WO2016064716A1 - Combination therapy of tshr antagonist and igfr inhibitor - Google Patents

Abstract

A method of treating Graves' disease in a subject, comprising co-administering to a subject in need thereof a therapeutically effective amount of: (a) an inverse agonist of TSHR and/or a neutral antagonist of TSHR; and (b) an IGFR inhibitor.

Description

COMBINATION THERAPY OF TSHR ANTAGONIST AND IGFR INHIBITOR

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the earlier filing date of U.S. Provisional Application No.

62/066,193, filed on October 20, 2014, the contents which is incorporated herein by reference in its entirety.

BACKGROUND Graves' disease (GD) is an autoimmune disease comprised of two major components - hyperthyroidism and ophthalmopathy (or orbitopathy, GO). It is clear that Graves' hyperthyroidism is caused by activation by circulating immunoglobulins (GD-IgG or thyroid-stimulating antibodies) of thyrotropin (thyroid-stimulating hormone, TSH) receptors (TSHR) on thyroid cells leading to stimulated synthesis and secretion of thyroid hormones. The pathogenesis of GO, however, is less clear. Although it appears that GD-IgG activation of TSHR on fibroblasts/preadipocytes and adipocytes in the soft tissue of the eye plays a role in GO pathogenesis, it has been proposed that GD-IgG may also directly activate insulinlike growth factor 1 (IGF-1) receptors (IGF-IR) on these cells to contribute to disease development. A functional relationship between TSHR and IGF-IR signaling has been previously established in thyroid cells wherein simultaneous activation of the two receptors leads to synergistic up-regulation of DNA synthesis and cell proliferation. In support of this idea in the pathogenesis of GO, patients with GO have been shown to have circulating antibodies which bind TSHR and IGF-IR, but whether IGF-IR is a secondary GO target has not been established. As GD-IgG are polyclonal, it is possible that different antibodies within a patient's GD-IgG may bind to and activate TSHR and IGF-IR. Recently, however, it was reported that a human monoclonal antibody M22, in addition to stimulating cAMP, also activates phosphatidylinositol 3-kinase (PBK)-Akt signaling, which is downstream of both TSHR and IGF-IR pathways.

A major component of GO is the excessive deposition of hyaluronan (hyaluronic acid, HA) in the extracellular matrix of orbital soft tissue. As attempts at generating and animal model for GO have yet to be reproduced, most research in this field has been performed in tissue culture using fibroblasts/preadipocytes (GOFs) and adipocytes obtained from GO patients at orbital decompression surgery. GOFs express TSHR and IGF-IR and selective activation of both receptors by their cognate ligands TSH and IGF-1, respectively, has been shown to stimulate HA secretion by these cells.

SUMMARY

It is proposed herein that cross-talk between TSHR and IGF-IR may occur in GOFs as has been shown for G protein-coupled receptors (GPCRs), including TSHR and receptor tyrosine kinases (RTKs) including IGF-IR. Disclosed herein is a method of treating Graves' disease in a subject, comprising co- administering to a subject in need thereof a therapeutically effective amount of: (a) an inverse agonist of TSHR and/or a neutral antagonist of TSHR; and (b) an IGFR inhibitor

Also disclosed herein is a method of inhibiting TSHR- and IGFR-stimulated hyaluronan secretion in a subject, comprising co-administering to the subject an inhibitory effective amount of: (a) an inverse agonist of TSHR and/or a neutral antagonist of TSHR; and (b) an IGFR inhibitor, thereby inhibiting TSH- and IGF- 1 -stimulated hyaluronan.

Also is disclosed is a pharmaceutical composition comprising: (a) a therapeutically effective amount of an inverse agonist of TSHR and/or a neutral antagonist of TSHR; (b) a therapeutically effective amount of an IGFR inhibitor; and (c) at least one pharmaceutically acceptable additive.

The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting data demonstrating that TSH and IGF-1 synergistically stimulate HA secretion.

FIG. 2 is a graph depicting data demonstrating inhibition of TSH- and IGF-1 -stimulated HA secretion by TSHR antagonist CI and IGF-1R antagonist linsitinib.

FIG. 3 is a graph depicting data demonstrating the effects of IGF-1 and the IGF-1R antagonist linsitinib on M22-stimulated HA secretion.

FIG. 4 is a graph depicting data demonstrating the effects of IGF-1 and M22 on IGF-1R stimulation. FIG. 5 is a graph depicting data demonstrating inhibition of M22-stimulated HA secretion by a TSHR antagonist CI and an IGF-1R antagonist linsitinib.

FIG. 6 is a graph depicting data demonstrating inhibition of HA secretion by IGF-1R antagonist Axlpq401 (also referred to as PQ401).

FIG. 7 is a graph of concentration of HA versus concentration of M22, depicting the M22 dose response curve of HA in primary Graves' orbital fibroblasts (GOFs).

FIG. 8 is a graph depicting the dose response curve of S2-7 in GOFs following stimulation by M22 at ltS ECmed.

FIG. 9 is a graph depicting the dose response curve of linsitinib in GOFs following stimulation by M22 at its EC_med.

FIG. 10 is an isobologram graph depicting the additive function of a combination of S2-7 and linsitinib in inhibiting HA production in GOFs.

FIG. 11 is a graph depicting the inhibition of HA production in GOFs stimulated by M22, by administration of S2-7, linsitinib and a 50:50 combination of S2-7 and linsitinib. FIG. 12 is a graph depicting the efficacies of various anti-IGF-lR antibodies in inhibiting M22- stimulated HA production.

DETAILED DESCRIPTION

Terminology

The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only and is not intended to be limiting.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B.

"Administration of and "administering a" compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to the subject (e.g., intravenously) or it can be self- administered by the subject (e.g., tablets).

By the term "co-administer" is meant that each of at least two compounds or agents be administered during a time frame wherein the respective periods of biological activity overlap. Thus, the term includes sequential as well as coextensive administration of two or more drug compounds. In certain embodiments, a first compound or agent and a second compound or agent may be administered via the same delivery route (e.g., both first and second are orally administered). In certain embodiments, a first compound or agent and a second compound or agent may be administered via different delivery routes (e.g, the first compound or agent is orally administered and the second compound or agent is parenterally administered).

"Optional" or "optionally" means that the subsequently described event or circumstance can but does not need to occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

"Derivative" refers to a compound or portion of a compound that is derived from or is theoretically derivable from a parent compound.

The term "subject" includes both human and veterinary subjects.

"Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. The phrase "treating a disease" refers to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a hormone receptor mediated disorder, particularly a thyroid disorder, such as a hyperthyroid or hypothyroid disorder. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

The term "pharmaceutically acceptable salt or ester" refers to salts or esters prepared by

conventional means that include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. "Pharmaceutically acceptable salts" of the presently disclosed compounds also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine,

tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When compounds disclosed herein include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. Such salts are known to those of skill in the art. For additional examples of "pharmacologically acceptable salts," see Berge et al., . Pharm. Sci. 66: 1

(1977). "Pharmaceutically acceptable esters" includes those derived from compounds described herein that are modified to include a hydroxy or a carboxyl group. An in vivo hydrolysable ester is an ester, which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for carboxy include Ci-6 alkoxymethyl esters for example methoxy-methyl, Ci-6 alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8 cycloalkoxycarbonyloxyCi- 6 alkyl esters for example 1-cyclohexylcarbonyl-oxyethyl; l,3-dioxolen-2-onylmethyl esters for example 5- methyl-l,3-dioxolen-2-onylmethyl; and Ci-6 alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyl- oxyethyl which may be formed at any carboxy group in the compounds.

An in vivo hydrolysable ester containing a hydroxy group includes inorganic esters such as phosphate esters and a-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group. Examples of a-acyloxyalkyl ethers include acetoxy-methoxy and 2,2-dimethylpropionyloxy-methoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N- alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. Examples of substituents on benzoyl include morpholino and piperazino linked from a ring nitrogen atom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.

ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p- aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

The compounds containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N- methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

The term "addition salt" as used hereinabove also comprises the solvates which the compounds described herein are able to form. Such solvates are for example hydrates, alcoholates and the like.

The term "quaternary amine" as used hereinbefore defines the quaternary ammonium salts which the compounds are able to form by reaction between a basic nitrogen of a compound and an appropriate quaternizing agent, such as, for example, an optionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulionates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be introduced using ion exchange resins.

It will be appreciated that the compounds described herein may have metal binding, chelating, complex forming properties and therefore may exist as metal complexes or metal chelates.

"Saturated or unsaturated" includes substituents saturated with hydrogens, substituents completely unsaturated with hydrogens and substituents partially saturated with hydrogens. The term "acyl" refers group of the formula RC(O)- wherein R is an organic group.

The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, w-propyl, isopropyl, «-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having from 1 to 10 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms. Alkyl groups may be "substituted alkyls" wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, or carboxyl.

The term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and structural formula containing at least one carbon-carbon double bond.

The term "alkynyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon triple bond.

The terms "halogenated alkyl" or "haloalkyl group" refer to an alkyl group as defined above with one or more hydrogen atoms present on these groups substituted with a halogen (F, CI, Br, I).

The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl group" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous.

The term "aliphatic" is defined as including alkyl, alkenyl, alkynyl, halogenated alkyl and cycloalkyl groups as described above. A "lower aliphatic" group is a cyclic, branched or unbranched aliphatic group having from 1 to 10 carbon atoms.

The term "alkoxy" refers to a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to 4 carbon atoms, that include an oxygen atom at the point of attachment. An example of an "alkoxy group" is represented by the formula -OR, where R can be an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group as described above. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec- butoxy, tert-butoxy cclopropoxy, cyclohexyloxy, and the like.

"Alkoxycarbonyl" refers to an alkoxy substituted carbonyl radical, -C(0)OR, wherein R represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety.

The term "alkyl amino" refers to alkyl groups as defined above where at least one hydrogen atom is replaced with an amino group.

"Aminocarbonyl" alone or in combination, means an amino substituted carbonyl (carbamoyl) radical, wherein the amino radical may optionally be mono- or di-substituted, such as with alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl and the like. An aminocarbonyl group may be -N(R)-C(0)-R (wherein R is a substituted group or H) or -C(0)-N(R). An "aminocarbonyl" is inclusive of an amido group. A suitable aminocarbonyl group is acetamido. The term "aryl" refers to any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aromatic" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can be unsubstituted.

"Carbonyl" refers to a radical of the formula -C(O)-. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C=0), including acyl groups, amides, carboxy groups, esters, ureas, carbamates, carbonates and ketones and aldehydes, such as substituents based on -COR or - RCHO where R is an aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine.

"Carboxyl" refers to a -COOH radical. Substituted carboxyl refers to -COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or a carboxylic acid or ester.

The term "hydroxyl" is represented by the formula -OH.

The term "hydroxyalkyl" refers to an alkyl group that has at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group that has at least one hydrogen atom substituted with an alkoxy group described above.

The term "amine" or "amino" refers to a group of the formula -NRR', where R and R' can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above.

The term "amide" or "amido" is represented by the formula -C(0)NRR', where R and R' independently can be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above. A suitable amido group is acetamido.

The term "aralkyl" refers to an aryl group having an alkyl group, as defined above, attached to the aryl group, as defined above. An example of an aralkyl group is a benzyl group.

Optionally substituted groups, such as "optionally substituted alkyl," refers to groups, such as an alkyl group, that when substituted, have from 1-5 substituents, typically 1, 2 or 3 substituents, selected from alkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted heterocyclyl, hydroxy, sulfonyl, thiol and thioalkoxy. In particular, optionally substituted alkyl groups include, by way of example, haloalkyl groups, such as fluoroalkyl groups, including, without limitation, trifluoromethyl groups.

A "therapeutically effective amount" or "diagnostically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount or diagnostically effective amount of an agent is an amount sufficient to inhibit or treat the disease without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount or diagnostically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition.

Prodrugs of the disclosed compounds also are contemplated herein. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into an active compound following administration of the prodrug to a subject. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).

Pharmaceutically acceptable prodrugs refer to compounds that are metabolized, for example, hydrolyzed or oxidized, in the subject to form an antiviral compound of the present disclosure. Typical examples of prodrugs include compounds that have one or more biologically labile protecting groups on or otherwise blocking a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound. In general the prodrug compounds disclosed herein possess hormone receptor modulating activity and/or are metabolized or otherwise processed in vivo to form a compound that exhibits such activity.

The term "prodrug" also is intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when the prodrug is administered to a subject. Since prodrugs often have enhanced properties relative to the active agent pharmaceutical, such as, solubility and bioavailability, the compounds disclosed herein can be delivered in prodrug form. Thus, also contemplated are prodrugs of the presently disclosed compounds, methods of delivering prodrugs and compositions containing such prodrugs. Prodrugs of the disclosed compounds typically are prepared by modifying one or more functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds having a phosphonate and/or amino group functionalized with any group that is cleaved in vivo to yield the corresponding amino and/or phosphonate group, respectively. Examples of prodrugs include, without limitation, compounds having an acylated amino group and/or a phosphonate ester or phosphonate amide group. In particular examples, a prodrug is a lower alkyl phosphonate ester, such as an isopropyl phosphonate ester.

Protected derivatives of the disclosed compound also are contemplated. A variety of suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis,

hydrogenolysis and the like. One preferred method involves the removal of an ester, such as cleavage of a phosphonate ester using Lewis acidic conditions, such as in TMS-Br mediated ester cleavage to yield the free phosphonate. A second preferred method involves removal of a protecting group, such as removal of a benzyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof. A t-butoxy-based group, including t-butoxy carbonyl protecting groups can be removed utilizing an inorganic or organic acid, such as HCl or trifluoroacetic acid, in a suitable solvent system, such as water, dioxane and/or methylene chloride. Another exemplary protecting group, suitable for protecting amino and hydroxy functions amino is trityl. Other conventional protecting groups are known and suitable protecting groups can be selected by those of skill in the art in consultation with Greene and Wuts Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When an amine is deprotected, the resulting salt can readily be neutralized to yield the free amine. Similarly, when an acid moiety, such as a phosphonic acid moiety is unveiled, the compound may be isolated as the acid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one or more asymmetric centers; thus these compounds can exist in different stereoisomeric forms. Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain embodiments the compounds disclosed herein are synthesized in or are purified to be in substantially enantiopure form, such as in a 90% enantiomeric excess, a 95% enantiomeric excess, a 97% enantiomeric excess or even in greater than a 99% enantiomeric excess, such as in enantiopure form.

It is understood that substituents and substitution patterns of the compounds described herein can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art and further by the methods set forth in this disclosure. Reference will now be made in detail to the presently preferred compounds.

Overview

Disclosed herein are results demonstrating that TSHR and IGF-1R on GOFs are more active when they interact with each other, a concept known as receptor cross-talk. Simultaneous treatment with TSH and IGF-1 synergistically increased HA secretion by GOFs, wherein increasing IGF-1 concentration augmented potency and efficacy of TSH on TSHR, and that dose-dependent stimulation of HA secretion by M22 was biphasic with the higher potency phase mediated in part by IGF-1R. These data provide evidence of M22- induced cross-talk between TSHR and IGF-1R to synergistically increase HA secretion. This suggests that GD-IgG-induced bidirectional cross-talk plays a pivotal role in the pathogenesis of GO. These results consequently strongly indicate that inhibiting both receptors simultaneously would lead to a more effective GO therapy than inhibiting TSHR or IGF-1R alone.

