WO2006085330A2 - Use of protein tyrosine kinase inhibitors for the treatment of leiomyomas - Google Patents

Abstract

Pharmaceutical compositions containing epidermal growth factor receptor tyrosine kinase (EGFR-TK) irreversible inhibitors or natural protein kinase inhibitors and use thereof for the treatment of leiomyomas are disclosed.

Description

USE OF PROTEIN TYROSINE KINASE INHIBITORS FOR THE TREATMENT

OF LEIOMYOMAS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to the field of pharmacology and more particularly to novel, non-surgical methods of treating leiomyomas.

Uterine leiomyomas or fibroids are the most common pelvic tumors in women, with a reported incidence of 30 % of women over 35 years. Leiomyomas are benign tumors which arise from the smooth muscle cells of the myometrium. Leiomyomas are clinically important because they are a major cause of abnormal uterine bleeding (menorrhagia), as well as causing infertility, miscarriage or pain, and are the most commonly cited reason for hysterectomy. Recently, it has been reported that the estimated cumulative incidence of uterine leiomyomas by age 50 was greater than 80 % for black women and nearly 70 % for white women. The precise pathophysiology of leiomyomas growth is still unresolved.

Chromosomal abnormalities, hormonal deregulation, growth and angiogenic factors, have all been implemented in the etiology of these clonal proliferations of smooth muscle cells that arise independently.

Surgical removal through hysterectomy or myomectomy has been the traditional treatment of leiomyomas. Presently, less invasive surgical techniques such as hysteroscopic removal are being tested. However, although the present surgical treatments are very effective in the short term, the long-term results are less satisfactory: 51 % recurrence in 5 years and 15-26 % need of second surgery.

Previous and present research has opened the way to new potential medical treatments. These are based on the understanding that leiomyomas are smooth muscle steroid responsive tumor cells. Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential (Vollenhoven et at, 1990). A body of research has demonstrated that myomas are affected by estrogen (E2) and progesterone (P4) and that their response is different compared to that of the normal myometrium (Nowak, 2000). Gonadotropin- releasing hormone (GnRH) agonists, which reduce ovarian steroid hormones are also widely in use (Carr et al, 1993). However, side effects such as premature menopause and associated symptoms, including hot flushes and vaginal dryness, as well as bone demineralization due to estrogen withdrawal prohibit the long-term use of these compounds. Since leiomyomas are chronic or recurrent, a therapy that allows for more prolonged treatment is highly recommended. Indeed GnRH agonists with add- back estrogen and progesterone therapy are among the most frequently studied medical therapy (Nowak, 2001; Nowak, 2000). Such a therapy, however, is still an expensive alternative when compared with definitive surgery.

Newer proposed and studied therapies for leiomyomas are based on the differential expression/production of growth factors between myomas and normal myometrium, e.g. heparin-binding growth factors, insulin-like growth factors and the Transforming Growth Factor (TGF)-β ligand-receptor system (Dixon et al, 2000). Interferon-α or interferon-β have already been suggested as possible treatment modalities, since they were shown to oppose the actions of b-Fibroblast Growth Factor (FGF) in a number of systems (Lee et al, 1998). Thus, targeting these growth factors and their receptors can contribute to development of potential targets for therapies, through more than one mechanism. However, interferon therapy has been associated with a number of severe side effects, including autoimmune diseases resulting from their immunostimulatory properties.

Thus, in view of the limitations associated with the presently known therapies for leiomyomas, there is a highly recognized need for and it would be highly advantageous to have a novel and efficient non-surgical treatment for leiomyomas.

As is well recognized in the art, cells typically undergo a four-phase replication cycle. The first phase, called Gl, is when the cell prepares to replicate its chromosomes. The second stage, called S, involves DNA synthesis and duplication. The next phase, called G2, involves duplication of RNA and proteins. The final stage is the M stage, in which actual cell division occurs. In this final stage, the duplicated DNA and RNA split and move to separate ends of the cell, and the cell actually divides into two identical, functional cells. The control of cell proliferation is intimately connected to apoptosis, a homeostatic process which ensures that abnormal cells (old, mutated or damaged) die or are killed. In cancer cells this mechanism appears frequently to be disrupted, such that malignant cells do not die but, instead, continue to proliferate. Accumulating data indicate that the effects of steroid hormones on proliferation of leiomyomas are mediated by the local production of endothelial growth factor (EGF). Heightened EGFR-mediated signaling in cancer cells may play a role in blocking the normal process of apoptosis, allowing abnormal cells to live on to replicate and spread. Therefore, selective inhibitors of the EGF-receptor (EGFR) might be useful in the treatment of leiomyomas.

The epidermal growth factor receptor (EGFR, Erb-Bl) belongs to a family of proteins that are involved in the proliferation of normal and malignant cells. Growth factors often mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic intracellular protein tyrosine kinase activity. Overexpression of Epidermal Growth Factor Receptor (EGFR) is present in at least 70 % of human cancers, such as, for example, non-small cell lung carcinomas (NSCLC), breast cancers, gliomas, squamous cell carcinoma of the head and neck, and prostate cancer. The EGFR is therefore widely recognized as an attractive target for the design and development of compounds that can specifically bind and inhibit tyrosine kinase activity and its signal transduction pathway in cancer cells, and thus can serve as either diagnostic or therapeutic agents. EGF has been shown to mediate estrogen action and to play a crucial role in regulating leiomyomas growth (Shushan, 2004).

Compounds belonging to the 4-amlinoquinazolines family, which are also referred to herein as 4-(phenylamino)quinazolines, have been shown to potently and selectively inhibit EGFR-TK activity by binding reversibly to an inner membrane ATP binding site on EGFR-TK, the prototype for such compounds being the small- molecule AG 1478, also known as PD 153035 (Fry et al., 1994; Levitzki and Gazit, 1995), which is presently in clinical development. Recent studies of the suppressive effect of AG 1478 on leiomyomas cells prior and subsequent to steroidal hormones treatment showed that leiomyoma cell growth is effectively blocked by AG 1478 and is unaffected by the presence of physiological concentrations of progesterone and 17-β estradiol (Shushan, 2004). These studies further showed that leiomyoma cell growth did not recover after cessation of treatment.

The potency of reversible EGFR-TK inhibitors in general and of AG1478 in particular, however, is limited by their non-specific binding and rapid blood clearance. Irreversible EGFR-TK inhibitors, which are based on the structure of AG

1478, have been described in the art (Smaill et al., 2000; and U.S. Patents Nos. 6,153,617 and 6,127,374). The irreversible binding of these inhibitors was achieved by substituting the 6 or 7 position of the quinazoline ring of an 4-(anilino)quinazolme derivative with an α,β-unsaturated carboxylic group, preferably an acrylamide group, which binds covalently to the Cys-773 at the EGFR-TK ATP binding site. Some of these compounds showed high potency toward EGFR inhibition in both in vitro and in vivo experiments (Smaill et al., 2000). However, more recent studies showed that these irreversible EGFR-TK inhibitors are limited by a relatively low accumulation at EGFR-expressing tumor cells (Ben David et al., 2003).

In the recently published International Patent Application WO 04/064718, which is incorporated by reference as if fully set forth herein, a novel class of irreversible EGFR-TK inhibitors characterized by reduced biodegradation, enhanced bioavailability and hence by improved in vivo performance as compared with the structurally related reversible EGFR-TK inhibitors described above, has been disclosed. The compounds belonging to this newly designed class have an α- chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring. According to the teachings of WO 04/064718, it was found that replacing the α,β- unsaturated side chain of the carboxylic moiety, which is a highly chemical reactive group, by the less reactive chloro and methoxy groups, which can further act as leaving groups and thus readily react so as to form a covalent bond with the cysteine moiety at the receptor binding site, resulted in potent irreversible inhibitors with enhanced biostability and bioavailability. It was thus found that such newly designed compounds, having an α-chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring, show high affinity toward EGFR and high ability to irreversibly bind to the receptor, thus indicating their potential as improved EGFR-TK irreversible inhibitors and as a result as improved therapeutic agents.

A representative member of this family of irreversible EGFR inhibitors is TK050, which has been described for use as a biomarker for positron emission tomography and for molecular imaging of EGFR in EGFR-positive tumors.

