WO2012125443A2

WO2012125443A2 - Methods and compositions for treating inflammatory dermatoses

Info

Publication number: WO2012125443A2
Application number: PCT/US2012/028430
Authority: WO
Inventors: Andrey Semechkin; Nikolay A. Turovets; Larisa S. Agapova; Ruslan A. Semechkin
Original assignee: International Stem Cell Corp
Current assignee: International Stem Cell Corp
Priority date: 2011-03-11
Filing date: 2012-03-09
Publication date: 2012-09-20
Anticipated expiration: 2013-09-11
Also published as: WO2012125443A3

Abstract

Disclosed are methods and compositions for the treatment of an inflammatory dermatosis selected from acne (especially, inflammatory acne), psoriasis, eczema and/or rosacea by topical application of a dermatologic composition consisting essentially of lysate of human parthenogenetic stem cells ("hpSC lysate"). The methods of the present invention include topical monotherapy of an inflammatory dermatosis with a dermatologic composition consisting essentially of hpSC lysate as well as co-therapy by topical application of dermatologic composition consisting essentially of hpSC lysate with other active pharmaceutical and/or cosmetic ingredients.

Description

Methods and Compositions for Treating Inflammatory Dermatoses

Cross-Reference to Related Applications

This application claims the priority benefit of U.S. Provisional Application Ser. No. 61/451 ,957 filed on March 11 , 2011 , the contents of which are incorporated herein by reference in their entirety.

Field of Invention

The present application is directed to methods and compositions for treating a dermatologic condition selected from the group of acne (especially, inflammatory acne), psoriasis, eczema and rosacea.

Summary of the Invention

The methods and compositions of the present invention are directed to treatment of acne (especially, inflammatory acne), psoriasis, eczema and/or rosacea by topical application of a dermatologic composition consisting essentially of lysate of human parthenogenetic stem cells (referred to as "hpSC lysate").

The methods of the present invention include topical monotherapy of an inflammatory dermatosis with a dermatologic composition consisting essentially of hpSC lysate. As will be understood by the skilled artisan, by topical monotherapy is meant administration of the dermatologic composition of the present invention (i.e., consisting essentially of hpSC lysate) without administration of another topical therapeutic product directed toward the treatment of an inflammatory dermatosis.

The methods of the present invention are also directed to co-therapy by topical application of dermatologic composition consisting essentially of hpSC lysate with other active pharmaceutical and/or cosmetic ingredients. "Co-therapy" is also referred to synonymously in the present application as "co-administration". Depending on the dermatologic condition(s) for which improvement is desired, the methods of the present invention include adding a condition-specific active ingredient or combination of active ingredients to the dermatologic composition. In other embodiments, the dermatologic composition consisting essentially of hpSC lysate is co -administered with a second separate topically-applied composition containing a different cosmetic or pharmaceutical active ingredient. Methods according to this embodiment are directed to improved and/or more rapid treatment of the dermatologic condition due to the co-administration of the topical composition consisting essentially of hpSC lysate.

Dermatologic Conditions Treated by Methods of the Present Invention

Acne is a disorder of the skin's sebaceous glands that results in clogged pores and lesions, namely comedones, papules and/or pustules. Acne may be further characterized by small nodulo-cystic lesions. Papules are typically less than 1 mm in diameter and may be further described as either (i) whiteheads, which are closed and contain sebum and keratotic debris or (ii) dark-gray, blackheads in which the core of debris is dark in color due to exposure to air and oxidation. Acne lesions usually occur on the face, neck, back, chest, and shoulders.

As used in the present application, by "inflammatory acne" is meant a

dermatologic condition in which a patient present with multiple inflammatory lesions as well as several to many comedones and papules/pustules. Patients with inflammatory acne may (or may not) present with small nodulo-cystic lesions.

Psoriasis appears in a variety of forms, each with distinct characteristics. Plaque psoriasis is the most prevalent form and is characterized by raised, inflamed, red lesions covered by silvery white scales or plaques. Psoriasis is typically found on the elbows, knees, scalp and lower back. Other types of psoriasis include (i) guttate psoriasis - small, red spots on the skin, usually on the trunk and limbs, which are not usually as thick as plaque lesions; (ii) inverse psoriasis - smooth and shiny bright-red lesions in skin folds (e.g., the armpits, groin, under the breasts); and pustular psoriasis - white blisters of surrounded by red skin.

