Kaempferia parviflora extract compositions and methods of treatment for reducing dermal adipocyte size and enhancing dermal adipocyte number

Abstract

Topical compositions and methods of treatment are provided that are effective for reducing the size of dermal adipocytes and increasing the regeneration of dermal adipocytes in vivo.

Description

Attorney Docket No.115061.PH590WO TOPICAL COMPOSITIONS AND METHODS OF TREATMENT FOR REDUCING DERMAL ADIPOCYTE SIZE AND ENHANCING DERMAL ADIPOCYTE NUMBER CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 641,040, filed May 1, 2024, which is expressly incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention generally relates to topical compositions and methods of treatment of reducing dermal adipocyte size and enhancing dermal adipocyte number. BACKGROUND

[0003] With age, and, independently with sun exposure, the number of dermal fat cells declines due to insufficient adipogenesis (e.g., new fat cell formation). This leads to volume deficits in the face and weak or saggy skin throughout the body. A reduction in the number of dermal fat cells causes the remaining cells to become larger in size. Bloated dermal fat cells cause dermal fibroblasts to produce significantly less collagen and elastin. One specific manifestation of this reduction in elastin is a crepe-like structure of the skin.

[0004] Cellulite is another manifestation of a hypertrophic fat cell phenotype. The adipocytes found in the biopsies of cellulite subjects were termed “giant” by Scarano et al. The hypertrophy is caused by insufficient regeneration. New fat cell formation is limited by insufficient nutrient supply due to poorly vascularized tissue. The lack of oxygenation also stimulates inflammation which further suppresses new fat cell formation. See, e.g., Scarano A, Petrini M, Sbarbati A, Amore R, Iorio EL, Marchetti M, Amuso D., Pilot study of histology aspect of cellulite in seventy patients who differ in BMI and cellulite grading. J CosmetAttorney Docket No.115061.PH590WO Dermatol.2021 Dec; 20(12):4024-4031.

[0005] The biopsies of cellulite subjects reveal a dermis structure that is different from normal skin. The collagen and elastin are fragmented and disordered (See, e.g., Scarano et al.). Hypertrophic fat cells weaken the skin by secreting inflammatory cytokines that suppress collagen and elastin production by resident fibroblasts. Hypertrophic fat cells also dramatically increase production of matrix metalloproteinase enzymes which degrade collagen and elastin. See, e.g., Ezure T, Amano S., Negative regulation of dermal fibroblasts by enlarged adipocytes through release of free fatty acids. J Invest Dermatol. 2011 Oct; 131(10):2004-9.

[0006] The biopsies of subjects with cellulite reveal fibrotic septa around the lobes of hypertrophic fat cells (See, e.g., Scarano et al.). Septa are bands of collagen, which are normally stretchy and elastic. In the context of cellulite, these bands connect the skin to the underlying structures, which can be fascia, muscle or bone. Under the type of inflammatory conditions that hypertrophic fat cells create, myofibroblasts – which specialize in making scar tissue – are recruited and restructure the septa to be thicker and stiffer. The connection between inflammation and fibrotic septa is illustrated by Humphrey S. et al. (Humphrey S, Munavalli GS, Yoelin SG, Friedmann DP, Kavali CM, Sangha S., Submental Area Treatment with ATX-101: Relationship of Mechanism of Action, Tissue Response, and Efficacy. Plast Reconstr Surg Glob Open.2022 Apr 27;10(4)).

[0007] Adipocyte size is closely correlated with body mass index (BMI). See, e.g., Honecker J, Weidlich D, Heisz S, Lindgren CM, Karampinos DC, Claussnitzer M, Hauner H., A distribution-centered approach for analyzing human adipocyte size estimates and their association with obesity-related traits and mitochondrial function. Int J Obes (Lond). 2021Attorney Docket No.115061.PH590WO Sep; 45(9):2108-2117.

[0008] Relatively small differences in average adipocyte size can lead to major differences in the inflammatory state of the adipose tissue. In one study, first degree relatives of subjects with type 1 diabetes had an average adipocyte size that was nine percent larger than that of the control population. This difference was associated with levels of inflammatory markers that were 100% higher. (See, e.g., Henninger AM, Eliasson B, Jenndahl LE, Hammarstedt A., Adipocyte hypertrophy, inflammation and fibrosis characterize subcutaneous adipose tissue of healthy, non-obese subjects predisposed to type 2 diabetes. PLoS One.2014 Aug 22; 9(8)).

[0009] Increases in fat cell size also occur during menopause, and other conditions like puberty and pregnancy, where hormone levels fluctuate. During menopause, for example, the mean adipocyte size increases by approximately 15%. As in the case of cellulite adipocyte hypertrophy, during menopause women experience rapid deterioration in skin integrity and a sagging phenotype. (See, e.g., Greendale GA, Sternfeld B, Huang M, Han W, Karvonen-Gutierrez C, Ruppert K, Cauley JA, Finkelstein JS, Jiang SF, Karlamangla AS., Changes in body composition and weight during the menopause transition. JCI Insight.2019 Mar 7; 4(5)). In the Greendale study the 15% increase in average adipocyte size is greater than the 12% increase in body fat volume, and much greater than the 4% increase in weight that occurred during the same period, suggesting that weight gain only partially explains fat cell size bloating.

[0010] The other key factor in the fat cell size equation is the rate of new fat cell formation or rate of adipogenesis. The greater the rate of adipogenesis, the smaller the average fat cell size will be. Lifestyle factors that suppress adipogenesis include protein-Attorney Docket No.115061.PH590WO deficient diet, high levels of stress, and chronic inflammation or frequent acute inflammation. One surprising source of inflammation is exercise. Post exercise, inflammatory markers are elevated for 1-4 hours, and there is a dose response. The greater the intensity of the exercise, the more inflammation markers are elevated. Dynamic movement also suppresses adipogenesis, and areas of the body subject to frequent movement, such as the neck, hands, elbows and sides of the mouth have weaker, thinner skin as a result of having fewer adipocytes, and thus more hypertrophic adipocytes.

[0011] Hypertrophic fat cells actively suppress new fat cell formation in part by releasing inflammatory mediators. See, e.g., Gustafson B, Gogg S, Hedjazifar S, Jenndahl L, Hammarstedt A, Smith U., Inflammation and impaired adipogenesis in hypertrophic obesity in man. Am J Physiol Endocrinol Metab.2009 Nov; 297(5). This reduction in regeneration is also seen with menopause-induced fat cell hypertrophy. See Greendale.

[0012] Moreover, loss of dermal fat cell numbers leads to impaired metabolic function in the body. The hypertrophy of the remaining dermal fat cells leads to production of inflammatory cytokines and further downregulation of adipogenesis, which creates a vicious cycle. As the health of the dermal adipocytes declines, this affects the health of the adipocytes in the adjacent subcutaneous area. Over time, the decline in the quality and quantity of the superficial fat compartment causes a compensatory increase in visceral fat, with negative consequences for the metabolic health of the subject.