Disclosed herein is a method of treating Graves' disease in a subject, comprising co-administering to a subject in need thereof a therapeutically effective amount of: (a) an inverse agonist of TSHR or a neutral antagonist of TSHR; and (b) an IGFR inhibitor. Also disclosed herein is a pharmaceutical composition comprising: (a) an inverse agonist of TSHR or a neutral antagonist of TSHR; (b) an IGFR inhibitor; and (c) at least one pharmaceutically acceptable additive.

The compounds that are neutral antagonists of TSHR inhibit signaling stimulated by TSH or TSAbs, and inverse agonists inhibit signaling stimulated by TSH and TSAbs and also inhibit basal signaling.

Activation of TSHR by its endogenous hormone TSH is required for normal thyroid homeostasis but may also regulate the function of extra-thyroidal cells including adipocyte (fat) precursor cells, adipocytes, fibroblasts, immune cells and bone. The TSHR also exhibits activity that does not depend on stimulation by TSH; this is termed agonist-independent, basal or constitutive activity. Agonist-independent signaling activity is thought to be important in some thyroid disease states.

TSHR in thyroid cells, and likely in fibroblasts and adipocytes in the supporting tissue behind the eye (in the retro-orbital space) and perhaps in the supporting tissues of the skin, also are stimulated by TSHR-stimulating antibodies (TSAbs), resulting in Graves' disease. Graves' disease, which is an autoimmune disease that occurs in 1% of the US population, has two important clinical components - 1) hyperthyroidism from stimulation of TSHR on thyroid cells and 2) Graves' orbitopathy (or Graves' ophthalmopathy or thyroid eye disease), which appears to result from stimulation of TSHR on retro-orbital fibroblasts and/or adipocytes, and 3) a less common component termed Graves' dermopathy.

Graves' hyperthyroidism is a hypermetabolic state that affects virtually every tissue/cell in the body and can lead to, in particular, cardiovascular dysfunction and death. Graves' hyperthyroidism can be treated by surgical resection, therapeutic doses of radioactive iodine, or pharmacologically (methimazole or propylthiouracil). However, each of these treatment modalities has side effects associated with it (Cooper DS, 2005 N Engl J Med, 352, 905-917).

Graves' orbitopathy occurs in 80% of Graves' hyperthyroid patients as diagnosed by computerized tomographic scan. Symptoms range from mild to moderate to severe to sight-threatening. Protrusion of the eyeball (proptosis) and varying degrees of extra-ocular muscle weakness or paralysis leading to double vision (diplopia) can be disfiguring and incapacitating. Treatment with glucocorticoids may give some improvement, but correction of the hyperthyroid state to normal has no effect. Vision can be threatened by corneal abrasion or pressure on the optic nerve, requiring emergency therapy using intravenous

glucocorticoids and orbital radiotherapy, and in some cases surgical decompression of the orbit (Bahn RS 2010, N Engl J Med 362, 726-738). There is no simple therapy without untoward side effects for Graves' orbitopathy.

Graves' dermopathy (also called pretibial myxedema) is a skin condition characterized by red, swollen skin, usually on the shins and tops of the feet. Topical steroid application is the usual treatment.

The treatment of Graves' hyperthyroidism by surgery, radioactive iodine or drugs that block thyroid hormone synthesis (methimazole or propylthiouracil) reduces or abolishes the hyperthyroid state but does not address the root cause of Graves' hyperthyroidism, Graves' orbitopathy, or the presence of TSHR activating antibodies stimulating thyroid, retro-orbital, or skin supporting cells (fibroblasts) and, therefore, does not treat Graves' orbitopathy or Graves' dermopathy.

An "inverse agonist" as used herein refers to an agent that inhibits basal or TSH-independent or constitutive TSHR activity. The inverse agonist may also be an antagonist that inhibits TSH activation. In particular, an "inverse agonist" as used herein refers to an agent that inhibits TSH- and thyroid-stimulating antibodies-independent (basal or constitutive) TSHR activity as well as inhibiting TSH- and thyroid- stimulating antibodies-dependent activation. By contrast, a "neutral antagonist" blocks the action of the agonists (TSH or thyroid-stimulating antibodies for TSHR), but does not inhibit basal/constitutive TSHR activity. Thus, inverse agonists and neutral antagonists both antagonize the activation of TSHR by TSH and thyroid-stimulating antibodies. Small-molecule ligands for the TSHR (agonists, inverse agonists, neutral antagonists) bind to an intra-membrane domain of the receptor, and act by inducing a conformational change rather than simply competing for TSH binding to its extracellular site on the receptor.

Small molecule (for example, less than 1000 daltons) inverse agonists and neutral antagonists are attractive agents because they are more easily employed as probes and drugs compared to TSH, its analogs or anti-TSHR antibodies, can be synthesized chemically in large amounts at moderate cost, and can be given orally because they are not degraded within, and can be absorbed from, the gastrointestinal tract. Disclosed herein are inverse agonists that inhibit basal signaling by wild-type TSHR and several constitutively active mutants receptors (CAMs) that may be used for probes of TSHR biology, treating subjects with thyroid cancer (especially TSH-independent thyroid cancer), treating subjects with hyperthyroidism (especially Graves' hyperthyroidism), or treating subjects with Graves' orbitopathy. Also disclosed are neutral antagonists that may be used for probes of TSHR biology or treating subjects with Graves' orbitopathy and/or Graves' hyperthyroidism. In certain embodiments, the inverse agonists and neutral antagonists may be selective inverse agonists or neutral antagonists for TSHR (i.e, the compounds do not activate or modulate other hormone receptors, particularly luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and follicle-stimulating hormone receptor (FSHR)).

Thus, in certain embodiments, the combination therapy disclosed herein may be used for treating Graves' hyperthyroidism, orbitopathy and dermopathy in a subject.

The combination therapy disclosed herein may be used to inhibit stimulation of thyroid, orbital, and supporting skin tissues by blocking TSAbs in Graves' hyperthyroidism, Graves' orbitopathy, and/or Graves' dermopathy.

Inverse Agonists and/or Neutral Antagonists

In one embodiment, the inverse agonists or neutral antagonists are 2,3-dihydroquinazolin-4-one compounds, particularly, 2-substituted, 3-substituted 2,3-dihydroquinazolin-4-one compounds. The substituent at the 2-position may be, for example, a furanyl-containing group, a pyridinyl-containing group, a thienyl-containing group, hydroxyalkyl, or alkoxyalkyl. The substituent at the 3-position may be, for example,

wherein Ar¹ is a substituted or unsubstituted arylene group (e.g., -C6H₄-); Ar² is a substituted or unsubstituted aryl group; and X is O or S. In certain embodiments, Ar² is 2,6-dialkyl phenyl, particularly 2,6-dimethyl. In certain embodiments, Ar¹ is methoxy-substituted phenylene.

In general, illustrative inverse agonists or neutral antagonists may have a structure of:

(Formula I) wherein R¹ is selected from a furanyl-containing group, a pyridinyl-containing group, a thienyl-containing group, hydroxyalkyl, or alkoxyalkyl;

R² is H, alkoxy, alkyl, substituted alkyl or halogen; and

R³ - R⁷ are each individually selected from H, alkyl, substituted alkyl, halogen, or aminocarbonyl; and X is O or S.

