WO2024182811A1 - Compositions and methods for treating uterine leiomyomas - Google Patents

Abstract

Described herein are methods of treating uterine leiomyoma by administering to a patient in need thereof a composition that increases adenosine levels.

Description

COMPOSITIONS AND METHODS FOR TREATING UTERINE LEIOMYOMAS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to US provisional application 63/488,013, filed March 2, 2023, and US provisional application 63/560,088, filed March 1, 2024, each of which are incorporated by reference herein in its entirety.

BACKGROUND

Uterine leiomyomas are proliferations of uterine smooth muscle found in as many as 80% of pre-menopausal women. Clinically, leiomyomas, also known as “fibroids,” are a frequent cause of abnormal uterine bleeding, pelvic pain, urinary symptoms and infertility. As a result, these benign tumors are the most common indication for hysterectomy in the United States.

Medical alternatives for managing symptoms associated with leiomyomas is essentially limited to the use of hormonal interventions (Donnez, J, et al. “Long-term treatment of uterine fibroids with ulipristal acetate.” Fertil Steril 2014:101(6): 1565-73; Williams, AR, et al. “The effects of the selective progesterone receptor modulator asoprisnil on the morphology of uterine tissues after 3 months treatment in patients with symptomatic uterine leiomyomata.” Hum Reprod 2007;22(6): 1696-704). Although often effective, use of these agents, such as leuprolide acetate, adversely impacts fertility options for women who may be seeking to bear children. Non-hormonal options for shrinking leiomyomas and controlling their symptoms are a critical unmet need in women’s health.

Biologically, leiomyomas are monoclonal tumors that arise from tissue-specific stem cells in the human myometrium. Pluripotent subpopulations of myometrial cells identified by CD 105 have been shown to repopulate tumors in vivo that histologically resemble leiomyomas when transplanted to the renal capsule. It is now well established that subsequent leiomyoma growth depends critically on circulating levels of estrogen and progesterone to fuel their growth (Kim, JJ, et al. “The role of progesterone signaling in the pathogenesis of uterine leiomyoma.” Mol Cell Endocrinol 2012;358(2):223-31). Progesterone receptor is highly expressed in uterine leiomyomas, and progesterone has been shown to stimulate proliferation and inhibit apoptosis in primary cultures derived from these tumors (Maruo, T, et al. “Effects of progesterone on uterine leiomyoma growth and apoptosis.” Steroids 2000;65(10-l 1 ) : 585-92) . Pathogenic mutations in DNA regulator complex MED12 have also been shown to be a common feature of uterine leiomyomas. However, for the most part, the signals that initiate and promote the growth of uterine leiomyomas remain poorly understood. What are thus needed are new compositions and methods for treating uterine leiomyomas. The subject matter disclosed herein addresses these and other needs.

SUMMARY

In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to compounds, compositions, and methods of making and using compounds and compositions.

The role of ecto-5 '-nucleotidase (NT5E: CD73) in uterine leiomyomas is disclosed. CD73 catalyzes the extracellular release of adenosine from adenosine monophosphate (AMP) and plays a key role in promoting the growth of new blood vessels. Overexpression of CD73 is a common feature of multiple cancers, where high levels of its expression play a key, non- redundant role in abrogating cytotoxic anti-tumoral immune responses.

Thus, in some aspects, described herein are methods of treating uterine leiomyoma by administering to a patient in need thereof a composition that increases adenosine levels. Compositions for use in the described methods, are also disclosed.

Additional advantages will be set forth in part in the following description and in part will be obvious from the description or may be learned by practicing the aspects described below. The advantages described below will be realized and attained by the elements and combinations pointed out in the appended claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

Figures 1A-1H show expression of CD73 in specimens of healthy myometrium and uterine leiomyomas. The mRNA expression for CD73 exon epitope 2-3 (Fig. 1A), exon epitope 6-7 (Fig. IB) and protein expression levels of CD73 were significantly lower in proliferative (Fig. 1C), luteal (Fig. ID) and post-menopausal (Fig. IE) Leio tissues than matched specimens of Myo tissues. Protein expression levels reported as mean intensity + SEM in proliferative (Fig. IF), luteal (Fig. 1G) and post-menopausal (Fig. 1H); n=5; * P<0.05 vs. control. ** P<0.01 vs. control.

Figures 2A-2C show that spatial distribution of CD73 expression in human myometrium and leiomyomas CD73 was highly expressed throughout healthy Myo (Fig. 2A). CD73 visualized using immunohistochemistry to interrogate hemotoxylin stained cross sections of FFPE specimens probed with antibody specific to CD73 (brown). Insets demonstrate specific areas of interest: l=leiomyoma 2=pseudocapsule and 3 =myometrium. In comparison, weak expression level of CD73 was observed in Leio (Fig. 2B). Fig. 1C: Quantified results were reported as expression scores ± SEM, n=6; *P<0.05 vs. control.

Figures 3A-3I show the biologic impact of a non-hydrolyzable adenosine analog (2- chloro-adenosine (2-CL-AD) on primary cultures derived from human leiomyomas. Proliferation assays showed that 2-CL-AD suppress the growth of Leio primary cells in a dose- and time- dependent manner in Leio42 (Fig. 3A), Leio44 (Fig. 3D) and Leio46 (Fig. 3G). Flow cytometry demonstrated that exposure to 2-CL-AD decreased the proportion of cells in both the S- and G2-phases of the cell cycle in Leio42 (Fig. 3B), Leio44 (Fig. 3E) and Leio46 (Fig. 3H), and increased the apoptosis in Leio42 (Fig. 3C), Leio44 (Fig. 3F) and Leio46 (Fig. 31).

Figures 4A-4I show that overexpression CD73 inhibits growth of primary Leio cells. Proliferation assays show that overexpression CD73 suppress the growth of Leio44 (Fig. 4A), Leio46 (Fig. 4B), and Leio58 (Fig. 4C) primary cells. The mRNA and protein expression level in Leio44 (Fig. 4D), Leio46 (Fig. 4E), and Leio58 (Fig. 4F) of CD73 were confirmed by qPCR and WB after transfection. Adenosine concentration in cell supernatant was measured by Elisa in Leio44 (Fig. 4G), Leio46 (Fig. 4H), and Leio58 (Fig. 41). CON: sham transfected controls; CD73, CD73 transfected. Bars represent mean ±SEM, n=3; * P<0.05 vs. control. ** P<0.01 vs. control.

Figures 5A-5I show that AD0RA2B agonist suppress leio primary cells proliferation but siRNA AD0RA2B blocked 2-CL-AD induced proliferation suppressing. Multiple subtype specific agonists were examined for known adenosine receptors revealed that only AD0RA2B agonist BAY60-6583 was able to suppress the proliferation of Leio42 (Fig. 5A), Leio44 (Fig. 5B), and Leio46 (Fig. 5C) primary cells. Knockdown AD0RA2B blocked 2-CL- AD induced growth suppress of Myo44 (Fig. 5D), Myo58 (Fig. 5E), and Myo60 (Fig. 5F) primary cells. Impact of transfecting primary cultures of Myo44 (Fig. 5G), Myo58 (Fig. 5H), and Myo60 (Fig. 51) with either siRNA targeting AD0RA2B expression or a scrambled nontargeting siRNA control (CON) on AD0RA2B expression. GAPDH: glyceraldehyde phosphate dehydrogenase. Bars represent mean ±SEM, n=3; * P<0.05 vs. control. ** P<0.01 vs. control.

Figures 6A-6F show that incubation with 2-CL-AD decreases levels of Phospho-Akt, Cyclin DI, Cdk2 pTyrl5 and Histones H3 pSerlO in primary cultures of both Leio and Myo. Western Blot showed the expression of Phospho-Akt, AKT, Cyclin DI (Fig. 6A) and Cdk2, Cdk2 pTyrl5 and Histones H3 and pSerlO (Fig. 6D) were significantly decrease in response to 2-CL-AD exposure. Quantified results for P-Akt (Fig. 6B), Cyclin DI (Fig. 6C), Cdk2 pTyrl5 (Fig. 6E) and Histones H3 pSerlO (Fig. 6F) reported as mean intensity ±SEM, n=3; * P<0.05 vs. control.

Figures 7A-7D show relative expression of CD73 in arterioles (Fig. 7A), venules (Fig. 7B), capillaries (Fig. 7C), and lymphatics (Fig. 7D) of healthy myometrium and leiomyomas determined by immunohistochemistry. Results reports as semi-quantiative HSCORES. Bars represent mean + SEM, n=3.

Figures 8 shows relative expression of CD73, as measured by RT-qPCR in matched primary cultures of healthy myometrium (Myo) and leiomyoma (leio) collected from 5 different pre-menopausal women (Pt#42, Pt#44, Pt#46, Pt#58, Pt#60).

