Patent Application
20230102941
The present invention relates generally to modulation of the Wht/β-catenin pathway in mammalian (both human and non-human) cells and tissues, and more particularly to novel CREB binding protein (CBP)/β-catenin inhibitors and prodrugs thereof, cosmetic, and therapeutic uses thereof (e.g., in dermatological applications), and methods of making the disclosed exemplary compounds.
Skin is the largest organ of human body, and along with hair and nails, skin acts as a boundary between the body and the external environment, protecting the body against pathogens and excessive water loss. Skin is also critical for insulation, temperature regulation, sensation, and the synthesis of molecules such as vitamin D. Skin and hair are additionally important in self-image and self-esteem. Skin care and dermatological treatments, including for healing of wounds, ulcers and burns, and treatment of acne, atopic dermatitis, psoriasis, alopecia, as well as for providing cosmetic, anti-aging effects, are thus needed or desired to improve health and appearance of the outer epidermis, the underlying dermis, and other tissues.
Wounds, ulcers and burns, which are either injury induced (e.g., cuts, abrasions either from injury or treatments such as laser mediated dermabrasion, blisters) or surgically induced (e.g., surgical incisions, ostomies) require localized treatment to remedy the affected area and to prevent further dermal damage. Most current medications for wounds, ulcers, burns, and skin diseases or disorders, however, merely focus on relief of the symptoms (e.g., inflammation), failing to target and address the underlying cause of the problem (see, e.g., Okur, M. E., et al., Asian J Pharm Sci. 2020; 10:661-684), and, therefore, do not speed the healing process. Similar challenges exist for cosmetic skin improvement and hair growth. For example, skin is the most visible reflection of human health and vitality, and wrinkles are an early sign of aging, which can have a negative effect on quality of life. Although Botox is one of the most common wrinkles treatments, it does not heal the skin and its use to reduce wrinkles must be under strict control due to its high toxicity (Benedetto, A. V., Clinics in dermatology. 1998; 16:129-139). Thus, novel safer and effective agents for skin care and dermatological treatments are needed.
As the largest organ in the human body, skin undergoes significant turnover and harbors multiple distinct stem and progenitor cells and yet routinely heals with a scar. The WNT signaling cascade has been implicated in skin development and maintenance (see: e.g., Weltri, A., et al., STEM CELLS. 2018; 36:22-35). Secreted Wht proteins can stimulate multiple intracellular signaling pathways and act as growth factors that regulate diverse processes, including cell proliferation, differentiation, migration, and polarity. Among the Wnt stimulated pathways, Wht/β-catenin signaling is known as an important regulatory pathway that governs developmental processes and fate choices during tissue morphogenesis. Wnt signaling is one of the major cues directing skin development and maintenance. Although Wht signaling has been mainly implicated in HF (Hair Follicle) induction during skin development, it has also been recently shown to regulate epidermal stratification. In primary human keratinocytes, Wht5a acts as an autocrine stimulus to promote extracellular calcium-induced keratinocyte differentiation by coupling with the Wt/β-catenin pathway. Throughout life the skin epidermis is regularly renewed. Skin epidermal stem cells (SC), capable of self-renewal and differentiation, provide unlimited sources of cells to maintain tissue homeostasis, as well as to regenerate HFs and repair the epidermis after injury. Wnt signaling, including the Wnt/CBP/β-catenin pathway, is critical in all of these processes and Wht dependent signaling plays crucial roles in the maintenance, activation, and fate determination of the SC populations. Thus, development of effective pharmacological Wht/CBP/β-catenin antagonists and delivery strategies that result in an appropriately calibrated Wnt signaling modulation are needed to provide new and effective agents for treating skin related diseases and disorders, and effective cosmeceutical agents.
It is known that selective antagonists of the Wht/CBP/β-catenin pathway (e.g., ICG-001) are extremely safe at effective levels based on animal toxicity studies and clinical studies, and it was found that CBP/β-catenin mediated transcription is critical for stem cell/progenitor cell maintenance and proliferation (Emami, K. H., et al., Proc. Natl. Acad. Sci. U.S.A. 2004; 101:12682-12687). However, in order to cross the predominant barrier function of stratum corneum in skin, a strategy for enhancing drug delivery (e.g., in a cream, lotion, ointment, spray, gel, powder, etc.) should be used in order to enhance topical delivery. In aspects of the present invention, esterified moieties (prodrugs) of the disclosed exemplary selective CBP/β-catenin inhibitors having enhanced lipophilicity are used to facilitate absorption or permeation across the stratum corneum into the viable epidermis and beyond. Such esterified drug moieties may be readily cleaved to release the corresponding non-esterified drug when exposed to skin microenvironments and the esterases contained within.
Additionally, a recent preclinical study indicated that ICG-001, a selective antagonist of the Wht/CBP/β-catenin pathway, prevents the emergence of hapten-induced atopic dermatitis-like dermatitis (Matsuda-Hirose, H., et al., Ann Dermatol. 2019; 31(4):631-619). It is also known that humans can synthesize many of the fatty acids, with the exception of long chain fatty acids omega-3 (n-3) and omega-(n-6), which are called essential. Omega-3 fatty acids, represented by eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), can reduce the severity of symptoms in many inflammatory skin diseases, including psoriasis (Balbas, G. M., et al., Clin. Cosmet. Investig. Dermatol. 2010; 28:615-26).
There is an unmet need in the art, and the disclosed CBP/β-catenin inhibitors, and the ester-containing analogs thereof (e.g., fatty acid esters, such as omega-3 and omega-fatty acid containing drug analogs, etc.) provide new and effective agents for treating skin related diseases or disorders, and aging related conditions, including for cosmetic purposes, preferably via topical delivery. Aspects of the present invention provide such compounds and salts thereof, along with pharmaceutical and/or cosmeceutical compositions comprising the compounds. Methods for synthesizing and using the compounds and compositions, both therapeutically and cosmeceutically, are also provided.
Aspects of the present invention may be described in the following clauses:
1. A compound of formula (I):
and pharmaceutically and/or cosmeceutically acceptable salts thereof, wherein:
R1 and R2 are independently selected from hydrogen or CH3;
R3 is hydrogen or deuterium;
Q is a phenyl group; a substituted phenyl group having one or more substituents wherein the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, optionally substituted C1-4alkyl, optionally substituted C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzyl group with one or more substituents where the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, optionally substituted C1-4alkylamino, optionally substituted C1-4dialkylamino, halogen, perfluoro C1-4alkyl, optionally substituted C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl group; or a optionally substituted bicyclic aryl group having 8 to 11 ring members, which may have 1 to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur;
L is —C(O)—, or —SO2—;
X is —C(O)—, or —CH2C(O)—;
Y is hydrogen, or —C(O)R, wherein R is optionally substituted C1-C30 alkyl, optionally substituted C1-C30 alkenyl, or optionally substituted C1-C30 alkylene.
2. The compound of clause 1, wherein the compound is of formula (Ia):
or a pharmaceutically and/or cosmeceutically acceptable salt thereof, wherein:
R1, R2, R3, X, and Y are as defined in claim 1; and wherein
Q is selected from naphthyl, quinolinyl, isoquinolinyl, phthalazine, quinazoline, cinnoline, or naphthyridine.
3. The compound of clause 2, wherein the compound is of the formula (Ib):
or a pharmaceutically and/or cosmeceutically acceptable salt thereof, wherein:
Ra is defined in claim 2:
X1 is N, or —CH—; and wherein
Y is hydrogen, or —C(O)R, wherein R is selected from:
wherein m is 1 to 10,
or
4. The compound of clause 2, wherein the compound is of the formula (Ic):
or a pharmaceutically and/or cosmeceutically acceptable salt thereof, wherein:
R3 is defined as in claim 2;
X1 is N, or —CH—, and wherein
Y is hydrogen, or —C(O)R, wherein R is selected from:
wherein m is 1 to 10,
or
5. The compound of any one of clauses 1 to 4, or a pharmaceutically and/or cosmeceutically acceptable salt thereof, wherein the compound is:
6. A composition comprising at least one compound of any one of clauses 1 to 5.
7. The composition of clause 6, wherein the composition is a pharmaceutical and/or a cosmeceutical composition comprising a pharmaceutically acceptable or cosmeceutically acceptable excipient, respectively.
8. A method for cosmetically treating a skin condition, comprising topically administering to a mammalian subject in need thereof, a cosmeceutically effective amount of a compound of any one of clauses 1-5.
