Pharmaceutical composition for use in medical and veterinary ophthalmology

Abstract

The invention relates to the manufacturing and use of pharmaceutical compositions of medicines (ophthalmic preparations) comprising a mitochondria-addressed antioxidant and a set of auxiliary substances providing effective treatment for ophtalmological diseases in humans and animals.

Claims

1. A pharmaceutical formulation for treatment or prophylaxis of eye pathology, comprising: (a) from 1 nm to 25000 nm of a mitochondria-addressed antioxidant comprising: ##STR00010## (b) from 0.01% to 0.2% of a lipophilic, cationic concentration stabilizer that stabilizes a concentration of the mitochondria-addressed antioxidant by preventing reversible and irreversible absorption of the antioxidant to walls of a vial containing the pharmaceutical composition, the stabilizer comprising a benzalkonium salt, berberine, palmatine, tetraphenylphosphonium, tetrabutyl ammonium, or combinations thereof; and (c) from 0.001% to 1% of a prolongator comprising a disaccharide, a trisaccharide, a polysaccharide, methylcellulose, hydroxyethylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, sodium chondroitin sulfate, sodium hyaluronate, carboxyvinyl polymer, polyvinyl ethanol, polyvinylpyrrolidone, macrogol, or combinations thereof.

2. The pharmaceutical formulation of claim 1, wherein the mitochondria-addressed antioxidant is SkQ1-Br, SkQ1-C1, or SkQ1.sub.2-SO.sub.4.

3. The pharmaceutical formulation of claim 1, further comprising a physiologically acceptable pH buffer comprising a phosphate buffer, an acetate buffer, a borate buffer, a carbonate buffer, a citrate buffer, a Tris buffer, a glutamine buffer, an epsilon-aminocaproic acid buffer, or combinations thereof.

4. The pharmaceutical formulation of claim claim 1, wherein the water soluble cellulose derivative comprises methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium chondroitin sulfate, sodium hyaluronate, or combinations thereof.

5. The pharmaceutical formulation of claim 1, further comprising an isotonic component comprising sodium chloride, calcium chloride, glycerol, mannitol, sorbitol, boric acid, glucose, propylene glycol, or combinations thereof.

6. The pharmaceutical formulation of claim 1, further comprising an active ingredient different than the mitochondria-addressed antioxidant, the additional active ingredient being an antioxidant, an antiseptic, an astringent, a cautery, a resorbent, a hyposensitizer, an anti-biotic, an anti-inflammatory preparation, an antibacterial, a sulfanilamide preparation, an antiviral preparation, a non-specific anti-inflammatory, a reparative preparation, a local anesthetic, a systemic ophthalmologic preparation, an anti-cataract preparation, a hypotensive anti-glaucoma medical preparation, a cholinomimetic preparation, an anticholinesterase preparation, a sympathomimetic, an adrenergic preparation, an antiadrenergic preparation, a beta-adrenergic blocker, a prostaglandin, a carbonic anhydrase inhibitor, a mydriatic, a vitamin, and a vitamin analog, a natural cornea moisture restorer, or combinations thereof.

7. The pharmaceutical formulation of claim 1 comprising, per 5 ml of solution: about 770 ng SkQ1 bromide; about 4.4 mg sodium dihydrophosphate; about 4.7 mg sodium hydrogen phosphate dodecahydrate; about 10 mg hydroxypropylmethylcellulose; about 5 μg benzalkonium chloride; about 45 mg sodium chloride; and purified water to 5.0 ml.

8. The pharmaceutical formulation of claim 1, wherein the lipophilic, cationic concentration stabilizer comprises benzalkonium chloride, benzethonium chloride, berberine, palmatine, tertraphenylphosphonium, tertrabutyl, ammonium, or combinations thereof.

