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WO2015017775A2 - Compositions contenant des cellules souches adultes dérivées de lignées embryonnaires et extraits en contenant - Google Patents

Compositions contenant des cellules souches adultes dérivées de lignées embryonnaires et extraits en contenant Download PDF

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Publication number
WO2015017775A2
WO2015017775A2 PCT/US2014/049401 US2014049401W WO2015017775A2 WO 2015017775 A2 WO2015017775 A2 WO 2015017775A2 US 2014049401 W US2014049401 W US 2014049401W WO 2015017775 A2 WO2015017775 A2 WO 2015017775A2
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WO
WIPO (PCT)
Prior art keywords
ela
formulation
cosmetic
cells
cell
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Ceased
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PCT/US2014/049401
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English (en)
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WO2015017775A3 (fr
Inventor
Keith D. Crawford
John GARVEY
Colin White
Pamela Layton
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PARCELL LABORATORIES LLC
PARCELL LABS LLC
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PARCELL LABORATORIES LLC
PARCELL LABS LLC
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Publication of WO2015017775A2 publication Critical patent/WO2015017775A2/fr
Publication of WO2015017775A3 publication Critical patent/WO2015017775A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • compositions having early lineage adult stem cells and extracts thereof having early lineage adult stem cells and extracts thereof
  • the present invention relates to formulations and methods for dermal and connective tissue rejuvenation.
  • Skin is subject to a number of conditions that can lead to loss of elasticity, wrinkling and age spots, and a resultant desire for cosmetic enhancement
  • Such conditions include aging due to chronological aging or photoaging from exposure to the sun, or both.
  • Skin aging results in wrinkling, the appearance of pigmented areas ("age spots"), thinning of the skin, loss of elasticity, and other undesirable characteristics.
  • Other skin conditions, which can be improved by cosmetic approaches, include reduction in visible scarring, for example, from acne or other causes, uneven pigmentation, and the like.
  • Examples of such procedures include treatment with Botulinum Toxin Type A (“botox”), retinoids and derivatives thereof and especially retinoic acid (all-trans and 13-cis) and retinol, and the use of hydroxy acids.
  • Botulinum Toxin Type A (“botox”)
  • retinoids and derivatives thereof and especially retinoic acid (all-trans and 13-cis) and retinol and the use of hydroxy acids.
  • An embodiment of the invention provides a formulation including, a cosmetic base having from about 0.01% to 10% w/w of an ELA cell preparation.
  • the ELA cell preparation is devitalized, extracted and or fractionated.
  • the ELA cell preparation is lyophilized.
  • Some embodiments further include a tissue growth factor.
  • Other embodiments further include a tissue growth factor and cell membrane fraction(s).
  • ELA cells Some embodiments of the invention further includes from about 0.01% to 10% w/w of cell-free medium conditioned by growth of ELA cells and/or ELA-lineage cells. In some embodiments the media is lyophilized. In some embodiments the invention is substantially free from non-human animal products.
  • Another embodiment of the invention provides use of ELA cells (ECCS), lysates
  • ECL ELA cell products
  • ELP ELA cell products
  • Another embodiment of the invention provides a method of treating a skin condition including: wrinkling, folds, sagging, age spots, uneven pigmentation, thinning, elasticity, scarring, surface roughness, surface roughness, surface vessels, redness, and pore size, including administering to a subject having one or more of the aforesaid conditions, a dose of a formulation including, a cosmetic base having from about 0.01% to 10% w/w of an ELA cell preparation.
  • Another embodiment of the invention provides a method of tissue augmentation, including inducing tissue growth and recovery from standard cosmetic procedures by administering to a subject subdermally, a preparation of 10 ⁇ 2 to 10 ⁇ 6 live ELA cells.
  • Another embodiment of the invention provides a method of tissue augmentation, including inducing tissue growth and recovery from standard cosmetic procedures by administering to a subject externally, the formulation including, a cosmetic base having from about 0.01% to 10% w/w of an ELA cell, cell extract, or cell-free media preparation.
  • Another embodiment of the invention provides a method of tissue augmentation, including inducing tissue growth and recovery from standard cosmetic procedures by administering to a subject subdermally, a preparation of 10 ⁇ 2 to 10 ⁇ 6 live ELA cells and subsequently administering externally one or more doses of the formulation including, a cosmetic base having from about 0.01 % to 10% w/w of an ELA cell, cell extract, or cell-free media preparation.
  • the mode of administration is topical, and optionally, other cosmetics or devices, may be in contact on the surface or below the dermal surface at the same time.
  • the cosmetic base is a lotion. In other embodiments the cosmetic base is a cream. In some embodiments the cosmetic base includes a pigment. In another embodiment the cosmetic base includes a sunscreen. In some embodiments the cosmetic base includes at least one anti-acne agent. In other embodiments the cosmetic base includes liposomes. In some embodiments the cosmetic base includes at least one antioxidant. In other embodiments die cosmetic base includes a platelet-rich fibrin matrix.
  • embodiment of the invention provides a composition including, a cosmetic base having from about 0.01% to 10% w/w of one or more of: an ELA cell preparation, ELA cell extract, and ELA cell-conditioned medium; the ELA cell-conditioned medium generated in an environmentally-closed cell culture system.
  • the medium is filter-sterilized.
  • the medium is concentrated.
  • Fig. 1 is a bar graph showing increased cytokine release from cultured ELA cells, into the conditioned media.
  • ELA cells were cultured and the conditioned medium was collected after 72 hours. The cells were harvested and counted and the supernatants were centrifuged at 14,000 ⁇ g for 5 min to remove cellular debris.
  • Immunoreactive VEGF, IL-6 and IL-8 protein levels were measured in the conditioned media using commercial ELISA kits in accordance with the manufacturer's instructions. Pre-conditioned medium was used as control and cytokine levels in the control were analyzed by ELISA. The data collected from
  • cytokine levels in ELA cell conditioned medium were observed to be significantly higher by at least about two-fold than cytokine levels in the pre-conditioned medium.
  • the present invention includes skin rejuvenating compositions that result in improvements to skin health and/or appearance, including but not limited to improvements with respect to wrinkling, folds, sagging, age spots, uneven pigmentation, thinning, elasticity, scarring, surface roughness, surface roughness, surface vessels, redness, and pore size.
  • a composition is achieved by incorporating a population of fresh or expanded ELA cells, or extracts or derivatives thereof.
  • ELA Early lineage adult stem cells
  • ELA stem cells are capable of differentiating into tissues of endodermal, mesodermal and ectodermal origin.
