SGLT2 antagonists to delay ovarian aging

Abstract

Provided herein are compositions and methods for reducing or preventing ovarian aging comprising: administering to a subject in need of a reduction or prevention of ovarian aging an effective amount of an SGLT2 antagonist. Examples of SGLT2 antagonists include canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof.

Description

SGLT2 ANTAGONISTS TO DELAY OVARIAN AGINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a PCT International patent application of and claims priority to U.S. provisional patent application serial number 63 / 680,775 filed on August 8, 2024, the contents of which are incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present invention relates in general to the field of preventing ovarian aging, and more particularly, to the use of SGLT2 antagonists such as canagliflozin to delay ovarian aging.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under R01 AG069742 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] Not applicable.BACKGROUND

[0005] Without limiting the scope of the invention, its background is described in connection with ovarian aging.

[0006] Aging in females in closely associated with ovarian function decline. Ovarian aging leads not only to reduced fertility but also systemic healthspan consequences [1, 2], For instance, early menopause is associated with a shorter lifespan and an increased risk of diseases in women [3, 4], A hallmark of ovarian aging is the decline observed in the numbers of follicles, which ultimately leads to exhaustion of the ovarian reserve and consequent menopause / estropause. Besides the loss of follicles, aged ovaries show collagen accumulation and a pro-fibrotic profile [5-8] combined with increased pro-inflammatory signaling and accumulation of immune cells [5, 8-11], Redox homeostasis also decreases in the aged ovary, which contributes to oocyte DNA damage, telomere shortening, and mitochondrial dysfunction, further drivers of ovarian functional declines and infertility [12-14],

[0007] Despite these advances, a need remains for novel therapeutic interventions that will slow, reduce, or prevent ovarian aging.SUMMARY

[0008] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for treatment, amelioration, or prophylaxis of conditions related to ovarian aging comprising: administering to a subject in need of treatment, amelioration, or prophylaxis of conditions related to ovarian aging an effective amount of a sodium-glucose co-transporter 2 (SGLT2) antagonist. In one aspect, the SGLT2 antagonist is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution. In another aspect, a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose. In another aspect, the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, or phlorizin. In another aspect, the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours. In one aspect, ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or blood hormone analyses. In one aspect, the blood hormone analyses is selected from at least one of: anti- mullerian hormone (AMH), Follicle-stimulating hormone (FSH), and 17b-estradiol. In one aspect, the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration. In one aspect, the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL 12 signaling, or both. In one aspect, the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4. In one aspect, the e canagliflozin exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function.

[0009] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for slowing or preventing ovarian aging comprising: administering to a subject in need of slowing or prevention of ovarian aging an effective amount of a sodium-glucose co-transporter 2 (SGLT2) antagonist, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose. In one aspect, the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, or phlorizin. In another aspect, the SGLT2 antagonist is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution. In another aspect, the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours. In another aspect, ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or bloodhormone analyses. In another aspect, the blood hormone analyses is selected from at least one of: anti -Mullerian hormone (AMH), Follicle-stimulating hormone (FSH), and 17b-estradiol. In one aspect, the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration. In another aspect, the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL 12 signaling, or both. In another aspect, the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4. In one aspect, the SGLT2 antagonist exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function. In one aspect, the subject is a human.

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a method to slow a rate of ovarian aging comprising: administering to a subject in need of slowing ovarian aging a composition comprising an effective amount of SGLT2 antagonist in a pharmaceutically acceptable carrier, wherein a dose of the canagliflozin is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose. In another aspect, the SGLT2 antagonist is canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof. In another aspect, the SGLT2 antagonist is canagliflozin.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which:

[0012] FIGS. 1 A to 1H show: (FIG. 1 A) Fasting plasma glucose levels; (FIG. IB) fasting plasma insulin levels; (FIG. 1C) plasma IGF1 levels; (FIG. ID) estimated number of primordial, transition / primary; secondary and tertiary follicles (FIG. IE) total estimated number of follicles; (FIG. IF) primary: primordial follicles ratio; (FIG. 1G) representative images of HE stained ovarian sections; (FIG. 1H) estimated number of zona pellucida remnants (ZPR) from 12 months- old control mice (n=6) and 12 months-old mice treated with canagliflozin (CANA) for 6 months (n=6). Data is presented as mean ± SEM. *<0.05, **<0.01, ***<0.001 by one-way t test or oneway ANOVA followed by Tukey post-hoc test.

