Methods of treating conditions with SIK3 inhibitors

Abstract

The present invention relates to methods, compositions and kits for treating obesity and / or an obesity-related disease or disorder and / or diabetes in an individual. The present invention also relates to methods, compositions and kits for treating fatty liver disease in an individual. In particular, the compositions, methods and kits are particularly useful, but not limited to, the treatment of obesity, treatment of adiposity, increasing white adipose tissue browning. The invention also provides a method of promoting blood glucose clearance and increased insulin secretion in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual. The present invention also provides a method of treating fatty liver disease in an individual in need thereof, the method comprising administering to the individual a SIK-3 inhibitor, thereby treating fatty liver disease in the individual in need thereof.

Description

Methods of treating conditions with SIK3 inhibitorsField of the invention

[0001] The present invention relates to methods for treating overweight or obese in individuals in need thereof.Related applications

[0002] This application claims priority from Australian provisional application nos. 2023903333 and 2024901647, the entire contents of each are in incorporated herein by reference.Background of the invention

[0003] More than 1.9 billion people are overweight or obese worldwide. The prevalence of obesity has reached epidemic levels and is increasing rapidly. Chronic diseases associated with obesity such as diabetes and cardiovascular disease are leading causes of morbidity and mortality. Weight loss strategies focused on lifestyle modifications are difficult to maintain due to rebound weight gain. Moreover, there are limited treatment options currently available.

[0004] The development of obesity is primarily due to an imbalance in energy homeostasis, whereby energy intake exceeds energy expenditure. The maintenance of energy balance is under strict control by a complex system involving endocrine and neural pathways. The hypothalamus is a critical regulator of energy balance as it senses information about peripheral energy availability, comparing it to the actual value of body fat and adjusting food intake or expenditure as required. The molecular mechanisms involved in hypothalamic nutrient sensing are poorly defined. A lack of understanding about the fundamental mechanisms that regulate energy balance is a critical roadblock to the development of effective anti-obesity treatments.

[0005] Body weight is tightly regulated within a specific range. In order to prevent excessive weight gain or chronic weight loss, a complex neuronal regulatory network has developed in the brain, specifically in the hypothalamus. This system has the capacity to sense the amount of fat accumulated in the body and adjust food intake and fuel consumption so that body weight stays reasonably constant despite variations in daily food intake and energy expenditure. To elicit appropriate responses, thehypothalamus, in particular the arcuate nucleus (ARC), needs to obtain accurate information about the body’s energy status from the periphery. The hypothalamic ARC contains two major neuronal populations responsible for control of energy homeostasis: neurons that express orexigenic (appetite-inducing) neuropeptide Y (NPY) / agouti- related protein (AgRP) and another set of neurons that produce anorexigenic (appetitesuppressing) pro-opiomelanocortin (POMC). When this system is disrupted, obesity or the other extreme, anorexia, can develop. Identifying endogenous regulators within the arcuate network that control body weight and energy balance is critical to understand the pathology of obesity. This may in turn provide the means for the development of more effective and safer therapeutic approaches.

[0006] Obesity and overconsumption of calories is known to lead to ectopic lipid deposition around and within tissue and organs, such as the muscle, heart, liver and pancreas.

[0007] Heavy alcohol use is known to lead to liver complications, including alcoholic hepatitis which is often characterized by fatty liver and inflammation. Alcoholic hepatitis can ultimately lead to cirrhosis of the liver (scarring) and hardening of the liver tissue. However, individuals that do not consume excessive amounts of alcohol can also be found to have liver disease complications. Non-alcoholic fatty liver disease (NAFLD) is understood to encompass a variety of liver diseases, including steatosis (simple fatty liver), non-alcoholic steatohepatitis (NASH) and advanced scarring of the liver (cirrhosis). NASH has traditionally been diagnosed by means of a liver biopsy to characterize the liver histology, particularly with respect to the characteristics of inflammation, fibrosis and steatosis (fat accumulation). NASH then generally refers to clinical findings based upon the liver biopsy of a patient with steatohepatitis, combined with the absence of significant alcohol consumption.

[0008] In NASH, fat accumulation is seen in varying degrees of inflammation (hepatitis) and may lead to more serious conditions involving scarring (fibrosis). Patients having NASH are also often characterized by abnormal levels of liver enzymes, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0009] There exists a need for new and / or improved treatments for overweight and / or obese individuals.

[0010] There exists a need for new and / or improved treatments for obese individuals having a further obesity-related disease or disorder.

[0011] Separately, or in addition to the above needs, there exists a need for new and / or improved treatments for fatty liver disease.

[0012] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0013] Surprisingly the inventors have found that administration of a SIK-3 inhibitor has numerous benefits in overweight or obese individuals including to resistance to weight gain, and promotion of weight loss. Further, the inventors have found that administration of a SIK-3 inhibitor has a beneficial effect on fatty liver disease, including NAFLD and NASH.

[0014] In an aspect of the disclosure there is provided a method of treating obesity in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby treating obesity in the individual.

[0015] In another aspect there is provided a method of treating, or minimising the risk of, an obesity-related disease or disorder, the method comprising administering to an individual suffering from or at risk of suffering from an obesity-related disease or disorder a SIK-3 inhibitor to the individual, thereby treating, or minimising the risk of, an obesity in the individual.

[0016] In a further aspect there is provided a method of reducing adiposity in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby reducing adiposity in the individual.

[0017] In a further aspect there is provided a method of reducing the body weight of an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby reducing body weight of the individual.

[0018] In a further aspect there is provided a method of increasing white adipose tissue browning in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby increasing white adipose tissue browning in the individual.

[0019] Advantageously, the methods of the present invention may result in reduction in body weight and adiposity without a corresponding decrease in lean mass, ie no or minimal reduction in muscle and / or bone mass.

[0020] In a further aspect there is provided a method of preventing or minimising the weight gain of an individual consuming a high energy / caloric diet, the method comprising administering a SIK-3 inhibitor to the individual, thereby preventing or minimising the weight gain of the individual consuming a high energy / caloric diet.

[0021] In a further aspect there is provided a method of preventing or minimising adiposity in an individual consuming a high energy / caloric diet, the method comprising administering a SIK-3 inhibitor to the individual, thereby preventing or minimising adiposity in the individual consuming a high energy / caloric diet.

[0022] In a further aspect there is provided a method of enhancing energy expenditure in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby enhancing energy expenditure in the individual.

[0023] In preferred embodiments the method of enhancing energy expenditure does not require the individual to increase their level of exercise. In embodiments the method of enhancing energy expenditure does not require the individual to modify their diet. Alternatively, in other embodiments the method includes modification to the exercise and diet regimen of the individual. Preferably the administration of a SIK-3 inhibitor in combination with modification to the exercise and diet regimen of the individual has an additive effect or a synergistic effect.

[0024] Advantageously in some embodiments the methods of the disclosure can contribute to a reduction in body weight and adiposity without requiring a change in activity level or caloric intake of the individual in need thereof.

[0025] In a further aspect there is provided a method of promoting weight loss in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting weight loss in the individual.

[0026] In a further aspect there is provided a method of promoting or improving insulin secretion in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby improving insulin secretion in the individual.

[0027] In a further aspect there is provided a method of promoting blood glucose clearance in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting blood glucose clearance in the individual.

[0028] In a further aspect there is provided a method of promoting or improving glycaemic control in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting glycaemic control in the individual.

[0029] In a further aspect there is provided a method of treating pre-diabetes or diabetes in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby treating pre-diabetes or diabetes in an overweight or obese individual.

[0030] In a further aspect there is provided a method of treating insulin resistance in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby treating the insulin resistance.

[0031] In a further aspect there is provided a method of decreasing food intake in an individual in need thereof comprising the step of administering to the individual a SIK-3 inhibitor. Preferably the individual is an overweight or obese individual.

[0032] In a further aspect there is provided a method of reducing caloric intake in an individual in need thereof comprising the step of administering to the individual a SIK-3 inhibitor. Preferably the individual is an overweight or obese individual.

[0033] In a further aspect there is provided a method of reducing or supressing appetite in an individual in need thereof, the method comprising administering to theindividual a SIK-3 inhibitor, thereby supressing appetite in the individual. Preferably the individual is an overweight or obese individual.

[0034] In a further aspect there is provide a method of treating fatty liver disease in an individual in need thereof, the method comprising administering to the individual a SIK-3 inhibitor, thereby treating fatty liver disease in the individual in need thereof. In one embodiment, the individual is an overweight or obese individual, for example an overweight or obese individual as defined herein. In another embodiment, the individual is not an overweight or obese individual, for example not an overweight or obese individual as defined herein. In embodiments the fatty liver disease may be any one described herein, including non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0035] The present disclosure also provides the use of a SIK-3 inhibitor in the manufacture of a medicament for: treating obesity; in an individual in need thereof; treating or minimising the risk of, an obesity-related disease or disorder in an individual suffering from or at risk of suffering from an obesity-related disease or disorder reducing the body weight of an individual in need thereof; preventing or minimising the weight gain of an individual consuming a high energy / caloric diet; preventing or minimising adiposity in individual consuming a high energy / caloric diet; enhancing energy expenditure; improving insulin secretion in an individual in need thereof; promoting blood glucose clearance in an overweight or obese individual; promoting or improving glycaemic control in an overweight or obese individual; treating pre-diabetes or diabetes in an overweight or obese individual; treating insulin resistance in an individual; decreasing food intake in an individual; decreasing caloric intake in an individual; supressing appetite in an individual; or treating fatty liver disease in an individual.

[0036] In another aspect of the present disclosure there is provided a SIK-3 inhibitor for use for: treating obesity; in an individual in need thereof; treating or minimising the risk of, an obesity-related disease or disorder in an individual suffering from or at risk of suffering from an obesity-related disease or disorder reducing the body weight of an individual in need thereof; preventing or minimising the weight gain of an individual consuming a high energy / caloric diet; preventing or minimising adiposity in individual consuming a high energy / caloric diet; enhancing energy expenditure; improving insulin secretion in an individual in need thereof; promoting blood glucose clearance in an overweight or obese individual; promoting or improving glycaemic control in an overweight or obese individual; treating pre-diabetes or diabetes in an overweight or obese individual; treating insulin resistance in an individual; decreasing food intake in an individual; decreasing caloric intake in an individual; supressing appetite in an individual; or treating fatty liver disease in an individual.

