WO2011094749A2

WO2011094749A2 - Small molecule inhibitors that block assembly of the tgf-beta signaling complex

Info

Publication number: WO2011094749A2
Application number: PCT/US2011/023380
Authority: WO
Inventors: Andrew Hinck; Luzhe Sun; York Tomita; Yumi Ueda
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2010-02-01
Filing date: 2011-02-01
Publication date: 2011-08-04
Also published as: WO2011094749A3

Abstract

A method of treating fibrotic disorders or cancer in a subject comprises administering to a subject who would benefit from such treatment a therapeutically effective amount of one or more TGF-β inhibitors. Methods for screening for compounds that inhibit assembly of the TGF-β ternary complex are also disclosed.

Description

TITLE: SMALL MOLECULE INHIBITORS THAT BLOCK ASSEMBLY OF THE

TGF-BETA SIGNALING COMPLEX

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to compounds for the treatment of fibrotic disorders and cancer. More particularly, the invention relates to the treatment of fibrotic disorders and cancer using inhibitors of the TGF-β signaling complex.

2. Description of the Relevant Art

TGF-β isoforms (TGF-βΙ, -β2, and -β3) are vertebrate signaling ligands that coordinate wound repair, suppress the immune system, and regulate the growth and differentiation of epithelial cells. TGF^s, as well as other ligands of the TGF-β superfamily, such as Activins, Bone Morphogenetic Proteins (BMPs), and Growth and Differentiation Factors (GDFs), signal by binding and bringing together two single-pass transmembrane receptor kinases, known as RI and RII. This triggers a phosphorylation cascade, wherein RII phosphorylates RI, leading to its activation, and the subsequent phosphorylation of cytoplasmic effectors, a major one of which are the Smads.

Seven RIs and five RIIs have been identified in humans. Specificities have been determined based on cell-based affinity labeling studies with radiolabeled ligands and have enabled the identification of major ligands for most receptors of the superfamily, including those specific for TGF-ps, Activins, BMPs, and GDFs. Such studies suggest that there appear to be two fundamental mechanisms of assembly, the interdependent one exemplified by TGF^s and Activins and the independent one exemplified by BMPs and GDFs. Structural studies of the TGF-β and BMP receptor extracellular domains complexed to their cognate ligands have revealed that although ligands and receptors of the different subfamilies share the same overall fold, they nevertheless bind and assemble their RIs and RIIs into complexes in ways that are entirely distinct. Such alternate modes of assembly arise from differences in the manner of RI and RII binding and are brought about by simple evolutionary modifications of the receptor three- finger toxin fold, including changes in the length of critical contact loops and terminal regions. Such results are significant since they provide a striking example of how simple evolutionary modifications of ligands and receptors of the superfamily have occurred to expand the range of specificity, and hence range of function within the superfamily. TGF-β isoforms play prominent roles in human disease, especially cancer. The loss of the tumor suppressive activity of TGF-β by mutational inactivation of the protein components of the TGF-β pathway leads to certain hereditary forms of colon and pancreatic cancer. These types of defects, however, are relatively rare and in most human cancers, including those of the breast, the TGF-β pathway typically remains intact. This usually has adverse consequences, as many cancer cells over express c-myc, which antagonizes TGF-βδ ability to inhibit cell growth. Furthermore, the tumor promoting activities of TGF-β, including its ability to function as a potent suppressor of the immune system, induce endothelial-tomesenchymal transitions, and promote angiogenesis, remain intact. It is this complex response, coupled with its overexpression in most cancer cells, that is thought to underlie TGF^'s demonstrated role in the growth and invasiveness of many cancers, including those of the breast and brain. For example, TGF-β has also been has been demonstrated to play an important role in the metastasis of breast cancer cells to the lungs and to bone, the leading cause of morbidity and mortality in patients with advanced breast cancer. In metastatic breast cancer, TGF-β is thought to drive a "vicious" cycle, whereby the breast cancer cells release osteolytic and pro-angiogenic factors, such IL11 and OPN and CTGF and FGF5, respectively. In turn, TGF-β, which is stored in bone, is released and acts on the cancer cells to induce pro-metastatic factors, such as CTGF and IL11 through the Smad pathway. In the brain, high level TGF-β expression has been shown to potently suppress immune surveillance, promoting tumor growth and metastasis. In clinical trials, antisense oligonucleotides that repress the expression of TGF^2 have proven to have significant efficacy in prolonging survival rates in patients with recurrent high-grade glioma and anaplastic astrocytoma.

