Treatment for metabolic syndrome

Abstract

Disclosed herein are methods of treating metabolic syndrome in a subject using vitamin D receptor agonists. A preferred vitamin D receptor agonist for this invention is elocalcitol.

Description

[Technical Field]

[0001] The invention disclosed herein generally relates to methods of treating metabolic syndrome in a subject using vitamin D receptor agonists. [Background technology]

[0002] Metabolic syndrome is a constellation of co-occurring conditions that increase the risk of heart disease, stroke, and type 2 diabetes. These conditions include excess body fat around the waist (visceral fat), abnormal cholesterol or triglyceride levels (dyslipidemia), high blood pressure (hypertension), insulin resistance or impaired glucose tolerance, high blood sugar (hyperglycemia), obesity, atherosclerosis, diabetes, and fatty liver. Activation of NF-κB signaling has been shown to play a central role in the development of metabolic syndrome; therefore, inhibiting NF-κB may be a therapeutic pathway to address metabolic syndrome. Vitamin D acts as an agonist of a nuclear receptor called the vitamin D receptor, which controls the expression of genes involved in regulating calcium homeostasis and other cellular functions (e.g., inhibiting elevated NF-κB signaling). In particular, elocalcitol, a vitamin D analog, is a high-affinity vitamin D receptor agonist that inhibits NF-κB, making it a promising treatment for metabolic syndrome.

[0003] The prevalence of metabolic syndrome is estimated to be at least 34% in the United States and at least 25% worldwide. The total medical costs of cardiovascular and musculoskeletal complications, cancer, and neurodegenerative diseases attributable to metabolic syndrome, together with lost economic activity, are estimated to be in the trillions of dollars (Van Saklayen, 2018).

[0004] The exact underlying causes of metabolic syndrome vary, but both genetic and lifestyle factors play a major role. Metabolic syndrome develops when chronic excess energy intake leads to an imbalance in energy metabolism, resulting in chronic inflammation and exacerbating underlying diseases and manifesting pathology (Chawla et al., 2011).

[0005] Activation of NF-κB signaling promotes the expression of proinflammatory cytokines such as TNFα, IL1β, IL18, and IL6, priming of the NRLP3 inflammasome, and the emergence of insulin resistance, playing a central role in the development of metabolic syndrome (Shi et al., 2006; Hotamisligil 2006; He et al., 2016; Hotamisligil, 2017). Therefore, NF-κB inhibition may be a therapeutic pathway to address metabolic syndrome (Baker et al., 2011).

[0006] Vitamin D is a steroid produced endogenously or ingested through diet that is converted into an active substance that regulates calcium absorption in the intestinal tract and maintains proper serum calcium and phosphate homeostasis, which is the basis for bone mineralization for bone growth and remodeling. Vitamin D has been shown to play a role in the development and function of multiple organs, including the immune and nervous systems. Vitamin D acts as an agonist at a nuclear receptor called the vitamin D receptor, which controls gene expression involved in regulating calcium homeostasis and other cellular functions (e.g., inhibiting elevated NF-κB signaling). The natural ligand for the vitamin D receptor is 1,25(OH)2D3, but its action as a vitamin D receptor can be mimicked by a number of synthetic vitamin D analogs. As with other receptors, agonists must mimic the action of the natural ligand on its cognate receptor but are not necessarily structural analogs, i.e., in the same chemical class as the natural agonist. The functional class of vitamin D agonists includes vitamin D analogs, including, but not limited to, compounds not classified as steroids, peptides, or nucleic acids.

[0007] Metabolic syndrome is becoming increasingly common, affecting up to one-third of adults in the United States, and therefore there is a need for effective treatments for metabolic syndrome. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the mean body weights (±SD) of experimental animals in the elocalcitol, vitamin D3, and vehicle groups. [Figure 2] FIG. 2 shows the mean relative weight loss or gain of experimental animals in the elocalcitol, vitamin D3, and vehicle groups. [Figure 3A-3B] Figures 3A-3B show the average body weight and weight gain data of the animals: comparison of the average body weight in four groups of mice (LFD, HFD, HFD+vitD, and HFD+Eloc) after 16 weeks of low- or high-fat diet and HFD-fed mice treated with 1,25(OH)2D3 or elocalcitol, data shown as mean ± SEM (**p<0.01; ****p<0.001 - compared to the LFD group; #p<0.05, ###p<0.001 - compared to the HFD group; one-way ANOVA, n=15 per group) (Figure 3A); kinetics of relative average body weight gain of animals in the various experimental groups, data shown as mean ± SEM (*p<0.05; **p<0.01 - HFD+vitD group compared to the HFD group; ##p<0.01 - HFD+Eloc group compared with HFD group; two-way RMANOVA followed by Tukey's multiple comparison post-hoc test, n = 15 per group) (Figure 3B). [Figures 4A-4D]Figures 4A-4C show fat distribution and changes in visceral fat volume. Figure 4D shows lean mass calculated from the volume of the back muscles. Figure 4A shows visceral and subcutaneous fat distribution as shown in axial sections of MRI at the kidney level. Figure 4B shows changes in visceral fat volume after 16 weeks of LFD or HFD diet and treatment with 1,25(OH)2D3 or elocalcitol. Figure 4C shows changes in subcutaneous fat volume after 16 weeks of LFD or HFD diet and treatment with 1,25(OH)2D3 or elocalcitol. Values ​​in Figures 4B and 4C are shown as mean ± SEM (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, two-way ANOVA followed by Tukey's multiple comparison post-hoc test; n=5 per group). [Figures 5A-5D] Figures 5A-5D show comparisons of time points: individual time points (Figures 5A and 5C) and AUC glucose (Figures 5B and 5D) at week 16 of the experiment. Values ​​in Figures 5A and 5C are means ± SEM (*p<0.05, **p<0.01, ***p<0.001 - HFD group compared to LFD group; ***p<0.001, **p<0.01 - HFD+vitD group compared to LFD group; #p<0.05, ##p<0.01, ###p<0.001 - HFD+Eloc group compared to HFD group; RM two-way ANOVA followed by Tukey's multiple comparisons post-hoc test; n=9-10 per group). Values ​​in Figures 5B and 5D are means ± SEM (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA followed by Tukey's multiple comparison post-hoc test; n=9–10 per group). DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of the Invention The present invention relates to methods of using vitamin D receptor agonists to treat metabolic syndrome.

