Treatment methods for breast tissue

Abstract

A method for the prevention or treatment of an abnormal mammary cell pathology characterized by cell proliferation in a subject is described. The method involves administering an effective amount of an androgenic agent and an effective amount of an aromatase inhibitor to the subject in combination to increase the serum concentration of the androgenic agent to a level that increases the concentration of intramammary androgens due to the action of 5α-reductase on the androgenic agent, the increased intramammary androgen concentration inducing suppression of c-Myc and / or mTOR expression or activity in the breast, which is associated with lower intramammary androgen concentrations. The increase in serum concentration of the androgenic agent and the increase in intramammary androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the subject's serum. Furthermore, the use of the above-described androgenic agents and aromatase inhibitors is described for the purpose of reducing the risk of breast cancer progressing to hormone-resistant breast cancer, for reducing the risk of HER2-positive breast cancer becoming unresponsive to HER2-targeted anticancer drug therapy, and for inducing the suppression of c-Myc and / or mTOR expression and / or activity. In embodiments, the androgenic agents may be testosterone and the androgen 5α-dihydrotestosterone (DHT).

Description

Technical Field

[0001] The present disclosure relates to a method for preventing or treating abnormal breast cell pathologies in a subject, including the treatment of the subject by the combined use of an androgenic agent and an aromatase inhibitor.

Background Art

[0002] Worldwide, breast cancer is the most common malignancy in women. The development stages of breast cancer are the initiation of the primary malignant cell mechanism, the progression to uncontrolled growth and undifferentiated state, the promotion of established malignant cells, and the progression to resistance to hormonal regulation. Attempts at breast cancer prevention and the most frequently used therapies for breast cancer are to manipulate the inherent hormonal sensitivity that most breast cancers have. Unfortunately, the most common cause of death from breast cancer is that the tumor grows despite anti-estrogen therapy and resistance to hormonal therapy develops.

[0003] HER2-positive breast cancer occurs in approximately 20% of breast cancer cases and is associated with high aggressiveness, poor prognosis, and shortened overall survival. Drug resistance in these cancers remains an ongoing issue, and the molecular mechanisms involved in the development of resistance to Her2-targeted therapy include the PI3K / Akt / mTOR pathway (Non-Patent Document 1).

[0004] Hormonal resistance in breast cancer refers to the phenomenon in which breast cancer cells acquire resistance to hormonal therapy, which is a common treatment for hormone receptor-positive breast cancer. Hormonal therapy targets the estrogen receptor (ER) and progesterone receptor (PR) pathways and delays or stops the growth of cancer cells that depend on these hormones. There are several reasons why hormonal resistance can occur, as follows. · Gene mutation: A change in a gene responsible for hormone production or signal transduction can cause resistance to hormonal therapy. For example, a mutation in the ESR1 gene encoding the estrogen receptor can cause resistance to anti-estrogen drugs. • Changes in receptor expression: Some breast cancer cells may stop expressing hormone receptors or reduce their expression levels, resulting in reduced responsiveness to hormone therapy. • Activation of alternative signaling pathways: Cancer cells may activate alternative pathways that promote cell growth and survival without requiring hormonal signaling. For example, overexpression or activation of growth factor receptors such as the HER2 / neu receptor can contribute to hormone resistance. • Crosstalk between signaling pathways: Complex interactions between different cellular pathways can also contribute to resistance. For example, signaling molecules from other pathways can enhance the function of hormone receptors, reducing dependence on hormones. • Changes in the tumor microenvironment: The tumor microenvironment, including factors such as surrounding cells, blood vessels, and immune cells, can influence the cancer cell's response to hormone therapy. Some factors secreted from the tumor microenvironment may contribute to the development of resistance. • Epigenetic changes: Epigenetic modifications such as DNA methylation and histone modifications can regulate gene expression and alter the responsiveness of cancer cells to hormone therapy.

[0005] Because these hormonal systems are inherent in the physiological functions of normal women, this therapy inevitably involves systemic hormonal side effects and requires long-term administration. Therefore, there has been a continuous need for therapeutic agents and therapies that can suppress the onset of cancer, the promotion of malignant phenotypes, and hormonal resistance to the therapy, without inducing hormonal physiological adverse events that would limit the application of therapeutic agents or therapies for long-term treatment.

[0006] Since the late 1800s, it has been known that removing the ovaries of women with advanced breast cancer yields a good therapeutic response. For the next 130 years, the focus has been on establishing therapeutics to mitigate the known stimuli of hormone-sensitive breast cancer by estrogen. During this time, intermittent research has been conducted to explore whether agonists, rather than antagonists, can be used as alternative hormone therapies. High-dose androgens and high-dose estrogens have both been shown to be effective in treating advanced metastatic hormone-sensitive breast cancer, but their use is limited due to their severe side effects. Therefore, in recent years, especially in the last 20 years, there has been a rapid increase in therapeutic agents that block the effects of estrogen and attempt to overcome the development of hormone resistance that inevitably occurs and leads to advanced breast cancer.

[0007] The mechanism by which high-dose androgens work in breast cancer treatment has been thought to be a secondary effect due to the blocking of the hypothalamic-pituitary-ovarian axis. However, this therapy was just as effective in women who underwent pituitary gland removal as in patients with intact pituitary glands, even when receiving androgen therapy upon recurrence.

[0008] In fact, there has been considerable ambiguity regarding whether testosterone (T), the major physiological androgen, is beneficial to breast cancer or a factor that induces malignancy. This is mainly because testosterone can be metabolized to either 5α-dihydrotestosterone (DHT) or estradiol (E), depending on the presence and levels of the enzymes 5α-reductase and aromatase. This has led to the noteworthy situation in the management of advanced breast cancer where both androgen receptor (AR) antagonists and agonists are being tested.

[0009] AR is almost universally present in hormone-sensitive invasive breast cancer and its precursor lesions, such as ductal carcinoma in situ, lobular carcinoma in situ, and atypical ductal hyperplasia, as well as in normal breast tissue.

[0010] Much of the prejudice against androgen therapy in breast cancer stems from the known effects of testosterone in promoting prostate cancer (PCa). However, it has also been reported that when stimulated with high concentrations of androgens, AR can function as a tumor suppressor of PCa cells, and high doses of testosterone are in clinical trials as a treatment for castration-resistant prostate cancer (CRPC) (Non-Patent Literature 2). These authors showed that retinoblastoma (Rb) family proteins play a central role in maintaining the inhibitory effect of AR, and that inactivation of Rb reduces the sensitivity of CRPC to high-dose testosterone therapy. These authors further showed that the efficacy of high-testosterone therapy is enhanced by CDK4 / 6 inhibitors, and they proposed that their study provides strong mechanistic and preclinical evidence for the combined use of high-testosterone administration and CDK4 / 6 inhibitors in the treatment of CRPC. Studies investigating the molecular effects of high-dose androgens (testosterone) on breast tissue in transgender men (those transitioning from female to male phenotypes) have shown reduction in ductal structure, induction of sex-biased transcriptional changes by testosterone, and remodeling of the breast's interstitial compartment. The observed changes have been reported to be induced either directly via AR or indirectly via parasecretory signaling to cells lacking hormone receptors (Non-Patent Literature 3).

[0011] Recently, it has been reported that in the presence of high concentrations of DHT, androgen receptors form AR dimers on prostate cancer (PCa) cells, and that these dimers are associated with decreased c-Myc expression. Conversely, in the presence of low concentrations of DHT, AR exists as a monomer on PCa cells, and the expression of monomeric AR is associated with increased intracellular mTOR activity (Non-Patent Literature 4). Another study using human explant tissue derived from breast cancer and breast cancer cell lines demonstrated that DHT can activate the endogenous AR pathway and inhibit ER-mediated transcriptional activity in tumors resistant to current endocrine therapy or tumors with genomic abnormalities in ESR1 or CCND1. Furthermore, it has been demonstrated that when radioactive DHT was injected into women before breast cancer surgery, the radioactive DHT first accumulated in breast fat and then strongly accumulated in malignant tumors.

[0012] Traditionally, breast cancer has been treated with 500-1800 mg of testosterone. High-dose testosterone and aromatase inhibitor combination therapy via subcutaneous implants in the breast has also long been recommended for the treatment of breast cancer, with typical testosterone doses ranging from approximately 130 mg to over 200 mg. For example, in the treatment of metastatic breast disease, a subcutaneous pellet implant containing 320 mg of testosterone and 24 mg of letrozole was administered every nine weeks, resulting in serum testosterone levels exceeding the male range of 20 nmol / L (i.e., 20 nM) (Non-Patent Literature 5).

[0013] However, in the case of long-term administration required for cancer treatment, a significant increase in serum testosterone levels is associated with an undesirable risk of masculinization in women. Similarly, long-term administration of high doses of aromatase inhibitors can also produce undesirable physiological effects.