Description

EXAMPLES

Example 1

Stability of Active Ingredient in Pharmaceutical Composition (Composition 1)

(1) In this stability study of aqueous solutions of SkQ1 (10 mM sodium phosphate, pH 6.5, 0.9% NaCl) in concentration ranges up to 250 nM, the addition of 0.01% benzalkonium chloride prevents a decrease in the concentration of active ingredient during storage for 24 hours at room temperature (see Table 2).

(2) TABLE-US-00002 TABLE 2 Stability of SkQ1 in Solutions Containing Benzalkonium Chloride Determined Concentration of Added concentration Relative benzalkonium concentration of SkQ1 in 24 content chloride, % of SkQ1, nM hours, nM of SkQ1, % 0 (control) 250 104 42 0.01 250 254 102 0.01 180 180 100 0.01 100 97 97 0.01 50 56 112

(3) These data show that in the absence of benzalkonium chloride, the concentration of SkQ1 decreases by 58%. This phenomenon is completely prevented by addition of 0.01% benzalkonium chloride, irrespective of the initial concentration of SkQ1.

(4) A comparison of the effect of the two prolongators used in eye drops, methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), on the stability of aqueous solutions of SkQ1 (10 mM sodium phosphate, pH 6.5, 0.9% NaCl, 0.01% benzalkonium chloride) shows that only the addition of 0.2% HPMC stabilizes the concentration of the active ingredient during storage for 50 days at room temperature (see Table 3).

(5) TABLE-US-00003 TABLE 3 Stability of SkQ1 in Solutions With Different Prolongators Relative content of Content of SkQ1 after incubation prolongators for 50 days, % 0.2% HPMC 93 0.2% MC 79 control 83
Materials and Method for Quantitative Detection of SkQ1

(6) Analysis is carried out by high-performance liquid chromatography with MS/MS detection.

(7) The following is placed in a flask with a capacity of 1000 ml: 0.78 ml (920 mg) of formic acid, 50 ml of acetonitrile, and the volume is adjusted to the mark with Milli-Q water and mixed thoroughly. The solution is effective for one week.

(8) The following is placed in a flask with a capacity of 1000 ml: 0.78 ml (920 mg) of formic acid, and the volume is adjusted to the mark with acetonitrile and is mixed thoroughly. The solution is effective for one week.

(9) A. 0.5 M Solution of 3-chloroperbenzoic Acid in Ethanol.

(10) 12.4 g of 3-chloroperbenzoic acid is placed in a flask with a capacity of 100 ml. 50 ml of 96% ethanol is added and the mixture stirred. The solution is brought to volume with 96% ethanol and stirred.

(11) B. Internal Standard Solution.

(12) 12.65 mg of SkQ1 labeled with 15 atoms of deuterium (SkQ1-d15) is dissolved in 1 ml of ethanol 96% and transferred quantitatively into a flask with a capacity of 100 ml. The volume is adjusted to the mark with 96% ethanol and mixed thoroughly (solution 1). The solution is stored at −80° C. for up to 1 year. 0.5 ml of the resulting solution is transferred to a flask with a capacity of 100 ml, and the volume is adjusted to the mark with ethanol 96%. The solution is stored at a temperature of −24° C. for up to 3 days.

(13) C. Original Standard Solution.

(14) 50 mg of SkQ1 bromide is dissolved in 1 ml of 96% ethanol and transferred quantitatively into a flask with a capacity of 50 ml. The volume of solution is adjusted to the mark with the same solvent (solution 1). This solution is stored at −80° C. for up to 3 months. 1 ml of the solution is transferred to a flask with a capacity of 10 ml, and the volume is adjusted to the mark with ethanol 96% (solution 2). 0.77 ml of solution 2 is transferred to a flask with a capacity of 50 ml, and the volume is adjusted to the mark with ethanol 96% (original standard solution). The solution 2 and original standard solution is stored at a temperature of −24° C. for up to 3 days.

(15) D. A Solution of Standard Sample (SS).

