[0001] This application claims priority to United States Provisional Patent Application No. 61/310,451 , filed March 4, 2010, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to the field of medicine. More particularly, the invention relates to compositions, kits and methods for predicting or assessing efficacy of a therapeutic agent (e.g., a drug) for a hair or skin disease in a subject, treating an inflammatory disease of skin or hair in a subject, and predicting baldness in a subject.

BACKGROUND

[0003] Recent experimental evidence suggests that inflammation is an important event in the regulation of the hair cycle mediating hair growth. When these inflammatory events are triggered, the hair cycle is disrupted and hair loss can take place. A host of other skin diseases may also be triggered by these same inflammatory events. One basic challenge is to define the location and contribution of inflammatory cells during hair follicle regression and to correlate this location with principal effectors of the inflammatory pathway. Increased production of cytokines of the inter leukin-l (IL-I) family, such as IL-Ι β, is well documented, providing clear evidence for a pivotal role of this cytokine in triggering inflammation. IL-Ι β and IL-18 are potent mediators of inflammation and initiate and/or amplify a wide variety of effects associated with innate immunity, host responses to tissue injury and microbial invasion. Although many important biological effects of IL-Ιβ and IL-18 have been described, key questions remain unresolved about the mechanisms by which the production of these cytokines are regulated, particularly in the most common form of hair loss called androgenetic alopecia (AGA), which effects both men and women. [0004] Both IL-Ιβ and IL-18 are synthesized as inactive cytoplasmic precursors that are proteo lyrically processed as biologically active fornis in response to proinflammatory stimuli by caspase-1, a cysteine protease (Bums et al,, Curr Opin Immunol vol. 15:26-30, 2003; Martinon and Tschopp, Cell vol. 1 17:561-574, 2004; Dmarello, CA J Exp Med vol. 201 : 1355-1359, 2005). The processing of pro-lL-Ιβ involves the activation of a multiprotein caspase-1 -activating complex termed the inflammasome (Martinon et al, Mol Cell vol. 10:417-426, 2002; Martinon and Tschopp, Cell Death Differ, vol. 14: 10-22, 2007; Ogura et al., Ceil vol. 126:659-662, 2006). The inflammasome in humans is formed by members of the NLR (nucleotide binding, leucine- rich repeat), pyrin domain containing protein (NLRP) family, such as NLRP1 , NLRP2 or NLRP3, and the adaptor protein apoptosis-associated speck-like protein containing a caspase- activating recruitment domain (C ARD) (ASC) that connects the NLRPs with caspase-1 (Tschopp et al., 2003; Agostini et al., 2004). However, the molecular composition of inflammasomes in the human hair follicle has not yet been determined. Activation of the inflammasome results in processing and secretion of proinflammatory IL-Ιβ and IL-18, thus triggering the inflammatory response (Martinon et al., 2002; Martinon and Tschopp, 2004; Miao et al., 2006; Ozoren et al,, 2006). Inflammasome components show a distinct and. non-overlapping tissue distribution (Kummer et al., 2007), suggesting different roles in separate cell types, but the signals and mechanisms leading to inflammasome activation, particularly in AGA hair loss (e.g., male pattern baldness), have been poorly defined.

[0005] The hair follicle is unique in that it continuously cycles, undergoing stages of growth, involution, and rest throughout human life. It is well known that the target tissue active androgen dihydrotestosterone (DHT) plays a key role in signaling anagen hair follicles into a miniaturized state and, with successive hair cycles, producing smaller, thinner, indeterminate hairs, hence signaling inflammation. DHT binds to the androgen receptor (AR), an intracellular transcription factor that belongs to the steroid/nuclear receptor superfamily (Sawaya & Price, J Invest Dermatol vol. 109:296-300, 1997). When DHT binds to AR, a nuclear translocation signal is triggered, which initiates the transport of the DHT-AR bound, complex to the nucleus to activate specific DNA sequences of androgen-regulated genes. The sensitivity of the hair follicle to androgens such as DHT is also regulated through the pre-receptor enzyme pathway in which testosterone, secreted in high levels by the gonads, is converted to the target tissue active androgen, DHT, by the 5a-reductase enzymes type I and type II. [0006] The development of AGA depends on several factors, including genetic predisposition, AR, secretion of DHT/androgens and AR coactivators, Other androgen-related disorders/diseases also work by a similar mechanism, such as acne vulgaris, seborrhea, hirsutism, polycystic ovarian disease, androgen-related infertility disorders, benign prostatic hypertrophy (BPH), as well as other systemic diseases such as coronary heart disease, insulin resistance, hypertension and prostate cancer which are associated, with androgen-dependent disorders (Sawaya & Price J Invest Dermatol vol. 109:296-300, 1997; Alsantali & Shapiro Curr Opin Endocrinol Diabetes Obes vol, 16:246-253, 2009).

[0007] Finasteride (Propecia™, Merck & Co, Inc., Whitehouse Station, NJ) is a specific 5a- reductase type II enzyme -blocking agent that inhibits the formation of Di i I and has been shown to be effective in treating men ages 18-41 years of age with AGA. Patients taking finasteride show stabilization and improvement of hair growth on the top of the scalp, producing thicker, longer hair follicles in 86% of men. The effect of finasteride on the regulation of inflammation commonly seen with AGA has not been determined. Similar findings are also found in older men taking 5a-reductase inhibitors such as finasteride and dutasteride (a dual 5a-reducatse inhibitor, inhibits both type I and II isoenzyme forms) for BPH. The role of androgens and how they propagate the inflammatory pathway in BPH is also not well understood.

[0008] Currently, there are no known tests to assess the therapeutic efficacy of drugs currently being used to treat chronic inflammatory-mediated skin diseases/disorders. It is very common for clinicians to treat patients with long term therapies with great uncertainty whether or not the drug is still having any beneficial effect. In addition, current treatments for chronic inflammatory-mediated skin diseases/disorders such as male pattern baldness, for example, fail to treat the majority of patients in need thereof and are often associated with undesirable side effects. There is thus a significant need for the development of a protocol for assessing the efficacy of a particular drug for treating a hair or skin disease and for improved treatments for chronic inflammatory mediated skin diseases/ disorders.

SUMMARY

[0009] The invention relates to the development of methods and kits for predicting or assessing efficacy of a drug for a hair or skin disease in a subject (e.g., human), methods of treating an inflammatory disease of skin or hair in a subject (e.g., human), and methods of predicting baldness in a subject (e.g., male pattern baldness in a human). The compositions, kits and methods described herein include reagents for analyzing expression and activity of NLRP1 inf!ammasome components, such as antibodies that specifically bind to at least one component (e.g., ASC, NLRPL NLRP1-4, NOD 1-5, NLRC4, NAIP, CIITA, caspase-1, caspase-5, XIAP, P2X7R, Pannexin-1 , P2X4, AR) of the mflanimasonie in the subject, as well as expression and activity of other proteins involved in inflammation.

