Methods of treating obesity with luteinizing hormone receptor agonists

Abstract

The present disclosure relates to methods of treating or preventing obesity with luteinizing hormone receptor agonists. More specifically, provided herein are methods of treating obesity in a subject in need thereof, comprising administering to the subject a thieno [2, 3-d] pyrimidine derivative.

Description

[0001] Claiming priority

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 366,060, filed on June 8, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Federally funded research or development

[0004] This invention was made with government support under Grant No. U19 AG060917 awarded by the National Institute on Aging of the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to methods for treating or preventing obesity using luteinizing hormone receptor agonists. More specifically, the present disclosure relates to methods for treating or preventing obesity using luteinizing hormone receptor agonists such as ORG 43553 and ORG 43902. Background Art

[0006] Obesity is a complex disease involving excess body fat. Obesity increases the risk of other diseases and health problems, including heart disease, diabetes, high blood pressure, and certain cancers. Obesity can occur at any age, however, advanced age may be accompanied by hormonal and lifestyle changes that increase the risk of obesity. In addition, muscle mass generally decreases with age, leading to a decrease in metabolism. These changes can reduce caloric needs and increase the risk of obesity.

[0007] Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are beneficial to mammalian reproduction and support pregnancy. The cessation of reproduction is consistent with the menopausal transition, which is associated with an early rise in gonadotropin levels, with changes in serum follicle-stimulating hormone (FSH) greater than luteinizing hormone (LH). This transition is not only accompanied by rapid bone loss, but also by visceral obesity, unbalanced energy balance and reduced physical activity. During menopause, the number and amplitude of mid-term LH surges decrease in response to ovarian aging, while visceral fat increases from about 5-8% of total body fat to about 15-20%. Epidemiological studies have also shown that in menopausal women and women with polycystic ovary syndrome (PCOS), body mass index (BMI) is negatively correlated with serum LH levels. Therefore, there is a need for compositions and methods for treating obesity, particularly in menopausal and postmenopausal women. Summary of the invention

[0008] Disclosed are methods for treating obesity in a subject in need thereof, comprising administering to the subject a luteinizing hormone (LH) receptor agonist and a human chorionic gonadotropin (hCG) receptor agonist (LH / CG receptor). The present disclosure is based, at least in part, on the following findings: (1) LH / CG receptor (LHCGR) transcripts and proteins are abundantly expressed in adipose tissue, with gonadal white adipose tissue levels in female mice approaching those of the ovary itself; (2) activation of LHCGR by LH, hCG, and a small molecule agonist ORG 43553, which has undergone clinical trials for infertility, results in less fat accumulation in mice on a high-fat diet independent of testosterone; and (3) ORG 43553 reduces adipocyte differentiation in organoid cultures and induces thermogenesis both in vitro and in vivo. In one embodiment, the LH / CG receptor agonist is ORG 43533 and / or ORG 43902. In one embodiment, ORG 43533 and / or ORG 43902 is administered at a dose of between 1 and 100 mg / kg. In one embodiment, ORG 43533 and / or ORG 43902 is provided as a pharmaceutical composition including a pharmaceutically acceptable carrier.

[0009] In one aspect, the present disclosure provides a method of treating obesity in a subject in need thereof, comprising administering to the subject in need thereof a thieno[2,3-d]pyrimidine derivative according to Formula I or a pharmaceutically acceptable salt thereof,

[0010]

[0011] wherein R1 and R2 together with the nitrogen atom to which they are bound form a ring having 2-6 carbon atoms, optionally containing one or more heteroatoms selected from N, O and / or S. In some embodiments, the thieno[2,3-d]pyrimidine derivative is a compound selected from the group consisting of tert-butyl 5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, In some embodiments, the thieno[2,3-d]pyrimidine derivative is ORG 43533.

[0012] In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 100 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 50 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 30 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 20 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 10 and 20 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of 17 mg / kg.

[0013] In some embodiments, the thieno[2,3-d]pyrimidine derivative is administered daily. In some embodiments, the thieno[2,3-d]pyrimidine derivative is administered weekly. In some embodiments, the thieno[2,3-d]pyrimidine derivative is administered for at least 26 weeks. In some embodiments, the thieno[2,3-d]pyrimidine derivative is provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering luteinizing hormone (LH) to a subject in need thereof.

[0014] In another aspect, the present disclosure provides a method of treating diet-induced obesity in a subject in need thereof, comprising administering to the adipose tissue of the subject in need thereof a thieno[2,3-d]pyrimidine derivative according to Formula I or a pharmaceutically acceptable salt thereof,

[0015]

[0016] wherein R1 and R2 together with the nitrogen atom to which they are bound form a ring having 2-6 carbon atoms, optionally containing one or more heteroatoms selected from N, O and / or S. In some embodiments, the thieno[2,3-d]pyrimidine derivative is a compound selected from the group consisting of tert-butyl 5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, In some embodiments, the thieno[2,3-d]pyrimidine derivative is ORG 43902.

[0017] In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 100 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 50 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 30 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 1 and 20 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of between 10 and 20 mg / kg. In some embodiments, the thieno [2,3-d] pyrimidine derivative is administered at a dose of 17 mg / kg.

[0018] In some embodiments, the thieno[2,3-d]pyrimidine derivative is administered daily. In some embodiments, the thieno[2,3-d]pyrimidine derivative is administered weekly. In some embodiments, the thieno[2,3-d]pyrimidine derivative agonist is administered for at least 26 weeks. In some embodiments, the thieno[2,3-d]pyrimidine derivative is provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering luteinizing hormone (LH) to a subject in need thereof.

[0019] In another aspect, the present disclosure provides a method of treating obesity in a subject in need thereof, comprising administering a combination of ORG 43553 and ORG 43902 to the subject in need thereof.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A is a graph of relative Lhcgr expression measured by qPCR in various male and female mouse fat depots.

[0023] Figure 1BChromatogram of Sanger sequencing of the Lhcgr extracellular domain sequence in mouse gonadal white adipose tissue (gWAT), which is identical to the ovarian receptor.

[0024] Figure 1C RNAscope TM A series of representative images of in situ hybridization and immunohistochemistry (IHC) showing the expression of LHCGR in various mouse fat depots and gonads of male and female C57BL / 6 mice.

[0025] Figure 1D The drug was injected intravenously into multiple organs including mesenteric and gonadal WAT in male and female C57BL / 6 mice. 89 Plot of the percentage of injected dose per gram of tissue measured after Zr-LH.

[0026] Figure 1E The C57BL / 6 and Lhcgr - / - A series of representative fluorescence microscopy images of AlexaFluor-488 labeled hCG in gonads, iWAT, and gWAT of mice.