[0014] Patent Document 1 describes that when mammography breast density (MBD) is high and magnetic resonance imaging (MRI) background enhancement (BPE) is high, administering testosterone and an aromatase inhibitor (Ai) such as anastrozole to breast tissue via subcutaneous implantation elicits a unique pharmacokinetic response in normal, non-malignant breast tissue. In particular, it has been found that treatment with testosterone and anastrozole leads to stabilization of AR expression, an increase in serum total cystic disease fluid protein 15 (GFP-15), an androgen-induced protein associated with a good prognosis, and a decrease in MBD and BPE. [Prior art documents] [Patent Documents]

[0015] [Patent Document 1] International Publication No. 2016 / 061615 [Non-patent literature]

[0016] [Non-Patent Document 1] Elshazly,AMand DAGewirtz,(2022).An overview of resistance to Human epidermal growth factor receptor 2(Her2)targeted therapies in breast cancer.Cancer Drug Resist 5(2):472-486. [Non-Patent Document 2] Han W et al.,(2022).Exploiting the tumor-suppresive activity of the androgen receptor by CDK4 / 6 inhibition in castration-resistant prostate cancer.Molecular Therapy Vol.30,No 4,April,pp.1628-1644. [Non-Patent Document 3] Raths,F.et al.,(2023).The molecular consequences of androgen activity in the human breast.Cell Genom 3(3):100272. [Non-Patent Document 4] Androgen Receptor in Breast Cancer / Prostate Cancer Symposium,Dec.5th,2022,UCSF;Qiu X.et al.,2022 [Non-Patent Document 5] Glaser R and Dimitrakakis C, (2021). Testosterone Implant Therapy in Women With and Without Breast Cancer: Rationale, Experience, Evidence. Androgens: Clinical Research and Therapeutics, Vol. 2.1, pp. 94-110. [Overview of the project]

Problems to be Solved by the Invention

[0017] There remains a continuing need for therapeutic agents and methods for treating breast tissue hyperplasia and breast cancer while reducing or avoiding the undesirable effects associated with the administration of high doses of testosterone and aromatase inhibitors.

Means for Solving the Problems

[0018] The present disclosure first stems from the finding that when an androgenic agent and an aromatase inhibitor (AI) are administered in combination for the prevention or treatment of breast cancer, serum testosterone shows a well-tolerated increase and there are hardly any physiologically relevant changes in serum DHT levels. Second, it stems from the finding that a low DHT concentration in breast tissue is associated with an increase in mTOR activity, while exposure to high concentrations of DHT can result in the suppression of the expression and / or activity of c-Myc and / or mTOR in breast tissue. Suppression of c-Myc in breast tissue is not observed upon exposure to low concentrations of DHT. Thus, by using the dosage-specific combination therapy described herein, the risk of developing invasive breast cancer or pre-invasive breast malignancies, and further the risk of progression to or occurrence of hormone-insensitive breast cancer, may be reduced. This reduction may be obtained by the combination therapy alone or in combination with other therapies targeting the hormone sensitivity of breast cancer. Similarly, the risk of progression to breast cancer insensitive to HER2-targeted therapies, which may result from resistance arising from the persistent activation of signaling pathways despite HER2 blockade, may also be reduced.

[0019] In the process leading to this disclosure, the inventors believe that the AR dimerization in PCa cells under recently demonstrated high-DHT conditions contributes to the tumor suppressor activity reported in the studies by Han W. et al. (2022) and Hickey TE. et al. (2021), and that this suppressive effect disappears when AR monomers are expressed at low DHT concentration levels. The combination therapy of androgens and aromatase inhibitors described herein makes it possible to provide breast tissue with an environment in which DHT or other androgens resulting from 5α-reductase activity are present in levels substantially related to AR dimerization and promote the tumor suppressor activity of AR without increasing systemic serum androgen concentrations and the risk of masculinization in the subject.

[0020] More specifically, in one aspect of the present disclosure, a method for the prevention or treatment of an abnormal mammary cell pathology characterized by cell proliferation in a subject, This method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject in combination to increase the serum concentration of the androgenic agent, thereby reaching a level that increases the concentration of breast androgens due to the action of 5α-reductase on the androgenic agent. Increased intramammary concentrations induce suppression of c-Myc and / or mTOR expression and / or activity associated with lower intramammary androgen concentrations. A method is provided in which an increase in the serum concentration of an androgenic agent and an increase in the intramammary androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

[0021] Typically, aromatase inhibitors block the aromatization of androgenic agents to estrogen. In a preferred embodiment, breast estrogen concentrations do not increase substantially during the extension period, and preferably, breast estrodiol concentrations do not increase substantially during the extension period.

[0022] Typically, androgenic agents are selected from testosterone and its physiologically active forms.

[0023] In preferred embodiments according to this disclosure, the androgen is 5α-dihydrotestosterone (DHT) or a physiologically active form of DHT.

[0024] Typically, intramammary androgen concentrations rise to over 1.0 nM.

[0025] Typically, androgen concentrations in the serum of the subject remain below 0.3 ng / ml (0.09 nM) over a given period, and in most cases, are zero or negligible.

[0026] Similar to the discovery in prostate cancer (PCa) that androgen receptors (ARs) can be expressed in monomeric and dimeric forms, the inventors believe that in the method described herein, androgen receptors (ARs) are expressed in the breast primarily as AR monomers below the lower limit of intramammary androgen concentrations, and primarily as AR dimers above lower intramammary androgen concentrations.

[0027] In a further aspect of this disclosure, a method for the prevention or treatment of an abnormal mammary cell pathology characterized by cell proliferation in a subject, The method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject to increase the serum concentration of the androgenic agent to a level that raises the intramammary concentration of 5α-dihydrotestosterone (DHT) or a physiologically active form of DHT to more than 1.0 nM for a predetermined period of time. The increase in the concentration of DHT or its physiologically active form results from the action of 5α-reductase activity on androgenic agents. A method is provided in which an increase in the serum concentration of an androgenic agent and an increase in the concentration of DHT or a physiologically active form thereof in the breast are maintained for a predetermined period, and the concentration of DHT or a physiologically active form thereof in the serum of the subject does not substantially increase over the predetermined period.

[0028] Typically, the methods described herein are methods for the prevention or treatment of breast cancer.

[0029] Typically, breast cancer is either preinvasive or malignant breast cancer. Preinvasive breast cancer in at least some forms of this disclosure is It may be non-invasive ductal carcinoma or lobular cell carcinoma (DCIS or LCIS).

[0030] In other embodiments, the method is for the prevention or treatment of mammary cell hyperplasia, typically ductal cell hyperplasia or lobular cell hyperplasia.

[0031] Particularly desirable is a combination therapy of an androgenic agent and an aromatase inhibitor, as described herein, which reduces the risk of breast cancer or breast hyperplasia progressing to hormone-resistant breast cancer or breast cancer that is not sensitive to HER2-targeted chemotherapy.

[0032] In particular, a method for reducing the risk of breast cancer progressing to hormone-resistant breast cancer in a subject, according to yet another aspect of this disclosure, This method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject in combination to increase the serum concentration of the androgenic agent, thereby reaching a level that increases the concentration of breast androgens due to the action of 5α-reductase on the androgenic agent. Increased intramammary concentrations induce suppression of c-Myc and / or mTOR activity in the breast, which is associated with lower intramammary androgen concentrations. A method is provided in which an increase in the serum concentration of an androgenic agent and an increase in the intramammary androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

[0033] In another embodiment of this disclosure, a method for reducing the risk of HER2-positive breast cancer in a subject becoming unresponsive to HER2-targeted anticancer drug therapy, This method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject in combination to increase the serum concentration of the androgenic agent, thereby reaching a level that increases the concentration of breast androgens due to the action of 5α-reductase on the androgenic agent. Increased intramammary concentrations induce suppression of c-Myc and / or mTOR activity in the breast, which is associated with lower intramammary androgen concentrations. A method is provided in which an increase in the serum concentration of an androgenic agent and an increase in the intramammary androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

[0034] In another aspect of this disclosure, a method for inducing suppression of c-Myc and / or mTOR activity in a subject breast, This method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject in combination to increase the serum concentration of the androgenic agent, thereby increasing the concentration of androgens in the breast due to the action of 5α-reductase on the androgenic agent. A method is provided in which increased intramammary concentrations induce suppression of c-Myc and / or mTOR activity in the breast, which is associated with lower intramammary androgen concentrations.

[0035] In another embodiment of the present invention, an androgenic agent for use in a method for the prevention or treatment of breast cancer or mammary cell hyperplasia in a subject, wherein the method The patient is administered an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor in combination. This includes increasing the serum concentration of an androgenic agent to a level that increases the concentration of mammary androgens due to the action of 5α-reductase on the androgenic agent, Increased intramammary concentrations induce suppression of c-Myc and / or mTOR activity in the breast, which is associated with lower intramammary androgen concentrations. An androgenic agent is provided in which an increase in the serum concentration of the androgenic agent and an increase in the intramammary androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the target serum.

[0036] In another embodiment of the present invention, the use of an androgenic agent in the manufacture of a medicament for the prevention or treatment of breast cancer or mammary cell hyperplasia in a subject, wherein the prevention or treatment is This method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject in combination to increase the serum concentration of the androgenic agent, thereby reaching a level that increases the concentration of breast androgens due to the action of 5α-reductase on the androgenic agent. Increased intramammary concentrations induce suppression of c-Myc and / or mTOR activity in the breast, which is associated with lower intramammary androgen concentrations. The use of an androgenic agent is provided such that the increase in serum concentration of the androgenic agent and the increase in breast androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the subject's serum.