9. The method of clause 8, wherein the skin condition is mediated by CREB binding protein (CBP)/β-catenin signaling, and wherein the amount is sufficient to inhibit the CBP/β-catenin signaling.
10. The method of clause 8 or 9, wherein the skin condition comprises one or more skin conditions selected from roughness, fine lines or wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation, redness, rosacea, dyschromias, dryness, cracking, loss of vibrance, loss of elasticity, loss of firmness, sagging, loss of density, thinning, scarring, acne, and/or sun or U.V. damage.
11. The method of clause 10, where the amount is sufficient to reduce at least one of wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation, redness, rosacea, dyschromias, dryness, cracking, loss of vibrance, loss of elasticity, loss of firmness, sagging, loss of density, thinning, scarring, acne, and/or sun or U.V. damage.
12. The method of clause 8 or 9, wherein the skin condition comprises one or more of hair loss, loss of hair coloration, reduced eyebrow or eyelash growth, reduced cuticle growth, and/or reduced nail growth.
13. The method of any one of clauses 8 to 11, wherein the amount is sufficient to increase one or more of glycosaminoglycans (GAGs), elastin fibers, and/or collagen in the skin.
14. The method of any one of clauses 8-13, wherein topically administering comprises topically administering the compound in an amount in a range 0.125 mg to 1.250 mg at least once per day.
15. The method of clause 14, wherein topically administering comprises topically administering at least 0.375 mg of the compound at least once per day.
16. The method of clause 15, wherein topically administering comprises topically administering twice per day.
17. The method of clause 16, wherein topically administering comprises topically administering for a period of at least 4 weeks.
18. The method of clause 17, wherein topically administering comprises topically administering for a period of at least 8 weeks.
19. The method of any one of clauses 8-18, wherein, for the compound, Y is not hydrogen.
20. The method of clause 19, wherein Y is —C(O)R, and wherein R is selected from:
wherein m is 1 to 10.
21. Use of a compound of any one of clauses 1 to 5 for manufacturing of a cosmeceutical for cosmetic treatment of a skin condition.
22. The use of clause 21, wherein the skin condition comprises one or more of roughness, fine lines or wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation, redness, rosacea, dyschromias, dryness, cracking, loss of vibrance, loss of elasticity, loss of firmness, sagging, loss of density, thinning, scarring, acne, and/or sun or U.V. damage, hair loss, loss of hair coloration, reduced eyebrow or eyelash growth, reduced cuticle growth, reduced nail growth.
23. The use of clause 21 or 22, wherein, for the compound, Y is not hydrogen.
24. Use of a compound of any one of clauses 1 to 5 for manufacturing of a medicament for treatment of a skin disease or disorder.
25. The use of clause 24, wherein the skin disease or disorder is mediated, at least in part, by CREB binding protein (CBP)/β-catenin signaling, and wherein the treatment is sufficient to inhibit, at least in part, the CBP/β-catenin signaling.
26. The use of clause 24 or 25, wherein the skin disease or disorder comprises one or more of atopic dermatitis, psoriasis, acne, fibrosis, wounding, scarring, post-laser surgical scarring, burns, sun or U.V. damage, actinic keratosis, diabetic ulceration, and/or alopecia.
27. The use of any one of clauses 24-26, wherein, for the compound, Y is hydrogen.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Provided are compounds of formula (I):
and solvates (e.g., hydrates), metabolites, isotopically-labeled derivatives, and any pharmaceutically and/or cosmeceutically acceptable salts thereof, wherein:
R1 and R2 are independently selected from: hydrogen or —CH3;
R3 is hydrogen or deuterium;
Q is a phenyl group; a substituted phenyl group having one or more substituents wherein the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzyl group with one or more substituents where the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl group; or a bicyclic aryl group having 8 to 11 ring members, which may have 1 to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur;
L is —C(O)—, or —SO2—;
X is —C(O)—, or —CH2C(O)—;
n is 1 or 2;
Y is hydrogen, or —C(O)R, wherein R is C1-C30 alkyl, C1-C30 alkenyl, or C1-C30 alkylene.
The compounds may be of formula (Ia):
and solvates (e.g., hydrates), metabolites, isotopically-labeled derivatives, and any salts including pharmaceutically and/or cosmeceutically acceptable salts thereof, wherein:
R1, R2, R3, X, and Y are as defined above for formula (I); and wherein
Q is selected from naphthyl, quinolinyl, isoquinolinyl, phthalazine, quinazoline, cinnoline, or naphthyridine.
Preferably, the provided compounds are of formula (Ib) and formula (Ic):
and solvates (e.g., hydrates), metabolites, isotopically-labeled derivatives, and any salts including pharmaceutically and/or cosmeceutically acceptable salts thereof, wherein:
X1 is N, or —CH,
R3 is hydrogen or deuterium;
Y is hydrogen, or —C(O)R, wherein R is C1-C30 alkyl, C1-C30 alkenyl, or C1-C30 alkylene.
In the compounds of formula (I), (Ia), (Ib), and (Ic), where Y is not hydrogen, R is preferably selected from:
wherein m is 1 to 10,
or
Particular preferred compounds are:
and compositions, and pharmaceutical and/or cosmeceutical compositions comprising the compounds disclosed herein, and optionally comprising a pharmaceutically or cosmeceutically acceptable excipient or carrier.
According to aspects of the present invention, compounds of formula (I), formula (Ia), formula (Ib) and formula (Ic) are potent modulators of the Wnt/β-catenin pathway that inhibit CBP/β-catenin mediated signaling.
Provided, therefore, are pharmaceutical and/or cosmeceutical compounds, and compositions comprising these compounds, and uses of these compounds and compositions for the treatment of any aberrant CBP/β-catenin signaling-mediated skin related diseases, conditions, or disorders (e.g., dermatitis, psoriasis, alopecia, etc), or aging related skin conditions selected from roughness, fine lines or wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation redness, rosacea, dyschromias, dryness, cracking, loss of vibrance, loss of elasticity, loss of firmness, sagging, loss of density, thinning, scarring, acne, sun damage, hair loss, loss of hair coloration, reduced cuticle growth, reduced nail growth, reduced eyelash or eyebrow growth.
The compounds of formula (I), formula (Ia), formula (Ib) and formula (Ic) of the present invention may contain chiral centers and therefore may exist in different enantiomeric and diastereomeric forms. This invention relates to all optical isomers and all stereoisomers of compounds having the structures as defined above, both as racemic mixtures and as individual enantiomers and diastereoisomers of such compounds, and mixtures thereof, and to all pharmaceutical compositions and cosmeceutical compositions and methods of treatment defined below that contain or employ them, respectively. In some embodiments, the preferred compounds are the (S)-enantiomer. In other embodiments, the preferred compounds are the (R)-enantiomer. Alternatively, for the disclosed methods, the compounds are racemic mixtures.
As the compounds of the present invention may possess at least two asymmetric centers, they are capable of occurring in various stereoisomeric forms or configurations. Hence, the compounds can exist in separated (+)- and (−)-optically active forms, as well as mixtures thereof. Compounds and methods of the present invention encompass all such forms within the scope of the disclosure. Individual isomers can be obtained by known methods, such as optical resolution, optically selective reaction, or chromatographic separation in the preparation of the final product or its intermediate.
Aspects of the present invention also include isotopically labeled compounds, which are identical to those recited in the present invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 11C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and MCI, respectively. Compounds according to formula (I), formula (Ia), formula (Ib), and formula (Ic) described herein or pharmaceutically acceptable salts, tautomers, isomers, prodrugs, or solvates of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are encompassed within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 4C are incorporated, are useful in drug and/or substrate tissue distribution assays. In addition, it is known that the deuterium atom (2H) is a non-radioactive isotope of the hydrogen atom. Such compounds may increase resistance to metabolism, and thus may be useful in the disclosed methods for increasing the half-life of the compounds of the present invention or pharmaceutically acceptable salts, isomers, prodrugs, or solvates thereof, when administered to a mammal (see: e.g., Foster, A. B., Trends Pharmacol. Sci. 1984; 5(12): 524-527). Isotopically labeled compounds of this invention can generally be prepared by carrying out the procedures disclosed in the Examples of Preparations below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
Also provided are pharmaceutical compositions and pharmaceutical and/or cosmeceutical dermatologically acceptable topical formulations comprising one or more of the disclosed compounds in an amount sufficient, when topically administered to a warm-blooded mammalian subject having a skin condition, or disease or disorder mediated by aberrant CBP/β-catenin signaling, to specifically inhibit the CBP/β-catenin mediated signaling within the subject. The amount of the topical administered compound preferably comprises a cosmeceutically and/or therapeutically effective amount, and in such cases the pharmaceutical and/or cosmeceutical dermatologically acceptable compositions and formulations may comprise a cosmeceutically and/or therapeutically effective amount of the compound having a structure disclosed herein or a pharmaceutically and/or cosmeceutically dermatologically acceptable salt thereof and a pharmaceutically and/or cosmeceutically dermatologically acceptable carrier, diluent, or excipient therefor. All of these formulations are encompassed within the scope of the present invention. In preferred embodiments, the amount of the topically administered compound in the pharmaceutically and/or cosmeceutically dermatologically acceptable formulations is in the range of 0.5% (w/w) to 0.05% (w/w).