While the effect of these irreversible EGFR-TK inhibitors on cell proliferative disorders such as specific types of cancer have been demonstrated in WO 04/064718, the use of these EGFR-TK inhibitors for treating leiomyomas in particular has not been practiced nor suggested hitherto.

Protein tyrosine kinases (PTKs) are enzymes which catalyze the phosphorylation of tyrosine residues. These enzymes are involved in cellular signaling pathways and regulate key cell functions such as proliferation, differentiation, anti-apoptotic signaling and neurite outgrowth. Unregulated activation of these enzymes, through mechanisms such as point mutations or over- expression, can lead to various forms of cancer as well as benign proliferative conditions. There are two main classes of PTKs: receptor PTKs and cellular, or nonreceptor, PTKs. Receptor PTKs possess an extracellular ligand binding domain, a transmembrane domain and an intracellular catalytic domain. The intracellular kinase domains of receptor PTKs can be divided into two classes: those containing a stretch of amino acids separating the kinase domain and those in which the kinase domain is continuous. Activation of the kinase is achieved by ligand binding to the extracellular domain, which induces dimerization of the receptors. Receptors thus activated are able to autophosphorylate tyrosine residues outside the catalytic domain via cross- phosphorylation. The results of this auto-phosphorylation are stabilization of the active receptor conformation and the creation of phosphotyrosine docking sites for proteins which transduce signals within the cell. Signaling proteins which bind to the intracellular domain of receptor tyrosine kinases in a phosphotyrosine-dependent manner include RasGAP, PI3-kinase, phospholipase C, phosphotyrosine phosphatase SHP and adaptor proteins such as She, Grb2 and Crk.

Genistein is a natural isoflavone compound with a potent activity against protein tyrosine kinases. It is also classified as a phytoestrogen. Phytoestrogens are plant-derived non-steroidal compounds that possess estrogen-like biological activity. Genistein has been found to have both weak estrogenic and weak anti-estrogenic effects. Genistein is the aglycone (aglucon) of genistin. The isoflavone is found naturally as the glycoside genistin and as the glycosides 6"-O-malonylgenistin and 6"- O-acetylgenistin. Genistein and its glycosides are mainly found in legumes, such as soybeans and chickpeas The use of natural PTK inhibitors such as genistein for treating leiomyomas has not been practiced nor suggested hitherto.

SUMMARY OF THE INVENTION The present inventors have now surprisingly found that EGFR-TK irreversible inhibitors of the 4-anilinoquinazolines family, which have an α-chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring, can be beneficially used in the treatment of leiomyomas. The present inventors have further uncovered that natural protein tyrosine kinase inhibitors (PTK), such as genistein, can also be beneficially utilized in the treatment of leiomyomas.

The present invention therefore provides novel non-surgical methods for treating leiomyomas.

Hence, according to one aspect of the present invention there is provided the use of a therapeutically effective amount of an irreversible inhibitor of EGFR-TK in the manufacture of a medicament for the treatment of leiomyomas.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient the compound described hereinabove and a pharmaceutical acceptable carrier, the composition being identified for use in the treatment of leiomyomas. The EGFR-TK irreversible inhibitor has the general Formula I:

Formula I wherein:

Ql is X-W(=Y)-Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino, or

Ql is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino and Q2 is X-W(=Y)-Z; X is selected from the group consisting of -NR¹-, -O-, -NH-NR¹-, -0-NR¹-,

NH-CHR¹-, -CHR^NH-, -CHR^O-, -O-CHR¹-, -CHR^CH₂- and -CHR^S- or absent;

W is carbon; Y is selected from the group consisting of oxygen and sulfur;

Z Is -CR²R³R⁴;

R^a is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;

A, B, C and D are each independently selected from the group consisting hydrogen and a first derivatizing group;

R¹ is selected from the group consisting of hydrogen, and substituted or non- substituted alkyl having 1-6 carbon atoms;

R² is a leaving group; and

R³ and R⁴ are each independently selected from the group consisting of hydrogen and a second derivatizing group.

According to further features in preferred embodiments of the invention described below, the first derivatizing group is selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carbalkoxy, thiocarboxy, thiohydroxy, thioalkoxy, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano.

According to still further features in the described preferred embodiments the second derivatizing group is selected from the group consisting of halogen, alkyl, haloalkyl, cycloalkyl, heteroalicyciic, aryl, heteroaryl, carboxy, hydroxy, alkoxy, aryloxy, carbonyl, thioalkoxy, thiohydroxy, thioaryloxy, thiocarboxy, thiocarbonyl, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano, or alternatively, R³ and R⁴ together form a five- or six-membered ring.

According to still further features in the described preferred embodiments the leaving group is selected from the group consisting of halogen, alkoxy, aryloxy, thioalkoxy, thioaryloxy, azide, sulfinyl, sulfonyl, sulfonamide, phosphonyl, phosphinyl, carboxy and carbamyl.

According to still further features in the described preferred embodiments the alkoxy comprises a morpholino group. According to still further features in the described preferred embodiments the alkylamino comprises an N-piperazinyl group.

According to still further features in the described preferred embodiments the Ql is X-W(=Y)-Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino. Preferably, Q2 is hydrogen, alkoxy or alkylamino, as described hereinabove. Further preferably, X is - NR¹- and Y is oxygen. Further preferably each of R¹, R³ and R⁴ is hydrogen. Further preferably, R is a leaving group selected from the group consisting of alkoxy and halogen. According to still further features in the described preferred embodiments at least one of A, B, C and D is fluorine. Preferably D is fluorine. More preferably, D is fluorine, A and B are each chlorine and C is hydrogen.

According to still further features in the described preferred embodiments A is bromine or iodine. Preferably, A is bromine or iodine and B, C and D are each hydrogen.

The preferred compound for use in the present invention is N-{4-[(3,4- dichloro-6-fluorophenyl) amino] quinazoline-6-yl}-2-chloroacetamide, which is also referred to herein interchangeably as TKS050 or ML05.

According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a natural PTK inhibitor in the manufacture of a medicament for the treatment of leiomyomas.

According to an additional aspect of the present invention, there is provided pharmaceutical composition comprising a natural PTK inhibitor, being identified for use in the treatment of leiomyomas. Preferably, the PTK inhibitor is a flavonoid, more preferably an isoflavone.

A preferred natural PTK inhibitor according to these aspects of the present invention is genistein.

According to further features in the described preferred embodiments, a therapeutically effective amount of the compound of formula I ranges from about 0.1 μM to about 50 μM, more preferably from about 1 μM to about 10 μM. A therapeutically effective amount of genistein ranges from about 10 μM to about 100 μM, more preferably from about 10 μM to about 50 μM. According to yet further features in the described preferred embodiments, any of the compounds of described herein optionally forms a part of a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier.

According to further features in the described preferred embodiments, the pharmaceutical composition further comprises a therapeutically effective amount of at least one agent that is capable of treating leiomyomas, such as, for example, an interferon, a reversible inhibitor of Epidermal Growth Factor Receptor (EGFR), a steroid hormone, an estrogen modulator, an anti-gonadotropic hormone, a gonadotropin-releasing hormone agonist, a gonadotropin-releasing hormone antagonist, an antiprogestin, and a somatostatin analogue. This agent may be administered prior to, concomitant with or subsequent to administration of the compound of Formula I or genistein.