[0027] Figure 2A is a graph of relative Lhcgr expression in mouse iWAT and 3T3-L1 adipocytes measured by qPCR.

[0028] Figure 2B A series of representative RNAscope TM In situ hybridization and immunohistochemistry (IHC) images showing LHCGR staining in differentiated 3T3-L1 murine adipocytes.

[0029] Figure 2C is a series of representative fluorescence microscopy images showing siRNA lentivirus-mediated knockdown of Lhcgr (Cy-3, red) in 3T3-L1 adipocytes. DAPI, siRNA (GFP), LHCGR (CY3) and merged images are shown for siControl and siLhcgr.

[0030] Figure 2D is a series of representative fluorescence microscopy images of the specific binding of AlexaFluor-488 labeled hCG to differentiated 3T3-L1 cells.

[0031] Figure 2E Is a series of Western blots probing ERK1 / 2 phosphorylation in differentiated 3T3L1 adipocytes after treatment with hCG, LH, ORG 43553.

[0032] Figure 2Fis a set of graphs of the log2 (fold change) of Lep, Cebpb, Cebpa, Ucp2 and Ppargc1b ​​adipogenic genes measured by RNAseq in differentiated 3T3-L1 adipocytes after treatment with 1 nM LH or hCG for 12 hours.

[0033] Figure 2G is a set of representative images and graphs of oil droplet accumulation (Oil Red O staining) in differentiated 3T3-L1 adipocytes.

[0034] Figure 3A It is a female Lhcgr fed normal chow. + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of fat mass (g) over time (weeks) compared to littermates.

[0035] Figure 3B It is a female Lhcgr fed a normal diet + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of lean mass (g) over time (weeks) compared to littermates.

[0036] Figure 3C It is a female Lhcgr fed a normal diet + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of total mass (g) versus time (weeks) compared to littermates.

[0037] Figure 3D Is with Lhcgr + / + Compared with littermates, Lhcgr - / - and Lhcgr + / - A series of representative images of H&E staining of adipocytes in iWAT and gWAT in mice.

[0038] Figure 3E is a graph of glucose tolerance in the three genotypes.

[0039] Figure 3F is a graph of serum estrogen levels in the three genotypes.

[0040] Figure 3G Male Lhcgr fed a high fat diet (HFD) + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of fat mass (g) over time (weeks) compared to littermates.

[0041] Figure 3H Male Lhcgr fed a high fat diet (HFD) + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of lean body mass (g) over time (weeks) compared to littermates.

[0042] Fig. 3I Male Lhcgr fed a high fat diet (HFD) + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of total mass (g) versus time (weeks) compared to littermates.

[0043] Figure 3J Is with Lhcgr + / + Compared with littermates, Lhcgr - / - and Lhcgr + / - A series of representative images of H&E staining of adipocytes in iWAT and gWAT in mice.

[0044] Figure 3K Male Lhcgr fed a high fat diet (HFD) + / - and Lhcgr - / - Mouse and Lhcgr + / + Plot of glucose tolerance compared to littermates.

[0045] Figure 4A is a graph of fat mass (g) in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0046] Figure 4B is a graph of lean body mass (g) in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0047] Figure 4C is a graph of total mass (g) of 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0048] Figure 4D is a graph of food intake (g) in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0049] Figure 4E is a graph of the weights (g) of various fat depots in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0050] Figure 4Fis a graph of glucose tolerance in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0051] Figure 4G is a graph of serum testosterone levels (ng / mL) in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0052] Figure 4H is a graph of serum leptin levels (ng / mL) in 14-week-old C57BL / 6 male mice fed a high-fat diet following twice-weekly injections of high-dose LH or hCG.

[0053] Figure 5A is a graph of fat mass (g) over time in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0054] Figure 5B is a graph of lean body mass (g) over time in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0055] Figure 5C is a graph of total mass (g) over time in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0056] Figure 5D is a graph of food intake (g) in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0057] Figure 5E is a graph of the weight (g) of various fat depots in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0058] Fig. 5F is a graph of glucose tolerance in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0059] Figure 5G is a graph of leptin levels (ng / ml) in mice treated with vehicle+placebo, vehicle+flutamide, hCG+placebo, or hCG+flutamide.

[0060] Fig. 6A is a graph of fat mass (g) of 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injection of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0061] Figure 6B is a graph of total mass (g) of 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0062] Figure 6C is a graph of lean body mass (g) of 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0063] Fig.6D is a graph of food intake (g) of 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injection of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0064] Fig. 6E is a graph of the weights (g) of various adipose depots in 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0065] Fig. 6F is a graph of glucose tolerance in 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0066] Figure 6G is a graph of serum testosterone levels (ng / mL) in 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0067] Figure 6H is a graph of serum leptin levels (ng / mL) in 14-week-old C57BL / 6 mice (25 mice / group) treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks.

[0068] Fig. 7A is a graph of relative Lhcgr expression levels measured by qPCR in white adipose tissue (WAT) depots in male and female C57BL / 6 mice.

[0069] Figure 7B RNAscope TM A series of representative images of in situ hybridization showing male and female Lhcgr - / -and Lhcgr + / + Expression of Lhcgr in various mouse fat depots and gonads in mice.

[0070] Figure 7C The polynucleotide and amino acid sequences of the LHCGR ectodomain (shaded) identical to the ovarian receptor, verified by Sanger sequencing.

[0071] Fig.7D Is to show male and female Lhcgr - / - and Lhcgr + / + A series of representative immunohistochemistry images of the expression of LHCGR in various mouse adipose depots and gonads in mice.

[0072] Fig. 7E In males and females Lhcgr - / - and Lhcgr + / + A series of representative fluorescence microscopy images of intraperitoneal AlexaFluor-488-labeled hCG binding to gonadal and subcutaneous fat pads in mice.

[0073] Figure 7F In male and female mice, the drug was injected intravenously in multiple organs including mesenteric WAT and gonadal WAT. 89 Plot of the percentage of injected dose per gram of tissue measured after Zr-LH.

[0074] Figure 7G Is a series of Western blots probing ERK1 / 2 phosphorylation in differentiated 3T3L1 adipocytes following treatment with hCG, LH, ORG 43553.

[0075] Fig. 8A Is a female Lhcgr on a normal diet + / - and Lhcgr + / + Plots of fat mass (g) of mice over time and serum estrogen levels at the same time intervals.

[0076] Figure 8B Is a high-fat diet male Lhcgr + / - and Lhcgr + / + Plot of fat mass (g) over time in mice and plot of serum testosterone levels over the same time intervals.