[0037] In a further aspect of this disclosure, an androgenic agent for use in a method for the prevention or treatment of breast cancer or mammary cell hyperplasia in a subject, wherein the method The method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject to increase the serum concentration of the androgenic agent to a level that raises the intramammary concentration of dihydrotestosterone (DHT) or a physiologically active form of DHT to more than 1.0 nM for a predetermined period of time. The increase in the concentration of DHT or its physiologically active form results from the action of 5α-reductase activity on androgenic agents. An androgenic agent is provided that maintains an increase in serum concentration of the androgenic agent and an increase in intramammary concentration of DHT or a physiologically acceptable form thereof for a predetermined period, without substantially increasing the concentration of DHT or a physiologically active form thereof in the serum of the subject over a predetermined period.

[0038] In a further aspect of this disclosure, the use of androgenic agents in the manufacture of a pharmaceutical product for the prevention or treatment of breast cancer or mammary cell hyperplasia in a subject, wherein the prevention or treatment is The method involves administering an effective dose of an androgenic agent and an effective dose of an aromatase inhibitor to a subject to increase the serum concentration of the androgenic agent to a level that raises the intramammary concentration of dihydrotestosterone (DHT) or a physiologically active form of DHT to more than 1.0 nM for a predetermined period of time. The increase in the concentration of DHT or its physiologically active form results from the action of 5α-reductase activity on androgenic agents. The use of an androgenic agent is provided such that an increase in the serum concentration of the androgenic agent and an increase in the intramammary concentration of DHT or a physiologically acceptable form thereof are maintained for a predetermined period, without substantially increasing the concentration of DHT or a physiologically active form thereof in the serum of the subject over a predetermined period.

[0039] Thus, methods for treating mammary cell hyperplasia, pre-invasive breast cancer, and malignant breast cancer by agonist activation of the androgen receptor (AR) are described. In embodiments, the intramammary androgen concentration is increased to a lower limit to induce or promote the tumor suppressor activity of AR, resulting in suppression of the expression and / or activity of the oncogene c-Myc, and further suppression, avoidance, or reduction of the expression and / or activity of mTOR, which is associated with intramammary androgen concentrations below the lower limit. Therefore, the embodiments described herein provide prevention or treatment in a dose-specific manner. The effect of increased intramammary androgen concentration is thought to be due to the formation of AR dimers when androgen concentrations are elevated, compared to the expression of AR monomers observed when intramammary androgen concentrations are lower.

[0040] Therefore, the present disclosure enables the induction of suppression of c-Myc and / or mTOR expression or activity, thereby inhibiting cell proliferation and inducing a differentiated phenotype. Furthermore, in one or more forms, the present disclosure enables the inhibition, blockade, or reduction of the risk of progression to drug-resistant breast cancer, such as hormone-resistant breast cancer, or HER2-positive breast cancer that is unresponsive to HER2-targeted anticancer therapies, all of which are expressly intended.

[0041] Advantageously, in the preferred embodiments described herein, the serum concentration of the administered androgenic agent in the patient is maintained for a given period at a level that does not substantially cause masculinization or changes in the physical characteristics of the subject that are typically associated with androgenic agents, or if such masculinization or changes occur, they are essentially negligible. Similarly, since intramammary androgen levels due to the action of 5α-reductase can be obtained substantially without an increase in serum androgen concentrations, in the embodiments described herein, essentially no masculinization of the subject occurs from androgens other than the effects exerted by intramammary androgens.

[0042] Furthermore, in one or more forms, the methods of the Disclosure provide treatment for breast cancer with lower doses of testosterone and aromatase inhibitors than conventionally used doses, as described herein. By enabling the use of lower doses of either testosterone or aromatase inhibitors, the risk of undesirable or undesirable systemic side effects caused by aromatase inhibitors may be reduced or avoided. In addition, since testosterone is a precursor of estrogen that promotes the proliferation of mammary cells, the risk of undesirable or undesirable estrogen synthesis is also reduced by providing lower doses of testosterone and a relatively low increase in serum testosterone concentration, as described herein. Moreover, methods according to one or more forms of the Disclosure may enable enhanced therapy for cancer compared to existing conventional therapies.

[0043] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” should be understood to mean including the element, integer, or step, or group of elements, integers, or steps described, but not to mean excluding any other element, integer, or step, or group of elements, integers, or steps.

[0044] Any discussion of documents, operations, materials, devices, articles, etc., contained herein is solely for the purpose of providing context for the present invention. None of these matters should be construed as forming part of the foundation of the prior art or being common general knowledge in the art relevant to the present invention, having existed in Australia or elsewhere prior to the priority date of this application. The features and advantages of the present invention will become even clearer from the following detailed description relating to exemplary embodiments of the invention, along with the accompanying drawings. [Modes for carrying out the invention]

[0045] Abnormal c-Myc expression and hyperactivation of mTOR signaling are associated with the development and progression of cancers, including breast cancer.

[0046] c-Myc is a proto-oncogene that codes for the transcription factor protein MYC. MYC plays a crucial role in regulating cell growth, division, and metabolism. It can also influence cell differentiation, apoptosis (programmed cell death), and the maintenance of stem cell-like properties in cancer cells. In breast cancer, c-Myc is often overexpressed or amplified. This means that the production of MYC protein is increased, which may contribute to the development and progression of cancer. In breast cancer, c-Myc plays several roles. • Promotes cell proliferation: Overexpression of c-Myc can promote cell growth and division by regulating the expression of genes involved in the cell cycle, thereby accelerating the cell cycle and increasing the rate of cell proliferation. • Suppression of apoptosis: c-Myc can suppress the process of programmed cell death (apoptosis) by downregulating the expression of apoptosis-promoting genes and upregulating anti-apoptotic genes. This allows cancer cells to survive and continue to grow despite unfavorable conditions. • Increased metabolic activity: c-Myc upregulates the expression of genes involved in metabolism, particularly those involved in glucose and glutamine metabolism. This increased metabolic activity provides the energy and components necessary for the rapid growth and proliferation of cancer cells. • Contribution to tumor heterogeneity: c-Myc may influence the differentiation and maintenance of cancer stem cells, which are part of a tumor cell population that possesses self-renewal ability and the capacity to generate diverse cell lineages within the tumor. This contributes to tumor heterogeneity, making cancer more aggressive and difficult to treat. • Promotion of angiogenesis: c-Myc may also contribute to the formation of new blood vessels (angiogenesis) within tumors, which is essential for supplying nutrients and oxygen to growing cancer cells. • Promotion of metastasis: c-Myc also promotes the invasion and spread of cancer cells to other parts of the body by regulating genes involved in cell adhesion, cell migration, and extracellular matrix remodeling.

[0047] mTOR is a protein kinase that plays a central role in regulating cell growth, proliferation, survival, metabolism, and angiogenesis, and is a key component of the PI3K / ACT / mTOR signaling pathway, which is frequently dysregulated in various cancers, including breast cancer. In breast cancer, activation or hyperactivation of the mTOR pathway contributes to cancer development and progression through multiple pathways. The following occurs by enhancing cell proliferation and survival: Activation of the mTOR pathway promotes cell growth and division by regulating protein synthesis, ribosome production, and cell cycle progression. Furthermore, mTOR signaling inhibits apoptosis, enabling the continued survival and growth of cancer cells. • By increasing metabolism and nutrient uptake: mTOR plays a role in regulating cellular metabolism, including glucose and lipid metabolism, to meet the energy and biosynthetic demands of rapidly dividing cancer cells. Activation of the mTOR pathway also increases nutrient uptake, supporting cancer cell growth and survival. • By contributing to tumor angiogenesis: mTOR signaling can promote the formation of new blood vessels (angiogenesis) within tumors by upregulating the expression of angiogenic factors such as vascular endothelial growth factor (VEGF). This ensures that growing tumors receive sufficient nutrients and oxygen. By influencing cancer cell migration and invasion: Activation of the mTOR pathway can affect the expression of proteins involved in cell adhesion, migration, and extracellular matrix remodeling, potentially promoting cancer cell invasion and metastasis. • By supporting cancer stem cells: mTOR signaling is involved in the maintenance and self-renewal of cancer stem cells (subpopulations of tumor cells that have the ability to produce diverse types of cells within a tumor). This contributes to tumor heterogeneity, making cancer more aggressive and difficult to treat.

[0048] As described herein, inhibiting c-Myc and suppressing the sustained activation of signaling pathways such as the PI3K / Akt / mTOR pathway may delay the onset and progression of hormone-resistant breast cancer, or delay the onset or progression of drug-resistant cancers such as HER2-positive cancers that are not sensitive to HER2-targeted anticancer therapy despite HER2 blockade, and / or reduce the risk of breast cancer onset or progression. For example, c-Myc has been reported to modulate the sensitivity of breast cancer cells to the selective CDK4 and CDK6 inhibitor palbociclib via the c-Myc / miR-29b-3p / CDK axis, and inactivation of c-Myc may enhance the efficacy of palbociclib (Ji W. et al, 2020).