The present invention successfully addresses the shortcomings of the presently known methods of treatment of leiomyomas by providing highly efficient non-surgical treatment methods which employ irreversible EGFR-TK inhibitors with improved biostability and bioavailability and/or natural PTK inhibitors.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION QF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIGS. IA-D present comparative plots showing the inhibitory effects of

TKS050 and genistein on the proliferation and rescue after treatment of leiomyoma and myometrium cell cultures (FIGS. IA and IB represent the effect of TK050 on leiomyoma cells and myometrium cells, respectively; FIGS. 1C and ID represent the effect of genistein on leiomyoma cells and myometrium cells, respectively); FIGS. 2A-D present comparative plots showing the effects of estradiol (E2) alone and in combination with genistein on the proliferation and rescue after treatment of leiomyoma and myometrium cell cultures (FIGS. 2 A and 2B represent the effects of estradiol alone on leiomyoma and myometrium cells, respectively; FIGS. 2C and 2D represent the effect of estradiol in combination with genistein on leiomyoma and myometrium cell cultures);

FIGS. 3A-D present bar charts showing the effects of TKS050 and genistein on the cell cycle phase distribution and apoptosis of leiomyoma and myometrium cell cultures (FIGS. 3A and 3B represent the effects of TKS050 on of leiomyoma and myometrium cells, respectively; FIGS. 3 C and 3D represent the effects of genistein on of leiomyoma and myometrium cells, respectively);

FIG. 4 presents an immunoblot analysis of the biochemical activity of TKS050 in myometrium cells;

FIG. 5 presents an immunoblot analysis of the biochemical activity of TKS050 in leiomyoma cells; FIG. 6 presents an immunoblot analysis of the biochemical activity of genistein in myometrium cells; and

FIG. 7 presents an immunoblot analysis of the biochemical activity of genistein in leiomyoma cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of treating leiomyomas using irreversible EGFR-TK inhibitors and/or natural PTK inhibitors. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

As is discussed in detail hereinabove, a novel class of A- (phenylamino)quinazoline, which acts as irreversible EGFR-TK inhibitors has recently been uncovered. These compounds are taught in WO 04/064718, which is incorporated by reference as if fully set forth herein. Preferred compounds in this class have an α-chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring and are characterized by reduced biodegradation, enhanced bioavailability and hence by improved in vivo performance as compared with other, structurally related irreversible EGFR-TK inhibitors (such as those described, for example, in Fry et al., 1998; Smaill et al., 2000; and U.S. Patents Nos. 6,153,617 and 6,127,374).

While conceiving the present invention, it was envisioned, based on the potent effect of reversible EGFR-TK inhibitors such as AG 1478, that the novel and improved irreversible EGFR-TK inhibitors described above could be useful in the treatment of leiomyomas.

At least two broad groups of leiomyomata have been previously identified which were characterized by the dysregulation of different groups of gene products. One of these groups is characterized by up-regulation of the EGFR. Thus, the effective inhibition of EGFR by irreversible EGFR inhibitors might improve the efficacy of current medical therapies, based on ligand-mediated apoptosis. Previous biochemical analyses of AG1478, an EGFR specific inhibitor, have shown that this tyrphostin effectively inhibits EGFR autophosphorylation in a dose dependent manner, with no effect on the level of EGFR expression. While reducing the present invention to practice it was indeed found that a representative member of a family of novel irreversible EGFR inhibitors described hereinabove, TKS050, efficiently blocks growth of leiomyoma cells.

It was further found that no recovery of the cells occurred after cessation of treatment, indicating that TKS050 provides an effective, long-term blockage of leiomyoma cell growth.

It was further found that TKS050 efficiently alters cell cycle distribution, by decreasing the proportion of cells in the Gi phase, and increasing the proportion of cells in the synthesis and mitotic phases S and G₂/M phases, with a significant increase in the apoptotic cell fraction.

It was further found that TKS050 specifically and potently induced a dose- dependent inhibition of EGFR autophosphorylation.

It was further found that the activity of TKS050 was not affected by estradiol, and thus, that a combined treatment that includes co-administration of TKS050 and GnRH agonists with add-back estradiol can be used for treating leiomyomas.

Herein, the term "add-back" with regard to estradiol, refers to physiological levels of the estradiol, which are administered following treatment with GnRH agonists_^ in order to suppress the adverse effects of hypoestrogenism commonly induced by GnRH agonists. TKS050 was therefore shown to have a marked antiproliferative effect on leiomyoma cell cultures and to cause change in their cell cycle distribution via increased apoptosis.

Thus, according to one aspect of the present invention there is provided a novel method of treating leiomyomas. The method is effected by administering to a subject in need thereof a therapeutically effective amount of a compound having the • general Formula I:

Formula I

wherein: Ql is X-W(=Y)-Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino, or Ql is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino and Q2 is X-W(=Y)-Z;

X is selected from the group consisting of -NR¹-, -O-, -NH-NR¹-, -ONR¹-, NH-CHR¹-, -CHR^NH-, -CHR^O-, -0-CHR¹-, -CHR¹-CH₂- and -CHR^S- or absent;

W is carbon;

Y is selected from the group consisting of oxygen and sulfur; Z is -CR²R³R⁴; R^a is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;

A, B, C and D are each independently selected from the group consisting hydrogen and a first derivatizing group;

R¹ is selected from the group consisting of hydrogen, and substituted or non- substituted alkyl having 1-6 carbon atoms; R² is a leaving group; and

R³ and R⁴ are each independently selected from the group consisting of hydrogen and a second derivatizing group.

Accordingly, compounds having Formula I above can be used in the manufacture of a medicament for treating lieomyomas.

As used herein, the phrase "derivatizing group" refers to a major portion of a chemical group which is covalently attached to another group. The term "halogen", which is also referred to herein as "halo", refers to fluorine, chlorine, bromine or iodine.

As used herein, the term "hydroxy" refers to an -OH group. As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group is a medium size alkyl having 1 to 10 carbon atoms. More preferably, it is a lower alkyl having 1 to 6 carbon atoms. Most preferably it is an alkyl having 1 to 4 carbon atoms. Representative examples of an alkyl group are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl and hexyl. The alkyl group, according to the present invention, may be substituted or non- substituted. When substituted, the substituent group can be, for example, cycloalkyl, aikenyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo, perhalo, trihalomethyl, carboxy, alkoxycarbonyl, thiocarboxy, carbamyl, cyano, nitro, N-piperidinyl, N-piperazinyl, NrpiperazinyMSU- alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N-thiomorpholino, N- hexahydroazepine, amino or NRbRc, wherein Rb and Rc are each independently hydrogen, alkyl, hydroxyalkyl, cycloalkyl, aryl, N-piperidinyl, N-piperazinyl, N₁- piperazinyl-Nt-alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N- thiomorpholino and N-hexahydroazepine, as these terms are defined herein. The term "haloalkyl" refers to an alkyl group, as defined hereinabove, which is substituted by one or more halogen atoms.

As used herein, the term "cycloalkyl" refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene and adamantane.

The term "alkoxy" refers to both an -O-alkyl and an -0-cycloalkyl group, as defined hereinabove. Representative examples of alkoxy groups include methoxy, ethoxy, propoxy and tert-butoxy.

The O-alkyl and the O-cycloalkyl groups, according to the present invention, may be substituted or non-substituted. When substituted, the substituent group can be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo, perhalo, trihalomethyl, carboxy, alkoxycarbonyl, thiocarboxy, carbamyl, cyano, nitro, N-piperidinyl, N-piperazinyl, N_t-piperazinyl-Nj-alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N- thiomorpholino, N-hexahydroazepine, amino or NRbRc, wherein Rb and Rc are each independently hydrogen, alkyl, hydroxyalkyl, N-piperidinyl, N-piperazinyi, N₁- piperazinyl-N₄-alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N- thiomorpholino and N-hexahydroazepine, as these terms are defined herein.

The term "thiohydroxy" refers to a -SH group. The term "thioalkoxy" refers to both an -S-alkyl group, and an -S-cycloaikyl group, as defined herein.

The term "amino" refers to a -NH₂ group.

The term "alkylamino" refers to a -NRbRc group wherein Rb and Rc are each independently hydrogen, alkyl, hydroxyalkyl, N-piperidinyl, N-piperazinyl, Ni- piperazinyl-Nj-alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N- thiomorpholino and N-hexahydroazepine, as these terms are defined herein, or, alternatively, Rb and Rc are covalently attached one to the other so as to form a cyclic amino compound such as, but not limited to, N-piperidinyl, N-piperazinyl, Ni- piperazinyl-N₄-alkyl, N-pyrrolidyl, pyridinyl, N-imidazoyl, N-morpholino, N- thiomorpholino and N-hexahydroazepine.

The term "carboxy" refers to a -C(=O)-OR' group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined herein.

The term "alkoxycarbonyl", which is also referred to herein interchangeably as "carbalkoxy", refers to a carboxy group, as defined hereinabove, where R' is not hydrogen.

The term "carbonyl" refers to a -C(=O)-R' group, where R' is as defined hereinabove.

