(12) United States Patent (10) Patent No.: US 8,748,464 B2 Verdin Et Al

USOO8748464B2

(12) United States Patent (10) Patent No.: US 8,748,464 B2 Verdin et al. (45) Date of Patent: Jun. 10, 2014

(54) USE OF SIRT1 ACTIVATORS OR INHIBITORS Ott et al., “Immune hyperactivation of HIV-1-infected T cells medi TO MODULATE AN IMMUNE RESPONSE ated by Tat and the CD28 pathway.” (1997) Science 275: 1481-1485. Nayagam et al., “SIRT1 modulating compounds from high-through (75) Inventors: Eric M. Verdin, San Francisco, CA put Screening as anti-inflammatory and insulin-sensitizing agents.” (US); Melanie Ott, San Francisco, CA (2006) J. Biomol. Screening 11:959-967. (US); Hye-Sook Kwon, San Mateo, CA Ott, M. “Role of SIRT1 in T cell Hyperacitivation during HIV infec (US); Hyungwook Lim, San Francisco, tion California HIV/AIDS Research Program.” 2008 http://chrp. ucop.edu/funded research/abstracts/2007 ott.html (2007), para CA (US) 1-3. Feige et al., “Specific SIRT1 Activation Mimics Low Energy Levels (73) Assignee: The J. David Gladstone Institutes, San and Protects against Diet-Induced Metabolic Disorders by Enhanc Francisco, CA (US) ing Fat Oxidation.” (2008) Cell Metabolism 8:347-358. Sakamoto, K. “Silencing Metabolic Disorders by Novel SIRT1 Acti (*) Notice: Subject to any disclaimer, the term of this vators.” (2008) Cell Metabolism 7:3-4. patent is extended or adjusted under 35 Pacholec et al., “SRT1720, SRT2183, SRT1460, and Resveratrol are U.S.C. 154(b) by 519 days. not Direct Activators of SIRT1. Published on Jan. 8, 2010 as Manu script M109.088682 The latest version is at http://www.jbc.org/cgi/ (21) Appl. No.: 12/838,247 doi/10.1074/bc.M109.088.682–25 pages. Sundrud et al., “Synergistic and combinatorial control of T cell (22) Filed: Jul. 16, 2010 activation and differentiation by transcription factors.” ScienceDirect www.sciencedirect.com, Current Opinion in Immunology (2010) (65) Prior Publication Data 22:1-7. Tao et al., “Deacetylase inhibition promotes the generation and func US 2010/0330114A1 Dec. 30, 2010 tion of regulatory T cells.” (2007) Nat. Med. 13 (11): 1299-1307. Saouaf et al., “Deacetylase inhibition increases regulatory T cell Related U.S. Application Data function and decreases incidence and severity of collagen-induced arthritis.” (2009) Exp. Mol. Pathol. 87:99-104. (63) Continuation-in-part of application No. Van Loosdregt et al., “Regulation of Treg functionality by acetyla PCT/US2009/000761, filed on Feb. 5, 2009. tion-mediated Foxp3 protein stabilization” (2010) Blood (60) Provisional application No. 61/026,997, filed on Feb. 115(5):965-974. 7, 2008. Wang et al., “Using histone deacetylase inhibitors to enhance Foxp3+ regulatory T-cell function and induce allograft tolerance.” (2009) Immunol. Cell Biol. 87: 195-202. (51) Int. Cl. Pagans, et al. “SIRT1 Regulates HIV Transcription via TAT AOIN 43/78 (2006.01) Deacetylation”. PLoS Biology, Feb. 2005, vol. 3, Issue 2, pp. 0210 A 6LX3/535 (2006.01) O220. (52) U.S. Cl. USPC ...... 514/366; 514/229.5: 514/233.2 * cited by examiner (58) Field of Classification Search USPC ...... 514/366 Primary Examiner — Sreeni Padmanabhan See application file for complete search history. Assistant Examiner — Timothy E. Betton (74) Attorney, Agent, or Firm — Paula A. Borden; (56) References Cited Bozicevic, Field & Francis LLP. U.S. PATENT DOCUMENTS (57) ABSTRACT 7,345,178 B2* 3/2008 Nunes et al...... 548.154 The present disclosure provides a method of increasing an 2005/02093OO A1 9/2005 Napper et al. 2007/0043050 A1 2/2007 Nunes et al. immune response in an individual, the method involving 2007/0105109 A1 5/2007 Geesaman et al. administering to an individual in need thereof an inhibitor of 2007/O1900.73 A1 8, 2007 Tucket al. SIRT1. The present disclosure provides a method of reducing 2008.O255382 A1 10/2008 Andrus et al. an immune response, e.g., to treat chronic immune hyperac 2009/0012080 A1 1/2009 Bemis et al. tivity, the method generally involving administering to an 2010 OO61984 A1 3/2010 Greene et al. individual in need thereofan activator of SIRT1. The present OTHER PUBLICATIONS disclosure provides a method of modulating activation and differentiation of CD4 T cells. Milne et al "Small rinoiecuie activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.” (2007) Nature 450:712-716. 7 Claims, 13 Drawing Sheets

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***************§…*,** US 8,748,464 B2 1. 2 USE OF SIRT1 ACTIVATORS OR INHIBITORS individual in need thereofan activator of SIRT1. The present TO MODULATE AN IMMUNE RESPONSE disclosure provides a method of modulating activation and differentiation of CD4 T cells. CROSS-REFERENCE BRIEF DESCRIPTION OF THE DRAWINGS This application is a continuation-in-part of International Patent Application No. PCT/US2009/000761, which claims FIGS. 1A-F depict data showing the effect of Tat and the benefit of U.S. Provisional Patent Application No. 61/026, nicotinamide on T cell hyperactivation. 997, filed Feb. 7, 2008, which applications are incorporated FIGS. 2A-D depict the Tat-mediated superinduction of herein by reference in their entirety. 10 NF-kB-responsive genes. FIGS. 3A-C depicts the effect of Tat on the inhibitory effect STATEMENT REGARDING FEDERALLY of SIRT1 on NK-K-B-responsive promoters. SPONSORED RESEARCH FIGS. 4A-E depict binding of Tat to the acetyl lysine binding site in SIRT1. This invention was made with government Support under 15 FIGS.5A-D depict the effect of Tat on SIRT1 deacetylase grant no. R01 A1067.118-01A awarded by the National Insti activity. tutes of Health. The government has certain rights in the FIGS. 6A-C depict the effect of hyperacetylation of p85 by invention. Tat on T-cell hyperactivation. BACKGROUND FIGS. 7A and 7B depict a model for Tat effects on SIRT1 deacetylase activity, T-cell activation, and HIV transcription. Immune activation is a hallmark of human immunodefi FIGS. 8A and 8B depict the effect of SIRT1 activators on ciency virus-1 (HIV-1) infection and a significant factor that Tat-mediated T cell hyperactivation. promotes continuous viral replication and CD4+ T-cell deple FIGS. 9A and 9B depict the effect of SIRT1 on induced tion. In HIV-infected individuals, levels of circulating activa 25 Treg (iTreg) differentiation. tion markers correlate with accelerated disease progression FIGS. 10A-D depict interaction of SIRT1 with FoxP3, and and shortened survival. HIV infection is critically dependent the effect of inhibition of SIRT1 deacetylase activity on on the activated state of CD4+ T cells since the virus cannot acetylation of FoxP3. replicate efficiently in resting T cells. Quiescent T cells in FIGS. 11A-D depict destabilization of FoxP3 protein via blood are refractory to infection because of blocks at the level 30 deacetylation by SIRT1. of reverse transcription and proviral integration. In addition, FIGS. 12A and 12B depict the effect of inhibition of SIRT1 T-cell activation enhances viral transcription through the acti activity on differentiation of iTreg cells and FoxP3 expression Vation of various transcription factors, notably nuclear factor in nTreg cells. KB (NF-kB). FIG. 13 depicts an alignment of amino acid sequences of SIRT1 is a mammalian homologue of the yeast transcrip 35 tional repressor silent information regulator 2 (Sir2), an human FoxP3 (GenBank Accession Nos. NP 054728 and important factor governing longevity in yeast. Like Sir2. NM 014009), mouse FoxP3 (GenBank Accession Nos. SIRT1 requires nicotinamide adenine dinucleotide (NAD') NM 054039 and NP 473380), and cow FoxP3 (GenBank as a cofactor, which links its activity to the metabolic state of Accession Nos. NM 001045933 and NP 001039398). the cell. In addition to its enzymatic activity on histone Sub 40 strates in vitro, recent experimental evidence suggests that DEFINITIONS