Figures 9A-9F show that adenosine inhibits growth of primary Myo and Leio cultures. Proliferation assays showed that AD suppress the growth of both Myo42 (Fig. 9A), Myo44 (Fig. 9B), Myo46 (Fig. 9C), and Leio42 (Fig. 9D), Leio44 (Fig. 9E), and Leio46 (Fig. 9F) primary cells in a dose-dependent manner. Bars represent mean ± SEM, n=3; * P<0.05 vs. control. ** P<0.01 vs. control.

Figures 10A-10K show the impact of 2-chloro-adenosine (2-CL-AD) on the proliferation of a primary culture derived from healthy human myometrium Myo42 (Fig. 10A), on the proportion of myometrial cells (Myo42) in Gl, S and G2 phases of the cell cycle (Fig. 10B), on rates of apoptosis in a primary culture derived from healthy myometrium (Myo42) (Fig. IOC), on the proliferation of a primary culture derived from healthy human myometrium Myo44 (Fig. 10D), on the proportion of myometrial cells (Myo44) in Gl, S and G2 phases of the cell cycle (Fig. 10E), on rates of apoptosis in a primary culture derived from healthy myometrium (Myo44) (Fig. 10F), on the proliferation of a primary culture derived from healthy human myometrium Myo46 (Fig. 10G), on the proportion of myometrial cells (Myo46) in Gl, S and G2 phases of the cell cycle (Fig. 10H), and on rates of apoptosis in a primary culture derived from healthy myometrium (Myo42) (Fig. 101). Quantitation of cells in different phases of the cell cycle in primary culture of myometrium treated with 2-CL-AD or vehicle alone (CON) (Fig. 10J). Proportion of cells undergoing early and late apoptosis in culture of myometrium treated with 2-CL- AD or vehicle (CON) (Fig. 10K). * P<0.05 vs. control. ** P<0.01 vs. control

Figures 11A-11L show the impact of targeting CD73 expression in primary cultures of myometrium Myo44 (Fig. 11A), Myo46 (Fig. HE), and Myo58 (Fig. Ill), and myometrium Myo44 (Fig. 1 IB), Myo46 (Fig. 1 IF), and Myo58 (Fig. 11J) transfected with an siRNA or a scrambled non-targeting control as measured by RT-PCR or Myo44 (Fig. 11C), Myo46 (Fig. 11G), and Myo58 (Fig. UK) as measured by Western blot. Adenosine concentrations in media collected from primary cultures of myometrium (Myo44) (Fig. 1 ID), Myo46 (Fig. 11H), or Myo58 (Fig. 11L) transfected with an siRNA or a scrambled nontargeting control. * P<0.05 vs. control. ** P<0.01 vs. control.

Figures 12A-12C show the impact of ADORA subtype-specific agonists on proliferation of Myo42 (Fig. 12A), Myo44 (Fig. 12B), and Myo46 (Fig. 12C), a primary cell line derived from healthy myometrium. Bars represent mean ± SEM, n=3; * P<0.05 vs. control. ** P<0.01 vs. control.

Figure 13 shows the expression of AKT, phosphor- Akt, Cyclin DI and actin in primary cultures of leiomyoma trasted with BAY60-6583, a selective AD0RA2B agonist or transfected with siRNAs targeting AD0RA2B expression or a non-targeting scrambled siRNA control. Bars represent mean +SEM. n=3; * P<0.05 vs. control. ** P<0.01 vs. control

Figures 14A-14I show the AD0RA1 expression in leiomyoma and myometrium. ADORA 1 mRNA transcripts were significantly higher in leiomyoma specimens collected from women in the proliferative (Fig. 14A) and luteal (Fig. 14D) phases as well as postmenopausal (Fig. 14G) women. A similar overexpression of ADORA 1 was also observed when leiomyoma specimens were compared to matched myometrium by Western blot. Quantified protein levels normalized to a standard gel-loading control are reported as mean intensity ± SEM in proliferative (Fig. 14C), luteal (Fig. 14F), and post-menopausal (Fig. 141) phases, n=5 each; *P<0.05 vs. control; **P<0.01 vs. control.

Figures 15A-15I show the AD0RA2A expression in matched specimens of leiomyoma and myometrium. AD0RA2A mRNA was measured in matched specimens of leiomyoma and myometrium collected from women in the proliferative (Fig. 15 A) or luteal (Fig. 15D) phases of the menstrual cycle as well as menopausal women (Fig. 15G). Levels of AD0RA2A protein were also compared by Western blot (Fig. 15B, Fig. 15E, Fig. 15H). Quantified protein levels are reported as mean intensity ± SEM in proliferative (Fig. 15C), luteal (Fig. 15F), and post-menopausal (Fig. 151) phases, n=5 each; *P<0.05 vs. control.

Figures 16A-16I show the expression of ADORA2B receptor in leiomyoma and myometrium. AD0RA2B expression in leiomyoma and myometrium tissues in proliferative (Fig. 16A), luteal (Fig. 16D) phases of the menstrual cycle and menopausal women was evaluated by RT-qPCR (Fig. 16G). Levels of AD0RA2B protein were compared by Western blot (Fig. 16B, Fig. 16E, Fig. 16H). Quantified protein levels are reported as mean intensity ± SEM in proliferative (Fig. 16C), luteal (Fig. 16F), and post-menopausal (Fig. 161) phases, n=5 each; *P<0.05 vs. control.

Figures 17A-17I show the expression of AD0RA3 receptor in leiomyoma and myometrium. AD0RA3 mRNA expression in matched specimens of leiomyoma and myometrium from proliferative (Fig. 17A), luteal (Fig. 17D) phases of the menstrual cycle, and post-menopausal women (Fig. 17G). Expression of AD0RA3 in leiomyoma and myometrium tissue from proliferative (Fig. 17B), luteal (Fig. 17E), and post-menopausal (Fig. 17H) specimens were also compared by Western blot. Quantified protein levels are reported as mean intensity ± SEM in proliferative (Fig. 17C), luteal (Fig. 17F), and postmenopausal (Fig. 171) specimens. n=5 each; *P<0.05 vs. control.

Figures 18A-18I show the impact of targeting AD0RA1 expression on proliferation. Rates of proliferation observed for primary cultures derived from human leiomyomas transfected with siRNAs targeting AD0RA1 expression were significantly slower than matched controls transfected with a scrambled nontargeting siRNA alone (Fig. 18A, Fig. 18D, Fig. 18G). Levels of AD0RA1 were significantly lower in cultures transfected with the AD0RA1 -siRNA in each of three patients when measured by rt-PCR (Fig. 18B, Fig. 18E, Fig. 18H) or Western blot (Fig. 18C, Fig. 18F, Fig. 181). Quantified protein expression was normalized to actin gel-loading control.

Figures 19A-19C show that targeting AD OR Al expression decreases levels of p-Akt and Cyclin DI. Significantly, decreased p-Akt and Cyclin DI expression was observed when primary leiomyoma cultures transfected with ADORAl-specific siRNA which were compared to matched cultures transfected with non-targeting scrambled control by Western blot (Fig. 19A). Quantified protein levels were normalized to actin gel-loading control and reported as mean intensity ± SEM for both p-Akt (Fig. 19B) and Cyclin DI (Fig. 19C). n=5 each; *P<0.05 vs. control; **P<0.01 vs. control.

Figures 20A-20C show that application of ADORA 1 -specific agonists significantly induces proliferation of primary cultures of myometrium in a dose dependent manner. Proliferation in primary cultured myometrium cells derived from 3 distinct patients Myo42 (Fig. 20A), Myo44 (Fig. 20B), and Myo46 (Fig. 20C) responded to the AD0RA1 agonist, CPA (N6-cyclopentyl adenosine), in a dose-dependent fashion.

Figures 21A-21C show that ADORAl-specific agonists significantly induce expression of p-Akt and Cyclin DI in vitro. Significantly increased levels of p-Akt and Cyclin DI (Fig. 21 A) were found in primary cultures of myometrium after administering the Al Agonist. Protein levels reported as mean intensity ± SEM in p-Akt (Fig. 2 IB) and Cyclin DI (Fig. 21C), n=5 each; *P<0.05 vs. control.

DETAILED DESCRIPTION

The materials, compounds, compositions, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

Before the present materials, compounds, compositions, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, which may, of course, vary. It is also to be understood that the terminology used herein describes particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and expressly incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

General Definitions

In this specification and in the claims that follow, reference will be made to many terms, which shall be defined to have the following meanings:

Throughout the specification and claims, the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agonist" includes mixtures of two or more such agonists and the like.

"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur and that the description includes instances where the event or circumstance occurs and instances where it does not.

An "agonist" is a molecule that interacts with a target to cause or promote increased activation of the target. An "antagonist" is a molecule that opposes the action of an agonist. Antagonists prevent, reduce, inhibit or neutralize the activity of agonists, and antagonists also prevent, inhibit constitutive activity of a target, e.g., a target receptor, even in the absence of an identified agonist, can be reduced or reduced.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has two strands, a 5'— >3' strand, referred to as the plus strand, and a 3'— 5' strand (the reverse compliment), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5'— 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T). Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA. Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Antigene molecules are either antisense or sense molecules directed to a dsDNA target.