The term “CBP protein” refers to the protein that is also known as CREB-binding protein, where CREB is an abbreviation for “cAMP-response element binding.” This protein is well known in the art (see, e.g., Takemaru, K I., et al., J. Cell Biol. 2000; 149:249-54; U.S. Pat. No. 6,063,583).
The phrase “Wnt pathway” refers to a signaling cascade that may be initiated by the binding of Wht proteins (secreted glycoproteins) to frizzled seven-transmembrane-spanning receptors. This pathway is known and characterized in the art and is the subject of numerous articles and reviews (see: e.g., Huelsken, J., et al., J. Cell Sci. 2002; 115:3977-8; Wodarz, A., et al., Annu. Rev. Cell Dev. Biol. 1998; 14:59-88; Morin, P. J., Bioessays 1999; 21:1021-30; Moon, R. T., et al., Science 2002; 296:1644-46; Oving, I. M., et al., Eur. J. Clin. Invest. 2002; 32:448-57; Sakanaka, C., et al., Recent Prog. Horm. Res. 2000; 55:225-36). Classical Wht signaling (termed canonical Wnt signaling or Wnt/beta-catenin signaling) has as its hallmark a soluble pool of cytoplasmic beta-catenin, associated with the degradation complex that consists of the core proteins Axin, APC, GSK3 (glycogen synthase kinase 3), and CK1-alpha (casein kinase 1- alpha). In the absence of Wnt ligand, β-catenin is phosphorylated within this complex thereby targeting it for ubiquitination and subsequent destruction by the proteasomal machinery (Kohn, A. D., et al., Cell Calcium 2005; 38:439-446). Activation of the Wht pathway triggers a series of events that disrupts the APC/Axin/GSK3 complex that is required for the targeted destruction of β-catenin, and thereby promotes the stabilization and accumulation of β-catenin in the cytoplasm. This build-up in the cytoplasm coincides with the translocation of β-catenin into the nucleus through a mechanism that is still not entirely defined. In the nucleus, β-catenin, in the classical definition of the Wht signaling cascade, forms a complex with members of the TCF/LEF family of transcription factors. To generate a transcriptionally active complex, 0-catenin recruits the transcriptional coactivators, cAMP response element-binding protein (CREB)-Binding Protein (CBP) or its closely related homolog, p300 (E1A-Binding Protein, 300-KD) as well as other components of the basal transcription machinery, leading to the expression of a host of downstream target genes (Logan, V. Y., et al., Ann Rev Cell Dev Biol 2004; 20:781-810; Angers, S., et al., Ann Rev Cell Dev Biol 2009; 10:468-477).
“Alkyl” means a linear or branched, saturated, aliphatic radical having a chain of carbon atoms.
“Alkenyl” means a linear or branched, carbon chain that contains at least one carbon-carbon double bond.
“Alkylene,” unless indicated otherwise, means a linear or branched, saturated or un-saturated, aliphatic, polyvalent carbon chain.
“Protected derivatives” means derivatives of compound in which a reactive site or sites are blocked with protecting groups. A comprehensive list of suitable protecting groups can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.
“Isomers” mean any compounds having identical molecular formulae but differing in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers” and stereoisomers that are non-superimposable mirror images are termed “enantiomers” or sometimes “optical isomers.” A carbon atom bonded to four non-identical substituents is termed a “chiral center.” A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of the two enantiomeric forms is termed a “racemic mixture.” A compound that has more than one chiral center has 2n−1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a “diastereomeric mixture.” When one chiral center is present a stereoisomer may be characterized by the absolute configuration of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (e.g., see “Advanced Organic Chemistry,” 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).
“Animal” includes humans, non-human mammals (e.g., mice, rats, dogs, cats, rabbits, cattle, horses, sheep, goats, swine, deer, apes, monkeys, and the like) and non-mammals (e.g., birds, and the like).
“Mammal” includes human and non-human mammals, preferably warm-blooded mammals.
“Disease” specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition that may be caused by, or incident to, medical or veterinary therapy applied to that animal, i.e., the “side effects” of such therapy.
“Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
“Cosmeceutically acceptable” means that which is useful in preparing a cosmeceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human cosmeceutical use.
“Pharmaceutically acceptable salt” or “salt” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartarc acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. Pharmaceutically acceptable salts also include base addition salts, which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
“Amount effective to treat” or “therapeutically effective amount” means that amount which, when administered to an animal for treating a disease, is sufficient to effect such treatment for the disease.
A “cosmeceutically effective amount” is amount which, when administered (e.g., transdermal, topical) is sufficient to affect cosmetic treatment of a cosmetic condition (e.g., roughness, fine lines or wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation, redness, rosacea, dyschromias, dryness, cracking, loss of vibrance, loss of elasticity, loss of firmness, sagging, loss of density, thinning, scarring, acne, sun damage, hair loss, loss of hair coloration, reduced cuticle growth, reduced nail growth).
“Amount effective to prevent” means that amount which, when administered to an animal for preventing a disease, condition or disorder, is sufficient to effect such prophylaxis for the disease, condition or disorder.
“Treatment” or “treat” means any administration of a compound of the present invention and includes: (i) preventing a disease, condition or disorder from occurring in an animal which may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, condition or disorder; (ii) inhibiting the disease, condition or disorder in an animal that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); or (iii) ameliorating the disease, condition or disorder in an animal that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
“Topical delivery” refers to dermal delivery (e.g., delivery into the skin).
The exemplary inventive compounds disclosed herein provide novel antagonists with unexpected potency to inhibit CBP/β-catenin signaling. Based on animal toxicity studies and human clinical studies, and as recognized in the art, known specific CBP/β-catenin antagonists (e.g., ICG-001) are extremely safe at effective dose levels. Since many skin and hair conditions may require long-term administration, a large safety margin is favorable to physicians and patients alike. Thus, according to aspects of the invention, since the disclosed exemplary inventive compounds are specific CBP/β-catenin antagonists, they are of value in the treatment of any aberrant CBP/β-catenin signaling mediated skin related conditions, or diseases or disorders, including aging related skin conditions.
The present invention also provides prodrugs using one or more compounds of formula (I), formula (Ia), formula (Ib), and/or formula (Ic). The term “prodrug” as used herein refers to compounds that are rapidly transformed in vivo to yield the active drug having structure as defined herein, for example, during or after absorption by enzymatic cleavage in blood or tissues. A thorough discussion is provided in Higuchi et al., Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Roche (ed.), “Bioreversible Carriers in Drug Design,” American Pharmaceutical Association and Pergamon Press, (1987), both of which are hereby incorporated by reference. In the present invention, the use, for example, of an alkyl or fatty acid ester-containing prodrug approach is to enhance topical delivery via increasing the lipophilicity of the parent drug, and such change is designed to facilitate absorption or permeation across the stratum corneum into the viable epidermis and beyond, and importantly, to be readily cleaved to release the parent drug when exposed to skin microenvironments and esterases.