SIRT1 predominantly targets nonhistone proteins for 99 &g deacetylation. It has been reported that Tat is a substrate for As used herein, the terms “treatment,” “treating, and the the deacetylase activity of SIRT1. Acetylation of lysine 50 like, refer to obtaining a desired pharmacologic and/or physi (K50) in Tat is mediated by the histone acetyltransferase 45 ologic effect. The effect may be prophylactic in terms of activities of p300 and human GCN5 and generates binding completely or partially preventing a disease or symptom sites for the bromodomains of PCAF and Brg1. SIRT1 binds thereof and/or may be therapeutic in terms of a partial or and deacetylates Tat at K50, a process necessary to recycle complete cure for a disease and/or adverse affect attributable nonacetylated Tat protein for binding to TAR RNA and the to the disease. “Treatment’, as used herein, covers any treat cellular positive transcription elongation factor b (P-TEFb). 50 ment of a disease in a mammal, e.g., in a human, and includes: Literature (a) preventing the disease from occurring in a Subject which Milne et al., (2007) Nature 450:712-716; U.S. Patent Pub may be predisposed to the disease but has not yet been diag lication No. 2007/0043050; U.S. Pat. No. 7,345,178; Ottet al. nosed as having it; (b) inhibiting the disease, i.e., arresting its (1997) Science 275:1481; U.S. Patent Publication No. 2009/ development; and (c) relieving the disease, e.g., causing 0012080; U.S. Patent Publication No. 2008/0255382: Naya 55 regression of the disease, e.g., to completely or partially gam et al. (2006) J. Biomol. Screening 11:959; U.S. Patent remove symptoms of the disease. Publication No. 2007/0105109; U.S. Patent Publication No. The term “effective amount’ or “therapeutically effective 2007/0190073: U.S. Patent Publication No. 2005/0209300. amount’ means a dosage Sufficient to provide for treatment for the disease state being treated or to otherwise provide the SUMMARY OF THE INVENTION 60 desired effect (e.g., induction of an effective immune response, reduction of chronic immune hyperactivity, etc.). The present disclosure provides a method of increasing an The precise dosage will vary according to a variety of factors immune response in an individual, the method involving Such as Subject-dependent variables (e.g., weight, age, etc.), administering to an individual in need thereof an inhibitor of the disease, and the treatment being effected. SIRT1. The present disclosure provides a method of reducing 65 The terms “individual,” “host,” “subject,” and “patient.” an immune response, e.g., to treat chronic immune hyperac used interchangeably herein, refer to a mammal, including, tivity, the method generally involving administering to an but not limited to, murines, felines, simians, humans, etc. In US 8,748,464 B2 3 4 Some embodiments, an individual is a human. In some coordinates with an organic base Such as ethanolamine, embodiments, an individual is a rodent (e.g., a mouse, a rat, diethanolamine, triethanolamine, tromethamine, N-methyl etc.) or a lagomorph. glucamine, and the like. A“pharmaceutically acceptable excipient,” “pharmaceuti Before the present invention is further described, it is to be cally acceptable diluent,” “pharmaceutically acceptable car understood that this invention is not limited to particular rier and pharmaceutically acceptable adjuvant’ means an embodiments described, as Such may, of course, vary. It is excipient, diluent, carrier, or adjuvant that is useful in prepar also to be understood that the terminology used herein is for ing a pharmaceutical composition that is generally safe, non the purpose of describing particular embodiments only, and is toxic and neither biologically nor otherwise undesirable, and not intended to be limiting, since the scope of the present include an excipient, diluent, carrier, or adjuvant that is 10 acceptable for veterinary use as well as human pharmaceuti invention will be limited only by the appended claims. cal use. A pharmaceutically acceptable excipient, diluent, Where a range of values is provided, it is understood that carrier and adjuvant as used in the specification and claims each intervening value, to the tenth of the unit of the lower includes one and more than one such excipient, diluent, car limit unless the context clearly dictates otherwise, between rier, and adjuvant. 15 the upper and lower limit of that range and any other stated or As used herein, a “pharmaceutical composition' is meant intervening value in that stated range, is encompassed within to encompass a composition Suitable for administration to a the invention. The upper and lower limits of these smaller Subject, Such as a mammal, e.g., a human. In general a “phar ranges may independently be included in the Smaller ranges, maceutical composition' is sterile, and is free of contami and are also encompassed within the invention, Subject to any nants that are capable of eliciting an undesirable response specifically excluded limit in the stated range. Where the within the Subject (e.g., the compound(s) in the pharmaceu stated range includes one or both of the limits, ranges exclud tical composition is pharmaceutical grade). Pharmaceutical ing either or both of those included limits are also included in compositions can be designed for administration to Subjects the invention. or patients in need thereof via a number of different routes of Unless defined otherwise, all technical and scientific terms administration including oral, buccal, rectal, parenteral, intra 25 used herein have the same meaning as commonly understood peritoneal, intradermal, intratracheal and the like. In some by one of ordinary skill in the art to which this invention embodiments the composition is Suitable for administration belongs. Although any methods and materials similar or by a transdermal route, using a penetration enhancer other equivalent to those described herein can also be used in the than dimethylsulfoxide (DMSO). In other embodiments, the practice or testing of the present invention, the preferred pharmaceutical compositions are Suitable for administration 30 methods and materials are now described. All publications by a route other than transdermal administration. A pharma mentioned herein are incorporated herein by reference to ceutical composition will in some embodiments include a disclose and describe the methods and/or materials in con compound and a pharmaceutically acceptable excipient. In nection with which the publications are cited. Some embodiments, a pharmaceutically acceptable excipient It must be noted that as used herein and in the appended is other than DMSO. 35 claims, the singular forms “a,” “an,” and “the include plural As used herein, “pharmaceutically acceptable derivatives' referents unless the context clearly dictates otherwise. Thus, of a compound of the invention include salts, esters, enol for example, reference to “a SIRT1 modulator includes a ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, plurality of such modulators and reference to “the SIRT1 hemiketals, acids, bases, Solvates, hydrates or prodrugs inhibitor” includes reference to one or more SIRT1 inhibitors thereof. Such derivatives may be readily prepared by those of 40 and equivalents thereof known to those skilled in the art, and skill in this art using known methods for Such derivatization. so forth. It is further noted that the claims may be drafted to The compounds produced may be administered to animals or exclude any optional element. As such, this statement is humans without substantial toxic effects and are either phar intended to serve as antecedent basis for use of such exclusive maceutically active or are prodrugs. terminology as “solely.” “only' and the like in connection A "pharmaceutically acceptable salt” of a compound 45 with the recitation of claim elements, or use of a “negative' means a salt that is pharmaceutically acceptable and that limitation. possesses the desired pharmacological activity of the parent The publications discussed herein are provided solely for compound. Such salts include: (1) acid addition salts, formed their disclosure prior to the filing date of the present applica with inorganic acids such as hydrochloric acid, hydrobromic tion. Nothing herein is to be construed as an admission that acid, Sulfuric acid, nitric acid, phosphoric acid, and the like; 50 the present invention is not entitled to antedate Such publica or formed with organic acids Such as acetic acid, propionic tion by virtue of prior invention. Further, the dates of publi acid, hexanoic acid, cyclopentanepropionic acid, glycolic cation provided may be different from the actual publication acid, pyruvic acid, lactic acid, malonic acid, Succinic acid, dates which may need to be independently confirmed. malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic 55 DETAILED DESCRIPTION acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1.2-ethanedisulfonic acid, 2-hydroxyethanesulfonic The present disclosure provides a method of increasing an acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, immune response in an individual, the method involving 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, cam administering to an individual in need thereof an inhibitor of phorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3- 60 SIRT1. The present disclosure provides a method of reducing hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, an immune response (e.g., to treat chronic immune hyperac trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric tivity, to treat an autoimmune disorder), the method generally acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, involving administering to an individual in need thereof an salicylic acid, Stearic acid, muconic acid, and the like; or (2) activator of SIRT1. The present disclosure provides a method salts formed when an acidic proton present in the parent 65 of modulating activation and differentiation of CD4 T cells, compound either is replaced by a metal ion, e.g., an alkali the method generally involving contacting the CD4 T cell metal ion, an alkaline earth ion, or an aluminum ion; or with a SIRT1 inhibitor. US 8,748,464 B2 5 6 As used herein, “SIRT1 refers to a polypeptide exhibiting 7,485,416 and U.S. Patent Publication No. 2005/0287597 for nicotinamide adenosine dinucleotide (NAD)-dependent descriptions of methods of determining SIRT1 Tat deacety deacetylase activity, and having an amino acid sequence that lase activity. is at least about 70%, at least about 75%, at least about 80%, In some embodiments, a suitable SIRT1 modulator (a at least about 85%, at least about 90%, at least about 95%, at SIRT1 activator or a SIRT1 inhibitor) is a selective SIRT1 least about 98%, at least about 99%, or 100%, identical to the modulator. For example, in some embodiments, a Suitable amino acid sequence of a known SIRT1 polypeptide. A SIRT1 activator is a selective SIRT1 activator; and in some SIRT1 protein includes yeast Sir2 (GenBank Accession No. embodiments, a suitable SIRT inhibitor is a selective SIRT1 P53685); C. elegans Sir-2.1 (GenBank Accession No. inhibitor. For example, in some embodiments, a suitable NP 501912); human SIRT1 (see, e.g., GenBank Accession 10 SIRT1 activator increases the enzymatic activity of a SIRT1 No. NM 012238 or NP 036370 (or AF083106); Frye polypeptide, but does not substantially increase the enzy ((1999) Biochem. Biophys. Res. Comm. 260:273); and Gen matic activity of any other sirtuin. For example, in some Bank Accession Nos. Q96EB6, AAH12499, NP 036370, embodiments, a suitable SIRT1 activator increases the enzy and AAD40849); mouse SIR1 (see, e.g., GenBank Accession matic activity of a SIRT1 polypeptide, but does not substan Nos. Q923E4 and NP 062786); and equivalents and frag 15 tially increase the enzymatic activity of SIRT2, SIRT4, or ments thereof. For example, a SIRT1 polypeptide comprises SIRT5. In some embodiments, a suitable SIRT1 inhibitor an amino acid sequence having at least about 70%, at least reduces the enzymatic activity of a SIRT1 polypeptide but about 75%, at least about 80%, at least about 85%, at least does not Substantially reduce the enzymatic activity of any about 90%, at least about 95%, at least about 98%, at least other sirtuin. For example, in some embodiments, a Suitable about 99%, or 100%, amino acid sequence identity with a SIRT1 inhibitor reduces the enzymatic activity of a SIRT1 contiguous stretch of from about 500 amino acids (aa) to polypeptide, but does not Substantially reduce the enzymatic about 550 aa, from about 550 aa to about 600 aa, from about activity of SIRT2, SIRT4, or SIRT5. 600 aa to about 650 aa, from about 650 aa to about 700 aa, or In some embodiments, a suitable SIRT1 modulator modu from about 700 aa to about 747 aa, of the amino acid sequence lates the activity of SIRT1, and can also modulate the activity set forth in SEQID NO:1. 25 of one or more additional sirtuins. For example, in some SIRT1 Modulators embodiments, a suitable SIRT1 activator increases the enzy A suitable SIRT1 modulator modulates an enzymatic matic activity of a SIRT1 polypeptide, and can also increase activity of a SIRT1 polypeptide. For example, a suitable the enzymatic activity of SIRT5. As another example, in some SIRT1 modulator modulates Tat deacetylase activity of embodiments, a suitable SIRT1 inhibitor reduces the enzy SIRT1. SIRT1 Tat deacetylase activity can be determined 30 matic activity of a SIRT1 polypeptide, and can also reduce the using known methods. For example, a Tat polypeptide (e.g., a enzymatic activity of SIRT5. synthetic Tat polypeptide) that is acetylated is used as a sub A suitable SIRT1 activator can increase the enzymatic strate. A reaction sample can include an acetylated Tat activity of SIRT1 by at least about 25%, at least about 50%, at polypeptide, NAD", a SIRT1 polypeptide, and a SIRT1 least about 75%, at least about 2-fold, at least about 2.5-fold, modulator. Acetylated Tat polypeptides include Tat polypep 35 at least about 5-fold, at least about 10-fold, at least about tides having one or more acetylated lysine residues, e.g., 20-fold, or more than 20-fold. A suitable SIRT1 activator can acetylated K28, acetylated K41, and acetylated K50. A suit increase the enzymatic activity of SIRT1 by from about 25% able acetylated Tat polypeptide includes the amino acid to about 50%, from about 50% to about 75%, from about 75% sequence Ser-Tyr-Gly-Arg-AcLys-Lys-Lys-Arg-Arg-Gln to about 2-fold, from about 2-fold to about 5-fold, from about Arg (SEQID NO:02), where AcLys is acetylated lysine. An 40 5-fold to about 10-fold, from about 10-fold to about 20-fold, acetylated Tat protein can be generated as described in, e.g., or more than 20-fold. Doerr et al. (2002) EMBO.J. 21:2715-2723; or Peloponese A suitable SIRT1 inhibitor can reduce the enzymatic activ (1999).J. Biol. Chem. 274:11473-11478. Suitable acetylated ity of SIRT1 by at least about 5%, at least about 10%, at least Tat polypeptides are described in, e.g., U.S. Pat. No. 7,485, about 15%, at least about 20%, at least about 25%, at least 416 and U.S. Patent Publication No. 2005/0287597. 45 about 30%, at least about 35%, at least about 40%, at least The effect of the SIRT1 modulator on SIRT1 Tat deacety about 45%, at least about 50%, at least about 60%, at least lase activity can be determined by measuring the amount of about 65%, at least about 70%, at least about 75%, or more deacetylated Tat polypeptide produced by action of the SIRT1 than 75%. A suitable SIRT1 inhibitor can reduce the enzy polypeptide in the presence of the SIRT1 modulator, com matic activity of SIRT1 by from about 5% to about 10%, from pared to the amount of deacetylated Tat polypeptide produced 50 about 10% to about 15%, from about 15% to about 20%, from by action of the SIRT1 polypeptide in a control reaction about 20% to about 25%, from about 25% to about 30%, from sample that does not include the SIRT1 modulator. As an about 35% to about 40%, from about 40% to about 50%, from alternative to, or in addition to, measuring the amount of about 50% to about 60%, from about 60% to about 75%, or deacetylated Tat polypeptide, the amount of remaining acety more than 75%. lated Tat can be measured. Methods of determining the level 55 A suitable SIRT1 activator can increase SIRT1 enzymatic of acetylated Tat in a sample include immunological assays activity at an ECso (half maximal effective concentration) of using antibody that is specific for acetylated form of Tat, and from about 1 nM to about 1 mM, e.g., from about 1 nM to that therefore distinguishes between acetylated Tat and about 10 nM, from about 10 nM to about 15 nM, from about deacetylated Tat. Any of a variety of immunological assays 15 nM to about 25 nM, from about 25 nM to about 50 nM, can be used, including, e.g., enzyme linked immunosorbent 60 from about 50 nM to about 75 nM, from about 75 nM to about assay (ELISA), radioimmunoassay (RIA), protein blot 100 nM, from about 100 nM to about 150 nM, from about 150 (“Western blot) assays, and the like. In some embodiments, nM to about 200 nM, from about 200 nM to about 250 nM, mass spectroscopy is used. SIRT1 activity can also be deter from about 250 nM to about 300 nM, from about 300 nM to mined by measuring the level of NAD in the test sample. The about 350 nM, from about 350 nM to about 400 nM, from action of SIRT1 on acetylated Tat can be coupled to a second 65 about 400 nM to about 450 nM, from about 450 nM to about enzymatic reaction that reduces NAD to NADH, and measur 500 nM, from about 500 nM to about 750 nM, from about 750 ing fluorescence of NADHat, e.g., 340 nm. See U.S. Pat. No. nM to about 1 uM, from about 1 uM to about 10 uM, from US 8,748,464 B2 7 8 about 10 uM to about 25uM, from about 25uM to about 50 Methods of Reducing an Immune Response uM, from about 50 uM to about 75uM, from about 75uM to In some embodiments, the present disclosure provides about 100 uM, from about 100 uM to about 250 uM, from methods of reducing an immune response, the methods gen about 250 uM to about 500 uM, or from about 500 uM to erally involving administering to an individual in need thereof about 1 mM. an effective amount of a SIRT1 activator. A suitable SIRT1 activator can increase SIRT1 enzymatic In some embodiments, an effective amount of a SIRT1 activity at an ECs (concentration of compound required to activator is an amount that, when administered to an indi increase enzyme activity by 50%) of from about 1 nM to vidual in one or more doses, is effective to reduce the level of about 1 mM, e.g., from about 1 nM to about 10 nM, from a cytokine associated with an immune response. Cytokines 10 associated with an immune response include, e.g., interleu about 10 nM to about 15 nM, from about 15 nM to about 25 kin-2 (IL-2), tumor necrosis factor-alpha (TNF-C.), interleu nM, from about 25 nM to about 50 nM, from about 50 nM to kin-4 (IL-4), interferon gamma (IFN-Y), and interleukin-12 about 75 nM, from about 75 nM to about 100 nM, from about (IL-12). In some embodiments, an effective amount of a 100 nM to about 150 nM, from about 150 nM to about 200 SIRT1 activator is an amount that, when administered to an nM, from about 200 nM to about 250 nM, from about 250 nM 15 individual in one or more doses, is effective to reduce the level to about 300 nM, from about 300 nM to about 350 nM, from in the individual of a cytokine associated with an immune about 350 nM to about 400 nM, from about 400 nM to about response by at least about 10%, at least about 15%, at least 450 nM, from about 450 nM to about 500 nM, from about 500 about 20%, at least about 25%, at least about 30%, at least nM to about 750 nM, from about 750 nM to about 1 uM, from about 35%, at least about 40%, at least about 50%, at least a bout 1 uM to about 10 uM, from about 10 uM to about 25 about 60%, at least about 70%, or at least about 80%, com M, from about 25 uM to about 50 uM, from about 50 uM to pared to the level of the cytokine in the absence of treatment bout 75uM, from about 75 uM to about 100 uM, from about with the SIRT1 activator. 00 uM to about 250 uM, from about 250 uM to about 500 In some embodiments, an effective amount of a SIRT1 M, or from about 500 uM to about 1 mM. For example, a activator is an amount that, when administered to an indi uitable SIRT1 activator can have an ECs of from about 0.01 25 vidual in one or more doses, is effective to reduce the level of M to about 100 uM, e.g., from about 0.01 uM to about 0.1 IL-2 (e.g., circulating IL-2, e.g., a serum level of IL-2) in the :M. from about 0.1 uM to about 0.5uM, from about 0.5uM individual by at least about 10%, at least about 15%, at least to about 1.0 LM, from about 1.0 uM to about 10 uM, from about 20%, at least about 25%, at least about 30%, at least about 10 uM to about 25uM, from about 25uM to about 50 about 35%, at least about 40%, at least about 50%, at least 30 about 60%, at least about 70%, or at least about 80%, com uM, or from about 50 uM to about 100 uM. pared to the level of IL-2 in the individual in the absence of A suitable SIRT1 inhibitor can have an ICso of less than 50 treatment with the SIRT1 activator. uM, e.g., a suitable SIRT1 inhibitor can have an ICs of from Methods of reducing an immune response are useful for about 50 uM to about 5 nm, or less than 5 nM. For example, treating chronic immune hyperactivation. Thus, the instant in some embodiments, a suitable SIRT1 inhibitor has an ICso 35 disclosure provides methods for treating chronic immune of from about 50 uM to about 25 uM, from about 25 M to hyperactivation, the methods generally involving administer about 10 M, from about 10 uM to about 5uM, from about 5 ing to an individual in need thereof an effective amount of a uM to about 1 uM, from about 1 uM to about 500 nM, from SIRT1 activator. In some embodiments, the instant disclosure about 500 nM to about 400 nM, from about 400 nM to about provides a method for treating chronic immune hyperactiva 300 nM, from about 300 nM to about 250 nM, from about 250 40 tion, the method comprising administering to an individual in nM to about 200 nM, from about 200 nM to about 150 nM, need thereof an effective amount of a selective SIRT1 activa from about 150 nM to about 100 nM, from about 100 nM to tor. In some embodiments, the instant disclosure provides a about 50 nM, from about 50 nM to about 30 nM, from about method for treating chronic immune hyperactivation that 30 nM to about 25 nM, from about 25 nM to about 20 nM, results from infection with an immunodeficiency virus, e.g., a from about 20 nM to about 15 nM, from about 15 nM to about 45 human immunodeficiency virus (e.g., HIV-1), the method 10 nM, from about 10 nM to about 5 nM, or less than about 5 comprising administering to an individual in need thereof an nM. effective amount of a SIRT1 activator, where in some embodi SIRT1 modulators are known in the art, and any modulator ments, the SIRT1 inhibitor is a selective SIRT1 activator. of SIRT1 can be used. For example, Ota et al. ((2006) Onco SIRT1 Activators gene 25:176) discusses sirtinol; resveratrol is discussed in, 50 Examples of SIRT1 activators that are suitable for use in a e.g., de la Lastra (2005) Mol. Nutr Food Res. 49:405; Napper subject method include, but are not limited to, resveratrol et al. ((2005).J. Med. Chem. 48:8045) discusses indole com ((E)-5-(p-Hydroxystyryfiresorcinol (E)-5-(4-hydroxystyryl) pounds that are SIRT1 inhibitors; Solomon et al. (2006) Mol. benzene-1,3-diol); or 3.5,4'-trihydroxy-trans-stilbene); Cell. Biol. 26:28) discusses EX-527, a SIRT1 inhibitor: U.S. butein (3.4.2',4'-tetrahydroxychalcone); piceatannol (3.5.3', Patent Publication Nos. 2007/0043050, 2007/0037865, and 55 4-tetrahydroxy-trans-Stilbene); isoliquiritigenin (4.2',4'-tri 2007/0037809 discuss SIRT1 modulators; and U.S. Patent hydroxychalcone); fisetin (37.3',4'-tetrahydroxyflavone); Publication No. 2008/0021063 discusses SIRT1 modulators. quercetin (3.5.7.3',4'-pentahydroxyflavone); a SIRT1 activa Whether a given agent (e.g., a SIRT1 modulator, such as a tor as described in U.S. Pat. No. 7,345,178; a SIRT1 activator SIRT1 inhibitor or a SIRT1 activator) is effective in modulat as described in U.S. Patent Publication No. 2008/02555382: ing an immune response (e.g., increasing an immune 60 and a SIRT1 activator as described in U.S. Patent Publication response; e.g., reducing an immune response, Such as reduc No. 2009/0012080. Pharmaceutically acceptable salts of any ing chronic immune hyperactivity) can be readily determined of the foregoing SIRT1 activators are also suitable for use in using well known methods to assess one or more immune a Subject method. response parameters. Immune response parameters include, For example, a suitable SIRT1 activator is a compound of but are not limited to, CD4 count; serum cytokine levels, e.g., 65 any one of Formulas I-XXVIII as described in U.S. Pat. No. serum levels of IL-2; serum antibody levels; levels of an 7,345,178, where substituents are as described in U.S. Pat. autoantibody; and the like. No. 7,345,178, or a pharmaceutically acceptable salt of a US 8,748,464 B2 9 10 compound of any one of Formulas I-XXVIII as described in TABLE 1-continued U.S. Pat. No. 7.345,178, provided that the compound acti vates SIRT1 activity. For example, a suitable SIRT1 activator includes a compound shown in Table 4 of U.S. Pat. No. 7,345,178. 5 O For example, suitable SIRT1 activators are shown in Table Br 1, below. O NH

TABLE 1. 10 OH O O O O Compound 5 OH HN 15 O O O NH

O NH O e O 2O O NH S. OH O Compound 6 Compound 1 25

OH O N NN a O OH O 30 NR O HN NH

NH O O 35 - O Compound 7 Compound 2 O

OH 40

O No NH

HN O O 45 O n-1

O NH O Compound 8 OH 50

Compound 3 O21 O 55 O W O N NH N O- H HN 60

O O HN -- Compound 4 Compound 9 65 US 8,748,464 B2 11 12 TABLE 1-continued TABLE 1-continued

N N N N- n O O

O 21 O H C NH Compound 29

O o 10 O O

N N OH n N Compound 10 2 N 15 O 21 f Compound 31 F N O O -(- F 2O O F NN N )-( N 2NOX- X- N-N N)-() Compound 19 21NO 25 Compound 32

O O N-N - N) ( )-(\ /) O 2No. H 30 N N V Compound 20 C2 NoX-()- O Compound 33 N-n - ) ( )-( ) 2No. O Compound 21 N N-N O

F 2 O O Compound 34

N N O O n N N \ 45 Nn N O 21 O H N N \ Compound 22 2 O Compound 35 O 50 N N N/ / Oy21 No ()Compound 24 OK 55 N y , -)- V - O 2 O H O Compound 36

N N 60 O

2. \ / (\ Cy-K)-21 No H Compound 27 65 Compound 37 US 8,748,464 B2 13 14 TABLE 1-continued TABLE 1-continued O \ () 5 N

21 No 15 Compound 43 Compound 38 () / N

H N 25 N N-y O 21 No Compound 45 30 Compound 39 O

N N N O y H O F F 2No. 40 CO Compound 46

Compound 40 O O F 45 Y, F H F N y” H N N 50 n N O O 21 No Compound 48

Compound 41 55 ( V 60 N O

21s No)-( s Compound 49 Compound 42 65 US 8,748,464 B2 15 16 TABLE 1-continued TABLE 1-continued

O o

O 5

H X-f N F F H N n N O N O N 10 N N O / 21 No n N Compound 50 2 O Compound 55 15

H y” 2O N N N O Nn N O C-321 O C-32 O Compound 51 25 Compound 56

O- NO ON

O 30 \ O O\

O N H sy 35 N-N N 2NO elN/N \ / Compound 52 Compound 57 40 O- O-N-

N y” 45 NHC.