In certain embodiments of Formula I, R¹ is selected from:

(a) a furanyl-containing group, wherein the furanyl-containing group is a furanylalkyl group having the structure -R¹⁰-furanyl, wherein R¹⁰ is a lower alkylene group (for example, having from 1 to 10 carbon atoms such as methylene (-CH2-), ethylene (-CH2CH2-), trimethylene (-CH2CH2CH2-), methylethylene (- CEb XCE )!!-), etc.). The furanyl ring may be unsubstituted or substituted with a lower alkyl. In certain embodiments, the furanyl ring is substituted at the 3 carbon position with a lower alkyl, particularly methyl. The furanyl may be 2-furanyl or 3-furanyl. In certain embodiments, the furanyl-containing group is 2- furanyl or furan-2-ylmethyl;

(b) a pyridinyl-containing group, wherein the pyridinyl-containing group is a pyridinylalkyl group having the structure -R¹⁰-pyridinyl, wherein R¹⁰ is a lower alkylene group (for example, having from 1 to 10 carbon atoms such as methylene (-CH2-), ethylene (-CH2CH2-), trimethylene (-CH2CH2CH2-),

methylethylene (-CH₂C(CH₃)H-), etc.). The pyridinyl ring may be unsubstituted or substituted with a lower alkyl. The pyridinyl may be 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl. In certain embodiments, the furanyl- containing group is 3-pyridinyl or pyridin-3-ylmethyl;

(c) a thienyl-containing group, wherein the thienyl-containing group is a thienylalkyl group having the structure -R¹⁰-thienyl, wherein R¹⁰ is a lower alkylene group (for example, having from 1 to 10 carbon atoms such as methylene (-CH2-), ethylene (-CH2CH2-), trimethylene (-CH2CH2CH2-), methylethylene (- CEb XCE )!!-), etc.). The thienyl ring may be unsubstituted or substituted with a lower alkyl. In certain embodiments, the thienyl ring is substituted at the 3 carbon position with a lower alkyl, particularly methyl. The thienyl may be 2-thienyl or 3-thienyl. In certain embodiments, the thienyl-containing group is 2-thienyl or thien-2-ylmethyl; or (d) an alkoxy alkyl having a structure of -R⁸OR⁹, wherein R⁸ is a lower alkylene group (for example, having from 1 to 10 carbon atoms such as methylene (-CH2-), ethylene (-CH2CH2-), trimethylene (- CH2CH2CH2-), methylethylene (-CH₂C(CH₃)H-), etc.), and R⁹ is a lower alkyl (particularly methyl); R² is a lower alkyl group; R³ and R⁷ are each a lower alkyl group; R⁴ and R⁶ are each H; and R⁵ is an

aminocarbonyl group (particularly acetamido (-NHAc or -NHC(0)CH3)) or H.

In particular embodiments of Formula I, R³ and R⁷ are each a lower alkyl, particularly methyl. In other embodiments of Formula I, R² is methoxy. In further embodiments of Formula I, -R⁸OR⁹ is - (CH2)20CH3. In additional embodiments of Formula I, X is S. In other embodiments of Formula I, R⁵ is an aminocarbonyl group. According to another embodiment of Formula I, R¹ is -R⁸OR⁹. In additional embodiments of Formula I, R³ and R⁷ are alkyl groups, particularly lower alkyl groups, other than methyl. In further embodiments of Formula I, one of R³ or R⁷ is a lower alkyl, and the other one of R³ or R⁷ is H.

In another embodiment, illustrative inverse a onists or neutral antagonists may have a structure of:

(Formula II) wherein R¹ is selected from:

R²-R⁶ are each individually selected from H, alkyl, substituted alkyl or halogen; provided that the compound is no

In certain embodiments of Formula II, R¹ is:

In other embodiments of Formula II, R¹ is:

In certain embodiments of Formula II, R²-R⁶ are each individually selected from H or alkyl (particularly lower alkyl). In one particular embodiment of Formula II, R³-R⁵ are each H and R² and R⁶ are lower alkyl, especially methyl.

Specific examples of inverse agonists are shown below:

Compound 1 (also referred to herein as compound S2) (NCGC00161856)

Compound S2-6 (NCGC00229600)

Compound S2-7 (NCGC00242364) Specific examples of neutral antagonists are shown below:

Compound l(also referred to herein as compound S2) (NCGC00161856)

Compound S2-6 (NCGC00229600)

Compound S2-7 (NCGC00242364)

Compound S2-8 (NCGC00242595)

Compound S2-17 (NCGC00242589-01)

Compound S2-29 (NCGC00242580-01)

IGFR Inhibitors

IGFR inhibitors are agents that target one or more members of the insulin-like growth factor (IGF) family (e.g. IGF1 and/or IGF2 and/or insulin), particularly of the IGFR family of tyrosine kinases, e.g. IGFR-1 (either as single kinase inhibitor or as multikinase inhibitor), and/or of insulin receptor pathways. In certain embodiments, the inhibitor inhibits signaling through an IGF-IR signaling pathway. In certain embodiments, the inhibitor inhibits signaling upstream of IGF-IR. In certain embodiments, the inhibitor inhibits IGF-1 or IGF-2. In certain embodiments, the inhibitor inhibits the transcription or translation of IGF-1 or IGF-2. In certain embodiments, the inhibitor inhibits the processing or secretion of IGF-1 or IGF- 2. In certain embodiments, the inhibitor binds to IGF-1 or IGF-2. In certain embodiments, the inhibitor inhibits the binding of IGF-1 or IGF-2 to IGF-IR. In certain embodiments, the inhibitor is an anti-IGF-1 or anti-IGF-2 antibody, antigen binding fragment of said antibody, isolated IGF-1 or IGF-2 binding protein, recombinant human IGF-1 or IGF-2 binding protein, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, or IGFBP7. In certain embodiments, the inhibitor is an inhibitor of IGF-IR. In certain embodiments, said inhibitor is a small molecule (for example, less than 1000 daltons) inhibitor of IGF-IR. In certain embodiments, the small molecule inhibitor of IGF-IR is a kinase inhibitor.

In certain embodiments, the small molecule inhibitor is linsitinib (OSI-906), BMS-754807, INSM- 18, XL228, AXL1717, BMS-536924, NVP-ADW742, GSK621659A, GSK1838705A, A-928605,

AZD4253, TAE226, AG1024, PQ401 (CAS 196868-63-0), or AG538 (CAS 133550-18-2).

In certain embodiments, the inhibitor is an anti-IGF-1 R antibody, or an antigen binding fragment of said antibody. In certain embodiments, said antibody inhibits binding of IGF-1 or IGF-2 to IGF-IR. In certain embodiments, the antibody inhibits binding of IGF-1 and IGF-2 to IGF-IR. In certain embodiments, the antibody downregulates IGF-IR. In certain embodiments, the antibody is fully human, humanized, or chimeric. In certain embodiments, the anti-IGF-1 R antibody is ganitumab, AMG 479, figitumumab, CP- 751,871, cixutumumab, IMC-A12, dalotuzumab, MK0646, teprotumumab (RG1507), robatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, LlHl, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52. In another embodiment, said inhibitor affects an IGF-1 or IGF-2 binding protein.

Pharmaceutical Compositions

Another aspect of the disclosure includes pharmaceutical compositions prepared for administration to a subject and which include (a) a therapeutically effective amount of an inverse agonist of TSHR or a neutral antagonist of TSHR; (b) a therapeutically effective amount of an IGFR inhibitor; and (c) at least one pharmaceutically acceptable additive. In certain embodiments, the pharmaceutical compositions are useful for treating hyperthyroidism (particularly Graves' hyperthyroidism), or Graves' orbitopathy. The therapeutically effective amount of a disclosed compound will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those of skill in the art.

Pharmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include

pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. The pharmaceutical compositions may be in a dosage unit form such as an injectable fluid, an oral delivery fluid (e.g., a solution or suspension), a nasal delivery fluid (e.g., for delivery as an aerosol or vapor), a semisolid form (e.g., a topical cream), or a solid form such as powder, pill, tablet, or capsule forms.

Pharmaceutical compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Particular disclosed compounds possess at least one basic group that can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and imino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2- acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2- hydroxymethanesulfonic acid, ethane- 1 ,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid, naphthalene -2- sulfonic acid, 2- or 3 -phosphogly cerate, glucose-6-phosphate or N-cyclohexylsulfamic acid (with formation of the cyclamates) or with other acidic organic compounds, such as ascorbic acid. In particular, suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the

pharmaceutical art.