Provided, therefore, are inventive compounds that are specific CBP/β-catenin antagonists having substantial utility for use in treating skin related diseases or disorders or conditions, and for cosmetic applications (e.g., in aging subjects). Therapeutically relevant exemplary skin related diseases, conditions or disorders include those that occur in skin structure, including but not limited to wounds, acne, sun damage, certain skin diseases or conditions for which there is currently no cure (e.g., latent viral infection of epidermal or mucosal tissues), ulcers (e.g., diabetic), burns, atopic dermatitis, psoriasis. Cosmeceutically relevant exemplary conditions include, but are not limited to, effects of aging, and including roughness, fine lines or wrinkles, hyperpigmentation, hypopigmentation, mottled hyperpigmentation, dryness, redness, cracking, rosacea, dyschromias, firmness, elasticity, loss of firmness, sagging, loss of density, thickness, appearance, etc. Cosmetic usage may include improvement and preventive applications for both skin, nail and hair structure. In addition, the compounds of the present invention can be used to promote skin stem cell differentiation, and display a broad range of beneficial effects, such as accelerating skin healing, promoting skin maintenance, and delaying skin aging. Aspects of the invention, therefore, provide therapeutic and/or cosmetic methods for treating skin related diseases, conditions, or disorders, comprising administering (e.g., topically or otherwise) a therapeutically and/or cosmeceutically effective amount of an inventive compound to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. Adjunctive and combination therapy embodiments are also encompassed and provided.
Additional aspects of the present invention provide pharmaceutical and/or cosmeceutical compositions, comprising at least one of the inventive compounds disclosed herein and/or pharmaceutical and/or cosmeceutically acceptable salts thereof, and optionally comprise a pharmaceutically and/or cosmeceutically acceptable carrier, diluent, or excipient. The pharmaceutical and/or cosmeceutical compositions of the present invention are formulated to be compatible with an intended route of administration (e.g., forms of local or systemic administration). Preferably, administration of a compound of the present invention comprises topical administration. As used herein, a pharmaceutically and/or cosmeceutically acceptable carrier or diluent refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. The pharmaceutical and/or cosmeceutical compositions may further comprise inert diluents such as additional solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Pharmaceutical and/or cosmeceutical compositions of the present invention may include, in addition to a least one inventive compound, one or more of: water caprylic/capric triglyceride, glycerin, dirnethicone, cetearyl alcohol, arachidyl alcohol, arachidyl glucoside, cetearyl glucoside, sclerotium gum, tetrasodium glutamate diacetate, behenyl alcohol, xanthan gum, sodium hydroxide, citric acid, hexylene glycol, ethylhexylglycerin, caprylyl glycol, phenoxyethanol, etc.
Techniques for formulation and administration of pharmaceutical compositions can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co, Easton, Pa., 1990; and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes, Eds.). The pharmaceutical compositions are formulated to contain suitable pharmaceutically acceptable carriers, and optionally can comprise excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The mode of administration generally determines the nature of the carrier. The pharmaceutical and/or cosmeceutical compositions of the present invention can be manufactured using any conventional method, e.g., mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, melt-spinning, spray-drying, or lyophilizing processes.
The compounds and pharmaceutical and/or cosmeceutical compositions of the present invention can be formulated and employed in combination therapies or treatments, that is, the compounds and pharmaceutical and/or cosmeceutical compositions of the present invention can be formulated with or administrated concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures (e.g., such as simultaneously or adjunctively treating the subject with an anti-skin aging agent).
An optimal pharmaceutical and/or cosmeceutical formulation can be determined by one of skill in the art depending on the route of administration and the desired dosage. The present invention encompasses pharmaceutically or cosmeceutically dermatological acceptable formulations (e.g., topical) of inventive compounds. The term “pharmaceutically or cosmeceutically dermatological acceptable topical formulations,” as used herein, refers to any formulation which is pharmaceutically or cosmeceutically dermatological acceptable to deliver a therapeutically and/or cosmeceutically effective amount of a compound of the present invention in skin layers, to exert a local effect. In certain embodiments, the amount of the topical administered inventive compound in the pharmaceutically and/or dermatologically acceptable formulations is in the range of 0.5% (w/w) to 0.05% (w/w). In certain other embodiments, the topical formulation comprises a carrier system. As used herein, “pharmaceutically acceptable carrier,” “cosmeceutically acceptable carrier” and “carrier” generally refer to components that are chemically inert to the therapeutic agents, and that have no detrimental side effects or toxicity under the conditions of use, i.e., non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type (e.g., including creams and lotions, emulsions, jellies, depot formulations). Pharmaceutically and/or cosmeceutically dermatological effective carriers include, but are not limited to, solvents (e.g., water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., buffered saline) or any other carrier known in the art for topically administering therapeutics and/or consmeceutical agents. The pharmaceutically and/or cosmeceutically acceptable carriers may include polymers and polymer matrices, nanoparticles, microbubbles, and the like. In certain other embodiments, the topical formulations of the invention may comprise excipients. Any pharmaceutically and/or cosmeceutically dermatological acceptable excipient known in the art may be used to prepare the inventive pharmaceutically and/or cosmeceutically dermatological acceptable topical formulations. Examples of excipients include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffered agents, solubilizing agents, other penetration agents, skin protectants, surfactants, propellants. In certain embodiments, the composition formulation may also comprise at least one additional agent selected from the group consisting of carriers, adjuvants, emulsifying agents, suspending agents, sweeteners, flavorings, perfumes, and binding agents.
In general, most suitable means of administration of a compound or composition for a particular subject will depend on the nature and severity of the disease, disorder or condition being treated or the nature of the therapy and/or cosmetic treatment being used, as well as the nature of the therapeutic and/or cosmetic composition or additional therapeutic and/or cosmetic agent. Examples of routes of administration include intradermal, subcutaneous, transdermal (topical), and transmucosal administration. The pharmaceutical and/or cosmeceutical composition of the present invention is formulated to be compatible with its intended route of administration. Preferably, topical administration is used.
Accordingly, the final dosage regimen will be determined by good medical practice, considering various factors that modify the action of drugs, e.g., the agent's specific activity, the identity and severity of the disease, disorder or condition state, the responsiveness of the patient, the age, condition, body weight, sex, and diet of the patient, and the severity (e.g., of any infection). Additional factors that can be considered include time and frequency of administration, drug combinations, reaction sensitivities, and tolerance/response to therapy. Further refinement of the dosage appropriate for treatment involving any of the formulations mentioned herein is done routinely by the skilled practitioner without undue experimentation, especially in light of the dosage information and assays used.
Yet further aspects provide kits comprising an inventive compound or composition, a container, and optionally a package insert or label indicating a treatment. In one embodiment, the container may be a vial, jar, ampoule, preloaded syringe, or an intravenous bag. The term “package insert” refers to instructions customarily included in commercial packages of therapeutic and/or cosmetic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic and/or cosmetic products. Suitable containers include, e.g., bottles, vials, syringes, blister pack, etc. The container may be formed from a variety of materials such as glass or plastic.
An illustration of the preparation of exemplary compounds of the present invention is provided in the representative Examples and Schemes below, wherein specific non-limiting examples of compounds are intended to illustrate particular embodiments of the present invention, and are not intended to limit the scope of the specification or the claims in any way. The compounds of the present invention may be prepared by the synthetic sequence shown in the non-limiting Examples and Schemes below. A skilled artisan will appreciate that other routes of synthesis may be employed as well. In particular, other routes of synthesis may in fact be applied to certain aspects of the present invention. For example, (R)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(tert-butyldimethylsilyloxy)butanoic acid can be prepared according to Step 1-2 of Example 3, and used for the synthesis of compound 2. The skilled artisan is referred to general textbooks, such as March's Advanced Organic Chemistry (Michael B. Smith & Jerry March, Wiley-Interscience, 2000), The Practice of Medicinal Chemistry (Camille G. Wermuth, Academia Press, 2003) and Protective Groups in Organic Synthesis (Theodora W. Greene & Peter G. M. Wuts; John Wiley & Sons Inc, 1999), all incorporated by reference herein for their respective teachings.
Unless otherwise noted, all reagents, starting materials and solvents were obtained from commercial suppliers and used without further purification. Concentration or evaporation refers to evaporation under vacuum using a Buchi rotatory evaporator. Reaction products were purified by silica-gel chromatography with the solvent system indicated, or by HPLC purification using a C18 reverse phase semi-preparative HPLC column with solvent A (0.1% of TFA in water) and solvent B (0.1% of TFA in CH3CN) as eluents. All final products have at least 95% purity as determined by analytical HPLC analysis with UV detection at 210 nm and/or 254 nm. Reported yields are isolated yields.
