Agonists Of Adiponectin

Abstract

The present invention is related to agonist of adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) for the treatment and/or prevention of polycystic ovary syndrome (PCOS).

Claims

1. An agonist of adiponectin receptor 1 (AdipoR1) protein activity and/or adiponectin receptor 2 (AdipoR2) protein activity for the treatment and/or prevention of polycystic ovary syndrome (PCOS).

2. The agonist according to claim 1, wherein the polycystic ovary syndrome (PCOS) is characterized by: a) underexpression or deficiency or inadequate activation of an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) gene product; b) deletion or loss of an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) gene; or c) underexpression or lack or deficiency or inadequate activation of Adiponectin.

3. The agonist according to claim 2, wherein the underexpression or deficiency of the adiponectin receptor 1 (AdipoR1) and/or the adiponectin receptor 2 (AdipoR2) gene product is at least partially age-related.

4. The agonist according to claim 1, wherein the polycystic ovary syndrome (PCOS) is characterized by at least one of: polycystic ovaries, with preferably 12 or more follicles in one ovary; increased size of one or both ovaries compared to a healthy patient; increased serum or blood levels of at least one of, as compared to a healthy patient: androgens, preferably testosterone (hyperandrogenism); Luteinizing hormone (LH); Estrogens; Androstenedione; and/or Anti-Mullerian hormone (AMH); decreased serum or blood levels of at least one of, as compared to a healthy patient: follicle-stimulating hormone (FSH); and/or sex hormone binding globulin SHBG); excess facial or body hair growth; scalp hair loss; acne; and/or menstrual dysfunction, such as, lack of periods or menses (menstrual flow), menstrual irregularity and/or lack of ovulation.

5. The agonist according to claim 1, wherein the agonist activates an adiponectin receptor 1 (AdipoR1) gene product and/or an adiponectin receptor 2 (AdipoR2) gene product.

6. The agonist according to claim 1, wherein the agonist is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic, which specifically binds to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein.

7. The agonist according to claim 1, wherein the agonist is an aptamer that specifically binds to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein.

8. The agonist according to claim 1, wherein the agonist is a peptide that specifically binds to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein.

9. The agonist according to claim 1, wherein the agonist is a small molecule that specifically binds to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein.

10. The agonist according to claim 9, wherein the agonist is 2-(4-Benzoylphenoxy)-N-(1-benzylpiperidin-4-yl)acetamide (AdipoRon) ##STR00003##

11. The agonist according to claim 1, wherein the agonist can be found by means of an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) assay.

12. The agonist according to claim 1, wherein an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein comprises SEQ ID NO 1, or SEQ ID NO 2, respectively, or a functional fragment thereof.

13. Use of the agonist according to claim 1 (for the manufacture of a medicament) in the treatment of a human or animal subject being diagnosed for, suffering from, or being at risk of developing polycystic ovary syndrome (PCOS), or for the prevention of such condition.

14. A pharmaceutical composition comprising the agonist according to claim 1.

15. A combination of the pharmaceutical composition according to claim 14 and one or more additional therapeutically active compounds.

16. A method for treating or preventing polycystic ovary syndrome (PCOS) in a human or animal subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 14.

17. A method for identifying a compound for use in the treatment and/or prevention of a patient suffering from, at risk of developing, and/or being diagnosed for polycystic ovary syndrome (PCOS), the method comprising screening one or more test compounds in an adiponectin receptor 1 (AdipoR1) and/or an adiponectin receptor 2 (AdipoR2) assay, to identify one or more candidate compounds.

18. The method according to claim 17, further comprising, prior to screening the one or more test compounds, creating and/or provisioning a library of test compounds.

19. The method according to claim 18, wherein the library is a DNA-encoded library (DEL).

20. The method according to claim 19, wherein excess DNA is applied to a medium to avoid unspecific binding of adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) to DNA tags of the DNA-encoded library.

