WO2015009884A1

WO2015009884A1 - Cell culture media formulations comprising an activator of nad-dependent protein deacteylase and ueses thereof

Info

Publication number: WO2015009884A1
Application number: PCT/US2014/046940
Authority: WO
Inventors: Bud M. NELSON
Original assignee: The Hamner Institutes
Priority date: 2013-07-18
Filing date: 2014-07-17
Publication date: 2015-01-22

Abstract

Methods, compositions, and formulations relating to the cultivation of stem cells in vitro are disclosed. A culture medium formulation comprises a nicotinamide antagonist, and may further comprise a sirtuin-activatiing compound. A method for culturing mammalian stem cells in vitro, comprising the steps of contacting the stem cells with a cell culture media formulation according to the invention, and cultivating the stem cells under conditions suitable to support their cultivation in vitro.

Description

CELL CULTURE MEDIA FORMULATIONS COMPRISING AN ACTIVATOR OF NAD- DEPENDENT PROTEIN DEACETYLASE AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to the discovery that certain compounds, including drugs and their metabolites, can activate NAD-dependent protein deacetylases. The invention also relates to cell culture media formulations which support the in vitro cultivation of stem cells (e.g., embryonic stems, or induced pluripotent stem cells). The media formulation comprises a nicotinamide antagonist (as described herein), and may further comprise a sirtuin-activatiing compound (as described herein) for modulating NAD-dependent protein deacetylase activity, as well as the activity of biological pathways affected by NAD- dependent protein deacetylase activity. The invention also provides for methods of cultivation of stem cells in vitro using the cell culture media formulations, kits comprising the cell culture media formulations, and cell culture compositions comprising the culture media and the stem cells.

BACKGROUND OF THE INVENTION

Post-translational modification of cellular proteins regulates a wide variety of cellular functions. One important mechanism for post-translational modification is by reversible acetylation of proteins at lysine residues, such as by the opposing activities of protein acetyltransferases and protein deacetylases which act on both histone and nonhistone proteins. One class of protein deacetylases is known as sirtuins. Some of the sirtuins, particularly sirtuin 1 (SIRT1 ), sirtuin 2 (SIRT2), sirtuin 3 (SIRT3), sirtuin 5 (SIRT5), sirtuin 6 (SIRT6), and sirtuin 7 (SIRT7), have protein deacetylase activity. Such sirtuins are typically described as NAD-dependent protein deacteylases because, unlike other known protein deacetylases in which acetyl-lysine residues are simply hydrolyzed, the sirtuin-mediated deacetylation reaction couples lysine deacetylation to NAD (nicotine adenine dinucleotide) hydrolysis. The sirtuins acting as NAD-dependent protein deacetylases, have an NAD-binding pocket. In this reaction, NAD, the acetylated protein substrate and the sirtuin enzyme form a complex, and as a result of the enzymatic reaction (i) NAD is cleaved into nicotinamide and an ADP-ribose product; and (ii) deacetylated protein. In the case of high concentrations of free nicotinamide, nicotinamide can (i) occupy the NAD-binding pocket of the NAD-dependent protein deacetylase and may block the conformational change and subsequent cleavage of NAD, (ii) react with a substrate intermediate (O-alkyl-amidate intermediate) in a process known as nicotinamide exchange or base exchange, and, hence (iii) slow or inhibit deacetylase activity or the rate thereof. Accordingly, dependence of NAD-dependent protein deacetylases on NAD links their enzymatic activity directly to the energy status of the cell via one or more of (a) the cellular NAD:NADH ratio, and (b) the absolute levels of NAD, NADH, or nicotinamide.

Sirtuins, as NAD-dependent protein deacetylases, are found in, and regulate important biological pathways in, organisms including bacteria and eukaryotes. In humans, sirtuins are called SIRT 1 -SIRT7 ("SIRT"- silent information regulators). In humans, SIRT1 (known in the art to comprise amino acid sequences selected from SEQ ID NO:1 and SEQ ID NO:2, and isoforms thereof having most, if not all, of the amino acids in sequence as shown in either SEQ ID NO:1 or SEQ ID NO:2) is an NAD- dependent protein deacetylase localized in the cytoplasm and nucleus, and can repress transcription in the nucleus by various different mechanisms. One way SIRT1 can repress transcription is through deacteylation of transcription factors. For example, it has been shown in human cells that SIRT1 decreased acetylation of transcription factor Nrf2 (nuclear factor erythroid-related factor 2, comprising an amino acid sequence of SEQ ID NO:4, including isoforms thereof having most, if not all, of the amino acids in sequence as shown in SEQ ID NO:4). Deacetylation of Nrf2 enhanced localization of Nrf2 to the cytoplasm, as well as decreased Nrf2-dependent gene transcription. When Nrf2 is acetylated, there is increased nuclear localization of Nrf2, and promoted is binding of Nrf2 to the response element specific for Nrf2 (ARE or Antioxidant Response Element), with resultant increased Nrf2-dependent gene transcription of genes containing ARE in their promoter region.

Genes having functional ARE include cytoprotective genes, such as genes encoding antioxidant enzymes, phase II detoxification enzymes, and multidrug resistant proteins. Thus, NRF2-mediated adaptive antioxidant response plays pivotal roles against oxidative/electrophilic stress, and in chemical detoxification (including drug metabolism). For example, Nrf2 has been shown to regulate the transcription of genes that encode drug metabolizing enzymes, including UGT (UDP-glucuronosyltransferase) and NQ01 (NAD(P)H quinone oxidoreductase 1 ), cytochrome P450 2A5 (CYP2A5), and glutathione S-transferase (GST). Nrf2 also regulates the expression of genes that are involved in direct reduction of reactive oxygen species (ROS), including superoxide dismutase, catalase, and glutathione peroxidases. Additionally, Nrf2 induces genes involved in reduction of oxidized proteins, such as thioredoxin-1 , thioredoxin reductase-1 , and sulfiredoxin, as well as genes encoding enzymes that synthesize glutathione (GSH); i.e., γ-glutamate-cysteine ligase catalyze subunit (Gclc) and the modifier subunit (Gclm).

SUMMARY OF THE INVENTION

Provided herein are novel composition comprising activators of NAD-dependent protein deacetylases, and methods of use thereof. The invention is based, in part, upon the discovery of compounds which can activate NAD-dependent protein deacetylases, and the correlation between the structure of these compounds and their function to activate NAD-dependent protein deacetylases by a mechanism comprising acting as an antagonist of nicotinamide. Without being bound to any particular mechanism, it is believed that antagonists of nicotinamide inhibit or reduce the base exchange process that slows or inhibits NAD-dependent protein deacetylase activity; i.e., thereby promoting NAD-dependent protein deacetylase activity. These compounds comprise a class of activators of NAD-dependent protein deacetylases ("Nicotinamide Antagonist") distinct in chemical structure and function as compared to the class of human SIRT1 activator compounds known as sirtuin activating compounds ("STACs") comprising polyphenols (e.g., resveratrol, fisetin, chlorogenic acid) and their analogs (e.g., SRT1720, SRT1460, SRT2183), quinoxaline compounds, stilbene compounds (ester analogs of resveratrol) and the like (see, e.g., Formulas ll-VI). For example, STACs are believed to work by an allosteric mechanism in which the binding of activator is enhanced for enzyme-substrate complexes or by the activator promotes a conformational change that produces enzyme- substrate complexes, in promoting NAD-dependent protein deacetylase activity.

In one aspect of the invention provided is a composition for activating NAD- dependent protein deacetylases comprising one or more Nicotinamide Antagonists (a first class of activators of NAD-dependent protein deacetylases), or one or more Nicotinamide Antagonists combined with one or more STACs (a second class of activators of NAD- dependent protein deacetylases). The composition of the invention may further comprise a physiologically acceptable carrier. Such composition may be added to become components of existing cell culture media in the production of a cell culture media formulation useful for cultivation of cells in vitro, wherein activation of a NAD-dependent protein deacetylase such as SIRT1 is desired. In another aspect, the composition may be used to coat a surface of a cell culture container or culture vessel which is then available for contacting cells added to the culture vessel.