Antisense oligonucleotide: A sequence of at least about 8 nucleotides, such as about at least 10, 12, 15, 20, 30 or 50 nucleotides, wherein the sequence is from a gene sequence (such as all or a portion of a cDNA or gene sequence, or the reverse complement thereof), arranged in reverse orientation relative to the promoter sequence in a transformation vector.

By "reduce" or other forms of the word, such as "reducing" or "reduction," it is meant lowering of an event or characteristic e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value. In other words, it is relative, but it is not always necessary for the standard or relative value to be referred to. For example, "reduces tumor growth" means decreasing the number of tumor cells relative to a standard or a control.

By "prevent" or other forms of the word, such as "preventing" or "prevention," is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

As used herein, "treatment" refers to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, any one or more of alleviation of one or more symptoms (such as tumor growth), diminishment of the extent of tumor growth, stabilized (i.e., not worsening) state of cell proliferation, preventing or delaying spread of tumors, delaying occurrence or recurrence of tumors, delay or slowing of tumor progression, and remission (whether partial or total).

The term "patient" refers to a human needing treatment for uterine leiomyomas or treatment for any purpose, and more preferably, a human needing such a treatment to treat cancer or a precancerous condition or lesion. However, the term "patient" can also refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep, and non-human primates, among others, that need treatment.

A "pharmaceutically acceptable" component is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include those that may be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g., sodium, potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids e.g., hydrochloric and hydrobromic acids) and organic acids e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene- sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). When two acidic groups are present, a pharmaceutically acceptable salt may be a mono-acid-mono-salt or a di-salt; similarly, where there are more than two acidic groups present, some or all of such groups can be converted into salts.

"Pharmaceutically acceptable excipient" refers to an excipient that is conventionally useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

A "pharmaceutically acceptable carrier" is a carrier, such as a solvent, suspending agent, or vehicle, for delivering the disclosed compounds to the patient. The carrier can be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a type of pharmaceutical carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

The term "therapeutically effective amount" means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human being sought by a researcher, veterinarian, medical doctor, or other clinician. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay occurrence and/or recurrence. An effective amount can be administered in one or more doses. The effective amount of the drug or composition may: (i) reduce the number of tumor cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop tumor cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the tumors.

Effective amounts of a compound or composition described herein for treating a mammalian subject can include about 0.1 to about 1000 mg/Kg of body weight of the subject/day, such as from about 1 to about 100 mg/Kg/day, especially from about 10 to about 100 mg/Kg/day. The doses can be acute or chronic. A broad range of disclosed composition dosages is believed to be safe and effective.

“Uterine leiomyomas” are also called “uterine fibroids” and are smooth mulscle tumors that develop in the uterus. Uterine fibroids are classified into several types, based on their location, including subserosal, intramural, submucosal, pedunculated submucosal, fibroid in statu nascendi, and fibroid of the broad ligament. Any and all of these uterine fibroids are contemplated for treatment using the invention. Myometrial Hyperplasia is a condition which can mimic uterine fibroid symptoms and may be a precursor lesion of these tumors. It is structural variation with irregular zones of hypercellularity and increased nucleus/cell ratio, causing a bulging, firm, enlarged uterus. The condition often leads to hysterectomy. Deeper MMH has lower cellularity, and tends to have increased collagen. Therefore, this condition also may be treated using the methods and compositions of the invention. Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and the Figures.

Methods

Each year, more than 300,000 hysterectomies are performed in the United States, most commonly to control symptoms related to the presence of uterine leiomyomas. It is well understood that both estrogen and progesterone play a role in driving the growth of uterine leiomyomas. More recently, pathogenic mutations in the MED 12 nuclear regulator complex have also been identified as a key driver for uterine leiomyomas. However, the mechanisms of the proliferation of myometrial stem cells into leiomyomas remain poorly understood.

As disclosed herein, restricted patterns of CD73 expression are a hallmark of uterine leiomyomas that are most robustly observed in the estrogen-rich, proliferative phase of the menstrual cycle. The data herein further indicate that a dramatic reduction in CD73 expression among myofibroblasts comprising the bulk of leiomyomas is associated with readily measurable decreases in intratumoral adenosine concentrations when compared to adjacent healthy myometrium. The data herein establish that alterations in adenosine potentially contribute to the growth of these tumors and are capable of regulating the proliferation of myofibroblasts, likely acting via type 2B adenosine receptors to promote enhanced activity in AKT signaling pathways known to be important for leiomyoma growth. Overexpression of CD73 has recently been identified as a feature of multiple cancers, where high concentrations in the tumor microenvironment. In the cancer context, driven by massive release of ATP from rapid cell turnover and its continuous degradation by CD73 and other ectoenzymes, sustained high concentrations of adenosine result in global suppression of T ceil functions as part of a feed-forward loop that involves the expression of adenosine type 2B receptors (ADORA2B) on cancer-associated fibroblasts (Yu, M, et al. “CD73 on cancer- associated fibroblasts enhanced by the A2B -mediated feedforward circuit enforces an immune checkpoint.” Nat Commun 2020; 11 (1): 515). CD73 has also been shown to influence the differentiation of other immune cells in the cancer complex, including growth-promoting M2 macrophages (Jacoberger-Foissac, C, et al. “CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.” Cancer Immunol Res 2023;! 1 (l):56-71 ).

These observations create multiple potential opportunities for non-hormonal options to regulate leiomyoma growth. Collectively, these observations confirm that modulating CD73 expression can alter ambient adenosine concentrations sufficiently to directly impact the rates at which smooth muscle cells from leiomyomas and myometrium proliferate in vitro.

Disclosed herein, in certain aspects, are methods of treating uterine leiomyoma, comprising administering to a patient in need thereof a composition that increases adenosine levels (in the patient, patient’s uterus, and/or intratumor environment of uterine fibroids). Compositions that increase adenosine levels and that can be used in the disclosed methods are disclosed herein. To summarize here, the compositions can be small molecules that increase adenosine, such as adenosine itself, or salts, analogs, or precursors of adenosine. Additionally or alternatively, the compositions can target one or more adenosine receptors to modulate the receptor. Examples include agonist that activate receptors (e.g., 2B) or antagonists that deactivate or inhibit receptors (e.g., Al). Still further, antibodies that bind to adenosine receptors, antisense oligolucleotides that modulate expression can also be used.

In the disclosed methods, the patient can be in the proliferative phase or luteal phase of the menstrual cycle. In other examples, the patient can be pre-menopausal or postmenopausal. In other examples, the patient can have a pathogenic mutation in the MED12 nuclear regulator complex. In other examples, the patient can have decreased levels of CD73 expression as compared to a control that does not have uterine leiomyoma. In other examples, the patient can have decreased levels of intratumoral adenosine concentrations as compared to a control that does not have uterine leiomyomas.

It can also be desired to determine whether the patient has decreased CD73, increased AD0RA1, or decreased adenosine concentration in the uterine environment. Thus, the disclosed methods can additionally include a step of measuring CD73 expression levels in the patient and, and when CD73 expression is less than a control that does not have uterine leiomyomas, administering the composition to the patient. The CD73 can be measured during estrogen-rich, proliferative phase of the menstrual cycle. In other examples, the disclosed methods can additionally include a step of measuring expression of adenosine receptor Al in the patient and, when expression is greater than a control that does not have uterine leiomyomas, administering the composition to the patient. Measuring CD73 and adenosine receptor Al can be done my methods known in the art, such as by MRI, using tracer ligands, labeled antibodies, metabolic assays, and the like.

In other examples, the disclosed methods can additionally include a step of measuring adenosine levels in the patient and, when adenosine levels are less than a control that does not have uterine leiomyomas, administering the composition to the patient. Adenosine levels can be measured by methods known in the art.

In other examples, further comprising treating the patient with hormonal treatments or surgically removing the leiomyoma.

The local treatment of uterine fibroids by injection of compositions that increase adenosine can be conducted in an office or clinic visit under ultrasound guidance with minimal chance for sequelae. This method can be used to treat small to moderate size fibroids or asymptomatic fibroids, which currently are not treated at all, allowing the clinician to prevent potentially debilitating symptoms and preservation of fertility in women of childbearing years, and also larger fibroids, eliminating the need for hysterectomy for this disease. Thus, the methods of this invention are contemplated to be useful to treat any stage or type of uterine fibroid disease.

Compositions

Disclosed herein are compositions that can be used in the disclosed methods of treating uterine leiomyomas. The disclosed compositions can be a single or combination of components that result in the increase of adenosine levels in the patients. The increased levels of adenosine can be systematic levels, uterine levels or levels inside a uterine fibroid.

Adenosine or adenosine analog or pharmaceutically acceptable salt thereof.