Analytical HPLC analysis was performed on an Agilent 1100 HPLC with a Phenomenex Luna C18 (2) column (3 micron, 150×4.6 mm id) at a flow rate of 0.6 mL/min, eluting with a binary solvent system A and B using a 35%-70% B in 20 min and then 70%-95% B in 5 min (gradient elution 1), or 70%-95% B in 25 min and then 95%-100% B in 3 min (gradient elution 2) (A: Milli-Q water with 0.1% TFA; B: CH3CN with 0.1% TFA). NMR spectra were recorded on a Bruker AV-300 300 MHz NMR instrument using DMSO-de or CDCl3 with TMS as an internal standard. Mass spectra data was obtained with Bruker Esquire Liquid Chromatography-Ion Trap Mass Spectrometer.
The following abbreviations are used in the synthetic examples: aq (aqueous), h (hour), min (minutes), sat'd (saturated), THF (tetrahydrofuran), rt (room temperature), Et3N (triethylamine), NaCl (sodium chloride), MgSO4 (magnesium sulfate), CDCl3 (deuterated chloroform), H2O (water), HCl (hydrochloric acid), MeOH (methanol), NaOH (sodium hydroxide), TFA (trifluoroacetic acid), Na2CO3 (sodium carbonate), CH2Cl2 (methylene chloride), EtOAC (ethyl acetate), DMF (dimethylformamide), EtOH (ethanol), DMSO (dimethyl sulfoxide), DMSO-d6 (dimethyl sulfoxide-de), NaHCO3 (sodium bicarbonate), HPLC (high performance liquid chromatography), ESI-MS or MS (ESI) (electrospray ionization-mass spectrometry), NMR (nuclear magnetic resonance), DIEA (diisopropylethylamine), brine (saturated aqueous NaCl solution), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate), and other similar standard abbreviations are used herein.
Biological characterization of exemplary compounds of the present invention was performed in at least the following assays:
Assay of Wnt/driven TCF/LEF reporter (Luc) activity in a stably transfected celline.
The Wht/driven TCF/LEF reporter (Luc) activity in a stably transfected cell line assay was performed at BPS Bioscience (San Diego, Calif. 92121, USA). In brief, TCF/LEF reporter (Luc)-HEK293 cells are harvested from culture in growth medium and seeded at a density of about 35,000 cells per well into a white clear-bottom 96-well microplate in 80 μL of assay medium. 20 μL of 50 mM LiCl solution in assay medium are added to each well (final concentration 10 mM), and cells are incubated at 37° C. in a CO2 incubator for 16 hours. 10 μL of threefold serial dilution of mouse Wht3a in assay medium are added to each stimulated well. The plate is incubated at 37° C. in a CO2 incubator for 6 hours. 100 μL of ONE-Step™ Luciferase Assay buffer per well are added and the plate is shaken for 30 min prior to measuring luminescence using a luminometer.
SuperTOPFLASH Cell-based Luciferase Assay. Hek-293, STF1.1 cells are maintained in DMEM, 10% FBS, Pen-Strep supplemented with 200 μg/mL G418. On the day prior to assay, cells are split into a white, opaque 96-well plate at 10,000 cells per well in 50 μL of complete medium without G418 (for screening of Wnt-signaling inhibitors, G418 can be left out during screening process). After allowing the cells to stabilize and attach overnight, 40 μL of complete medium (without G418) containing 2.5× final concentration of compound or DMSO control is added to the cells and allowed to incubate for 1 hour at 37° C., 5% CO2 prior to adding 10 μL of a 100 mM LiCl solution prepared in complete medium (without G418). After 24 hours, 100 μL of BrightGlo™ (Promega, Cat. #: G7573) is added to each well and the plate is shaken for 5 minutes prior to reading on the Perkin-Elmer EnVision™ Plate Reader. For example, on the day prior to assay: cells are split into a white opaque 96-well plate at 10,000 cells per well in 50 μL of complete growth medium; the plate is incubated overnight at 37° C., 5% CO2 and the cells allowed to attach; the next day inhibitors to be tested are prepared in complete growth medium at 2.5× the desired final concentration (all conditions are done in duplicates), and 40 μL of the medium containing the 2.5× concentration of compound is added to each well (include 2 wells for stimulation control, 2 wells for DMSO control; once all inhibitors and controls are added, incubate the plate for 1 hour at 37° C., 5% CO2 (while plate is incubating, prepare fresh 100 mM LiCl in complete growth medium); after 1 hour, the plate is removed from the incubator and 10 μL of the medium containing 100 mM LiCl are added to each well (except for the two wells of the unstimulated control, to which 50 microliters of just complete medium is added); the plate is incubated for 24 hours at 37° C., 5% CO2; after 24 hours, 100 microliters of BrightGlo™ (Promega, Cat. #: G7573) is added to each well, the plate is shaken for 5 minutes to ensure complete lysis, and the plate is then read on a Perkin-Elmer EnVision™ 96-well plate reader.
Compound 1 was prepared according to the procedures disclosed in U.S. Pat. No. 7,671,054, as shown in Scheme 1, starting from 1-naphthaldehyde. The title compound 1 as a colorless oil. MS (ESI): m/z 493.3 (M+H)+; analytical HPLC: 15.3 min.
Compound 2 was prepared according to the procedures set forth in steps 1-6 of Scheme 2 below:
The compound was prepared according to the procedures reported (Andres J. M., et. al., Eur. J. Org. Chem. 2003; 17: 3387-3397; Luppi, G., et al., Synlett. 2003; 6:797-800), using (R)-3-amino-4-hydroxybutanoic acid as the starting material.
To a solution of (S)-2-amino-3-(4-tert-butoxyphenyl)-N-(2,2-diethoxyethyl)-N-(naphthalen-1-ylmethyl)propanamide (268 mg, 0.544 mmol) and (R)-3-(benzyloxycarbonylamino)-4-(tert-butyldimethylsilyloxy)butanoic acid (200 mg, 0.544 mmol) in CH2Cl2 (4.1 mL) and DMF (0.41 mL) at 0° C. was added HATU (207 mg, 0.544 mmol) and DIEA (0.174 mL, 1.09 mmol). The reaction mixture was stirred at 0° C. for 0.5 hour and at room temperature overnight under argon, and concentrated under reduced pressure, and then taken into ethyl acetate (25 mL) and water (25 mL). The organic layer was washed with brine (15 mL), dried (Na2SO4), and evaporated. The crude material was purified by silica-gel chromatography using 25% and 35% EtOAc/Hexane to give the desired product (0.246 g, 54%) as a colorless oil. MS (ESI): m/z 842.4 (M+H)+.
To a solution of benzyl (6R,10S)-10-(4-tert-butoxybenzyl)-14-ethoxy-2,2,3,3-tetramethyl-12-(naphthalen-1-ylmethyl)-8,11-dioxo-4,15-dioxa-9,12-diaza-3-silaheptadecan-6-ylcarbamate (246 mg, 0.29 mmol) in CH3OH (6 mL) was added Pd/C (26 mg). The reaction mixture was stirred under H2 atmosphere at room temperature overnight. Filtration via Celite and evaporation afforded the desired product (0.20 g, 95%) as a pale-yellow oil, which was used in the next step without further purification. Analytical HPLC: 23.7 min.
To a solution of (R)-3-amino-N-((S)-3-(4-tert-butoxyphenyl)-1-((2,2-diethoxyethyl) (naphthalen-1-ylmethyl)amino)-1-oxopropan-2-yl)-4-(tert-butyldimethylsilyloxy)butanamide (196 mg, 0.23 mmol) in CH2Cl2 (2.7 mL) was added benzyl isocyanate (54 μL, 0.39 mmol). The reaction mixture was stirred at room temperature overnight, and concentrated under reduced pressure. The resulting residues were purified on silica gel chromatography with 50% EtOAc/hexane to afford the title compound (0.16 g, 83%) as a white residue. MS (ESI): m/z 842.0 (M+H)+.
A solution of crude of (R)-3-(3-benzylureido)-N-((S)-3-(4-tert-butoxyphenyl)-1-((2,2-diethoxyethyl)(naphthalen-1-ylmethyl)amino)-1-oxopropan-2-yl)-4-(tert-butyldimethylsilyloxy)butanamide (0.156 g) in formic acid (8 mL) was stirred at room temperature over 20 hours and evaporated to dryness. The resulting residues were treated with conc. NH40H in CH3OH at room temperature over 2 hours and evaporated to dryness to give a crude product (128 mg). The crude material (33 mg) was purified by preparative HPLC to give the desired product (13 mg) as a white solid. MS (ESI): m/z 579.2 (M+H)+; analytical HPLC: 13.4 min (98% pure).