[0013] FIGS. 2A to 2H show: (FIG. 2A) Fluorescence intensity of phosphorylated Forkhead box O3a protein (pFoxO3a) in primordial follicles; (FIG. 2B) representative images of pFoxO3a and DDX4 fluorescence in primordial follicles; (FIG. 2C) quantification of ovarian collagen accumulation by picrosirius-red (PSR) staining; (FIG. 2D) representative images of PSR stainedovarian sections; (FIG. 2E) quantification of ovarian of Sudan Black staining; (FIG. 2F) representative images of Sudan Black stained ovarian sections from 6 months-old control mice (n=5-6), 12 months-old control mice (n=5-6) and 12 months-old mice treated with canagliflozin for 6 months (n=5-6). Data is presented as mean ± SEM. *<0.05, **<0.01, ***<0.001 by one-way ANOVA followed by Tukey post-hoc test. Yellow arrows are pointing to Sudan Black positive areas. White scale bar represents 10 pm and black scale bar represents 500pm. FIG. 2G shows representative CD3 (green) and DAPI (blue) immunostained images (magnification = lOx; scale bar = 200 pm); and FIG. 2H quantified immunofluorescent intensity of CD3 (n = 5-6 / group) in 6-month-old control, 12-month-old control, and 12-month-old CANA-treated mice. All data are presented as mean + / - SEM and were analyzed by one-way ANOVA with Tukey post hoc analyses. *p < 0.05; **p < 0.01; ***p < 0.001.

[0014] FIGS. 3 A to 3D show: (FIG. 3 A) Volcano plot showing up and downregulated transcripts; (FIG. 3B) principal components analysis (PCA) of transcriptomic data; (FIG. 3C) IPA canonical pathway analysis, (FIG. 3D) IPA upstream regulator analysis showing inhibition or activation of regulators associated to mitochondrial activity and cell proliferation from ovarian RNAseq from 12 months-old control mice (n=6) and 12 months-old mice treated with canagliflozin for 6 months (n=6). Statistical analysis was performed by Z-test. P-values <0.05 and log2 change >1 and <-l were considered significant.

[0015] FIGS. 4A to 4D show: (FIG. 4A) Volcano plot showing up and downregulated protein reads; (FIG. 4B) principal components analysis (PCA) of proteomic data; (FIG. 4C) IPA canonical pathway analysis, (FIG. 4D) IPA upstream regulator analysis showing inhibition or activation of regulators associated to cell proliferation, metabolism, endoplasmic reticulum (ER) stress and inflammation / immune response from ovarian proteomic analysis by data-independent acquisition (DIA) from 12 months-old control mice (n=7, each sample is a pool of ovaries from 2 mice) and 12 months-old mice treated with canagliflozin for 6 months (n=6, each sample is a pool of ovaries from 2 mice). Statistical analysis was performed by Z-test. P-values <0.05 and log2 change >1 and <-l were considered significant.

[0016] FIGS. 5A to 5E show: (FIG. 5A) NADH oxidase activity normalized by from 12 months- old control mice (n=7, each sample is a pool of ovaries from 2 mice) and 12 months-old mice treated with canagliflozin for 6 months (n=6, each sample is a pool of ovaries from 2 mice). Heatmaps showing very modest to no changes in protein panels associated with (FIG. 5B) beta oxidation; (FIG. 5C) glycolysis; (FIG. 5D) mitochondrial markers, and (FIG. 5E) antioxidants and stress response from ovarian proteomic analysis by data-independent acquisition (DIA) from 12months-old control mice (n=7, each sample is a pool of ovaries from 2 mice) and 12 months-old mice treated with canagliflozin for 6 months (n=6, each sample is a pool of ovaries from 2 mice). Data is presented as mean ± SEM. *<0.05, **<0.01, ***<0.001 by / -test.DETAILED DESCRIPTION

[0017] While the making and using of various aspects of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific aspects discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the disclosure.

[0018] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims.