[0010] metabolic syndrome Metabolic syndrome is a collection of co-occurring conditions that increase the risk of heart disease, stroke, and type 2 diabetes. These conditions include excess body fat around the waist (visceral fat), abnormal cholesterol or triglyceride levels (dyslipidemia), high blood pressure (hypertension), insulin resistance or impaired glucose tolerance, high blood sugar (hyperglycemia), obesity, atherosclerosis, diabetes, and fatty liver. Metabolic syndrome is the result of chronic overnutrition. According to the definition of the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III; 2011), metabolic syndrome is present when at least three of the following criteria are met: (i) visceral fat, reflected in disproportionate adipose tissue in and around the abdomen; (ii) atherogenic dyslipidemia (high triglycerides (≥150 mg / dL), high LDL cholesterol, and low HDL cholesterol (≤50 mg / dL)); (iv) high blood pressure (≥130 / 85 mmHg); and insulin resistance or impaired glucose tolerance (inability to properly use insulin or blood glucose, respectively).

[0011] These symptoms of metabolic syndrome are accompanied by a chronic pro-inflammatory state reflected by high levels of high-sensitivity C-reactive protein (CRP) in the blood (Ridker et al., 2003). Individuals with metabolic syndrome are at increased risk for type 2 diabetes, coronary heart disease, heart failure, other diseases associated with plaque buildup in arterial walls (e.g., stroke, myocardial infarction, and peripheral vascular disease), and nonalcoholic steatohepatitis (NASH), the main cause of liver cirrhosis and hepatocellular carcinoma.

[0012] Vitamin D receptor agonists Elocalcitol is a non-hypercalcemic vitamin D analogue and a high-affinity vitamin D receptor agonist that increases bone metabolism (Peleg et al., 2002) and has antiproliferative and anti-inflammatory effects. It inhibits NF-κB by blocking the nuclear translocation of the p65 subunit (Penna et al., 2009). Consequently, elocalcitol has been studied in humans as an experimental drug for a variety of conditions, including overactive bladder, male infertility, chronic nonbacterial prostatitis, benign prostatic hyperplasia, and adult osteoporosis. Elocalcitol is one of the vitamin D receptor agonists with the lowest hypercalcemic potential (Nagpal et al., 2005) and is generally recognized as safe for long-term treatment in adults at oral doses up to 300 μg daily (Montorsi et al., 2008).

[0013] This specification discloses a method of using a vitamin D receptor agonist to treat metabolic syndrome. In some embodiments, one or more symptoms of metabolic syndrome may be improved by administering a vitamin D receptor agonist or a pharmaceutical composition containing a vitamin D receptor agonist. Also provided is the use of a vitamin D receptor agonist in the manufacture of a medicament for treating metabolic syndrome. In some embodiments, the vitamin D receptor agonist of the present invention is elocalcitol.

[0014] In some embodiments, the vitamin D receptor agonist useful in the present invention is the vitamin D analog elocalcitol or a pharmaceutically acceptable salt thereof. Elocalcitol is a synthetic, biologically active vitamin D analog with side chain and A-ring modifications.

[0015] [ka]

[0016] Examples of vitamin D receptor agonists suitable for the present invention include those represented by formulae I-01 to I-57; I-57 is preferred.

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] [ka]

[0021] Another preferred vitamin D receptor agonist suitable for the present invention comprises inecalcitol, which has the International Nonproprietary Name 19-nor-9,10-seco-14βH-cholesta-5(Z),7(E)-dien-23-yno-1α,3β,25-triol-23-yne (Formula II), or a pharmaceutically acceptable salt thereof:

[0022] [ka]

[0023] Metal salts can be formed by adding an inorganic base to the compounds described herein. Examples of suitable metals include lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, and zinc. Examples of suitable metal salts include lithium salts, sodium salts, potassium salts, cesium salts, cerium salts, magnesium salts, manganese salts, iron salts, calcium salts, strontium salts, cobalt salts, titanium salts, aluminum salts, copper salts, cadmium salts, and zinc salts.

[0024] Examples of suitable ammonium salts include triethylamine salts, diisopropylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, morpholine salts, N-methylmorpholine salts, piperidine salts, N-methylpiperidine salts, N-ethylpiperidine salts, dibenzylamine salts, piperazine salts, pyridine salts, pyrazole salts, piperazole salts, imidazole salts, pyrazine salts, piperazine salts, ethylenediamine salts, N,N'-dibenzylethylenediamine salts, procaine salts, chloroprocaine salts, choline salts, dicyclohexylamine salts, and N-methylglucamine salts.