[0037] In another aspect of the present disclosure there is provided a SIK-3 inhibitor when used for: treating obesity; in an individual in need thereof; treating or minimising the risk of, an obesity-related disease or disorder in an individual suffering from or at risk of suffering from an obesity-related disease or disorder; reducing the body weight of an individual in need thereof; preventing or minimising the weight gain of an individual consuming a high energy / caloric diet; preventing or minimising adiposity in individual consuming a high energy / caloric diet; enhancing energy expenditure;improving insulin secretion in an individual in need thereof; promoting blood glucose clearance in an overweight or obese individual; promoting or improving glycaemic control in an overweight or obese individual; treating pre-diabetes or diabetes in an overweight or obese individual; treating insulin resistance in an individual; decreasing food intake in an individual; decreasing caloric intake in an individual; supressing appetite in an individual; or treating fatty liver disease in an individual.

[0038] In any aspect of the present invention, the individuals in need thereof are typically those that are overweight, obese, morbidly obese or extremely obese. Preferably, the individual has a BMI greater than 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 kg / m2. The individual may have a BMI of between 25 kg / m2to 29.9 kg / m2. The individual may have a BMI of 30 kg / m2or greater, or a BMI of 40 kg / m2or greater.

[0039] In any aspect of the present invention, the individuals in need thereof may have been diagnosed with insulin deficiency. In embodiments the individuals have an insulin resistance. In embodiments the individuals have both insulin deficiency and insulin resistance. In embodiments the individuals are pre-diabetic or may have hyperinsulinemia.

[0040] In embodiments the individuals may have mild insulin deficiency, and not require specific treatment with insulin. In embodiments the individuals may have moderate insulin deficiency. In embodiments the individuals may have severe insulin deficiency. In embodiments the individuals have absolute insulin deficiency.

[0041] In any aspects of the present invention the SIK-3 inhibitor is administered to the individual in need thereof after disease onset or diagnosis.

[0042] In any aspects of the present invention, the individual in need thereof may be one who has been identified as being in need of treatment for a prescribed period of time prior to starting treatment according to the methods as described herein. In some embodiments the individual in need thereof has been obese for a prescribed period prior to starting treatment.

[0043] In some embodiments the individual in need thereof has been treated by other methods for a prescribed period prior to starting treatment with the methods as described herein.

[0044] In some embodiments the prescribed period is at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months or at least 12 months. In some embodiments the prescribed period is from about 6 months to about 2 years, or from about 1 year to about 5 years. In some embodiments the prescribed period is more than 5 years.

[0045] In an embodiment of any one of the aspects as herein described the obesity- related disease or disorder is selected from the group consisting of obesity, pre-obesity, morbid obesity, Prader-Willi Syndrome, Hypothalamic Injury Associated Obesity, hyperlipidemia, hypertension, pre-diabetes, diabetes, lipodystrophy, lipodema, Bardet- Biedl Syndrome, Cohen Syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, hypothyroid and MOMO Syndrome.

[0046] In an embodiment of any one of the aspects as herein described the obesity- related disease or disorder is a disorder the causes or is concomitant with increased adiposity or obesity. For example the obesity-related disease or disorder may be a thyroid condition.

[0047] In an embodiment of any one of the aspects as herein described the individual is an obese and diabetic individual.

[0048] In any method of the disclosure, the obesity is treated, adiposity is reduced, weight gain may be prevented or minimised, or body weight may be reduced without an additional change in diet, or an increase in exercise. However, in any method of the disclosure, the method may further comprise the step of reducing the caloric intake in the individual and / or increasing the level of exercise undertaken by the individual.

[0049] In embodiments of the aspects as herein described the administration of the SIK-3 inhibitor to an individual may supress appetite in the individual, thereby leading to a reduction of caloric and / or food intake, contributing to treatment of obesity, reduction of adiposity or prevention or minimisation of body weight gain.

[0050] In any aspect or embodiment, treating fatty liver disease, such as NAFLD or NASH, may be the partial or complete alleviation, suppression, delay (onset), prevention, amelioration and / or alleviation of fatty liver disease (such as NAFLD or NASH), or one or more symptoms of fatty liver disease (such as NAFLD or NASH).

[0051] In one embodiment, the individual who receives the SIK-3 inhibitor for treating fatty liver disease is an overweight or obese individual, for example an overweight or obese individual as defined herein.

[0052] In another embodiment, the individual who receives the SIK-3 inhibitor for treating fatty liver disease is not an overweight or obese individual, for example not an overweight or obese individual as defined herein.

[0053] In any aspect or embodiment, the fatty liver disease may be any one described herein, including steatosis, nonalcoholic fatty liver disease (NAFLD), Alcoholic fatty liver disease (AFLD), Nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), liver fibrosis caused by fatty liver disease typically NASH, cirrhosis caused by fatty liver disease typically NASH, or hepatocellular carcinoma (HCC) caused by fatty liver disease typically NASH. Preferably the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0054] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0055] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0056] Figure 1: Expression of SIK3 in relation to metabolic stress in the hypothalamic ARC. mRNA levels of SIK1, SIK2, and SIK3 in the hypothalamus of chow- and HFD-fed mice, leptin receptor deficient db / db mice and their heterozygous db / + counterpart. Immunostaining for SIK3 in the mediobasal hypothalamus of Chow vsHFD-fed 16-week-old male mice and 8-10-week-old male lean db / + and obese db / db mice. Data are mean ± s.e.m, *p < 0.05, **p < 0.01 , ***p<0.001 by two-tailed t-test.

[0057] Figure 2: Diagram of the generation of neuronal specific SIK3 knockout mice (SIK3-NPYKO). Exon 5 was flanked by loxP sites, and a neomycin resistance cassette flanked by FRT sites was inserted upstream from the 5’ loxP site. The neomycin resistance cassette was excised by the expression of the FLP recombinase in vivo. Live disruption of exon 5 flanked by loxP sites was achieved by crossing SIK3 floxed mice with NPY-Cre transgenic mice. DNA was extracted from different tissues (HY: Hypothalamus, HIP: Hippocampus, BS: Brain Stem, CER: Cerebrum, OB: Olfactory Bulb, PG: Pituitary gland, BR: Whole brain, LIV: Liver). Recombination of the floxed SIK3 allele was assessed by PCR. Recombination was detected in the hypothalamus, hippocampus, cerebrum, brain stem, olfactory bulb, and brain of SIK3-NPYKO mice. Immunofluorescence analysis showing colocalization of SIK3 (green) and NPY;Ai9- expression (Red) in the central ARC (Bregma 1.58-1.94) of WT vs KO mice.

[0058] Figure 3: WT and SIK3-NPYKO mice were fed on a high fat diet for 10 weeks. Weekly body weight were recorded and fat mass and lean mass were determined by EchoMRI. Dissected weights of individual fat pads from epididymal (eWAT) and inguinal (iWAT) were measured. Data are mean ± s.e.m. *p < 0.05, **p < 0.01 , ***p<0.001 by two-tailed t-test.

[0059] Figure 4: WT and SIK3-NPYKO mice were fed on a high fat diet for 10 weeks. Mice were placed into the Promethion metabolic cages and energy expenditure, daily food intake, RER and Pedestrian meters were recorded. Hypothalamic neuropeptide (AgRP, NPY and POMC) were assessed by qRTPCR. Data are mean ± s.e.m. *p < 0.05, **p < 0.01, ***p<0.001 by two-tailed t-test.

[0060] Figure 5: WT and SIK3-NPYKO mice were fed on a high fat diet for 10 weeks. Intraperitoneal (i.p.) glucose tolerance test (2g / kg body weight) on 6 h fasted 16- week-old mice fed with HFD. i.p. pyruvate tolerance test (1 g / kg body weight) on 16 h fasted 18-week-old mice fed with HFD. Intraperitoneal insulin tolerance test (0.75 lU / kg body weight) on 6 h fasted 17-week-old mice fed with HFD. Blood glucose levels during each tolerance tests were monitored at 0, 15, 30, 16, 90, and 120 minutes. Results are expressed over the time course as the area under the curve. 16 h / overnight fasted, and 30 minutes re-fed blood glucose levels were measured from WT v SIK3-NPYKO HFDfed mice. Data ± s.e.m. P values by two-way repeated ANOVA or student t test. *p < 0.05, **p < 0.01, ***p<0.001

[0061] Figure 6: Following SIK3 overexpression in SHSY5Y cells for 72 hours and overnight serum starvation, cells were treated with 100 nm insulin for 15 minutes. Phosphorylated IRS1 (Ser789), Phosphorylated AKT were assessed by Western blot analysis. Western blot analysis showing overexpression of SIK3 in SHSY5Y cells. In p- AKT (S473) densitometry analysis, total-AKT was used as loading control. In p-IRS1 (S789) densitometry analysis, beta-actin was used as a loading control. Data ± s.e.m., n = 3-per conditions. P values by student t test. Experiments were repeated in 3 independent biological replicates. *p < 0.05, **p < 0.01, ***p<0.001

[0062] Figure 7: 8-10-week-old male chow-fed WT v SIK3-NPYKO mice on NPY Ai9TdTomato background were fasted overnight and intraperitoneally injected with 2.5 mll / g insulin for 15 minutes. Consequently, brains were extracted for ARC p-AKT (Ser 473) immunohistochemistry and p-AKT positive NPY neurons quantified across the rostral-caudal extent of the hypothalamus. Data ± s.e.m., P values by student t test. *p < 0.05, **p < 0.01, ***p<0.001

[0063] Figure 8: WT and SIK3-NPYKO mice were fed on a high fat diet for 10 weeks. Representative infrared thermal image of the shaved interscapular BAT, lumbar back and tail regions of a mouse used in the study. BAT temperature, Lumbar temperature, Tail temperature, Tail corrected BAT temperature values from the FLIR infrared camera at 16 weeks after 8-10 weeks of HFD feeding. mRNA expression levels of thermogenic and mitochondrial function / biogenesis markers in intrascapular BAT tissue of HFD-fed WT v SIK3-NPYKO mice Representative H&E and UCP-1 immunohistochemistry staining of intrascapular BAT in WT and SIK3-NPYKO mice. Western blot analyses of UCP-1 protein levels in iBAT of WT and SIK3-NPYKO mice. Alpha-tubulin was used as a control. Data ± s.e.m., P values by student t test. *p < 0.05, **p < 0.01 , ***p<0.001

[0064] Figure 9: WT and SIK3-NPYKO mice were fed on a high fat diet for 10 weeks. mRNA expression levels of thermogenic and mitochondrial function / biogenesis markers in inguinal WAT of HFD-fed WT v SIK3-NPYKO mice. Representative H&E and UCP-1 immunohistochemistry staining of inguinal WAT in WT and SIK3-NPYKO mice fed with HFD. Mean adipocyte size and Frequency distribution of adipocyte size iniWAT of WT v SIK3-NPYK0 HFD-fed mice. Data ± s.e.m., P values by student t test. *p < 0.05, **p < 0.01 , ***p<0.001

[0065] Figure 10: C57BL6 mice were fed on a high fat diet for 4 weeks and subsequently injected with multiple low doses of streptozotocin to induced diabetes. Obese and diabetic mice were either treated with YKL-05-099 (18mg / kg of body weight) or placebo for 4 weeks. Body weights were measured and fat and lean mass were determined using EchoMRI imaging. Intraperitoneal glucose tolerance tests (1 g / kg of body weight) were performed and fasted, and postprandial induced insulin secretion were determined.