In another aspect of the invention, provided is defined cell culture media capable of supporting the in vitro cultivation of stem cells such as embryonic stems, or induced pluripotent stem cells, wherein the culture medium comprises, as ingredients, a

Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, in an amount effective to induce SIRT1 activity. The medium provided by the invention can be a 1 X formulation (ready for use without further dilution) or may be in a concentrated form, such as 10X formulation (e.g., each ingredient in the formulation is 10 times more concentrated than the same ingredient when used for cultivation of cells) or higher concentrate. The basal medium useful to produce the cell culture formulation comprises ingredients that may be selected from amino acids, salts (e.g., one or more of inorganic salts and organic salts), vitamins, sugars, growth factors, and other components, or various combinations thereof, with each ingredient being present in an amount which supports the in vitro cultivation of stem cells such as human embryonic stems, or human induced pluripotent stem cells or stem cells of mammalian origin.

Also provided is a method for activating NAD-dependent protein deacetylase, SIRT1 , in cells (e.g., stem cells such as human embryonic stems, or human induced pluripotent stem cells) in culture, comprising contacting the cells with a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, in an amount effective to induce SIRT1 activity. In the method, the combination comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, comprise an ingredient in a cell culture medium formulation, or may comprise an additive or supplement that is added to a cell culture in vitro comprising cell culture medium and cells to form the cell culture medium formulation. As an additive or supplement, the Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, may further comprise one or more physiologically acceptable carriers. As an additive or supplement, the Nicotinamide Antagonist and a STAC may each comprise separate compositions, which can be administered simultaneously or sequentially, or the Nicotinamide Antagonist and a STAC may together comprise a single composition. With this aspect, provided is a cell culture supplement comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC; and may further comprise one or more physiologically acceptable carriers.

Also provided are methods for culturing mammalian stem cells in vitro using the culture medium formulations or cell culture supplement as disclosed herein, comprising the steps of (a) contacting the stem cells with the cell culture media formulation of the invention, and (b) cultivating the stem cells in vitro under conditions suitable to support their cultivation in vitro (as known to those skilled in the art suitable conditions comprise factors such as the appropriate temperature and atmosphere for cultivation). Also provided are methods for culturing mammalian stem cells in vitro using the composition of the invention as disclosed herein (comprising one or more Nicotinamide Antagonists, or one or more Nicotinamide Antagonists combined with one or more STACs), comprising the steps of (a) contacting the stem cells with the composition of the invention, and (b) cultivating the stem cells in vitro under conditions suitable to support their cultivation in vitro (as known to those skilled in the art suitable conditions comprise factors such as the appropriate temperature and atmosphere for cultivation).

In another aspect, provided is a kit comprising a carrier or container for confinement of the cell culture media formulation or composition of the invention. The kit may also be configured to comprise a first container containing a cell culture medium for cultivating the stem cells in vitro, and one or more containers containing one or more cell culture supplements or compositions comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC; and may further comprise one or more

physiologically acceptable carriers. The kit may further comprise a culture vessel. The culture vessel may further comprise one or more surfaces coated with a composition of the invention.

In another aspect of the invention, the cell culture medium formulation is suitable for one or more in vitro uses comprising the initiation of the stem cell culture, growth of established stem cell cultures, and expansion of established stem cell cultures (one or more of which comprises cultivation of stem cells).

Other aspects, objects and features of the invention will be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.1 A is a graph showing that isoniazid ("INH") suppresses Nrf2-ARE activity in 3T3-L1 preadipocytes in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2- ARE activity by isoniazid.

FIG.1 B is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of glutamate-cysteine ligase catalytic subunit ("Gclc") in 3T3- L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG.1 C is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of NAD(P)H dehydrogenase [quinone] 1 ("Nqo1 ") activity in 3T3-L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid. FIG.1 D is a graph showing that isoniazid ("INH", 10 mM) suppresses Nrf2-ARE- dependent gene expression of Heme oxygenase ("Ho1 ") activity in 3T3-L1 preadipocytes under basal ("Veh") conditions, and that treatment with an Nrf2 activator (tBHQ, 50 μΜ) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG 2A is a graph showing that isoniazid ("INH") inhibits Nrf2-ARE activity in human hepatocellular liver carcinoma HepG2 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG. 2B is a graph showing that isoniazid ("INH") suppresses Nrf2-ARE-dependent gene expression of Heme oxygenase ("Ho1 ") activity in HepG2 cells in a concentration- dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by isoniazid.

FIG. 3 is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE activity in HepG2 cells in a concentration-dependent manner under basal ("Vehicle") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG.4A is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of Heme oxygenase ("HO ') activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG.4B is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of glutamate-cysteine ligase catalytic subunit ("GCLM") activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide. FIG.4C is a graph showing that ethionamide (ETH) suppresses Nrf2-ARE-dependent gene expression (mRNA expression as a percent of the Control with Vehicle) of sulfiredoxin ("SRX") activity in THP-1 cells in a concentration-dependent manner under basal ("Veh") conditions, and that treatment with an Nrf2 activator (iAs³⁺) modulated the inhibition of Nrf2-ARE activity by ethionamide.

FIG. 5 is an illustration of chemical structures of compounds having a formula of Formula I identified as Nrf2-ARE-inhibitors. FIG. 6A is a graph showing the effects of rifampicin ("Rif"), isoniazid ("INH"), or isoniazid together with rifampicin, on PPARYI mRNA levels (expressed as percent of Control) as compared to the vehicle assay control ("Control").

FIG. 6B is a graph showing the effects of rifampicin ("Rif"), isoniazid ("INH"), or isoniazid together with rifampicin, on PPARy2 mRNA levels (expressed as percent of Control) as compared to the vehicle assay control ("Control").

FIG. 7 is a schematic showing representation of an activated SIRT1 and effects on downregulating PPARy and Nrf2 activity (black arrow), and of dysregulation of NAD- dependent protein acetylase as shown with increased activation of PPARy and effects on downregulating SI RT1 (white arrow).

DETAILED DESCRIPTION OF THE INVENTION

Provided are compositions comprising activators of NAD-dependent protein deacetylases comprising one or more Nicotinamide Antagonists, or one or more

Nicotinamide Antagonists and one or more STACs in forming a composition comprising a combination. The composition of the invention may further comprise a physiologically acceptable carrier. The compositions may be used to activate a NAD-dependent protein deacetylase, and are useful as components for cultivating stem cells in vitro. It was unexpectedly discovered that heterocyclic compounds used in the treatment of tuberculosis can activate NAD- dependent protein deacetylases by acting as nicotinamide antagonists. As shown herein, a Nicotinamide Antagonist comprises a heterocyclic compound having a hydrazide moiety or carboxamide moiety (typically, as a side chain), and is selected from a compound represented by Formula I. As illustrative, non-limiting examples, such compounds comprise ethionamide (2-ethylpyridine-4-carbothioamide), pyrazinamide (pyrazine-2-carboxamide), and isoniazid (isonicotinohydrazide). In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds represented by Formula I, including Formula IA and Formula IB.

In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds represented by Formula IA, and a pharmaceutically acceptable salt thereof.

Formula IA

wherein: A is N or C;

B is N or C;

R1 or R2 or R3 are each independently selected from H, (Ci-C₆)alkyl, CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, CHCHCONHNH₂, or COCH₃;

wherein at least one of R1 , R2, and R3 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂; and

the dashed lines represent optional double bonds; with the proviso that the compound of Formula IA is not nicotinamide (also known as 3-pyridinecarboxamide), isonicotinamide (also known as pyridine-4-carboxamide), or nicotinamide adenine dinucleotide (NAD) (i.e., excluded from a compound represented by Formula IA, and thus excluded from being a Nicotinamide Antagonist in methods and compositions of the invention, is nicotinamide; NAD+; and isonicotinamide).

In one aspect of the invention, a Nicotinamide Antagonist is selected from compounds represented by Formula IB, and a pharmaceutically acceptable salt thereof.

Formula IB

wherein:

A is O or N;

B is N or C;

R1 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂;

R2 is absent if B is NH;

if B is C, R2 is absent or selected from CH₃, CH₂CH₃, NH₂, or NHNH₂; and

the dashed lines represent optional double bonds; with the proviso that the compound of Formula IB is not nicotinamide (also known as 3-pyridinecarboxamide), isonicotinamide (also known as pyridine-4-carboxamide), or nicotinamide adenine dinucleotide (NAD) (i.e., excluded from a compound represented by Formula IB, and thus excluded from being a Nicotinamide Antagonist in methods and compositions of the invention, is nicotinamide; NAD+; and isonicotinamide).