[0019] To date, some interventions capable of improving health span and lifespan have also been shown to delay ovarian aging

[0015] , Interventions that modulate nutrient-sensing pathways are especially good candidates for preserving of ovarian reserve. Activation of the phosphoinositide 3 -kinase (PI3K) / protein kinase B (Akt) signaling pathway results in phosphorylation of forkhead box protein O 3a (FoxO3a) in the nucleus of the quiescent oocyte, which is the ultimate signaling for the activation of primordial follicles

[0016] , Hence, suppression of molecules upstream of the PI3K / Akt pathways can decrease the rate of primordial follicles activation and consequently delay the exhaustion of the reserve. This is observed with inhibition of Insulin / Insulin-like growth factor 1 (IGF-1) and / or Mammalian target of rapamycin (mTOR) signaling pathways. For instance, calorie restriction (CR) starting early in the reproductive window is able to decrease follicle activation in mice [17-19], Also, in mouse models with growth hormone deficiency, such as the Growth Hormone Receptor Knockout mice and the Ames dwarf mice, reduced IGF1 and mTOR signaling result in preservation of ovarian reserve [20, 21], Likewise, pharmacological interventions that modulate insulin / IGFl pathways interventions such as rapamycin

[0019] , and metformin

[0022] also have positive effects on ovarian reserve.

[0020] The present inventors found that sodium-glucose co-transporter 2 (SGLT2) antagonists, such as canagliflozin (CANA), (IS)-Hydrate (2: 1), l,5-anhydro-l-C-[3-[[5-(4-fhiorophenyl)-2- thienyl]methyl]-4-methylphenyl]-d-glucitol, administration significantly delays ovarian aging.As modulation of glycemia, insulin, IGF1 and mTOR is associated with follicular activation, the inventors evaluated the effects of CANA on ovarian aging. By way of explanation, but not a limitation of the present invention, the inventors hypothesized that CANA treatment during the second half of the reproductive window would exert positive effects on ovarian reserve and slow ovarian aging. Other SGLT2 antagonist(s) for use herein include bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, and / or phlorizin.

[0021] Canagliflozin is generally dosed orally. Canagliflozin can be provided with an initial dose of 100 mg once daily, taken before first meal of the day. If the initial dose is well tolerated, the dose can be increased to 300 mg once daily.

[0022] FIGS. 1 A to 1H show: (FIG. 1 A) Fasting plasma glucose levels; (FIG. IB) fasting plasma insulin levels; (FIG. 1C) plasma IGF1 levels; (FIG. ID) estimated number of primordial, transition / primary; secondary and tertiary follicles (FIG. IE) total estimated number of follicles; (FIG. IF) primary: primordial follicles ratio; (FIG. 1G) representative images of HE stained ovarian sections; (FIG. 1H) estimated number of zona pellucida remnants (ZPR) from 12 months- old control mice (n=6) and 12 months-old mice treated with canagliflozin for 6 months (n=6). Data is presented as mean ± SEM. *<0.05, **<0.01, ***<0.001 by one-way t test or one-way ANOVA followed by Tukey post-hoc test.

[0023] Next, the inventors determined if the CANA-mediated preservation of ovarian reserve was associated with changes in age-related ovarian hallmarks. The inventors evaluated ovarian fibrosis, MNGC formation, and T cell accumulation. (FIG. 2A) Fluorescence intensity of phosphorylated Forkhead box O3a protein (pFoxO3a) in primordial follicles; (FIG. 2B) representative images of pFoxO3a and DDX4 fluorescence in primordial follicles. It was found that CANA treatment attenuated age-related ovarian fibrosis by approximately 50% (FIGS. 2C and 2D). CANA treatment also significantly reduced age-related lipofuscin accumulation in the ovary (FIGS. 2E and 2F), which we recently reported to be an excellent marker of ovarian MNGCs

[0014] , Lastly, it was also found that CANA treatment reduced the age-related increase in ovarian T cells (CD3), which are known to accumulate in the aging ovary [11, 14, 15] (FIGS. 2G and 2H).