  • the source, isolation, characterization and certain uses and formulations of ELA cells are described in patent applications: U.S. provisional application serial numbers: 60/927,596, 61/247,236, 61/247,242, 61/249,172, and 61/501,846, as well as U.S. patent application serial number 12/598,047 and PCT applications serial numbers PCT/US2008/005742 and
  • ELA cells are described by their expression of certain stem cell specific pluripotency genes (e.g., Oct-4, KFL-4, Nanog, Sox-2, Rex-1, GDF-3 and Stella).
  • ELA cells are distinct from embryonic stem cells (ESC) and other types of early lineage adult cells such as MSC, VSEL, MAPC and cord blood derived progenitor cells because the ELA stem cells do not appear to detectibly express the various stem cell markers CD34, CD44, CD45, CD49a, CD66A,
  • ELA cells are may be derived from multiple sources including, bone marrow stroma, blood, dermis, periosteum and tissues, but a useful exemplary source is synovial fluid (SF).
  • SF synovial fluid
  • ELA cells are extracted from patient intended to receive the cellular therapy (i.e., an autologous transplant). ELA cells can be culture expanded prior to their introduction into the patient. Growth of ELA cells in vitro can be used, for example, to increase the number of ELA cells available for implantation or injection.
  • ELA cell numbers are increased about two fold or greater, about ten fold or greater, or about twenty fold or greater or more, depending on the desired number of cells.
  • Patient-specific master cell banks for example can include ten or more passages of expanded autologous cells, which increases ELA cell counts by many orders of magnitude in comparison to primary isolates.
  • growing ELA cells in vitro can include expansion in a cell culture medium, which includes nutrients, buffers, salts, proteins, vitamins and/or growth factors, which promote ELA cell growth.
  • a useful cell culture medium is RPMI 1680 supplemented with 10% serum, and appropriate antibiotics such as penicillin/streptomycin and G418.
  • Human serum is preferred for expanding the ELA cells for human transplant preparations, but fetal bovine serum has produced acceptable yields of the ELA cells in culture.
  • a currently preferred culture system is an environmentally controlled, closed-culture system, commonly known as a bioreactor, which is particularly useful for collection of ELA cell-conditioned media.
  • Other systems are suitable for culture of ELA cells, the expansion of donor cells, and the creation of master ELA cell banks. These should comply with Good Tissue Practices and ideally should be cGMP.
  • the expanded cells are commonly but not necessarily frozen prior to implantation, and a suitable cryogenic medium that is acceptable to the FDA for such purposes is
  • the cosmetic composition having ELA cells is substantially free from non-human animal products.
  • Another embodiment of the invention provides for the use of a medium conditioned (ELA cellular cytokine suspension or ECCS) by growth of ELA cells to prepare a medicament or cosmetic for administration to skin of a subject as a cellular rejuvenation compound.
  • Another embodiment of the invention provides for the use of lysates from devitalized ELA cells to prepare a medicament for a cosmetic or for the administration to a subject as a cellular rejuvenation compound.
  • Another embodiment of the invention provides for the use of ELA cell derived products from primary, expanded or transgene expressing ELA cells to prepare a medicament for the administration to a subject as a cellular rejuvenation compound. Methods of expressing transgene products from transfected ELA cells are described in PCT application serial number PCT/US2008/005742.
  • the present invention provides various dermatological and cosmetic compositions for topical application to an individual that includes ELA cell, ECCS, ELA cell lysates derived (ECL), and ELA cell products (ECP), each alone or in combination.
  • ELA cell ELA cell lysates derived
  • ECP ELA cell products
  • the composition is formulated for application to the skin of an individual.
  • the amount of ELA cells, ECCS, ECL, and ECP in this dermatological or cosmetic composition is an amount capable of providing a detectable improvement in the health or appearance of an individual's skin compared to untreated skin of the individual.
  • a 0.01% to 10% w/w of cell extract, cell conditioned media or lyophyilized ELA cells, applied over a 30 day period results in detectable improvement.
  • compositions of the invention include lotions, creams, oils, gels, including hydrogels, powders, serums, salves, foundations, facial masks, lip care products, sunscreens, hair care products, such as shampoos, conditioners, including deep conditioners, hair care treatments, hot oil treatments, skin cleansers, exfoliants, ointments, compact formulations, or the like.
  • cosmeceutical refers to a formulation or composition including at least one biologically active ingredient that has an effect on the user of the product and at least one cosmeceutically-acceptable carrier. Cosmeceuticals may be viewed as cosmetics that, in certain applications and under appropriate conditions, provide medicinal or drug-like benefits.
  • cosmeceuticals affect the underlying structure of the skin, decrease wrinkle depth, or reverse or ameliorate the effect of photooxidation or aging on the skin.
  • Cosmeceuticals are particularly useful as skin care products, hair care products, and sun care products.
  • cosmeceutical compositions include delivery systems including at least one of liposomes, cyclodextrins, polymer systems, or hyaluronic acid or related compounds.
  • Cosmeceutical compositions include cosmeceutically- acceptable carriers.
  • a topical cosmetic or cosmeceutical ointment, lotion, or gel composition typically contains an effective amount of EL A cells, ECCS, ECL cells, and cell products derived therefrom.
  • the cosmeceutical ointment may also include other active and inert ingredients in a cosmetically- or a cosmeceutically-acceptable carrier, such as a pharmaceutical cream base, an oil-in-water emulsion, a water-in-oil emulsion, or a gel.
  • a cosmetically- or a cosmeceutically-acceptable carrier such as a pharmaceutical cream base, an oil-in-water emulsion, a water-in-oil emulsion, or a gel.
  • compositions for topical use include drops, tinctures, lotions, creams, salves, serums, solutions, and ointments containing conditioned media or extracts, and an appropriate carrier.
  • the optimal percentage of the conditioned media or extract in each composition varies according to the composition's formulation and the therapeutic effect desired.
  • inventive compositions may include any of a number of cosmetically-, cosmeceutically-, or pharmaceutically-acceptable formulations, depending on the type of product, the nature of the composition, the location of composition's use, the desired effect, and the like. While proprietary formulations are common in the formulation arts, fbrmulators of ordinary skill will be able to determine or readily select appropriate formulations for specific applications without undue
  • the methods of the invention have wide applicability to cosmetic conditions.
  • the mode of administration for cosmetic applications is typically topical, but administration and dosage regimens vary depending on the cosmetic condition for which modulation is sought.