A preferred Nicotinamide Antagonist may be used as an activator of NAD-dependent protein deacetylase in accordance with the invention to the exclusion of an Nicotinamide Antagonist other than the preferred Nicotinamide Antagonist. Many compounds of Formula I are commercially available, as well as chemicals used as starting materials in their chemical syntheses. Many synthesis methods are known for preparing compounds of Formula I according to the invention such as by using standard organic chemical synthesis methods well known in the art. These and/or other well-known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional compounds within the scope of the present invention. Compounds as described herein may be purified by any of the means known in the art, including but not limited to chromatographic means, such as high pressure liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. From chemical libraries or collections of chemical compounds, a chemical compound can be screened for identifying a Nicotinamide Antagonist by using assays for determining inhibition of NAD-dependent protein deacetylase activity as known in the art, including those described herein, as well as selecting compounds to test in the assays which have a chemical structure represented by Formula I. A compound of Formula I can be tested (in the presence of nicotinamide) for the level of activation of NAD-dependent protein deacetylase such as by measuring the rate of fluorescent-free activation substrate deacetylation, or by measuring the rate of NAD+ hydrolysis, by using methods known to those skilled in the art. In another example, and with respect to SIRT1 , the level of activation of NAD-dependent protein deacetylase may be measured by determining the effects of deacetylation of Nrf2 (e.g., by degree of inhibition of Nrf2 activity) as described herein in more detail.

A sirtuin-activating compound ("STAC") has a core structure centering around a bicyclic heterocycle (as shown by Formulas II, IV, V, Va, and VI), and is typically selected from either a benzimidazole, imidazothiazole, quinoxaline, and thiazolopyridine; or a core structure comprising a stilbene (as shown by Formula III).

In one aspect of the invention, a sirtuin activating compound ("STAC") is selected from compounds of the formula of any one of Formula II, Formula III

wherein is selected from -(CH₂)₃— CH₃, and -(CH₂)CH(CH₃)₂; and R₂ is piperidine or — (CH₂)₂— NH— CH₃; or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Published Patent Appl. No. US 20130102009; the contents of which are herein incorporated by reference).

Formula I II

wherein

AT is selected from H and (CO)P ;

A₂ is selected from H and (CO)R₂;

A₃ is selected from H and (CO)R₃;

and wherein at least one of A^ A₂, and A₃ is different from another;

RT and R₂ when present are each independently selected from alkyl with at least two carbon atoms, unsubstituted aryl, and aralkyi;

R₃ when present is selected from alkyl with at least two carbon atoms, aryl, and aralkyi, and when R₃ is alkyl, it is unsubstituted straight or branched alkyl;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.

Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent 7,714,161 ; the contents of which are herein incorporated by reference).

Formula IV

wherein

R is H or CH3;

R¹ is selected from a substituted or unsubstituted nitrogen-containing heterocyclmethyl group containing a second heteroatom selected from a nitrogen or oxygen, or a morpholine, or a morpholinomethl group, or a 1 ,2,4-triazolylmethyl group;

R² is H or CH3; and

R³ is an unsubstituted pyridyl group;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent Nos. 7,829,556 and 8,247,565; the contents of which are herein incorporated by reference).

Formula V

wherein

1⁹ is

wherein each of Z₁₀, Z , Z₁₂, and Z₁₃ is independently selected from CR²⁰ or CR¹ ; wherein zero to one R²⁰ is a solubilizing group; and

zero to one R¹ is an optionally substituted C1 -C3 straight or branched alkyl;

each R²⁰ is independently selected from H or a solubilizing group;

R²¹ is -NR¹-C(0)-; each R¹ is independently selected from H, or an optionally substituted C1 -C3 straight or branched alkyl;

R³¹ is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally substituted monocyclic or bicyclic heteroaryl;

or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent No. 7,345,178; the contents of which are herein incorporated by reference). Some examples of compounds of Formula V

Formula Va

wherein

RT is selected from a bicyclic heterocycle that is selected from a naphthalene and a quinoxaline, and a benzene ring substituted with between 2 and 3 methoxy groups; R₂ is selected from a piperazine, and a pyrrole substituted with an OH group, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof. Examples of compounds with this formula, and methods of making them are known to those skilled in the art (for example, see U.S. Patent No. 7,345,178) and include selective SIRT1 inhibitors known as SRT1720, SRT1460, and SRT2183, one or more of which is commercially available.

Other quinoxaline compounds that are STACs include 3-benzenesulfonyl-1 -(4-fluro- phenyl)-1 H-pyrrolo[2,3-b]quinoxalin-2-ylamine; 2 -amino-1 -(2-ethyl-phenyl)-1 H- pyrrolo[2,3b]quinoxaliine-3-carboxylic acid (tetrahydro-furan-2-ylmethyl)-amine; and 2 - amino-1 -(3-methoxy-propyl)-1 H-pyrrolo[2,3b]quinoxaliine-3-carboxylic acid

cyclopentamide.

Formula VI

wherein

ring A is selected from

wherein "^*" represents a portion of ring A bound to phenyl, and

" J^⁴^^ " represents a portion of ring A bound to C=0 in the compound;

1 is selected from hydrogen, or

R³ is selected from h drogen, methoxypropyl, methox rop-1 -ynyl,

; and

at least one of R¹ or R³ comprises a nitrogen-containing saturated heterocyclyl portion. Examples of compounds with this formula, and methods of making them, are known to those skilled in the art (for example, see U.S. Patent No. 8,343,997; the contents of which are herein incorporated by reference).

Other known STACs include naturally-occurring compounds such as a polyphenol, methylxanthine, or stilbene, illustrative examples of which may include resveratrol, fisetin, chlorogenic acid, or ester analogs thereof such as 3,5,4'-trihydroxy-trans-stilbene.

While the terms used in the description of the invention are believed to be well understood by one of ordinary skill in the pharmaceutical arts, definitions, where provided herein, are set forth to facilitate description of the invention, and to provide illustrative examples for use of the terms.

As used herein, the terms "a", "an", and "the" mean "one or more", unless the singular is expressly specified (e.g., singular is expressly specified, for example, in the phrase "a single formulation").

The term 'alkyl" is used herein to refer to a hydrocarbon containing normal, secondary, tertiary, or cyclic carbon atoms (e.g., linear saturated aliphatic hydrocarbon groups, branched saturated aliphatic hydrocarbon groups, or a saturated or unsaturated non- aromatic hydrocarbon mono or multi-ring system (e.g., cycloalkyl)). When the term "alkyl" is used without reference to a number of carbon atoms, it is to be understood to refer to a C(i)-C(io >alkyl ; e.g., a C(i), C^, Cpj, C(4), C(5), C(6), Cpj, C^), C(g ₎Or C(io >alkyl.

The term "aryl" is used herein to refer to cyclic, aromatic hydrocarbon groups which have 1 to 3 aromatic rings, for example phenyl or naphthyl. The aryl group may have fused thereto a second or third ring which is a heterocyclo, cycloalkyl, or heteroaryl ring, provided in that case the point of attachment will be to the aryl portion of the ring system. "Heteroaryl" refers to an aryl group in which at least one of the carbon atoms in the aromatic ring has been replaced by a heteroatom selected from oxygen, nitrogen and sulphur. The nitrogen and/or sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heteroaryl group may be a 5 to 6 membered monocyclic, 7 to 1 1 membered bicyclic, or 10 to 16 membered tricyclic ring system.

The term "aralkyl" is used herein to refer to an aryl-alkyl- group in which the aryl and alkyl are as defined herein. In one aspect of the aspects of the invention, preferably an aralkyl comprises a lower alkyl group.

The terms "first", "second", and "additional", are used herein for purposes of distinguishing between two compounds, or between two or more compositions, or between two or more steps of a method, as will be clearer from the description.

The term "stem cell" is used herein to mean a mammalian cell capable of self- replication, and pluripotency. Preferably, the stem cell is a human stem cell. Stem cells may comprise, but are not limited to, embryonic stem cells (ESCs) or tissue-specific stem cells. Also included in the definition of stem cells are artificially produced cells that have stem-like abilities (e.g., self- replication, and pluripotency), such as induced pluripotent stem cells (iPSCs). Pluripotent stem cells are cells that can differentiate (by using the appropriate stimuli) into cells derived from any one of the three germ layers (e.g., endoderm, mesoderm, or ectoderm).