N N O 50 N N3-) 2NO Compound 53 C O \ / Compound 58

60 NH N N s N NS 2 O lus2 V / Compound 54 65 Compound 59 US 8,748,464 B2 17 18 TABLE 1-continued TABLE 1-continued

O-N- No

O 'ss \ o21 O NH 10 N NH n N N N elN/ \ / Compound 60 21 O \ N/ 15 C-K. Compound 66 O-N- NMe2 O s \ o?e NH O N n N - 25 NH 2No. \ / N Compound 61 2 O \ N/ N N 30 Compound 68 n N NN 21 No N NMe2 CF 35 O F Compound 62 NH N O-N- 40 n N N

O 2N/ \ / O \ Compound 70 45 NH N N - g-C> O C-K.21 No \ / 50 Compound 63 . . . . .

NMe2 Compound 71

Compound 64 65 Compound 72 US 8,748,464 B2 19 20 TABLE 1-continued TABLE 1-continued

O ( ) /\

O | 10 O O) \ / O)D)- \ ^ Compound 78 Compound 73 15 p\ u)-()N O 25 Compound 79 Compound 74 Y, / 30 O

H 35 V N s us 40 O)DX \ / Compound 80 Compound 75 OMe

V N H N OMe 50 O)-4. / \ A N N1\ O Compound 76 -- Compound 82 55

N W N "V 60 Pi—{S o1

1. O C-3 65 Compound 84 Compound 77 US 8,748,464 B2 21 22 TABLE 1-continued TABLE 1-continued O)-N /N r 5 O-N-

Compound 85 . . . . 15 Compound 69 O)- Compound 86 COO O r O /N C 25 Compound 89 On N

N N - 30 y N O 2NO \ / Compound 65 35 S Compound 90 - 40 v () O C-K.)N-N N O O)-Y-N C, C O/ Compound 87 45 O f Compound 91

O O \ 50 O

NH N lsN N N-N - C-2NO N N)-(3 Compound 67 Compound 92 CC-ClNv () 6 -CIOO

/ 65 Compound 93 Compound 88 US 8,748,464 B2 23 24 TABLE 1-continued TABLE 1-continued

ls N

10 O Compound 94 HN S

15 N N-K M n \ N C4N. Compound 97 2No. Compound 95

H N OESEO

S N O 30 HN us N Compound 81 M N S C.4N 35

Compound 98

O 40

H N O N NN-( 45 N1-S-S O / 2N.

Compound 83 50 Compound 99 / Y,

O O

HN 60 H O)lel () C4. / Compound 96 65 Compound 100 US 8,748,464 B2 25 26 TABLE 1-continued TABLE 1-continued

O N H 10 H / N O O

N C ompound 101 2 / - 15 N N NH - O O 4 \ / / Compound 107 O 20 O NH2 N Sl-O N O 25 2 / l? N / \ N N N NH Compound 102 O N / Compound 108 s 30 O O

N H H Sl-O 35 V M N N N N N O O Compound 103 / O 40 2 S N N N Q Compound 109 N n NE N Cl O 21 No \ / 45 Compound 104

O Fl N

N n -CIO - - - - - N - Y-N- Ces? 2 O \ / Compound 110 Compound 105 55 O- / O \ / HV 60 N N N O O N-O O N / M 2 / 2NN N N 65 Compound 111 Compound 106 US 8,748,464 B2 27 28 TABLE 1-continued TABLE 1-continued Y,

/ 1. 10 colo Compound 117

Compound 112 15 roc1. Compound 118

or CompoundOS 119 30

or CompoundOS 120

40 C O O O N N N 45 C Compound 121

O 50 sC. Cl o1 -O 55 Compound 122 Compound 115 Cl O OMe O O 60 N N OMe Cl O 1. O 1. O cCompound 116 65 Compound 123 US 8,748,464 B2 29 30 TABLE 1-continued TABLE 1-continued

O o1 O O S N 5 2n N1 n N N N N 1. N O 1. O Compound 124 10 -O Compound 131 O N O O n N N S N O 15 Y N o1 |-- No O 1. O Compound 132 Compound 125 2O N - O Y, O O / N O

N No N C ompoun d 126 N S O 30 M O 21 N O O N N V N O o1 35 Compound 133 1. O - O Y, Compound 127

O o1 40 O uCCN N o1 45 N1s-S N O Compound 128 A 2N. O OH

O 50 O uCC N N Compound 134 S N o1 \ - O O O 1 55 / Compound 129 O

O N

N A N O 2NN

OH Compound 130 65 Compound 135 US 8,748,464 B2 31 32 TABLE 1-continued TABLE 1-continued

OMe

H H N N O r N Sl-O O N 4N H 15 O / \ Compound 141 Compound 136

MeO

O MeO

25 H N 2. N-O O 4N 30 Nv OH O Compound 137 Compound 142 35 / N w raO 40 s Ks Sl-O O HN

N 45 N N NH O \ / N Compound 138

50 Compound 143 O MeO OMe

H N-O O N 60 O rS N 7 S N N / le O \ / Compound 144 Compound 139 65 US 8,748,464 B2 33 34 TABLE 1-continued TABLE 1-continued MeO O MeO 5 Sl-S O )-( )- N N N - O H Compound 152 N O 10 S C C 7 le S Compound 145 p 15 O OMe

MeO NH N-O O 2O H 2 M N N N O S O M / le> 25 \ \ N N Compound 146 / Compound 153 H O N > 30 -( / N \ NE S N CC \ / 35 Compound 147 N O O N O O CO

H 1SN - O O ( \ . Compound 148 y O 45 Compound 154 N N N)-( )- N Y, N N -O H 50 Compound 149

O N-N O H

O) NH \ ss O N O O 4NN O- N H M Compound 150 2 N N O H 60 N N O ( \- Cy-K)-4. N - O) / \ /N Compound 151 65 Compound 155 US 8,748,464 B2 35 36 TABLE 1-continued TABLE 1-continued

H N 10 O O)-()ll 15 v (i. H

Compound 159 Compound 156 25 ( ( H 30 N H N O O

N -N ( 40 H

45 Compound 160

Compound 157 O

H N O 55 C4N. O 60 C. H Compound 158 65 Compound 161 US 8,748,464 B2 37 38 TABLE 1-continued TABLE 1-continued

- O Y, 5 (

HN n O O O 10 CuO A-() HN O)O) () 15 Compound 166 Compound 162 / –o Y 2O V

\O N O v.

25 2 / O N N HN

M 30 Compound 167 O2 N No Compound 163 35 O

O / 40 O O HN

O)-()ll C-)N N N NH Compound 168 4 \ / 50

Compound 164

/ 55 N\ s NMe HN H N O O 60 N 1. N u 4. / ^ Compound 165 Compound 169 65 US 8,748,464 B2 39 40 TABLE 1-continued TABLE 1-continued

-O O - O O 5

H N 10 O N1S-S O HN S N / e 21NN 15 -N7–C N N / -v Compound 178 Compound 174 \ 2O - O O

MeO O MeO / 25 O HN H N SN2N. O N 2 30 N N / N -N S. \ Compound 179 35 \ Compound 175 21 N N

N )-( )-N MeO N N OMe 40 O MeO Compound 180

- O O

45 O O N 2 N H N N S. O

Compound 176 p s N H MeO Compound 181 55 MeO

60 H 1. O N O 2N 21 N O N N

Compound 177 65 N H Compound 182 US 8,748,464 B2 41 42 TABLE 1-continued TABLE 1-continued \- / 5 \ H s O N H CO-O-, . O Compound 188 4N Compound 183

O- 15

Compound 189 O

H 2O N H C X-K s O N N 25 Compound 190 Compound 184

- O o1

H N

C O 35 N O s ) N} { s Compound 191 N H Compound 185 40 No

O O N H 45 N

O N 50 O Compound 192

Compound 186

O CO 60 N

Sn O Compound 187 65 Compound 193 US 8,748,464 B2 43 44 TABLE 1-continued TABLE 1-continued

/ \ -N 21 S H O O 5

O \ H Compound 194 10 N N-S O

2 N O)sul- ()- -)- 15 V Compound 200 Compound 195 - O O

21 S

25 O)s N ()- H N O Compound 196 S O

30 \ N 21 S H O

Compound 201 O \ 35 O Compound 197 O O- 40 H N O S O

H 45 N N Sl-Sl? N N 50 Compound 202

Compound 198 -O O

H O N 60 HN N S

N l?N Compound 199 65 Compound 203 US 8,748,464 B2 45 46 TABLE 1-continued TABLE 1-continued

- O O - O O

O / O 10 HN S N N KI) () 15 SC) ()

Compound 204

Compound 207 25

40 SI) ()

Compound 205 Compound 208

60 \ SC) () / \ SC) () / d \ / 65 Compound 209 US 8,748,464 B2 47 48 TABLE 1-continued TABLE 1-continued

O 10

15 Compound 215 O N SC) () \ / Compound 210

25 H N O

30 N Compound 211 Compound 216 / 35

H N N O r } { s 40 21 N Compound 212

Y, 45

Compound 217

50 N O rN- O CI) () 55 Compound 213 / 60 H N O N-S O

CI) () 65 Compound 218 Compound 214 US 8,748,464 B2 49 50 TABLE 1-continued TABLE 1-continued

--O/ N H N H N 10 O O)-(3 Z-S Compound 226 Cy O SN 15 H Compound 219 N \ - O O 21 / NF O/ Compound 227 O H N NH y 25 O N N1 y ( ) 21 / 2 N \ N

Compound 228 O O Compound 220 R Y N O H 35 N 2 4 Compound 229 2 / O 2 s - to 40 O O Compound 221

O H N Cl 45 N C-32 / -s Compound 222 Compound 230 50 O H H V/ N N-S NN S S O)-()2 4. t2 55 2N, Compound 223 Compound 231

O 60 N-SHV/

rryM (S O 21 N S Compound 225 65 Compound 232 US 8,748,464 B2 51 52 TABLE 1-continued TABLE 1-continued

O O N-SHV/ \/ 5 N1S-S O K 21 / N N O)21 / () Compound 234 10 NC Compound 240

O O N

\ O \/ 15 H N C O)-()2 / N1S-S O C 2O O) Compound 235 Compound 241

O HV/O N-S 25 N N1S-S N M 2 O)-()2 / 2 N Compound 244 30 O H Compound 236 N O

O O H. V/ 35 / N-S O-K2NN s Compound 245 21 / O)-() 40 CF Compound 237 N N S M 2NN 45 N

2 S-O Compound 246 H N CF O 50 H N N1 N 2-3 O O) A 21 Compound 238 55 2NN Compound 247 O O O H. V/ H N-S N CF 2N. C)2 / () O-CF FC Compound 239 65 Compound 248 US 8,748,464 B2 53 54 TABLE 1-continued TABLE 1-continued - O

10 Compound 249

30 Compound 255

Compound 256 Compound 252 50

OMe 55 MeO

Compound 253 65 Compound 257 US 8,748,464 B2 55 56 TABLE 1-continued TABLE 1-continued

MeO O MeO 5 M / N O O O N 10 / Q O- Compound 263

H \ N N Compound 258 15 H M N N

O O O Compound 264 2O O\ / / NS O 25 O /A \ A O Compound 259 30 HN OMe 21Ne.N. MeO N-N / 35 Compound 265

/ O N O N N 40 N / \ A Compound 260 O 45 HN MeO N 21 Ne. N-N / 50 Compound 266 N O N N / O N / \ A 55 O Compound 261 /

/ 60 HN

O)O ) / C.N-N/ Compound 262 65 Compound 267 US 8,748,464 B2 57 58 TABLE 1-continued TABLE 1-continued

HN 21 2N. 10 N-N/ N-S O Compound 268 15 Compound 273

-O MeO

MeO

H 25 N O N N

H/ \ A 30 Compound 276

Compound 270 O H 35 N N1 N -() ( ) 40 21 N ( )

Compound 280

45 O O H. V/ N-S N S f \ 50 M N Compound 271 2N. o1 Compound 282

55 O N-SH 2 N1 N

60 2N. O

Compound 272 65 Compound 283 US 8,748,464 B2 59 60 TABLE 1-continued TABLE 1-continued O H. S21leo O.\/ O 5 S

21 N 2 / ON- O 10 /soS a" Compound 284 Compound 290

O O H V M 15 N-S O N1S-S W S O O / Compoundi? 285 o- O)N O () O 25 Compound 292

- O O O

HN O

C / O 4N 35 HN Compound 286 N O H. leO N 4 H N S N S 40 Compound 293 A W 2 S YN O 2N. \ { O Compound 287 45 N O O. O N N

OCXH ( )- O S 50 Compound 294 2N? O / / \ O N O H-( \ / Compound 288 55

O O H. V/ N N1-S-S 60 HN

O)-(3) O)O)N () Compound 289 65 Compound 295 US 8,748,464 B2 61 62 TABLE 1-continued TABLE 1-continued Y, r) (3) Compound 304 10

N O N

Compound 296 15 N1,N-S M 2N.

Compound 305

25 O O)-( ) N Compound 297 N S / \ M 30 2NN N - o YO O / Compound 306 O 35 O H R N N-O O N1,N-S / \ 40 M N 2 N - Compound 298

45 Compound 307 N O H N

H 50 Sl-S N M Sl-O O 2NN C C Na2 K s 55 Compound 308 Compound 299 O H O N F N

O)-(321 / y--() C-3 Compound 303 F 65 Compound 309 US 8,748,464 B2 63 64 TABLE 1-continued TABLE 1-continued

O - O

R 5

M O2NN O/ 10 HN Compound 310 Nues?N -) N/ 15 Compound 317

\ O)O)-(Sl-O ) O 2O Y, O Compound 311 25 MeO O OMe HN MeO S 30 rs M N 2 N Compound 318 O N N 35 \ NMe - O O Compound 313

O MeO 40 /

MeO O

45 S ON NO 21 N k ) N Compound 319

NMe 50 Compound 314 / -N MeO

OMe O H N 60 HN N-S

NMe N 2 N Compound 315 65 Compound 320 US 8,748,464 B2 65 66 TABLE 1-continued TABLE 1-continued

/ 5

O HN N S 10 O O) SN lN HN Compound 321 e / 15 S- / O HN N O / Sl-S O 2O 2 ^ N Compound 327 Compound 322

O- 25 O N S

Na2N. X- X- O 3O Compound 323 O O- HN O 35 S 2N. S O N M H Nine1O N. N O \ HN N )--/ / 40 N / Compound 324 Compound 328

N S ') { ) N/ 45 N M N 21 N Compound 325 HO 50 1No

SC HN N Sir O "V / \ / Compound 326 65 Compound 329 US 8,748,464 B2 67 68 TABLE 1-continued TABLE 1-continued

A. / O

10 C-K.)

HN N \ / / O Compound 330

O o1

25 N 2 CON o1 N Compound 331 30

N N 35 2 CON Compound 332 40

N N O H 45 2 N O SI) () 1. O Compound 337 Compound 333 50 1N

HN \ / Compound 334 65 Compound 338 US 8,748,464 B2 69 70 TABLE 1-continued TABLE 1-continued / O 5 O O / N1S-O O N-O O

10 Compound 339 Compound 345

O- O O O 15 N N O N O

O)2 N ()- O- ul N V i O Compound 340 2O Compound 346

O- O O O O O N N O O 25 N \ N \ A N A H N 21 N 2 N O / Compound 341 Compound 347 p 30 O O / / N O N N1 2NN O\ N N 35 Na2N.X- X- H \ N C d 342 Compound 348 ompoun

\ N O 40 O S M H H 45 Na2N.CO-O- N O- Compound 349 N O O / \

O)N 50 O O Compound 343

60 N-N/

Compound 344 65 Compound 350 US 8,748,464 B2 71 72 TABLE 1-continued TABLE 1-continued / Y, / Y,

O 10 O HN HN 21 2N. 21 2N. N-N / 15 N-N /

Compound 351 2O A-2 Compound 364

/ 25 V O 30

HN N O o O 21 Ne.N 2 4 \ / N / 35 N N-N Compound 367

HNO 40 C Compound 359

Y, 4N./ \ / N 50 Compound 369

O HN OCF 21 2N. 55 N-N/ N-s r N 60 Sl-O o /\, A-2 CO \ / N Compound 362 N Compound 370 65 US 8,748,464 B2 73 74 TABLE 1-continued TABLE 1-continued

HN)-( )—c

10 2 ^ O)N () Compound 376

Compound 378

35 O Os / HN 40

Compound 373 C4S. M Compound 379 45

N-S CF

Compound 374

HN 60

C4N. / Compound 375 65 Compound 381 US 8,748,464 B2 75 76 TABLE 1-continued TABLE 1-continued

FC O 5

s/ O N-N HN W \ O 2N r -) 10 HN N 2N, N1S-S Compound 382 /

15 Compound 390 FCO O O N

/ 2O HN FN S-O-HN ) ( ) O)2N? () 25 Compound 391 Compound 383 O

CF3 N 30 2 O HN sM N1 N S HN 21 / N O - ) CF 35 N Compound 392 2 N N Cl Compound 384 O 40 C HN O N1N S HN CO - C2N, - ) Compound 393

O O SN 2 ^ 50 \/ S h O Compound 385 in? \ f N N

Cl 55 O)2 / ) ) ) Compound 394 O C -HN \ / 60 C-3 -( ) Compound 387 65 Compound 396 US 8,748,464 B2 77 78 TABLE 1-continued TABLE 1-continued / N -N D)DX ( ) ( is) { / 10 O NH Compound 398 15 Nues??) { s s O Compound 402 O 2O \ N N-s

N S / \, 25

21 N O Compound 399 NH

30 Nuel?rs Compound 403

35 \ O \ O O 40 NH N S O HN 45 Compound 400 N O Nues? Compound 404 50 / O O \ 55 O

O O NH HN N S O N O Compound 401 Nues? Compound 405 65 US 8,748,464 B2 79 80 TABLE 1-continued TABLE 1-continued \ - O / 5 \ O O HN - O O

Na2 M N S N / Compound 406 N 2 N 15 Compound 411

N M)-( )-NH 2O O21 N O Compound 412 25 HN - O ) O Na2N,O. \ Compound 407 30 O

\ 35 N O Compound 413 N N O

2N,\N N 40 Compound 408

45 O

O N S V 50 M N Compound 414 N 2 N Compound 409 O O

Y 60 O)O ()- Compound 410 65 Compound 415 US 8,748,464 B2 81 82 TABLE 1-continued TABLE 1-continued

H Sl-O S N 10 O) { O Compound 423 Compound 416 15 1.