[0024] FIGS. 2A to 2H show: (FIG. 2A) Fluorescence intensity of phosphorylated Forkhead box O3a protein (pFoxO3a) in primordial follicles; (FIG. 2B) representative images of pFoxO3a and DDX4 fluorescence in primordial follicles; (FIG. 2C) quantification of ovarian collagen accumulation by picrosirius-red (PSR) staining; (FIG. 2D) representative images of PSR stained ovarian sections; (FIG. 2E) quantification of ovarian of Sudan Black staining; (FIG. 2F) representative images of Sudan Black stained ovarian sections from 6 months-old control mice(n=5-6), 12 months-old control mice (n=5-6) and 12 months-old mice treated with canagliflozin for 6 months (n=5-6). Data is presented as mean ± SEM. *<0.05, **<0.01, ***<0.001 by one-way ANOVA followed by Tukey post-hoc test. Yellow arrows are pointing to Sudan Black positive areas. White scale bar represents 10 pm and black scale bar represents 500pm. FIG. 2G shows representative CD3 (green) and DAPI (blue) immunostained images (magnification = lOx; scale bar = 200 pm); and FIG. 2H quantified immunofluorescent intensity of CD3 (n = 5-6 / group) in 6-month-old control, 12-month-old control, and 12-month-old CANA-treated mice. All data are presented as mean + / - SEM and were analyzed by one-way ANOVA with Tukey post hoc analyses. *p < 0.05; **p < 0.01; ***p < 0.001.

[0025] CANA promotes transcriptomic changes associated to mitochondrial function in the ovary. To better understand the protective effects of CANA in the aging ovary, RNAseq was performed comparing the 12mo CANA treated mice and the age-matched controls. The dispersion of the genes is displayed in a volcano plot (FIG. 3 A). Among the DEGs, 42 genes were downregulated and 95 were upregulated (data not show). Interestingly, the transcriptomic differences between the groups were mild, as evidenced by the subtle separation of the groups in the principal components analysis (PCA) (FIG. 3B). The list of DEGs was uploaded into Ingenuity Pathway analysis (IP A) in order to evaluate CANA modulation of canonical pathways and upstream regulators in the ovary at transcriptomic level. The IP A analysis showed that the estrogen receptor signaling pathway was upregulated by CANA. Estrogen signaling is associated with improved ovarian function, as decreases in this pathway have been reported in aged ovaries[8]. The pathway associated to mitochondrial dysfunction was downregulated in the CANA treated ovaries, which is likely a result of upregulation of several mitochondrial genes. Also, the oxidative phosphorylation pathway was upregulated suggesting more mitochondrial activity. Similarly, in the analysis of upstream regulators, upregulation of genes associated with mitochondrial health and function such as PHGDH, SLC25A5, and RABGEF1 was observed in the CANA -treated ovaries. Downregulation of regulators associated with cell proliferation such as RICTOR, FLCN, and EIF6 and upregulation of cell proliferation inhibitors such as LIF, FBXW7, and STK11 was also observed in the CANA mice. These regulators are associated with the decreased follicular activation observed in the CANA group. (FIG. 3C) IPA canonical pathway analysis, (FIG. 3D) IPA upstream regulator analysis showing inhibition or activation of regulators associated to mitochondrial activity and cell proliferation from ovarian RNAseq from 12 months-old control mice (n=6) and 12 months-old mice treated with canagliflozin for 6 months (n=6). Statistical analysis was performed by / -test. P-values <0.05 and log2 change >1 and <-l were considered significant.

[0026] CANA treatment upregulates pathways associated to decreased PI3K / Akt signaling in the ovary. To evaluate potential effects of CANA treatment in the ovary at protein level, proteomics were performed of ovarian tissue from 12mo CANA treated mice and age-matched controls using the Data-independent acquisition (DIA) system

