COMPOUNDS AND COMPOSITIONS FOR USE

Abstract

The present disclosure provides compounds and compositions which may be used to augment, supplement and/or replace the products of certain UV sensitive/dependent processes. In particular, the disclosure provides sunscreen compositions which avoid problems associated with prior art sunscreen compositions which inhibit the natural progression of certain in vivo sunlight/UV dependent processes including processes which result in Nitric Oxide/Vitamin D production in the skin.

Claims

1. A method of treating or preventing diseases associated with reduced, impaired or inhibited nitric oxide (NO) and/or vitamin D production, said method comprising administering to a subject in need thereof a composition comprising: (i) a thiol compound and/or a disulfide; and (ii) a NO-precursor compound.

2. The method of claim 1, wherein the composition further comprises: (iii) 7-dehydrocholesterol.

3. The method of claim 1, wherein the thiol and/or disulfide compound comprises a compound capable of forming a photolytically cleavable nitrosothiol compound.

4. The method of claim 1, wherein the thiol compound comprises a —S—H moiety, optionally wherein the thiol compound is selected from the group consisting of: glutathione, cysteine, homocysteine, cysteamine and thiolactate.

5. The method of claim 1, wherein the disulfide compound comprises a —S—S— or persulfide (R—SSH) moiety, optionally wherein the disulfide compound is selected from the group consisting of: dithioglycolate; lipoic acid (oxidised) and cystine.

6. The method of claim 1, wherein the NO-precursor compound is selected from the group consisting of: nitrite-containing compounds; nitrate-containing compounds; and nitro-containing organic compounds, optionally wherein the NO-precursor compound is selected from the group consisting of: metal nitrites; sodium nitrite (NaNO.sub.2); potassium nitrite; dinitrosyl-iron complexes; iron sulfur compounds; sodium nitrate; potassium nitrate; carboxylic acid comprising a nitro (—NO.sub.2) group; nitropropionic acid; nitrooleate; 3-nitropropionic acid; 9-nitrooleate; and 10-nitrooleate.

7. The method of claim 1, wherein the subject is a subject exposed or to be exposed to the sun and requiring the use of a sunscreen composition.

8. The method of claim 1, wherein the composition is administered to, applied to or used by a subject: (i) to be exposed to the sun; or (ii) a subject susceptible, predisposed to or at risk of developing a disease or condition associated with reduced NO and/or vitamin D(3) production.

9. A method of augmenting or stimulating nitric oxide (NO) and/or vitamin D3 production in a subject wearing or using a sunscreen composition, said method comprising using, applying or administering a sunscreen composition comprising: (i) a thiol compound and/or a disulfide; and (ii) a NO-precursor compound.

10. The method of claim 9, wherein the composition further comprises: (iii) 7-dehydrocholesterol.

11. The method of claim 9, wherein the thiol and/or disulfide compound comprises a compound capable of forming a photolytically cleavable nitrosothiol compound.

12. The method of claim 9, wherein the thiol compound comprises a —S—H moiety, optionally wherein the thiol compound is selected from the group consisting of: glutathione, cysteine, homocysteine, cysteamine and thiolactate.

13. The method of claim 9, wherein the disulfide compound comprises a —S—S— or persulfide (R—SSH) moiety, optionally wherein the disulfide compound is selected from the group consisting of: dithioglycolate; lipoic acid (oxidised) and cystine.

14. The method of claim 9, wherein the NO-precursor compound is selected from the group consisting of: nitrite-containing compounds; nitrate-containing compounds; and nitro-containing organic compounds, optionally wherein the NO-precursor compound is selected from the group consisting of: metal nitrites; sodium nitrite (NaNO.sub.2); potassium nitrite; dinitrosyl-iron complexes; iron sulfur compounds; sodium nitrate; potassium nitrate; carboxylic acid comprising a nitro (—NO.sub.2) group; nitropropionic acid; nitrooleate; 3-nitropropionic acid; 9-nitrooleate; and 10-nitrooleate.

15. The method of claim 9, wherein the subject is a subject exposed or to be exposed to the sun and requiring the use of a sunscreen composition.

16. The method of claim 9, wherein the composition is administered to, applied to or used by a subject: (i) to be exposed to the sun; or (ii) a subject susceptible, predisposed to or at risk of developing a disease or condition associated with reduced NO and/or vitamin D(3) production.