[0049] The method of this disclosure stems from the observation that when the intramammary concentration of 5α-dihydrotestosterone (DHT) exceeds the lower limit of 1 nM, c-Myc and mTOR signaling and / or activity may be suppressed or blocked in the breast. Without being bound by theory, the inventors believe that elevated intramammary DHT levels induce the formation of androgen receptor (AR) dimers, leading to their transcriptional activation, and consequently suppressing the c-Myc and / or mTOR activity observed when intramammary DHT concentrations are less than 1 nM, and that this c-Myc and mTOR activity is associated with the expression of monomeric AR at intramammary DHT concentrations less than 1 nM.

[0050] Breast cancer is rare in men, and when it does occur, tumors are generally androgen receptor (AR) positive and hormone receptor positive for estrogen and progesterone. Although male breast malignancies are likely to occur secondary to genetic predisposition, these malignancies typically retain hormone sensitivity, suggesting a better response to therapy. Men are prone to elevated DHT levels in breast tissue, which is the most likely reason why men do not usually develop breast cancer. Under normal circumstances, DHT levels in women's breast tissue do not rise, and consequently, AR receptor-induced downstream genetic changes in breast tissue associated with elevated DHT generally do not occur.

[0051] Recently, it has been reported that the androgen receptor (AR) can interact with the DNA-binding factor GATA3 (GATA-binding protein 3), and that DHT induces AR / GATA3 binding, thereby promoting the tumor suppressor function of AR. GATA3 binding to AR is associated with AR-mediated growth inhibition in breast cancer cells and a better overall survival prognosis for breast cancer, and this association is independent of the cell's estrogen receptor (ER) expression status (Hosseinzadeh, L. et al., 2024).

[0052] In embodiments, an increase in DHT (or other androgens) levels in the breast resulting from 5α-reductase activity as described herein may promote the tumor suppressor activity of AR associated with AR dimerization and promote the binding of GATA3 to AR. It has recently been shown that GATA3 binding to the AR receptor promotes the tumor suppressor activity of AR (Hosseinzadeh, L. et al., 2024), and consequently, this binding is a crucial element associated with breast cells that are least likely to progress malignantly or become hormone-insensitive and are treatable with hormone therapy. Therefore, inducing the tumor suppressor activity of AR can suppress or prevent the formation or progression of ER-negative breast cancer. Furthermore, combination therapy with the androgenic agents described herein and aromatase inhibitors may provide a treatment that reprograms ER-negative breast cancer cells or tumors into hormone-sensitive breast cancer cells or tumors (e.g., cancers expressing ER and / or progesterone receptors (PR), and / or cancers treatable with hormone therapy as described herein). All such embodiments are expressly encompassed.

[0053] In particular, in at least some form, the disclosure provides a means of suppressing, preventing, or reducing the risk of progression to hormone-sensitive or hormone-insensitive breast cancer, and, if malignant breast cancer develops, the cancer is hormone-sensitive, improving prognosis and / or response to hormone therapy.

[0054] Testosterone is converted to DHT by the action of 5α-reductase. This enzyme is present in the breast, and its expression has been reported to be upregulated by DHT. Testosterone is typically administered according to the methods of this disclosure, but other androgenic agents that can be reduced by 5α-reductase in the breast can also be used. DHT itself is an agonist of the androgen receptor (AR) and has been reported to have 2 to 3 times higher affinity and 2.5 to 10 times stronger than testosterone. Testosterone is also converted to estradiol in the breast by the aromatase enzyme. As described herein, in preferred embodiments, by administering testosterone or other androgenic agents to a subject in combination with an aromatase inhibitor, DHT concentrations can be increased in the breast without increasing breast estradiol to harmful levels, thereby obtaining an anti-estrogen effect in breast tissue.

[0055] As used herein, “androgenic agents” means testosterone and steroid and nonsteroidal hormones that bind to androgen receptors (e.g., in the nM range) and stimulate transactivation activity, thereby modulating the expression of target genes, and further include hormones that can be 5α-reduced by 5α-reductase in the breast of the subject, and encompass synthetic and non-synthetic hormones, as well as physiologically active forms of testosterone. Non-limiting examples include precursors, derivatives, isomers, and esters of testosterone.

[0056] Examples of androgenic agents include methyltestosterone, a synthetic testosterone in which a methyl group is introduced at the 17th carbon position and can be reduced to 17α-methyldihydrotestosterone (17α-MDHT) by 5α-reductase; fluoxymesterone, another synthetic androgen that can be converted to dihydrofluoxymesterone (DHF); metadienone (also known as methandrostenolone), a steroid derived from testosterone that can be reduced to dihydromethandrostenolone (also known as 17α-methyl-5α-dihydrotestosterone or 17α-methyl-DHT); androstenedione, a precursor of testosterone that can be converted to 5α-androstanedione by 5α-reductase; and dehydroepiandrosterone (DHEA), which can be converted to 5α-androstanediol by 5α-reductase. Physiologically acceptable forms of testosterone available through the methods described herein include testosterone enanthate, testosterone propionate, testosterone cypionate, and testosterone undecanoate.

[0057] As used herein in the context of the preventive or therapeutic effects of this disclosure, “androgen” means an androgen that results from the action of 5α-reductase on an androgenic agent in the breast of the subject and binds to an androgen receptor, and includes DHT and physiologically active forms of DHT.

[0058] As used herein, "aromatase inhibitor" means an aromatase enzyme inhibitor that inhibits the aromatization of androgenic agents into estrogens such as estradiol.

[0059] The aromatase inhibitors used in the methods of this disclosure may be selected from nonsteroidal aromatase inhibitors, steroidal aromatase inhibitors, and mechanism-based steroidal inhibitors. Nonsteroidal aromatase inhibitors can be classified into three categories: aminoglutethimide-like molecules, imidazole / triazole derivatives, and flavonoid analogs. Examples of nonsteroidal aromatase inhibitors include, but are not limited to, anastrozole (Arimidex®; 2,2′-[5-(1H-1,2,4-triazole-1-ylmethyl)-1,3-phenylene]bis(2-methylpropanenitrile)), letrozole (Femara®; 4,4′-((1H-1,2,4-triazole-1-yl)methylene)dibenzonitrile), borozole (Rivizor®), and phadrozole (Afema®).

[0060] Exemestane (Aromasin®; 6-methyleneandrost-1,4-diene-3,17-dione) and formestane (Lentaron®; 4-hydroxyandrost-4-ene-3,17-dione) are examples of steroidal aromatase inhibitors that can be used. Typically, the aromatase inhibitor used in the methods of this disclosure is selected from the group consisting of anastrozole and letrozole, most commonly anastrozole.

[0061] As used herein, “effective dose” or “pharmaceutically effective dose” means a sufficient amount of the active compound or drug to produce the desired therapeutic effect, which is non-toxic and has an acceptable toxicity profile and / or an acceptable side effect profile. The amount may vary from person to person, depending, for example, on the subject’s age, overall physiological state, severity of the condition being treated, the specific therapeutic agent administered, and combinations of these factors. The appropriate “effective dose” can usually be determined in individual cases by the attending physician or healthcare professional, using routine experiments and evaluations based on relevant texts and literature and / or established principles.

[0062] The dosage of the selected aromatase inhibitor will generally be determined to avoid an increase in the intramammary concentration of naturally occurring or synthetic estrogen (particularly estradiol, which is well known to promote the proliferation and differentiation of mammary epithelial and stromal cells) caused by the administered androgenic agent.

[0063] In the methods described herein, an androgenic agent is administered to a subject to increase the serum concentration of the androgenic agent to a level corresponding to the increase in intramammary androgen concentration resulting from the action of 5α-reductase on the androgenic agent in the breast, such that the level is sufficient to obtain or maintain inhibition of c-Myc and / or mTOR in the breast. Inhibition of c-Myc and / or mTOR activity may be at least half of the c-Myc and / or mTOR activity (e.g., greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than or equal to), or complete or substantially complete, in which case the detectable c-Myc and / or mTOR activity is at a negligible level or below a physiologically insignificant level. These can be determined by polymerase chain reaction (PCR) protocols, ELISA, cell assays, or other appropriate assays.

[0064] Repression of c-Myc may be achieved by downregulating or blocking the activation of the c-Myc oncogene and / or downregulating the expression and / or activity of the encoded MYC protein. Similarly, repression of mTOR includes downregulating or blocking the expression and / or activity of the kinase.

[0065] Abnormal mammary cell conditions that may be treated in accordance with this disclosure include mammary cell hyperplasia (such as atypical mammary cell hyperplasia, ductal cell hyperplasia, and lobular cell hyperplasia), ductal carcinoma in situ (DCIS), lobular cell carcinoma in situ (LCIS), cancer, pre-invasive cancer, and malignant cancer of the breast. “Pre-invasive cancer” means cancer or carcinoma in which cell proliferation is uncontrolled but there is no spread or metastasis from the primary site to surrounding breast structures, or from the ducts, lobules, or glandular tissue of the breast. In contrast, malignant cancer is cancer that has metastasized to the breast or other parts of the body, or has metastasized from the ducts, lobules, or glands of the breast to surrounding breast tissue.