21. The method according to claim 17, wherein cells or tissues of the adiponectin receptor 1 (AdipoR1) and/or the adiponectin receptor 2 (AdipoR2) assay have a DNA binding deficient mutant of adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2).

22. A method for determining whether a human or animal subject is suitable of being treated with the agonist according to claim 1, a pharmaceutical composition comprising the agonist according to claim 1, or a pharmaceutical composition comprising the agonist according to claim 1 and one or more additional therapeutically active compounds, said method comprising: providing a tissue or liquid sample from said subject; and determining whether or not said sample is characterized by underexpression or deficiency of an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) gene product, or a deletion or loss of the adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) gene.

23. The method according to claim 22, wherein the expression of adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) is determined: on an mRNA level (e.g., RT-PCR, in situ PCR and/or Fluorescence in situ hybridization (FISH); or on a protein level.

24. A companion diagnostic for use in the method according to claim 22, wherein the companion diagnostic comprises at least one agent that/is selected from the group consisting of: a nucleic acid probe or primer capable of hybridizing to a nucleic acid (DNA or RNA) that encodes an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein; an antibody that is capable of binding to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein; and/or an aptamer that is capable of binding to an adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) protein.

25. A method for treating or preventing polycystic ovary syndrome (PCOS) in a human or animal subject, comprising administering to a subject in need thereof an effective amount of the combination according to claim 15.

Description

EXAMPLES

[0137] The experiments shown herein clearly support adiponectin receptor 1 (AdipoR1) and/or adiponectin receptor 2 (AdipoR2) as a target whose activation provides a therapy option for different types of polycystic ovary syndrome (PCOS).

[0138] Adiponectin activates the AMPK (AMP-activated protein kinase) signaling pathway to regulate lipid metabolism in bovine hepatocytes. Adiponectin also stimulates a significant increase in cortisol production, together with increases in mRNA levels of key steroidogenic genes including, inter alia, CYP11B1 (Steroid-11(3-Hydroxylase).

FIGURES

[0139] FIG. 1: A) shows the expression of the mRNA for Adiponectin receptors AdipoR1 (grey columns) and AdipoR2 (black columns) in MCF-7 cells (solid column) and Y-1 cells (hatched column), as done with quantitative PCR performed with suitable primers on AdipoR1 and AdipoR2 mRNA. For this experiments, and the experiments below, MCF-7 cells were cultivated in RPMI/10% FCS medium with insulin (10 μg/ml); and Y-1 cells (epithelial cells of mouse adrenal gland origin) were cultivated in F12-K medium with 10% FBS and 15% donor horse serum. Both cell lines express substantial amounts of AdipoR1 and AdipoR2 receptors.

[0140] FIG. 1: B) shows the ratio of p-AMPK to total AMPK in MCF-7 cells which were treated with different concentrations of the agonist AdipoRon (2-(4-Benzoylphenoxy)-N-(1-benzylpiperidin-4-yl)acetamide). The ratio of p-AMPK to total AMPPK is a marker for the activity of AdipoRon on Adiponectin receptors in MCF-7 cells. Cells were starved for three hours in medium w/o FCS, and subsequently treated with AdipoRon at a final concentration of 0 μM, 0.1 μM, 5 μM, 10 μM, 20 μM, 40 μM for one hour. Cells were harvested, and pAMPK and total AMPK were determined by a commercial HTRF assay (CisBio). AMPK (adenosine monophosphate activated kinase) is activated by phosphorylation and stimulates synthesis of ATP and thus energy metabolism. AdipoRon stimulates phosphorylation, and in consequence the activation of AMPK. The EC.sub.50 of activation is approximately 5-10 μM in this assay. These data confirm literature data shown in FIG. 1 C (Okada-Iwabu et al. 2013) and validate AdipoRon for further in vitro and in vivo assays.

