometric analysis data allowed to obtain objective biomicroscopic examination results (see Fig.6, 7). In animals of the 1st group (irradiation and lack of treatment), there was a progressive increase in the optical density index (POP) of the lens layers during the entire observation period, and the growth of this indicator was most pronounced in the first 3 months. Within 3-10 months from the start of the experiment, the optical density growth curve has a linear function graph. By the end of the observation period, the maximum POP values were noted in the nuclear layers (an increase of 117 ± 5 units). In the cortical layers of the lens, the growth rate was slightly less and reached 97 ± 3 units.

During the entire observation period, the 2nd (control) group was diagnosed with a slight uniform increase in POP in all layers (an increase of 37 ± 4 units), which may be evidence of the natural process of development of age-related lens changes in Wistar rats. Compared with the 1st group, differences in indices are highly significant at each observation (p <0.001).

In the groups of animals receiving the proposed composition, the dynamics of changes in the optical density index was as follows (see Fig.6, 7):

Group 3 (irradiation and drug installations) - the growth schedule of POP in the cortical layers and the lens nucleus almost completely repeats the curve of the 2nd group (control). Group 4 (irradiation and administration of 25 mg / kg of the drug intraperitoneally) - the values of the optical density index in dynamics exceed the corresponding values in the control group. For all observations, the value of POP with a high degree of confidence is lower than in group 1 (irradiation and lack of treatment) and higher than in the control (p <0.001).

Group 5 (irradiation and administration of 150 mg / kg of the drug intraperitoneally) - the characteristic curve of the change in POP corresponds to the 4th group. Although the POP value exceeds the value of this indicator in animals of the 4th group, however, intergroup differences at all points are statistically unreliable (p> 0.05).

Thus, experiments using the developed pharmaceutical composition, carried out both in laboratory conditions and in experimental animals, showed a more effective slowdown in the aggregation of crystallines of the lens of the eye than using known analogues.

Claims (2)

1. A pharmaceutical composition for preventing the development and treatment of the initial stage of age-related cataract, containing at least one short-chain peptide, characterized in that, as an active principle, it contains a mixture of two short-chain peptides D-pantetin and N-acetylcarnosine at the following concentration of components:
D-pantetin not less than 0.001% N-acetylcarnosine not less than 0.001% saline up to 100% 2. A pharmaceutical composition for preventing the development and treatment of the initial stage of age-related cataract, containing at least one short-chain peptide, characterized in that as an active principle it contains a mixture of two short-chain peptides D-pantetin and L-carnosine at the following concentration of components:
D-pantetin not less than 0.001% L-carnosine not less than 0.001% saline up to 100%

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