Novel, long-lasting compositions for modulating gene expression in human skin and methods for their preparation

Abstract

Formulations containing biologically produced, high-purity R-mevalonolactone are provided for regulating gene expression and metabolism related to epidermal turnover, hyaluronic acid synthesis, barrier function, anti-aging elasticity, extracellular matrix degradation, inflammation, stress, and pigmentation in human skin. Biologically produced, high-purity R-mevalonolactone can be used as an ingredient in pharmaceuticals, foods, and cosmetics and can be applied topically or ingested for the regulation of gene expression and metabolism.

Description

[Technical field]

[0001] The present invention generally relates to a formulation containing mevalonic acid derivatives. More specifically, the present invention generally relates to a healthcare formulation containing mevalonolactone with high purity, low odor, and low coloring, which can regulate the expression of various epithelial tissue-related genes. [Background technology]

[0002] Like skin, human epithelial cells begin to age from the moment of birth, both as a result of intrinsic biological aging and external factors such as exposure to toxins and UV radiation. This process accelerates after puberty, with visible effects often appearing after the age of 20 and continuing until death. Visible effects of skin aging include, for example, the development of fine wrinkles, dryness, loss of elasticity (sagging), and discoloration or a "mottled" appearance.

[0003] At the molecular level, these effects of skin aging are characterized by the progressive breakdown of the epidermal extracellular matrix ("ECM"). The ECM is the largest component of human skin and is composed primarily of fibrous structural proteins such as collagen and elastin, which give the network its strength and elasticity. Glycosaminoglycans ("GAGs"), such as hyaluronic acid (also known as proteoglycans), keep connective tissue moist and promote the proliferation of new epithelial cells, among other important cellular functions.

[0004] Exposure to external factors that lead to skin aging (extrinsic aging) can be at least partially mitigated by lifestyle choices, including limiting skin exposure to sunlight or using sunscreens, making informed dietary choices, and quitting smoking.

[0005] To maintain strong, healthy skin, these ECM components must be continually produced by epithelial cells to counteract the breakdown of natural tissue and damage caused by oxygen radicals and UV rays. However, production of ECM components naturally declines after puberty, with production of collagen, elastin, and hyaluronic acid declining from the age of 20. As the desire for healthy, youthful skin has become widespread, a number of products have been developed that aim to increase levels of these ECM components.

[0006] Despite the large market for such products, the effectiveness of oral collagen supplements has been questioned by many scientists due to the lack of comprehensive research and the basic anatomical fact that orally ingested proteins are broken down in the stomach. Furthermore, because collagen is the major connective tissue component found throughout the body, such supplements cannot be "targeted" to the skin, especially facial skin.

[0007] Topical compositions containing hyaluronic acid (“HA”) and other high molecular weight ECM components suffer from similar failures of the supplement to reach the target tissue. HA has a molecular weight in the range of 5 million daltons, and the average HA molecule has a diameter of 3,000 nm. In contrast to the average intercellular space of epithelium, which is 15 to 50 nm, such compositions may face a significant challenge in penetrating beyond the skin surface to which they are applied. Other active ingredients, such as retinol derivatives, are known to stimulate cell turnover, but can be complicated to formulate and deliver. They may also cause side effects such as increased skin sensitivity and dryness.

[0008] Considering these issues, it is clear that promoting the synthesis of these large molecular weight ECM components along with cell growth in the target tissue is preferable to exogenous supplementation. Genes associated with collagen, elastin, and hyaluronan synthesis in humans have been well characterized and show age-related decreases in expression, coinciding with the loss of extracellular matrix and the visible progression of skin aging. Genes associated with stimulation of cell growth and differentiation have also been well characterized and show age-related decreases in expression, coinciding with the visible progression of skin aging.

[0009] Furthermore, there is an increasing need for more sustainable solutions that address the challenges highlighted above. Conventional processes for producing ingredients like retinol rely on fossil fuel feedstocks such as coal and petroleum. They also require multiple steps in synthesis, generating significant amounts of waste and carbon emissions per unit of product. It is estimated that the production of 1 kg of retinol results in the equivalent of 40 kg of CO2 greenhouse gas emissions. Furthermore, even when renewable feedstocks are used, the resulting products typically suffer from process inefficiencies, high levels of undesirable by-products, and the use of organic solvents and petrochemicals in processing, negatively impacting the sustainability benefits of using renewable starting materials. Therefore, new, more sustainable ingredients and methods continue to be needed in the production of sustainable healthcare formulations.

[0010] Japanese Patent Application Laid-Open No. 09-221406A discloses a cosmetic skin treatment composition in which 0.001 to 10% by weight of mevalonolactone is blended with amino acids to impart a skin-beautifying effect.

[0011] DE19918761 discloses cosmetic preparations containing mevalonolactone to enhance the barrier function of the skin.

[0012] DE10148266 discloses a cosmetic composition containing mevalonolactone for treating skin damage caused by ultraviolet light.

[0013] JP4854110 discloses the use of mevalonolactone as an active ingredient for anti-inflammatory and anti-allergic effects via inhibition of histamine release.

[0014] It has now been found that health care formulations containing high-purity mevalonolactone can regulate the expression of various epithelial tissue-related genes. The present invention is based on the discovery that mevalonolactone acts to regulate gene expression in a manner not previously known to the inventors' knowledge. Another aspect of the present invention is the previously unknown high-purity, low-odor, low-color mevalonolactone. Properties such as low odor are important for the use of mevalonolactone in health care applications such as sustainable, fragrance-free topical compositions for use on the face. Summary of the Invention [Problem to be solved by the invention]

[0015] One or more embodiments generally relate to a gene regulation formulation for regulating the expression of one or more genes. Generally, the formulation comprises mevalonolactone, wherein the mevalonolactone exhibits an APHA color of less than 500 units / million ppm of platinum-cobalt in water as measured by ASTM D1209. Additionally, the mevalonolactone contains less than 5% by weight of fermentation by-products, malodor-causing agents, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the mevalonolactone.

[0016] One or more embodiments generally relate to a method of forming a dermatological formulation. In general, the method includes the steps of: (a) providing an aqueous solution of mevalonolactone, the aqueous solution exhibiting an APHA color of less than 500 units / million ppm of platinum-cobalt in water as measured by ASTM D1209, and containing less than 5% by weight of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the aqueous solution; and (b) combining the aqueous solution with at least one additive to form a dermatological formulation.

[0017] 1 One or more embodiments generally relate to a purified mevalonolactone solution that includes: (a) mevalonolactone, where at least 95 weight percent of the mevalonolactone comprises at least 95 weight percent R-mevalonolactone, based on the total weight of the mevalonolactone; and (b) water. Further, the solution exhibits an APHA color of less than 500 units / million ppm of platinum-cobalt on water as measured by ASTM D1209, and contains less than 5 weight percent of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the solution. [Brief description of the drawings]

[0018] Embodiments of the invention are described herein with reference to the following drawings:

[0019] [Figure 1] FIG. 1 shows an HPLC chromatogram from the end of the fermentation process described herein.

[0020] [Diagram 2] FIG. 2 is a graph showing gene expression levels (modulation) of genes related to anti-aging elasticity when the model was treated with MVL solution.

[0021] [Diagram 3]FIG. 3 is a graph showing the change (modulation) in gene expression levels for genes related to moisturizing elasticity when the model was treated with MVL solution.

[0022] [Figure 4] FIG. 4 is a graph showing the change (modulation) in gene expression levels for genes associated with epidermal turnover when the model was treated with MVL solution.

[0023] [Diagram 5] FIG. 5 is a graph showing the change (modulation) in gene expression levels for genes related to barrier function when the model was treated with MVL solution.

[0024] [Figure 6] FIG. 6 is a graph showing the change (modulation) in gene expression levels for genes associated with extracellular matrix degradation when the model was treated with MVL solution.

[0025] [Figure 7] FIG. 7 is a graph showing the change (modulation) in gene expression levels of genes associated with skin inflammation when the model was treated with MVL solution.

[0026] [Figure 8] FIG. 8 is a graph showing the change (modulation) in gene expression levels for genes related to skin pigmentation when the model was treated with MVL solution.

[0027] [Figure 9] FIG. 9 is a graph comparing the effect on skin hydration levels between MVL solution and placebo.

[0028] [Figure 10] FIG. 10 is a graph comparing the effect on skin roughness between MVL solution and placebo.

[0029] [Figure 11] FIG. 11 is a graph comparing the effect on skin blemishes between MVL solution and placebo.

[0030] [Figure 12] FIG. 12 is a graph comparing the effect on skin redness between MVL solution and placebo.

[0031] [Figure 13] FIG. 13 is a graph showing the effect of MVL concentration on the regulation of cortisol production.

[0032] [Figure 14] FIG. 14 shows two GC-FID chromatograms comparing the presence of impurities before and after the electrodialysis step.