Compound 3 was prepared according to the procedures described in Example 3, using (S)-3-amino-4-hydroxybutanoic acid as the starting material. MS (ESI): m/z 579.2 (M+H)+; analytical HPLC: 12.0 min (99% pure).
Compound 4 was prepared according to the procedures described in Example 3, using (R)-2-amino-3-hydroxypropanoic acid as the starting material. MS (ESI): m/z 565.3 (M+H)+; analytical HPLC: 14.0 min (98% pure).
The compound (2R,5S,8aS)-N-benzyl-5-(4-hydroxybenzyl)-2-(hydroxymethyl)-7-(naphthalen-1-ylmethyl)-3,6-dioxohexahydroimidazo[1,2-a]pyrazine-1(5H)-carboxamide was synthesized, according to the procedures described in Example 3, using (S)-2-amino-3-hydroxypropanoic acid as the starting material. The desired product was obtained with a low yield.
Compound 5 is prepared according to the procedures described in Example 3, using (R)-2-amino-4-hydroxybutanoic acid as the starting material. MS (ESI): m/z 579.2 (M+H)+; analytical HPLC: 13.6 min (99% pure).
wherein m is 1 to 10
Exemplary compound 6, (2S,6S,9aS)-1-(benzylcarbamoyl)-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidin-2-yl)methyl dodecanoate, was prepared according to the procedures set forth in steps 1-3 of Scheme 3 below:
To a solution of (S)-3-(3-benzylureido)-N-((S)-3-(4-tert-butoxyphenyl)-1-((2,2-diethoxyethyl)(naphthalen-1-ylethyl)amino)-1-oxopropan-2-yl)-4-(tert-butyldimethylsilyloxy)butanamide (3.5 g, 4.16 mmol) in THF (62 mL) at 0° C. was added TBAF (1 N, 8.3 mL) dropwise. The reaction mixture was stirred at room temperature overnight under argon, and evaporated to dryness. The crude material was purified by silica-gel chromatography using 5% CH3OH/CH2Cl2 to give the desired product (3.3 g, 100%) as a white foam. MS (ESI): m/z 727.8 (M+H)+.
To a solution of (S)-3-(3-benzylureido)-N-((S)-3-(4-tert-butoxyphenyl)-1-((2,2-diethoxyethyl)(naphthalen-1-ylmethyl)amino)-1-oxopropan-2-yl)-4-hydroxybutanamide (1.98 g, 2.72 mmol) in dry CH2C2 (48 mL) at 0° C. was added pyridine (0.44 mL, 5.4 mmol) and lauroyl chloride (0.82 mL, 3.54 mmol). The reaction mixture was stirred under argon at 0° C. for 1 hour and at room temperature overnight. After addition of water (4.0 mL) at 0° C., the reaction mixture was evaporated to dryness. The crude material was purified by silica-gel chromatography using 5% CH3OH/CH2Cl2 to give the desired product (2.3 g, 93%) as a white solid. MS (ESI): m/z 910.4 (M+H)+.
A solution of (S)-2-(3-benzyureido)-4-((S)-3-(4-tert-butoxyphenyl)-1-((2,2-diethoxyethyl)(naphthalen-1-ylmethyl)amino)-1-oxopropan-2-ylamino)-4-oxobutyl dodecanoate (2.3 g, 2.5 mmol) in formic acid (80 mL) was stirred at room temperature over 24 hours and evaporated to dryness. The resulting residues were purified by silica-gel chromatography using 25% and 50% EtOAc/CH2Cl2 to give the desired product (1.28 g, 67%) as a white foam. MS (ESI): m/z 761.6 (M+H)+; analytical HPLC: 16.9 min (98% pure; gradient elution 2).
Exemplary compound 7, (4Z,7Z,10Z,13Z,16Z,19Z)-((2S,6S,9aS)-1-(benzylcarbamoyl)-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidin-2-yl)methyl docosa-4,7,10,13,16,19-hexaenoate, was prepared according to the procedures as described in Example 7, using docosahexaenoic acid chloride instead, as a pale-yellow foam. MS (ESI): m/z 889.7 (M+H)+; analytical HPLC: 22.1 min (96% pure; gradient elution 2).
Exemplary compounds, wherein Y is hydrogen, of the present invention were tested for Wnt/driven TCF/LEF reporter (Luc) activity in HEK293 cell line assay. The IC50 values of the compounds against TCF/LEF reporter activity are summarized in Table 1. As demonstrated in Table 1, the unexpected inhibitory activity or potency of the exemplary compounds of formula (Ib) and formula (Ic), wherein Y is hydrogen, is attributed to the optimal selection of the chirality at 2 positions of the bicyclic rings, and n equal 1 is required for optimal inhibition. In both bicyclo[5,6] and bicyclo[6,6] systems, the S-configuration of —CH2OH is needed to most potently inhibit the TCF/LEF reporter activity. The positive control compound for this assay is IWR-1-endo, which is known in the art as a non-CBP/β-catenin Wht antagonist, which stabilizes Axin-dependent β-catenin destruction (Chen, B., et al., Nat. Chem. Biol. 2009; 5(2):100-107).
For cosmetic evaluation, compound 6 (0.15% wt./wt.) formulated in an aqueous based vehicle was topically applied twice daily (0.25 g formulation per application) during an 8-week testing period according to the following daytime/nighttime regimen.
During the study, subjects used no other skin care regimen. Prior to application of the test formulation, subjects washed their faces with Purpose cleanser, rinsed and then dry patted their faces. For each daytime and each nighttime application, 0.25 g of the test formulation (2 pumps from an applicator) was then applied to the entire face, around the eyes and onto the neck. Photographs (without any face and eye make-up), and self-assessment was performed before and after the 8-week period.
Overview. Skin aging is a ubiquitous phenomenon affecting all individuals from birth. Aging is characterized by intrinsic aging, caused by metabolism and other internal body factors, and extrinsic aging, caused by external environmental factors such as solar exposure and pollution. This study examined the use of a novel agent for the improvement of facial appearance.
Objective. For cosmetic evaluation, a clinical study was performed to investigate the efficacy of compound 6 (0.15% wt./wt.) on clinical benefits associated with fine lines and wrinkles, uneven skin tone (dyschromias), sagging, firming, and contouring on the face and neck over the course of 8 weeks.
Design. In compliance with 21CFR Part 50), consenting healthy subjects, 35-65 years of age (20 female and 5 males) of Fitzpatrick skin types I-V (15 subjects with type I, 8 subjects with type II and 2 subjects with type V) with mild to moderate facial skin sagging, firming, contouring, nasolabial folds, and expression lines on the face (4-6 on a 10-point photoaging scale with rating options of 0-9) were enrolled in a single site 8-week study on facial photoaging.
Subjects received the study treatment product (0.15% wt./wt.NP002 in vehicle) to be applied (0.25 g formulation per application) to the face following cleansing, every morning and evening. If desired, SPF30 sunscreen was applied in the morning after the study product had dried on the face for 15 minutes. No other products was applied to the face.
Subjects were seen at baseline, week 4, and week 8. The dermatologist investigator and subjects assessed efficacy and tolerability at each visit on a 5-point ordinal scale. Photographs were taken of all subjects at baseline, week 4, and week 8 with the Visia CR 4.3 using standard lighting of the central, right, and left face.
Noninvasive assessments consisting of facial trans-epidermal water loss (TEWL), corneometry, ultrasound, and elasticity were conducted at baseline, week 4, and week 8.
Ten (10) volunteer subjects were selected to undergo skin biopsies from the upper inner arms. These 10 subjects in the biopsy sub-study applied 0.15% wt./wt. NP002 in vehicle (the study test product; 0.25 g formulation per application) and vehicle alone (the control; (0.25 g control per application)) to the randomized left and right upper inner arms each time they applied the study test product to the face. At week 8, the subjects had a 3 mm punch biopsy taken from the right and left upper inner arms where the test product and vehicle control had been applied. These biopsy sites were closed with sutures, which were removed after 2 weeks. The biopsy specimens were split, with half (1.5 mm) placed in formalin for H&E processing and laboratory analyses for collagen; elastin; and glycosaminoglycans (GAGs). Stains were performed with: H&E, elastic stain, and Alcian blue stain. The other 1.5 mm half of the biopsy was subjected to PCR analysis (see Example 14 below).