[0025] Treatment for metabolic syndrome Disclosed herein are methods for treating metabolic syndrome using vitamin D receptor agonists, such as elocalcitol, inecalcitol, and compounds of formulas I-01 through I-57. The methods comprise administering to a subject in need thereof an effective amount of a vitamin D receptor agonist or a pharmaceutically acceptable salt thereof. As used herein, an "effective amount" is an amount effective to treat metabolic syndrome by ameliorating the pathology or reducing the symptoms of metabolic syndrome. In some embodiments, the effectiveness of treatment may be measured by reducing excess body fat around the waist, reducing high cholesterol or triglyceride levels, lowering blood pressure, alleviating insulin resistance or impaired glucose tolerance, reducing hyperglycemia, and reducing body weight in obese individuals.

[0026] Also disclosed herein are methods for providing preventative treatment of metabolic syndrome in a patient in need thereof, comprising administering to the patient an effective amount of a vitamin D analog. In some embodiments, the efficacy of the preventative treatment may be measured by fasting blood glucose level, AUC insulin level, fasting insulin level, cholesterol level, AUC glucose level, triglyceride level, and / or body weight.

[0027] Also disclosed herein is a method for treating existing abnormal levels of fasting blood glucose, AUC insulin, fasting insulin, cholesterol, AUC glucose, and / or body weight in a patient in need thereof, comprising administering an effective amount of a vitamin D receptor agonist to the patient. In some embodiments, the abnormal levels may be determined as levels higher than those detected in healthy individuals. In certain other embodiments, the abnormal levels of fasting blood glucose, AUC insulin, fasting insulin, cholesterol, AUC glucose, and / or body weight are associated with metabolic syndrome. Methods for measuring the level or extent of fasting blood glucose, AUC insulin, fasting insulin, cholesterol, AUC glucose, and / or body weight are well known in the art.

[0028] In some embodiments, the level or extent of fasting blood glucose, AUC insulin, fasting insulin, cholesterol, AUC glucose and / or body weight is reduced by about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%.

[0029] In some embodiments, metabolic disorders may be associated with type 2 diabetes. In certain embodiments, metabolic syndrome may be regulated by reducing visceral fat, fasting glucose, HbAlc, non-fasting glucose, improving insulin resistance, and / or reducing weight gain / weight loss. Regulation of specific glucose parameters (i.e., fasting glucose levels) may be determined using a suitable method, such as an oral glucose tolerance test.

[0030] In some embodiments, the methods of the present invention can be implemented, guided, and modified based on a personalized medicine approach. In some embodiments, personalized medicine provides medical care tailored to the needs of a specific subject, as opposed to methods that are established through medical cohort or epidemiological studies and then applied to an individual. In some embodiments, personalized medicine allows a healthcare provider to optimize treatment for a specific subject based on a variety of factors, such as genetics, metabolism, family history, personal history, environment, behavior, diet, lifestyle, social trends, and personal goals. In some embodiments, a healthcare provider can investigate relevant factors before or at any time during treatment and use the information obtained to design or refine a treatment regimen. Investigations can include assays described herein or individualized counseling between a healthcare provider and a subject. In some embodiments, personalized medicine allows for the selection of patient populations that will benefit from the treatments described herein in combination with a companion diagnostic test (such as genotype assessment of genes involved in energy metabolism) (e.g., leptin, leptin receptor, genes involved in immunometabolism, or any other gene linked to a clinical phenotype of metabolic syndrome).

[0031] The subject of the treatment methods disclosed herein may be a subject in need of treatment for one or more of the conditions disclosed herein. The subject may be a human. Other non-limiting examples of subjects include non-human mammals such as companion animals, pets, livestock, working animals, guard animals, labor animals, and zoo animals.

[0032] In some embodiments, metabolic syndrome comprises at least one symptom selected from the group consisting of excess body fat around the waist (visceral fat), abnormal cholesterol or triglyceride levels (dyslipidemia), high blood pressure (hypertension), insulin resistance or impaired glucose tolerance, high blood sugar (hyperglycemia), obesity, atherosclerosis, diabetes, and fatty liver. In some embodiments, the symptom is excess body fat around the waist (visceral fat). In some embodiments, the symptom is abnormal cholesterol or triglyceride levels (dyslipidemia). In some embodiments, the symptom is high blood pressure (hypertension). In some embodiments, the symptom is high blood sugar (hyperglycemia). In some embodiments, the symptom is diabetes. In some embodiments, the symptom is obesity.

[0033] In some embodiments, the vitamin D receptor agonist is administered subcutaneously, orally, topically, transdermally, intradermally, parenterally, intravenously, intraarterially, intramuscularly, intracolonically, intracerebroventricularly, intraspinally, intraperitoneally, intranasally, intramuscularly, sublingually, buccally, mucosally, by aerosol, or by suppository. In preferred embodiments, the vitamin D receptor agonist is administered subcutaneously or orally.

[0034] In some embodiments, the dose of vitamin D receptor agonist is administered at about 0.1 mg / day to about 10 mg / day. In some embodiments, the dose is about 0.1 mg / day to about 1 mg / day by oral administration. In some embodiments, the dose is about 0.1 mg / week to about 10 mg / week. In some embodiments, the dose is a weekly extended release dose, e.g., by extended release subcutaneous injection. In some embodiments, the dose is about 0.1 mg / month to about 300 mg / month. In some embodiments, the dose is a monthly extended release dose, e.g., by extended release subcutaneous injection. In some embodiments, the dose is about 0.1 mg every 6 months to about 2 g every 6 months.