[0066] DCIS is a non-invasive form of breast cancer in which abnormal cells are found on the inner wall of the milk ducts, but they do not spread beyond the ducts. It is considered an early form of breast cancer. DCIS has a high chance of being treated, but if not properly managed, it can progress to invasive breast cancer.

[0067] GATA3 is a transcription factor that plays a crucial role in the development and differentiation of various tissues, including mammary gland tissue. It is one of the major regulators of luminal epithelial cell differentiation in the breast, and is involved in maintaining the function and integrity of these cells. GATA3 is a marker of luminal epithelial cells in the breast, and its expression in DCIS indicates that tumor cells possess luminal characteristics. This is typical of many breast cancers, particularly those classified as luminal A or luminal B subtypes. Therefore, this marker is widely used in pathology, contributing to the differentiation of various breast lesions, its presence helping to confirm the diagnosis of DCIS, and is also useful in differentiating it from other breast conditions such as invasive cancer and benign lesions. Studies have shown that GATA3 expression is associated with a favorable prognosis in breast cancer, and high levels of GATA3 expression in DCIS may correlate with a reduced risk of progression to invasive disease and improved overall prognosis. Furthermore, GATA3 plays a role in regulating the estrogen receptor (ER) signaling pathway, making it suitable for treating patients with hormone therapy. For example, tumors expressing GATA3 are often ER-positive, which means they may respond well to anti-estrogen therapies such as tamoxifen or aromatase inhibitors. Furthermore, GATA3 influences cell proliferation, differentiation, and apoptosis, which are crucial processes in cancer development, progression, and response to treatment. Therefore, GATA3 is an important biomarker from the perspective of DCIS, providing insights into diagnosis, prognosis, and treatment. Luminous mammary cell differentiation with GATA3 expression is associated with a favorable prognosis and responsiveness to hormone therapy, as described above, and this disclosure extends to the determination of induction of GATA3 expression and / or activity by combination therapy with aromatase inhibitors and androgenic agents as described herein for prognostic assessment and / or selection or suitability of cancer cell therapy (e.g., hormone therapy such as tamoxifen or other hormone therapies described herein), which is expressly intended herein.For example, elevated GATA3 expression and / or activity in mammary cell samples from subjects treated with androgenic agents and aromatase inhibitors may be evaluated by appropriate assays to determine whether GATA3 activity is above or below baseline levels. If the expression or activity of a marker exceeds the baseline level, it suggests a better prognosis and / or suitability for hormone therapy compared to evaluation levels where GATA3 expression and / or activity in the test sample is below the baseline level. DCIS can be further classified into two distinct subgroups: "immune-hot" and "immune-cold." The former is characterized by increased lymphocyte and macrophage infiltration associated with a "hot" immune response and indicates a poor prognosis. It is accompanied by elevated expression of PDCD1 and CTLA4 markers and decreased GATA3 expression (Hong R. et al., 2024). Mutation analysis of GATA3 in breast cancer has shown relatively high mutation rates in women and men with early-onset IDS (Hong R. et al., 2024; Li Y. et al., 2024). Therefore, evaluation of GATA3 expression and / or activity can be combined with evaluation of the immune response status of cancer and / or GATA3 mutation analysis of cell samples for prognosis assessment and / or evaluation of the selection or suitability of cancer cell therapy, as described herein.

[0068] Typically, when administered to a subject in accordance with this disclosure, androgenic agents raise serum concentrations to 0.2 ng / ml or higher, usually 0.25 ng / ml, 0.3 ng / ml, 0.35 ng / ml, 0.4 ng / ml, 0.45 ng / ml, 0.5 ng / ml, 0.55 ng / ml, 0.60 ng / ml, 0.65 ng / ml, 0.70 ng / ml, 0.75 ng / ml, 0.8 ng / ml, 0.85 ng / ml, and 0.9 ng / ml. The concentration is increased to g / ml, 0.95 ng / ml, 1 ng / ml, 1.25 ng / ml, 1.5 ng / ml, 1.75 ng / ml, 2 ng / ml, 2.25 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 3.75 ng / ml, 4 ng / ml, 4.25 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, or 7 ng / ml or higher. In most cases, the serum concentration of the androgenic agent is increased from approximately 0.2 ng / ml, 0.25 ng / ml, or 0.5 ng / ml or higher to approximately 7 ng / ml, 6.5 ng / ml, 6 ng / ml, 5.75 ng / ml, or 5.5 ng / ml or lower, explicitly encompassing all serum concentrations of the androgenic agent within the specified ranges above. Typically, serum concentrations of androgenic agents are increased from approximately 4 ng / ml to approximately 6 ng / ml, and then typically to approximately 5 ng / ml.

[0069] In embodiments, androgenic agents are administered in doses that increase the intramammary androgen concentration resulting from the action of 5α-reductase in the breast to a level of 1 nM or higher, in order to obtain inhibition of c-Myc and / or mTOR activity, which can generally be achieved through induction or activation of AR-mediated inhibitory activity in the breast. Generally, administered androgenic agents increase serum concentrations of the androgenic agent, causing the breast concentration of 5α-reductive androgen to exceed 1 nM. Most commonly, breast androgen concentrations are approximately 1.2 nM, 1.25 nM, 1.3 nM, 1.35 nM, 1.4 nM, 1.45 nM, 1.5 nM, 1.55 nM, 1.6 nM, 1.65 nM, 1.7 nM, 1.75 nM, 1.8 nM, 1.85 nM, 1.9 nM, 1.95 nM, 2 nM, 2.25 nM, 2.5 nM, 3 nM, and 3. The androgen concentrations in the breast defined above are 5nM, 4nM, 4.5nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, or 14nM, or higher, preferably in the range of 1nM or more to about 12nM, more typically in the range of 1nM to about 10nM, most preferably in the range of about 1.5nM, 1.75nM, or 2nM, or higher to about 10nM, and all of the above defined intramammary androgen concentrations are expressly provided herein.

[0070] The androgenic agent is typically testosterone or its physiologically active form, and therefore, the 5α-reductase androgenic agent in the breast is typically DHT or its physiologically active form. Most typically, the androgenic agent is testosterone, and the androgen obtained by the action of 5α-reductase in the breast is DHT.

[0071] If the androgenic agent is other than testosterone, the androgenic agent may be administered to the subject in a dose that increases the intramammary concentration of the 5α-reduced form of the androgenic agent in the breast. The increase corresponds to at least a lower intramammary concentration of DHT necessary to obtain the androgenic effect resulting in the suppression of c-Myc and / or mTOR activity described herein, and may be evaluated by an appropriate assay.

[0072] Androgenic agents are typically administered to subjects in doses dependent on the mode of administration, selected to be sufficient to increase and maintain serum concentrations of the androgenic agent within a range that increases intramammary androgen concentrations resulting from the action of 5α-reductase on the androgenic agent in the breast. The intramammary concentration is above the level required to induce both the inhibition of c-Myc and / or mTOR activity as described herein for a predetermined period, which is at least one week, more commonly at least two or three weeks, and preferably at least one, two, or three months. In a particularly preferred embodiment, the increase in serum concentration of the androgenic agent is maintained for a period of about three months depending on the dose of the androgenic agent. As understood, by maintaining elevated serum concentrations of the administered androgenic agent over a predetermined period in accordance with this disclosure, the intramammary androgen concentration required to exert a therapeutic effect is also maintained over a predetermined period above the level required to induce the inhibition of c-Myc and / or mTOR.

[0073] The dosage of the selected aromatase inhibitor is similarly chosen to be sufficient to block the aromatization of androgenic agents in the breast, thereby inhibiting the production of estrogen, particularly estradiol, in the breast tissue.

[0074] Continuous treatment for the subject can be achieved by repeatedly administering androgenic agents and aromatase inhibitors at regular intervals (e.g., every three months) corresponding to the end of a predetermined period. This repeated administration is performed, as described herein, to maintain, or within the required range, an increase in serum levels of androgenic agents and an increase in intramammary androgen concentration derived from 5α-reductase activity in the breast over a predetermined period.

[0075] The combination therapy of androgenic agents and aromatases of this disclosure can be administered to a subject by any suitable method of application, such as subcutaneous implantation, transdermal delivery, or injection (e.g., subcutaneous injection, intramuscular injection, and intravenous injection), typically for continuous or sustained delivery of the androgenic agent and aromatase.

[0076] Furthermore, androgenic agents and aromatase inhibitors may be administered simultaneously or sequentially in the same or different pharmaceutical or veterinary compositions to exert their effects in the same or overlapping therapeutic range. Pharmaceutical or veterinary compositions useful herein may include one or more binders, fillers, preservatives, and pharmaceutically acceptable or physiologically acceptable carriers and excipients conventionally used in pharmaceutical or veterinary formulations and preparations. The terms “pharmaceutically acceptable” or “physiologically acceptable” herein are understood to mean compounds, agonists, substances, compositions, excipients, carriers and / or dosage forms suitable, within the bounds of medically reasonable judgment, for contact with human or animal tissue without causing excessive toxicity, irritation, allergic reactions or other problems or complications. Such suitability is determined to the extent that it is commensurate with a reasonable benefit and / or risk ratio.