Endpoints. The study was IRB approved. Exclusion criteria included: no anticancer or immunosuppressive agents, no radiation; no facial retinoids, neurotoxins or fillers within 3 months; no history of immunosuppression/immune deficiency disorders; no cosmetic surgery within 12 months; etc. The tolerability endpoint was the investigator-assessed absence of skin irritation from the study product at any time during the 8-week study. The safety endpoint was the overall incidence of all adverse events reported during the study. The primary efficacy endpoint was statistically significant improvement in the dermatologist investigator's assessment of overall facial appearance in subjects using the study product for 8 weeks as compared to baseline.
Measures. Dermatologist investigator assessed efficacy parameters included: fine lines, wrinkles, uneven skin tone (dyschromias), radiance/brightness, skin roughness (tactile), skin roughness (visual), mottled hyperpigmentation, discrete hyperpigmentation, skin firmness, facial sagging, and overall appearance. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
Subject assessed efficacy parameters: Fine lines, wrinkles, radiance/brightness, skin roughness (tactile), skin roughness (visual), mottled hyperpigmentation, discrete hyperpigmentation, skin firmness, facial sagging, and overall appearance. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
Dermatologist investigator assessed tolerability parameters included: dryness, peeling, erythema, edema. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
Subject assessed tolerability parameters included: dryness, peeling, stinging, and itching. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
Noninvasive biomeasurements included: Ultrasound, TEWL, corneometry, and elasticity measurements on the face were conducted at baseline, week 4, week 8.
Photography: Color photographs were taken at each time point with standard 1 lighting of the central, right, and left face with a Visia CR4.3. Photographs were performed at the following time points: baseline, week 4, week 8.
Statistical methods. Along with descriptive statistics (means, standard deviations and percentages), investigator ordinal nonparametric data were analyzed using Wilcoxon signed rank test and sign test for paired comparison at different time points. The noninvasive parametric data were analyzed using paired t-test. Change was considered significant at the alpha level of 0.05.
Code Disclosure: A code was not maintained for facial application, as all subjects received the active study product. The 10 biopsy subjects were randomized to receive vehicle or vehicle plus active for application to the inner arms. The specimens were submitted for PCR analysis unblinded.
Adverse Events and Adverse Experiences. No adverse events or adverse experiences occurred during the conduct of the study. The study was run during the COVID-19 pandemic, but no cases were reported during the study.
Corneometry. Corneometry results are shown in Table 2.
Corneometry is a measure of the water in the skin. A higher number is indicative of superior moisturization. Measurements were taken from the same target area on the cheek at each visit. There was a 25% increase in skin water content at week 4 (p=0.004) and a 35% increase in water content at week 8 (p=0.003). This is a substantial increase in skin water indicating superior moisturization properties for the study product relative to the vehicle control.
Elasticity. Elasticity results are shown in Table 3.
Elasticity was measured from the face with a suction probe at the same target site on the cheek at each visit. Three values were obtained: VE representing viscoelastic properties, E representing Young's modulus, and R representing retraction time. Retraction time is the amount of time for the skin to snap back to its original conformation after distention with a negative pressure suction cup. Young's modulus represents the ability of the skin to stretch. The viscoelastic property is a calculated number based on the retraction time and Young's modulus. There were no statistically significant changes in skin elasticity.
Investigator Efficacy. As shown in Table 4, the dermatologist investigator assessed fine lines, wrinkles, uneven skin tone (dyschromias), radiance/brightness, skin roughness (tactile), skin roughness (visual), mottled hyperpigmentation, discrete hyperpigmentation, skin firmness, facial sagging, and overall appearance. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
After 4 weeks of use, there was a statistically significant 29% improvement in radiance (p<0.001), 21% improvement in tactile roughness (p<0.001), and 21% improvement in visual roughness (p<0.001). Improvement continued on into week 8 with an 18% improvement in fine lines (p=0.002), a 17% improvement in uneven skin tone (dyschromias)(p=0.002), a 43% improvement in radiance/brightness (p<0.001), a 41% improvement in skin roughness (tactile (p<0.001), a 39% improvement in skin roughness (visual)(p<0.001), a 12% improvement in mottled hyperpigmentation (p=0.008), a 24% improvement in skin firmness (p<0.001), and a 25% improvement in overall facial appearance (p<0.001). These findings demonstrate the strong anti-aging performance of the product with 8 weeks of use.
Investigator tolerability. As shown in Table 5, the dermatologist investigator assessed tolerability in terms of dryness, peeling, erythema, edema. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8. No tolerability issues were noted.
Subject efficacy. As shown in Table 6, the subjects were asked to assess fine lines, wrinkles, radiance/brightness, skin roughness (tactile), skin roughness (visual), mottled hyperpigmentation, discrete hyperpigmentation, skin firmness, facial sagging, and overall appearance. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
The subjects rated strong appearance improvement attributes after using the product for 4 weeks. There was a 22% improvement in fine lines (p<0.001), a 19% improvement in wrinkles (p=0.002), a 19% improvement in uneven skin tone (dyschromias)(p=0.001), a 24% improvement in radiance/brightness (p<0.001), a 24% improvement in skin roughness (tactile)(p=0.002), a 21% improvement in skin roughness (visual)(p=0.002), a 15% improvement in mottled hyperpigmentation (p=0.009), a 19% improvement in skin firmness (p=0.004), and a 22% improvement in overall facial appearance (p=0.001).
This improvement continued and increased into week 8 with a 34% improvement in fine lines (p<0.001), a 32% improvement in wrinkles (p<0.001), a 35% improvement in uneven skin tone (dyschromias)(p<0.001), a 41% improvement in radiance/brightness (p<0.001), a 46% improvement in skin roughness (tactile)(p<0.001), a 49% improvement in skin roughness (visual)(p<0.001), a 30% improvement in mottled hyperpigmentation (p=0.001), a 34% improvement in skin firmness (p=0.004), a 25% improvement in skin sagging (p=0.001), and a 35% improvement in overall facial appearance (p<0.001).
Thus, the subjects felt the study treatment product was highly effective at improving a variety of anti-aging parameters.
Subject Tolerability. As shown in Table 7, the subjects assessed tolerability in terms of dryness, peeling, stinging, and itching. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4, week 8.
There was a statistically significant increase in facial dryness at week 4 (p=0.004) and week 8 (p=0.016), indicating that subjects with dry skin might desire to use an additional moisturizer on top of the study treatment product. No other tolerability issues were identified.
Transepidermal Water Loss (TEWL). As shown in Table 8, TWEL measurements were taken from a target area on the cheek. TEWL was measured with 2 humidity meters spaced at a known distance from the skin surface that sensed water vapor. The amount of water vapor escaping from the skin is directly related to the strength of the skin barrier. A lower TEWL is indicative of a stronger skin barrier.
There was no statistically significant change in the TEWL indicating that the subject treatment product did not irritate or damage the skin barrier, consistent with the favorable investigator and subject tolerability assessments.
Ultrasound. As shown in Table 9, ultrasound measurements were taken to evaluate the skin thickness and intensity. The LEB, which is a line found in younger skin, was not assessed due to the age of the participants.
There was a statistically significant 23% increase in skin intensity at week 8, which correlates with an increase in skin density (p<0.001). The skin thickness increased 15% at week 8, but it was not statistically significant. The skin thickness likely would have become significant had the study been extended.
In conclusion, the primary efficacy endpoint was statistically significant improvement in the dermatologist investigator's assessment of overall facial appearance in subjects using the study treatment product (compound 6) for 8 weeks as compared to baseline. The primary efficacy endpoint was therefore met.
The tolerability endpoint was the investigator-assessed absence of skin irritation from the facial study treatment product at any time during the 8-week study. The tolerability endpoint was therefore met.
The safety endpoint was the overall incidence of all adverse events reported during the study. No adverse events or adverse experiences occurred during the conduct of the study. The study was run during the COVID-19 pandemic, but no cases were reported during the study. The safety endpoint was therefore met.
