Multiphasic compositions

Abstract

The present invention relates to compositions comprising a colloidal dispersion and an Agent of Interest (“AOI”), wherein the colloidal dispersion comprises deformable colloidal particles and wherein the AOI is not associated with the deformable colloidal particles. The present invention also provides kits and transdermal drug release devices comprising the compositions of the present invention, and the use of these compositions in medicine, skin care and cosmetics.

Claims

1. A composition comprising a colloidal dispersion and an Agent of Interest (“AOI”), wherein said colloidal dispersion comprises deformable colloidal particles comprising a surfactant and a phospholipid and wherein the AOI is not associated with the deformable colloidal particles, wherein the AOI comprises a biologically active agent, wherein the AOI comprises capsaicin.

2. The composition of claim 1 wherein the deformable colloidal particles comprise vesicles.

3. The composition of claim 1, wherein the capsaicin is present in the composition in the form of aggregates or wherein the capsaicin is associated with liposomes comprising one or more phospholipids.

4. The composition of claim 1, wherein the deformable colloidal particles comprise menthol in their membranes.

5. The composition of claim 1 wherein the phospholipid is selected from the group consisting of sphingomyelin, sphingomyelin lauroyl, phosphatidyl choline and phosphatidyl glycerol.

6. The composition of claim 1 wherein the surfactant is a non-ionic surfactant, optionally wherein the surfactant is polyoxyethylene (20) sorbitan monooleate (Polysorbate 80) or 2-[(Z)-octadec-9-enoxy]ethanol.

7. The composition of claim 1 wherein the composition comprises a phospholipid and a surfactant in a molar ratio of 1:30 to 30:1.

8. A kit comprising a container comprising the composition of claim 1 and instructions for administration of the composition to a patient in need thereof, wherein the composition is contained within a single compartment of said container.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram of the movement of the deformable colloidal particles and AOI of the compositions of the present invention through the skin.

(2) FIG. 2 shows a simplified generic manufacturing process for compositions of the present invention.

(3) FIG. 3 is a graph illustrating the change in sebum levels over time after application of two compositions of the present invention comprising chlorhexidine digluconate to the skin of subjects, compared to a control composition.

(4) FIG. 4 is a graph illustrating the change in the number of comedones over time after application of two compositions of the present invention comprising chlorhexidine digluconate to the skin of subjects, compared to a control composition.

(5) FIG. 5 is a graph illustrating the change in the number of pustules over time after application of two compositions of the present invention comprising chlorhexidine digluconate to the skin of subjects, compared to a control composition.

EXAMPLE 1: METHOD OF MANUFACTURE

(6) A simplified generic manufacturing process of compositions in accordance with the present invention is illustrated in FIG. 2 and can be summarised as follows. Firstly, the colloidal dispersion comprising the deformable colloidal particles (for example, the Sequessome™ intermediate) is made. This colloidal dispersion is formed from an ‘organic phase’ containing alcohol-soluble components and an ‘aqueous phase’ consisting of water-soluble components. Secondly, the gel phase (a thickener) is formed within which the colloidal dispersion is dispersed. During this secondary stage, more than one kind of colloidal dispersion may be introduced such that the final composition comprises more than one kind of colloidal particle. During secondary manufacture, the AOI is added to the gel phase.

EXAMPLE 2: AN IN-USE STUDY IN HEALTHY VOLUNTEERS TO INVESTIGATE THE ANTI-SPOT EFFICACY OF TWO TEST ARTICLES COMPRISING CHLORHEXIDINE, USING OBJECTIVE INSTRUMENTAL ASSESSMENTS OF SKIN SEBUM LEVELS AND SUBJECTIVE VISUAL ASSESSMENTS OF LESION PREVALENCE, AGAINST A PLACEBO FOLLOWING A 3-WEEK USE PERIOD

(7) Summary

(8) This study compared two variants of a multiphasic Sequessome™-based mixed product with a control product in their ability to control sebum and reduce the incidence of acne in acne-prone individuals.

(9) The test products (n=36 and n=37) contained two types of deformable colloidal particle, namely Sequessomes™ (empty SPC/Tween (polysorbate) vesicles) and Tethersomes comprising tethered zinc, in addition to other excipients, including chlorhexidine as an anti-microbial. The Sequessomes™ and Tethersomes in the two products contained different ratios of SPC:Tween (polysorbate) to assess if this affected the efficacy of the product. The control product contained no vesicles, zinc or chlorhexidine (n=20).

(10) The objectively measured results demonstrated that against the control product the test products: Significantly reduced surface sebum (by up to 50%).sup.1 .sup.1 Published data demonstrate that a reduction in sebum production of 30-50% correlates with reduced acne symptoms (Janiczek-Dolphin N1, Cook J, Thiboutot D, Harness J, Clucas A: Can sebum reduction predict acne outcome? Br J Dermatol. 2010 October; 163(4):683-8) Significantly reduced comedones Significantly reduced the numbers of papules and pustules

(11) TABLE-US-00005 TABLE 1 Summary of results Objective assessment Test Article 1 Test Article 2 Reduction in sebum after. . . 1 week >20%; 1 week >20%; 3 weeks >50% 3 weeks >50% Reduction in comedones after. . . 1 week >40%; 1 week = 50%; 3 weeks >90% 3 weeks >80% Reduction in pustules after . . . 1 week = 90%; 1 week >90%; 3 weeks >98% 3 weeks >98% Subjective assessment: % agreeing/strongly agreeing Product reduced blackheads 64.7% 76.5% Product stopped spots recurring 69.6% 74.5% Spots less painful (tender to touch) 66.7% 82.4% Spots not as swollen 73.5% 75.5% Skin less shinySkin less oily 59.8% 80.4% Skin less greasy 67.6% 77.5% 69.6% 75.5%

(12) The results showed the multifunctional nature of the product. We hypothesise that the Sequessomes™ both assisted in the clearing of the excess sebum from the skin surface, whilst their penetration into the skin dragged the chlorhexidine in solution into the skin structure, killing bacteria. The tethered zinc may also have had a role in both reducing sebum production and/or having an anti-microbial function.