[0033] [Figure 15] FIG. 15 is a graph showing the effect of MVL concentration on regulating hyaluronic acid levels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure relates generally to the manufacture of sustainable formulations comprising biologically produced, high purity mevalonolactone ("MVL"), particularly R-mevalonolactone ("R-MVL"), for regulating gene expression and metabolism related to epidermal turnover, hyaluronic acid synthesis, barrier function, anti-aging elasticity, extracellular matrix degradation, inflammation, stress, energy metabolism, and / or pigmentation in human epithelial cells. As described below, this biologically produced, high purity R-MVL can be used as an ingredient in pharmaceutical, food, and / or cosmetic products and can be applied topically or ingested for the regulation of gene expression and metabolism.

[0035] It should be noted that R-mevalonolactone can exist in equilibrium with its open-ring form, R-mevalonic acid, and the corresponding anionic form, R-mevalonate, depending on pH and water concentration. As used herein, these terms may be used interchangeably and collectively as R-mevalonolactone or R-MVL. Similarly, the term "mevalonolactone" or MVL may also be used interchangeably with its open-ring form, mevalonic acid, and the corresponding anionic form, mevalonate.

[0036] As described in more detail below, a process has been developed for the sustainable biological production of high purity R-mevalonolactone from bio-based feedstocks (i.e., MVL products). It has been discovered that the R-MVL products can be used to manufacture and provide topical (i.e., skin) compositions that can increase the expression of various genes or gene families by at least 1, 5, 10, 20, 30, 40, 50, 75, or 100% to enhance the local synthesis of key ECM components such as collagen, elastin, and hyaluronic acid.

[0037] [Gene Regulatory Agents] It has been found that sustainable formulations containing MVL products, such as formulations containing 0.05-10% by weight of MVL products, exhibit potent broad-spectrum modulation of gene expression in human epithelial skin cells. Specifically, in certain embodiments, compositions containing 0.1-1.0% by weight of MVL products have been found to modulate gene expression in human epithelial cells by increasing expression of genes associated with epithelial turnover, hyaluronic acid synthesis, barrier function, and anti-aging elasticity by at least 10%, and decreasing expression of genes associated with ECM degradation, inflammation, and pigmentation by at least 10%. These gene-modulating formulations and the MVL formulations used to make the formulations are described in more detail below.

[0038] It should be noted that any of the following properties and ranges are relevant: (i) a process for producing an MVL product, (ii) a process for producing a gene regulatory formulation containing the MVL product, (iii) the MVL product itself, and (iv) the formulation itself, even if listed separately, are not mutually exclusive and may be combined in any combination so long as such combination does not create a contradiction between any of the properties or ranges.

[0039] In one or more embodiments, the MVL product may be solvent-free, optically pure, and highly pure MVL. As described in more detail below, these characteristics and properties derive in large part from the processes and materials used to make the MVL.

[0040] In one or more embodiments, the gene regulatory formulations of the present disclosure may comprise at least 0.01, 0.1, or 0.05 weight percent of a MVL product, such as solvent-free, optically pure high purity MVL, based on the total weight of the formulation. Generally, in one or more embodiments, the gene regulatory formulations will comprise at least 0.01, 0.05, 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 weight percent, and / or less than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 weight percent of MVL, such as mevalonolactone, mevalonic acid, and / or salts of mevalonic acid, based on the total weight of the formulation. It should be noted that these weight percents of mevalonolactone, mevalonic acid, and / or salts of mevalonic acid relate to the colorless aqueous MVL solutions described below and may be produced according to the purification techniques described herein. Additionally, these weight percentages of mevalonolactone, mevalonic acid, and / or salts of mevalonic acid may also be applied to solvent-free, optically pure, high purity MVL.

[0041] In one or more embodiments, the MVL product in a gene regulatory formulation of the present disclosure may be predominantly R-MVL (versus the S version), the biologically active isomer of MVL. In various embodiments, the MVL product in the gene regulatory formulation comprises at least 95, 99, 99.9, 99.99, or 99.999 percent by weight R-MVL, based on the total weight of the MVL product.

[0042] Furthermore, in various embodiments, the gene regulatory formulations of the present disclosure can be substantially free of undesirable by-products. In various embodiments, the gene regulatory formulations produced herein can contain less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 weight percent of one or more undesirable by-products based on the total weight of the formulation. Exemplary undesirable by-products include fermentation by-products (e.g., acetic acid, citric acid, glucose, pyruvic acid, ethanol, pyruvic acid, glycerol, and / or lactic acid), organic solvents (e.g., ethyl acetate, methyl ethyl ketone, dibasic acid esters, dichloromethane, tetrahydrofuran, and / or isopropanol), petrochemicals, color bodies, odor bodies, inorganic salts, and / or organic salts. It should be noted that the weight ranges for the undesirable by-products above can be applied to any individual by-products above or any combination thereof. As used herein, "petrochemical" refers to substances obtained by the refining and processing of petroleum or natural gas.

[0043] Additionally or alternatively, in various embodiments, the gene regulatory formulation of the present disclosure can be substantially free of ceramide, glucosylceramide, galactosylceramide, and / or sphingomyelin.In various embodiments, the gene regulatory formulation produced herein can contain less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 weight percent of ceramide, glucosylceramide, galactosylceramide, and / or sphingomyelin based on the total weight of the formulation.It should be noted that the weight ranges above for the above products can apply to the above individual products or any combination thereof.

[0044] Gene-modulating formulations for topical use containing the long-lasting and high-purity MVL of the present disclosure may be developed in various forms, including but not limited to mists, lotions, emulsions, milks, milky lotions, milky lotions, packs, gels, creams, ointments, granules, powders, foams, etc. In one or more embodiments, such formulations of the present disclosure may contain at least one, two, three, or four of the following additives without departing from the scope of achieving the objectives of the present disclosure: carotenoid-type coloring elements, such as lutein, astaxanthin, and fucoxanthin; vegetable oils, such as olive squalane, rice squalane, rice germ glyceride, jojoba oil, castor oil, safflower oil, olive oil, macadamia nut oil, and sunflower oil; waxes, such as beeswax, fruit wax, and carnauba wax; ester oils include octyldodecyl myristate, cetyl palmitate, isostearyl isostearate, and isopropyl myristate. Lower alcohols such as ethanol. Higher alcohols, such as cetanol, behenyl alcohol, stearyl alcohol, branched long-chain aliphatic alcohols; sterols and their derivatives, such as cholesterol, phytosterol, branched fatty acid cholesterol ester, macadamia nut fatty acid phytosteryl ester; processed oils such as hardened oils. Higher fatty acids: stearic acid, myristic acid, isostearic acid, oleic acid, iso-type long-chain fatty acids, anteiso-long-chain fatty acids, etc. Terpenes such as bactiol, limonene, and hydrogenated bisabolol. Examples of triglycerides include glyceryl tricaprylcaprate, glyceryl 2-ethylhexanoate, triiso-type long-chain fatty acid glyceryl, and glyceryl tripalmitate. Examples of anionic surfactants include sodium cetyl sulfate and N-stearoyl-L-glutamate. Examples of the nonionic surfactant include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene polyhydric alcohol fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyhydric alcohol fatty acid esters.Modified silicones such as polyoxyethylene-modified silicones, polyglycerol fatty acid esters, and sucrose esters; cationic surfactants, for example, tetraalkylammonium salts; amphoteric surfactants such as betaine-based, sulfobetaine-based, and sulfoamino acid-based surfactants; natural surfactants such as lecithin, lysophosphatidylcholine, ceramide, and cerebroside; pigments such as titanium oxide and zinc oxide; color pigments such as iron oxide; tar-based color additives; silicone oils such as dimethylpolysiloxane, methylphenylpolysiloxane, and cyclic silicone; preservatives such as paraben and phenoxyethanol; hydrocarbons such as paraffin and petrolatum; antioxidants such as dibutylhydroxytoluene; inorganic salts such as sodium chloride, magnesium chloride, sodium sulfate, potassium nitrate, sodium sulfate, sodium metasilicate, and calcium chloride. Organic acids and their salts such as sodium citrate, potassium acetate, sodium succinate, sodium aspartate, sodium lactate, dichloroacetic acid, and glycyrrhizic acid; organic amines and their salts such as ethanolamine hydrochloride, ammonium nitrate, arginine hydrochloride, diisopropylamine salt, urea, and decarboxycarnosine; and chelating agents such as edetic acid. Examples of thickening agents include xanthan gum, carboxyvinyl polymer, carrageenan, pectin, alkyl-modified carboxyvinyl polymer, and agar. Examples of neutralizing agents include potassium hydroxide, diisopropanolamine, and triethanolamine. Ultraviolet absorbing agents such as hydroxymethoxybenzophenone sulfonate. Polyhydric alcohols such as dipropylene glycol, malbitol, 1,3-butylene glycol, glycerin, propylene glycol, sorbitol, diglycerin, and raffinose. Vitamins such as various amino acids, ascorbic acid, biotin, and tocopherol. Vitamin derivatives include ascorbic acid sulfate, ascorbic acid phosphate, tocopherol nicotinate, and combinations thereof.In various embodiments, the gene regulatory formulations produced herein contain at least 0.1, 0.5, 1, 2, 3, 4 or 5, and / or less than 99, 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 weight percent of one or more additives based on the total weight of the formulation.

