TECHNOLOGICAL FIELD

The invention generally pertains to the field of cosmetics, and specifically to cosmetic formulations using advanced liposomal constructs to provide improved topical and dermal delivery of lipophilic and non-lipophilic actives so as to enhance their benefits to the skin.

BACKGROUND

Medical and cosmetic benefits of minerals have been known for millennia. Modem cosmetic products are made to incorporate various sorts of minerals and mixtures of minerals, some examples are silicates (e.g., smectite, kaolinite), sodium, potassium and magnesium salts and magnesium silicates (talc). Scientifically, studies have shown that Mg ⁺² , Ca ²⁺ , Zn ²⁺ and K ⁺ ions are key elements for a range of enzymatic activities partaking in normal metabolism and permeability of the skin, or in the maintenance of healthy skin homeostasis. Due to their hygroscopic properties, minerals further contribute to the restoration of skin moisture. Upon absorption, they enhance humectant properties of the skin by retaining the intracellular water capacity of the skin tissue and supplying humidity from within. Deficiencies of specific minerals have been related to specific skin conditions, such as zinc deficiency in alopecia, hair discoloration and dermatitis.

Dead Sea Water (DSW) is known for its richness in natural minerals, with a total content reaching up to 34% w/v. Compared to Mediterranean and typical ocean water, DSW is further distinct by exceptionally high and unique composition of specific electrolytes. Some examples are concentrations of Cl’ (224,900 mg/1 in DSW vs. 22,900 and 19,000 mg/1 in Mediterranean and ocean water, respectively), Mg ²⁺ (44,000 mg/1 vs. 1,490 and 1,350 mg/1), Na ⁺ (40,100 mg/1 vs. 12,700 and 10,500 mg/1), Ca ²⁺ (17,200 mg/1 vs. 470 and 400 mg/1), K ⁺ (7,650 mg/1 vs. 470 and 390 mg/1), and Br (5,300 mg/1 vs. 76 and 65 mg/1). Electrolytes content in DSW is subjected to fluctuations, depending on season, annual rainfall, surface water inflow, DSW source, its proximity to mineral rich hot springs, surface springs and groundwater, and other factors such as water pollution. In general, the salinity of DSW tends to increase over time due to climate change. DSW extracts have a special interest for cosmetics. One example has been provided by AHAVA under the brand name “Osmoter” ™, listed as "Maris Aqua (Dead Sea Water)" in INCI (International Book of Cosmetic Ingredients), which has become a globally approved active ingredient for cosmetics. Osmoter per se is considered a natural product with a distinct content of naturally occurring minerals characteristic of a specific location and season at the Dead Sea, which is further boosted by solar evaporation. It has been related to a series of molecular changes involving mediators of signal transduction, anti-inflammatory and red-ox pathways, and modulation of specific genes and gene products related to skin homeostasis, with demonstrable impact on improved recovery and functionality of the skin barrier, rejuvenation and hydration of the skin and improved protection of the skin layers against external and internal insults [1-2].

Despite the strong incentive to use Osmoter and other DSW extracts in cosmetics, their translation to specific cosmetic products has been extremely difficult. For example, the cosmetic products by AHAVA generally contain very low concentration of DSW (about 1% w/w). The main reason is that compositions with high mineral content are generally unstable and lose some of their effectiveness over time. Another reason is rooted in the relatively poor absorption of minerals into the skin. Altogether, achieving successful formulations of DSW and DSW extracts has proved to be very challenging in terms desired homogeneity, translucency, texture, effectiveness in vivo and long-term stability that would be expected for quality cosmetic products.

In other words, there is an obvious need to find new approaches to formulation of mineral rich compositions, and specifically DSW and DSW extracts, that will meet the requirements of consistency, stability and effectiveness in vivo, and that in time can be expanded to a commercial scale. Several examples of such formulations were reported in the patent literature [3-7] .

Liposome-based technology has set many precedents of formulations with improved delivery of lipophilic and hydrophilic actives, especially for oral applications. Applications of liposomes-based formulations in cosmetics goes back to 1987 with the launch of Capture™ by Christian Dior. Many additional brands are currently using liposomal formulations in lotions, creams, and other cosmetic products. In skin models in vitro, liposomal formulations were consistently associated with benefits of improved protectivity, bioavailability and controlled release of actives, and moisturization [8-9]. However, more recent studies in vivo applying liposomes with their cargo of actives directly onto the skin suggested otherwise. Some of these studies have shown that upon contact with the skin surface, the liposomes are prone to breaking, and factually are less effective than expected for topical delivery of actives into the skin [10]. More generally, liposomes have a general tendency to leak and fuse when in suspension, which results in loss of encapsulated material and gradual shift towards larger liposomes. In addition, the phospholipids which constitute an essential part of the liposomal core structure tend to degrade due to oxidation and hydrolysis, which in turn compromises this structure and leads to shortened half-life. In other words, the implementation of liposomal technology, especially in the context of skin, still remains elusive and requires specific solutions to the problems of liposomal instability, lack of intactness and inconsistency of structure and size, to support their incorporation into specific products on a commercial scale.

REFERENCES

1. Portugal-Cohen et al. Dear Sea minerals: new findings on skin and the biology beyond. Exp Biol 2019, 28(5):585-592

2. Cohen et al. Nrf-2 pathway involvement in the beneficial skin effects of moderate ionic osmotic stress - the case of the Dead Sea water. J Cosmet Dermatol Sci Appl 2022, 12:109-130

3. US 6,248,340

4. US 6,582,709

5. US 7,687,065

6. US 9,693,936

7. WO 2020/208628

8. Hofland et al. Interactions between liposomes and human stratum corneum in vitro: freeze fracture electron microscopical visualization and small angle X-ray scattering studies. Br J Dermatol 1995, 132(6):853-866.

9. Kirjavainen et al. Interaction of liposomes with human skin in vitro - The influence of lipid composition and structure. Biochim Biophys Acta - Lipids and Lipid Metabolism 1996, 1304(3): 179-189.

10. Dreier et al. Super-resolution and fluorescence dynamics evidence reveal that intact liposomes do not cross the human skin barrier. PLOS One 2016; 11(1): e0146514. GENERAL DESCRIPTION

Tranexamic acid (TXA/TA) is a known antifibrinolytic, which is currently its only approved indication. More recently, there is a growing body of evidence that it may have additional anti-inflammatory and skin lightening properties, which in turn make it an attractive active for cosmetic products. In terms of skin lightening, it has been shown that TXA interferes with the interaction between keratinocytes and melanocytes that underlies the appearance of pigmentation, dark spots and uneven skin tone. Studies have shown that for treating melasma (black spots) TXA is as effective as hydroquinone (the gold standard for reducing hyperpigmentation), and that it may be further effective against sun spots, age spots and other damages of UV exposure of the skin. Altogether, owing to its intrinsic anti-inflammatory, antifibrinolytic and skin lightening activities, TXA seemed to be a good candidate drug to address common skin conditions such as imperfect skin tones, skin redness, skin inflammation and acne, and also more severe conditions such as rosacea, erythema, eczema, dermatitis and other inflammatory and red skin conditions. In addition, TXA is generally safe and well-tolerated by all skin types, which again makes it an attractive candidate for cosmetics, cosmeceutical, and OTC skin care products.

The main problem, however, with the use of TXA in cosmetics is it being zwitterionic and therefore incompatible with the native negative charge and slight acidity of the skin. As a result, topical adsorption and absorption of a “naked” TXA, without permeation enhancers, is relatively limited and is highly dependent on the formulation context.