The term "thiocarbonyl" refers to a -C(=S)-R' group, where R' is as defined hereinabove.

An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl.

A "phenyl" group, according to the present invention can be substituted by one to three substituents or non-substituted. When substituted, the substituent group may be, for example, halogen, alkyl, alkoxy, nitro, cyano, trihalomethyl, alkylamino or monocyclic heteroaryl.

The term "heteroaryl" group includes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.

A "heteroalicyclic" group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.

An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.

A "thioaryloxy" group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein.

A "trihalomethyl" group refers to a -CX₃ group, wherein X is a halogen as defined herein. A representative example of a trihalomethyl group is a -CF₃ group.

A "perhalo" group refers to a group in which all the hydrogen atoms thereof have been replaced by halogen atoms. A "thiocarboxy" group refers to a -C(=S)-OR' group, where R' is as defined herein.

A "sulfmyl" group refers to an -S(=O)-R' group, where R' is as defined herein.

A "sulfonyl" group refers to an -S(^O)₂ -R' group, where R' is as defined herein. A "carbamyl" group refers to an -OC(=O)-NRbRc group, where Rb and Rc are as defined herein.

A "nitro" group refers to a -NO₂ group. A "cyano" group refers to a -C≡N group.

The term "N-piperazinyl", which is also referred to herein as "N-piperazino"

refers to

group.

The term "N-piperidinyl" refers to a

group.

-R' The term "Nrpiperazinyl-N^alkyl" refers to a N / , where R' is an alkyl, as defined hereinabove.

The term "N-pyrrolidyl" refers to a

group

The term "pyridinyl" refers to a Λ v — !/ group.

The term "N-imidazoyl" refers to

group

The term "N-morpholino" refers to a

group

The term "N-thiomorpholino" refers to a

group.

The term "N-hexahydroazepine" refers to

The compounds utilized in this and other aspects of the present invention are derivatized 4-(phenylamino)quinazolines, substituted at position 6 or 7 of the quinazoline ring by a carboxylic or thiocarboxylic group that is substituted at the α position by a leaving group. This substituted carboxylic/thiocarboxylic group is also defined herein as a X-W(=Y)-Z group.

As used herein throughout, and is well known in the art, the phrase "leaving group" refers to a chemical moiety that can be easily replaced by a nucleophilic moiety in a nucleophilic reaction. Representative examples of leaving groups include, without limitation, halogen, alkoxy, aryloxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, carboxy and carbamyl, as these terms are defined herein, with halogen and alkoxy being the presently most preferred. Additional examples of leaving groups include, without limitation, azide, sulfonamide, phosphonyl and phosphinyl. As used herein, the term "azide" refers to a -N₃ group. The term "sulfonamide" refers to a -S(=O)₂-NR'R" group, with R' as defined hereinabove and R" as defined herein for R'.

The term "phosphonyl" describes an -O-P(=O)(OR')2 group, with R' as defined hereinabove.

The term "phosphinyl" describes a -PR'R" group, with R' and R" as defined hereinabove.

As is described in the art (see, for example, U.S. Patent No. 6,126,917 and Smaill et al., 2000), the level of the biological activity of 4-(phenylamino)quinazoline EGFR-TK inhibitors, whether reversible or irreversible, is influenced by the nature of the derivatizing groups at both the anilino ring and the quinazoline ring thereof. The nature of these derivatizing groups may affect the binding affinity of the compound to the receptor as well as other biological activity parameters such as specificity, metabolism of the compound and kinetic rates. Thus, according to a preferred embodiment of the present invention, the derivatizing group is attached to the aniline ring (as is represented in Formula I hereinabove by A, B, C and D as a first derivatizing group) and includes, for example, hydrogen, halogen, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carbalkoxy, thiohydroxy, thiocarboxy, thioalkoxy, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano, as these terms are defined hereinabove.

According to another preferred embodiment of the invention, a derivatizing group is attached to the quinazoline group (as is represented in Formula I hereinabove by either Ql or Q2) and includes, for example, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino. Preferably, this derivatizing group is an alkoxy group and, more preferably, it is an alkoxy group that comprises a morpholino group such as, but not limited to, a 3-(4-morpholinyl)propoxy group. Further preferably, the derivatizing group is a substituted or non-substituted morpholino group or a substituted or non-substituted piperazino group. The presence of a morpholino or piperazino group in this class of compounds in known to increase their biological availability (Smaill et al., 2000).

Another factor which influences the binding potency of the compounds of the present invention is the position of which the carboxylic group is attached to the quinazoline ring. A 6-position carboxylic group has higher binding potency to the EGFR-TK ATP site (Smaill et al, 1999, Smaill et al., 2000 and U.S. Pat. Nos. 6,153,617 and 6,127,374). Thus, according to another preferred embodiment of the present invention, the X-W(=Y)-Z group of the compound is attached to position 6 of. the quinazoline ring, such that Ql in Formula I above is X-W(=Y)-Z.

According to still another preferred embodiment of the invention, the 6- position carboxylic group substituted by a leaving group is an α-chloroacetamide or α-methoxyacetamide group. Thus, preferred compounds according to the present invention are N-[4-(phenylamino)quinazolin-6-yl]-2-chloroacetamide and N-[4- (phenylamino)quinazolin-6-yl]-2-methoxyacetamide, derivatized by the R^a, A, B, C and D as these symbols are defined above, with the first being more active and therefore presently more preferred. These compounds are represented by Formula I hereinabove, wherein Ql is X-W(=Y)-Z, X is -NH-, Y is oxygen, and Z is -CH₂Cl or CH₂OCH₃, respectively. As is taught, for example, in U.S. Patent No. 6,126,917, 4-

(phenylarnino)quinazolines that are derivatized at position 6 of the anilino group by fluorine are potent inhibitors of EGFR-TK. The highest affinity toward the receptor is achieved using 4-[(3,4-dichloro-6-fluorophenyl)- amino]quinazolines.

Thus, preferred compounds for use in this and other aspects of the present invention are those in which R^a is hydrogen, A and B are each chlorine, C is hydrogen and D is fluorine. More preferred compounds are the N-[4-(phenylamino)quinazolin- 6-yl]-2-chloroacetamide and N-[4-(phenylamino)quinazolin-6-yl]-2- methoxyacetamide described hereinabove, in which. R^a is hydrogen, A and B are each chlorine, C is hydrogen and D is fluorine. As is taught in U.S. Patent No. 6,562,319, in U.S. Application No.

20020128553 and in WO 04/064718, 4-(phenylamino)quinazolines that are derivatized at position 3 of the anilino group by bromine or iodine are also potent inhibitors of EGFR-TK. These compounds further serve as precursors for radioactive bromine or radioactive iodine labeled compounds, which, as is detailed hereinbelow, are highly potent radiolabeled compounds.

Hence, additional preferred compounds for use according to the method of the present invention are those in which R^a is hydrogen, A is bromine or iodine and B, C and D are each hydrogen. More preferred compounds are the N- [4- (phenylamino)quinazolin-6-yl]-2-chloroacetamide and N-[4-

(phenylamino)quinazolin-6-yl]-2-methoxyacetamide described hereinabove, in which R^a is hydrogen, is bromine or iodine and B, C and D are each hydrogen. As is discussed hereinabove, each of the preferred compounds described above may be further advantageously derivatized by an alkoxy (e.g., a 3-(4- morpholinyl)propoxy group) or an alkylamino group (e.g., a piperazino group) at position 7 of the quinazoline ring.

The carboxylic group substituted by a leaving group (represented by X- W(=Y)-Z in Formula I hereinabove) can be further substituted by one or more derivatizing groups (as is represented in Formula I hereinabove by R³ and/or R⁴ as a second derivatizing group).

Such derivatizing groups can be, for example, halogen, alkyl, haloalkyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl, carboxy, hydroxy, alkoxy, aryloxy, carbonyl, thioalkoxy, thiohydroxy, thioaryloxy, thiocarboxy, thiocarbonyl, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano, as these terms are defined hereinabove. Alternatively, R³ and R⁴ can together form a five- or six-membered ring, such as, for example, cycloalkyl, heteroalicyclic, phenyl or heteroaryl, as these terms are defined hereinabove. While further conceiving the present invention, it was envisioned, that natural

PTK inhibitors, such as flavanoids, could also be useful in the treatment of leiomyomas.