17. A method of providing a sunscreen composition which: (i) does not substantially inhibit NO and/or vitamin D production in a user; and/or (ii) which facilitates, enhances, induces and/or stimulates a level of vitamin D and/or NO production in a user; said method comprising supplementing a sunscreen composition with one or more compounds selected from the group consisting of: (i) a compound which upon exposure to sunlight (or the UV component thereof), generates NO or an intermediate or metabolite which can be used to generate NO in vivo; (ii) a thiol compound; (iii) a disulfide; (iv) a compound which upon exposure to sunlight (or the UV component thereof), generates vitamin D or an intermediate or metabolite which can be used to generate active vitamin D in vivo; and (iv) 7-dehydrocholesterol.

18. The method of claim 17, wherein the sunscreen composition is supplemented with: (i) (a) a thiol compound and/or a disulfide; and (b) a NO-precursor compound; and optionally (ii) 7-dehydrocholesterol.

Description

DETAILED DESCRIPTION

[0148] The present invention will now be described in detail by reference to the following figures which show:

[0149] FIG. 1: Generation of NO by skin samples following exposure to UV light

[0150] FIG. 2: Topical application of devised formulation can enhance NO generation in response to UVA.

[0151] FIG. 3: Effects of UV on NO generation in the presence of SPF 50 sunscreen and SPF 50 sunscreen mixed with formulation 1.

[0152] FIG. 4: Use of the Clarke electrode method in the determination of the effects of exogenous compounds on NO generation from skin homogenates in response to UVA.

[0153] FIG. 5: Examples of the effects of “S” compounds on UV induced generation of NO from nitrite.

[0154] FIG. 6: The effects of organic sunscreens containing NO generating components on skin microcirculation in response to UV radiation.

[0155] FIG. 7: The effects of physical sunscreens containing NO generating components on skin microcirculation in response to UV radiation.

[0156] FIG. 8: The effects of NO generating components on the formation of Vitamin D3 from 7-DHC induced by UV light.

MATERIALS & METHODS

[0157] Histological Method for NO Determination in Skin.

[0158] Redundant skin from surgical procedures was snap frozen and microtomed to 5 mm sections. Sections were later incubated with 10 mM of the NO fluorochrome DAF-2DA for 1 hour at room temperature (Rodriguez et al., 2005). The applied DAF-2DA spontaneously crosses the plasma membrane into the skin cells and is cleaved by esterases to generate intracellular DAF-FM which cannot exit the cell. Skin sections were then irradiated with the light sources below for differing periods of time. On exposure to UV light NO generated oxidises the DAB-FM to a form a triazole product resulting in increased fluorescence.

[0159] A xenon arc lamp (Model 66021, Thermo Oriel) was used as light source

[0160] A monochromator (Model 77200, Thermo Oriel) with a xenon arc lamp (Model 66921, Thermo Oriel) was used to produce narrowband UV (280-400 nm, half bandwidth 0.2 nm).

[0161] Broadband UVB 290-310 nm (300FS10-50 filter, L.O.T Oriel)

[0162] Broadband UVA 320-420 nm, (WG320+MUG2 filters, Schott)

[0163] Sections were then examined using a Leica SPSC spectral confocal laser scanning microscope (Wetzlar, Germany) and fluorescence intensities quantified using Image-Pro Plus (Media Cybernetics, Rockville, Md.).

[0164] For the investigation of the enhancing effects of exogenously applied formulations (as identified by the NO electrode method below) compounds were applied to the surface of the skin samples prior to irradiation. Typical results are shown in FIG. 2. Topical formulations enhancing NO generation increased NO production in the stratum corneum and epidermis.

[0165] For the investigation of the effects of sunscreen SPF factor 50 on UV radiation induced NO generation, commercially available sunscreen comprising both chemical and physical sunscreen barriers was applied to the surface of the skin prior to irradiation. Physical and chemical sunscreen ingredients were Zinc Oxide and Avobenzone. Exogenously added NO enhancing components were added directly to the sunscreen prior to application.

[0166] While sunscreen prevented the induction of NO production in skin in response to UV irradiation (FIG. 3) inclusion of NO enhancing formulation re-enabled the generation of NO in the presence of sunscreen.