The present invention stems from a general need to find successful formulations of actives or combinations of actives that are relatively new to cosmetics, or those which use was impeded by difficulties in finding the right formulations. Both TXA and Osmoter, in view of the problems of instability and/or incompatibility of charge and reduced topical absorption and ineffectiveness in vivo, seemed to be likely candidates for the invention.

The first step forward in this invention was driven by the finding that TXA and cetyltrimethylammonium chloride (CTAC) can form a stable self-assembling non- covalent complex. CTAC is a long-chain quaternary ammonium surfactant (a cationic surfactant). It is frequently used in hair conditioners and shampoos, predominantly in combination with long-chain fatty alcohols, and is considered generally safe. It is also used as a topical antiseptic and dispersant. The molecular structures of TXA and CTAC and the formation of TXA-CTAC complex is illustrated in Scheme 1. The complex is essentially a salt wherein the amino ethylene moiety of TXA is protonated.

N, N,N -trimethylhexadecan- 1 -aminium chloride

4-(Aminomethyl)cyclohexanecarboxylic acid

TA-CTAC complex

Scheme 1

It has been presently shown that the tendency to form of TXA-CTAC complex in the presence of its respective components is relatively high and occurs even at low CTAC concentrations (EXAMPLE 1), and further that the tendency to form this complex was distinctive of CTAC and TXA and could not be reproduced to the same extent with other tested complexants and cationic agents (EXAMPLE 2). More specifically, it was shown that the complexing capacity of CT AC with TXA was superior to other complexing agents in terms of efficiency and charge. About 0.25% CTAC (w/w) was sufficient to produce a sufficiently high yield of a positively charged TXA-CTAC complex. The aspect of charge is critical in the context of skin in view of a high content of negatively charged lipids in the external layers of the stratum corneum, which precludes the absorption of negatively charged molecules.

The importance of finding of the positively charged self-assembling non-covalent TXA-CTAC complex is two-fold:

1 ^st , the TXA-CTAC complex per se is an attractive topical prototype formulation due to a series of valuable cosmetic properties, i.e., the TXA-related anti-inflammatory, antifibrinolytic and skin lightening effects, and

2 ^nd , it provides an initial framework for introducing other actives of interest to produce pluripotent topical formulations for cosmetic and dermatological purposes.

The next step forward was made with the attempt to combine the two actives of choice, TXA-CTAC and Osmoter, in the same formulation construct in the presence additional co-formulation agents, of which phospholipid(s) and surfactant(s) proved to be obligatory. To that end, the invention proposes two prototype formulations, each incorporates TXA and an Osmoter - the Crystal (Crys) or the Aqueous (Aq) Osmoters (EXAMPLE 3), and further proposes methods for making such formulations by organic to aqueous (ORG to Aq) phase and Aq to ORG phase methods (EXAMPLES 4-5).

Both types of the formulations, with Crys and Aq Osmoters, exhibited a characteristic morphology resembling unilamellar liposomes, i.e., spherical vesicles with a unilamellar bilayer of phospholipids and a hydrophilic core, owing to which they were named LipOsmoter. In terms of particle size, the average diameter of these liposomal structures was estimated at 50-100 nm by Cryo-TEM images and at 20-30 nm by Dynamic Light Scattering (DLS), thus classifying them as nanoliposomal delivery systems with TXA-CTAC and Osmoter as optional actives (EXAMPLE 4).

Subsequent studies of surface charge of LipOsmoter showed that the two actives are compartmentalized, with the Osmoter residing in the hydrophilic core and the TXA- CTAC always associated with the outer surface, thus yielding an overall positive charge of the entire construct (net charge of >+l). Further studies showed that the positive net charge can be mitigated or masked and brought closer to neutral by the addition of various non-active components such as Guar Gum (EXAMPLE 4.4). In essence, LipOsmoter constitutes a multi-layered structure that compartmentalizes various active and non-active ingredients so as to produce an overall neutral or positive charge. In the context of skin, a formulation with such properties should be advantageous and has the potential of a better adsorption and absorption into the skin.

Additional studies showed that the physical properties of LipOsmoter, the particle size and surface charge, can be further modified by the choice of phospholipids (lecithin) and other non-active ingredients. Phospholipids with lower Phosphatidylcholine (PC) content were generally associated with smaller particle size (EXAMPLE 5). Current studies focusing on advanced versions of LipOsmoter, branded Multi Benefit LipOsmoter Ampule, have succeeded to produce formulations with an average surface charge that is sufficiently close to neutral (3.2 ±1.6 mV), which makes them especially advantageous for topical cosmetic and dermal applications (EXAMPLE 6).

More generally, both ORG to Aq and Aq to ORG phase methods yielded Crys and Aq LipOsmoters with very similar properties. Both types of products were characterized by a unique nano-liposomal layer-by-layer encapsulation structure that confers surprising properties of charge distribution, protectivity of actives and long-term stability (EXAMPLE 7).

Taken together, these properties provide significant advantages to producing effective pluripotent topical formulations with a combination of two important actives, TXA and Osmoter, which previously resisted formulation efforts. In the context of cosmetic and dermatological applications, combined presentation of both these actives in a liposomal construct that is specifically adapted to the physical properties of the skin holds promise of effective topical delivery and performance of these actives in the skin.

Nanoliposomal delivery systems, and especially those in the range of 20-100 nm, have many advantages, most prominently an increased surface area (increased drug exposure) and an improved stability profile. The presence of phospholipids allows them to act as effective drug penetration enhancers. They have the potential to entrap a wide range of hydrophilic and hydrophobic actives. Unlike other nanodelivery systems, nanoliposomes can be produced from natural and inexpensive ingredients. In other words, they are biocompatible and biodegradable and ecofriendly, and thus constitute highly adaptable and “smart” delivery systems. There are specific advantages in the context of skin. Apart from improved delivery and protection of actives, liposomes have been shown to improve skin hydration by surface adhesion, increase dermal bioavailability, and to protect the skin from external stressors like sweat or sun. They allow to combine various types of actives, nutrients and vitamins with specific benefits to the skin. In themselves, they are biodegradable and nontoxic and thus can be readily incorporated into cosmetic and OTC skin care products.

Several LipOsmoter forms are now being designed and developed with specific combinations of actives for providing specific cosmetic effects. Some examples are:

A. Formulations for effective skin brightening/even-tone/translucency

- TXA-CTAC complex

- Arbutin

- Dipotassium glycyrrhizate

- Lactic acid

B. Formulations for lasting anti-ageing effect

- Acetyl hexapeptide 8

- Copper peptide (GHK2-Cu)

C. Formulation for prolonged skin hydration /moisturization

- Trehalose

- Methyl gluceth-20

- Panthenol (Provitamin B5)

D. Formulations for effective skin firming/tightening

- TXA-CTAC complex

- Calcium lactate

Additional formulations can include amino acids, vitamins, and herbal extracts. Examples of such formulations are provided in this application (EXAMPLES 6-7).

Ultimately, the presently proposed LipOsmoter formulations serve as a basis for producing a variety of advanced cosmetic products with one or a combination of attributes of skin brightening, skin hydration/moisturization, skin anti-aging and/or skin firming/tightening, which are more improved and more long-lasting. One example of such products is a Multi Benefits Facial Ampules described in this application. BRIEF DECSRIPTION OF DRAWINGS

To better understand the subject matter and to exemplify how it may be carried out in practice certain embodiments of the invention are described by way of examples with reference to the following figures.