[0077] Figure 8C Graphs of fat mass (g) and food intake (g) of 14-week-old male C57BL / 6 mice (N=20 / group) fed a high-fat diet and injected intraperitoneally with LH (250 ng), hCG (250 ng), or vehicle (200 μl saline) twice a week for 6 weeks.

[0078] Fig.8D Male Lhcgr were fed a high-fat diet and injected intraperitoneally with LH (250 ng) or vehicle (200 μl saline) twice a week for 6 weeks. - / - Plot of fat mass (g) in mice.

[0079] Fig. 8E Graphs are of fat mass (g) and food intake (g) of 14-week-old male C57BL / 6 mice (N=20 / group) fed a high-fat diet and injected intraperitoneally twice weekly for three weeks with vehicle, vehicle and flutamide, hCG, or hCG and flutamide.

[0080] Fig.8F Is a graph of fat mass (g) and food intake (g) of 14-week-old male C57BL / 6 mice injected with LHCGR agonist ORG 43553 (17 mg / kg, subcutaneously, twice a week) or vehicle for 9 weeks (left). In the middle is a graph of tissue weight (g) of various fat depots of 14-week-old male C57BL / 6 mice injected with LHCGR agonist ORG 43553 (17 mg / kg, subcutaneously, twice a week) or vehicle for 9 weeks. On the right is a graph of serum testosterone levels (ng / mL) of the same mice.

[0081] Figure 8G is a graph of adipocyte distribution in iWAT of 14-week-old male C57BL / 6 mice treated with ORG 43553 (17 mg / kg, subcutaneously, twice a week) or vehicle for 9 weeks. Cell size ≤ 4000 μm is also shown. 2 and cell size <4000 μm 2 Representative images of total adipocyte area and H&E staining of adipocytes.

[0082] Figure 8H Figures are graphs of fat mass (g) (top), total mass (g) (middle), tissue weight (g) (lower left), and testosterone levels (ng / mL) (lower right) in 14-week-old male C57BL / 6 mice injected with ORG 43553 (17 mg / kg, subcutaneously, twice weekly) or vehicle for 9 weeks.

[0083] Fig. 9A A series of representative microscopic images of Oil Red O staining of 3T3.L1 adipocytes treated with ORG 43553, hCG, or vehicle, and quantification of Oil Red O staining.

[0084] Fig. 9B A series of representative images of paraffin sections of organoids derived from 3T3-L1 cells. H&E staining, RNAscopeTM Images of in situ hybridization and immunohistochemistry. Quantification of the thickness (μm) of the adipocyte layer is also shown.

[0085] Fig. 9C Figure 1 is a graph of oxygen consumption rate (OCR) of 3T3-L1 adipocytes pretreated with ORG 43553 for 1 hour prior to measuring OCR on a Seahorse Xf96 analyzer (left). Figure 2 is a graph of OCR of 3T3-L1 adipocytes treated with ORG43553, LH or hCG at basal measurement and after addition of oligomycin.

[0086] Fig.9D On the right is a graph of oxygen consumption (VO2) in high fat diet mice treated with ORG 43553. On the right is a table of VO2 and energy expenditure in mice treated with ORG43553 or vehicle.

[0087] Fig. 10A It is the chemical structure of ORG 43553 [5-amino-2-methylthio-4-[3-(2-morpholin-4-yl-acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-butylamide].

[0088] Fig. 10B is a schematic diagram of the binding of ORG 43553 to mouse LHCGR.

[0089] Fig. 10C Schematic diagram of the side view (left) and top view (right) of ORG 43553 in complex with LHCGR at 280 ns molecular dynamics. DETAILED DESCRIPTION

[0090] Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) receptors

[0091] The luteinizing hormone / chorionic gonadotropin receptor (LHCGR) is a member of the G protein-coupled receptor (GPCR) subfamily, characterized by the presence of a large N-terminal extracellular domain containing several leucine-rich repeats (LRRs). This family of glycoprotein hormone receptors is designated the LRR-containing GPCR (LGR) family. The human LHCGR protein is encoded by the LHCGR gene at the cytogenetic location 2p16.3, genomic coordinates (GRCh38): 2: 48,686,774-48,755,724. The amino acid sequence of the human LHCGR precursor protein is provided below as SEQ ID NO: 1.

[0092] (SEQ ID NO:1)

[0093] In some embodiments, the methods disclosed herein comprise administering to a subject one or more LHCGR agonists. In some embodiments, the LHCGR agonist is a thieno[2,3-d]pyrimidine derivative. In some embodiments, the thieno[2,3-d]pyrimidine derivative is a compound according to Formula I or a pharmaceutically acceptable salt thereof.

[0094]

[0095] In some embodiments, R1 and R2 together with the nitrogen atom to which they are bound form a ring having 2-6 carbon atoms, optionally containing one or more heteroatoms selected from N, O and / or S. In some embodiments, the thieno [2,3-d] pyrimidine derivative is a compound selected from the group consisting of tert-butyl 5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetylamino)-phenyl)-thieno [2,3-d] pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetylamino)-phenyl)-thieno [2,3-d] pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetylamino)-phenyl)-thieno [2,3-d] pyrimidine-6-carboxamide, tert-Butyl 5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-Butyl 5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide or tert-Butyl 5-amino-2-methylthio-4-(3-(2-(piperazin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide.

[0096] In some embodiments, the thieno[2,3-d]pyrimidine derivative is ORG 43533. ORG 43553 is an orally active, low molecular weight (LMW) LHCGR agonist. ORG 43553 exhibits agonist activity on human LHCGR, EC 50 The value is 3.7 nM. The structure of ORG 43553 is provided below:

[0097]

[0098] Pharmaceutical composition

[0099] In some aspects, the compositions and methods described herein include making and using pharmaceutical compositions and medicaments that include one or more compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.

[0100] In some aspects, compositions disclosed herein may include other compounds, drugs or agents for treatment. For example, in some cases, pharmaceutical compositions disclosed herein may be combined with one or more (e.g., one, two, three, four, five or less than ten) compounds.

[0101] In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the compound or its delivery form.

[0102] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant or solvent. As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are generally considered to be physiologically tolerable and generally do not produce allergic or similar adverse reactions, such as stomach discomfort and dizziness, etc. when applied to humans. Pharmaceutically acceptable carriers, adjuvants or solvents are compositions that can be applied to patients together with the compounds of the present invention and do not destroy their pharmacological activity and are nontoxic when applied in a dose sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants and solvents include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc. that are compatible with drug administration.

[0103] In particular, pharmaceutically acceptable carriers, adjuvants and solvents that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopheryl polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrins can also be advantageously used to enhance the delivery of compounds of the formula described herein.