[0010] The experiments described herein show that the NLRP1 inflammasome regulates inflammation in patients with skin and hair diseases. The NLRPl inflammasome consists of a multiprotein complex containing NLRPl , ASC, caspase-1 and XIA P. Hair and skin samples from patients with AGA, alopecia areata and scarring alopecia and from adult male patients treated with finasteride (Propecia™) were analyzed. The results show that NLRPl inflammasome proteins are elevated in these diseases and that treatment with finasteride decreases inflammasome protein expression. Finasteride resistant patients showed, increased NLRPl inflammasome protein expression, indicating that these proteins may be used as a diagnostic screen for the efficacy of treatment for male pattern baldness.

[0011] Presently, there is no diagnostic test or screening procedure on the market for assessing if chronic long-term treatments will be potentially efficacious for inflammatory skin diseases. Usually, patients are prescribed long-term therapies for treating chronic skin conditions, such as treatment in AGA with Propecia™, or topical corticosteroids for a host of other skin diseases. Yet after a year or so, the therapy has lost its effectiveness and it is not always obvious if it is still having benefits to the patient, such as men taking Propecia™ for AGA, or topical therapies for inflammatory skin diseases. Clinicians keep patients on these medications with great uncertainty whether or not they are still having any therapeutic benefit. The methods and kits described herein can provide valuable information to the clinician and patient to assess if continuing prescribed medications is still beneficial or if the patient has become tolerant or there is no current benefit. This could benefit patients financially, as well as improve health care so that patients are not kept on prolonged therapies that have no further benefit and may limit side-effects due to prolonged and unnecessary treatment.

[0012] In one embodiment, a skin biopsy is submitted for analysis to detect expression and/or activity of one or more components of the NLRPl inflammasome while patients are on current prolonged medications to treat chronic skin diseases/disorders, such as AGA, and a host of other inflammatory mediated skin diseases, In this embodiment, a small skin biopsy is submitted for analysis to detect expression and/or activity of one or more components of the NLRP! inflammasome before and during treatment regimens. Often, men with AGA are undergoing elective, cosmetic hair transplant surgery, while they have been on Propecia™ for at least a year or longer. In this procedure, a small piece (4 mm) scalp section which is usually discarded in the trash is obtained and is used to assess whether the patient should continue Propecia™ treatment. Presently, men are placed on the medication for years, not knowing if it is really having any beneficial effects. Because biopsies are routinely performed in most dermatologists' offices to obtain confirmation of inflammatory skin diseases, a small piece may be submitted for assessing if specific therapies which effect NLRP1 inflammasome activity would be beneficial to the patient. This is a unique way to assess treatment status and. assess if continued therapy should be prescribed or discontinued. This could save patients money by obviating the use of medications that are no longer beneficial. This may also be helpful to assess if other treatment options should be considered. Therefore, the methods and kits described herein are novel therapeutic tools for the clinician and patient to assess treatment efficacy as well as pro vide financial savings, as many patients pay out of pocket for some current therapies such as Propecia™, which is not covered by insurance.

[0013] Accordingly, described herein is a method of predicting or assessing efficacy of a therapeutic agent for a hair or skin disease in a subject. The method includes the steps of: obtaining a first sample from the subject having the hair or skin disease; analyzing the first sample for expression of at least one protein selected from the group consisting of: NLRP! , ASC, androgen receptor, caspase-1, XIAP and IL-Ιβ; determining the level of expression of the at least one protein in the first sample; obtaining at least a second sample from the subject; analyzing the second sample for expression of the at least one protein; determining the level of expression of the at least one protein in the second sample; comparing the level of expression of the at least one protein in the first sample with the level of expression of the at least one protein in the second sample; and correlating a decreased level of expression of the at least one protein in the second sample relative to the level of expression of the at least one protein in the first sample with the therapeutic agent's ability to treat the hair or skin disease, and correlating an equal or increased level of expression of the at least one protein in the second sample relative to the level of expression of the at least one protein in the first sample with an inability of the therapeutic agent to treat the hair or skin disease,

[0014] In one embodiment of the method, the first sample is obtained from the subject prior to the subject receiving the therapeutic agent, and the at least second, sample is obtained from the subject subsequent to the subject receiving at least one dose of the therapeutic agent (e.g., the at least second sample is obtained approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 months after receiving the at least one dose of the therapeutic agent for a hair or skin disease). In another embodiment of the method, the first sample and the at least second sample are obtained from the subject subsequent to the subject receiving at least one dose of the therapeutic agent. The sample can be, for example, a skin biopsy, scalp biopsy, hair sample, hair pluck, hair root, portion of a hair root, hair root-associated sheath or fibers, urine, blood, plasma, saliva, serum, or epithelial cells from skin, mouth, scalp or other body tissue. The hair or skin disease can be, for example, AGA, alopecia areata and cicatricial scarring alopecia's. The therapeutic agent can inhibit the formation of DHT. Examples of therapeutic agents include 5a-reductase type II enzyme- blocking agents (e.g., finasteride, dutasteride), steroids, and retinoids. Determining that the therapeutic agent is not efficacious for the hair or skin disease in the subject may indicate that the subject is resistant to the therapeutic agent, and may assist a physician in determining if the subject should continue receiving the therapeutic agent.

[0015] In another embodiment, a kit for determining if a therapeutic agent for a hair or skin disease has efficacy for the hair or skin disease in a human subject is described herein. The kit includes: at least one reagent for detecting expression of at least one of the following proteins: NLRP1, ASC, androgen receptor, caspase-1, XIAP, and IL-l β, in a sample from a human subject having the hair or skin disease; instructions for use; and packaging. The sample can be, for example, a skin biopsy, scalp biopsy, hair sample, hair pluck, hair root, portion of a hair root, hair root-associated sheath or fibers, urine, blood, plasma, saliva, serum, or epithelial cells from skin, mouth, scalp or other body tissue. The hair or skin disease can be, for example, AGA, alopecia areata and cicatricial scarring alopecia's. Examples of therapeutic agents include 5 a- reductase type II enzyme-blocking agents (e.g., finasteride, dutasteride), steroids, and retinoids. In one embodiment of the kit, the at least one reagent for detecting expression is an antibody specific for one of the following proteins: NLRP1 , ASC, androgen receptor, caspase-1 , XIAP, and IL-Ιβ.

[0016] In a farther embodiment, a kit for determining if a therapeutic agent for a hair or skin disease has efficacy for the hair or skin disease in a human subject includes: at least one reagent for determining the level of caspase-1 activation, I L-Ιβ activation, and/or XIAP cleavage in a sample from a human subject having the hair or skin disease; instructions for use; and packaging. The sample can be, for example, a skin biopsy, scalp biopsy, hair sample, hair root, portion of a hair root, hair root-associated sheath or fibers, urine, blood, plasma, saliva, serum, or epithelial cells from skin, mouth, scalp or other body tissue. The hair or skin disease can be, for example, AGA, alopecia areata and cicatricial scarring alopecia's. The therapeutic agent can be, for example, a 5a-reductase type II enzyme-blocking agent (e.g., finasteride, dutasteride), a steroid, or a retinoid.

