RU2694226C1 - Pharmaceutical composition for treating eye diseases accompanied by oxidative stress, and a method for use thereof - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: group of inventions refers to topical drugs for mono-and complex therapy of eye diseases accompanied by oxidative stress. Pharmaceutical composition for local application in the form of a suspension contains an active substance in form of nanoparticles of superoxide dismutase included in glutaraldehyde cross-linked, the activity of which is 50–500 kED/ml, in a buffer solution, as well as target additives and/or medicinal agents, wherein in nanoparticles, the enzyme is coated with an inner shell of protamine and an outer shell from a poly(glutamic acid) and polyethylene glycol block copolymer, wherein hydrodynamic radius of enzyme nanoparticles is 40–80 nm, concentration of nanoparticles in suspension is 0.5–5⋅10¹⁴ particles/ml, the buffer solution is isotonic with physiologic saline and has pH 7.4. Also disclosed is a method of using said composition, which consists in instillation in the conjunctival cavity of eye daily 2-3 times a day, duration of 1 week to the moment of completion of the inflammatory process.

EFFECT: group of inventions provides intensified antioxidative activity of superoxide dismutase.

5 cl, 6 dwg, 1 tbl, 5 ex

Description

The invention relates to topical drugs for mono - and complex therapy of eye diseases, accompanied by oxidative stress, and containing as an active substance the enzyme superoxide dismutase.

Under the oxidative stress understand the strengthening of free radical processes against the background of the weakening of its own antioxidant system with the depletion of a pool of endogenous antioxidants, which leads to damage to almost all structural and functional components of cells [Zenkov NK, Lankin VZ, Menshchikova EB Oxidative stress: Biochemical and pathophysiological aspects. 2001, M .: MAIK "Science / Interperiodica"]. At the same time, free radicals initiate lipid peroxidation, promote the formation of toxic substances, aggravate microcirculation disorders in the tissues, leading to the ischemia of tissues.

The development of almost all ocular pathologies is accompanied by the development of oxidative stress. [Ziangirova G.G., Antonova O.V. Lipid peroxidation in the pathogenesis of primary open-angle glaucoma (2003) Vestn. Ophthalmol., 4, 54-55; Yang X., Hondur G., Tezel G. Antioxidant treatment limits neuroinflammation in experimental glaucoma (2016); Beishenova G.A. Chesnokova N.B. The role of free radical oxidation in the pathogenesis of uveitis (Literature review) (2015) ROZH, 8, 99-106].

For the treatment of eye diseases accompanied by oxidative stress, antioxidants, antihypoxants, agents that have a regenerative effect and improve microcirculation are used [Rational pharmacotherapy in ophthalmology (2006) (edited by EA Egorov), Litterra Publishing House, Moscow, p. 652-657; Morozov V.I., Yakovlev A.A. (2009) Pharmacotherapy of eye diseases, MED Press Inform Publishing House, Moscow, p. 169-179], in particular, preparations of emoxipin, pyridoxine, echinochrome ("Histochrome").

There is a method of treating diseases of the eye, accompanied by oxidative stress (primary open-angle glaucoma, anterior uveitis, herpetic and adenoviral damage to the eyes, secondary keratopathy, injuries and burns of the eye), which involves instillation into the conjunctival cavity of an antioxidant preparation based on recombinant superoxide dismountase vectors (a group of atoxicoptomaceae). , Registration certificate LSR-006689/10 dated 15.07.10, and Erisod [Patent RU 2144343 C1]). However, the known drugs superoxide dismutase poorly penetrate into the internal structures of the eye.

