METHODS AND COMPOSITIONS FOR PREVENTION OF ANAPHYLAXIS

Abstract

Embodiments of the invention include methods of preventing and/or reducing the risk or severity of an allergic reaction in an individual. In some embodiments, particular small molecules are employed for prevention and/or reduction in the risk or severity of anaphylaxis. In at least particular cases, the small molecules are inhibitors of STAT3. In some cases, the small molecule comprises N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide.

Description

TECHNICAL FIELD

The present invention generally concerns at least the fields of cell biology, molecular biology, and medicine.

BACKGROUND

Anaphylaxis is a systemic hyperacute allergic reaction that causes more than 1,500 deaths per year in the United States. It is associated with intense vasodilatation and bronchoconstriction, severe laryngeal edema, drop of cardiac pressure, and hypothermia.

Anaphylaxis can occur in response to almost any foreign substance, although usual triggers include insect venom, foods, medication, and in some cases semen, latex, hormonal changes, or food additives. Physical factors, including exercise or temperature (either hot or cold) may also act as triggers because of their direct effects on mast cells. Exercise induced events are frequently associated with the ingestion of certain foods. In some cases, the cause is idiopathic.

The present disclosure satisfies a need in the art to provide novel compounds and methods for treating and/or preventing anaphylaxis or any mast-cell mediated allergic disorder in individuals.

SUMMARY

Embodiments of the disclosure include methods and compositions for the prevention and/or reduction in the risk or severity of an allergic reaction. In alternative embodiments, one or more compositions herein are useful for the treatment of allergic reaction. In embodiments of the invention, there are methods and compositions for the prevention and/or reduction in the risk or severity of any medical condition associated with mast cell degranulation. In specific embodiments, there are methods and compositions for the prevention and/or reduction in the risk or severity of anaphylaxis, anaphylactic shock, allergic rhinitis (hay fever), urticaria (hives), food allergy, drug allergy, hymenoptera allerga, bronchial constriction, asthma, eczema, and so forth.

Embodiments of the disclosure include methods and/or compositions for the prevention of an allergic reaction in an individual known to have the allergy, suspected of having the allergy, or at risk for having the allergy. The compositions include small molecules and functional derivatives as described herein. In some embodiments, the individual is receiving an additional therapy for the prevention and/or treatment of allergic reaction, including anaphylaxis.

In at least certain embodiments, an individual receives an effective amount of the composition for the inhibition of mast cell activity, such as the inhibition of mast cell degranulation.

In at least certain embodiments, an individual receives an effective amount of the composition as a preventative indication. The composition may be administered continually through the life of the patient following a realization of a need thereof. The composition may be administered only in anticipation of being in an environment that puts the individual at risk for being in need thereof. For example, the individual may be susceptible for allergic reaction (including anaphylaxis) from a particular food allergen but may be administered the composition prior to consumption of the food (days, hours, or minutes before consumption, for example). An individual with a susceptibility to allergic reaction to insect stings may be administered the composition prior to exposure to an environment or situation where the individual is at risk of being stung by the insect. An individual may be susceptible to allergic reaction because the allergen is only present in an environment of the individual in a seasonal pattern, and in such cases the individual may be administered the composition prior to and/or during the season.

In embodiments of the disclosure, an individual is given more than one dose of one or more compositions described herein or functional derivatives thereof. The dosing regimen may be separated in time by minutes, hours, days, months or years.

An individual in need thereof may be an individual that has at least one symptom of allergic reaction, is susceptible to having allergic reaction, has a biological marker for having allergic reaction but never been exposed to the allergen in a natural environment, or has had allergic reaction in the past. In certain cases, the individual has a family history of allergic reaction, including a family history of anaphylaxis; in such cases, the individual may or may not be known to have allergic reaction, including anaphylaxis.

Delivery of the composition of the invention may occur by any suitable route, including systemic or local, although in specific embodiments, the delivery route is oral, intravenous, topical, subcutaneous, intraarterial, intraperitoneal, buccal, and so forth, for example.

In particular embodiments, there is a method of inhibiting mast cell degranulation comprising exposing the mast cell(s) to one or more of the compositions disclosed herein or functional derivatives thereof. The mast cell(s) may be in vitro, ex vivo, or in vivo. The inhibition may be complete or may be reduced compared to the cells in the absence of exposure to the composition. In particular embodiments, the mast cells are in vivo in an individual known to have allergic reaction, at risk for allergic reaction, or that is susceptible to allergic reaction.

In some embodiments of the invention, the methods and/or compositions of the invention are useful for preventing and/or reducing the risk of or severity of allergic reaction (such as anaphylaxis), and in specific cases such action occurs by inhibiting Stat3 and/or Stat1 activity. In some embodiments of the invention, the methods and/or compositions of the invention are useful for preventing and/or reducing the risk of or severity of allergic reaction (such as anaphylaxis), and in specific cases such action occurs by inhibiting or reducing mast cell degranulation. In certain embodiments, the compositions inhibit Stat3 but fail to inhibit Stat1. In some embodiments, compounds of the invention interact with the Stat3 SH2 domain, competitively inhibit recombinant Stat3 binding to its immobilized pY-peptide ligand, and/or inhibit IL-6-mediated tyrosine phosphorylation of Stat3, for example. In particular embodiments, the compositions of the invention fulfills the criteria of interaction analysis (CIA): 1) global minimum energy score ≤−30; 2) formation of a salt-bridge and/or H-bond network within the pY-residue binding site of Stat3; and/or 3) formation of a H-bond with or blocking access to the amide hydrogen of E638 of Stat3, for example. In some embodiments, the composition(s) interacts with a hydrophobic binding pocket with the Stat3 SH2 domain. In some embodiments, the composition(s) inhibit the binding of Stat3 to its cognate phosphopeptide ligand. In some embodiments, the composition(s) inhibit cytokine-mediated Stat3 phosphorylation within cells. In some embodiments, the composition(s) inhibit nuclear translocation of Stat3 within cells.

In a specific embodiment of the invention, there is a method of preventing and/or reducing the risk or severity of allergic reaction (such as anaphylaxis) in an individual comprising delivering to the individual a therapeutically effective amount of a compound selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide (which may be referred to as Cpd 188-9), N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, a functionally active derivative thereof, and a mixture thereof.

In a specific embodiment of the invention, there is a method of preventing and/or reducing the risk or severity of allergic reaction (such as anaphylaxis) in an individual comprising delivering to the individual a therapeutically effective amount of a compound selected from the group consisting of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoic acid; 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid; 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid; methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate; 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid; a functionally active derivative thereof; and a mixture thereof. In a specific embodiment, any of the compounds disclosed herein are suitable to treat and/or prevent allergic reaction, for example.

In another embodiment, the inhibitor comprises the general formula:

wherein R₁ and R₂ may be the same or different and are selected from the group consisting of hydrogen, carbon, sulfur, nitrogen, oxygen, flourine, chlorine, bromine, iodine, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises the general formula:

wherein R₁, and R₃ may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flouring, chlorine, bromine, iodine, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives; and R₂ and R₄ may be the same or different and are selected from the group consisting of hydrogen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises the general formula:

wherein R₁, R₂, and R₃ may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

In other embodiments of the invention, there are methods of treating anaphylaxis in an individual wherein the composition(s) is an inhibitor of any members of the STAT protein family, including STAT1. STAT2. STAT3, STAT4, STAT5 (STAT5A and STAT5B), or STAT6, for example.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IG demonstrates inhibition of Stat3 binding to immobilized phosphopeptide ligand by compounds. Binding of recombinant Stat3 (500 nM) to a BiaCore sensor chip coated with a phosphododecapeptide based on the amino acid sequence surrounding Y1068 within the EGFR was measured in real time by SPR (Response Units) in the absence (0 μM) or presence of increasing concentrations (0.1 to 1,000 μM) of Cpd3 (FIG. 1A), Cpd30 (FIG. 1B), Cpd188 (FIG. 1C), Cpd3-2 (FIG. 1D), Cpd3-7 (FIG. 1E) and Cpd30-12 (FIG. 1F). Data shown are representative of 2 or more experiments. The equilibrium binding levels obtained in the absence or presence of compounds were normalized (response obtained in the presence of compound÷the response obtained in the absence of compound×100), plotted against the log concentration (nM) of the compounds (FIG. 1G). The experimental points fit to a competitive binding curve that uses a four-parameter logistic equation (see exemplary methods for details). These curves were used to calculate ICs % (Table 4).

FIGS. 2A-2F demonstrates inhibition of IL-6-mediated activation of Stat3 by compounds. HepG2 cells were pretreated with DMSO alone or DMSO containing Cpd3 (FIG. 2A), Cpd188 (FIG. 2B), Cpd30 (FIG. 2C), Cpd3-2 (FIG. 2D), Cpd3-7 (FIG. 2E) or Cpd30-12 (FIG. 2F) at the indicated concentration for 60 min. Cells were then stimulated with IL-6 (30 ng/ml) for 30 min. Protein extracts of cells were separated by SDS-PAGE, blotted and developed serially with antibodies to pStat3, total Stat3 and β-actin. Blots were stripped between each antibody probing. The bands intensities of immunoblot were quantified by densitometry. The value of each pStat3 band's intensity was divided by each corresponding value of total Stat3 band intensity and the results normalized to the DMSO-treated control value and plotted as a function of the log compound concentration. The best-fit curves were generated based on 4 Parameter Logistic Model/Dose Response One Site/XLfit 4.2, IDBS. Each panel is representative of 3 or more experiments.

FIGS. 3A-3F provides exemplary chemical formulas and names of compounds. The chemical formulas and names are indicated for Cpd3 (FIG. 3A), Cpd30 (FIG. 3B), Cpd188 (FIG. 3C), Cpd3-2 (FIG. 3D), Cpd3-7 (FIG. 3E) and Cpd30-12 (FIG. 3F).

FIG. 4 shows effect of compounds on Stat1 activation. HepG2 cells were pretreated with DMSO alone or DMSO containing each of the compounds at a concentration of 300 μM for 60 min. Cells were then stimulated with IFN-γ (30 ng/ml) for 30 min. Protein extracts of cells were separated by SDS-PAGE and immunoblotted serially with antibodies to pStat1, total Stat1 and β-actin. Blots were stripped between each immunoblotting. The results shown are representative of 2 or more experiments.

FIGS. 5A-5C provides comparisons of the Stat3 and Stat1 SH2 domain sequences, 3-D structures and van der Waals energies of compound binding. Sequence alignment of Stat3 and Stat1 SH2 domains is shown in FIG. 5A. The residues that bind the pY residue are highlighted in and pointed to by a solid arrow, the residue (E638) that binds to the +3 residue highlighted and pointed to by a dotted arrow and Loop_βC-βD and Loop_αB-αC, which comprise the hydrophobic binding site consisting, are highlighted and pointed to by dot-dashed and dashed arrows, respectively. FIG. 5B shows an overlay of a tube-and-fog van der Waals surface model of the Stat3 SH2 domain and a tube-and-fog van der Waals surface model of the Stat1 SH2. The residues of the Stat3 SH2 domain represents Loop_βC-βD are highlighted and shown by dotted circles and the residues represent Loop_αB-αC are highlighted and shown by a dotted-dashed circle; the corresponding loop residues within the Stat1 SH2 domain are shown in a light fog surrounding the circles. This overlay is shown bound by Cpd3-7 as it would bind to the Stat3 SH2 domain. The van der Waals energy of each compound bound to the Stat1 SH2 domain or the Stat3 SH2 domain was calculated, normalized to the value for Stat1 and depicted in FIG. 5C.

FIGS. 6A-6F shows a computer model of each compound bound by the Stat3 SH2 domain. The results of computer docking to the Stat3 SH2 domain is shown for Cpd3 (FIG. 6A), Cpd30 (FIG. 6B), Cpd188 (FIG. 6C), Cpd3-2 (FIG. 6D), Cpd3-7 (FIG. 6E) and Cpd30-12 (FIG. 6F). The image on the left of each panel shows the compound binding to a spacefilling model of the Stat3 SH2 domain. The pY-residue binding site is represented by dashed circle, the +3 residue binding site is represented by a solid circle, loop Loop_βC-βD is represented by dotted circle and loop Loop_αB-αC is represented by dot-dashed circle. Residues R609 and K591 critical for binding pY are shown within a dashed circle, residue E638 that binds the +3 residue shown within a solid circle and the hydrophobic binding site consisting of Loop_βC-βD and LoopαB-αC is shown within a dash-dot and dotted circle, respectively. The image on the right side of each panel is a closer view of this interaction with hydrogen bonds indicated by dotted lines. In FIG. 6A the negatively charged benzoic acid moiety of Cpd3 has electrostatic interactions with the positively-charge pYresidue binding site consisting mainly of the guanidinium cation group of R609 and the basic ammonium group of K591. The benzoic acid group also forms a hydrogen-bond network consisting of double H-bonds between the carboxylic oxygen and the ammonium hydrogen of R609 and the amide hydrogen of E612. H-bond formation also occurs between the benzoic acid carbonyl oxygen and the side chain hydroxyl hydrogen of Serine 611. Within the +3 residue-binding site, the oxygen atom of 1,4-benzodioxin forms a hydrogen bond with the amide hydrogen of E638. In addition, the 2,3-dihydro-1,4-benzodioxin of Cpd3 interacts with the loops forming the hydrophobic binding site. In FIG. 6B the carboxylic terminus of the benzoic acid moiety of Cpd30, which is negatively charged under physiological conditions, forms a salt bridge with the guanidinium group of R609 within the pYresidue binding site. Within the +3 residue-binding site, the oxygen of the thiazolidin group forms a H-bond with the peptide backbone amide hydrogen of E638. In addition, the thiazolidin moiety plunges into the hydrophobic binding site. In FIG. 6C there is an electrostatic interaction between the (carboxymethyl) thio moiety of Cpd188 carrying a negative charge and the pY-residue binding site consisting of R609 and K591 carrying positive charge under physiological conditions. There are H-bonds between the hydroxyloxygen of the (carboxymethyl) thio group of Cpd188 and the guanidinium hydrogen of R609, between the hydroxyl-oxygen of the (carboxymethyl) thio group and the backbone amide hydrogen of E612, and between the carboxyl-oxygen of the (carboxymethyl) thio group of Cpd188 and the hydroxyl-hydrogen of S611. Within the +3 residue-binding site, there is a H-bond between the hydroxyl-oxygen of benzoic acid group of Cpd188 and the amide-hydrogen of E638. In addition, the benzoic acid group extends and interacts with the hydrophobic binding site. In FIG. 6D the benzoic acid group of Cpd3-2 has significant electrostatic interactions with the pY-residue binding site pocket, mainly contributed by R609 and K591, and forms two H bonds; the carboxylic oxygen of the benzoic acid group binds the guanidinium hydrogen of R609, and the carbonyl oxygen of the benzoic acid group binds to the carbonyl hydrogen of S611. Within the +3 residue-binding site, oxygen within the 1,3-dihydro-2H-inden-2-ylidene group forms an H bond to the backbone amide-hydrogen of E638. In addition, the 1,3-dihydro-2H-inden-2-ylidene group plunges into the hydrophobic binding site. In FIG. 6E H-bonds are formed between the carbonyl-oxygen of the methyl 4-benzoate moiety of Cpd 3-7 and the side chain guanidinium of R609 and between the methoxy-oxygen and the hydrogen of the ammonium terminus of K591. The (2-methoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen group of Cpd3-7 blocks access to the amide hydrogen of E638 within the +3 residue-binding site. In addition, this group plunges into the hydrophobic binding site. In FIG. 6F there are electrostatic interactions between the benzoic acid derivative group of Cpd30-12 and R609 and 591 within the pY-residue binding site. Also, H-bonds are formed between the hydroxyl-oxygen of Cpd30-12 and the guanidinium-hydrogen of R609, between the carboxyl-oxygen of Cpd30-12 and the hydroxyl-hydrogen of S611 and between the furyl group of Cpd30-12 and the hydrogen of ammonium of K591. The 1,3-diethyl-4, 6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene groups blocks access to the +3 residue binding site; however, it extends into the groove between the pY-residue binding site and LoopβC-βD, while sparing the hydrophobic binding site.