O Sl-O S so, O-K O N N O Compound 424 )-( )- NH 2 / 25 Compound 419 | 1 - O O On H 30 Sl-O S N Na2 X-C? O Compound 425

35 o1 ON

40 H uN-O \JS N O Compound 426 45 O N 1 N

- O

HN 50 H N O N-O S N N M 2 N Compound 421 55 Compound 427

O NS 60 Sl-O S H O

Compound 422 65 Compound 428 US 8,748,464 B2 83 84 TABLE 1-continued TABLE 1-continued

O HN N 21 N 15 N-N/ Compound 437

? O

O HN N

30 21 N Compound 430 N-N/ Compound 438

35 / O y 40 O HN N

45 2 N O)-() N-N/ Compound 431 Compound 439

50 O- -

O HN 60 - / SI)- ) Compound 436 65 Compound 440 US 8,748,464 B2 85 86 TABLE 1-continued TABLE 1-continued

N() N

15 HN N HN N SI)- ) SI) () \ / \ / Compound 445 Compound 441 2 S S.

O O HN S. 25 NS N- /

HN N 30 \ / Compound 446 HN N \ / - O Compound 442 35

SO/ 40 / HN

NS N- / 45 HN N \ / Compound 447

Compound 443 50

-S

55 SO/ / HN

60 NS S- /

HN N HN N SI) () \ / \ / 65 Compound 448 Compound 444 US 8,748,464 B2 87 88 TABLE 1-continued TABLE 1-continued

O 5 N O V A N NaNea S. H Compound 449 10 N N1,N-O O

O- O) C d 454 ompoun O 15 N S V N Na2 X- X- H o O Compound 450 2O / O

O -O 25 HN

A H / 30 O.2N. N Compound 455 Sl-O O 21 / 35 \ Compound 451 O Y, \ 40 O O / HN O O 45 N1 N O- ul N O O Compound 456 2 / 50 Compound 452 \

55 / ( O 60 HN

Compound 453 65 Compound 457 US 8,748,464 B2 89 90 TABLE 1-continued TABLE 1-continued \ N- 5 O NN N

HN) {

HN O 21 \ N N Compound 462 2 ? 15 Compound 458 O y (

HN O

o N ul Compound 463

25 O

O HN O HN O N 21 / C 21 N Compound 465 Compound 459 35 O O O in? \ • C)2 \ ) Compound 466

O 45 O.\/ O / HN O N-S HN N O 2 N 50 21 M / Compound 460 N Compound 467

55 F O

HN 60 N s N 2 2-)^ O)-()4. / \ A Compound 461 65 Compound 468

US 8,748,464 B2 93 94 TABLE 1-continued TABLE 1-continued

S O H O 5 N o / N1 N S N \ / M N 21 N Compound 485 N-S 10 N O o / \, O

ON \ / C Compound 479 15 C

S 4 / H 2O Compound 486 N S N1s-S NH NM la / A N O 21N O Compound 481 25 sy M N 30 y C.4S. Compound 487

O 35 O N Os N l? N O Compound 482 4N.A / Compound 488 O F 45 O O Os

O 50 ? N N O O / N C4. N 55 2 4 Compound 489 Compound 483 O Sa

N 60 N -( )- - / \, 21NN O)4. ^ () Compound 484 65 Compound 490 US 8,748,464 B2 95 96 TABLE 1-continued TABLE 1-continued

5 N1O) N -() () N OX-d ON 10 N Compound 491 C O 2 M N N C 15 Compound 496 O 1.

onM / aN N 2O

C.4N.}- ) NOX-d Compound 492 25 C.% \ O O Compound 497 sy O 30 M

DXK) S O CO 35 N Compound 493 N-( N N N S H A O 40 21 N O Compound 498 Ossy N M 45 CCM O 4 N Compound 494 50 X-d

55 Compound 499 )- p-()—a Sl-O 60 N-( 4N. 4. / \ A Compound 495 65 Compound 490 US 8,748,464 B2 97 98 TABLE 1-continued TABLE 1-continued

10 Compound 501

2 M CN N Compound 506 N -O- 15 / ONDX \ / Compound 502 /

O : N N

30 O)N Compound 507 O N 35 N O)-H ) Compound 503 ) 40 O N 45 3. N 21 N N-N/ CSR)Compound 508 Compound 504 50 YNo 55 C O N 60 / \ SI)- ) \ / Compound 505 65 Compound 510 US 8,748,464 B2 99 100 TABLE 1-continued TABLE 1-continued

N1 O YN \ f 5 --> H N S S N

O 10 HN Compound 515 S 2N 1. c) () 15 O HN N H Compound 511 N S S N O * NuN/ \l HN-N Compound 516 N S. 25 o1 O O O HN N H

se 30 N S S N / \ S-N - / Nel/OC) {\l 8 N Compound 517

Compound 512 35 N1

H 40 N-S S N N M / N OC) { O O Compound 518 HN 45 Nn

() Compound 513 corrCompound 519 55 N o1 W On

N- N-O2 / O Na2O) / {N O O Compound 520 Compound 514 65 US 8,748,464 B2 101 102 TABLE 1-continued TABLE 1-continued

- O O

O O 10 HN HN

O)4. N () C4SN 15 Compound 525 Compound 521

()N

O 25 O O HN HN

30 O)% N () CN N Compound 526 Compound 522 35 ()

40 O

O O HN HN 45

O)% N () O)% N () Compound 527 Compound 523 50

O O

HN 60 HN

O)4. N () O)4. N () Compound 524 65 Compound 528 US 8,748,464 B2 103 104 TABLE 1-continued TABLE 1-continued F-XF F O

HN 10 N N O HN O)4SN Compound 529 15 O)% N () Y, Compound 534

O HN O N N N 4SN 25 / Compound 530 O HN

/ 30 O)4. N ()

O 35 Compound 535 HN N N N O 40 4NN O Compound 531 / Y, O 45 HN N O HN O)4SN () N N 50 N Compound 536 4SN Compound 532 O N 55 \ / O /

O O 60 HN HN N N N N 2 N 4NN 65 C)N Compound 533 Compound 537 US 8,748,464 B2 105 106 TABLE 1-continued TABLE 1-continued

H-N N / \, O \ / y

HN N 2. 2N. Cy-K) 15 N-N N C-K.) Compound 538 Compound 542

1. 25

HN N 30 \ / d O)N () Compound 539

O / 40 O HN

45 O)% N () / \ Compound 540 \ / 50

O/ \ O \ /

O HN 60 CN-N/ Compound 541 Compound 545 65 US 8,748,464 B2 107 108 TABLE 1-continued TABLE 1-continued

\ O

O 10 HN 2N. N / 15 Compound 546

N \ / 25

Compound 547 CSR)Compound 558 35 /

40 () sk 45 Compound 559

Compound 548 50

55 NS is Compound. 560 .

60 ON H N N1-S-O S O CSR) 65 Compound 556 Compound 561 US 8,748,464 B2 109 110 TABLE 1-continued TABLE 1-continued - - H 1N-O S N 10 CO \ / Compound 568 Compound 562

H N O N1N-O S N CO \ / N Compound 569 Compound 563 25

N O

35 O \ / N Compound 570 O \ / N 40 / Compound 565 O)

H 45 N H N N O O N O o O N N elN^ \ / 2 4 \ / 50 N N Compound 571 Compound 566 / O 55 O

H 60 N N O o O O \ / 2 4 \ / N N N Compound 567 65 Compound 572 US 8,748,464 B2 111 112 TABLE 1-continued TABLE 1-continued

N O H 5 N F ( N F F N 4 / \ / O O)-(3 10 Compound 573 HN C 15 4N. H N Compound 578

N O S N

N elN/ \ / Compound 574

O O HN y 30 O)% / () C.le / C.\ / Compound 579 Compound 575 35

O 40 ( O

O O HN 45 HN N O C.4N. Compound 576 4N. 50 Compound 580

F 55 1N N V NF

O O HN HN 60 N O O)lel () 4N. Compound 577 65 Compound 581 US 8,748,464 B2 113 114 TABLE 1-continued TABLE 1-continued

F F F 5 N / O 10

X-d N HN O N-O N O)% N () ... ON Compound 582 Compound 587

2O \ - O O

O O 25 / ) O O HN HN N

N% 2-)NA 30 N-1. N uC.2 N2 k ) Compound 583 Compound 588 35 Y, 40 y

HN 45 HN N O)N-O () HNCuO2- N-S ) N 50 Compound 589 1N

O- 55

HN)-3- N O 2) O N S HN 2N, N- N 2 / / Compound 585 CuO2-)uCC 65 Compound 590 US 8,748,464 B2 115 116 TABLE 1-continued TABLE 1-continued ON

5 O

O X- O HN HN 10 S

N-S2 M O)2 4 () N Compound 597 Compound 591 15 S NN-( NE N1,N-S N V 2O M O 2N. O HN \ N S Compound 599

M 25 H S 21 N N / C 592

- N 30 2 ^ C.O / O Compound 600 HN S N-s 35 N-(H / N N N M Compound 593 21 N F F 40 Compound 601

O S-(H

O HN City2N? (, ) O N S Compound 604 n 50 O 2N'? S-( Compound 594

F F / p F 55 2 N O. Compound 605

F F

60 O F F S-(H / 65 2 \ Compound 596 Compound 607 US 8,748,464 B2 117 118 TABLE 1-continued TABLE 1-continued O S) - \, )-K)N 5 HN HN N N N1,N-S A O)21 N () N4NCompound 615 Compound 608

H O N C ON N 15 HN N1-S-S C S 21 ? 2O X- OCH Compound 609 S N S H N-4 25 2N,/ N N S N Compound 616 A 2NN Compound 610 30 OMe

S ) NH --O OM 35 N N / N HN

N N S HN O

l?N 40 S 2N. Compound 611 CO-) NH (S O\ /N N 21N SX- s i-Co 45 Compound 617 Compound 612 50 () N-N

55 O y-N HN H Sl-S HN 60 S-DC-) /

Compound 614 65 Compound 618 US 8,748,464 B2 119 120 TABLE 1-continued TABLE 1-continued

N M / N \ /

O 10 O

Compound 619 2O \ f

/ \ 25 SN2N.

30 w Compound 623

So- 40 /

/ \ \ / d

O N SI) () 60 Compound 621 Compound 625 65 US 8,748,464 B2 121 122 TABLE 1-continued TABLE 1-continued -

10 C-COCompound 633 ( St 15

O Compound 628

O Compound 634 O

25 HN N N-( / O)4. / () C) 30 n-O / N O Compound 629 O) () /O Compound 635 O 35

HN N

40 O)4. ? () Compound 630

O 4. ^ \ / V 45 O

HN N Compound 636 50 O)4. ? ) -O Compound 631 55 O

)-(3- 60 ()

O) Compound 632 65 Compound 637 US 8,748,464 B2 123 124 TABLE 1-continued TABLE 1-continued

O O

5 el? \ / N O\ ll. V / N O\ 10

()N ?N Compound 638 15 Pi—/ Compound 643 O 2O -SN

N O O 4. / \ / \ 25 N Compound o se 30 A v S-C O Compound 644

N-O o O)-(4N\, \ / ) ) Compound 640 NF \ / 40 O

O N H S N N 45 Sl N-O o =y N - / 2 / \ / N N-N N N 2 N N \ / Compound 641 50 Compound 645

elN( \ / \ O () CuO2- ) Compound 642 65 Compound 646 US 8,748,464 B2 125 126 TABLE 1-continued TABLE 1-continued

'( ) \ / O 10 O N HN S 2N. S 2N. o / O O NN

Compound 647 2O Y () 25 S \ / 30 \ / . O N HN SN2.N. 35 se S- / Sl / O Compound 648 40 Oy- ?yN Compound 651 s 45 O \ / HN 2 / \ / - CN S 2N. Compound 655 CO-) 55 O O N - - - -

O Compound 649 65 O)-(3.Compound 656 US 8,748,464 B2 127 128 TABLE 1-continued TABLE 1-continued

H N 5 O-( - Sl-O S O CO \ / - 10 Compound 662 Compound 657 H / 15 H H N-( }-Cl N O o -K O N

O \ / N Compound 663 Compound 658

25 Br H N Br H 30 S F -( X N HN ON \ / 35 Compound 664 O)4. ^ () Compound 659 40 - )

Compound 665 45 N O H N --/ H N N N -( )-( N 50 Sl-O { C.% / C.\ / Compound 660 O \ / N Compound 666 55 O F F F

N N 60 H N-( N N O o S O o -( C O) \ / N O N Compound 661 65 ON \ / Compound 667 US 8,748,464 B2 129 130 TABLE 1-continued TABLE 1-continued

O F HN) ()-) -k O)% / ) -- 10 O)- Compound 668

15 4 / / Compound 672 v /

25 S ( ), n- HN) -? /Y

O)Compound { 669 30 O)4. / () Compound 673

O (S/ O / X-d 40 S HN O)O () R N N2 / Compound 670 45 C.% / Compound 674 50 / / O O- O

55 O V S V X-d )- HN 60 HN

M % 4. 4. N Compound 671 65 Compound 675 US 8,748,464 B2 131 132 TABLE 1-continued TABLE 1-continued

F O

o O N N HN N M / S N e O 10 O) N ?\,

Compound 679 - S M / Compound 676 O 25

N. . ... O Compound 680 \ S se - O O N -- / 40 O N /

45 O HN N S Compound 677 50 CuO2-Nu 2 / ) Compound 681 \ Y, NNN 55

65 O l- / Compound 678 Compound 682 US 8,748,464 B2 133 134 TABLE 1-continued TABLE 1-continued

F F 1No

O 10 HN N HN O N-S CuO2-N ) re2 / - ) Compound 687 Compound 683

25 \ /

HN HN N-S n S

ul Compound 688 Compound 684 35 - O Y,

NE 40

s-s ul - ) -N 21N, Compound 685 Compound 689 50

N() 55 M /

HN 60 N-S S ul Compound 686 Compound 690 65 US 8,748,464 B2 135 136 TABLE 1-continued TABLE 1-continued

F F

15 HN N \ / Compound 698 Compound 692

25 \ /

30 N.

HN N \ / Compound 695 40

Compound 699

OH HO-B 50

55 y V \/ /\ Compound 697 65 Compound 700 US 8,748,464 B2 137 138 TABLE 1-continued TABLE 1-continued

N-s 10 M \, 4. \ \ / N Compound 705 15 Compound 701

- O O K y 25 4. / Compound 706 CuO2-N -S )