The signal transducer and activator of transcription 3 (Stat3), which is a latent transcription factor required in proliferation and differentiation, is activated constitutively in a number of cancers. Upon stimulation, Stat3 is phosphorylated on a specific tyrosine residue by protein tyrosine kinases (PTKs). It was therefore considered that decreasing the level of phosphorylated Stat3 by use of a natural protein tyrosine kinase inhibitor might thus contribute to the shrinkage of leiomyomas.

While reducing the present invention to practice it was indeed found that a representative flavanoid, the isoflavanoid genistein, efficiently blocks growth of leiomyoma cells.

It was further found that no recovery of the cells occurred after cessation of treatment, indicating that genistein provides an effective, long-term blockage of leiomyoma cell growth.

It was further found that genistein significantly suppresses the level of pStat3 in leiomyomas.

Thus, according to a further aspect of the present invention there is provided a method of treating leiomyomas, which is effected by administering to a subject in need thereof a therapeutically effective amount of a natural PTK inhibitor.

The use of a natural PTK inhibitor was considered to be particularly advantageous, since natural agents are usually safe, often having little or no toxicity, and few if any significant adverse effects.

Accordingly, a natural PTK inhibitor can be used in the manufacture of a medicament for treating lieomyomas.

As used herein, the phrase "natural PTK inhibitor" describes an inhibitor of the protein-tyrosine kinase (PTK) enzyme, which occurs naturally in plants.

Examples of natural PTK inhibitors include, but are not limited to phenylpropanes, chalcones, flavonoids, coumarins, styrenes, quinones and terpenes.

Preferably, the natural PTK inhibitor is a flavanoid.

As used herein, the term "flavanoid" describes phenylbenzopyrone compounds. Examples include flavonols (such as quercetin, kaempferol, myricetin, and isorhamnetic); flavones (such as luteolin and apigenin); flavanones (such as hesperetin, naringenin, and eriodictyol); flavon-3-ols (such as (+)-catechin, (+)- gallocatechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin 3-gallate, (-)- epigallocatechin 3-gallate, theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3' digallate, thearubigins); isoflavones (such as genistein, daidzein and glycitein); and anthocyanidins (such as cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin) Preferably, the flavonoid is an isoflavone. As used herein, the term

"isoflavone" refers to a polyphenols flavonoid, produced almost exclusively by the members of the Leuguminosae family (bean-family), such as soybeans and chickpeas. A preferred compound according to this embodiment of the present invention is genistein, an iso-flavanoid phytoestrogen, having the following structural formula:

a pharmaceutically acceptable salt thereof or a prodrug thereof . As used herein, the phrase "pharmaceutically acceptable salt" describes a charged species of the parent compound and its counter ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound.

The term "prodrug" refers to an agent, which is converted into the active compound (the active parent drug) in vivo. Prodrugs are typically useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions.

There is a growing body of in vitro and animal studies suggesting that genistein may be helpful in preventing and treating some cancers, principally breast and prostate cancers. Several mechanisms have been proposed for genistein's putative anticarcinogenic activity. These include upregulation of apoptosis, inhibition of angiogenesis, inhibition of DNA topoisomerase II and inhibition of protein tyrosine kinases. Genistein has been found to have a number of antioxidant activities. It is a scavenger of reactive oxygen species and inhibits lipid peroxidation. It also inhibits superoxide anion generation by the enzyme xanthine oxidase. In addition, genistein, in animal experiments, has been found to increase the activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase.

Genistein's weak estrogenic activity has been suggested as another mechanism for genistein's putative anti-prostate cancer activity. In addition to the above mechanisms, other mechanisms of genistein's putative anti-prostate cancer activity include inhibition of nuclear factor (NF)-Kappa B in prostate cancer cells, downregulation of TGF (transforming growth factor)-beta and inhibition of EGF (epidermal growth factor)-stimulated growth. Genistein's anti-estrogenic action may be another possible mechanism to explain its putative anti-breast cancer activity. While reducing the present invention to practice it was indeed found that genistein efficiently blocks growth of leiomyoma cells. As used herein throughout, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

The term "administering" as used herein refers to a method for bringing a compound of the present invention and a target tyrosine kinase (e.g., EGFR-TK or PTK) together in such a manner that the compound can affect the catalytic activity of the kinase either directly; i.e., by interacting with the kinase itself or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent. As used herein, administration can be accomplished either in vitro, i.e. in a test tube, or in vivo, i.e., in cells or tissues of a living organism.

In vivo administration can be effected via oral, rectal, transmucosal, intestinal, parenteral, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, and intraocular administration routes.

Herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease or disorder, substantially ameliorating clinical symptoms of a disease or disorder or substantially preventing the appearance of clinical symptoms of a disease or disorder.

Herein, the term "preventing" refers to a method for barring an organism from acquiring a disorder or disease in the first place. The term "therapeutically effective amount" refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease or disorder being treated.

For any compound utilized in this and other aspects of the invention, a therapeutically effective amount, also referred to herein as a therapeutically effective dose, can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ or the IC₁₀O as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data. Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, chapter 1, page 1). Suitable routes of administration may, for example, include oral, rectal, transmucosal, transdermal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect.

Preferably, a therapeutically effective amount of TK050 ranges between about 0.1 μM and about 50 μM, more preferably between about 1 μM and about 10 μM, and more preferably between about 1 μM and about 5 μM.

Preferably, a therapeutically effective amount of genistein ranges between about 10 μM and about 50 μM.

Preferably, therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

The method of treating leiomyomas, as described herein, can be further effected by co-administering to a treated subject, along with administration of the EGFR-TK inhibitors or natural PTK-inhibitors presented herein, one or more agents that are capable of treating leiomyomas, such as, for example, an interferon (such as interferon-α and interferon-β, which oppose the actions of b-Fibroblast Growth Factor (FGF)); a reversible inhibitor of Epidermal Growth Factor Receptor (EGFR), such as AG 1478 (which blocks leiomyoma cell growth); a steroid hormone (such as estrogen, progesterone, aldosterone, androsetenediol, androstenedione, corticosterone, Cortisol, dehydroepiandrosterone, estradiol, estriol, estrone, pregnenolone, progesterone, testosterone, 11 -deoxycorticosterone, 11-deoxycortisol, 17-hydroxypregnenolone, 17- hydroxyprogesterone, and 18-hydroxycorticosterone); an estrogen modulator (such as raloxefine, clomifene, tamoxifen, toremifene, bazedoxifene, and lasofoxifene); an anti-gonadotropic hormone (such as danazol or melatonin); a gonadotropin-releasing hormone agonist, such as nafarelin, leuprolide, buserelin, historelin, goserelin and deslorelin (which act by initially increasing the release of gonadotropins, followed by desensitization and downregulation to a hypogonadotropic, hypogonadal state clinically resembling menopause); a gonadotropin-releasing hormone antagonist (such as cetrorelix acetate and ganirelix acetate); an antiprogestin (such as mifepristone (RU-486)); and a somatostatin analogue (such as octreotide).

These agents may be administered prior to, concomitant with or subsequent to administration of the active ingredient, namely, the irreversible EGFR-TK inhibitor represented by Formula I hereinabove or a natural PTK inhibitor.

Similarly, when used in the manufacture of a medicament, the irreversible EGFR-TK inhibitor represented by Formula I hereinabove or the natural PTK inhibitor can be utilized in combination with an additional active agent, as described hereinabove. Any of the compounds described herein may be utilized in the treatment of leiomyomas either per se or as a part of a pharmaceutical composition.

Thus, according to another aspect of the present invention there is provided a pharmaceutical composition, which is identified for use in the treatment of leiomyomas, and which comprises, as an active ingredient, any of the compounds described herein and a pharmaceutically acceptable carrier.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the compounds described herein, with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein, the term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

The pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol. For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprise suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols. The pharmaceutical compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of leiomyomas. Hence, according to a preferred embodiment of the present invention, the pharmaceutical composition described hereinabove is packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of leiomyomas, as is described hereinabove.