[0066] Figure 11 : Qualitative chromatograms of YKL-05-099 found in mouse brain tissue by LC-MS. A dose of placebo or YKL-05-099 (18mg / kg) was orally administered to mice 2 hours before sacrifice. (A) YKL-05-099 was detected in the mediobasal hypothalamus of the brains from treated mice (green peak). (B) YKL-05-099 was detected in the midline coronal cross sections of the brains from treated mice (green peak). The smaller peaks in black correspond to the brain tissues from placebo mice. Representative chromatograms shown (n = 3 placebo vs n = 3 treated with YKL-05- 099).

[0067] Figure 12: WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 treatment began at 20 weeks of age. (A-B) Body weights and percentage body weight loss from baseline of placebo or YKL- 05-099 treatment mice. (C) Body weight measured 5 weeks after placebo or YKL-05- 099 treatment. Data presented as mean ± SEM (n = 9 placebo vs n = 7 treated with YKL-05-099), p-values by Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

[0068] Figure 13: Body compositions from obese mice treated with placebo or YKL- 05-099 were measured by Echo MRI. (A) Fat mass and (B) lean mass. (C-D) Mass of fat depots (iWAT = inguinal white adipose tissue; eWAT = epidydimal adipose tissue). (E) Representative photo of dissected tissue is shown. Data presented as mean ± SEM (n = 9 placebo vs n = 7 treated with YKL-05-099), p-values by Student’s t test. *p < 0.05, **p < 0.01.

[0069] Figure 14: Brown adipose tissue (BAT) from obese mice treated with placebo or YKL-05-099 were isolated. Western blot analysis for UCP-1 protein levels in BATnormalised to Tubulin. Data presented as mean ± SEM (n = 7 placebo vs n = 7 treated with YKL-05-099), p-values by Student’s t test. *p < 0.05.

[0070] Figure 15: Metabolic parameters, hypothalamic neuropeptide expressions and serum leptin levels of obese mice treated with placebo or YKL-05-099. (A) Daily food intake from placebo and YKL-05-099 treated mice. (B-C) Relative hypothalamic mRNA expression of Agrp and Npy. Results were normalised to the house keeping gene Ppia and shown as fold change relative to the placebo group. (D) Fasted serum leptin levels. Data presented as mean ± SEM (n = 7-9 placebo vs n = 7 treated with YKL-05-099), p-values by Student’s t test. *p < 0.05.

[0071] Figure 16: WT C57BL / 6J mice were fed a high fat diet for 12 weeks starting from 8 weeks of age, and placebo or GLPG3970 treatment (50mg / kg or 25mg / kg) began at 20 weeks of age. (A-B) Body weights and percentage body weight loss from baseline of placebo or GLPG3970 treatment. (C) Mice body weight 19 days after placebo or GLPG3970 treatment. Data presented as mean ± SEM (n= 5 placebo, n = 5 GLPG3970 50mg / kg, n = 5 GLPG397025mg / kg), p-values by Student’s t test. *p < 0.05, **p <0.01, ***p < 0.001.

[0072] Figure 17: Body compositions from obese mice treated with placebo or GLPG3970 were measured by Echo MRI. (A) Fat mass and (B) lean mass. (C) Mass of fat depots (iWAT =inguinal white adipose tissue; eWAT = epidydimal adipose tissue).(D) Representative photo of dissected tissue is shown. Data presented as mean ± SEM (n = 5 placebo, n = 5 GLPG3970 50mg / kg, n = 5 GLPG397025mg / kg), p-values by Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

[0073] Figure 18: (A) Daily food intake from placebo and GLPG3970 (50mg / kg) treated mice. (B) Fasted blood glucose levels from placebo and GLPG3970 treated mice. (C-D) Intraperitoneal glucose tolerance tests (1mg / g) from placebo and GLPG3970 treated mice and expressed as area under the curve (AUG). Data presented as mean ± SEM (n = 5 placebo, n = 5 GLPG3970 50mg / kg, n = 5 GLPG397025mg / kg), p-values by Student’s t test. *p < 0.05, **p < 0.01.

[0074] Figure 19: (A) Primary hepatocytes were harvested from WT (Sik3lox / lox) and liver-specific SIK3-KO (AlbCre / +Sik3lox / lox) mice. Lipogenesis (lipid production) in WT and liver-specific SIK3-KO primary hepatocytes in response to basal and SIK3inhibitor Pterosin B was determined by measuring the incorporation of radiolabelled acetate in the primary hepatocytes. Data presented as mean ± SEM, n = 3 independent experiments (each experiment contains at least three replicates), p-values by two-way ANOVA with Sidak post-hoc test: *p < 0.05 for the difference between genotypes. (B) Primary hepatocytes were harvested from WT (Sik3lox / lox) and liver-specific SIK3-KO (AlbCre / +Sik3lox / lox) mice. Rates of oxygen consumption (JO2) were determined in permeabilised primary hepatocytes isolated from WT and liver-specific SIK3-KO mice. Hepatocytes were incubated in the presence of buffer alone (basal) and 25 or 50 pM palmitoyl-CoA (P-CoA). All data are shown as the mean ± SEM, n = 3-4 independent experiment per genotype, p-values by Student’s t-test: *p< 0.05.

[0075] Figure 20: Photographs of liver and Hematoxylin and eosin (H&E) stain of liver tissue from high fat-fed obese mice treated with placebo or YKL-05-099. WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 treatment began at 20 weeks of age.

[0076] Figure 21: Photographs of liver and Hematoxylin and eosin (H&E) stain of liver tissue from high fat-fed obese mice treated with placebo or GLPG-3970. WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 treatment began at 20 weeks of age.

[0077] Figure 22:(A) WT C57BL / 6J mice were fed a high fat diet for 12 weeks starting from 8 weeks of age, and placebo or GLPG3970 treatment (25mg or 50mg / kg) began at 20 weeks of age. Liver triglyceride levels from high fat-fed obese mice treated with placebo or GLPG3970; and (B) WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 treatment began at 20 weeks of age. Liver triglyceride levels from high fat-fed obese mice treated with placebo or YKL-05-099.Detailed description of the embodiments

[0078] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0079] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0080] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0081] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0082] The present disclosure relates to methods of treating obesity, reducing body weight and increasing energy expenditure in an individual in need thereof, the method comprising administering a SIK-3 inhibitor. Surprisingly the inventors have found that administration of a SIK-3 inhibitor to an individual suffering from obesity or an obesity related disorder positively affects the individual through one or more mechanisms, thereby treating the obesity or obesity related disorder.Definitions

[0083] As used herein, the term “condition” refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.

[0084] As used herein, ‘preventing’ or ‘prevention’ is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians.

[0085] The terms ‘treatment’ or ‘treating’ of a subject includes the application or administration of an inhibitor or composition comprising the inhibitor, as described herein, to a subject (or application or administration of inhibitor or composition comprising the inhibitor to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, lessworsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.

[0086] As used herein, the term “subject” or “individual” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0087] It will be clearly understood that, although this specification refers specifically to applications in humans, the invention is also useful for veterinary purposes. Thus in all aspects the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term "individual” or “individual to be I being treated" is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows).Overweight and obesity

[0088] In any method of the disclosure, the method may further comprise identifying an individual in need thereof. Typically the individual is in need of a reduction in body weight, or reduction in adiposity. The individual may be one who has not displayed a significant reduction in body weight after consuming a reduced caloric diet. The individual may have one or more conditions requiring treatment. For example, the individual may overweight or obese, and also have an impaired ability to clear glucose from the blood. The individual may need a reduction in adiposity and also have insulin resistance.

[0089] In any aspect of the present disclosure, the individuals in need thereof are typically those that are overweight, obese, morbidly obese or extremely obese. Unless the context suggests otherwise, any statement herein referring to “obese” may also equally refer to “morbidly obese” or “extremely obese”. Preferably, the individual has aBMI greater than 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 kg / m2. The individual may have a BMI of between 25 kg / m2to 29.9 kg / m2. The individual may have a BMI of 30 kg / m2or greater, or a BMI of 40 kg / m2or greater.

[0090] In any aspect of the present disclosure, the individuals may have been diagnosed as overweight or obese by a physician.

[0091] In any aspect of the present disclosure, the individuals in need thereof may also have an impaired ability to clear glucose from the blood, display a level of insulin resistance and / or be diagnosed with pre-diabetes or type 2 diabetes.

[0092] The term “glycaemic control” refers to maintaining, restoring, or achieving normal or near normal blood glucose levels.

[0093] In any aspect of the present disclosure the method of treatment comprising administration of a SIK-3 inhibitor may additionally lead to improved glycaemic control in the individual.

[0094] In any aspect of the present disclosure the individual may be one that has been identified as having an elevated level of circulating insulin. Preferably the level of circulating insulin that is elevated is at a fasting state or is a basal level.

[0095] In any aspect of the present disclosure the individual may be one that has been identified as having an insulin deficiency.

[0096] In embodiments the individuals may have mild insulin deficiency, and not require specific treatment with insulin. In embodiments the individuals may have moderate insulin deficiency. In embodiments the individuals may have severe insulin deficiency. In embodiments the individuals have absolute insulin deficiency. In embodiments the individual may be being administered insulin.