FIGS. 7A-7B shows inhibition of cytoplasmic-to-nuclear translocation of Stat3 assessed by confocal and high-throughput fluorescence microscopy. In FIG. 7A. MEF/GFP-Stat3 cells grown on coverslips were pretreated with DMSO that either contained (row four) or did not contain (row three) Cpd3 (300 μM) for 60 min before being stimulated without (row one) or with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes (rows two, three and four). Coverslips were examined by confocal fluorescent microscopy using filters to detect GFP (column one), DAPI (column two) or both (merge; column three). In FIG. 7B, MEF-GFP-Stat3 cells were grown in 96-well plates with optical glass bottoms and pretreated with the indicated compound at the indicated concentrations in quadruplicate for 1 hour then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes. Cells were fixed and the plates were examined by high-throughput microscopy to determine the fluorescence intensity in the nucleus (FLIN) and the % ΔFLIN_Max was calculated as described in Example 1. Data shown are mean±SD and are representative of 2 or more studies. Best-fit curves were generated based on 4 Parameter Logistic Model/Dose Response One Site/XLfit 4.2. IDBS and were used to calculate IC₅₀ (Table 1).

FIG. 8 demonstrates inhibition of Stat3 DNA binding by compounds. Electrophoretic mobility shift assays were performed using whole-cell extracts prepared from HepG2 cells without and with stimulation with IL-6 (30 ng/ml) for 30 min. Protein (20 μg) was incubated with radiolabeled duplex oligonucleotide (hSIE) and DMSO without or with the indicated compounds (300 uM) for 60 minutes at 37° C. then separated by PAGE. The gel was dried and autoradiographed; the portion of the gel corresponding to the Stat3-bound hSIE band is shown. Data shown are representative of 2 studies.

FIG. 9 shows Cpd3, Cpd30 and Cpd188 and the hydrophobicity or hydrophilicity of the surface of the molecule. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces.

FIG. 10 illustrates exemplary compound 3 (Cpd3). The top-left picture of FIG. 11 shows Cpd3 docked into Stat3 and the interaction between Cpd3 and the surface of the protein and derivatives of Cpd3 that can fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one or more functional derivatives. The hydrophobic/hydrophilic surfaces of Cpd3 are also demonstrated on the top-right picture. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. R₁ and R2 could be identical or different and may comprise hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.

FIG. 11 illustrates exemplary compound 30 (Cpd30). The top-left picture of FIG. 12 shows Cpd30 docked into Stat3 and the interaction between Cpd30 and the surface of the protein, and derivatives of Cpd30 that fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one or more functional derivatives. The hydrophobic/hydrophilic surfaces of Cpd30 are also demonstrated on the top-right picture. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. 2-D structure of Cpd30 shown on the bottom picture. R₁, R₂ R₃ and R₄ could identical or different and may comprise be hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.

FIG. 12 illustrates exemplary compound 188 (Cpd188). The top picture of FIG. 12 shows Cpd188 docked into Stat3 SH2 domain and the interaction between Cpd188 and the surface of the protein, and derivatives of Cpd188 that fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one ore more functional derivative. The hydrophobic/hydrophilic surfaces of Cpd188 are also demonstrated on the left picture on the bottom. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. Shown on the right bottom picture, R₁ and R₂ could be identical or different and may comprise hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.

FIG. 13 illustrates schematic diagrams of Stat1 and Stat3.

FIG. 14 demonstrates that SPR IC₅₀ of 2nd generation Stat3 chemical probes is inversely correlated with 3-D pharmacophore score.

FIG. 15, shows SPR IC₅₀ and AML apoptosis EC₅₀ of parent Cpd188 and two 2nd generation 188-like Stat3 chemical probes.

FIG. 16 provides an illustration of structure-activity relationships of 38 Cpd188-like, 2nd generation Stat3 probes.

FIG. 17 shows an exemplary modification scheme for 3rd generation Stat3 probe development using Cpd188-15 as a scaffold.

FIG. 18 provides illustration of the electrostatic surface of Stat3 SH2 domain (positive area in blue, neutral in white and negative in red in a color figure) and 20 docking poses of 5 (R=CH₂PO₃²⁻), showing strong interactions between phosphonate groups (in purple and red) and K591/R609.

FIGS. 19A and 19B. FIG. 19A shows physician diagnosed food allergies in healthy volunteers, AD-HIES, and atopic control patients were determined by interview. FIG. 19B demonstrates incidence of physician diagnosed anaphylaxis in AD-HIES and atopic control patients. Significance determined by a two-tailed Chi-squared test.

FIGS. 20A and 20B. FIG. 20A shows that mast cell degranulation was measured by FcεRI crosslinking and subsequent β-hexosaminidase release in LAD2 cells transduced with five different shRNAs against STAT3. Data representative of two independent experiments. LAD2=unstimulated control, LAD+=FcεRI crosslinking. FIG. 20B shows that mast cell degranulation was measured by FcεRI crosslinking and subsequent β-hexosaminidase release in primary human mast cells transduced with two different shRNAs against STAT3. HuMC=unstimulated control, HuMC+=FcεRI crosslinking.

FIG. 21 demonstrates correlation between STAT23 knockdown and inhibition of mast cell degranulation (r²=0.9463).

FIG. 22 demonstrates effective treatment in an anaphylaxis model using Cpd188-9.

FIG. 23 provides a dose response curve using different dosages of Cpd188-9 in an anaphylaxis model utilizing beta-hexosaminidase (% release) as a measure of mas cell degranulation.

FIG. 24 shows that systemic anaphylaxis was prevented in vivo with an exemplary STAT3 inhibitor.

FIG. 25 demonstrates that peripheral and central vascular leakage is decreased by Cpd 188-9.

FIG. 26 illustrates the effect of Cpd188-9 is not because of a decreased mast cell degranulation in vivo.

FIG. 27 demonstrates the effect of Cpd 188-9 on Ag-induced degranulation in murine mast cells.

FIG. 28 illustrates an exemplary transwell permeability assay.

FIG. 29 shows that Cpd188-9 pretreated (as an example, for 7 days) HUVECS resistant to histamine-induced permeability.

FIG. 30 shows HIES mouse is resistant to anaphylaxis (Siegel et al., JACI, 2013).

FIG. 31 demonstrates STAT3 mutant (HIES6) HUVECS resistant to histamine-induced permeability.

DETAILED DESCRIPTION OF THE INVENTION

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

In some embodiments, there is a method of preventing, and/or reducing the risk or severity of allergic reaction (such as anaphylaxis) in an individual, comprising delivering to the individual one or more particular compounds. In some embodiments, the compound(s) is a STAT3 inhibitor. In certain embodiments the compound(s) is not a STAT3 inhibitor. In particular cases, the compound(s) is a STAT1 inhibitor, but in particular cases it is not a STAT1 inhibitor. In certain aspects, there are some compounds that are both STAT3 and STAT1 inhibitors or is neither a STAT3 or STAT1 inhibitor. In some cases, the composition is a mast cell inhibitor, including a mast cell degranulation inhibitor.

In certain embodiments of the invention, there is a compound for use in the prevention and/or reduction in risk or severity of allergic reaction (such as anaphylaxis), wherein the compound is selected from the group consisting of N-(1,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, or a combination thereof, a functionally active derivative, and a mixture thereof.

In certain embodiments of the invention, there is a compound for use in the prevention and/or reduction in risk of allergic reaction (such as anaphylaxis), wherein the compound is selected from the group consisting of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoic acid; 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl] benzoic acid; 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid; methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate; 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid; a functionally active derivative and a mixture thereof. In a specific embodiment of the invention, the composition is a Stat3 inhibitor but does not inhibit Stat1. The composition may be a mast cell degranulation inhibitor.

In a specific embodiment of the invention, the composition is delivered in vivo in a mammal. In another embodiment the mammal is a human. In another specific embodiment the human is known to have anaphylaxis, is suspected of having anaphylaxis, or is at risk for developing anaphylaxis. In another embodiment, the human is known to have anaphylaxis and is receiving an additional therapy for the anaphylaxis. Composition(s) of the disclosure prevent and/or reduce the risk or severity of allergic reaction, in particular embodiments.

I. Definitions

As used herein the specification. “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Still further, the terms “having”, “including”, “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

The term “inhibitor” as used herein refers to one or more molecules that interfere at least in part with the activity of Stat3 to perform one or more activities, including the ability of Stat3 to bind to a molecule and/or the ability to be phosphorylated. In alternative embodiments, an inhibitor reduces the level of degranulation of mast cells, which may be measured in vitro by % release of beta-hexosaminidase or other mast cell mediators such as cytokines, histamine, leukotrienes, etc. The level of degranulation of mast cells may be measured in vivo in a multitude of allergy and anaphylaxis models that primarily measure core temperature reductions with acute challenge, vascular permeability, inflammation, or systemic mast cell mediators, such as histamine or tryptase.

The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention that is effective for producing some desired therapeutic effect, e.g., treating (i.e., preventing and/or ameliorating) allergic reaction in a subject, or inhibiting protein-protein interactions mediated by an SH2 domain in a subject, at a reasonable benefit/risk ratio applicable to any medical treatment. In one embodiment, the therapeutically effective amount is enough to reduce or eliminate at least one symptom. One of skill in the art recognizes that an amount may be considered therapeutically effective even if the allergic reaction is not totally eradicated but improved partially. For example, a symptom from the allergic reaction may be partially reduced or completed eliminated, and so forth.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “at risk for having allergic reaction” as used herein refers to an individual that has had an allergic reaction before, has one or more family members with allergic reaction history, or is a child.

As used herein. “binding affinity” refers to the strength of an interaction between two entities, such as a protein-protein interaction. Binding affinity is sometimes referred to as the K_a, or association constant, which describes the likelihood of the two separate entities to be in the bound state. Generally, the association constant is determined by a variety of methods in which two separate entities are mixed together, the unbound portion is separated from the bound portion, and concentrations of unbound and bound are measured. One of skill in the art realizes that there are a variety of methods for measuring association constants. For example, the unbound and bound portions may be separated from one another through adsorption, precipitation, gel filtration, dialysis, or centrifugation, for example. The measurement of the concentrations of bound and unbound portions may be accomplished, for example, by measuring radioactivity or fluorescence, for example. K_a also can be inferred indirectly through determination of the K_i or inhibitory constant. Determination of the K_i can be made several ways for example by measuring the K_a of STAT3 binding to its phosphopeptide ligand within the EGFR at position Y1068 and by measuring the concentration of a molecule that reduces binding of STAT3 by 50%. In certain embodiments of the invention, the binding affinity of a Stat3 inhibitor for the SH2 domain of Stat3 is similar to or greater than the affinity of the compounds listed herein.

The term “domain” as used herein refers to a subsection of a polypeptide that possesses a unique structural and/or functional characteristic; typically, this characteristic is similar across diverse polypeptides. The subsection typically comprises contiguous amino acids, although it may also comprise amino acids that act in concert or that are in close proximity due to folding or other configurations. An example of a protein domain is the Src homology 2 (SH2) domain of Stat3. The term “SH2 domain” is art-recognized, and, as used herein, refers to a protein domain involved in protein-protein interactions, such as a domain within the Src tyrosine kinase that regulates kinase activity. The invention contemplates modulation of activity, such as activity dependent upon protein-protein interactions, mediated by SH2 domains of proteins (e.g., tyrosine kinases such as Src) or proteins involved with transmission of a tyrosine kinase signal in organisms including mammals, such as humans.

As used herein, a “mammal” is an appropriate subject for the method of the present invention. A mammal may be any member of the higher vertebrate class Mammalia, including humans; characterized by live birth, body hair, and mammary glands in the female that secrete milk for feeding the young. Additionally, mammals are characterized by their ability to maintain a constant body temperature despite changing climatic conditions. Examples of mammals are humans, cats, dogs, cows, mice, rats, and chimpanzees. Mammals may be referred to as “patients” or “subjects” or “individuals”.

II. General Embodiments

General embodiments include one or more compositions for the prevention of allergic reaction and methods of their use. An individual in need of allergic reaction prevention, including reduction in the severity of at least one symptom of allergic reaction, is provided with an effective amount of one or more compositions as disclosed herein. Although any composition disclosed herein may be suitable, in specific embodiments the composition is N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide (Cpd 188-9) or a functional derivative thereof.

In some cases an individual prevents the allergic reaction or reduces the severity of the allergic reaction with one or more compositions as disclosed herein by intaking the composition routinely, such as routinely after having a first allergic reaction or after identifying the risk of having an allergic reaction (such as by a standard allergy test, for example). The term “routinely” may be described as a regular course of procedure, such as once or more than once daily, biweekly, weekly, monthly, and so forth, for example.

In some cases, an individual prevents the allergic reaction or reduces the severity of the allergic reaction with one or more compositions as disclosed herein by intaking the composition periodically, such as periodically after having a first allergic reaction or after identifying the risk of having an allergic reaction (such as by a standard allergy test, for example). The period may be one or more seasons of the year. The period may be one or more periods of time for one or more increased allergens in an environment, such as during pollination of one or more types of plants, for example.

In some cases, an individual prevents the allergic reaction or reduces the severity of the allergic reaction with one or more compositions as disclosed herein by intaking the composition prior to an event or environment or condition where the individual is likely to be or known to be exposed to the allergen. For example, the individual may be administered the composition prior to consumption of a particular food allergen, prior to close proximity to or exposure to a particular plant allergen, prior to exposure to an environment having stinging insects, prior to an exposure to latex, prior to sexual intercourse, and so forth.

In some cases, an individual may intake the composition routinely but may take an increased dosage of the composition prior to an event or environment or condition where the individual is likely to be or known to be exposed to the allergen.

In some cases, an individual may intake the composition periodically but may take an increased dosage of the composition prior to an event or environment or condition where the individual is likely to be or known to be exposed to the allergen.

In certain cases, an individual is receiving, has received, and/or will receive an effective dosage of one or more compositions of the disclosure, but the individual will also receive another medical composition for the allergic reaction. In some cases, the other medical composition may be one or more doses of an antihistamine, steroids epinephrine, or a combination thereof, for example.

In cases wherein an individual has at least one symptom of allergic reaction, the individual may be provided with an effective amount of one or more compositions of the invention prior to and/or after the appearance of allergic reaction. When the individual is provided one of more compositions prior to the appearance of allergic reaction, the onset of allergic reaction may be delayed or completely inhibited and/or the severity of the allergic reaction may be reduced, compared to the condition of the individual without having received the composition(s), for example.

In particular embodiments, an individual has been diagnosed with allergic reaction, and methods of the invention may include steps of diagnosing of the allergic reaction in the individual. An individual may be tested for allergic reaction by standard means in the art. For example, one can perform skin tests (where a small amount of a suspected allergen is placed on or below the skin to see if a reaction develops) and/or blood tests for antibodies to the allergen, such as using ELISA to measure IgE. Such test may be performed before or after it is known that the individual has one or more allergies or that the individual has had an allergic reaction.

III. Allergic Reaction

Embodiments of the invention concern compositions and methods for treatment and/or prevention or reduction in the risk of any kind of allergic reaction. An allergic reaction is a hypersensitivity disorder of the immune system in which a person's immune system reacts to a normally harmless substance (an allergen), such as from the environment. Allergic reactions are characterized by excessive activation of mast cells and basophils by Immunoglobulin E (IgE), and the reaction results in an inflammatory response with a range from discomfort to being fatal.

Any type of allergic reaction may be addressed with one or more compositions as disclosed herein. The allergic reaction may be anaphylaxis, anaphylactic shock, allergic rhinitis (hay fever), urticaria (hives), food allergy, drug allergy, hymenoptera allerga, bronchial constriction, asthma, eczema, and so forth. The compositions as disclosed herein are useful for prevention of one or more of these allergic reactions or for the reduction in the risk or severity of one or more of these allergic reactions.

In specific embodiments, the allergic reaction is anaphylaxis, which is characterized by rapid onset and can be fatal. It typically causes a number of symptoms including an itchy rash, throat swelling, and low blood pressure, for example. Common causes include insect bites/stings, foods, and medications.