(16) 1 ml of the original standard solution is placed into a flask with a capacity of 10 ml and the volume is adjusted to the mark with water-ethanol solution (solution A). 0.1 ml of the original standard solution is placed into a flask with a capacity of 10 ml. 5 ml of the water-ethanol solution is added and stirred for 11 min. 0.5 ml of solution A is added to 0.1 ml of an 0.5 M solution of 3-chloroperbenzoic acid in ethanol, and the volume is adjusted to the mark with ethanol-water solution and stirred for 30 min. 1 ml of the derived solution is transferred into a chromatographic vial. The solution must be freshly prepared before use.

(17) E. Water-ethanol Solution.

(18) Equal volumes of 96% ethanol and water are mixed.

(19) F. Test Solution.

(20) 100 μL of the internal standard solution is placed into a 10 ml flask. 5 ml of the water-ethanol solution is added and stirred for 11 min. 500 μL of sample solution is added to 0.1 ml of a 0.5 M solution of 3-chloroperbenzoic acid in ethanol, and the volume is adjusted to the mark with ethanol-water solution and stirred for 30 min. 1 ml of the resulting solution is transferred into a chromatographic vial.

(21) The chromatographic conditions are as follows: The device used is a Waters ACQUITY HPLC®. The column used is steel, 2.1×50 mm, filled with sorbent Acquity BEH C18, 1.7 μm (Waters, P/N 186002350) or equivalent. The column temperature is 35° C. The mobile phase is eluent A: 20 mM solution of formic acid in 5% aqueous acetonitrile and eluent B: 20 mM solution of formic acid in aqueous acetonitrile. The flow rate is 0.5 ml/min. The volume of injection is the total loop (about 11 μL). The elution mode is a gradient of eluents A and B according to Table 4.

(22) TABLE-US-00004 TABLE 4 Time (min.): % eluent A: % eluent B: 0.0 60 40 4.0 20 80 4.1 60 40 5.0 60 40

(23) The time of the analysis is about 5 min. The detector is a Waters TQD (tri-quadrupole) for MS/MS.

(24) The conditions of detection is as follows: The electrospraying mode is positive (ES+). The working mode is monitoring of reactions of settled ions (MRM). The temperature of the ion source is 120° C. The vaporization temperature is 450° C. The cone voltage is 55 V. The capillary voltage is 3.0 kV. The collision gas (CID) is argon. The collision gas flow rate is 0.18 ml/min. The collision gas pressure (CID) is 4.5×10.sup.−3 mbar. The transitions of scans and collision energy are according to Table 5.

(25) TABLE-US-00005 TABLE 5 Transitions Collision No. scan (daltons) energies, eV Substance: 1 537.30 > 262.13 50 SkQ1 2 537.30 > 289.12 50 SkQ1 3 552.40 > 274.20 50 SkQ1-d15 4 552.40 > 304.30 50 SkQ1-d15

(26) Solution of SS and the test solution should be chromatographed.

(27) G. Suitability of the Chromatographic System.

(28) The chromatographic system is suitable if: 1) the MRM chromatogram of SkQ1-d15 asymmetry factor of the main peak is not more than 2; 2) the effectiveness of the chromatographic column on the main peak is not less than 3000 theoretical plates; and 4) the relative standard deviation of the ratio of SkQ1 and SkQ1-d15 peak areas on the chromatograms of the solution is no more than 15%.

(29) The SkQ1 content in the product (μg/ml) was calculated using the equation:

(30) $X=\frac{a.Math.\left(100-W\right).Math.P.Math.S/S_{\mathrm{smpl}}}{1000000.Math.S/S_{\mathrm{std}}}\mathrm{wherein}$ “S/S.sub.smpl” is the ratio of peak areas and SkQ1 and SkQ1-d15 in chromatograms of the sample solution; “S/S.sub.std” is the ratio of SkQ1 and SkQ1-d15 peak areas in chromatograms of the SS solution; “a” is the amount of SkQ1 taken to prepare the original standard solution, in mgs; “W” is the water content and organic solvents in SkQ1 substance taken to prepare the original standard solution, in percent; and “P” is the SkQ1 content in SkQ1 substance taken to prepare the original standard solution, in percent.