(13) The test articles were both well tolerated with no adverse effects (AEs) reported and no erythema at the test site.

(14) These results demonstrate that the test compositions are a very effective treatment for acne. The reduction in sebum and both spots and blackheads by 21 days supports the role of these compositions in preventing future recurrences of the symptoms of acne.

(15) Methods

(16) Summary Protocol

(17) TABLE-US-00006 Study design: Single-blind, within-subject comparison. Test Groups: Test article 1. CBL-DERM-14-005-E Test Article 2. CBL-DERM-14-006-F Control: CBL-DERM-14-008-P Dose regime: The test groups followed a 12-week usage schedule according to treatment specific in-use regimes. Duration of 21 Days. study: Number of 36 subjects completed the active phase for subjects: group 1, 37 subjects for group 2 and 20 subjects completed assessments for group 3. Duration of Study Started: w/c 26 Jan. 2015 study: Study Ended: w/e 20 Feb. 2015 Location: Princeton Consumer Research Ltd. 307 College Road East Princeton New Jersey 08540
Test Articles—Summary Formulae:
Quantities required per 100 g final product:

(18) TABLE-US-00007 CBL-DERM- CBL-DERM- CBL-DERM- 14-006-F 14-005-E 14-008-P SPC (dry mass)  6.870 g  7.146 g Polysorbate 80  0.850 g  0.472 g Benzylalcohol  0.525 g  0.525 g Methyl-4-hydroxybenzoate  0.250 g  0.250 g  0.250 g Ethyl-4-hydroxybenzoate  0.250 g  0.250 g  0.250 g Butylhydroxyanisol  0.020 g  0.020 g Linalool  0.100 g  0.100 g Disodium hydrogen  0.530 g  0.530 g  0.530 g phosphate 12 H.sub.20 Citric acid monohydrate  0.128 g  0.128 g  0.128 g Glycerol  3.000 g  3.000 g Ethanol  3.569 g  3.418 g Sodium hydroxide  0.113 g  0.113 g  0.150 g Carbopol 947P NF  0.750 g  0.750 g  1.000 g Water 81.463 g 81.716 g 97.692 g Agent of Interest-present in continuous phase Chlorhexidine digluconate  1.500 g  1.500 g (20% solution)** Agent of Interest-tethered to Tethersomes Zinc Stearate  0.082 g  0.082 g pH 5.5 5.5 5.5 **% with respect to the salt
Test Articles—Detailed Formulation Information:
CBL-DERM-14-006-F:

(19) TABLE-US-00008 Quantity Organic Phase- Required Empty Sequessome Per 100 g Intermediate Final Product (g) SPC  3.43500 Ethanol  1.82550 BHA  0.01000 Methyl-4-hydroxybenzoate  0.12500 Ethyl-4-hydroxybenzoate  0.12500 Benzyl alcohol  0.26250 Polysorbate 80  0.42500 Linalool  0.05000 Organic Phase Sub-Total  6.25800 Aqueous Phase-Empty Sequessome Intermediate Citric acid monohydrate  0.03600 diNa hydrogen phos 12 H20  0.14400 Water  22.72850 Aqueous Phase Sub-Total  22.90850 Empty Sequessome Intermediate Total  29.16650 Organic Phase-Zinc Stearate Intermediate SPC  3.43500 Ethanol  1.74350 BHA  0.01000 Methyl-4-hydroxybenzoate  0.12500 Ethyl-4-hydroxybenzoate  0.12500 Benzyl alcohol  0.26250 Polysorbate 80  0.42500 Zinc stearate  0.08200 Linalool  0.05000 Organic Phase Sub-Total  6.25800 Aqueous Phase-Zinc Stearate Intermediate Citric acid monohydrate  0.03600 diNa hydrogen phos 12 H20  0.14400 Water  22.72850 Aqueous Phase Sub-Total  22.90850 Zinc Tethersomes Intermediate Total  29.16650 Sum of Sequessome and Tethersome  58.33300 Intermediates Gel Phase-Final Product Citric acid monohydrate  0.05600 diNa hydrogen phos 12 H20  0.24200 Sodium hydroxide  0.11300 Carbopol 974P  0.75000 Glycerol  3.00000 Water  36.00600 Gel Phase Sub-Total  40.16700 Chlorhexidine digluconate  1.50000 Overall Total 100.00000
CBL-DERM-14-005-E:

(20) TABLE-US-00009 Quantity Organic Phase- Required Empty Sequessome Per 100 g Intermediate Final Product (g) SPC  3.57300 Ethanol  1.75000 BHA  0.01000 Methyl-4-hydroxybenzoate  0.12500 Ethyl-4-hydroxybenzoate  0.12500 Benzyl alcohol  0.26250 Polysorbate 80  0.23600 Linalool  0.05000 Organic Phase Sub-Total  6.13150 Aqueous Phase-Empty Sequessome Intermediate Citric acid monohydrate  0.06400 diNa hydrogen phos 12 H20  0.26500 Water  34.30000 Aqueous Phase Sub-Total  34.62900 Empty Sequessome Intermediate Total  40.76050 Organic Phase-Zinc Stearate Intermediate SPC  3.57300 Ethanol  1.66800 BHA  0.01000 Methyl-4-hydroxybenzoate  0.12500 Ethyl-4-hydroxybenzoate  0.12500 Benzyl alcohol  0.26250 Polysorbate 80  0.23600 Zinc stearate  0.08200 Linalool  0.05000 Organic Phase Sub-Total  6.13150 Aqueous Phase-Zinc Stearate Intermediate Citric acid monohydrate  0.06400 diNa hydrogen phos 12 H20  0.26500 Water  34.30000 Aqueous Phase Sub-Total  34.62900 Zinc Tethersome Intermediate Total  40.76050 Sum of Sequessome and Tethersome  81.52100 Intermediates Gel Phase-Final Product Sodium hydroxide  0.11300 Carbopol 974P  0.75000 Glycerol  3.00000 Water  13.11600 Gel Phase Sub-Total  16.97900 Chlorhexidine digluconate  1.50000 Overall Total 100.00000
Results
1. Sebum Reduction

(21) TABLE-US-00010 TABLE 2 Average reduction in Sebum score from Day 0. A negative score indicates an increase in sebum. n Day 3 Day 7 Day 14 Day 21 Test article 1 36 20.47 34.94 71.64 90.11 Test article 2 37 14.51 36.30 85.35 98.49 Control 20 −21.30 −48.60 −40.35 −44.95 1 vs. 2 t test p- 0.0163 0.6767 0.2065 0.3936 1 vs. Control values 7.04E−16 5.27E−23 9.74E−11 5.69E−16 2 vs. Control 1.06E−14 1.62E−24 1.10E−14 4.63E−17

(22) As illustrated in FIG. 3, at all time points both test articles reduced sebum significantly more than the control at p<0.1%. On day 3 Test Article 1 was better than Test Article 2 at p<0.02. There was no significant difference in the effectiveness of the two test articles at all later time points.

(23) By Day 21, both test articles had reduced the average sebum levels to less than 50% of the starting value.

(24) 2. Comedone Reduction

(25) TABLE-US-00011 TABLE 3 Average reduction in occurrence of comedones from Day 0. A positive score indicates an increase in comedones. n Day 3 Day 7 Day 14 Day 21 Test article 1 36 −1.44 −2.31 −2.92 −3.44 Test article 2 37 −1.14 −1.97 −2.84 −3.38 Control 20 1.20 1.30 1.25 0.35 1 vs. 2 t test p- 0.414 0.468 0.903 0.934 1 vs. Control values 2.35E−06 2.98E−06 4.66E−05 6.63E−04 2 vs. Control 1.08E−07 2.93E−05 7.20E−05 8.97E−05

(26) As illustrated in FIG. 4, at all time points both test articles reduced comedones significantly more than the control at p<0.1%. There was no significant difference in the effectiveness of the two test articles at all time points.

(27) 3. Papule Reduction

(28) TABLE-US-00012 TABLE 4 Analysis of progression participants who had blemishes at day 0 n Day 0 Day 3 Day 7 Day 14 Day 21 Test article 1 5 13 5 1 0 0 Test article 2 7 16 9 5 0 0 Control 4 10 9 8 5 3

(29) In those participants who began the trial with at least one papule, all treatments showed a reduction in papule numbers over 21 days.

(30) TABLE-US-00013 TABLE 5 Probabilities of U-statistic (Mann-Whitney) calculated on lesion reductions from Day 0 D14 vs D21 vs D3 v D0 D7 vs D0 D0 D0 1 vs. 2 0.146 0.044 0.373 0.373 1 vs. Control 0.043 0.019 0.056 0.089 2 vs. Control 0.110 0.093 0.110 0.254

(31) The test statistic (U) shows that Test article 1 was better than Control @<5% on Day 3 and Day 7 and @<10% on Day 14 and Day 21 and better that test article 2 @<5% on Day 7. Test article 2 was better than Control @<10% on Day 7

(32) 4. Pustule Reduction

(33) TABLE-US-00014 TABLE 6 Reduction in pustule numbers from day 0. A positive score indicates an increase in numbers of pustules. n Day 3 Day 7 Day 14 Day 21 Test article 1 36 −1.28 −1.97 −2.11 −2.17 Test article 2 37 −0.92 −1.49 −1.57 −1.59 Control 20 0.60 0.05 0.15 −0.75 1 vs. 2 t test p- 0.127 0.221 0.228 0.226 1 vs. Control values 2.71E−08 2.00E−05 9.67E−05 1.37E−02 2 vs. Control 8.60E−07 7.48E−04 1.04E−03 9.15E−02

(34) As illustrated in FIG. 5, at all time points from Day 3 to Day 14 both test articles reduced pustule numbers significantly more than the control at p<0.1%. At Day 21, test article 1 still outperformed control at p<2.0%; test article 2 outperformed control at p<10%. There was no significant difference in the effectiveness of the two test articles at all time points.