The other major pathological response associated with TGF-β is tissue fibrosis. TGF^'s role in fibrosis of organs such as the kidney and lung has been ascribed to the coordinate effect that TGF-β has in promoting the accumulation of protein components of the extracellular matrix, such as collagen and fibronectin.

Though TGF-p's role as a tumor-promoter and as a promoter of fibrosis had been clearly established, it was less clear whether TGF-β could be safely inhibited due to its established role as a tumor suppressor. To address this, a transgenic mouse that expressed a soluble form of the TGF-β type II receptor (sRII) at high levels has been studied. The results showed that sRII expressing mice, an established TGF- β antagonist, rendered the mice no more prone to tumorigenesis than normal mice. The mice were also found to be much less prone to undergo metastasis in an MMTV-neu model of metastatic breast cancer. These results showed that tumor- promoting activities of TGF-β can indeed be inhibited without significantly interfering with its tumor suppressor activity. Through other studies, it has also been established that many tumor cells produce TGF-β at high-levels, and hence it has been proposed that this "excessive" pool of TGF-β drives tumor progression and metastasis.

A number of strategies have been proposed for inhibiting TGF-β, including TGF-β monoclonal antibodies, soluble forms of the TGF-β receptors, TGF-β antisense oligonucleotides, and small molecules that target the ATP binding site of the ΤβΜ kinase. Although, each offers potential for treating fibrotic disorders and cancer, none have been approved for clinical use. Additionally, each has specific disadvantages, such as the antisense oligonucleotides, monoclonal antibodies, and soluble receptors that have limited stabilities. Additionally, in some cases, specificities are either too limited, such as the ΤβΙ Ι kinase inhibitors, which target a number of other related kinases, including the TGF-β superfamily type I receptor, ActRIb, or too narrow, such as the soluble TGF-β type III receptor that targets predominantly TGF-P2, but not TGF^s 1 and 3, or the soluble TGF-β type II receptor that targets predominantly TGF-Ps 1 and 3, but not TGF-P2.

SUMMARY OF THE INVENTION

A method of treating fibrotic disorders or cancer in a subject comprises administering to a subject who would benefit from such treatment a therapeutically effective amount of one or more TGF-β inhibitors.

In one embodiment, the TGF-β inhibitor has the structure:

(I)

where X is

where L¹ is -NHC(O)-, -N=N- -NH-, -OC(O)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR^², -SO3R¹, -NC(0)-R³, -OC(0)-R³,

N=CR¹R², at least one R being -S0₃R^!; where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

In another embodiment, the TGF-β inhibitor has the structure:

(IV)

where X

or

where L² is -NHC(O)-, -N=N- -NH-, -OC(O)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR^², -SO3R¹, -NC(0)-R³, -OC(0)-R³, or - N=CR¹R², at least one R being -SC^R¹;

where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

In an embodiment, a phamiaceutical composition for treating fibrotic disorders or cancer in a subject comprises one or more TGF-β inhibitors and a pharmaceutically acceptable carrier.

In an embodiment, a method of screening for compounds that inhibit assembly of the TGF-β ternary complex includes measuring the degree of polarization exhibited by fluorescently- tagged TfiRl extracellular domain as its binds to TβRII:TGF-β3 complex in the presence of one or more compounds. The measured change in the degree of polarization may be used to determine the inhibitory effect of the one or more compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which: FIG. 1 depicts graphical data from a rapid fluorescence polarization (FP) assay used to screen for compounds that inhibit assembly of the TGF-β ternary complex;

FIG. 2 depicts fluorescence polarization (FP) assay results monitored when a ternary complex of TGF-β is formed or it is reversed by the novel TGF-β inhibitors;

FIGS. 3A-3C depict the results of SPR experiments to detect direct binding of TGF-β inhibitors to immobilized ΤβΡνΙ;

FIGS. 4A-4C depicts the results of the effect of TGF-β inhibitors on binding of ΤβΡνΙ to TGF^3 in the presence of a saturating concentration of ΤβΡνΙΙ (4 μΜ); and

FIG. 5 depicts data collected from Cell-based Smad phosphorylation assays to evaluate the biological activity of the novel TGF-β inhibitors.