[0017] In still a further embodiment, a method of determining if a therapeutic agent is efficacious for treating a hair or skin disease in a subject is described herein. The method includes the steps of: obtaining a first sample from the subject having the hair or skin disease; analyzing the first sample for caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample; obtaining at least a second sample from the subject; analyzing the second sample for caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; comparing the level of caspase-1 activation, IL-ϊβ activation, and/or XIAP cleavage in the first sample with the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; and correlating a decreased level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample with efficacy of the therapeutic agent for treating the hair or skin disease, and an equal or increased level of caspase- 1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ι β activation, and/or XI AP cleavage in the first sample with no or limited efficacy of the therapeutic agent for treating the hair or skin disease.

[0018] In one embodiment of this method, the first sample is obtained from the subject prior to the subject receiving the therapeutic agent, and the at least second, sample is obtained from the subject subsequent to the subject receiving at least one dose of the therapeutic agent (e.g., the at least second sample is obtained approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, I I , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 months after receiving the at least one dose of die therapeutic agent for a hair or skin disease). In an alternative embodiment of this method, the first sample and the at least second sample are obtained from the subject subsequent to the subject receiving at least one dose of the therapeutic agent. The sample can be, for example, a skin biopsy, scalp biopsy, hair sample, hair pluck, hair root, portion of a hair root, hair root-associated sheath or fibers, urine, blood, plasma, saliva, serum, or epithelial cells from skin, mouth, scalp or other body tissue. The hair or skin disease can be, for example, AGA, alopecia areata or cicatricial scarring alopecia's. In the method, the therapeutic agent can inhibit the formation of DHT. The therapeutic agent can be, for example, a 5a-reductase type Π enzyme-blocking agent (e.g., finasteride, dutasteride), a steroid, and a retinoid.

[0019] Yet further described herein is a method of predicting male pattern baldness in a subject (e.g., a human). The method includes the steps of: obtaining a sample from the subject; analyzing the sample for expression of at least one protein selected from the group consisting of: NLRP1 , ASC, androgen receptor, caspase-1, XIAP and IL-Ιβ, and/or caspase-1 activation, IL- 1β activation, and/or XIAP cleavage in the sample; and determining the le vel of expression of the at least one protein, and/or the level of caspase-1 activation, IL- Ιβ activation, or XIAP cleavage in the sample, wherein elevation or upregulation of expression of die at least one protein, and/or the level of caspase-1 activation, IL-Ι β activation, or XIAP cleavage in the sample indicates a predisposition to male pattern baldness (e.g., male pattern baldness caused by AGA) in the subject. The step of analyzing the sample for expression of the at least one protein and/or caspase- 1 activation, IL-Ιβ activation, and/or XIAP cleavage in the sample can be performed by any suitable method, e.g., one or more of ELISAs, radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence assays, immunoprecipitation, protein A assays, Immunoelectrophoresis assays, enzymatic assays, fluorometric assays, colorometric assays, and photometric assays.

[0020] Yet further described, herein is a method of treating an inflamniatoiy disease of skin or hair in a subject. The method includes the steps of: providing a therapeutically effective amount of a composition including at least one antibody that specifically binds to a component of a mammalian inflammasome; administering the composition to the subject, wherein administering the composition to the subject results in a reduction of inflammation in the subject; obtaining a sample from the subject after the composition has been administered to the subject; and analyzing the sample for inflammation levels, The inflammasome component can be, for example, ASC, and the at least one antibody binds to an amino acid sequence having at least 85% sequence identity with amino acid sequence SEQ ID NO:l . The inflammasome component can be, as another example, NLRP-1 , and the at least one antibody binds to an amino acid sequence having at least 85%> sequence identity with amino acid sequence SEQ ID NO:2. Administration of the composition results in decreased hair loss in the subject. The composition can include at least one pharmaceutically acceptable carrier or diluent and can be administered topically.

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translationai modification, e.g., glycosyiation, phosphorylation, or sulfated.

[0023] By the terms "Apoptosis-associated Speck-like protein containing a Caspase Activating Recruitment Domain (C ARD)" and " ASC" is meant an expression product of an ASC gene or isoforms thereof, or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with ASC (e.g., accession number Q9ULZ3 in humans) and displays a functional activity of ASC. A "functional activity" of a protein is any activity associated with the physiological function of the protein. Functional activities of ASC include, for example, recruitment of proteins for activation of caspase- 1 and initiation of cell death.

[0024] By the term "ASC gene," or "ASC nucleic acid" is meant a native ASC-encoding nucleic acid sequence, genomic sequences from which ASC cDNA can be transcribed, and/or allelic variants and homologues of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0025] As interchangeably used herein, "NALP1" and "NLRP1" mean an expression product of a NALP1 or NLRPl gene or isoforms thereof; or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with NALP1 (e.g., accession number(s) AAH51787, NP001028225, NP127500, NP 127499, NP127497, NP055737) and displays a functional activity of NALPl ,

[0026] As used herein, the term "mflammasome" means a multi-protein (e.g., at least two proteins) complex that activates caspase-1. The terms "NLRP1 mflammasome" and "NALPl mflammasome" are used interchangeably and mean a protein complex of at least caspase-1 and one adaptor protein, e.g., ASC. For example, the terms "NLRP1 inflammasome" and "NALPl inflammasome" can mean a raultiprotein complex containing NLRP1, ASC, caspase-1, caspase- 1 1 , XIAP, and pannexin-1 for activation of caspase-1 and processing of interleukin-ΐβ, interleukin-18 or interleukin-33.

[0027] As used herein, the phrase "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, CA).

[0028] By the phrases "therapeutically effective amount" and "effective dosage" is meant an amount sufficient to produce a therapeutically (e.g., clinically) desirable result; the exact nature of the result will vary depending on the nature of the disorder being treated. For example, where the disorder to be treated is AGA, the result can be a reduction in hair loss or prevention of hair loss. The compositions described herein can be administered from one or more times per day to one or more times per week. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions of the invention can include a single treatment or a series of treatments,

[0029] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent described herein, or identified by a method described herein, to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease. [0030] By the phrase "therapeutic agent" is meant any composition that cures, heals, alleviates, relieves, ameliorates or mitigates a disease or disorder or at least one symptom(s) of a disease or disorder. Examples of therapeutic agents include compounds, drags, and biologies (e.g., antibodies, nucleic acids), etc.

[0031 ] The terms "patient" "subject" and "individual" are used interchangeably herein, and mean a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary appl ications, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, as well as non-human primates.

[0032] The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques. For example, the anti-ASC and anti- NLRP1 antibodies described herein are capable of binding portions of ASC and NLRP1, respectively that interfere with caspase-1 activation.

