Anti-radical agents

Abstract

Compounds of the formulae ##STR00001##
and selected hindered nitroxyl, hydroxylamine and hydroxylamine salt compounds such as the compound of the formula ##STR00002## wherein G.sub.1 is hydrogen; C.sub.1-C.sub.22alkyl; C.sub.1-C.sub.22alkylthio; C.sub.2-C.sub.22alkylthioalkyl; C.sub.5-C.sub.7cycloalkyl; phenyl; C.sub.7-C.sub.9-phenylalkyl; or SO.sub.3M; G.sub.2 is C.sub.1-C.sub.22alkyl; C.sub.5-C.sub.7cycloalkyl; phenyl; or C.sub.7-C.sub.9-phenylalkyl; E is oxyl or hydroxyl; V is O; or NH; a is 0 or 1 or 2; b, c and d and g are each independently of one another 0 or 1; e is an integer from 1 to 4; f, m, n and p are each independently of one another an integer from 1 to 3; q is 0 or an integer from 1 to 3; Q, T and G.sub.3 are as defined in claim 1; G.sub.4 and G.sub.5 are each independently of the other hydrogen; or C.sub.1-C.sub.22alkyl; exhibit marked antiinflammatory action.

Claims

1. A method for the local treatment or prevention of inflammatory conditions comprising: administering to a patient in need of such treatment an effective amount of a pharmaceutical composition comprising at least one of the following compounds of formulae (7) to (35) ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## the reaction product of glycerine, coconut oil and a compound of the formula ##STR00095## together with a pharmaceutically acceptable carrier or adjuvant, wherein the pharmaceutical composition is free from skin-lightening agents.

2. The method according to claim 1, wherein the compounds (7) to (35) are present in a total amount of 0.001 to 10% by weight of the total composition.

3. The method according to claim 1, which additionally comprises at least one substance selected from antiphlogistic agents, antiinflammatory agents, vitamins, antipsoriatic agents, further skin actives, cell proliferation regulators, antiallergic, UV protecting, moisturizing, antiageing agents, DNA-protectants.

4. The method according to claim 1, which contains the compounds (7) to (35), and optionally a further active ingredient, in nanodispersed or encapsulated form.

5. The method according to claim 1, wherein the following compounds are administered to a patient in need are selected from the group consisting of AO3: a reaction product of glycerine, coconut oil and a compound of the formula ##STR00096## (mixture of mono-, di- and triglycerides of -(3,5-di-tert.butyl-4-hydroxyphenyl) propanoic acid and fatty acids, CAS Reg.-No. 179986-09-5) AO5 a mixture of the compounds of the formulae: ##STR00097## and AO7: a pentaerythritol-tetrakis(3-[3,5-di-tert.butyl-4-hydroxyphenyl]-propionate) ##STR00098##

6. The method according to claim 1, wherein the compounds administered to the patient in need is selected from the group consisting of AO3: a reaction product of glycerine, coconut oil and a compound of the formula ##STR00099## a mixture of the compounds of the formulae: ##STR00100## AO7: a pentaerythritol-tetrakis(3-[3,5-di-tert.butyl-4-hydroxyphenyl]-propionate) ##STR00101##

7. The method according to claim 1, comprising administering the pharmaceutical composition to the patient in need of local treatment or prevention of inflammatory conditions for other than an after-sun inflammatory condition.

8. The method according to claim 1, wherein the inflammatory condition is an adverse radical induced inflammatory condition selected from the group consisting of collagen damage inflammation, reperfusion damage inflammation, DNA-damage inflammation, and combinations thereof.

9. The method according to claim 1, wherein the inflammatory condition is an adverse radical induced inflammatory condition selected from the group consisting of reperfusion damage inflammation, DNA-damage inflammation, and combinations thereof.

Description

EXAMPLE 1

Cell Toxicity

(1) Cell cultures: Primary human dermal fibroblasts (HDF-N2) are maintained in Dulbecco's culture media (DMEM; 1 g/l glucose), supplemented with 10% of fetal calf serum (FCS), 100 g/ml of streptomycin sulphate, 100 ml of penicillin and 2 mM of L-glutamine, all of them from Biowhitaker, USA. Confluent cells are detached by incubation for 3 minutes in 0.055 trypsin/0.53 mM EDTA at 37 C. and plated at the established cell density (normally, 1510.sup.4 cells/cm.sup.2).