In some examples, the compositions can comprise adenosine or adenosine analogs, including pharmaceutically acceptable salts thereof. Specific examples of adenosine analogs include, but are not limited to, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, 2-chloroadenosine, R-phenylisopropyladenosine, 2-alkyl substituted adenosine, 2-alkylamino substituted adenosine, 2-propynyl adenosine, cladribine, pentostatin, 2'-deoxyadenosine, 2',3'-isopropylidene adenosine, toyokamycin, 1- methyladenosine, 5N-carboxamides of adenosine, N-6-methyladenosine, adenosine N- oxide, 2-alklylhydrazino adenosine, 6-methylmercaptopurine riboside, and the like.

Modulators of adenosine receptor activity.

In one example, compositions that can be administered herein modulate one or more adenosine receptors, such as the Al, A2A, A2B, or A3 receptor. By “modulators” is meant a composition that can alter, increase, activate, turn on, decrease, suppress, knock down the activity or expression of an adenosine receptor, either specifically or non-specifically.

There has been progressive development of compounds that are more and more potent and/or selective as agonists of adenosine receptors based on radioligand binding assays and physiological responses. Compounds with little or no selectivity for adenosine receptors, which can be used herein, include adenosine itself or 5N-carboxamides of adenosine, such as 5N — N-ethylcarboxamidoadenosine (NECA) (Cronstein, BN, et al., J. Immunol., 135, 1366 (1985). Additional examples include 2-alkylamino substituents that have increased potency and selectivity for A2A, e.g., CV1808 and CGS21680 (M. F. Jarvis et al., J. Pharmacol. Exp. Then, 251, 888 (1989)). 2-Alkoxy-substituted adenosine analogs such as WRC-0090 are even more potent and selective as agonists (M. Ueeda et al., J. Med. Chem., 34, 1334 (1991)). The 2-alklylhydrazino adenosine derivatives, e.g., SHA 211 (also called WRC-0474) have also been evaluated as agonists for A2A receptor (K. Niiya et al., J. Med. Chem., 35, 4557 (1992)). Each of these can be used in the methods disclosed herein.

In other examples, a modulator of an adenosine receptor can be an antisense sequence. The sequence can be an adenosine receptor sequence (e.g. Genbank accession number L22214, AH003248, NM000676, and AH003597). Where the reverse complement of an adenosine receptor sequence is used to suppress expression of proteins from the adenosine receptor locus, the sense strand of adenosine or adenosine receptor locus or cDNA is inserted into the antisense construct. A reduction of adenosine receptor protein expression in a transgenic cell can be obtained by introducing into cells an antisense oligonucleotide based on an adenosine receptor locus, e.g. the adenosine receptor Al, A2A, A2B, or A3 locus, including the reverse complement of the adenosine receptor cDNA coding sequence, the adenosine receptor cDNA or gene sequence or flanking regions thereof.

The introduced sequence need not be the full-length human adenosine receptor cDNA or gene or reverse complement thereof, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native adenosine or adenosine receptor locus sequence will be needed for effective antisense suppression. The introduced antisense sequence in the vector can be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases, such as when the sequence is greater than 100 nucleotides. For suppression of the adenosine receptor gene itself, transcription of an antisense construct results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous adenosine receptor gene in the cell. For suppression of protein expression from the opposite strand of the adenosine receptor locus, transcription of an antisense construct results in the production of RNA molecules that are identical to the mRNA molecules transcribed from the endogenous adenosine or adenosine receptor gene, assuming the antisense construct was generated from sequence within the adenosine receptor gene rather than in a flanking region. Antisense molecules made to target the sequence that is the reverse complement of the adenosine receptor locus will serve to suppress any abnormal expression of proteins or peptides from the strand of the locus not encoding the adenosine receptor cDNA.

For suppression of an adenosine receptor gene, transcription of an antisense construct results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous adenosine receptor gene in the cell. The introduced sequence need not be the full-length human adenosine receptor cDNA or gene or reverse complement thereof, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native adenosine or adenosine receptor locus sequence is needed for effective antisense suppression. In one example, the introduced antisense sequence in the vector is at least 10, such as at least 30 nucleotides in length. Improved antisense suppression is typically observed as the length of the antisense sequence increases. Shorter polynucleotide (oligonucleotides) can conveniently be produced synthetically as well as in vivo. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 30, at least 100 nucleotides, or at least 200 nucleotides.

To inhibit the translation of the target RNA molecule, such as an adenosine receptor, the antisense molecule will ideally persist in the cell for a sufficient period to contact the target RNA. However, cells contains enzymes and other components that cause polynucleotides (such as an antisense molecule) to degrade. The antisense molecule can be engineered such that it is not degraded in the cell. This can be done, for example, by substituting the normally occurring phosphodiester linkage which connects the individual bases of the antisense molecule with modified linkages. These modified linkages can, for example, be a phosphorothioate, methylphosphonate, phosphodithioate, or phosphoselenate. Furthermore, a single antisense molecule can contain multiple substitutions in various combinations.

The antisense molecule can also be designed to contain different sugar molecules. For example the molecule can contain the sugars ribose, deoxyribose or mixtures thereof, which are linked to a base. The bases give rise to the molecules' ability to bind complementarily to the target RNA. Complementary binding occurs when the base of one molecule forms a hydrogen bond with another molecule. Normally the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). Therefore, the sequence ATCG of the antisense molecule will bond to TAGC of the target RNA. Additionally, the antisense molecule does not have to be 100% complementary to the target RNA to be effective.

The oligonucleotides can be DNA or RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, and can include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., PNAS USA 86:6553-6, 1989; Lemaitre et al., PNAS USA 84:648- 52, 1987; PCT Publication No. WO 88/09810) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134), hybridization triggered cleavage agents (see, e.g., Krol et al., BioTechniques 6:958-76, 1988) or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539-49, 1988).

In a particular example, an adenosine receptor antisense polynucleotide is provided, for example as a single-stranded DNA. Such a polynucleotide can include a sequence antisense to a sequence encoding an Al, A2A, A2B, or A3 receptor. The oligonucleotide can be modified at any position on its structure with substituents generally known in the art. For example, a modified base moiety can be 5-fluorouracil, 5 -bromouracil, 5 -chlorouracil, 5- iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6-sopentenyladenine, 1- methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5 -oxy acetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5 -oxyacetic acid methylester, uracil-S- oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6- diaminopurine.

In another example, the polynucleotide includes at least one modified sugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof. The antisense polynucleotide can be conjugated to another molecule, for example a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization- triggered cleavage agent. A targeting moiety can also be included that enhances uptake of the molecule by tumor cells. The targeting moiety can be a specific binding molecule, such as an antibody or fragment thereof that recognizes a molecule present on the surface of the tumor cell.

Suppression of endogenous adenosine receptor locus expression can also be achieved using catalytic nucleic acids such as ribozymes. Ribozymes are synthetic RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No. 5,543,508 to Haselhoff. Ribozymes can be synthesized and administered to a cell or a subject, or can be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (as in PCT publication WO 9523225, and Beigelman et al. Nucl. Acids Res. 23:4434-42, 1995). Examples of oligonucleotides with catalytic activity are described in WO 9506764, WO 9011364, and Sarver et al. (Science 247: 1222-5, 1990). The inclusion of ribozyme sequences within antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

Antibodies specific for adenosine receptor Al , A2A, A2B, or A3 can also be used.

Inhibitors or Antagonist of Adenosine receptor Al

In a specific aspect, the administered composition can comprise an inhibitor or antagonist of adenosine receptor Al. Adenosine receptor antagonist refers to a molecule that inhibits activity of adenosine receptors (e.g., Al receptors). Adenosine receptors antagonists can be small or large molecule antagonists. Examples of adenosine receptor antagonists include, but are not limited to: CPI-444, ZM241385; MRS1220; 1,7, methylxantine (caffeine); theophylline; theobromine; SCH 58261 [7-(2-phenylethyl)-5-amino-2-(2-furyl)- pyrazolo-[4,3-e]-l,2, 4-triazolo[l,5-c]pyrimidine] (Schering-Plough Research Institute, Milan, Italy); KW-6002 RE)-l,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-lH- purine-2, 6-dione] (Kyowa Hakko Kogyo Co. Ltd., Shizuoka, Japan); and ADA-PEG. Particular non-limiting examples of antagonists are described in U.S. Pat. Nos. 5,565,566; 5,545,627, 5,981,524; 5,861,405; 6,066,642; 6,326,390; 5,670,501; 6,117,998; 6,232,297; 5,786.360; 5,424,297; 6,313,131, 5,504,090; and 6,322,771. Inhibitors of adenosine receptor Al can also be selected from the group consisting of a ribozyme, an antisense oligonucleotide, a catalytic nucleic acid that selectively binds mRNA encoding the adenosine receptor, an antibody specific for the adenosine receptor, an agent that increases endogenous adenosine kinase activity, an agent that increases endogenous adenosine deaminase activity, an oxygenation agent, a redox -potential changing agent, an adenosine-accumulation-reducing agent, adenosine deaminase, adenosine kinase an adenosine kinase enhancer, and oxygenation of the subject.