The term "induced pluripotent stem cell" or abbreviations thereof (e.g., iPS cells or iPSCs) refers to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell. The non-pluripotent cell is typically an adult somatic cell or terminally differentiated cell such as a fibroblast, hematopoietic cell, an epidermal cell, and the like. There are several known methods of reprogramming cells to generate iPSCs. One well known method is to introduce (e.g., by episomal expression, retroviral expression, or Sendai virus expression) OCT4, SOX2, KLF4, and MYC genes (commonly known as the "Yamanaka factiors") to convert cells from post-natal tissue cells into iPSCs.

The term "contacting" refers to placing of cells to be cultivated in physical presence of or with the cell culture medium formulation or supplement of the invention in vitro. Typically, the contacting takes place in a culture vessel (e.g., plate, flask, chamber, bioreactor, cassette, gas- permeable bag, roller bottle, culture dish, slide, tube, tray, cartridge, etc., which is capable of culturing stem cells therein) in which the cells are to be cultivated in vitro. Contacting can also be performed in a container or device (e.g., pipette) in which cells and formulation or supplement are mixed with subsequent introduction into a culture vessel for culturing.

The term "defined media" is used herein to mean a culture medium which is specifically formulated to support the cultivation of one or more desired cell types, and contains no undefined supplements, but rather comprises defined amounts of amino acids, vitamins, growth factors, lipids, sugars, salts, buffering agents, dyes (e.g., phenol red) or other substances included as components in the medium. A "basal medium" is known to those skilled in the art as an aqueous-based defined medium, or alternatively may be in dried form. To a basal medium is added a Nicotinamide Antagonist, or a Nicotinamide

Antagonist and a STAC, to produce a cell culture medium formulation of the invention. If in dried form, the cell culture medium formulation would then need to be dissolved or reconstituted with a liquid such as a physiologically acceptable carrier. The cell culture medium formulation is typically sterilized to prevent contamination of the cell culture to which it is contacted. Sterilization can be by any means known in the art which include but are not limited to filter sterilization, and production under aseptic conditions.

The term "NAD-dependent protein deacetylase" means a protein deacetylase in which the protein-mediated deacetylation reaction couples lysine deacetylation to NAD. In one aspect of aspects of the invention, NAD-dependent protein deacetylase is used to preferably refer to one or more of human SIRT1 (substantially comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2), human SIRT2 (substantially comprising an amino acid sequence of SEQ ID NO:3).

The term "activating NAD-dependent protein deacetylase" refers to the inducing effect of an activator of NAD-dependent protein deacetylase (e.g., an NAD Antagonist or sirtuin activating compounds ("STAC") or a combination thereof) on level or activity of an NAD- dependent protein deacetylase. Thus, an activator of NAD-dependent protein deacetylase has the ability to increase the level or activity of NAD-dependent protein deacetylase, resulting in increased deacetylase activity, as compared to activity of NAD- dependent protein deacetylase in the absence of such activator. A Nicotinamide

Antagonist, or a Nicotinamide Antagonist and a STAC, (optionally, further comprising a physiologically acceptable carrier) according to the invention, for activating NAD- dependent protein deacetylase, may result in an increase in the level or activity of an NAD-dependent protein deacetylase by a factor of at least about 2, 5, 10, 20, 50, 100, or greater fold (or any range derivable therefrom), as compared to the level or activity of the NAD-dependent protein deacetylase in the absence of adding a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, to the cell culture . Such increase can be measured in vitro such as in a cellular assay or in a cell-based assay, and compared to a basal level (e.g., measured in the absence of the activator of NAD-dependent protein deacetylase in the same assay system), using methods known to those skilled in the art. For example, a compound's ability to activate NAD-dependent protein deacetylase can be assessed and quantitated using commercially available fluorescence-based assay, such as the "SIRT1 fluorometric drug discovery assay kit" (Enzo Life Sciences), using the manufacturer's directions. In the assay, a fluorescent-labeled substrate of the NAD- dependent protein deacetylase is mixed in the presence of the NAD-dependent protein deacetylase and in the presence of the compound(s) being assessed for its ability to activate NAD-dependent protein deacetylase. However, if assessing for a compound as a Nicotinamide Antagonist, nicotinamide is added to the reaction (also provided in the kit). The assay is run in parallel (e.g., separate microtiter well) but in the absence of the compound(s) ("control well"). Deacetylation of the substrate sensitizes the substrate to the addition of a detection reagent. The reaction is subjected to excitation at 360 nanometers (nm), and detection at 460 nm. Detection of increased NAD-dependent protein deacetylase activity in the presence of the compound(s) as compared to the absence of the compound(s) (control well) is an indication that the compound(s) has the ability to activate NAD-dependent protein deacetylase.

The term "non-naturally occurring" used in reference to a compound means that the compound is not known to exist in nature or that does not exist in nature. The term "naturally occurring" when used in connection with compounds refers to a compound which is found in nature. It is apparent to those skilled in the art that a naturally occurring compound can be modified or engineered by a human or by an engineered organism to be structurally or chemical different to form a non-naturally occurring compound.

Prodrugs of the compounds of Formula I, or salts thereof, are included within the scope of the invention. The term "prodrug", as used herein, refers to a compound that is transformed in vivo (e.g., by a metabolic, physiological, or chemical process) to yield a compounds of Formula I, or a pharmaceutically acceptable salt, hydrate or solvate of the compound. Prodrugs, made by synthesizing one or more prodrug moieties as part of an active compound, can serve to enhance one or more of solubility, absorption, lipophilicity, pharmacodynamics, pharmacokinetics, and efficacy, as compared to the active compound without the one or more prodrug moieties. Various forms of prodrugs are known in the art. Examples of prodrugs of the compounds of the invention include an in vivo cleavable ester of a carboxy group (e.g., lower alkyl esters, cycloalkyl esters, lower alkenyl esters, benzyl esters, mono-or di-substituted lower alkyl esters, and the like); or S-acyl and O-acyl derivatives of thiols, alcohols, or phenols. "Prodrug moiety" refers to a labile functional group, including but not limited to a protective group, which can be removed or reduced from the active compound during a process elected from one or more of metabolism, systemic circulation, intracellular, hydrolysis, or enzymatic cleavage. Enzymes which are capable of an enzymatically activating a phosphonate prodrug include, but are not limited to, amidases, esterases, phospholipases, cholinesterases, and phosphases. Prodrug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. A prodrug moiety may include an active metabolite or drug itself. Exemplary prodrug moieties include the hydrolytically sensitive or labile acyloxymethyl esters -CH₂OC(=0)R³ and acyloxymethyl carbonates esters -CH₂OC(=0)OR³ where R³ is d-C₆ alkyl, d-C₆ substituted alkyl, C₆- C₂₀ aryl or C₆-C₂₀ substituted aryl. Other examples of prodrug moieties include addition of a halogen (e.g., fluoro group), carbon replacement with nitrogen ("aza" compounds), carbocyclic analogs, and chloroacetyl (2b-d) derivatives.

The terms "purified" or "isolated" for a compound or composition refers to the physical state of the compound or composition following isolation from a synthetic process or purification step described herein or well known to those in the art, and in sufficient purity to be characterizable by standard analytical methods described herein or well known in the art.

The terms "salt" or pharmaceutically acceptable salt", as used herein, refers to inorganic or organic salts of a compound. These salts can be prepared, for example, by reacting a compound of Formula I, or a compound of Formulas ll-VI disclosed herein, with an amount of acid or base, such as an equivalent amount, and in a medium such as one in which the salt formed then precipitates, or in an aqueous medium followed by lyophilization. Representative salts include bisulfate, sulfate, benzene sulfonate, camphorsulfonate, laurylsulphonate, methanesulfonate, toluenesulfonate,

naphthalenesulformate, acetate, trifluoracetate, benzoate, borate, butyrate, citrate, formate, fumarate, hydorbromide, hydrochloride, hydroiodide, lactate, laurate, maleate, malonate, mesylate, nitrate, oxalate, phosphate, hexafluorophosphate, propionate, salicylate, stearate, succinate, tartrate, thiocyanate, and the like. The salts may include base salts based on the alkali and alkaline earth metals, such as calcium, sodium, lithium, magnesium, and potassium; or with organic bases such as with organic amines (e.g., dicyclohexylamine, t-butyl amine, methylamine, dimethylamine, triethylamine, ethylamine, procaine, morpholine, N-methylpiperidine, dibenzylamine, and the like); or as an ammonium salt.