Certain compounds include at least one acidic group that can form an acid-base salt with an inorganic or organic base. Examples of salts formed from inorganic bases include salts of the presently disclosed compounds with alkali metals such as potassium and sodium, alkaline earth metals, including calcium and magnesium and the like. Similarly, salts of acidic compounds with an organic base, such as an amine (as used herein terms that refer to amines should be understood to include their conjugate acids unless the context clearly indicates that the free amine is intended) are contemplated, including salts formed with basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines and amidines. Of the aliphatic amines, the acyclic aliphatic amines, and cyclic and acyclic di- and tri- alkyl amines are particularly suitable for use in the disclosed compounds. In addition, quaternary ammonium counterions also can be used.

Particular examples of suitable amine bases (and their corresponding ammonium ions) for use in the present compounds include, without limitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris- (2-hydroxy ethyl) amine, 2-hydroxy-teri-butylamine, tris(hydroxymethyl)methylamine, N,N- dimethyl-N-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additional examples of "pharmacologically acceptable salts," see Berge et al., /. Pharm. Sci. 66: 1 (1977).

Compounds disclosed herein can be crystallized and can be provided in a single crystalline form or as a combination of different crystal polymorphs. As such, the compounds can be provided in one or more physical form, such as different crystal forms, crystalline, liquid crystalline or non-crystalline (amorphous) forms. Such different physical forms of the compounds can be prepared using, for example different solvents or different mixtures of solvents for recrystallization. Alternatively or additionally, different polymorphs can be prepared, for example, by performing recrystallizations at different temperatures and/or by altering cooling rates during recrystallization. The presence of polymorphs can be determined by X-ray crystallography, or in some cases by another spectroscopic technique, such as solid phase NMR

spectroscopy, IR spectroscopy, or by differential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the compositions can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes. In other alternative embodiments, the compound can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the compounds can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration.

Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The compounds can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl

(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid- glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.

The compounds can be combined with the base or vehicle according to a variety of methods, and release of the compound can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the compound is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al., . Pharmacy Pharmacol. 43: 1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the compound can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the compound and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL- lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly( amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677,191 and

4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile -filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In accordance with the various treatment methods of the disclosure, the compounds can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a

prophylactically or therapeutically effective amount of the compounds is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof.

The administration of the compounds of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the compounds are provided in advance of any symptom. The prophylactic administration of the compound serves to prevent or ameliorate any subsequent disease process. When provided therapeutically, the compounds are provided at (or shortly after) the onset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the compounds can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the compounds can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, avian, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the compound (for example, amounts that are effective to alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the compound may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the compounds will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the compound for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the compounds is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of compounds within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation that contains one or more of the compounds described herein. In one example, this component is formulated in a pharmaceutical preparation for delivery to a subject. The compound is optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator. Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.

Examples

Example 1

Materials and Methods

Materials. Thyrotropin from bovine pituitary (TSH) was purchased from Sigma- Aldrich.

Recombinant human IGF-1 was purchased from PeproTech (Rocky Hill, NJ). Thyroid stimulating human monoclonal autoantibody (M22) was purchased from Kronus (Star, ID). Thyroid stimulating hamster monoclonal antibody (MSI) was kindly provided by Dr. Terry Davies (Mount Sinai Hospital, New York, NY). TSHR antagonist NCGC00229600 (CI) was synthesized by the National Center for Advancing

Translational Science (NCATS), National Institutes of Health as previously reported (Neumann, S., et al. J Clin Endocrinol Metab 2011;96(2):548-554.). IGF-1R receptor kinase inhibitors linsitinib and

Axlpq401 were purchased from Selleckchem (Houston, TX). HA ELISA kits were purchased from

Corgenix (Broomfield, CO). One MDa HA was purchased from Lifecore Biomedical (Chaska, MN).

Cell Culture. Retro-oribital adipose tissue from two female patients and one male patient with GD was generously supplied by Drs. Neil Miller, Prem Subramanian and Shannath Merbs (Johns Hopkins School of Medicine, Baltimore, MD). Adherent cells were isolated from tissue by standard methods. Cells were maintained in F-media containing DMEM with FBS (10% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2mM), Ham's F-12 Nutrient Mixture (25% vol/vol), hydrocortisone (25 ng/mL), EGF (0.125 ng/mL), insulin (5 μg/mL), cholera toxin (11.7 nM), gentamicin (10 μg/ml), Fungizone (250 ng/mL), and Y-27632 (5 μΜ) in an humidified 7%-C0₂ incubator at 37 °C. Measurement of HA secretion in orbital fibroblasts. To measure HA secretion, cells were grown to confluence then pre -treated with hyaluronidase (lU/mL in HBSS) for 1 hour, 37°C. Following digestion of pre-existing HA, GOF were switched to DMEM with FBS (10% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL) with individual or combination treatments of TSH, IGF-1, M22, PDGF, FGF-2, TGF i, or IL1 β and incubated for 5 days in 7%-C0₂ at 37 °C. For experiments inhibiting TSHR or IGF-1R, cells were pre -treated with antagonist in low-serum DMEM (1% FBS) at 37 °C for 1 hour before addition of FBS (final concentration 10%) and TSH, IGF-1 or M22. Conditioned media were collected and stored at - 20 °C. Conditioned media were assayed using a modified Corgenix HA ELISA kit as previously described (Krieger, 2014).

Measurement of IGF-1 R phosphorylation. GOF were grown to confluence in 6-well plates, then serum-starved in DMEM with 2% BSA for 24 hours. Cells were pre -treated with antagonist in HBSS at 37 °C for 1 hour, then incubated at 37 °C for 30 minutes with maximally effective doses of IGF-1 or M22 in HBSS with or without antagonist. Lysates were prepared using the Bio-Plex Pro Cell Signaling Kit (Bio- Rad, Cat # 171-304006M) according to manufacturer's directions, and adjusted so that all samples had equal protein concentrations. Phosphorylated IGF-1R levels were measured with a Bio-Plex MAGPIX multiplex reader (Bio-Rad, Cat # 171-015001) using the Phospho-IGF-IR (Tyrl l31) Set (Bio-Rad, Cat # 171- V50009M) according to manufacturer's directions.

Statistical Analysis. Statistical analysis was performed by GraphPad Prism version 6.04 for Windows, GraphPad Software, San Diego California USA. Significance was determined using Student's t- test. Model discrimination for dose-response curves was conducted using the Extra sum-of-squares F-test.

Results

TSH and IGF-1 synergistically stimulate HA secretion. To assess possible TSHR and IGF-1R crosstalk, effects of individual receptor activation by their cognate ligands with simultaneous activation of the two receptors with concurrent treatment by TSH and IGF-1 were compared (FIG. 1). Strains 1 and 2 GOFs were stimulated to secrete HA as described in the Materials and Methods. The cells were incubated in medium containing various concentrations of TSH and IGF-1. After 5 days, the mediums were collected, and HA was assayed by our modified ELISA. The data points represent the mean+SE of four independent experiments. Open and closed symbols indicate different IGF-1 concentrations for the TSH dose-response curves. Solid and dotted lines show curve fits to a monophasic sigmoidal dose -response. The EC50S for TSH decreased from 150+21 nM in control cultures to 7.8+1.9 nM in cultures exposed to the highest concentrations of IGF-1 (P<0.001). The EC₅₀s for IGF-1 did not change - 0.45+0.15 nM. Total number of samples, N = 192.

Both TSH and IGF-1 stimulated monophasic dose-dependent increases in HA secretion when GOFs were treated with them alone - the EC₅₀s were 150+21 nM (mean+SD) and 0.45+0.15 nM for TSH and IGF- 1 , respectively, and the maximal increases over basal in HA secretion were 320+52 and 430+65 μg/ml for TSH and IGF-1, respectively (FIG. 1). Combined TSH and IGF-1 treatments synergistically upregulated HA secretion. Compared to TSH alone, the presence of increasing doses of IGF-1 increased the potency of TSH to a maximal 19-fold (EC5₀ was 7.8+1.9 nM) in the presence of the highest doses of IGF-1. There was no effect of TSH on the potency of IGF-1. Moreover, there was a synergistic increase in the maximal stimulation of HA secretion when the highest doses of TSH and IGF-1 were combined to 1,300+95 μg/ml. Experiments were done in two strains of GOFs for these experiments, and no differences were found in the responses to TSH and maximally effective IGF-1 when three strains of GOFs were studied individually.