A promising approach to solving the problem of drug delivery to intraocular tissues is the use of nanoparticles as drug delivery systems, since they significantly increase the bioavailability of the drug and allow you to create the necessary concentration of drug substance in the tissues. [Patent RU 2508123; Patent RU 2577236; Chesnokova N.B., Galitsky V.A., Beznos O.V., Beishenova G.A., Kost O.A., Nikolskaya I.I. Preparation of calcium-phosphate particles containing superoxide dismutase, and their effect on the inflammatory process in the eye in experimental uveitis in rabbits (2015) РЖ, 8, 31-36; Nikolskaya I.I., Beznos O.V., Galitsky V.A., Chesnokova N.B., Kost O.A. Calcium-phosphate particles containing superoxide dismutase - a promising drug for the treatment of eye diseases accompanied by oxidative stress (2016) Vestn. Mosk. Unta Ser. Chem., 57, 138-144; Kost OA, Beznos O.V., Davydova N.G., Manickam D.S., Nikolskaya I.I., Guller A.E., Binevski P.V., Chesnokova N..B., Shekhter A.B., Klyachko N.L., Kabanov A.V. Superoxide dismutase I nanozyme for treatment of eye inflammation (2016) Oxidative Medicine and Cellular Longevity, 2016, 5194239doi].

However, the cationic polymer used as a matrix in [Patent RU 2508123], namely, methoxy-poly (ethylene glycol) -poly (L-lysine) block copolymer, is potentially toxic, and biodegradable and biocompatible calcium phosphate particles [Patent RU 2577236] have relatively large hydrodynamic radii up to 350 nm, while for effective penetration into tissues particles up to 100 nm in radius are considered optimal.

As mentioned above, currently, for the treatment of eye diseases accompanied by oxidative stress (primary open-angle glaucoma, anterior uveitis, herpetic and adenoviral eye damage, secondary keratopathy, injuries and burns of the eye, pharmaceutical preparations are used to instil them into the conjunctival cavity, based on superoxide dismissed - “Reksod-OF”, (registration certificate LSR-006689/10 dated 07.15.10,) and “Erisod” [Patent RU 2144343 C1]).

The drug Rexod-OF and the method of its use we have chosen as a prototype both in the “composition” part and in the “method” part. The drug Rexod-OB is produced in the form of a lyophilisate of recombinant human superoxide dismutase. Before use, the contents of the bottle of 0.8 million ED diluted in 2 ml of water and the resulting solution (eye drops) stored in a refrigerator at 4-10 ° C for no more than 3 days. Treatment with this drug includes installing it into the conjunctival cavity in case of primary open-angle glaucoma, 6-7 times for 30 minutes twice a day, for adenoviral eye damage 6-8 times a day for at least 10 days.

The disadvantage of this drug and method of treatment with it is poor penetration into the internal structures of the eye. It is known that, on average, only about 5% of the injected drug substance is able to penetrate the cornea and reach the intraocular tissues [Ahmad I., Patton T. Importance of the ophthalmic drug delivery. (1985) Invest. Ophtalm. Vis. Sci., 26, 584-587]. As a result, the required concentration of the drug substance in the drug must either be increased or often produced by the drug installation (up to 8 times a day), which increases the undesirable side effects (both local and systemic) associated with the subsequent ingress of these drugs into the bloodstream.

The technical result of the claimed invention, both in the “composition” part and in the “method” part, is the enhancement of the antioxidant activity of superoxide dismutase and the enhancement of the anti-inflammatory therapeutic effect of superoxide dismutase in eye diseases accompanied by oxidative stress. This is expressed in reducing the healing time of inflammation, reducing the manifestations of the inflammatory process in both external and internal parts of the eye. In addition, the use of the proposed composition allows to reduce the frequency of drug administration.

This technical result in the “composition” part is achieved by the fact that a pharmaceutical composition for topical use in inflammatory eye diseases accompanied by oxidative stress is a suspension in a buffer, isotonic saline with a pH of 7.4, the active substance in the form of superoxide dismutase with activity 50- 500 kUd / ml incorporated into nanoparticles cross-linked with glutaraldehyde, in which the enzyme is coated with an inner shell of protamine and an outer shell of a poly block copolymer (glutamine to slots) and polyethylene glycol (PEG-SG), the hydrodynamic radius of the nanoparticles of the enzyme is 40-80 nm and the concentration of nanoparticles in the suspension is 0,5-5 ^⋅1014 particles / ml, the composition also comprises special additives and / or medicaments.