(2) Testing: Stock solutions of each test compound (active) in DMSO or in ethanol (5%) are diluted into the cell culture medium containing 10% FCS (see above) as indicated in table 1. Culture media of negative controls are containing 10 mMol NaCl or 0.05% DMSO or 0.05% ethanol, respectively. Positive control is a culture medium containing 0.02% of Sodium Lauryl Sulfate (SDS).

(3) For comparative purposes, a further culture medium is used containing the compound of the formula

(4) ##STR00088##
in the same dilution.

(5) The test media thus obtained are added for 24 h to 48 h-cultures of Human Dermal Fibroblast (HDF) described above, and the rate of survival is measured via a specific color marker penetrating only into live-cells (The more absorption at OD.sub.450, the more viable cells: Living dermal fibroblast possess an esterase that liberates a color, which absorbs at 450 nm. Dead cells do not. Measuring in cell cultures in presence of test substances the OD450 and comparing it to non-treated controls yields thus a measure for cell toxicity). Results are compiled in the following table.

(6) TABLE-US-00010 TABLE 1 Absorbance (OD.sub.450) after 24 h treatment active OD.sub.450 none (NaCl) 2.2 none (DMSO) 2.1 control (SDS) 0.25 AO3 1:500 1.55 AO4 1:500 1.0 AO5 1:500 1.45 AO6 1:500 1.9 AO7 1:500 2.0 AO8 1:500 1.65 comparison 1:500 0.25

(7) Cytotoxicity of the present test compounds is in an acceptable range.

EXAMPLE 2

Anti-Inflammatory Activity (In Vitro)

(8) Compounds are tested for anti-inflammatory activities via their capability of modifying the basal PGE.sub.2 release in HDF-N2 cultivated as described in example 1.

(9) For this purpose, cells are cultured in 24 well culture plates for 3 days. Before treatment confluent cells are arrested for 24 h with culture media containing 1% FCS and then treated for 18-24 h with selected concentrations of the test compounds. Each of the test compounds AO3, AO5, AO6 and AO7, respectively, is applied in dilutions 1:500, 1:1000 and 1:1500, each prepared by adding appropriate amounts of the stock solution described in example 1 to the culture medium.

(10) After treatment, cells supernatants are removed and aliquots stored at 20 C. until analysis of PGE.sub.2 using an ELISA kit from Amersham Biosciences. The PGE.sub.2 values (pg/well) are individually corrected for the total protein, measured by the BCA Method (Pierce) and expressed as PGE.sub.2 (pg/mg protein or ng/mg protein).

(11) Morphological control of culture at 24 h of product application is performed by phase contrast microscopy.

(12) Results: The test compounds show good PGE.sub.2 inhibition activity.

EXAMPLE 3

Evaluation of the Efficacy of Present Compounds Against Photooxidative Stress in the Skin Induced by UVA Irradiation

(13) Anti-oxidant activity in vivo is measured via the capacity of a test compound to reduce radical induced lipid-peroxidation. Persons tested: 10 individuals (4 female, 6 male); age range: 18 to 32 years Body region tested: inner forearm Application phase: once a day Test period 7 days Evaluation method: Determination of squalene Determination of squalenehydroperoxide Time of Evaluation: after UVA radiation Evaluation: Descriptive statistics: average, median, minimum, maximum, variance, standard error, standard deviation; Multiple range test.
Test Method

(14) Each test compound (active) is applied as a 1% b.w. solution in ethanol, except for AO7 where a 0.1% b.w. solution in ethanol is applied; in the following, these test solutions are also recalled as formulation. On the inner forearm of each subject symmetrically opposed areas are defined. The different formulations are applied once a day at a dose of about 2 mg of test solution/cm.sup.2 for one week (application with a syringe for fine dosage: Omnifix-F 1 ml; manufacturer: Braun Melsungen AG, Germany).

(15) Two areas remain untreated.

(16) The unique application of a solution of tocopherol in ethanol (0.2%) before irradiation serves as control.