Fig. 1 illustrates the effect of CTCA concentration on the formation of TXA- CTAC complex with a fixed concentration of TXA/TA, using modified ninhydrin assay and UV spectroscopy (Sunrise absorbance reader at 575 nm). Figure shows that the formation of TXA-CTAC was independent of CTAC concentration and was equally effective with high and low concentrations of CTAC (CTAC 5% TA 5% vs. CTAC 0.25% TA 5% w/w).

Fig. 2 illustrates morphological properties of two LipOsmoter prototypes, Crystal (Crys) and Aqueous (Aq) Osmoters, produced by the ORG to Aq method. Figure shows Cryo-TEM images of Crys (left) and Aq (right) LipOsmoters, with spherical vesicles constructed of a unilamellar phospholipid bilayer and a hydrophilic core with an overall size (average diameter) in the range of about 50-100 nm.

Figs 3A-3B illustrate the effect of various formulation components, i.e., TXA- CTAC complex, Guar Gum and other additives, on the surface charge (zeta potential) of the resulting LipOsmoter, including Crys (3A) and Aq (3B) prototypes. Figures show that the initial charge of the constructs without TXA-CTC (LipOsmoters) was close to neutral or slightly negative, and with the addition TXA-CTAC (LipOsmoters +Complex) has become positive, suggesting that TXA-CTAC is associated with the surface of liposomes. The addition of Guar Gum and other additives (LipOsmoters+Complex+additives/Guar) masked the positive charge bringing it closer to neutral or about >+l, suggesting that they produce another layer of encapsulation.

Fig. 4 illustrates the effect of different types of lecithin on particle size (Zetasizer Nano ZS, AVR+SD, n=3). Figure shows mean particle size of LipOsmoters produced by Aq to ORG (RP) and ORG to Aq (NP) methods with Aq Osmoter and lecithin Emtk300 and lecithin Emtk900 (23% and 50% Phosphatidylcholine (PC) content, respectively). All preparations had mean particle size of < 100 nm. The method of preparation had some effect of particle size (RP vs. NP). Lecithin with reduced PC content related to smaller particle size (Emtk300 vs. Emtk 900). Fig. 5 illustrates the effect of different types of lecithin on surface charge in the same experiment (as in Fig.4). Figure shows that reduced PC content had neutralizing effect on surface charge.

Fig. 6 illustrates morphological properties of LipOsmoter with Aq Osmoter prepared by Aq to ORG phase method. Figure shows Cryo-TEM images in two magnifications, with similar spherical vesicles constructed of a unilamellar phospholipid bilayer and a hydrophilic core and an overall size of about 50-100 nm, suggesting that the physical properties of LipOsmoters produced by the ORG to Aq and Aq to ORG phase methods are essentially the same.

Fig. 7 reproduces the morphological analysis of the same preparation as above by Cryo-SEM (7A) and Cryo-TEM (7B) microscopy, showing particles with liposomal morphology and nanometric size.

Fig. 8 illustrates the feature of long-term stability (at 40°C) overtime (7 months) as revealed by the effect of various components (5%, 3% or 2% TXA/TA complex; 0.3% Guar gum; 0.5% Ziboxan; concentrations are in w/w) on particle size. LipOsmoters were prepared by ORG to Aq phase method The bar chart shows distribution of particles populations (%), and the line chart mean size of particles populations. The construct with 5% TXA/TA complex+0.3% Guar gum (light grey bars) was the most resilient in terms of preservation of particle size over the entire study period.

Fig. 9 presents partial data from the same experiment in relation to changes of net charge (mean zeta potential) overtime. Figure shows that the construct with 5% TXA/TA complex+0.3% Guar gum (light grey dotted line) was also resilient in terms of preservation of net charge over the entire study period.

Figs. 10A-10D present data from the same experiment in relation to changes in sensory properties of the formulations, i.e., opacity (A), color (B), odor (C) and pH (D). The construct with 5% TXA/TA complex+0.3% Guar gum (light grey dotted line) showed relatively good preservation of all these properties for the longest period time.

Fig. 11 reproduces the experiment of Figs 8-10 with LipOsmoters prepared by Aq to ORG phase method, and with additional variations of components. Long-term stability was assessed in relation to particle size. The results are similar to Fig. 8, with 5% TXA/TA complex+0.3% Guar gum (light grey bars) showing superiority in terms of preservation of particle size, by both methods of preparation. Fig. 12 presents analysis of net charge in the same experiment (as in Fig. 9). The construct with 5% TXA/TA complex+0.3% Guar gum (light grey dotted line) prepared by Aq to ORG phase method was also superior in terms of preservation of net charge.

Figs. 13A-13D present analyses of sensory properties in the same experiment (as in Figs 10A-10D). The construct with 5% TXA/TA complex+0.3% Guar gum (light grey dotted) had relatively good performance in terms preservation sensory properties and pH.

Fig. 14 reproduces the same experiment with LipOsmoters produced with varying concentrations of CTAC (0.25% or 0.5% w/w), TXA/TA (2% or 3% w/w); and Guar gum (0.3% w/w). Long-term stability was assessed in relation to particle size (as in Fig. 8). The constructs with the lower concentration of CTAC (0.25%, grey and dashed pattern bars) proved to be superior in terms of preservation of particle size.

Fig. 15 presents analysis of net charge in the same experiment (as in Fig. 9). The constructs with the higher CTAC (0.5%, black and light grey lines) had a better performance in terms of preservation of net charge.

Figs. 16A-16D present analyses of sensory properties in the same experiment (as in Figs 10A-10D). The construct with 3% TXA/TA complex+0.25% CTAC+0.3% Guar gum (dark grey) had relatively good performance for sufficient time, suggesting that low CTAC does not interfere with the preservation of sensory properties and pH.

DETAILED DESCRIPTION OF EMBODIMENTS

The primary goal of this invention has been to provide successful formulations of actives that are relatively new in the field of cosmetics or which use has been limited because of past difficulties with finding the right formulations. Examples of such actives are tranexamic acid (TXA or TA) and extracts Dead Sea Water (DSW), both of which suffer from significant limitations of absorption into the skin, in part, due to incompatibility with the native skin surface charge.

The terms “tranexamic acid' - TXA, TXA/TA or TA, these terms herein are interchangeable, refers herein to synthetic derivatives of lysine or substances under names of trans-4-(Aminomethyl)cyclohexanecarboxylic acid, 4-(Aminomethyl)cyclohexane carboxylic acid, 4-(aminomethyl)cyclohexane-l -carboxylic acid (IUPAC name) and Cyklokapron. More generally, it encompasses substances with the general formula C8H15NO2 and the chemical structure in Scheme 2 below.

Scheme 2

This term encompasses derivatives of tranexamic acid [trans-4-(aminomethyl) cyclohexanecarboxylic acid], including those containing one or more than one (e.g., two) tranexamic acid moieties. It further encompasses a monocarboxylic acid derived from a cyclohexanecarboxylic acid and all synthetic derivatives of lysine, including those used as anti-fibrinolytic and hematologic agent.

The term “DSW extract” broadly refers herein to inorganic and organic materials obtained from DSW, including natural minerals and nutrients, the mud and the soil bed. This term encompasses herein a natural DSW obtained from the Dead Sea region, or a solution prepared by dissolving minerals obtained from DSW in an aqueous medium, or, an aqueous solution that simulates the natural DSW by at least one parameter, e.g., salt content, mineral content, salt concentration, and content and concentration of a particular element, mineral, ion, a soluble natural substance, etc.