[0013] Thus, the scientific literature describes a clear connection between hypertrophic adipocytes, inflammation, suppressed adipogenesis, further hypertrophy and consequently metabolic dysfunction and weak, saggy skin. The concept of restoring adipogenesis by reducing adipocyte size is attractive but to our knowledge this has neverAttorney Docket No.115061.PH590WO been achieved with either drug therapy or non-drug natural compositions, delivered orally or topically. Thus, it is not known to one skilled in the art how much fat cell size reduction would be sufficient to reverse the hypertrophy vicious cycle and enable a virtuous cycle of increased adipogenesis leading to smaller cell size, leading to more effective adipogenesis, nor what drugs or non-drug natural compositions could achieve the goal in vivo.

[0014] Kaempferia Parviflora Extract (KPE) has been shown to have lipolytic properties. Okabe et al. demonstrated that KPE extract could decrease the size of mature murine adipocytes in 2D culture. (Okabe Y, Shimada T, Horikawa T, Kinoshita K, Koyama K, Ichinose K, Aburada M, Takahashi K., Suppression of adipocyte hypertrophy by polymethoxyflavonoids isolated from Kaempferia parviflora. Phytomedicine. 2014 May 15;21(6):800-6). In this experiment, the adipocytes were treated at day 8, a time point where they are mature but have not yet become hypertrophic. The experiment did not evaluate whether treatment with KPE extract could increase the number of adipocytes.

[0015] The lipolytic properties of KPE have been shown in a human clinical study with KPE taken orally. Compared to subjects taking a placebo supplement, Yoshino et al. demonstrated that subjects taking 150 mg of a KPE daily had significant reductions in subcutaneous and visceral fat volume as measured by CT scanning. See, e.g., Yoshino S, Awa R, Miyake Y, Fukuhara I, Sato H, Ashino T, Tomita S, Kuwahara H., Daily intake of Kaempferia parviflora extract decreases abdominal fat in overweight and preobese subjects: a randomized, double-blind, placebo-controlled clinical study. Diabetes Metab Syndr Obes. 2018 Aug 28; 11:447-458. Erratum in: Diabetes Metab Syndr Obes. 2020 May 12; 13:1609. This study did not assess fat cell size or numbers in the adipose tissue of the subjects. The study also did not assess the skin quality of the subjects.Attorney Docket No.115061.PH590WO

[0016] KPE was also known to have adipogenic properties in murine adipocytes. Horikawa et al. conducted an experiment in 2D culture with murine fibroblasts and found that the KPE could increase PPAR gamma activity without directly binding to PPAR gamma. Treatment with KPE starting at the beginning of the differentiation protocol resulted in a larger number of mature adipocytes when measured after 8 days. See, e.g., Horikawa T, Shimada T, Okabe Y, Kinoshita K, Koyama K, Miyamoto K, Ichinose K, Takahashi K, Aburada M., Polymethoxyflavonoids from Kaempferia parviflora induce adipogenesis on 3T3-L1 preadipocytes by regulating transcription factors at an early stage of differentiation. Biol Pharm Bull. 2012;35(5):686-92. In this experiment, KPE treated a population of 100 immature cells. There were no mature adipocytes and no hypertrophic adipocytes.

[0017] The inventors previously disclosed an invention that restored dermal fat volume or reduced dermal fat volume as needed. This invention required an adipogenic agent, a lipolytic agent and a penetrant to function. The inventors defined an adipogenic agent as one that stimulated PPAR gamma, the master regulator of pre-adipocyte differentiation. The inventors exemplified a composition where the minimum weight percentage of the adipogenic agent (15%) was more than twice the maximum weight percentage of the lipolytic agent (7%). The inventors also described the use of adipogenic agents comprising long chain fatty acids. Long chain fatty acids are lipogenic, meaning they increase the size of adipocytes by increasing the size of their lipid droplets.

[0018] Many subjects have bloated or excessively sized dermal fat cells. This excessive size can be approximated by body mass index (BMI) in the case of subjects who are overweight or observed from the state of loose, saggy or deeply wrinkled skin or overt volume loss in subjects who are not overweight but have a deficiency in dermal adipocyteAttorney Docket No.115061.PH590WO numbers. Loose, saggy or deeply wrinkled skin reflects disordered and deficient collagen and elastin in the dermis caused by hypertrophic fat cells. This condition is distinct from fine lines or wrinkles which may reflect a more superficial condition, caused for example, by insufficient water content. Because of they have different etiologies than superficial skin conditions such as shallow wrinkles and fine lines, laxity and deep wrinkles are evaluated in clinical practice using metrics that are used for these superficial conditions. For example, in the FDA evaluation of retinoic acid, improvement in the appearance of “fine lines” was considered a separate analysis from improvement in the appearance of deep wrinkles or laxity. Retinoic acid was found to be effective for improving the appearance of fine lines but was not found to be effective for the appearance of deep wrinkles or laxity.

[0019] In order for Kaempferia Parviflora Extract to positively modulate the dermal adipocytes, it must be delivered by a topical formulation to the dermal white adipose tissue in a sufficient concentration. The most effective path for reaching the dermal white adipose tissue is the transfollicular route. The inter-cellular and intra-cellular routes through the stratum corneum provide alternative ways for reaching the dermal white adipose tissue but they are highly disadvantageous as the stratum corneum represents a significant barrier and allows only a very small percentage of the active ingredients in a topical composition to pass through to the lower dermis. The percentage of an active ingredient in a topical composition that penetrates to the lower dermis is variable and depends on several formulation, skin, and compound-specific factors. However, in general, less than 1% of the applied dose reaches the deep dermis, with most estimates ranging from 0.01% to 1% under typical conditions. To increase the penetration of active ingredients, some formulations employ barrier disruptors such as hyaluronic acid, ethanol, and polyoxyethylene, among others. UseAttorney Docket No.115061.PH590WO of such barrier disruptors long term can lead to barrier dysfunction and treatment limiting irritation. It may also lead to less of the active ingredients reaching the dermal white adipose tissue because the disrupted barrier absorbs the active ingredients before they can funnel to the hair follicle and go down the transfollicular route.

[0020] For subjects with hypertrophic dermal fat cells, employing a topical composition with a large concentration of adipogenic agents comprising long chain fatty acids may not be effective as such a composition is likely to either fail to decrease the size of the dermal fat cells or may actually increase the size of the dermal fat cells. Similarly, employing compositions with lipogenic ingredients such as squalane, stearic acid, jojoba oil and sesamum indicum seed oil would cause the mature adipocytes to bloat and suppress new fat cell formation.

[0021] The use of anti-adipogenic ingredients in a formulation comprising Kaempferia Parviflora Extract could also undermine the positive adipocyte modulating effects of KPE. Examples of anti-adipogenic ingredients include retinal palmitate, L-arginine, Tea seed oil, licorice extract and perilla extract. SUMMARY OF THE INVENTION

[0022] In one embodiment of the invention, a topical composition is provided that contains Kampferia Parviflora Extract, an emulsifying agent or other agent that serves to suspend KPE on top of the skin and optionally, an adipogenic agent; wherein a ratio of adipogenic agent to lipolytic agent, as measured by weight by volume, is less than 2 to 1, and wherein the composition visibly improves the health of the dermal white adipose tissue.