Compound 707 45

60 HN N SI) () \ / Compound 704 Compound 708 65 US 8,748,464 B2 139 140 TABLE 1-continued TABLE 1-continued

c) () 10 N \ / 15

Compound 714

35 / \ SI)- ) "V / Compound 710 40

Compound 715

50 \ / N

) / 65 Compound 716 US 8,748,464 B2 141 142 TABLE 1-continued TABLE 1-continued

\ /

10 N.

Compound 717 Compound 720

Compound 718

45 Compound 721

55 O / O

60 HN 1 luUC) () Compound 719 65 Compound 722 US 8,748,464 B2 143 144 TABLE 1-continued TABLE 1-continued

15 Compound 727

Compound 723

Compound 728

Compound 726 65 Compound 730 US 8,748,464 B2 145 146 TABLE 1-continued TABLE 1-continued

F F F

10 Compound 737

CucCompound 731 R) O OMe 15 \ S y=n N 2- \ O \U/ S. Compound 738

25 O OMe

CuO2- ) 30 Compound 732 Sl N OMe Na OMe

Compound 739 O / 35

CoCompound 733

45 Compound 740

& n S N \ f \ S=N N N \U/ \ / OMe O Compound 735 55 N

60 \ K St Co O Compound 736 65 Compound 741 US 8,748,464 B2 147 148 TABLE 1-continued Non-limiting examples of suitable SIRT1 activators include, e.g.,

SN, HO N

OH 15 BML-230 S N S-C \ / Compound 742 HO N

OH 25 se HN F BML-225 Cl; and IO SO-) 30 HO N O Compound 743 OH 35 BML-217

45 HN scoOH Compound 744 Pinosylvin

50 Other suitable SIRT1 activators include, e.g., N / 55 O O

H 60 C’sNo % NH; N N

Compound 745 65 US 8,748,464 B2 149 150 -continued include, e.g., 3-benzenesulfonyl-1-(4-fluoro-phenyl)-1H pyrrolo2.3-b-quinoxalin-2-ylamine; 2-amino-1-(2-ethyl SR1460 phenyl)-1H-pyrrolo2,3-biquinoxaline-3-carboxylic acid / (tetrahydro-furan-2-ylmethyl)-amine; 2-amino-1-(3-meth O O oxy-propyl)-1H-pyrrolo2,3-bquinoxaline-3-carboxylic \ acid cyclopentylamide. See, e.g., Nayagam et al. (2006) J. O Biolmolec. Screening 11:959. Other suitable SIRT1 activators include, e.g., stilbene com 10 pounds, e.g., ester analogs of resveratrol, e.g., as described in O U.S. Patent Publication No. 2008/0255382. For example, HN suitable SIRT1 activators include, e.g., ester analogs of 3.5, 4'-trihydroxy-trans-stilbene.

se 15 N - / s OH.

HO HNO

SR1720 OH (resveratrol) 25 Ester analogs include compounds of the formula: N N N / 30 O HN T- ZmBm,

Nse - / s 8. d 35 ?\, "Nu 40 where each Y and each Z is independently —O (ethers), —O—C—O. —C—O—O (esters); —O—C=O O (car bonates); O C-O NH; O-O NR; NH SRT-21.83 C=O O: NR C=O O (carbamates); NH–C–P: NR-C—O; -C=O NH; -C=O NR (primary and 45 secondary amides)-NH; —NR (primary and secondary amines); —N (heterocyclic rings); —S (thiol ethers); and halogen; where each n and each m is independently 1, 2, 3, 4, or 5: 50 where each A and each B is independently H. R. or absent; O where each V and each W is independently H, straight or branched alkyl of from 1 to 6 carbon atoms, cycloalkyl of from 3 to 8 carbon atoms, alkoxy, phenyl, benzyl, or halogen, 55 and where R is an alkyl with at least one carbon atom, an aryl, or an aralkyl. Suitable SIRT1 activators include, e.g., 4'-acetoxy-3,5-bis (methoxymethoxy)stilbene; 4'-acetoxy-3,5-dihydroxy stil bene; 3,5-diacetoxy-4'-chloroacetoxy stilbene; 3,5-diac 60 etoxy-4-hydroxy stilbene: 3,4'-diacetoxy-5- hydroxyStilbene: 3-acetoxy-4,5-dihydroxy stilbene; and 3,4, 5'-triacetoxystilbene. SRT1720, SRT1460, and SRT2183 are selective SIRT1 Suitable SIRT1 activators include compounds of any one activators. See, e.g., Milne et al. (2007) Nature 450:712. 65 of Formulas I-VI as described in U.S. Patent Publication No. Also suitable for use are SIRT1 activators that are quinoxa 2009/0012080. For example, a suitable SIRT1 activator is a line compounds. Suitable quinoxaline SIRT1 activators compound of the formula: US 8,748,464 B2 151 152 Thelper cells, and (b) returning the CD4 T helper cells from N step (a) to the donor individual or to a recipient individual Y \ other than the donor. S. s O In some embodiments, an effective amount of a SIRT1 O S 5 inhibitor is an amount that, when contacted with a starting population of CD4 T helper cells, increases the number of CD4"Thelpercells by at least about 10%, at least about 15%, ) (O. at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold, or more, compared to the number of CD4 T helper cells in the starting population before contact ON- 15 ing with the SIRT1 inhibitor. As noted above, in some embodiments, the contacting occurs invitro. In other embodi ments, the contacting occurs ex vivo. In other embodiments, the contacting occurs in vivo. In some embodiments, an effective amount of a SIRT1 inhibitor is an amount that is effective to reduce deacetylation O 2O of NF-kB in a CD4 T helper cell by at least about 10%, at N least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80%, compared to the level of deacetylation of NF-kB in the CD4 T helper cell in the absence of the SIRT1 inhibitor. In some embodiments, an effective amount of a SIRT1 inhibitor is an amount that, when administered to an indi 30 vidual, increases an immune response in the individual by at least about 10%, at least about 15%, at least about 20%, at For example, a suitable SIRT1 activator is 4-methyl-N-(2 least about 25%, at least about 30%, at least about 35%, at (3-morpholinomethyl)imidazo[2.1-bithiazol-6-yl)phenyl)- least about 40%, at least about 50%, at least about 2-fold, at 2-(pyridin-3-yl)thiazol-5-carboxamide, or a pharmaceuti least about 2.5-fold, at least about 5-fold, at least about 35 10-fold, at least about 25-fold, at least about 50-fold, or at cally acceptable salt thereof. least about 100-fold, compared to the immune response in the Methods of Modulating Activation and Differentiation of a absence of treatment with the SIRT1 inhibitor. CD4 T Cell In some embodiments, an effective amount of a SIRT1 The present disclosure provides a method of modulating inhibitor is an amount that increases the number of CD4 cells activation and differentiation of a CD4 T cell, the method a in an individual by at least about 10%, at least about 15%, at generally involving contacting the CD4 T cell (in vitro, ex least about 20%, at least about 25%, at least about 30%, at vivo, or in vivo) with a SIRT1 inhibitor. In some embodi least about 35%, at least about 40%, at least about 50%, at ments, the CD4 T cell is a helper T cell (e.g., a Th1. Th2, or least about 2-fold, at least about 2.5-fold, at least about 5-fold, Th17 cell), and the SIRT1 inhibitor inhibits deacetylation of at least about 10-fold, at least about 25-fold, at least about NF-kB in the CD4" T cell, thereby increasing CD4 T helper 45 50-fold, or at least about 100-fold, compared to the number of T cell activity and/or numbers. In other embodiments, the CD4 T cells in the individual in the absence of treatment with CD4 T cell is a CD4"/CD25"/FoxP3 Treg cell, and the the SIRT1 inhibitor. SIRT1 inhibitor reduces deacetylation of FoxP3 in the CD4"/ In some embodiments, an effective amount of a SIRT1 CD25"/FoxP3' Treg cell, thereby increasing Treg activity inhibitor is an amount that increases the number of cytotoxic and/or numbers. 50 T cells in an individual by at least about 10%, at least about Increasing CD4 T Helper Cell Activity and/or Numbers 15%, at least about 20%, at least about 25%, at least about In some embodiments, the CD4 T cell is a helper T cell 30%, at least about 35%, at least about 40%, at least about (e.g., a Th1, a Th2, or a Th17 cell), and the SIRT1 inhibitor 50%, at least about 2-fold, at least about 2.5-fold, at least inhibits deacetylation of NF-kB in the CD4" T cell, thereby about 5-fold, at least about 10-fold, at least about 25-fold, at 55 least about 50-fold, or at least about 100-fold, compared to the increasing CD4 T helper cell activity and/or numbers. CD4" number of cytotoxic T cells in the individual in the absence of T helper cell activity and/or numbers can be increased in treatment with the SIRT1 inhibitor. vitro, in vivo, or ex vivo. Increasing CD4 T helper cell In some embodiments, an effective amount of a SIRT1 activity and/or numbers is useful for increasing an immune inhibitor is an amount that is effective to reduce the number of response, e.g., increasing an immune response to an antigen. 60 cancer cells and/or to reduce the Volume of a tumor in an Thus, the present disclosure provides a method of increasing individual by at least about 10%, at least about 15%, at least an immune response in an individual, the method involving about 20%, at least about 25%, at least about 30%, at least administering to an individual in need thereof an effective about 35%, at least about 40%, at least about 50%, at least amount of an inhibitor of SIRT1. The method can also be about 60%, at least about 70%, at least about 80%, at least carried out ex vivo, e.g., by (a) contacting CD4 T helper cells 65 about 90%, or more than 90%, compared to the number of obtained from a donor individual with a SIRT1 inhibitor ex cancer cells or compared to the tumor Volume in the indi vivo, thereby increasing the activity and/or numbers of CD4" vidual in the absence of treatment with the SIRT1 inhibitor. In US 8,748,464 B2 153 154 some embodiment, a SIRT1 inhibitor is administered in con (b) bacterial diseases Such as, for example, diseases result junction with administration of a tumor-specific antigen. ing from infection by bacteria of for example, the genus In some embodiments, a SIRT1 inhibitor is administered in Escherichia, Enterobacter, Salmonella, Staphylococcus, Shi conjunction with administration of an antigen that induces an gella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, immune response to a pathological microorganism. In some 5 Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, embodiments, an effective amount of a SIRT1 inhibitor is an Pneumococcus, Neisseria, Clostridium, Bacillus, Coryne amount that is effective to reduce the number of pathological bacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, microorganisms in an individual by at least about 10%, at Providencia, Chromobacterium, Brucella, Yersinia, Haemo least about 15%, at least about 20%, at least about 25%, at philus, and Bordetella, least about 30%, at least about 35%, at least about 40%, at 10 (c) other infectious diseases, such Chlamydia infection, least about 50%, at least about 60%, at least about 70%, at fungal diseases including but not limited to candidiasis, least about 80%, at least about 90%, or more than 90%, aspergillosis, histoplasmosis, cryptococcal meningitis, and compared to the number of pathological microorganisms in parasitic diseases including but not limited to malaria, Pneu the individual in the absence of treatment with the SIRT1 mocystis carinii pneumonia, leishmaniasis, cryptosporidi inhibitor. In some embodiment, a SIRT1 inhibitor is admin 15 osis, toxoplasmosis, and trypanosome infection; and istered in conjunction with administration of an antigen that (d) neoplastic diseases, such as, for example, intraepithe induces an immune response to a pathological microorgan lial neoplasias, cervical dysplasia, actinic keratosis, basal cell ism. carcinoma, squamous cell carcinoma, renal cell carcinoma, In some embodiments, an effective amount of a SIRT1 Kaposi's sarcoma, melanoma, renal cell carcinoma, leuke inhibitor is an amount that increases the level of one or more mias including but not limited to myelogeous leukemia, antigen-specific antibodies in an individual by at least about chronic lymphocytic leukemia, multiple myeloma, non 10%, at least about 15%, at least about 20%, at least about Hodgkin’s lymphoma, cutaneous T-cell lymphoma, B-cell 25%, at least about 30%, at least about 35%, at least about lymphoma, and hairy cell leukemia, and other cancers. 40%, at least about 50%, at least about 2-fold, at least about Increasing Treg Activity and/or Numbers 2.5-fold, at least about 5-fold, at least about 10-fold, at least 25 In other embodiments, the CD4 T cell is a CD4"/CD25"/ about 25-fold, at least about 50-fold, or at least about 100 FoxP3." Treg cell, and the SIRT1 inhibitor reduces deacety fold, compared to the level of the one or more antigen-specific lation of FoxP3 in the CD4"/CD25"/FoxP3." Treg cell, antibodies in the individual in the absence of treatment with thereby increasing Treg activity and/or numbers. Thus, in the SIRT1 inhibitor. Some embodiments, the present disclosure provides a method Analysis (both qualitative and quantitative) an immune 30 of increasing Treg activity and/or numbers, the method gen response can be by any method known in the art, including, erally involving contacting a naive CD4 T cell and/or a Treg but not limited to, measuring antigen-specific antibody pro cell (a CD4"/CD25"/FoxP3." T cell) with a SIRT1 modulator, duction, activation of specific populations of lymphocytes where the contacting can take place in vitro, ex vivo, or in such as CD4 T cells or NK cells, and/or production of cytok vivo. ines such as IFN, IL-2, IL-4, or IL-12. Methods for measuring 35 Increasing Treg activity and/or numbers is useful for reduc specific antibody responses include enzyme-linked immun ing an undesirable immune response Such as an autoimmune osorbent assay (ELISA) and are well known in the art. Mea response, an allergic response, graft rejection, or a graft Surement of numbers of specific types of lymphocytes such as Versus-host response. Thus, in Some embodiments, the CD4 T cells can be achieved, for example, with fluores present disclosure provides a method of treating an autoim cence-activated cell sorting (FACS). Cytotoxicity assays can 40 mune disorder, a method of treating an allergic disorder, a be performed for instance as described in Raz et al. (1994) method of reducing graft rejection, and a method of reducing Proc. Natl. Acad. Sci. USA 91: 9519-9523. Serum concen graft-Versus-host disease, in an individual, the methods gen trations of cytokines can be measured, for example, by erally involving administering to an individual in need thereof ELISA. These and other assays to evaluate the immune (e.g., an individual having an autoimmune disorder, an indi response are well known in the art. In some embodiments, a 45 vidual having an allergic disorder, a graft recipient) an effec Th1-type immune response is increased. tive amount of an SIRT1 inhibitor. A subject method of increasing an immune response in an In some embodiments, an autoimmune response, an aller individual is useful for treating various disorders including, gic response, graft rejection, or a graft-versus-host response, e.g., treating an infectious disease (e.g., a disease caused by a can be reduced by contacting naive CD4 T cells and/or Treg pathological microorganism Such as a virus, a bacterium, 50 cells obtained from an individual (e.g., an individual having etc.); increasing an immune response to a cancer cell in an an autoimmune disorder, an individual having an allergic individual; enhancing an immune response in an individual disorder, a graft recipient) with an SIRT1 inhibitor ex vivo, who is immunodeficient or immunocompromised; etc. and returning the Treg cells to the individual. For example, an Thus, for example, a Subject method can be used to ex vivo method can involve: (a) contacting naive CD4 T cells increase an immune response for treating: 55 and/or Tregs obtained from a donor individual with a SIRT1 (a) viral diseases Such as, for example, diseases resulting inhibitor ex vivo, thereby increasing the activity and/or num from infection by an adenovirus, a herpesvirus (e.g., HSV-I. bers of Tregs, and (b) returning the Tregs from step (a) to the HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus donor individual or to a recipient individual other than the Such as variola or vaccinia), a picornavirus (e.g., rhinovirus or donor. enterovirus), an orthomyxovirus (e.g., influenzavirus), a 60 In some embodiments, an effective amount of a SIRT1 paramyxovirus (e.g., parainfluenzavirus, mumps virus, inhibitor is an amount that, when contacted with a starting measles virus, and respiratory syncytial virus (RSV)), a coro population of Tregs (or a starting population that is a mixture navirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, of naive CDC T cells and Tregs), increases the number of Such as those that cause genital warts, common warts, or Tregs by at least about 10%, at least about 15%, at least about plantar warts), a hepadnavirus (e.g., hepatitis B virus), a fla 65 20%, at least about 25%, at least about 30%, at least about Vivirus (e.g., hepatitis C virus or Dengue virus), or a retrovi 35%, at least about 40%, at least about 50%, at least about rus (e.g., a lentivirus Such as HIV); 2-fold, at least about 2.5-fold, at least about 5-fold, at least US 8,748,464 B2 155 156 about 10-fold, at least about 25-fold, at least about 50-fold, at about 50%, at least about 60%, at least about 70%, at least least about 100-fold, at least about 500-fold, or at least about about 80%, or at least about 90%, or more, when compared to 1000-fold, or more, compared to the number of Tregs in the the number and/or level of autoreactive cells in the individual starting population before contacting with the SIRT1 inhibi not treated with a SIRT1 inhibitor. tor. As noted above, in some embodiments, the contacting Whether administration of a SIRT1 inhibitor is effective to occurs in vitro. In other embodiments, the contacting occurs reduce the number and/or activity of an autoreactive T lym ex vivo. In other embodiments, the contacting occurs in vivo. phocyte in an individual is readily determined using known assays. For example, where the autoreactive T lymphocytes In some embodiments, an effective amount of a SIRT1 are specific for an autoantigen, the number and activity level inhibitor is an amount that is effective to reduce deacetylation 10 of autoantigen-specific T lymphocytes is determined using, of FoxP3 in a Treg by at least about 10%, at least about 15%, e.g., a mixed lymphocyte reaction in which irradiated cells at least about 20%, at least about 25%, at least about 30%, at comprising a detectable label in the cytoplasm and displaying least about 35%, at least about 40%, at least about 50%, at the autoantigen are mixed with lymphocytes from the indi least about 60%, at least about 70%, at least about 80%, or vidual. Release of detectable label from the cytoplasm of the more than 80%, compared to the level of deacetylation of 15 autoantigen-displaying cells indicates the presence in the FoxP3 in the Treg in the absence of the SIRT1 inhibitor. For individual of autoreactive lymphocytes. Methods of detecting example, in Some embodiments, an effective amount of a autoreactive T lymphocytes associated with Type 1 diabetes SIRT1 inhibitor is an amount that is effective to reduce are known in the art; and any such methods can be used. See, e.g., U.S. Pat. No. 6,022,697 for a discussion of a method of deacetylation of K31, K263, and K268 (or an equivalent detecting autoreactive T lymphocytes associated with Type 1 lysine residue) in a FoxP3 polypeptide in a Treg cell by at least diabetes. about 10%, at least about 15%, at least about 20%, at least A subject method is useful for treating an autoimmune about 25%, at least about 30%, at least about 35%, at least disease, e.g., for reducing or ameliorating at least one symp about 40%, at least about 50%, at least about 60%, at least tom of an autoimmune disease in an individual having an about 70%, at least about 80%, or more than 80%, compared 25 autoimmune disease (e.g., an individual who has been diag to the level of deacetylation of K31, K263, and K268 residues nosed as having an autoimmune diseases). Autoimmune dis in FoxP3 in the Treg in the absence of the SIRT1 inhibitor. eases include but are not limited to rheumatoid arthritis, Amino acid sequences of FoxP3 polypeptides are known in Crohn's disease, multiple Sclerosis, systemic lupus erythe the art. Examples of FoxP3 amino acid sequences are pre matosus (SLE), autoimmune encephalomyelitis, myasthenia sented in FIG. 13, which shows an alignment of human, 30 gravis (MG), Hashimoto's thyroiditis, Goodpasture’s syn mouse, and bovine FoxP3, and which shows the K31, K263, drome, pemphigus (e.g., pemphigus Vulgaris), Grave's dis and K268 residues. A FoxP3 polypeptide can comprise an ease, autoimmune hemolytic anemia, autoimmune thromb amino acid sequence having at least about 70%, at least about ocytopenic purpura, Scleroderma with anti-collagen 80%, at least about 90%, at least about 95%, at least about antibodies, mixed connective tissue disease, polymyositis, 98%, at least about 99%, or 100%, with the human FoxP3 35 pernicious anemia, idiopathic Addison's disease, autoim amino acid sequence depicted in FIG. 13. mune-associated infertility, glomerulonephritis (e.g., cres In some embodiments, an “effective amount of a SIRT1 centic glomerulonephritis, proliferative glomerulonephritis), inhibitor is an amount that achieves a degree of immunosup bullous pemphigoid, Sjogren's syndrome, insulin resistance, pression Sufficient to delay, inhibit, Suppress or moderate and autoimmune diabetes mellitus (type 1 diabetes mellitus: tissue transplant rejection and/or delay, inhibit, Suppress or 40 insulin-dependent diabetes mellitus). moderate one or more symptoms of an autoimmune disease A subject method can be used to increase transplantation and/or delay, inhibit, Suppress or moderate an undesired tolerance in a subject (e.g., to reduce the likelihood of rejec immune response to a foreign antigen Such as a therapeutic tion of the transplant by the transplant recipient). In some protein, a viral vector, an allergen, a Venom and the like. embodiments, the Subject is a recipient of an allogeneic trans In some embodiments, an “effective amount of a SIRT1 45 plant (e.g., the graft is an allograft). Cells, tissues or organs (or inhibitor is an amount that reduces an undesired immune parts thereof) that can be transplanted between members of the same species include, but are not limited, to heart, lung, response (e.g., an autoimmune response, an allergic response, kidney, liver, pancreas, pancreatic islets, brain tissue, cornea, a graft-versus-host response, a graft rejection) by at least stomach, bone, bone marrow, muscle, intestine, bladder, skin about 10%, at least about 20%, at least about 30%, at least 50 and stem cells. Optionally, the transplanted tissue or organ is about 40%, at least about 50%, at least about 60%, at least bio-engineered, e.g., when the transplanted tissue or organ is about 70%, at least about 80%, or more than 80%, compared grown from a stem cell or other type of precursor cell(s). to the degree of the undesired immune response in the Bio-engineered tissue or organ can be grown outside of the absence of treatment with the SIRT1 inhibitor. Whether an body and transplanted directly into the host. Alternatively, the SIRT1 inhibitor is effective in reducing an undesired immune 55 precursor cells or immature organ or tissue is transplanted response (e.g., an autoimmune response, an allergic response, into the host to grow and mature. a graft-Versus-host response, a graft rejection) can be deter The transplanted organ, tissue or cell(s) can also be a Xenograft, i.e., the donor is a member of a species different mined by measuring one or more well-known parameters than the recipient. Xenografts are advantageously used with a associated with the undesired immune response (e.g., the 60 bio-engineered tissue or organ, which, instead of being trans level of autoantibody; the level of auto-reactive T cells; the planted directly into the recipient in need of the tissue or level of allergen-specific IgE, a symptom of allergy; the level organ, can be transplanted into a Surrogate host Such as non of antibody specific for a graft tissue; etc.). human mammal until a suitable human recipient in need of In some embodiments, a subject method is effective to the bio-engineered tissue or organ is identified. Alternatively, reduce the number and/or activity of an autoreactive cell in an 65 the tissue or organ can be transplanted into the Surrogate to individual by at least about 5%, at least about 10%, at least allow the bio-engineered tissue or organ to mature. Use of about 25%, at least about 30%, at least about 40%, at least Surrogate hosts may be preferred in instances where further US 8,748,464 B2 157 158 development of the tissue or organ is required before trans plantation into a human recipient. In another alternative, a (Compound 13) Xenograft is used when a Suitable allograft donor is unavail able. When a transplanting into a different a species, it is OH S N 5 desirable to select a host such that the size of the organs in the O S. host and donor are similar. In addition, the host is selected to O O minimize transmission of communicable diseases.