On a pathophysiologic level, anaphylaxis is caused by the release of mediators from mast cells, such as by the release of inflammatory mediators and cytokines from mast cells and basophils, typically due to an immunologic reaction but sometimes non-immunologic mechanism. In the immunologic mechanism, immunoglobulin E (IgE) binds to the antigen (the foreign material that provokes the allergic reaction). Antigen-bound IgE then activates FcεRI receptors on mast cells and basophils. This leads to the release of inflammatory mediators such as histamine. These mediators subsequently increase the contraction of bronchial smooth muscles, trigger vasodilation, increase the leakage of fluid from blood vessels, and cause heart muscle depression. Non-immunologic mechanisms involve substances that directly cause the degranulation of mast cells and basophils.

Anaphylaxis typically presents with many different symptoms over minutes or hours. The most common affected areas include the skin, respiratory system, gastrointestinal system, heart and vasculature, and central nervous system and often include more than one system or organ.

Skin symptoms usually include generalized hives, itchiness, flushing, and/or swelling of the afflicted tissues. The tongue may swell, and some experience a runny nose and swelling of the conjunctiva. The skin may also be blue tinted because of reduced oxygen. Respiratory symptoms include shortness of breath, wheezing, or stridor, hoarseness, pain with swallowing, and/or a cough. Cardiac symptoms include coronary artery spasm, myocardial infarction, dysrhythmia, cardiac arrest, changes in heart rate, and/or a drop in blood pressure or shock. Gastrointestinal symptoms may include crampy abdominal pain, diarrhea, vomiting, confusion, a loss of bladder control and/or pelvic pain similar to that of uterine cramps.

Anaphylaxis may be diagnosed based on clinical criteria, such as when within minutes or hours of exposure to an allergen there is involvement of the skin or mucosal tissue in addition to either respiratory difficulty or a low blood pressure. In certain cases anaphylaxis is diagnosed with two or more of the following symptoms: a. involvement of the skin or mucosa; b. respiratory difficulties; c. low blood pressure; and d. gastrointestinal symptoms. Low blood pressure after exposure to a known allergen may be involved. Diagnosis may include blood tests for tryptase or histamine (released from mast cells) for anaphylaxis because of insect stings or medications.

There are three main classifications of anaphylaxis, all of which may be preventable or reduced in severity with one or more compositions as disclosed herein: anaphylactic shock; biphasic anaphylaxis; and pseudoanaphylaxis or anaphylactoid reactions (which are a type of anaphylaxis that does not involve an allergic reaction but is due to direct mast cell degranulation and may be referred to as non-immune anaphylaxis).

In certain embodiments, an individual in need thereof is provided one or more compositions as disclosed herein but also is exposed to desensitization.

Individual with food allergies are suitable for exposure to one or more compositions as disclosed herein. Typical food allergens include milk, legumes (such as peanuts), shellfish, tree nuts, eggs, fish, soy, and wheat, for example. Severe cases are usually caused by ingesting the allergen, but some people experience a severe reaction upon contact and/or close proximity.

Individual with medication allergies are suitable for exposure to one or more compositions as disclosed herein. Any medication may potentially trigger allergic reaction, including anesthetics. β-lactam antibiotics, aspirin, NSAIDs, chemotherapy, vaccines, protamine and herbal preparations. Some medications (such as vancomycin, morphine, or x-ray contrast) cause anaphylaxis by directly triggering mast cell degranulation.

Individuals with venom allergies are suitable for exposure to one or more compositions as disclosed herein. Venom from stinging or biting animals (such as Hymenoptera (bees and wasps), jellyfish, sting ray) may induce anaphylaxis in susceptible people. Individuals with previous systemic reactions (anything more than a local reaction around the site of the sting) are at risk for future anaphylaxis, although some individuals have had no previous systemic reaction.

In some cases, people that have had one type of allergic reaction are also susceptible to having another type of allergic reaction, and these individual may receive effective amounts of one or more compositions as disclosed herein.

Allergic reaction symptoms can develop quickly, often within seconds or minutes. They may include the following: abdominal pain; abnormal (high-pitched) breathing sounds; anxiety; chest discomfort or tightness; cough; diarrhea; difficulty breathing; difficulty swallowing; dizziness or light-headedness; hives; itchiness; nasal congestion; nausea or vomiting; palpitations; skin redness; slurred speech; swelling of the face, eyes, or tongue; unconsciousness; wheezing; rapid pulse, arrhythmia; pulmonary edema; low blood pressure; blue skin; weakness; and/or wheezing.

U.S. Pat. No. 8,099,167, which is incorporated by reference herein, describes methods and devices for treating anaphylaxis, anaphylactic shock, bronchial constriction, and/or asthma.

IV. Compositions

Embodiments of the invention encompass compositions that are useful for preventing and/or reducing the risk or severity of allergic reaction (such as anaphylaxis). Specific compositions are disclosed herein, but one of skill in the art recognizes that functional derivatives of such compositions are also encompassed by the invention. The term “derivative” as used herein is a compound that is formed from a similar compound or a compound that can be considered to arise from another compound, if one atom is replaced with another atom or group of atoms. Derivative can also refer to compounds that at least theoretically can be formed from the precursor compound.

In particular embodiments, compositions and functionally active derivatives as described herein are utilized in treatment and/or prevention of anaphylaxis. Specific but nonlimiting examples of different R groups for the compositions are provided in Tables 1, 2, and 3.

The term “functionally active derivative” or “functional derivative” is a derivative as previously defined that retains the function of the compound from which it is derived. In one embodiment of the invention, a derivative of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoic acid, 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl] benzoic acid, 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid, methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy)methyl)benzoate, or 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl)benzoic acid retains Stat3 inhibitory activity. In another embodiment of the invention, a derivative of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoic acid, 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid, 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid, methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate, or 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid retains Stat3 inhibitory activity and, in specific embodiments, also retains non-inhibition of Stat1, although in some cases it may also inhibit Stat1.

In a specific embodiment of the invention, there is a method of preventing or reducing the risk or severity of allergic reaction (such as anaphylaxis) in an individual comprising delivering to the individual a compound selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl] benzoic acid 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl] benzoic acid, 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid, methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate, 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, and a mixture thereof.

In another embodiment, the composition comprises the general formula:

wherein R₁ and R₂ may be the same or different and are selected from the group consisting of hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises the general formula:

wherein R₁, and R₃ may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives, and R₂ and R₄ may be the same or different and are selected from the group consisting of hydrogen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

In another embodiment of the invention, the composition comprises the general formula:

wherein R₁, R₂, and R₃ may be the same or different and are selected from the group consisting of hydrogen, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

An exemplary and illustrative list of alkanes, cyclic alkanes, and alkane-based derivates are described herein. Non-limiting examples of ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives; carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amino-based derivatives, amides, and amide-based derivatives are listed herein. Exemplary monocyclic or polycyclic arene, heteroarenes, arene-based or heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid and benzoic acid-based derivatives are described herein.

TABLE 1
Chemical names  Formulas
Methyl          CH3
Ethyl           C2H5
Vinyl (ethenyl) C2H3
Ethynyl         C2H
Cyclopropyl     C3H5
Cyclobutyl      C4H7
Cyclopentyl     C5H9
Cyclohexyl      C6H11

TABLE 2
Chemical names          Chemical formulas
Acetonyl                C3H5O
Methanal (formaldehyde) CH2O
Paraldehyde             C6H12O3
Ethanoic acid           CH3COOH
Diethyl ether           C4H10O
Trimethylamine          C3H9N
Acetamide               C2H5NO
Ethanol                 C2H5OH
Methanol                CH3OH

TABLE 3
Chemical names Chemical formulas
Benzol         C6H6
Phenol         C6H6O
Benzoic acid   C7H6O2
Aniline        C6H7N
Toluene        C7H8
Pyridazine     C4H4N2
Pyrimidine     C4H4N2
Pyrazine       C4H4N2
Biphenyl       C12H10

The compositions of the present invention and any functionally active derivatives thereof may be obtained by any suitable means. In specific embodiments, the derivatives of the invention are provided commercially, although in alternate embodiments the derivatives are synthesized. The chemical synthesis of the derivatives may employ well known techniques from readily available starting materials. Such synthetic transformations may include, but are not limited to protection, de-protection, oxidation, reduction, metal catalyzed C—C cross coupling, Heck coupling or Suzuki coupling steps (see for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structures. 5^th Edition John Wiley and Sons by Michael B. Smith and Jerry March, incorporated here in full by reference).

V. Embodiments for Targeting Stat3

STAT proteins, of which there are seven (1, 2, 3, 4, 5A, 5B and 6), transmit peptide hormone signals from the cell surface to the nucleus. Detailed structural information of STAT proteins currently is limited to Stat1 and Stat3. Stat1 was the first STAT to be discovered (Fu et al., 1992) and is required for signaling by the Type I and II IFNs (Meraz et al., 1996; Wiederkehr-Adam et al., 2003; Durbin et al., 1996; Haan et al., 1999). Studies in Stat1-deficient mice (Meraz et al., 1996; Durbin et al., 1996; Ryan et al., 1998) support an essential role for Stat1 in innate immunity, notably against viral pathogens. In addition, Stat1 is a potent inhibitor of growth and promoter of apoptosis (Bromberg and Darnell, 2000). Also, because tumors from carcinogen-treated wild-type animals grow more rapidly when transplanted into the Stat1-deficient animals than they do in a wild-type host, Stat1 contributes to tumor surveillance (Kaplan et al., 1998).

Stat3 was originally termed acute-phase response factor (APRF) because it was first identified as a transcription factor that bound to IL-6-response elements within the enhancer-promoter region of various acute-phase protein genes (Akira, 1997). In addition to receptors for the IL-6 cytokine family, other signaling pathways are linked to Stat3 activation include receptors for other type I and type II cytokine receptors, receptor tyrosine kinases, G-protein-coupled receptors and Src kinases (Schindler and Darnell, 1995; Turkson et al., 1998). Targeted disruption of the mouse Stat3 gene leads to embryonic lethality at 6.5 to 7.5 days (Takeda et al., 1997) indicating that Stat3 is essential for early embryonic development possibly gastrulation or visceral endoderm function (Akira, 2000). Tissue-specific deletion of Stat3 using Cre-lox technology has revealed decreased mammary epithelial cell apoptosis resulting in delayed breast involution during weaning (Chapman et al., 1999). Recent findings indicate that switching of the predominant STAT protein activated by a given receptor can occur when a STAT downstream of that receptor is genetically deleted (Costa-Pereira et al, 2002; Qing and Stark, 2004). These findings suggest the possibility that the effect of Stat3 deletion in breast tissue may be mediated indirectly by increased activation of other STAT proteins, especially Stat5.

Stat1 and Stat3 isoforms. Two isoforms of Stat1 and Stat3 have been identified-α (p91 and p92, respectively) and β (p84 and p83, respectively) (Schindler et al., 1992; Schaefer et al., 1995; Caldenhoven et al., 1996; Chakraborty et al., 1996)—that arise due to alternative mRNA splicing (FIG. 13). In contrast to Stat1β (712 aa), in which the C-terminal transactivation is simply deleted, the 55 amino acid residues of Stat3α are replaced in Stat3β by 7 unique amino acid residues at its C-terminus. Unlike Stat1β, Stat3β is not simply a dominant-negative of Stat3α (Maritano et al., 2004) and regulates gene targets in a manner distinct from Stat3β (Maritano et al., 2004; Yoo et al., 2002). Stat3α has been demonstrated to contribute to transformation in cell models and many human cancers including breast cancer. Stat3α was shown to be constitutively activated in fibroblasts transformed by oncoproteins such as v-Src (Yu et al., 1995; Garcia and Jove, 1998) and to be essential for v-Src-mediated transformation (Turkson et al., 1998; Costa-Pereira et al., 2002). In contrast to Stat3α, Stat3β antagonized v-Src transformation mediated through Stat3α (Turkson et al., 1998). Overexpression of a constitutively active form of Stat3α in immortalized rat or mouse fibroblasts induced their transformation and conferred the ability to form tumors in nude mice (Bromberg et al., 1999). Stat3 has been shown to be constitutively activated in a variety of hematological and solid tumors including breast cancer (Dong et al., 2003; Redell and Tweardy, 2003) as a result of either autocrine growth factor production or dysregulation of protein tyrosine kinases. In virtually all cases, the isoform demonstrating increased activity is Stat3α.

Targeting Stat3α while sparing Stat1. Given its multiple contributory roles to oncogenesis, Stat3 has recently gained attention as a potential target for cancer therapy (Bromberg, 2002; Turkson, 2004). While several methods of Stat3 inhibition have been employed successfully and have established proof-of-principle that targeting Stat3 is potentially beneficial in a variety of tumor systems including breast cancer in which Stat3 is constitutively activated (Epling-Burnette et al., 2001; Yoshikawa et al., 2001; Li and Shaw, 2002; Catlett-Falcone et al., 1999; Mora et al., 2002; Grandis et al., 2000; Leong et al., 2003; Jing et al., 2003; Jing et al., 2004; Turkson et al., 2001; Ren et al., 2003; Shao et al., 2003; Turkson et al., 2004; Uddin et al., 2005); all have potential limitations for translation to clinical use for cancer therapy related to issues regarding delivery, specificity or toxicity.

Specific strategies that target Stat3 by identifying inhibitors of Stat3 recruitment and/or dimerization have been pursued by several groups (Turkson et al., 2001; Ren et al., 2003; Shao et al., 2003; Uddin et al., 2005; Song et al., 2005; Schust et al., 2006). As outlined below, this strategy has the potential to achieve specificity based on the observation that the preferred pY peptide motif of each STAT protein is distinct. When coupled to a small molecule approach, this strategy has the potential to overcome issues of delivery and toxicity.

Targeting Stat3α while sparing Stat3β. Some of the distinct biochemical features of Stat3β vs. Stat3α, notably constitutive activation and a 10-to-20 fold increased DNA binding affinity, have been attributed to the absence of the C-terminal transactivation domain (TAD) resulting in increased Stat3p dimer stability (Park et al., 1996; Park et al., 2000). Increased dimer stability likely results from higher binding affinity of the SH2 domain to pY peptide motifs when in the context of Stat3p compared to Stat3α because of reduced steric hindrance conferred by removal of the TAD. These differential biochemical features between Stat3α and Stat3β are exploited to develop a chemical compound that selectively targets Stat3α, in some embodiments. This selectivity enhances the anti-tumor effect of such compounds, in certain cases, because they would spare Stat3β, which functions to antagonize the oncogenic functions of Stat3α.

In certain embodiments of the invention, specific therapies targeting Stat3 signaling are useful for treatment of allergic reaction.

VI. Combination Therapy

It is an aspect of this invention that a composition as disclosed herein is used in combination with another agent or therapy method, such as allergic reaction treatment. The composition(s) (which may or may not be a Stat3 inhibitor) may precede or follow the other agent treatment by intervals ranging from minutes to weeks, for example. In embodiments where the other agent and the composition of the invention are applied separately to an individual with anaphylaxis, such as upon delivery to an individual suspected of having anaphylaxis, known to have anaphylaxis, or at risk for having anaphylaxis, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and composition of the invention would still be able to exert an advantageously combined effect on the individual.

For example, in such instances, it is contemplated that one may contact the individual with one, two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the composition of the invention. In other aspects, one or more agents may be administered within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, to about 48 hours or more prior to and/or after administering the composition of the invention. In certain other embodiments, an agent may be administered within of from about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20, to about 21 days prior to and/or after administering the composition of the invention, for example. In some situations, it may be desirable to extend the time period for treatment significantly, such as where several weeks (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more) lapse between the respective administrations. In some situations, it may be desirable to extend the time period for treatment significantly, such as where several months (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more) lapse between the respective administrations.

Various combinations may be employed, the composition of the invention is “A” and the secondary agent, which can be any other cancer therapeutic agent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the therapeutic compositions of the present invention to a patient will follow general protocols for the administration of drugs, taking into account the toxicity. It is expected that the treatment cycles would be repeated as necessary.

Exemplary combination therapies include antihistamines, steroids, epinephrine, and so on.