[0097] The terms “obesity” and “being overweight” refers to an excess of fat in proportion to lean body mass. Excess fat accumulation is associated with an increase in size (hypertrophy) as well as number (hyperplasia) of adipose tissue cells. Obesity is variously measured in terms of absolute weight, weight: height ratio, degree of excess body fat, distribution of subcutaneous fat, and societal and aesthetic norms. A common measure of body fat is Body Mass Index (BMI). The BMI refers to the ratio of body weight (expressed in kilograms) to the square of height (expressed in meters). Bodymass index may be accurately calculated using the formulas: SI units: BMI=weight(kg) / (height2(m2), or US units: BMI=(weight(lb)*703) / (height2(in2).

[0098] In accordance with the U.S. Centers for Disease Control and Prevention (CDC), an overweight adult has a BMI of 25 kg / m2to 29.9 kg / m2, and an obese adult has a BMI of 30 kg / m2or greater. A BMI of 40 kg / m2or greater is indicative of morbid obesity or extreme obesity. A BMI of 40 kg / m2 or greater may be indicative of morbid obesity or extreme obesity. The individual may be unable to conduct any exercise or be limited in their capacity to do so. In any aspect of the invention, any degree of obesity is diet-induced. For children, the definitions of overweight and obese take into account age and gender effects on body fat.

[0099] BMI does not account for the fact that excess adipose can occur selectively in different parts of the body, and development of adipose tissue can be more dangerous to health in some parts of the body rather than in other parts of the body. For example, “central obesity”, typically associated with an “apple-shaped” body, results from excess adiposity especially in the abdominal region, including belly fat and visceral fat, and carries higher risk of co-morbidity than “peripheral obesity”, which is typically associated with a “pear-shaped” body resulting from excess adiposity especially on the hips. Measurement of waist / hip circumference ratio (WHR) can be used as an indicator of central obesity. A minimum WHR indicative of central obesity has been variously set, and a centrally obese adult typically has a WHR of about 0.85 or greater if female and about 0.9 or greater if male.

[0100] In any method of the invention, a reduction in adiposity may be a reduction in central obesity and / or peripheral obesity. In particular, the reduction in adiposity may be a reduction in subcutaneous adiposity. The subcutaneous adiposity may be abdominally located. Alternatively, the reduction in adiposity may be a reduction in visceral adiposity.

[0101] Methods of determining whether a subject is overweight or obese that account for the ratio of excess adipose tissue to lean body mass may involve obtaining a body composition of the subject. Body composition can be obtained by measuring the thickness of subcutaneous fat in multiple places on the body, such as the abdominal area, the subscapular region, arms, buttocks and thighs. These measurements are then used to estimate total body fat with a margin of error of approximately four percentage points. Another method is bioelectrical impedance analysis (BIA), which uses theresistance of electrical flow through the body to estimate body fat. Another method is using a large tank of water to measure body buoyancy. Increased body fat will result in greater buoyancy, while greater muscle mass will result in a tendency to sink. Another method is fan-beam dual energy X-ray absorptiometry (DEXA). DEXA allows body composition, particularly total body fat and / or regional fat mass, to be determined non- invasively.

[0102] The existence of, improvement in, treatment of or prevention of obesity may be by any clinically or biochemically relevant method (e.g. as described herein) of the subject or a biopsy therefrom. For example, treatment may result in a reduction in excess fat, either a reduction in size (hypertrophy) or number (hyperplasia) of adipose tissue cells. In addition, or alternatively, there may be an improvement in various measurements of absolute weight, weightheight ratio, degree of excess body fat, distribution of subcutaneous fat, and societal and aesthetic norms. Further, there may be a reduction in Body Mass Index (BMI). Treatment may result in a reduction in adiposity throughout the entire individual or at certain sites. There may be a reduction in central obesity and / or peripheral obesity. In particular, the reduction in adiposity may be a reduction in subcutaneous adiposity. The subcutaneous adiposity may be abdominally located. Alternatively, the reduction in adiposity may be a reduction in visceral adiposity.

[0103] In addition, an improvement in exercise capacity or mobility may be observed. The improvement in exercise capacity or mobility may be a result of a reduction in adiposity and / or body weight.

[0104] An advantage of an aspect of the present invention is that the reduction in body weight and adiposity does not result in a substantial decrease in lean muscle mass nor a substantial reduction in bone density. In one embodiment, the individual retains substantially more muscle mass as compared to body fat reduction in a subject using an energy restricted diet alone. Typically for an effective therapy for treating a subject having an overweight or obese condition, the treatment should reduce adipose tissue without resulting in substantial deleterious side effects, for example, significant wasting. Wasting is characterized by degradation and loss of a substantial amount of lean body mass (muscle tissue, bones, and / or organs) in addition to adipose tissue. In particular, lean body mass refers to structural and functional elements in cells, body water, muscle, bones, and other body organs such as the heart, liver, and kidneys. Although weight loss may involve loss of fat along with slight loss of muscle or fluid,weight loss for the purposes of maintaining health should aim to lose fat while conserving lean body mass. Wasting involves uncontrollable weight loss.

[0105] In an embodiment of any one of the aspects as herein described the obesity- related disease or disorder is selected from the group consisting of obesity, pre-obesity, morbid obesity, Prader-Willi Syndrome, Hypothalamic Injury Associated Obesity, hyperlipidemia, hypertension, pre-diabetes, diabetes, lipodystrophy, lipodema, Bardet- Biedl Syndrome, Cohen Syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, hypothyroid and MOMO Syndrome.Browning of white adipose tissue

[0106] Brown adipose tissue is a type of fat that is activated in response to cold temperature. The heat produced by brown and beige adipocytes is essential for the survival of small mammals in cold environments, clothing and adequate shelter in modern humans have largely diminished the need for cold-induced brown and beige thermogenesis.

[0107] White adipose tissue is a fat tissue which stores extra energy. In contrast brown or beige adipose tissue breaks down blood sugar and fat molecules to create heat. The capacity of brown / beige adipocytes to utilise lipids and glucose as a fuel source, and to expend the energy as heat, accompanied by their decreased abundance in older and overweight individuals, has garnered interest in promoting brown and beige fat thermogenesis to combat the obesity epidemic.

[0108] In embodiments of the disclosure, administration of a SIK-3 inhibitor may lead to increased browning of white adipose tissue. In embodiments of the disclosure, administration of a SIK-3 inhibitor may lead to increase in thermogenic gene markers, for example llcp-1 , Dio2, Cidea, Prdm16, Pgc-1a and / or PPARy.

[0109] Advantageously increasing the browning of white adipose tissue in an individual is proposed to increase the energy expenditure of the individual when exposed to cold, subsequently leading to a decrease in body weight.

[0110] In embodiments of the disclosure the method of treatment may increase the energy expenditure of the individual.

[0111] Without wishing to be bound by theory is it proposed that administration of a SIK-3 inhibitor can result in increased energy expenditure due increased browning of white fat and increased thermogenesis of brown fat.Insulin resistance and diabetes

[0112] In another embodiment, the individual in need thereof also has insulin resistance, pre-diabetes or Type II diabetes.

[0113] Insulin resistance (IR) is a condition in which the body's cells become less sensitive to the glucose-lowering effects of the hormone insulin. Environmental factors such as physical inactivity, abdominal obesity, diet (e.g., high calorie intake), medications (e.g., Cortisol), hyperglycaemia (glucose toxicity), increased free fatty acids, and the aging process may also contribute. The most common type of insulin resistance is associated with obesity resulting in a condition known as metabolic syndrome.

[0114] In an insulin-resistant person, normal levels of insulin do not have the same effect in controlling blood glucose levels. During the compensated phase in insulin resistance insulin levels are higher, and blood glucose levels are largely maintained. Therefore, in most people with insulin resistance there are normal levels of glucose in the blood but high levels of insulin in the blood. If compensatory insulin secretion fails, then either fasting (impaired fasting glucose) or postprandial (impaired glucose tolerance) blood glucose concentrations increase. Eventually, Type 2 diabetes occurs when blood glucose levels become higher throughout the day as the resistance increases and compensatory insulin secretion fails.

[0115] Type 2 diabetes mellitus is generally characterized by the body's resistance to insulin, caused by the loss or diminished function of insulin receptors that mediate the entrance of insulin into the body's cells. Type 2 diabetes occurs commonly in association with other disorders such as hypertension, dyslipidaemia (includes high LD1 cholesterol, low HDL cholesterol, and high triglycerides) and hypercoagulability. All these problems usually occur in association with obesity, especially abdominal obesity.

[0116] Without wishing to be bound by theory it is proposed that SIK-3 may be a negative regulator of insulin signalling in the brain. Accordingly, SIK-3 inhibition maylead to increased insulin signalling thereby potentially improving glucose regulation, and energy homeostasis.

[0117] Disruption in the normal regulation of glucose can lead to blood glucose levels deviating from, i.e. , elevated or low compared to, normal blood glucose levels. Chronically elevated blood glucose levels, characteristic, for example, of hyperglycaemia, diabetes, can impose multiple detrimental effects on various organs, tissue, and systems of the body. Diabetes, hyperglycaemia, or elevated blood glucose levels are associated with numerous disorders and conditions, including accelerated atherosclerosis, increased chronic heart disease, myocardial infarction, stroke, microangiopathy, damage to blood vasculature, peripheral vascular disease leading to decreased circulation in the arms and legs, macrovascular complication, ocular disorders, such as, for example, diabetic retinopathy, macular degeneration, cataracts, etc., kidney disorders, including, diabetic nephropathy, kidney damage, etc., damage to nerves and other neuropathies, including diabetic neuropathy, peripheral neuropathy, damage to nerves of the autonomic nervous system, etc., hyperinsulinaemia, hyperlipidaemia, insulin resistance, skin and connective tissue disorders, foot wounds and ulcerations, diabetic ketoacidosis, etc.

[0118] Altered or impaired glucose regulation, and the presence of or risk for development of disorders including diabetes, hyperglycaemia, etc., can be identified by measurement of circulating glucose or determination of blood / plasma glucose levels. Blood glucose levels are most often measured by a fasting blood glucose test, a random blood glucose test, or an oral glucose tolerance test.