The composition may optionally further include at least one agent that is capable of treating leiomyomas, as described in detail hereinabove.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as defined hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion. MATERIALS AND EXPERIMENTAL METHODS

Materials:

TKS050 (N-{4-[(3,4-dichloro-6-fluorophenyl) amino]quinazoline-6-yl}-2- chloroacetamide) was prepared as described in WO 04/064718.

Genistein was purchased from LC Laboratories, Woburn, MA, USA (Cat. No. G-6055).

Stock solutions of 10 mM TKS050 or genistein in dimethylsulfoxide (DMSO) were stored at -70 °C. For the experiments, the agent was diluted with Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum. The highest concentration of DMSO was 0.1 %.

Fetal bovine serum (USA origin) was obtained from Gibco-BRL (Life Technologies, Inc. Grand Island, NY, USA).

Tissue culture media, trypsin-EDTA solution, and antibiotics were from Biological Industries (Beit Haemek, Israel).

Tissue culture reagents, growth supplements and the ovarian steroid hormones 17β-estradiol (E2) and progesterone (P4) were obtained from Sigma Chemical Company (St. Louis, Mo₃ USA).

Antibodies: The following antibodies were used for monitoring signaling proteins:

Anti-EGFR external domain: (clone F4) monoclonal antibody (Boehringer Mannheim, Germany; Cat. No. 1428551).

Anti-phosphotyrosine: 4G10 mouse monoclonal antibody (Upstate Biotechnology UBI, Lake Placid N. Y, USA; Cat. No. 05321). Bcl-2: mouse monoclonal anti-human antibody (Upstate Biotechnology UBI;

Cat. No. 05-341).

Stat3: rabbit polyclonal IgG, anti-human antibody (Upstate Biotechnology UBI; Cat. No. 50906).

Phosphorylated Stat3: mouse anti-phospho Stat3-Tyr 705 synthetic peptide (Upstate Biotechnology UBI; Y704 Cat. No. 06-596).

FITC-conjugated goat anti-mouse antibody (Cat. No. F2653; Sigma Chemical Company, St. Louis, Mo, USA) was used for immunofluorescence staining. Cell cultures;

Paired cell cultures of leiomyoma and adjacent normal myometrium tissue samples were established from pre-menopausal women following hysterectomy conducted for benign disease, after Helsinki approval and informed consent. Primary cell cultures were initiated in HAM/F12: Dulbecco's modified Eagle's medium (DMEM) 1:1 supplemented with 20 % FBS and antibiotics (penicillin 100 U/πύ and streptomycin 100 μg/ml). Thereafter, the cell cultures were propogated in phenol red- free DMEM and 10 % charcoal-treated FBS, specifically for the experiments with ovarian steroid hormones. The experiments were done on secondary and/or tertiary cultures. Cells were maintained at 37 ⁰C in a humidified incubator containing 5-8 % CO₂. Exponentially growing cells were used in the experiments.

Immunocytochemical staining with α-actin was routinely performed to verify the SMC origin of the cells in culture. Experimental design: Cells were seeded at 1 x 10⁴/well in 96-well microtitre plates and were grown for 2-3 days, as described above. Thereafter, the medium was replaced with phenol red-free DMEM and 10 % charcoal-treated FBS medium containing 0.1-10 μM TKS050 or 0.1-100 μM genistein. After two days, the medium was replaced with a new TKS050- or genistein-containing medium. Control cells were grown in medium alone and in medium containing various concentrations of dimethylsulfoxide (DMSO. Two days after the second treatment (4 days treatment), the medium was removed, a new medium without TKS050 or genistein was added, and the cells were grown for another 3 days, to evaluate rescue/recovery of the cells after treatment. The cells were thereafter subjected to determination of growth, using the colorimetric methylene blue assay described hereinbelow.

Calculation of growth inhibition:

For each concentration of TKS050 or genistein used, medium containing only DMSO was used for the control. Thus, for each concentration, the control was taken as 100 % growth. Automated microculture methylene blue assay:

Cell growth was determined by the automated microculture methylene blue assay: the TKS050- or genistein-treated cultures and controls were fixed in glutaraldehyde, at a 0.05 % final concentration, for 10 minutes at room temperature.

After washing, the microplates were stained with 0.01 % methylene blue in 0.1 mol/liter borate buffer for 60 minutes at room temperature. Then the microplates were washed extensively and rigorously to remove excess dye, and dried. The dye taken up by cells was eluted in 0.1 mol/liter HCl for 60 minutes at 37 ⁰C, and the optical density was measures at 620 nm. In preliminary titration experiments, linear readings were obtained for 1 x 10³ — 4 x 10⁴ cells/well. Each point of the growth curve was calculated from 8 wells.

Fluorescence-activated cell sorter (FACS) analysis of DNA content and determination ofapoptotic cells:

Selected samples of cell cultures were FACS analysed for DNA content. Samples of cells treated with various concentrations of TKS050 or genistein for predetermined periods were dispersed with. 1:1 trypsin 0.25 %: EDTA 0.05 % and stained with ethidium iodide in suspension. Cell cycle analysis and determination of the apoptotic cell fraction of the cell populations were carried out with FACS FPAR PLUS (Becton-Dickinson, Inc., Mountain View, CA).

Effects of estrogen on the inhibitory capacity of genistein on cell growth of paired leiomyoma and normal myometrium cell cultures:

Cell cultures were seeded 5 x 10³ cells/35 mm plates in DMEM with 10 % FCS medium and cultured for 2 days. Dose-response experiments were then performed using 0.1 or 1 μM estrogen alone, or 0.01 μM estrogen together with various concentrations of genistein (0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM and 100 μM). The cells were grown for another 7 days, followed by determination of growth using the colorimetric methylene blue assay. Effects of TKOSO and genistein on cell cycle phase distribution and apoptosis of leiomyoma and myometrium cell cultures:

Cell cultures were seeded at 5 x 10³ cells/35 mm plates in DMEM with 10% FCS and grown for 2 days. The cells were then treated with 0.5 μM, 1 μM or 5 μM TK050, or with 5 μM, 10 μM, or 25 μM genistein and harvested after a further 2 days. Cell cycle distribution and apoptosis were determined by FACS analysis. Effects of estradiol on the biochemical activities ofTKS050 and genistein:

Myometrium and leiomyoma paired cells were seeded at 2 x 10⁵ cells/35 mm plates in DMEM with 10% FCS and grown for 2 days. Thereafter, the cultures were washed, fed with medium containing 10 ng/ml estradiol without serum and starved for 48 hours. TKS050 or genistein at the appropriate concentration (0, 0.1 μM, 1 μM or 5 μM TK050; or 0, 0.1 μM, 1 μM, 10 μM or 50 μM genistein) was added for 4 hours. Cells were then stimulated with 30 ng/ml EGF for 10 minutes. The reaction was stopped by placing the cultures on ice and washed with ice-cold PBS. Immunoblot analysis of relevant proteins was performed on whole cell-lysates. Statistical analysis:

Data obtained from determination of growth, FACS analysis and densitometry studies were expressed as mean ± SD. Statistical analysis was conducted using the student-t test. Statistical significance was established at P values of less than 0.05.

EXPERIMENTAL RESULTS

Inhibition of leiomyoma growth by TKS050 and genistein:

The growth patterns of secondary and tertiary actively proliferating cells of paired leiomyoma and normal myometrium, cultured for up to 21 days, have previously been shown to be similar (Shushan, 2004). In some cases, the growth rate of primary leiomyoma cells was slower than that of the myometrium cells.

Since the effects of ovarian steroid hormones on leiomyoma growth are mediated by local production of growth factors such as EGF, the potency of the selective EGFR-inhibitor, TKS050, and the natural protein tyrosine kinase (PTK) inhibitor, genistein to suppress the proliferation of leiomyoma cell cultures was tested by treatment of the cells with medium containing TKS050 or genistein for 4 days. In parallel, the effects of these compounds on the growth of normal myometrium cell cultures were tested. For each concentration of TKS050 and genistein used, medium containing only DMSO was used for the control. The obtained results are presented in Figures 1 and 2. As can be seen in Figure 1, TKS 050 very effectively inhibited the growth of leiomyoma (at IC50 of 0.7 μM) and myometrium cell cultures (at IC50 of 1.1 μM), at concentrations of greater than 1.0 μM.