The composition may be in the form of eye drops or spray.

The composition may contain as a target additive a preservative.

The composition as a drug may contain antibiotics, and / or steroids and / or blockers.

Specified technical result in part "method" is achieved by producing installations in the conjunctival cavity of the eye of the pharmaceutical composition according to paragraphs. 1-4, containing an effective amount of superoxide dismutase at a dose of 1.5 to 15 cED daily, 2-3 times a day, lasting from 1 week until the end of the inflammatory process.

The invention is illustrated in table. 1 "The antioxidant activity of the drug Rexod, pharmaceutical composition and nanoparticles that do not contain an enzyme", which demonstrated that, while the drug Rexod had antioxidant activity only in relation to the superoxide anion radical, superoxide dismutase in the composition of the proposed pharmaceutical composition also shows activity in relation to hydroxyl radical. It is shown that polymers forming nanoparticles exhibit their own antioxidant activity with respect to both active forms of oxygen. However, the antioxidant activity of the pharmaceutical composition is even higher than the total activity of superoxide dismutase and empty particles. Table 1 also shows that the antioxidant activity of the claimed pharmaceutical composition is significantly higher than that of the Rexod preparation.

The invention is also illustrated in FIG. 1, which demonstrates the effect of superoxide dismutase (Rexod preparation) and the claimed pharmaceutical composition on the antioxidant activity in the tear fluid of rabbits (rabbit No. 2 - untreated; rabbit No. 6 - treated with superoxide dismutase; rabbit No. 12 - treated with the claimed pharmaceutical composition) after an alkaline cornea burn. Antioxidant activity in tears was determined by the chemiluminescent method in the model system hemoglobin - hydrogen peroxide - luminol, in which luminol acts as the oxidation substrate. The magnitude of antioxidant activity was expressed in Trolox equivalents, for which purpose a calibration curve was constructed for the dependence of the latent period of chemiluminescence on Trolox concentration according to [Vladimirov Yu.A. Free radicals and antioxidants (1998) Vestn. RAMS, No. 7, 43-51]. Statistical processing of the results was performed using the Mann-Whitney U-test. It is important that, unlike the superoxide dismutase solution, the antioxidant potential of a tear reduced in the acute period of the burn process is restored under the action of the declared pharmaceutical composition earlier (14 days against 21) and remains higher than in untreated animals. This suggests that superoxide dismutase in the composition of the pharmaceutical composition more effectively suppresses oxidative stress, which is the cause of increased formation of pro-inflammatory metabolites and damage to the structural and functional components of eye tissue.

Preparation of the pharmaceutical composition according to the invention: The pharmaceutical composition contains as an active substance an effective amount of superoxide dismutase incorporated into nanoparticles based on crosslinked protamine and poly (glutamic acid) and polyethylene glycol block copolymer according to the method [Patent RU 2575836; Application RU 2013 153 179] in the form of a suspension in an aqueous buffer solution, an isotonic saline solution with a pH of 7.4, which may also contain targeted supplements and drugs. The solution has a colorless color.

Eye drops were prepared as follows: The resulting suspension of nanoparticles was transferred to the required aqueous solution by repeated dilution and centrifugation on 50 kDa membrane systems; prior to use, the solution was sterile filtered through 0.22 μm Millipor filters.

Compliance was checked by:

1. The activity of superoxide dismutase, determined by the method [Kostyuk VA, Potapovich AI, Kovaleva Zh.V. A simple and sensitive method for determining the activity of superoxide dismutase, based on the reaction of oxidation of quercetin (1990) Top. Honey. Chemistry, 36, 88-91]. The method is based on determining the degree of inhibition of the oxidation of quercetin in the presence of superoxide dismutase. Per unit of superoxide dismutase activity, such an amount of enzyme in 1 ml of the reaction mixture was taken, which causes inhibition of the oxidation reaction of quercetin by 50%.