Tn another example, the antagonist of Al can be a peptide, or a pepidomimetic, that binds the adenosine receptor but does not trigger a G1 protein dependent intracellular pathway. Suitable antagonists are described in U.S. Pat. Nos. 5,565,566; 5, 545, 627, 5,981.524; 5,861,405: 6,066,642; 6,326,390; 5.670,501; 6.117,998; 6,232.297; 5,786,360; 5,424,297; 6,313,131, 5,504,090; and 6,322,771.

In another example, the antagonist of Al is an antisense molecule or catalytic nucleic acid molecule (e.g. a ribozyme) that specifically binds mRNA encoding an adenosine receptor. In a further example, an antisense molecule or catalytic nucleic acid targets biochemical pathways downstream of the adenosine receptor. For example, the antisense molecule or catalytic nucleic acid can inhibit an enzyme involved in the Gs protein- or Gi protein-dependent intracellular pathway.

Activator or Agonist of Adenosine receptor 2B

In a specific aspect, the administered composition comprises an inhibitor or antagonist of adenosine receptor 2B. Adenosine receptor agonist refers to a molecule that activates adenosine receptors (e.g. 2B receptors). Adenosine receptors agonists can be small or large molecule agonists. Examples adenosine receptors agonists include adenosine, NECA, or analogs thereof.

Modulators of CD73 expression

In other examples, the compounds that can increase adenosine levels in the patients can be modulators of CD73 or CD73 expression. In particular, compositions that increase CD73 expression or activity can be administered to treat uterine leiomyomas.

Administration

The disclosed compounds can be administered sequentially or simultaneously in separate or combined pharmaceutical formulations. When one or more of the disclosed compounds is combined with a second therapeutic agent, the dose of each compound can be either the same or differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is combined with one or more other active agents (e.g., a cytotoxic agent, etc.), "administration" and its variants are each understood to include the concurrent and sequential introduction of the compound or prodrug thereof and other agents.

In vivo application of the disclosed compounds and compositions containing them can he accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art, including oral, vaginal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrauterine administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration or at continuous or distinct intervals as readily determined by a person skilled in the art.

The compounds disclosed herein and compositions comprising them can also be administered utilizing liposome technology, slow-release capsules, slow release implants, implantable pumps, and biodegradable containers. These delivery methods can provide a uniform dosage over an extended period. The compounds can also be administered in their salt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier to facilitate the effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semisolid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99%, and especially, 1 and 15% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials. The formulations can be stored in a freeze-dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art regarding the type of formulation in question.

Compounds disclosed herein and compositions comprising them can be delivered to a cell either through direct contact with the cell or via a carrier. Carriers for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivering compounds and compositions disclosed herein to a cell comprises attaching the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition, allowing the composition to be translocated across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Compounds can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymers; poly[bis(p- carboxyphenoxy) propane: sebacic acidj (as used in GLIADEL); chondroitin; chitin; and chitosan.

For the treatment of uterine leiomyomas, the compounds disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances and/or with radiation and/or photodynamic therapy and/or with surgical treatment to remove a tumor. These other substances or treatments can be given simultaneously or at different times from the compounds disclosed herein. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies.

Therapeutic application of compounds and/or compositions containing them can be accomplished by any suitable therapeutic method and technique presently or prospectively known to those skilled in the art. Further, compounds and compositions disclosed herein have use as starting materials or intermediates for the preparation of other useful compounds and compositions.

Compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used as ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, algimc acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to modify the solid unit dosage form's physical form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, sugar, and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavorings such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceutically acceptable salts, hydrates, or analogs thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, oils, and mixtures thereof. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile- filtered solutions. For topical administration, compounds and agents disclosed herein can be applied as a liquid or solid. However, it will generally be desirable to administer them topically to the skin as compositions, combined with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds, agents, and compositions disclosed herein can be applied topically to a subject's skin to reduce the size (and can include complete removal) of malignant or benign growths or treat an infection site. Compounds and agents disclosed herein can be applied directly to the growth or infection site. Preferably, the compounds and agents are applied to the growth site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, alcohols or glycols, or water-alcohol/glycol blends, where the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver a compound to the skin are disclosed in U.S. Patent No. 4,608,392; U.S. Patent No. 4,992,478; U.S. Patent No. 4,559,157; and U.S. Patent No. 4,820,508.

Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949.

Also disclosed are pharmaceutical compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical, or parenteral administration, comprising an amount of a compound, constitute a preferred aspect. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

For the treatment of uterine leiomyomas, compounds and agents and compositions disclosed herein can be administered to a patient in need of treatment before, after, or in combination with other antitumor or anti-cancer agents or substances (e.g., chemotherapeutic agents, immunotherapeutic agents, radiotherapeutic agents, cytotoxic agents, etc.) and/or with radiation therapy and/or with surgical treatment to remove a tumor. For example, compounds and agents and compositions disclosed herein can be used in methods of treating cancer wherein the patient is to be treated or is or has been treated with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively. These other substances or radiation treatments can be given at the same or different times from the compounds disclosed herein. Examples of other suitable chemotherapeutic agents include, but are not limited to, altretamine, bleomycin, bortezomib (VELCADE), busulphan, calcium folinate, capeci tabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gefitinib (IRESSA), gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib (GLEEVEC), irinotecan, liposomal doxorubicin, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, raltitrexed, streptozocin, tegafur-uracil, temozolomide, thiotepa, tioguanine/thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine. In an exemplified embodiment, the chemotherapeutic agent is melphalan. Examples of suitable immunotherapeutic agents include, but are not limited to, alemtuzumab, cetuximab (ERBITUX), gemtuzumab, iodine 131 tositumomab, rituximab, trastuzamab (HERCEPTIN). Cytotoxic agents include, for example, radioactive isotopes (e.g., I¹³¹, 1¹²⁵, Y⁹⁰, P³², etc.), and toxins of bacterial, fungal, plant, or animal origin (e.g. , ricin, botulinum toxin, anthrax toxin, aflatoxin, jellyfish venoms (e.g., box jellyfish), etc.) Also disclosed are methods for treating an oncological disorder comprising administering an effective amount of a compound and/or agent disclosed herein before, after, and/or in combination with administration of a chemotherapeutic agent, an immunotherapeutic agent, a radiotherapeutic agent, or radiotherapy.

In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg-200 mg).

EXAMPLES

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, the temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. Numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions, can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions. Materials and Methods

Tissue Specimens and Primary Cell Culture

Human tissue specimens and matched demographic data were obtained from the USF Health/Tampa General Hospital Precision Medicine Biorepository. All specimens were obtained from women undergoing hysterectomy for benign indications. Myometrial specimens were obtained from healthy tissue at least 2 cm from the closest adjacent leiomyoma. Menstrual phase was determined from the date of last menses reported by each subject prior to surgery. Menstrual phase was further confirmed using accepted criteria to histologically date matched endometrial specimens (Noyes, RW, et al. “Dating the endometrial biopsy.” Am J Obstet Gynecol 1975;122(2):262-3). Only specimens collected from pre-menopausal women during the mid-proliferative (cycle day 10-14) or mid- secretory (cycle day 22-26) phase of the menstrual cycle were utilized. Explicit written consent that included permission to generate primary cell lines was documented for all subjects from whom specimens were used.

Primary cultures of myometrium and leiomyoma were generated as previously described (Delaney, MA, et al. “A Role for Progesterone-Regulated sFRP4 Expression in Uterine Leiomyomas.” J Clin Endocrinol Metab 2017; 102(9): 3316-3326). In brief, fresh tissue specimens were minced under sterile conditions, and suspended in 5 ml 1: 1 mix of Dulbecco’s Modified Eagle Medium/F-12 (ThermoFisher, Waltham, MA) supplemented with 1 mg/ml collagenase type II (Worthington Biochemical, Lakewood, NJ) and 0.1 mg/ml deoxyribonuclease I (Sigma-Aldrich, St. Louis, MO). Tissue slurries were incubated at 37°C overnight with gentle agitation, after which, the resulting digest was filtered through a 70 pm strainer, washed once, and resuspended in fresh media. All cultures were maintained at 37°C under 5% CO2 and used within 8 passages of their origination.

RNA Isolation and Real Time Quantitative PCR (qPCR)

Total RNA was prepared using the mirVana™ miRNA Isolation Kit (Invitrogen™, Waltham, MA). qScript cDNA SuperMix (Quantabio, Beverly, MA) was used to perform reverse transcription, after which, relative transcript levels were quantified using the following TaqMan gene expression assays (Applied Biosystems, Foster City, CA): CD73 Exon 2-3: Hs01554888_ml: CD73 Exon 6-7: Hs01573922_ml; ADORA2B: Hs00386497_ml 18S: Hs99999901_sl. All assays were run in triplicate. Mean CT values were normalized to relative expression of 18S mRNA levels. The AACT method was used to calculate relative fold-change in mean levels of gene expression. (Livak, KJ, et al. “Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.” Methods 2001;25(4):402-8).

