METHODS AND COMPOSITIONS FOR TREATMENT OF DISORDERS ASSOCIATED WITH DAMAGE INDUCED BY FREE RADICALS

Abstract

The present invention relates to treatment of various disorders, diseases or conditions associated with or mediated by oxidative stress arising from an imbalance between the production of and the ability to neutralize reactive free radicals. The present invention is directed to methods and compositions capable of scavenging reactive free radicals for therapeutically or prophylactically treating disorders associated with free radical-induced damage, said compositions comprising an amount of 2-(3-chlorophenylamino) phenylacetic acid (23CPPA) or a pharmaceutically acceptable salt thereof that interacts with free radicals generated by cellular metabolism or ultraviolet radiation. In some embodiments, the composition comprises pharmaceutically or cosmetically acceptable adjuncts. The present invention further provides one or more kits that are useful for delaying, treating or preventing the consequences of aging and free radical damage on the condition or appearance of the skin.

Claims

1. A method of treating disorders associated with free radical damage, said method comprising administering an effective amount of the compound 2-(3-chlorophenylamino) phenylacetic acid or a pharmaceutically or cosmetically acceptable salt thereof.

2. The method of claim 1 wherein said disease or disorder is selected from the group consisting of skin aging, skin wrinkling, hyperkeratosis, and skin solar damage.

3. The method of claim 1 wherein said disease or disorder is selected from the group consisting of scleroderma, rheumatoid arthritis, and auto-immune disease.

4. The method of claim 1 wherein said disease or disorder is selected from the group consisting of Parkinson's disease and neurodegenerative disease.

5. The method of claim 1 wherein said compound is administered from the group consisting of topical, oral, intramuscular, intravenous and subcutaneous dosage forms.

6. The method of claim 5 wherein said topical dosage form is selected from the group consisting of a lotion, a cream, a gel, a liniment, an ointment, a paste, a solution, a powder and a suspension.

7. The method of claim 5 wherein said oral dosage form is selected from the group consisting of a tablet, a capsule, a pill, powder and granules.

8. The method of claim 5 wherein said intramuscular, intravenous and subcutaneous dosage forms are selected from the group consisting of physiologically acceptable sterile aqueous or nonaqueous solutions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.

9. The method of claim 6 wherein said topical dosage form contains a pharmaceutically acceptable carrier.

10. The method of claim 7 wherein said oral dosage form contains a pharmaceutically acceptable carrier.

11. The method of claim 8 wherein said intramuscular, intravenous and subcutaneous dosage forms contain a pharmaceutically acceptable carrier.

12. The method of claim 9 wherein said pharmaceutically acceptable carrier further comprises selections from the group consisting of a moisturizer, a humectant, a demulcent, oil, water, an emulsifier, a thickener, a surface active agent, a fragrance, a preservative, an antioxidant, a hydrotropic agent, a chelating agent, a vitamin, a mineral, a permeation enhancer, a cosmetic adjuvant, a depigmentation agent, a foaming agent, a conditioner, a viscosifier a buffering agent and a sunscreen.

13. The method of claim 10 wherein said pharmaceutically acceptable carrier further comprises selections from the group consisting of inert excipients, fillers, extenders, binders, humectants, disintegrating agents, solution retarders, wetting agents, adsorbents, lubricants and buffering agents.

14. The method of claim 11 wherein said pharmaceutically acceptable carrier further comprises selections from the group consisting of water, ethanol, polyols, oils, organic esters, lecithin, and suitable mixtures thereof.

15. A kit for treating disorders of the skin associated with free radical damage, said kit comprising an effective amount of the compound 2-(3-chlorophenylamino) phenylacetic acid or a pharmaceutically or cosmetically acceptable salt thereof and a dermatologically acceptable medium.

16. The kit of claim 15 wherein said dermatological acceptable medium comprises one or more cosmetic or manufacturing adjuncts selected from the group consisting of a sunscreen, a skin-lightening agent, a skin-tanning agent, a perfume, an opacifier, a preservative, a colorant and a buffer.

17. The kit of claim 15 wherein said dermatologically acceptable medium further comprises one or more selections from the group consisting of water, a buffered aqueous solution, a liquid emollient, or solid emollient, a silicone oil, an emulsifier, a solvent, a humectant, a thickener, a powder, or a propellant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0020] It has been unexpectedly discovered as described in the present invention that the compound 23CPPA and its pharmaceutically acceptable salts possess the intrinsic ability to scavenge reactive free radicals and thereby modulate the imbalance between the production of reactive oxygen species and physiological antioxidant defenses to neutralize these radicals.

[0021] It has been unexpectedly discovered as described in the present invention that the compound 23CPPA and its pharmaceutically acceptable salts possess the intrinsic ability to absorb ultraviolet radiation and thereby modulate damage to the skin arising from exposure to ultraviolet radiation from the sun, which generates highly reactive chemical intermediates, such as hydroxyl and oxygen radicals, which injure components of the skin and can damage DNA.