The clinical presentation of atopic dermatitis, also referred to as eczema, varies among patients. The most common symptoms are dry, itchy skin and rashes on the face, inside the elbows and behind the knees, and on the hands and feet. Scratching leads to redness, swelling, cracking, "weeping" clear fluid, and finally, crusting and scaling.

Rosacea is a dermatologic condition characterized by livid red erythema and telangiectasias, principally localized on nose, cheeks, chin, forehead and glabella.

Patients having rosacea also present with episodes of flushing, papules and pustules. Dermato logic Compositions Consisting Essentially of hpSC Lysate

Human oocytes parthenogenetically activated stem cells ("hpSCs") differ from human embryonic stem cells from normally-fertilized oocytes. The alleles found near the centromeres of the DNA and at the distal ends of the DNA of heterozygous hpSCs exhibit a larger than average homozygosity than do somatic cells derived from the oocyte donor. Comparative analysis of single nucleotide polymorph markers shows that hpSCs display a lower proportion of heterozygosity near both the centromeres and the teleomeres in comparison with the heterozygosity in the intervening regions, as compared to human embryonic stem cells activated with contribution of a male gamete. Because there is no male gamete, there are no imprinting patterns donated by the male genome (i.e., no "paternal imprinting").

Parthenogenetic activation of human oocytes to obtain hpSCs can be carried-out according to the method described in United States Patent No. 7,732,202, incorporated herein in its entirety by reference. However, other methods of parthenogenetic activation of human oocytes are known and can be employed to obtain the hpSCs useful to make the hpSC lysate essential to the composition and method of the present invention. For example, the oocyte may be activated by chemical, mechanical, and/or electrical methods known in the art. Important is that the activation of the oocyte be accomplished without participation of a male gamete. However, the parthenogenetic oocyte activation methods in United States Patent No. 7,732,202 are preferred methods for parthenogenetic activation of human oocytes to obtain the hpSCs useful in obtaining the hpSC lysate of the compositions and methods of the present invention. The hpSC lysate can be derived from one or more human parthenogenetic stem cell lines.

Parthenogenetically activated human oocytes are cultured, preferably in a non- xenogenic medium, until a quantity of cells sufficient to an isolate inner cell mass (ICM) that, in turn, provides the hpSCs that are cultured to obtain the cells from which the hpSC lysate useful in the practice of the present invention is obtained. Isolation of the ICM can be done mechanically, or by immune surgery to remove the trophectoderm, to provide the hpSCs for further propagation and processing according to processes described below to obtain the hpSC lysate useful in the practice of the present invention. Importantly, the hpSCs from which the hpSC lysate is obtained are cultured in a culture vessel in feeder-free medium that, in preferred embodiments, is non-xenogenic medium. That is, hpSCs are cultivated without feeder cells, for example fibroblasts, in a medium that preferably does not include compositions from a mammal that is other than a human, i.e., in non-xenogenic medium. A combination of Dulbecco's modified Eagle medium and Ham's medium, supplemented with minimal essential medium (including non-essential amino acids), further augmented with Ham's fibroblast growth factor and human Actavin A is an example of a non-xenogenic medium useful in cultivating the hpSCs employed to obtain the hpSC lysate useful in the practice of the present invention. Reference to hpSC medium in the remainder of this description will be understood to mean culture medium that, in preferred embodiments, is free of xenoreagents.

To obtain a volume of hpSCs sufficient to obtain the desired quantity of hpSC lysate, preferably in non-xenogenic medium, equilibrated before use at 37°C in an atmosphere of 5% C0₂ / 5% 0₂ or under ambient conditions (about 21% 0₂) is used. Culture media, preferably free of xenoreagents, useful in cultivating hpSCs from which the lysate can be obtained are known in the art. An example of a suitable cell culture medium is as follows:

Knockout™ D-MEM/F12 (Gibco)

15% Knockout™ Serum Replacement XenoFree (Gibco)

GlutaMAX-1 (Gibco) lOOx

MEM NEAA (Gibco) 200x

2-Mercaptoethanol (Gibco) lOOOx

5 ng/ml Human FGF-basic (PeproTech)