[0077] Typically, combination therapies are administered percutaneously, such as by subcutaneous implants (e.g., subcutaneous pellets) or percutaneous patches, most commonly by subcutaneous pellets inserted into the lower abdomen, upper buttocks, or other locations deemed appropriate. Subcutaneous pellets useful for administering the androgenic agents and aromatases of the combination therapies of the present invention are described, for example, in international patent applications WO2016 / 061615 and WO2020 / 243777, the contents of both of which are incorporated herein in their entirety. In particular, the androgenic agents and aromatase inhibitors are preferably administered in the form of multiphase compositions described in WO2020 / 243777, and the use of all such compositions, dosage forms, implants, and pellets described therein is expressly encompassed.

[0078] In a particularly preferred embodiment, the combination therapy is administered to the subject by subcutaneous pellets containing about 40 mg to about 120 mg of testosterone and 2 mg to about 6 mg of anastrozole. Typically, the pellets contain about 60 mg to about 100 mg, most preferably about 80 mg, of testosterone or about 100 mg of testosterone, and about 3 mg to about 5 mg, most preferably about 4 mg, of anastrozole.

[0079] In the methods described herein, when letrozole or borozole is used as an aromatase inhibitor, the dosage used in the pellet is typically in the range of 8 mg to 16 mg.

[0080] To ensure continuous treatment for the subjects, pellets will be administered every three months, as long as deemed appropriate.

[0081] The methods described herein can maintain low serum levels of administered androgenic agents without substantially increasing serum concentrations of androgens derived from 5α-reductase activity in the breast. Therefore, subjects can be treated for several months to several years (e.g., 1, 2, or 3 years or more) as described herein, without substantially causing masculinization of the subjects, and without avoiding the use of high doses of testosterone and aromatase enzymes conventionally administered in breast cancer treatment.

[0082] As described herein, by inducing long-term suppression of c-Myc and / or mTOR activity, the progression of abnormal mammary cell pathologies to breast cancer, pre-invasive breast cancer, malignant breast cancer, hormone-resistant breast cancer, or, for example, the progression of HER2-positive breast cancer to cancer unresponsive to HER2-targeted drug therapy despite HER2 blockade, may be delayed, blocked, or the risk of such progression may be reduced. Hormone therapies for breast cancer that respond to such therapies include treatment with androgens, selective estrogen receptor modulators (SERMS), aromatase inhibitors, estrogen blockers, and anti-estrogen drugs that target the estrogen receptor (ER) to block or downregulate ER signaling. SERMs are a class of drugs that selectively interact with the ER, including tamoxifen, raloxifene, toremifene, clomiphene, bazedoxifene, and rasofoxifene. Selective estrogen receptor degraders (SERDs) are other drugs that target the estrogen receptor (ER) by inducing its degradation and thereby more potently and sustainably inhibiting ER signaling. Examples of SERDs that can be used include fulvestrant, GDC-0810 (buparlisib), GDC-0927, AZD9496, elastrant (RAD1901), and LSZ102.

[0083] Therapies used in targeted therapy for HER2-positive cancers include monoclonal antibodies such as trastuzumab, pertuzumab, and margetuximab-cmkb, antibody-drug conjugates, pan-HER inhibitors such as neratinib, tyrosine kinase inhibitors such as tucatinib, and signal transduction inhibitors such as lapatinib.

[0084] The subjects may be any mammal treatable according to the methods disclosed herein, and may be selected from, for example, rodents, felines, canines, equids, pigs, goats, sheep, rabbits, and primates, including domesticated animals such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, horses, goats, and sheep, as well as monkeys, baboons, and apes. The subjects are women, most typically humans. The subjects may be, for example, premenopausal, perimenopausal, or postmenopausal women, but any woman deemed a suitable candidate may be treated by the methods described herein. "Perimenopausal" refers to the period in which a woman is in the transition towards menopause and may be aware of signs of progression, such as irregular menstruation. During perimenopausal, estrogen levels may fluctuate erratically, menstrual cycles may become longer or shorter, and menstrual cycles without ovulation may begin. Additionally, symptoms similar to those of menopause may appear, such as hot flashes, sleep disturbances, and mood swings. A "postmenopausal" woman refers to a woman who has gone through menopause naturally, a woman who has had her ovaries removed, or a woman whose estrogen production is permanently suppressed and who has not had a menstrual period for 12 months.

[0085] The methods used in accordance with this disclosure are further described below by non-limiting examples. [Examples]

[0086] 1. Effects of testosterone and aromatase inhibitors 1.2 Method A single-dose, single-center, open-label, non-randomized trial investigating the efficacy of combination therapy with testosterone and an aromatase inhibitor in premenopausal women was conducted at Wellend Health Clinic (Nyroca House, North Adelaide, SA, Australia).

[0087] Premenopausal women are typically between 35 and 55 years old, with a BMI of 20-30 kg / m². 2 The range was as follows: A T+Ai® pellet implant containing 80 mg of testosterone (T) and 4 mg of the aromatase inhibitor (Ai) anastrozole was subcutaneously inserted into the lower abdomen or upper buttocks between 8:00 AM and 10:00 AM on the day of administration, i.e., day 1. The implant was intended to remain in place and was expected to be almost completely absorbed within 3 months after insertion.

[0088] A total of 11 subjects received the implant and were included in the safety analysis population. All 11 subjects were included in the pharmacokinetic (PK) analysis population.

[0089] Serum concentrations of testosterone, dihydrotestosterone (DHT), and anastrozole were measured on day 1 before administration (predose), and at 1, 2, 4, 6, 8, and 12 hours after administration. Further measurements were taken on days 2, 3, 4, 5, 8, 15, 22, 29, 43, 57, 71, and 85 using single samples. (Note: The 6-hour post-administration sample collection point was added for the third and subsequent subjects; therefore, serum measurements were not performed for the first two subjects, as shown below.) Patients who desired treatment with T+Ai to alleviate high MBD were included in this study. Those who passed screening visited the Wellend Health Clinic for administration. This study was designed to administer the drug to all subjects during the luteal phase of the menstrual cycle to avoid significant changes in the hormonal environment during the follicular phase. PK blood collection was performed for 3 months after implant insertion (see below). Briefly, samples were repeatedly collected on the day of administration, daily for the first week, weekly for the following 4 weeks, and every 2 weeks for the last 8 weeks, for a total sampling period of 3 months. After all evaluations were completed on day 85, the study concluded, and subjects continued testosterone and aromatase inhibitor therapy, if desired, according to agreed clinical practice.

[0090] To determine whether the selected PK sampling schedule adequately described the serum / plasma concentration-time profiles of testosterone and aromatase inhibitor therapy, blood samples were collected from the first two participants on day 29, after which the samples were analyzed for testosterone, dihydrotestosterone, and anastrozole. The sampling schedule was revised by adding one blood collection point (6 hours post-administration, as described above) without changing the timing of existing collection points. The sampling period was not extended.

[0091] Blood samples were taken on day 1, before administration (within 1 hour after administration), and at 1, 2, 4, 6, 8, and 12 hours. Participants came to the hospital for blood sampling at 9:00 AM on days 2, 3, 4, 5, 8, 15, 22, 29, 43, 57, 71, and 85.

[0092] 1.3 Results Median T value of anastrozole in plasma max The average C level was 2 days (range 1-3 days) after administration. max The concentration was 5.30 ng / mL, and the mean terminal half-life was 14 days. After 12 weeks, the concentration was below the limit of quantification (0.1 ng / mL) in most participants.

[0093] Regarding serum testosterone, the median T value was reached 8 hours after administration (range 6-12 hours). max This was observed, and the average C max The average serum testosterone level was 4.1 ng / mL. In contrast, the mean serum testosterone level at baseline was 0.2 ng / mL. The observed increase in serum testosterone compared to baseline persisted in all subjects after 12 weeks, with a mean serum concentration of 0.8 ng / mL.

[0094] Serum dihydrotestosterone (DHT) was unmeasurable in most samples, and in participants who showed the highest measurable value, T max These were observed 2-3 days after administration. Serum concentrations of anastrozole, testosterone, and DHT are shown in Tables 1-3 below.

[0095] [Table 1] [Table 2] [Table 3-1]

[0096] Serum estradiol levels did not decrease in the testosterone and anastrozole treatment groups. This is consistent with the low concentration of anastrozole used in this study, which is equivalent to one-fifth of the concentration achieved with conventional total oral administration.

[0097] 2. Effects of DHT on breast cancer tissue To evaluate the effects of DHT on breast cancer tissue, we modified the method described in Jankovic-Karasoulos T. et al., 2008, and examined three primary tumors in the presence of a low concentration of anastrozole (5 nM).

[0098] In short, the tumor tissue is 3 mm 2 The tissue was cut into sections and arranged on gelatin sponges as three replicates under identical conditions. These sponges were immersed in a medium supplemented with 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FCS) and contained either vehicle (control) or 0.1, 1, or 10 nM (nmol / l) of DHT. These DHT concentrations were set to replicate DHT levels in mammary tissue that correlated with increased serum testosterone levels.