[0119] The methods and compositions of the invention described herein are for promoting, accelerating, increasing the rate of, or improving the clearance, depletion or reduction in blood glucose. Typically, that means that at each time point after an event that leads to an increase in the fasting or basal glucose level of an individual, the level of blood glucose in an individual who has received a SIK3 inhibitor is less than in an individual that has not received a SIK3 inhibitor (i.e. a control individual).

[0120] The term "hyperglycaemia" as used herein refers generally to blood glucose concentrations or levels that are above normal. Hyperglycaemia can be determined by any measure accepted and utilized by those of skill in the art. Currently, in humans, normal blood glucose is considered to be between about 70 and 120 mg / dl (3.9 - 6.6mmol / L), but varies depending on the fasting state. Before a meal, blood glucose can range from about 80 to 120 mg / dl (4.4 - 6.6 mmol / L), whereas two hours after a meal, blood glucose can be at or below about 180 mg / dl (10 mmol / L). Additionally, in fasted individuals, normal blood glucose is below about 110 mg / dl (6.1 mmol / L). A subject having a blood glucose value of about 126 mg / dl (7 mmol / L) or greater is generally considered hyperglycaemic, and a subject whose blood glucose is above about 200 mg / dl (11.1 mmol / L) is generally considered diabetic.

[0121] The existence of, improvement in, treatment of or prevention of a disease associated with, or arising from, an impaired ability to clear glucose from the blood may be determined by any clinically or biochemically relevant method of the subject or a biopsy therefrom. For example, a parameter measured may be the presence of a certain level of glucose in the blood or rate or degree of decline of glucose in the blood after a glucose challenge. Typically, that includes blood glucose levels measured by a fasting blood glucose test, a random blood glucose test, or an oral glucose tolerance test.

[0122] A method of treating insulin resistance may result in reversing one or more clinical or biochemical characteristics, or symptoms, of insulin resistance.

[0123] A subject or individual “in need thereof” includes a subject or individual that has (a) an impaired ability to clear glucose from the blood, (b) an elevated fasting level of glucose or insulin, (c) any other condition or disease described herein including, but not limited to, insulin resistance, pre-diabetes, diabetes (preferably type 2 diabetes). Typically, an individual that has (a), (b) and / or (c) is overweight or obese.Food intake reduction and Appetite suppression

[0124] The inventors have shown that obese mice treated with a SIK-3 inhibitor reduce their caloric intake (food intake). The reduction in food intake is proposed to contribute to weight loss in an individual administered with a SIK-3 inhibitor. A reduction in caloric intake may be caused by appetite suppression, increased appetite regulation or inducing satiety in the individual. Administration of a SIK-3 inhibitor may also treat or prevent an eating disorder such as binge eating disorder.Fatty liver disease

[0125] Fatty liver disease encompasses a spectrum of liver conditions and is typically classified as either alcoholic or nonalcoholic. In either case, fatty liver disease ranges from simple hepatic steatosis (lipid accumulation and deposition) to steatohepatitis (ASH or NASH), which often progresses to hepatic fibrosis, cirrhosis, and probably hepatocellular carcinoma. Alcoholic fatty liver disease (AFLD) and nonalcoholic fatty liver disease (NAFLD) are histologically indistinguishable; however, by definition NAFLD develops in patients who consume little or no alcohol. Nonalcoholic fatty liver disease (NAFLD) consists of a spectrum of conditions ranging from relatively benign steatosis to more severe non-alcoholic steatohepatitis (NASH), which when untreated can lead to fibrosis, cirrhosis, liver failure, or hepatocellular carcinoma.

[0126] As used herein, fatty liver disease means any disease or other deleterious condition characterized by and / or caused by the accumulation of excess liver fat, including (but not limited to) steatosis, nonalcoholic fatty liver disease (NAFLD), Alcoholic fatty liver disease (AFLD), Nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), liver fibrosis caused by fatty liver disease typically NASH, cirrhosis caused by fatty liver disease typically NASH, or hepatocellular carcinoma (HCC) caused by fatty liver disease typically NASH.

[0127] In some embodiments, the NAFLD is steatosis. In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the NAFLD is liver fibrosis caused by NASH. In some embodiments, the NAFLD is liver cirrhosis caused by NASH. In some embodiments, the NAFLD is hepatocellular carcinoma (HCC) caused by NASH.

[0128] Non-alcoholic steatohepatitis (NASH) is usually a silent disease with few or no symptoms. Patients generally feel well in the early stages and only begin to have symptoms — such as fatigue, weight loss, and weakness — once the disease is more advanced or cirrhosis develops. The progression of NASH can take years, even decades. NASH can slowly worsen, causing scarring or “fibrosis” to appear and accumulate in the liver. As fibrosis worsens, cirrhosis develops; the liver becomes seriously scarred, hardened, and unable to function normally. A person with cirrhosis experiences fluid retention, muscle wasting, bleeding from the intestines, and liver failure.

[0129] The term "liver fibrosis" means the formation or development of excess fibrous connective tissue (fibrosis) in the liver thereby resulting in the development of scarred (fibrotic) tissue. The scarred tissue replaces healthy tissue by the process of fibrosis and leads to subsequent cirrhosis of the liver.

[0130] The invention includes methods of delaying or reducing progression to liver fibrosis caused by or associated with fatty liver diseases. The liver fibrosis may be associated with or co-morbid to cirrhosis, and associated conditions, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH).

[0131] Severe liver fibrosis, if untreated, may lead to cirrhosis. Cirrhosis may be compensated cirrhosis, where the liver is able to continue working more of less normally, and decompensated cirrhosis, where the liver is failing. Symptoms of liver fibrosis may include appetite loss, difficulty thinking clearly, fluid buildup in the legs of stomach, jaundice, nausea, unexplained weight loss and / or weakness. Symptoms of decompensated cirrhosis include ascites, hepatic encephalopathy, bleeding, and jaundice.

[0132] The terms "treatment, treat and treating" of fatty liver disease, such as NAFLD or NASH, refers to the partial or complete alleviation, suppression, delay (onset), prevention, stabilization, amelioration and / or alleviation of fatty liver disease (such as NAFLD or NASH), or one or more symptoms of fatty liver disease (such as NAFLD or NASH). In some embodiments, treatment may be administered after one or more symptoms have occurred.

[0133] In some embodiments, the term "treating" includes preventing or halting the progression of fatty liver disease, such as NAFLD or NASH. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals prior to the onset of symptoms (eg, due to history of symptoms and / or due to genetic or other predisposition factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. Thus, in some embodiments, the term "treating" includes preventing the recurrence of fatty liver disease, such as NAFLD or NASH.

[0134] In one embodiment, the present invention provides a method of reducing or delaying progression of, or worsening of, fatty liver disease in an individual comprisingadministering a SIK-3 inhibitor to the individual, thereby reducing or delaying progression of, or worsening of, fatty liver disease in the individual. In this embodiment, the progression or worsening may be:NAFLD to NASH;AFLD to ASH;NAFLD or NASH to liver fibrosis;AFLD to ASH to liver fibrosis;NAFLD or NASH to liver cirrhosis;AFLD or ASH to liver cirrhosis;NAFLD or NASH to hepatocellular carcinoma; orAFLD or ASH to hepatocellular carcinoma.

[0135] In an aspect of the invention there is provided a method of treating nonalcoholic fatty liver disease in an individual in need thereof, the method comprising administering to the individual a SIK-3 inhibitor.

[0136] In any method of the invention, the method optionally further comprises the step of identifying or diagnosing an individual having fatty liver disease, preferably a fatty liver disease described herein, including NAFLD or NASH.

[0137] In embodiments of the invention the individual in need of treatment has been diagnosed with NAFLD. In embodiments the individual in need of treatment has been diagnosed with NASH. In embodiments the individual in need of treatment has been diagnosed with liver fibrosis. In embodiments the individual in need of treatment has been diagnosed with liver cirrhosis.

[0138] NAFLD can be differentiated from NASH by the assessment of NAFLD activity (NAS), the sum of the histopathology ratings of a liver biopsy for steatosis (0 to 3), lobular inflammation (0 to 2), and hepatocellular embolism (0 to 2). A NAS of <3 corresponds to NAFLD, 3-4 corresponds to bordering NASH, and> 5 corresponds to NASH. Biopsy is also evaluated for fibrosis (0 to 4).

[0139] Fibrosis may be characterized by a liver biopsy in 5 stages (0 to 4). The biopsy sample is evaluated and given a score with 0 being no fibrosis, 1 being portal fibrosis without septa, 2 being portal fibrosis with few septa, 3 being numerous septa without cirrhosis and 4 being cirrhosis.

[0140] In embodiments the individual in need of treatment has been diagnosed with a fatty liver disease, such as NAFLD or NASH, and at least one other obesity related disorder. In embodiments the individual is obese.

[0141] In some embodiments, the individual with a fatty liver disease, such as NAFLD or NASH, has pre-diabetes or diabetes, preferably type 2 diabetes.

[0142] In some embodiments, the individual with a fatty liver disease also has insulin resistance.

[0143] There are instances where individuals who are not obese have fatty liver disease, such as those fatty liver diseases defined herein, for example NAFLD or NASH. The administration of a SIK-3 inhibitor is also beneficial for these individuals.

[0144] In alternative embodiments, the individual in need of treatment for a fatty liver disease, such as NAFLD or NASH, is not obese.

[0145] In alternative embodiments, the individual with a fatty liver disease, such as NAFLD or NASH, does not have pre-diabetes or diabetes, preferably type 2 diabetes.

[0146] In alternative embodiments, the individual with a fatty liver disease does not have insulin resistance.Salt-inducible kinase 3

[0147] Salt-inducible kinase 3 (SIK3 or SIK-3) is a serine / threonine protein kinase that regulates multiple signalling pathways involved in metabolic regulation in response to changes in hormonal and nutrient status. SIK3 is widely expressed in the brain, particularly in the arcuate nucleus (ARC) of the hypothalamus, in subsets of anorexigenic pro-opiomelanocortin (POMC) and orexigenic neuropeptide Y (NPY) / agouti-related protein (AgRP)-expressing neurons. Although these two populations of neurons play critical roles in the coordination of energy balance, the physiological role of SIK3 in regulating energy balance in NPY / AgRP and POMC neurons remains unknown.

[0148] The inventors have shown that hypothalamic SIK-3 expression is significantly upregulated in diet-induced and genetically obese mice. And that genetic ablation of SIK3 in orexigenic NPY neurons leads to reduced diet-induced weight gain attributed toan increase in energy expenditure. It is therefore proposed that increased SIK-3 may be a key factor contributing to obesity development.