[0023] In another embodiment of the invention, a method is provided of increasing the number of dermal adipocytes and also decreasing the average size of dermal adipocytes,Attorney Docket No.115061.PH590WO such method consisting of applying a topical composition comprising Kaempferia Parviflora Extract and an emulsifying agent or other agent to suspend the KPE on top of the skin to a subject with dermal adipocytes of excessive size, as evidenced by a BMI in excess of 20 or alternatively as evidenced by visibly weak or saggy skin or skin manifesting deep wrinkles or cellulite, where such composition is delivered effectively to the dermal white adipose tissue via the transfollicular route Such compositions would not employ ingredients capable of disrupting the stratum corneum with long term use.

[0024] In another embodiment of the invention, a topical composition effective for delivering agents to the skin is provided, the agent comprising a silicone elastomer superstructure comprising: dimethicone, silicone crosspolymers, and / or dimethiconol, wherein the viscosity of the resulting composition is at least 8,000 centipoise (cP) and no greater than 15,000 cP.

[0025] Dimethicone is the International Nomenclature for Cosmetic Ingredients (INCI) name for a series of basic silicones used as the base fluid in personal care formulations. These inert polymers are optically clear, odorless and non-flammable. They are often referred to as silicone oil, dimethyl polysiloxane, and polydimethylsiloxane, and take viscosities within a broad range from 5 centistokes (cst) to 1 million cst.

[0026] Dimethiconol, also referred to as silicone gum, is a polymer similar to dimethicone where two chain-end methyl groups have been replaced by hydroxyl (-OH) groups.

[0027] Dimethicone crosspolymer is classified as a silicone elastomer gel. It comprises crosslinked dimethicone molecules, chemically bonded by covalent bonds.

[0028] Other objects, advantages and novel features of the present invention will beAttorney Docket No.115061.PH590WO readily ascertainable to persons of ordinary skill in the art. Other objects and features of the invention will be ascertainable from the following detailed description of one or more preferred embodiments when considered in conjunction with the figures presented. It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present invention is intended to encompass variations and equivalents of these examples. The disclosure is written for those skilled in the art. Although the disclosure uses terminology and acronyms that may not be familiar to the layperson, those skilled in the art will be familiar with the terminology and acronyms used herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The description herein will be more fully understood in view of the following drawings.

[0030] FIG. 1 shows the average lipid droplet (LD) size after human pre-adipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Parviflora Extract (KPRE) and the combination (KPSS) of safflower seed oil and KPRE from the day 0 to day 7, measured at day 7.

[0031] FIG.2 shows the number of nuclei after human pre-adipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Parviflora Extract (KPRE) and the combination (KPSS) of safflower seed oil and Kaempferia Parviflora Extract from the day 0 to day 7 and measured at day 7.

[0032] FIG. 3 shows the average lipid droplet (LD) size after human preadipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Parviflora Extract (KPRE) and the combination (KPSS) of safflower seed oil and KPRE from the day 0 to day 14, measured at day 14.Attorney Docket No.115061.PH590WO

[0033] FIG. 4 shows the number of nuclei after human preadipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Parviflora Extract (KPRE) and the combination (KPSS) of safflower seed oil and KPRE from the day 0 to day 7 and measured at day 7.

[0034] FIG. 5 shows the average lipid droplet (LD) size after a mix human preadipocytes and mature adipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Extract (KPRE) and the combination (KPSS) of safflower seed oil and KPRE from the day 14 to day 21, measured at day 21.

[0035] FIG. 6 shows the number of nuclei after a mix of human preadipocytes and mature adipocytes growing in 3D culture medium were treated with control medium, safflower seed oil (SSO), Kaempferia Parviflora Extract (KPRE) and the combination (KPSS) of safflower seed oil and KPRE from the day 14 to day 21 and measured at day 21.

[0036] FIG. 7 shows the changes in transcription levels of the master regulator of adipogenesis, PPAR gamma, in human preadipocytes growing in 3D culture medium treated with control medium, safflower seed oil (SSO) or Kaempferia Parviflora Extract (KPRE) from day 0 to day 14 measured on day 7 and also on day 14 (“pre-differentiation” experiment).

[0037] FIG. 8 shows the before (in FIG. 8A) and after (in FIG. 8B) photo close up images of the arm skin of a female subject with a BMI in excess of 25 who applied a composition according to an embodiment of the invention for fewer than 30 days.

[0038] FIG.9 shows the before (in FIG.9A) and after (in FIG.9B) of a photo close up images of the arm skin of a different female subject with a BMI in excess of 25 who applied a composition according to an embodiment of the invention for fewer than 30 days.

[0039] FIG.10 shows the before (in FIG.10A) and after (in FIG.10B) of a photo closeAttorney Docket No.115061.PH590WO up images of the right hemiface of a woman with a BMI of less than 25 and with an overt volume deficit in the mid-cheek who used a composition according to an embodiment of the invention for 1 month after previously using a composition according to the embodiment of the invention disclosed in the previous patent for more than 3 months.

[0040] FIG.11 shows the before (in FIG.11A) and after (in FIG.11B) of a photo close up images of the left hemiface of a woman with a BMI less than 25 and with an overt volume deficit in the mid-cheek who used a composition according to an embodiment of the invention for 1 month after previously using a composition according to the embodiment of the invention disclosed in the previous patent for more than 3 months.

[0041] FIG.12 shows the before (in FIG.12A) and after (in FIG.12B) of a photo close up images of the right hemiface of a woman with a BMI less than 25 and with an overt volume deficit under the eye and an overt saggy nasolabial fold who used a composition according to an embodiment of the invention for 1 month after previously using a composition according to the embodiment of the invention disclosed in the previous patent for more than 2 months.

[0042] FIG.13 shows the before (in FIG.13A) and after (in FIG.13B) of a photo close up images of the left hemiface of a woman with a BMI less than 25 and with an overt volume deficit under the eye and also in the mid cheek who used a composition according to an embodiment of the invention for 1 month after previously using a composition according to the embodiment of the invention disclosed in the previous patent for more than 2 months.

[0043] FIG.14 shows the primary endpoint analysis of a clinical trial that evaluated a composition according to an embodiment of the invention in subjects with mild, moderate and severe cellulite over a period of three months. The subjects applied the compositionAttorney Docket No.115061.PH590WO two times per day and took photos of the cellulite conditions. The baseline and three-month photos were presented in random order and with random codes to four blinded evaluators. The evaluators were asked which of the photos, if any, reflected a greater cellulite severity. The primary endpoint evaluated all grades of cellulite severity. On the primary endpoint, three or more evaluators identified the “before” image as having greater cellulite severity 75% of the time.

[0044] FIG.15 shows the secondary endpoint analysis of a clinical trial that evaluated a composition according to an embodiment of the invention in subjects with mild, moderate and severe cellulite over a period of three months. The subjects applied the composition two times per day and took photos of the cellulite conditions. The baseline and three-month photos were presented in random order and with random codes to four blinded evaluators. The evaluators were asked which of the photos, if any, reflected a greater cellulite severity. The secondary endpoint evaluated moderate and severe baseline cellulite severity. On the secondary endpoint, three or more evaluators identified the “before” image as having greater cellulite severity 100% of the time.