While the invention may be susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. The drawings may not be to scale. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood the present invention is not limited to particular devices or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include singular and plural referents unless the content clearly dictates otherwise.

The terms used throughout this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the devices and methods of the invention and how to make and use them. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed in greater detail herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term.

As used herein the terms "administration," "administering," or the like, when used in the context of providing a pharmaceutical or nutraceutical composition to a subject generally refers to providing to the subject one or more pharmaceutical, "over-the-counter" (OTC) or

nutraceutical compositions in combination with an appropriate delivery vehicle by any means such that the administered compound achieves one or more of the intended biological effects for which the compound was administered. By way of non-limiting example, a composition may be administered by parenteral, subcutaneous, intravenous, intracoronary, rectal, intramuscular, intra- peritoneal, transdermal, or buccal routes of delivery. Alternatively, or concurrently,

administration may be by the oral route. The dosage administered will be dependent upon the age, health, weight, and/or disease state of the recipient, kind of concurrent treatment, if any, frequency of treatment, and/or the nature of the effect desired. The dosage of pharmacologically active compound that is administered will be dependent upon multiple factors, such as the age, health, weight, and/or disease state of the recipient, concurrent treatments, if any, the frequency of treatment, and/or the nature and magnitude of the biological effect that is desired.

As used herein, terms such as "pharmaceutical composition," "pharmaceutical formulation," "pharmaceutical preparation," or the like, generally refer to formulations that are adapted to deliver a prescribed dosage of one or more pharmacologically active compounds to a cell, a group of cells, an organ or tissue, an animal or a human. Methods of incorporating pharmacologically active compounds into pharmaceutical preparations are widely known in the art. The determination of an appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical composition in order to achieve a desired biological outcome is within the skill level of an ordinary practitioner of the art. A pharmaceutical composition may be provided as sustained-release or timed-release formulations. Such formulations may release a bolus of a compound from the formulation at a desired time, or may ensure a relatively constant amount of the compound present in the dosage is released over a given period of time. Terms such as "sustained release" or "timed release" and the like are widely used in the pharmaceutical arts and are readily understood by a practitioner of ordinary skill in the art. Pharmaceutical preparations may be prepared as solids, semi-solids, gels, hydrogels, liquids, solutions, suspensions, emulsions, aerosols, powders, or combinations thereof. Included in a pharmaceutical preparation may be one or more carriers, preservatives, flavorings, excipients, coatings, stabilizers, binders, solvents and/or auxiliaries that are, typically, pharmacologically inert. It will be readily appreciated by an ordinary practitioner of the art that, pharmaceutical compositions, formulations and preparations may include pharmaceutically acceptable salts of compounds. It will further be appreciated by an ordinary practitioner of the art that the term also encompasses those pharmaceutical compositions that contain an admixture of two or more pharmacologically active compounds, such compounds being administered, for example, as a combination therapy.

As used herein the term "pharmaceutically acceptable salts" includes salts prepared from by reacting pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases, with inorganic or organic acids. Pharmaceutically acceptable salts may include salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, etc. Examples include the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'- dibenzylethylenediamine, diethylamine, 2-dibenzylethylenediamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

The terms "reducing," "inhibiting" and "ameliorating," as used herein, when used in the context of modulating a pathological or disease state, generally refers to the prevention and/or reduction of at least a portion of the negative consequences of the disease state. When used in the context of an adverse side effect associated with the administration of a drug to a subject, the term(s) generally refer to a net reduction in the severity or seriousness of said adverse side effects.

As used herein the term "subject" generally refers to a mammal, and in particular to a human.

As used herein, the term "treat" generally refers to an action taken by a caregiver that involves substantially inhibiting, slowing or reversing the progression of a disease, disorder or condition, substantially ameliorating clinical symptoms of a disease disorder or condition, or substantially preventing the appearance of clinical symptoms of a disease, disorder or condition.