[0033] , The dispersion of the proteins detected is displayed in a volcano plot (FIG. 4A). Forty proteins were downregulated and 37 were upregulated. The differences in protein levels were also mild, as evidenced by the unclear separation of the groups in the principal components analysis (PCA) (FIG. 4B). The list of differential proteins was uploaded into IPA in order to evaluate CANA modulation of canonical pathways in the ovary at the protein level. Similar to the transcriptomic results, proteomic data also shows upregulation of Estrogen signaling pathway. Interestingly, IL12 signaling pathway was downregulated in the CANA treated ovaries. IL12 signaling pathway has been previously reported as upregulated in the stoma of aged ovaries [8], Pathways associated with Pi3K / AKT activation such were also downregulated by the CANA treatment. Whereas pathways associated to inhibition of the Pi3K / AKT pathway, such as PTEN and Cachexia signaling, were upregulated by CANA. PTEN as an upstream regulator was also downregulated by CANA, showing the downregulation of molecules downstream from PTEN signaling. Similar to the RNAseq observations, proteomics also revealed downregulation of upstream regulators associated with cell proliferation, such as RICTOR and SMAD3 and upregulation of cell proliferation inhibitors, such as KLF15. Insulin as an upstream regulator was also downregulated along with other regulators associated to glucose metabolism such as HNF4A. Upregulation of regulators associated with antioxidant function, such as FOXA2 and NFE2L2 was also observed in the CANA -treated ovaries. Endoplasmic reticulum stress also appears to be reduced by CANA in the ovarian cells, as evidenced by downregulation of regulators ATF4, ERN1 and XBP1. CANA was also shown to be a modulator of inflammation and immune response in the ovary as it modulates upstream regulators associated with these processes at protein level. Upstream regulators associated with lymphocyte accumulation such as CD38, NFAT5 and STAT 4 were downregulated by CANA. Lymphocytes have been shown to accumulate in aged ovaries[8, 10, 11], (FIG. 4C) IPA canonical pathway analysis, (FIG. 4D) IPA upstream regulator analysis showing inhibition or activation of regulators associated to cell proliferation, metabolism, endoplasmic reticulum (ER) stress and inflammation / immune response from ovarian proteomic analysis by data-independent acquisition (DIA) from 12 months-old control mice (n=7, each sample is a pool of ovaries from 2 mice) and 12 months-old mice treated with canagliflozin for 6 months (n=6, each sample is a pool of ovaries from 2 mice). Statistical analysis was performed by Ltest. P-values <0.05 and log2 change >1 and <-l were considered significant.

[0027] CANA exerts no effects on NADH oxidase activity and proteins associated with oxidation and mitochondrial function. To further investigate the modulation of mitochondrial function by CANA observed in the transcriptomic data, a NADH oxidase activity assay was performed

[0031] and a targeted proteomics analysis using the DIA system. The protein panels analyzed were for proteins associated with beta oxidation, glycolysis, mitochondrial function, antioxidants and stress response

[0031] , Surprisingly, no differences were observed in the NADH oxidase assay (FIG. 5A). Similarly, no differences were observed for most of the proteins selected for the panels with the exception of acadvl (Very long-chain specific acyl-CoA dehydrogenase), ubb / c (Bbiquitin C), hsp90bl (heat shock protein 90 beta family member 1), hspa5 (heat shock protein family A member 5), which showed statistical significance (p<0.05) (FIG. 5B).

[0028] Control and Experimental Diets: TestDiet, a division of Purina Mills (Richmond, IN), prepared all the diets for the studies performed herein. LabDiet 5LG6 (62.2% CHO, 21.7% PRO, 12.1% FAT) was provided as the control (Con) diet and was supplemented with CANA (180 ppm) for the treatment diet.

[0029] Animals: UM-HET3 female mice were purchased from The Jackson Laboratory (strain #: 036603; N=44) at 5 months of age and were acclimated to their new environment for a month. Mice were group-housed at 22 ± 0.5°C on a 12: 12-hour light-dark cycle. Unless otherwise noted, mice had ad libitum access to food and water throughout the experimental timeframe. At 6 months of age, mice were randomly assigned to one of three groups: [1] 6-month-old control mice (n=6), [2] 12-month-old control mice (n=18), or [3] 12-month-old mice that were treated with CANA from 6 to 12 months of age (n=20). The 6-month-old control mice were immediately anesthetized in the fasted state (5-6 hours) with isoflurane and euthanized by exsanguination due to cardiac puncture prior to tissue collection and the remaining mice were aged to 12 months under their respective treatment conditions. At 12 months of age, the remaining mice were anesthetized in the fasted state (5-6 hours) with isoflurane and euthanized by exsanguination due to cardiac puncture prior to tissue collection. Blood was collected into EDTA-lined tubes and plasma was collected and frozen. One ovary from each animal was excised, flash frozen, and stored at -80°C. The other ovary from each animal was collected into 4% PF A, processed, and serially sectioned. Ovaries from six animals per group were used for histological assays, while the opposing ovary from these mice were used for bulk RNA sequencing. Frozen ovaries from the remaining 12-month-old control (n=12) and 12-month-old CANA-treated groups (n=14) were pooled together (2 ovaries / sample) to provide enough tissue for proteomic and NADH oxidase activity assays. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Wayne State University.