Generally, a similar pattern of growth inhibition was obtained for normal myometrium cell cultures and leiomyomas. It is of interest that in some myometrium cultures, complete growth inhibition was obtained only with TKS050 at greater than_2 μM, suggesting that myometrium cells might be less sensitive to the suppressive effect of TKS050 (Figures IA and IB).

Genistein was effective in inhibiting growth at concentrations greater than 10 μM (Figure 1C, ID). Complete growth suppression was obtained with 50 μM genistein, as shown in Figures 2C and 2D.

The data obtained indicate that leiomyoma cell growth is effectively blocked by TKS050 and genistein. 2 μM TKS050 or 50 μM genistein suppressed proliferation completely and the cells did not recover after cessation of treatment. TK050 induced cell cycle arrest and apoptosis in a dose- and time-dependent manner.

Recovery of cells after treatment:

Following treatment of cells with medium containing TKS050 or genistein for 4 days, the medium containing the agent was removed and new medium without the compound was added. The cells were grown further for another 4 days. The period between day 4 and day 8 indicates recovery of the proliferation activity, i.e. rescue of the cells after cessation of treatment. As can be seen in Figure IA and IB, TKS050 at

1 μm or higher exerted 100 % inhibition and no recovery of cells after cessation of treatment.

Complete growth suppression was obtained with greater than 50 μM genistein, and the cells remained arrested after withdrawal of the compound on day 4 as monitored on day 7 (Figures 1C and ID).

Rescue experiments in which the self-renewal capacity of the cells was measured after removal of the agent on day 4, thus confirm the growth inhibition experiments. Ovarian steroids do not affect genistein inhibitory activities:

The ovarian steroid-dependent growth potential of leiomyomas in vivo is well established (Stewart, 2001). It has also been suggested that estrogen and progesterone may act in combination to stimulate the proliferation of leiomyoma cell cultures. The potency of the EGF receptor inhibitor, AG1478, in suppressing the proliferation of leiomyomas cultured with estradiol and progesterone, separately and combined has previously been studied by the present inventors (Shushan, 2004). The results of the dose-response experiments with estradiol, progesterone and combined estradiol plus progesterone did not indicate any significant effect. In the present study the effect of estrogen alone, and in combination with genistein, on the growth of leiomyoma and myometrium cell cultures was studied. The results are presented in Figure 2. The concentrations chosen, 0.1 and 1 μM are within the physiological tissue concentrations found in leiomyomas and myometrium. The results indicated that estrogen alone had no effect on the growth of leiomyoma or myometrium cell cultures (Figures 2 A and 2B), and the combination of estrogen with genistein did not affect the growth suppressive effect of genistein (Figures 2A and 2B), as compared to the effect of genistein alone.

Points of growth arrest in the cell cycle and apoptosis:

To explore more directly the mechanism of growth suppression by TKS050 and genistein, post-treatment cell cycle analysis was performed on leiomyoma and myometrium cell cultures. Cells exposed to TKS050 or genistein were assessed by FACS analysis on day 2 after initiating treatment. The results are presented in Figure 3.

These results clearly indicate that TKS050 alters the cell cycle distribution, by reducing the proportion of cells in the G₀ZG₁ phase, and concomitantly increasing the number of apoptotic cells (Figures 3 A and 3B).

In contrast, genistein induced cell cycle alteration in a dose- and time- dependent manner, with no apoptosis. After a single treatment with a concentration of greater than 25 μM genistein for 2 days the proportion of cells in the Gl phase is slightly decreased and the proportion of cells in the G2/M phase is increased, with no effect on the apoptotic cell fraction (Figures 3 C and 3D), indicating that the mechanism of action of genistein is different from that of TK050, and thus that the effects of genistein are not exerted via stimulation of apoptosis. Biochemical activities ofTKSOSO and genistein:

Since treatment of the leiomyoma and normal myometrium cell cultures with TKS050 and genistein induced growth arrest and changes in the cell cycle, relevant proteins that could be linked to these processes were examined. On the basis of previous dose-response experiments with estradiol and progesterone by the present inventors (Shushan, 2004), Western Blot analysis was performed on leiomyomas and myometrium cells cultured with 10 ng/ml of estradiol. As shown in Figures 4 and 5, TKS050 induced a dose-dependent inhibition of

EGFR-autophosphorylation that is enhanced greatly in the presence of the ligand. TKS050 was already effective at 0.1 μM, giving at least about 51 % inhibition of leiomyoma and myometrium cultures in the presence of the ligand. With TKS050 concentrations of at least 1.0 μM, inhibition of autophosphorylation was slightly increased (at least 59 % inhibition of leiomyoma cultures, and at least 61 % inhibition of myometrium cultures) in the presence of the ligand. The results also indicate that TKSO 50 did not alter the level of EGFR in the cells. These findings are in accordance with conclusive evidence that TKS050 specifically and potently inhibits ligand- induced autophosphorylation of the EGFR. The effects of TKS050 on protein expression of two major regulatory proteins of apoptosis, Bcl2 (anti-apoptotic) and Bax (pro-apoptotic) were examined. TKS050 had no effect on the expression of the Bc 12 or Bax proteins of either cell culture.

Concentrations of greater than 5μM TKS050 did not alter the level of Stat3 or pStat3 of the leiomyoma cell cultures (Figure 5), but a slight dose-dependent inhibition of pStat3 was detected in the myometrium cell cultures (greater than 15.5 % without EGF stimulation, and greater than 26 % inhibition after EGF stimulation; Figure 4). This inhibitory effect of TKS050 was unaffected by the presence of physiological concentrations of E₂.

Genistein at 50 μM significantly suppressed the level of pStat3 protein in leiomyoma cells (Figure 7).

The above results show that unlike AG1478, which blocked the growth of leiomyoma cell cultures without inducing apoptosis, TKS050 increased the fraction of apoptotic cells. Nevertheless, TKS050 did not alter the expression of the apoptosis related proteins Bcl2 (anti-apoptotic) and Bax (pro-apoptotic) in the leiomyoma and myometrium cultures. Further, TKS050 had no effect on the expression of the Bcl2 and Bax proteins in leiomyoma and myometrium cells cultured with E2 or P4. WB analysis of the Bcl2 and Bax proteins in matched leiomyoma and myometrium cell cultures did not demonstrate consistent and/or clearly defined differential expression level of these proteins between the two types of cells.

In contrast to these findings, previous reports concerning Bcl2 expression in leiomyomas (Matsuo, 1997), demonstrated over-expression of this protein in leiomyomas as compared to normal myometrium. Matsuo et al. have also suggested that since Bcl2 protein expression was up-regulated by P4, but down-regulated by E2, P4 might have participated in leiomyoma development through induction of Bcl2. A possible reason for the discrepancy between the present findings and those of Matsuo could be the monoclonal origin of individual fibroids leading to differences between fibroids, even from the same individual uterus.

In summary, TKS050 and genistein are shown to be inhibitors of leiomyoma and myometrium cell cultures. TKS050 inhibits selectively autophosphorylation of EGFR and down stream signal tranduction events, including suppression of cell proliferation and cell cycle progression at micromolar concentrations. The inhibitory effect of TKS 050 is unaffected by the presence of physiological concentrations of 17-β estradiol, such as are commonly administered following treatment with gonadotropin-releasing hormone agonist. Hence a combined treatment comprising TKS050 with GnRH agonists and add-back estradiol may prove to be particularly effective. Therefore, the growth arresting properties of TKS050 and genistein identifies these as new potential agents for the non-surgical management of leiomyomas.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

LIST OF REFERENCES CITED

Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black and white women: Ultrasound evidence. Am J Obstet Gynecol 2003;188:100-7. Ben-David, L, Rozen, Y., Ortu, G., and Mishani, E. (2003). Radiosynthesis of

ML03, a novel positron emission tomography biomarker for targeting epidermal growth factor receptor via the labeling synthon: [C-ll]Acryloyl chloride. Appl. Rad. Isotp., 58 (2), 209-217.

Dixon, D., He, H., and Haseman, J. Immunohistochemical localization of growth factors and their receptors in uterine leiomyomas and matched myometrium. Environ Health Perspect 2000;108:795-802.