[0167] Due to its small size and lipophilic behaviour, generated NO was transported from the stratum corneum, to the epidermis even in the presence of sunscreen.

[0168] Estimation of radiation transmitted through the sunscreen barrier in the presence and absence of NO enhancing formulations was made by measuring radiation transmission through a film of sunscreen on a microscope slide. Results shown in Table 1 show that the addition of NO enhancing formulation did not impair the radiation screening capacity of sunscreen.

[0169] Measurement of Vitamin D3 Production in Response to UV-B Irradiation in the Presence of Sunscreens.

[0170] Vitamin D3 (Cholecalciferol) was measured using an ELISA kit (AMS Biotechnology, 184 Millton Park, Abingdon, Oxford. OX14 4SE, UK) The ELISA is based on the competitive binding enzyme immunoassay technique. Skin samples were irradiated with UV-B radiation in the presence and absence of sunscreen products. In the presence of sunscreen UV-B irradiation reduced the production of Vitamin D3 in the skin. Sunscreen products supplemented with 7-dehydrocholesterol produced increased amounts of Vitamin D3 which was detected in the lower epidermal sections.

[0171] In Vitro Measurement of NO Generation as Determined by the NO Specific Clarke-Type Electrode Method.

[0172] Electrochemical sensors are widely used for the measurement of NO generation and Clark-type electrodes are most widely used as they are commercially available and easy to handle. The principle of these sensors is that NO diffuses through a gas-permeable membrane and a thin film of electrolyte, followed by oxidation on the working electrode. This oxidation creates a current that is proportional to the concentration of NO outside the membrane. The advantage of electrochemical NO sensors is the ability to directly detect NO concentration in solution or in biological samples with a low nanomolar detection limit. This makes NO electrodes an excellent tool for studying NO generation especially in biological samples.

[0173] Current theories of UV induced NO generation propose that UVA induced decomposition of nitrite (NO2-; equations 1-5 in Table 2) is self-limiting due to reaction with NO2. (equation 4 in Table 2). However it is proposed that in the presence of reduced thiols such as reduced glutathione, nitrosothiols are formed which then decompose on UVA challenge producing high levels of NO (equations 6-9 in Table 2).

[0174] To date the only “thiol” reported to influence skin NO generation in response to UVA is reduced glutathione and cosmetically approved reagents have not been identified.

[0175] Similarly, “N” sources identified as being involved in UVA induced NO generation are limited to nitrite and others have not been identified.

[0176] Likewise cosmetically acceptable facilitators and excipients to expedite NO formation in response to UVA have not been identified.

[0177] We used the Clarke electrode technique to investigate the mechanism of UVA light dependent NO generation in skin and cosmetically acceptable components which could facilitate augment the endogenous process. (FIG. 4).

[0178] Method

[0179] A 10% (g/mL) full thickness human skin homogenate in phosphate buffer was prepared using a glass/glass homogenizer. The homogenate was preheated at 56° C. for 1 hour to inactivate enzymatic generation of NO.

[0180] 200 ul of homogenate was placed in a quartz cuvette in a thermostatically controlled environment and irradiated with broadband (320-400 nm) UVA radiation. Baseline signals were recorded.

[0181] Various sequential additions were made to the homogenate as indicated in FIG. 1 in order to identify

[0182] (i) “N” donors able to contribute to the UV induced NO generation pathway.

[0183] (ii) Potential Thiol “S” participants in the UV induced NO generation pathway.

[0184] (iii) Facilitators and excipients able to enhance and sustain the NO generation pathway.

[0185] Results

[0186] Results are summarised in FIG. 5.

[0187] Unexpectedly both reduced (—SH) and oxidised (S—S) were effective in sustaining UV induced NO generation from a nitrite source.

[0188] In addition oxidised thiols (disulphide) such as lipoic acid and cystine were unexpectedly able to participate in sustained NO generation from nitrite in response to UVA radiation. However, not all disulphides sustained UV induced NO generation with Pantethine for example being ineffective. Oxidised Glutathione was effective, however.

[0189] We hypothesise that the capacity to participate in sustained NO generation in response to UV radiation is related to:

[0190] (i) the propensity of “S” containing compound to form the thiyl radical RS(dot) in response to UV irradiation. Once an RS (dot) is formed it will immediately trap NO (at trace level from nitrite or other “N” donor), forming a nitrosothiol.