DSW is generally characterized by a high content of Mg, Ca, Str, Br, B and Li, and a high ratio of divalent cations (Ca ²⁺ , Mg ²⁺ ) relative to monovalent cations (Na ⁺ , K ⁺ ).

The term DSW extract encompasses herein any mixture of natural materials obtained from DSW, including DSW extracts consisting of filtered or unfiltered DSW obtained from various locations and depth of the Dead Sea, and DSW treated by any known method (e.g., removal of organic matter and residual contaminants), and artificially or naturally evaporated DSW and then reconstituted in an aqueous solution.

More specifically:

In some embodiments the DSW extract can be an aqueous solution simulating DSW, or which is substantially similar to DSW in terms of salts content, minerals content, salts concentration, minerals concentrations, concentration of a particular cation or anion, ratio of divalent cations to monovalent cations, soluble natural substances and other parameters known to define or characterize natural DSW.

In some embodiments the DSW extract can be an aqueous solution simulating DSW or which is substantially similar to DSW in terms of salt content (a hypersaline concentration) and or mineral content.

The term “substantially similar" encompasses herein deviations from a measurable characteristic of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20% or more.

In numerous embodiments the DSW extract can be characterized as a clear* colorless viscous liquid at 25°C.

In some embodiments the DSW extract can be characterized as having a specific density of 1.25-1.35 g/ml, pH = 4.6-5.6 (at 25°C), and/or less than 100 cfu/g of non- pathogenic microbes.

In some embodiments the DSW extract can be characterized as having specific content of Ca, Mg, Na and K cations and Cl and Br anions, and other electrolytes.

In some embodiments, the concentrations of specific electrolytes can be in the ranges of: Ca ²⁺ 35,000-40,000, CT 320,000-370,000, Mg ²⁺ 90,000-150,000, Na ⁺ 1,500- 3,200, K ⁺ 1,000-2,500, and Br" 5,000-15,000 (mg/L).

In some embodiments, the concentration of Ca ²⁺ can be in the range of 5,000- 10,000 (mg/L).

In some embodiments DSW extract can comprise Sr ² * at the concentration of about 800 mg/L.

In some embodiments, the DSW extract can be obtained from natural DSW which was allowed to evaporate, naturally or artificially, and then reconstituted in a aqueous solution.

In some embodiments the DSW extract can be a specific DSW extract referred to herein as “Osmoter (also known as "Maris Sal and Aqua", “Maris Aqua” commercially available by AHAVA, Israel), which has distinguishing characteristics as to the precise location and season for collecting this specific sample of DSW with the particular mineral content that can elicit optimal benefits to the skin. In some embodiments Osmoter can comprise Ca ²⁺ 35,000-40,000, Cl' 320,000- 370,000, Mg ²⁺ 92.000-95,000, Na ⁺ 1,800-3,200, K ⁺ 2,100-2,500 and Br 10,000-12,000 (mg/L).

The term “Osmoter” encompasses herein the two available forms, the Crystal (Crys) and Aqueous (Aq) Osmoters.

Regarding the specific formulations provided by the invention, in one of its first aspects the invention provides an advantageous complex of tranexamic acid (TXA) and cetyltrimethylammonium chloride (CTAC), referred to herein as a TXA-CTAC complex, which has a positive charge and therefore is more adapted to the native charge of the skin.

The term “ cetyltrimethylammonium chloride (CTAC)” encompasses herein substances named hexadecyltrimethylammonium chloride, cetrimonium chloride, N,N,N-trimethylhexadecan-l-aminium chloride and hexadecyl(trimethyl)azanium; chloride (IUPAC name). It generally refers herein to substances with the molecular formula C19H42N.CI or C19H42CIN and the chemical structure in Scheme 3 below.

Scheme 3

This term encompasses herein an organic chloride salt of cetyltrimethylammonium, also a quaternary ammonium salt and an organic chloride salt, which is used as a cationic surfactant.

In numerous embodiments the TXA-CTAC complex is in a salt form wherein the aminomethylene moiety of TXA is protonated.

In some embodiments the TXA-CTAC complex is self-assembled.

In some embodiments the interaction between TXA and CTAC in the TXA- CTAC complex can be defined as a stable ionic or non-covalent association.

In some embodiments the self-assembly can involve heating of the source solutions of TXA and CTAC. Due to the non-covalent nature of this complex, it expected that TXA can form analogous complexes with other complexants. Several alternative complexants that can form non-covalent complexes with TXA have been presently exemplified.

Thus, in some embodiments CTAC can be substituted with other cationic complexants such as SrCh and polycationic complexants such as Sharomix ™ AM24 (methylpropanediol, caprylyl glycol, Polyquaternium-80 and didecyldimonium chloride), EUDRAGIT® El 00 (dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate), Dehyquart® Guar HP, Polyquat-4, and others.

In some embodiments CTAC can be substituted with cetrimonium bromide (C19H42N.Br or Ci9H ₄₂ BrN).

It is another important objective of the invention to provide specific formulations of the TXA-CTAC complex. One example of such formulations are liposomal formulations or liposomes comprising TXA-CTAC complex or analogous complexes as referred to above.

The term “liposome” broadly refers herein to vesicles consisting of one or more bilayers of amphiphilic lipids or a mixture of such lipids, known examples of which are phospholipids. This term encompasses herein a wide range of liposomal preparations produced by various methods, with varying lipid composition, surface charge and size.

Under the broadest definition herein, liposomes are spherical vesicles with particle sizes ranging from xlO nm and xlOO nm to several pm. They consist of one or more amphiphilic lipid bilayers surrounding aqueous units, with the polar head groups facing the interior and exterior aqueous phases. It should be noted that self-aggregation of polar lipids is a process dependent on molecular shape, temperature, and environmental and preparation conditions, and therefore is not limited to conventional bilayer structures but may result in various types of colloidal particles. Liposomes can entrap both hydrophobic and hydrophilic compounds and release the entrapped compounds under specific conditions.

In numerous embodiments the liposomes of the invention are unilamellar liposomes with a single phospholipid bilayer with a size in a nanometric range.

In some embodiments the liposomes of the invention can be composed of comprise at least one phospholipid, a DSW extract. In such cases the DSW extract can be encapsulated or entrapped inside the liposomes. The term “encapsulated” is interpreted herein the broadest sense, without being bound to a specific physical/chemical model or theory. Encapsulated is meant to convey compartmentalization of the liposomal particle, with certain components entrapped or residing, placed or located in the inner compartment of the liposome vis-a-vis components located on the liposomal surface and affecting its surface charge (see below).

In some embodiments the liposomes can further comprise a TXA-CTAC complex or an analog of such complex, the TXA-CTAC complex tends to be associated with the surface of the liposomes.

In some embodiments the liposomes can be composed of or comprise at least one phospholipid, a DSW extract and a TXA-CTAC complex, wherein the at least one phospholipid can form a unilamellar layer of the liposomes and the DSW extract can be encapsulated inside the liposomes and the TXA-CTAC complex can be associates with the surface of the liposomes.

In numerous embodiments the phospholipid components can be natural phospholipids obtained from plant or animal sources. Of special relevance are natural phospholipids described in Pharmacopeias and Regulatory Guidance by the Food and Drug Administration (FDA) and European Medicines Agency (EMA), and phospholipids recognized as GRAS (Generally Safe).