  • the present invention provides methods, compositions, and kits for cosmetic use with individuals.
  • the term "individual” as used herein includes humans as well as other mammals.
  • the compositions, methods, and/or kits are used to provide a cosmetic treatment to an individual desiring and/or in need of cosmetic treatment (e.g., young children subject to burn or other scarring may not desire treatment but may nonetheless be in need of treatment).
  • the term “treating” or “treatment” as used herein includes achieving a cosmetic benefit.
  • cosmetic benefit is meant any desired modulation of the cosmetic condition being treated. For example, in an individual with wrinkling, cosmetic benefit includes eradication or lessening of the appearance of wrinkling.
  • a cosmetic benefit is achieved with the eradication or amelioration of one or more of the psychological symptoms associated with the underlying condition such that an improvement is observed in the patient, notwithstanding the fact that the patient may still be affected by the cosmetic condition.
  • ELA cells, ECCS, ECL, ECP or combination thereof provides cosmetic benefit not only when a cosmetic defect is eradicated, but also when an improvement is observed in the individual with respect to the cosmetic defect and its attendant consequences, such as psychological consequences.
  • methods and compositions of the invention may be directed at achieving a prophylactic benefit.
  • a "prophylactic,” or “preventive” effect includes prevention of a condition, retarding the progress of a condition (e.g., skin aging), or decreasing the likelihood of occurrence of a condition.
  • “treating” or “treatment” includes prophylaxis.
  • an effective amount encompasses an amount sufficient to effect beneficial or desired cosmetic results.
  • An effective amount is administered in one or more administrations.
  • an "effective amount" of ELA cells, ECCS, ECL, ECP or a combination thereof of the invention is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of a cosmetic condition, or to provide a desired effect such as cosmetic augmentation of a soft tissue.
  • An "effective amount” may be of a ELA cells, ECCS, ECL, and ECP or a combination thereof used alone or in conjunction with one or more agents used to modulate a cosmetic condition.
  • the skin is subject to a number of cosmetic conditions that result in alterations of function and/or appearance mat are considered undesirable, and the manifestation of these conditions can lead to psychological discomfort or harm. In some cases, no defect may be present however, it may be desirable to the subject to augment or alter the skin in such a way as to produce a cosmetically pleasing effect.
  • cosmetic conditions include, but are not limited to, skin aging, cosmetic defect, undesired pigmentation, and post-cosmetic procedure damage.
  • Skin aging includes chronological aging as well as photoaging, and may appear as wrinkling, lack of elasticity (for example sagging), uneven pigmentation, thinning of the skin and/or collagen so that veins and other underlying structures become more prominent, and the like.
  • Skin aging is a major example of a skin condition that involves a decrease in cell proliferation and in cell function.
  • skin aging refers to alterations in the appearance and function of skin that occur with aging, such as wrinkling, loss of elasticity, sagging, uneven pigmentation (for example, "age spots” or “liver spots”), and loss of underlying tissue mass. Such conditions may be accelerated and/or exacerbated by exposure to ultraviolet radiation ("photoaging”) and other environmental conditions.
  • ELA cells, ECCS, ECL, and ECP therefrom can be used cosmetically to counteract, at least for a time, the effects of aging on skin.
  • a formulation containing ELA cells, ECCS, ECL, and ECP or a combination thereof may be applied topically in areas where it is desired to counteract skin aging.
  • cosmetic defects that, while not pathological or physiologically harmful, may nonetheless cause psychological distress, in some cases to the extreme. In these cases it is desirable to correct a particular feature or features causing distress or, alternatively, enhance a feature considered desirable.
  • skin aging such conditions include, e.g., striae gravidarum and striae distensiae ("stretch marks), atrophic scarring (e.g., acne scarring), wound (e.g., traumatic wounds, chronic wounds, or burn wounds) or surgical scarring, thickened and cracked skin (especially on the feet), and hair loss.
  • the invention relates to the use of preparations mat comprise ELA cells, ECCS, ECL, and ECP or combination thereof to enhance hair growth.
  • Cells from which the hair is produced grow in die bulb of the follicle. They are extruded in the form of fibers as the cells proliferate in the follicle.
  • Hair "growth” refers to the formation and elongation of the hair fiber by the dividing cells.
  • the methods of the invention provide a means for altering the dynamics of the hair growth cycle to induce proliferation of hair follicle cells, particularly stem cells of the hair follicle.
  • compositions and method can be used to increase hair follicle size and the rate of hair growth in individuals, such as humans, e.g., by promoting proliferation of hair follicle stem cells.
  • the method comprises administering to the skin in the area in which hair growth is desired an amount of ELA cells, ECCS, ECL, and ECP or combination thereof sufficient to increase hair follicle size and/or the rate of hair growth in the animal, e.g., human.
  • the composition is administered topically as a cream or lotion, and is applied on a daily basis until hair growth is observed and for a time thereafter sufficient to maintain the desired amount of hair growth.
  • Undesired pigmentation includes pigmentation over an area of the body that is different than the pigmentation desired by the individual. Undesired pigmentation can be the result of, e.g., photoaging, reaction to inflammation, or reation to trauma such as surgical or accidental skin breakage, and the like. Undesired pigmentation includes altered or undesired pigmentation over small areas such as freckles, as well as altered or undesired pigmentation over larger areas, such as, for example, uneven pigmentation or larger areas of undesired pigmentation.
  • Further cosmetic uses of the methods of the invention include tissue augmentation through, generally, topical application, such as for lip enhancement. By “augment” is meant to include giving the appearance of greater fullness, generally through an increase in the tissue of the skin or underlying tissue. Any suitable skin area may be selected for
  • the methods of the invention may be employed to enhance and/or accelerate recovery from standard cosmetic procedures, which are damaging to skin and/or underlying tissues.
  • these procedures may be associated with lengthly recovery time and suboptimal results.
  • Such procedures include chemical peel, dermabrasion, laser resurfacing, ablative resurfacing, nonablative resurfacing, photodynamic therapy, noncoherent light phototherapy, breast lift, face lift, eyelid lift, forehead lift, neck lift, thigh lift, buttock lift, tummy tuck, and scar revision.
  • the methods of the invention provide a method for achieving firming and lifting of the eyelids; this may be done either in place of or in conjunction with a conventional eyelid lift procedure.
  • the methods of the invention may be used in conjunction with both types of procedures to enhance and/or accelerate healing and recovery.
  • ELA cells, ECCS, ECL, and ECP or combination thereof may be administered in any cosmetically acceptable carrier, as described in more detail below.