[0030] Plasma Analyses: Fasting glucose was measured in whole blood at the time of euthanasia using Accu-Chek Aviva Plus glucometers (Roche, Basel, CH). Fasting insulin and IGF-1 levels were measured in plasma collected during the terminal harvests using kits from Alpco using ELISA kits from Alpco (cat #: 80-INSMSU-E01) and R&D Systems (Cat #: MG100), respectively, as previously described [40, 41],

[0031] Histological Analyses: Ovarian reserve was determined by counting follicles in H&E- stained slides as previously described

[0014] , Zona Pellucida remnants were also counted and calculated a previously described

[0021] , Piero-Sirius Red staining for collagen deposition and Sudan Black staining for lipofuscin accumulation were performed on one randomly assigned section from the middle of each ovary and analyzed as previously described

[0014] , Immunofluorescence was performed as previously described

[0014] , with minor adaptations. In brief, slides were incubated with primary rabbit anti-pFoxO3a antibody (cat #: 9465; Cell Signaling; 1 :200) and chicken anti-DDX4 antibody (cat #: ab314236; Abeam; 1 :200) or rabbit anti-CD45 antibody (cat #: abl0558; Abeam; 1 :200), or rabbit anti-CD3 antibody (cat #: 17617- 1; ProteinTech; 1 :200), or rabbit anti-F4 / 80 antibody (cat #: ab300421; Abeam; 1 : 1000). Primary antibodies were incubated overnight at 4C, followed by secondary goat anti-rabbit IgG Alexa Fluor 488 antibody (cat #: 111-545-003; Jackson ImmunoResearch Laboratories; 1 : 1000) and Rhodamine Red donkey anti-chicken (cat #: 703-295-155; Jackson ImmunoResearch Laboratories; 1 : 1000) for 1 h at room temperature and DAPI for 5 min. Sudan Black staining was performed during the immunofluorescence protocol in order to quench the autofluorescence from the ovaries. Images were captured on a confocal microscope (Zeiss LSM 880 w / Airyscan) at lOOx magnification in the green (pFoxO3a), red (DDX4), and blue (DAPI) channels and at lOx magnification in the green (CD45, CD3 & F480) and blue (DAPI) channels. The mean fluorescent intensities of pFoxO3a from 5-10 primordial follicles were quantified using Image J and averaged. CD45, CD3 & F480 fluorescent intensities were measured in the whole ovarian section.

[0032] RNA Sequencing: RNA was extracted from whole ovaries as previously described

[0042] , cDNA Libraries were constructed from 40 ng total RNA using the NEB Ultra II Directional Library preparation kit (cat # NEBE7760L; New England Biolabs) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (cat #: NEBE7490L; New England Biolabs), as previously described [43, 44], Library sizing was performed with HSD1000 ScreenTape (cat #: 5067-5584; Agilent Technologies) and quantified by Qubit dsDNA HS Assay Kit (cat #: Q32851; ThermoFisher Scientific) on a Qubit 4 Fluorometer (cat #: Q33226; ThermoFisher Scientific). The libraries for each sample were pooled at 4 nM concentration and sequenced using an IlluminaNovaSeq 6000 system. The sequence depth obtained was -20,000 reads. Data analysis was performed in Partek.

[0033] Protein & Mitochondria Isolation: Protein and mitochondria were isolated from the same samples. As addressed above, each sample consisted of two ovaries pooled from two mice. During the process of mitochondrial isolation, an aliquot containing protein was used for proteomic assays. Mitochondria were isolated from the remaining volume for assessment of electron transport chain activity.

[0034] NADH Oxidase / Activity Assay: To determine the electron transport chain activity through complexes I-III-IV, NADH oxidase activity was evaluated as previously described

[0045] , In brief, ovarian homogenates were diluted to 0.01 mg / mL in 25 mM MOPS buffer (pH 7.4). Complex I activity was measured spectrophotometrically (Agilent 8453 diode array UV-Vis spectrophotometer) as the rotenone-sensitive rate of NADH oxidation in the presence of 50 nM antimycin A and 100 pM ubiquinone-1, subsequent to the addition of 150 pM NADH. Activity was dependent on the presence of ubiquinone-1 and was sensitive to inhibition by rotenone (100 nM), indicating the necessity of complex I function for NADH utilization.