In numerous embodiments the liposomes can contain different types of phospholipids that have different head groups modified by choline, ethanolamine, serine, or inositol, referred to as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) and sphingomyelin.

In some embodiments the liposomes can contain synthetic phospholipids synthesized from glycerophosphocholine (GPC), which is obtained from natural phospholipids using acylation and enzyme catalyzed reactions.

In some embodiments the liposomes can contain hydrogenated phospholipids.

In numerous embodiments the liposomes can comprise a mixture of phospholipids referred to as a lecithin. The term “Lecithin” generally refers herein to a mixture of phospholipids from animal or plant source, typically derived from eggs, soy and sunflower, and also from canola, cottonseed, or animal fats. The exact composition of lecithin can vary depending on its origin. A typical lecithin is composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid.

In some embodiments the liposomes of the invention can comprise vegetable lecithin or lecithin from a plant source to provide the basis for “green” cosmetic products.

In some embodiments the liposomes can contain lecithin with a relatively low Phosphatidylcholine (PC) content.

In some embodiments the lecithin can have PC content of less than about 80%, 75%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% and 5% wt%.

In numerous embodiments the liposomes can have a nanometric particle size in the range of up to about 500 nm, or more specifically, a mean particle size of less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nm, and further, less than about 150, 200, 250, 300, 350, 400, 450, and 500 nm, or particle size in the range of 10-50 nm, 50-100 nm, 100-150 nm, 150-200 nm, 200-250 nm, 250-300 nm, 300-350 nm, 350-400 nm, 400- 450 nm and 450-500 nm or more.

In some embodiments the liposomes can have a mean particle size of in the range of less than about 200 nm, or more specifically, a particle size in the range of about 10- 50, 50-100, 100-150 and 150-200 nm.

In some embodiments the liposomes can have a particle size in the range of up to 100 nm, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nm, or in the range of about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 nm.

In numerous embodiments the liposomes can have a surface charge of about neutral or about >+l, or more specifically, a surface charge of 0, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10 or more.

As the term “ surface charge" also implies herein “ surface charge density’’’, the liposomes of the invention can have a surface charge density in the range of between about 0 to +1, 0 to +2, 0 to +3, 0 to +4, 0 to +5, to +6, to +7, to +8, to +9, to +10 or more, and generally a surface charge or a surface charge density between 0 to about >+l.

In some embodiments the liposomes can comprise a specific type of DSW extract referred to herein as an “Osmoter”, which can be an Aqueous Osmoter or a Crystal Osmote according to the definition of the branding company Ahava, Israel. In numerous embodiments the liposomes can comprise or be associated with at least one surfactant. The term “surfactant” broadly refers herein to organic amphiphilic compounds that contain oil- soluble and water-soluble components and can act as surface active (or surface tension lowering) agents. It generally encompasses agents that act as detergents, wetting agents, emulsifiers, foaming agents or dispersants.

The term “surfactants” broadly encompasses herein reagents referred to as nonionic (e.g., fatty alcohol ethoxylate, alkyl phenol ethoxylate and fatty acid alkoxylate), anionic (e.g., alkyl sulfates, sodium lauryl sulfate and ammonium lauryl sulfate), cationic (stearalkonium chloride, dicetyldimonium chloride, and behentrimonium chloride) and amphoteric surfactants (e.g., coco betaine, lauryl betaine, and hydroxysultaines).

In some embodiments the surfactants can be non-ionic surfactants. The term “non -ionic surfactant” broadly refers herein to non-charged surfactant molecules, which are the primary surfactants used to create emulsions. Some types include fatty alcohols and fatty alkanolamides, including lauramide diethanolamine (DEA) and cocamide DEA. Other nonionic surfactants found in cosmetics include amine oxides such as lauramine oxide or stearamine oxide.

In numerous embodiments the applicable surfactants can be polyol esters, polyoxyethylene esters, poloxamers. Polyol esters include glycol and glycerol esters and sorbitan derivatives. Fatty acid esters of sorbitan (Spans) and their ethoxylated derivatives (Tweens, e.g., Tween 20 or 80).

In some embodiments the applicable surfactants can be monoglyceride of long- chain fatty acids, polyoxyethylenated alkylphenol, and polyoxyethylenated alcohol.

In some embodiments the applicable surfactants can be ethoxylated castor oil or esters of polyglycerol-4 and sebacic and lauryl acids.

In some embodiments at least one surfactant can be polyoxyethylene sorbitan monooleate, also referred to as Polysorbate 80 (Tween 80).

Some additional examples of suitable non-ionic surfactants are fatty alcohol ethoxylates, fatty alcohol ethoxylates, ethoxylated esters of fatty acids, glycol esters, glyceryl fatty acid esters, transesters, sorbitan esters, polyalkoxylated sorbitan esters, glucoside esters, sucrose esters, poly acrylates, dimethicone copolyols and polaxomers. In numerous embodiments the liposomes can comprise or be associated with at least one co-surfactant. The term “ co-surfactant” broadly refers herein to a chemical used in combination with a surfactant to enrich the emulsifying properties of the surfactant.

In some embodiments the co-surfactants can be alcoholic co- surfactants. Some examples are provitamin B5 (D-panthenol), which is a salt of pantothenic acid, glycerin, propylene glycol, butylene glycol and/or PEG-400. Additional optional co- surfactants can be n-pentanol, alkyl alcohol, and cholesterol.

In some embodiments the liposomes of the invention can comprise or be associated with more than one surfactant and more than one co- surfactant.

In some embodiments the liposomes can comprise or be associated with one or more natural or synthetic amino acids or amino acid analogs. Amino acids and their salts are widely used in cosmetics as natural moisturizing factors regulating skin hydration and skin pH. Certain examples are arginine that helps to restore skin damage; histidine that soothes the skin and has antioxidant properties; methionine that protects the skin; lysine that strengthens the skin's surface; and proline, leucine and glycine that reduce fine lines and wrinkles.

In numerous embodiments the liposomes can comprise or be associated with one or more natural or synthetic polysaccharides. Polysaccharides are widely used in natural cosmetics, predominantly as thickeners, emulsifiers and stabilizers. In addition to natural polysaccharides, chemical industry has modified and customized the natural substances. Notable examples of both types are agar, alginic acid (algin), carrageen (carrageenan), chitin, CM-glucan, CMC (carboxy methyl cellulose), dextrins, glycogen, Guar gum, Gum Arabic, HSP (hydroxypropyl starch phosphate), hyaluronic acid, HEC (hydroxyethyl cellulose), MC (Methyl Cellulose), mucopolysaccharides (glycosaminoglycans), pectin, sugar tensides, Tragant (E 413), Xanthan gum.

In numerous embodiments the liposomes can comprise or be associated with one or more natural or synthetic lipids. Lipids perform different functions in cosmetic formulations. There is wide range of vegetable and animal oils and fats that can be used as neutral bases and bioactive ingredients, but today the lipids and their derivatives in cosmetic formulations are of plant or biotechnological origin. The types of lipids commonly used in cosmetics include triacylglycerides, waxes, ceramides, glycerophospholipids, sterols, hydrogenated, esterified, and oxidized lipids. In some embodiments the liposomes can be multilamellar liposomes comprising more than one liposomal structure. A multilamellar liposome refers herein to a vesicle having an onion structure, typically composed of several unilamellar vesicles making a multilamellar structure of concentric phospholipid spheres separated by layers of water.