  • the concentration of ELA cells, ECCS, cell lysates derived therefrom, and cell products derived therefrom or a combination thereof used may be more than about 0.00001, 0.00005, 0.0001, 0.001, 0.01, 0.1, 1, S, 10 or 20% on a w/w basis.
  • the concentration of ELA cells, ECCS, ECL, and ECP or combination thereof is more than about 0.00005%.
  • the concentration of ELA cells, ECCS, ECL, and ECP or combination thereof may be less than about 0.0001, 0.001, 0.001, 0.01, 0.1 , 1, 5, 10, or 20% (all concentration percentages given herein are w/w unless otherwise indicated).
  • the ELA cells, ECCS, ECL, and ECP or a combination thereof are used at a concentration of about 0.00001% to about 1%; or about 0.00001% to about 0.1%; or about 0.0001% to about 0.01%; or about 0.0005% to about 0.005%; or about 0.0005% to about 0.002%; or about 0.001%. In some embodiments, lower and higher concentrations are contemplated.
  • Skin coverage may also be described in terms of total uL of ELA cells, ECCS, ECL, and ECP or combination thereof7cm2 of skin; in these terms, a typical coverage per administration would be more than about 5, 10, 100, 1000, 10,000, 50,000, or 500,000 uL/ cm2 of skin; less man about 1,000,000, 500,000, 50,000, 5000, 500, 50, or 5 ⁇ L / cm2of skin; or about 5 uL / cm2 to about 500 uL / cm2 of skin; or about 50 uL / cm2 of skin.
  • the methods of the invention typically utilize topical administration, which may be by any suitable means mat brings the ELA cells, ECCS, ECL, and ECP or combination thereof and, optionally, other cosmetic or dermatological agents, in contact with the surface of the skin.
  • topical administration may be by any suitable means mat brings the ELA cells, ECCS, ECL, and ECP or combination thereof and, optionally, other cosmetic or dermatological agents, in contact with the surface of the skin.
  • the frequency and duration of administration of a formulation comprising a ELA cells, ECCS, ECL, and ECP or combination thereof is dependent on factors including the nature of the formulation (e.g., concentration, presence or lack of other cosmetic or dermatological agents, vehicle type), the severity and extent of the condition, and in some cases the judgment of a skin care professional, e.g., a health care professional such as a dermatologist, or a cosmetologist.
  • a skin care professional e.g., a health care professional such as a dermatologist, or a cosmetologist.
  • Topical application may be applied more than once, twice, three times, four times, five times, or six times per week, or more than once, twice, three times, four times, five times, or six times per day. Frequency of application may be less than about twice, three times, four times, five times, or six times per week, or less man about once, twice, three times, four times, five times, or six times per day.
  • Some embodiments of the invention provide a method for cosmetic treatment of the skin of an individual by topical administration of an effective amount of a ELA cells, ECCS, ECL, and ECP therefrom or a combination thereof.
  • the formulation is administered an average of about once per day; in some embodiments, the formulation is administered an average of about once or twice per day; in some embodiments, the formulation is administered an average of about once to three times per day; in some embodiments, the formulation is administered an average of more than about three times per day. In one embodiment, the formulation is administered an average of about twice per day, typically in the morning upon rising and in the evening before retiring.
  • Topical administration may be without a covering.
  • topical administration may include the use of a covering over the formulation, which may be occlusive or non-occlusive.
  • administration in the evening before retiring may include covering the administered area with an occlusive or non-occlusive covering, which may remain in place during sleep.
  • the duration of treatment generally will depend on the response of the skin to cosmetic treatment. Treatment may continue at the discretion of the individual being treated. In some cases, administration or application of a formulation containing ELA cells, ECCS, ECL, and ECP or combination thereof may be more frequent at the beginning of treatment and less frequent as treatment continues and the condition is ameliorated or the desired effect is achieved. In some cases, treatment may continue indefinitely in order to maintain a condition in abeyance or in an improved state, to delay onset of a cosmetic condition (e.g., skin aging), or to slow the progression of a cosmetic condition. These modifications of frequency and duration are easily accomplished by the individual being treated.
  • a cosmetic condition e.g., skin aging
  • Some embodiments of cosmetic treatment of skin employ topical administration of a lotion, in some embodiments a mixture of emulsifying lanolin alcohols, waxes, and oils (e.g., EUCERINTM) or a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CUREL, as described below, comprising ELA cells, ECCS, ECL, and ECP or combination thereof.
  • a lotion in some embodiments a mixture of emulsifying lanolin alcohols, waxes, and oils
  • EUCERINTM e.g., EUCERINTM
  • a mixture of petrolatum or mineral oil e.g., a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CUREL, as described below, comprising ELA cells
  • the lotion containing the ELA cells, ECCS, ECL, and ECP or combination thereof is applied at a frequency of once to three times per day, in some embodiments once per day, until the desired result, e.g., reduction or elimination of wrinkling, sagging, and the like, is observed, followed by topical application once to three times per week, in some embodiments once per week, thereafter.
  • Some embodiments of cosmetic treatment of skin employ topical administration of a cream, in some embodiments a mixture of emulsifying lanolin alcohols, Water/Aqua/Eau, Petrolatum, Glycerin, Isostearyl Palmitate, Butylene Glycol, or Glyceryl Stearate (e.g., Creme LuXuryTM) or a mixture of, comprising of ELA cells, ECCS, ECL, and ECP or combination thereof.
  • a cream in some embodiments a mixture of emulsifying lanolin alcohols, Water/Aqua/Eau, Petrolatum, Glycerin, Isostearyl Palmitate, Butylene Glycol, or Glyceryl Stearate (e.g., Creme LuXuryTM) or a mixture of, comprising of ELA cells, ECCS, ECL, and ECP or combination thereof.
  • This composition is applied at a frequency of once to three times per day, in some embodiments once per day, until the desired result, e.g., reduction or elimination of wrinkling, sagging, and the like, is observed, followed by topical application once to three times per week, in some embodiments once per week, thereafter.
  • Some embodiments involves the cosmetic treatment of the eyelashes by employing a mascara in some embodiments a mixture of mineral oils, linseed oil, castor, eucalyptus, lanolin, or beeswax (e.g. COVERGIRL® NatureLuxe Mascara) or a mixture of, comprising of ELA cells, ECCS, ECL, and ECP or combination thereof.
  • This composition is applied at a frequency of once to three-times per day, in some embodiments once per day, until the desired result, e.g. lengthening, thicken and/or curling of the eyelashes.