VII. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise an effective amount of a composition as disclosed herein dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that in some cases contains at least one Stat3 inhibitor of the invention, and in some cases an additional active ingredient, will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein. “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The composition(s) may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration such as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularily, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), as an aerosol, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example. Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present invention administered to an individual can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, and the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a composition. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight, 0.5 mg/kg/body weight, 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 20 mg/kg/body weight, about 30 mg/kg/body weight, about 40 mg/kg/body weight, about 50 mg/kg/body weight, about 75 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, about 750 mg/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 10 mg/kg/body weight to about 100 mg/kg/body weight, etc., can be administered, based on the numbers described above. In certain embodiments of the invention, various dosing mechanisms are contemplated. For example, the composition may be given one or more times a day, one or more times a week, or one or more times a month, and so forth.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The composition may be formulated in a free base, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example, liquid polyol or lipids; by the use of surfactants such as, for example, hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the instant invention in the required amount of the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

VIII. Kits of the Invention

Any of the compositions described herein may be comprised in a kit, and they are housed in a suitable container. The kits will thus comprise, in suitable container means, one or more compositions and, in some cases, an additional agent of the present invention. In some cases, there are one or more agents other than the composition of the disclosure that are included in the kit, such as one or more other agents for the treatment of anaphylaxis. In particular embodiments, there is an apparatus or any kind of means for the diagnosing of anaphylaxis.

The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the composition, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

Compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Exemplary Materials and Methods

Virtual ligand screening. The inventors isolated the three-dimensional structure of the Stat3 SH2 domain from the core fragment structure of phosphorylated Stat3 homodimers bound to DNA (Becker el al., 1998) deposited in the RCSB Protein Data Bank (PDB) databank (PDB code 1BG1) and converted it to be an Internal Coordinate Mechanics (ICM)-compatible system by adding hydrogen atoms, modifying unusual amino acids, making charge adjustments and performing additional cleanup steps. In addition, the inventors retrieved the coordinates of the Stat1 SH2 domain from the PDB databank (PDB code 1BF5) for use in computational selectivity analysis (Chen et al., 1998). Commercial chemical databases (Chembridge, Asinex, ChemDiv. Enamine, Keyorganics and Life Chemicals) were chosen as sources of compounds for screening in silico. Selection was of the amide hydrogen of E638 within the site that binds the +3 residue (Q, C or T) within the pY-peptide ligand (Shao et al., 2006) as the central point of the binding pocket, which consisted of a cube with dimensions 16.0×16.9×13.7 angstrom. In addition to the +3 binding site, this cube contained the pY residue binding site consisting mainly of R609 and K591 (Shao et al., 2006) and a hydrophobic binding site consisting of Loop_βC-βD and Loop_αB-αC. Sequence alignment and overlay of the Stat3 and Stat1 structures revealed substantial differences in sequence of these loops; lack of their superimposition indicated that this region might serve as a selectivity filter (Cohen et al., 2005). A flexible docking calculation (Totrov and Abagyan 1997) was performed in order to determine the global minimum energy score and thereby predict the optimum conformation of the compound within the pocket. A compound was selected for purchase and biochemical testing based on fulfilling the criteria of interaction analysis (CIA): 1) global minimum energy score ≤−30, 2) formation of a salt-bridge and/or H-bond network within the pY-residue binding site and 3) formation of a H-bond with or blocking access to the amide hydrogen of E638. Most, but not all, compounds also interacted with the hydrophobic binding site.

Stat3 SH2/pY-peptide binding assay. Stat3 binding assays were performed at 25° C. with a BIAcore 3000 biosensor using 20 mM Tris buffer pH 8 containing 2 mM mercaptoethanol and 5% DMSO as the running buffer (Kim et al., 2005). Phosphorylated and control non-phosphorylated biotinylated EGFR derived dodecapeptides based on the sequence surrounding Y1068 (Shao et al., 2004) were immobilized on a streptavidin coated sensor chip (BIAcore inc., Piscataway N.J.). The binding of Stat3 was conducted in 20 mM Tris buffer pH 8 containing 2 mM β-mercaptoethanol at a flow rate of 10 uL/min for 1-2 minute. Aliquots of Stat3 at 500 nM were premixed with compound to achieve a final concentration of 1-1.000 uM and incubated at 4° C. prior to being injected onto the sensor chip. The chip was regenerated by injecting 10 uL of 100 mM glycine at pH 1.5 after each sample injection. A control (Stat3 with DMSO but without compound) was run at the beginning and the end of each cycle (40 sample injections) to ensure that the integrity of the sensor chip was maintained throughout the cycle run. The average of the two controls was normalized to 100% and used to evaluate the effect of each compound on Stat3 binding. Responses were normalized by dividing the value at 2 min by the response obtained in the absence of compounds at 2 min and multiplying by 100. IC₅₀ values were determined by plotting % maximum response as a function of log concentration of compound and fitting the experimental points to a competitive binding model using a four parameter logistic equation: R=R_high−(R_high−R_low)/(1+conc/A1){circumflex over ( )}A2, where R=percent response at inhibitor concentration, R_high=percent response with no compound. R_low=percent response at highest compound concentration. A2=fitting parameter (slope) and A1=IC₅₀ (BIAevaluation Software version 4.1).

Immunoblot assay. The human hepatocellular carcinoma cell line (HepG2) was grown in 6-well plates under standard conditions. Cells were pretreated with compounds (0, 1, 3, 10, 30, 100 and 300 uM) for 1 hour then stimulated under optimal conditions with either interferon gamma (IFN-γ; 30 ng/ml for 30 min) to activate Stat1 or interleukin-6 (IL-6; 30 ng/ml for 30 min) to activate Stat3 (30-31). Cultures were then harvested and proteins extracted using high-salt buffer, as described (Shao et al., 2006). Briefly, extracts were mixed with 2× sodium dodecyl sulfate (SDS) sample buffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20% glycerol; 10%2-mercaptoethanol) at a 1:1 ratio and heated for 5 minutes at 100° C. Proteins (20 μg) were separated by 7.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Waltham, Mass.) and immunoblotted. Prestained molecular weight markers (Biorad, Hercules, Calif.) were included in each gel. Membranes were probed serially with antibody against Stat1 pY⁷⁰¹ or Stat3 pY⁷⁰⁵ followed by antibody against Stat1 or Stat3 (Transduction labs. Lexington, Ky.) then antibody against β-actin (Abcam, Cambridge, Mass.). Membranes were stripped between antibody probing using Restorer-Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, Mass.) per the manufacturer's instructions. Horseradish peroxidase-conjugated goat-anti-mouse IgG was used as the secondary antibody (Invitrogen Carlsbad, Calif.) and the membranes were developed with enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc.; Arlington Heights, Ill.).

Similarity screen. Three compounds identified in the initial virtual ligand screening (VLS)—Cpd3, Cpd30 and Cpd188—inhibited Stat3 SH2/pY-peptide binding and IL-6-mediated Stat3 phosphorylation and were chosen as reference molecules for similarity screening. A fingerprint similarity query for each reference compound was submitted to Molcart/ICM (Max Distance, 0.4). Similarity between each reference molecule and each database molecule was computed and the similarity results were ranked in decreasing order of ICM similarity score (Eckert and Bajorath 2007). The databases searched included ChemBridge, LifeChemicals, Enamine, ChemDiv, Asinex, AcbBlocks, KeyOrganics and PubChem for a total of 2.47 million compounds. All compounds identified were docked into the binding pocket of Stat3 SH2 domain in silico. Compounds that fulfilled CIA criteria were purchased and tested as described for compounds identified in the primary screen.

Electrophoretic Mobility Shift Assay (EMSA): EMSA was performed using the hSIE radiolabeled duplex oligonucleotide as a probe as described (Tweardy et al., 1995). Briefly, high salt extracts were prepared from HepG2 cells incubated without or with IL-6 (30 ng/ml) for 30 minutes. Protein concentration was determined by Bradford Assay and 20 ug of extract was incubated with compound (300 uM) for 60 minutes at 37° C. Bound and unbound hSIE probe was separated by polyacrylamide gel electrophoresis (4.5%). Gels were dried and autoradiographed.

Molecular modeling. All 3-D configurations of the Stat3 SH2 domain complexed with compounds were determined by global energy optimization that involves multiple steps: 1) location of organic molecules were adjusted as a whole in 2 Å amplitude by pseudo-Brownian random translations and rotations around the molecular center of gravity, 2) the internal variables of organic molecules were randomly changed. 3) coupled groups within the Stat3 SH2 domain side-chain torsion angles were sampled with biased probability shaking while the remaining variables of the protein were fixed, 4) local energy minimizations were performed using the Empirical Conformation Energy Program for Peptides type-3 (ECEPP3) in a vacuum (Nemethy et al., 1992) with distance-dependent dielectric constant ε=4r, surface-based solvent energy and entropic contributions from the protein side chains evaluated added and 5) conformations of the complex, which were determined by Metropolis criteria, were selected for the next conformation-scanning circle. The initial 3-dimensional configuration of the Stat1 SH2 domain in a complex with each compound was predicted and generated by superimposing, within the computational model, the 3-dimensional features of the Stat1 SH2 onto the 3-dimensional configuration of the Stat3 SH2 domain in a complex with each compound. The final computational model of Stat1 SH2 in a complex with each compound was determined by local minimization using Internal Coordinate Force Field (ICFF)-based molecular mechanics (Totrov and Abagyan 1997). The inventors computed the van der Waals energy of the complex of Stat1 or 3-SH2 bound with each compound using Lennard-Jones potential with ECEPP/3 force field (Nemethy et al., 1992).

Confocal and high-throughput fluorescence microscopy. Confocal and highthroughput fluorescence microscopy (HTFM) of MEF/GFP-Stat3α cells were performed as described (Huang et al., 2007). Briefly, for confocal fluorescence microscopy, cells were grown in 6-well plates containing a cover slip. For HTFM, cells were seeded into 96-well CC3 plates at a density of 5.000 cells/well using an automated plating system. Cells were cultured under standard conditions until 85-90% confluent. Cells were pretreated with compound for 1 hour at 37° C. then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes. Cells were fixed with 4% formaldehyde in PEM Buffer (80 mM Potassium PIPES, pH 6.8, 5 mM EGTA pH 7.0, 2 mM MgCl₂) for 30 minutes at 4° C., quenched in 1 mg/ml of NaBH4 (Sigma) in PEM buffer and counterstained for 1 min in 4,6-diamidino-2-phenylindole (DAPI; Sigma; 1 mg/ml) in PEM buffer. Cover slips were examined by confocal fluorescent microscopy. Plates were analyzed by automated HTFM using the Cell Lab IC Image Cytometer (IC100) platform and CytoshopVersion 2.1 analysis software (Beckman Coulter). Nuclear translocation is quantified by using the fraction localized in the nucleus (FLIN) measurement (Sharp et al., 2006).

Example 2

Identification by VLS of Compounds that Blocked Stat3 Binding to its Phosphopeptide Ligand and Inhibited IL-6-Mediated Phosphorylation of Stat3

The VLS protocol was used to evaluate a total of 920,000 drug-like compounds. Of these, 142 compounds fulfilled CIA criteria. These compounds were purchased and tested for their ability to block Stat3 binding to its phosphopeptide ligand in a surface plasmon resonance (SPR)-based binding assay and to inhibit IL-6-mediated phosphorylation of Stat3. SPR competition experiments showed that of the 142 compounds tested, 3 compounds—Cpd3, Cpd30 and Cpd188—were able to directly compete with pY-peptide for binding to Stat3 with IC₅₀ values of 447, 30, and 20 μM, respectively (FIGS. 1 and 3; Table 4).

TABLE 4
IC50 values (μM) of 6 active compounds
Assay	Cpd3	Cpd30	Cpd188	Cpd3-2	Cpd3-7	Cpd30-12
SPR	4471	30	20	256	137	114
pStat3	91	18	73	144	63	60
HTM	131	77	39	150	20	>300
1Data presented are the mean or mean ± SD; ND = not determined.

In addition, each compound inhibited IL-6-mediated phosphorylation of Stat3 with IC50 values of 91, 18 and 73 μM respectively (FIG. 2; Table 4).

Similarity screening with Cpd3, Cpd30 and Cpd188 identified 4,302 additional compounds. VLS screening was performed with each of these compounds, which identified 41 compounds that fulfilled CIA criteria; these were purchased and tested. SPR competition experiments showed that of these 41 compounds, 3 compounds—Cpd3-2, Cpd3-7 and Cpd30-12—were able to directly compete with pY-peptide for binding to Stat3 with IC₅₀ values of 256, 137 and 114 μM, respectively (FIGS. 1 and 3; Table 4). In addition, each compound inhibited IL-6-mediated phosphorylation of Stat3 with IC50 values of 144, 63 and 60 μM, respectively (FIG. 2; Table 4).

Example 3

Compound-Mediated Inhibition of Ligand-Stimulated Phosphorylation of Stat3 is Specific for Stat3 Vs. Stat1

While Stat3 contributes to oncogenesis, in part, through inhibition of apoptosis, Stat1 is anti-oncogenic; it mediates the apoptotic effects of interferons and contributes to tumor surveillance (Kaplan et al., 1998; Ramana et al., 2000). Consequently, compounds that target Stat3 while sparing Stat1, leaving its anti-oncogenic functions unopposed, may result in a synergistic anti-tumor effect. To assess the selectivity of the compounds for Stat3 vs. Stat1, HepG2 cells were incubated with Cpd3, Cpd30, Cpd188, Cpd3-2, Cpd3-7, and Cpd30-12 (300 μM) for 1 hour at 37° C. before IFN-γ stimulation (FIG. 4). Only treatment with Cpd30-12 blocked Stat1 phosphorylation while each of the other five compounds—Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7—did not. Thus, five of the six exemplary compounds identified were selective and inhibited ligand-stimulated phosphorylation of Stat3 but not Stat1.

Example 4

Sequence Analysis and Molecular Modeling of the Interaction of Each Compound with the Stat3 Vs. Stat1 SH2 Domain

To understand at the molecular level the basis for the selectivity of Cpds 3, 30, 188, 3-2 and 3-7 and the absence of selectivity in the case of Cpd 30-12, the amino acid sequence and available structures of the Stat1 and Stat3 SH2 domain were compared and also it was examined how each compound interacted with both. Sequence alignment revealed identity in the residues within Stat3 and Stat1 corresponding to the binding site for the pYresidue and the +3 residue (FIG. 5A). In addition, overlay of the Stat3 and Stat1 SH2 structures revealed that the loops that contained these binding sites were superimposed (FIG. 5B). In contrast, sequence alignment revealed substantial differences in the sequence of the regions of the SH2 domain corresponding to the loops forming the hydrophobic binding site (FIG. 5A). In addition, review of the overlay of Stat3 and Stat1 SH2 domains revealed that, in contrast to the close apposition of the two loops of Stat3 that form the hydrophobic binding site, the corresponding two loops of Stat1 are not closely apposed to form a pocket (FIG. 5B).

Review of computational models of Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7 in a complex with the Stat3 SH2 domain revealed that each has significant interactions with the Stat3 SH2 domain binding pocket at all three binding sites, the pY-residue binding site, the +3 residue binding site and the hydrophobic binding site (FIGS. 6A, B, C, D, and E). In contrast, Cpd30-12 interacts with the pY-residue binding site and blocks access to the +3 residue-binding site but does not interact with or block access to the hydrophobic binding site (FIG. 6F). In addition, van der Waals energies of the 5 selective compounds were much more favorable for their interaction with the loops of Stat3 forming the hydrophobic binding site than with corresponding loops of Stat1 (FIG. 5C). Thus, computer modeling indicated that activity of compounds against Stat3 derives from their ability to interact with the binding sites for the pY and the +3 residues within the binding pocket, while selectivity for Stat3 vs. Stat1 derives from the ability of compounds to interact with the hydrophobic binding site within the Stat3 SH2 binding pocket, which served as a selectivity filter.

