SOLUTE AND SOLUTE MIXTURE, AS WELL AS A COMPOSITION COMPRISING AT LEAST ONE SOLUTE, FOR USE IN THE PREVENTION OR TREATMENT OF COSMETIC OR PATHOLOGIC EFFLORESCENCES CAUSED BY AIRBORNE PARTICLES

Abstract

The invention relates to a compatible solute or a solute mixture, as well as to a composition comprising the solute or solute mixture, for use in the prevention or treatment of cosmetic or pathologic efflorescences caused by airborne particles, such as pigmentation accompanying with skin ageing, and de- or hypopigmentation, hyperpigmentation, as well as alterations accompanying with atrophy in the broadest sense. The solute or solute mixture, as well as the composition comprising the solute or solute mixture comprises at least one compound according to formula I, formula II, physiologically compatible salts of formula I and formula II, stereoisomeric forms of the compound of formula I, formula II, and physiologically compatible salts of the stereoisomeric forms, and the solute mixture comprises at least two of the afore-mentioned compounds, wherein in formula I

##STR00001##

and in formula II

##STR00002##

R1=H or alkyl; R2=H, COOH, COO-alkyl or CO—NH—R5; R3 and R4 each independently H or OH; R5=H, alkyl, an amino acid residue, dipeptide residue or tripeptide residue and n=1, 2 or 3 and alkyl corresponds to an alkyl group having C.sub.1-C.sub.4 carbon atoms.

Claims

1. A method for preventing or treating cosmetic or pathologic efflorescences in a subject caused by airborne particles, the method comprising administering to the subject an effective amount of a composition comprising at least one compound selected from the group consisting of compounds of formula I, compounds of formula II, physiologically compatible salts thereof, stereoisomeric forms thereof, physiologically compatible salts of the stereoisomeric forms thereof, and combinations thereof, ##STR00005## wherein: R1 is H or C.sub.1-C.sub.4 alkyl; R2 is H, COOH, COO—[C.sub.1-C.sub.4 alkyl], or CO—NH—R5; R3 and R4 are each independently H or OH; R5 is H, C.sub.1-C.sub.4 alkyl, an amino acid residue, a dipeptide residue, or a tripeptide residue; and n is 1, 2 or 3, thereby preventing or treating cosmetic or pathologic efflorescences in the subject.

2. The method according to claim 1, wherein the at least one compound is selected from the group consisting of S-ectoine, R-ectoine, (S,S)-hydroxyectoine, (S,R)-hydroxyectoine, (R,S)-hydroxyectoine, (R,R)-hydroxyectoine S-homoectoine, physiologically compatible salts thereof, amides thereof, esters thereof, and combinations thereof.

3. The method according to claim 1, wherein the at least one compound is present in enantiopure form with a purity of greater than or equal to 90%.

4. The method according to claim 1, wherein the at least one compound or the solute mixture is an expression inhibitor of genes associated with efflorescences.

5. The method according to claim 4, wherein the at least one compound or the solute mixture inhibits the expression of a gene selected from the group consisting of POMC, Cyp1a1, and MMP1, which is induced by airborne particles.

6. The method according to claim 1, wherein the cosmetic or pathologic efflorescence is an efflorescence of melanocytes and/or keratinocytes.

7. The method according to claim 1, wherein the cosmetic or pathologic efflorescence comprises an alteration of at least one cell layer in at least one part of the skin, being phenotypically visible on the skin surface,

8. The method according to claim 1, wherein the solute mixture comprises at least two compounds selected from the group consisting of compounds of formula I, compounds of formula II, physiologically compatible salts thereof, stereoisomeric forms thereof, physiologically compatible salts of the stereoisomeric forms thereof, and combinations thereof.

9. The method according to claim 1, wherein the solute mixture comprises, based on the sum of all compounds having a total content of 100% by weight, greater than or equal to 85% by weight S-ectoine, and less than or equal to 15% by weight (S,S)-hydroxyectoine.

10. The method according to claim 1, wherein the airborne particles that cause the cosmetic or pathologic efflorescences comprise particulate and/or fibrous environmental noxae according to specification of DIN EN 481, WHO/SDE/OEH/99.14 or ISO 4225.