Overview. As described in Example 13, ten (10) volunteer subjects of the clinical study using compound 6 (coded as NP002) were selected to undergo skin biopsies from the upper inner arms. During the clinical study of Example 13, these 10 biopsy sub-study subjects applied 0.15% NP002 in vehicle (the study test product) and vehicle alone (the control) to the randomized left and right upper inner arms each time they applied the study test product to the face. At week 8, these subjects had a 3 mm punch biopsy taken from the right and left upper inner arms where the test product and vehicle control had been applied. These biopsy sites were closed with sutures that were removed after 2 weeks. The biopsy specimens were split, with half (1.5 mm) placed in formalin for H&E processing and laboratory analyses for collagen, elastin, and glycosaminoglycans (GAGs) (stains were performed with: H&E, elastic stain, and Alcian blue stain), as described in Example 15 (Histology) below. The other 1.5 mm half of the biopsy was subjected to gene expression (PCR)s analysis as described below in this Gene Expression Study Example.
Gene Expression Study. Gene expression of the individual sub-study subject biopsies was assessed using a standard qPCR-based gene expression panel (standard skin panel (SSP)) containing 107 target genes known to play important roles in skin biology, and 5 endogenous control genes.
RNA Isolation: RNA was isolated from each tissue using an RNeasy Mini kit {Qiagen) following the manufacturers instructions for fibrous samples. Each tissue was first cut into pieces of ˜30 mg, with 2 pieces isolated from each tissue. RNA from the 2 pieces was pooled together for analysis. RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer, RNA integrity was measured using a Bioanalyzer 2100.
cDNA Synthesis & Preamplification: cDNA was generated from 140 ng of total RNA per sample using a Superscript Vilo RT Kit and a custom primer pool according to the manufacturers instructions (Thermo Fisher). RT samples were preamplified for 14 cycles and diluted 1:20 with I×TE Buffer for qPCR processing.
qPCR Processing: qPCR reactions were run using validated Taqman® gene expression assays in an OpenArray format and in a 384-well format. All assays were run in a Life Technologies QuantStudio 12K Flex instrument. Each gene was assayed in duplicate.
Data Analysis: qPCR data quality and statistical analysis was assessed and performed on the raw data files using ThermoFisher Connect Software (Life Technologies). Statistical analysis was performed using the relative quantitation (RQ) method. In the first step of an RQ analysis, the Cq value of the target gene is normalized to the Cq value of an endogenous control gene to generate the delta Cq (dCq). dCq values are calculated in order to normalize for variability between the samples that may occur during the experimental procedures. Five candidate control genes (GAPDH, GUSB, HPRTI, PPIA, and UBC) were analyzed with the Standard Skin panel (OpenArray (OA)), and the most consistent endogenous control gene was chosen based on the stability score generated by the ThermoFisher Connect software. Statistics (unpaired t-tests) were carried out for each comparison using dCq values normalized to the endogenous control gene. PPIA was selected as the endogenous control gene for the All Donars comparisons presented herein (Table 9).
Statistical Data Analysis Using ThermoFisher Connect Software: Unpaired t-tests were carried out using Thermo Fisher Connect software. The statistical comparison generated delta delta Cq [dd Cq] values (the mean d Cq of the treated group—the mean d Cq of the control group). The statistical software converts the dd Cq values into log and linear RQ values for export [RQ=2−ddCq]. The linear RQ values were converted to linear fold-change values to simplify data interpretation; linear fold-change data was calculated from exported linear RQ values using Microsoft Excel: for RQ values ˜1.0, linear fold-change value=RQ value; and for RQ values <1.0, linear fold-change value=−1/RQ value.
RNA quality RNA quality was assessed using A260/280 and A260/230 readings, coupled with determining RIN (RNA integrity number) scores using an Agilent Bioanalyzer. The RIN scores (not shown) were determined to be adequate for the qPCR analysis.
qPCR Data Quality and Statistical Data Analysis: qPCR data quality is assessed using a combination of factors, including visual analysis of the shape of the qPCR curve and the Cq value. Cq values are an indication of the total amount of transcript present in the sample and can impact the quality of the qPCR data. qPCR amplification takes place over a total of 40 cycles, and typically occurs before cycle 28. The relative amount of the gene transcript level is associated with the Cq value of the PCR reaction. Cq values less than 28 are typically associated with high transcript levels and robust, high quality PCR data, whereas Cq values greater than 28 are typically associated lower level transcripts and less robust qPCR data. In this study, genes of the standard skin panel (SSP) that showed poor quality PCR amplification for all Donors were removed from the dataset (resulting in a SSP′ dataset) prior to performing the statistical analysis.
The changes in skin panel gene expression with linear fold-change values (FC) ≥1.5 for all donors is shown in Table 10.
Table 10 shows the overall changes in expression for this analysis. There were statistically significant changes (p<0.05). Amongst these the gene for Interleukin 1 alpha (II1A), a key inflammatory cytokine was significantly reduced (62%). II1A expression is associated with senescence (e.g., see Wiggins et al., Aging Cell, 2019 June; 18(3): e1294627; published online 2019 March 27, doi.org/10.1111/acel.12946), which involves the gradual deterioration of functional characteristics in living organisms with aging.
Although not reaching statistical, several other inflammatory cytokines were reduced including Interleukin 1 beta (IL1B) 56%, Interleukin 6 (IL6) 54%, both of which are also associated with the senescence-associated secretory phenotype or SASP, a hallmark of aging.
Caspase 3 (CASP3) has been found to be downregulated in three different in-vitro skin aging models, indicating that this gene is an important biomarker for aging studies (see, e.g., PLOS ONE|https://doi.org/10.1371/journal.pone.0219165 Jul. 3, 2019; and see also Eskandari & Eaves, J. Cell Biol. 2022 Vol. 221 No. 6 e202201159, doi.org/10.1083/jcb.202201159). Overall, CASP3 was upregulated 321% in this gene expression study.
Although compound 6 (NP002) is not per se an anti-inflammatory, it likely exerts an anti-inflammatory response via two mechanisms: 1) increasing skin barrier function by for example upregulation of Desmoglein-3 (DSG3), a gene involved in cell/cell junctions and also by 2) increasing NRF2 (gene NFE2L2) expression (see, e.g., Schafer et al., EMBO Mol Med 4, 364-379, 2012). NRF2 is a transcription factor is the master regulator of cellular redox homeostasis, which declines with age. The increased expression of bona fide downstream targets of NRF2, including Heme Oxygenase 1 (HMOX1) 201% and NAD(P)H dehydrogenase [quinone] 1 (NQO1) 166%, is consistent with a beneficial effect on oxidative stress and reduced inflammation.
An increase in the expression of Collagen 1A1 (Col1A1) 137% was also observed. Col1A1 expression decreases with age and decreased collagen is associated with the “old appearance” of skin as represented by wrinkles and sagging (see, e.g., PLOS ONE|https://doi.org/10.1371/journal.pone.0219165 Jul. 3, 2019)
Overview. As described in Example 13, ten (10) volunteer subjects of the clinical study using compound 6 (coded as NP002) were selected to undergo skin biopsies from the upper inner arms for analysis of collagen, elastin, and glycosaminoglycans (GAGs).
Methods. During the clinical study of Example 13, these 10 biopsy sub-study subjects applied (0.25 g formulation per application) 0.15% NP002 in vehicle (the study test product) and vehicle alone (the control) to the randomized left and right upper inner arms each time they applied the study test product to the face. At week 8, these subjects had a 3 mm punch biopsy taken from the right and left upper inner arms where the test product and vehicle control had been applied. These biopsy sites were closed with sutures that were removed after 2 weeks. The biopsy specimens were split, with half (1.5 mm) placed in formalin for H&E processing and laboratory analyses for collagen, elastin, and glycosaminoglycans (GAGs) (stains were performed with: Alcian blue stain; hematoxylin and eosin (H&E); and Verhoeff-Van Gieson (Verhoeff's elastic stain (VEG)). The other 1.5 mm half of the biopsy was subjected to gene expression (PCR)s analysis as described above in the Gene Expression Study of Example 14.
Glycosaminoglycans (GAGs).
Epidermal thickness.
Elastin fibers.
The invention and the manner and process of making and using it, are now described in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same.
It will be appreciated that, although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.
All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification, are incorporated herein by reference.
This application is a continuation-in-part of U.S. patent application Ser. No. 17/492,514, filed Oct. 1, 2021, entitled “TOPICAL AGENTS FOR DERMATOLOGICAL APPLICATIONS,” which claims the benefit of U.S. Provisional Patent Application No. 63/087,670, filed Oct. 5, 2020, the disclosures of which are hereby incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63087670 | Oct 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17492514 | Oct 2021 | US |
| Child | 17984200 | US |