  • mascara may contain nanofibers, nanotubals, ELA cells, ECCS, ECL, and ECP or combination thereof to further lengthen, thicken, and/or curl the eyelashes.
  • oils, waxes or other standard fatty substances, or conventional gelling agents and/or thickeners oils, waxes or other standard fatty substances, or conventional gelling agents and/or thickeners; emulsifiers; moisturizing agents; emollients; sunscreens;
  • hydrophilic or lipophilic active agents such as ceramides; agents for combating free radicals; bactericides; sequestering agents; preservatives; basifying or acidifying agents; fragrances; surfactants; fillers; natural products or extracts of natural product, such as aloe or green tea extract; vitamins; or coloring materials.
  • active agents such as ceramides; agents for combating free radicals; bactericides; sequestering agents; preservatives; basifying or acidifying agents; fragrances; surfactants; fillers; natural products or extracts of natural product, such as aloe or green tea extract; vitamins; or coloring materials.
  • ELA cells, ECCS, ECL, and ECP or combination thereof is used in combination with another skin care method or composition
  • any suitable combination of the ELA cells, ECCS, ECL, and ECP or combination thereof and the additional method or composition may be used.
  • the two may be administered simultaneously, consecutively, in overlapping durations, in similar, the same, or different frequencies, etc.
  • a composition will be used that contains a ELA cells, ECCS, ECL, and ECP or combination thereof in combination with one or more other cosmetic or dermatological agents.
  • compositions are for topical use, they need not be sterile; however, if sterility is desired it is readily accomplished by filtration through sterile filtration (0.22 micron) membranes, or by other art-accepted means.
  • compositions may be incorporated in the formulations.
  • the nature of the other ingredient(s) will depend on the cosmetic condition to be modulated and/or cosmetic result desired. These are described more fully below.
  • the ELA cells, ECCS, ECL, and ECP or combination thereof may be used neat (e.g., with an occlusive dressing so that the ELA cells, ELA cells, ECCS, ECL, and ECP or combination thereof is dissolved or dispersed in perspiration at the site), but generally is prepared in a vehicle suitable for topical administration.
  • Compositions of the invention include ELA cells, ECCS, ECL, and ECP or combination thereof in a vehicle suitable for topical administration.
  • compositions usually employed for topically administering cosmetic compositions may be used, e.g., creams, lotions, gels, dressings, shampoos, tinctures, pastes, ointments, salves, powders, liquid or semi-liquid formulation, patches, liposomal preparations, and the like.
  • Application of said compositions may, if appropriate, be by aerosol e.g.
  • compositions such as salves, creams, lotions, pastes, gels, ointments and the like will conveniently be used.
  • the ELA cells, ECCS, ECL, and ECP or combination thereof may optionally be dissolved or diluted in a small amount of an appropriate solvent, such as ethanol or DMSO, before dispersion in the vehicle; however, this is not required.
  • compositions known in the art are especially preferred for topical administration, and include toilet waters, packs, lotions, skin milks or milky lotions.
  • the preparations contain, besides the ELA cells, ECCS, ECL, and ECP or combination thereof and, optionally, other active ingredients, components usually employed in such preparations.
  • components are oils, fats, waxes, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, perfumes, dyestuffs, lower alkanols, and the like.
  • oils examples include fats and oils such as olive oil and hydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons such as liquid paraffin, ceresin, and squalene; fatty acids such as stearic acid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and esters such as isopropyl myri state, isopropyl palmitate and butyl stearate.
  • oils include fats and oils such as olive oil and hydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons such as liquid paraffin, ceresin, and squalene; fatty acids such as stearic acid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and esters such as isopropyl myri state, isopropyl palmitate and but
  • anionic surfactants such as sodium stearite, sodium cetylsulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; cationic surfactants such as stearyldimethylDenzylammonium chloride and stearyltrimethylarnmonium chloride; ampholytic surfactants such as alkylaminoethylglycine hydrochloride solutions and lecithin; and nonionic surfactants such as glycerin monostearate, sorbitan monostearate, sucrose fatty acid esters, propylene glycol monostearate,
  • polyoxyethylene oleylether polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropylene glycol (e.g. the materials sold under the trademark "Plutonic"), polyoxyemylene castor oil, and polyoxyethylene lanolin.
  • humectants include glycerin, 1,3-butylene glycol, and propylene glycol
  • examples of lower alcohols include ethanol and isopropanol
  • examples of thickening agents include xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol and sodium carboxymethyl cellulose
  • examples of antioxidants include butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid and ethoxyquin
  • examples of chelating agents include disodium edetate and ethanehydroxy diphosphate
  • examples of buffers include citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate
  • examples of preservatives are methyl
  • the concentration of ELA cells, ECCS, ECL, and ECP or combination thereof may be more man about 0.00001, 0.00005, 0.0001, 0.001, 0.01, 0.1, 1, 5 or 10% by weight. In some embodiments, the concentration of the ELA cells, ECCS, ECL, and ECP or combination thereof is more than about 0.00005%. The concentration of ELA cells, ECCS, ECL, and ECP or combination thereof may be less than about 0.0001, 0.001, 0.001, 0.01, 0.1, 1, 5, 10 or 20% (all concentration percentages given herein are w/w unless otherwise indicated). In some embodiments, lower and higher concentrations are contemplated.
  • the pharmaceutically acceptable "base” is a carrier for example, mat can contain 1 to 20%, in particular 5 to 15% of a humectant, 0.1 to 10% in particular from 0.5 to 5% of a thickener and water; or said carrier may consist of 70 to 99%, in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5 to 15% of a fat; or 80 to 99.9% in particular 90 to 99% of a thickener; or 5 to 15% of a surfactant, 2- 15% of a humectant, 0 to 80% of an oil, very small ( ⁇ 2%) amounts of preservative, coloring agent and/or perfume, and water.
  • a carrier for example, mat can contain 1 to 20%, in particular 5 to 15% of a humectant, 0.1 to 10% in particular from 0.5 to 5% of a thickener and water; or said carrier may consist of 70 to 99%, in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5 to 15% of
  • the carrier for example consists of 2 to 10% of a lower alcohol, 0.1 to 10% or in particular 0.5 to 1% of a surfactant, 1 to 20%, in particular 3 to 7% of a humectant, 0 to S% of a buffer, water and small amounts ( ⁇ 2%) of preservative, dyestuff and/or perfume.