11. The method according to claim 1, wherein the airborne particles comprise nanoparticles, carbon nanoparticles, particles containing polycyclic aromatic hydrocarbons, and/or diesel soot particles.

12. A composition comprising at least one compatible solute or one solute mixture comprising at least two compatible solutes for use in the prevention or treatment of cosmetic or pathologic efflorescences caused by airborne particles, wherein the compatible solute or the solute mixture comprises at least one compound selected from the group consisting of compounds of formula I, compounds of formula II, physiologically compatible salts thereof, stereoisomeric forms thereof, physiologically compatible salts of the stereoisomeric forms thereof, and combinations thereof, ##STR00006## and further wherein: R1 is H or C.sub.1-C.sub.4 alkyl; R2 is H, COOH, COO—[C.sub.1-C.sub.4 alkyl], or CO—NH—R5; R3 and R4 are each independently H or OH; R5 is H, C.sub.1-C.sub.4 alkyl, an amino acid residue, a dipeptide residue, or a tripeptide residue; and n is 1, 2 or 3.

13. The composition according to claim 12, wherein the at least one compatible solute or the solute mixture is present in the composition in an amount of 0.0001% by weight to 50% by weight based on the total content of the composition.

14. The composition according to claim 12, wherein the at least one compatible solute or the solute mixture is present in the composition in an amount of 0.0001% by weight to 10% by weight based on the total content of the composition.

15. The composition according to claim 12, wherein the airborne particles that cause the cosmetic or pathologic efflorescences comprise particulate and/or fibrous environmental noxae according to specification of DIN EN 481, WHO/SDE/OEH/99.14 or ISO 4225.

16. The composition according to claim 12, wherein the composition is present in a form selected from the group consisting of: i) solid forms comprising one or more powders, tablets, granules, film-coated tablets, dragees, capsules, effervescent tablets, powders, soaps, and combinations thereof; ii) liquid forms comprising one or more solutions, injections, infusions, tinctures, infusion solutions, suspensions, syrups, juices, emulsions, applications, foams, creams, lotions, surfactant-containing cleaning preparations, oils, and combinations thereof and iii) mixtures comprising one or more sprays, aerosols, inhalants, ointments, pastes, and combinations thereof.

17. The composition according to claim 12, wherein the cosmetic efflorescence is a senescence of skin exposed to airborne particles, and further wherein the skin exhibits deviating pigmentation, deviating expression of at least one gene, and/or deviating production of at least one biomolecule as compared to skin that has not been exposed to the airborne particles.

18. The composition according to claim 12, wherein the cosmetic efflorescence caused by airborne particles is a skin aging and/or pigmentation.

19. The composition according to claim 12, wherein the pathologic efflorescence comprises depigmentation, hypopigmentation, hyperpigmentation, acquired melanotic and non-melanotic hyperpigmentation, incontinentia pigmenti, dermatoses, dermatoses caused by building materials or cement, pigmented spots, anuli pigmentosi, prurigo pigmentosa, peribuccal pigmentation, carcinomas, spinocellular coarcinoma, pigmented spindle-cell tumor, skin diseases caused by airborne particles containing polycyclic aromatic hydrocarbons, benign or malign tumor formation, Leschke syndrome, skin metastases, Becker's nevus, malignant melanoma, pigmented neurofibroma, urticarial pigmentosa, Peutz-Jeghers syndrome, epidermodysplasia verruciformis, tacrolimus, pimecrolimus, vitiligo, atrophy-associated diseases comprising exsiccation eczemas, elastoses, purpura senilis, lentigo solaris, or Favre-Racouchot syndrome.

Description

DESCRIPTION OF THE FIGURES

[0134] FIG. 1 shows the expression of Cyp1a1 gene relative to the total content of 18s-rRNA of keratinocytes. Keratinocytes incubated with Printex90, Huber 990, SRM1650 and SRM2975 exhibit significantly increased Cyp1a1 expression compared with untreated Keratinocytes (=blank). Cyp1a1 expression could be inhibited by pretreatment with 2 mM ectoine (model particles/ectoine). (p<0.05 and comparison to blank or p<0.05 as treated).