[0045] Additionally or alternatively, in various embodiments, the gene regulatory formulation of the present disclosure may contain bactiol and / or lactic acid. In such embodiments, the gene regulatory formulation produced herein may contain at least 0.1, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, or 3.25, and / or less than 40, 30, 20, 15, 10, or 5 weight percent bactiol and / or lactic acid based on the total weight of the formulation. It has been observed that bactiol, lactic acid, and MVL may have a synergistic effect on the expression of certain genes related to skin care.

[0046] Additionally or alternatively, in various embodiments, the gene regulatory formulations of the present disclosure may comprise bactiol, lactic acid, ceramide, hyaluronic acid, retinal, retinol, vitamin C, peptides, or combinations thereof. In such embodiments, the gene regulatory formulations produced herein may comprise at least 0.1, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, or 3.25, and / or less than 40, 30, 20, 15, 10, or 5 weight percent bactiol, lactic acid, ceramide, hyaluronic acid, retinal, retinol, vitamin C, peptides, or combinations thereof, based on the total weight of the formulation.

[0047] In one or more embodiments, the gene regulatory formulations described herein may comprise a pH of at least 1.5, 2.0, or 2.5, and / or no greater than 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, or 4.9.

[0048] In one or more embodiments, the gene regulatory formulations described herein may comprise a viscosity at 25° C. of at least 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 centipoise.

[0049] As described above, the gene regulatory formulations described herein can simultaneously regulate the expression of at least two or more genes involved in collagen synthesis, elascin synthesis, hyaluronic acid synthesis, skin barrier function, cell turnover regulation, skin pigmentation, skin inflammation, and / or cortisol production.

[0050] In one or more embodiments, the gene regulatory formulations produced herein can be used as topical (i.e., applied to the skin) compositions that can increase or decrease the expression of various genes or gene families by at least 1, 5, 10, 20, 30, 40, 50, 75, or 100%, promoting the local synthesis of important ECM components such as collagen, elastin, and hyaluronic acid, and regulating metabolism involved in the stress response by reducing the production of cortisol in human epithelial skin cells. These genes include, for example, COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, DEFA1, MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2 (COX2), POMC, or combinations thereof.

[0051] In one or more embodiments, the gene regulatory formulations produced herein can be used as topical (i.e., external skin) compositions that can increase expression of at least 1, 5, 10, 20, 30, 50, 75, or 100 percent when measured 24 hours after application of at least 1, 2, 3, 4, 5, 6, 7, or 8 of the following genes: COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, and DEFA1. For example, an increase in gene expression of at least 10 percent refers to a positive change in expression of 0.1 or greater in magnitude compared to a negative control.

[0052] In one or more embodiments, the gene regulatory formulations produced herein can be used as topical (i.e., external skin) compositions that may decrease expression by at least 1, 5, 10, 20, 30, 50, 75, or 100 percent when measured 24 hours after application of at least 1, 2, 3, 4, 5, 6, 7, or 8 of the following genes: MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2 (COX2), and POMC. For example, a decrease in gene expression of at least 10 percent refers to a negative change in expression of 0.1 or greater in magnitude compared to a negative control.

[0053] In one or more embodiments, the gene regulatory formulations produced herein can be used as topical (i.e., external to the skin) compositions capable of reducing the production of cortisol in human epithelial skin cells by at least 1-, 5-, 10-, 20-, 30-fold, 40-, 50-, 75-, or 100-percent when measured 24 hours after application.

[0054] In one or more embodiments, the gene regulatory formulations produced herein can be used as topical (i.e., external to the skin) compositions that can increase skin hydration levels, reduce skin roughness levels, and / or reduce skin blemish levels, improving human goals by at least 1, 5, 10, 20, 30, 40, 50, 75, or 100 percent over a four-week period with daily application (once or twice daily) compared to a baseline without treatment.

[0055] In one or more embodiments, the gene regulatory formulations produced herein can be used as disinfectants to disinfect surfaces capable of killing at least 99.9 percent of model bacteria present on the treated surface.

[0056] [MVL manufacturing process] MVL has traditionally been prepared by either chemical catalysis or fermentation followed by solvent processing. Generally, catalytic processes result in a mixture of R and S configurations of mevalonolactone, which is not ideal for use in the formulations of the present disclosure, since only the R version is biologically active. Furthermore, both traditional processes utilize petrochemical inputs and are therefore not environmentally sustainable.

[0057] Furthermore, conventional fermentation techniques utilize complex media for the propagation of microorganisms, which may add residual impurities at the end of fermentation, thereby necessitating the use of techniques such as solvent treatment that are not sustainable. For example, a process first disclosed by Tokyo University of Agriculture and Technology and subsequently utilized by ADEKA for the production of MVL utilizes the microorganism Saccharomycopsis fibuligera for fermentation. In MVL production, S. fibuligera is generally grown in complex media containing peptone, malt extract, yeast extract, or a combination thereof. At the end of such fermentation, many impurities from these complex organic reagents remain in the broth. Furthermore, MVL concentrations are very low, less than 10 g / l even after 12 days of fermentation. One method of purifying this low-concentration MVL from the ADEKA process is by acidifying the broth, followed by solvent extraction using petrochemicals such as ethyl acetate and methyl ethyl ketone, which are volatile organic compounds (VOCs) of fossil origin. The MVL-rich solvent layer is then separated and the solvent evaporated, leaving behind MVL with a very high boiling point (>260°C) along with other impurities, including residual solvent. Such a process and final composition are not sustainable or environmentally friendly.

[0058] In contrast to the conventional methods discussed above, the highly sustainable, high purity, solvent-free MVL products used in the formulations described herein are produced through a novel fermentation of renewable feedstocks by microorganisms in a defined minimal medium. This is followed by downstream processing methods that do not generally involve a solvent extraction step that involves the use of petrochemicals. As described in more detail below, purified mevalonolactone, mevalonic acid, and / or salts of mevalonic acid can be produced by the following methods: (a) fermenting an initial feedstock to form a crude solution containing mevalonolactone, mevalonic acid, and / or salts of mevalonic acid; (b) optionally contacting the crude solution with an acid, thereby forming an acidic mixture; (c) purifying the mixture using a wiped film evaporator, a falling film evaporator, a rotary evaporator, an electrodialysis device, and / or an electrodeionization device, thereby forming a purified solution containing mevalonolactone, mevalonic acid, and / or salts of mevalonic acid.

[0059] In one or more embodiments, the fermentation feedstock providing the carbon source includes, but is not limited to, at least one or more starch-based glucose, and at least one or more glucose; cellulose hydrolysates containing one or more sugars including glucose, xylose, arabinose, mannose, and rhamnose; glycerol; anaerobic digestates of food and / or agricultural wastes containing one or more short chain acids such as acetic acid, lactic acid, propionic acid, butyric acid, isovaleric acid, etc.; ethanol; and / or disaccharides such as sucrose. Other sustainable inputs may include carbon dioxide and / or carbon monoxide.

[0060] Fermenting microorganisms include, but are not limited to, K-12 E. coli strains expressing the genes required for the synthesis of MVL, and yeasts such as S. cerevisiae and I. orientalis. The microorganisms may also exhibit properties such as minimal flux to by-products such as acetate, pyruvate, ethanol, glycerol, and lactate due to reduced or eliminated activity in the corresponding pathways via gene, transcript, and / or protein levels.

[0061] In one or more embodiments, the fermentation process is carried out in batch or fed-batch mode using a defined growth medium containing inorganic salts with one or more renewable carbon sources listed above, preferably glucose. In various embodiments, the defined growth medium can include glucose, ammonium sulfate, citric acid, monobasic potassium phosphate, anhydrous magnesium sulfate, calcium sulfate dihydrate, ferrous sulfate heptahydrate, thiamine hydrochloride, and a trace metal solution. For example, 1 L of batch medium may contain 20 g glucose, 7.5 g ammonium sulfate, 9.2 g triammonium citrate, 1.361 g potassium dihydrogen phosphate, 0.602 g anhydrous magnesium sulfate, 21.52 mg calcium sulfate dihydrate, 0.267 g ferrous sulfate heptahydrate, 20 mg thiamine hydrochloride, and 8 mL of trace metal solution. In a particular embodiment, 1 L of trace metals solution contains 10 mL of concentrated sulfuric acid, 0.6 g CoSO4·7H2O, 0.6 g ZnSO4·7H2O, 0.2 g Na2MoO4·7H2O, 0.1 g H3BO3, 0.3 g MnSO4·H2O, and 5 g CuSO4·5H2O. The advantage of such a batch medium is that residual organic matter, undefined impurities, and / or trace ionic impurities are minimized and can be easily separated from the MVL at the end of the fermentation.

[0062] The temperature during fermentation may be at least 20°C, 30°C or 35°C, and / or less than 50°C, 45°C or 40°C. In certain embodiments, the temperature during fermentation is between 20 and 50°C, and most preferably about 37°C. The oxygen content of the medium may be maintained at at least 1, 5, 10, or 15 percent saturation in the medium, and / or less than 50, 40, 30, or 20 percent saturation. In certain embodiments, the oxygen content of the medium may be maintained at a rate between 1 and 50 percent of saturation in the medium, and most preferably at 20 percent of saturation in the medium.