[0045] FIG.16 shows the before (in FIG.16A) and after (in FIG.16B) of a photo close up images of an area of cellulite on the leg of a subject who used a composition according to an embodiment of the invention for 3 months as part of a clinical study.

[0046] FIG.17 shows the before (in FIG.17A) and after (in FIG.17B) of a photo close up images of an area of cellulite on the leg in another subject who used a composition according to an embodiment of the invention for 3 months as part of a clinical study.

[0047] FIG.18 shows the before (in FIG.18A) and after (in FIG.18B) of a photo close up images of an area of cellulite on the other leg of a subject shown in FIG 15. who used aAttorney Docket No.115061.PH590WO composition according to an embodiment of the invention for 3 months as part of a clinical study.

[0048] FIG.19 shows the before (in FIG.19A) and after (in FIG.19B) of a photo close up images of an area of cellulite on the leg in another subject who used a composition according to an embodiment of the invention for 3 months as part of a clinical study.

[0049] FIG. 20 is a depiction of how liposome carriers deliver ingredients to the skin compared with unencapsulated delivery.

[0050] FIG.21 shows the absorption kinetics of a silicon elastomer composition according to an embodiment of the invention. DETAILED DESCRIPTION

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] As used herein, the terms “invention” or “present invention” are non-limiting terms and not intended to refer to any single aspect of the particular invention but encompass all possible aspects as described in the specification and the claims.

[0053] The inventors now disclose new methods of treating dermal white adipose tissue where a lipogenic PPAR gamma agonist such as a long-chain free fatty acid found in oils such as sunflower seed oil and cotton seed oil is dispensable for stimulating adipogenesis and instead restoration of defective adipogenesis can be accomplished by delivering an agent that either decreases the size of existing mature adipocytes or sustains pre-adipocyte proliferation without inhibiting the ultimate maturation of pre-adipocytes.

[0054] The inventors disclose for the first time that Kaempferia Parviflora Extract isAttorney Docket No.115061.PH590WO capable of sustaining the proliferation of pre-adipocytes without inhibiting their ultimate maturation.

[0055] The inventors also disclose for the first time that Kaempferia Parviflora Extract can enable adipogenesis in the context of hypertrophic adipocytes having a suppressive effect on adipogenesis.

[0056] Japanese patent JP 5863721 B2 teaches that Kaempferia Parviflora Extract can increase lipolysis in mature adipocytes but does not teach whether this effect is sufficient to enhance the maturation of immature adipocytes.

[0057] Japanese patent JP 2016056163 A teaches that a combination of Kaempferia Parviflora Extract and at least one component selected from the group consisting of Zingiber officinale, Cinnamomum cassia, medicinal ginseng, and vitamin B. can promote adipogenesis by stimulating PPAR gamma activity but does not teach that Kaempferia Parviflora Extract is capable of acting on its own or that it can enable adipogenesis in subjects with hypertrophic adipocytes without activating PPAR gamma.

[0058] Here the inventors also disclose new compositions and methods of treatment where the concentration of adipogenic agent is significantly less than previously exemplified compositions. Preferentially, the ratio of the weight by volume of the adipogenic agent to the weight by volume of the lipolytic agent is less than 2 to 1, and more preferably, less than 0.5 to 1, and more preferably, less than 0.1 to 1.

[0059] Compositions with a lower ratio of adipogenic to lipolytic agent weight by volume are capable of improving the health of dermal white adipose tissue and consequently the health of the skin. Specifically, such compositions can reduce the crepe- like structure of the skin.Attorney Docket No.115061.PH590WO

[0060] U.S. Patent No.6,361,806 B1 teaches that a topical composition that contains cottonseed oil is capable of increasing subcutaneous fat tissue mass. Cottonseed oil contains a preponderance of adipogenic and lipogenic agents (linoleic acid, oleic acid, palmitic acid) to a small concentration of lipolytic agent (palmitoleic acid). U.S. Patent No. 6,361,806 B1 does not teach that cottonseed oil is capable of increasing the number of adipocytes or decreasing the size of adipocytes in a mixed population of pre-adipocytes and adipocytes as would be found in the skin of subjects. One publication estimated the ratio of adipogenic / lipogenic agents to lipolytic agents as greater than 150:1. (See e.g., Yang A, Qi M, Wang X, Wang S, Sun L, Qi D, Zhu L, Duan Y, Gao X, Ali Rajput S, Zhang N., Refined cottonseed oil as a replacement for soybean oil in broiler diet. Food Sci Nutr. 2019 Feb 5;7(3):1027-1034).

[0061] The inventors also now disclose a new delivery approach that utilizes a combination of a liposome carrier and a silicone elastomer super structure to deliver the active ingredients to the dermal white adipose tissue. The use of liposome to deliver active ingredients to the skin is known. Liposomes aide the delivery of active ingredients in a number of ways as depicted in figure 5. A silicone elastomer superstructure suspends the liposome on the top of the skin, allowing the actives more time to enter the pore structures of the skin where they can access the dermal white adipose tissue more effectively. This is represented by element “E” in Figure 5. A silicone elastomer superstructure refers to a composition created by combining silicone polymers of different molecular weights.

[0062] The theory of combining a liposome carrier and a silicone elastomer superstructure to target the dermal white adipose tissue is known (See e.g., US Patent No. 11,103,455 B2). The practice of creating such a combination is not trivial as there are manyAttorney Docket No.115061.PH590WO varieties of silicone elastomers that can be employed in combination with varied concentrations and types of liposome constituents, as well as various other components used routinely in topical preparations (e.g., solvents, emulsifiers, thickening agents). Extensive testing and experimentation were required to identify a working combination.

[0063] The useability of a topical formulation is impacted by the amount and nature of the silicone components, the ratio of silicone components to the liposome components, as well as the ratio of the silicone and liposome components to the emulsifier components, among other factors. To attain a composition that has desirable useability characteristics (e.g., spreadable, fast absorbing, minimal oily residue), we tested a wide variety of ingredients to identify an optimal silicone / liposome superstructure.

[0064] The inventors disclose a silicone / liposome superstructure with a target viscosity of at least 8,000 centipoise (cP) more preferentially, at least 10,000 cP to a maximum of 15,000 cP.

[0065] Useability is achieved by including total silicone components (e.g., dimethicone, dimethicone crosspolymers, dimethiconol and vinyl dimethicone crosspolymers) at a minimum of 6 percent of the weight by volume of the topical composition, more preferably at range of 8 to 12 percent of the weight by volume of the topical composition, most preferably at a range of 8 to 9.5 percent of the weight by volume of the topical composition.

[0066] Useability is further achieved by including a concentration of higher viscosity crosspolymer components at 8% or more of the total silicone components and more preferentially at a minimum concentration of 10% of the total silicone components.