[0141] FIG. 2: shows the fold induction of StAR mRNA relative to vehicle control (A) and the fold induction of Cyp11b1 mRNA relative to vehicle control (B) as effects of AdipoRon in the adrenal cell line Y-1 treated with 10 nM Adrenocorticotropin hormone (ACTH). Y-1 cells (epithelial cells of mouse adrenal gland origin) were cultivated in F12-K medium with 10% FBS and 15% donor horse serum. Cells were treated with 10 nM ACTH, and 30 min later the Adipor1/2 agonist AdipoRon was added in a final concentration of 0.1 μM, 1 μM, 10 μM for two and a half hours. Cells were harvested, mRNA isolated and Cyp1b1 and Star mRNA quantified relative to β-actin (Actb) mRNA. Hatched column: vehicle control, grey column: 10 nM ACTH, white columns: 10 nM ACTH+0.1 μM, 1 μM, or 10 μM AdipoRon.

[0142] ACTH is a hormone of the Hypothalamic-pituitary-adrenal axis and stimulates the expression of Steroidogenic acute regulatory protein (StAR) and Cyp1b1 (steroid-1113-hydroxylase), which then can lead to cortisol production and, as a consequence, insulin resistance. Insulin insensitivity is observed in women diagnosed with PCOS. AdipoRon dose-dependently reduces the induction of StAR and Cyp11b1 by ACTH in Y-1 adrenal cell line and, as such, cortisol synthesis. EC.sub.50 for AdipoRon for this effect is around 10 μM for StAR, and around 0.1 to 1 μM for Cyp11b1, but seems to be saturated at higher concentrations.

[0143] FIG. 3: A) shows the peak of LH release in the afternoon at 7 p.m. of d3 (proestrous) of the rat estrous cycle. For this, the cycle phase of untreated naive female Han-Wistar rats was staged by vaginal smears. Rat plasma was taken at different days and time points of the cycle (d3=proestrous, d4=estrous), and LH in plasma determined by a rat pituitary magnetic bead panel (Milliplex, Cat No RPTMAG-86K). LH peak could be detected at d3 at 7 pm in the afternoon as described previously (Smith et al. 1975). Thus, this time point is well suited for measurement of the effect of AdipoR1 and/or AdipoR2 receptors activation by AdipoRon. FIG. 3: B) shows the LH concentration [pg/ml] in plasma of female rats treated with vehicle (circle), 50 mg/kg (upwards triangle) or 100 mg/kg (downwards triangle) AdipoRon. AdipoRon dose-dependently reduces the induction of LH in proestrous in the afternoon of d3 in the rat. For this, the cycle phase of untreated naive female Han-Wistar rats was staged by vaginal smears. Rats were treated for one estrous cycle, beginning with metestrous, with 50 mg/kg or 100 mg/kg of Adiporon in a suitable vehicle twice a day. Rat plasma was taken at d3=proestrous at 7 pm, and LH was determined by a rat pituitary magnetic bead panel (Milliplex Cat No RPTMAG-86K).

[0144] Increased LH is a hallmark and diagnostic criterion of PCOS in women. This experiment shows that AdipoRon dose-dependently reduces the induction of LH in proestrous.

[0145] FIG. 4: shows tissue sections of the ovaries of naïve female wt (A) and db/db mice (B). Wt mice show corpus luteae after ovulation (see arrows in A). db/db mice show reduced or lacking corpora luteae, and many non-developing follicles in the ovaries (see arrows in B). Wt mice show a corpus luteae after ovulation. Such mice also show other typical symptoms of PCOS, as follicular maturation goes down and ovulation stops, and plasma testosterone increases (Garris et al. 1985). db/db mice are leptin receptor deficient and a known model for diabetes with increased body weight, increased insulin, decreased plasma adiponectin.

[0146] FIG. 5: shows a single dose exposure experiment of AdipoRon in mice. At t=0, a dose of 50 mg/kg (circle) or 100 mg/kg (squares) was given p.o. to adult female db/db mice. At each time point (0.5 h, 1 h, and 4 h) three mice were killed and the plasma concentration of unbound AdipoRon was determined by LC/MS. Dosage of 100 mg/kg in mice reaches the EC.sub.50 value (dotted line) for stimulatory effects of this compound in Y-1 cell, which was found to be at ˜100 nM (see FIG. 2).