[0063] Once the initial batched glucose is exhausted, glucose can be continuously fed to the fermentation vessel to maintain the concentration below 1 g / L. The pH during fermentation can be maintained at a pH of at least 3, 4, 5, 6 or 6.5, and / or below 7.5, 7 or 6.9. In certain embodiments, the pH is maintained between 3 and 7.5, most preferably 6.8. The pH can be maintained by adding a suitable base, such as, but not limited to, ammonium hydroxide, calcium carbonate, sodium hydroxide, or calcium hydroxide, to neutralize acidic fermentation products, including MVL.

[0064] The fermentation can be carried out for 60 to 150 hours, resulting in an MVL concentration of at least 20, 50, 70, or 100 g / L of the final fermentation volume. In certain embodiments, the final MVL concentration is at least 50 g / L, preferably at least 100 g / L.

[0065] FIG. 1 provides a representative high pressure liquid chromatography (HPLC) chromatogram from the end of a typical fermentation of the present disclosure utilizing an E. coli based engineered microorganism reaching a concentration of 79 g / l MVL. As is evident from the chromatogram in FIG. 1, there are no detectable peaks at retention times corresponding to metabolic products such as acetate, pyruvate, glycerol, and ethanol. Other molecules such as citrate and glucose that are added to the fermentation medium are also completely consumed by the end of the fermentation. There are no detectable peaks at corresponding retention times. As described below, this chromatogram can be used to measure the purity of the MVL by analyzing and measuring the peaks present in the chromatogram. The presence of nearly isolated peaks, such as those shown in FIG. 1, indicates high purity MVL.

[0066] Downstream processing used to obtain solvent-free optically pure R-MVL begins at the end of fermentation with a "broth" containing microbial cells, water, residual salts, and at least 0.05, 0.1, 0.5, 1 or 2, and / or less than 25, 20, or 15 weight percent MVL based on the total weight of the broth. Under these conditions, in one or more embodiments, the primary form of MVL is in the form of mevalonate anion along with the corresponding cation, such as calcium (Ca(MVL)2), which is two deprotonated mevalonate molecules in solution with one calcium as the counterion.

[0067] At the end of fermentation, the resulting broth may contain trace amounts of aggregation by-products (e.g., acetate, pyruvate, ethanol, glycerol, lactate, etc.) relative to the MVL concentration in the broth. In one or more embodiments, the broth after fermentation may contain an aggregation by-product concentration that is less than 0.2, 0.1, 0.02, or 0.01 times the MVL concentration in the broth.

[0068] The resulting broth can be further purified and concentrated by contacting the broth with one or more absorbents and / or concentrated acids (e.g., sulfuric acid). Exemplary absorbents can include, for example, cation exchange resins, anion exchange resins, activated carbon, charcoal, or combinations thereof. In various embodiments, the broth obtained after fermentation can be concentrated by evaporation, reverse osmosis, forward osmosis, electrodialysis, or combinations thereof.

[0069] In one or more embodiments, the resulting broth may first be acidified by adding sulfuric acid until the pH reaches 3.0, well below the pKa of MVL (approximately 4.2). Thus, the predominant form of MVL under these conditions is the protonated form, which exists in equilibrium with the lactone form, with sulfate being the counterion to calcium. Highly insoluble CaSO4 precipitates from the solution. CaSO4 is a well-known aggregate of microbial cells. Interactions between CaSO4 and bacterial membranes cause cells and cell debris to aggregate, and these aggregates are then aggregated with CaSO4 salts to form a "cake". This causes the majority of cells and other solids to form a cake, with over 90 percent of the produced MVL remaining in the upper aqueous layer (also called the supernatant). The cake may be removed by filtration and / or centrifugation. The remaining solids may be removed by filtration, for example, through cross-flow or tangential flow filtration units using membranes with a molecular weight cutoff of 1 to 100 kDa.

[0070] Subsequently, the remaining MVL-containing solution can be further concentrated to the desired level by evaporation or filtration techniques, such as rotary evaporators, falling film evaporators, and / or wiped film evaporators. For example, the remaining solution can be treated in a standard laboratory rotary evaporator and / or falling film evaporator at temperatures ranging from 40°C to 70°C and pressures ranging from 40 to 200 millibars to remove at least 99% of the amount of water remaining in the solution. If necessary, after evaporation and / or filtration, the remaining crude MVL solution can be further treated in a wiped film evaporator at less stringent conditions, such as temperatures ranging from 50 to 120°C and pressures ranging from 0.1 to 10 mmHg. More preferably, the temperature is about 70°C and the pressure is 0.3 mmHg. This optional additional treatment can remove compositions with a boiling point higher than water but lower than MVL.

[0071] The remaining crude MVL solution can then be subsequently distilled in a wiped film evaporator at low temperatures, such as in the range of 90-200°C, 100-150°C, 110-130°C, or about 120°C, and high vacuum pressures, such as in the range of 0.1-10 mmHg, 0.2-5 mmHg, or about 0.3 mmHg. Furthermore, the residence time in the wiped film evaporator may be in the range of 20-1000 seconds, 20-500 seconds, 20-200 seconds, 20-100 seconds, or preferably about 30 seconds. Thus, with the wiped film evaporator and the above conditions, high purity MVL may be obtained.

[0072] In contrast to prior art techniques, the use of wiped film evaporators for distillation allows for the formation of high purity MVL with fewer undesirable by-products. Furthermore, the inventive purification techniques described herein minimize the production of by-products such as anhydromevalonolactone due to dehydration of MVL during distillation.

[0073] This high purity MVL formed from the purification techniques described above can be diluted back with water to obtain a colorless aqueous solution of MVL at the desired concentration for various applications. For example, water can be added to the high purity MVL to obtain a 10% solution. If the purity is not as desired, any of the steps described above including a wiped film evaporator may be utilized. Thus, this allows for the creation of any purification loop that uses a wiped film evaporator as described herein. In such embodiments, this optional purification loop may include at least 1, 2, 3, or 4 passes through a wiped film evaporator under the conditions described herein.

[0074] Another possible purification method for producing high purity MVL involves taking advantage of the charged nature of MVL through the use of ion-selective membranes to selectively recover high purity MVL. Such recovery can be performed using an electrodialysis and / or electrodeionization device. For example, fermentation can be carried out as described above by utilizing sodium hydroxide as a base to maintain the pH at 6.1-6.9. At the end of the fermentation, centrifugation and / or filtration can be used to remove various solids such as cells and cell debris. Exemplary filtration can include cross-flow and / or tangential flow filtration units using membranes with a molecular weight cutoff of 1-100 kDa. The clarified broth can then be passed through an electrodialysis and / or electrodeionization device to remove inorganic salts, resulting in a concentrate stream rich in sodium sulfate. This low inorganic salt MVL solution can then be passed again through a second device and / or electrodeionization device to obtain high purity mevalonate in the concentrate stream. Alternatively, the clarified broth can be passed once through an electrodialysis device and / or an electrodeionization device to obtain a substantially decolorized and substantially deodorized mevalonate solution in a retentate stream.

[0075] Treatment in the electrodialysis and / or electrodeionization apparatus can be performed for at least 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 minutes, and / or less than 500 minutes. Further, in certain embodiments, the electrodialysis and / or electrodeionization apparatus can be operated at a voltage between 1 and 10 volts, preferably about 5 volts.

[0076] It should be noted that in certain embodiments, the methods for producing the MVL solutions and formulations do not include columns packed with ion exchange resins and / or crystallization techniques or equipment. Such processes may not reduce odor-causing substances to the desired levels to achieve the required purity. In other words, crystallization columns and / or ion exchange columns cannot be used to produce the MVL solutions and formulations described herein.

[0077] In one or more embodiments, the product of the above step is a relatively colorless aqueous solution containing about 2-15 weight percent MVL, such as mevalonic acid, based on the total weight of the solution, and containing impurities such as color bodies and other organic species that remain in the original feed stream. Generally, in one or more embodiments, the resulting relatively colorless aqueous solution has at least 0.5, 1, 1.5, or 2 weight percent, and / or less than 40, 30, 25, 20, or 15 weight percent mevalonolactone, mevalonic acid, and / or salts of mevalonic acid based on the total weight of the solution.

[0078] In one or more embodiments, the mevalonic acid solution can be further concentrated to a desired level by evaporation or filtration techniques. For example, processing the MVL solution in a standard rotary evaporator at 50° C. and 40 mbar can remove over 99 percent of the water from the solution. In certain embodiments, the MVL solution is preferably concentrated to 10 percent by weight, which is high enough for use in a variety of formulations.

[0079] The resulting MVL solution may be substantially free of traces of petrochemical solvents and other organic or inorganic impurities. In certain embodiments, the MVL solution is at least 95, 96, 97, 97.5, 98, 98.5, 99, or 99.5 percent pure in aqueous solution as measured by HPLC. The absence of long-chain "polymeric" species in the resulting MVL solution can also be confirmed by gel permeation chromatography (GPC). The resulting MVL solution is believed to be long-lasting and of high purity, making it suitable for high performance formulations for topical application and for ingestion at higher concentrations than previously possible.