[0149] Without wishing to be bound by theory it is proposed that SIK3 in NPY and POMC neurons is critical for the regulation of body weight through control of energy balance. It is further proposed that inhibition of hypothalamic SIK-3 increases energy expenditure and BAT thermogenesis, resulting in decreasing adiposity and promoting weight loss.SIK-3 inhibitors

[0150] In any aspect of the disclosure, the SIK-3 inhibitor is any inhibitor as described herein. Typically, the inhibitor of SIK-3 is any compound, e.g. small molecule, that inhibits the activity of SIK-3. Preferably, the inhibitor binds to the active of site of SIK-3. Typically SIK-3 inhibitors may also inhibit SIK-1 and / or SIK-2.

[0151] The inhibitor may be specific for SIK family (eg SIK-1, SIK-2 and SIK-3), and in particular SIK-3, and only have some low level inhibitory activity against other kinases (for example, an Ki of more than about 100pM, preferably more than 1mM against other kinases as measured using an assay as described herein, or otherwise known in the art). Preferably the inhibitor of SIK-3 is a substance that limits the activity of SIK-3 to 25% or less in comparison with control. Control is a solvent, in which the inhibitor is tested, used at the same quantity however without the inhibitor.

[0152] In embodiments of the disclosure the SIK-3 inhibitor has an Ki value of less than 1mM, less than 100pM, less than 10pM, or less than 1pM.

[0153] In preferred embodiments of the disclosure the SIK-3 inhibitor has an Ki value of less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM less than 50nM, or less than 40nM.

[0154] In preferred embodiments of the disclosure the SIK-3 inhibitor has a Ki for SIK-2 and / or SIK-1 of less than 1mM, less than 100pM, less than 10pM, less than 1pM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM less than 50nM, or less than 40nM.

[0155] In some embodiments of the disclosure the SIK-3 inhibitor has an Ki of about 30nM. In some embodiments the SIK-3 inhibitor also inhibitors SIK-1 and SIK-2 with a nanomolar Ki.

[0156] In embodiments the potency of the SIK-3 inhibitor is measured in a binding assay measuring the binding to hSIK3. The binding assay may be a commercially available binding assay, for example LanthaScreen® kinase assay. The binding assay may be determined by measuring the binding and displacement of a fluorescence label with a labelled anti-tag antibody which binds to the kinase of interest. Simultaneous binding of both the tracer and antibody to the kinase results in a high degree of FRET (fluorescence resonance energy transfer). Binding of the inhibitor to the kinase competes for binding with the tracer resulting in a loss of FRET. Detailed protocol information may be found from the manufacturer (Life Technologies, catalogue numbers PV6403, PV6404 and PV6405 for example https: / / tools.thermofisher.com / content / sfs / manuals / PV6403_PV6404_PV6405_SIK3_PI. pdf).

[0157] Alternatively, the potency of the SIK-3 inhibitor may be determined by any known assay that measured SIK-3 activity. For example, the SIK3 Kinase System (Promega) contains SIK3 Kinase, 10pg (Human, recombinant; amino acids 1-307). MW: ~62kDa, AMARA Peptide Substrate (1mg / ml), (AMARAASAAALARRR), and 5X Reaction Buffer, 0.1M DTT. It can be used with the ADP-Glo™ kinase assay (Promega). The ADP-Glo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is a substrate in a reaction catalyzed by Ultra-Gio™ Luciferase that produces light. The luminescent signal positively correlates with ADP amount and kinase activity.

[0158] Alternatively, SIK-3 enzymatic activity may be assessed by transfecting cells with PCDNA5-Flag SIK3 vector and cultured for 48 hr to produce SIK3 overexpressed cells. After 48h, SIK3 overexpressed cells are treated with SIK3 inhibitors. Following FLAG immunoprecipitation, purified SIK3 proteins are subjected to kinase assay for 10 minutes at 32°C in the presence of [y-32P]-ATP using AMARAASAAALARRR peptide as a substrate. The reaction is stopped by spotting 15 pL of reaction mix onto p81 phosphocellulose paper and placing it in 1% phosphoric acid and measuring using a scintillation counter.

[0159] In a further alternative SIK-3 inhibitor activity may be measured by western blotting with a phosphorylation SIK-3 antibody (eg Invitrogen, Cat# PA5-105914) in the catalytic site.

[0160] In some embodiments the inhibitor is selective for SIK-3 and demonstrates higher inhibitory activity for SIK-3 over SIK-1 or SIK-2 or both. For example, the SIK-3 inhibitor may have higher inhibitory activity for SIK-3 and SIK-2 over SIK-1 , or the SIK-3 inhibitor may have higher inhibitory activity for SIK-3 and SIK-1 over SIK-2.

[0161] In some embodiments the SIK-3 inhibitor has at least the same inhibitory activity for SIK-3 as for SIK-1 and / or SIK-2. In embodiments ICso of the SIK-3 inhibitor for SIK-3 inhibition is the same order of magnitude compared to the ICso for SIK-1 and / or SIK-2.

[0162] In some embodiments the SIK-3 inhibitor has at least 2, or at least 5 or at least 10 or at least 20 or at least 50 times higher inhibitory activity for SIK-3 over SIK-1. Alternatively or additionally, the SIK-3 inhibitor has at least 2, or at least 5, or at least 10 times high inhibitory activity for SIK-3 over SIK-2.

[0163] In embodiments the SIK-3 inhibitor is not bosutinib. In embodiments the SIK- 3 inhibitor is not dasatinib.

[0164] In alternative embodiments the SIK-3 inhibitor may also be an inhibitor of related family kinases, for example SIK-1 or SIK-2 or both. In alternative embodiments the SIK-3 inhibitor may inhibit SIK-1, SIK-2 and SIK-3. In one embodiment, the SIK-3 inhibitor has the same, or similar, selectivity for SIK-3 compared to SIK-1 and / or SIK-2 as any one of the inhibitors described herein, preferably YKL-05-099 or GLPG3970.

[0165] In some embodiments, the SIK-3 inhibitor shows selectivity for SIK family kinases, ie the SIK-3 inhibitor is more potent for inhibition of SIK family kinases, preferably SIK-3 kinase, compared inhibition of other kinases. For example, the SIK-3 inhibitor does not exhibit significant inhibitory activity for kinase ABL (or tyrosine kinase ABL). In some embodiments the SIK-3 inhibitor does not show significant activity for BTK (Bruton’s tyrosine kinase) or Src kinase. For example in some embodiments, the SIK-3 inhibitor is more than 50 times, 100 times, 200 times or 250 times less potent against ABL, BTK and / or Src compared to SIK-3. Alternatively, the SIK-3 inhibitor exhibits similar potency as any inhibitor described herein (e.g. GLPG-4399, GLPG-3970, OMX-0407, Tenalsib, Pterosin B, YKL-05-099, Dasatinib, Bosutinib, Crenolanib, HG-9-91-01, YKL-06-061, MRT- 199665, ARN-3261, GLPG 3312, MRIA9, ARN-3236, and YKL-06-062) against ABL, BTK and / or Src compared to SIK-3.

[0166] In some instances, administration of kinase inhibitors with broad activities may have off-target effects. Without wishing to be bound by theory, it is proposed that a SIK-3 inhibitor with a high degree of selectivity for SIK family kinases and particularly SIK-3 may have reduced side effects compared to an inhibitor of multiple kinases.

[0167] The SIK-3 inhibitor may be any inhibitor that inhibits the activity of SIK-3 or reduces the level of SIK-3 in a cell. The inhibitor may be a direct inhibitor of the SIK-3 active site, may act allosterically to inhibit SIK-3 activity, inhibit interaction of SIK-3 with its substrate, or reducing the amount of SIK-3 mRNA or protein present in the cell. For example, the amount of SIK-3 protein levels can be reduced through ubiquitin degradation.

[0168] An inhibitor of SIK-3 may be selected from the group consisting of a small molecule, an antibody, a peptide, an interfering RNA (including an antisense RNA, siRNA, microRNA, or shRNA or a gRNA), a PROTAC, or may be a gRNA for gene editing (for example using CRISPR-Cas9 genome editing methods).

[0169] In any aspect of the present disclosure, the SIK-3 inhibitor is a small molecule. Examples of small molecule SIK-3 inhibitors can be found, for example, as described in US11339166B2, US2022 / 0402911 A1 , US10954242B2, US 20230167116 and US9914735B2.

[0170] Further and non-limiting examples of SIK-3 inhibitors include GLPG-4399, GLPG-3970, OMX-0407, Tenalsib, Pterosin B, YKL-05-099, Dasatinib, Bosutinib, Crenolanib, HG-9-91-01, YKL-06-061 , MRT- 199665, ARN-3261 , GLPG 3312, MRIA9, ARN-3236, and YKL-06-062.Chemical structure of Pterosin BChemical structure of YKL-06-061Chemical structure of DasatinibChemical structure of BosutinibChemical structure of CrenolanibChemical structure of TenalisibChemical structure of GLPC-3970Chemical structure of MRIA9Chemical structure of HG-9-91-01Chemical structure of ARN-3236Chemical structure of YKL-06-062Administration

[0171] The term ‘administered’ means administration of a therapeutically effective dose of the aforementioned inhibitor(s) or composition(s) to an individual. By ‘therapeutically effective amount’ is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0172] The skilled person will appreciate that varying doses of SIK-3 will be effective at treating individuals in need thereof, depending on, for example the level of severity of the obesity, or adiposity in the individual being treated. The skilled person will be readily able to determine the appropriate dose of SIK-3 for promoting reduction in body weight or increased energy expenditure in an individual in need thereof.

[0173] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific SIK-3 inhibitor employed, the metabolic stability and length of action of that SIK-3 inhibitor, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0174] Moreover, the skilled person will be familiar with methods for determining an appropriate human dose based on the disclosure provided herein in relation to therapeutically effective doses for use in mice. For example, Human dose conversion can be by any of the relevant methods described in U.S. Department of Health andHuman Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Rockville, MD 2005, incorporated herein by reference.