[0104] As used herein, the compounds disclosed herein are defined as including pharmaceutically acceptable derivatives or prodrugs thereof. "Pharmaceutically acceptable derivatives" means any pharmaceutically acceptable salt, solvate or prodrug of a compound or medicament disclosed herein, such as a salt or other derivative of a carbamate, ester, phosphate, ester, which can (directly or indirectly) provide a compound or its active metabolite or residue as described herein when applied to a recipient. Particularly advantageous derivatives and prodrugs are those that increase the bioavailability of compounds disclosed herein when such compounds are applied to mammals (e.g., by making the orally administered compound more easily absorbed into the blood) or enhance the delivery of the parent compound to biological compartments (e.g., brain or lymphatic system) relative to the parent substance. Preferred prodrugs include derivatives of the structure of formulas described herein that are attached to groups that enhance water solubility or active transport by intestinal membranes. Such derivatives are recognized by those skilled in the art without excessive experiments. Nonetheless, reference is made to the teachings of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Volume 1: Principles and Practice, which is incorporated herein by reference to the extent it teaches such derivatives.

[0105] The compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates, as well as the meso form and pharmaceutically acceptable salts, solvent complexes, morphological forms or deuterated derivatives thereof.

[0106] In particular, pharmaceutically acceptable salts of compounds disclosed herein include, for example, those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecyl sulfate, formates, fumarates, glycolates, hemisulfates, enanthate, caproates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, palmitates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, tartrates, toluenesulfonates, trifluoromethylsulfonates, and undecanoates. Salts derived from suitable bases include, for example, alkali metal (e.g., sodium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts. The present invention also contemplates the quaternization of any basic nitrogen-containing groups of compounds disclosed herein. Water-soluble or oil-soluble or dispersible products can be obtained by such quaternization.

[0107] In some aspects, pharmaceutical compositions disclosed herein may include one or more compounds of an effective amount. As used herein, the terms "effective amount" and "effective treatment" refer to the amount or concentration of one or more compounds or pharmaceutical compositions described herein that effectively cause a desired effect or physiological result, for a period of time (including acute or chronic administration and periodic or continuous administration) in the context of its administration. In some aspects, pharmaceutical compositions may further include one or more other compounds, drugs or medicaments for treatment in an amount that effectively causes a desired effect or physiological result.

[0108] In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in, imported into, or exported from the United States.

[0109] The present disclosure also encompasses the therapeutic combinations disclosed herein in the form of kits or packaged preparations. Kits or packaged preparations as used herein include one or more doses of the subject peptide and its salt in a container, and the container packs the dosage and the instructions for simultaneous or sequential administration to the patient together. For example, packaging can include peptides and drug carriers combined in powder form for mixing in an aqueous solution that can be taken in by a sick subject. Packaging or kits include suitable instructions, which include charts, records (e.g., audio, video, CDs) and computer programs, providing guidance for the use of conjoint therapy. The aforementioned description of the specific embodiments of the present disclosure is given for the purpose of illustration and description. They are not intended to be exhaustive or limit the present disclosure to the disclosed precise form, and it is apparent that many modifications and variations are possible according to the above teachings.

[0110] Administration of pharmaceutical compositions

[0111] Pharmaceutical compositions disclosed herein can be formulated or adapted to be administered to a subject via any route (e.g., any route approved by the Food and Drug Administration (FDA)). Exemplary methods are described in the FDA Data Standards Manual (DSM). In particular, pharmaceutical compositions can be formulated for oral, parenteral or transdermal delivery and administered via oral, parenteral or transdermal delivery. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0112] For example, a pharmaceutical composition disclosed herein can be administered, for example, topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subcutaneously (e.g., by injection or via an implanted reservoir), or ophthalmically.

[0113] For example, the pharmaceutical composition of the present invention can be orally administered in any oral acceptable dosage form (including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersants and solutions). In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When an aqueous suspension or emulsion is administered orally, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If necessary, certain sweeteners, flavorings or coloring agents can be added.

[0114] For example, the pharmaceutical compositions of the present invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0115] For example, the pharmaceutical compositions of the invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing agents or dispersants known in the art.

[0116] For example, the pharmaceutical composition of the present invention can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated using suitable dispersants or wetting agents (e.g., such as Tween 80) and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable solvents and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used to prepare injections, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions, or carboxymethyl cellulose or similar dispersants. Other commonly used surfactants commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms such as Tweens, Spans or other similar emulsifiers or bioavailability enhancers may also be used for formulation purposes.

[0117] In some aspects, the effective dose of the pharmaceutical composition of the present invention can include, but is not limited to, for example, about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8 .8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000 or 10000 mg / kg / day, or as required for a particular pharmaceutical composition.

[0118] When the pharmaceutical compositions disclosed herein include a compound of the formula described herein and one or more additional compounds (e.g., for the treatment of obesity or any other obesity-related condition or disease, including one or more additional compounds, drugs or agents known to be associated with or caused by obesity) in combination, both the compound and the additional compound should be present at a dosage level of between about 1% and 100% of the dose typically administered in a monotherapy regimen, and more preferably between about 5% and 95%. Additional agents can be administered separately from the compounds of the present invention as part of a multiple dose regimen. Alternatively, these agents can be part of a single dosage form, mixed together with the compounds of the present invention in a single composition.

[0119] In some aspects, a pharmaceutical composition disclosed herein may be included in a container, pack, or dispenser together with instructions for administration.

[0120] Combination therapy

[0121] In various embodiments, the compounds described herein or compositions described herein can be administered to a subject in combination with one or more other therapies (e.g., biotherapy or antibody therapy). In some embodiments, pharmaceutical compositions described herein can be administered to a subject in combination with one or more therapies. One or more other therapies can be in the same composition or in different compositions with the compounds described herein.

[0122] In certain embodiments, therapy is administered less than 5 minutes interval, less than 30 minutes interval, 1 hour interval, about 1 hour interval, about 1 to about 2 hours interval, about 2 hours to about 3 hours interval, about 3 hours to about 4 hours interval, about 4 hours to about 5 hours interval, about 5 hours to about 6 hours interval, about 6 hours to about 7 hours interval, about 7 hours to about 8 hours interval, about 8 hours to about 9 hours interval, about 9 hours to about 10 hours interval, about 10 hours to about 11 hours interval, about 11 hours to about 12 hours interval, about 12 hours to 18 hours interval, 18 hours to 24 hours interval, 24 hours to 36 hours interval, 36 hours to 48 hours interval, 48 hours to 52 hours interval, 52 hours to 60 hours interval, 60 hours to 72 hours interval, 72 hours to 84 hours interval, 84 hours to 96 hours interval, or 96 hours to 120 hours interval. In some embodiments, two or more therapies are administered simultaneously. Two or more therapies can be administered with the same composition or different compositions. Furthermore, the two or more therapies can be administered by the same route of administration or by different routes of administration.