20 ng/ml Recombinant human Activin A (R&D Systems) Human parthenogenetic stem cells hpSCs from blastocyst ICM are passaged into hpSC culture medium contained in a culture vessel. The culture vessel in this step and in all steps in the method is passivated with human serum, removed from the culture vessel before passaging. Prior to first passaging, the culture vessel and hpSC are incubated at 37°C (5%) C0₂ / 5%> 0₂). The first passage cells are incubated ca. 30 min, the medium is separated, and the cells washed in situ with calcium- and magnesium-free PBS. The cells are then treated with collagenase type IV at 37°C (5% C0₂ / 5% 0₂). The collagenase is separated and the remaining cells washed with calcium- and magnesium-free PBS, after which hpSC is introduced to the culture vessel. Cells are divided 1 :3, passaged into passivated culture vessels, and incubated (ca. 7 days) in hpSC medium at 37°C (5% C02 / 5% 02). Cells are divided, passaged, and cultivated (ca. 7 days) in hpSC medium until a desired volume of hpSC, free of xenoreagents, is obtained.

To harvest and lyse the hpSCs, hpSC culture medium is removed from the culture vessel(s) and cells are trypsinized with non-animal origin trypsin replacement enzyme. TrypLE™ available from Gibco (LifeTechnologies Corp., Carlsbad CA). The replacement enzyme is subsequently neutralized with hpSC culture medium that, importantly, is free of proteinase inhibitors. Harvesting and lysing of hpSCs without use of proteinase inhibitors is an important feature of the present invention.

Following neutralization of the trypsin replacement enzyme, the hpSCs and conditioned medium are centrifuged to pellet the cells and, importantly, supernatant conditioned medium is separated from the pellet.

The pelleted cells are suspended in isotonic solution, incubated, and subjected to three or more, preferably 4 or more freeze (liq. N2) - thaw cycles. The resulting suspension is centrifuged and the supernatant that is the hpSC lysate of the present invention is separated, free of conditioned medium. The hpSC lysate can be used immediately, or cryostored (e.g., -80°C) until used.

While the lysate can be applied directly to the skin to treat dermatologic conditions within the scope for the present invention, in preferred embodiments the hpSC lysate is, encapsulated or contained within liposomes.

Liposomes are microscopic spherical vesicles formed by hydrating phospholipids, including lecithins, phosphatidyl ethanol amines, or sphingomyelins. In the compositions of the present invention, liposomes based on lecithins are preferred

Methods of preparing liposomes are well known in the art. Generally, under low- sheer mixing in water, the phospholipids arrange themselves in multi-lamellar sheets of concentric phospholipid spheres in a hydrophilic head-to-head and hydrophobic tail-to- tail configuration. It is well-known to persons having skill in the art that liposomal vesicles can be designed to entrap and deliver (at desired rates of release) specific active ingredients by varying the lipid content, size, surface charge and method of preparation. Liposomes can be sized within a desired particle size range and particle size distribution by methods known in the art.

Preferably liposomes of the present invention are less than one micron.

With respect to surface charge, cationic charged liposomes may be prepared by incorporating stearylamine as a charged lipid into a formulation. Nonionic and anionic charged liposomes may be prepared by altering the molarity of dipalmitoylphosphatidyl- glycerol as the charged lipid.

Liposomes of the present invention can be made of a single type of phospholipid or by mixtures of different phospholipids. Non-phospholipid components such as cholesterol, fatty acids and other lipid soluble molecules including, preferably, vitamin E may be added to the liposomes.

In preferred embodiments, the liposomes serve as carriers for both water-soluble conditioned cell media and hydrophobic active ingredients.

In certain embodiments the hpSC lysate is cultivated without feeder cells in a culture medium that, in more preferred embodiments, is free of xenoreagents.

As used in the present application, by the term "consisting essentially of is meant that additional active pharmaceutical ingredients and/or cosmetic ingredient may be added to, or co-administered with the dermatologic composition containing hpSC lysate, provided that the additional ingredient(s) do/does not negatively impact the efficacy of the method of treatment. By "negative impactly" is meant causing one or more adverse clinical events, including, for example, skin irritation, erythema, increasing the number of lesions associated with inflammatory dermatoses (comedones, papules and/or pustules), increasing the time to clearance or remission of inflammatory dermatoses, causing skin dryness, loss of skin moisture, impairment of skin barrier function, or otherwise exacerbating a pre-existing dermatologic condition.