[0035] In some embodiments, vitamin D receptor agonists such as elocalcitol may be administered at therapeutic doses for long periods without affecting calcium levels. In some embodiments, elocalcitol has been administered to adults for long periods of treatment at oral doses of up to 150 μg per day, which has been shown to be safe (Montorsi et al., 2008). Oral doses above 300 μg per day can cause hypercalcemia, a common side effect of long-term use of vitamin D analogs.

[0036] In some embodiments, the method further comprises administering to the subject at least one additional therapeutic agent, wherein the at least one therapeutic agent is selected from the group consisting of a glucagon-like peptide-1 (GLP-1) receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a glucagon agonist, an amylin agonist, a farnesoid X receptor (FXR) agonist, a liver X receptor (LXR) agonist, a melanocortin 4 receptor (MC4R) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, a thyroid hormone receptor-β (TRβ) agonist, a fibroblast growth factor 21 (FGF21) analog, an activin type II receptor (ActRII) blocker, a statin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, and a dipeptidyl peptidase 4 (DPP4) inhibitor.

[0037] In some embodiments, the at least one therapeutic agent is selected from the group consisting of semaglutide, liraglutide, tirzepatide, caglilintide, lixisenatide, exenatide, albiglutide, dulaglutide, rosiglitazone, empagliflozin, dapagliflozin, canagliflozin, orlistat, bimagrumab, resmetirom, pegosafermin, setomelanotide, and metformin. In some embodiments, the therapeutic agent is semaglutide.

[0038] In the combined treatment of metabolic syndrome with a vitamin D receptor analogue of the present invention and another therapeutic agent, the standard dosage of the other therapeutic agent is used, e.g., semaglutide is administered by weekly infusion of 1 to 2.4 mg, and tirzepatide is administered by weekly infusion of 2.5 to 15 mg.

[0039] In some embodiments, the subject has been diagnosed with metabolic syndrome. In some embodiments, the subject has three or more symptoms selected from the group consisting of: (i) visceral fat, reflected by disproportionate adipose tissue in and around the abdomen; (ii) atherogenic dyslipidemia (high triglycerides (≧150 mg / dL), high LDL cholesterol, and low HDL cholesterol (≦50 mg / dL)); (iv) high blood pressure (≧130 / 85 mmHg); and (v) insulin resistance or impaired glucose tolerance (the inability to properly use insulin or blood glucose, respectively).

[0040] This specification discloses the use of a vitamin D receptor agonist in the manufacture of a medicament for treating metabolic syndrome in a subject in need thereof. This specification also discloses a vitamin D receptor agonist, or a pharmaceutically acceptable salt thereof, as a pharmaceutical compound for use in a method for preventing and / or treating metabolic syndrome in a subject. This specification further discloses a compound selected from the group consisting of compounds of Formula I (elocalcitol), Formula II (inecalcitol), and Formulas I-01 to I-57, for use in a method for treating, preventing, and / or reducing metabolic syndrome in a subject.

[0041] Pharmaceutical Composition The pharmaceutical composition of vitamin D receptor agonist can be administered to a subject together with pharmaceutical excipients or diluents.These compositions can be in the form of drops, liquids, suspensions, tablets, pills, capsules, powders, sustained-release, controlled-release or immediate-release formulations and other formulations known in the art.The pharmaceutical composition of the present invention can be prepared using suitable excipients and diluents.

[0042] The pharmaceutical compositions of the present invention can be formulated in unit dosage form, with each dosage containing, for example, about 0.01 mg to 10 g of vitamin D receptor agonist.

[0043] In some aspects, the unit dosage form is administered to a human, domestic pet, livestock, or other animal with a pharmaceutically acceptable diluent or excipient, hi some aspects, administration is topical, parenteral, intravenous, intraarterial, intracerebroventricular, intraperitoneal, intranasal, intramuscular, subcutaneous, aerosol, oral, or by suppository.

[0044] In some aspects, the dosage of the pharmaceutical composition of the present invention varies depending on the condition, age and weight of the subject, the nature and severity of the disorder being treated, the route of administration, and the form of the composition. In some aspects, the pharmaceutical composition of the present invention is administered in a single dose or in divided doses.

[0045] The combination of multiple compounds in the pharmaceutical composition of the present invention can reduce the required dosage of any individual compound. In such combination therapy, the compounds can be delivered together or separately, at the same time or at different times.

[0046] The pharmaceutical composition of the present invention can be administered by various means known in the art. For oral administration, the pharmaceutical composition of the present invention can be formulated as tablets, capsules, granules, powders, or syrups. The pharmaceutical composition of the present invention can be administered parenterally as an injection (intravenous, intramuscular, or subcutaneous), a drip infusion preparation, or a suppository.

[0047] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. [Example]

[0048] Example 1 Weight Gain or Loss in Animals Upon Administration of Elocalcitol Abbreviations used in the specification are listed in Table 1.

[0049] [Table 1]

[0050] Two-month-old male C57BL / 6J mice were housed (4–5 per cage) at 24–26°C with a 12-hour light / dark cycle and food and water available ad libitum. Three to four days before the start of the experiment, mice were randomized into three experimental groups (N = 10 per group) using an online randomization tool (Graph Pad, USA). During the experiment, each animal's weight was measured once daily, and their health status was also assessed. Elocalcitol (100 μg / kg), vitamin D3 (cholecalciferol, 100 μg / kg), or vehicle control was administered intraperitoneally once daily for 6 days.