[0123] Treatment

[0124] Methods disclosed herein contemplate the administration of an effective amount of a compound or composition to achieve a desired or described effect. Typically, the compound or composition of the present invention will be administered about 1 time to about 6 times a day, or alternatively or additionally as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the treated host and the specific mode of administration. Typical preparations will contain about 5% to about 95% (w / w) of active compound. Alternatively, such preparations can contain about 20% to about 80% of active compound.

[0125] In some aspects, the present disclosure provides methods of using compositions comprising compounds, including a pharmaceutical composition disclosed herein (hereinafter denoted as "X") in the following methods:

[0126] Substance X for use as a medicament for treating one or more diseases or conditions disclosed herein. Use of substance X in the manufacture of a medicament for treating Y; and Substance X for use in treating Y.

[0127] In some aspects, the disclosed methods include administering a therapeutically effective amount of one or more compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) that is in need or has been determined to need such treatment. In some aspects, the disclosed methods include selecting a subject and administering an effective amount of one or more compounds or compositions described herein to the subject, and optionally repeating the administration as needed to prevent or treat obesity or obesity-related diseases.

[0128] In some respects, subject selection can include obtaining sample from subject (for example, candidate subject) and test sample to indicate that subject is suitable for selection.In some respects, subject can be confirmed or identified as once suffered from or suffering from the patient's condition or disease by health care professional, for example.In some respects, suitable subject includes for example suffering from or once suffered from the patient's condition or disease but disease or one of its aspects disappears, presents the disease symptoms of alleviation (for example, relative to other subjects (for example, most subjects) suffering from the same patient's condition or disease), or survives the extended time period (for example, relative to other subjects (for example, most subjects) suffering from the same patient's condition or disease) in the case of suffering from the patient's condition or disease, for example, in asymptomatic state (for example, relative to other subjects (for example, most subjects) suffering from the same patient's condition or disease).In some respects, the positive immune response for the patient's condition or disease can be shown from the indication of patient record, family history or detection positive immune response.In some respects, subject selection can include multiple parties.For example, the first party can obtain sample from candidate subject and the second party can test sample.In some respects, subject can be selected or mentioned by medical practitioner (for example, general practitioner). In some aspects, subject selection can include obtaining a sample from the selected subject and storing the sample or using the sample in the methods disclosed herein.The sample can include, for example, a cell or a cell population.

[0129] In some aspects, the method of treatment can include single administration, multiple administration and repeated administration of one or more compounds disclosed herein to prevent or treat the disease or condition suffered by the subject as needed. In some aspects, the method of treatment can include assessing the disease level of the subject before, during or after treatment. In some aspects, treatment can continue until a reduction in the disease level of the subject is detected.

[0130] As used herein, the term "subject" refers to any animal. In some cases, the subject is a mammal. In some cases, as used herein, the term "subject" refers to a human (e.g., a male, female, or child).

[0131] As used herein, the terms "administer," "administering," or "administration" refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of its form. For example, the methods disclosed herein include administering an effective amount of a compound or composition to achieve a desired or described effect.

[0132] As used herein, the terms "treat," "treating," or "treatment" refer to partially or completely alleviating, inhibiting, ameliorating, or alleviating a disease or condition suffered by a subject. This means any manner in which one or more symptoms of a disease or condition are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular condition refers to any relief, whether permanent or temporary, lasting or transient, attributable to or associated with treatment by the compositions and methods of the invention.

[0133] As used herein, the terms "prevent", "preventing" and "prevention" shall refer to a reduction in the occurrence of a disease in a subject or a reduction in the risk of developing a disease or its associated symptoms. Prevention can be complete, such as the complete absence of disease or pathological cells in a subject. Prevention can also be partial, such that the occurrence of a disease or pathological cells in a subject is less, occurs later, or develops more slowly than would occur in the absence of the present invention.

[0134] As used herein, the term "preventing a disease" in a subject means, for example, preventing the development of one or more symptoms of a disease in a subject before they occur or are detectable, for example, by the patient or the patient's physician. Preferably, the disease does not develop at all, i.e., no symptoms of the disease are detectable. However, it may also mean delaying or slowing the development of one or more symptoms of the disease. Alternatively or additionally, it may mean reducing the severity of one or more symptoms that subsequently develop.

[0135] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom, and the judgment of the treating physician.

[0136] The effective amount can be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., effective dose) of the therapeutic compound depends on the selected therapeutic compound. In addition, treating a subject with a therapeutically effective amount of a compound or composition as described herein can include a single treatment or a series of treatments. For example, at least one effective amount can be administered. The composition can be administered once or more per day to once or more per week, including once every other day. It will be appreciated by those skilled in the art that certain factors can affect the dosage and timing required for the effective treatment of a subject, including but not limited to the severity of the disease or condition, previous treatment, the general health or age of the subject, and other diseases present.

[0137] After administration, the subject can be evaluated to detect, assess or determine its disease level. In some cases, treatment can be continued until a change (e.g., reduction) in the disease level in the subject is detected. When the patient's condition improves (e.g., a change (e.g., reduction) in the subject's disease level), if necessary, a maintenance dose of a compound or composition disclosed herein can be administered. Subsequently, the dosage or frequency of administration or both can be reduced to the level of the improved condition, for example, as a function of symptoms. However, once there is any recurrence of disease symptoms, the patient may need long-term intermittent treatment.

[0138] The publications disclosed herein are provided only for disclosure prior to the date of submission of the present disclosure. Anything herein should not be construed as admitting that the present disclosure is not entitled to precede such publications. In addition, the publication date provided may be different from the actual publication date, and the actual publication date may need to be independently confirmed.

[0139] Each application and patent cited herein, and each document or reference, patent or non-patent document cited in each application and patent (including during the prosecution of each issued patent; "application cited documents"), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each application cited document, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited herein (either in the reference list preceding the claims or in the present document itself); and each of these documents or references ("herein cited references") and each document or reference cited in each reference cited herein (including any manufacturer's specifications, instructions, etc.) are hereby expressly incorporated herein by reference.

[0140] The following non-limiting examples are provided to further illustrate the present disclosure.

[0141] Example

[0142] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0143] method

[0144] The following materials and methods were used in the following examples.

[0145] qPCR: Total RNA was isolated using TRIzol reagent (Invitrogen, 15596018) and reverse transcribed (Invitrogen, 18064014). qPCR was performed using SYBR Green Master Mix (Applied Biosystems 4472918).