The compounds disclosed herein may exist in a solvated form or unsolvated form. Solvates of a compound disclosed in the invention may be formed in the synthetic process in which the compound becomes physically associated with one or more solvent molecules (e.g., such as by ionic and/or covalent bonding) or, optionally, may be converted to a solvate such as by dissolving the compound in desired amounts of a solvent of choice (e.g., organic solvent, water, or mixtures thereof) in forming a solution, heating the solution to a temperature higher that ambient temperature, and cooling the solution at a rate sufficient to form crystals of the solvate, which may then be further isolated using methods known the art. Examples of suitable solvents include

methanolates, ethanolates, hydrates (where the solvent molecule is water), and the like. The compounds of Formulas l-VI may contain asymmetric or chiral centers, and thus exist in different stereoisomeric forms. All stereoisomers (e.g., geometric isomers, optical isomers, and the like), enantiomeric forms, diastereomeric forms, tautomeric forms, positional isomers, of the compounds disclosed in the invention are embraced within the scope of the invention. A first conformational form of a compound can be separated from a second and different conformational form of the compound using methods well known in the chemical arts such as by chromatography, crystallization, and methods of synthesis which selectively result in a particular desired conformational form.

The term "physiologically acceptable carrier" is used herein to mean any compound or composition or carrier medium useful in any one or more of administration, delivery, storage, stability of a composition or compound described herein. These carriers are known in the art to include, but are not limited to, a diluent, water, saline, suitable vehicle (e.g., one or more of liposome, microparticle, nanoparticle, emulsion, polymer, or capsule), buffer, medical parenteral vehicle, excipient, aqueous solution, suspension, solvent, emulsions, detergent, chelating agent, solubilizing agent, salt, colorant, polymer, hydrogel, surfactant, emulsifier, adjuvant, filler, preservative, stabilizer, oil, binder, disintegrant, absorbant, or combinations thereof, and the like as broadly known in the pharmaceutical art or tissue culture art.

The phrase "effective amount" generally means an amount of a composition comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, (and optionally, further comprising a physiologically acceptable carrier) effective to induce NAD-dependent protein deacetylase (e.g., SIRT1 ) activity in a cell contacted by the composition, as compared to the level of activity in the absence of the composition. In one aspect, the NAD-dependent protein deacetylase activity induced comprises human SIRT1 . Use of a composition or formulation of the invention to induce or increase NAD- dependent protein deacetylase activity can be determined by methods known in the art (as will also be apparent from the description and figures herein).

An effective amount of a composition comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, (and optionally, further comprising a physiologically acceptable carrier) for use in a supplement or formulation of the invention will depend on such factors as the type of stem cell to be cultivated, the length of cultivation time, the conditions of cultivation, and other factors which can be taken into consideration by a researcher whom is skilled in the art of determining appropriate dosages for use in cell or tissue culture. An amount of compound used in the invention in a composition or formulation may vary from 0.001 nM to about 10 mM or any range derivable therefrom, and more typically from about 0.1 nM about 2 mM, or any range derivable therefrom. A physiologically acceptable carrier, used in a composition according to the invention, may facilitate one or more of storage, stability, administration, and delivery, of the composition. EXAMPLE 1

Discovered and identified is a novel class of compounds with strong inhibitory effects on Nrf2-ARE activity (including downstream genes having an ARE). These compounds include a panel of antitubercular agents, such as isoniazid, ethionamide, ethambutol dihydrochloride, rifampicin, ethionamide, and sparfloxacin; and other chemicals, including 4-aminobenzoic hydrazide, aminopyrazine,

cyclohexanecarboxamide, 2-furoic hydrazide, phenylhydrazine, phenylacetic hydrazide, pyrazinecarboxamide, p-toluic hydrazide, and 4-(aminomethyl) piperidine (see, e.g., Table 1 ). These compounds decrease ARE-luciferase activity, in a concentration- dependent manner in treated cells, under basal and arsenite-treated conditions. These Nrf2-ARE-inhibitors suppress ARE activity, and suppress induction of ARE-driven gene expression. However, no change in Nrf2 protein level was observed.

These chemical modulators of Nrf2-ARE activity were identified by a series of chemical screenings using an assay in which an ARE-luciferase reporter is stably expressed in cells in which there is confirmed constitutive activation of Nrf2-ARE activity. These cells include mouse preadipocyte 3T3-L1 cell line; mouse insulinoma MIN6 cell line; human keratinocyte HaCaT cell line; and human hepatocellular cancer cell line, HepG2 cells. A commercially available ARE-luciferase reporter, in ready-to-transduce lentiviral particles, was used for assessing when the Nrf2 pathway is activated or inhibited by a drug or chemical, via detection of any modulation of luciferase reporter activity which can then be measured quantitatively. This type of assay has been validated in the art to measure and represent induction or inhibition of Nrf2-ARE activity taking place in cells in the body.

Lentiviral transduction of 3T3-L1 , HaCaT and HepG2 cells was performed based on manufacturer's protocol. Briefly, 24 hours before transduction, the cells to be transduced were plated in 6-well plates at 40-50% confluency in complete cell culture medium. The following day, hexadimethrine bromide, a transduction enhancer, was added to each well at a concentration of 8 \g/m\, and viral particles were added to each well at a concentration of 2 x 10⁵ transducing units/ml. After overnight incubation, medium containing viral particles was removed and replaced with fresh medium containing 2 μg/ml puromycin. Cells were grown to -90% confluence and sub-cultured in medium containing puromycin. The 3T3-L1 cells, HaCaT cells, MIN6 cells, and HepG2 cells, with stable expression of ARE-luciferase reporter, were used to identify ARE activators and inhibitors, and more particularly, Nrf2 inhibitors.

Assessed was the ability of a chemical compound (including drugs) to inhibit Nrf2- ARE activity under basal conditions (i.e., no added exogenous Nrf2 activator; absence of exogenous Nrf2 activator). For those chemical compounds identified as having Nrf2- ARE-inhibitory activity, also assessed was the ability of an Nrf2 activator to modulate the Nrf2-ARE-inhibitory activity of the compound (e.g., induce Nrf2 activation which then lessens, prevents or inhibits (in "modulating") Nrf2-ARE-inhibition). Briefly, chemical compounds were individually added to the cells, and incubated for 24 hours under basal conditions (no added Nrf2 activator), or with tBHQ-treated or sodium arsenite-treated cells (6 hour treatment, 5-10 μΜ iAs³⁺ or 50 μΜ tBHQ, each a known Nrf2 activator), and measured was luciferase activity, as compared to assay controls. The luciferase activity was measured by a commercially available luciferase reporter assay system according to the manufacturer's protocol. The luciferase activity was normalized to protein content or cell viability. To confirm an inhibitory effect on ARE as observed by a decrease in luciferase activity (as compared to the assay control), the compound was also tested for its ability to inhibit, in a concentration dependent manner, cell expression of multiple ARE- dependent genes, including one or more of H01 (Heme oxygenase 1), GCLC

(Glutamate— cysteine ligase catalytic subunit), GCLM (Glutamate— cysteine ligase modifier subunit), Nqo1 (NAD(P)H dehydrogenase [quinone] 1) and SRX (sulfiredoxin 1) by using real-time quantitative reverse transcription polymerase chain reaction (RT- qPCR) and commercially available primers. An inhibitory effect on expression of multiple ARE-dependent genes was used as an indication of inhibition of Nrf2-ARE activity.