It is another objective of the invention to provide cosmetic formulations incorporating a plurality of liposomal structures as described above, and additional cosmetically beneficial components.

In the most simple version the cosmetic formulations of the invention can comprise a TXA-CTAC complex, and optionally further comprise a cosmetically acceptable carrier or excipient.

In some embodiments the cosmetic formulations can comprise one or more phospholipids and a DSW extract.

In some embodiments the DSW extract can be a mineral extract of DSW which is “Osmoter”, either an Aqueous Osmoter or a Crystal Osmoter.

In some embodiments the cosmetic formulations can comprise liposomes of the invention, with the phospholipid forming a unilamellar layer of the liposome thereby encapsulating the DSW extract and wherein TXA-CTAC complex remaining associated with the surface of the liposome.

In some embodiments the phospholipid can be a mixture of phospholipids referred to as lecithin, and different types of lecithin.

In some embodiments the lecithin can be vegetable lecithin.

In some embodiments the lecithin can have a low or a reduced PC content, as defined above.

In some embodiments the TXA can be present in the formulation at a concentration in the range between about 1% to about 10% wt%, or more specifically at a concentration in the range between about 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10% wt%, and more.

In some embodiments the TXA can be present in the formulation at a concentration of up to about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% wt% of the formulation. In some embodiments the CTAC can be present in the formulation at a concentration of in the range between about 0.1%-5% wt%, or more specifically at a concentration the range between about 0.1-0.5%, 0.5-1%, 1-1.5%. 1.5-2%. 2-2.5%, 2.5- 3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5% wt% of the formulation.

In some embodiments the phospholipids can be present in the formulation at a concentration of in the range between about 0.1%-2% wt%, or more specifically at a concentration in the range between about 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%, 0.5- 0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-1.1%, 1.1-1.2%, 1.2-1.3%, 1.3-1.4%, 1.4- 1.5%, 1.5-1.6%, 1.6-1.7%, 1.7-1.8%, 1.8-1.9%, 1.9-2% wt% of the formulation.

In some embodiments the phospholipids can be present in the formulation at a concentration of in the range between about 2%-10% wt%, or more specifically at a concentration in the range between about 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5- 5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, 9.5-10% wt% of the formulation.

In some embodiments the DSW extract can be present in the formulation at a concentration of in the range between about 0.1%-2% wt%, or more specifically at a concentration in the range between about 0.1%-0.2%, 0.2%-0.3%, 0.3%-0.4%, 0.4%- 0.5%, 0.5%-0.6%, 0.6%-0.7%, 0.7%-0.8%, 0.8%-0.9%, 0.9%-l% 1.1%-1.2%, 1.2%- 1.3%, 1.3%-1.4%, 1.4%-1.5%, 1.5%-1.6%, 1.6%-1.7%, 1.7%-1.8%, 1.8%-1.9% and 1.9%-2% wt% of the formulation.

In some embodiments the DSW extract can be present in the formulation at a concentration of in the range between about 2%-10% wt%, or more specifically at a concentration in the range between about 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5- 5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, 9.5-10% wt% of the formulation.

In numerous embodiments the cosmetic formulations can comprise liposomes having a nanometric particle size in the range of less than about 500 nm, or more specifically, mean particle size of less than about 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 nm.

In some embodiments the formulations can comprise liposomes having a particles size in the range of less than about 200 nm, or more specifically, mean particle size in the range of about 10-50, 50-100, 100-150 and 150-200 nm. In some embodiments the formulations can comprise liposomes having mean particle size in the range of less than about 100 nm, or less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nm, or in the range of about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 nm.

In some embodiments the liposomes comprised in the cosmetic formulations can have a net charge of about neutral or about >+l as defined above.

In some embodiments the cosmetic formulations can comprise one or more cosmetically acceptable surfactants as defined above.

In some embodiments the cosmetic formulations can further comprise one or more cosmetically acceptable co- surfactants as defined above.

In some embodiments said cosmetically acceptable surfactants can be cosmetically acceptable non-ionic surfactants.

In some embodiments said cosmetically acceptable co- surfactants can be cosmetically acceptable alcoholic co-surfactants

In some embodiments the formulations can further comprise one or more natural or synthetic amino acids or amino acid analogs as defined above.

In some embodiments the formulations can further one or more cosmetically acceptable natural or synthetic polysaccharides as defined above.

In numerous embodiments the formulations can further comprise at least one cosmetically acceptable additive from the groups of diluents, preservatives, abrasives, anti-caking agents, antistatic agents, binders, buffers, dispersants, emollients, emulsifiers, co-emulsifiers, fibrous materials, film forming agents, fixatives, foaming agents, foam stabilizers, foam boosters, gallants, lubricants, moisture barrier agents, opacifiers, plasticizers, propellants, stabilizers, surfactants, suspending agents, thickeners, wetting agents and liquefiers.

In some embodiments the formulations can comprise one or more agents from the groups of emulsifiers, humectants, thickening agents, fillers, stabilizers or preservatives.

In some embodiments the formulations of the invention can further comprise one or more additional cosmetic active ingredients related to skin brightening, skin hydration/moisturization, skin anti-aging, skin firming/tightening, skin photoprotection, and skin rejuvenation. In some embodiments the formulations of the invention can further comprise various vitamins (e.g., Vit A and E), enzymes and antioxidants, organic UV filters, herbal extracts, animal extracts and other types nutraceuticals, and perfumes.

In numerous embodiments the formulations of the invention can have liquid, semi-liquid, or solid consistency.

In numerous embodiments the formulations can be transparent, semi-transparent or opaque.

In numerous embodiments the formulations can be provided in an ampule, a vial or a container.

It is another objective of the invention to provide methods for making such cosmetic formulations, with characteristic steps of: i. preparing an organic (ORG) phase comprising the at least one phospholipid, the DSW extract and the at least one non-ionic surfactant, ii. preparing an aqueous (Aq) phase comprising the TXA-CTAC complex and optionally at least additional surfactant and/or co-surfactant, iii. adding the ORG phase into the Aq phase, or vice versa, and mixing.

In some embodiments steps (i), (ii) and/or (iii) further comprise heating the ORG phase and/or the Aq phase.

The invention can be further articulated in the terms of a topical or dermal delivery system comprising a cosmetic formulation or a liposomal construct as described above.

It can be further articulated in the terms of cosmetic formulations as above for use in skin brightening, skin hydration/moisturization, skin anti-aging, skin firming/ tightening, skin photoprotection and/or skin rejuvenation.

It can be articulated in the terms of methods for cosmetic improvement of the skin comprising skin brightening, skin hydration/moisturization, skin anti-aging, skin firming/tightening, skin photoprotection and/or and skin rejuvenation, comprising topical administering to a subject one or more of the above-described formulations.

It can be in terms of use of the cosmetic formulations as above in cosmetic improvement of the skin comprising at least one of skin brightening, skin hydration/moisturization, skin anti-aging, skin firming/tightening, skin photoprotection, and/or skin rejuvenation. Ultimately, the invention intends to provide a series of cometic products incorporating or comprising one or more of the above-described cosmetic formulations or liposomal constructs. The term “ cosmetic products" encompasses herein the entire range of articles defined as cosmetics under the Federal Food, Drug & Cosmetic Act (FD&C Act), i.e., “articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body...for cleansing, beautifying, promoting attractiveness, or altering the appearance”.