[0191] (ii) In a subsequent step the nitrosothiol is then photolytically cleaved to release NO. The relative stability of the formed nitrosothiol and propensity of the thiol/disulfide to form RS(dot) will determine outcome.

[0192] Thiyl radicals are formed from one-electron oxidation of thiols. Thiol ionization, dissociation of S—H, is the most important single property of thiols governing reactivity. The S—H bond of thiols dissociates with pKa in the range ˜7-10. However the response of Thiols to UV radiation will strongly depend on the chemical structures involved and the groups adjacent to the Thiol moiety.

[0193] Similarly the ability of disulphide bonds to form RS(dot) or dithiyl radical by the photolytic rupture of disulphide (S—S) bond will be highly dependent on the individual chemical structures involved.

[0194] The ability of a series of thiols RSH and disulfides RS—SR to generate RS(dot) and initiate sustained NO generation process will therefore strongly depend on the chemical structures involved.

[0195] Further use of the Clarke electrode method shown in FIG. 4 has identified other “N” donors that can participate in the UV radiation induced formation of NO (Table 3).

[0196] In addition factors and excipients which facilitate these reactions have also been identified (Table 3).

[0197] Tables

TABLE-US-00001 TABLE 1 Radiation Transmitted UVA BB-UVA 290 nm (mW/cm.sup.2) (mW/cm.sup.2) Vehicle Control 106.6 0.4 Sunscreen alone 11.5 0 (SPF50) Sunscreen with 12.6 0 Formulation 1

TABLE-US-00002 TABLE 2 NO.sub.2.sup.− + hv .fwdarw. NO.sup.• + O.sup.•− (1) O.sup.•−+ H.sub.2O .fwdarw. OH.sup.• + OH.sup.− (2) NO.sub.2.sup.− + OH.sup.• .fwdarw. NO.sub.2.sup.•+ OH.sup.− (3) NO.sub.2.sup.•+ NO.sup.• .fwdarw. N.sub.2O.sub.3 (4) N.sub.2O.sub.3 + H.sub.2O .fwdarw. 2NO.sub.2.sup.− + 2H.sup.+ (5) N.sub.2O.sub.3 + GS.sup.− .fwdarw. NO.sub.2.sup.− + GSNO (6) GSNO + hv .fwdarw. NO.sup.• + GS.sup.• (7) NO.sub.2.sup.• + GS.sup.− .fwdarw. NO.sub.2.sup.− + GS.sup.• (8) NO.sup.• + GS.sup.• .fwdarw. GSNO (9)

TABLE-US-00003 TABLE 3 Active “S” donors Active “Facilitators” Active “N” donors Glutathione Ascorbate Nitrite Thiolactate Ceramides Nitrate Dithioglycolate Erucic Acid 3-Nitropropionic acid Lipoic acid Homocysteine 9-Nitrooleate (Oxidised) Cystine 10-Nitrooleate

Example 2

[0198] To further examine the effects of enhancing formulations in sunscreens, on UV induced nitric oxide and vitamin D production, physical and chemical sunscreen formulations were formulated using the INCI listed compounds show in the tables below. For organic chemical sunscreens a generic oil in water formulation with known stability profile was selected for the base formulation using INCI ingredients shown in Table 4.

TABLE-US-00004 TABLE 4 INCI ingredients used in formulation of organic sunscreen base. Aqua Octocrylene C12-15 alcohols benzoate Butyl methoxydibenzoylmethane Bis-ethylhexyloxyphenol methoxyphenyl Glycerin Stearyl alcohol Potassium cetyl phosphate Coco-Caprylate Nylon-12 Diethylhexyl butamido triazone Phenoxyethanol Polyacrylate-13 Polyisobutene Disodium EDTA Polysorbate 20

[0199] For physical sunscreen formulations a generic water-in-oil formulation containing both

[0200] Titanium Dioxide and Zinc Oxide for sun protection was for the base formulation using INCI ingredients shown in Table 5.