[0175] The human equivalent doses described herein are derived using the methods described in the CDER ‘Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers’ for dose conversion based on body surface area. It will be appreciated that any variation on the methods described in the CDER document may also be utilised for determining an appropriate human dose from the dosages administered to mice, as described herein (for example, in some circumstances it may be appropriate to dose scaling based on body weight rather than on body surface area). Moreover, the skilled person will also be familiar with methods for determining the appropriate dose for a juvenile human (i.e. , non-adult human), as well as methods for determining the appropriate dose in a non-human organism, to which the methods of the present invention may be applied. The skilled person will also be familiar with methods for adjusting the appropriate dose depending on the intended method of administration (for example, intravenous, intramuscular, subcutaneous, topical, oral or other method of administration).

[0176] Human Equivalent Dose (HED in mg / kg) = Animal Dose (mg / kg) x Animal K Human K, where K is a correction factor reflecting the relationship between body weight and body surface area.

[0177] For a typical adult (body weight 60 kg, body surface area 1.6m2), K is 37 and mouse K is 3.

[0178] An adult suffering from obesity will likely weigh more than 60 kg and the skilled person will be familiar with appropriate methods for adjusting the dose of SIK-3 accordingly to account for the increased body surface area as compared with a 60 kg adult.

[0179] For example in some embodiments the SIK-3 inhibitor may be administered as an injection, and the injection dose levels may range from about 0.1 mg / kg / h to at least 10 mg / kg / h, all for from about 1 to about 120 h and especially 24 to 96 h. Inembodiments a preloading bolus of from about 0.1 mg / kg to about 10 mg / kg or more may also be administered to achieve adequate steady state levels.

[0180] For the prophylaxis and / or treatment of long-term conditions, such as degenerative conditions, the regimen for treatment usually stretches over many months or years so oral dosing is preferred for patient convenience and tolerance.

[0181] With oral dosing, one to four (1-4) regular doses daily, especially one to three (1-3) regular doses daily, typically one to two (1-2) regular doses daily, and most typically one (1) regular dose daily are representative regimens. Alternatively for long lasting effect drugs, with oral dosing, once every other week, once weekly, and once a day are representative regimens. In particular, dosage regimen can be every 1-14 days, more particularly 1-10 days, even more particularly 1-7 days, and most particularly 1-3 days.

[0182] Using these dosing patterns, each dose provides from about 1 to about 1000 mg of a compound of the invention, with particular doses each providing from about 10 to about 500 mg and especially about 30 to about 250 mg.

[0183] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.Pharmaceutical composition

[0184] In embodiments of the disclosure the SIK-3 inhibitor may be provided as a pharmaceutical composition, comprising a SIK-3 inhibitor and a pharmaceutically acceptable diluent, excipient or carrier.

[0185] In any method or use of the disclosure, SIK-3 inhibition may be achieved by administering a composition or pharmaceutical composition as described herein.

[0186] Generally, a composition of this invention may be administered orally or parenterally (eg subcutaneously), however, any other suitable route of administration is contemplated, for example topical (for example, transdermal or ocular), oral, buccal, nasal, vaginal or rectal administration.

[0187] The term parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal,intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.

[0188] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

[0189] In certain embodiments, compositions in a form suitable for oral use or parenteral use are preferred. Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilisate.

[0190] A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatine-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

[0191] In embodiments the pharmaceutical composition may comprise a further active ingredient. The further active ingredient may contribute towards the methods of the invention by an alternative mechanistic pathway. In embodiments the pharmaceutical composition may be a synergistic composition.Kits

[0192] In another embodiment there is provided a kit or article of manufacture comprising: a container holding a SIK-3 inhibitor, or a pharmaceutical composition comprising a SIK-3 inhibitor; and a label or package insert with instructions for use.

[0193] In certain embodiments the present disclosure provides a kit for use in: treating obesity; in an individual in need thereof; treating or minimising the risk of, an obesity-related disease or disorder in an individual suffering from or at risk of suffering from an obesity-related disease or disorder reducing the body weight of an individual in need thereof; preventing or minimising the weight gain of an individual consuming a high energy / caloric diet preventing or minimising adiposity in individual consuming a high energy / caloric diet enhancing energy expenditure improving insulin secretion in an individual in need thereof promoting blood glucose clearance in an obese individual; promoting or improving glycaemic control in an obese individual; treating pre-diabetes or diabetes in an obese individual; treating insulin resistance in an individual,; or treating a fatty liver disease in an individual, the kit comprising a SIK-3 inhibitor, preferably a SIK-3 inhibitor as described herein.

[0194] Preferably, the kit also comprises written instructions for use of the kit in a method of the disclosure as described herein.

[0195] The kit or “article of manufacture” may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a therapeutic composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat a disease described herein.

[0196] The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second container with a second active principle or ingredient contained therein. The kit in this embodiment of the invention may further comprise a package insert indicating the composition and other active principle can be used to treat a disorder or prevent a complication stemming from obesity or an obesity related disorder. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0197] In certain embodiments the therapeutic composition may be provided in the form of a device, disposable or reusable, including a receptacle for holding the therapeutic, prophylactic or pharmaceutical composition. In one embodiment, the device is a syringe. The device may hold 1-2 mL of the therapeutic composition. The therapeutic or prophylactic composition may be provided in the device in a state that is ready for use or in a state requiring mixing or addition of further components.

[0198] The present disclosure also provide for the use of agent in the manufacture of a kit as described herein.Examples

[0199] The inventors have shown the therapeutic potential of SIK-3 inhibition in promoting weight loss and improving glucose and insulin tolerance in an in vivo preclinical model through two distinct inhibitory mechanisms - (1) genetic manipulation of SIK-3 and separately through (2) pharmacological inhibition. Relevantly, multiple structurally distinct SIK-3 inhibitors have been tested and beneficial effects observed.

[0200] Furthermore, the inventors have shown the therapeutic potential of SIK-3 inhibition in inhibiting hepatic lipid production and promoting hepatic lipid utilisation, and reducing lipid accumulation in the liver in a preclinical model through two distinct inhibitory mechanisms - (1) genetic manipulation of SIK-3 and separately through (2) pharmacological inhibition. Relevantly, multiple structurally distinct SIK-3 inhibitors have been tested and beneficial effects observed.Example (1) Genetic manipulation of SIK-3 inhibition

[0201] In vivo NPY neuron specific SIK-3 knock-out studies were performed according to the methods explained below.Animal and Experimental Design

[0202] All animal care and experiments were performed in accordance with protocols approved by the St Vincent’s institute Animal ethics committee (AEC No. 015 / 18, 017 / 19, and GBNML849). All mice were housed in a temperature-controlled room of 22 °C with a 12-hour light / dark cycle (lights on from 0700-1900 hours) and were allowed free access to food and water. All experimental mice used in this study were on a C57NL / 6J background. Age- and sex- matched mice were used for all experiments. Male mice were used in all studies. Mice were fed a standard chow (19% protein, 4.6% fat and 4.8% crude fibre) or a high-fat diet (23%fat, 45% of total energy from fat; SF04- 027; Specialty Feeds, Australia) for the entirety of the experiments except during experiments where fasting is required.

[0203] Mouse body weight is dependent on mouse age and sex. For the mice used in this study, a mouse having a body weight of about 35g or more is considered an overweight mouse, and a mouse having a body weight of more than about 40g is considered an obese mouse. More specifically a mouse having a total body fat mass of about 5-7g and / or 15-20% of fat mass / body weight is considered an overweight mouse, and a mouse having a total body fat mass of >7g and / or >20% of fat mass / body weight is considered an obese mouse.Generation of NPY neuron specific SIK3 KO mice

[0204] To generate NPY neuron-specific SIK3 deficient mice, SI K3fl / fl were mated with NPY-cre mice to generate NPYcre / +; SI K3fl / fl mice. NPYcre / +;SIK3fl / fl mice were then subsequently crossed with Gt(ROSA)26Sortm9 to generate NPYcre / +; SI K3fl / fl;Ai9 mice. SI K3fl / fl mice were used as control.Isolation of genomic DNA

[0205] Mouse tail tips and tissues (Hypothalamus, hippocampus, brain stem, cerebrum, olfactory Bulb, pituitary gland, and liver) were digested in 250 pL tail digestion buffer (100 mM Tris-HCI, pH 8.5, 5 mM EDTA, 0.2% (w / v) SDS, 200 mMNaCI) containing 20 g proteinase K and incubated overnight at 55 C. Digested tissues were centrifuged at room temperature at 13 000 rpm for 5 mins. Resulting supernatant were transferred to fresh tubes. 100 % isopropanol were added and mixed gently by inverting. 100% isopropanol were removed from the precipitated genomic DNA by centrifugation at 13 000 for 3 mins. Clinging isopropanol were removed by pipette then samples were left to dry for 30 minutes. 250 pL of 1x TE buffer were added. Routine PCR was conducted to confirm recombination of SIK3 allele in these tissues using the following primers:Genotyping and primers

[0206] Routine genotyping was carried out to confirm the presence of SIK3 floxed alleles and presence of NPY ere transgenes. PCR reactions were conducted using PCR master mix and protocol as indicated in the table below:Table 1. Primer sequenceTable 2. PCR Master MixTable 3. PCR Reaction ProtocolMetabolic Measurements and Body Composition

[0207] Body composition such as whole-body fat mass and lean mass were measured at 8, and 16 weeks of age using nuclear magnetic resonance imaging (Whole Body Composition Analyzer, Echo MRI, Houston, USA). Blood glucose levels were determined using blood collected from tail tip and measured with Accu-check Performa glucose meter (Roche, Switzerland). These measurements were taken at the same time of the day between 9:00 h - 11 :00 h. Physical activity, food intake and energy expenditure measures were determined using a computer-controlled indirect calorimetry system (Promethion®, Sable Systems, Las Vegas, NV). The calorimetry system consists of 16 metabolic cages, reflecting their home cages, which are equipped with indirect open circuit calorimetry, food consumption & activity monitors. Mice were put in the cages for 72 hours. Data presented were averaged for two dark and two light cycles. Energy expenditure and the respiratory exchange ratio were calculated from the gas exchange data (Rate of oxygen consumption (VO2) in ml / min and Rate of carbon dioxide emission (VCO2) in ml / min). Gas exchange data was used to calculate carbohydrate and fat oxidation rates. Respiratory exchange ratio (RER) reflects the percentage use of fuel substrate at the cellular level at any time (carbohydrate or lipids). RER of 0.7 for fats, 0.8 for proteins, 1.0 for carbohydrates. Ambulatory movements were measured by pedestrian meters, which sums all of the directed ambulatory locomotionof 1 cm / sec or above within the x,y,z beam-break system, excluding the wheel activity. Feeding was determined by measuring the mass of food consumed.Glucose tolerance test (GTT)