(17) Use of other cosmetic products is restricted on the test areas throughout the whole study. The areas (exception: one of the two untreated areas was not irradiated and can be attributed to environmental UVA radiation during the test) of the subject's back were then irradiated with UVA light (10 joule/cm.sup.2). The lipids present on the test areas are harvested via a solvent extraction (4 ml ethanol for 2 minutes). The samples are first filtered through hydrophobic polypropylene filters to decant squames and other insoluble material, then dried under nitrogen at room temperature and taken up in 1 ml ethanol. Squalene (SQ) and squalenehydroperoxide (SQOOH) are then analysed by High Performance Liquid Chromatography (HPLC).

(18) The results are expressed as the rate of inhibition relative to the untreated area:
% inhibition=100[SQOOH(untreated)SQOOH(active)]/SQOOH(untreated)
(SQOOH in pmoles hydrogen peroxide per pg squalene)
HPLC Analysis for SQ column: LiChrospher 100 RP-18 (5 m, 1254 mm) Merck-Germany mobile phase: acetonitrile/isopropanol (1/1; V/V) detection: UV 210 nm flow rate: 1 ml/min injection volume: 20 l equipment: Beckman System Gold (USA) with Programmable Solvent Module 126 and Programmable Detector Module 166
HPLC Analysis for SQOOH column: LiChrospher 100 RP-Select B (5 m, 1254 mm) Merck-Germany mobile phase: methanol detection: Chemiluminescence (post column detection) flow rate: 1 ml/min injection volume: 20 l reaction solution: Luminol (1 g/ml) and Cytochrom C (10 g/ml solved in 50 mM Borate-buffer, pH 10) equipment: Beckman System Gold (USA) with Programmable Solvent Module 126 and fluorometer RF-551 (Shimadzu, Japan)

(19) The fluorometer is used as a photon detector with the excitation source turned off.

(20) This assay measures the hydroperoxy groups themselves and not indirect indices of lipid peroxidation such as conjugated dienes or breakdown products of lipid hydroperoxides. Chemiluminescence also detects ubiquinols. To confirm that any chemiluminescence observed in this assay was due to a hydroperoxide, not a hydroquinone, some samples were reduced with triphenyl phosphine and rerun. Since the chemiluminescence response of hydroperoxides, but not of hydroquinones, is eliminated by triphenyl phosphine, the disappearance of chemiluminescence peaks in the treated samples indicated that chemiluminescence observed in this assay was due to hydroperoxides and not hydroquinones.

(21) Biometry

(22) Measuring data are centrally computerised after validity check and quality assurance. Evaluation is done using the software Statgraphics for Windows, Version 5.0Manugistics, USA. Data are analysed by multiple range test (LSD: Least Significant Differences). The 0.05 level is selected as the point of minimal acceptance statistical significance.

(23) Results

(24) The present skincare formulations significantly (p<0.05) reduce squalene peroxidation, compared to UV radiated non-treated control.

(25) In the untreated, non-irradiated area the formation of ethanol extractable squalenehydroperoxide is not detected.

(26) The average inhibition in % of peroxidation is:

(27) TABLE-US-00011 Formulation Inhibition AO4 28% AO5 26.5% AO6 28% AO7 21.5% AO8 21% Control 22.5%
Conclusion:

(28) Pretreatment of the epidermal surface before exposure to UVA irradiation with the formulations according to the present invention significantly reduces UV-induced sebum peroxidation. These studies provide compelling evidence for the free radical hypothesis of UVA light induced cutaneous pathology: Lipid peroxidation increased on irradiation, and the topical application with an antioxidant system protected from this damage.