To determine whether the receptors stimulated HA secretion using parallel pathways or whether TSHR or IGF-IR was a downstream signaling partner of the other, GOFs were pre-treated with either TSHR or IGF-IR small molecule antagonists before exposure to maximally effective doses of TSH and/or IGF-1. Strains 1, 2 and 3 GOFs were cultured as described above with respect to FIG. 1 in medium containing TSH (1.8 μΜ), IGF-1 (13 nM), CI (10 μΜ) and/or linsitinib (Lins) (10 μΜ). Bars represent mean+SEM for six independent experiments. *P<0.03, **P<0.001 compared to Control. Total number of samples, N = 146.

The TSHR antagonist CI (NCGC00229600) had no effect on basal secretion whereas the IGF-IR antagonist linsitinib had a small inhibitory effect (FIG. 2). CI fully inhibited HA secretion stimulated by TSH (100+1.5%) and had a partial inhibitory effect on IGF-1 (32+1.5%). Similarly, linsitinib completely inhibited IGF-1 stimulation (94+1.2%) and only partially inhibited TSH induction (44+4.0%). CI

(63+1.0%) and linsitinib (67+1.0%) partially inhibited combined TSH and IGF-1 treatment. Neither CI nor linsitinib affected secretion of HA stimulated by other cytokines that also signal via receptor tyrosine kinases, showing that the TSHR and IGF-IR antagonists we used were specific inhibitors of these receptors (although linsitinib is known to inhibit the insulin receptor tyrosine kinase also). These data are consistent with the idea that both TSHR activated by TSH and IGF-IR activated by IGF-1 exhibit bidirectional crosstalk but that both receptors when activated by their cognate ligands stimulate HA secretion by independent pathways as well.

M22 stimulation of HA secretion is mediated by both TSHR and IGF-IR. Though it was possible for TSHR and IGF-IR to work together to regulate HA, whether this is the case for GD-IgG activation in the pathogenesis of GO is not known. To this end, the stimulatory monoclonal antibody M22 was used to model the effects of GD-IgG. In a typical experiment, HA induction by maximally effective doses of M22 (540+64 μg/ml) approximated the additive effects of TSH alone (330+90 μg/ml) and IGF-1 alone (350+60 μg/ml) and was similar to that of combined TSH plus IGF-1 (650+58 μg/ml). This could be interpreted as simultaneous, parallel activation of TSHR and IGF-IR pathways. However, unlike combined TSH and IGF- 1, M22 stimulated HA secretion by GOFs in a biphasic dose-dependent manner with EC50S of 0.010 nM and 0.78 nM (FIG. 3). Strains 1, 2 and 3 GOFs were cultured as described above with respect to FIG. 1 in medium containing various concentrations of M22, M22 and IGF-1 (13 nM), or M22, IGF-1 and linsitinib (Lins) (10 μΜ). The best fit curve for M22 was biphasic, F-test P < 0.0001, EC₅₀s 0.010 nM and 0.78 nM. The best fit curves for M22+IGF-1 and M22+IGF-1+Lins were monophasic - EC₅₀s 0.16 nM and 0.93 nM, respectively. Data points represent mean+SEM. For M22+IGF-1 and M22+IGF-1+Lins, data were from six independent experiments. For M22-Control, data were from twelve independent experiments. Total number of samples, N = 328. The high potency phase accounted for approximately 30% of the maximal response and was eliminated with linsitinib pretreatment (FIG. 3). The resulting monophasic dose-response had an EC5₀ of 0.93 nM. Simultaneous treatment with IGF-1 also resulted in a monophasic dose-response to M22 with an EC5₀ of 0.16 nM and baseline 3-fold higher than control. Maximal stimulation, however, was not different than M22 alone. Hence, the M22 effect appears to be mediated by TSHR and IGF-1R.

Because previous reports assert that M22 binds to and activates IGF-1R, the ability of M22 to stimulate IGF-1R autophosphorylation was tested, a primary effect of IGF-1R activation. As expected, IGF- 1 increased IGF-1R phosphorylation 6.4±l-fold and linsitinib abolished this effect. Unlike with HA secretion, CI did not significantly change the IGF-1 effect. In contrast, M22 treatment had no effect on IGF-1R phosphorylation (FIG. 4). Strains 1 and 2 were treated with IGF-1 (13 nM) or M22 (2 nM) for 30 minutes with or without CI or linsitinib (Lins). M22 did not affect IGF-1R phosphorylation in contrast to IGF-1, which increased IGF-1R phosphorylation approximately 6.4-fold. IGF-1 stimulation was not affected by CI but was completely inhibited by Lins. Bars represent mean±SEM from six independent experiments, except for Lins, which was from two. Total number of samples, N = 42. Therefore, the linsitinib-sensitive phase of the M22 dose-response was not the result of direct activation of IGF-1R by M22.

The effects of TSHR antagonist CI on M22 stimulation of HA secretion by GOFs was examined (FIG. 5). Strains 1, 2 and 3 GOFs were cultured as described above with respect to FIG. 1 in medium containing M22 (2 nM) (Control), CI (10 μΜ) and/or linsitinib (10 μΜ). Bars represent mean±SEM from at least four independent experiments. *P<0.01, **P<0.001 compared to Control. Total number of samples, N = 60. Compared to linsitinib, which inhibited M22 stimulation by only 27+9.7%, CI abolished M22 stimulation (inhibition was 100±4%). These data contradict a process where TSHR and IGF-1R are simultaneously activated. Rather, M22 activation of TSHR most likely initiates two signaling pathways - a major one that relies solely on TSHR and a minor one based on TSHR-dependent activation of IGF-1R. HA secretion by a second monoclonal TSHR stimulating antibody MSI (19) was also partially inhibited by linsitinib and totally inhibited by CI indicating that indirect IGF-1R activation by M22 is not unique.

Discussion

The goal of this study was to determine the roles of TSHR and IGF-1R in the stimulated secretion of HA by GOFs so as to gain insight into the mechanism by which GD-IgG cause GO. HA secretion by GOFs was used as an endpoint as it is a major component in the pathogenesis of GO. GOFs were treated with TSH or IGF-1 to selectively activate TSHR or IGF-1R, respectively, and with a combination of both to determine whether cross-talk between these receptors occurred. Treatment of GOFs with TSH or IGF-1 stimulated HA secretion and simultaneous treatment with TSH and IGF-1 acted synergistically, wherein increasing IGF-1 concentrations augmented the potency and efficacy of TSH. It is believed that synergism where the presence of a growth factor increases the effective potency of a ligand to its cognate receptor has not been previously described. Of note, full HA induction only occurred in metabolically active cells, requiring the use of media containing 10% FBS. This media likely contains nanomolar concentrations of IGF-1 ; therefore it is possible that basal IGF-IR activity is necessary for TSHR to induce HA. Given the ubiquitous expression of IGF-IR, cooperative signaling may occur in every tissue where these two receptors are present with tissue-specific biological outcomes. In thyroid tissue, this outcome is increased proliferation, for orbital tissue, HA secretion.

The monoclonal TSHR stimulatory antibody M22 was used as a surrogate for polyclonal GD-IgGs so as to determine whether a single antibody could activate both receptors. This was not the case, as M22 did not change the phosphorylation state of IGF-IR. Prior studies indirectly demonstrated IGF-IR activation by M22 by studying IGF-IR downstream signaling events rather than the receptor itself.