[0051] By the fourth day of treatment, the mean weight loss in the elocalcitol group was 10.7%. By the sixth day of treatment, the mean weight loss in the elocalcitol group reached 18.6%. When elocalcitol administration was stopped on the sixth day, the mean weight of the mice returned to normal within two days. No weight loss was observed in the vitamin D3 group or the vehicle control group. No adverse changes in the health status of the mice were observed in either group.

[0052] [Table 2]

[0053] Example 2 Prevention of total weight gain Six-week-old male C57BL / 6J mice were divided into four experimental groups (N = 15 per group) fed a low-fat diet (LFD) or a high-fat diet (HFD), providing 10% (3.61 kcal / kg) or 45% (4.65 kcal / kg) of total energy as fat, respectively (the main fat source was lard; ssniff Spezialdiaeten GmbH): (Group 1) LFD-fed mice, (Group 2) HFD-fed mice, (Group 3) HFD-fed mice and treated with 1,25(OH)2D3 (HFD + VitD), and (Group 4) HFD-fed mice and treated with elocalcitol (HFD + Eloc). Mice were group-housed (4–5 mice per cage) under a 12-h light / dark cycle at 24–26 °C with food and water available ad libitum. 1,25(OH)2D3 (1,25-dihydroxycholecalciferol, 15 μg / kg) or elocalcitol (15 μg / kg) was administered intraperitoneally twice weekly for 16 weeks to the HFD + VitD and HFD + Eloc groups, respectively, while the LFD and HFD groups received vehicle solution. Body weight was measured weekly, and the amounts of food and water consumed by the animals were measured monthly.

[0054] The mean weight gain of HFD mice was significantly higher than that of LFD mice at week 16 of the study (Figure 3A). The relative weight gain of elocalcitol and 1,25(OH)2D3 treatments was 20% and 10% less, respectively, compared to vehicle-treated HFD mice (Figures 3A and 3B). Inhibition of HFD-induced weight gain by elocalcitol reached statistical significance much earlier than in 1,25(OH)2D3-treated mice (weeks 7 vs. 12, respectively, Figure 3B).

[0055] Example 3 Normalization of visceral and subcutaneous fat volume Six-week-old male C57BL / 6J mice were divided into four experimental groups (N = 15 per group) receiving a low-fat diet (LFD) or a high-fat diet (HFD) providing 10% (3.61 kcal / kg) or 45% (4.65 kcal / kg) of total energy as fat, respectively (the main fat source was lard; ssniff Spezialdiaeten GmbH): (Group 1) LFD-fed mice, (Group 2) HFD-fed mice, (Group 3) HFD-fed mice and treated with 1,25(OH)2D3 (HFD + VitD), and (Group 4) HFD-fed mice and treated with elocalcitol (HFD + Eloc). Mice were group-housed (4–5 mice per cage) under a 12-h light / dark cycle at 24–26 °C with food and water available ad libitum. 1,25(OH)2D3 (1,25-dihydroxycholecalciferol, 15 μg / kg) or elocalcitol (15 μg / kg) was administered intraperitoneally twice a week for 16 weeks to the HFD+VitD and HFD+Eloc groups, respectively, while the LFD and HFD groups were administered vehicle solution.

[0056] To assess visceral and subcutaneous fat load, animals were scanned in the abdominal region using a magnetic resonance imaging system (MRI, Bruker BioSpin Group, Bruker Corporations, Germany) before and monthly during the experiment. Experiments were performed as follows: Mice (n = 5 per group) were anesthetized with isoflurane (1.5–2.5% in medical oxygen at 1.5 L / min) and placed on a heated animal bed during MRI measurements. Scans were performed using a 9.4T Bruker BioSpec 94 / 21 USR system connected to a 1H circularly polarized transmit / receive coil and running ParaVision 6.0.1® software (Bruker BioSpin Group, Bruker Corporation, Germany). Respiration was monitored using a breathing pillow (SA Instruments Inc., Stony Brook, USA), and the respiratory rate was maintained at 35–70 breaths per minute. Two adaptive pilot scans were performed to stabilize the animal's position and identify appropriate anatomical landmarks for subsequent scans. A final T1-weighted Bruker:RARE sequence was performed using the following parameters: repetition time (TR) 1164 ms, echo time (TE) 6 ms, flip angle 90°, number of averages 2, image matrix 320 × 320 × 40, spatial resolution 0.125 × 0.125 × 0.5 mm. Volumes were manually segmented by a blinded observer using ITK-SNAP (v3.8.0). Visceral and subcutaneous fat volumes were measured using 40x magnification, and renal involvement was the starting point for fat volume analysis.

[0057] Representative images in Figure 4 for the analysis of visceral and subcutaneous fat volume by MRI show that the visceral fat level in the HFD group was significantly higher compared to the LFD group at week 16 of the experiment ( ****p<0.0001, two-way ANOVA followed by Tukey's multiple comparison post-hoc test, n=5 per group). Treatment with elocalcitol significantly reduced visceral and subcutaneous fat levels in the HFD+Eloc group compared with the HFD group (Figure 4; A, B, and C). In fact, elocalcitol treatment restored visceral and subcutaneous fat in HFD mice to the levels observed in LFD mice (Figure 4A-C). 1,25(OH)2D3 treatment had no statistically significant effect on visceral fat volume or subcutaneous fat distribution in HFD-fed mice (Figure 4A and 4B). Lean mass, calculated from back muscle volume, was preserved (Figure 4D). Analysis of epididymal fat deposit weight at the end of the experiment confirmed the MRI results, showing that the weight of the epididymal fat deposit in the LFD and HFD+Eloc groups was significantly lower than that in the HFD and HFD+vitD groups (Table 3).