Western Blot

Protein extracts were prepared using a standard lysis buffer supplemented with protease inhibitor cocktail (ThermoFisher). Protein concentration was determined using the BCA protein assay (ThermoFisher). A total of 15 pg protein was electrophoresed using NuPAGE™ 4 to 12% mini protean gels (Invitrogen) and blotted onto poly vinylidene fluoride (PVDF) membrane (Invitrogen). Non-specific binding was blocked using 5% nonfat dried milk resuspended in Tris-buffered saline supplemented with 0.1% Polysorbate- 20 (TBS-T) for 2 hours at room temperature. All primary antibodies were diluted in TBS-T supplemented with 2% non-fat dried milk and incubated overnight at 4°C. Primary antibodies for CD73 (1: 1,000; Cell Signaling #13160S), AD0RA2B (1 : 1,000; Invitrogen #PA5-96008), Phospho -Akt (1 : 1,000; ABclonal #AP0637), Akt (1: 1,000; ABclonal #A21393), Cyclin Dl(l: 1,000; ABclonal #A19038), Cdk2 pTyrl5 and Histone H3 pSerlO (1 : 1,000; Abeam #Abl 39417), Cdk2 (1: 1,000; Novus Biologicals #3560007106), Histone H3 (1: 1,000; Invitrogen #PA5-121930) were used as described. Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:3,000; Cell Signaling #7074S) was used to visualize immunoreactivity by chemiluminescence (ThermoFisher) using an Odyssey imaging system (LI-COR, Lincoln, NE). Band intensities were quantified by densitometry and normalized to GAPDH (1 :2,000; Sigma-Millipore #G9545-200pl) or Actin (1: 1,000; Abcam#Abl39417) using Image STUDIO Lite Ver 5.2 (LI-COR).

Immunohistochemistry’

Formalin-fixed paraffin-embedded specimens were evaluated by immunohistochemistry as previously described (Mahtani, RL, et al. “A prospective cohort study to evaluate the incidence of febrile neutropenia in patients receiving pegfilgrastim on- body injector versus other options for prophylaxis of febrile neutropenia: breast cancer subgroup analysis.” Support Care Cancer 2022;30(7):6135-6144). In brief, goat polyclonal antibody specific to CD73 was used (R&D Labs, Minneapolis, MN). Biotinylated horse anti-goat IgG (1:500; Vector Labs, Newark, CA) was used for secondary antibody. Antigenantibody complexes were visualized using by incubating tissue cross sections with avidin- biotin-peroxidase complex (Elite ABC kit; Vector Labs) followed by 3,3-diaminobenzidine (DAB; Vector Labs). A rabbit polyclonal antibody was used to visualize smooth muscle actin (1 :400; Abeam, Cambridge. MA). Immunofluorescence was visualized using an avidin-biotin kit with phosphatase-based detection (Vector Labs). Vector Red (Vector Labs) was used to detect alpha smooth muscle actin. For negative controls, an equivalent concentration of non-specific IgG matched to the species of primary antibody was used (Sigma). CD73-specific staining was evaluated in at least 3 sequential high power (40x) fields from each cross-section and scored by a semi quantitative histologic score (HSCORE) as previously described (Delaney, MA, et al. J Clin Endocrinol Metab 2017;102(9):3316- 3326). All cross-sections were evaluated by two investigators blinded to tissue type. Results are reported as an average of both investigators’ scores for each specimen.

In Vitro Assays

Adenosine concentration in tissue specimens was measured by enzyme-linked assay (Cell Biolabs, Inc, San Diego, CA) according to the manufacturer’s instructions. Briefly, equal masses of myometrium and leiomyoma were sonicated in 300 pl PBS and centrifuged at 10,000g for 10 minutes at 4°C and assayed immediately. Fluorescence was measured at Ex/Em of 535 nm/587nm. Adenosine concentrations in culture media were measured using a fluorometric assay (Abeam, #211094) read at OD 450 nM.

To assess the biologic impact of adenosine exposure, 2.5 x 10^s cells were serum- starved for 36 hours using media supplemented 0.4% bovine serum albumin (ThermoFisher). After replating in media containing 2.5% FBS, media were supplemented with either adenosine (ThermoFisher), a hydrolysis-resistant stable adenosine analog, 2- chloro-adenosine (2-CL-AD, ThermoFisher) or 5 pM adenosine receptor subtype-specific agonists: GS21680 (CGS; #1063); BAY60-6583 (BAY #4472); N6-cyclopentyladenosine (CPA; #1702); 1 -Deoxy- l-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-P- D-ribofuranuronamide (IB-MECA; #1066) as specified. All agonists were purchased from Tocris, Inc. (Minneapolis, MN).

In separate experiments, CD73 expression was induced by transfecting 2.5 x 10⁵ cells/well with a plasmid containing a cDNA expression vector containing a clone for the open reading frame of human NT5E (Sino Biological, Houston, TX). The pCMV3 backbone plasmid was used as a negative control for all experiments. ADORA2B expression was targeted by transfecting cells with either 20 nM human ADORA2B siRNA (Dharmacon, Inc., Lafayette, CO) or an ON-TARGETplus non-targeting control pool (siRNA control) using Lipofectamine RNAiMax reagent (Invitrogen)) according to the manufacturer’s instructions. Twenty-four hours after transfection or treatment, cells were seeded to 96 well plates (2500 cells/well); proliferation was measured using the CellTiter 96™ AQeous One assay (Promega, Inc., Madison, WI). To confirm transfection efficiency, cells were collected 24 hours after transfection for total RNA extraction and 72 h after transfection for protein extraction.

To assess cell cycle distribution, leiomyoma cultures (5 x 10⁵ cells) were seeded into 10 cm dishes and serum starved for 36 hours using media supplemented with 0.4% BSA. Cultures were treated with 50 pM 2-CL-AD in DMEM/F-12 supplemented with 2.5% FBS for 48 hours, after which, cells were collected, washed twice in phosphate -buffered saline (PBS) and fixed overnight in 70% ethanol. Fixed cells were centrifuged at 1500 g for 8 min, washed once in PBS, resuspended in 500pl FxCycle™ PI/RNAse Solution (Invitrogen) and incubated for 30 minutes at room temperature in the dark. Labeled cells were sorted using BD FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA). DNA content was analyzed using FlowJo software (vl 0.8.0).

To assess apoptosis, 5 x 10⁵ cells were seeded into 10 cm dishes and growth arrested in media supplemented with 0.4% BSA for 36 hours. Cultures were treated with 50 pM 2- CL-AD in DMEM/F-12 supplemented with 2.5% FBS for 48 hours, harvested, washed twice with sterile PBS and stained for Annexin V-APC and DAPI (Elabscience, Houston, TX). Treated cells were gently vortexed and incubated at room temperature for 15-20 min in the dark, after which, 400 pL of lx Annexin V Binding Buffer was added. Flow cytometry was used to evaluate labeled cells

Statistical Analyses

Paired and unpaired Student t-test was used to assess the statistical significance of comparisons made with a threshold for significance set at < 0.05. All analyses were performed using GraphPad Prism 8 software (GraphPad, Inc, San Diego, CA) Loss Of CD73 Promotes the Growth of Uterine Leiomyomas Via ADORA2B- Regulated Akt Signaling Pathways

Restricted CD73 expression and reduced adenosine concentrations are a feature of uterine leiomyomas

To dissect the role of CD73 in uterine leiomyomas, patterns of its expression were first examined using a series of tissue specimens collected from pre- and post-menopausal women undergoing hysterectomy. Significantly lower levels of CD73 expression were consistently observed in leiomyoma specimens collected during the proliferative phase of the menstrual cycle (n = 18) when assessed by RT-qPCR, regardless of whether primers specific to a portion of the CD73 coding region spanning Exon 2 and 3 or Exons 6 and 7 were utilized (Fig. 1A, Fig. IB). Although lower levels of CD73 transcript were also observed in leiomyoma specimens collected during the luteal phase of the menstrual cycle (n=ll) and from post-menopausal women (n=13), these differences were not consistently observed with both sets of qPCR primers.

CD73 expression was also evaluated in a second set of matched specimens by Western blot. Significantly lower levels of CD73 expression were typically observed in leiomyomas collected during the proliferative phase of the menstrual cycle than the corresponding specimens of myometrium from the same subject (0.37 ± 0.14 vs. 0.90 ± 0.29, p=0.0003, n=5, Fig. 1C, Fig. IF) as well as specimens of leiomyoma and myometrium collected during the secretory phase (0.35 ± 0.06 vs. 0.52 ± 0.11 , p=0.046; Fig ID, Fig. 1 G). However, it was not possible to identify any consistent trend in CD73 expression in postmenopausal specimens (0.49 ± 0.06 vs. 0.28 ± 0.13, p=0. 16; Fig. IE).