First, cytotoxicity of isoniazid (a widely used antitubercular drug) in 3T3-L1 cells and HepG2 cells was determined by exposing the cells to various concentrations of isoniazid, ranging from 1 mM to 200 mM, for 24 hours, and subsequently determining cell viability by a commercially available MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. From this determination, non-cytotoxic

concentrations of isoniazid ranging from 1 mM to 50 mM were tested, as was an equal volume of cell culture without isoniazid ("Vehicle") for comparison, in the ARE-luciferase reporter assay. As shown in FIGs. 1 A and 2A, isoniazid ("INH") exhibited a

concentration-dependent inhibitory effect on ARE-luciferase activity in 3T3-L1 cells and HepG2 cells, respectively, under basal conditions. However, as shown in FIGs. 1 A and 2A, cells treated with both isoniazid and Nrf2 activator (iAs³⁺-treated) exhibited substantial ARE-luciferase activity, indication that the Nrf2 activator was capable of modulating the Nrf2-ARE-inhibitory effect of isoniazid. The Nrf2-ARE-inhibitory effect of isoniazid was also observed in HaCaT cells stably expressing the same ARE-luciferase reporter assay. The inhibitory activity of isoniazid was confirmed by decreased expression of multiple ARE-dependent genes, including GCLC (FIG. 1 B), NQ01 (FIG. 1 C) and H01 (FIG. 1 D, and FIG. 2B) under basal conditions. The ability of an Nrf2 activator (as illustrated by fert-butylhydroquinone (tBHQ) or iAs³⁺) to modulate the Nrf2-ARE-inhibitory activity of isoniazid was confirmed by an increased expression of multiple ARE-dependent genes, including GCLC (FIG. 1 B), NQ01 (FIG. 1 C) and H01 (FIG. 1 D, and FIG. 2B) in cells treated with both the Nrf2 activator and the compound having Nrf2-ARE-inhibitory activity, as compared to expression of the ARE-dependent genes in cells treated with the compound having Nrf2-ARE-inhibitor activity alone (e.g., under basal conditions).

Using these methods and the ARE-luciferase reporter assay described herein, another antitubercular agent ethionamide (ETH), in non-cytotoxic concentrations, also displayed a concentration-dependent inhibitory effect on ARE-luciferase activity under basal conditions in HepG2 cells (FIG. 3). Additionally, as shown in FIG. 3, HepG2 cells treated with both ethionamide (ETH) and an Nrf2 activator ("iAs³⁺") exhibited substantial ARE-luciferase activity, indicating that the Nrf2 activator was capable of modulating the Nrf2-ARE-inhibitory effect of the compound having Nrf2-ARE-inhibitory activity.

Confirmation of the Nrf2-ARE-inhibitory effect mediated by ETH was shown by the ability of ETH treatment to significantly decrease the expression of multiple ARE-dependent genes, including H01 (FIG. 4A), GCLM (FIG. 4B), and sulfiredoxin (SRX, FIG. 4C) in THP-1 cells under basal conditions. Likewise, confirmation of the ability of an Nrf2 activator to modulate the Nrf2-ARE-inhibitory activity of a compound having Nrf2-ARE- inhibitory activity (e.g., ETH) was shown by an increased expression of multiple ARE- dependent genes, including H01 (FIG. 4A), GCLM (FIG. 4B), and sulfiredoxin 1 (SRX, FIG. 4C) in THP-1 cells treated with both the Nrf2 activator and the drug having Nrf2- ARE-inhibitory activity, as compared to expression of the ARE-dependent genes in cells treated with the drug having Nrf2-ARE-inhibitor activity alone (e.g., under basal conditions). By using the same methods, it was demonstrated that (a) antitubercular agents other than isoniazid and ethionamide, including ethambutol dihydrochloride, rifampicin, and sparfloxacin, are Nrf2-ARE-inhibitors as demonstrated by the concentration- dependent inhibitory effect on ARE-luciferase activity under basal conditions (Table 1 ); (b) that an Nrf2 activator can modulate the Nrf2-ARE-inhibitory activity of a compound having Nrf2-ARE-inhibitory activity, including antitubercular agents; and (c) additionally, a number of other compounds represented by either Formula I, particularly heterocyclic compounds having a hydrazide side chain or carboxamide side chain, including 4- aminobenzoic hydrazide, aminopyrazine, 2-furoic hydrazide, cyclohexane-carboxamide, phenylhydrazine, phenylacetic hydrazide, pyrazinecarboxamide, and p-toluic hydrazide, were discovered inhibit Nrf2 activity as demonstrated by the concentration-dependent inhibitory effect on ARE-luciferase activity under basal conditions and iAs³⁺-treated or tBHQ-treated conditions (Table 1 ). As apparent from FIG. 5 showing the chemical structure of these newly discovered Nrf2 inhibitors, many of these compounds are represented by Formula I, particularly heterocyclic compounds having a hydrazide side chain or carboxamide side chain, suggesting a structure-function relationship between such chemical representation and the ability to inhibit Nrf2-ARE activity. Based on this discovery, and structure-function relationship, additional compounds represented by Formula I may be screened for activity for Nrf2 inhibitory activity. Illustrative examples of such compounds may include pyrrole-2 carboxamide, and pyrrole-2 hydrazide, or other compounds consisting of five- or six-membered rings that comprise a hydrazide side chain or carboxamide side chain. . As apparent from FIG. 5 showing the chemical structure of compounds that have Nrf2-ARE-inhibitory activity, these compounds comprise a formula of Formula I, an indication of a structure-function relationship between compounds of Formula I, and the ability to inhibit Nrf2-ARE activity.

Table 1

2- Amino-isonicotinamide 13538-42-6 1 -50 mM 1 -50 mM

Phenylacetic hydrazide 937-39-3 10 mM 1 -10 mM

Pyrazinecarboxamide 98-96-4 1 -10 mM 1 -10 mM

(including pyrazinamide)

EXAMPLE 2

In this Example, the mechanism of action by which compounds of Formula I inhibit Nrf2-ARE activity was further explored. Unexpectedly, compounds of Formula I inhibited Nrf2-ARE activity. It is known that Nrf2 undergoes acetylation to enhance the binding of Nrf2 to the ARE in promoting Nrf2-induced transcription of genes containing an ARE in their promoter region. Human NAD-dependent protein deacetylase SIRT1 has been shown to deacetylate Nrf2, resulting in decreased Nrf2-dependent gene transcription. Deacetylation of Nrf2 by SIRT1 resulted in primarily cytoplasmic localization of Nrf2 with a resultant decrease in Nrf2 transcription in the nucleus of the cell. The mechanisms by which NAD-dependent protein deacetylases can be activated (e.g., induction of NAD- dependent protein deacetylase activity) was examined. As previously described herein, changes in nicotinamide and NAD+ concentrations occur in response to altered cell physiology or stress factors. Nicotinamide is believed to be involved in a chemical competition between base-exchange and deacetylation involving NAD-dependent protein deacetylases, thereby inhibiting deacetylation. Isonicotinamide has been described as binding to the site at which nicotinamide binds in the enzyme complex, and inhibits the base exchange reaction, thereby increasing the deacetylation rate (e.g., relieves nicotinamide inhibition of deacetylation by acting as a nicotinamide antagonist). Thus, for example, SIRT1 is an NAD-dependent protein deacetylase that can be inhibited by nicotinamide, but activated by isonicotinamide. Exemplary compounds of Formula I, discovered to inhibit Nrf2-ARE activity as described herein, were compared to

isonicotinamide in a structural analysis. As shown below in Table 2, exemplary compounds of Formula I, discovered to inhibit Nrf2-ARE activity as described herein, are structurally similar to isonicotinamide, an indication that these compounds can also serve as nicotinamide antagonists in increasing the rate of deacetylation by NAD-dependent protein deacetylases.

Table 2

Based on the discovery of the structural likeness between compounds of Formula I discovered to have Nrf2-ARE inhibitory activity and that of isonicotinamide,

isonicotinamide was tested in the ARE-luciferase reporter assay using the methods described herein in Example 1 . As shown in Table 3, surprisingly, isonicotinamide shared the same ability (e.g., at the same concentrations) as isoniazid to inhibit Nrf2-ARE activity. The structural resemblances of compounds of Formula I discovered to have Nrf2-ARE inhibitory activity with isonicotinamide, the shared ability of isonicotinamide and compounds of Formula I to inhibit Nrf2-ARE activity, and that NAD-dependent protein deacetylase can act to deacetylate Nrf2 in reducing Nrf2-mediated transcription, are all indications that compounds of Formula I act as nicotinamide antagonists which increase NAD-dependent protein deacetylase activity (e.g., are activators of NAD-dependent protein deacetylase) that can subsequently modulate Nrf2-mediated transcription.