The hpSC lysate, whether or not within a liposomal dispersion or other delivery system, is present in an amount that is therapeutically effective to reduce the clinical manifestations of an inflammatory dermatosis and/or the frequency of recurrence of the inflammatory dermatosis and/or the time of clearance or remission of the inflammatory dermatosis. In embodiments where the inflammatory dermatosis is acne, the hpSC lysate is present in the dermatologic composition at a concentration such that when applied to skin manifesting acne, the number of comedones, papules and/or pustules associated with acne are reduced.

In embodiments where the inflammatory dermatosis is psoriasis, the hpSC lysate is present in the dermatologic composition at a concentration such that when applied to skin manifesting psoriasis the number of plaques and scales associated with psoriasis are reduced.

In embodiments where the inflammatory dermatosis is rosacea, the hpSC lysate is present in the dermatologic composition at a concentration such that when applied to skin manifesting rosacea the degree of erythema associated with rosacea is reduced.

In embodiments where the hpSC lysate is in a liposomal dispersion, the dispersion is present at a concentration of from about 0.1 to about 10.0% wt/wt of the composition, preferably from about 1% to about 8% wt/wt of the composition, still more preferably from about 5% to about 8% wt/wt. As used herein in connection with a measured quantity, "about" refers to a variation in the measured quantity that would be expected by the skilled artisan performing the measurement and exercising a level of care

commensurate with the objective of the measurement and the equipment used; and includes rounding errors.

Co-Administered Cosmetic and/or Pharmaceutical Active Ingredients

Certain embodiments of the present invention are directed to methods of treating a dermatologic condition selected from the group of acne, psoriasis, eczema and rosacea, where the method involves topical application of a "retinoid". While the retinoid may be added to the dermatologic composition of the present invention, the methods of the present invention are also directed to co-administration of two dermatologic

compositions, a first composition according to the present invention (i.e., consisting essentially of hpSC lysate) and a second composition comprising a retinoid.

As used in the present invention by the term "retinoid" is meant a natural or synthetic analog of vitamin A, including geometric isomers and stereoisomers, and includes the following compounds: retinol; retinal; C₂ - C₂₂ alkyl esters of retinol;

including retinyl palmitate, retinyl acetate, retinyl propionate; retinoic acid (including all- trans retinoic acid and/or 13-cis-retinoic acid); as well as compounds described as retinoids in U.S. Patent Nos. 4,677,120; 4,885,311; 5,049,584; and 5,124,356.

Retinoic acid is preferably present at a concentration of from about 0.05% to about 0.1%, more preferably from about 0.01 to about 0.1%.

Retinol is preferably present at a concentration of from about 0.125% to about

1.0%, more preferably from about 0.25 to about 1.0%.

Retinoids are preferably contained within a time-release delivery system, including liposomal dispersions and micron-sized spherical particles having a continuous non-collapsible network of pores open to the exterior of the particles as described, for example, in U.S. Patent No. 5,955,109.

A non-limiting example of a commercially available dermatologic composition for the treatment of acne vulgaris containing a retinoid is Tazorac^® (cream or gel) containing 0.05% wt/wt or 0.1% wt/wt of the active pharmaceutical retinoid Tazarotene.

Other embodiments of the present invention are directed to methods of treating a dermatologic condition selected from the group of acne, psoriasis, eczema and rosacea, where the method involves topical application of one or a mixture of hydroxy acids selected from the group consisting of alpha hydroxy acids (AHAs), beta hydroxy acids, and/or polyhydroxy acids, as well as their salts, esters, and stereoisomers.

In one aspect of this embodiment, the hydroxy acid (or mixture of hydroxy acids) is/are added to the dermatologic composition of the present invention.

In another aspect of this embodiment, the method of the present invention is directed to co-administration of a two dermatologic compositions, a first composition according to the present invention (i.e., consisting essentially of hpSC lysate) and a second composition comprising a hydroxy acid or a mixture of hydroxy acids.

AHAs may be used in the methods of the present invention at concentrations ranging from about 0.1% to about 10%, more preferably from about 0.2% to about 5%, also preferably from about 0.5% to about 2%.