Terms such as "in need of treatment," "in need thereof," "benefit from such treatment," and the like, when used in the context of a subject being administered a pharmacologically active composition, generally refers to a judgment made by an appropriate healthcare provider that an individual or animal requires or will benefit from a specified treatment or medical intervention. Such judgments may be made based on a variety of factors that are in the realm of expertise of healthcare providers, but include knowledge that the individual or animal is ill, will be ill, or is at risk of becoming ill, as the result of a condition that may be ameliorated or treated with the specified medical intervention.

By "therapeutically effective amount" is meant an amount of a drug or pharmaceutical composition that will elicit at least one desired biological or physiological response of a cell, a tissue, a system, animal or human that is being sought by a researcher, veterinarian, physician or other caregiver.

In one embodiment, structure-based inhibitors that interfere with assembly of the TGF-β signaling complex have been developed. This approach for inhibiting TGF-β has only recently become feasible based on the structure the TGF-β signaling complex (TGF^3 bound to the extracellular domains of ΤβΜ and ΤβΜ), which we have recently determined. There are multiple advantages to such "assembly" inhibitors. First, these assembly inhibitors are highly specific and as such have a major advantage over TGF-β kinase inhibitors, the only other class of small molecules known to target TGF-β. The underlying reason for the expected high specificity relates to the distinct manner by which TGF-β has been shown to assemble its signaling complex. Thus, not only does TGF-β assemble its signaling complex in a distinct manner relative to that of other grown factors that effect tumor cell growth, but also from that of other members of the TGF-β superfamily. Second, the compounds we have identified have excellent bioavailability since they are small and designed to function extracellularly. This represents an important advantage compared to both the macromolecular ligand traps that function extracellularly, but which diffuse slowly, as well as the kinase inhibitors, which act intracellularly. Third, assembly inhibitors that bind to a pocket on one of the receptors, also equally inhibit all TGF-β isoforms, not just one or two, such as the antisense oligos or soluble type II receptor, which preferentially binds and antagonizes TGF-βΙ and TGF^3, but not TGF^2, or the soluble type III receptor, which preferentially binds and antagonizes TGF^2, but not TGF-βΙ and TGF-^3. This could be very important as previous studies have shown that inhibitors that target all three TGF-β isoforms are more effective than inhibitors that target just one or two isoforms.

In one embodiment, TGF-β inhibitors include compounds having the general structure (I):

(I)

where X is

where L¹ is -NHC(O)-, -N=N- -NH-, -OC(0)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR^!R², -SO3R¹, -NC(0)-R³, -OC(0)-R³, or - N=CR'R², at least one R being -SO3R¹;

where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

As used herein the term amino-acid residue refers to the side chain of an amino acid. Natural and unnatural side chains may be present. Examples of side chains of amino acids include, but are not limited to, H (glycine); -CH₃ (alanine); -CH(C]¾)-CH₃ (valine); -CH₂- CH(CH₃)-C¾ (leucine); -CH(CH₃)-CH₂-CH₃ (isoleucine); -CH₂-Ph (phenylalanine); -CH₂-C¾- S-CH₃ (methionine); -CH₂-OH (serine); -CH(CH₃)-OH (threonine); -CH₂-SH (cysteine); -CH₂- Ph-OH (tyrosine); -CH₂-C(0)-NH₂ (aspargine); -CH₂-CH₂-C(0)-NH₂ (glutamine); -CH₂-C0₂H (aspartic acid); -CH₂-CH₂-C0₂H (glutamic acid); -CH₂-CH₂-CH₂-CH₂-NH₂ (lysine); -CH₂-CH₂-

- -CH₂-(/ J

rnithine); -CH₂-CH₂-CH₂-NH-C(NH)-NH₂ (arginine); ^ξ (histidine); and

(tryptophan).

In one embodiment, a compound of structure (I) is a pharmaceutically acceptable salt. In a specific embodiment, the compound of structure (I) is a sodium salt.

In one embodiment, TGF-β inhibitors include compounds having the general structure

(IA):

where X is

where L¹ is -NHC(O)-, -N=N- or -OC(O)-;

where R is -OR¹ or -NR^!R²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and

where R² is H, alkyl, phenyl, or aryl.

In one embodiment, TGF-β inhibitors include compounds having the general structure (IB):

where L¹ is -NHC(O)-, -N=N- or -OC(O)-;

where R is -OR¹ or -NR*R²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and

where R² is H, alkyl, phenyl, or aryl.