EXAMPLE 3: A STUDY IN VOLUNTEERS TO INVESTIGATE THE SENSATION PRODUCED USING THREE FORMULATIONS COMPRISING CAPSAICIN

(35) Method

(36) Three formulations comprising capsaicin at a concentration of 0.025% by weight were applied to the skin of three individuals. The first formulation (“TP1”) comprised the insoluble aggregates of capsaicin in combination with Transfersomes™ comprising menthol. The second (“TP2”) comprised insoluble and inflexible liposomes comprising capsaicin and menthol in combination with Transfersomes™ comprising menthol. The third formulation (“TP3”) comprised the insoluble aggregates of capsaicin in combination with empty, fragrance-free Sequessomes™

(37) The three starting materials used to formulate TP1, TP2 and TP3 are set out below.

(38) TP1 (PD-14-0072) was formed by taking 100 g of Start Material 1, and adding/mixing in 0.25 g of a 100 mg/g capsaicin solution in ethanol.

(39) TP2 (PD-14-0073) was formed by taking 90 g of Start Material 1 and adding/mixing in 10 g of Start Material 2.

(40) TP3 (PD-14-0074) was formed by taking 100 g of Start Material 3, and adding/mixing in 0.25 g of a 100 mg/g capsaicin solution in ethanol.

(41) Start Materials

(42) Start Material 1 (Flexible, Menthol-Fragranced Vesicles):

(43) TABLE-US-00015 Quantity Required Per 100 g Final Product (g) Organic Phase - Empty Transfersome Intermediate SPC (Dry Mass) 6.87000 Ethanol 3.65100 BHT 0.02000 Methyl-4-hydroxybenzoate 0.25000 Ethyl-4-hydroxybenzoate 0.25000 Benzyl alcohol 0.52500 Polysorbate 80 0.85000 Capsaicin 0.00000 Menthol 0.10000 Organic Phase Sub-Total 12.51600 Aqueous Phase - Empty Transfersome Intermediate Sodium dihydrogen orthophosphate 2 H.sub.2O 0.03700 disodium hydrogen orthophosphate 12 H.sub.2O 0.45300 Sodium EDTA 0.10000 Water 45.22700 Aqueous Phase Sub-Total 45.81700 Transfersome Intermediate Total 58.33300 Gel Phase - Final Product Sodium dihydrogen orthophosphate 2 H.sub.2O 0.02400 disodium hydrogen orthophosphate 12 H.sub.2O 0.30200 Sodium hydroxide 0.63000 Carbopol 974P NF 1.25000 Glycerol 3.00000 Water 36.46100 Gel Phase Sub-Total 41.66700 Overall Total 100.00000
Start Material 2 (Capsaisin Liposomes):

(44) TABLE-US-00016 Quantity Required Per 100 g Final Product (g) Organic Phase - Liposome Intermediate SPC (Dry Mass) 6.87000 Ethanol 4.25100 BHT 0.02000 Methyl-4-hydroxybenzoate 0.25000 Ethyl-4-hydroxybenzoate 0.25000 Benzyl alcohol 0.52500 Polysorbate 80 0.00000 Capsaicin 0.25000 Menthol 0.10000 Organic Phase Sub-Total 12.51600 Aqueous Phase - Liposome Intermediate Sodium dihydrogen orthophosphate 2 H.sub.2O 0.03700 disodium hydrogen orthophosphate 12 H.sub.2O 0.45300 Sodium EDTA 0.10000 Water 45.22700 Aqueous Phase Sub-Total 45.81700 Liposome Intermediate Total 58.33300 Gel Phase - Final Product Sodium dihydrogen orthophosphate 2 H.sub.2O 0.02400 disodium hydrogen orthophosphate 12 H.sub.2O 0.30200 Sodium hydroxide 0.63000 Carbopol 974P NF 1.25000 Glycerol 3.00000 Water 36.46100 Gel Phase Sub-Total 41.66700 Overall Total 100.00000
Start Material 3 (Flexible, Empty, Fragrance-Free Vesicles):

(45) TABLE-US-00017 Quantity Required Per 100 g Final Product (g) Organic Phase - Sequessome Intermediate SPC (Dry Mass) 6.87000 Ethanol 3.75100 BHT 0.02000 Methyl-4-hydroxybenzoate 0.25000 Ethyl-4-hydroxybenzoate 0.25000 Benzyl alcohol 0.52500 Polysorbate 80 0.85000 Capsaicin 0.00000 Menthol 0.00000 Organic Phase Sub-Total 12.51600 Aqueous Phase - Empty Sequessome Intermediate Sodium dihydrogen orthophosphate 2 H.sub.2O 0.03700 disodium hydrogen orthophosphate 12 H.sub.2O 0.45300 Sodium EDTA 0.10000 Water 45.22700 Aqueous Phase Sub-Total 45.81700 Sequessome Intermediate Total 58.33300 Gel Phase - Final Product Sodium dihydrogen orthophosphate 2 H.sub.2O 0.02400 disodium hydrogen orthophosphate 12 H.sub.2O 0.30200 Sodium hydroxide 0.63000 Carbopol 974P NF 1.25000 Glycerol 3.00000 Water 36.46100 Gel Phase Sub-Total 41.66700 Overall Total 100.00000
Summary Formulae:

(46) TABLE-US-00018 PD-14-0072 PD-14-0073 PD-14-0074 (Start (90% Start (Start Material 1 Material 1 Material 3 plus 0.025% plus 10% plus 0.025% Title Start Start Start capsaicin in Start capsaicin in Ingredient Material 1 Material 2 Material 3 ethanol) Material 2 ethanol) SPC (dry mass) 6.870 g 6.870 g 6.870 g 6.870 g 6.870 g 6.870 g Polysorbate 80 0.850 g — 0.850 g 0.850 g 0.765 g 0.850 g Benzylalcohol 0.525 g 0.525 g 0.525 g 0.525 g 0.525 g 0.525 g Methyl-4-hydroxybenzoate 0.250 g 0.250 g 0.250 g 0.250 g 0.250 g 0.250 g Ethyl-4-hydroxybenzoate 0.250 g 0.250 g 0.250 g 0.250 g 0.250 g 0.250 g Butylhydroxytoluene 0.020 g 0.020 g 0.020 g 0.020 g 0.020 g 0.020 g Disodium EDTA 0.100 g 0.100 g 0.100 g 0.100 g 0.100 g 0.100 g Disodium hydrogen 0.755 g 0.755 g 0.755 g 0.755 g 0.755 g 0.755 g phosphate 12 H.sub.2O Sodium dihydrogen 0.061 g 0.061 g 0.061 g 0.061 g 0.061 g 0.061 g phosphate 2 H.sub.2O Glycerol 3.000 g 3.000 g 3.000 g 3.000 g 3.000 g 3.000 g Ethanol 3.651 g 4.251 g 3.751 g 3.876 g 3.711 g 3.976 g Sodium hydroxide 0.630 g 0.630 g 0.630 g 0.630 g 0.630 g 0.630 g Carbopol 974P NF 1.250 g 1.250 g 1.250 g 1.250 g 1.250 g 1.250 g Water 81.688 g 81.688 g 81.688 g 81.688 g 81.688 g 81.688 g Agent of Interest in continuous phase Capsaicin — 0.250 g — 0.025 g 0.025 g 0.025 g Agent of Interest in Transfersomes Menthol 0.100 g 0.100 g — 0.100 g 0.100 g Total mass 100.000 g 100.000 g 100.000 g 100.250 g 100.000 g 100.250 g
Results

(47) Following the application of the formulations to the skin of three individuals, the following combined results were recorded regarding the onset, duration and intensity of the warming sensation produced by the capsaicin.

(48) TABLE-US-00019 TABLE 8 Combined results of three individuals testing “TP1”, “TP2” and “TP3” TP1 TP2 TP3 Product code (PD-14-0072) (PD-14-0073) (PD-14-0074) Capsaicin Insoluble In liposomes Insoluble aggregate aggregate Menthol In membranes of In membranes of None Transfersomes ™ Transfersomes ™ and of liposomes Sequessomes No No Yes Test product TP1 TP2 TP3 Onset of sensation  5 to 30 mins  5 to 30 mins 15-30 mins Onset of optimal 15 to 60 mins 15 to 60 mins 30-60 mins sensation Duration of 60 to 120 mins 45 to 180 mins 45 to 60 mins sensation (capsaicin) Intensity Pleasant Pleasant Pleasant (capsaicin)

EXAMPLE 4: EXEMPLARY COMPOSITIONS COMPRISING (I) GLUCOSAMINE HYDROCHLORIDE AND CHONDROITIN SULPHATE (CBL-LS-15-002) AND (II)N-ACETYL GLUCOSAMINE SULPHATE AND CHONDROITIN SULPHATE (CBL-LS-15-003)

(49) Below is provided two exemplary formulations of chondroitin and glucosamine. The first (CBL-LS-15-002) comprises glucosamine hydrochloride and chondroitin sulphate as the “free” AOIs in the continuous phase and palmitoyl ascorbate and tocopheryl lineoleate tethered to Tethersomes. The second (CBL-LS-15-00) comprises N-acetyl glucosamine sulphate and chondroitin sulphate as the “free” AOIs in the continuous phase and palmitoyl ascorbate and tocopheryl lineoleate tethered to Tethersomes.

(50) TABLE-US-00020 CBL-LS-15-002 CBL-LS-15-003 Ingredient Supplement Gel Supplement Gel SPC (dry mass)  6.870 g  6.870 g Polysorbate 80  0.850 g  0.850 g Benzylalcohol  0.525 g  0.525 g Methyl-4-hydroxybenzoate  0.250 g  0.250 g Ethyl-4-hydroxybenzoate  0.250 g  0.250 g Butylhydroxyanisole  0.020 g  0.020 g Linalool  0.100 g  0.100 g Sodium metabisulphite  0.050 g  0.050 g Disodium EDTA  0.100 g  0.100 g Disodium hydrogen phosphate 12  0.530 g  0.530 g H.sub.2O Citric acid monohydrate  0.128 g  0.128 Glycerol  3.000 g  3.000 g Ethanol  3.455 g  3.455 g Sodium hydroxide  0.160 g  0.160 g Carbopol 974P NF  0.750 g  0.750 g Water 82.366 g 82.366 g Agents of Interest in Continuous Phase Chondroitin Sulphate  0.200 g  0.200 g Glucosamine •HCl  0.200 g N-Acetyl glucosamine sulphate  0.200 g Agents of Interest - Tethered to Tethersomes Palmitoyl ascorbate  0.096 g  0.096 g Tocopheryl linoleate  0.100 g  0.100 g