In some embodiments, a Subject method can be used to N H reduce the incidence and/or severity of an allergic reaction in 10 an individual. Thus, a Subject method can be used to reduce cr" adverse reaction in an individual to an allergen. Allergens of interest include antigens found in food, such as Strawberries, (Compound 14) peanuts, milk polypeptides, egg whites, etc. Other allergens of interest include various airborne antigens, such as grass 15 pollens, animal danders, house mite feces, etc. Molecularly NY cloned allergens include Dermatophagoides pteryonyssinus O (Der P1); Lol pl-V from rye grass pollen; a number of insect O Venoms, including venom from jumperant Myrmecia pilo O sula, Apis melliferabee venom phospholipase A2 (PLA and antigen 5S, phospholipases from the yellow jacket Vespula HN maculiffons and white faced hornet Dolichovespula macu O OH: lata; a large number of pollen polypeptides, including birch 25 pollen, ragweed pollen, Parol (the major allergen of Pari O N etaria officinalis) and the cross-reactive allergen NO (from us H Parietaria judaica), and other atmospheric pollens including O Olea europaea, Artemisia sp., gramineae, etc. Otherallergens of interest are those responsible for allergic dermatitis caused 30 (Compound 15) by blood sucking arthropods, e.g. Diptera, including mosqui tos (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.) particularly black flies, deer flies and biting midges; ticks (Dermacenter sp., Orni 35 O thodoros sp., Otobius sp.); fleas, e.g. the order Siphonaptera, including the genera Xenopsylla, Pulex and Ctenocephalides felis. Other allergens of interest include drug allergens. SIRT1 Inhibitors 40 C. orsC. SIRT1 inhibitors that are suitable for use in a subject method include, but are not limited to, sirtinol (2-(2-hydrox (Compound 23) ynaphthalen-1-ylmethylene)amino-N-(1-phenyl-ethyl)ben Zamide); a compound as described in U.S. Pat. No. 7,345,178, Y provided the compound is an SIRT1 inhibitor, e.g., a com 45 pound as shown in Table 5 of U.S. Pat. No. 7,345,178; a compound as described in U.S. Patent Publication No. 2008/ 0255382; and the like; or a pharmaceutically acceptable salt of any of the foregoing compounds. 2NO H 50 Suitable SIRT1 inhibitors include, e.g., (Compound 25) N: C / O N N N N O NH2. 2NO (Ex-527) 60 (Compound 26) O Ex-527 is 6-chloro-2.3.4.9-tetrahydro-1H-carbazole-1- N NT. carboxamide. Ex-527 is a selective SIRT1 inhibitor. See, e.g., n N )-()- N: Napper et al. (2005) J. Med. Chem. 48:8045. 65 | | Y N Suitable SIRT1 inhibitors include, e.g., compounds of 2NO Table 5 of U.S. Pat. No. 7,345,178, e.g.: US 8,748,464 B2 159 160 -continued -continued (Compound 28) (Compound 653)

O ; and ()N H 7v N N ) ) 10 N 21 No CO \ / (Compound 30) (Compound 654) C 15 O N N O N S so 2NO H S O (Compound 44) H 7v EN: N N S O O 25 O)-() N N O Suitable SIRT1 inhibitors include a compound as disclosed in U.S. Patent Publication No. 20008/0214800, or a pharma C-32NO 30 ceutically acceptable salt of Such a compound. Examples of (Compound 47) suitable SIRT1 inhibitors include, e.g.:

35 C Y-4

40 HN N-(. O; 45 N (Compound 291) H HN

C 50 N-(. O; and S S F N H HN O)N ()-AF F; (Compound 652) Br 55 N-(. O. N H HN 60 Suitable SIRT1 inhibitors include, e.g., 7-chloro-1,2,3,4- tetrahydro-cyclopentablindole-3-carboxylic acid amide: 6-bromo-2.3.4.9-tetrahydro-1H-carbazole-2-carboxylic acid 65 amide; 1,2,3,4-tetrahydro-cyclopentablindole-3-carboxylic acid amide; and 6-chloro-2.3.4.9-tetrahydro-1H-carbazole 1-carboxylic acid (5-chloro-pyridin-2-yl)-amide. US 8,748,464 B2 161 162 Combination Therapy (e.g., IL-2); and the like. Suitable ISS include polynucle In some embodiments, a Subject treatment method com otides that include an unmethylated 5'-CG-3' sequence. ISS prises administering to an individual in need thereof an effec can be from about 6 nucleotides in length to about 200 nucle tive amount of a SIRT1 inhibitor or a SIRT1 activator, and at otides in length, or longer than 200 nucleotides in length. An least one additional therapeutic agent. ISS can comprise a phosphate backbone modification, where HIV-associated Immune Hyperactivation backbone phosphate group modifications include, e.g., meth In some embodiments, as discussed above, a subject ylphosphonate, phosphorothioate, phosphoroamidate and method provides for treating immune hyperactivation that is phosphorodithioate internucleotide linkages associated with an immunodeficiency virus infection. In TLR agonists include, e.g., TLR1, TLR2, TLR3, TLR4, Some embodiments, a subject method for reducing immuno 10 TLR5, TLR6, TLR7, TLR8 and TLR9 agonists. Suitable TLR deficiency virus infection-associated immune hyperactiva agonists include, e.g., an imidazoquinoline amine, a tetrahy tion involves administering a SIRT1 activator alone, e.g., as droimidazoquinoline amine, an imidazopyridine amine, a monotherapy. In other embodiments, a Subject method for 1.2-bridged imidazoquinoline amine, a 6.7-fused cycloalky reducing immunodeficiency virus infection-associated limidazopyridine amine, an imidazonaphthyridine amine, a immune hyperactivation involves administering a SIRT1 acti 15 tetrahydroimidazonaphthyridine amine, an oxazoloquinoline vator, and at least one additional therapeutic agent, in com amine, a thiazoloquinolineamine, an oxazolopyridine amine, bination therapy. a thiazolopyridine amine, an oxazolonaphthyridine amine, Suitable additional therapeutic agents include, e.g., thera and a thiazolonaphthyridine amine. peutic agents for the treatment of an immunodeficiency virus Reducing an Undesired Immune Response infection, or for the treatment of a disorder that may accom In some embodiments, a Subject method of reducing an pany an immunodeficiency virus infection (e.g., a bacterial undesired immune response (e.g., an autoimmune response; infection, a fungal infection, and the like). Suitable additional an allergic response; a graft-Versus-host response; rejection therapeutic agents include, e.g., beta-lactam antibiotics, tet of a transplanted organ, tissue, or cell) involves administering racyclines, chloramphenicol, neomycin, gramicidin, bacitra a SIRT1 inhibitor alone, e.g., as monotherapy. In other cin, Sulfonamides, nitrofuraZone, nalidixic acid, cortisone, 25 embodiments, a Subject method of reducing an undesired hydrocortisone, betamethasone, dexamethasone, fluocor immune response (e.g., an autoimmune response; an allergic tolone, prednisolone, triamcinolone, indomethacin, Sulindac, response; a graft-versus-host response; rejection of a trans acyclovir, amantadine, rimantadine, recombinant soluble planted organ, tissue, or cell) involves administering a SIRT1 CD4 (rsCD4), anti-receptor antibodies (e.g., for rhinovi inhibitor in combination therapy with one or more additional ruses), nevirapine, cidofovir (Vistide TM), trisodium phospho 30 therapeutic agents. noformate (FoscarnetTM), famcyclovir, pencyclovir, Valacy For the treatment of autoimmune disorders, a SIRT1 clovir, nucleic acid/replication inhibitors, interferon, inhibitor can be administered in combination therapy with zidovudine (AZT, RetrovirTM), didanosine (dideoxyinosine, one or more agents for treating an autoimmune disorder. ddI, VidexTM), stavudine (d4T, ZeritTM), Zalcitabine Those skilled in the art are aware of agents that are suitable for (dideoxycytosine, ddC, HividTM), nevirapine (ViramuneTM), 35 treating autoimmune disorders. For example, agents that are lamivudine (EpivirTM, 3TC), protease inhibitors, saquinavir Suitable for treating Type 1 diabetes include insulin, including (InviraseTM, FortovaseTM), ritonavir (NorvirTM), nelfinavir naturally occurring insulin, insulin analogs, and the like. (ViraceptTM), efavirenz (SustivaTM), abacavir (ZiagenTM), Interferon-alpha is an agent that is currently in use for treating amprenavir (AgeneraseTM) indinavir (CrixivanTM), ganciclo multiple Sclerosis, and can be used in combination therapy vir, AZDU, delavirdine (RescriptorTM), kaletra, trizivir, 40 with a SIRT1 inhibitor. Other agents that are currently in use rifampin, clathiromycin, erythropoietin, colony stimulating for treating autoimmune disorders include corticosteroid factors (G-CSF and GM-CSF), non-nucleoside reverse tran drugs; non-steroidal anti-inflammatory drugs (NSAIDs); and Scriptase inhibitors, nucleoside inhibitors, adriamycin, fluo immunosuppressant drugs such as cyclophosphamide, meth rouracil, methotrexate, asparaginase and combinations otrexate, and azathioprine. thereof. 45 For the treatment of allergies, a SIRT1 inhibitor can be Increasing an Immune Response administered in combination therapy with one or more agents In some embodiments, a Subject method of increasing an for treating an allergic disorder. Suitable agents include, but immune response comprises administering to an individual in are not limited to, antihistamines such as diphenhydramine need thereof a SIRT1 inhibitor and at least one additional (Benadryl); epinephrine; decongestants; and the like. therapeutic agent in combined effective amounts to increase 50 For reduction of graft rejection or for reduction of GVHD, an immune response in the individual. In some embodiments, a SIRT1 inhibitor can be administered in combination therapy a Subject method of increasing an immune response com with one or more agents for reducing graft rejection or prises administering to an individual in need thereof a SIRT1 GVHD. Agents suitable for reducing graft rejection or GVHD inhibitor and an antigen in combined effective amounts to include, but are not limited to, glucocorticoids (e.g., cortisol; increase an immune response to the antigen in the individual. 55 dexamethasone; etc.); cytostatic agents (e.g., cyclophospha In some embodiments, a Subject method of increasing an mide; nitrosoureas; platinum compounds; methotrexate, aza immune response comprises administering to an individual in thioprine; mercaptopurine; dactinomycin; anthracyclines; need thereof a SIRT1 inhibitor and at least one additional mitomycin C: bleomycin; mitramycin; etc.); an anti-CD20 therapeutic agent in combined effective amounts to increase monoclonal antibody; cyclosporin; tacrolimus; Voclosporin; an immune response in the individual; and administering an 60 Sirolimus; and the like. antigen to the individual, where an immune response to the Formulations, Dosages, Routes of Administration antigen is increased. A SIRT1 activator or a SIRT1 inhibitor can be formulated Suitable additional therapeutic agents include, e.g., immu with one or more pharmaceutically acceptable excipients. nostimulatory polynucleotides (“ISS”; see, e.g., U.S. Pat. No. SIRT1 activators and SIRT1 inhibitors are referred to collec 7,479.285); an ISS conjugated to an antigen (see, e.g., U.S. 65 tively below as “active agent' or “drug. A wide variety of Pat. No. 6,610,661); a Toll-like receptor agonist (see, e.g., pharmaceutically acceptable excipients are known in the art U.S. Pat. No. 7,387,271); an immunostimulatory cytokine and need not be discussed in detail herein. Pharmaceutically US 8,748,464 B2 163 164 acceptable excipients have been amply described in a variety An active agent can be formulated into preparations for of publications, including, for example, A. Gennaro (2000) injection by dissolving, Suspending or emulsifying them in an “Remington: The Science and Practice of Pharmacy.” 20th aqueous or nonaqueous solvent, Such as Vegetable or other edition, Lippincott, Williams, & Wilkins; Pharmaceutical similar oils, synthetic aliphatic acid glycerides, esters of Dosage Forms and Drug Delivery Systems (1999) H. C. higheraliphatic acids or propylene glycol; and if desired, with Ansel et al., eds. 7" ed., Lippincott, Williams, & Wilkins; and conventional additives such as solubilizers, isotonic agents, Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe Suspending agents, emulsifying agents, stabilizers and pre et al., eds., 3" ed. Amer. Pharmaceutical Assoc. servatives. The pharmaceutically acceptable excipients, such as An active agent can be utilized in aerosol formulation to be 10 administered via inhalation. An active agent can be formu vehicles, adjuvants, carriers or diluents, are readily available lated into pressurized acceptable propellants such as dichlo to the public. Moreover, pharmaceutically acceptable auxil rodifluoromethane, propane, nitrogen and the like. iary Substances, such as pH adjusting and buffering agents, Furthermore, an active agent can be made into Supposito tonicity adjusting agents, stabilizers, wetting agents and the ries by mixing with a variety of bases Such as emulsifying like, are readily available to the public. 15 bases or water-soluble bases. An active agent can be admin The term “unit dosage form, as used herein, refers to istered rectally via a Suppository. The Suppository can include physically discrete units Suitable as unitary dosages for vehicles such as cocoa butter, carbowaxes and polyethylene human and animal Subjects, each unit containing a predeter glycol monomethyl ethers, which melt at body temperature, mined quantity of a Subject active agent calculated in an yet are solidified at room temperature. amount Sufficient to produce the desired effect in association Unit dosage forms for oral or rectal administration Such as with a pharmaceutically acceptable diluent, carrier or vehicle. syrups, elixirs, and Suspensions may be provided wherein The specifications for an active agent depend on the particular each dosage unit, for example, teaspoonful, tablespoonful, compound employed and the effect to be achieved, and the tablet or Suppository, contains a predetermined amount of the pharmacodynamics associated with each compound in the Subject active agent. Similarly, unit dosage forms for injec host. 25 tion or intravenous administration may comprise a subject In the subject methods, a SIRT1 activator or a SIRT1 active agent in a composition as a solution in sterile water, inhibitor may be administered to the host using any conve normal saline or another pharmaceutically acceptable carrier. nient means capable of resulting in the desired outcome, e.g., An active agent can be formulated for administration by reduction of disease, reduction of a symptom of a disease, etc. injection. Typically, injectable compositions are prepared as Thus, a SIRT1 activator or a SIRT1 inhibitor can be incorpo 30 liquid solutions or Suspensions; solid forms suitable for solu rated into a variety of formulations for therapeutic adminis tion in, or Suspensionin, liquid vehicles prior to injection may tration. More particularly, a SIRT1 activator or a SIRT1 also be prepared. The preparation may also be emulsified or inhibitor can be formulated into pharmaceutical composi the active ingredient encapsulated in liposome vehicles. tions by combination with appropriate, pharmaceutically In some embodiments, an active agent is delivered by a acceptable carriers or diluents, and may be formulated into 35 continuous delivery system. The term “continuous delivery preparations in Solid, semi-solid, liquid or gaseous forms, system” is used interchangeably herein with “controlled Such as tablets, capsules, powders, granules, ointments, solu delivery system’’ and encompasses continuous (e.g., con tions, Suppositories, injections, inhalants and aerosols. trolled) delivery devices (e.g., pumps) in combination with Suitable excipient vehicles are, for example, water, Saline, catheters, injection devices, and the like, a wide variety of dextrose, glycerol, ethanol, or the like, and combinations 40 which are known in the art. thereof. In addition, if desired, the vehicle may contain minor Mechanical or electromechanical infusion pumps can also amounts of auxiliary Substances such as wetting or emulsify be suitable for use with the present invention. Examples of ing agents or pH buffering agents. Actual methods of prepar such devices include those described in, for example, U.S. ing Such dosage forms are known, or will be apparent, to those Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725, skilled in the art. See, e.g., Remington’s Pharmaceutical Sci 45 852; 5,820,589; 5,643.207; 6,198.966; and the like. In gen ences, Mack Publishing Company, Easton, Pa., 17th edition, eral, delivery of active agent can be accomplished using any 1985. The composition or formulation to be administered of a variety of refillable, pump systems. Pumps provide con will, in any event, contain a quantity of the agent adequate to sistent, controlled release over time. In some embodiments, achieve the desired state in the subject being treated. the active agent is in a liquid formulation in a drug-imperme In pharmaceutical dosage forms, a SIRT1 activator or a 50 able reservoir, and is delivered in a continuous fashion to the SIRT1 inhibitor (“active agent’) may be administered in the individual. form of its pharmaceutically acceptable salts, or an active In one embodiment, the drug delivery system is an at least agent may be used alone or in appropriate association, as well partially implantable device. The implantable device can be as in combination, with other pharmaceutically active com implanted at any suitable implantation site using methods and pounds. The following methods and excipients are merely 55 devices well known in the art. An implantation site is a site exemplary and are in no way limiting. within the body of a subject at which a drug delivery device is For oral preparations, an active agent (a SIRT1 activator or introduced and positioned. Implantation sites include, but are a SIRT1 inhibitor) can be used alone or in combination with not necessarily limited to a Subdermal, Subcutaneous, intra appropriate additives to make tablets, powders, granules or muscular, or other suitable site within a subjects body. Sub capsules, for example, with conventional additives, such as 60 cutaneous implantation sites are used in Some embodiments lactose, mannitol, corn starch or potato starch; with binders, because of convenience in implantation and removal of the Such as crystalline cellulose, cellulose derivatives, acacia, drug delivery device. corn starch or gelatins; with disintegrators, such as corn Drug release devices suitable for use in the invention may starch, potato starch or sodium carboxymethylcellulose; with be based on any of a variety of modes of operation. For lubricants, such as talc or magnesium Stearate; and if desired, 65 example, the drug release device can be based upon a diffu with diluents, buffering agents, moistening agents, preserva sive system, a convective system, or an erodible system (e.g., tives and flavoring agents. an erosion-based system). For example, the drug release US 8,748,464 B2 165 166 device can be an electrochemical pump, osmotic pump, an Oral Formulations electroosmotic pump, a vapor pressure pump, or osmotic In some embodiments, an active agent (a SIRT1 activator bursting matrix, e.g., where the drug (“active agent') is incor or a SIRT1 inhibitor) is formulated for oral delivery to an porated into a polymer and the polymer provides for release individual in need of Such an agent. of drug formulation concomitant with degradation of a drug For oral delivery, a formulation comprising an active agent impregnated polymeric material (e.g., a biodegradable, drug will in some embodiments include an enteric-soluble coating impregnated polymeric material). In other embodiments, the material. Suitable enteric-soluble coating material include drug release device is based upon an electrodiffusion system, hydroxypropyl methylcellulose acetate succinate (HPM an electrolytic pump, an effervescent pump, a piezoelectric CAS), hydroxypropyl methyl cellulose phthalate (HPMCP), pump, a hydrolytic system, etc. 10 cellulose acetate phthalate (CAP), polyvinyl phthalic acetate (PVPA), EudragitTM, and shellac. Drug release devices based upon a mechanical or electro As one non-limiting example of a suitable oral formula mechanical infusion pump can also be suitable for use with tion, an active agent is formulated with one or more pharma the present invention. Examples of such devices include those ceutical excipients and coated with an enteric coating, as described in, for example, U.S. Pat. Nos. 4,692,147; 4.360, 15 described in U.S. Pat. No. 6,346.269. For example, a solution 019; 4.487,603: 4.360,019; 4,725,852, and the like. In gen comprising an active agent and a stabilizer is coated onto a eral, a Subject treatment method can be accomplished using core comprising pharmaceutically acceptable excipients, to any of a variety of refilable, non-exchangeable pump sys form an active agent-coated core; a Sub-coating layer is tems. Pumps and other convective systems are generally pre applied to the active agent-coated core, which is then coated ferred due to their generally more consistent, controlled with an enteric coating layer. The core generally includes release over time. Osmotic pumps are used in Some embodi pharmaceutically inactive components such as lactose, a ments due to their combined advantages of more consistent starch, mannitol, Sodium carboxymethyl cellulose, sodium controlled release and relatively small size (see, e.g., PCT starch glycolate, sodium chloride, potassium chloride, pig published application no. WO 97/27840 and U.S. Pat. Nos. ments, salts of alginic acid, talc, titanium dioxide, Stearic 5,985.305 and 5,728,396)). Exemplary osmotically-driven 25 acid, Stearate, micro-crystalline cellulose, glycerin, polyeth devices suitable for use in the invention include, but are not ylene glycol, triethyl citrate, tributyl citrate, propanyl triac necessarily limited to, those described in U.S. Pat. Nos. etate, dibasic calcium phosphate, tribasic sodium phosphate, 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790: calcium sulfate, cyclodextrin, and castor oil. Suitable sol 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202: vents for an active agent include aqueous solvents. Suitable 30 stabilizers include alkali-metals and alkaline earth metals, 4,111.203; 4,203,440; 4,203,442; 4,210,139; 4,327,725: bases of phosphates and organic acid salts and organic 4,627,850: 4,865,845; 5,057,318; 5,059,423: 5,112,614; amines. The sub-coating layer comprises one or more of an 5,137,727: 5,234,692: 5,234,693; 5,728,396; and the like. adhesive, a plasticizer, and an anti-tackiness agent. Suitable In some embodiments, the drug delivery device is an anti-tackiness agents include talc, Stearic acid, Stearate, implantable device. The drug delivery device can be 35 Sodium Stearyl fumarate, glyceryl behenate, kaolin and aero implanted at any suitable implantation site using methods and sil. Suitable adhesives include polyvinyl pyrrolidone (PVP), devices well known in the art. As noted infra, an implantation gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellu site is a site within the body of a subject at which a drug lose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl delivery device is introduced and positioned. Implantation acetate (VA), polyvinyl alcohol (PVA), methyl cellulose sites include, but are not necessarily limited to a Subdermal, 40 (MC), ethyl cellulose (EC), hydroxypropyl methyl cellulose Subcutaneous, intramuscular, or other Suitable site within a phthalate (HPMCP), cellulose acetate phthalates (CAP), xan subjects body. than gum, alginic acid, salts of alginic acid, EudragitTM, In some embodiments, an active agent (a SIRT1 activator copolymer of methyl acrylic acid/methyl methacrylate with or a SIRT1 inhibitor) is delivered using an implantable drug polyvinyl acetate phthalate (PVAP). Suitable plasticizers delivery system, e.g., a system that is programmable to pro 45 include glycerin, polyethylene glycol, triethylcitrate, tributyl vide for administration of an active agent. Exemplary pro citrate, propanyl triacetate and castor oil. Suitable enteric grammable, implantable systems include implantable infu soluble coating material include hydroxypropyl methylcellu sion pumps. Exemplary implantable infusion pumps, or lose acetate succinate (HPMCAS), hydroxypropyl methyl devices useful in connection with Such pumps, are described cellulose phthalate(HPMCP), cellulose acetate phthalate 50 (CAP), polyvinyl phthalic acetate (PVPA), EudragitTM and in, for example, U.S. Pat. Nos. 4.350,155; 5,443,450; 5,814, shellac. 019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687: Suitable oral formulations also include an active agent 6,475,180; and 6.512,954. A further exemplary device that formulated with any of the following: microgranules (see, can be adapted for the present invention is the Synchromed e.g., U.S. Pat. No. 6,458,398); biodegradable macromers infusion pump (Medtronic). 