  • the carrier typically consists of 10-50% of oil, 1 to 10% of surfactant, 50-80% of water and 0 to 3% of preservative and/or perfume.
  • ELA cells, ECCS, ECL, and ECP or combinations thereof, optionally with other active ingredients are dissolved, mixed, or suspended in a mixture of emulsifying lanolin alcohols, waxes, and oils (e.g., EUCERINTM Lotion) or a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CURELTM Lotion), or lotions that are substantially similar in composition.
  • a mixture of emulsifying lanolin alcohols, waxes, and oils e.g., EUCERINTM Lotion
  • petrolatum or mineral oil e.g., a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CURELTM Lotion)
  • CURELTM Lotion fatty ester emollient
  • EUCERINTM Dry Skin Therapy Original Moisturizing Lotion comprises water, mineral oil, isopropyl myristate, PEG-40 sorbitan peroleate, glyceryl lanolate, sorbitol, propylene glycol, cetyl palmitate, magnesium sulfate, aluminum stearate, lanolin alcohol, BHT, methylchloroisothiazolinone, and methylisothiazolinone.
  • CURELTM Fragrance Free Daily Moisturizing Lotion comprises water, glycerin, distearyldimonium chloride, petrolatum, isopropyl palmitate, cetyl alcohol, dimethicone, sodium chloride, methylparaben. and propylparaben.
  • compositions of the invention comprise ELA cells, ECCS,
  • compositions of the invention comprise ELA cells, ECCS, ECL, and ECP or combinations thereof in a lotion comprising a mixture of emulsifying lanolin alcohols, waxes, and oils (e.g., EUCERINTM Dry Skin Therapy Original Moisturizing Lotion) at a concentration greater than about 0.00005%.
  • Some embodiments of compositions of the invention comprise ELA cells, ECCS, ECL, and ECP or combinations thereof in a lotion comprising a mixture of emulsifying lanolin alcohols, waxes, and oils (e.g., EUCERINTM Dry Skin Therapy Original Moisturizing Lotion) at a concentration of about 0.0001% to about 0.01%.
  • compositions of the invention comprise ELA cells, ECCS, ECL, and ECP or combinations thereof in a lotion comprising a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CURELTM Fragrance Free Daily Moisturizing Lotion) at a concentration greater than about 0.00005%.
  • a lotion comprising a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CURELTM Fragrance Free Daily Moisturizing Lotion) at a concentration greater than about 0.00005%.
  • compositions of the invention comprise a ELA cells, ECCS, ECL, and ECP or combination thereof in a lotion comprising a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty ester emollient (e.g., CURELTM Fragrance Free Daily Moisturizing Lotion) at a concentration of about 0.0001% to about 0.01%.
  • a fatty ester emollient e.g., CURELTM Fragrance Free Daily Moisturizing Lotion
  • ELA cells may be formulated in liposome-containing compositions.
  • Liposomes are artificial vesicles formed by amphipathic molecules such as polar lipids, for example, phosphatidyl cholines, ethanolamines and serines, sphingomyelins, cardiolipins,
  • Liposomes are formed when suitable amphipathic molecules are allowed to swell in water or aqueous solutions to form liquid crystals usually of multilayer structure comprised of many bilayers separated from each other by aqueous material (also referred to as coarse liposomes).
  • aqueous material also referred to as coarse liposomes.
  • Another type of liposome known to be consisting of a single bilayer encapsulating aqueous material is referred to as a unilamellar vesicle. If water-soluble materials are included in the aqueous phase during the swelling of the lipids they become entrapped in the aqueous layer between the lipid bilayers.
  • EL A cells, ECCS, ECL, and ECP or combination thereof into liposomal preparations suitable for topical application can be achieved by a number of methods.
  • liposomal preparations any known methods for preparing liposomes for treatment of a condition may be used. See, for example, Bangham et al., J. MoL Biol, 23: 238-2S2 (1965) and Szoka et al., Proc. Natl Acad. Sci. 75: 4194-4198 (1978).
  • Ligands may also be attached to the liposomes to direct these compositions to particular sites of action.
  • Liposomes containing ELA cells, ECCS, ECL, and ECP or combination thereof and, optionally, other ingredients can be employed directly or they can be employed in a suitable pharmaceutically acceptable carrier for topical administration.
  • the viscosity of the liposomes can be increased by the addition of one or more suitable thickening agents such as, for example xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and mixtures thereof.
  • the aqueous component may consist of water alone or it may contain electrolytes, buffered systems and other ingredients, such as, for example, preservatives.
  • Suitable electrolytes which can be employed include metal salts such as alkali metal and alkaline earth metal salts.
  • Exemplary metal salts are calcium chloride, sodium chloride and potassium chloride.
  • concentration of the electrolyte may vary from zero to 260 mM, preferably from 5 mM to 160 mM.
  • the aqueous component is placed in a suitable vessel which can be adapted to effect homogenization by effecting great turbulence during the injection of the organic component. Homogenization of the two components can be accomplished within the vessel, or, alternatively, the aqueous and organic components may be injected separately into a mixing means which is located outside the vessel. In the latter case, the liposomes are formed in the mixing means and then transferred to another vessel for collection purpose.
  • the organic component consists of a suitable non-toxic, cosmetically acceptable solvent such as, for example ethanol, glycerol, propylene glycol and polyethylene glycol, and a suitable phospholipid which is soluble in the solvent.
  • suitable phospholipids which can be employed include lecithin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. phosphatidylinositol, lysophosphatidylcholine and phosphatidyl glycerol, for example.
  • Other lipophilic additives may be employed in order to selectively modify the characteristics of the liposomes.
  • additives examples include stearylamine, phosphatide acid, tocopherol, cholesterol and lanolin extracts.
  • other ingredients which can prevent oxidation of the phospholipids may be added to the organic component
  • examples of such other ingredients include tocopherol, butyl ated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.
  • Preservatives such as benzoic acid, methyl paraben and propyl paraben may also be added.
  • ELA cells, ECCS, ECL, and ECP or combinations thereof are generally capable of penetrating cell membranes and reaching the deep layers of skin, it may be useful in some embodiments to also include a penetration enhancer in the formulations of the invention.
  • a penetration enhancer is a substance that improves cutaneous penetration of a bioactive substance. Suitable penetration enhancers include, for example, dimethyl sulfoxide (DMSO), DMSO-like compounds, ethanolic compounds, pyroglutamic acid esters and other solvents or compounds known to those skilled in the pharmaceutical art which facilitate dermal penetration of the drugs or chemicals chosen for the pharmaceutical composition.