[0147] FIGS. 6A and 6B Lhcgr mRNA expression (6A) and Cyp17a1 mRNA expression (6B) in ovaries of untreated db/db mice (black column) or db/db mice treated with 50 mg/kg Adiporon (grey column) is shown (n=3 biological replicates; error bars show SD). Expression of Cyp17a1 and Lhrgr mRNA in ovaries of adult female db/db mice were analyzed for untreated mice or mice treated with 50 mg/kg Adiporon as a single po dose given at t=0 h. At 0.5 h, 1 h, 2 h, 4 h, 24 h, mice were killed, and ovaries were taken, mRNA isolated (Qiagen, RNeasy Mini kit #74106) and mRNA quantified by Q-PCR (ThermoFisher Scientific Assay on demand #Mm00484040_ml for Cyp17a1 and Mm00442931_ml for Lhcgr).

[0148] The experiment shows that in db/db mice, AdipoRon decreases the expression of the LH receptor (LHR) and of CYP17A1 (Steroid-17α-Hydroxylase). Reduction of Lhrgr and Cyp17a1 mRNA expression is highest immediately after p.o. treatment due to the kinetics of AdipoRon (as described in FIG. 5). Lhcgr is the LH receptor, its reduction decreases the sensitivity of the ovaries for LH. Increased LH is a hallmark of PCOS. Furthermore, the rate limiting enzyme for testosterone synthesis in the ovaries, Cyp17a1, is also reduced.

[0149] FIG. 7: shows the plasma AUC [ng×ml/h] of testosterone (7A) and progesterone (7B) in female untreated db/db mice (grey column) or db/db mice treated with 50 mg/kg Adiporon (upwards hatched column) or 100 mg/kg Adiporon (downwards hatched column). It shows the effect of stimulation of adiponectin receptors on steroid synthesis in db/db mice. Female db/db mice, which were untreated, or treated with Adiporon at 50 mg/kg and 100 mg/kg with a single po dose, were analysed. Blood was taken at 0.5, 1, 2, 4, and 24 hours, and plasma concentrations of progesterone (ibl/Tecan Order No RE52231, Hamburg) and testosterone (Demeditec Order No DEV9911, Kiel) were determined by commercial ELISAs. Area under the curve (AUC) of total steroids was determined by an algorithm according to Gagnon et al. (1998) by GraphPadPrism software. AdipoRon stimulates the AdipoR1 and/or AdipoR2 receptor in these mice, and thus reduces Testosterone plasma concentration (FIG. 7 A) and increases Progesterone plasma concentration (FIG. 7 B). The effects are significant at the higher dosage of AdipoRon of 100 mg/kg after a single application (* t-test AUC versus 100 mg/kg Adiporon (n=5 time points), double sided; p<0.1).

[0150] FIG. 8: Plasma testosterone concentration [nM] (8A) and adiponectin concentration [ng/mL] in a DHEA-induced PCOS Model in rats (Anderson et al. 1997) untreated (8A,B: squares) or treated with mg/kg AdipoRon (8A,B triangles and controls (8A,B: open circle) are shown. Dehydroepiandrosterone (DHEA) has been used to induce a PCOS phenotype, which is manifest by an increase of Testosterone and a decrease of Adiponectin in the plasma of the rats. Female rats were treated with 60 mg/kg/d DHEA sc, as described for the established PCOS model, for 20 days. For the last 10 days, the rats were additionally treated with 50 mg/kg/d Adiporon. Stimulation of the AdipoR1 and/or AdipoR2 receptor by AdipoRon significantly reduces the increase in Testosterone (FIG. 8 A; * p<0.05 single sided t-test) as measured by an ELISA (see FIG. 7), and normalizes plasma adiponectin (FIG. 8 B; ** p<0.01; ****p<0.001 Dunnett's t-test), as measured by an electrochemiluminescence assay (Mesoscale). An autostimulation of adiponectin by stimulation of AdipoR1 and/or AdipoR2 has been described before and validates the activity of AdipoRon on the AdipoR1 and/or AdipoR2 receptors (Jardé et al. 2009).