[0080] Samples to be analyzed by HPLC are eluted on a Bio-Rad Aminex HPX-87H column (i.e., a stainless steel column with an inner diameter of 7.8 mm and a length of 30 cm, packed with polystyrene divinylbenzene (particle size 5 μm) for liquid chromatography). HPLC samples can be run at a constant temperature of approximately 50 °C and a flow rate of 0.6 mL / min using 5 mM sulfuric acid as the eluent. MVLs can be detected with a retention time of approximately 17-18 min.

[0081] Alternatively, purity analysis by HPLC can be performed on a Beckman Coulter Gold-168 system equipped with a photodiode array detector using an Alltech reversed-phase Econosil C-18 column (10 μm, 10×250 mm) with a flow rate of 1.5 mL / min. This alternative test is described in "Bioactive sesquiterpene lactones and other complex isosoled from Vernonia cinerea" by Youn et al., the disclosure of which is incorporated herein by reference in its entirety.

[0082] Additionally or alternatively, purity analysis by HPLC can be performed using an Agilent 1200 HPLC system (Santa Clara, USA) which may be equipped with a pump, an autosampler (ALS) (model G1329A), and a Hypersil Gold Thermo Scientific C18 (250 cm x 4.6 mm) 5 μm column (Paisley, UK). The detector consists of a UV / VIS operating at 277 nm. Chemstation software (version Rev B.04.03 (16)) can be used for data processing and evaluation. The method is further described in "Development and Validation of an HPLC Method for the Determination of the Antidiabetic Drug Alogliptin Benzoate in Bulk and Tablets" by Naseef et al., the disclosure of which is incorporated herein by reference in its entirety.

[0083] In one or more embodiments, the resulting MVL solution may be substantially free of anions, cations, or trace metals. In certain embodiments, the MVL solution may contain less than 10,000 ppm, 5,000 ppm, 1,000 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, or 10 ppm of cation, anion, and / or metal content, as measured by ICP-OES.

[0084] Trace metal analysis can be performed using a Thermo Scientific iCAP 6000 Series ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometer). This can be done by utilizing a 100-fold dilution of the sample, which can be prepared by diluting and mixing 100 μL of sample with 9.9 mL of 2% nitric acid solution. Generally, calibration concentrations for all elements analyzed will be in the range of 0-10 ppm.

[0085] Alternatively, ICP-OES can also be performed with a PQ 9000 Elite ICP-OES instrument, specifically for metal analysis. Generally, calibration, which involves the generation of a linear response between the concentration of a particular element and the ICP-OES instrument, can be performed using a set of standards and blanks created using deionized water and the reagents used for acid digestion (HNO3, HCl, H2O2). In ICP-OES, a matrix-matched solution of 1% nitric acid can be used as the calibration solution, with calibration concentrations for all analytical elements ranging from 0-5 ppm.

[0086] ICP-OES analysis for anions and cations may be performed using an iCAP 6500 ICP-OES spectrometer in radial view mode using a quartz torch and a quartz injector tube with an inner diameter of 2 mm (Thermo Scientific, USA). Laser ablation (LA) is performed using a NWR 213 nm solid-state Nd:YAG laser. During LA, a helium gas flow of 0.9 L min-1 was flowed through the cell. After the ablation chamber, a gas flow of 0.4 L min-1 Ar was added through a Y connector. The LA system was directly connected to the ICP torch via 1 m of PTFE tubing with an inner diameter of 4 mm.

[0087] As used herein, "high purity" MVL refers to MVL or an MVL solution that contains at least 95 percent purity as measured by HPLC and / or a cation content of less than 10,000 ppm as measured by ICP-OES.

[0088] In various embodiments, the resulting colorless aqueous MVL solution, before or after further evaporation / concentration, contains less than at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90, and / or 99, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, or 8 weight percent mevalonolactone, mevalonic acid, and / or salts of mevalonic acid, based on the total weight of the solution.

[0089] The fermentation process described herein may result in an MVL product that is almost entirely made up of the R version (i.e., R-MVL). In various embodiments, the resulting colorless aqueous MVL solution, before or after further evaporation / concentration, contains at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.9, or 99.99 percent by weight of R-MVL, based on the total weight of the solution. In certain embodiments, the fermentation process described herein may result in the MVL present in the solution or formulation containing at least 95, 99, 99.9, or 99.99 percent by weight of R-MVL, based on the total weight of the MVL. In such embodiments, the remaining MVL may correspond to S-MVL.

[0090] In various embodiments, the resulting colorless aqueous MVL solution, before or after further evaporation / concentration, contains at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90, and / or 99, 90, 80, 70, 60, 50, 40, 30, 25, 20, or less than 15 percent water by weight, based on the total weight of the solution.

[0091] As mentioned above, the resulting MVL solution is considered to be "high purity" and therefore may contain little or no undesirable by-products. In various embodiments, the resulting MVL solution may contain less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 weight percent of one or more undesirable by-products based on the total weight of the solution, before or after further evaporation / concentration. Exemplary undesirable by-products include fermentation by-products (e.g., acetic acid, citric acid, glucose, pyruvic acid, ethanol, pyruvate, glycerol, and / or lactic acid), solvents (e.g., ethyl acetate, dichloromethane, tetrahydrofuran, and / or isopropanol), petrochemicals, color bodies, odorous bodies, inorganic salts, and / or organic salts. It should be noted that the weight ranges above for the undesirable by-products above can apply to any individual by-product listed above, or any combination thereof.

[0092] In various embodiments, the resulting MVL solution may contain at least 0.001 parts petrochemical or organic solvent (e.g., ethyl acetate, dichloromethane, tetrahydrofuran, and / or isopropanol) content before or after further evaporation / concentration. For mevalonic acid, parts per million and / or less than 10 ppm or less than 10 ppm. The petrochemical origin of the impurities may be as low as 1:1.35x10 C-14 to C-12 ratio of petrochemical impurities. -12 This can be confirmed by radioisotope dating, which is less than 100%.

[0093] In various embodiments, due to the substantial absence of impurities such as color bodies, the resulting MVL solution, before or after further evaporation / concentration, may exhibit an APHA color of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 1, 0.1 or 0.01 parts per million of platinum cobalt to water as measured by ASTM D1209.

[0094] Additionally, it should be noted that fermentation products such as mevalonate broth typically contain high amounts of odor-causing substances. Such odor-causing substances are difficult to remove, and formulations used in end uses may exhibit undesirable odors. Thus, it is undesirable to use conventional fermentation products in topical skin care formulations due to the presence of large amounts of odor-causing substances in the fermentation products. However, in contrast to previous MVL products produced by conventional methods, the inventive high purity MVL solutions of the present disclosure contain very small amounts of odor-causing substances, which are generally removed using the inventive purification methods described herein. In one or more embodiments, the resulting MVL solution contains less than 10,000 ppm, 5,000 ppm, 1,000 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, 10 ppm, or 1 ppm of odor-causing substances as measured by gas chromatography (GC) before or after further evaporation / concentration. Exemplary odor-causing substances include aldehydes (e.g., acetaldehyde and / or butyraldehyde), ketones (e.g., diacetyl), volatile fatty acids (e.g., propionic acid, butyric acid, valeric acid, isovaleric acid), esters (e.g., ethyl acetate), amines (e.g., trimethylamine, indole, and / or pyridine), and / or sulfur compounds (e.g., dimethyl sulfide and / or mercaptans).

[0095] The gene regulatory formulations described herein can be produced using pre-concentrated or post-concentrated MVL solutions.

[0096] The present invention can be further illustrated by the following examples of embodiments thereof, it will be understood that these examples are included for illustrative purposes only and are not intended to limit the scope of the invention unless specifically stated.

[0097] [Example] [Example 1: Formulation Example] A topical skin care composition (500 grams) for application to the face or body was prepared according to the following process. First, 416.0 grams of Nikumurese, a commercial product containing behenyl alcohol, polyglyceryl-10 pentastearate, and sodium stearoyl lactylate, was mixed with 2.5 grams of hydrogenated lecithin, 15.0 grams of squalene, 25.0 grams of triethylhexanoin, 5.0 grams of Simmondsia chinensis (jojoba) seed oil, 5.0 grams of Macadamia ternifolia seed oil, 5.0 grams of cyclopentasiloxane, and 0.5 grams of tocopherol in a homomixer and heated to 80°C with continuous mixing. This mixture was named fraction A. In a separate container, 2.0 grams of phenoxyethanol were mixed with 15.0 grams of butylene glycol, 20.0 grams of pentylene glycol, 25.0 grams of 2% by weight xanthan gum in water, 20.0 grams of 2% by weight NTC-carbomer.-381 aqueous solution, 15.0 grams of 1% by weight potassium hydroxide solution, and 289 grams of deionized water were heated to 80°C while continuing to mix. This mixture was named fraction B. The two fractions were combined by gradually decanting fraction A into fraction B while both were continuously mixed at 80°C. The mixture was then cooled to 40°C, after which 2.5 grams of a 2% by weight sodium hyaluronate aqueous solution, 2.5 grams of a 2% by weight phenoxyethanol aqueous solution, and 25.0 grams of an aqueous solution containing 10% of long-lasting, high-purity mevalonolactone by weight were added. The pH of the resulting composition was 4.9, and the final mevalonolactone concentration was 0.5% by weight.