[0035] Proteomic Analyses: Samples were digested using previously described methods [46, 47], In brief, 20 pg of each sample were immobilized in a short run 12.5% SDS-PAGE gel (Criterion, Bio-rad) followed by fixation and staining with Coomassie blue (Pierce). Each gel lane was cut out as a single sample, cut into smaller pieces and washed / de-stained. After the proteins were reduced, alkylated, and digested with trypsin, they were subjected to extraction using 70% methanol / 5% acetic acid in water, evaporation to dryness, and reconstitution with 1% acetic acid. Samples were then analyzed using the TSQ Quantiva system (Thermo Scientific). The instrumental and experimental parameters were identical to those used previously

[0045] , Data were acquired in the selected reaction monitoring (SRM) mode for the standard targeted quantitative proteomics panels [48-50], Additionally, data was also acquired using high-resolution accurate mass (HRAM) for potential re-interrogation in the future as needed. Data were analyzed using established Skyline methods

[0051] , Normalization was performed using BSA as the nonendogenous internal standard and housekeeping proteins (Hspdl, Mdhl, and Gpil). The proteomics assay development and data processing followed the inventors’ established protocol [46, 47], For data and pathway analyses, proteins with statistical differences across experimental groups were identified and subjected to Ingenuity Pathway Analysis (IP A). All raw proteomics data are available upon request.

[0036] Statistical Analyses: Results are presented as mean ± SEM with ^ values less than 0.05 considered to be significantly different. Analyses of differences between groups in plasma, histological assays, and NADH oxidase activity were performed by Student’s t-tests or one-way ANOVA with Tukey post-hoc analyses where appropriate using Graphpad Prism 9.0 Software. For RNA sequencing and proteomics, differentially expressed gene (DEGs) lists were imported into IPA software and filtered on FDR<0.1 and logFC>0.25 for pathway analyses. Pathways with p values less than 0.05 were considered statistically significant and the activation z-scores are reported by heatmap or bar charts. Corrections for multiple comparisons were made, where appropriate, using the Benjamini, Krieger, and Yekutieli correction for multiple comparisons. Significant differences were defined at P<0.05 or FDR<0.05 (for multiple comparisons).

[0037] Embodiment:

[0038] Embodiment 1. A method for treatment, amelioration, or prophylaxis of conditions related to ovarian aging comprising:

[0039] administering to a subject in need of treatment, amelioration, or prophylaxis of conditions related to ovarian aging an effective amount of a sodium-glucose co-transporter 2 (SGLT2) antagonist.

[0040] Embodiment 2. The method of embodiment 1, wherein the canagliflozin is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution.

[0041] Embodiment 3. The method of embodiments 1 or 2, wherein the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, sotagliflozin, or ertugliflozin.

[0042] Embodiment 4. The method of any one of embodiments 1 to 3, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

[0043] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours.

[0044] Embodiment 6. The method of any one of embodiments 1 to 5, wherein ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or blood hormone analyses.

[0045] Embodiment 7. The method of any one of embodiment 6, wherein the blood hormone analyses is selected from at least one of: anti-mullerian hormone (AMH), Follicle- stimulating hormone (FSH), and 17b -estradiol.

[0046] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration.

[0047] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL12 signaling, or both.

[0048] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4.

[0049] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the canagliflozin exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function.

[0050] Embodiment 12. A method for slowing or preventing ovarian aging comprising:

[0051] administering to a subject in need of slowing or prevention of ovarian aging an effective amount of an SGLT2 antagonist, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

[0052] Embodiment 13. The method of embodiment 12, wherein the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, sotagliflozin, or ertugliflozin.

[0053] Embodiment 14. The method of embodiments 12 or 13, wherein the SGLT2 antagonist is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution.

[0054] Embodiment 15. The method of any one of embodiments 12 to 14, wherein the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours.

[0055] Embodiment 16. The method of any one of embodiments 12 to 15, wherein ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or blood hormone analyses.

[0056] Embodiment 17. The method of embodiment 16, wherein the blood hormone analyses is selected from at least one of: anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), and 17b -estradiol.

[0057] Embodiment 18. The method of any one of embodiments 12 to 17, wherein the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration.

[0058] Embodiment 19. The method of any one of embodiments 12 to 18, wherein the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL12 signaling, or both.

[0059] Embodiment 20. The method of any one of embodiments 12 to 19, wherein the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4.

[0060] Embodiment 21. The method of any one of embodiments 12 to 20, wherein the SGLT2 antagonist exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function.