[0067] Useability is further achieved by including a concentration of higher viscosityAttorney Docket No.115061.PH590WO dimethiconol components at 0.5% or more of the total silicone components and more preferentially at a minimum concentration of 1% of the total silicone components.

[0068] Liposomes are highly viscous components and so the ratio of silicone components to liposome components is also important in determining product useability. The inventors disclose that the optimal ratio of silicone components to liposome components is at least 2:1 and more preferentially at least 2.5: 1 and more preferentially still at least 3.0: 1.

[0069] Emulsifying agents, as distinct from thickening agents, are also critical to determining product useability. The inventors disclose that an optimal ratio of silicone components to emulsifying agents is at least 2:1 and more preferentially at least 2.5:1 and more preferentially still at least 3.0:1. The inventors use the term emulsifier to describe agents whose primarily role is to suspend, solubilize and emulsify and / or incorporate the lipophilic ingredients into the main blend. s. For clarification, in the exemplified composition (table 1) sorbitan olivate and cetearyl olivate are emulsifying agents, while carbomer is a thickening agent.

[0070] Thickening agents like carbomer are typically added to the water phase in the production process, are neutralized to a pH of 7.0 and then blended without delay. The inventors found surprisingly that the saturation level of the carbomer significantly affected the viscosity of the silicone-liposome formulation. The inventors disclose that keeping the carbomer in the water phase for at least 12 hours and more effectively for at least 24 hours improved the useability properties of the silicone-liposome formulation. The inventors also found surprisingly that neutralizing the carbomer blend to a pH less than 7 resulted in superior useability characteristics.Attorney Docket No.115061.PH590WO

[0071] Table A exemplifies a composition where a ratio of adipogenic agents to lipolytic agents is less than 2 to 1. Table A: Percentage weight by Ingredients volume Ingredient type Water 65 to 80 Solvent Dimethicone 5 to 10 Silicon component Dimethicone cross polymer 0.1 to 3 Silicon component Vinyl dimethicone 0.1 to 3 crosspolymer Silicon component Dimethiconol 0.0001 to 1 Silicon component Solvent, Preservative Propanediol 1 to 10 enhancer Glycerin 1 to 10 Emollient Butylene Glycol 0.1 to 5 Naturally derived solvent Cetearyl Olivate 0.5 to 5 Emulsifier Lecithin 1 to 5 Liposome component Sorbitan olivate 1 to 5 Emulsifier Macadamia ternifolia seed 1,0.1 to 5 oil2* Active Oleic acid20.1 to 5ActiveKaempferia parviflora extract1.001 to 5ActiveArtemesia capillaris flower 1 0.1 to 5 extract Active Evodia fruit extract10.1 to 5ActivePolyacrylamide 0.1 to 2 Neutralized polymer C13-14 Isoalkane 0.1 to 2 Stabilizer Laureth-7 0.1 to 2 Emulsifier Polysorbate 20 0.001 to 1 Oil in water emulsifier Beeswax 0.1 to 1 Thickening agent Pentylene glycol 0.1 to 1.5 Preservative Phenethyl alcohol 0.01 to 1 Preservative C12-14 Alketh -12 .001 to 1 Emulsifier Propylene Glycol 0.01 to 1 Solvent Ethylhexylglycerin 0.001 to 1 Preservative Sodium Benzoate 0.001 to 1 Preservative Cucumis Sativus Seed Oil 0.001 to 1 Active Carbomer 0.1 to 1 Thickening agent1denotes lipolytic agent2denotes adipogenic agent*Macadamia ternifolia seed oil contains approximately 60% adipogenic agents and 20%Attorney Docket No.115061.PH590WO lipolytic agents (e.g., palmitoleic acid)

[0072] Figures 1 and 2 show the results of an experiment where an extract of Kaempferia Parviflora, was added to the 3D culture of human preadipocytes prior to their differentiation into mature adipocytes. The addition of Kaempferia Parviflora Extract (KPE) prolonged the proliferation phase for the pre-adipocytes but did not inhibit the maturation of the adipocytes. This resulted in a state where the number of adipocytes increased, and the average size of the adipocytes decreased. Note that KPE is the only treatment paradigm that increased the number of adipocytes relative to control after 7 days of treatment. This is surprising and not expected given our data showing that KPE suppresses PPAR gamma signaling. After 14 days of treatment, the combination of KPE and safflower seed oil (KPSS) could also increase the number of adipocytes relative to control.

[0073] In the pre-differentiation setting where initially there are only immature pre- adipocytes, the combination of safflower seed oil and KPE (KPSS) constrained the increase in adipocyte size through day 7. In the population of cells treated with the KPSS combination, the distribution of cell size is narrower than in the population of control treated cells. By day 14, the lipogenic effects of the safflower seed oil, even in combination with KPE, is noted. The distribution of adipocyte size in the KPSS combination treatment outflanks the control treatment, with the largest cells of the combination-treated group more than double the size of the control-treated group. The mean adipocyte size in the control treated population is 5.49 micrometers at day 14 but 8.98 micrometers in the KPSS-treated population. Hypertrophic adipocytes start to form at day 10 in the culture process. The fact that KPSS treatment cannot contain mature adipocyte size at day 14 reflects the presence and impact of hypertrophic adipocytes.Attorney Docket No.115061.PH590WO

[0074] It is also notable that despite the reduction in cell size seen with KPE treatment, that the size of the KPE-treated spheroids is the same as the KPSS treated spheroids, with both being significantly larger than the control and SSO treated spheroids at day 14. This shows that in a population of generally small sized mature adipocytes that KPE treatment can increase fat tissue volume something that has not been shown in vivo prior.

[0075] In the pre-differentiation setting it is important to note that the average cell size increases significantly in the control population, from a mean of less than 5.07 micrometers at day 7 to more than 5.49 micrometers at day 14. This reflects the appearance of hypertrophic cells adipocytes starting at day 10 that add a suppressive effect on incremental adipogenesis. This will be seen in the post-differentiation experiment where treatment begins only on day 14 and addresses a mixed population of immature and mature cells, with a larger pool of hypertrophic adipocytes.

[0076] Figures 3 and 4 show the results of an experiment where Kaempferia Parviflora Extract was added to the 3D culture of a mix of human pre-adipocytes and mature adipocytes after 14 days of differentiation and maturation. By day 14, the mature adipocyte population has developed a pool of hypertrophic adipocytes which retard new adipogenesis.

[0077] The suppressive effect of hypertrophic adipocytes can be seen by the results of adding SSO from day 14 on. SSO is lipogenic for mature adipocytes and thus increases their size. With the addition of SSO there is a significant drop in the number of total adipocytes at day 21. (See, Figures 5 and 6).

[0078] When KPE was added together with SSO to a population of mature adipocytes including hypertrophic adipocytes, it could prevent the SSO-enhanced hypertrophy and resultant reduction in adipogenesis. KPSS treatment however was not capable of increasingAttorney Docket No.115061.PH590WO adipogenesis relative to the control treatment.

[0079] When KPE was added on its own to a population of mature adipocytes including hypertrophic adipocytes, it could significantly reduce the average size of the mature adipocytes and enabled adipogenesis to the point where the number of cells doubled relative to control.