EXAMPLE 3

Dermatological Formulations

(29) General Preparation:

(30) A clear homogenous prephase is obtained by mixing

(31) (a) 5 to 20% by weight of a phospholipid (e.g. lecithin),

(32) (b) 15 to 40% by weight of a coemulsifier (e.g. a polyethoxylated sorbitan fatty acid ester, polyethoxylated fatty alcohol, polyethoxylated fatty acid, polyethoxylated vitamin E derivative, polyethoxylated lanoline or lanoline derivative, polyethoxylated fatty acid glyceride or partial glyceride, polyethoxylated alkylphenol, sulfuric acid semiester of a polyethoxylated fatty alcohol or salt thereof, polyethoxylated fatty amine or amide, polyethoxylated carbon hydrate),
(c) 30 to 70% by weight of a lipophilic component consisting of a triglyceride, a compound of the invention such as AO3-AO8, and optionally a further active agent, where the weight ratio of active agent(s):triglyceride usually ranges from 1:5 to 5:1, and
(d) 3 to 30% by weight of an alkohol such as ethanol,
with the sum of percentages of the components (a), (b), (c) and (d) adding to 100%, and
adding the liquid obtained to a water phase. The water phase (e.g. 90 kg) is placed, with stirring (e.g. magnetic agitator), at 50 C. in a vessel. The liquid prephase (e.g. 10 kg) is added to the water phase with stirring (e.g. with a magnetic agitator). The formulation thus obtained may be further diluted or admixed.
Vitamin a Palmitate Cream

(33) A dispersion is obtained by mixing a clear prephase containing:

(34) TABLE-US-00012 vitamin A palmitate (1.7 10.sup.6 IU/g) 0.45% compound AO7 0.45% soybean lecithin 1.73% miglyol 812 3.00% polysorbate 80 3.40% ethanol 1.42% to a water phase: 10 mm phosphate buffer, pH 6 ad 100.00%

(35) Final preparation contains:

(36) TABLE-US-00013 cetyl alcohol 10.00% hydrogenated groundnut oil 20.00% polysorbate 60 5.00% propylene glycol 20.00% phenoxyethanol 0.50% dispersion shown above 23.00% aqua purificata ad 100.00%

(37) Good results are also achieved when compound AO7 is replaced by AO3, AO4 or AO5.

(38) Solcoseryl 0.5% Hydrogel

(39) A dispersion is obtained by mixing a clear prephase containing:

(40) TABLE-US-00014 solcoseryl 1.00% compound AO7 1.00% soybean lecithin 1.73% polysorbate 80 3.40% miglyol 812 3.45% ethanol 1.42% to a water phase: 10 mm phosphate buffer, pH 6 ad 100.00%

(41) Final preparation contains:

(42) TABLE-US-00015 sodium carboxymethylcellulose 450 cP 3.50% dispersion shown above 50.00% aqua purificata ad 100.00%

(43) The preparation is pleasantly cooling and has good antiphlogistic action.

(44) Skin Protecting W/O Lotion

(45) A dispersion is obtained by mixing a clear prephase containing:

(46) TABLE-US-00016 vitamin E acetate 2.00% compound AO3 1.00% soybean lecithin 0.49% polysorbate 80 1.86% miglyol 812 0.71% ethanol 0.63% to a water phase: aqua purificata ad 100.00%

(47) Final preparation contains:

(48) TABLE-US-00017 glycerol sorbitan fatty acid ester 2.0% polyethoxy fatty acid ester 2.0% isopropylisostearate 5.0% mineral oil 7.0% isopropylpalmitate 4.0% wheat germ oil 3.0% propylene glycol 3.8% dispersion shown above 5.0% MgSO.sub.4 7H2O 0.7% perfume 0.5% perservative, water

(49) Good results are also achieved when compound AO3 is replaced by AO7, AO6 or AO8.

(50) Day Cream with UV Protection (O/W)

(51) A dispersion is obtained by mixing a clear prephase containing:

(52) TABLE-US-00018 parsol MCX 2.59% (octyl methoxycinnamate) Parsol 5000 1.11% (4-methylbenzylidene camphor) compound AO7 1.00% miglyol 812 1.30% soybean lecithin 0.50% polysorbate 80 3.40% ethanol 1.10% to a water phase: aqua purificata ad 100.00%

(53) Final preparation contains:

(54) TABLE-US-00019 PEG-5 glycerol stearate 5.0% steareth-21 2.0% mineral oil 30.0% cetyl alcohol 2.0% microcrystalline wax 1.0% propylene glycol 6.0% dispersion shown above 10.0% phenoxyethanol + methyl-, ethyl-, propyl-, butylparabene 0.3% water ad 100.0%