  • DMSO dimethyl sulfoxide
  • DMSO-like compounds ethanolic compounds
  • pyroglutamic acid esters and other solvents or compounds known to those skilled in the pharmaceutical art which facilitate dermal penetration of the drugs or chemicals chosen for the pharmaceutical composition.
  • penetration enhancers include amphiphiles such as L-amino acids, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, fatty acids and alcohols. Additional penetration enhancers are disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition (1995) on page 1583.
  • the penetration enhancer chosen and the relative proportion of the penetration enhancer with respect to the active drugs or chemicals depends on the desired rate of delivery of the drugs or chemicals into the skin, which in turn depends on the condition being treated and the outcome sought More specifically, the type and amount of enhancer is chosen so that a sufficiently high concentration of active drugs or chemicals is attained in the skin to treat the condition within the time period considered desirable.
  • compositions use may be made of covers, e.g. plasters, bandages, dressings, gauze pads, patches and the like, containing an appropriate amount of ELA cells, ECCS, ECL, and ECP or a combination thereof and, optionally, other ingredients.
  • covers e.g. plasters, bandages, dressings, gauze pads, patches and the like, containing an appropriate amount of ELA cells, ECCS, ECL, and ECP or a combination thereof and, optionally, other ingredients.
  • plasters, bandages, dressings, gauze pads, patches and the like which have been impregnated with a topical formulation containing the therapeutic formulation.
  • Additional cosmetic and dermatological agents may be included in methods and formulations of the invention.
  • Anti-wrinkling agents are one form of cosmetic or dermatological agents.
  • Anti- skin wrinkling agents include a variety of agents, often in combination, that prevent or treat wrinkling through a variety of actions. Many approaches are taken to reduce the appearance of facial and other wrinkles based on the understanding of the molecular basis of wrinkle formation.
  • Such treatments include cosmetic products, drug therapy and surgical procedures. For example, many cosmetic products contain hydroxy acids, which may stimulate collagen synthesis.
  • Another common treatment utilizes retinol, retinoic acid, retinol pahnitate, a derivative of vitamin A, (or prescribed versions, Retin-A and Renova) which may directly or indirectly stimulate collagen production or retard collagen degradation.
  • Bicyclic aromatic compounds with retinoid-type activity which are useful in particular in preventing or treating various keratinization disorders, are described in EP 679630. These compounds are particularly active for repairing or combating chronological or actinic ageing of the skin, for example such as in anti- wrinkle products. Antioxidants such as vitamin C and E and coenzyme Q-10 are believed to counteract free radicals, which damage cells and cause aging and have been used in treatments of wrinkles. Recently, the FDA approved cosmetic use of Botox (an extremely purified form of botulinum toxin) to treat glabella frown lines.
  • Botox an extremely purified form of botulinum toxin
  • free-radical scavenger refers to, for example, alpha-tocopherol, superoxide dismutase.
  • ubiquinol e.g., coenzyme QIO
  • certain metal-chelating agents include, e.g., alpha-hydroxy acids such as lactic acid and glycolic acid or beta-hydroxy acids such as salicylic acid and salicylic acid derivatives such as the octanoyl derivative; other hydroxy acids and keto acids include malic, citric, mandelic, tartaric or glyceric acids or the salts, amides or esters thereof.
  • anti-wrinkling agents and anti-skin aging agents useful in the invention include sulfur-containing D and L amino acids and their derivatives and salts, particularly the N-acetyl derivatives, a preferred example of which is N-acetyl-L- cysteine; thiols, e.g. ethane thiol; fat-soluble vitamins, ascorbyl palmitate, ceramides, pseudoceramides (e.g., pseudoceramides described in U.S. Pat. Nos. 5,198,210; 4,778,823; 4,985,547;
  • phospholipids e.g., distearoyl lecithin phospholipid
  • fatty acids e.g., distearoyl lecithin phospholipid
  • fatty alcohols e.g., distearoyl lecithin
  • cholesterol e.g., glycerol
  • phytic acid e.g., glycerol
  • lipoic acid e.g., lysophosphatidic acid
  • skin peel agents e.g., phenol and the like
  • Preferred fatty acids or alcohols are those that have straight or branched alkyl chains containing 12-20 carbon atoms.
  • a particularly preferred fatty acid is linoleic acid since linoleic acid assists in the absorption of ultraviolet light and furthermore is a vital component of the natural skin lipids.
  • suitable anti-wrinkle actives for use herein are described in U.S. Pat No. 6,217,888, which description is incorporated herein by reference.
  • Compositions for cosmetic treatment of skin may further include sunscreens to lower skin's exposure to harmful UV rays.
  • Sunscreens include those materials commonly employed to absorb or block ultraviolet light.
  • Illustrative compounds are the derivatives of PABA, cinnamate and derivatives of salicylate (other than ferulyl salicylate).
  • octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone also known as oxybenzone
  • Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trademarks, PARSOL MCX and BENZOPHENONE-3, respectively. Dermascreen may also be used.
  • sunscreens are FDA-approved or approved for use in the European Union.
  • FDA-approved sunscreens may be used, singly or, preferably, in combination. See, e.g., U.S. Pat.Nos. 5,169,624; 5,543,136; 5,849,273; 5,904,917; 6,224,852; 6,217,852; and Segarinet al., chapter Vil, pages 189 of Cosmetics Science and Technology, and Final Over-the-
  • Cosmetic compositions of the invention may further include anti-acne agents.
  • Antiacne agents include benzoyl peroxide, antibiotics, e.g., erythromycin, clindamycin phosphate, 5,7-dichloro-8-hydroxyquinoline, resorcinol, resorcinol acetate, salicylic acid, azaleic acid, long chain dicarboxylic acids, sulfur, zinc, retinoids, antiandrogens, and various natural agents such as those derived from green tea, tea tree oil, and mixtures thereof.
  • antibiotics e.g., erythromycin, clindamycin phosphate, 5,7-dichloro-8-hydroxyquinoline
  • resorcinol resorcinol acetate
  • salicylic acid e.g., erythromycin, clindamycin phosphate, 5,7-dichloro-8-hydroxyquinoline
  • resorcinol resorcinol acetate
  • Anticellulite agents include isobutylmethylxanthine, caffeine, theophylline, theobromine, aminophylline, yohimbine, and mixtures thereof.
  • Yet other cosmetic or dermatological agents that complement cosmetic treatment of skin include .alpha, -interferon, estradiol; progesterone; pregnanalone; methylsolanomethane (MSM); copper peptide (copper extract); plankton extract (phytosome); broparoestrol; estrone; adrostenedione; androstanediols; etc.