Preferred AHAs for use in the methods of the present invention are selected from the group of: 2-hydroxyethanoic acid (glycolic acid, hydroxyacetic acid); 2- hydroxy- propanoic acid (lactic acid); 2-hydroxybutane-l,4-dioic acid (malic acid); 2,3-dihydroxy- butane- 1,4-dioc acid (tartaric acid); 2-hydroxy-2-carboxypentane- 1,5-dioic acid (citric acid).

Salicylic acid is a preferred beta hydroxy acid, and is preferably included in dermatologic compositions used in methods of the present invention at concentrations of from 0.01% to about 10%, still more preferably from about 0.1% to about 5%, and even more preferably from about 0.2% to about 2%, by weight of the composition.

In methods of the present invention directed to methods of treating acne or rosacea, the following active pharmaceutical ingredients may be co-administered a dermatologic composition comprising, and preferably consisting essentially of, hpSC lysate: topical antibiotics - preferably, clindamycin, erythromycin, sodium sulfacetamide; benzoyl peroxide, alone or in combination with topical antibiotics, preferably

clindamycin; metronidazole; and azelaic acid.

In certain embodiments the co-administered active pharmaceutical ingredient is encapsulated in a microporous particle, for example sold under the tradename

Microsponge^®, a liposome, or other controlled release topical delivery system known in the art.

Hydrocortisone may be co-administered topically in the form a cream or ointment at a concentration of 2.5% wt/wt.

Betamethasone dipropionate is the 17,21 -dipropionate ester of Betamethasone and may be co-administered topically in the form of a lotion at a concentration of 0.05% wt/wt.

In embodiments of the present invention directed to the treatment of atopic dermatitis (eczema), either Tacrolimus or Pimecrolimus may be co-administered topically (e.g., in an ointment), respectively at a concentration of 0.03 or 0.1% wt/wt (Tacrolimus) or 1%) wt/wt (Pimecrolimus).

In embodiments of the present invention directed to the treatment of psoriasis, the combination of two active pharmaceutical ingredients calcipotriene (0.005%) and betamethasone dipropionate (0.064%) may also be topically co-administered (e.g., in the form of gel or ointment), including for scalp psoriasis and psoriasis vulgaris of the trunk and/or limbs. Topical products containing coal tars or synthetic derivatives thereof, typically at concentrations of from about 0.5% to about 5% wt/wt, may also be co-administered in treating eczema, psoriasis or seborrheic dermatitis.

Another aspect of the present invention is directed to improved methods for treating an inflammatory dermatosis by co-administration of (i) an orally-administered antibiotic or retinoid and (ii) a topical dermatologic composition consisting essentially of hpSC lystate. A non-limiting example of an orally-administered retinoid is Accutane^®. Formulation Example

The compositions of the present invention can be provided and used in any physical dosage form known in the dermatological and cosmetic arts for topical administration of active ingredients or medicaments. For example, the composition of the present invention can be provided and applied to skin in the form of a cream, a lotion, a gel, a serum or a spray that can be in the form of a single -phase dispersion, preferably a thickened aqueous dispersion, or an emulsion (e.g., oil-in-water, water-in-oil, silicone-in- water, water-in-silicone). Formulation and compounding of these forms is well known in the art and described, for example, in ML Schlossman, ed. Chemistry and Manufacture of Cosmetics, 4th Ed.

The dermatologic composition consisting essentially of hpSC lysate used in the monotherapy and co-therapy methods of treating inflammatory dermatoses of the present invention, in certain of its embodiments, is illustrated by the following non- limiting example.