[0058] [Table 3]

[0059] Example 4 Reversal of Impaired Glucose Tolerance and Insulin Resistance Six-week-old male C57BL / 6J mice were divided into four experimental groups (N = 15 per group) receiving a low-fat diet (LFD) or a high-fat diet (HFD) providing 10% (3.61 kcal / kg) or 45% (4.65 kcal / kg) of total energy as fat, respectively (the main fat source was lard; ssniff Spezialdiaeten GmbH): (Group 1) LFD-fed mice, (Group 2) HFD-fed mice, (Group 3) HFD-fed mice and treated with 1,25(OH)2D3 (HFD + VitD), and (Group 4) HFD-fed mice and treated with elocalcitol (HFD + Eloc). Mice were group-housed (4–5 mice per cage) under a 12-h light / dark cycle at 24–26 °C with food and water available ad libitum. 1,25(OH)2D3 (1,25-dihydroxycholecalciferol, 15 μg / kg) or elocalcitol (15 μg / kg) was administered intraperitoneally (ip) to the HFD+VitD and HFD+Eloc groups, while the LFD and HFD groups received vehicle solution twice weekly for 16 weeks.

[0060] To determine the effects of elocalcitol and 1,25(OH)2D3 on hyperglycemia and insulin sensitivity in mice, a glucose tolerance test (GTT) was performed at baseline and monthly throughout the experiment. For the GTT, mice were fasted overnight for 10–12 h, and tail blood glucose was measured using an Accu-Chek Performa system glucose meter (Roche, Germany). Baseline glucose values ​​(0 min) were recorded. Glucose (2 g / kg) was then injected intraperitoneally into the animals, and tail blood glucose was measured at 15, 30, 60, 90, and 120 min.

[0061] An insulin tolerance test (ITT) was performed at the end of the experiment (week 16). For the ITT, mice were fasted for 6 hours, and tail blood glucose was measured using an Accu-Chek Performa system glucose meter (Roche, Germany). Baseline glucose values ​​(0 min) were recorded. Animals were then intraperitoneally injected with insulin (0.5 U / kg; diluted with 0.9% saline at 100 U / ml (Novorapid, Novo Nordisk A / S)). Tail blood glucose was measured at 15, 30, 60, 90, and 120 min.

[0062] After glucose administration (2 g / kg, i.p.) at week 16 of the experiment, glucose tolerance in HFD-fed mice was significantly impaired compared with LFD controls, as determined by comparison of individual time points and area-under-the-curve analysis (Figures 5A and 5B). Analysis of the glucose area under the curve (glucose AUC), an index of total glucose excursion after glucose loading, revealed that treatment with elocalcitol, but not 1,25(OH)2D3, statistically significantly improved glucose tolerance in HFD-fed mice (Figure 5B). Insulin tolerance test results indicate that treatment with elocalcitol, but not 1,25(OH)2D3, statistically significantly reversed the increased insulin resistance observed with HFD (Figures 5C and 5D).

[0063] Example 5. Normalization of triglyceride and cholesterol levels Six-week-old male C57BL / 6J mice were divided into four experimental groups (N = 15 per group) receiving a low-fat diet (LFD) or a high-fat diet (HFD) providing 10% (3.61 kcal / kg) or 45% (4.65 kcal / kg) of total energy as fat, respectively (the main fat source was lard; ssniff Spezialdiaeten GmbH): (Group 1) LFD-fed mice, (Group 2) HFD-fed mice, (Group 3) HFD-fed mice and treated with 1,25(OH)2D3 (HFD + VitD), and (Group 4) HFD-fed mice and treated with elocalcitol (HFD + Eloc). Mice were group-housed (4–5 mice per cage) under a 12-h light / dark cycle at 24–26 °C with food and water available ad libitum. 1,25(OH)2D3 (1,25-dihydroxycholecalciferol, 15 μg / kg) or elocalcitol (15 μg / kg) was administered intraperitoneally to the HFD+VitD and HFD+Eloc groups, and vehicle solution was administered to the LFD and HFD groups twice a week for 16 weeks.

[0064] Twenty-four hours after the last dose, mice were deeply anesthetized with phenobarbital (200–300 mg / kg), and blood was collected from all mice by cardiac puncture according to standard protocols. Blood was allowed to clot and centrifuged at 2000 × g for 20 minutes. Serum was then stored at -80°C until analysis.

[0065] Triglyceride levels were significantly elevated in the HFD group compared with the LFD group (p<0.05, one-way ANOVA followed by Tukey's multiple comparison post-hoc test, Table 3). Both elocalcitol and 1,25(OH)2D3 treatments reduced triglyceride levels to levels similar to those in the LFD group, but the cholesterol-lowering effect of elocalcitol (combined HDL and LDL) was statistically significant (Table 4).