EXAMPLE 5: A USER TRIAL STUDY IN HEALTHY VOLUNTEERS TO INVESTIGATE THE EFFICACY OF A TEST ARTICLE COMPRISING CAFFEINE IN IMPROVING THE APPEARANCE OF PERIORBITAL SKIN

(51) This study tested the efficacy of a formulation comprising caffeine and tocopherol in the continuous phase and three types of deformable colloidal particles: 1) Tethersomes to which palmitoyl ascorbate is tethered, 2) Tethersomes to which palmitoyl tripeptide-1 is tethered, and 3) Tethersomes to which palmitoyl tetrapeptide-7 is tethered.

(52) Methods

(53) Summary Protocol

(54) TABLE-US-00021 Study design: Single-blind Test article: CBL-DERM-15-013 Periorbital skin serum Duration of treatment: 4 weeks Number of subjects: 102 Type of subiects: Healthy male and female subjects (from a breadth of ethnicities) aged between 40 and 70 years old (in equal proportions, 33% each 10 year age group), from a breadth of ethnicities and with a variety of skin types (normal, combination, dry, oily, sensitive) who suffer from the appearance of two of the following characteristics: Periorbital puffiness - swelling around the eyes Puffiness below the lower eyelids - ‘eye bags’ Periorbital dark circles caused by a) aging b) heredity and c) hyperpigmentation (dark skinned people) (There must be an equal representation of all three characteristics to ensure claims objectivity). All subjects must have visible wrinkles, fine lines and crow's feet around the eye area Observations: Subjects were asked to apply the product as per usage instructions. They were then asked to complete a Self-Perception Questionnaire (SPQ) after two weeks and at the end of the study after four weeks. Treatment: Subjects were issued with samples of the test article and directions for use following standard in-use application regime. Location Princeton Consumer Research Harbour House 23 Chandlers Quay Maldon CM9 4LF United Kingdom
Summary Formula for CBL-DERM-15-013:

(55) TABLE-US-00022 CBL-DERM-15-013 Ingredient Periorbital Skin SPC (dry mass)  6.870 g Polysorbate 80  0.714 g Benzylalcohol  0.525 g Methyl-4-hydroxybenzoate  0.250 g Ethyl-4-hydroxybenzoate  0.250 g Butylhydroxyanisole  0.020 g Linalool  0.100 g Sodium metabisulphite  0.050 g Disodium EDTA  0.100 g Disodium hydrogen phosphate 12 H.sub.2O  0.530 g Citric acid monohydrate  0.128 g Glycerol  3.000 g Ethanol  3.479 g Sodium hydroxide  0.160 g Carbopol 974P NF  0.750 g Water 82.716 g Agent of Interest in Continuous Phase Caffeine  0.050 g Tocopherol  0.200 g Agents of Interest - tethered to Tethersomes Palmitoyl tripeptide-1  0.006 g Palmitoyl tetrapeptide-7  0.006 g Palmitoyl ascorbate  0.096 g
Detailed Formulation Information for CBL-DERM-15-013:

(56) TABLE-US-00023 Quantity Required Per 100 g Final Product (g) Organic Phase - 1600 ppm PAA Intermediate Soy phosphatidylcholine (SPC) 4.12200 Ethanol 1.99660 Butylhydroxyanisole (BHA) 0.01200 Methyl-4-hydroxybenzoate 0.15000 Ethyl-4-hydroxybenzoate 0.15000 Benzyl alcohol 0.31500 Polysorbate 80 0.40800 Palmitoyl ascorbic acid 0.09600 +/− alpha tocopherol 0.20000 Linalool 0.06000 Organic Phase Sub-Total 7.50960 Aqueous Phase - 1600 ppm PAA intermediate Sodium metabisulphite 0.03000 Citric acid monohydrate 0.04320 Disodium EDTA 0.06000 Disodium hydrogen orthophosphate 0.17280 dodecahydrate Caffeine 0.05000 Water 27.13420 Aqueous Phase Sub-Total 27.49020 PAA Tethersome Intermediate Total 34.99980 Organic Phase - 300 ppm Tetrapeptide Intermediate Soy phosphatidylcholine (SPC) 1.37400 Ethanol 0.74120 Butylhydroxyanisole (BHA) 0.00400 Methyl-4-hydroxybenzoate 0.05000 Ethyl-4-hydroxybenzoate 0.05000 Benzyl alcohol 0.10500 Polysorbate 80 0.15300 Palmitoyl peptide 0.00600 Linalool 0.02000 Organic Phase Sub-Total 2.50320 Aqueous Phase - 300 ppm Tetrapeptide Intermediate Sodium metabisulphite 0.01000 Citric acid monohydrate 0.01440 Disodium EDTA 0.02000 Disodium hydrogen orthophosphate 0.05760 dodecahydrate Water 9.06140 Aqueous Phase Sub-Total 9.16340 Tetrapeptide Tethersome Intermediate Total 11.66660 Organic Phase - 300 ppm Tripeptide Intermediate Soy phosphatidylcholine (SPC) 1.37400 Ethanol 0.74120 Butylhydroxyanisole (BHA) 0.00400 Methyl-4-hydroxybenzoate 0.05000 Ethyl-4-hydroxybenzoate 0.05000 Benzyl alcohol 0.10500 Polysorbate 80 0.15300 Palmitoyl peptide 0.00600 Linalool 0.02000 Organic Phase Sub-Total 2.50320 Aqueous Phase - 300 ppm Tripeptide Intermediate Sodium metabisulphite 0.01000 Citric acid monohydrate 0.01440 Disodium EDTA 0.02000 Disodium hydrogen orthophosphate 0.05760 dodecahydrate Water 9.06140 Aqueous Phase Sub-Total 9.16340 Tripeptide Tethersome Intermediate Total 11.66660 Sum of Tethersome Intermediates 58.33300 Gel Phase - Final Product Sodium hydroxide 0.16000 Carbopol 974P NF 0.75000 Glycerol 3.00000 Citric acid monohydrate 0.05600 Disodium hydrogen orthophosphate 0.24200 dodecahydrate Water 37.45900 Gel Phase Sub-Total 41.66700 Overall Total 100.00000
Results