[0135] FIG. 2 shows the expression of POMC gene relative to the total content of 18s-rRNA of keratinocytes. Keratinocytes incubated with Printex90, Huber 990, SRM1650 and SRM2975 exhibit significantly increased POMC expression compared with untreated Keratinocytes (=blank). Cyp1a1 expression could be inhibited by pretreatment with 2 mM ectoine (model particles/ectoine). (p<0.05 and comparison to blank or p<0.05 as treated).

[0136] FIG. 3 shows the expression of MMP1 gene relative to the total content of 18s-rRNA of keratinocytes. Keratinocytes incubated with Printex90, Huber 990, SRM1650 and SRM2975 exhibit significantly increased MMP1 expression compared with untreated Keratinocytes (=blank). Cyp1a1 expression could be inhibited by pretreatment with 2 mM ectoine (model particles/ectoine). (p<0.05 and comparison to blank or p<0.05 as treated).

EXAMPLES

Example 1: Model Particles

[0137]

TABLE-US-00001 TABLE 1 Used model particles: Model particles Name Source ultra-fine soot particles Printex90 Evonik Industries, Essen, Germany fine soot particles Huber 990 Evonik Industries, Essen, Germany diesel exhaust particles SRM1650 National Institute of Standards and Technology, Gaithersburg, MD, USA diesel exhaust particles SRM2975 National Institute of Standards and Technology, Gaithersburg, MD, USA

[0138] The toxicity of soot particles, such as Printex90 and Huber990, has been extensively studied, and summarized by the European Commission of consumer protection in a meeting on 12 Dec. 2013. The revised report has been published as SCCS/1515/13 on 27 Mar. 2014 (ISBN 978-92-79-30120-9). Reference is made to this report and its content is incorporated by reference into the disclosure of the present invention.

Printex90 Particle (CAS Number 1333-86-4) has the Following Composition:

[0139] Printex90 particle are ultra-fine amorphous carbon soot particles insoluble in water (>99% pure soot, PAH=0.039 ppm) having a molecular weight of 12, a density at 20° C. of 1.7 to 1.9 g/cm, melting point 3000° C., pH according to ASTM 1512 in a range of 4 to 11 (at 50 g/L water, 20° C.). The particles have a bulk density of 20-640 kg/m.sup.3. Particles having a pH value of greater than 7 correspond to furnace soot rubber.

[0140] The particle size is 7.7 nm to 28.2 nm, and has a mean particle diameter of 14.3±0.6 nm (surface 250 to 337 m.sup.2/g).

[0141] After exposition of Printex90 particles acute toxicity in rats by oral intake has been shown (LD50 at least 8000 mg/kg).

Huber990 Particles:

[0142] Huber 990 particles are fine carbon soot particles insoluble in water, having comparable constitution to Printex90. Huber990 particles have a mean particle diameter of 260.2 nm (surface 7.9 m.sup.2/g).

SRM1650 Particles have the Following Composition:

[0143] SRM particles are complex diesel soot particles which, after a total of 200 operating hours, are obtained from heat exchangers of four diesel engines, operating and being loaded to a different degree. The obtained particles correspond to diesel soot particles occurring in environment, and have the composition described below. In salmonella mutagenesis assay (Maron, D., and Ames, B., “Revised Methods for Salmonella Mutagenicity Test”, Mutation Research, 113: 1723-212) mutagenic activity of 17.5±1.5% has been shown for SRM1650 particles, based on the average methylene chloride content which was isolated from SRM1650 particles.