[0066] [Table 4]

[0067] Example 6 Biphasic Weight Loss Treatment Using Variable Strength Extended Release Elocalcitol Formulations Subjects with symptoms of metabolic disease and diagnosed obesity were treated with monotherapy with the vitamin D receptor agonist elocalcitol. Elocalcitol was administered subcutaneously weekly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase consisting of phospholipids, diacylglycerol, ethanol, and buffer, releasing an effective daily dose of approximately 0.6-1.2 mg. Initial elocalcitol treatment was continued for a maximum of six months. Thereafter, elocalcitol was administered subcutaneously quarterly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase consisting of phospholipids, diacylglycerol, ethanol, and citrate buffer or in an in-situ polymer gel extended-delivery system, releasing an effective daily dose of approximately 0.3 mg. Treatment was continued for as long as efficacy was observed. Subjects are monitored during treatment to determine therapeutic effectiveness, minimization of side effects (e.g., hypercalcemia), and the need for modifications in dosage or treatment regimen.

[0068] Example 7 Biphasic Weight Loss Treatment Using a Variable Strength Extended Release Elocalcitol Formulation in Combination with a GLP-1 Agonist Subjects with symptoms of metabolic disease and diagnosed with obesity are prescribed vitamin D receptor agonist monotherapy, where the vitamin D receptor agonist is elocalcitol. Elocalcitol is administered subcutaneously weekly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase consisting of phospholipids, diacylglycerol, ethanol, and buffer, which delivers an effective daily dose of approximately 0.6-1.2 mg of elocalcitol. Concurrently, the subject is treated with weekly subcutaneous injections of semaglutide 2.4 mg. This initial treatment with the combination of elocalcitol and semaglutide is continued for a maximum of six months. Thereafter, elocalcitol is administered subcutaneously quarterly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase or in-situ polymer gel extended-release delivery system composed of phospholipids, diacylglycerol, ethanol, and citrate buffer, which releases an effective daily dose of approximately 0.3 mg of elocalcitol. Treatment is continued for as long as efficacy is observed. Subjects are monitored during treatment to determine therapeutic efficacy, minimization of side effects (e.g., hypercalcemia), and the need for changes in dosage or treatment regimen.

[0069] Example 8 Biphasic Treatment of Impaired Glucose Tolerance Using Variable Strength Extended Release Elocalcitol Formulations Subjects with metabolic disease symptoms and diagnosed with type 2 diabetes are prescribed vitamin D receptor agonist monotherapy, where the vitamin D receptor agonist is elocalcitol. Elocalcitol is administered subcutaneously weekly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase consisting of phospholipids, diacylglycerol, ethanol, and buffer, which releases an effective daily dose of approximately 0.6-1.2 mg. Initial elocalcitol treatment is continued for a maximum of six months. Thereafter, elocalcitol is administered subcutaneously quarterly using a prefilled syringe containing a sterile, liquid, extended-release formulation of elocalcitol in a polar lipid phase consisting of phospholipids, diacylglycerol, ethanol, and citrate buffer or in an in-situ polymer gel extended-delivery system, which releases an effective daily dose of approximately 0.3 mg. Treatment is continued for as long as efficacy is observed. Subjects are monitored during treatment to determine therapeutic effectiveness, minimization of side effects (e.g., hypercalcemia), and the need for modifications in dosage or treatment regimen.

[0070] Representative aspects In one embodiment, there is provided a method of treating or ameliorating the effects of metabolic syndrome in a subject, the method comprising administering to the subject in need thereof an effective amount of a vitamin D receptor agonist or a pharmaceutically acceptable salt thereof, wherein the vitamin D receptor agonist is selected from the group consisting of elocalcitol, inecalcitol, and compounds of formula I-01, I-02, I-03, I-04, I-05, I-06, I-07, I-08, I-09, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47, I-48, I-49, I-50, I-51, I-52, I-53, I-54, I-55, I-56, I-57, I-58, I-59, I-60, I-61, I-62, I-63, I-64, I-65, I-66, I-67, I-68, I-69, I-70, I-71, I-72, I-73, I-74, I-75, I-76, I-77, I-78, I-79, I-80, I-81, I-82, I-83, I-84, I-85, and I-57, wherein the effective amount of the vitamin D receptor agonist is selected from the group consisting of compounds represented by the formula I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47, I-48, I-49, I-50, I-51, I-52, I-53, I-54, I-55, I-56, and I-57, wherein the effective amount of the vitamin D receptor agonist does not exceed the threshold for causing hypercalcemia. In one embodiment, the vitamin D receptor agonist is elocalcitol, inecalcitol, or a compound of formula I-57. In one embodiment, the metabolic syndrome comprises at least one condition selected from the group consisting of excess body fat around the waist (visceral fat), abnormal levels of cholesterol or triglycerides (dyslipidemia), hypertension, insulin resistance or impaired glucose tolerance, hyperglycemia, obesity, atherosclerosis, diabetes, and fatty liver. In one embodiment, the condition is excess body fat around the waist (visceral fat). In one embodiment, the condition is abnormal cholesterol or triglyceride levels (dyslipidemia). In one embodiment, the condition is high blood pressure (hypertension). In one embodiment, the condition is high blood sugar (hyperglycemia). In one embodiment, the condition is diabetes. In one embodiment, the condition is obesity. In one embodiment, the threshold is 1,200 μg or less per day. In one embodiment, the threshold is from about 150 μg to about 1,200 μg per day. In certain embodiments, the vitamin D receptor agonist is administered topically, transdermally, intradermally, parenterally, intravenously, intraarterially, subcutaneously, intramuscularly, intracranially, intracolicly, intraorbitally, ophthalmically, intracerebroventricularly, intracapsularly, intrathecally, intracisternally, intraperitoneally, intranasally, intramuscularly, subcutaneously, sublingually, buccally, by aerosol, orally, orally, or by suppository. In some embodiments, the vitamin D receptor agonist is administered subcutaneously. In certain embodiments, the method further comprises administering to the subject one or more pharmaceutically acceptable excipients. In certain embodiments, the method further comprises administering to the subject at least one therapeutic agent or a pharmaceutically acceptable salt thereof. In certain embodiments, the at least one therapeutic agent is selected from the group consisting of glucagon-like peptide-1 (GLP-1) receptor agonists, glucose-dependent insulinotropic polypeptide (GIP) agonists, glucagon agonists, amylin agonists, farnesoid X receptor (FXR) agonists, liver X receptor (LXR) agonists, melanocortin 4 receptor (MC4R) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, thyroid hormone receptor-β (TRβ) agonists, fibroblast growth factor 21 (FGF21) analogs, activin type II receptor (ActRII) blockers, statins, sodium glucose cotransporter 2 (SGLT2) inhibitors, and dipeptidyl peptidase 4 (DPP4) inhibitors. In certain embodiments, the at least one therapeutic agent is selected from the group consisting of semaglutide, liraglutide, tirzepatide, caglilintide, lixisenatide, exenatide, albiglutide, dulaglutide, rosiglitazone, empagliflozin, dapagliflozin, canagliflozin, orlistat, bimagrumab, resmetirom, pegosafermin, setomelanotide, and metformin. In certain embodiments, the at least one therapeutic agent is administered to the subject at a dose of about 1000 mg or less. In some embodiments, the vitamin D receptor agonist is administered to the subject at a dose of about 1000 mg or less. In some embodiments, the subject has metabolic syndrome.