However, PBK-Akt and mitogen-activated protein kinase (MAPK) signaling pathways activated by IGF-IR are also downstream of TSHR, and M22 effects could have been mediated through that receptor. Because earlier experiments were conducted in systems where both receptors were present and M22 undeniably binds to TSHR, whether its effects are in part mediated by IGF-IR could not have been determined. In this investigation, the biphasic dose-dependence of M22 stimulation of HA secretion was shown to be in part dependent on IGF-IR activation. It was found that an IGF-lR-selective kinase antagonist linsitinib, which abolished IGF-1 stimulation of HA secretion, inhibited the higher potency component of the biphasic dose- response to M22, but did not inhibit lower potency HA secretion. By contrast, a selective TSHR antagonist CI abolished M22 stimulation. These data support a process wherein M22 activates TSHR, leading to stimulation of IGF-IR pathways and synergistic increase HA secretion but does not address whether M22 directly binds to and activates IGF-IR.

To address the question as to whether M22 binds to and thereby activates IGF-IR, IGF-IR phosphorylation was measured, which is the initial step in activation of IGF-IR by IGF-1. As expected, IGF-1 stimulated IGF-IR phosphorylation in GOFs and linsitinib abolished this effect, but CI did not inhibit IGF-1 -stimulated IFG-1R phosphorylation. By contrast, M22 did not stimulate IGF-IR phosphorylation. It can be concluded therefore that M22 does not directly activate IGF-IR. Since IGF-1 -stimulated IGF-IR phosphorylation was not inhibited by CI but IGF-1 stimulation of HA secretion was partially inhibited by the TSHR-selective antagonist, it appears that cross-talk between TSHR and IGF-IR by M22 in GOFs does not require direct activation of IGF-IR.

Cross-talk between GPCRs and RTKs is well-documented with multiple mechanisms occurring between different receptors in different cells including one-way models in which GPCRs influence RTKs or vice versa, and more rarely bidirectional systems in which both receptors affect each other. The results are consistent with a bidirectional model between TSHR and IGF1-R based on the findings that (i) stimulation of HA secretion by TSH or M22 was partially inhibited by linsitinib and (ii) IGF-1 stimulation was similarly suppressed by the TSHR antagonist CI. The data also supports a model by which IGF-IR and TSHR can independently stimulate HA production in GOFs as both linsitinib and CI completely abolished HA secretion when only their cognate receptors were activated. Previous studies have reported stimulation of HA secretion by IGF-1 in cells that do not express TSHR, and it is likely that IGF-IR in GOFs may use a similar pathway. In addition, the similar results seen with another, albeit less effective, stimulatory antibody MSI demonstrate that this pathway may be common to stimulatory GD-IgG. It has already been shown by co-immunoprecipitation that TSHR and IGF-IR are present in close proximity in the cell surface membrane. As it is unlikely both M22 and MSI bind to TSHR the same way, the plausible explanation is that TSHR and IGF-IR are in the same signaling complex.

Previous investigators have interpreted the finding that an antibody 1H7, which inhibits IGF-1 binding to the alpha subunit of IGF1-R, inhibited activation of IGF1-R by GD-IgG as evidence of GD-IgG directly binding to and activating IGF1-R. These findings, however, do not allow for this conclusion. 1H7 is an inverse agonist that inhibits IGF-1 -independent (or basal) activity as well as IGF-1 -dependent activation and therefore the effect of 1H7 to apparently inhibit GD-IgG activation of IGF-IR may have been caused by 1H7 inhibition of the agonist-independent activity of IGF1-R or activation mediated by GD-IgG- activated TSHR. Also, it is possible that binding of 1H7 to IGF-IR might sterically interfere with M22 binding to TSHR. A second monoclonal antibody that inhibits binding of IGF-1 to the IGF-IR has been shown to inhibit TSH stimulation also but whether it is an inverse agonist has not been reported. The IGF- IR antagonist that was used is a small molecule that binds to the active tyrosine kinase site of the intracellular portion of IGF-IR and therefore would not interfere with GD-IgG or M22 binding. It is important to note that the findings do not exclude the possibility that there are antibodies within GD-IgGs that bind to and activate IGF-lRs. However, it is clear that binding to IGF-IR is not necessary for its involvement in the pathogenesis of GO.

This data shows that activation of both TSHR and IGF-IR leads to synergistic stimulation of HA secretion by GOFs and that activation by a monoclonal stimulatory antibody M22 generated from a patient with GD uses this dual signaling cascade. GD-IgG, like M22 and MSI, appear to activate TSHR and IGF- IR cross-talk and that this cross-talk plays a major role in the pathogenesis of GO.

Example 2

M22 Dose Response Curve

Previous data have revealed that stimulation of hyaluronan (hyaluronic acid, HA) production by M22, a monoclonal TSAb, by GOFs is biphasic in regards to its dose response curve with EC5₀ values roughly equivalent to 0.001 nM (EC50-I) and 0.42 nM (EC₅o-2), respectively.

Cultured GOFs (at passage 3) were stimulated with increasing concentrations of M22 for 4 days. Total HA was measured in culture media utilizing a modified ELISA assay. Data represents the mean ± SE. The curve fits to a biphasic model with an ECmed concentration estimated at about 0.3 nM (FIG. 7). The grey dotted line corresponds to a forced-fit monophasic model in comparison to the best-fit biphasic curve (solid black line).

Results: The EC_med concentration for M22 in GOF cells is about 0.3 nM, which will be implemented in downstream assays to determine drug profiles for TSHR and IGF-IR inhibitors. S2-7 and Linsitinib Dose Response Curves

Linsitinib is a small molecule IGF-1R kinase inhibitor previously shown to inhibit HA production by GOFs.

Cultured GOFs (at passage 3) were stimulated with M22 EC_med (0.3 nM) and co-treated with increasing doses of S2-7 for 4 days. Total HA was measured in culture media utilizing a modified ELISA assay. FIG. 8 provides data representing the mean ± SE of 4 different GOF cell lines and plotted as percent HA levels relative to maximal response. IC5₀ concentration (about 3.34 μΜ) is indicated by the dashed line.

Cultured GOFs (at passage 3) were stimulated with M22 EC_med (0.3 nM) and co-treated with increasing doses of linsitinib for 4 days. Total HA was measured in culture media utilizing a modified ELISA assay as described above (FIG. 9). The dashed line indicates the Linsitinib IC5₀ concentration of about 151 nM.

Results: Both the TSHR inhibitor S2-7 and IGF-1R kinase inhibitor linsitinib were full antagonists for HA production stimulated by M22 at its EC_med with potencies of 3.34 μΜ and 151 nM, respectively. These data were used in subsequent studies to construct isobolograms.

S2-7 and Linsitinib Isobologram

The above dose response data were used to generate an isobologram to empirically define whether the inhibitory effects of S2-7 and linsitinib could function cooperatively to inhibit HA production by GOFs. The IC5₀ isobologram in FIG. 10 illustrates the combinatorial effect of S2-7 and linsitinib on HA production. Cultured GOFs (at passage 3) were stimulated with M22 EC_med (0.3 nM) and co-treated with S2-7 and linsitinib at the indicated constant dose ratios (f) for 4 days. The Chou-Talalay theorem (indicated below) was used to define dosing pairs based on different combination index (CI) values at intersecting f ratios.

Chou-Talalay Theorem

_{CI =} C(S2-7 ₊ CfLinsit)

IC₅₀(S2-7) IC₅₀(Linsit)

C(S2-7) and C(Linsit) refer to the concentrations of S2-7 and linsitinib used in combination. All defined dosing pairs were evaluated for effects on HA production by ELISA analysis. Only those dosing pairs with activity equivalent to S2-7 and linsitinib IC50 activity were plotted on the isobologram. The experimental values for S2-7 and linsitinib co-treatment were equal to 1 (CI=1) defining Loewe additivity in context to inhibiting M22 ECmed stimulation.

Results: These data quantitatively revealed that S2-7 and linsitinib acted additively to inhibit HA production as a result of simulation by M22.