2. The pH of the solution (7.4), which was checked using a pH meter.

3. Electrophoretic mobility of the drug under non-denaturing electrophoresis in a 10% polyacrylamide gel according to the method [Laemmli U.K. Cleavage of Bacteriophage T4 (1970) Nature, 227, 680-685]. Coomassie blue R-250 was used for protein staining.

4. Nanoparticles in the composition were characterized by the method of analyzing the trajectories of nanoparticles. This method allows you to determine both the size and concentration of particles.

A comparative study of the therapeutic effect of superoxide dismutase in solution and superoxide dismutase in the composition of nanoparticles was carried out on models of the inflammatory process, accompanied by oxidative stress, in the internal (immunogenic uveitis - inflammation of the choroid eye) of the rabbits. There is a high degree of correlation between the severity of inflammation and oxidative stress. It is shown that superoxide dismutase significantly reduces the level of oxidative stress in the internal media of the eye while uvehte [Chesnokova NB, Neroev VV, Beznos OV, Beishenova GA, Nikolskaya II, Kost O .A., Binevsky P.V., Shekhter AB Oxidative stress in uveta and its correction with antioxidant enzyme superoxide dismutase (2014) Vestn. Ophthalmol., 30, 30-36]. When an eye burns, an inflammatory process develops, accompanied by a significant decrease in the antioxidant potential, which indicates the depletion of its own antioxidant system during oxidative stress [Gulidova OV, Lyubitsky OB, Klebanov GI, Chesnokova NB Changes in the antioxidant activity of the tear fluid in experimental burn eye disease (1999) Byul. Exp. Biol. Med., 128, 571-574].

The experiment involved 73 rabbits of the chinchilla breed weighing 2-2.5 kg. Work with experimental animals was carried out in accordance with the provisions of the Helsinki Declaration. The animals were kept under standard conditions. Laboratory animals before the start of the study contained 14 days to adapt. During this period, animals were monitored for signs of deviation from their health. Each animal was assigned an individual number (in accordance with the group number and sequence number). In the experimental groups, animals were randomly selected without external deviations.

Simulation of experimental uveitis.

In the study of the claimed pharmaceutical composition for the course of experimental uveitis of burn eye disease in rabbits, 38 animals (76 eyes) were used. In animals, immunogenic uveitis was reproduced by the method [Neroev VV, Davydova GA, Perova TS Simulation of immunogenic uveitis in rabbits (2006) Bull. Experiment. Biol. Honey., 142, 598-600]. Normal equine serum was administered twice to each rabbit, first subcutaneously in an amount of 5 ml, then, on day 10 after subcutaneous injection, a resolving dose of serum was administered in an amount of 70 μl to both eyes.

For treatment, the drug Reksod-OF (OOO NPP Enzyme Technologies, Russia) was used, which is a recombinant human superoxide dismutase 1 (400 cED in 1 mg) and the declared pharmaceutical composition with equal enzyme activity as monotherapy. The first group received Rexod instillations, the second group received the instillations of the declared pharmaceutical composition, the third group received a placebo, the fourth group received intact rabbits (for control indicators). The drugs were injected 3 times a day, 30 μl in both eyes for 8 days.

Clinical signs of inflammation were assessed by sequential analysis of external examination of the eye, biomicroscopy using a slit lamp, and ophthalmoscopy. Evaluation of the clinical manifestations of uveitis (conjunctival hyperemia, discharge, swelling of the eyelids, corneas and irises, fibrin and total protein content in the anterior chamber moisture) were carried out in conventional points on a four-point scale: no sign - 0, manifestations of a weak degree - 1, medium - 2 , strong - 3.