Specific examples of compounds having general stmcture (I) include NSC 37176 (II) and NSC 47715 (III).

(Π) (III)

NSC37176 NSC 47715

In another embodiment, the compound of structure I is not NSC37176.

In another embodiment, TGF-β inhibitors include compounds having the general structure (IV):

(IV)

where X is

or

where L² is -NHC(O)-, -N=N- -NH-, -OC(O)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR^!R², -SO3R¹, -NC(0)-R³, -OC(0)-R³, or - N=CR^!R², at least one R being -SO₃R¹;

where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

In one embodiment, a compound of structure (IV) is a pharmaceutically acceptable salt.

In a specific embodiment, the compound of structure (IV) is a sodium salt. In another embodiment, TGF-β inhibitors include compounds having the general structure (IV):

where X is

where L² is -NH-, -C(O)-, or -0-;

where each R is, independently, -OR¹, or -NR R²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and

where R² is H, alkyl, phenyl, or aryl;

A specific example of a compound having general structure (IV) is NSC 115372 (V).

(V)

NSC 115372

In an embodiment, a method of treating fibrotic disorders or cancer in a subject includes administering to a subject who would benefit from such treatment a therapeutically effective amount of one or more TGF-β inhibitors. One or more TGF-β inhibitors, as described in Formulas (I) - (V) may be used to treat fibrotic disorders or cancer in a subject. In an embodiment, one or more TGF-β inhibitors (e.g., any of the compounds of Formulas (I) - (V) may be formulated in a pharmaceutical composition.

Any suitable route of administration may be employed for providing a subject with an effective dosage of the TGF-β inhibitors described herein. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.

The compositions may include those compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy .

In practical use, compositions may be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid

pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.

The pharmaceutical preparations may be manufactured in a manner which is itself known to one skilled in the art, for example, by means of conventional mixing, granulating, dragee- making, softgel encapsulation, dissolving, extracting, or lyophilizing processes. Thus, pharmaceutical preparations for oral use may be obtained by combining the compositions with solid and semi-solid excipients and suitable preservatives, and/or co-antioxidants. Optionally, the resulting mixture may be ground and processed. The resulting mixture of granules may be used, after adding suitable auxiliaries, if desired or necessary, to obtain tablets, softgels, lozenges, capsules, or dragee cores.

Suitable excipients may be fillers such as saccharides (e.g., lactose, sucrose, or mannose), sugar alcohols (e.g., mannitol or sorbitol), cellulose preparations and/or calcium phosphates (e.g., tricalcium phosphate or calcium hydrogen phosphate). In addition binders may be used such as starch paste (e.g., maize or corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone). Disintegrating agents may be added (e.g., the above-mentioned starches) as well as carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate). Auxiliaries are, above all, flow-regulating agents and lubricants (e.g., silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol, or PEG). Dragee cores are provided with suitable coatings, which, if desired, are resistant to gastric juices. Soft gelatin capsules ("softgels") are provided with suitable coatings, which, typically, contain gelatin and/or suitable edible dye(s). Animal component- free and kosher gelatin capsules may be particularly suitable for the embodiments described herein for wide availability of usage and consumption. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol (PEG) and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures, including dimethylsulfoxide (DMSO),

tetrahydrofuran (THE), acetone, ethanol, or other suitable solvents and co-solvents. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, may be used. Dye stuffs or pigments may be added to the tablets or dragee coatings or soft gelatin capsules, for example, for identification or in order to characterize combinations of active compound doses, or to disguise the capsule contents for usage in clinical or other studies.

In some embodiments, TGF-β inhibitors will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

For the prevention or treatment of disease, the appropriate dosage of the composition will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the compositions are administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition, and the discretion of the attending physician. The composition is suitably administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 0.015 to 15 mg/kg of TGF-β inhibitor is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful.

According to another embodiment of the invention, the effectiveness of the composition in preventing or treating disease may be improved by administering the composition serially or in combination with another agent that is effective for those purposes, such as another anti-cancer agent. Such other agents may be present in the composition being administered or may be administered separately. The composition may be suitably administered serially or in

combination with radiological treatments, whether involving irradiation or administration of radioactive substances.