[0146] SANGER sequencing: RNA from mouse genital adipose tissue was reverse transcribed (Invitrogen 18080093). A fragment of Lhcgr was amplified by PCR and sequenced.

[0147] RNASCOPE TM :Using RNAscope TM Probe-Mm-Lhcgr (ACD 408171) and RNAscope TM Paraffin sections were processed and stained using 2.5 HD Reagent Kit-Brown (ACD 322300). IHC: Paraffin sections were stained with rabbit LHCGR polyclonal antibody (Invitrogen, PA5-79598) and anti-rabbit HRP-IgG (Leica RE7161).

[0148] Biodistribution: Male and female C57BL / 6 mice (5 mice / group) were injected intravenously 89 Zr-LH, individual tissues were dissected 2 h later for gamma counting.

[0149] hCG conjugation: Recombinant hCG was conjugated with Alexa Fluor 488 Micro Protein Labeling Kit (Invitrogen A30006) and injected intraperitoneally (0.45 μg).

[0150] Cell culture: Differentiated 3T3-L1 preadipocytes (Zen-Bio) were treated with hCG or LH for 20 minutes.

[0151] siRNA knockdown: Lentiviral vectors expressing Lhcgr siRNA (ABM 2649609) or siControl (ABM LVP015-G) were used according to the manufacturer's recommendations.

[0152] Western blotting: Proteins were blotted using commercial antibodies (Cell Signaling, phosphorylated ERK1 / 2, 9101S; ERK1 / 2, 4695S, 1:5000).

[0153] Statistics: Two-tailed Student's t-test or two-way ANOVA was used (*p<0.05; **p<0.01).

[0154] Example 1: Lhcgr expression, binding and activation in mouse adipocytes

[0155] qPCR was used to measure relative expression in various male and female mouse adipose depots and the results are plotted in Figure 1A In. Figure 1BAs shown, the extracellular domain sequence of Lhcgr in mouse gonadal white adipose tissue (gWAT) is identical to that in the ovarian receptor. Figure 1C As shown, using RNAscope TM In situ hybridization and immunohistochemistry (IHC) were used to visualize the expression of LHCGR in various mouse fat depots and gonads of male and female C57BL / 6 mice. - / - No staining was detected in mice, indicating the specificity of the LHCGR probe and antibody. Figure 1D As shown, significant concentrations of intravenously injected WT were detected in multiple organs including mesenteric WAT and gonadal WAT. 89 Zr-LH. Figure 1E As shown, intraperitoneally injected AlexaFluor-488-labeled hCG localizes in mouse iWAT and gWAT. This hCG binding further confirms the presence of LHCGR in adipose tissue.

[0156] Example 2: LHCGR is expressed in adipocytes and is fully functional

[0157] like Figure 2A As shown, the relative Lhcgr expression in mouse iWAT and 3T3-L1 adipocytes measured by qPCR indicated that Lhcgr is expressed in mouse iWAT and 3T3-L1 adipocytes. Figure 2B As shown, RNAscope TM In situ hybridization and immunohistochemistry (IHC) showed LHCGR staining in differentiated 3T3-L1 mouse adipocytes. Figure 2C As shown, siRNA-lentivirus-mediated knockdown of Lhcgr (Cy-3, red) in 3T3-L1 adipocytes confirmed the antibody specificity. The GFP reporter gene (green) showed the high efficiency of lentiviral infection. Figure 2D As shown, the binding of AlexaFluor-488-labeled hCG to differentiated 3T3-L1 cells is specific, as this binding can be competitively displaced by pretreatment with unlabeled hCG. Figure 2EAs shown, LH and hCG induced ERK1 / 2 phosphorylation in differentiated 3T3L1 adipocytes in a dose-dependent manner, indicating that adipose LHCGR is fully functional. hCG-induced ERK1 / 2 phosphorylation was abolished by LHCGR siRNA knockdown, further indicating that ERK1 / 2 phosphorylation by LH in adipocytes acts through LHCGR. RNAseq was performed after treatment with 1 nM LH or hCG for 12 hours to measure the log2 (fold change) of Lep, Cebpb, Cebpa, Ucp2, and Ppargc1b ​​adipogenic genes in differentiated 3T3-L1 adipocytes. As Figure 2F As shown in Figure 2, several adipose genes were downregulated, including leptin (Lep). Figure 2G As shown, hCG treatment reduced oil droplet accumulation (Oil Red O staining) in differentiated 3T3-L1 adipocytes.

[0158] Example 3: Lhcgr deficiency causes weight gain in chow-fed female mice and high-fat diet (HFD) male mice

[0159] Male and female Lhcgr + / + 、Lhcgr + / - and Lhcgr - / - The mice were fed either a normal chow diet or a high-fat diet for 40 weeks. Figure 3A As shown, with Lhcgr + / + Compared with their littermates, female mice fed a normal diet had significantly higher Lhcgr + / - and Lhcgr - / - The mice showed higher fat mass, suggesting an anti-obesity effect of LH. Figure 3C As shown, with Lhcgr + / + Compared with mice, Lhcgr - / - The mice also showed a higher total mass. Figure 3D As shown, H&E staining showed that relative to Lhcgr + / + Littermates, Lhcgr - / - and Lhcgr + / - The adipocytes in iWAT and gWAT of mice were larger. Figure 3B , Figure 3E and Figure 3F As shown, there were no differences in lean body mass, glucose tolerance, or serum estrogen between the three genotypes. Figure 3G As shown, in a high-fat diet, + / + Compared with mice, male Lhcgr + / - and Lhcgr - / - The mice showed higher fat mass. Fig. 3I As shown, there was no difference in total mass among the three genotypes. Figure 3JAs shown, H&E staining showed that Lhcgr - / - and Lhcgr + / - Adipocytes in iWAT and gWAT are larger in mice. Figure 3H As shown, with Lhcgr + / + and Lhcgr + / - Compared with littermates, male mice on HFD had increased Lhcgr - / - The mice showed lower lean body mass. Figure 3K As shown, glucose tolerance was the same between the different genotypes.

[0160] Example 4: LH and hCG reduce HFD-induced weight gain in C57BL / 6 mice

[0161] 14-week-old C57BL / 6 mice (20 mice / group) were treated with intraperitoneal injections of vehicle (200 μL saline), LH (250 ng / mouse), or hCG (250 ng / mouse) and fed a high-fat diet (TestDiet0056833) for 6 weeks. Figure 4A and Figure 4E As shown in Figure 2, high doses of LH injected twice weekly into 14-week-old C57BL / 6 male mice fed a high-fat diet significantly reduced body fat mass. Figure 4B As shown, the same treatment increased lean body mass, and as Figure 4C As shown, this treatment reduces the overall mass. Figure 4D As shown, food intake was similar in all groups. Figure 4A As shown, hCG-treated mice showed lower fat mass and Figure 4E As shown, hCG-treated mice showed lower iWAT weight. Figure 4F As shown, there was no difference in glucose tolerance among the three treatment groups. Figure 4G As shown, mice treated with LH or hCG showed elevated testosterone. Figure 4H As shown, mice treated with hCG and LH had lower leptin levels.