Example 2

Use of Pharmaceutical Composition in Veterinary Practice

(31) The species and breed of animal is a dog, Zverg Schnauzer (miniature schnauzer), male, born in 2005 (4 years old). It was admitted on Dec. 4, 2008.

(32) The case history is as follows. The dog has been blind since November 2008. Since 2007, the dog could not see in the darkness. Now the dog can only keep itself oriented by sound. The owner believes that the animal has been ill for 1 year. The owner drew attention to changes in the animal's behavior during an evening stroll. The dog does not recognize the owner in a group of 5-6 people. The dog keeps itself poorly oriented during the daytime and stumbles upon objects. The dog recognizes the owner only by voice. In the dark the dog keeps itself oriented by sound.

(33) This was the primary visit. Investigations have not been conducted earlier. The exact diagnosis was not given in a previous diagnosis. There was no previous treatment. This clinical case is hereditary.

(34) The general condition of the animal at admission is satisfactory. The dog does not see. External examination shows that the eyelids have normal volume. The palpebral fissure is normal, the conjunctiva is pale pink, the cornea is shiny, spherical, transparent, moist, and contains no blood vessels. The anterior chamber is deep. The reaction of iris to light is absent.

(35) The preliminary diagnosis is generalized progressive retinal atrophy, with no complications of the disease, and no concomitant diseases.

(36) The examination results were as follows: Specific tests used were labyrinth, reaction to a cotton ball, and reaction to light. In the test with a labyrinth, the dog stumbled into obstacles both in darkness and in the light; it did not react to a cotton ball falling nearby; and the reaction of the iris to light was absent. The instrumental tests used were tests for leptospirosis, retinography and retino-photography. The test for leptospirosis produced negative results. For retinography, results see Table 4. Retino-photography revealed color change and hyper-reflection of the tapetum lucidum, and thinning of optic disc vessels. The optic disc color was white.

(37) The clinical diagnosis is generalized progressive retinal atrophy, stage 2. Major symptoms are: blindness; the surface of t. lucidum is altered and hyper-reflective; small vessels of the retina are absent; and thinning of large vessels of the retina. The type of the disease is chronic. There is vision dysfunction in darkness and on light. The animal does not see large stationary or moving objects.

(38) The prescribed treatment is the pharmaceutical composition (Composition 1) in a dose of 1 drop twice a day. The record of the treatment is shown in Table 6.

(39) TABLE-US-00006 TABLE 6 Clinical Records of Clinical Case 1 Methods of Prescribed Date investigation (tests) Test resutls therapy Dec. 4, 2008 1. Labyrinth Negative Pharmaceutical 2. Reaction to a Negative composition cotton ball 3. Reaction to light Negative (mydriasis). (composition 1) 4. Retinography See Appendix in a dose of 1 5. Retino-photo- Carpet area of t. Lucidum is drop once a day. graphy hyper-reflex, small vessels of retina are absent, thinning of great vessels, OD is white. Retinogram Right eye: A-wave - 56.5 micro Volts. B-wave - 9.5 micro Volts. Left eye: A-wave - 9.4 micro Volts, B-wave - 15 micro Volts Dec. 24, 2008 1. Labyrinth Negative 2. Reaction to a +Negative cotton ball 3. Reaction to light Positive 1 drop once a Fundus of eye is unchanged. day. The dog no longer stumbled upon objects during the daytime, at night the dog does not see. According to the words of the mistress, the dog began to keep itself oriented better, especially during the daytime. Apr. 13, 2009 1. Labyrinth Positive 2. Reaction to a Positive cotton ball 3. Reaction to light Positive Vision is preserved, the dog sees, keeps itself well-oriented in any time of day. Tests are positive. Vision is preserved. Retinography Right eye: A-wave - 56.5 micro Volts. B-wave - 203.5 micro Volts. Left eye: A-wave - 45 micro Volts, B-wave - 45.5 micro Volts

(40) A dog, Zverg Schnauzer (miniature schnauzer) breed, nickname Vintik, age of 4 years, was admitted to the clinic Apr. 12, 2008. As a result of the acquisition of anamnesis data, clinical examination revealed a generalized progressive retinal atrophy. The dog cannot see. Blindness is observed for 1 year, carpet area of t. lucidum is hyper-reflective, small vessels of retina are absent, thinning of great vessels, OD is white.