[0033] Although compositions, kits and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and. not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a series of photographs of electrophoretic gels showing that Propecia™ treatment decreases NLRP1 and ASC expression in patients with AGA. Immunoblots show that NLRP1 and ASC are expressed in patients with AGA (1-1 1 ). Patients with AGA were treated with Propecia™ (6-1 1). Propecia™ treatment resulted in decreased expression of NLRP1 and ASC irtflammasome proteins (9-11). b-actin was used as a standard and protein loading control. Data is shown as mean t-/'- SEM *P < 0.05. FIG. 1 also depicts a series of photographs of electrophoretic gels showing Caspase-1 activation and cleavage of XIAP are associated with poor outcomes in patients with AGA, Caspase-1 cleavage correlates with disease severity in patients with AGA (1-5). Increased caspase-1 activation and XIAP cleavage are seen in patients that were diagnosed with AGA and were treated with Propecia™ but responded poorly to treatment (6-8). b-actin was used as a protein standard and protein loading control. FIG. 1 also shows that AR protein expression is upregulated in patients diagnosed with AGA (1 -5). Immunoblot analysis shows that patients diagnosed with AGA (1-5) had increased expression of the AR compared to those treated with Propecia™ (6-11).

[0035] FIG. 2 is a series of photographs of electrophoretic gels that show patients (1 and 2) with AGA have increased expression of ARs in the crown balding area (A) when compared to the occipital donor area (B), b-actin was used as a protein standard and protein loading control.

DETAILED DESCRI PTION

[0036] Described herein are compositions, kits and methods for assessing the efficacy of therapies (e.g., therapeutic agents) used in chronic treatment situations, as it is commonly known that not all patients will respond to a particular therapy, and of those that do, some plateau or become less responsive to therapies over time. The compositions, kits and methods are also used for treating an inflammatory disease of skin or hair in a subject and predicting baldness in a subject. The below described preferred embodiments illustrate adaptations of these compositions, kits and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based, on the description provided below.

Biological Methods

[0037] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1 -3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al, Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard, and Matthew R. Kaser, CRC Press, Boca Raton, FL 2006; Medical Immunology, 6 ^th ed., edited by Gabriel Virella, Inforrna Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

Methods of Assessing or Predicting Efficacy of a Therapeutic Agent for Treating An

Inflammatory Disease of Skin or Hair In a Subject

[0038] Described herein are methods of predicting or assessing efficacy of a therapeutic agent (e.g., drug, biologic, etc.) for a hair or skin disease in a subject (e.g., dog, cat, pig, horse, rodent, non-human primate, human). Examples of inflammatory diseases of skin and hair include AGA, alopecia areata and cicatricial scarring alopecia's, acne vulgaris, seborrhea, hirsutism, polycystic ovarian disease, androgen-related infertility disorders, BPH, as well as other systemic diseases such as coronary heart disease, insulin resistance, hypertension and prostate cancer that are associated with androgen-dependent disorders. One embodiment of a method of predicting or assessing efficacy of a therapeutic agent (e.g., drug) for a hair or skin disease in a subject includes the steps of: obtaining a first sample from the subject having the hair or skin disease, wherein the subject has not received the therapeutic agent; analyzing the first sample for expression of at least one of the proteins NLRP1, ASC, androgen receptor, caspase-1, XIAP and IL-Ι β; determining the level of expression of the at least one protein in the first sample; obtaining at least a second sample from the subject after the subject has received at least one dose of the therapeutic agent for a hair or skin disease; analyzing the second sample for expression of the at least one protein; determining the level of expression of the at least one protein in the second sample; comparing the level of expression of the at least one protein in the first sample with the level of expression of the at least one protein in the second sample; and correlating a decreased level of expression of the at least one protein in the second sample relative to the level of expression of the at least one protein in the first sample with the therapeutic agent's ability to treat the hair or skin disease, and correlating an equal or increased level of expression of at least one protein in the second sample relative to the level of expression of at least one protein in the first sample with an inability of the therapeutic agent to treat the hair or skin disease.

[0039] In another embodiment, the efficacy of a therapeutic agent can be assessed in a subject who has already begun receiving the therapeutic agent (e.g., drug, biologic, etc.). In this embodiment, the at least first and second samples are obtained from the subject after the subject has received at least a first dose (e.g., first and second doses, respectively), of the therapeutic agent. In one example of such an embodiment, a method of predicting or assessing efficacy of a therapeutic agent (e.g., drug) for a hair or skin disease in a subject includes the steps of: obtaining a first sample from the subject having the hair or skin disease, wherein the subject has received at least one dose of the therapeutic agent; analyzing the first sample for expression of at least one of the proteins NLRP1 , ASC, androgen receptor, caspase-1 , XIAP and IL-Ιβ; determining the level of expression of the at least one protein in the first sample; obtaining at least a second sample from the subject after the subject has received at least a second dose of the therapeutic agent (e.g., drag) for a hair or skin disease; analyzing the second sample for expression of the at least one protein; determining the level of expression of the at least one protein in the second sample; comparing the level of expression of the at least one protein in the first sample with the level of expression of the at least one protein in the second sample; and correlating a decreased level of expression of the at least one protem in the second sample relative to the level of expression of the at least one protein in the first sample with the therapeutic agent's ability to treat the hair or skin disease, and correlating an equal or increased level of expression of at least one protein in the second sample relative to the level of expression of at least one protein in the first sample with an inability of the therapeutic agent to treat the hair or skin disease.

[0040] In these embodiments, any suitable method for analyzing a sample for expression of and determining the level of expression of at least one of the proteins NLRP1, ASC, androgen receptor, caspase-1, XIAP and IL-Ιβ (or any other protein involved in inflammation) can be used. Numerous antibody-based detection formats are well known in the art, and include ELISA (enzyme linked immunosorbent assay), radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence assays, immunoprecipitation, protein A assays, immunoelectrophoresis assays, enzymatic assays, fluorometric assays, colorometric assays, photometric assays, and other related techniques. In some embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the compositions, kits and methods described herein. In addition to or instead of NLRPl, ASC, AR, caspase-1, XIAP and IL-Ιβ, expression of any other protein involved in inflammation can be analyzed.

[0041] In another embodiment of a method of predicting or assessing efficacy of a therapeutic agent (e.g., drug) for a hair or skin disease in a subject, the method includes the steps of: obtaining a first sample from the subject having the hair or skin disease, wherein the subject has not received the therapeutic agent: analyzing the first sample for caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample; obtaining at least a second sample from the subject after the subject has received at least one dose of the therapeutic agent; analyzing the second sample for caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; comparing the level of caspase-1 activation, IL-Ι β activation, and/or XIAP cleavage in the first sample with the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; and correlating a decreased level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample with efficacy of the therapeutic agent for treating the hair or skin disease, and an equal or increased level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample with no or little efficacy of the therapeutic agent for treating the hair or skin disease.