[0080] Tables B and C below show the changes in transcription of selected genes induced by treatment of either human pre-adipocytes (“pre-differentiation” setting) or a mix of human pre-adipocytes and mature adipocytes (“post-differentiation” setting) with safflower seed oil (SSO) or Kaempferia Parviflora Extract (KPRE) relative to treatment with control medium. It is notable that SSO and KPE diverge far more in the pre-differentiation setting than in the post-differentiation setting. In the post differentiation setting, where the presence and impact of hypertrophic adipocytes can be seen, it is important to note that SSO and KPE have strongly differential effects on genes which play major roles in fat metabolism. For example, KPE down regulates and SSO upregulates RBP4 (retinol binding protein 4). RBP4 is an adipokine that promotes low grade inflammation and could be significant in suppressing adipogenesis. Also, KPE down regulates and SSO upregulates LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1). LRP1 assists with lipid uptake and could be significant in enhancing lipogenesis. Also, KPE upregulates and SSO downregulates ADIPOQ (Adiponectin). ADIPOQ is an important anti-inflammatory adipokine, and its upregulation by KPE can be expected to promote adipogenesis. Surprisingly, KPE downregulates and SSO upregulates AQP7 (Aquaporin 7). AQP7 limits adipocyte hypertrophy. The difference in genes regulation induced by KPE in the post-differentiation setting where hypertrophic adipocytes are present clarifies that the two settings are veryAttorney Docket No.115061.PH590WO different and underscores that it would not be possible to predict the activity of KPE in a post-differentiation setting based on data collected in the pre-differentiation setting, especially if a pre-differentiation setting did not allow for the development of hypertrophic adipocytes.

[0081] Table B: Pre-differenPaPon seQng SSO KRPE TRARG1 226% 108% ANGPTL4 217% -167% GHITM 207% -276% LRP1 188% 100% LIPE 168% -286% AQP7 162% -293% RBP4 147% -293% G0S2 144% -299% CAT 142% -289% ADIPOQ 141% -293% INSIG1 138% -293% UCP2 138% -133% LPL 134% -292% GPD1 132% -299% PCK1 132% -298% MRAP 126% -300% PFKFB3 124% -182% PLIN1 121% -295% PLAAT3 119% -300% VLDLR 118% 291% CIDEC 114% -300% CD36 110% -298% FABP4 109% -300% PC 102% 192% SERPINE1 -102% 225% DBI -109% -289% SLC27A1 -112% 208% SORBS1 -113% 153% AACS -117% 133% HP -159% -273% NR1D1 -205% 225%Attorney Docket No.115061.PH590WO

[0082] Table C: Post-differenPaPon seQng SSO KRPE VLDLR 150% 187% PFKFB3 149% 150% PC 144% 146% UCP2 138% 139% SORBS1 126% 100% LPL 126% 143% CD36 124% 125% AACS 120% 128% CIDEC 118% 110% SLC27A1 112% 111% GHITM 111% 119% PCK1 111% 117% ANGPTL4 108% 137% GPD1 107% 101% AQP7 105% -106% MRAP 105% 111% LRP1 104% -110% CAT 101% 108% RBP4 101% -115% INSIG1 100% 105% ADIPOQ -100% 110% PLAAT3 -102% -104% LIPE -103% -105% SERPINE1 -106% -153% PLIN1 -107% -109% DBI -107% -144% HP -108% -102% FABP4 -109% -116% TRARG1 -113% -117% NR1D1 -118% -145% G0S2 -124% -173%

[0083] Figure 7 shows the differential effects of KPE and SSO on transcription of PPAR gamma, the master regulator of adipogenesis. In the pre-differentiation setting, measured at both day 7 and day 14, KPE downregulates PPAR gamma transcription. This activity of KPE is fundamentally different from what was shown by Horikawa et al. in theAttorney Docket No.115061.PH590WO context of immature murine fibroblast cells. It underscores that our findings could not be predicted by what was known about KPE and that the adipogenic activity observed by Horikawa et al. in pre-adipocytes cultured through day 8 and not including any hypertrophic adipocytes could not inform on what activity KPE might have in a setting where hypertrophic adipocytes were present.

[0084] Figures 8A and 8B show the before and after images of the arm skin of a subject who applied the composition of Table A. The subject had a BMI in excess of 25 and had loose skin with a crepe-like appearance and deep-set wrinkles. The application of the test composition produced a significant reduction in the looseness of the skin, a reduction in the crepe-like structure of the skin and a significant improvement in the deep-set wrinkles. The improvement in deep-set wrinkles is consistent with an underlying improvement in collagen and elastin production caused by reducing dermal adipocyte size.

[0085] Figures 9A and 9B show the before and after images of the arm skin of a second subject who applied the composition of Table A. The subject had a BMI in excess of 25 and similarly to the subject photographed in Figures 6A and 6B had loose skin with a crepe-like appearance and deep-set wrinkles. The application of the test composition produced a significant reduction in the looseness of the skin, a reduction in the crepe-like structure of the skin and a significant improvement in the deep-set wrinkles. The improvement in deep-set wrinkles is consistent with an underlying improvement in collagen and elastin production caused by reducing dermal adipocyte size.

[0086] Figures 10 and 10B show the before and after images of the right hemiface of a subject who applied the composition of Table A. The subject had a BMI less than 25 and a pronounced volume deficit in the mid cheek. She had used a composition with a high ratioAttorney Docket No.115061.PH590WO of SS0 to KPE (greater than 5 to 1) for several months and had not achieved any volume changes. Within a month of using the composition of Table A, the subject saw a significant restoration of volume selectively in the area where the volume deficit had been. The fact that this subject achieved a selective restoration of volume is consistent with the fact that KPE can increase adipocyte number in the context of localized dermal adipocyte hypertrophy.

[0087] Figures 11A and 11B show the before and after images of the left hemiface of a subject who applied the composition of Table A. The subject had a BMI less than 25 and a pronounced volume deficit in the mid-cheek. She had used a composition with a high ratio of SS0 to KPE (greater than 5 to 1) for several months and had not achieved any volume changes. Within a month of using the composition of Table A, the subject saw a significant restoration of volume selectively in the area where the volume deficit had been. The fact that this subject achieved a selective restoration of volume is consistent with the fact that KPE can increase adipocyte number in the context of localized dermal adipocyte hypertrophy.

[0088] Figures 12A and 12B show the before and after images of the right hemiface of a subject who applied the composition of Table A. The subject had a BMI less than 25 and a pronounced volume deficit under the eye and overt loose skin encompassing the nasolabial fold. She had used a composition with a high ratio of SS0 to KPE (greater than 5 to 1) for more than 2 months and had not achieved any volume changes. Within a month of using the composition of Table A, the subject saw a significant restoration of volume selectively in the area where the volume deficit had been and a visible strengthening of the skin encompassing the nasolabial fold. The fact that this subject achieved both a restorationAttorney Docket No.115061.PH590WO of volume and a strengthening of the skin comprising the nasolabial fold is consistent with the dual actions of KPE of reducing cell size and increasing cell number in the context of dermal adipocyte hypertrophy.