Composition According to Available Certificate (See Source, Table 1)

[0144]

TABLE-US-00002 Component Concentration [μg] Standard deviation (±) 1-Nitropyrene 19 2 Benz[a]anthracene 6.5 1.1 Benzo[a]pyrene 1.2 0.3 Benzo[ghi]perylene 2.4 0.6 Fluoranthene 51 4 Pyrene 48 4

Non-Certified Composition Available at Afore-Mentioned Source (Table 1):

[0145]

TABLE-US-00003 Component Concentration [μg] 2-Nitrofluorene 0.2 6-Nitrobenzo[a]pyrene 1.6 7-Nitrobenz[a]anthracene 2.8 9-Fluorenone 33 Benzo[k]fluoranthene 2.1 Benzo[e]pyrene 9.6 Chrysene 22 Indeno[1,2,3-cd]pyrene 2.3 Perylene 0.13 Phenanthrene 71

SRM2975 Particles:

[0146] SRM2975 particles have comparable constitution to SRM1650 particles, but have another particle size. SRM2975 particles have a particle size of 10 μm to 50 μm.

[0147] The model particles were suspended in phosphate buffered saline solution and treated with ultrasound for 1 minute (Printex90, SRM1650 and SRM2975) or 90 seconds (Huber990). Subsequently, addition to keratinocytes was done immediately.

[0148] The concentration of the respective model particles used for treatment of keratinocytes was 1.5 μg cm.sup.−2, respectively, based on the total area of the keratinocytes surface in subconfluent cell layer.

[0149] The incubation time of keratinocytes under the influence of model particles was 24 hours.

Example 2: Cell Cultures

[0150] Human epidermal keratinocytes were taken from healthy patients during abdominoplasty or breast reduction with the consent of the patients. The tissue donors originate in Asia or were of Caucasian origin.

[0151] The keratinocytes isolated from the tissues were cultivated in Keratinocyte-SFM (Gibco®, Life Technologies GmbH, Darmstadt, Germany). The medium was enriched by 5 mg/L of human recombinant epidermal growth factor (EGF), 50 mg/L pituitarium which is an extract from pituitary gland from bovine (Bovine Pituitary Extract, BPE), 1% L-glutamine, 1% streptomycin/amphotericin B (Invitrogen, Karlsruhe, Germany). The cultivation was done at humid atmosphere in the presence of 5% CO.sub.2.

[0152] Subconfluent keratinocytes were transferred to sample vessels having 12 sample wells (12-well plates), and were each cultivated in the sample wells. Treatment with the model particles was also done in the sample wells.

Example 3: Treatment of Cell Cultures with Model Particles

[0153] The cultivated keratinocytes (Example 2) were subsequently incubated for 24 hours in the media of example 2 lacking BPE and EFG in the presence of compatible solute S-ectoine having a final concentration of 2 mM.

[0154] After incubation with S-ectoine, the suspended and ultrasonically treated model particles (see example 1) were immediately added to the keratinocytes. After an incubation time of 24 hours in the presence of the model particles, RNA was isolated from the keratinocytes and relative RNA content was determined.

Example 4: Gene Markers

[0155] The expression of proinflammatory gene ICAM-1, POMC gene encoding for proopiomelanocortin, MMP1 gene encoding for a human collagenase, and Cyp1a1 encoding for a phase-I-enzyme of Cytochrome P450 superfamily was investigated as a marker for the influence of the model particles on skin condition.

[0156] Proopiomelanocortin (POMC) is a precursor protein made of 241 amino acid residues. It is synthesized from pre-proopiomelanocortin (pre-POMC) consisting of a polypeptide being 285 amino acid in length, wherein a signaling peptide made of 44 amino acids is cleaved off during translation. POMC in turn can enzymatically be cleaved of in peptides, such as ACTH, α-MSH, β-MSH, and γ-MSH. The afore-mentioned peptides are peptide hormones and are summarized under the group of melanotropins or hormones stimulating melanocytes (MSH). One exception is ACTH which ranks among the group of melanocortins and is referred to as adrenocorticotropin. Melanotropins regulate the melanin synthesis in pigment forming melanocytes, as well as melanocytes expansion and pigment dispersion, thus having significant influence on pigmentation of the skin. Increased expression of POMC thus indicates increased melanotropin content and is thus an indicator for increased pigmentation of the skin.