[0022] It is a novel and unanticipated finding of the present invention that the free radical scavenging activity of the active agent 23CPPA is an inherent property of this compound.

[0023] It is a novel and unanticipated finding of the present invention that the ultraviolet radiation absorbing properties of the active agent 23CPPA is an inherent property of this compound.

[0024] It is an object of this invention to provide a method for treating disorders in which free radical damage is causally contributory, such disorders including skin damage due to aging and solar radiation, auto-immune disease such as scleroderma and rheumatoid arthritis and neurodegenerative disease such as Parkinson's disease.

[0025] It is another object of this invention to provide a method for treating disorders of the skin associated with damage from solar radiation.

[0026] It is another object of this invention to provide a method for treating disorders of the skin associated with damage to the skin from aging.

[0027] This invention also provides therapeutic compositions comprising the above described compound.

[0028] This invention further provides a method for treating disorders associated with free radical damage comprising administering to a patients with such a disorder an effective amount of a therapeutic composition comprised of 23CPPA capable of scavenging reactive free radicals and a pharmaceutically acceptable carrier.

[0029] It is also an object of this invention to provide a method for treating disorders associated with solar radiation comprising an effective amount of a therapeutic composition comprised of 23CPPA capable of absorbing ultraviolet radiation and a pharmaceutically acceptable carrier.

[0030] The present invention further provides one or more kits that are useful for delaying, treating or preventing the consequences of aging and free radical damage on the condition or appearance of the skin.

[0031] The present invention comprises one or more compositions containing 23CPPA or a pharmaceutically acceptable salt thereof formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection, for oral administration in solid or liquid form, and for topical administration.

[0032] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for examples, by use of coating such as lecithin, by maintenance of the required particle size in the case of dispersions and by use of surfactants. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispensing agents.

[0033] Solid dosage forms for oral administration include capsules, pills, tablets, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, as for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, as for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (d) solution retarders, as for example paraffin, absorption accelerators, as for example quaternary ammonium compounds; (e) wetting agents, as for example cetyl alcohol and glycerol monostearate; (f) adsorbents, as for examples kaolin and bentonite; and (g) lubricants, as for example talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsule, tablets and pills, the dosage forms may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like. Solid dosage forms such as tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coating and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.

[0034] Dosage forms for topical administration of the method of this invention include lotions, ointments, creams, gels, liniments, pastes, solutions, powders suspensions and sprays. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Topical compositions may also contain moisturizers, humectants, demulcents, oils, water, emulsifiers, thickeners, surface active agents, fragrances, preservatives, hydrotropic agents, chelating agents, vitamins, minerals, permeation enhancers, cosmetic adjuvants, depigmentation agents, foaming agents, conditioners, viscosifiers, buffering agents and sunscreens.

[0035] Actual dosage levels of active ingredients in the compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level depends on the desired therapeutic effect, route of administration, duration of treatment and other factors. The total daily dose of the method and compositions of this invention administered to a host in single or divided dose may be in amounts of 1 mg/kg to 20 mg/kg of body weight when administered parenteral or orally and may be from lower to higher values when administered topically. Dosage unit compositions may contain such amounts or such submultiples therefor as may be used to make the daily dose. It will be understood, however, that the specified dose level for any particular patient will depend on a variety of factors including the body weight, general health, gender, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. The dosage level may also depend on patient response as determined by symptoms and signs of the disease for which the treatment is administered.

[0036] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

223CPPA Scavenges Free Radicals

[0037] A radical cation of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) was generated by oxidation with manganese dioxide, forming the blue-green radical cation ABTSY+ with spectrophotometric absorbance at 734 nanommeters (nm). The reaction was initiated by the addition of 23CPPA (5-40 micromolar) to ABTSY+ (450 micromolar) in phosphate buffer (75 millimolar, pH 7.4) and decay of the blue-green radical cation absorbance was monitored over time. Absorbance profiles were obtained with incubation in phosphate buffer (75 millimolar, pH 7.4) as control. The reaction of anti-oxidants with stable free radicals such as ABTSY+ that is generated from ABTS measures their free radical scavenging capacities. The Table depicts change with time of the absorbance at 734 nm of ABTSY+ after addition of 23CPPA to buffer compared to buffer alone, demonstrating progressive bleaching of ABTSY+ by 23CPPA and, therefore, free radical scavenging activity of 23CPPA.

TABLE-US-00001 Decay (% of starting absorbance) 23CPPA (micromolar) Minutes 0 5 10 20 40 1 99 74 57 37 20 2 97 72 56 27 20 3 95 69 53 23 3 4 94 67 52 20 1 5 94 65 51 17 0 6 93 63 50 14 0 7 93 61 48 13 0 8 92 60 47 12 0 9 92 58 46 12 0 10 92 56 44 11 0 11 91 55 44 10 0 12 91 53 43 10 0 13 90 52 43 9 0 14 90 51 42 9 0 15 89 49 42 9 0

Example 2

23CPPA Absorbs Ultraviolet Radiation

[0038] Ultraviolet (UV) absorption peaks of 2-(3-chlorophenylamino)phenylacetic acid at 211 and 283 nm, encompassing wavelength range of medium wave UVB (315-280 nm) and short wave UVC (280-190 nm).