[0162] Example 5: The anti-obesity effect of LH / hCG is independent of testosterone

[0163] To evaluate the anti-obesity effects of LH / hCG relative to testosterone levels, the androgen receptor antagonist flutamide was used to block the increase in testosterone after LH / hCG treatment in mice. 14-week-old C57BL / 6 mice (15 mice / group) underwent the following intraperitoneal injections - Group I: vehicle and placebo (placebo 60-day release pellets); Group II: vehicle and flutamide (7.5 mg 60-day release pellets); Group III: hCG (250 ng / mouse) and placebo; Group IV: hCG and flutamide. The mice were fed a high-fat diet for 6 weeks. Figures 5A-5C As shown, in the presence of flutamide, hCG reduced diet-induced fat mass (A), but did not reduce lean body mass (B) or total mass (C), indicating a lack of a role for testosterone in the effect of hCG on reducing body fat. Figure 5E As shown in Figure 2, mice treated with hCG had lower iWAT and gWAT weights in both the flutamide and placebo groups. Figure 5D and Fig. 5F As shown, food intake (D) and glucose tolerance (F) were similar in all groups. Figure 5G As shown, mice treated with hCG had lower leptin levels in both the flutamide group and the placebo group.

[0164] Example 6: LHCGR agonist ORG 43553 reduces body fat

[0165] To evaluate the effect of treatment with the LHCGR agonist ORG 43553 on body fat in mice, 14-week-old C57BL / 6 mice (25 mice / group) were treated with subcutaneous injections of vehicle or ORG 43553 (17 mg / kg) and fed a high-fat diet for 9 weeks. Figures 6A-6C As shown, ORG 43553 reduced diet-induced fat mass (A), total mass (B), but not lean mass (C) in mice. Fig. 6E As shown, mice treated with ORG 43553 had lower iWAT, gWAT, and mWAT weights. Food intake ( Fig.6D ), glucose tolerance ( Fig. 6F ), testosterone ( Figure 6G ) and leptin levels ( Figure 6H )resemblance.

[0166] Example 7: LHCGR expression in adipose tissue

[0167] To characterize Lhcgr expression in adipose tissue, qPCR, RNAscope and PCR were performed on Lhcgr transcripts in mouse adipocytes. TMIn situ hybridization and immunohistochemistry (IHC). The relative expression of Lhcgr transcripts in white adipose tissue (WAT) depots in male and female C57BL / 6 mice measured by qPCR is shown in Fig. 7A In female mice, expression in gonadal WAT (gWAT) is close to that in the ovary itself (gonad). Figure 7B As shown, RNAscope TM In situ hybridization confirmed Lhcgr transcripts (dark spots) in gonads, gWAT, and subcutaneous WAT (sWAT) of wild-type mice of both sexes, but Lhcgr - / - Not in mice. Figure 7C Expression was verified by Sanger sequencing of the LHCGR extracellular domain, which is identical to the ovarian receptor. Fig.7D As shown, immunohistochemistry demonstrated LHCGR expression in gWAT and sWAT.

[0168] The binding of labeled hCG was studied in vivo. Fig. 7E As shown, intraperitoneal AlexaFluor-488-labeled hCG bound to gonadal and subcutaneous fat pads in wild-type mice but not in LHCGR-deficient mice (LHCGR-null mice). 89 Zr-LH, then sacrificed two hours later and perfused with PBS before organ removal and gamma counting. Figure 7F As shown in Figure 2, radioactivity was detected in mesenteric WAT (mWAT), inguinal WAT (iWAT), and gWAT, as well as other organs including the gonads. Figure 7G As shown, LH, hCG and ORG 43553 rapidly induced ERK1 / 2 phosphorylation in a concentration-dependent manner in differentiated 3T3.L1 cells, as expected, with no signal in siLhcgr cells. These results suggest that adipose LHCGR is functional. In summary, these results suggest that adipose tissue LHCGR is a target of LH action.

[0169] Example 8: LH-induced weight loss

[0170] like Fig. 8A As shown by EchoMRI TM -100 measured Lhcgr in females fed a normal diet + / - The mice gained fat without a decrease in serum estrogen. Figure 8B As shown, male Lhcgr + / -Mice became obese on a high-fat diet (TestDiet0056833) and, importantly, serum testosterone did not decrease. This gene-dosage effect of Lhcgr on obesity suggests a significant physiological role for LH on body composition.

[0171] like Figure 8C In gain-of-function studies, 14-week-old male C57BL / 6 mice (N=20 / group) were fed a high-fat diet and injected intraperitoneally with LH (250 ng), hCG (250 ng), or vehicle (200 μl saline) twice a week for 6 weeks. TM Both LH and hCG significantly reduce body fat accumulation but induce an increase in serum testosterone, as measured by 100-100. Fig.8D As shown, LH (250ng) could not inhibit Lhcgr - / - Fat accumulation in mice, demonstrating that it acts via LHCGR—however, this experiment did not separate the effects of LH on adipose tissue LHCGR from the effects on Leydig cells that produce testosterone. To address the confounding effects of high testosterone on body fat, the study was repeated in the presence of flutamide (7.5 mg, 60-day low-release pellets). Fig. 8E As shown, despite blockade of the androgen receptor by flutamide, the hCG-induced lean phenotype persisted, indicating that the anti-obesity effects of LH and hCG are largely independent of testosterone.

[0172] Next, the LHCGR agonist ORG 43553 (17 mg / kg, subcutaneously, twice a week) was injected into male C57BL / 6 mice (N=24 / group) for 9 weeks. Fig.8F As shown, a significant reduction in fat mass was observed, as well as reduced iWAT, gWAT, and renal WAT (rWAT). Serum testosterone remained unchanged, indicating that the anti-obesity effect of ORG 43553 is independent of testosterone. Figure 8G As shown, the distribution of adipocytes in iWAT showed that in mice treated with ORG 43553, the area was less than 4000 μm 2 The number of adipocytes is large and the area is larger than 4000 μm 2 There are fewer fat cells. Figure 8H As shown, injection of ORG 43553 into the replication cohort resulted in a significant reduction in fat mass and total mass, as well as reduced iWAT, gWAT, and mWAT, independent of testosterone. These data suggest that LHCGR activation induces a lean phenotype. Furthermore, male and female homozygous Lhcgr - / -Mice exhibited more pronounced obesity (data not shown), but this effect is likely confounded by concomitant hypogonadism, especially in old age.