Example 1

Rabbits with immunogenic uveitis were instilled with solutions of superoxide dismutase (Rexod preparation) at a concentration of 50 kU / ml in phosphate buffer, pH 7.4, containing 0.15 M NaCl, suspension of the declared pharmaceutical ^composition with a concentration of nanoparticles of 0.5 ⁰¹⁰¹⁴ particles / ml and the same enzyme activity in phosphate buffer, pH 7.4, containing 0.15 M NaCl, as well as placebo-phosphate buffer, pH 7.4, containing 0.15 M NaCl.

Edema of the iris was evaluated on different days after the initiation of uveitis.

The data is presented in FIG. 2. Iris edema in the group that received instillations with the declared pharmaceutical composition decreased faster than in other groups.

Example 2

Rabbits with immunogenic uveitis were instilled with superoxide dismutase solutions (Rexod preparation) at a concentration of 50 kU / ml in phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride, suspension of the claimed pharmaceutical composition with a concentration of nanoparticles 0 , 5 ^⋅1014 particles / ml and with the same enzyme activity in phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride, and placebo - phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride.

The fibrin content in the inner chamber of the eye was evaluated on different days after the initiation of uveitis.

The data is presented in FIG. 3. The number of fibrin clots in the anterior chamber in animals treated with the instillations of the claimed composition was significantly less than in those that received instillations of the superoxide dismutase solution.

Example 3

Rabbits with immunogenic uveitis were instilled with superoxide dismutase solutions (Rexod preparation) at a concentration of 50 kU / ml in phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride, suspension of the claimed pharmaceutical composition with a concentration of nanoparticles 0 , 5 ^⋅1014 particles / ml and with the same enzyme activity in phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride, and placebo - phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.3 mg / ml benzalkonium chloride.

The total protein content in the inner chamber of the eye on the 8th day of uveitis was assessed.

The data is presented in FIG. 4. The total protein content in the acute period of uveitis in the anterior chamber in animals treated with the instillations of the claimed composition was significantly less than in those that received instillations of the superoxide dismutase solution. This indicates a decrease in the permeability of the blood vessels of the uveal tract and, consequently, a lesser severity of inflammation.

Simulation of the burn of the central part of the cornea.

When simulating an alkaline cornea burned in area and depth, d = 7 mm circles from thin cotton fabric were used, soaked in 10% NaOH solution, which were applied to the central area of the cornea for 40 seconds, then washed with physiological saline in 20 ml portions. For anesthesia, a 2.5% solution of aminazine was used in combination with a 0.5% solution of seduxen (1: 1), which was administered subcutaneously (0.5 ml / kg of weight). At the same time, local anesthesia was performed by instillation of a 0.5% solution of dikain on the cornea. In the experiment, 35 rabbits (70 eyes) were used.

For treatment, the drug Rexod-OF and the obtained supercomplex-based pharmaceutical composition in nanoparticles obtained by us were used as complex therapy with simultaneous topical application of eye drops based on a 2.5% solution of the antibiotic levomycetin 5 times a day. The first group received Reksod instillations on the background of local treatment with chloramphenicol, the second group received instillations of the declared pharmaceutical composition on the background of local treatment with chloramphenicol, the third group received a placebo on the background of treatment with chloramphenicol, the fourth group - intact rabbits (for control indicators). The drugs were injected 2 times a day, 30 μl in both eyes for 21 days.

Example 4

Rabbits with an alkaline corneal burn were instilled with superoxide dismutase solutions (Rexod preparation) at a concentration of 500 kU / ml in phosphate buffer, pH 7.4, containing 0.15 M NaCl, suspension of the claimed pharmaceutical ^composition with a concentration of nanoparticles of 5 ^⋅014 particles / ml and with the same enzyme activity in phosphate buffer, pH 7.4, containing 0.15 M NaCl, as well as placebo - phosphate buffer, pH 7.4, containing 0.15 M NaCl. All rabbits also received instillation of chloramphenicol solution.