Next, CD73 expression was localized in healthy myometrium and leiomyomas. When formalin-fixed tissue specimens were evaluated by immunohistochemstiry (IHC), it was found that of the most robust expression of CD73 was observed in the mix of vascular structures and connective tissues that comprise the pseudocapsule of leiomyomas (Fig. 1A). CD73 expression was observed in nearly all other tissue structures in myometrium, including arterioles, venules and lymphatics. Lymphatic structures, identified by their coexpression with CD31, expressed CD73 at particularly robust levels, as much as 10-fold higher than the levels observed in arterioles (Figs. 7A-7D). In contrast, only very low levels of CD73 expression were observed in the myofibroblasts that comprise the bulk of a leiomyoma (Fig. 2A). In leiomyomas, CD73 expression was largely restricted to thin-walled vascular structures between whorls of myofibroblasts that comprise the bulk of these tumors. Semi-quantitative HSCOREs for leiomyoma pseudocapsule (independent of cell type) were more than 30% higher than myometrium within the same uterus and nearly 5- fold higher than the corresponding leiomyoma (Fig. 2B). HSCORES for leiomyoma were significantly lower than myometrium when only cells with a smooth muscle phenotype were considered (n=I0; p=0.0015; Fig. 2C). Lastly, the intensity of CD73 expression by HSCORE was significantly lower in venules, capillaries and lymphatics in leiomyomas when compared to the analogous structures in healthy myometrium (Figs. 9A-9F). No differences in levels of CD73 expression was observed in the arterioles found in myometrium and leiomyoma.

Leiomyomas are characterized by reduced adenosine levels

As an ecto-enzyme, CD73 play an important role in regulating ambient concentrations of adenosine by catalyzing the dephosphorylation of extracellular adenosine monophosphate, often released by adjacent cells (Colgan, SP, et al. “Physiological roles for ecto-5'-nucleotidase (CD73).” Purinergic Signal 2006;2(2):351-60). To determine whether the restricted patterns of CD73 expression observed in uterine leiomyomas are accompanied by lower ambient concentrations of adenosine, levels of this nucleotide were measured in homogenates prepared from fresh specimens of both myometrium and leiomyoma. It was found that the concentration of adenosine in leiomyomas was significantly lower than homogenates prepared from corresponding healthy myometrium in both pre- (8.89 ± 4. 13 vs. 15.03 + 5.98, n=6; p= 0.013) and post-menopausal (3.10 ± 1.14 vs. 12.06+2.41, n=6) subjects.

Decreased CD73 expression is a durable feature of myofibroblasts cultured from leiomyomas in vitro

CD73 expression was evaluated in a series of primary cultures derived from healthy myometrium and leiomyomas from the same subject. As shown in Fig. 8, CD73 expression in cultures derived from leiomyoma were significantly lower than those originating in myometrium..

Adenosine inhibits proliferation and induces apoptosis in leiomyoma cultures in vitro

The media used to maintain primary cultures contained only very low concentrations of adenosine, and that these concentrations changed only minimally over time. To assess the biologic impact of altered ambient concentrations of adenosine, media bathing primary leiomyoma cultures was supplemented with either adenosine or its non-hydrolyzable analog, 2-chloro adenosine (2-CL-AD), after which, the biologic impact of these exposures were assessed in a number of ways. First, both 2-CL-AD (Fig 3A, 3D, 3G) and adenosine (Figs. 10A-10F) significantly attenuated cell proliferation in a dose- and time- dependent manner. As shown in Figs. 3A-3I, incubation of primary cultures derived from leiomyomas with 2-CL-AD resulted in a greater proportion of cells accumulating at Gl-S interface of the cell cycle (77.32 + 7.62% vs. 72.43 + 7.61%, n=3; p=0.008) with corresponding decreases in the proportion of cells in S (5.50 + 1.13 vs. 7.37 + 1.57%, n=3, p=0.029) and G2-M phases (17.17 + 6.52% vs. 20.17 + 6.43%, n=3, p=0.017). Exposure to either adenosine or 2-CL-AD increased rates of apoptosis (12.23 + 3.87 vs. 20.35 + 7.73, n=3; p=0.04; (Figs. 3A-3I and Figs. 10A-10K). Similar proliferation and flow cytometry results were observed when primary myometrial cultures were incubated in adenosine or 2-CL-AD. Targeting CD73 expression impacts ambient adenosine concentrations and regulates uterine smooth muscle proliferation in vitro

Next, to determine whether altering levels of CD73 expression are sufficient to influence the behavior of uterine myocytes, primary cultures derived from leiomyomas were transfected with either a vector driving the overexpression of a full-length cDNA clone for CD73 or empty vector. Enforced CD73 expression not only resulted in significantly higher concentrations of adenosine in the media of transfected cells (Fig. 4G, Fig. 4H, and Fig. 41), but also resulted in slower proliferation when compared to cultures transfected with empty vector (Fig. 4A, Fig. 6B, and Fig. 6C). Consistent with these observations, knock down of CD73 expression in primary cultures derived from myometrium lowered ambient adenosine concentrations and increased rates of proliferation when compared to controls (Figs. HAUL).

Activity at Type 2B Adenosine Receptors suppresses myofibroblast proliferation

At least four distinct transmembrane receptors (Al, 2A, 2B and 3) have been previously identified for adenosine (Borea, PA, et al. “Pharmacology of Adenosine Receptors: The State of the Art.” Physiol Rev 2018;98(3): 1591-1625). The initial survey of uterine tissues by qPCR and Western blot found that all 4 adenosine receptor subtypes are expressed in human leiomyomas. To gain insight into which of these receptors, if any, mediates the impact of adenosine on uterine leiomyomas, primary cultures derived from leiomyomas were treated with adenosine receptor subtype- specific agonists for 72 hours. As shown in Fig. 5A, Fig. 5B and Fig. 5C, only BAY60-583, an ADORA2B-selective agonist, mimicked the impact of 2-CL-AD and inhibited the proliferation of uterine leiomyoma cells in vitro. In contrast, agonists for multiple other receptor subtypes, CPA (AD ORAL selective agonist), CGS21680 (AD0RA2A- selective agonist), and IB-MECA (ADORA3-selective agonist) had little detectable impact on the growth of these cultures. Similar results were observed when primary cultures derived from matched specimens of healthy myometrium were incubated with each agonist (Figs. 12A-12C).

Next, whether knockdown of AD0RA2B would be used to block the inhibitory effects of BAY60-583 in vitro was tested. Myometrial cultures (n=3) were transfected with siRNAs targeting AD0RA2B expression or a scrambled non-targeting controlled and transferred into media with and without agonist. In each culture, successful AD0RA2B knockdown abrogated the inhibitory effects of BAY60-583 (Fig. 5D, Fig. 5E, and Fig. 5F) on myofibroblast proliferation. 2-CL-AD inhibits phosphorylation of Akt and decreases levels of Cyclin DI, Cdk2 pTyrl 5 and Histones H3 pSerl 0

Activation of the Akt-regulated signaling pathways have been previously shown to play an important role in promoting leiomyoma growth. (Ben-Sasson, H, et al. “All-trans- retinoic acid mediates changes in PI3K and retinoic acid signaling proteins of leiomyomas.” Fertil Steril 2011;95(6):2080-6). To determine alterations in extracellular adenosine impacts Akt signaling, primary cultures derived from leiomyomas were incubated with 2-CL-AD and assessed the impact of this exposure activity in Akt signaling pathways as well as markers associated with both the Gl-S (cyclin DI, cdk2) and G2-M cell cycle checkpoint (phospho-Histone H3). Cultures were incubated with media supplemented with 50 pM 2- CL-AD 48 hours, after which, total protein was collected and used to perform Western blots. As shown in Figs. 6A-6F, administration of 2-CL-AD resulted in lower levels of Akt phosphorylation (Fig. 6B) which were accompanied by lower levels of cyclin DI (Fig. 6C) and lower levels of phosphorylation at both Cdk2 (pTyrl 5; Fig. 6E) and Histone H3 (pSerlO; Fig. 6F) in primary cultures derived from both healthy myometrium and leiomyomas. Similar changes were observed when primary cultures of leiomyomas were incubated with ADORA2B agonist (BAY60-6583; Fig 13). However, transfection with siRNAs targeting ADORA2B expression resulted in increased cyclin D expression and phosphorylation of AKT when compared to cultures transfected with scrambled nontargeting controls (Fig. 13).

Cyclin DI and Cdk2 are key regulators of Gl-S cell cycle progression and are involved in proliferation of Leio cells (Shime, H, et al. “Tranilast Inhibits the Proliferation of Uterine Leiomyoma Cells in Vitro through G1 Arrest Associated with the Induction of p21wafl and p53.” J Clin Endocrinol & Metabol 2002;87(12):5610-5617). Phosphorylated histones H3 at serine site 10 (H3 pSerlO) is a marker of cell proliferation in Leio (Yu, L, et al., “Metalloestrogenic” effects of cadmium downstream of G protein-coupled estrogen receptor and mitogen-activated protein kinase pathways in human uterine fibroid cells.” Arch Toxicol 2021;95:1995-2006).