In one embodiment, a method of screening for compounds that inhibit assembly of the TGF-β ternary complex includes:

measuring the degree of polarization exhibited that occurs when a TGF-β receptor complex is assembled from a mixture containing purified one of the TGF-β isoforms (TGF-β 1 , TGF^2, or TGF-p3), the ΤβΡν-ΙΙ extracellular domain, and the ΤβΙ -Ι extracellular domain, one of which is fluorescently labeled; and

determining the inhibitory effect of the one or more compounds based on the measured change in the degree of polarization as a function of compound concentration.

In one embodiment, a dimeric form of the ΤβΡν-Ι extracellular domain, such as Fc-ΤβΜ, is used in the method of screening compounds that inhibit assembly of the TGF-β ternary complex. In one embodiment, a dimeric form of the ΤβΡν-Π extracellular domain, such as Fc- ΤβΙΙ, is used in the method of screening compounds that inhibit assembly of the TGF-β ternary complex.

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 which follow represent techniques discovered by the inventor 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.

Virtual Screening of Candidates

Novel "assembly" inhibitor leads were initially determined using computer-based target site analysis of the TGF-β receptor complex coupled with computer-based virtual screening of the National Cancer Institute Developmental Therapeutics Program (NCI-DTP) repository. The advantage of employing this computational approach is that it greatly enhanced the hit rate, allowing us to identify novel TGF-β inhibitors by screening only a very limited number of compounds in the laboratory. We initially identified a pocket for the potential inhibitor. The pocket was characterized as having the following properties: it is large and deep enough to accommodate drug-like molecules; it is located on the surface of one of the receptors (e.g., ΤβΜ), and hence once filled with an inhibitor should equally block complex assembly and signaling by all three TGF-β isoforms; and it is involved in crucial interactions required for complex assembly; in particular it functions as the acceptor pocket for V22 and F24 from the N- terminal tail of the ΤβΜΙ. This interaction has previously been shown to be critical for drawing in and stabilizing the low affinity receptor, ΤβΜ, into the complex; specifically, elimination of the N-terminal tail of ΤβΜΙ (residues 21-25) diminishes the affinity of the ΤβΜΙιΤϋΡ-β complex for binding and recruiting ΤβΜ by nearly 80-fold; this modification also renders cultured cells insensitive to the effects of TGF-β.

To carry out the virtual screening, the freely available 140,000 NCI-DTP library was scanned using the program AUTODOCK4. The inhibitory activities of the 40 top-ranked compounds selected by virtual screening were assessed using a fluorescent polarization (FP) assay developed specifically for this purpose.

Fluorescent polarization assay

The principle of this assay is that the degree of polarization exhibited by fiuorescently- tagged ΤβΚΙ extracellular domain (11 kDa) will increase as its binds to the ΤβΡΙΙ:ΤΟΡ-β3 complex (58 kDa) and that this increase will be specifically blocked by small molecules that bind into the critical pocket on the surface of ΤβΡνΙ. To ensure the largest change in polarization possible, Alexa488 was chosen, as this has a short excited state lifetime (τ = 4ns; polarization is inversely proportional to lifetime). The flourophore was attached on lysine residues using a commercially available succinimidyl ester-Alexa488 conjugate, which is ideal as the structure of the TGF-β ternary complex showed that there are no lysine residues in ΤβΡνΓε binding interface with either TGF-β or ΤβΡνΙΙ. To validate the assay, the polarization of fluorescently labeled ΤβΜ was shown to increase as the concentration of added ΤβΜΙ:ΤΟΡ-β binary complex increases and that this increase can be reversed by the addition of unlabeled ΤβΡνΙ.

Data generated from a rapid fluorescence polarization (FP) assay used to screen for compounds that inhibit assembly of the TGF-β ternary complex is shown in FIG. 1. As shown in the left panel, the polarization of Alexa488 labeled ΤβΜ increases as the concentration of the ΤβΜΤΤΰΡ-β binary complex is increased. This increase can be reversed by a competitor, such as unlabeled ΤβΜ as shown in the right panel.