[0173] Example 9: LHCGR agonism inhibits adipocyte differentiation and induces thermogenesis

[0174] To investigate the mechanism of LHCGR activation on body fat, 3T3.L1 preadipocytes and adipose organoids were used. Oil Red O staining can be used to label lipid-containing vacuoles and quantify cellular lipid content. Fig. 9A As shown, treatment of 3T3.L1 adipocytes with hCG (1 nM) or ORG 43553 (6 nM) for 12 hours resulted in reduced oil droplet accumulation (Oil Red O staining), indicating reduced lipid content. Organoids were derived from 3T3-L1 cells cultured on Nunclon Sphera dishes for 8 days and exposed to LH, hCG or ORG 43553 for 3 days. They were further differentiated for 7 days, after which paraffin sections were stained with H&E and analyzed by immunostaining and RNAscope. TM Confirm LHCGR expression. Measure the thickness of the “differentiated cell layer” using QuPath-0.2.3. Fig. 9B As shown in the Figure, organoids treated with LH, hCG or ORG 43553 showed a substantial reduction in the differentiation layer compared to vehicle-treated organoids.

[0175] Next, 3T3-L1 adipocytes were treated with ORG 43553 to determine whether LHCGR agonism also enhances thermogenesis in addition to reducing adipocyte differentiation. Therefore, 3T3-L1 adipocytes were pretreated with ORG 43553 for 1 hour before measuring oxygen consumption rate (OCR) on a Seahorse Xf96 analyzer. Fig. 9C Oligo, FCCP, and rotenone / antimycin-A were added at the indicated times as shown. ORG 43553 increased OCR at baseline and at oligomycin exposure, indicating mitochondrial proton leak, an alternative mode of heat production. To confirm increased energy expenditure in vivo, mice on a high-fat diet (N=6 / group) were acclimated in metabolic cages for 1 day and then injected subcutaneously with ORG 43553 (17 mg / kg, blue line) or vehicle and monitored for 48 hours. Fig.9D As shown, oxygen consumption (VO2) and energy expenditure (EE) increased dramatically in ORG 43553-treated mice, while locomotor activity (distance) did not change. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. These results suggest that the anti-obesity effects of LHCGR agonism arise from reduced adipocyte differentiation and increased thermogenesis.

[0176] Example 10: Structure of ORG 43553 and binding to LHCGR

[0177] Fig. 10A The chemical structure of ORG 43553 [5-amino-2-methylthio-4-[3-(2-morpholin-4-yl-acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-butylamide] is shown. Binding of ORG 43553 to mouse LHCGR was computationally modeled. Unlike LH, which binds to the extracellular domain, ORG 43553 binds to the transmembrane domain (TMD) ( Fig. 10B ). Fig. 10C Shown are the side view (left) and top view (right) of ORG 43553 in complex with LHCGR at 280 ns molecular dynamics.

[0178] Other Implementations

[0179] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A method for treating obesity in a subject in need thereof, comprising administering to the subject in need thereof a thieno[2,3-d]pyrimidine derivative according to formula I or a pharmaceutically acceptable salt thereof, wherein R1 and R2 together with the nitrogen atom to which they are bound form a ring having 2 to 6 carbon atoms, optionally containing one or more heteroatoms selected from N, O and / or S.

2. The method according to claim 1, wherein the thieno[2,3-d]pyrimidine derivative is a compound selected from the group consisting of tert-butyl 5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide or tert-butyl 5-amino-2-methylthio-4-(3-(2-(piperazin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide.

3. The method of claim 1 or claim 2, wherein the thieno[2,3-d]pyrimidine derivative is ORG43533.

4. The method according to any one of claims 1 to 3, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 100 mg / kg.

5. The method of claim 4, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 50 mg / kg.

6. The method of claim 5, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose between 1 and 30 mg / kg.

7. The method of claim 6, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose between 1 and 20 mg / kg.

8. The method of claim 7, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 10 and 20 mg / kg.

9. The method of claim 8, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of 17 mg / kg.

10. The method of any one of claims 1-9, wherein the thieno[2,3-d]pyrimidine derivative is administered daily.

11. The method of any one of claims 1-9, wherein the thieno[2,3-d]pyrimidine derivative is administered weekly.

12. The method of any one of claims 1-11, wherein the thieno[2,3-d]pyrimidine derivative is administered for at least 26 weeks.

13. The method according to any one of claims 1 to 12, wherein the thieno[2,3-d]pyrimidine derivative is provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

14. The method of any one of claims 1-13, further comprising administering luteinizing hormone (LH) to the subject in need thereof.

15. A method for treating diet-induced obesity in a subject in need thereof, comprising administering to the adipose tissue of a subject in need thereof a thieno[2,3-d]pyrimidine derivative according to formula I or a pharmaceutically acceptable salt thereof, wherein R1 and R2 together with the nitrogen atom to which they are bound form a ring having 2 to 6 carbon atoms, optionally containing one or more heteroatoms selected from N, O and / or S.

16. The method according to claim 15, wherein the thieno[2,3-d]pyrimidine derivative is a compound selected from the group consisting of tert-butyl 5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide, tert-butyl 5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide or tert-butyl 5-amino-2-methylthio-4-(3-(2-(piperazin-1-yl)-acetylamino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide.

17. The method of claim 15 or 16, wherein the thieno[2,3-d]pyrimidine derivative is ORG43902.

18. The method of any one of claims 15-17, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 100 mg / kg.

19. The method of claim 18, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 50 mg / kg.

20. The method of claim 19, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 30 mg / kg.

21. The method of claim 20, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 1 and 20 mg / kg.

22. The method of claim 21, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of between 10 and 20 mg / kg.

23. The method of claim 22, wherein the thieno[2,3-d]pyrimidine derivative is administered at a dose of 17 mg / kg.

24. The method of any one of claims 15-23, wherein the thieno[2,3-d]pyrimidine derivative is administered daily.

25. The method of any one of claims 15-23, wherein the thieno[2,3-d]pyrimidine derivative is administered weekly.

26. The method of any one of claims 15-25, wherein the thieno[2,3-d]pyrimidine derivative agonist is administered for at least 26 weeks.

27. The method of any one of claims 15-26, wherein the thieno[2,3-d]pyrimidine derivative is provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

28. The method of any one of claims 15-27, further comprising administering luteinizing hormone (LH) to the subject in need thereof.

29. A method of treating obesity in a subject in need thereof comprising administering to the subject in need thereof a combination of ORG 43553 and ORG 43902.

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