[0099] In three excised invasive breast cancers, mTOR and c-Myc expression and response to DHT exposure were investigated by Western blotting. Whole cell lysates from tumor tissue and control adipose tissue were sonicated at 48°C and prepared using lysis buffer (1% Triton X-100, 50 mM KCl, 25 mM HEPES [pH 7.8], 10 mg / ml leupeptin, 20 mg / ml aprotinin, 125 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). These were then analyzed on the same Western blot. A 50 mg total protein sample was mixed with 50 ml of sodium lauryl sulfate (SDS)-mercaptoethanol sample buffer and boiled for 10 minutes. The proteins were then separated on a 7.5% SDS gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked at room temperature for 1 hour with a solution of phosphate-buffered saline (PBS) containing 0.5% Tween-20 and 5% skim milk powder. Immunoblotting was then performed using primary antibodies (anti-human mTOR or MYC antibodies diluted in PBS) and secondary antibodies (horseradish peroxidase (HRP) labeled in PBS, diluted in PBS, from Novus Biologicals LLC, CO, USA). Detection was further performed using a chemiluminescent reagent (Amersham Bioscience, Buckinghamshire, England). Band density was measured by densitometry and analyzed using Image Master VDS and ImageQuant analysis software (Amersham Pharmacia Biotech, Hong Kong). The relative protein amounts of mTOR or MYC and β-actin in the original total protein lysates obtained from breast tissue preparations were calculated. The protein expression levels of mTOR or MYC were normalized relative to the β-actin expression level. Immune host antibodies were prepared using synthetic peptides derived from human mTOR or MYC sequences, purified by peptide affinity chromatography, and confirmed using control peptides. The results of Western blot analysis of mTOR or MYC proteins in three excised samples at baseline and 24 hours after culture are shown in Tables 3 and 4 for mTOR and MYC, respectively.Cell lysates were immunoblotted with antibodies against mTOR or MYC. The experiment was performed twice, and similar results were obtained. Data were expressed as relative values ​​to β-actin.

[0100] [Table 3-2] [Table 4]

[0101] Recently, it was shown that androgen receptors (ARs) can act as tumor suppressors in estrogen receptor (ER)-positive breast cancer when intratumoral DHT concentrations range from 1 nM to an assessed 10 nM (Hickey TE. et al., 2021). This study, using a diverse and clinically relevant panel of cell lines and patient-derived models, demonstrated that AR activation, rather than suppression, exerts potent antitumor effects in multiple disease states, including resistance to standard treatments such as ER inhibitors and CDK4 / 6 inhibitors. In particular, the combination of AR agonists with standard therapies was found to improve the treatment response. Mechanism of action, AR agonist activation alters the genomic distribution of estrogen receptors (ERs) and essential co-activators (p300, SRC-3), resulting in suppression of ER-regulated cell cycle-related gene expression while upregulating AR target genes, including known tumor suppressors.

[0102] In this study, mTOR and Myc levels were significantly elevated in the presence of 0.1 nM DHT, only slightly detected in breast cancer excised tissue treated with 1 nM DHT, and essentially undetectable in tissue treated with 10 nM DHT.

[0103] 3. Discussion The results of this study demonstrate that a unique combination of moderate-dose testosterone and low-dose aromatase inhibitor can induce dihydrotestosterone concentrations in both the breast and tumors. These concentrations avoid or minimize the induction of the mTOR pathway associated with 0.1 nM DHT treatment, which is known to be linked to the promotion of malignant tumor growth and dedifferentiation. In relation to this, the results further showed that c-Myc expression was suppressed when tumor dihydrotestosterone concentrations were in the range of 1–10 nM. c-Myc is a major promoter of cancer progression and is known to be insensitive not only to classical hormone therapy but also to therapies not classified as hormone-based breast cancer therapy, such as the HER2 pathway and the nitric oxide pathway. In contrast, 0.1 nM DHT did not suppress c-Myc.

[0104] Increased DHT levels in the breast downregulate aromatase in breast tissue (Perel E. et al.) and upregulate 5α-reductase (McNamara KM. et al., 2014), resulting in anti-estrogenic effects. However, our results indicate that this only occurs when the breast concentration of DHT is sufficient to induce c-Myc suppression without inducing mTOR. While not limited to theory, the inventors believe this effect is due to AR dimerization and transcriptional activation occurring at the evaluated high concentrations of DHT (i.e., 1–10 nM), while the activation of c-Myc and mTOR activity is associated with the expression of AR in monomeric form under low DHT conditions of 0.1 nM.

[0105] As a result of the combination therapy described herein (either alone or in combination with other breast cancer drugs), a differentiated or latent malignant phenotype that is responsive to malignant tumors persists throughout the disease spectrum, from the onset of cancer to the accelerated and advanced stages of breast cancer. This reduces the risk of progression to hormone-resistant cancer or cancer that is unresponsive to targeted drug therapy, as described herein.

[0106] While intramammary and intratumoral testosterone concentrations are equilibrium with serum concentrations, previous studies have shown that DHT concentrations in tumor tissue are at least three times higher than those observed in serum due to 5α-reductase induction (Recchione C. et al, 1995). Our results suggest that at least 1 nM of DHT is required to suppress cMyc and inhibit the induction of mTOR activity via non-genomic interactions. Furthermore, intratumoral DHT concentrations in women in this pharmacokinetic study are expected to reach a peak of 14 nM in a relatively short timeframe without increasing serum DHT concentrations, thus achieving intramammary androgenesis while avoiding masculinization, as reported by Raths F. et al. (2023).

[0107] The method disclosed herein enables continuous treatment of subjects by regularly administering the combination therapy of the androgenic agent and aromatase inhibitor described herein via subcutaneous pellets, typically at 3-month intervals. Even 3 months after administration, when the subject's testosterone concentration reaches its lowest point, the intramammary DHT concentration remains at approximately 2 nM or higher, and the suppression of c-Myc expression and mTOR activity associated with low DHT concentrations persists as described above. Therefore, by repeatedly administering the combination via subcutaneous implants described herein, the intramammary and tumor testosterone and DHT concentrations of subjects are maintained within a therapeutic range suitable for the prevention or treatment of pre-malignant or malignant breast tissue pathologies and for inhibiting progression to hormone-resistant breast cancer or breast cancer unresponsive to targeted drug therapy. Moreover, this maintenance is achieved without substantially increasing serum DHT concentrations in the systemic circulation.

[0108] The embodiments described herein can be modified in various ways without departing from the scope of this disclosure, and the above examples are for illustrative purposes only and are not limiting.

[0109] References Deshpander,N.,P.Carson,I.Mitchell and B.Berry,(1977).The accumulation of 5alpha-dihydrotesterone(5alpha-DHT)by human breast tissue.J Steroid Biochem,8(1):105-106. Elshazly,A.M.and D.A.Gewirtz,(2022).An overview of resistance to Human epidermal growth factor receptor 2(Her2)targeted therapies in breast cancer.Cancer Drug Resist 5(2):472-486. Glaser R and Dimitrakakis C,(2021).Testosterone Implant Therapy in Women With and Without Breast Cancer:Rationale,Experience,Evidence.Androgens:Clinical Research and Therapeutics,Vol.2.1,pp.94-110. Han W et al.,(2022).Exploiting the tumor-suppresive activity of the androgen receptor by CDK4 / 6 inhibition in castration-resistant prostate cancer.Molecular Therapy Vol.30,No 4,April,pp.1628-1644. Hickey,T.E.,L.A.Selth,K.M.Chia,G.Laven-Law,H.H.Milioli,D.Roden,S.Jindal,M.Hui,J.Finlay-Schultz,E.Ebrahimie,S.N.Birrell,S.Stelloo,R.Iggo,S.Alexandrou,C.E.Caldon,T.M.Abdel-Fatah,I.O.Ellis,W.Zwart,C.Palmieri,C.A.Sartorius,A.Swarbrick,E.Lim,J.S.Carroll and W.D.Tilley,(2021).The androgen receptor is a tumor suppressor in estrogen receptor-positive breast cancer.Nat Med 27(2):310-320. Hong,R.,Cao,B.,Chen,D.,Wu,W.,Luo,T.,Lv,D.,Zhang,W.,Wang,S.and Shao,K(2024).Multi-omics portrait of ductal cell carcinoma in situ in young women.Breast Cancer Res and Treatment.Springer.Published on-line 5 May 2024.doi.org / 10.1007 / s10549-024-07254-5. Hosseinzadeh,L.,Z.Kikhtyak,G.Laven-Law,S.M.Pederson,C.G.Puiu,C.S.D’Santos,E.Lim,J.S.Carroll,W.D.Tilley,A.R.Dwyer and T.E.Hickey(2024).The androgen receptor interacts with GATA3 to transcriptionalluy regulate a luminal epithelial cell phenotype in breast cancer.Genome Biol 25(1):44,pp 1-28. Jankovic-Karasoulos,T.,S.Birrell,E.Cops,S.Jindal,A.Ochnik,M.Thomas,W.Tilley and T.Hickey,(2009).Evaluation of Testosterone Supplementation during Anastrozole Therapy in a Breast Explant Model.Cancer Research 69(24_Supplement):4089-4089. Ji W et al.,(2020).c-myc regulates the sensitivity of breast cancer cells to palbociclib via c-myc / miR-29b-3p / CDK6 axis.Cell Death and Disease.,11:760,pp 1-13. Li,Y.,Y.Guo,F.Chen,Y.Cui,X.Chen and G.Shi,(2024).Male breast cancer differs from female breast cancer in molecular features that affect prognoses and drug responses.Transl Oncol 45:101980,pp.1-18. McNamara,K.M.,T.Yoda,A.M.Nurani,Y.Shibahara,Y.Miki,L.Wang,Y.Nakamura,K.Suzuki,Y.Yang,E.Abe,H.Hirakawa,T.Suzuki,N.Nemoto,M.Miyashita,K.Tamaki,T.Ishida,K.A.Brown,N.Ohuchi and H.Sasano,(2014).Androgenic pathways in the progression of triple-negative breast carcinoma:a comparison between aggressive and non-aggressive subtypes.Breast Cancer Res Treat 145(2):281-293. Perel,E.,K.H.Stolee,L.Kharlip,M.E.Blackstein and D.W.Killinger,(1984).The intracellular control of aromatase activity by 5 alpha-reduced androgens in human breast carcinoma cells in culture.J Clin Endocrinol Metab 58(3):467-472. Raths,F.,M.Karimzadeh,N.Ing,A.Martinez,Y.Yang,Y.Qu,T.Y.Lee,B.Mulligan,S.Devkota,W.T.Tilley,T.E.Hickey,B.Wang,A.E.Giuliano,S.Bose,H.Goodarzi,E.C.Ray,X.Cui and S.R.V.Knott,(2023).The molecular consequences of androgen activity in the human breast.Cell Genom 3(3):100272. Recchione,C.,E.Venturelli,A.Manzari,A.Cavalleri,A.Martinetti and G.Secreto,(1995).Testosterone,dihydrotestosterone and oestradiol levels in postmenopausal breast cancer tissues.J Steroid Biochem Mol Biol 52(6):541-546. Qiu,X et al.,(2022).MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets.Nature Communications,13:2559,pp.1-17.

