COMPOSITIONS THAT PROTECT CELLS FROM OXIDATIVE AND MITOCHONDRIAL STRESS

Abstract

There are described compositions comprising an effective amount of a combination of two or more components, said components selected from acacetin, ACT1 peptide, alpha-lipolic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-beta-methyl-butyrate, Bacopa monnieri, catechin, catechol, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (−)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercitrin (EMIQ), kaempferol, kuromanin, leucine, lithium, luteolin, luteolin, luteolinidin, melatonin, menadione, 1-methylnicotinamide (MNA), methyl salicylate, myricetin, nadide, niacin (vitamin B.sub.3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (Na AD), nicotinic acid mononucleotide (Na MN), parsley (Petroselinium crispum), phenylephrine, pokeweed mitogen, 15-Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxrutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin and zinc (including salts thereof).

Claims

1. A composition comprising an effective amount of a combination of two or more components, said components selected from acacetin, ACT1 peptide, alpha-lipolic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-beta-methyl-butyrate, Bacopa monnieri, catechin, catechol, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (−)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercitrin (EMIQ), kaempferol, kuromanin, leucine, lithium, luteolin, luteolin, luteolinidin, melatonin, menadione, 1-methylnicotinamide (MNA), methyl salicylate, myricetin, nadide, niacin (vitamin B3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid mononucleotide (NaMN), parsley (Petroselinium crispum), phenylephrine, pokeweed mitogen, 15-Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxrutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin and zinc (including salts thereof); and derivatives thereof and any combination thereof.

2-28. (canceled)

29. A method of enhancing cell resistance to DNA damage, oxidative stress, mitochondrial dysfunction, or improving DNA repair capacity, said method comprising the administration of an effective amount of a composition comprising an effective amount of one or more components, said components selected from acacetin, ACT1 peptide, alpha-lipolic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-beta-methyl-butyrate, Bacopa monnieri, catechin, catechol, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (−)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercitrin (EMIQ), kaempferol, kuromanin, leucine, lithium, luteolin, luteolin, luteolinidin, melatonin, menadione, 1-methylnicotinamide (MNA), methyl salicylate, myricetin, nadide, niacin (vitamin B.sub.3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid mononucleotide (NaMN), parsley (Petroselinium crispum), phenylephrine, pokeweed mitogen, 15-Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxrutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin and zinc (including salts thereof); and derivatives thereof and any combination thereof.

30. A method according to claim 29 for use in enhancing cell resistance to DNA damage.

31. A method according to claim 29 for use in enhancing cell resistance to oxidative stress.

32. A method according to claim 29 for use in enhancing cell resistance to mitochondrial dysfunction.

33. A method according to claim 29 for use in improving a cell's DNA repair capacity.

34. A method according to claim 29 wherein the method comprises enhancing cell resistance to DNA damage, oxidative stress, mitochondrial dysfunction, and improving DNA repair capacity.

35. A method according to claim 29 wherein the amount of the active component administered in the method is from about 1 mg to about 1000 mg.

36. A method according to claim 29 wherein the method comprises the mitigation, alleviation or improvement of the effects of ageing in a host.

37. A method according to claim 36 wherein the method of mitigation, alleviation or improvement of the effects of ageing in a host is by improving a cell's resistance to DNA damage and/or enhancing the cell's DNA repair capacity.

38. (canceled)

39. A method according to claim 29 wherein the daily dosage of the composition is from about 0.1 to about 500 mg/kg body weight.

40. (canceled)

41. A method according to claim 36 wherein the effects of ageing include age related skin conditions, skin conditions related to sun exposure, skin conditions related to pollution exposure, skin conditions related to oxidative stress, and skin conditions related to lifestyle choices, such as diet, alcohol and/or smoking.

42. A method according to claim 36 wherein the effects of ageing include skin conditions related to inflammatory skin disorders and skin conditions related autoimmune disease skin disorders.

43-44. (canceled)

45. A method according to claim 42 wherein the skin condition is an age related skin condition.

46. A method according to claim 45 wherein the age related skin condition includes one or more of sagging, wrinkles, skin elasticity, skin ageing, skin moisture, wounds, acne, skin darkening, skin whitening, pigmentation, age-spots, loss of radiance, puffiness, uneven skin tone, redness, rosacea, loss of barrier function, loss of skin resilience, loss of firmness, stretch-marks, cellulite and dryness.