(57) Within-treatment analysis (p<0.05) of periorbital clinical assessment shows there to be a statistically significant reduction in wrinkles (15.69%), fine lines (31.22%), crow's feet (21.47%), puffiness (38.07%), dark circles (26.01%) and eye bags (38.94%) after 4 weeks of product usage.

(58) The product performed statistically favourably over the 4 week study in the majority of attributes under Clearcast guidelines of advertising. The product showed a preference or favourability in the majority of attributes: After using the product, 96.08% of users agreed, or strongly agreed the product reduced the appearance of puffiness and swelling around my eyes. After using the product, 96.08% of users agreed, or strongly agreed their skin felt and looked less thin. After using the product, 93.14% of users agreed, or strongly agreed their skin felt more elastic. After using the product, 96.08% of users agreed, or strongly agreed the product reduced the appearance of swelling and puffiness below the lower eyelids (eye bags). After using the product, 90.20% of users agreed, or strongly agreed the product lifted sagged skin (reduction in skin droopiness and sagging). After using the product, 92.16% of users agreed, or strongly agreed the product reduced the appearance of under eye dark circles. After using the product, 95.10% of users agreed, or strongly agreed their skin looked and felt more firm. After using the product, 90.20% of users agreed, or strongly agreed there was a reduction in fine lines (inc. crow's feet) around the eye area. After using the product, 87.25% of users agreed, or strongly agreed there was a reduction in deep wrinkles (inc. crow's feet) around the eye area. After using the product, 93.14% of users agreed, or strongly agreed their skin tone looked more even. After using the product, 90.20% of users agreed, or strongly agreed their eye contour looked and felt tighter. After using the product, 90.20% of users agreed, or strongly agreed their eye contour looked more toned/lifted. After using the product, 96.08% of users agreed, or strongly agreed their eyes looked rested/less tired. After using the product, 97.06% of users agreed, or strongly agreed their eye skin was more hydrated. After using the product, 95.10% of users agreed, or strongly agreed their skin looked smoother. After using the product, 99.02% of users agreed, or strongly agreed the product was gentle and well tolerated. After using the product, 75.49% of users stated, yes, they would buy this product instead of their usual product. After using the product, 86.27% of users stated, yes, they would recommend this product to a friend. After using the product, 46.08% of users stated, they looked at least 5 years younger.

EXAMPLE 6: A USER TRIAL STUDY IN HEALTHY VOLUNTEERS TO INVESTIGATE THE EFFICACY OF A TEST ARTICLE COMPRISING TOCOPHEROL AND TRIDECYL SALICYLATE IN IMPROVING SKIN TONE

(59) This study tested the efficacy of a formulation comprising a racemic mixture of alpha tocopherol in the continuous phase and two types of colloidal particles: 1) Tethersomes to which tridecyl salicylate is tethered and 2) Tethersomes to which palmitoyl ascorbate and tocopheryl linoleate are tethered.

(60) Methods

(61) Summary Protocol

(62) TABLE-US-00024 Study design: Single-blind Test article: CBL-DERM-15-014 Skin tone serum Duration of treatment: 4 weeks Number of subjects: 110 Type of subjects: Healthy male (20%) and female (80%) participants aged between 20 and 70 years (equal proportions of each 10 year age group; 20's, 30's, 40's, 50's, 60's, 70's), from a Breadth of Ethnicities and with a Variety of Skin Types. Subjects suffer from one well pronounced or two out of the following six conditions; Melasma or Chloasma spots, Uneven skin tone, Age, sun or “liver” spots, Freckles, Post- inflammatory hyperpigmentation or Periorbital hyperpigmentation (dark circles). Observations: Subjects were asked to apply the product as per usage instructions. They were asked to complete a Self-Perception Questionnaire (SPQ) at the end of Weeks 3 and 6. Treatment: Subjects were issued with samples of each test article and directions for use following standard in-use application regime. Location Princeton Consumer Research Harbour House 23 Chandlers Quay Maldon CM9 4LF United Kingdom
Summary Formula for CBL-DERM-15-014:

(63) TABLE-US-00025 CBL-DERM- 15-014 Uneven Skin Ingredient Tone SPC (dry mass)  6.870 g Polysorbate 80  0.850 g Benzylalcohol  0.525 g Methyl-4-hydroxybenzoate  0.250 g Ethyl-4-hydroxybenzoate  0.250 g Butylhydroxyanisole  0.020 g Linalool  0.100 g Sodium metabisulphite  0.050 g Disodium EDTA  0.100 g Disodium hydrogen phosphate 12  0.530 g H.sub.2O Citric acid monohydrate  0.128 g Glycerol  3.000 g Ethanol  3.255 g Sodium hydroxide  0.160 g Carbopol 974P NF  0.750 g Water 82.766 g Agent of Interest in Continuous Phase Tocopherol  0.100 g Agents of Interest - tethered to Tethersomes Palmitoyl ascorbate  0.096 g Tocopheryl Linoleate  0.100 g Tridecyl Salicylate  0.100 g pH* 5.5 *Final pH approximate; to be confirmed **The source of SC dry mass for Uneven Skin Tone will be lipoid Phospholipon 90K (P90K). The quantity of P90K and ethanol to be added should be calculated accordingly.
Detailed Formulation Information for CBL-DERM-15-014:

(64) TABLE-US-00026 Quantity Required Per 100 g Final Product (g) Organic Phase - Tridecyl Salicylate Intermediate Soy phosphatidylcholine (SPC) 3.43500 Ethanol 1.67550 Butylhydroxyanisole (BHA) 0.01000 Methyl-4-hydroxybenzoate 0.12500 Ethyl-4-hydroxybenzoate 0.12500 Benzyl alcohol 0.26250 Polysorbate 80 0.42500 Tridecyl Salicylate 0.10000 +/− alpha tocopherol 0.05000 Linalool 0.05000 Organic Phase Sub-Total 6.25800 Aqueous Phase - Tridecyl Salicylate Intermediate Citric acid monohydrate 0.03600 Disodium EDTA 0.05000 Sodium metabisulphite 0.02500 Disodium hydrogen orthophosphate 0.14400 dodecahydrate Water 22.65350 Aqueous Phase Sub-Total 22.90850 Tridecyl Salicylate Tethersome 29.16650 Intermediate Total Organic Phase - PAA/Tocopheryl Linoleate Intermediate Soy phosphatidylcholine (SPC) 3.43500 Ethanol 1.57950 Butylhydroxyanisole (BHA) 0.01000 Methyl-4-hydroxybenzoate 0.12500 Ethyl-4-hydroxybenzoate 0.12500 Benzyl alcohol 0.26250 Polysorbate 80 0.42500 Palmitoyl ascorbic acid 0.09600 Tocopheryl linoleate 0.10000 +/− alpha tocopherol 0.05000 Linalool 0.05000 Organic Phase Sub-Total 6.25800 Aqueous Phase - PAA/Tocopheryl Linoleate Intermediate Citric acid monohydrate 0.03600 Disodium EDTA 0.05000 Sodium metabisulphite 0.02500 Disodium hydrogen orthophosphate 0.14400 dodecahydrate Water 22.65350 Aqueous Phase Sub-Total 22.90850 PAA/Tocopheryl Linoleate Tethersome 29.16650 Intermediate Total Sum of Tethersome Intermediates 58.33300 Gel Phase - Final Product Sodium hydroxide 0.16000 Carbopol 974P NF 0.75000 Glycerol 3.00000 Citric acid monohydrate 0.05600 Disodium hydrogen orthophosphate 0.24200 dodecahydrate Water 37.45900 Gel Phase Sub-Total 41.66700 Overall Total 100.00000
Results

(65) The product did perform statistically favourably over the 6 week study in the majority of attributes under Clearcast guidelines of advertising. The product did show a preference or favourability in the majority of attributes: After using the product, 100% of users agreed, or strongly agreed the product reduced the appearance of fine lines and wrinkles. After using the product, 100% of users agreed, or strongly agreed their skin looked significantly smoother. After using the product, 100% of users agreed, or strongly agreed their skin felt significantly more healthy. After using the product, 96.36% of users agreed, or strongly agreed their skin felt more elastic After using the product, 99.09% of users agreed, or strongly agreed their complexion looks younger. After using the product, 99.09% of users agreed, or strongly agreed their complexion is visibly improved. After using the product, 100% of users agreed, or strongly agreed their complexion was visibly more radiant. After using the product, 100% of users agreed, or strongly agreed their complexion looked significantly healthier. After using the product, 98.18% of users agreed, or strongly agreed their skin toned looked significantly more even After using the product, 96.36% of users agreed, or strongly agreed that the amount of hyperpigmentation/pigmented skin blemishes were significantly reduced. After using the product, 97.27% of users agreed, or strongly agreed that the size of hyperpigmentation/pigmented skin blemishes were significantly reduced. After using the product, 99.09% of users agreed, or strongly agreed that hyperpigmentation/pigmented skin blemishes were significantly lighter. After using the product, 20.00% of users agreed, or strongly agreed that hyperpigmented/pigmented skin blemishes totally disappeared. After using the product, 100% of users agreed, or strongly agreed their periorbital dark circles got significantly lighter. After using the product, 95.45% of users agreed, or strongly agreed this was the most effective hyperpigmentation solution they had used. After using the product, 92.73% of users stated, yes, they would buy this product instead of their usual product. After using the product, 100% of users stated, yes, they would recommend this product to a friend.

(66) With reference to packaging/promotional claims, any results with a top 3 & 4 scoring greater than 80% are highly favourable.