[0042] In another embodiment, the efficacy of a therapeutic agent can be assessed in a subject who has already begun receiving the therapeutic agent (e.g., drug, biologic, etc.). In this embodiment, the at least first and second samples are obtained from the subject after the subject has received at least a first dose (e.g., first and second doses, respectively), of the therapeutic agen t. In one example of such an embodiment, a method of predicting or assessing efficacy of a therapeutic agent (e.g., drug) for a hair or skin disease in a subject includes the steps of: obtaining a first sample from the subject having the hair or skin disease, wherein the subject has received at least one dose of the therapeutic agent; analyzing the first sample for caspase-I activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ι β activation, and/or XIAP cleavage in the first sample; obtaining at least a second sample from the subject after the subject has received at least a second dose of the therapeutic agent; analyzing the second sample for caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage; determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; comparing the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP clea vage in the first sample with the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample; and correlating a decreased level of caspase-1 activation, I L-Ι β activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample with efficacy of the therapeutic agent for treating the hair or skin disease, and an equal or increased level of caspase- 1 activation, IL-Ιβ activation, and/or XIAP cleavage in the second sample relative to the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the first sample with no or little efficacy of the therapeutic agent for trea ting the hair or skin disease,

[0043] In these embodiments, any suitable method for analyzing caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage (e.g., determining the level of caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage) in a sample can be used. Examples of suitable assays include ELISA (enzyme linked immunosorbent assay), radioimmunoassays, imniunoblots, Western blots, flow cytometry, immunofluorescence assays, immunoprecipitation, protein A assays, Immunoelectrophoresis assays, enzymatic assays, fluorometric assays, colorometric assays, photometric assays, and other related techniques.

[0044] In a method of predicting or assessing efficacy of a therapeutic agent for a hair or skin disease in a subject, any suitable sample can be used. Examples of samples that can be used include skin biopsy, scalp biopsy, hair sample by hair pluck (sometimes referred to as "hair pull"), hair root, portion of a hair root, hair root-associated sheath or fibers, urine, blood, plasma, saliva, serum, and epithelial cells from skin, mouth, scalp or other body tissue. The methods described herein can be used to assess the efficacy of a therapeutic agent intended or used for treatment of any skin or hair disease that is caused by or associated with inflammation. Examples of such hair and skin diseases include AGA, alopecia areata and cicatricial scarring alopecia's, acne vulgaris, seborrhea, hirsutism, polycystic ovarian disease, androgen-related infertility disorders, BPH, as well as other systemic diseases such as coronary heart disease, insulin resistance, hypertension and. prostate cancer that are associated with elevated androgens and androgen receptor disorders. [0045] The efficacy of any therapeutic agent used for or intended to be used for a skin or hair disease thai is caused by or associated with inflammation can be assessed and/or predicted by the methods described herein. For example, the methods can be used for any drug that is used to treat or intended to treat inflammation in a subject. Examples include 5a-reductase type II enzyme-blocking agents (5a-reductase inhibitors) such as finasteride and dutasteride, anti- androgeris which specifically block or inhibit the androgen receptor, including retinoids, and. other steroids. Steroids that are often used to treat inflammation are glucocorticosteroids. Examples of additional steroids include mineralcorticoids, androgens, estrogens, progestins, and retinoids.

[0046] In a method of predicting or assessing efficacy of a drug for a hair or skin disease in a subject, determining that the drug is not efficacious for the hair or skin disease (e.g., does not alleviate or mitigate the hair or skin disease) in the subject may indicate that the subject is resistant to the drug. Determining whether or not the therapeutic agent is efficacious for the hair or skin disease (e.g., alleviates or mitigates the hair or skin disease) in the subject assists a physician in determining if the subject should begin and/or continue receiving the therapeutic agent.

[0047] in those embodiments where the at least first sample is obtained from a subject prior to the subject receiving a therapeutic agent as described herein, the at least second sample can be obtained at any time subsequent to administration of the at least one therapeutic agent to the subject e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 months after receiving the at least one dose of the drug. In those embodiments where the at least first and second, samples are obtained from a subject subsequent to the subject receiving a therapeutic agent as described herein, the at least second sample can be obtained at any appropriate time subsequent to obtaining the at least first sample from the subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 months. Some studies indicate that finasteride, for example, begins to show results at approximately 3 months after the first dose, and that in many cases, the results begin to plateau at approximately 2 years. Some subjects cease to show any results at approximately two years. Thus, many subjects (patients) would be interested in getting tested according to the methods described herein so that they can determine if the drug (e.g., finasteride) is still efficacious, or if it has ceased to work. Finasteride is a drug that is administered for the treatment of hair loss in men and women (e.g., AG A and other similar diseases). Generally, there are three types of hair loss patients: those that are balding but not receiving finasteride or other similar drug, those that are balding and are receiving finasteride or other similar drug and have shown results (i.e., reduced hair loss) at some point or continuously during the drug treatment, and those that are balding and are receiving finasteride or other similar drug but are showing no results (i.e., no reduction in hair loss). In the first group of patients (i.e., those that are balding but not receiving finasteride or other similar drug), the methods and kits described herein can be used to predict whether or not a drag such as finasteride could be expected to work, In the second group of patients (i.e., those that are balding and are receiving finasteride or other similar drag and have shown results (i.e., reduced hair loss) at some point or continuously during the drag treatment), the methods and kits described herein can be used to determine whether or not the patient should continue taking the drug or if the drug treatment should be discontinued.

[0048] As mentioned above, steroids are administered for treatment of many inflammatory diseases. The methods described herein can be used to determine if a subject receiving a steroid for an inflammatory disease is responding to the steroid treatment. Because steroids often cause undesirable side effects, it would benefit the subject (patient) if a determination could be made prior to starting steroid treatment as to whether or not the particular steroid is expected to be efficacious. Similarly, the methods described herein can be used to determine if a subject currently receiving steroid treatment for an inflammatory disease should continue receiving the steroid treatment. In this example, if it is found that the subject is no longer benefitting from or responding to the steroid treatment, the steroid treatment could be discontinued.

[0049] A subject includes any mammal, including humans, rodents, horses, pigs, cows, non-human primates, etc. In the experiments described below, ASC expression, NLRP1 expression, caspase-1 activation, IL-Ιβ activation, and XIAP cleavage were examined in AGA patients. However, the expression or activity of any one or more of the NL.RP1 inflammasome components and/or other proteins associated with inflammation can be examined in a method of assessing or predicting efficacy of a therapeutic agent (e.g., drug) for treating a hair or skin disease. Treating An Inflammatory Disease of Skin or Hair In a Subject

[0050] Described herein are methods of treating an inflammatory disease of skin or hair in a subject (e.g., dog, cat, pig, horse, rodent, non-human primate, human). Examples of inflammatory diseases of skin and hair include AGA, alopecia areata and cicatricial scarring alopecia's, acne vulgaris, seborrhea, hirsutism, polycystic ovarian disease, androgen-re!ated infertility disorders, BPH, as well as other systemic diseases such as coronary heart disease, insulin resistance, hypertension and prostate cancer that are associated with elevated androgens and androgen receptor disorders. A typical method of treating an inflammatory disease of skin or hair in a subject includes the steps of providing a therapeutically effective amount of a composition including at least one antibody that specifically binds to a component of a mammalian inflammasome; administering the composition to the subject, wherein administering the composition to the subject results in a reduction of inflammation in the subject; obtaining a sample from the subject after the composition has been administered to the subject; and analyzing the sample for inflammation levels. In one embodiment, in which the inflammatory disease of skin or hair is hair loss from the scalp, administration of the composition results in reduced inflammation (e.g., in the hair, hair follicle, hair root, scalp, etc.) and reduced hair loss (i.e., halting further hair loss) in the subject. In some, administration of the composition may result in reduced inflammation which may prevent hair loss.