Example 5

Inhibition of Nuclear Translocation of Phosphorylated Stat3 by Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7 Assessed by HTFM

Following its phosphorylation on Y705. Stat3 undergoes a change in conformation from head-to-head dimerization mediated through its N-terminal oligomerization domain to tail-to-tail dimerization mediated by reciprocal SH2/pY705-peptide ligand interactions. This conformational change is followed by nuclear accumulation. Compounds that targeted SH2/pY-peptide ligand interactions of Stat3 would be expected to inhibit nuclear accumulation of Stat3. To determine if this was the case with the compounds herein, a nuclear translocation assay (FIG. 7) was employed using murine embryonic fibroblast (MEF) cells that are deficient in endogenous Stat3 but constitutively express GFP-tagged Stat3α at endogenous levels. MEF/GFP-Stat3α (Huang el al., 2007). Preincubation of MEF/GFP-Stat3α cells with Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7, but not Cpd30-12, blocked ligand-mediated nuclear translocation of GFP-Stat3α with IC₅₀ values of 131, 77, 39, 150 and 20 μM (FIG. 7 and Table 4).

Example 6

Destabilization of Stat3-DNA Complexes by Cpd3 and Cpd3-7

Once in the nucleus. Stat3 dimers bind to specific DNA elements to activate and, in some instances, repress gene transcription. Tyrosine-phosphorylated dodecapeptides based on motifs within receptors that recruit Stat3 have previously been shown to destabilize Stat3 (Chakraborty et al., 1999; Shao et al., 2003). Compounds that bind to the phosphopeptide-binding site of Stat3 might be expected to do the same. To determine if this was the case for any of the identified compounds, extracts of IL-6-stimulated HepG2 cells were incubated in binding reactions containing radiolabeled hSIE (FIG. 8) and each of the five selective compounds (300 μM). Incubation with Cpd3 or Cpd3-7 reduced the amount of hSIE shifted by half or greater. The other compounds did not have a detectable effect on the Stat3:hSIE band intensity. Thus, 2 of the 5 selective compounds destabilized Stat3:hSIE complexes.

Example 7

Exemplary Approach for Stat3 Inhibitors for Cancer Stem Cells

In the field of Stat3 probe development the inventors have focused on small molecule Stat3 probes (Xu et al., 2009), and several features of the small molecule program are useful, including: 1) a clearly defined mode of action of these probes: they target the Stat3 Src-homology (SH) 2 domain that is involved in 2 steps in the Stat3 activation pathway; 2) their specificity of action; and 3) the potential for using lead probes identified so far to identify probes with 2-to-3 logs greater activity based on recent and exemplary SAR analysis and medicinal chemistry considerations outlined below.

In specific embodiments, compound affinity is improved upon gaining a log greater affinity upon moving from 1^st generation to 2nd generation probes using 3-D pharmacophore analysis. In addition, selectivity is improved through modeling embodiments, in particular through identification of a distinct hydrophobic binding domain in the phosphopeptide binding pocket of Stat3 SH2 vs. the Stat1 SH2 (Xu et al., 2009).

Identification of 1st generation Stat3 chemical probes. To develop chemical probes that selectively target Stat3, the inventors virtually screened 920,000 small drug-like compounds by docking each into the peptide-binding pocket of the Stat3 SH2 domain, which consists of three sites—the pY-residue binding site, the +3 residue-binding site and a hydrophobic binding site, which served as a selectivity filter (Xu et al., 2009). Three compounds (Cpd3, Cpd30 and Cpd188) satisfied criteria of interaction analysis, competitively inhibited recombinant Stat3 binding to its immobilized pY-peptide ligand and inhibited IL-6-mediated tyrosine phosphorylation of Stat3. These compounds were used in a similarity screen of 2.47 million compounds, which identified 3 more compounds (Cpd3-2, Cpd3-7 and Cpd30-12) with similar activities. Examinations of the 6 active compounds for the ability to inhibit IFN-T-mediated Stat1 phosphorylation revealed that all but Cpd30-12 were selective for Stat3. Molecular modeling of the SH2 domains of Stat3 and Stat1 bound to compound revealed that compound interaction with the hydrophobic binding site was the basis for selectivity. All 5 selective compounds inhibited nuclear-tocytoplasmic translocation of Stat3, while 3 of 5 compounds (Cpd3, Cpd30 and Cpd188) induced apoptosis preferentially of exemplary breast cancer cell lines with constitutive Stat3 activation.

Identification of 2nd generation Stat3 chemical probes. The similarity screening described above did not yield any hits using Cpd188, the most active of the 3 lead compounds, as the query compound. Consequently, the inventors repeated 2-D similarity screening using the scaffold of Cpd188 as the query structure and the Life Chemicals library, which yielded 207 hits. 3-D pharmacophore analysis was performed on these 207 compounds using Ligand Scout and the top 39 scoring compounds were purchased and tested for inhibition of Stat3 binding to its phosphopeptide ligand by SPR. All but six of these 39 compounds have measurable SPR IC50s, with 19 having IC50 values equal to or less than the parent compound and 2 (Cpd188-9 and Cpd188-15) having IC50 values one log lower. Examination of these 19 compounds has revealed a statistically significant correlation between 3-D pharmacophore scores and SPR IC50s and as well as 3-D pharmacophore score and IC50s for inhibition of ligand-mediated cytoplasmic-to-nuclear translocation. In addition, both Cpd188-9 and Cpd188-15 exhibited a log greater activity in inducing human leukemic cell line apoptosis than the parent Cpd188 (FIG. 15). In addition, Cpd188-38 exhibited a 2 logs greater activity than parent Cpd188 in inhibiting cytoplasmic-to-nuclear translocation in HTFM assay, while Cpd188-15 exhibited a 1 log greater activity than parent Cpd188 in decreasing MSFE (Table 5). Furthermore, several of the second-generation 188-like compounds represent a substantial improvement over Cpd188 from a medicinal chemistry, metabolism and bioavailability standpoint. In particular. Cpd188-9 lacked both carboxyl groups, which in particular cases improves cell permeability and/or the thioether group, which is subject to oxidation. R2=0.2 P=0.013 (μM)

TABLE 5
Summary of Certain 2nd Generation 188-like Compounds
		SPR IC50,	HTFM IC50,	Mammosphere
	Compound	μM*	μM*	~IC50, μM***
	188	20**	32 ± 4	30-100
	188-1	6 ± 2	26 ± 4	30
	188-9	3 ± 2	47 ± 21	10
	188-10	8 ± 3	22 ± 19	30
	188-15	2 ± 1	49
	188-16	4 ± 0	9 ± 5	30
	188-17	4 ± 2	76	30
	188-18	4 ± 1	27 ± 8	30
	188-38	19 ± 9		10-30
	*mean ± SD
	**Xu et al PLoS ONE
	***SUM159PT and HS578T cells plated (6 wells per test) without or with compound at 1, 10 or 100 μM, incubated 3 d; spheres counted on day 3.

Structure-activity relationship (SAR) analysis of 2nd generation Stat3 probes. All of the 39 second generation compounds described above, plus Cpd188 itself, are derivatives of N-naphth-1-yl benzenesulfamide. Upon careful analysis of their structure-activity relationships (SAR), the inventors found that most of these Cpd188-like compounds (38 out of 40: the rest of 2 are weak and will be described below in EXP ID) can be divided into three structural groups in a general trend of decreased activity, as shown in FIG. 16. Five compounds in Group III are actually the parents of compounds in Groups I and II. Addition of a variety of groups (the —R group highlighted in red in the general structure of Group I in FIG. 16), such as a triazole-3-yl-mercapto (188-15) or a chloro (188-10) group, to the 3-position of the naphthylamine ring led to the Group I compounds, which are the most potent series of Stat3 probes. In a specific embodiment, this is the most important contributor to the inhibitory activity: a total of eight 3-substituents are found in Group I compounds, which invariably enhance the activity by several orders of magnitude.

Most Stat3 probes in Group II contain a 5-membered ring that combines the 3-R and 4-OR2 groups, such as a furan (188-11). However, the compounds in this group are, in average, ˜5× less active than the Group I compounds, which indicates that in certain aspects the H atom of the 4-hydroxy group (highlighted in blue in the general structure of Group I in FIG. 16) is important, e.g., involved in a favorable H-bond with the protein. Lacking the ability to form the H-bond attributes to the weaker activities of Group II probes, in particular cases. These considerations underlie a medicinal chemistry approach outlined below.

Example 8

Medicinal Chemistry for Synthesis of 3^rd Generation 188-Like Sulfamide Stat3 Probes

The crystal structure of Stat3 shows that the SH2 domain has a large, widely dispersed and generally shallow binding area with several valleys and hills that recognize the pY-peptide ligand (FIG. 18). Structure-based molecular modeling (docking) was useful in identifying the contribution of the hydrophobic binding surface of the Stat3 SH2 domain as a selectivity filter (Xu et al., 2009). However, different docking programs gave distinct binding poses for the same probe over the binding surface with similar predicted binding affinities. The inventors therefore in particular embodiments, based on initial SAR results outlined above, use traditional medicinal chemistry to further carry out an exemplary comprehensive structure activity relationship study, to optimize the activity as well as the selectivity of this novel class of sulfamide probes of Stat3. Compound 188-15 serves as a scaffold for making the new generation compounds, as shown schematically (FIG. 16).

In addition, chemistry for making these compounds is straightforward with a good yield, involving the reaction of a sulfonyl chloride with an aniline/amine, which can be either obtained commercially or synthesized readily.

For the proposed modifications described below, one can consult FIG. 17. EXP IA. Modification 1. Since almost all of the 2^nd generation probes contain a phenylsulfonyl group, the first step towards activity optimization focuses on synthesizing a series of compounds that have a larger (e.g., bicyclic or tricyclic) or an alkyl sulfonyl group. The general synthetic route is shown as follows:

There are about 4.300 commercially available sulfonyl chlorides, among which 25, such as those shown above, are selected to make probes. Aniline 2, which is the amine component of compound 188-10 (FIG. 16), one the most active probes, is readily made in a simple two step reaction from nitro compound 1. One can first make 25 (for example) compounds and test their activities in an in vitro rapid throughput SPR and in vivo HTFM assays. Based on the outcomes of structure-activity relationship study, more compounds can be designed and synthesized and tested in an iterative manner until optimization of this modification.

EXP IB. Modification 2. Next, one can modify the 3-substituent of the naphthylamine ring, based on either the structure of compound 188-15, for example. Prior SAR studies demonstrated this substituent is useful to the activity of this class of probes, in certain embodiments. However, a total of 8 groups at this position with a huge difference in size, from a single atom Cl to a large, bicyclic benzothiazole-2-ylmercapto group, showed similar activities. This feature indicates that in certain embodiments modifications at this position should be more focused on other properties, such as electrostatic interactions with the protein, as exemplified below. In addition, many of these groups are thioethers, which may be subjected to oxidation/degradation in vivo and lead to an unfavorable pharmacokinetic profile, in particular aspects. The central —S— atom is changed to a more metabolically stable isosteres, such as —CH₂—, —NH—, and —O—, in certain cases. In certain aspects one can synthesize the following compounds to optimize the 3-substituent:

The synthesis is also started from 1, in certain cases. Regio-selective halogenation and formylation at the 3-position gives rise to two compounds, i.e., bromo- or iodo-compound 3 and aldehyde 4, which are versatile, common starting compounds for introducing a wide range of substituents at this position (e.g., those listed above).

Moreover, the crystal structure of Stat3 SH2 domain also provides strong evidence that more compounds with different electrostatic properties are useful for characterization. The electrostatic molecular surface of the protein shows two distinct features, as shown in FIG. 18. The first one is the negatively charged Glu638 surface stands out in the center. Next, of particular interest is a positively charged area, composed of Arg609 and Lys591 located in the edge of the domain, which is actually the pY (phosphorylated tyrosine) binding site of the receptor. The inventors also found that introducing a negatively charged group targeting the pY binding site leads to particularly active probes, in certain embodiments. For example, the docking study of the 3-phosphomethyl compound 5 (R=CH₂PO₃²⁻) showed all of the phosphonate groups of the 20 docking poses are tightly clustered together and located in the pY binding site, indicating strong electrostatic and H-bond interactions with the residues Arg609 and Lys591 (FIG. 18).

EXP IC. Modifications 3 and 4. Collectively, Modifications 3 and 4 test the effects of changing the substituents at the 4, 5, and 6-positions. The —OH at 4-position may be superior to —OR, in certain aspects. One can test whether the H atom in —OH is responsible for a better activity by synthesizing compounds 6 (acylated or alkylated 5), as schematically shown below. In addition, dehydroxy compounds 7 may also be made, starting from 3-bromonaphthyl-1-amine.

Regarding the general synthetic methods for modifying positions 5 and 6, one can first synthesize about a dozen of these compounds in this category and if very active compounds emerge, one can make more compounds to optimize the activity for these two positions.

EXP ID. Modification 5. The only two compounds not included in the SAR analysis (due to a different 4-substituent) are shown here, as well as their inhibitory activities against Stat3:

Despite the weak activity, masking the polar H of the sulfamide for the second compound is favorable, in certain aspects, which provides an easy route to making more potent probes. One can therefore use the following method to make a series of N-acyl or N-alkyl sulfamides 5:

Example 9

Identification of Stat3-Selective Chemical Probes from Sulfamide Compounds Synthesized in Example 11

Each novel sulfamide compound is tested for the ability to inhibit Stat3 binding to its phosphopeptide ligand by SPR and the ability to block IL-6-stimulated cytoplasmic-to-nuclear translocation in the HTFM assay. Probes with activity in these assays equivalent to or greater than the most active 2nd generation compounds are tested for inhibition of IL-6-stimulated Stat3 phosphorylation and lack of ability to inhibit IFN-γ-stimulated Stat1 phosphorylation as outlined below.

EXP IIA. Stat3/pY-peptide SPR binding inhibition assay. Stat3 pY-peptide binding assays is performed at 25° C. using a BIAcore 3000 biosensor as described (Xu et al., 2009). Briefly, phosphorylated and control nonphosphorylated biotinylated EGFR derived dodecapeptides based on the sequence surrounding Y1068 are immobilized on a streptavidin coated sensor chip (BIAcore Inc., Piscataway N.J.). The binding of Stat3 is performed in 20 mM Tris buffer pH 8 containing 2 mM β-mercaptoethanol at a flow rate of 10 uL/min for 1-2 minute. Aliquots of Stat3 at 500 nM are premixed with compound to achieve a final concentration of 1-1,000 uM and incubated at 4° C. prior to being injected onto the sensor chip. The chip is regenerated by injecting 10 uL of 100 mM glycine at pH 1.5 after each sample injection. A control (Stat3 with DMSO but without compound) is run at the beginning and the end of each cycle (40 sample injections) to ensure that the integrity of the sensor chip is maintained throughout the cycle run. The average of the two controls is normalized to 100% and used to evaluate the effect of each compound on Stat3 binding. Responses are normalized by dividing the value at 2 min by the response obtained in the absence of compounds at 2 min and multiplying by 100. IC₅₀ values are determined by plotting % maximum response as a function of log concentration of compound and fitting the experimental points to a competitive binding model using a four parameter logistic equation: R=R_high−(R_high−R_low)/(1+conc/A1)^A2, where R=percent response at inhibitor concentration. R_high=percent response with no compound, R_low=percent response at highest compound concentration. A2=fitting parameter (slope) and A1=IC₅₀ (BIAevaluation Software version 4.1).

EXP IIB. High throughput fluorescence microscopy (HTFM), cytoplasm-to-nucleus translocation inhibition assays. HTFM of MEF/GFP-Stat3α cells is performed to assess the ability of probes to inhibit GFP-Stat3 cytoplasmic-to-nuclear translocation, as described (Xu et al., 2009), using the robotic system available as part of the John S. Dunn Gulf Coast Consortium for Chemical Genomics at the University of Texas-Houston School of Medicine. Briefly, cells are seeded into 96-well CC3 plates at a density of 5,000 cells/well and cultured under standard conditions until 85-90% confluent. Cells are pre-treated with compound for 1 hour at 37° C. then stimulated with IL-6 (100 ng/ml) and IL-6sR (150 ng/ml) for 30 minutes. Cells are fixed with 4% formaldehyde in PEM Buffer (80 mM Potassium PIPES, pH 6.8, 5 mM EGTA pH 7.0, 2 mM MgCl₂) for 30 minutes at 4° C., quenched in 1 mg/ml of NaBH₄ (Sigma) in PEM buffer and counterstained for 1 min in 4,6-diamidino-2-phenylindole (DAPI; Sigma; 1 mg/ml) in PEM buffer. Plates are analyzed by automated HTFM using the Cell Lab IC Image Cytometer (IC100) platform and CytoshopVersion 2.1 analysis software (Beckman Coulter).