The application of the FP assay to assess the inhibitors selected from computational screening showed that more than 25% exhibited detectable inhibition. The compounds with the greatest inhibitory potential (NSC 37176, 47715, and 115372) had ¾s of 5.8, 5.1, and 4.4 μΜ respectively (See FIG. 2). These three compounds were found to share a common structural core, including a central sulfated aromatic ring with a large substituent at either the meta or para position. To confirm that the compounds indeed inhibited receptor complex assembly, the three lead compounds were further characterized using surface plasmon resonance (SPR). This was accomplished by first immobilizing ΤβΜ onto the sensor chip surface using standard

carbodiimide-based (EDC/NHS) coupling and then by injecting a fixed concentration of

TPRJI:TGF-P3 binary complex (0.5 μΜ) in the presence of increasing concentrations of inhibitor. The results showed that the TpRJI:TGF- 3 binary complex yielded a readily detectable response and that this was inhibited by approximately 30% and over 95% of its maximum value when NSC 11372 was included at concentrations of 1 and 10 μΜ, respectively (FIG. 3). To rule out the possibility that the decrease was caused by the DMSO in which the compound was dissolved, a control injection was performed in which the binary complex was injected in the presence of DMSO. The sensorgram from this control was coincident with the sensorgram when the binary complex alone was injected, showing that the decrease was not caused by the DMSO. Though too few points were collected to determine a ¾, the results are clearly consistent with the ¾ = 4.4 μΜ obtained from the FP assay shown in FIG. 2.

SPR experiments were performed to detect effects of the three compounds on binding of

TpRI to TGF-P3 in the presence of a saturating concentration of ΤβΚΠ (4 μΜ). The results obtained for NSC 37176, 47715, and 115372 are shown in panels depicted in FIG. 4 and demonstrate a dose-dependent decrease in response as a function of increasing compound concentration. Note, the offset of the curves when 20 μΜ compound was used is due to changes in refractive index caused by the high concentrations of DMSO that have not been properly subtracted out.

Inhibition of Smad Phosphorylation

To assess biological activity, we tested the ability of the compounds to inhibit Smad2 and Smad3 phosphorylation in an immortalized human mammary epithelial cell line (Smad2 and Smad3 are the cytoplasmic mediators of the TGF-β signaling pathway and are phosphorylated directly by the TGF-β receptors after the ligand binds to them and assembles them into a signaling complex). These experiments showed that these three compounds inhibit TGF-P3 signaling in a dose dependent manner. EC50 values for this effect were in the low μΜ range. Results of these experiments are depicted in FIG. 5.

In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for treating fibrotic disorders or cancer in a subject comprising one or more TGF-β inhibitors and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

where L¹ is -NHC(O)- -N=N- -NH-, -00(0)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR^JR²,

-NC(0)-R³, -OC(0)-R³, or - N=CR¹R², at least one R being -SO3R¹;

where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

The composition of claim 1, wherein the TGF-β inhibitor has the structure:

where X is

where L¹ is -NHC(O)-, -N=N- or -OC(O)-;

where R is -OR¹ or -NR^²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and

where R² is H, alkyl, phenyl, or aryl.

4. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

5. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

where L¹ is -NHC(O)-, -N=N- or -OC(O)-;

where R is -OR¹ or -NR R²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and where R² is H, alkyl, phenyl, or aryl.

6. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

7. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

where X is

where L² is -NHC(O)-, -N=N- -NH- -OC(O)-, -C(O)-, or -0-;

where each R is, independently, H, -OR¹, -NR*R²,

-OC(0)-R³, or - N=CR¹R², at least one R being -SO3R¹;

where each R¹ is, independently, H, alkyl, phenyl, or aryl;

where R² is H, alkyl, phenyl, or aryl; and

where R³ is alkyl, phenyl, aryl, or an amino-acid residue.

8. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

where X is

where L² is -NH-, -C(0)-, or -0-;

where each R is, independently, -OR¹, or -NR^!R²;

where each R¹ is, independently, H, alkyl, phenyl, or aryl; and

where R² is H, alkyl, phenyl, or aryl;

9. The composition of claim 1, wherein the TGF-β inhibitor has the structure:

10. A method of treating fibrotic disorders or cancer in a subject comprising administering to a subject who would benefit from such treatment a therapeutically effective amount of a pharmaceutical composition as described in any one of claims 1-9.