TABLE-US-00005 TABLE 5 INCI ingredients used in formulation of physical sunscreen base. Aqua Isohexadecane Propylheptyl Caprylate Cyclopentasiloxane Titanium Dioxide Zinc Oxide C12-15 alkyl benzoate Triethylhexanoin Polyglyceryl-3 Diisostearate Euphorbia Cerifera Glycerin Magnesium Sulphate Heptahydrate Aluminium Stearate Alumina Polyhydroxystearic acid Phenoxyethanol Ethyihexylglycerin

[0201] Additions to the base formulations were made as follows.

[0202] Glutathione (GSH), cysteine (CYS), sodium nitrite (NO2) and sodium nitrate (NO3) were dissolved in a small amount of water and stirred into the finished bulk formulation after cooling to 30° C. 7-dehydrocholesterol (DHC) was dissolved in the hot oil phase just prior to emulsification. Lipoic acid (LA) was dissolved in a small amount of ethanol and stirred into the finished bulk after cooling below 30° C.

[0203] Effects of No Generating Sunscreens on Skin Microcirculation in Response to UV Light.

[0204] Topical application of nitric oxide to the skin causes immediate vasodilation of the capillaries of the papillary plexus resulting in increased blood flow to the skin. This vasodilation results in an immediate transient localised erythema and dermal blood flow correlates directly with the concentration of NO delivered. This can be measured by Laser Doppler, a standard technique for the non-invasive blood flow monitoring and measurement of blood flow in the microcirculation (Seabra et al., British Journal of Dermatology 2004; 151: 977).

[0205] A laser Doppler perfusion monitor was used (Moor Instruments Ltd, Axminster, U.K.) with one satellite unit connected to the server allowed flux readings from two laser probes to be recorded simultaneously.

[0206] To measure the effects of sunscreen formulations on microcirculation control base sunscreen formulation was applied to one site on the forearm and sunscreen containing nitric oxide generating additives to the other site. A baseline recording was made in real time until readings had stabilised. In the absence of UV light no increase of blood flow flux was detected in response to the sunscreens. Recording was then paused, probes detached and the forearm area irradiated with broadband UV light for 5 minutes. Probes were then re-attached to the treated sites and recording of blood flow resumed. Cutaneous blood flow, measured as red blood cell flux, was used as an index of erythema.

[0207] Results are shown in FIGS. 6 and 7. Sunscreen formulations with nitric oxide generating components added produced an increase in flux in comparison to control sunscreen.

[0208] Enhancing Effects of No Generating Components on the Formation of Vitamin D3 from 7-Dehydro Cholesterol.

[0209] Vitamin D3 is synthesised in skin from 7-Dehydro Cholesterol (7-DHC) following exposure to UV-B. Sunscreens block UV-B from impinging on the skin and inhibit the formation of Vitamin D3 from 7-DHC (J Reichrath. British Journal of Dermatology 2009 161 (Suppl. 3), pp 54-60). Since NO generation and 7-DHC conversion occur in the same cutaneous environment in response to sunlight and are both blocked by sunscreen use we investigated the effects of NO generating components on formation of Vitamin D3 from 7-DHC.

[0210] Sunscreen was applied to skin samples over an area of 10 mm.sup.2 and irradiated with broadband UV-B (280-320 nm) radiation for 3 hours.

[0211] Skin samples were thoroughly wiped free of creams and 4 mm diameter punch biopsies on full thickness skin were taken and weighed. Skin specimens were then homogenized within 0.5 ml PBS buffer (pH7.2) and dispersed by vortexing and ultrasonication for 15 mins. 1 ml Hexane was added for lipid extraction. Samples were kept under 4 C overnight for Vitamin D3 extraction. Following centrifugation at 12000 rpm for 10 mins the upper Hexane phase was aspirated and dried under nitrogen. The nitrogen dried sample was re-suspended in aqueous buffer and Vitamin D3 quantified by ELISA (BioVision Vitamin D3 ELISA Cat #K4806-100).

[0212] Results are shown in FIG. 8. UV-B irradiation of skin alone did not result in Vitamin D3 production probably as a result of low endogenous levels of 7-DHC. Skin treated with organic or physical sunscreen supplemented with 7-DHC responded to UV-B irradiation by producing detectable levels of Vitamin D3. Unexpectedly, addition of NO generating components to 7-DHC supplemented sunscreens greatly enhanced the production of Vitamin D3 in response to UV irradiation.