The area of the cornea defect was evaluated on different days after the burn.

The data is presented in FIG. 5. Under the action of the claimed pharmaceutical composition, the area of the cornea defect is smaller and shrinks faster than with treatment with superoxide dismutase.

Example 5

Rabbits with an alkaline corneal burn were instilled with superoxide dismutase solutions (Rexod preparation) at a concentration of 500 kU / ml in phosphate buffer, pH 7.4, containing 0.15 M NaCl, suspension of the claimed pharmaceutical ^composition with a concentration of nanoparticles of 5 ^⋅014 particles / ml and with the same enzyme activity in phosphate buffer, pH 7.4, containing 0.15 M NaCl, and placebo - phosphate buffer, pH 7.4, containing 0.15 M NaCl. All rabbits also received instillation of chloramphenicol solution.

The intensity of corneal neovascularization was evaluated on different days after the burn.

The data is presented in FIG. 6. It is important that under the action of the proposed pharmaceutical composition, faster and more intensive neovascularization of the cornea occurs in the acute stage of the process, which ensures that the blood components necessary for tissue repair are brought into the affected area. It is important that after the drug is discontinued, the desolation of newly formed vessels occurs more quickly, which is necessary for the formation of less coarse scar tissue.

Thus, the examples show that the introduction of superoxide dismutase into nanoparticles and the local application of the claimed pharmaceutical composition enhances the positive effect of the enzyme on the inflammatory process in both external and internal structures of the eye, and also more effectively reduces the number of free radicals during inflammation.

Considering that the physiological features of the rabbit and human eyes are identical, this pharmaceutical composition can be recommended for use both in veterinary medicine and in the clinic.

In this study, we compared the same modes and doses of superoxide dismutase in solution and superoxide dismutase in the composition of nanoparticles. The data obtained, indicating a greater therapeutic efficacy of the enzyme in the composition of nanoparticles, suggest that it can be used less often and in a lower dose than the drug in a simple solution. At the same time, the choice of dose and mode of administration should depend on the severity, location, nature of the pathology. The indications for the use of superoxide dismutase in the composition of the proposed pharmaceutical composition is a wide range of eye diseases, in the pathogenesis of which an important role is played by the enhancement of free radical oxidation processes. These are inflammatory processes of any etiology and localization, neurodegenerative processes in the retina, including glaucoma and age-related dystrophy of the retina, diabetic retinopathy, prevention of cataracts.

Claims (5)

1. Pharmaceutical composition for topical use in inflammatory diseases of the eye, accompanied by oxidative stress, in the form of a suspension containing the active substance in the form of superoxide dismutase nanoparticles incorporated into glutaraldehyde-crosslinked nanoparticles, whose activity is 50-500 cED / ml, in a buffer solution, as well as targeted additives and / or drugs, while in nanoparticles the enzyme is coated with an inner shell of protamine and an outer shell of a block copolymer of poly (glutamic acid) and polyethyleneglass glycol (SG-PEG), the hydrodynamic radius of the enzyme is 40-80 nm nanoparticles, the nanoparticle concentration in the suspension is 0,5-5⋅10 ¹⁴ particles / ml, a buffer solution and isotonic physiological solution has a pH of 7.4. 2. The composition according to p. 1, characterized in that it is made in the form of eye drops or spray. 3. The composition according to p. 1, characterized in that as the target additive it contains a preservative. 4. The composition according to p. 1, characterized in that as a medicine it contains antibiotics and / or steroids and / or blockers. 5. The method of applying the composition according to claim 1, consisting in instillation in the conjunctival cavity of the eye of the pharmaceutical composition according to claims. 1-4, containing an effective amount of superoxide dismutase at a dose of 1.5 to 15 cED daily, 2-3 times a day, lasting from 1 week until the end of the inflammatory process.

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