[0098] [Example 2: Formulation example] A disinfectant skin topical composition (500 grams) for application to the hands was prepared according to the following process. First, 361.95 grams of SD Alcohol 40-B (190 proof) was mixed with 5.5 grams of hydroxypropyl cellulose (from Ashland) and 5.0 grams of a 10% by weight aqueous solution of high purity mevalonolactone at room temperature with continuous stirring on a stir plate. Next, 121.3 grams of deionized water was gradually added to the mixture with stirring. Once the mixture was visually uniform, 1.25 grams of squalene and 5.0 grams of safflower seed oil were added. The final mevalonolactone concentration of the composition was 0.1% by weight. The pH of the resulting solution was 4.5 and the viscosity was 4000 cps. All ingredients in this formula were substantially vegetable based.

[0099] As a result, this formed an innovative formulation of an alcohol-based disinfectant gel with a pH of less than 4.9, i.e., slightly acidic, to support the skin's natural acidic mantle. Furthermore, such gel formulation retained a good viscosity of at least 1000 cps. Common alcohol-based hand sanitizer gels (60-80% alcohol) are formulated with carbomer and have a neutral to slightly basic pH value. However, the slightly acidic pH profile of the formulation of this invention is at odds with typical carbomer-based formulations. In addition to the pH balance, another advantage of the formulation of this invention is the overall improvement of skin hydration with topical application of R-MVL, which can prevent the dryness caused by alcohol-based disinfectants.

[0100] [Example 3: Formulation example] A long-lasting, 100% plant-based topical skin care composition was prepared for application on the face or body for gene regulation. More specifically, 1,000 grams of the composition was prepared according to the following method. First, 25 grams of a mixture of lecithin, sclerotium gum, xanthan gum, and pullulan (ratio 40:35:15:10) was sprinkled into 662.8 grams of water and mixed at room temperature for 10 minutes. This mixture was labeled as fraction A. In a separate container, 70 grams of sunflower oil was mixed with 10 grams of lauroyl lysine, 10 grams of Bioxan SF T50 (tocopherol-enriched sunflower oil from Quimica Masso), and 5 grams of bactiol and stirred until completely homogenized. This mixture was then named fraction B. Then, 50 grams of water was mixed with 30 grams of 80% lactic acid in water, 50 grams of 10% mevalonolactone in water, and 50 grams of pentylene glycol under stirring until completely homogenized. This mixture was fraction C. Fraction B was then added to fraction A under stirring for 10 minutes at room temperature to obtain an emulsion. Fraction C was then added to the mixture under stirring for 10 minutes. Finally, about 37.2 grams of 20% sodium hydroxide in water was added to the mixture under stirring until completely homogenized. The pH of the resulting composition was 4.2, and the final mevalonolactone concentration was 0.5% by weight.

[0101] The advantage of combining multiple gene expression modulating active ingredients such as MVL and bactiol is the synergistic effect on the overall gene expression profile of the formulation. Such a formulation may reduce hyperpigmentation and rough skin and improve the skin barrier in patients using the formulation.

[0102] [Example 4: Evaluation of the effect of highly purified MVL] Gene expression studies were performed using EpiSkin-LM, a commercially available 3-dimensional in vitro human skin model consisting of normal human skin cells in a collagen matrix. TMThe test samples contained 0.5% or 1.0% by weight of long-lasting, high-purity MVL in phosphate-buffered saline (PBS) solution. The negative control sample was PBS only.

[0103] Reconstructed 3D skin epidermis model (EpiSkin-LM) TM ) were preincubated for 24 hours with 5 mL of maintenance medium according to the manufacturer's instructions. 2 mL of test sample or negative control was applied to the top of the epidermis. The treated models were incubated for 24 hours. After incubation, the models were rinsed with PBS and then treated again with 2 mL of test sample or negative control and incubated for another 24 hours.

[0104] After the second incubation, mRNA was extracted from the models by miRNeasy® Mini Kit (QIAGEN). The mRNA expression levels in the models were measured by Genopal® DNA microarray chips. The regulation of gene expression levels was expressed as the ratio of mRNA transcripts in the test sample-treated models to those in the negative control model. Positive values ​​indicated increased gene expression, and negative values ​​indicated decreased gene expression compared to the negative control.

[0105] Figure 2 shows the change in gene expression level (regulation) of genes related to anti-aging elasticity when the model is treated with a solution containing 0.5% mevalolactone. In the context of the present invention, "anti-aging elasticity" refers to the maintenance and construction of extracellular matrix. The following Table 1 provides the function of the regulated genes shown in Figure 1 on anti-aging elasticity. [Table 1]

[0106] Figure 3 shows the gene expression changes (regulation) of genes related to moisture elasticity when the model is treated with a solution containing 0.5% mevalolactone. In the context of the present invention, "moisture elasticity" refers to the synthesis and incorporation of hyaluronic acid or hyaluronan into the extracellular matrix. The following Table 2 shows the functions of the regulated genes shown in Figure 1 with respect to moisture elasticity. [Table 2]

[0107] Figure 4 shows the change (modulation) in gene expression levels of genes related to epidermal turnover when the model is treated with a solution containing 0.5% mevalolactone. In the context of the present invention, "epidermal turnover" refers to the process by which new keratinocyte skin cells are formed in the epidermis. Table 3 below shows the function of the regulated genes shown in Figure 1 related to epidermal turnover. [Table 3]

[0108] Figure 5 shows the change in gene expression levels (regulation) of genes related to barrier function upon treatment of the model with a solution containing 0.5% mevalolactone. In the context of the present invention, "barrier function" refers to the generation and maintenance of the epithelial layer of the skin that separates internal organs and tissues from the external environment and stimuli. Processes related to barrier function include, but are not limited to, keratinocyte proliferation and differentiation, and the generation and maintenance of the keratin network. Table 4 below shows the functions related to barrier function of the regulated genes shown in Figure 1. [Table 4]

[0109] Figure 6 shows the change in gene expression levels (regulation) of genes related to extracellular matrix degradation when the model was treated with a solution containing 1.0% mevalolactone. In the context of the present invention, "extracellular matrix degradation" refers to the degradation of major ECM components, including but not limited to glycoproteins such as collagen, elastin, fibronectin, laminin; proteoglycans such as hyaluronic acid; hyaluronic acid; and other components of the ECM such as casein and gelatin. Table 5 below shows the functions related to barrier function of the regulated genes shown in Figure 1. [Table 5]

[0110] Figure 7 shows the change (modulation) in gene expression levels of genes related to skin inflammation when the model is treated with a solution containing 1.0% mevalolactone. In the context of the present invention, "skin inflammation" refers to an innate immune response that causes heat, pain, swelling, redness and / or loss of function in the skin. Table 6 below shows the functions of the regulated genes shown in Figure 1 related to skin inflammation. [Table 6]

[0111] Figure 8 shows the change (modulation) in gene expression levels of genes related to skin pigmentation when the model is treated with a solution containing 1.0% mevalolactone. In the context of the present invention, "skin pigmentation" refers to the production of melanin in the skin or the proliferation of melanocytes, the skin cells responsible for melanin production. Table 7 below shows the functions of the regulated genes shown in Figure 1 related to skin inflammation. [Table 7]

[0112] The data in Figures 2-5 show that a composition containing 0.5 wt% of sustained high purity R-MVL potently regulates the expression of genes related to anti-aging elasticity, moisturizing elasticity, epidermal turnover, and barrier function, including COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, and DEFA1, with increased gene expression levels of at least 10%. An increase in gene expression of at least 10% refers to a positive change in expression of a magnitude of 0.1 or greater compared to the negative control.

[0113] The data in Figures 6-8 show that a composition containing 1.0 wt% sustained high purity R-MVL reduces gene expression levels of MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2 (COX2), and POMC by at least 10%. A reduction in gene expression of at least 10% refers to a negative change in expression of a magnitude of 0.1 or greater compared to the negative control.

[0114] [Example 5: Evaluation of gene regulatory effects] In vivo human studies were conducted to investigate whether the observed modulation of genes shown in Tables 1-7 corresponded to desirable physical and visible changes in relevant aspects of human skin. Nine women aged 25-48 years (mean=34.1, standard deviation=8.1) participated in the 4-week study. The test sample corresponded to the composition detailed in Example 1 and contained 0.5% long-lasting optically pure R-MVL by weight. The placebo preparation was the same composition without MVL. Each sample was applied twice a day (morning and evening) to half of each subject's face daily for 4 weeks. Three measurements of skin characteristics were made during the test: one at baseline, one after 2 weeks, and one after 4 weeks. Skin moisture levels were measured with a SKICON-200EX instrument. Visual skin parameters such as skin texture (roughness), mottling, and redness were quantified with a VISIA Image Complexion Analysis System. Measurements at weeks 2 and 4 were quantified against baseline measurements, and testing was conducted during the winter months when dry skin is most prevalent.

[0115] 9-12 show the effect on skin hydration levels, roughness, skin blemishes, and redness caused by the test sample (0.5% MVL) and the placebo preparation (no MVL) as a percentage of baseline measurements.