[0061] Embodiment 22. The method of any one of embodiments 12 to 21, wherein the subject is a human.

[0062] Embodiment 23. A method to slow a rate of ovarian aging comprising: administering to a subject in need of slowing ovarian aging a composition comprising an effective amount of an SGLT2 antagonist in a pharmaceutically acceptable carrier, wherein a dose of the canagliflozin is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

[0063] Embodiment 24. The method of embodiment 23, wherein the SGLT2 antagonist is canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof.

[0064] Embodiment 25. The method of embodiment 23 or 24, wherein the SGLT2 antagonist is canagliflozin.

[0065] It is contemplated that any aspects of the disclosure discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0066] It will be understood that particular aspects described herein are shown by way of illustration and not as limitations of the disclosure. The principal features of this disclosure can be employed in various aspects without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims.

[0067] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0068] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0069] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In aspects of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0070] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinationsthereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0071] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0072] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the disclosure(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any disclosure(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the disclosure(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure but should not be constrained by the headings set forth herein.

[0073] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the compositionsand / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0074] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

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Claims

What is claimed is:

1. A method for treatment, amelioration, or prophylaxis of conditions related to ovarian aging comprising: administering to a subject in need of treatment, amelioration, or prophylaxis of conditions related to ovarian aging an effective amount of a sodium-glucose co-transporter 2 (SGLT2) antagonist.

2. The method of claim 1, wherein the canagliflozin is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution.

3. The method of claim 1, wherein the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof.

4. The method of claim 1, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

5. The method of claim 1, wherein the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours.

6. The method of claim 1, wherein ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or blood hormone analyses.

7. The method of claim 5, wherein the blood hormone analyses is selected from at least one of: anti-mullerian hormone (AMH), Follicle-stimulating hormone (FSH), and 17b-estradiol.

8. The method of claim 1, wherein the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration.

9. The method of claim 1, wherein the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL 12 signaling, or both.

10. The method of claim 1, wherein the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4.

11. The method of claim 1, wherein the canagliflozin exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function.

12. A method for slowing or preventing ovarian aging comprising:administering to a subject in need of slowing or prevention of ovarian aging an effective amount of an SGLT2 antagonist, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

13. The method of claim 12, wherein the SGLT2 antagonist is selected from at least one of: canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof.

14. The method of claim 12, wherein the SGLT2 antagonist is administered in conjunction with a pharmaceutically acceptable carrier, buffer, salt, binder, lubricant, excipient, filler, or solution.

15. The method of claim 12, wherein the SGLT2 antagonist is formulated for extended release over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours.

16. The method of claim 12, wherein ovarian aging is measured by at least one of: a decrease in primordial follicle loss, maintenance of ovarian reserve, vaginal ultrasound to assesses follicular reserve, or blood hormone analyses.

17. The method of claim 14, wherein the blood hormone analyses is selected from at least one of: anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), and 17b -estradiol.

18. The method of claim 12, wherein the SGLT2 antagonist is formulated for oral, enteral, parenteral, intravenous, rectal, vaginal, subcutaneous, or topical administration.

19. The method of claim 12, wherein the SGLT2 antagonist at least one of: upregulates estrogen receptor, PTEN, and Cachexia signaling, downregulates IL 12 signaling, or both.

20. The method of claim 12, wherein the SGLT2 antagonist downregulates at least one of: ATF4, ERN1, XBP1, CD38, NFAT5, and STAT4.

21. The method of claim 12, wherein the SGLT2 antagonist exerts no effects on at least one of: NADH oxidase activity, protein oxidation, or mitochondrial function.

22. The method of claim 12, wherein the subject is a human.

23. A method to slow a rate of ovarian aging comprising: administering to a subject in need of slowing ovarian aging a composition comprising an effective amount of an SGLT2 antagonist in a pharmaceutically acceptable carrier, wherein a dose of the SGLT2 antagonist is 10, 20, 25, 30, 40, 50, 60, 70, 75, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 milligrams per dose.

24. The method of claim 23, wherein the SGLT2 antagonist is selected from canagliflozin, bexagliflozin, dapagliflozin, empagliflozin, ertugliflozin, sotagliflozin, phlorizin, or combinations thereof.

25. The method of claim 23, wherein the SGLT2 antagonist is canagliflozin.

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