(41) The prescribed treatment is Composition 1 eye-drops, 1 drop twice a day. After 20 days of treatment the dog began to see better during the daytime. After 2 months of treatment the dog began to see during the daytime and in the darkness. Tests became positive.

(42) The clinical picture of the eye fundus is unchanged. For retinography results see Table 7.

(43) TABLE-US-00007 TABLE 7 Retinography Results Date Right Eye Left Eye Dec. 4, 2008 A-wave - 56.5 micro Volts. A-wave - 9.4 micro Volts, B-wave - 9.5 micro Volts. B-wave - 15 micro Volts Apr. 13, 2009 A-wave - 56.5 micro Volts. A-wave - 45 micro Volts, B-wave - 203.5 micro Volts. B-wave - 45.5 micro Volts

Example 3

Use of Pharmaceutical Composition in Combination Therapy of Eye Diseases of Domestic Animals

(44) The following testing was done on dogs treated with Composition 1.

(45) Test 1

(46) A dog, 9 years old, nonpedigreed, was admitted to the clinic in late January 2009 with signs of hemorrhagic chorioretinitis and papillitis. At the time of examination the animal cannot see, and persistent mydriasis has been detected.

(47) Since January 2009, the following treatment was applied: Dexamethasone (methylated fluoroprednisolone) 0.1% eye drops, 1 drop 4 times a day; after 10 minutes, pharmaceutical composition (Composition 1), eye drops, 1 drop once a day; after 10 minutes Emoxypine (Methylethylpiridinol) 1%, eye drops, 1 drop 3 times a day.

(48) On Mar. 16, 2009, that the animal began to see was confirmed by the appearance of an electroretinogram signal. After that, only the pharmaceutical composition (Composition 1) (without additional preparations) was used as the maintenance therapy.

(49) Test 2

(50) A dog, 11 years old, Labrador (retriever) was admitted to the clinic in September 2008 with a diagnosis of uveodermatological syndrome (endogenous uveitis induced by autoimmune dermatitis). At the time of examination the animal cannot see.

(51) The following treatment was applied: Cyclomed, eye drops, 1 drop twice a day; after 10 minutes Prenacid, eye drops, 1 drop once a day; after 10 minutes Indocollyre, eye drops, 1 drop twice a day, after 10 minutes Betoptic, eye drops, 1 drop 3 times a day; after 10 minutes Pharmaceutical composition (composition 1), eye drops, 1 drop once a day; after 10 minutes Emoxypine 1% eye drops, 1 drop 3 times a day.

(52) On 30 Jan. 2009, electroretinography showed that the functional activity of the retina was very good. The animal can see. In the treatment of uveitis, significant remission of the disease was observed, but full recovery cannot be achieved due to the chronic type of the disease. Maintenance therapy with the aid of the pharmaceutical composition (Composition 1) was continued.

(53) Test 3

(54) A dog, 7 years old, German Shepherd, was admitted to the clinic in September 2008 with a diagnosis of endogenous uveitis induced by leptospirosis. Eyesight was preserved.

(55) The following treatment was administered: Prenacid, eye drops, 1 drop once a day; after 10 minutes Indocollyre, eye drops, 1 drop twice a day; after 10 minutes Betoptic, eye drops, 1 drop 3 times a day; after 10 minutes Pharmaceutical composition (composition 1), eye drops, 1 drop once a day; after 10 minutes Emoxypine 1%, eye drops, 1 drop 3 times a day.

(56) The treatment was completed in November 2008 and resulted in full recovery.

EQUIVALENTS

(57) Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific composition and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.