[0051] In a typical embodiment, the at least one antibody specifically binds to ASC or NALP1 (i.e., NLRP1). However, an antibody against any other component of a mammalian inflammasome (e.g., the NALP1 or NLRP1 inflammasome) may be used. A composition of two or more (e.g., 2, 3, 4, 5, etc.) antibodies (e.g., anti-ASC and anti-NLRPl) that each bind a different component of the NLRP1 inflammasome can be used. It may be found that administering antibodies to two or more inflammasome components or other proteins involved in inflammation may have a significant effect, An antibody as described herein can be a monoclonal or polyclonal antibody. Any suitable antibody that specifically binds a component of the NLRPl inflammasome (e.g., anti-ASC antibody) or other protein involved in inflammation can be used to inhibit or reduce inflammation in a subject, thus treating or preventing a hair or skin disease. In one example, the at least one antibody specifically binds to an amino acid sequence having at least 85% sequence identity with amino acid sequence SEQ ID NO:l (ASC). Similarly, in another embodiment, the at least one antibody specifically binds to an amino acid sequence having at least 85% sequence identity with amino acid sequence SEQ ID NO: 2 (NLRP-1),

[0052] By measuring inflammation levels after the composition has been administered to the subject, one can determine if the treatment was successful, By "inflammation levels" is meant the level of expression or activity of one or more components of the NLRP1 inflammasome and/or proteins processed upon inflammasome activation. If inflammation levels are high, the treatment did not work or did not work as well as expected or hoped. If inflammation levels, however, are normal or low, this finding indicates that the treatment was successful in at least reducing inflammation in the mammal, and possibly in treating the disease.

[0053] Typically, the composition includes at least one pharmaceutically acceptable carrier or diluent (e.g., physiological saline or buffer) and is administered topically, orally, intravenously, intrarterially, intrathecal!} ⁷ , intra lesionaliy, subcutaneously, intraperitoneally, rectally, or intranasally. Any suitable form of administration can be used, however. A subject includes any mammal, including humans, rodents, horses, pigs, cows, non-human primates, etc.

Method of Predicting Male Pattern Baldness In a Subject

[0054] Methods of detecting a predisposition to male pattern baldness in a subject generally include correlating overexpression of at least one protein associated with inflammation (e.g., NLRP1, ASC, androgen receptor, caspase-1, XI AP and IL-Ιβ) and/or caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage with a predisposition to male pattern baldness in the subject. Whether or not the at least one protein is overexpressed in the biological sample can be determined by comparing the level of expression of the at least one protein in the biological sample to a baseline level (also known as a control level) of expression of the at least one protein. A "baseline level" is a control level, and in some embodiments a normal level or a level not observed in subjects who are predisposed to male pattern baldness. Therefore, it can be determined, based on the control or baseline level of expression of the at least one protein, whether a sample to be evaluated for male pattern baldness development has a measurable increase (i.e., overexpression, upregulation), decrease, or substantially no change in expression, as compared to the baseline level.

[0055] A typical method of detecting a predisposition to male pattern baldness in a subject includes the steps of: obtaining a sample from the subject; analyzing the sample for expression of at least one of the proteins NLRPl , ASC, androgen receptor, caspase-1, XIAP and IL-Ιβ, and/or caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage in the sample; and determining the level of expression of the at least one protein, and/or the level of caspase-1 activation, IL-Ιβ activation, or XIAP cleavage in the sample, wherein elevated, levels of at least one of the proteins NLRP1, ASC, androgen receptor, caspase-1 , XIAP and IL-Ιβ, and/or elevated caspase-1 activation, IL-Ιβ activation, and/or XIAP cleavage indicates a predisposition to male pattern baldness in the subject, In some embodiments, the expression or activity of two or more (e.g., 2, 3 4, 5, 6, etc.) protems associated with inflammation is analyzed. For example, tissue samples or bodily fluids are extracted and analyzed for expression of at least one of the proteins NLRP1, ASC, androgen receptor, caspase-1, XIAP and IL- Ι β, and/or caspase-1 activation, IL-Ιβ activation, and/ or XIAP cleavage in the sample by one or several of the following procedures: ELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence assays, immunoprecipitation, protein A assays, Immunoelectrophoresis assays, enzymatic assays, fSuorometric assays, colorometric assays, photometric assays, and other related techniques.

Antibodies That Bind Specifically to At Least One Component of a

Mammalian Inflammasome

[0056] In some embodiments of the methods described herein for reducing inflammation in a subject and treating hair or skin diseases in a subject, the methods include compositions including at least one antibody that specifically binds to at least one component (e.g., ASC, NLRPl) of a mammalian inflammasome (e.g., the NALP1 inflammasome). A composition for treating a hair or skin disease (e.g., AGA, alopecia areata and cicatricial scamng alopecia's) and/or reducing inflammation in a subject can further include at least one pharmaceutically acceptable carrier or diluent.

[0057] In one embodiment, a composition for treating a hair or skin disease and/or reducing inflammation in a subject includes an antibody that typically specifically binds to a region of human ASC, e.g., amino acid sequence RESQSYLVEDLERS (SEQ ID NO: l). In this embodiment, an antibody as described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with amino acid sequence SEQ ID NO: l of human ASC. Such an antibody inhibits ASC activity in a subject (e.g., in hair, hair follicle, and skin cells of the mammal). [0058] In another embodiment, a composition for reducing inflammation and treating hair or skin diseases includes an antibody that specifically binds to NLRPL Any suitable anti-NLRPl antibody can be used, and several are commercially available. In this embodiment, an antibody as described herein typically specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with the amino acid sequence CEYYTEIREREREKSEKGR (SEQ ID NO:2) of human NLRPl . Such an antibody inhibits NLRPl activity in a subject (e.g., in hair, hair follicle, and skin cells of the mammal).

[0059] Antibodies that specifically bind to at least one component of the NLRPl inflammasome (e.g., anti-ASC and anti-NLRPl antibodies) or other protein involved in inflammation as described herein include polyclonal and monoclonal antibodies, or any portions • hereof, having at least one antigen binding region of an immunoglobulin variable region, which antibody specifically binds to at least one component of the NLRPl inflammasome (e.g., ASC or NLRPl) or other protein involved in inflammation. An antibody is specific for a polypeptide if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant polypeptide.