S2-7 and Linsitinib Dose Reduction

Cultured GOFs (at passage 3) were stimulated with M22 EC_med (0.3 nM) and co-treated with S2-7 IC5₀ (3.34 μΜ), Linsitinib IC5₀ (151 nM), or both in combination (50:50 Combo) for 4 days. Total HA was measured in culture media utilizing a modified ELISA assay (FIG. 11). Data represent the mean ± SD of 4 different GOF cell strains and plotted as percent HA levels relative to maximal response.

Results: These data indicate that dosage reductions equivalent to 50% activity for both drugs are fully efficacious in combination improving their therapeutic indexes.

Some anti-IGF-lR antibodies can act similarly to linsitinib

Anti-IGF-lR antibodies have been proposed as GO therapy by blocking IGF-1R stimulation. In addition, an anti-IGF-lR antibody (Teprotumumab) is currently in clinical development for GO. Assays were performed to determine if anti-IGF-lR antibodies can function similarly to linsitinib in context to HA production by M22 EC_med, and also to determine if anti-IGF-lR antibodies could function similarly to linsitinib in the context of HA production by M22 EC_med- The assays were designed to determine if a combinatorial approach for anti-IGF-lR antibodies and TSHR inhibitors (for example S2-7) also had potential as cooperative drugs.

Cultured GOFs (at passage 3) were stimulated with M22 EC_med (0.3 nM) and co-treated with anti- IGF-1R blocking antibodies AF305 or 1H7 at the indicated concentrations for 4 days. Total HA was measured in culture media utilizing a modified ELISA assay (FIG. 12). Data represents the mean ± SD and plotted as percent HA levels relative to maximal response.

Results: These data indicate that some IGF-1R blocking antibodies (i.e. 1H7) have efficacy in inhibiting HA production in GOFs by M22 EC_med, however, this effect is not indicative for all IGF-1R blocking antibodies (i.e. AF305). It also reveals that 1H7 functions similarly to linsitinib in its ability to fully inhibit HA production by M22 EC_med supporting the idea that TSHR inhibitors may also have function to improve the therapeutic index of anti-IGF-lR antibodies in the treatment of GO.

Conclusion

The results indicated that in combination, small molecule TSHR inhibitors (for example, S2-7) and

IGF-1R kinase inhibitors (for example, linsitinib) function cooperatively for dosage reduction. Linsitinib can be combined with S2-7 to allow for lower doses of both drugs, thereby improving their therapeutic indices. For example, linsitinib is also an inhibitor of the insulin receptor but at a lower potency. Lower doses of linsitinib can be compensated for by S2-7 (or other related compounds) potentially reducing the frequency of side-effects caused by insulin receptor inhibition. Additionally, small TSHR inhibitors (e.g. S2-7) can also be used in combination with some anti-IGF-lR blocking antibodies (e.g. 1H7 or

Teprotumumab) for dosage reduction to improve their therapeutic indices and reduce side effects.

In view of the many possible embodiments to which the principles of the disclosed methods and compositions may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.

Claims

What is claimed is:

1. A method of treating Graves' disease in a subject, comprising co-administering to a subject in need thereof a therapeutically effective amount of: (a) an inverse agonist of TSHR and/or a neutral antagonist of TSHR; and (b) an IGFR inhibitor.

2. The method of claim 1, wherein the Graves' disease is Graves' hyperthyroidism, Graves' orbitopathy, or Graves' dermopathy.

3. The method of claim 1, wherein the Graves' disease is Graves' orbitopathy,

4. The method of claim 1, wherein the method inhibits hyaluronan production in the subject.

5. A method of inhibiting TSHR- and IGFR-stimulated hyaluronan secretion in a subject, comprising co-administering to the subject an inhibitory effective amount of: (a) an inverse agonist of TSHR and/or a neutral antagonist of TSHR; and (b) an IGFR inhibitor, thereby inhibiting TSH- and IGF-1- stimulated hyaluronan.

6. The method of claim 5, wherein the inhibition of hyaluronan secretion is inhibition of hyaluronan secretion by Graves' orbitopathy fibroblasts.

7. The method of claim 2, wherein the Graves' disease is Graves' dermopathy and at least one of (a) or (b) is topically administered to the subject.

8. The method of any one of claims 1 to 9, wherein the method comprises co-administering (a) an inverse agonist of TSHR and (b) an IGFR inhibitor.

9. The method of any one of claims 1 to 9, wherein the method comprises co-administering (a) a neutral antagonist of TSHR and (b) an IGFR inhibitor.

10. The method of claim 1, wherein (a) and (b) are simultaneously administered to the subject.

11. The method of claim 1, wherein (a) and (b) are sequentially administered to the subject.

12. The method of claim 1, wherein the inverse agonist of TSHR or the neutral antagonist of

TSHR comprises a 2,3-dihydroquinazolin-4-one compound.

13. The method of claim 1, wherein the inverse agonist of TSHR or the neutral antagonist of is a compound, or a pharmaceutically acceptable salt or ester thereof, having a structure of:

(Formula I) wherein R¹ is selected from a furanyl-containing group, a pyridinyl-containing group, a thienyl-containing group, hydroxyalkyl, or alkoxyalkyl;

R² is H, alkoxy, alkyl, substituted alkyl or halogen; and

R³ - R⁷ are each individually selected from H, alkyl, substituted alkyl, halogen, or aminocarbonyl; and X is O or S.

14. The method of claim 13, wherein R¹ is selected from furan-2-ylmethyl, pyridin-3-ylmethyl, thien-2-ylmethyl or methoxyethyl.

15. The method of claim 13, wherein R² is me thoxy

16. The method of claim 13, wherein R³ and R⁷ are each methyl.

17. The method of claim 13, wherein X is O.

18. The method of claim 13, wherein X is S.

19. The method of claim 13, wherein R⁵ is an aminocarbonyl group.

20. The method of claim 14, wherein R² is methoxy, and R³ and R⁷ are each methyl.

21. The method of claim 20, wherein X is O.

22. The method of claim 21, wherein R⁵ is an aminocarbonyl group.

23. The method of claim 20, wherein X is S.

24. The method of claim 23, wherein R⁵ is an aminocarbonyl group

25. The method of claim 13,

26. The method of claim 13, wherein the compound is:

27. The method of claim 13, wherein the compound is:

O!vle

28. The method of claim 13, wherein the compound is:

29. The method of claim 13, wherein the compound is:

30. The method of any one of claims 1 to 29, wherein the IGFR inhibitor is linsitinib (OSI-906) , BMS-754807, INSM-18, XL228, AXL1717, BMS-536924, NVP-ADW742, GSK621659A,

GSK1838705A, A-928605, AZD4253, TAE226, AG1024, PQ401 (CAS 196868-63-0), or AG538 (CAS 133550-18-2).

31. The method of any one of claims 1 to 29, wherein the IGFR inhibitor is an anti-IGF-lR antibody selected from ganitumab, AMG 479, figitumumab, CP-751,871, cixutumumab, IMC-A12, dalotuzumab, MK0646, teprotumumab (RG1507), robatumumab, SCH 717454, AVE-1642a, MEDI-573, BIIB022, rhuMab IGFR, LlHl, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, LIOHIO, Ll lHl l, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51, or L52H52.

32. The method of any one of claims 1 to 10, or 12 to 31, wherein a pharmaceutical composition comprising: (a) a therapeutically effective amount of an inverse agonist of TSHR and/or a neutral antagonist of TSHR; (b) a therapeutically effective amount of an IGFR inhibitor; and (c) at least one pharmaceutically acceptable additive is administered to the subject.

33. A pharmaceutical composition comprising: (a) a therapeutically effective amount of an inverse agonist of TSHR and/or a neutral antagonist of TSHR; (b) a therapeutically effective amount of an IGFR inhibitor; and (c) at least one pharmaceutically acceptable additive.

34. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition is in a dosage unit form.