Table 3

In addition to human NAD-dependent protein deacetylase SIRT1 having the ability to regulate Nrf2-dependent gene transcription by deacetylation, SIRT1 also represses peroxisome proliferator-activated receptor gamma (PPARy), a protein known by those skilled in the art to comprise an amino acid sequence selected from SEQ ID NO:5 (PPARY isoform 1 , or "PPARYI "), or SEQ ID NO:6 (PPARY isoform 2, or "PPARY2"), and isoforms thereof. PPARyI is mostly expressed in hematopoietic cells; and PPARy2 is primarily expressed in adipose tissue, and hence is the most important isoform in adipogenesis. Thus, a Nicotinamide Antagonist, represented by Formula I, which activates NAD-dependent protein deacetylase SIRT1 should also repress PPARy.

PPARy is a transcription factor known to regulate adipogenesis; i.e., the differentiation of preadipocytes to adipocytes. Thus, repression of PPARy can be demonstrated by impairment of adipogenesis. It has been demonstrated that upregulation (activation) of SIRT1 attenuates adipogenesis, RNA interference of SIRT enhances adipogeneis, and these observations can be demonstrated in differentiation of 3T3-L1 preadipocytes. In this experiment, 3T3-L1 preadipocytes were treated with differentiation medium (e.g., cell culture medium with 10% fetal bovine serum containing 1 μΜ dexamethasone, 0.5 mM IBMX (3-isobutyl- 1 -methylxanthine), and 1 Mg/ml insulin). After 48 hours, the medium was changed to cell culture medium with 10% fetal bovine serum containing 1 μg/ml insulin, followed by additional 3 days of culture. Differentiation of preadipocytes to adipocytes was confirmed by Oil Red O staining (a fat soluble dye that stains neutral trigylcerides and lipids a red color), and detection was quantitated using spectral and software analysis using standard methods known in the art. Also, total RNA was isolated from the treated cells, and standard reverse transcription, quantitative real-time polymerase chain reaction was performed to amplify mRNA levels of PPARy isoforms PPARyI and PPARy2, followed by fluorescent detection for quantitation. For illustration purposes, isoniazid (in non-cytotoxic concentrations) was used as the Nicotinamide Antagonist, and was added to 4 hours prior to the addition of the differentiation medium, and kept in the medium during the differentiation process. A vehicle control was used in the assay as a negative control (i.e., would expect no effect on adipogenesis nor PPARy mRNA levels) for comparison purposes ("Control"). Oil Red O staining showed a decrease (approximately a one third reduction, as compared to the Control) in adipogenesis in the presence of 2.5 mM isoniazid. As shown in Figures 6 A and 6B, this correlates with an observed, similar reduction mediated by 2.5 mM isoniazid ("INH") in the level of mRNA (expressed as percent of Control) for both PPARyI (FIG. 6A) and PPARy2 (FIG. 6B). Similar studies using human adipose tissue-derived stem cells also showed that isoniazid and isonicotinamide each inhibited adipogenesis. These results showing repression of PPARy are further indications that isoniazid and other compounds represented by Formula I are activators of NAD-dependent protein deacetylase SIRT1 (See also FIG. 7). As noted above, a composition of the invention comprises a combination comprising an Nicotinamide Antagonist and a STAC. The unexpected discovery that compounds of Formula I can activate NAD-dependent protein deacetylase as

Nicotinamide Antgaonists provides a method by which an NAD-dependent protein deacetylase can now be dually activated by a Nicotinamide Antagonist represented by Formula I and a STAC, since a Nicotinamide Antagonist and a STAC each work by a different mechanism to activate NAD-dependent protein deacetylase, as compared to the other. In one aspect of the invention, a resultant effect of such combined or dual activation (i.e., activation by a Nicotinamide Antagonist, and activation by an STAC, each a separate class of activator based on different structure and function relationships) of NAD-dependent protein deacetylase may be a synergistic effect or an amplified effect (e.g., a greater effect than what is expected to be the total effect from addition of the separate effects of each class of activator of NAD-dependent protein deacetylase) which can potentiate the ability of such dually activated NAD-dependent protein deacetylase to regulate cell processes, physiological processes and disease by increased deacetylase activity. In this regard, in screening antitubercular drugs for Nrf2-ARE activity, it was noted that both rifampicin and sparfloxacin also demonstrate Nrf2-ARE inhibitory activity. Further, rifampicin demonstrates the ability to inhibit PPARy. Interestingly, rifampicin and sparfloxacin more resemble the structure of an STAC (compare, for example, to the chemical structure of fisetin, a known STAC; a heterocycle core structure including substitution with a plurality of oxygen or hydroxyl groups).

EXAMPLE 3

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are hold promise as therapeutic tools for regenerative medicine. hiPSCs are derived from human somatic cells; e.g, human somatic cells are genetically

reprogrammed to become embryonic-like stem cells known as hiPSCs. Once the hiPSCs have been derived from human somatic cells, hiPSCs are cultured for maintenance and expansion.

There are several examples of cell culture medium in which iPSCs are cultivated.

For example, ESSENTIAL 8 medium (Life Technologies) is a medium used in feeder free systems for the growth and expansion of hiPSCs. It contains eight components required for culturing hiPSCs: DMEM F-12 (Dulbecco's Modified Eagle's Medium F-12), L-ascorbic acid, selenium, transferrin, NaHC0₃, insulin, basic fibroblast growth factor 2 (FGF2), and transforming growth factor Beta 1 (TGF31 ). Another medium used for culturing iPSCs includes as components: DMEM F-12, L-ascorbic acid, selenium, transferrin, NaHC0₃, insulin, FGF2), TGF31 , glutathione, L-glutamine, defined lipids, thiamine, trace elements, β-mercaptoethanol, bovine serum albumin, pipecolic acid, LiCI, water, and gamma- aminobutyric acid (e.g., mTESR medium). Other commercially available cell culture media for growth and expansion of hiPSCs and hESCs includes STEMFIT medium,

STEMIUM medium, and PSGRO medium). From these, and other commercially available cell culture media, may be selected a defined medium or basal cell culture medium to which is added a composition comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, to produce a cell culture medium formulation of the invention.

In using a feeder-free system for cultivating stem cells such as iPSCs or ESCs, the cells are grown in the appropriate cell culture medium (examples mentioned above) on plates coated with extracellular matrix, or components thereof. The matrix is immobilized onto the surface of the culture vessel to provide a support surface for cell cultivation. It is recognized in the art that extracellular matrix is made up of components that include one or more of laminin, vitronectin, fibronectin, collagen, elastin, gelatin, hyaluronectin, chondronection, osteopontin, fibrillin, sialoprotein, tenascin, and other proteins or peptides. These components may be used alone or in combination, and in isolated and purified from natural sources (tissue) or recombinantly produced or synthesized. Typical examples of extracellular matrix used in cultivating iPSCs or ESCs include but are not limited to basement membrane extracts, recombinant human vitronectin or fragments thereof (vitronectin-N), laminin, collagen I, collagen IV, fibronectin or fragments thereof (commercially available as RETRONECTIN), and MATRIGEL. In using a feeder system for cultivating cells, the cells are grown in the appropriate cell culture medium on coated plates with a supporting layer of feeder cells such as fibroblasts (e.g., mouse embryonic fibroblasts, or human foreskin fibroblasts).

Recently, it was discovered that SIRT1 expression was induced in reprogramming of somatic cells into hiPSCs. Further, it was found that SIRT1 drives telomere elongation with passages of hiPSCs in culture. SIRT1 appears to slow the degradation of c-MYC which in turn promotes telomerase expression. As a result, SIRT1 expression in hiSPCs helps to maintain the genomic stability and integrity of hiPSCs, in maintaining the therapeutic usefulness of the hiPSCs. There are concerns about genomic integrity and stability of iPSCs, as well as hESCs, during their prolonged culture. Not only can genomic or epigenomic abnormalities compromise the cells' differentiation potential, but also can cause tumorigenesis in the recipients of iPSC-based therapies. Thus, provided as an aspect of the invention is a composition comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, to produce a cell culture medium formulation or cell culture supplement of the invention. The ceil culture medium formulation or cell culture supplement of the invention may be used to cultivate cells, such as stem cells, in vitro, which cultivated cells would benefit from activation of SIRT1 . in this aspect, the cell culture medium formulation comprises a Nicotinamide Antagonist, or a Nicotinamide

Antagonist and a STAC, in an amount effective to induce SIRT1 activity. With this aspect, provided is a cell culture medium formulation for cultivation of iPSCs or ESCs (e.g., hiPSCs and hESCs).