Nuclear translocation is quantified by using the fraction localized in the nucleus (FLIN) measurement. FLIN values are normalized by subtracting the FLIN for unstimulated cells then dividing this difference by the maximum difference (delta, Δ) in FLIN (FLIN in cells stimulated with IL-6/sIL-6R in the absence of compound minus FLIN of unstimulated cells). This ratio is multiplied by 100 to obtain the percentage of maximum difference in FLIN and is plotted as a function of the log compound concentration. The best-fitting curve and IC₅₀ value are determined using 4-Parameter LogisticModel/Dose Response/XLfit 4.2, IDBS software.

EXP IIC. Ligand-mediated pStat3 and pStat1 inhibition assays. Newly synthesized Stat3 probes with activity equivalent to or greater than parent compound 188 in the SPR and HTFM assays will be tested for the ability to selectively inhibit ligand-mediated phosphorylation of Stat3 as described (Xu et al., 2009). Briefly, human hepatocellular carcinoma cells (HepG2) are grown in 6-well plates and pretreated with compounds (0, 0.1, 0.3, 1, 3, 10, 30, 100 IM) for 1 hour then stimulated under optimal conditions with either interleukin-6 (IL-6; 30 ng/ml for 30 min) to activate Stat3 or interferon gamma (IFN-γ; 30 ng/ml for 30 min) to activate Stat1. Cells are harvested and proteins extracted using high-salt buffer, mixed with 2× sodium dodecyl sulfate (SDS) sample buffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20% glycerol; 10%2-mercaptoethanol) at a 1:1 ratio then heated for 5 minutes at 100° C. Proteins (20 μg) are separated by 7.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Waltham, Mass.) and immunoblotted. Membranes are probed serially with antibody against Stat1 pY701 or Stat3 pY705 followed by antibody against Stat1 or Stat3 (Transduction labs. Lexington, Ky.) then antibody against β-actin (Abcam, Cambridge, Mass.). Membranes are stripped between antibody probings using Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, Mass.) per the manufacturer's instructions. Horseradish peroxidase-conjugated goat-anti-mouse IgG is used as the secondary antibody (Invitrogen Carlsbad, Calif.) and the membranes are developed with enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc.; Arlington Heights, Ill.). Band intensities are quantified by densitometry. The value of each pStat3 band is divided by its corresponding total Stat3 band intensity; the results are normalized to the DMSO-treated control value. This value was plotted as a function of the log compound concentration. The best-fitting curve is determined using 4-Parameter Logistic Model/Dose Response/XLfit 4.2, IDBS software and was used to calculate the IC₅₀ value.

EXP IID. Molecular modeling of probe-Stat3 interactions. The results of modeling of the binding of the first generation probe to the Stat3 vs. Stat1 SH2 domains suggested that the basis for experimental selectivity of probes for Stat3 vs. Stat1 rested on the ability of the probes to have greater interaction with the hydrophobic binding site within the pY-peptide binding pocket of Stat3 compared to Stat1. Thus, the hydrophobic binding site served as a selectivity filter. To test if this remains the case for newly synthesized 3rd generation probes, one can use 2 complementary docking programs GLIDE (Schrödinger) and ICM (MolSoft) to determine the lowest energy docking configuration of each probe within the pY-peptide binding domain of Stat3 and Stat1 SH2 domain. One can review the computational models of each probe in a complex with the Stat3 vs. Stat1 SH2 domain and, in particular, compare the van der Waals energies and determine if they are equivalent for their interaction with the Stat3 SH2 domain vs. the Stat1 SH2 domain. It was this calculation that determined the selectivity of 1st generation probes for Stat3 vs. Stat1. In particular, van der Waals energy calculations implicated residues that form the hydrophobic binding site (W623, Q635, V637, Y640 and Y657) as critical for this selectivity.

In specific embodiments of the invention, there is identification of probes with one log or greater activity than 2^nd generation probes in SPR, HTFM and pStat3 assays. Furthermore, in certain aspects some of the most active 3^rd generation probes that emerge from this analysis are selective for Stat3 vs. Stat1 based on their greater interaction with the hydrophobic binding site within the Stat3 vs. Stat1 SH2 pY-peptide binding pocket.

Example 10

Exemplary Compositions of the Disclosure

Exemplary composition(s) of the disclosure are provided in Tables 6-11 below.

TABLE 6
IDNUMBER	Structure	Formula structure	MW	LogP
F1566-0306		C22H17NO3S2	407.5137	5.846
F1566-0318		C23H19NO3S2	421.5408	6.144
F1566-0330		C22H16ClNO3S2	441.9587	6.438
F1566-0342		C22H16BrNO3S2	486.4097	6.644
F1566-0366		C24H21NO3S2	435.5679	6.477
F1566-0414		C24H21NO3S2	435.5679	6.477
F1566-0438		C24H21NO3S2	435.5679	6.619
F1566-0450		C23H19NO4S2	437.5402	5.802
F1566-0462		C24H21NO4S2	451.5673	6.143
F1566-0486		C26H25NO3S2	463.6221	7.345
F1566-0510		C26H19NO3S2	457.5742	7.105
F1566-0546		C22H16N2O5S2	452.5112	5.818
F1566-0558		C23H18N2O5S2	466.5383	6.114
F1566-0618		C20H15NO3S3	413.5395	5.359
F1566-1606		C25H18N2O3S2	458.5618	6.046
F1566-1818		C18H17NO3S2	359.4691	4.705
F1566-1832		C19H19NO3S2	373.4962	5.147
F1566-1846		C20H21NO3S2	387.5233	5.589
F1566-1860		C17H15NO3S2	345.442	4.192
F5749-0371		C22H16N2O5S2	452.5112	5.781
F5749-0372		C22H23NO3S2	413.5615	6.171
F5749-0373		C25H23NO4S2	465.5944	6.468
F5749-0374		C23H18ClNO4S2	471.9852	6.429
F5749-0375		C24H21NO3S2	435.5679	6.438
F5749-0376		C24H19NO5S2	465.5507	5.787
F5749-0377		C24H20N2O4S2	464.566	5.137
F5749-0378		C24H21NO5S2	467.5667	5.54474
F5749-0379		C24H19NO5S2	465.5507	5.441
F5749-0380		C21H16N2O3S2	408.5013	4.613
F5749-0381		C18H18N2O3S2	374.4838	3.74
F5749-0382		C24H21NO3S2	435.5679	6.477
F5749-0383		C22H16N2O5S2	452.5112	5.779
F5749-0384		C23H19NO3S2	421.5408	5.98
F5749-0385		C20H14ClNO3S3	447.9845	6.649
F5749-0386		C22H15F2NO3S2	443.4946	6.187
F5749-0387		C21H19N3O3S2	425.5319	4.956
F5749-0388		C21H18N2O4S2	426.5166	4.99
F5749-0389		C23H22N2O5S2	470.5702	3.633
F5749-0390		C23H18FNO4S2	455.5306	5.99
F5749-0391		C24H21NO4S2	451.5673	6.135
F5749-0392		C26H20N2O3S2	472.5889	6.305
F5749-0393		C22H19NO3S3	441.5936	6.497
F5749-0394		C21H17NO3S3	427.5665	6.022
F5749-0395		C24H19NO3S2	433.5519	6.204
F5749-0396		C22H16FNO3S2	425.5041	5.997
F5749-0397		C23H19NO4S2	437.5402	5.839
F5749-0398		C22H16FNO3S2	425.5041	6.036
F5749-0399		C22H15ClFNO3S2	459.9492	6.626
F5749-0400		C23H16F3NO4S2	491.5115	7.24476
F5749-0401		C23H18ClNO3S2	455.9858	6.771
F5749-0402		C24H19NO4S2	449.5513	5.736
F5749-0403		C24H19NO4S2	449.5513	5.699
F5749-0404		C23H18ClNO3S2	455.9858	6.732
F5749-0405		C23H19NO4S2	437.5402	5.8
F5749-0406		C24H21NO4S2	451.5673	6.141
F5749-0407		C22H15F2NO3S2	443.4946	6.148
F5749-0408		C19H19NO3S2	373.4962	5.339
F5749-0409		C23H16F3NO3S2	475.5121	6.81776
F5749-0410		C23H16F3NO3S2	475.5121	6.78076
F5749-0411		C22H16ClNO3S2	441.9587	6.475
F5749-0412		C23H17Cl2NO3S2	490.4308	7.398
F5749-0413		C22H15F2NO3S2	443.4946	6.187
F5749-0414		C25H23NO3S2	449.595	7.061
F5749-0415		C26H23NO3S2	461.6061	6.933
F5749-0416		C26H20N2O5S2	504.5877	4.973
F5749-0417		C27H22N2O5S2	518.6148	5.415
F5749-0418		C23H20N2O4S3	484.6189	5.149
F5749-0419		C20H15N3O5S2	441.4877	2.891
F5749-0420		C25H20N2O4S2	476.5772	5.042
F5749-0421		C24H18N2O4S2	462.5501	4.954
F5749-0422		C22H19N3O5S2	469.5418	2.955
F5749-0423		C26H22N2O4S2	490.6042	5.277
F5749-0424		C23H18FNO3S2	439.5312	6.133
F5749-0425		C23H18FNO3S2	439.5312	6.17
F5749-0426		C25H23NO4S2	465.5944	6.206
F5749-0427		C28H25N3O3S2	515.6578	6.125
F5749-0428		C19H15N3O3S2	397.4777	3.986
F5749-0429		C27H23N3O3S2	501.6307	5.991
F5749-0430		C29H23NO5S2	529.6384	7.16174
F5749-0431		C28H20ClNO4S2	534.0569	8.046
F5749-0432		C29H23NO4S2	513.639	7.754
F5749-0433		C23H15ClF3NO3S2	509.9571	7.40776
F5749-0434		C28H21NO4S2	499.6119	7.456
F5749-0435		C22H16BrNO3S2	486.4097	6.642
F5749-0436		C22H16BrNO3S2	486.4097	6.681
F5749-0437		C22H15BrFNO3S2	504.4002	6.832
F5749-0438		C23H15BrF3NO3S2	554.4081	7.61376
F5749-0439		C22H16ClNO3S2	441.9587	6.436
F5749-0440		C22H17NO5S3	471.5765	5.046
F5749-0441		C23H16F3NO4S2	491.5115	7.24276

TABLE 7
IDNUMBER	Structure	Formula structure	MW	LogP
F0808-0081		C28H23NO45	469.5638	7.101
F0808-0084		C28H23NO5S	485.5632	6.767
F0808-0085		C26H18BrNO4S	520.4057	7.268
F0808-0086		C28H23NO4S	469.5638	7.243
F0808-0089		C30H21NO4S	491.5702	7.729
F0808-0091		C26H18FNO4S	459.5001	6.623
F0808-0092		C28H23NO4S	469.5638	7.101
F0808-0094		C26H18ClNO4S	475.9547	7.062
F1269-0222		C24H17NO4S2	447.5354	5.983
F1269-2003		C27H20N2O6S	500.5343	6.738
F1566-1138		C29H20N2O4S	492.5578	6.67
F5749-0001		C21H17NO4S	379.4379	4.816
F5749-0002		C26H18N2O6S	486.5072	6.405
F5749-0003		C26H25NO4S	447.5575	6.795
F5749-0004		C29H25NO5S	499.5903	7.092
F5749-0005		C27H20ClNO5S	505.9812	7.053
F5749-0006		C28H23NO4S	469.5638	7.062
F5749-0007		C28H21NO6S	499.5467	6.411
F5749-0008		C28H22N2O5S	498.5619	5.761
F5749-0009		C28H23NO6S	501.5626	6.16874
F5749-0010		C28H21NO6S	499.5467	6.065
F5749-0011		C25H18N2O4S	442.4972	5.237
F5749-0012		C22H19NO4S	393.465	5.329
F5749-0013		C28H23NO6S	501.5626	6.417
F5749-0014		C22H20N2O4S	408.4797	4.364
F5749-0015		C28H23NO4S	469.5638	7.101
F5749-0016		C26H18N2O6S	486.5072	6.403
F5749-0017		C23H21NO4S	407.4921	5.771
F5749-0018		C27H21NO4S	455.5367	6.604
F5749-0019		C24H23NO4S	421.5192	6.213
F5749-0020		C24H16ClNO4S2	481.9804	7.273
F5749-0021		C26H17F2NO4S	477.4905	6.811
F5749-0022		C25H21N3O4S	459.5278	5.58
F5749-0023		C25H20N2O5S	460.5126	5.614
F5749-0024		C27H24N2O6S	504.5661	4.257
F5749-0025		C27H20FNO5S	489.5266	6.614
F5749-0026		C28H23NO5S	485.5632	6.759
F5749-0027		C30H22N2O4S	506.5848	6.929
F5749-0028		C26H21NO4S2	475.5896	7.121
F5749-0029		C25H19NO4S2	461.5625	6.646
F5749-0030		C28H21NO4S	467.5479	6.828
F5749-0031		C26H18FNO4S	459.5001	6.621
F5749-0032		C27H21NO5S	471.5361	6.463
F5749-0033		C26H18FNO4S	459.5001	6.66
F5749-0034		C26H17ClFNO4S	493.9451	7.25
F5749-0035		C27H18F3NO5S	525.5074	7.86876
F5749-0036		C27H20ClNO4S	489.9818	7.395
F5749-0037		C28H21NO5S	483.5473	6.36
F5749-0038		C28H21NO5S	483.5473	6.323
F5749-0039		C27H20ClNO4S	489.9813	7.356
F5749-0040		C27H21NO5S	471.5361	6.424
F5749-0041		C28H23NO5S	485.5632	6.765
F5749-0042		C26H17F2NO4S	477.4905	6.772
F5749-0043		C23H21NO4S	407.4921	5.963
F5749-0044		C27H18F3NO4S	509.508	7.44176
F5749-0045		C27H18F3NO4S	509.508	7.40476
F5749-0046		C26H18ClNO4S	475.9547	7.099
F5749-0047		C27H19Cl2NO4S	524.4268	8.022
F5749-0048		C26H17F2NO4S	477.4905	6.811
F5749-0049		C29H25NO4S	483.5909	7.685
F5749-0050		C30H25NO4S	495.6021	7.557
F5749-0051		C30H22N2O6S	538.5836	5.597
F5749-0052		C31H24N2O6S	552.6107	6.039
F5749-0053		C27H22N2O5S2	518.6148	5.773
F5749-0054		C24H17N3O6S	475.4836	3.515
F5749-0055		C29H22N2O5S	510.5731	5.666
F5749-0056		C28H20N2O5S	496.546	5.578
F5749-0057		C26H21N3O6S	503.5378	3.579
F5749-0058		C30H24N2O5S	524.6002	5.901
F5749-0059		C27H20FNO4S	473.5272	6.757
F5749-0060		C27H20FNO4S	473.5272	6.794
F5749-0061		C29H25NO5S	499.5903	6.83
F5749-0062		C32H27N3O4S	549.6537	6.749
F5749-0063		C23H17N3O4S	431.4736	4.61
F5749-0064		C31H25N3O4S	535.6266	6.615
F5749-0065		C33H25NO6S	563.6343	7.78574
F5749-0066		C32H22ClNO5S	568.0528	8.67
F5749-0067		C33H25NO5S	547.6349	8.378
F5749-0068		C27H17ClF3NO4S	543.953	8.03176
F5749-0069		C32H23NO5S	533.6078	8.08
F5749-0070		C26H18BrNO4S	520.4057	7.266
F5749-0071		C26H18BrNO4S	520.4057	7.305
F5749-0072		C26H17BrFNO4S	538.3961	7.456
F5749-0073		C27H17BrF3NO4S	588.404	8.23776
F5749-0074		C26H18ClNO4S	475.9547	7.06
F5749-0075		C26H19NO6S2	505.5724	5.67
F5749-0076		C27H18F3NO5S	525.5074	7.86676