47. A method according to claim 45 wherein the skin condition is caused by sun exposure.

48. A method according to claim 45 wherein the skin condition includes one or more of actinic keratoses, freckles, lentigines or age spots, moles, photosensitivity, polymorphous light eruption, seborrheic keratoses, skin cancer (such as melanoma, squamous cell carcinoma, basal cell carcinoma), solar elastosis or wrinkles and sun burn.

49. A method according to claim 42 wherein the skin condition is caused by inflammation.

50. A method according to claim 42 wherein the skin condition includes one or more of acne, asteatotic eczema, atopic dermatitis, contact dermatitis, discoid eczema, eczematous drug eruptions, erythema multiforme, erythroderma, gravitational/varicose eczema, hand eczema, keratosis lichenoides chronica, lichen nitidus, lichen planus, lichen simplex, lichen striatus, mycosis fungoides, pityriasis lichenoides, psoriasis, seborrheic dermatitis, Stevens-Johnson Syndrome, toxic epidermal necrolysis and vasculitis.

51. A method according to claim 42 wherein the skin condition is caused by an autoimmune disease.

52-54. (canceled)

55. A method according to claim 29 for topical, transdermal, oral or parenteral administration.

56. A method according to claim 55 for topical administration.

57. A method according to claim 56 in the form of an aqueous solution, suspension, serum, ointment, cream, gel, sprayable formulation, transdermal patch or bandage.

58-60. (canceled)

61. A method according to claim 55 for parenteral administration.

62. A method according to claim 61 for parenteral administration in the form of an intramuscular, intravenous, subcutaneous, intraperitoneal, local or transdermal injections.

63. A method according to claim 55 for transdermal administration.

64. A method according to claim 63 in the form of an aqueous solution, suspension, ointment, cream, gel, sprayable formulation, transdermal patch or bandage.

65. (canceled)

Description

[0121] The present invention will now be described by way of example only and with reference to the accompanying figures, in which:

[0122] FIG. 1 illustrates potentiation of SIRT1 expression in human PBMCs from blood samples in a clinical trial over 16 days with exposure to the combination;

[0123] FIG. 2 illustrates HDF results for protection from oxidative stress; and

[0124] FIGS. 3(a) and (b) illustrate HDF results for protection from mitochondrial stress.

EXPERIMENTAL

[0125] The measurement of DNA damage was performed using the ‘Fluorimetric Detection of Alkaline DNA Unwinding (FADU)’ method [Birnboim H C, Jevcak J J. Cancer Res (1981) 41:1889-1892]. This is a sensitive procedure to quantify DNA strand breaks and is based on the partial denaturation (“unwinding”) of double-stranded DNA under controlled alkaline conditions. Briefly, after infliction of DNA damage by irradiation, cell lysis was performed. Controlled unwinding of DNA was then performed under controlled conditions of pH and temperature. DNA strand breaks are sites susceptible to unwinding, thus, more DNA damage will result in more DNA unwinding. To terminate unwinding, a neutralising solution was added. To quantify the amount of DNA remaining double-stranded after the alkali incubation, a fluorescent probe was added that specifically binds to double stranded DNA. Low fluorescence intensities indicated a large number of DNA strand breaks present at the time of lysis. DNA repair was measured by allowing the cells to recover post irradiation (1 hour) and then measuring fluorescent intensity.

[0126] Human dermal fibroblasts were treated with our composition comprising a combination, which was niacinamide, quercetin, zinc citrate, ascorbic acid, apigenin and alpha-lipoic acid, for 24 hours prior to performing the FADU assay and DNA damage and repair levels were measured and compared to values from untreated (control) cells. Results were as follows:

TABLE-US-00001 Damage repair average % cv average % cv Composition 0.00 8.51 100.00 8.91 Control 28.50 8.95 0.00 5.98

[0127] Values are average (and coefficient of variation as %) of at least 3 repeats for each condition. Results are in % and show: [0128] DNA Damage (%)=fluorescence signal intensity lost following 5 Gy irradiation [0129] DNA Repair (%)=% of damage recovered after allowing for 1 h repair at 37° C.