The term “about” herein denotes up to a ±10% deviation from the specified values and/or ranges, more specifically, up to ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ± 10% deviation therefrom.

EXAMPLES

The experimental data and figures provided herein serve only illustrative purposes and are not intended to be limiting in terms of scope.

EXAMPLE 1: Formation of the TXA-CTAC complex

TXA-CTAC complex was prepared from TXA solution (5% TXA w/w and 80°C) and CTAC solution (0.25% or 5% CTAC w/w). Complex formation was measured by ninhydrin assay using UV spectrometer at 575 nm, the results are shown in Fig. 1.

The results suggest that the formation of TXA-CTAC was independent of CTAC concentration and was equally effective with high and low concentrations of CTAC (CTAC 5% TA 5% vs. CTAC 0.25% TA 5% w/w).

EXAMPLE 2: TXA complexes with other complexants

Other complexants were evaluated for their ability to form TXA complex under the same conditions as in EXAMPLE 1, i.e., a fixed TXA concentration (5% w/w) and low and high concentrations of candidate complexants (0.25% and 5% w/w). Complex formation was determined as above. The results are shown in Table 1, expressed as percent of complex formation (AVR+SD, n=3). Table 1. TXA complex formation with various complexants

Complexant, % Charge Percent of TXA complex formation

0.25 5

AVE ± sd AVE ± sd

Cetrimonium chloride CTAC 32.1 ± 2.6 37.6 ± 1.6

SrCl ₂ +2 24.3 ± 1.8 33.1 ± 1.2

Guar HP 0.25% Polycationic 25.3 ± 1.4 10.8 ± 3.1

Polyquat-4 Polycationic 21.4 ± 1.5 35.1 ± 1.1

Sharomix™ AM24 Polycationic 16.1 ± 1.1 38.6 ± 0.2

EUDRAGIT® E100 Polycationic 25.8 ± 0.6 32.0 ± 0.3

Sorbitol 0 0.0 ± 0.7 2.6 ± 0.7

Osmoter® Electrolytes 15.7 ± 1.8 24.5 ± 0.7

Dipotassium glycyrrhizate -2 7.7 ± 1.2 20.4 ± 1.7

EDTA -4 20.3 ± 0.6 74.4 ± 0.3

Alginate Polyanionic 8.1 ± 0.3 26.5 ± 0.6

EUDRAGIT® L100 Polyanionic 9.0 ± 0.3 35.4 ± 0.5

The results show that TXA-CTAC complex was superior to the other complexants by efficiency of complex formation and charge. With CT AC, the efficiency of complex formation was independent of CTAC concentrations, and low CTAC concentrations (0.25%w/w) were sufficient to produce high yield of TXA-CTAC. More generally, apart from EDTA, cationic agents had better ability to form TXA complexes.

EXAMPLE 3: Prototype LipOsmoter formulations

Two prototypes of liposomal Osmoter formulations (LipOsmoter) were produced:

A. LipOsmoter formulations with Crystal (Crys) Osmoter, and

B. LipOsmoter formulations with Aqueous (Aq) Osmoter.

Examples of ingredients included in the two types of formulations are shown in Table 2.

Table 2. Examples of LipOsmoter formulations

Ingredients % w/w Function 1 Function 2 Trade name

Crys or Aq Osmoter Dead Sea minerals

Lecithin 0.4-0.8 Liposomes *EMULMETIK™

300 or 900

Polysorbate 80 0.4-1.6 Surfactant Tween 80

Tranexamic acid TXA Whitening Astringent SpecWhite® TA Arbutin Whitening Spec White® ABT

Methyl gluceth-20 Hydration Glucam™ E-20

Trehalose Hydration AHAVA

Calcium lactate Astringent, firming PURACAL

Lactic acid Lactic acid

Acetyl hexapeptide 8 Anti-wrinkle SpecPed® AH8P

Copper peptide (GHK2-Cu) Anti-wrinkle, anti-aging, SpecPed® GCu21 P skin repairing, wound healing

Allantoin Soothing Allantoin

Dipotassium

Dipotassium Glycyrrhizate Soothing glycyrrhizate

Panthenol Skin conditioning Panthenol

Propanediol Solvent AHAVA

Cetrimonium chloride CTAC Complexant

Preservative Preservative Sharomix™ AM24

Guar hydroxypropyl Viscosity agent Jaguar® Excel trimonium chloride Xanthan gum Viscosity agent

Herbal extracts

Spring water

Total 100.00

** EMULMETIK™ 300: 97% Phospholipids and glycolipids (23% Phosphatidylcholine)

EMULMETIK™ 900: 97% Phospholipids and glycolipids (50% Phosphatidylcholine)

EXAMPLE 4: LipOsmoter prepared by ORG to Aq method

4.1 Method of preparation

LipOsmoter formulations with Crys or Aq Osmoters were prepared by the organic to aqueous phase (ORG to Aq) method with the main steps of:

(i) Organic phase (ORG) was prepared from the following ingredients:

- Lecithin (Emulmetik 300 or 900),

- Tween 80 (or another non-ionic hydrophilic surfactant), and

- Osmo ter (Crys or Aq)

(ii) Aqueous phase (Aq) was prepared from the following ingredients:

- D-panthenol (or another water-miscible co- surfactant), and

Water (iii) Liposomes were prepared by mixing ORG with Aq (at 40°C and 95°C, respectively), during homogenization at 20K RPM for 3 min.

(iv) Additional ingredients were added in the following order:

- TA-CTAC complex at 80°C,

- Powders (Arbutin Allantoin, DPG, Ca lactate) dissolved in water at 60°C,

- Methyl gluceth-20 and lactic acid,

- Preservative,

- Jaguar® Excel (or another viscosity agent e.g., Xanthan gum), during homogenization at 13K RPM for 3 min.

(v) After the foam was reduced, Glycerin and herbal extracts were added.

4.2 Morphological evaluation

Morphological evaluation of LipOsmoters was performed using Cryo-TEM. In brief, LipOsmoter solution (2.5 uL) was placed on carbon lacey film supported on 300 mesh Cu grid (Ted Pella Ltd), excess liquid was blotted. The specimen was vitrified (liquid ethane precooled with liquid nitrogen) using controlled environment automatic vitrification system (Leica EM GP). Samples were examined at -178 C using ThermoFisher Scientific (FEI) Tecnai 12 G2 TWIN TEM operating at 120 kV and equipped with Gatan 626 cold stage. Images were taken with Gatan 794 MultiScan CCD camera. Fig 2 shows micrograph images of LipOsmoters with Crys and Aq Osmoters.

The results show that both types of LipOsmoters comprise spherical vesicles constructed of a unilamellar phospholipid bilayer and a hydrophilic core, with an overall average size (diameter) in the range of about 50-100 nm.

4.3 Mean particle size

Size evaluation of the liposomal particles was performed using Dynamic Light Scattering (DLS) (data not shown). Both Crys and Aq LipOsmoter formulations had similar average diameters in a nanoscale range, 20.0 ±0.3 nm and 24.2 ±0.1, respectively. Previous studies have shown advantages of liposomal delivery systems with nanometric particle size-in terms of improved delivery of actives to the skin.

4.4 Particles surface charge

Surface charge (zeta potential) studies were performed using known methods (Zetasizer Nano ZS, Malvern instrument). The results are shown in Figs 3A-3B, for LipOsmoters of Crys (3A) and Aq (3B) types with and without TXA-CTAC complex, Guar Gum and other additives.