TABLE 8
IDNUMBER	Structure	Formula structure	MW	LogP
F1566-0329		C26H20N2O3S2	472.5889	6.344
F1566-0341		C25H17ClN2O3S2	493.0068	6.638
F1566-0353		C25H17BrN2O3S2	537.4578	6.844
F1566-0377		C27H22N2O3S2	486.616	6.677
F1566-0425		C27H22N2O3S2	486.616	6.677
F1566-0449		C27H22N2O3S2	486.616	6.819
F1566-0473		C27H22N2O4S2	502.6154	6.343
F1566-0497		C29H26N2O3S2	514.6702	7.545
F1566-0521		C29H20N2O3S2	508.6224	7.305
F1566-0557		C25H17N3O5S2	503.5593	6.018
F1566-0569		C26H19N3O5S2	517.5864	6.314
F1566-0617		C27H22N2O5S2	518.6148	5.993
F1566-0629		C23H16N2O3S3	464.5876	5.559
F1566-1608		C28H19N3O3S2	509.6099	6.246
F1566-1821		C21H18N2O3S2	410.5172	4.905
F1566-1835		C22H20N2O3S2	424.5443	5.347
F1566-1849		C23H22N2O3S2	438.5714	5.789
F1566-1863		C20H16N2O3S2	396.4901	4.392
F5749-0077		C25H17N3O5S2	503.5593	5.981
F5749-0078		C25H24N2O3S2	464.6096	6.371
F5749-0079		C28H24N2O4S2	516.6425	6.668
F5749-0080		C26H19ClN2O4S2	523.0333	6.629
F5749-0081		C27H22N2O3S2	486.616	6.638
F5749-0082		C27H20N2O5S2	516.5989	5.987
F5749-0083		C27H21N3O4S2	515.6141	5.337
F5749-0084		C27H22N2O5S2	518.6148	5.74474
F5749-0085		C27H20N2O5S2	516.5989	5.641
F5749-0086		C24H17N3O3S2	459.5494	4.813
F5749-0087		C21H19N3O3S2	425.5319	3.94
F5749-0088		C27H22N2O3S2	486.616	6.677
F5749-0089		C25H17N3O5S2	503.5593	5.979
F5749-0090		C26H20N2O3S2	472.5889	6.18
F5749-0091		C23H15ClN2O3S3	499.0326	6.849
F5749-0092		C25H16F2N2O3S2	494.5427	6.387
F5749-0093		C24H20N403S2	476.58	5.156
F5749-0094		C24H19N3O4S2	477.5647	5.19
F5749-0095		C26H23N3O5S2	521.6183	3.833
F5749-0096		C26H19FN2O4S2	506.5787	6.19
F5749-0097		C27H22N2O4S2	502.6154	6.335
F5749-0098		C29H216N3O3S2	523.637	6.505
F5749-0099		C25H20N2O3S3	492.6418	6.697
F5749-0100		C24H18N2O3S3	478.6147	6.222
F5749-0101		C27H20N2O3S2	484.6001	6.404
F5749-0102		C25H17FN2O3S2	476.5522	6.197
F5749-0103		C26H20N2O4S2	488.5883	6.039
F5749-0104		C25H17FN2O3S2	476.5522	6.236
F5749-0105		C25H16ClFN2O3S2	510.9973	6.826
F5749-0106		C26H17F3N2O4S2	542.5596	7.44476
F5749-0107		C26H19ClN2O3S2	507.0339	6.971
F5749-0108		C27H20N2O4S2	500.5995	5.936
F5749-0109		C27H20N2O4S2	500.5995	5.899
F5749-0110		C26H19ClN2O3S2	507.0339	6.932
F5749-0111		C26H20N2O4S2	488.5883	6
F5749-0112		C27H22N2O4S2	502.6154	6.341
F5749-0113		C25H16F2N2O3S2	494.5427	6.348
F5749-0114		C22H20N2O3S2	424.5443	5.539
F5749-0115		C26H17F3N2O3S2	526.5602	7.01776
F5749-0116		C26H17F3N2O3S2	526.5602	6.98076
F5749-0117		C25H17ClN2O3S2	493.0068	6.675
F5749-0118		C26H18Cl2N2O3S2	541.479	7.598
F5749-0119		C25H16F2N2O3S2	494.5427	6.387
F5749-0120		C28H24N2O3S2	500.6431	7.261
F5749-0121		C29H24N2O3S2	512.6542	7.133
F5749-0122		C29H21N3O5S2	555.6358	5.173
F5749-0123		C30H23N3O5S2	569.6629	5.615
F5749-0124		C26H21N3O4S3	535.667	5.349
F5749-0125		C23H16N4O5S2	492.5358	3.091
F5749-0126		C28H21N3O4S2	527.6253	5.242
F5749-0127		C27H19N3O4S2	513.5982	5.154
F5749-0128		C25H20N4O5S2	520.59	3.155
F5749-0129		C29H23N3O4S2	541.6524	5.477
F5749-0130		C26H19FN2O3S2	490.5793	6.333
F5749-0131		C26H19FN2O3S2	490.5793	6.37
F5749-0132		C28H24N2O4S2	516.6425	6.406
F5749-0133		C31H26N4O3S2	566.7059	6.325
F5749-0134		C22H16N4O3S2	448.5258	4.186
F5749-0135		C30H24N4O3S2	552.6788	6.191
F5749-0136		C32H24N2O5S2	580.6865	7.36174
F5749-0137		C31H21ClN2O4S2	585.105	8.246
F5749-0138		C32H24N2O4S2	564.6871	7.954
F5749-0139		C26H16ClF3N2O3S2	561.0052	7.60776
F5749-0140		C31H22N2O4S2	550.66	7.656
F5749-0141		C25H17BrN2O3S2	537.4578	6.842
F5749-0142		C25H17BrN2O3S2	537.4578	6.881
F5749-0143		C25H16BrFN2O3S2	555.4483	7.032
F5749-0144		C26H16BrF3N2O3S2	605.4562	7.81376
F5749-0145		C25H17ClN2O3S2	493.0068	6.636
F5749-0146		C25H18N2O5S3	522.6246	5.246
F5749-0147		C26H17F3N2O4S2	542.5596	7.44276

TABLE 9
IDNUMBER	Structure	Formula structure	MW	LogP
F1565-0253		C18H14N4O3S2	398.4653	3.698
F1566-0328		C19H16N4O3S2	412.4924	3.996
F1566-0340		C18H13ClN4O3S2	432.9103	4.29
F1566-0520		C22H16N4O3S2	448.5258	4.957
F1566-0556		C18H13N5O5S2	443.4628	3.67
F1566-0568		C19H15N5O5S2	457.4899	3.966
F1566-0616		C20H18N4O5S2	458.5183	3.645
F1566-0628		C16H12N4O3S3	404.491	3.211
F1566-0148		C13H12N4O3S2	336.3936	2.044
F5749-0149		C18H13N5O5S2	443.4628	3.633
F5749-0150		C18H20N4O3S2	404.5131	4.023
F5749-0151		C21H20N4O4S2	456.546	4.32
F5749-0152		C19H15ClN4O4S2	462.9368	4.281
F5749-0153		C20H18N4O3S2	426.5195	4.29
F5749-0154		C20H16N4O5S2	456.5023	3.639
F5749-0155		C20H17N5O4S2	455.5176	2.989
F5749-0156		C20H18N4O5S2	458.5183	3.39674
F5749-0157		C20H16N4O5S2	456.5023	3.293
F5749-0158		C17H13N5O3S2	399.4529	2.465
F5749-0159		C14H14N4O3S2	350.4207	2.557
F5749-0160		C14H15N5O3S2	365.4354	1.592
F5749-0161		C20H18N4O3S2	426.5195	4.329
F5749-0162		C18H13N5O5S2	443.4628	3.631
F5749-0163		C15H16N4O3S2	364.4478	2.999
F5749-0164		C19H16N4O3S2	412.4924	3.832
F5749-0165		C16H18N4O3S2	378.4749	3.441
F5749-0166		C16H11ClN4O3S3	438.9361	4.501
F5749-0167		C18H12F2N4O3S2	434.4461	4.039
F5749-0168		C17H16N6O3S2	416.4835	2.808
F5749-0169		C17H15N5O4S2	417.4682	2.842
F5749-0170		C19H19N5O5S2	461.5218	1.485
F5749-0171		C19H15FN4O4S2	446.4822	3.842
F5749-0172		C20H18N4O4S2	442.5189	3.987
F5749-0173		C22H17N5O3S2	463.5405	4.157
F5749-0174		C21H15N5O3S2	449.5134	3.898
F5749-0175		C18H16N4O3S3	432.5452	4.349
F5749-0176		C17H14N4O3S3	418.5181	3.874
F5749-0177		C20H16N4O3S2	424.5035	4.056
F5749-0178		C18H13FN4O3S2	416.4557	3.849
F5749-0179		C19H16N4O4S2	428.4918	3.691
F5749-0180		C18H13FN4O3S2	416.4557	3.888
F5749-0181		C18H12ClFN4O3S2	450.9007	4.478
F5749-0182		C19H13F3N4O4S2	482.4631	5.09676
F5749-0183		C19H15ClN4O3S2	446.9374	4.623
F5749-0184		C20H16N4O4S2	440.5029	3.588
F5749-0185		C20H16N4O4S2	440.5029	3.551
F5749-0186		C19H15ClN4O3S2	446.9374	4.584
F5749-0187		C19H16N4O4S2	428.4918	3.652
F5749-0188		C20H18N4O4S2	442.5189	3.993
F5749-0189		C18H12F2N4O3S2	434.4461	4
F5749-0190		C15H16N4O3S2	364.4478	3.191
F5749-0191		C19H13F3N4O3S2	466.4637	4.66976
F5749-0192		C19H13F3N4O3S2	466.4637	4.63276
F5749-0193		C18H13ClN4O3S2	432.9103	4.327
F5749-0194		C19H14Cl2N4O3S2	481.3824	5.25
F5749-0195		C18H12F2N4O3S2	434.4461	4.039
F5749-0196		C21H20N4O3S2	440.5466	4.913
F5749-0197		C22H20N4O3S2	452.5577	4.785
F5749-0198		C22H17N5O5S2	495.5393	2.825
F5749-0199		C23H19N5O5S2	509.5664	3.267
F5749-0200		C19H17N5O4S3	475.5704	3.001
F5749-0201		C16H12N6O5S2	432.4392	0.743
F5749-0202		C21H17N5O4S2	467.5287	2.894
F5749-0203		C20H15N5O4S2	453.5017	2.806
F5749-0204		C18H16N6O5S2	460.4934	0.807
F5749-0205		C22H19N5O4S2	481.5558	3.129
F5749-0206		C19H15FN4O3S2	430.4828	3.985
F5749-0207		C19H15FN4O3S2	430.4828	4.022
F5749-0208		C21H20N4O4S2	456.546	4.058
F5749-0209		C24H22N6O3S2	506.6093	3.977
F5749-0210		C15H12N6O3S2	388.4293	1.838
F5749-0211		C23H20N6O3S2	492.5823	3.843
F5749-0212		C25H20N4O5S2	520.59	5.01374
F5749-0213		C24H17ClN4O4S2	525.0085	5.898
F5749-0214		C25H20N4O4S2	504.5906	5.606
F5749-0215		C19H12ClF3N4O3S2	500.9087	5.25976
F5749-0216		C24H18N4O4S2	490.5635	5.308
F5749-0217		C18H13BrN4O3S2	477.3613	4.494
F5749-0218		C18H13BrN4O3S2	477.3613	4.533
F5749-0219		C18H12BrFN4O3S2	495.3517	4.684
F5749-0220		C19H12BrFN4O3S2	545.3597	5.46576
F5749-0221		C18H13ClN4O3S2	432.9103	4.288
F5749-0222		C18H14N4O5S3	462.5281	2.898
F5749-0223		C19H13F3N4O4S2	482.4631	5.09476

TABLE 10
IDNUMBER	Structure	Formula structure	MW	LogP
F0808-0128		C25H20N2O3S3	492.6418	6.892
F0808-0132		C23H16N2O3S3	464.5876	6.261
F0808-0133		C23H15ClN2O3S3	499.0326	6.853
F0808-0134		C24H18N2O3S3	478.6147	6.559
F0808-0136		C25H20N2O3S3	492.6418	7.034
F0808-0137		C23H15BrN2O3S3	543.4836	7.059
F1269-0225		C21H14N2O3S4	470.6133	5.774
F1269-1420		C24H18N2O4S3	494.6141	6.217
F1566-1144		C26H17N3O3S3	515.6357	6.461
F1566-1584		C24H17N3O5S3	523.6122	6.529
F1566-1596		C25H20N2O5S3	524.6406	6.208
F1566-1816		C19H16N2O3S3	416.543	5.12
F1566-1830		C20H18N2O3S3	430.5701	5.562
F1566-1844		C21H20N2O3S3	444.5972	6.004
F1566-1858		C18H14N2O3S3	402.5159	4.607
F5749-0224		C23H15N3O5S3	509.5851	6.196
F5749-0225		C23H22N2O3S3	470.6354	6.586
F5749-0226		C26H22N2O4S3	522.6682	6.883
F5749-0227		C24H17ClN2O4S3	529.0591	6.844
F5749-0228		C25H20N2O3S3	492.6418	6.853
F5749-0229		C25H18N2O5S3	522.6246	6.202
F5749-0230		C25H19N3O4S3	521.6399	5.552
F5749-0231		C25H20N2O5S3	524.6406	5.95974
F5749-0232		C25H18N2O5S3	522.6246	5.856
F5749-0233		C22H15N3O3S3	465.5752	5.028
F5749-0234		C19H17N3O3S3	431.5576	4.155
F5749-0235		C25H20N2O3S3	492.6418	6.892
F5749-0236		C23H15N3O5S3	509.5851	6.194
F5749-0237		C24H18N2O3S3	478.6147	6.395
F5749-0238		C21H13ClN2O3S4	505.0584	7.064
F5749-0239		C23H14F2N2O3S3	500.5684	6.602
F5749-0240		C22H18N4O3S3	482.6058	5.371
F5749-0241		C22H17N3O4S3	483.5905	5.405
F5749-0242		C24H21N3O5S3	527.6441	4.048
F5749-0243		C24H17FN2O4S3	512.6045	6.405
F5749-0244		C25H20N2O4S3	508.6412	6.55
F5749-0245		C27H19N3O3S3	529.6628	6.72
F5749-0246		C23H18N2O3S4	498.6675	6.912
F5749-0247		C22H16N2O3S4	484.6404	6.437
F5749-0248		C25H18N2O3S3	490.6258	6.619
F5749-0249		C23H15FN2O3S3	482.578	6.412
F5749-0250		C24H18N2O4S3	494.6141	6.254
F5749-0251		C23H15FN2O3S3	482.578	6.451
F5749-0252		C23H14ClFN2O3S3	517.023	7.041
F5749-0253		C24H15F3N2O4S3	548.5854	7.65976
F5749-0254		C24H17ClN2O3S3	513.0597	7.186
F5749-0255		C25H18N2O4S3	506.6252	6.151
F5749-0256		C25H18N2O4S3	506.6252	6.114
F5749-0257		C24H17ClN2O3S3	513.0597	7.147
F5749-0258		C24H18N2O4S3	494.6141	6.215
F5749-0259		C25H20N2O4S3	508.6412	6.556
F5749-0260		C23H14F2N2O3S3	500.5684	6.563
F5749-0261		C20H18N2O3S3	430.5701	5.754
F5749-0262		C24H15F3N2O3S3	532.586	7.23276
F5749-0263		C24H15F3N2O3S3	532.586	7.19576
F5749-0264		C23H15ClN2O3S3	499.0326	6.89
F5749-0265		C24H16Cl2N2O3S3	547.5047	7.813
F5749-0266		C23H14F2N2O3S3	500.5684	6.602
F5749-0267		C26H22N2O3S3	506.6688	7.476
F5749-0268		C27H22N2O3S3	518.68	7.348
F5749-0269		C27H19N3O5S3	561.6616	5.388
F5749-0270		C28H21N3O5S3	575.6887	5.83
F5749-0271		C24H19N3O4S4	541.6927	5.564
F5749-0272		C21H14N4O5S3	498.5615	3.306
F5749-0273		C26H19N3O4S3	533.651	5.457
F5749-0274		C25H17N3O4S3	519.6239	5.369
F5749-0275		C23H18N4O5S3	526.6157	3.37
F5749-0276		C27H21N3O4S3	547.6781	5.692
F5749-0277		C24H17FN2O3S3	496.6051	6.548
F5749-0278		C24H17FN2O3S3	496.6051	6.585
F5749-0279		C26H22N2O4S3	522.6682	6.621
F5749-0280		C29H24N4O3S3	572.7316	6.54
F5749-0281		C20H14N4O3S3	454.5516	4.401
F5749-0282		C28H22N4O3S3	558.7045	6.406
F5749-0283		C30H22N2O5S3	586.7122	7.57674
F5749-0284		C29H19ClN2O4S3	591.1308	8.461
F5749-0285		C30H22N2O4S3	570.7128	8.169
F5749-0286		C24H14ClF3N2O3S3	567.031	7.82276
F5749-0287		C29H20N2O4S3	556.6858	7.871
F5749-0288		C23H15BrN2O3S3	543.4836	7.057
F5749-0289		C23H15BrN2O3S3	543.4836	7.096
F5749-0290		C23H14BrFN2O3S3	561.474	7.247
F5749-0291		C24H14BrF3N2O3S3	611.482	8.02876
F5749-0292		C23H15ClN2O3S3	499.0326	6.851
F5749-0293		C23H16N2O5S4	528.6504	5.461
F5749-0294		C24H15F3N2O4S3	548.5854	7.65776