Overexpression of Adenosine Receptor Al (ADORA1) Modulates AKT Signaling to Promote Growth of Uterine Leiomyomas

Leiomyomas are benign tumors found in as many as 80% of women that frequently cause pelvic pain, abnormal bleeding, and problems with infertility. Recently, it was reported that leiomyomas are characterized by reduced levels of extracellular adenosine due to the loss of ecto-5 '-nucleotidase (CD73), a glycosyl-phosphatidylinositol-linked cell membrane-bound enzyme. However, the mechanisms by which suppressed extracellular concentrations of adenosine promote leiomyoma growth remain poorly understood. It is shown that dysregulated activity at one or more adenosine receptors directly mediates proliferation within the leiomyoma complex.

After obtaining IRB permission, flash frozen specimens of uterine leiomyoma and adjacent normal myometrium were obtained from women undergoing hysterectomy for benign indications. Subjects receiving hormonal medication at the time of surgery were excluded. Total RNA and protein were prepared as previously described. Expression of known adenosine receptors Al, 2 A, 2B and A3 were measured by semi-quantitative realtime PCR (RT-qPCR) and Western blot. Expression of AD0RA1 in primary leiomyoma cultures was targeted by using transcript-specific siRNAs (Dharmacon). AD0RA1 -specific agonist N6-cyclopentyladenosine (CPA) was purchased from R&D. Cell proliferation was measured by using a commercially available assay (Promega). Antibodies for Akt (pSer473) and CyclinDl were obtained from Abclonal. Statistical significance was assessed using paired Student’s t-tests.

Robust overexpression of the high-affinity adenosine receptor AD0RA1 is a robust feature of uterine leiomyoma, where it may be preferentially activated by residual levels of extracellular adenosine to promote growth. Data is shown in Figs. 14A-21C.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Specific embodiments

In some aspects, the techniques described herein relate to a method of treating uterine leiomyoma, including: administering to a patient in need thereof a composition that increases adenosine levels.

In some aspects, the techniques described herein relate to a method, wherein the composition is adenosine, adenosine analog, or a pharmaceutically acceptable salt thereof.

In some aspects, the techniques described herein relate to a method, wherein the composition includes one or more of adenosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, 2-chloroadenosine, R-phenylisopropyladenosine, 2- alkyl substituted adenosine, 2-alkylamino substituted adenosine, 2-propynyl adenosine, cladribine, pentostatin, 2'-deoxyadenosine, 2',3'-isopropylidene adenosine, toyokamycin, 1- methyladenosine, 5N-carboxamides of adenosine, N-6-methyladenosine, adenosine N- oxide, 2-alklylhydrazino adenosine, or 6-methylmercaptopurine riboside.

In some aspects, the techniques described herein relate to a method, wherein the composition includes a modulator of an adenosine receptor.

In some aspects, the techniques described herein relate to a method, wherein the adenosine receptor is one or more of adenosine receptor A 1 , A2A, A2B , or A3.

In some aspects, the techniques described herein relate to a method, wherein the composition is an antisense sequence for adenosine receptor A 1 , A2A, A2B, or A3; a ribozy me; or antibody specific for adenosine receptor Al, A2A, A2B, or A3.

In some aspects, the techniques described herein relate to a method, wherein the composition is an inhibitor or antagonist of adenosine receptor Al.

In some aspects, the techniques described herein relate to a method, wherein the inhibitor or antagonist of adenosine receptor Al is antisense oligonucleotide, ribozyme that selectively binds mRNA encoding adenosine receptor Al, an antibody specific for adenosine receptor A 1 , or peptide or a pepidomimetic that binds adenosine receptor A 1 but does not trigger a G 1 protein dependent intracellular pathway.

In some aspects, the techniques described herein relate to a method, wherein the composition is an activator or agonist of adenosine receptor A2B.

In some aspects, the techniques described herein relate to a method, wherein the composition is N6-cyclopentyladenosine (CPA), BAY60-583, CGS21680, or IB-MECA.

In some aspects, the techniques described herein relate to a method, wherein the composition is a modulator of CD73.

In some aspects, the techniques described herein relate to a method, wherein the patient is in a proliferative phase or luteal phase of their menstrual cycle.

In some aspects, the techniques described herein relate to the method of any one of preceding claims, wherein the patient is pre-menopausal or post-menopausal.

In some aspects, the techniques described herein relate to the method of any one of preceding claims, wherein the patient has a pathogenic mutation in a MED 12 nuclear regulator complex.

In some aspects, the techniques described herein relate to the method of any one of preceding claims, wherein the patient has decreased levels of CD73 expression as compared to a control that does not have uterine leiomyoma. In some aspects, the techniques described herein relate to the method of any one of preceding claims, wherein the patient has decreased levels of intratumoral adenosine concentrations as compared to a control that does not have uterine leiomyomas.

In some aspects, the techniques described herein relate to a method, further including measuring CD73 expression levels in the patient and, and when CD73 expression is less than a control that does not have uterine leiomyomas, administering the composition to the patient.

In some aspects, the techniques described herein relate to the method of any one of the previous claims, further including measuring expression of adenosine receptor Al in the patient and, when expression is greater than a control that does not have uterine leiomyomas, administering the composition to the patient.

In some aspects, the techniques described herein relate to a method, further including measuring adenosine levels in the patient and, when adenosine levels are less than a control that does not have uterine leiomyomas, administering the composition to the patient.

In some aspects, the techniques described herein relate to a method, further including treating the patient with hormonal treatments or surgically removing the leiomyoma.

Claims

CLAIMS What is claimed is:

1. A method of treating uterine leiomyoma, comprising: administering to a patient in need thereof a composition that increases adenosine levels.

2. The method of claim 1 , wherein the composition is adenosine, adenosine analog, or a pharmaceutically acceptable salt thereof.

3. The method of claim 2, wherein the composition comprises one or more of adenosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, 2- chloroadenosine, R-phenylisopropyladenosine, 2-alkyl substituted adenosine, 2-alkylamino substituted adenosine, 2-propynyl adenosine, cladribine, pentostatin, 2'-deoxyadenosine, 2',3'-isopropylidene adenosine, toyokamycin, 1 -methyladenosine, 5N-carboxamides of adenosine, N-6-methyladenosine, adenosine N-oxide, 2-alklylhydrazino adenosine, or 6- methylmercaptopurine riboside.

4. The method of claim 1 , wherein the composition comprises a modulator of an adenosine receptor.

5. The method of claim 4, wherein the adenosine receptor is one or more of adenosine receptor Al, A2A, A2B, or A3.

6. The method of claim 5, wherein the composition is an antisense sequence for adenosine receptor Al, A2A, A2B, or A3; a ribozyme; or antibody specific for adenosine receptor Al, A2A, A2B, or A3.

7. The method of claim 1 , wherein the composition is an inhibitor or antagonist of adenosine receptor Al .

8. The method of claim 7, wherein the inhibitor or antagonist of adenosine receptor Al is antisense oligonucleotide, ribozyme that selectively binds mRNA encoding adenosine receptor Al, an antibody specific for adenosine receptor Al, or peptide or a pepidomimetic that binds adenosine receptor Al but does not trigger a G1 protein dependent intracellular pathway.

9. The method of claim 1 , wherein the composition is an activator or agonist of adenosine receptor A2B.

10. The method of claim 1, wherein the composition is N6-cyclopentyladenosine (CPA), BAY60-583, CGS21680, or IB-MECA.

11. The method of claim 1 , wherein the composition is a modulator of CD73.

12. The method of any one of the preceding claims, wherein the patient is in a proliferative phase or luteal phase of their menstrual cycle.

13. The method of any one of preceding claims, wherein the patient is pre-menopausal or post-menopausal.

14. The method of any one of preceding claims, wherein the patient has a pathogenic mutation in a MED12 nuclear regulator complex.

15. The method of any one of preceding claims, wherein the patient has decreased levels of CD73 expression as compared to a control that does not have uterine leiomyoma.

16. The method of any one of preceding claims, wherein the patient has decreased levels of intratumoral adenosine concentrations as compared to a control that does not have uterine leiomyomas.

17. The method of any one of the preceding claims, further comprising measuring CD73 expression levels in the patient and, and when CD73 expression is less than a control that does not have uterine leiomyomas, administering the composition to the patient.

18. The method of any one of the preceding claims, further comprising measuring expression of adenosine receptor Al in the patient and, when expression is greater than a control that does not have uterine leiomyomas, administering the composition to the patient.

19. The method of anyone of the preceding claims, further comprising measuring adenosine levels in the patient and, when adenosine levels are less than a control that does not have uterine leiomyomas, administering the composition to the patient.

20. The method of anyone of the preceding claims, further comprising treating the patient with hormonal treatments or surgically removing the leiomyoma.