[0060] Methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow, et al,, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ( ^" old Spring Harbor, N.Y., 1988, Colligan et al,, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601, 1983, which references are entirely incorporated herein by reference.

[0061] Antibodies against one or more components of the NLRPl inflammasome (e.g., anti- ASC and anti-NLRPl antibodies) or other protein involved in inflammation as described herein can be routinely made according to methods such as, but not limited to, inoculation of an appropriate animal with the polypeptide or an antigenic fragment, in vitro stimulation of lymphocyte populations, synthetic methods, hybridomas, and/or recombinant cells expressing nucleic acid encoding such antibodies, Immunization of an animal using purified recombinant ASC or peptide fragments thereof, e.g., SEQ ID NO: l of human ASC, is an example of a method of preparing anti-ASC antibodies. Similarly, immunization of an animal using purified, recombinant NLRPl or peptide fragments thereof, e.g., SEQ ID NO:2 of human NLRPl, is an example of a method of preparing anti-NLRPl antibodies. [0062] Monoclonal antibodies that specifically bind at least one component of the NLRP1 inflammasome (e.g., ASC or NLRPl) or other protein involved in inflammation may be obtained by methods known to those skilled in the art. See, for example Kohler and Mil stein, Nature 256:495-497, 1975; U.S. Pat. No. 4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colli gan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ or in vivo.

Kits For Determining Efficacy of A Therapeutic Agent

[0063] Described herein are kits for determining if a therapeutic agent (e.g., drug, antibody, nucleic acid, etc.) for a hair or skin disease has efficacy for the hair or skin disease in a subject. A typical kit includes at least one reagent for detecting expression of at least one protein such as NLRP1 , ASC, androgen receptor, caspase-l, XIAP, and IL-Ιβ and/or at least one reagent for determining the level of caspase-l activation, IL-Ιβ activation, and/or XIAP cleavage in a sample from a subject having the hair or skin disease; instructions for use; and packaging. An example of a reagent for detecting expression of at least one protein such as NLRP1, ASC, androgen receptor, caspase-l , XIAP, and IL-Ιβ includes antibodies. An example of a reagent that determines the level of caspase-l activation, IL-Ιβ activation and/ or XIAP cleavage includes antibodies. However, any suitable reagents can be included in the kits described herein. Kits may include additional reagents or components including buffers, controls, etc.

[0064] Any suitable sample can be analyzed using the kits described herein. Examples of samples include DNA, RNA, cell extracts, skin biopsy, scalp biopsy, hair sample, hair root, portion of a hair root, hair pluck (sometimes referred to as "hair pull"), hair root- associated sheath or fibers, urine, blood, plasma, saliva, serum, and epithelial ceils from skin, mouth, scalp or other body tissue. Examples of hair or skin diseases a subject may have who will find use for the kits described herein include AGA, alopecia areata and cicatricial scarring alopecia's, acne vulgaris, seborrhea, hirsutism, polycystic ovarian disease, androgen-related infertility disorders, BPH, as well as other systemic diseases such as coronary heart disease, insulin resistance, hypertension and prostate cancer that are associated with elevated androgens and androgen receptor disorders. The efficacy of any therapeutic agent intended for or used for treatment of a skin or hair disease can be determined using the kits described herein. For example, the therapeutic agent may be one that inhibits the formation of DHT, e.g., a 5a-reductase type Π enzyme-blocking agent such as finasteride or dutasteride. Additional examples of therapeutic agents whose efficacy for a skin or hair disease can be determined include steroids and retinoids.

Administration of Compositions

[0065] The compositions of the invention may be administered to mammals (e.g., dog, cat, pig, horse, rodent, non-human primate, human) in any suitable formulation. For example, antibodies directed against one or more components of the inflammasome (e.g., anti-ASC antibodies, anti-NLRPl antibodies, etc.) may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. A description of exemplar}' pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in U8P/NF, Other substances may be added to the compositions to stabilize and/or preserve the compositions.

[0066] The compositions of the invention may be administered to mammals by any conventional technique. Such administration routes include topical, oral and parenteral (e.g., intravenous, subcutaneous, intratumorai, intramuscular, intraperitoneal, or intrathecal introduction). The compositions may be administered directly to a target site by, for example, topical, subcutaneous, intravenous, intramuscular, intraperitoneal or intrathecal routes. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously, by peritoneal dialysis, pump infusion). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form.

Effective Doses

[0067] The compositions described above are preferably administered to a mammal (e.g., dog, cat, pig, horse, rodent, non-human primate, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal (e.g., reducing inflammation and decreasing or preventing hair loss in a mammal). Such a therapeutically effective amount can be determined as described below.

[0068] Toxicity and therapeutic efficacy of the compositions utilized in me thods of the invention can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD ₅₀ (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a deliver}' system that minimizes the potential damage of such side effects. The dosage of preferred compositions lies preferably within a range that includes an ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0069] As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.

EXAMPLES

[0070] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Regulation of Inflammation in Hair and Skin Diseases

[0071] The expression patterns of key parameters of mflammasome signaling in scalp of normal human and AGA male subjects were examined by using a panel of anti-inflammasome antibodies to characterize expression in AGA and finasteride-treated individuals. The results described herein show that AGA involves activation of the NLRP1 inflammasome and XIAP in discrete areas of the scalp of men with AGA. Finasteride appears to influence the expression of these parameters and may be useful in treatment strategies targeting hair, skin and other systemic androgen-related diseases. Materials and Methods

[0072] Patients and hair samples: Small pieces (3 mm) of human scalp tissue were obtained during routine hair transplant surgery for an elective cosmetic procedure to aesthetically correct for hair loss. Adult males, age 25 to 68 yrs of age with mild to moderate hair loss on the front and vertex of the scalp signed informed consents to undergo hair transplant surgery . Patients were males diagnosed with AGA between the ages of 37 to 68 for non-treated patients and 25 to 61 for patients treated with finasteride (Propecia™). The l ength of treatment was in the range of 4 months to 10 years. According to Norwood's classification of male pattern baldness, AGA patients were between IB and IV. Routinely, a large scalp donor strip (2 cm wide x 6 to 8 cm long) is excised from the occipital area of scalp, where the hairs are thick and unaffected by the balding miniaturization process. Hairs from die donor strip are microdissected and replanted into the balding frontal and vertex areas of scalp. Any residual small pieces of tissue that would normally be thrown away or discarded (usually 2 to 3 mm) were used, for research analysis. Small excised pieces were obtained from the balding frontal or vertex areas of scalp as new hair follicles were implanted from the donor strip. These small residual pieces of tissue (1 to 2 mm) were used for research analysis.