55 (see, e.g., U.S. Pat. No. 6,703,037); biodegradable hydrogels Suitable excipient vehicles are, for example, water, Saline, (see, e.g., Graham and McNeill (1989) Biomaterials 5:27 dextrose, glycerol, ethanol, or the like, and combinations 36); biodegradable particulate vectors (see, e.g., U.S. Pat. No. thereof. In addition, if desired, the vehicle may contain minor 5,736,371); bioabsorbable lactone polymers (see, e.g., U.S. amounts of auxiliary Substances such as wetting or emulsify Pat. No. 5,631,015); slow release protein polymers (see, e.g., ing agents or pH buffering agents. Actual methods of prepar 60 U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); a poly ing Such dosage forms are known, or will be apparent, to those (lactide-co-glycolide/polyethylene glycol block copolymer skilled in the art. See, e.g., Remington’s Pharmaceutical Sci (see, e.g., U.S. Pat. No. 6,630,155; Atrix Laboratories, Inc.); ences, Mack Publishing Company, Easton, Pa., 17th edition, a composition comprising a biocompatible polymer and par 1985. The composition or formulation to be administered ticles of metal cation-stabilized agent dispersed within the will, in any event, contain a quantity of an active agent 65 polymer (see, e.g., U.S. Pat. No. 6,379,701: Alkermes Con adequate to achieve the desired State in the Subject being trolled Therapeutics, Inc.); and microspheres (see, e.g., U.S. treated. Pat. No. 6,303,148; Octoplus, B.V.). US 8,748,464 B2 167 168 Suitable oral formulations also include an active agent with cartridges, e.g. gelatin, or blister packs, from which the pow any of the following: a carrier such as Emisphere(R) (Emi der may be administered with the aid of an inhaler. sphere Technologies, Inc.); TIMERX, a hydrophilic matrix There are several different types of inhalation methodolo combining Xanthan and locust bean gums which, in the pres gies which can be employed in connection with the present ence of dextrose, form a strong binder gel in water (Penwest); invention. An active agent can be formulated in basically GeminexTM (Penwest): ProciseTM (GlaxoSmithKline); three different types of formulations for inhalation. First, an SAVITTM (Mistral Pharma Inc.); RingCapTM (Alza Corp.); active agent can be formulated with low boiling point propel Smartrix R (Smartrix Technologies, Inc.); SQZgelTM (Mac lants. Such formulations are generally administered by con roMed, Inc.); GeomatrixTM (Skye Pharma, Inc.); Oros(R Tri ventional meter dose inhalers (MDIs). However, conven layer (Alza Corporation); and the like. 10 tional MDI’s can be modified so as to increase the ability to Also Suitable for use are formulations such as those obtain repeatable dosing by utilizing technology which mea described in U.S. Pat. No. 6.296,842 (Alkermes Controlled Sures the inspiratory Volume and flow rate of the patient as Therapeutics, Inc.); U.S. Pat. No. 6,187,330 (Scios, Inc.); and discussed within U.S. Pat. Nos. 5,404.871 and 5,542,410. the like. 15 Alternatively, an active agent can beformulated in aqueous Also suitable for use hereinareformulations comprising an or ethanolic solutions and delivered by conventional nebuliz intestinal absorption enhancing agent, and an active agent. ers. In some embodiments, such solution formulations are Suitable intestinal absorption enhancers include, but are not aerosolized using devices and systems such as disclosed limited to, calcium chelators (e.g., citrate, ethylenediamine within U.S. Pat. Nos. 5,497,763; 5,544,646; 5,718,222; and tetracetic acid); Surfactants (e.g., Sodium dodecyl sulfate, bile 5,660,166. salts, palmitoylcarnitine, and sodium salts of fatty acids); An active agent can be formulated into dry powder formu toxins (e.g., Zonula occludens toxin); and the like. lations. Such formulations can be administered by simply Inhalational Formulations inhaling the dry powder formulation after creating an aerosol An active agent (a SIRT1 activator or a SIRT1 inhibitor) mist of the powder. Technology for carrying Such out is will in some embodiments be administered to a patient by 25 described within U.S. Pat. Nos. 5,775,320 and 5,740,794. means of a pharmaceutical delivery system for the inhalation Dosages and Dosing route. An active agent can beformulated in a form Suitable for Depending on the Subject and condition being treated and administration by inhalation. The inhalational route of on the administration route, an active agent can be adminis administration provides the advantage that the inhaled drug tered in dosages of for example, 0.1 g to 500 mg/kg body can bypass the blood-brain barrier. The pharmaceutical deliv 30 weight per day, e.g., from about 0.1 g/kg body weight per ery system is one that is Suitable for respiratory therapy by day to about 1 ug/kg body weight per day, from about 1 ug/kg delivery of an active agent to mucosal linings of the bronchi. body weight per day to about 25 ug/kg body weight per day, This invention can utilize a system that depends on the power from about 25 ug/kg body weight per day to about 50 g/kg of a compressed gas to expel the active agent from a container. body weight per day, from about 50 ug/kg body weight per An aerosol or pressurized package can be employed for this 35 day to about 100 ug/kg body weight per day, from about 100 purpose. ug/kg body weight per day to about 500 ug/kg body weight As used herein, the term “aerosol is used in its conven per day, from about 500 g/kg body weight per day to about tional sense as referring to very fine liquid or Solid particles 1 mg/kg body weight per day, from about 1 mg/kg body carries by a propellant gas under pressure to a site of thera weight per day to about 25 mg/kg body weight per day, from peutic application. When a pharmaceutical aerosol is 40 about 25 mg/kg body weight per day to about 50 mg/kg body employed in this invention, the aerosol contains an active weight per day, from about 50 mg/kg body weight per day to agent, which can be dissolved, Suspended, or emulsified in a about 100 mg/kg body weight per day, from about 100 mg/kg mixture of a fluid carrier and a propellant. The aerosol can be body weight per day to about 250 mg/kg body weight per day, in the form of a solution, Suspension, emulsion, powder, or or from about 250 mg/kg body weight per day to about 500 semi-solid preparation. Aerosols employed in the present 45 mg/kg body weight per day. The range is broad, since in invention are intended for administration as fine, Solid par general the efficacy of a therapeutic effect for different mam ticles or as liquid mists via the respiratory tract of a patient. mals varies widely with doses typically being 20, 30 or even Various types of propellants known to one of skill in the art 40 times Smaller (per unit body weight) in manthan in the rat. can be utilized. Suitable propellants include, but are not lim Similarly the mode of administration can have a large effect ited to, hydrocarbons or other suitable gas. In the case of the 50 on dosage. Thus, for example, oral dosages may be about ten pressurized aerosol, the dosage unit may be determined by times the injection dose. Higher doses may be used for local providing a value to deliver a metered amount. ized routes of delivery. An active agent can also be formulated for delivery with a For example, an active agent can be administered in an nebulizer, which is an instrument that generates very fine amount of from about 1 mg to about 1000 mg per dose, e.g., liquid particles of Substantially uniform size in a gas. For 55 from about 1 mg to about 5 mg, from about 5 mg to about 10 example, a liquid containing the active agent is dispersed as mg, from about 10 mg to about 20 mg, from about 20 mg to droplets. The small droplets can be carried by a current of air about 25 mg, from about 25 mg to about 50 mg, from about 50 through an outlet tube of the nebulizer. The resulting mist mg to about 75 mg, from about 75 mg to about 100 mg, from penetrates into the respiratory tract of the patient. about 100 mg to about 125 mg, from about 125 mg to about A powder composition containing an active agent, with or 60 150 mg, from about 150 mg to about 175 mg, from about 175 without a lubricant, carrier, or propellant, can be administered mg to about 200 mg, from about 200 mg to about 225 mg. to a mammal in need of therapy. This embodiment of the from about 225 mg to about 250 mg, from about 250 mg to invention can be carried out with a conventional device for about 300 mg, from about 300 mg to about 350 mg, from administering a powderpharmaceutical composition by inha about 350 mg to about 400 mg, from about 400 mg to about lation. For example, a powder mixture of the compound and 65 450 mg, from about 450 mg to about 500 mg, from about 500 a suitable powder base such as lactose or starch may be mg to about 750 mg. or from about 750 mg to about 1000 mg presented in unit dosage form in for example capsular or per dose. US 8,748,464 B2 169 170 An exemplary dosage may be a solution Suitable for intra cheal, Subcutaneous, intradermal, transdermal, Sublingual, venous administration; a tablet taken from two to six times topical application, intravenous, ocular (e.g., topically to the daily, or one time-release capsule or tablet taken once a day eye, intravitreal, etc.), rectal, nasal, oral, and other enteral and and containing a proportionally higher content of active parenteral routes of administration. Routes of administration agent, etc. The time-release effect may be obtained by capsule may be combined, if desired, or adjusted depending upon the materials that dissolve at different pH values, by capsules that agent and/or the desired effect. The active agent can be release slowly by osmotic pressure, or by any other known administered in a single dose or in multiple doses. means of controlled release. An active agent can be administered to a host using any Those of skill in the art will readily appreciate that dose available conventional methods and routes suitable for deliv levels can vary as a function of the specific compound, the 10 ery of conventional drugs, including systemic or localized severity of the symptoms and the susceptibility of the subject routes. In general, routes of administration contemplated by to side effects. Preferred dosages for a given compound are the invention include, but are not necessarily limited to, readily determinable by those of skill in the art by a variety of enteral, parenteral, and inhalational routes. CaS. Parenteral routes of administration other than inhalation Although the dosage used will vary depending on the clini 15 administration include, but are not necessarily limited to, cal goals to be achieved, a Suitable dosage range is in some topical, transdermal, Subcutaneous, intramuscular, intraor embodiments one which provides up to about 1 lug to about bital, intracapsular, intraspinal, intrasternal, ocular, and intra 1,000ug or about 10,000 ug of active agent in a blood sample venous routes, i.e., any route of administration other than taken from the individual being treated, about 24 hours after through the alimentary canal. Parenteral administration can administration of the active agent to the individual. be carried to effect systemic or local delivery of the agent. Unit dosage forms for oral or rectal administration Such as Where systemic delivery is desired, administration typically syrups, elixirs, and Suspensions may be provided wherein involves invasive or systemically absorbed topical or mucosal each dosage unit, for example, teaspoonful, tablespoonful, administration of pharmaceutical preparations. tablet or Suppository, contains a predetermined amount of the An active agent can also be delivered to the subject by composition containing one or more active agents. Similarly, 25 enteral administration. Enteral routes of administration unit dosage forms for injection or intravenous administration include, but are not necessarily limited to, oral and rectal (e.g., may comprise the active agent(s) in a composition as a solu using a Suppository) delivery. tion in sterile water, normal saline or another pharmaceuti Methods of administration of an active agent through the cally acceptable carrier. skin or mucosa include, but are not necessarily limited to, In some embodiments, multiple doses of an active agent are 30 topical application of a suitable pharmaceutical preparation, administered. The frequency of administration of an active transdermal transmission, injection and epidermal adminis agent can vary depending on any of a variety of factors, e.g., tration. For transdermal transmission, absorption promoters severity of the symptoms, etc. For example, in some embodi or iontophoresis are Suitable methods. Iontophoretic trans ments, an active agent is administered once per month, twice mission may be accomplished using commercially available per month, three times per month, every other week (qow), 35 “patches” which deliver their product continuously via elec once per week (qw), twice per week (biw), three times per tric pulses through unbroken skin for periods of several days week (tiw), four times per week, five times per week, six O. O. times per week, every other day (qod), daily (qd), twice a day Subjects Suitable for Treatment (bid), or three times a day (tid). As discussed above, in some Subjects suitable for treatment with a subject method embodiments, an active agent is administered continuously. 40 include individuals in need of such treatment. The duration of administration of an active agent, e.g., the Methods of Reducing Chronic Immune Hyperactivity period of time over which an active agent is administered, can Subjects suitable for treatment with a subject method of vary, depending on any of a variety of factors, e.g., patient reducing chronic immune hyperactivity include individuals response, etc. For example, an active agent can be adminis who have chronic immune hyperactivity due to an infection tered over a period of time ranging from about one day to 45 with an immunodeficiency virus, e.g., human immunodefi about one week, from about two weeks to about four weeks, ciency virus-1, human immunodeficiency virus-2. from about one month to about two months, from about two Methods of Increasing an Immune Response months to about four months, from about four months to Subjects suitable for treatment a subject method of increas about six months, from about six months to about eight ing an immune response include an individual who has been months, from about eight months to about 1 year, from about 50 infected with a pathogenic microorganism; an individual who 1 year to about 2 years, or from about 2 years to about 4 years, is susceptible to infection by a pathogenic microorganism, or more. In some embodiments, an active agent is adminis but who has not yet been infected; and an individual who has tered for the lifetime of the individual. CaCC. Routes of Administration Subjects suitable for treatment with a subject method of An active agent is administered to an individual using any 55 increasing an immune response include pediatric target popu available method and route suitable for drug delivery, includ lations, e.g., individuals between about 1 year of age and ing in vivo and ex vivo methods, as well as Systemic and about 17 years of age, including infants (e.g., from about 1 localized routes of administration. Administration can be month old to about 1 year old); children (e.g., from about 1 acute (e.g., of short duration, e.g., a single administration, year old to about 12 years old); and adolescents (e.g., from administration for one day to one week), or chronic (e.g., of 60 about 13 years old to about 17 years old). Subjects suitable for long duration, e.g., administration for longer than one week, treatment with a Subject method of increasing an immune e.g., administration over a period of time of from about 2 response include adult individuals. weeks to about one month, from about one month to about 3 Subjects suitable for treatment with a subject method of months, from about 3 months to about 6 months, from about increasing an immune response include immunodeficient 6 months to about 1 year, or longer than one year). 65 individuals, e.g., individuals with an acquired immunodefi Conventional and pharmaceutically acceptable routes of ciency, e.g., as a results of radiation therapy for cancer, as a administration include intranasal, intramuscular, intratra result of corticosteroid treatment, as a result of cancer che US 8,748,464 B2 171 172 motherapy, etc. Also suitable for treatment with a subject were preincubated with nicotinamide (10 mM, Sigma) for 1 h method of increasing an immune response are individuals before treatment with C.-CD3/28 antibodies. Flow cytometry who have undergone bone marrow transplantation or any analysis of GFP expression (FACSCalibur, BD Bioscience) other organ transplantation, e.g., immunosuppressed indi showed that infection efficiencies ranged from 70-98% GFP viduals. cells in individual experiments. Tat expression was visualized by western blotting with polyclonal C.-FLAG and C-3-actin EXAMPLES (Sigma) antibodies. Generation of SIRT1- and Tat-expressing MEF Cell Lines The following examples are put forth so as to provide those Open reading frames corresponding to Myc-tagged human of ordinary skill in the art with a complete disclosure and 10 SIRT1 and T7-tagged HIV-1 Tat (101 aa) were inserted into description of how to make and use the present invention, and the murine stem cell virus (MSCV)-based retroviral vectors are not intended to limit the scope of what the inventors regard MSCV-puromycin and MSCV-Zeocin, respectively (Clon as their invention nor are they intended to represent that the tech). To obtain recombinant virus, 10ug of each constructor experiments below are all or the only experiments performed. empty control vectors were transfected into BOSC23 cells, a Efforts have been made to ensure accuracy with respect to 15 retroviral packaging cell line derived from 293 cells. The numbers used (e.g. amounts, temperature, etc.) but some supernatants were collected 48 h after transfection and fil experimental errors and deviations should be accounted for. tered through a 0.45-lum membrane. SIRT1-/- MEF cells Unless indicated otherwise, parts are parts by weight, (2x10) were incubated with 2 ml of the supernatant contain molecular weight is weight average molecular weight, tem ing SIRT1-expressing retrovirus or control virus together perature is in degrees Celsius, and pressure is at or near with polybrene (8 g/ml, Sigma). Cells were selected after 48 atmospheric. Standard abbreviations may be used, e.g., bp. h with puromycin (2.5 ug/ml. Invitrogen). Polyclonal puro base pair(s): kb, kilobase(s); pl. picoliter(s); S or sec, mycin-resistant MEF cells were reinfected with Tat-express second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s): ing or empty control retroviruses and selected in the presence kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intra of both puromycin (2.5ug/ml) and Zeocin (100 g/ml. Invit muscular(ly); i.p., intraperitoneal(ly); s.c., Subcutaneous(ly); 25 rogen). and the like. “C.” preceding a protein denotes an antibody to RNA Purification, cDNA Synthesis, and Real-time RT-PCR that protein; for example, C.-p65 is an antibody to p65. Total RNA was extracted using RNA STAT-60 reagent (Tel-Test) according to the manufacturer's instruction. The Example 1 first strand cDNA was generated using 2 ug of total RNA and 30 SuperScript reverse transcriptase (Invitrogen). Human IL-2 Human Immunodeficiency Virus Type 1 Tat Protein and murine E-selectin mRNAs were quantified by QuantiTect Inhibits the SIRT1 Deacetylase and Induces T Cell gene expression assays (Qiagen). Murine IKBO. and murine/ Hyperactivation human GAPDH mRNA concentrations were determined with SYBR Green I master mix (MCLab). Relative gene expres Experimental Procedures 35 sion ratios between stimulated and nonstimulated conditions were calculated by the equation, ratio=2^^, comparative Cells and Plasmids Ct method (Applied Biosystems). HeLa, 293, 293T and Jurkat cells were cultured under Transfections and Coimmunoprecipitations standard tissue culture conditions. MEF cells derived from Luciferase reporter constructs and protein expression vec SIRT1-/- cells were grown as described (Pagans et al., 2005). 40 tors were transiently cotransfected into HeLa cells using Mutant constructs for SIRT1 HDAC domain were prepared Lipofectamine (Invitrogen).24hafter transfection, cells were by site-directed mutagenesis using FLAG-tagged SIRT1 as a lysed and processed for luciferase assays (Promega). PValues template. The Sir2a deletion constructs were kindly provided (paired t-test) were used for statistical analysis. by V. Sartorelli, NIH, the 3x-KB luciferase reporter by N. 293 cells grown in 6-well plates at 70% confluency were Chirmule, Merck Inc, the IKBO. luciferase reporter by K. 45 cotransfected with expression vectors indicated in the Figure Yamamoto, UCSF, and the E-selectin luciferase reporter by J. legends. Transfection, cell lysis, immunoprecipitation, and Pober, Yale University. western blotting were previously described (Pagans et al., Infection with Lentiviral Vectors 2005). LTR-GFP and LTR-Tat-GFP are HIV-based vectors Fluorescent Deacetylase Assay derived from the pHR' series, in which GFP alone or Tat (101 50 Full-length human SIRT1 (His-tagged) was purified using aa) and GFP are under the control of the HIV-1 LTR through Ni-NTA agarose (Qiagen) and Superdex 200 gel filtration. the use of an internal ribosomal entry site (Jordan et al., 2001). The rate of 1 uM SIRT1 was calculated for 2001 uM fluoro For experiments with mutant Tat, the EF-1C. promoter was genic deacetylase substrate (BioMol) in the presence of inserted into the LTR-GFP vector upstream of wild type or twelve concentrations (1,2,3,4, 5, 6, 10, 15, 25, 50, 126,252 mutant Tat (EF1C.-Tat-GFP). Each vector was cotransfected 55 uM) of synthetic Tat (72 aa; Peptide Specialty Laboratory, into 293T cells together with a packaging construct (pCMV Heidelberg, Germany). R8.91) that provides all HIV genes required for production of Radioactive HDAC Assays infective particles and a plasmid encoding the vesicular sto Expression plasmids for FLAG-tagged wild type and matitis virus envelope G protein (VSV-G) to produce mutants SIRT1 (1 lug) were transfected in 293 cells with pseudotyped viral particles with broad host range and high 60 lipofectamine reagent. Equal amounts of whole cell extracts infectivity. Viral supernatant containing 1500 ng of p24 was (2 mg) were immunoprecipitated and in vitro HDAC assays used to infect 18x10 JurkatT cells. Infections were carried were performed as previously described (Pagans et al., 2005). out in 6-well plates at 2400 rpm in a Beckman-Coulter cen Recombinant p65 protein prepared from baculovirus-in trifuge for 2 hat 32°C. in the presence of polybrene (1 g/ml. fected Sf9 insect cells (BD Biosciences) was acetylated by Sigma). Jurkat T cells (10) were stimulated with plate bound 65 immunoprecipitated p300 overexpressed in 293 cells as pre C.-CD3 (3 ug/ml) and soluble C.-CD28 (1 lug/ml) antibodies 36 viously described (Chen et al., 2005). Acetylated p65 protein hafter infection as previously described (Ott et al., 1998) or was incubated with recombinant SIRT1 (1 lug/5U: Biomol) in