TABLE 11
ID NUMBER	Structure	Formula structure	MW	LogP
F0433-0038		C16H12ClNO3S	333.7959	4.192
F0433-0041		C17H14ClNO3S	347.823	4.49
F0433-0044		C16H11Cl2NO3S	368.241	4.784
F0433-0047		C17H14ClNO4S	363.8224	4.148
F0433-0050		C20H14ClNO3S	383.8565	5.451
F0808-1895		C18H16ClNO3S	361.8501	4.823
F0808-1902		C16H11BrClNO3S	412.692	4.99
F0808-1909		C16H11ClN2O5S	378.7935	4.164
F0808-1913		C18H16ClNO3S	361.8501	4.823
F0808-1914		C20H20ClNO3S	389.9043	5.691
F1269-0272		C14H10ClNO3S2	339.8217	3.705
F1269-1995		C17H13ClN2O5S	392.8206	4.46
F1566-1223		C19H13ClN2O3S	384.8441	4.392
F5749-0295		C11H10ClNO3S	271.7243	2.538
F5749-0296		C16H11ClN2O5S	378.7935	4.127
F5749-0297		C16H18ClNO3S	339.8438	4.517
F5749-0298		C19H18ClNO4S	391.8766	4.814
F5749-0299		C17H13Cl2NO4S	398.2675	4.775
F5479-0300		C18H16ClNO3S	361.8501	4.784
F5749-0301		C18H14ClNO5S	391.833	4.133
F5749-0302		C18H15ClN2O4S	390.8483	3.483
F5749-0303		C18H16ClNO5S	393.8489	3.89074
F5749-0304		C18H14ClNO5S	391.833	3.787
F5749-0305		C15H11ClN2O3S	334.7835	2.959
F5749-0306		C12H12ClNO3S	285.7513	3.051
F5749-0307		C18H16ClNO5S	393.8489	4.139
F5749-0308		C12H13ClN2O3S	300.766	2.086
F5749-0309		C18H16ClNO3S	361.8501	4.823
F5749-0310		C16H11ClN2O5S	378.7935	4.125
F5749-0311		C13H14ClNO3S	299.7784	3.493
F5749-0312		C17H14ClNO3S	347.823	4.326
F5749-0313		C14H16ClNO3S	313.8055	3.935
F5749-0314		C14H9Cl2NO3S2	374.2667	4.995
F5749-0315		C16H10ClF2NO3S	369.7768	4.533
F5749-0316		C15H14ClN3O3S	351.8141	3.302
F5749-0317		C15H13ClN2O4S	352.7989	3.336
F5749-0318		C17H17ClN2O5S	396.8524	1.979
F5749-0319		C17H13ClFNO4S	381.8129	4.336
F5749-0320		C18H16ClNO4S	377.8495	4.481
F5749-0321		C20H15ClN2O3S	398.8712	4.651
F5749-0322		C16H14ClNO3S2	367.8759	4.843
F5749-0323		C15H12ClNO3S2	353.8488	4.368
F5749-0324		C18H14ClNO3S	359.8342	4.55
F5749-0325		C16H11ClFNO3S	351.7864	4.343
F5749-0326		C17H14ClNO4S	363.8224	4.185
F5749-0327		C16H11ClFNO3S	351.7864	4.382
F5749-0328		C16H10Cl2FNO3S	386.2314	4.972
F5749-0329		C17H11ClF3NO4S	417.7937	5.59076
F5749-0330		C17H13Cl2NO3S	382.2681	5.117
F5749-0331		C18H14ClNO4S	375.8336	4.082
F5749-0332		C18H14ClNO4S	375.8336	4.045
F5749-0333		C17H13Cl2NO3S	382.2681	5.078
F5749-0334		C17H14ClNO4S	363.8224	4.146
F5749-0335		C18H16ClNO4S	377.8495	4.487
F5749-0336		C16H10ClF2NO3S	369.7768	4.494
F5749-0337		C13H14ClNO3S	299.7784	3.685
F5749-0338		C17H11ClF3NO3S	401.7943	5.16376
F5749-0339		C17H11ClF3NO3S	401.7943	5.12676
F5749-0340		C16H11Cl2NO3S	368.241	4.821
F5749-0341		C17H12Cl3NO3S	416.7131	5.744
F5749-0342		C16H10ClF2NO3S	369.7768	4.533
F5749-0343		C19H18ClNO3S	375.8772	5.407
F5749-0344		C20H18ClNO3S	387.8884	5.279
F5749-0345		C20H15ClN2O5S	430.87	3.319
F5749-0346		C21H17ClN2O5S	444.897	3.761
F5749-0347		C17H15ClN2O4S2	410.9011	3.495
F5749-0348		C14H10ClN3O5S	367.7699	1.237
F5749-0349		C19H15ClN2O4S	402.8594	3.388
F5749-0350		C18H13ClN2O4S	388.8323	3.3
F5749-0351		C16H14ClN3O5S	395.8241	1.301
F5749-0352		C20H17ClN2O4S	416.8865	3.623
F5749-0353		C17H13ClFNO3S	365.8135	4.479
F5749-0354		C17H13ClFNO3S	365.8135	4.516
F5749-0355		C19H18ClNO4S	391.8766	4.552
F5749-0356		C22H20ClN3O3S	441.94	4.471
F5749-0357		C13H10ClN3O3S	323.76	2.332
F5749-0358		C21H18ClN3O3S	427.9129	4.337
F5749-0359		C23H18ClNO5S	455.9206	5.50774
F5749-0360		C22H15Cl2NO4S	460.3392	6.392
F5749-0361		C23H18ClNO4S	439.9212	6.1
F5749-0362		C17H10Cl2F3NO3S	436.2394	5.75376
F5749-0363		C22H16ClNO4S	425.8941	5.802
F5749-0364		C16H11BrClNO3S	412.692	4.988
F5749-0365		C16H11BrClNO3S	412.692	5.027
F5749-0366		C16H10BrClFNO3S	430.6824	5.178
F5749-0367		C17H10BrClF3NO3S	480.6904	5.95976
F5749-0368		C16H11Cl2NO3S	368.241	4.782
F5749-0369		C16H12ClNO5S2	397.8587	3.392
F5749-0370		C17H11ClF3NO4S	417.7937	5.58876

Example 11

A Role for Stat3 Signaling in Mast Cell Degranulation

Autosomal-dominant hyper-IgE syndrome (AD-HIES) patients carry dominant-negative STAT3 mutations, develop frequent skin and lung infections, and also have a variety of non-immunologic manifestations affecting bones and connective tissue. In addition, almost all have an eczematous rash present very early in life, as well as the markedly elevated serum IgE levels which give the disease its name. Of note, one-third of patients in the broader population with atopic dermatitis develop food allergies. Despite these observations, the susceptibility of AD-HIES patients to specific food allergies has not been carefully examined. The inventors have found that fewer AD-HIES patients develop food allergies and anaphylaxis than patients with marked IgE elevations and eczema without STAT3 mutations. In embodiments of the invention, this is due at least in part to the effects of defective STAT3 signaling on mast cell degranulation.

Thirty eight percent of STAT3-mutant patients had immediate hypersensitivity to food, significantly less than the 58.3% observed in atopic patients without a STAT3 mutation (FIG. 19A). Far fewer AD-HIES patients had anaphylaxis to a food allergen than atopic controls (8.5% vs. 33.3%) (FIG. 19B).

Furthermore, silencing of STAT3 expression inhibited mast cell degranulation following IgE crosslinking in direct proportion to the degree of silencing of STAT3 in LAD2 cells (FIG. 21). Similarly, silencing of STAT3 in primary human mast cells lead to decreased IgE-mediated mast cell degranulation (FIG. 20B).

Example 12

Examples of Compositions for Anaphylaxis Treatment

One or more compositions are characterized as anaphylaxis treatment and/or prevention using standard means in the art. In certain cases, a rodent model of anaphylaxis is employed to test one or more compositions of interest for effectiveness in anaphylaxis. In at least some aspects, a temperature drop in the rodent is used as a measure of anaphylaxis.

Systemic Anaphylaxis Assay

Any suitable in vivo model of anaphylaxis may be employed. Mice may be sensitized (i.v., for example) with an effective amount of DNP-specific IgE (H1-DNP-e_-26) in an appropriate buffer and challenged (i.v., for example) after an appropriate amount of time (24 h, for example) with an effective amount of rat anti-mouse IgE.

Alternatively, anaphylaxis may be induced by injection (i.v.) of compound 48/80 (Sigma Aldrich) at a sub-lethal concentration (for example, concentrations less than 100 μg in 200 μl of buffer were lethal). Implantable electronic transponders (Bio Medic Data Systems) may be inserted under the dorsal skin of anesthetized mice at least 24 hrs before the start of the anaphylaxis studies. Basal body temperatures before induction of anaphylaxis and temperature changes during anaphylaxis may be monitored using an electronic scanner (Bio Medic Data Systems).

Therapeutic Assay

In certain embodiments, the composition being tested (for example, Cpd 188-9) is provided to the individual for a period of at least 1, 2, 3, 4, 5, 6, 7, or more days; the administration may be by any suitable route, including intravenous, subcutaneous, aerosolization, inhalation, orally, and so forth. Following this period of time, anaphylaxis may be induced in the model system.

FIG. 22 demonstrates effective treatment in an anaphylaxis model using Cpd188-9. Mice as an anaphylaxis model were pre-treated with 50 mg/kg Cpd 188-9 or which vehicle for one week, after which anaphylaxis was induced via systemic IgE cross-linking at time 0. Detectably within at least 40 minutes the reverse in temperature drop occurred, demonstrating effective use in anaphylaxis conditions.

FIG. 23 illustrates dose response using different examples of dosages of Cpd188-9 on normal human mast cells in vitro. Beta-hexosaminidase (% release) is used as an example of a measure of mast cell degranulation, which reflects the intensity of anaphylaxis. With increasing amounts of Cpd 188-9 administered for at least three days prior, mast cell degranulation was reduced.

FIG. 24 shows that systemic anaphylaxis was prevented in vivo with an exemplary STAT3 inhibitor. Healthy wild-type mice were pretreated for either one day (top panel) or one week (bottom panel) with C188-9 at 50 mg/kg. Mice were then injected with IgE specific for an antigen, and the following day the antigen was injected and drop in body temperature recorded as a measure of anaphylaxis. Inhibition of the drop is shown only in the bottom panel, when mice were pretreated (in red) for one week with C188-9.

FIG. 25 demonstrates that peripheral and central vascular leakage is decreased by Cpd 188-9. Mice were pretreated with C188-9 for one week as in FIG. 24, then injected with a dye to measure the inhibition by C188-9 of locally induced IgE-mediated vascular leakage (top left) or mast cell secretagogue C48/80-induced vascular leakage (top right) or platelet activating factor-induced drop in hematocrit—a measure of vascular leakage (bottom).

FIG. 26 illustrates that the effect of Cpd188-9 is not because of a decreased mast cell degranulation in vivo. Serum histamine and MCPT-1 levels are shown at 90 seconds (left panels) or 30 minutes (right panels) after Ag-challenge after 1 week C188-9 treatment as in FIG. 24. There was no statistical difference, suggesting that mast cell degranulation was not a factor in C188-9 mediated inhibition in mice.

FIG. 27 demonstrates the effect of Cpd 188-9 on Ag-induced degranulation in murine mast cells. Pretreatment of murine bone marrow derived mast cells or peritoneal derived mast cells with C188-9 does not lead to inhibition of mast cell degranulation, in contrast to human mast cells as in FIG. 23. Only incubation of mouse peritoneal mast cells with IL-6 (left panel) enables mast cells to be mildly inhibited by C188-9.

FIG. 28 shows a schematic representation of transwell permeability assay to measure vascular endothelial cell permeability in response to soluble factors and inhibitors.

FIG. 29 demonstrates inhibition of vascular permeability of human umbilical vein endothelial cells (HUVECs) by C188-9 pretreated for one week+/−IL-6. DMSO was used as a control for C188-9. Transwell assay was performed in response to 100 um of histamine. Maximal inhibition was seen with 1 ug C188-9 (on right).

FIG. 30 shows that Hyper-IgE syndrome mouse is resistant to anaphylaxis (Siegel et al., JACI, 2013). Systemic anaphylaxis is induced as in FIG. 24 in a mouse model of the Hyper-IgE syndrome. Mice with dominant negative STAT3 mutations were less prone to severe temperature drop with IgE crosslinking than littermate controls.

FIG. 31 demonstrates STAT3 mutant (HIES6) HUVECS resistant to histamine-induced permeability. Human umbilical vein endothelial cells derived from patients with dominant negative STAT3 mutations (HIES6) were less responsive to histamine induced vascular permeability than healthy controls (labeled HUVECS). This is direct evidence that impaired STAT3 signaling leads to impaired vascular permeability responses to histamine.

Thus, Cpd188-9 pretreatment of 7 days (but under at least some conditions not one) inhibits systemic anaphylaxis in vivo. In specific embodiments, the action is more in vascular endothelial responses to STAT3 than in mast cells, whereas human mast cell and endothelial responses are both highly affected.

REFERENCES

All patents and publications cited herein are hereby incorporated by reference in their entirety herein. Full citations for the references cited herein are provided in the following list.

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Claims

1. A method of treating anaphylaxis or anaphylactic shock in a human individual, comprising administering to the individual having anaphylaxis or anaphylactic shock a therapeutically effective amount of a Stat3 inhibitor, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, a functional derivative thereof, or a salt thereof, wherein the anaphylaxis or anaphylactic shock is induced by one or more allergens selected from the group consisting of food, venom, medication, an environmental allergen or seasonal allergen, and latex, wherein the therapeutically effect amount is an amount of at most about 100 mg/kg to inhibit Stat3 in the human individual and does not induce toxicity in the human individual.

2. The method of claim 1, wherein the individual is provided the composition in multiple doses.

3. The method of claim 2, wherein the multiple doses are separated by minutes, hours, days, or weeks.

4. The method of claim 1, wherein the individual is provided with an additional therapy for the anaphylaxis.

5. The method of claim 1, wherein the composition is delivered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, sublingually, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion, via a catheter, via a lavage, in lipid compositions, in liposome compositions, or as an aerosol.

6. The method of claim 1, wherein the therapeutically effective amount is an amount of about 10 mg/kg to about 100 mg/kg.