[0073] immunoblotting: Hair/scalp tissue was homogenized in skin lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na ₃ V0 ₄ , ) with protease coctail inhibitor (Sigma), Proteins were resolved in 10-20% Tris-HCl Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Applied Biosystems) and placed in blocking buffer (PBS, 0.1 % Tween-20, 0.4% I-Block (Applied Biosystems) and then incubated for 1 h with: monoclonal antibody to caspase-1 (1:1000, Imgenex), antibody to androgen receptor (1 : 1000, Santa Cruz), ASC (1 :5000, AnaSpec), NLRP-1 (1 :5000, Cell Signaling) and X1AP (1 : 1000 Cell Signaling). Membranes were incubated for 1 h with primary antibodies followed by appropriate secondary horseradish peroxidase (HRP)-l inked antibodies (Cell Signaling). Visualization of signal was enhanced by chemilluminescence using a phototope-HRP detection kit (Cell Signaling), To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce) and blotted for β-actin using monoclonal anti-p-actin antibody (1 : 10000, Chemicon). Quantification of band density was performed using the UN- SCAN-IT gel software, and data was normalized to β-actin. [0074] Statistical Analysis: Data are expressed as mean +/- standard error of the mean (+/- s.e.m.), Statistical comparisons between treated and untreated groups were made using two-tailed Student's /-test and a one-way ANOVA followed by Tukey's multiple comparison tests. P -value of significance was *P < 0,05.

Results

[0075] The data indicate that the inflammasome proteins ASC and NLRP1 are upregulated. in patients that have been diagnosed with androgenetic alopecia (Figure 1). ASC serves as a scaffold protein involved in the activation of the NLRP1 inflammasome. Densitometric analysis of immunoblots indicate that treatment with Propecia™ (finasteride) resulted in decreased NLRP1 and ASC protein expression. In addition, cleavage of caspase-1 is present in these patients signifying activation of caspase-1 and activation of the inflammasome complex (Figure !). These findings correlate with cleavage of the inhibitor of apoptosis X-linked inhibitor of apoptosis protein (XIAP) (Figure 1).

[0076] It has been previously shown that in the spinal cord, XI AP (full length) functions as an inhibitory protein that maintains the NLRPl inflammasome in an inactive state. After injury to the cord, XIA P is cleaved and results in decreased inhibitory capacity; thus leading to inflammasome activation. Figure 2 shows that the androgen receptor (AR) is increased in balding areas, whereas occipital donor areas containing healthy hair had lower levels of this receptor. Similar findings were found in patients that were treated with Propecia™ in which patients that responded, well to treatment had decreased levels of these receptors (Figure 2).

[0077] In the experiments described herein, the expression of infianimasome proteins was examined, to determine whether these molecules play a role in the regulation of hair loss and treatment of patients with Propecia™. The results indicate that the NLRPl inflammasome pathway is activated in patients with AGA and that Propecia™ treatment results in decreased expression of NLRP1 and the scaffolding protein ASC. Gene targeting studies have revealed that caspase-1 plays an important role in the regulation of the immune response and previous work has shown that caspase-1 is expressed in epidermis, hair bulb, and hair shaft suggesting that these structures play an important role in cytokine regulation and/or inflammatory reactions (Sawaya et al, J Invest Dermatol vol. 109:296-300, 2002). Since there is perifollicular lympho- histiocytic inflammatory pattern in the infundibular-isthmus area of the hair follicle in 40% of scalp biopsies with AGA, it is possible that caspase-1 and the NLRPl inflammasome play an important role in this process.

[0078] The NLRPl inflammasome is a multiprotein complex involved in the initiation of innate inflammatory responses. The results described herein are the first to show that an innate inflammatory component is involved with AGA and that NLRPl inflammasome proteins are involved in dictating disease severity and effectiveness of Propecia™ treatment. It has been previously shown that in the spinal cord of rats, this complex includes NLRPl, caspases-1 and - 1 1 , ASC and the inhibitory protein XIAP. In addition, the NLRPl inflammasome can be used as a therapeutic target to improve histopathologicai and functional outcomes after injury to the spinal cord. Promoting the inhibition of this complex decreases the inflammatory response by decreasing the levels of pro-inflammatory cytokines IL-Ιβ and IL-18. These cytokines are responsible for the deleterious effects that are seen during many inflammatory responses in conditions such as trauma to the central nervous system, neurodegenerative diseases or autoimmune diseases. Thus decreasing the activation of these cytokines results in improved outcomes following these detrimental conditions.

[0079] In protein lysates obtained from patients that had been diagnosed with AGA, inimunobiot analysis revealed inflammasome proteins caspase- 1, ASC, NLRPl and XIAP in hair follicles. Patients treated with Propecia™ demonstrated down regulation of ASC and NLRPl expression. In addition, caspase-1 activation and cleavage of XIAP were inhibited by Propecia™ treatment. Non-responsive patients showed increased, cleavage/activation of caspase- 1 and cleavage of the inhibitory protein XIAP. These findings suggest that the inflammasome can be used as a therapeutic target to improve treatment outcomes in patients and. importantly used to de velop diagnostics for the determination of efficacy of hair loss treatments and diseases of the skin and scalp.

[0080] Current treatment options for AGA are topical minoxidil therapy and oral finasteride (Propecia™), The mode of action for minoxidil topical therapy is still uncertain but it is well established that it does not give cosmetically acceptable results for the majority of patients. For patients taking oral finasteride as Propecia™, results from the 5-year clinical studies published by Merck & Co*, indicate that if men see any optimal hair regrowth, it will occur in the first year of therapy. After the second year of therapy, hair growth plateaus and hair counts decline with some seeing stabilization of the hair cycle and only a few percent continuing to see hair growth at a cosmetically acceptable level. While oral finasteride must continuously be taken, it is uncertain if it is having any effect at all for the majority of men who may be having minimal to no effective hair regrowth. For many men, they continue to take oral finasteride in hopes that it is stabilizing their hair loss and inhibiting the continual miniaturization that usually takes place with AG A.

[0081] The cost of taking oral finasteride as Propecia™, can be approximately $600.00 per year, if not more. It may be more cost effective for many patients to assess their hair growth potential by assessing the infiammasome complex for predicting hair regrowth potential. Finasteride has also been shown to have adverse side effects for some men and it would be of great benefit to those taking the drug to discontinue the therapy if it is not having any hair growth effect. Therefore, assessing the patient's potential for hair regrowth due to AGA by examining the infiammasome complex may be beneficial for several reasons. First, hair regrowth potential can be assessed and whether or not finasteride will be effective can be determined. Second, finasteride therapy can be discontinued to avoid any side effects if results show an upregulation of infiammasome associated factors. Third, cost effectiveness and savings are provided to the patient. Thus far, assessing the role of the infiammasome and associated factors and how they effect the hair growth cycle in humans has generated much knowledge in this field. From this, more effective preventative treatments can be generated, and new treatments for those already affected by AGA can be established.

Other Embodiments

[0082] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. For example, although the experiments described herein involve subjects diagnosed with AGA, the compositions and methods described herein can find use in a number of other diseases and disorders, including alopecia areata and cicatricial scarring alopecia's, acne, seborrhea, hirsutism, polycystic ovarian disease, androgen- reiated infertility disorders, BPH, coronary artery disease, insulin resistance, hypertension and. prostate cancer. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abo ve-described elements in all possib le variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.