[0151] FIG. 9: shows results of an oral glucose tolerance test in transgenic mice overexpressing LH (“tgLH”) with increased pituitary LH secretion (Risma et al. 1995). 9A: Plasma insulin concentration [mg/L] in wt-mice (light grey column) and untreated tgLH mice (dark grey column) or tgLH mice treated with 100 mg/kg Adiporon (black column) is shown. 9B: Insulin concentrations [ng/mL] during OGTT in wt (left diagram) and Adipor1−/−Adipor2−/−tg mice (right diagram) treated with vehicle (open circle) or treated with 100 mg/kg (circle) are shown.

[0152] Increased LH secretion is causal for the development of PCOS in women, and tgLH overexpression in mice recapitulates the human PCOS phenotype in women. For this experiment, an oral glucose tolerance test was performed in control, transgenic LH overexpressing mice, and tgLH mice treated with Adiporon at 100 mg/kg/d for four weeks. To determine insulin sensitivity, plasma was taken at 15 min after giving an oral glucose gavage of 2 g/kg after fasting of the mice for 6 h in the last two days of the experiment (Andrikopoulos et al. 2008). FIG. 9A shows a significant increase of plasma insulin after glucose challenge in fasted tgLH mice but not in AdipoRon-treated tgLH mice, meaning that the insulin sensitivity is increased by AdipoRon in the tgLH mice as a model for human PCOS.

[0153] FIG. 9B shows comparable data in wild-type mice, while no effect on plasma insulin could be shown in mice being double negative for Adiponectin Receptors 1 and 2 (Okada-Iwabu et al. 2013). These data confirm the specific effect of AdipoRon on insulin sensitivity via its activity on the AdipoR1 and/or AdipoR2 receptor.

[0154] FIG. 10: shows the Ceramide 42:1 (Acyl-C24, 10A) and Ceramide 42:2 (Acyl C24:1, 10B) concentration [μM] in plasma in a rat PCOS model induced by DHEA (grey columns, controls: circle; DHEA treated: squares) and wt/db compared to db/db mice (hatched columns, wt/db: circles; db/db mice: squares). FIG. 10A,B hatched grey bars: Comparison of the ceramide levels in plasma of db/db versus db/+ mice. Adult female mice were killed, and plasma was taken and ceramide content analysed by LC/MS (t-test of db/+ versus db/db mice; two sided; *** p<0.005). FIG. 10 A,B grey bars: Comparison of the ceramide levels in rat plasma from the experiment described in FIG. 8 from the control versus the DHEA treated PCOS group in rats. Plasma from rats of the two experimental groups were taken at necropsy at the end of the experiment analyzed (t-test of control versus DHEA rats; two sided; * p<0.05).

[0155] Ceramide lipids were determined by LC/MS using a commercial kit (Biocrates, Innsbruck). The data show that the concentration of certain plasma ceramides is significantly increased in models for PCOS. AdipoR1 and/or AdipoR2 receptors have been described to show ceramidase activity after their stimulation by an activating ligand such as AdipoRon or Adiponectin (Vasiliauskaite-Brooks et al. 2017). Increase of ceramides in plasma of these models indirectly shows a decreased activity of the AdipoR1 and/or AdipoR2 receptors, thus proofing a decreased activity of AdipoR1 and/or AdipoR2 in PCOS.

[0156] FIG. 11 A shows Biochemical and Biophysical Assay Options to determine agonist binding to AdipoR1/AdipoR2 (Nevola and Giralt 2015). FIG. 11 B shows results from such an assay and proofs it's functionality (Sun Y et al. 2013; circle AdipoR1; open circle AdipoR2). See more explanations in the text.

REFERENCES

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