[0157] The enzyme encoded by Cyp1a1 gene ranks among the group of phase-I-P450-Cytochrome enzymes and mediates metabolism of drugs, foreign substances and environmental toxins. The Cy1a1 enzyme is a monooxygenase and responsible for metabolizing the afore-mentioned compounds. The protein is encoded in endoplasmic reticulum and its expression can severely be induced by polycyclic aromatic hydrocarbons. The enzyme encoded by Cyp1a1 metabolizes environmental noxae, in particular polycyclic aromatic hydrocarbons, into cancer-causing intermediates. Cyp1a2 is a family member related to Cyp1a1. Increased Cyp1a1 expression, and, in particular, increased Cyp1a2 expression, thus proves detoxification reaction.

[0158] The collagenase encoded by MMP1 gene ranks among the matrix metalloproteases and cleaves the peptide bond between proline and other amino acids of collagen I, II, III, VII and X, thereby degrading the collagen. Collagenases and, in particular, the MMP1 collagenase are involved in tissue remodeling and biological processes of the skin, such ach morphogenesis or tumor growth (benign or malign tumor formation). Thus, increased expression of MMP1 gene is a proof for increased activity of the skin cells, in particular keratinocytes. Increased MMP1 gene expression can be an indication of excessive cell growth and, thereby, of benign or malign carcinogenesis.

Example 5: RNA Isolation and PCR

[0159] The total RNA content was determined according to Grether-Bech et al. (Grether-Beck S, Mühlberg K, Brenden H, Felsner I, Brynjólfsdóttir A, Einarsson S, Krutmann J. “Bioactive molecules from the Blue Lagoon: in vitro and in vivo assessment of silica mud and microalgae extracts for their effects on skin barrier function and prevention of skin ageing”. Exp Dermatol 2008 17:771.).

[0160] For this purpose, the total RNA was isolated using RNeasy Total RNA Kits (Qiagen, Hilden; Germany). The RNA concentration was photometrically determined at 260/280 nm (Biophotometer, Eppendorf, Hamburg, Germany). Aliquots of total RNA of 100 ng were used for first strand cDNA synthesis using Superscript™ III for the transcription step using random primers (Invitrogen, Karlsruhe, Germany).

[0161] A specific, complementary primer was designed for each gene, Cyp1a1, MMP1 and POMC, based on the cDNA sequence using Primer Express™ 2.0 software. For the total RNA content a specific primer for the proof of 18s rRNA was designed.

[0162] Independent experiments were performed with a triple determination in each case, and the mean was determined. PCR reaction was done using CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, München, Germany). In order to fluorescent marking SybrGreen-Mix was used (Thermo Fisher Scientific, St. Leon-Rot, Germany). For each experiment three samples, respectively, were determined in duplicate, and the mean was determined.

[0163] The PCR protocol comprised 46 cycles, each cycle 15 minutes at 94° C. (activation of heat-activating taq polymerase) 10 seconds at 95° C. (denaturation), 20 seconds at 55° C. (annealing), 20 seconds at 72° C. (elongation).

[0164] Likewise, for comparison of relative expression the expression of untreated keratinocytes was investigated. The expression of untreated keratinocytes was set to 1 and the changes in expression were determined according to Livak and Schmittgen of 2001 (Livak K J, Schmittgen TD (2001) “Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method”. Methods 25: 402-408).

Example 6: Statistics

[0165] One-way analysis of variance was used as non-parametric test for comparison of the differences between the measurements, and a p-value of less than 0.05 was specified as being statistically significant (SigmaPlot 12.5). The data underlying the statistical analysis are not shown here.

Example 7: Protective Effect of a Solute According to the Invention, Here S-Ectoine, on the Skin

[0166] In the following table 3, the effects of all experiments are summarized, as also shown in FIGS. 1, 2, and 3.