[0130] Results show no loss in fluorescence intensity in cells treated with our composition compared to 28% loss in control cells, indicating that our composition protects the cells from DNA damage.

Example 1

[0131] SIRT1 Expression

[0132] Clinical participants' PBMCs (peripheral blood mononuclear cells) were analysed for changes in the expression of SIRT1 using Western blot analysis. SIRT1 expression increased markedly over a 16-day exposure to our composition. Increased SIRT1 expression indicates that beneficial downstream pathways associated with enhanced DNA repair were activated by our composition.

[0133] See FIG. 1.

[0134] Clinical Results: Proteomics

[0135] Mass spectrometry was used to determine changes with the composition in levels of proteins associated with ageing.

[0136] Increased abundance of mitochondrial proteins was detected in volunteers' samples including: [0137] Fumarate hydratase: Catalyses the hydration of fumarate to L-malate in the tricarboxylic acid (TCA) cycle to facilitate a transition step in the production of energy in the form of NADH. [0138] Aspartate aminotransferase: Catalyses the transamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid (KA) as part of the De Novo pathway of NAD synthesis [0139] VDAC2: A mitochondrial membrane channel [0140] Complex IV subunits: Specifically, COXSA which is shown to decrease in skin with age, reducing mitochondrial function [0141] Complex V subunits: Also found to decrease with age resulting in mitochondrial dysfunction.

[0142] Increased abundance of proteasome proteins were detected in volunteers' samples including:

[0143] 19S and 20S proteasomal subunits: These subunits complex together to form the 26S proteasome which degrades damaged and unfolded proteins which are known to accumulate with age.

[0144] The proteasome is also important during mitochondrial biogenesis. This is because many mitochondrial proteins are synthesised in the cytoplasm and then need to be transported (unfolded) across the mitochondrial membrane. The proteasome ensures any damaged proteins are removed during this process. The 20S subunit is specifically implicated in the degradation of mitochondrial unfolded precursor proteins before their mitochondrial import, suggesting increased mitochondrial biogenesis with our composition.

[0145] Increased abundance of telomere-associated proteins was detected in volunteers' samples including:

[0146] RIF1: Telomere associated protein RIF-1 has been shown to be involved in the maintaining telomere length and in coordinating repair of telomere DNA damage.

[0147] We tested combination interventions in Human Dermal Fibroblasts (HDFs) against a variety of cellular stressors.

Example 2

[0148] HDF Results: Protection from Oxidative Stress [0149] Pre-treatment with NAM or combinations [0150] Followed by exposure to oxidative stress (tbh7ox) [0151] Cell viability measured to assess protection from oxidative stress [0152] Our composition provides much greater protection than niacinamide [0153] The composition is required to yield this enhanced protection, as predicted by systems pharmacology [0154] The composition was alpha lipoic acid, apigenin, ascorbic acid, zinc citrate, niacinamide and quercetin.

[0155] The results are illustrated in FIG. 2.

Example 3

[0156] HDF Results: Protection from Mitochondrial Stress

Example 3(a)

[0157] Pre-treatment with NAM or our composition [0158] Followed by exposure to mitochondrial stress (Rotanone—a Complex I inhibitor) [0159] Cell viability measured to assess protection from mitochondrial dysfunction [0160] Our composition provides much greater protection than niacinamide [0161] The composition was alpha lipoic acid, apigenin, ascorbic acid, zinc citrate, and niacinamide.

[0162] The results are illustrated in FIG. 3(a).

Example 3(b)

[0163] Pre-treatment with NAM or our composition [0164] Followed by exposure to mitochondrial stress (NaN.sub.3—a Complex IV inhibitor) [0165] Cell viability measured to assess protection from mitochondrial dysfunction [0166] Our composition provides much greater protection than niacinamide [0167] The composition was alpha lipoic acid, apigenin, ascorbic acid, zinc citrate, and niacinamide.

[0168] The results are illustrated in FIG. 3(b).