[0116] FIG. 9 shows the effect of MVL composition (0.5% MVL) and placebo composition (0% MVL) on skin hydration levels. The data from FIG. 6 are as follows: FIG. 9 shows that application of the test composition containing 0.5% by weight of long-lasting optically pure MVL increased the mean skin hydration level compared to placebo at both 2 and 4 weeks, with a 33% increase at week 2. This data highlights the hydration-boosting effect of high-purity MVL in an exemplary commercial formulation containing other skin-improving agents such as jojoba seed oil and hyaluronic acid as shown in Example 1. After 2 weeks of winter season, skin treated with the placebo (no MVL) formulation showed a slight decrease in hydration level compared to the start of the study, while skin treated with the same formulation additionally containing 0.5% MVL showed a slight increase in hydration level over baseline during the same period.

[0117] FIG. 10 shows the effect of an MVL composition (0.5% MVL) and a placebo composition (0% MVL) on rough skin.

[0118] FIG. 11 shows the effect of an MVL composition (0.5% MVL) and a placebo composition (0% MVL) on skin blemishes.

[0119] FIG. 12 shows the effect of an MVL composition (0.5% MVL) and a placebo composition (0% MVL) on skin redness.

[0120] Thus, Figures 10-12 show that application of a test composition containing 0.5% long-lasting optically pure R-MVL reduced roughness, spotting, and redness of the skin compared to placebo after 2 and 4 weeks.

[0121] [Example 6 Evaluation of the effect of MVL on cortisol] The effect of MVL on metabolism, especially cortisol production, was investigated in keratinocytes. In general, 11β-HSD1 (hydroxysteroid dehydrogenase type 1) converts costizon to cortisol, which induces a stress response. The effect of MVL on the conversion of cortisone to cortisol was evaluated. Cortisol production tests were performed on normal human epidermal keratinocyte cells. Test samples contained 0–0.5% by weight of long-lasting, highly pure MVL in phosphate-buffered saline (PBS) solution, as well as cortisone, an inactive form of cortisol. Negative control samples contained only PBS. Cortisol production was then measured via the Alpha LISA system.

[0122] The data in Figure 13 show that compositions containing 0.125-0.5% by weight of sustained, highly pure MVL potently modulate the conversion of cortisone to cortisol, with higher MVL concentrations resulting in greater reductions in cortisol levels. A 0.5% concentration of MVL reduced cortisol production by over 15%.

[0123] [Example 7: Production of mevalonolactone] An E. coli strain exhibiting properties of minimal flux into by-products such as acetate, citrate, pyruvate, ethanol, glycerol, and lactate due to reduced or eliminated activity in the corresponding cells. Pathways via alterations in gene, transcript, and / or protein levels are constructed from strains disclosed in prior art U.S. Pat. No. 10,807,963. Genes including ldhA, adhE, gltA, poxB, and pta-ack are deleted using methods disclosed in the prior art. This mevalonate-producing strain is grown in a 1-liter benchtop bioreactor in minimal defined medium. 1 L of minimal defined medium contains 20 g glucose, 9.2 g triammonium citrate, 1.361 g potassium dihydrogen phosphate, 0.602 g anhydrous magnesium sulfate, 21.52 mg calcium sulfate dihydrate, 0.267 g ferrous sulfate heptahydrate, 20 mg thiamine hydrochloride, and 8 mL of trace metals solution. 1L of trace metals solution is formulated with 10 mL of concentrated sulfuric acid, 0.6 g CoSO4*7H2O, 0.6 g ZnSO4*7H2O, 0.2 g Na2MoO4*7H2O, 0.1 g H3BO3, 0.3 g MnSO4*H2O, and 5 g CuSO4*5H2O. The final pH of the medium is adjusted to 6.8 and maintained using 5M NaOH during fermentation. Temperature is kept at 37°C. Dissolved oxygen is maintained above 20% of saturation level by sparging with air in the range of 0.5-1 liters per minute (LPM) and agitation in the range of 600-1100 rpm. 15 hours after mevalonate production is induced. Antifoam is added as needed. Glucose concentration is maintained between 1-3 g / l by adding 600 g / l glucose at hourly intervals. The bioreactor run is stopped at 96 hours. The cells are separated from the broth using a 0.22 micron filter to obtain a clear broth. The mevalonic acid concentration was found to be 79 g / l at the end of the fermentation, as shown in Figure 1.

[0124] This clarified broth was circulated in an electrodialysis device similar to the Acilyzer system from Astom Corporation to selectively isolate charged products such as mevalonate. The system was operated in recirculation mode at 5 V / cell pair for 5 hours. The feed was 79 g / l mevalonic acid at the start. The product stream obtained at the end of the first cycle was 40 g / l pure mevalonate, with higher purity, lower odor and color compared to the feed as characterized by GC-FID shown in Figure 14.

[0125] [Example 8 Evaluation of the effect of MVL on hyaluronic acid] The effect of MVL on hyaluronic acid was performed on keratinocytes. Hyaluronic acid is an important component of skin. The effect of MVL on the conversion of hyaluronic acid was evaluated. Hyaluronic acid tests were performed on normal human epidermal keratinocyte (NHEK) cells. Test samples contained 0–0.25% by weight of long-acting, highly pure MVL in phosphate-buffered saline (PBS) solution. Negative control samples contained PBS only. Hyaluronic acid levels in the medium were then measured by ELISA after 24 and 48 h and normalized to protein levels in NHEK cells.

[0126] The data in Figure 15 show that compositions containing 0.13-0.25 wt% of long-lasting, highly pure MVL strongly modulate hyaluronic acid levels, with higher MVL concentrations leading to higher hyaluronic acid levels. Both 0.13% and 0.25% concentrations of MVL increased hyaluronic acid levels by more than 5% at 48 hours (p-value: p<0.05).

[0127] [Definition] It should be understood that the following is not intended to be an exhaustive list of defined terms. Other definitions may be provided in the preceding description, for example, with the use of the defined terms within the context.

[0128] As used herein, the terms "a," "an," and "the" mean one or more.

[0129] As used herein, the term "about" refers to a value within 10 percent of the stated value. For example, "about 10" corresponds to a value in the range of 9 to 11.

[0130] As used herein, the term "and / or," when used in a list of two or more items, may be employed to mean that any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if a composition is described as containing components A, B, and / or C, the composition may contain A alone; B alone; C alone; a combination of A and B; a combination of A and C, a combination of B and C; or a combination of A, B, and C.

[0131] As used herein, the terms "comprises," "including," and "comprising" are open-ended transitional terms used to transition from the subject matter listed before that term to one or more elements listed after that term, and the elements or elements listed after the transitional period are not necessarily the only elements that make up the subject matter.

[0132] As used herein, the terms "have", "having", and "having" have the same open-ended meaning as "include", "including", and "including" above.

[0133] As used herein, the terms "comprise", "including", and "including" have the same open-ended meaning as "comprise", "includes", and "including" above.

[0134] [Numeric range] In this description, numerical ranges are used to quantify certain parameters related to the present invention. When numerical ranges are provided, it should be understood that such ranges should be interpreted as literally supporting the limitations of claims that recite only the lower limit of the range and the limitations of claims that recite only the upper limit of the range. For example, a disclosed numerical range of 10 to 100 provides literal support for claims that recite "greater than 10" (without upper limit) and claims that recite "less than 100" (without lower limit).

[0135] Additionally, terms introducing ranges containing multiple numbers, such as "at least," "less than," or "less than," apply to all of the numbers in the range listing. For example, "at least 1, 2, 3, or 4" should be interpreted to cover the ranges "at least 1, at least 2, at least 3, or at least 4."

[0136] [Claims not limited to the disclosed embodiments] The above-mentioned preferred embodiments of the present invention should be used only as examples, and should not be used in a limiting sense to interpret the scope of the present invention. Modifications to the above-mentioned exemplary embodiments can be easily made by those skilled in the art without departing from the spirit of the present invention.

[0137] The inventors express herein their intention to rely on the doctrine of equivalents to determine and evaluate the reasonably fair scope of the invention as it pertains to any device that falls outside the scope but does not materially depart from the literal scope of the invention as set forth below.

Claims

1. A gene regulatory formulation for regulating the expression of one or more genes, said formulation comprising mevalonolactone; Here, the mevalonolactone is (a) is at least 97.5 percent pure in aqueous solution as determined by HPLC; (b) contains less than 1 weight percent of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the mevalonolactone; and (c) Contains less than 10,000 ppm of odor-causing substances as determined by gas chromatography (GC).

2. 2. The gene regulatory formulation of claim 1, wherein said mevalonolactone is at least 98.5 or 99.5 percent pure in aqueous solution as measured by HPLC.

3. 2. The gene regulatory formulation of claim 1, wherein said gene regulatory formulation comprises 0.1 to 10 weight percent of said mevalonolactone.

4. The gene regulatory formulation of claim 1 , wherein the mevalonolactone is in the form of an aqueous solution containing the mevalonolactone.

5. The gene modulator formulation reduced COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, and DEFA1 by at least 10% when measured 24 hours after application.

6. The gene-modulating formulation reduced MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2(COX2), and POMC by at least 10% when measured 24 hours after application.