TABLE-US-00004 Expression ±S-ectoine Remaining inhibition mean ± SD mean ± SD activity % % Cyp1a1 Printex90 1.64 ± 0.43  1.19 ± 0.29.sup.  30 70 Huber990 5.17 ± 48*.sup.  2.80 ± 0.14.sup.+ 43 57 SRM1650 33.20 ± 4.64*  18.26 ± 1.72.sup.+  54 46 SRM2975 9.46 ± 1.90* 6.03 ± 0.52.sup.+ 59 41 POMC Printex90 1.61 ± 0.23* 0.88 ± 0.05.sup.+ 0 100 Huber990 1.94 ± 0.30* 0.83 ± 0.04.sup.+ 0 100 SRM1650 1.72 ± 0.20* 1.01 ± 0.05.sup.+ 1 99 SRM2975 1.51 ± 0.20* 0.99 ± 0.08.sup.+ 0 100 MMP1 Printex90 0.88 ± 0.08  0.82 ± 0.10.sup.  — — Huber990 1.91 ± 0.13* 1.53 ± 0.13.sup.+ 58 42 SRM1650 3.19 ± 0.22* 2.62 ± 0.17.sup.+ 74 26 SRM2975 1.27 ± 0.21  1.28 ± 0.22.sup.  — —

[0167] Ultra-fine Printex90 particles enhance the expression of Cyp1a1 mRNA by a factor of 1.64 (mean±0.43) (see table 3, column 2 “Expression”), which is reduced by adding S-ectoine by a factor of 1.19 (mean±0.29).

[0168] When treated with Huber990 particles that enhance Cyp1a1 expression by a factor of 5.17, the effect is significantly stronger. It has surprisingly been found that pretreating with S-ectoine results in significant reduction of Cyp1a1 expression by a factor of 2.80. This shows the preventive effect of S-ectoine in prevention of the formation of cancer-causing intermediates from environmental noxae.

[0169] Experiments with polycyclic aromatic hydrocarbons, such as SRM1650 and SRM2975, show an even stronger effect. Both particles can be converted into cancer-causing intermediates by Cyp1a1 mediated metabolizing, which may cause skin diseases right up to skin cancer, as described above.

[0170] SRM2975 particles enhance Cyp1a1 expression by a factor of 9.46. Pretreating with a solute according to the invention of formula I or of formula II, here S-ectoine, results in inhibition of Cyp1a1 expression by a factor of 6.03.

[0171] SRM1650 particles enhance Cyp1a1 expression by a factor of 33.20. Pretreating with a solute according to the invention of formula I or of formula II, here S-ectoine, results in significant inhibition of Cyp1a1 expression by a factor of 18.25. This proves the protective effect of the solutes according to the invention of formula I and of formula II in the prevention of pathologic efflorescences induced by polycyclic aromatic hydrocarbons, in particular of those comprising an alteration of keratinocytes.

[0172] All of the tested particles Printex90, Huber990, SRM1650, as well as SRM2975 resulted in significantly enhanced POMC expression after incubation of keratinocytes in the presence of the afore-mentioned particles for 24 hours.

[0173] S-ectoine pretreatment for 24 hours resulted in significant inhibition of POMC expression, nearly to the initial level of untreated keratinocytes, for all of the tested germs, as summarized in table 3.

[0174] The protective and inhibitory effect of S-ectoine on POMC expression induced by airborne particles proves the effectiveness of the solutes according to the invention of formula I and of formula II in the prevention or treatment of cosmetic or pathologic efflorescences, which exhibit phenotypic alteration in pigmentation, as well as against skin ageing.

[0175] Due to reduced POMC expression less proopiomelanocortin is synthesized. Consequently, fewer melanotropins are cleaved off, thereby in turn stimulating fewer pigment forming cells, melanocytes. This result in reduced pigment formation (melanin) compared to untreated cells exposed to airborne particles. Phenotypically, for example, hypopigmentation, hyperpigmentation, cosmetic alteration in pigmentation and senile atrophy, micro- and macroscopically visible, are thus reduced by pretreatment with the solute according to the invention of formula I and of formula II, or the solute mixture.

[0176] Similar effects could be shown in the proof of inhibition of the solute according to the invention, here S-ectoine, on MMP1 expression induced by Huber990 and SRM1650. Inhibition of the expression of MMP1 gene proves downregulation of collagenase, hence reduced collagen degradation, and thus an inhibitory effect against skin ageing. In this way the solute according to the invention of formula I and of formula II, as well as the mixture, preferably comprising S-ectoine and (S,S)-hydroxyectoine, shows positive effect in the prevention or treatment of skin ageing, senescence of the skin or elasticity displacement, caused by airborne particles.