Fry, D. W., Kraker, A. J., McMichael, A., Ambroso, L. A., Nelson, J. M., Leopold, W. R., Connors, R. W., and Bridges, A. J. (1994). A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265, 1093-5. Lee B., Stewart E., Sahakian M., and Nowak R. Interferon-alpha is a potent inhibitor of basic fibroblast growth factor-stimulated cell proliferation in human uterine cells. Am J Reprod Immunol 1998;40:19-25.

Levitzki, A.; Gazit, A. (1995). Science 267, 1782-1788. Matsuo H, Maruo T and Samoto T. Increased expression of Bcl-2 protein in human uterine leiomyoma and its up-regulation by progesterone. J Clin Endocrinol Metab 1997;82:293-299.

Miyaji, K., Tani, E., Shindo, H., Nakano, A., and Tokunaga, T. (1994). Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas. J Neurosurg 81, 411-9. Nowak, R. Novel therapeutic strategies for leiomyomas: targeting growth factors and their receptors. Environ Health Perspect 2000;108:849-53.

Shushan A., Rojansky N., Laufer N., Klein B.Y., Shlomai Z., Levitzki R., Hartzstark, Z., and Ben-Bassat, H. The AGl 478 tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth. Human Reprod 2004; 19: 1957-1967. Vollenhoven, B., Lawrence A., and Healy D. Uterine fibroids: a clinical review. Br J Obstet Gynaecol 1990;97:285-98.

Claims

WHAT IS CLAIMED IS:

1. A method of treating leiomyomas, the method comprising administering to a subject in need thereof a therapeutically effective amount of an irreversible epidermal growth factor inhibitor.

2. Use of a therapeutically effective amount of an irreversible epidermal growth factor inhibitor in the manufacture of a medicament for the treatment of leiomyomas

3. A pharmaceutical composition being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of leiomyomas, the composition comprising a therapeutically effective amount of an irreversible epidermal growth factor inhibitor and a pharmaceutically acceptable carrier.

4. The method, use or composition of any of claims 1 to 3, wherein said irreversible epidermal growth factor inhibitor comprises a compound having the general Formula I:

Formula I wherein:

Ql is X-W(=Y)-Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino, or

Ql is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino and Q2 is X-W(=Y)-Z; X is selected from the group consisting of -NR¹-, -O-, -NH-NR¹-, -O-NR¹-,

NH-CHR¹-, -CHR^L-NH-, -CHR^O-, -O-CHR¹-, -CHR'-CHa- and -CHR^S- or absent;

W is carbon;

Y is selected from the group consisting of oxygen and sulfur;

Z is -CR²R³R⁴;

R^a is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;

A, B, C and D are each independently selected from the group consisting hydrogen and a first derivatizing group;

R¹ is selected from the group consisting of hydrogen, and substituted or non- substituted alkyl having 1-6 carbon atoms;

R² is a leaving group; and

R³ and R⁴ are each independently selected from the group consisting of hydrogen and a second derivatizing group.

5. The method, use or composition of claim 4, wherein said first derivatizing group is selected from the group consisting of halogen, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carbalkoxy, thiocarboxy, thiohydroxy, thioalkoxy, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano.

6. The method, use or composition of claim 4, wherein said second derivatizing group is selected from the group consisting of halogen, alkyl, haloalkyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl, carboxy, hydroxy, alkoxy, aryloxy, carbonyl, thioalkoxy, thiohydroxy, thioaryloxy, thiocarboxy, thiocarbonyl, sulfinyl, sulfonyl, amino, alkylamino, carbamyl, nitro and cyano, or alternatively, said R³ and R⁴ together form a five- or six-membered ring.

7. The method, use or composition of claim 4, wherein said leaving group is selected from the group consisting of halogen, alkoxy, aryloxy, thioalkoxy, thioaryloxy, azide, sulfinyl, sulfonyl, sulfonamide, phosphonyl, phosphinyl, carboxy and carbamyl.

8. The method, use or composition of claim 4, wherein said alkoxy comprises a morpholino group.

9. The method, use or composition of claim 4, wherein said alkylamino comprises a N-piperazinyl group.

10. The method, use or composition of claim 4, wherein Ql is X-W(=Y)-Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino.

11. The method, use or composition of claim 4, wherein Ql is X-W(=Y)-Z and Q2 is hydrogen.

12. The method, use or composition of claim 4, wherein Ql is X-W(=Y)-Z and Q2 is alkoxy.

13. The method, use or composition of claim 12, wherein said alkoxy comprises a morpholino group.

14. The method, use or composition of claim 4, wherein Ql is X-W(=Y)-Z and Q2 is alkylamino.

15. The method, use or composition of claim 14, wherein said alkylamino comprises a N-piperazinyl group.

16. The method, use or composition of claim 11, wherein X is said -NR¹- and Y is oxygen.

17. The method, use or composition of claim 16, wherein each of R¹, R³ and R⁴ is hydrogen.

18. The method, use or composition of claim 11, wherein R² is a leaving group selected from the group consisting of alkoxy and halogen.

19. The method, use or composition of claim 4, wherein at least one of A, B, C and D is fluorine.

20. The method, use or composition of claim 4, wherein D is fluorine.

21. The method, use or composition of claim 20, wherein A and B are each chlorine and C is hydrogen.

22. The method, use or composition of claim 4, wherein A is bromine.

23. The method, use or composition of claim 4, wherein A is iodine.

24. The method, use or composition of claim 11, wherein A and B are each chlorine, C is hydrogen and D is fluorine.

25. The method, use or composition of claim 11, wherein A is bromine and B, C and D are each hydrogen.

26. The method, use or composition of claim 11, wherein A is iodine and B, C and D are each hydrogen.

27. The method, use or composition of any of claims 1 to 3, wherein said therapeutically effective amount ranges between about 0.1 μM and about 50 μM.

28. The method, use or composition of any of claims 1 to 3, wherein said therapeutically effective amount ranges between about 1 μM and about 10 μM.

29. The method or use of any of claims 1 to 2, wherein said irreversible epidermal growth factor inhibitor is used in combination with at least one agent that is capable of treating leiomyomas.

30. The composition of claim 3, further comprising at least one agent that is capable of treating leiomyomas.

31. The method, use or pharmaceutical composition of any of claims 29 and 30, wherein said agent is selected from the group consisting of an interferon, a reversible inhibitor of Epidermal Growth Factor Receptor (EGFR), a steroid hormone, an estrogen modulator, an anti-gonadotropic hormone, a gonadotropin-releasing hormone agonist, a gonadotropin-releasing hormone antagonist, an antiprogestin, and a somatostatin analogue.

32. A method of treating leiomyomas, the method comprising administering to a subject in need thereof a therapeutically effective amount of a natural protein tyrosine kinase inhibitor.

33. Use of a natural protein tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of leiomyomas

34. A pharmaceutical composition being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of leiomyomas, the composition comprising a therapeutically effective amount of a natural protein tyrosine kinase inhibitor.

35. The method, use or composition of any of claims 32 to 34, wherein said natural protein tyrosine kinase inhibitor is selected from the group consisting of a phenylpropane, a chalcone, a flavonoid, a coumarin, a styrene, a quinone and a terpene.

36. The method, use or composition of claim 35, wherein said flavonoid is selected from the group consisting of a flavonol, a flavone, a flavanone, a flavon-3-ol, an isoflavone, and an anthocyanidin.

37. The method, use or composition of claim 36, wherein said isoflavone comprises genistein.

38. The method, use of any of claims 32 to 34, wherein said therapeutically effective amount ranges between about 10 μm and about 100 μm.

39. The method or use of any of claims 32 to 33, wherein said natural protein tyrosine kinase inhibitor is used in combination with at least one agent that is capable of treating leiomyomas.

40. The composition of claim 34, further comprising at least one agent that is capable of treating leiomyomas.

41. The method, use or pharmaceutical composition of any of claims 39 and 40, wherein said agent is selected from the group consisting of an interferon, a reversible inhibitor of Epidermal Growth Factor Receptor (EGFR), a steroid hormone, an estrogen modulator, an anti-gonadotropic hormone, a gonadotropin-releasing hormone agonist, a gonadotropin-releasing hormone antagonist, an antiprogestin, and a somatostatin analogue.