  • compositions of the present invention may contain a wide range of additional components.
  • CTFA Cosmetic Ingredient Handbook Seventh Edition, 1997 and the Eighth Edition, 2000, which are incorporated by reference herein in their entirety, describes a wide variety of ingredients commonly used in skin care compositions, which are suitable for use in the compositions of the present invention.
  • Other topically-applied compounds are listed in Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Witkins, Baltimore, Md. (2000) (hereinafter Remington's), U.S.
  • concentration of the other active ingredient in formulations provided by the invention is that which provides an effective amount of the other active ingredient; these concentrations are well-known in the art.
  • ELA* cells are early lineage adult stem cells that are isolated in an undifferentiated and highly plastic state. ELA ® cells were cultured in TheraPEAKTM MSCGM-CDTM
  • ELA stem cells are obtained from blood of mammalian subjects, for example human individual which is harvested, diluted in serum-free medium (GIBCOTM AIM V Medium liquid; Invitrogen, Carlsbad, CA), and centrifuged three times at 200 g for ten minutes at room temperature. The cells are then re-suspended in serum-free medium, and are layered over a combined gradient of Ficoll-Paque and Stem Cells Technologies Granulocyte gradient commercially available from ROSETTE SEP DM-M; Stem Cell Technologies, Vancouver, British Columbia. Granulocytes are prevented from entering the gradient and red blood cells and stem cells are obtained in to pellet. The sample is centrifuged at 500 g for 30 minutes at room temperature. The Ficoll-Stem Cell Technology gradients separate the cells into a buffy layer, an intermediate layer, and pelleted layer.
  • GEBCOTM AIM V Medium liquid Invitrogen, Carlsbad, CA
  • the cells are then re-suspended in serum-free medium, and are
  • ELA stem cell population is isolated from the pelleted layer, washed in phosphate-buffered saline, and resuspended in culture medium. See U.S. utility application serial number 12/598,047 filed October 29, 2009, publication US 2010/0291042 published November 18, 2010.
  • Example 3 ELA cells stimulate cytokine production
  • ELA cells were cultured and cell-free conditioned media were collected after an period of cell growth sufficient to permit expression and production of secreted cell factors (e.g., 24-72 hours). Cells were harvested by centrifugation at 72 hours, counted and supernatants were centrifuged at 14,000 ⁇ g for 5 min to remove cellular debris.
  • VEGF Vascular Endothelial Derived Growth Factor
  • IL-6 Interleukin-6
  • IL-8 Interleukin-8 protein levels were measured in the conditioned medium using commercial Enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturer's instructions.
  • the cytokine levels in ELA cell conditioned medium were observed to be consistently and significantly higher than cytokine levels in unconditioned media.
  • the cytokines VEGF, IL-6 and IL-8 play a role in anti-aging. VEGF stimulates angiogenesis and IL-6 and IL-8 reduce inflammation. It is here envisioned mat ELA cells combat symptoms of aging by stimulating increase in production of cytokines and that ELA cell conditioned media might be used either as a source for production or directly following sterilization by filtration in a cosmetic preparation.
  • Example 4 Assay of additional growth factors including keratinocyte growth factor, dermal fibroblasts growth factor, and endothelial cells growth factor
  • ELA cells are cultured and the cell-free ELA-condittoned media is collected after an period of cell growth sufficient to permit expression and production of secreted cell factors (e.g., 24-72 hours). Cells are harvested by centrifugation at 72 hours, counted and supernatants are centrifuged at 14,000 ⁇ g for 5 min to remove cellular debris.
  • VEGF Vascular Endothelial Derived Growth Factor
  • ELISA Enzyme-linked immunosorbent assay
  • VEGF vascular endothelial growth factor
  • Keratinocyte growth factor and dermal fibroblasts growth factor etc. The protein levels of VEGF, Keratinocyte growth factor and dermal fibroblasts growth factor etc. is greater than control unconditioned medium. It is here envisioned that increased production of e.g., Keratinocyte growth factor and dermal fibroblast and other growth factors produced therein stimulates increase in tissue production of endogenous cells, collagen and elastin. It is also envisioned that increased production of VEGF stimulates angiogenesis. Other cytokines have immune-mediating effects.
  • ELA cells combat aging symptoms by stimulating increase in production of e.g., VEGF, Keratinocyte growth factor and dermal fibroblasts growth factor and other secreted soluble growth factors and cytokines, and that ELA cell- conditioned media is useful as a source of these factors, or directly in a cosmetic preparation
  • ELA cell extracts and fractions are useful as such membrane fractions add fatty acids and membrane receptor proteins, and microsomal fractions contain bioactive polypeptides and microRNAs, which have further tissue regenerative effects on target tissues.
  • Such ELA cell extracts and/or ELA cell-conditioned media are effective at ranges of 0.01% to 10% w/w. Specific concentrations will depend on the fraction, e.g., growth factors are effective in lower concentrations than fatty acids, as will be apparent to those of skill in the art.

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Abstract

La présente invention concerne diverses compositions dermatologiques et cosmétiques destinées à être appliquées par voie topique chez un sujet et contenant des cellules souches adultes dérivées de lignées embryonnaires (cellules ELA), ainsi que des extraits ou des formulations en contenant. Lesdites compositions peuvent améliorer efficacement la santé ou l'aspect de la peau d'un sujet.
PCT/US2014/049401 2013-08-01 2014-08-01 Compositions contenant des cellules souches adultes dérivées de lignées embryonnaires et extraits en contenant Ceased WO2015017775A2 (fr)

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US11535825B2 (en) 2015-08-28 2022-12-27 TACS Bio Inc. Adult stem cell compositions and methods of identification and isolation

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WO2011041240A1 (fr) * 2009-09-30 2011-04-07 The Brigham And Women's Hospital, Inc. Compositions de greffe tissulaire et méthodes d'utilisation
EP2039348A1 (fr) * 2007-09-21 2009-03-25 Jürgen Schliefelbein Préparation cosmétique et procédé pour obtenir une préparation de cellule souche somatique
EP2379089B1 (fr) * 2008-12-19 2019-04-17 DePuy Synthes Products, Inc. Regeneration et reparation du tissu nerveux suite à une blessure
WO2013148334A1 (fr) * 2012-03-27 2013-10-03 Parcell Laboratories, Llc Compositions et méthodes de réparation de disque intervertébral

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