Also provided is use of a composition comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, in an amount effective to induce SIRT1 activity, as applied to one or more surfaces of a culture vessel. In this aspect, the composition can be used to coat a surface onto which stem cells are cultivated (e.g., the bottom surface of a microtiter plate or flask or other container, for growing cells). A number of plastics can be used in the construction of a culture vessel, particularly surfaces suitable for contacting cells. Most typically, polystyrene is used. These plastics typically can bind molecules based on hydrophobic interactions, or can be treated (e.g., by irradiation or treatment with plasma gas) to promote binding of molecules based on both hydrophobic and polar interactions. In each case, the molecules are adsorbed to the surface in a process called coating. In the coating process, the composition is provided in a physiologically acceptable carrier or buffered solution (examples of buffers include but are not limited to phosphate-buffered saline, and carbonate-bicarbonate buffer) in forming a coating solution. The coating solution is then contacted with a surface of the culture vessel to be coated, and the culture vessel is left to incubate for several hours to overnight at 4°C to 37°C. The coating solution is then removed, and a blocking buffer is added to ensure that binding sites remaining on the coated surface are covered. After removal of the blocking buffer, the coated culture vessel may then be used immediately, or dried and stored for later use. To coated culture vessel may be added a culture medium for cultivating stem cells, and the stem cells to be cultivated.

Provided is a method for activating NAD-dependent protein deacetylase, SIRT1 , in cells (e.g., stem cells such as human embryonic stems, or human induced pluripotent cells) in culture, comprising contacting the cells with a composition, cell culture medium formulation, or cell culture supplement of the invention. Also provided is a method for culturing mammalian stem cells in vitro using the composition, culture medium

formulations or cell culture supplement as disclosed herein, comprising the steps of (a) contacting the stem cells with the composition, cell culture media formulation or cell culture supplement of the invention, and (b) cultivating the stem cells in vitro under conditions suitable to support their cultivation in vitro (as known to those skilled in the art suitable conditions comprise factors such as the appropriate temperature and atmosphere for cultivation).

Conditions suitable to support cell cultivation in vitro using the methods, compositions, and formulations of the invention are known in the art. For example, such conditions typically comprise culturing cells at 37 °C in a suitable incubator. Typically such incubation is performed with 5% C0₂ in air atmosphere. Other culturing conditions, where appropriate, can be used. For example, the temperature used in cultivation may be in a range from about 30°C to about 40°C (e.g., 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C). The C0₂ concentration in the air atmosphere used for cultivation may be in a range from 1 % to 10%, but more preferably from 2% to 5%. The oxygen tension in the air atmosphere used for cultivation may be in a range from 5% to 20%, or any range derivable therefrom.

In another aspect, a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a

STAC, may comprise a cell culture supplement which is introduced into cell culture medium containing the cells being cultured (e.g., human embryonic stems, or human induced pluripotent stem cells). The composition may further comprise one or more physiologically acceptable carriers. With this aspect, provided is a cell culture reagent comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC; and may further comprise one or more physiologically acceptable carriers.

What is claimed is:

Claims

1. Use of a composition comprising a compound represented by Formula I in an effective amount to activate a NAD-dependent protein deacteylase, SIRT1 , wherein:

(a) Formula I is represented by Formula IA and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C;

R1 or R2 or R3 are each independently selected from H, (C₁-C₆)alkyl, CONH₂,

CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, CHCHCONHNH₂, or COCH₃; wherein at least one of R1 , R2, and R3 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or CHCHCONHNH₂; a pharmaceutically acceptable salt thereof, and the dashed lines represent optional double bonds;

as

Formula IB

wherein:

A is O or N;

B is N or C;

R2 is absent if B is NH;

if B is C, R2 is absent or selected from CH₃, CH₂CH₃, NH₂, or NHNH₂; a pharmaceutically acceptable salt thereof; and the dashed lines represent optional double bonds;

(iii) with the proviso that the compound of Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD); and

(b) the use is for contacting stem cells in vitro.

2. The use of claim 1 , wherein the composition comprises more than one compound represented by Formula I, a physiologically acceptable carrier, or a combination thereof.

3. The use of claim 1 , wherein the composition comprises a component included in, or supplement added to, a cell culture medium.

4. The use of claim 1 , wherein the composition is added to a culture vessel for culturing cells.

5. The use of claim 1 , wherein the composition further comprises a sirtuin-activating compound ("STAC").

6. The use of claim 5, wherein the STAC comprises a structure comprising a benzimidazole, imidazothiazole, quinoxaline, thiazolopyridine, stilbene, polyphenol, or methylxanthine.

7. The use of claim 1 , wherein the stem cells comprise cells selected from the group consisting of human embryomic stem cells, and human induced pluripotent stem cells.

8. A cell culture medium formulation comprising:

(a) a defined cell culture medium capable of cultivation of stem cells; and

(b) a Nicotinamide Antagonist comprising a compound represented by Formula I, wherein the Nicotinamide Antagonist is in an amount effective to activate a NAD-dependent protein deacteylase, SIRT1 , wherein Formula I is represented by Formula I A and Formula IB;

(i) with Formula IA as

Formula IA

wherein:

A is N or C;

B is N or C; R1 or R2 or R3 are each independently selected from H, (C₁-C₆)alkyl, CONH₂,

as

Formula IB

wherein:

A is O or N;

B is N or C;

R1 is selected from CONH₂, CONHNH₂, CSNH₂, S0₂NH₂, NH₂, NHNH₂, CHCHCONH₂, or

CHCHCONHNH₂;

R2 is absent if B is NH;

(iii) with the proviso that the compound represented by Formula I is not nicotinamide, isonicotinamide, or nicotinamide adenine dinucleotide (NAD).

9. The cell culture medium formulation of claim 8, wherein the composition comprises more than one compound represented by Formula I.

10. The cell culture medium formulation of claim 8, further comprising a STAC selected from the group consisting of a benzimidazole, imidazothiazole, quinoxaline,

thiazolopyridine, stilbene, polyphenol, or methylxanthine.

1 1 . An in vitro cell culture composition comprising stem cells, a defined extracellular matrix, and a cell culture medium formulation according to claim 8.

12. An in ^'fro cell culture composition comprising stem cells, a defined extracellular matrix, and a cell culture medium formulation according to claim 9.

13. An in vitro cell culture composition comprising stem cells, a defined extracellular matrix, and a cell culture medium formulation according to claim 10.

14. The cell culture composition of claim 8, wherein the stem cells comprise cells selected from the group consisting of human embryonic stem cells, and human induced pluripotent stem cells.

15. The cell culture composition of claim 9, wherein the stem cells comprise cells selected from the group consisting of human embryonic stem cells, and human induced pluripotent stem cells.

16. The cell culture composition of claim 10, wherein the stem cells comprise cells selected from the group consisting of human embryonic stem cells, and human induced pluripotent stem cells.

17. A method for activating NAD-dependent protein deacetylase, SIRT1 , in stem cells in culture in vitro, comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC, in an amount effective to induce SIRT1 activity

18. The method of claim 17, comprising more than one Nicotinamide Antagonist, a physiologically acceptable carrier, or a combination thereof.

19. The method of claim 17, wherein the stem cells comprise cells selected from the group consisting of human embryonic stem cells, and human induced pluripotent stem cells.

20. A cell culture reagent comprising a Nicotinamide Antagonist, or a Nicotinamide Antagonist and a STAC.

21 . The cell culture reagent of claim 13, comprising more than one Nicotinamide Antagonist, a physiologically acceptable carrier, or a combination thereof.

22. A method for culturing mammalian stem cells in vitro, comprising the steps of (a) contacting the stem cells with a cell culture media formulation according to claim 8, and (b) cultivating the stem cells under conditions suitable to support their cultivation in vitro.

23. A method for culturing mammalian stem cells in vitro, comprising the steps of

(a) contacting the stem cells with a cell culture media formulation according to claim 9, and

(b) cultivating the stem cells under conditions suitable to support their cultivation in vitro.

24. A method for culturing mammalian stem cells in vitro, comprising the steps of

(a) contacting the stem cells with a cell culture media formulation according to claim 10, and