Claims

1. A method for the prevention or treatment of abnormal mammary cell pathology characterized by cell proliferation in a subject, This includes administering an effective amount of an androgenic agent and an effective amount of an aromatase inhibitor in combination to the subject to increase the serum concentration of the androgenic agent to a level that increases the concentration of breast androgens due to the action of 5α-reductase on the androgenic agent, The increased intramammary androgen concentration induces suppression of intramammary c-Myc and / or mTOR activity associated with lower intramammary androgen concentrations. A method wherein the increase in the serum concentration of the androgenic agent and the increase in the breast androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

2. The method according to claim 1, wherein the aromatase inhibitor blocks the aromatization of the androgenic agent to estrogen.

3. The method according to claim 1 or 2, wherein the estrogen concentration in the breast does not substantially increase during the predetermined period.

4. The method according to claim 3, wherein the concentration of estrodiol in the breast does not substantially increase during the predetermined period.

5. The method according to any one of claims 1 to 4, wherein the androgen is 5α-dihydrotestosterone (DHT) or a physiologically active form of DHT.

6. The method according to any one of claims 1 to 5, wherein the intramammary androgen concentration is 1 nM, which is lower than the above-mentioned concentration.

7. The method according to any one of claims 1 to 6, wherein the intramammary androgen concentration rises to a level in the range of more than 1 nM to about 12 nM.

8. The method according to claim 7, wherein the intramammary androgen concentration rises to a level in the range of about 2 nM to about 10 nM.

9. The method according to any one of claims 1 to 8, wherein the androgenic agent is selected from testosterone and physiologically active forms of testosterone.

10. The method according to any one of claims 1 to 9, wherein the androgenic agent is testosterone.

11. The method according to any one of claims 1 to 10, wherein the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole, fadrozole, volozole, exemestane, and formestane.

12. The method according to any one of claims 1 to 11, wherein the androgen is DHT.

13. The method according to any one of claims 1 to 12, wherein the androgenic agent is testosterone and the aromatase inhibitor is anastrozole.

14. The method according to any one of claims 1 to 13, wherein the androgenic agent is testosterone, and the increased concentration of testosterone in the serum of the subject is at least 1 ng / ml.

15. The method according to claim 14, wherein the increased level of testosterone in the subject is in the range of about 1 ng / ml to about 5 ng / ml.

16. The method according to any one of claims 1 to 15, wherein the androgenic agent and the aromatase inhibitor are administered to the subject by subcutaneous, intramuscular, and / or transdermal application in order to continuously deliver the androgenic agent and the aromatase inhibitor to the subject.

17. The method according to any one of claims 1 to 16, wherein the androgenic agent and the aromatase inhibitor are administered to the subject by a subcutaneous implant.

18. The method according to any one of claims 1 to 17, wherein the increase in the serum concentration of the androgenic agent is maintained for the predetermined period of at least one week.

19. The method according to claim 18, wherein the predetermined period is at least one month.

20. The method according to claim 19, wherein the predetermined period is three months.

21. The method according to any one of claims 1 to 20, wherein the inhibition of c-Myc activity and / or mTOR activity includes substantially complete inhibition of c-Myc activity and / or mTOR activity.

22. The method according to any one of claims 1 to 21, wherein the androgen receptor (AR) is expressed in the breast at least mainly as an AR monomer when the breast androgen concentration is below the lower than stated above, and is expressed at least mainly as an AR dimer when the breast androgen concentration exceeds the lower than stated above.

23. The method according to any one of claims 1 to 22, wherein the abnormal breast cell pathology is breast cancer.

24. The method according to claim 23, wherein the breast cancer is pre-invasive breast cancer or malignant breast cancer.

25. The method according to claim 24, wherein the breast cancer is malignant breast cancer.

26. The method according to claim 24, wherein the breast cancer is ductal carcinoma in situ or lobular cell carcinoma in situ.

27. The method according to any one of claims 1 to 22, wherein the abnormal mammary cell pathology is mammary cell hyperplasia.

28. The method according to any one of claims 1 to 27, wherein the mammary cell hyperplasia is lobular cell hyperplasia.

29. The method according to any one of claims 1 to 28, wherein the risk of the abnormal breast cell pathology progressing to hormone-resistant breast cancer or breast cancer that is not sensitive to HER2-targeted anticancer drug therapy is reduced.

30. A method for reducing the risk of breast cancer progressing to hormone-resistant breast cancer in the target population, This includes administering an effective amount of an androgenic agent and an effective amount of an aromatase inhibitor in combination to the subject to increase the serum concentration of the androgenic agent to a level that increases the concentration of the breast androgen due to the action of 5α-reductase on the androgenic agent, The increased intramammary androgen concentration induces suppression of intramammary c-Myc and / or mTOR activity associated with lower intramammary androgen concentrations. A method wherein the increase in the serum concentration of the androgenic agent and the increase in the breast androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

31. A method to reduce the risk of HER2-positive breast cancer in the target population becoming unresponsive to HER2-targeted anticancer drug therapy, This includes administering an effective amount of an androgenic agent and an effective amount of an aromatase inhibitor in combination to the subject to increase the serum concentration of the androgenic agent to a level that increases the concentration of the breast androgen due to the action of 5α-reductase on the androgenic agent, The increased intramammary androgen concentration induces suppression of intramammary c-Myc and / or mTOR activity associated with lower intramammary androgen concentrations. A method wherein the increase in the serum concentration of the androgenic agent and the increase in the breast androgen concentration are maintained for a predetermined period without substantially increasing the androgen concentration in the serum of the subject.

32. A method for the prevention or treatment of abnormal mammary cell pathology characterized by cell proliferation in a subject, The subject is administered a combination of an effective amount of androgenic agent and an effective amount of aromatase inhibitor. This includes increasing the serum concentration of the androgenic agent to a level that raises the intramammary concentration of 5-dihydrotestosterone (DHT) or a physiologically active form of DHT to more than 1.0 nM for a predetermined period of time. The increase in the concentration of DHT or its physiologically active form results from the action of 5α-reductase activity on the androgenic agent, A method wherein the increase in the serum concentration of the androgenic agent and the increase in the intramammary concentration of DHT or its physiological form are maintained for a predetermined period, and the concentration of DHT or its physiologically active form in the serum of the subject does not substantially increase over the predetermined period.

33. A method for the prevention or treatment of abnormal mammary cell pathology characterized by cell proliferation in a subject, The subject is administered a combination of an effective amount of androgenic agent and an effective amount of aromatase inhibitor. A method comprising increasing the intramammary concentration of dihydrotestosterone (DHT) or a physiologically active form of DHT to more than 1.0 nM for a predetermined period, wherein the increase in the concentration of DHT or its physiologically active form is due to the action of 5α-reductase activity on the androgenic agent, and the concentration of DHT or its physiologically active form in the serum of the subject does not substantially increase over the predetermined period.

34. A method for inducing suppression of c-Myc and / or mTOR activity in the breast of a target, The subject is administered a combination of an effective amount of androgenic agent and an effective amount of aromatase inhibitor. A method comprising increasing the serum concentration of the androgenic agent, thereby increasing the intramammary androgen concentration, inducing the suppression of c-Myc and / or mTOR activity in the breast associated with lower intramammary androgen concentrations, wherein the increase in the androgen concentration is attributable to the action of 5α-reductase on the androgenic agent.

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