TABLE-US-00002 Wavelength (nm) Absorbance 190 0.5 195 0.75 197 1.0 198 1.25 199 1.5 205 2.0 208 2.1 211 2.2 212 2.0 220 1.25 222 1.0 232 0.5 235 0.45 239 0.43 243 0.43 249 0.43 252 0.43 256 0.43 260 0.5 272 0.75 283 0.93 291 0.75 300 0.5 308 0.25 325 0

Example 3

Bioavailability of 23CPPA

[0039] Male rats were given a single dose of 23CPPA by the oral route (30 mg/kg) or by the intravenous route (3.0 mg/kg). Timed samples of blood were collected before and after dosing, and plasma concentrations of the compound were determined with liquid chromatography mass spectrometry (LC-MS-MS) analysis. Oral bioavailability, calculated from the plasma concentrations after oral versus intravenous administration, was 85%, indicating that the drug is absorbed from the gastrointestinal tract and enters the circulation for systemic delivery.

Example 4

23CPPA Penetrates Brain Following Oral Administration in Rats

[0040] Rat hemi brains were obtained 1,4 and 6 hours after administration by gavage of the sodium salt of 23CPPA at doses of 15 and 60 mg/kg and were mixed with two equivalents of water and homogenized. The resulting homogenate was extracted with acetonitrile containing internal standard and concentrations of the compound were analyzed by high pressure liquid chromatography/two stage mass spectrometry (HPLC/MS/MS). Plasma samples also were collected from the same rats at these time points for measurement of plasma concentrations of the compound. Brain concentrations were dose proportional and showed an approximately 3:1 molar ratio with plasma concentrations, indicating excellent penetration of the compound into the brain.

TABLE-US-00003 Dose Time Brain Blood (mg/kg) after Dose (micromolar) (micromolar) 15 1 hour 76 27 15 4 hours 12.4 4 15 6 hours 11.9 3 60 1 hour 148 57 60 4 hours 61 28 60 6 hours 45 22

Example 5

23CPPA Reduces UV-Induced Collagen Cross Linking

[0041] Oxidative change of type I collagen was induced by exposure to ultraviolet (UV) radiation (325 nm) conducted for 24 hours at 25 C. in the absence or presence of 23CPPA (10:1 molar ratio of 23CPPA to type I collagen). Samples were frozen and lyophilized, the residue was dissolved in 0.1 ml of 70% formic acid, cyanogen bromide (CnBr) (20:1 volume:volume) was added, and the solutions were incubated at 30 C. for 18 hours. Samples were spin filtered into 0.125M Tris, pH 6.8, containing 2% sodium dodecyl sulfate (SDS) and 2% glycerol using a filter with a molecular weight cutoff of 10,000. The extent of collagen cross linking was determined by analyzing equal volumes of samples subjected to SDS-polyacrylamide gel electrophoreses (SDS-PAGE) (15% Tris-HCl gel) and assessing migration of the CnBr peptides. CnBr peptides less than 10,000 molecular weight, prepared from UV-treated collagen in either the absence or presence of 23CPPA, escaped from the samples during the spin filter due to the molecular weight cut-off limits of the filter, with retention of CnBr peptides >10,000 molecular weight. SDS-PAGE analysis showed that CnBr peptides of higher molecular weight were produced when 23CPPA was not present during exposure to UV radiation. CnBr peptides of type I collagen treated with UV radiation in the presence of 23CPPA acid were of lower molecular weight than CnBr peptides of collagen treated with UV radiation in the absence of 23CPPA.

SDS PAGE Electrophoresis

[0042]

TABLE-US-00004 CnBr Molecular Sample UV Digestion 23CPPA Weight Type I collagen Yes No No 180,000 Type I Collagen Yes Yes No 85,000 Type I Collagen Yes Yes Yes 65,000

Example 6

23CPPA Decreases UV-Induced Changes in Type I Collagen Fluorescence Emission.

[0043] Type I collagen was solubilized in 1.5% acetic acid in the absence or presence of 23CPPA (10:1 molar ratio) and was subjected to UV radiation at for 3 to 24 hours at 25 C. The intensity of the fluorescence emission peak at excitation 380 nm, which is due to pepsin-digestible cross-links, decreases with UV radiation; this decrease was lessened in the presence of 23CPPA during exposure to the UV radiation.

TABLE-US-00005 UV Exposure Relative fluorescence (hours) 23CPPA Absent 23CPPA Present 0 100% 100% 3 95% 120% 6 75% 120% 24 60% 85%