The results show that both types of LipOsmoters exhibit unique pattern of electric change distribution. Specifically, with the initial surface charge (net charge) of the core construct (without TXA-CTAC) was slightly negative or close to neutral and with the addition of TXA-CTAC has become positive, suggesting that TXA-CTAC was associated with the outer layer or the surface of liposome. The addition of other non-actgive components (e.g., Guar gum) masked the positive charge bringing it closer to neutral (about >+l), suggesting that they produce yet another outer encapsulation layer. Overall, the studies suggest that LipOsmoter constitutes a multi-layered structure that compartmentalizes various active and non-active ingredients so as to produce an overall neutral or positive charge which is compatible with the native negative charge of the skin. This unique structure of layer-by-layer encapsulation, with the Osmoter in the core and the TXA-CTAC complex and other additives in the outer layers, makes LipOsmoters likely candidates for improved delivery and enhanced performance of actives in the skin.

EXAMPLE 5: LipOsmoter prepared by the Aq to ORG method

Analogous LipOsmoter formulations were prepared by Aq to ORG phase method (see EXAMPLE 4.1 above), using Aq Osmoter and various types of lecithin by PC content. Aq phase was added to the ORG phase (at 95°C and 40°C, respectively), during homogenization at 20K RPM for 3 min. Morphology, particle size and surface charge were evaluated as above. The characteristics of the LipOsmoter formulations prepared by this method are shown in Figs 4-6 and Tables 3-4 below.

The results show that different types of lecithin, EMULMETIK™ 300 (Emtk300) and EMULMETIK™ 900 (Emtk900) with 23% and 50% PC respectively, produced liposomes with different particle size by inverse relationship, with a reduced PC content having a reducing effect on particle size (Fig. 4). In terms of surface charge, lecithin with a reduced PC content had a neutralizing effect in producing liposomes with a net charge closer to neutral (Fig. 5). Morphologically, LipOsmoters produced by Aq to ORG phase method were similar to the those in ORG to Aq phase method, with spherical vesicles constructed of a unilamellar phospholipid bilayer and a hydrophilic core, and an average size of about 50-100 nm (Figs. 6-7). The effect of different types of lecithin on the physical properties of LipOsmoter was further investigated, using Aq Osmoter containing constructs produced by Aq to ORG phase method. The results are summarized in Tables 3-4.

Table 3. Particle size of LipOsmoter formulations with different types of lecithin

Commercial name Description Preliminary size (nm)

PHOSPHOLIPON® 90 G About 90% PC 123.0 ±21.6

Soy lecithin granules About 21% PC 20.9 ±0.4

Heelfeel™ Glyceryl Stearate Citrate (and) Polyglyceryl-3 N/A (Sediment)

Stearate and Hydrogenated Lecithin

Proliponeo™ Propanediol and Lecithin 13.2 ±0.2

Lysofix™ Glycerin and Glycine Soja (Soybean) seed extract 12.1 ±0.1

Soy lecithin 258.3 ±41.0

Table 4. Surface charge of LipOsmoter formulations with different types of lecithin

Commercial name Mean zeta potetial, mV

A A d jd jed j _t Li ■nO-Asmoters A „dded LipOsm ,ote .r, TA .-C.. T. AC,

Guar gum and other additives

PHOSPHOLIPON® 90 G 0.657 ±1.16 5.28 ±1.42

Soy lecithin granules -1.29 ±0.42 4.31 ±0.45

Heelfeel™ N/A (Sediment)

Proliponeo™ -1.72 ±0.29 6.24 ±0.85

Lysofix™ -1.29 ±0.42 5.6 ±0.72

Soy lecithin -1.05 ±0.41 2.72 ±0.63

The results show that different types of lecithin yielded liposomal formulations with markedly different particle size, ranging from xlO nm to x 100 nm. Lecithin with a lower PC content, as in soy lecithin granules (21% PC) vs. PHOSPHOLIPON® 90 G (90% PC), produced smaller liposomes. Apart from PHOSPHOLIPON® 90 G, all other types of lecithin produced negatively charged liposomes. In all cases, the surface change changed to positive with the incorporation of TXA-CTAC and other additives. Overall, both preparation methods produced LipOsmoters with very similar physical properties by morphological characteristics, average particle size and net charge. The results also show that these physical properties can be further modified by inclusion of lecithin with a lower PC content, thus producing more advantageous formulations in terms of particle size and net charge compatibility with the skin.

EXAMPLE 6: Advanced LipOsmoter formulations with beneficial properties

Current efforts are directed towards design and development of more advanced LipOsmoters formulations with Aq or Crys Osmoters and other agents with the aim to obtain improved effects of skin brightening, moisturization, even-toning, translucency and skin anti-aging, and rejuvenating skin appearance overall. Some examples are provided in Table 5.

Table 5. Candidate actives in advanced LipOsmoter formulations

Desired cosmetic effects Candidate actives

Skin brightening/ even-toning/translucency TXA-CTAC complex

Arbutin

Dipotassium glycyrrhizate

Lactic acid

Skin anti-ageing Acetyl hexapeptide 8

Copper peptide (GHK2-Cu)

Skin hydration /moisturization (> 12 hr) Trehalose

Methyl gluceth-20

Panthenol (Provitamin B5)

Firm skin TXA (Tranexamic acid)

Calcium lactate

One of the leading products is Multi Benefit Ampule. Studies of net charge of this type of LipOsmoter show that it is sufficiently close to neutral (about 3.2 ±1.6 mV), which makes it compatible with the native charge of the skin and more suitable for topical and dermal applications. More recently, additional formulations were designed and developed based on the same technology by modifying the type of and concentrations of specific active and nonactive components, e.g., concentrations of TXA complex (2% or 5%), concentrations and types of thickening agents (0.3% to 1% Guar or Xanthan/Ziboxan gums), inclusion of Glycerin (0.25% or 0.5%), soluble Argan oil ester (0.5% orl%) and Betaine (0.5% or 1% OSMS) so as to produce products with various textures, hydration and sensory properties (concentrations are in w/w).

Overall, LipOsmoters with 5% TXA complex+0.3% Guar gum (w/w) proved to be advantageous in terms of preservation of physicochemical properties, pH, consistency, homogeneity, and inherent odor quality.

EXAMPLE 7: Stability studies of elected LipOsmoter formulations

LipOsmoters with various composition of components (5%, 3% or 2% TXA complex; 0.3%, 0.45%, 0.6%, 0.8%, 1% or no Guar gum; 0.2% or 0.5% Ziboxan; 0.25% or 0.5% CTAC; concentrations are in w/w) were prepared by ORG to Aq or Aq to ORG phase methods. Stability studies were performed over a period of at least 7 months at 40°C (long-term stability) considering changes (quantitative or qualitative evaluation) of the following parameters:

— particle size,

— net charge,

— pH

— opacity (loss of transparency)

— color (the original color is buttermilk yellow), and

— odor (the formulations are originally fragrance free).

The results are shown in Figs 8-16.

Overall, LipOsmoters prepared by both preparation methods showed relatively good preservation of core properties (particle size, net charge, sensory properties, and pH) during the entire study period. The best performance was observed with the liposomal construct comprising 5% TXA/TA complex+0.3% Guar gum w/w. Relatively good preservation of particle size was also observed in constructs comprising low CTAC (0.25% w/w). Additional studies are required, using additional preservative agents, excipients, buffers, etc., to improve the aspect of preservation of sensory properties.