[0089] Figures 13A and 13B show the before and after images of the left hemiface of a subject who applied the composition of Table A. The subject had a BMI less than 25 and pronounced volume deficits under the eye and in the mid-cheek. She had used a composition with a high ratio of SS0 to KPE (greater than 5 to 1) for more than 2 months and had not achieved any improvements in the volume. Within a month of using the composition of Table A, the subject saw a significant restoration of volume selectively in the two areas where the volume deficit had been. The fact that this subject achieved a selective restoration of volume is consistent with the fact that KPE can increase adipocyte number in the context of localized dermal adipocyte hypertrophy.

[0090] Figure 14 shows the results of a clinical study where subjects with mild, moderate and severe cellulite were supplied with the composition of Table A and were asked to apply it two times per day for three months. The subjects took photographs of areas of cellulite. Baseline and 3-month photographs were cropped to show the same areas of cellulite. The photos were put in random order and assigned random codes. Four independent blinded unpaid evaluators assessed the photographs for cellulite severity, noting which of the two photos if any showed a greater cellulite severity. For the primary endpoint, the evaluators rated all 32 sets of images. At least three evaluators correctly identified the “before” image as having greater cellulite severity 75% of the time with 95% confidence intervals.

[0091] Figure 15 shows the results of a clinical study where subjects with mild,Attorney Docket No.115061.PH590WO moderate and severe cellulite were supplied with the composition of Table A and were asked to apply it two times per day for three months. The subjects took photographs of areas of cellulite. Baseline and 3-month photographs were cropped to show the same areas of cellulite. The photos were put in random order and assigned random codes. Four independent blinded unpaid evaluators assessed the photographs for cellulite severity, noting which of the two photos if any showed a greater cellulite severity. For the secondary endpoint, the evaluators rated only the sets of images where the baseline cellulite severity was either moderate or severe. At least three evaluators correctly identified the “before” image as having greater cellulite severity 100% of the time with 95% confidence intervals.

[0092] Figures 16A and 16B show the before and after photo close up images, respectively, of an area of cellulite on the leg of a subject who used a composition of Table A for 3 months as part of the clinical study. Relative to the before image, the after image shows a significant reduction in the pitting and dimpling phenotypes that are characteristic of cellulite, and which derive from low grade inflammation generated by hypertrophic fat cells.

[0093] Figures 17A and 17B show the before and after photo close up images, respectively, of an area of cellulite on the leg of a different subject who used a composition of Table A for 3 months as part of the clinical study. Relative to the before image, the after image shows a significant reduction in the pitting and dimpling phenotypes that are characteristic of cellulite, and which derive from low grade inflammation generated by hypertrophic fat cells.

[0094] Figures 18A and 18B show the before and after photo close up images, respectively, of an area of cellulite on the other leg of the same subject as is depicted inAttorney Docket No.115061.PH590WO Figures 15A and 15B who used a composition of Table A for 3 months as part of the clinical study. Relative to the before image, the after image shows a significant reduction in the pitting and dimpling phenotypes that are characteristic of cellulite, and which derive from low grade inflammation generated by hypertrophic fat cells.

[0095] Figures 19A and 19B show the before and after photo close up images, respectively, of an area of cellulite on the leg of a different subject who used a composition of Table A for 3 months as part of the clinical study. Relative to the before image, the after image shows a significant reduction in the pitting and dimpling phenotypes that are characteristic of cellulite, and which derive from low grade inflammation generated by hypertrophic fat cells.

[0096] Figure 20 shows how a liposome can direct the delivery of active agents to various parts of the skin compared with unencapsulated delivery. In FIG.18, A denotes non- encapsulated ingredient penetration. B denotes non-encapsulated ingredient penetration when a penetrant is added. C denotes transcellular penetration (e.g., fusion of liposome with skin and diffusion of ingredients through skin layers). D denotes intercellular penetration (e.g., diffusion of liposome through skin layers). E denotes transfollicular penetration (e.g., penetration of liposome through hair follicle). The formulations described in Examples 1-5 foster effective delivery to the dermal adipocytes via the transfollicular path E. By suspending the liposomes containing the active agents in a silicone elastomer super structure, the transfollicular pathway E is advantaged over the transcellular and intercellular pathways C and D, which would be far less efficient paths for reaching the dermal fat cells which reside at the bottom third of the hair follicle..

[0097] Figure 21 shows the absorption kinetics of a topical composition of table 1Attorney Docket No.115061.PH590WO compared to a topical composition without a delivery system comprised of a liposome and silicone elastomer super structure. The silicone elastomer super structure effectively funnels the liposomes to the hair follicles and enables very rapid absorption.

[0098] It should be understood by a skilled artisan that, while skin care systems will be discussed herein, regimes using the compositions of the present disclosure can be used for various other daily regimens comprising steps to cleanse, treat, moisturize, and protect the skin. It is understood that skin care regimens can combine all of these steps, some of these steps, or have multiple iterations of the same steps so as to provide desired benefits to the skin.