Phase A Stearic Acid Soap 0.50 - 5.00

Fatty Alcohol 0.50 - 4.00

Glyceryl Monostearate 1.00 - 4.00

Sorbitan Oleate 0.20 - 2.50

Polysorbate 80 0.20 - 2.50

Safflower Oil 0.50 - 2.00

Retinyl Palmitate 0.10 - 0.30

Phase B Carbomer 0.10 - 0.70

Preservatives 0.50 - 3.00

Glycerin 0.50 - 5.00

Chelating Agent (e.g., EDTA) 0.02 - 0.08

Hyaluronic Acid 0.05 - 1.00

Sodium PCA 0.10 - 1.00 Phase C Liposomal Dispersion of hpSC Lysate 5.00 - 8.00

Hydrocortisone 0.50 - 1.00

Vitamin E 1.00 - 4.00

Aloe Vera Extract 2.00 - 6.00

Bioflavonoids 1.00 - 3.00

B Vitamin Complex 2.00 - 8.00

Clinical Testing Examples

The topical composition of the present invention (consisting essentially of hpSC lysate), alone or in co-therapy (i.e., co-administration with a second medicament) may be used to reduce the clinical manifestations of inflammatory dermatoses. This reduction may be expressed in terms of the number of lesions (comedones, papules and/or pustules), the degree and extent of erythema, as well as the frequency of recurrence of lesions.

Additionally, the methods and compositions of the present invention, both as monotherapy and co-therapy, may be used to reduce the time needed to observe clearance or remission of the inflammatory dermatosis. Observation of improvement (reduction in clinical manifestations) may be self-assessed or by a clinician (e.g. a dermatologist).

The efficacy of methods of the present invention in treating acne may be assessed by one or more methods known to the skilled artisan, including is measured based on lesion count and the acne classification scheme described by PE Pochi et al. "Report of the consensus conference on acne classification" J. Am. Acad. Dermatol., Vol. 24, pp. 495-500 (1991) as well as based on clinical photography as described in C.H. Cook et al. "An Acne Grading Method Using Photographic Standards" Arch. Dermatology, 571-575 (1979).

The efficacy of methods of the present invention in treating rosacea may be assessed by a dermatologist as described in "Standard Grading System for Rosacea: Report of the National Rosacea Society Expert Committee on the Classification and Staging of Rosacea" published in Journal of the American Academy of Dermatology, Volume 50, Issue 6 (June 2004).

The efficacy of methods of the present invention in treating atopic dermatitis, including specifically, eczema may be assessed clinically using the Eczema Area and Severity Index (EASI) as further described in J.M. Hanifin et al. Experimental

Dermatology, Vol. 10, pp. 11-18 (2001).

Among the improvements that may be measured by the methods of the present invention are one or more of (i) reduction in transepidermal water loss, (ii) improved skin hydration, (iii) improved skin elasticity (e.g., as measured with a ballistometer), and/or (iv) reduced erythema (e.g., as measured with a colorimeter).

Claims

We claim:

1. A method for reducing the clinical manifestations of an inflammatory dermatosis, the inflammatory dermatosis being one or more of (i) eczema (ii) psoriasis or (iii) acne, the method comprising the step of applying a dermatologic composition consisting essentially of hpSC lysate to the face or portion of the body having clinical

manifestations of the inflammatory dermatosis.

2. The method for claim 1 whereby the frequency of recurrence of the inflammatory dermatosis is reduced.

3. The method for claim 1 whereby the time of clearance or remission of the inflammatory dermatosis is reduced.

4. The method of claim 1 wherein the inflammatory dermatosis is acne and whereby the number of comedones, papules and/or pustules associated with acne are reduced.

5. The method of claim 1 wherein the inflammatory dermatosis is psoriasis and whereby the number of plaques and scales associated with psoriasis are reduced.

6. The method of claim 1 wherein the inflammatory dermatosis is rosacea and whereby the degree of erythema associated with rosacea is reduced.

7. An improved method for treating an inflammatory dermatosis by topical administration of a therapeutically-effective amount of an active pharmaceutical ingredient, the inflammatory dermatosis being selected from the group consisting of psoriasis, eczema and acne, the active pharmaceutical ingredient being selected from retinoids, corticosteroids, topical antibiotic ingredients, and hydroxy acids, wherein the improvement comprises topical co-administration of a dermatologic composition consisting essentially of hpSC lysate.

8. The method of claim 7 wherein the hpSC lysate is in the form a dispersion and is present at a concentration of from 0.1% to 10% based on the total weight of the dermatologic composition.

9. An improved method for treating inflammatory dermatosis by oral administration of a therapeutically-effective amount of an active pharmaceutical ingredient, the inflammatory dermatosis being selected from the group consisting of psoriasis, eczema and acne, the active pharmaceutical ingredient being selected from retinoids and antibiotic ingredients, wherein the improvement comprises topical co-administration of a dermato logic composition consisting essentially of hpSC lysate.