7. 10. The gene regulatory formulation of claim 1, wherein said gene regulatory formulation reduces cortisol production in human epithelial skin cells by at least 10 percent as measured 24 hours after application.

8. 2. The gene regulatory formulation of claim 1, wherein said mevalonolactone comprises at least 95 percent by weight R-mevalonolactone based on the total weight of said mevalonolactone.

9. 10. The gene regulatory formulation of claim 1, wherein the gene regulatory formulation is a topical skin care composition in the form of a mist, lotion, emulsion, gel, cream, or ointment.

10. The mevalonolactone is a fermentation by-product, ethyl acetate, dichloromethane, n, isopropanol, a petrochemical based on the total weight of the mevalonolactone, or a combination thereof.

11. 2. The gene regulatory formulation of claim 1, wherein said mevalonolactone comprises a cation, anion, and / or metal content of 10,000 ppm, 5,000 ppm, 1,000 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, or less than 100 ppm as measured by ICP-OES.

12. The formulation comprises at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 weight percent, and / or less than 99, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, or 8 weight percent mevalonolactone, mevalonic acid, and / or salts of mevalonic acid based on the total weight of the formulation.

13. 2. The gene regulatory formulation of claim 1, wherein the mevalonolactone exhibits an APHA color of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, or 20, 10, 1, 0.1, or 0.01 parts per million of platinum cobalt to water as measured by ASTM D1209.

14. 10. The gene regulatory formulation of claim 1, wherein the formulation comprises bactiol, lactic acid, or a combination thereof.

15. 10. The gene regulatory formulation of claim 1, wherein the formulation contains less than 5,000 ppm, 1,000 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, or 10 ppm of odor-causing substances as measured by gas chromatography (GC).

16. 2. The gene regulatory formulation of claim 1, wherein the mevalonolactone is purified in a wiped film evaporator, a rotary evaporator, and / or a falling film evaporator.

17. 1. A method of forming a dermatological formulation, the method comprising: (a) providing an aqueous solution of mevalonolactone, said mevalonolactone comprising (i) At least 97.5 percent pure in aqueous solution, as determined by HPLC; (ii) contains less than 1 weight percent of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the mevalonolactone; and (iii) Contains less than 10,000 ppm of odor-causing substances, as determined by gas chromatography (GC); and (b) combining said aqueous solution with at least one excipient to form said dermatological formulation.

18. 18. The method of claim 17, wherein the mevalonolactone is at least 98.5 or 99.5 percent pure in aqueous solution as measured by HPLC.

19. 18. The method of claim 17, wherein the dermatological formulation comprises 0.1 to 10% by weight of the mevalonolactone.

20. 18. The method of claim 17, wherein the mevalonolactone is in the form of an aqueous solution comprising the mevalonolactone.

21. 18. The method of claim 17, wherein the dermatological preparation increases expression of at least two genes selected from the group consisting of:

18. The method of claim 17, wherein expression of at least two genes selected from the group consisting of COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, and DEFA1 is increased by at least 10% as measured 24 hours after application.

22. 18. The method of claim 17, wherein the dermatological preparation reduces the expression of at least two genes selected from the group consisting of: MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2 (COX2), and POMC by at least 10% as measured 24 hours after application.

23. 18. The method of claim 17, wherein the dermatological preparation reduces cortisol production in human epithelial skin cells by at least 10% as measured 24 hours after application.

24. 18. The method of claim 17, wherein the mevalonolactone comprises at least 95% by weight R-mevalonolactone, based on the total weight of the mevalonolactone.

25. 18. The method of claim 17, wherein the dermatological formulation is a topical skin care composition in the form of a mist, lotion, milk, gel, cream, or ointment.

26. 18. The method of claim 17, wherein the mevalonolactone comprises less than 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 weight percent of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the mevalonolactone.

27. 18. The method of claim 17, wherein the mevalonolactone comprises a cation, anion, and / or metal content of less than 10,000 ppm, less than 5,000 ppm, less than 1,000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, or less than 10 ppm as measured by ICP-OES.

28. 18. The method of claim 17, wherein the dermatological formulation comprises at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90, and / or less than 99, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, or 8 weight percent mevalonolactone, mevalonic acid, and / or salts of mevalonic acid based on the total weight of the formulation.

29. 18. The method of claim 17, wherein the mevalonolactone exhibits an APHA color of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 1, 0.1 or 0.01 parts per million of platinum cobalt on water as measured by ASTM D1209.

30. 18. The method of claim 17, wherein the dermatological preparation comprises bactiol, lactic acid, or a combination thereof.

31. 18. The method of claim 17, wherein the dermatological preparation contains less than 5,000 ppm, less than 1,000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, or less than 10 ppm of odorants as measured by gas chromatography (GC).

32. 18. The method of claim 17, wherein the mevalonolactone is purified in a wiped film evaporator, a rotary evaporator, and / or a falling film evaporator.

33. 18. The method of claim 17, wherein said providing comprises: fermenting the initial feedstock to form a crude solution comprising mevalonolactone, mevalonic acid, and / or salts of mevalonic acid; contacting the raw solution with an acid to form an acidic mixture; purifying the acidic mixture using a wiped film evaporator, a falling film evaporator, a rotary evaporator, an electrodialysis apparatus, and / or an electrodeionization apparatus to form a purified solution of mevalonolactone, mevalonic acid, and / or a salt of mevalonic acid.

34. 18. The method of claim 17, wherein said providing comprises fermenting an initial feedstock to form a crude solution comprising mevalonolactone, mevalonic acid, and / or a salt of mevalonic acid; contacting the crude solution with an acid to thereby form an acidic mixture; purifying the acidic mixture using a falling film evaporator and / or a rotary evaporator to form a purified solution; and optionally further purifying the purified solution in a wiped film evaporator.

35. 18. The method of claim 17, wherein said providing comprises fermenting the initial feedstock to form a crude solution comprising mevalonolactone, mevalonic acid, and / or a salt of mevalonic acid, contacting the crude solution with an acid to thereby form an acidic mixture, and purifying the acidic mixture in an electrodialysis and / or electrodeionization device.

36. A purified mevalonolactone solution containing: (a) mevalonolactone, wherein said mevalonolactone: (i) is at least 97.5% pure in aqueous solution as measured by HPLC; (ii) contains less than 1% by weight, based on the total weight of the mevalonolactone, of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof; and (iii) Contains less than 10,000 parts per million (ppm) of odor-causing substances, as determined by gas chromatography (GC); and (b) Water.

37. 37. The method of purifying mevalonolactone according to claim 36, wherein the mevalonolactone is at least 99 or 99 in aqueous solution as measured by HPLC.

38. 37. The purified mevalonolactone method of claim 36, wherein the solution increases expression of at least two genes selected from the group consisting of: COL1A1, COL1A2, COL4A1, ELN, EMILIN1, EMILIN2, MFAP5, FN1, LAMA1, TIMP1, TIMP2, TIMP3, TIMP4, HAS1, HAS2, HAS3, HABP4, HAPLN1, EGF, FGF7, HBEGF, KRT1, KRT2, KRT4, and DEFA1 by at least 10% as measured 24 hours after application.

39. 37. The purified mevalonolactone method of claim 36, wherein the solution reduces expression of at least two genes selected from the group consisting of: MMP1, MMP2, MMP3, MMP7, MMP9, NFATC1, IL1A, IL1B, TNFA, SELE, SELL, EDN1, EDN2, EDN3, PTGS2 (COX2), and POMC by at least 10% as measured 24 hours after application.

40. 37. The method of claim 36, wherein the solution reduces cortisol production in human epithelial skin cells by at least 10% as measured 24 hours after application.

41. 37. The method of purifying mevalonolactone of claim 36, wherein the mevalonolactone comprises at least 95% by weight R-mevalonolactone based on the total weight of the mevalonolactone.

42. 37. The method of claim 36, wherein the mevalonolactone comprises less than 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 weight percent of fermentation by-products, ethyl acetate, dichloromethane, tetrahydrofuran, isopropanol, petrochemicals, or combinations thereof, based on the total weight of the mevalonolactone.

43. 37. The method of purifying mevalonolactone of claim 36, wherein the mevalonolactone comprises a cation, anion, and / or metal content of less than 10,000 ppm, less than 5,000 ppm, less than 1,000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, or less than 10 ppm as measured by ICP-OES.

44. 37. The method of claim 36, wherein the solution comprises at least 0.5, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90, and / or less than 99, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, or 8 weight percent mevalonolactone, mevalonic acid, and / or salts of mevalonic acid based on the total weight of the formulation.

45. 37. The method of purifying mevalonolactone of claim 36, wherein the mevalonolactone exhibits an APHA color of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 1, 0.1 or 0.01 parts per million of platinum cobalt on water as measured by ASTM D1209.

46. 37. The method of purifying mevalonolactone of claim 36, wherein the preparation contains less than 5,000 ppm, less than 1,000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, or less than 10 ppm odorants as measured by gas chromatography (GC).

47. 37. The process for purifying mevalonolactone of claim 36, wherein the mevalonolactone is purified in a wiped film evaporator, a rotary evaporator, and / or a falling film evaporator.

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