[0099] Other objects, advantages and novel features of the present invention are apparent from the foregoing detailed description of the one or more preferred embodiments, examples and aspects. It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present invention is intended to encompass variations and equivalents of these examples. EXAMPLES [000100] All example formulations were prepared using the following silicon elastomer compositions: [000101] Composition A [000102] CHT Beausil Gel 8014: dimethicone (and) dimethicone crosspolymer (approximately 9 percent), a clear gel with a viscosity of 400,000 cP. This blend has high molecular weight specialty silicone elastomer carried in the dimethicone. [000103] Composition B [000104] CHT Beausil Gum 8547: dimethicone (and) dimethiconol, a clear liquid with aAttorney Docket No.115061.PH590WO viscosity of 5,000 cP. This blend contains approximately 15% of ultra-high viscosity dimethiconol in the dimethicone fluid. [000105] Composition C [000106] CHT Beausil Bead 81000 EM: dimethicone / vinyldimethicone crosspolymer (approximately 68%), a white emulsion with a viscosity of 5,000 cP (C). [000107] Example 1 [000108] Example 1 formulation was prepared by combing Compositions A, B, and C, at 2.83% each to obtain provisional starting point in order to assess final viscosity. Resultant product viscosity was at 2060 cP. Observation: Unacceptable product characteristics. [000109] Example 2 [000110] Example 2 formulation was prepared by combining Composition A at 3.85%, Composition B at 2.85%, Composition C at 1.8%. Resultant product viscosity was at 4130 cP. Observation: Unacceptable product characteristics. [000111] Example 3 [000112] Example 3 formulation was prepared by combining Composition A at 4.85%, Composition B at 1.85%, Composition C at 1.8%. Resultant product viscosity was at 6780 cP. Observation: Unacceptable product characteristics. [000113] Example 4 [000114] Example 4 formulation was prepared by combining Composition A at 6.0%, Composition B at 0.7%, Composition C at 1.8%. Resultant product viscosity was at 8635 cP. Observation: Acceptable product characteristics. [000115] Example 5 [000116] Example 5 formulation was prepared by combining Composition A at 6.90%,Attorney Docket No.115061.PH590WO Composition B at 0.80%, Composition C at 0.80%. Resultant product viscosity was at 12,440 cP. Observation: Superior product characteristics. [000117] Methods [000118] Description of the methods used to conduct experiments exemplified in Figures 1A, 1B, 2A, and 2B. [000119] 3D human adipocyte spheroid culture [000120] Primary human subcutaneous SVF cells from a healthy 60-year-old male donor with a BMI of 30 (obtained from Lonza Bioscience, Switzerland) were initially seeded in DMEM / F-12 GlutaMAX containing 10% FBS and 1% penicillin-streptomycin, in culture flasks. After 48 hours, the medium was replaced, and the cells were incubated at 37 °C with 5% CO2 until they reached approximately 70% confluency. The cells were then trypsinized, counted and transferred to 96-well ultra-low attachment microplates (Corning, Costar no. CLS7007) at a density of 5,000 cells per well and centrifuged at 150 g for 2 minutes. Four days post-seeding into spheroids, the culture medium was switched to a serum-free differentiation medium consisting of William’s E medium supplemented with 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL insulin, 10 μg / mL transferrin, 6.7 ng / mL sodium selenite, and 100 nM dexamethasone, 500 μM IBMX, 10 nM hydrocortisone, 2 nM 3,3′,5- Triiodo-L-thyronine, 10 μM rosiglitazone, 33 μM biotin, and 17 μM pantothenic acid. The medium was refreshed after 48 hours and then every 3–4 days over a 17-day differentiation period. After differentiation, cells were switched to a serum-free maintenance medium (William’s E medium supplemented with 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL insulin, 10 μg / mL transferrin, 6.7 ng / mL sodium selenite, and 100 nM dexamethasone). Supplements including safflower seed oil (SSO) and KaempferiaAttorney Docket No.115061.PH590WO parviflora root extract (KPRE) were added to the medium either during differentiation (Figure 1A and 1B) or post-differentiation (Figure 2A and 2B) according to Table 1. The SSO was conjugated with 10% bovine serum albumin (BSA) at a molar ratio of 1:5 and incubated for 2 hours at 40 °C before being added to the culture as specified. [000121] Brightfield and fluorescence confocal imaging [000122] Total spheroid area was analyzed from light microscopy pictures and measured using the Fiji ImageJ software. For fluorescent microscopic analysis, spheroids were washed in Dulbecco’s phosphate buffered saline (DPBS, Gibco) after harvesting and subsequently fixed for 30 min in 10% formalin (Sigma). Fixed spheroids were stained overnight protected from light in 1:2000 Bodipy-FL (1 mg / ml, Invitrogen) and 1:500 DAPI (final concentration 2μg / ml, Thermo Scientific) diluted in PBS-0.1% Tween. After staining, the spheroids were washed twice for five minutes with DPBS and mounted on microscopy slides using an iSpacer (SunJin LabCo., Taiwan) in mounting medium. Imaging was performed on a Zeiss LSM 880 confocal microscope using consistent lighting settings for all samples within the experiment. Measurements of lipid droplet diameter and nuclei count were carried out using the integrated tools in Fiji ImageJ software on stacked images. Data analysis was subsequently conducted using GraphPad Prism. [000123] Table 1: Medium additives Cocktail Compound (s) Concentration (s) oleic acid 160 µM SSO linoleic acid 160 µM oleic acid 160 µM SSO / KPRE linoleic acid 160 µM KPRE (5%) 1:100 (v / v) Kaempferia parviflora root 1:100 (v / v) KPRE extract (5%) 1:1000 (v / v) Placebo - 1:100 (v / v)Attorney Docket No.115061.PH590WO [000124] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Claims

Attorney Docket No.115061.PH590WO CLAIMS What is claimed is:

1. A topical composition comprising kaempferia parviflora extract that can visibly strengthen the skin and restore volume in subjects having a BMI in excess of 20, wherein the composition contains a ratio of adipogenic to lipolytic ingredients by weight of less than 2:

1.

2. A topical composition comprising kaempferia parviflora extract that can visibly strengthen the skin and restore volume in subjects who have achieved rapid weight loss, wherein the composition contains a ratio of adipogenic to lipolytic ingredients by weight of less than 2:

1.

3. A topical composition comprising kaempferia parviflora extract that can visibly strengthen the skin in subjects who have regional adipocyte hypertrophy manifesting as cellulite, wherein the composition contains a ratio of adipogenic to lipolytic ingredients by weight of less than 2:

1.

4. A method of reducing skin weakness or skin sag or the crepe-like structure of the skin of a subject by administering a topical composition of claim 1 to the subject.

5. A method of increasing the number of dermal adipocytes and decreasing the size of dermal adipocytes of a subject, the method comprising applying topically to the subject a composition of claim 1.

6. A method of improving the metabolic health of a subject, the method comprising applying a topical composition of claim 1 to the subject.

7. A method of increasing the number of dermal adipocytes and decreasing theAttorney Docket No.115061.PH590WO average size of dermal adipocytes of a subject, the method comprising applying a topical composition comprising a lipolytic agent which effects lipolysis without inhibiting adipogenesis to the subject with dermal adipocytes of excessive size, as evidenced by a BMI in excess of 20 or alternatively as evidenced by visibly weak or saggy skin, wherein the composition is delivered effectively to the subject’s dermal white adipose tissue.

8. A method of increasing the number of dermal adipocytes and decreasing the average size of dermal adipocytes of a subject, the method comprising applying a topical composition comprising an agent that increases pre-adipocyte proliferation without also inhibiting pre-adipocyte maturation to the subject, wherein the composition is delivered effectively to the subject’s dermal white adipose tissue.

9. The method of claim 8, wherein the agent is an extract of Kaempferia Parviflora extract.

10. A method of improving the metabolic health of an individual, the method comprising practicing the method of claim 3 combined with treating the individual with a weight loss agent comprising a GLP-1 antagonist.

11. A method of improving the metabolic health of an individual, the method comprising practicing the method of claim 6 combined with treating the individual with a weight loss agent comprising a GLP-1 antagonist.

12. A topical composition effective for delivering agents to a subject’s skin, wherein the delivering agent comprises a silicone elastomer superstructure selected from the group consisting of dimethicone, silicone crosspolymers, and dimethiconol, wherein the viscosity of the composition is at least 8,000 centipoise and less than 15,000Attorney Docket No.115061.PH590WO centipoise.

13. The topical composition of claim 12, wherein the silicon elastomer superstructure comprises at least 6 percent of the composition weight by volume, and wherein at least 8 percent of the silicone elastomer superstructure components includes crosspolymers or dimethiconol.

14. The topical composition of claim 12, wherein the agents to be delivered are carried in liposomes.

15. The topical composition of claim 12, wherein the agents to be delivered target the subject’s dermal white adipose tissue.

16. The topical composition of claim 12, wherein the agents to be delivered are fatty acids or oils.

17. The topical composition of claim 12, wherein the viscosity of the composition is at least 10,000 centipoise and no more than 15,000 centipoise.

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