[0071] References Baker RG et al., Cell Metab. 2011; 13(1): 11-22. Chawla A. et al., Nat. Rev. Immunol. 2011; 11(11): 738-749. He Y. et al., Trends Biochem. Sci. 2016; 41(12):1012-1021. Hotamisligil GS, Immunity 2017; 47(3): 406-420. Hotamisligil GS, Nature 2006; 444(7121): 860-867. Montorsi F. et al., J. Urol. 2008; 179(suppl): 700; Abstract 2035. Nagpal S. et al., Endocrine Rev. 2005; 26 (5): 662-687. National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III), JAMA 2001; 285(19): 2486-2497. Peleg S. et al., Endocrinology 2002;143(5): 1625-1636. Penna G. et al., Prostate 2009;69(5): 480-493. Ridker PM et al., Circulation 2003; 107(3): 391-397. Shi H. et al., J. Clin. Invest. 2006; 116(11): 3015-3025. Van Saklayen M.G., Curr. Hypertens. Rep. 2018; 20(2): 12.

Claims

1. 1. A method for treating metabolic syndrome in a subject, comprising administering to a subject in need thereof an effective amount of a vitamin D receptor agonist or a pharmaceutically acceptable salt thereof; The vitamin D receptor agonist may be elocalcitol, inecalcitol, or a compound of formula I-01, I-02, I-03, I-04, I-05, I-06, I-07, I-08, I-09, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47, I-48, I-49, I-50, I-51, I-52, I-53, I-54, I-55, I-56, I-57, I-58, I-59, I-60, I-61, I-62, I-63, I-64, I-65, I-66, I-67, I-68, I-69, I-70, I-71, I-72, I-73, I-74, I-75, I-76, I-77, I-78, I-79, I-80, I-81, I-82, I-83, I-84, I-85, I-86, I-87, I-88, I-89, I-90, I-91, I-92, I-93, I-94, I-95, I-96, I-97, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, and 1-57.

2. 10. The method of claim 1, wherein the vitamin D receptor agonist is elocalcitol.

3. 2. The method of claim 1, wherein the vitamin D receptor agonist is inecalcitol.

4. 2. The method of claim 1, wherein the vitamin D receptor agonist is a compound of formula I-57.

5. 5. The method of any one of claims 1 to 4, wherein the metabolic syndrome comprises at least one symptom selected from the group consisting of excess body fat around the waist, abnormal cholesterol or triglyceride levels, high blood pressure, insulin resistance or impaired glucose tolerance, hyperglycemia, obesity, atherosclerosis, diabetes, and fatty liver.

6. 6. The method of claim 5, wherein the condition is excess body fat around the waist.

7. 6. The method of claim 5, wherein the symptom is abnormal cholesterol or triglyceride levels.

8. 6. The method of claim 5, wherein the condition is high blood pressure.

9. 6. The method of claim 5, wherein the symptom is hyperglycemia.

10. 6. The method of claim 5, wherein the condition is diabetes.

11. 6. The method of claim 5, wherein the condition is obesity.

12. The method of any one of claims 1 to 11, wherein the vitamin D receptor agonist is administered orally or subcutaneously.

13. 13. The method of any one of claims 1 to 12, further comprising administering at least one therapeutic agent selected from the group consisting of semaglutide, liraglutide, tirzepatide, caglilintide, lixisenatide, exenatide, albiglutide, dulaglutide, rosiglitazone, empagliflozin, dapagliflozin, canagliflozin, orlistat, bimagrumab, resmetirom, pegosafermin, setomelanotide, and metformin.

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