[0208] Whole body glucose clearance was assessed by intraperitoneal glucose tolerance test (IPGTT) on 6 h fasted conscious mice at 8- and 16-weeks of age. Briefly, mice were weighed and after taking basal blood glucose levels, injected intraperitoneally with 1 g / kg body weight of D-glucose. Blood glucose levels were determined at basal and, 15, 30, 60, 90- and 120-minutes following glucose administration, using blood collected from tail tip and measured with Accu-check Performa glucose meter (Roche Diagnostics, Switzerland). Area under the curve was calculated using prism software to determine glucose tolerance.Insulin tolerance test (ITT)

[0209] Whole body insulin sensitivity was assessed by intraperitoneal insulin tolerance test (I PITT) on 6 h fasted conscious mice at 9- and 17-weeks of age. Briefly, mice were weighed and after taking basal blood glucose levels, injected intraperitoneally with 0.75U / kg body weight human insulin (Actrapid, Novo Nordisk Pharmaceuticals). Blood glucose levels were determined at basal and, 15, 30, 60, 90- and 120-minutes following insulin administration, using blood collected from tail tip and measured with Accu-check Performa glucose meter (Roche Diagnostics, Switzerland). Area under the curve was calculated using prism software to determine insulin sensitivity.Pyruvate tolerance test (PTT)

[0210] Pyruvate tolerance test was conducted to test the hepatic gluconeogenesis, in which the intraperitoneal pyruvate injection was administered on 16 h fasted 10- and 19 weeks old mice. Briefly, mice were weighed and after taking basal blood glucose levels, injected intraperitoneally with 1g / kg body weight sodium pyruvate. Blood glucose levels were determined at basal and, 15, 30, 60, 90- and 120-minutes following pyruvate administration, using blood collected from tail tip and measured with Accu- check Performa glucose meter (Roche Diagnostics, Switzerland). Area under the curve was calculated using prism software.Fed and fasted serum collection

[0211] Overnight (16 h) fasted and 30-miuntes re-fed blood were collected by retro- orbital bleeding (Performed by Ms Rhiannon Walder from Bioresource Centre Animal Facility. Briefly, a small amount of blood collected was used to measure fasting and refeeding blood glucose levels using Accu-check Performa glucose meter (Roche Diagnostics, Switzerland). The remainder blood, approximately 200pL, were put on heparinised 1.5 mL Eppendorf tubes, stored on ice then centrifuged at 13,000 rpm for 10 minutes at 4°C. Serum was collected and stored at -20° C.Insulin Concentration Determination

[0212] Plasma insulin levels were measured in duplicates from serum obtained from the 16 h fasted and 30 mins re-fed mice using a commercially available sandwich type insulin ELISA assay: mouse specific ultrasensitive insulin ELISA (ALPCO Diagnostics, Salem, NH, USA).Tissue isolation and Homogenisation

[0213] After completing all metabolic characterizations, mice at 16-20 weeks of age were fasted for 3-4 hours then sacrificed by cervical dislocation. Tissues were collected. The skull was then pierced and dissected exposing the brain. The brain was carefully flipped to expose the hypothalamus located on the underside of the brain.Hypothalamus were excised and snap frozen in liquid nitrogen and stored at -80 °C for storage. Other tissues including gastrocnemius, soleus and quadricep muscles, liver, and individual fat pads were weighed and either put into 1.5 mL Eppendorf tubes, snap frozen in liquid nitrogen, and stored at -80 °C or put into histology cassettes and fixed in 4% PBS-buffered paraformaldehyde for 72 h before processing and embedded in paraffin. Tissues were mechanically homogenised. For protein extraction, ice cold RIPA lysis buffer containing 20 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 pg / ml leupeptin, supplemented with protease and phosphatase inhibitors (Cell Signalling Technology) was used. For RNA extraction, RNAzol® RT (Sigma, St. Louise, MO) was used.RNA extraction, reverse transcription, and quantitative real-time PCR

[0214] Total RNA of hypothalamic tissues was extracted using RNAzol reagent (Sigma, St. Louis, MO) following the manufacturer’s instructions. Whole hypothalamic was resuspended in 250 pL RNAzol followed by vigorous homogenisation using beads and TissueLyser LT (QIAGEN). Homogenates were centrifuge at 12,000 x g for 5 minutes at 4°C. Supernatant containing RNA were transferred into a fresh tube. 100 pL of RNase-free water were added to the homogenates. Samples were mixed vigorously for 15 seconds. Samples were allowed to stand for 15 minutes at RT. The resulting mixture was centrifuged at 12,000 x g for 15 minutes at 4°C. Supernatant was transferred to a new tube then equal volumes of 100% isopropanol was added to precipitate 100%. Samples were centrifuged at 12,000 x g for 10 minutes. RNA pellets were washed twice with 75% ethanol. RNA pellets were solubilised in 50uL RNAse-free water. RNA quality and concentration was determined using nanodrop. Purified RNA was stored at -20 C. Isolated RNA was reverse transcribed into cDNA using the Superscript IV First-Strand Synthesis System (Invitrogen, Australia). In a 20 pL reaction, RNA samples were incubated with 1 pL of random hexamers, 1 pl of 10 mM dNTP mix plus 2-5 pg of the template RNA. To facilitate annealing of primers to the template RNA, the above mixture was incubated at 65°C for 5 minutes, and then incubate on ice for at least 1 minute. Subsequently, annealed template RNA was combined with 4 pL of 5x SSI buffer, 1 pL of 100mM DTT, 1 pL of ribonuclease inhibitor, and 1 pL of Superscript Reverse Transcriptase (Invitrogen, Australia). Annealed RNA and reverse transcription reaction mix were combined and incubated at 23 °C for 10 mins, 50 °C for 10 mins and 80 °C for 10 mins. Primer annealing and reverse transcription was carried out in a thermocycler (Bio-Rad). A control for the genomic DNA contamination was prepared as a reaction without the addition of reverse transcriptase. The resulting cDNA samples were stored at -20 °C for subsequent quantitative real-time PCR analysis. Quantitative Real-Time PCR was conducted using TaqMan® Gene Expression System (Thermo Fisher). Quantitative real-time PCR was performed using the Light-Cycler 480 Real- Time PCR system (Roche, Switzerland). Reactions were set up in a 384-well PCR plate. Each reaction performed in a 10 pL volume containing 5 pL of 2x TaqMan® Universal PCR Master Mix, 0.5 pL 1x TaqMan Gene Expression Assay, 3.5 pL RNAse free H2O, and 1 pL cDNA template. GAPDH was utilised as internal control to evaluate the relative amount of target gene expression levels.

[0215] The cycling conditions used in all the RT-qPCR experiments were as follows: 95 °C for 10min, 95 °C for 15s, and 60 °C for 60s for 40 cycles. The relative gene expression of SIK3 was performed under the assumption that the binding efficiency of the probes are equal. GAPDH was used as the housekeeping gene for the normalization of SIK3 expression. Prism software was utilised to quantify the comparative Ct (AACt) method and the results were reported as fold change from the controls.Immunohistochemistry

[0216] For brain immunohistochemistry, mice were anaesthetised. Incisions were made through both sides of the rib cage up to the collarbone, exposing the lung and the heart. Trimmed needle was then inserted towards the tip of the left ventricle at an angle parallel to the midline of the heart. Needle is then clamped to secure position within the left aorta. A small incision was then made on the right atrium to let blood flow out. Immediately, heparinised saline [10,000 units / l heparin in 0.9% (w / v) NaCI] was perfused at a constant speed (10 rpm) using a peristaltic pump. After blood is flushed out, indicated by the liver gradually turning pale (approximately 20 mL of heparinised saline), 4% (w / v) paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) is subsequently perfused (approximately 50 mL). Mice were then decapacitated, the skull was pierced and dissected exposing the brain. The brain was carefully removed from the skull and post fixed in 4% (w / v) paraformaldehyde in phosphate buffer for 24 hours at 4 C. Following post-fixation, brains were then washed twice for 15 minutes in 1x PBS.Subsequently, brains were transferred to 30% sucrose (w / v) in 1x PBS for four days until they sink at the bottom for cryoprotection. Brains were frozen and stored at -80 C. Fixed brains were embedded in Tissue-Tek O.C.T. compound (ProScieTech) and allowed to acclimatise in the cryostat for 1-2hours prior to sectioning. Using cryostat, brains were cut in the coronal plane and sectioned into 30 urn thickness 120 urn apart. Brains were collected into 12 well plate containing anti-freeze media (for 500 mL: mix 250 mL of 0.05 M sodium phosphate buffer, 150 mL ethylene glycol, and 100 mL glycerol). Brains can be stored at -20 °C until ready for staining. For detection of SIK3, samples were removed from the freezer and allowed to equilibrate at room temperature for 30 minutes. Sections were transferred into fresh 12 well plate containing 1xPBS. Sections were washed with 1x PBS 3x 10 minutes each on an orbital shaker using low speed at room temperature. Sections were then placed in freshly prepared 1%NaOH,1%H2O2 in 1X PBS for 20 min. Sections were then washed 3x for 5 mins in 1X PBS. Subsequently, sections were incubated in 0.3% glycine in PBS for 10 min then washed 3x for 5 mins in 1x PBS. After, sections were placed into 0.73ul of 10% SDS to 25ml 1X PBS for another 10 minutes. Sections were washed 3x for 5 minutes. Sections were then blocked for 2 hours in blocking-permeabilising solution (1x PBS, 0.3% Triton X- 100, and 3% normal goat serum). For primary antibody incubation, sections were incubated in 800 pL of 1 :300 SIK3 primary antibody diluted appropriately in 1x PBS with 0.3% Triton X-100 and 3% normal goat serum on an orbital shaker rotating at low speed (e.g., speed 7 rpm) overnight at 4C. The following day, brains were poured into well inserts. Sections were then washed once for 30 secs and 2x for 10 minutes with 1xPBS with 0.3% Triton X-100, and 3% normal goat serum. Sections were then incubated in secondary antibody solution (1 :250 Goat anti-rabbit alexa-fluor488) for 2 hours shaking on an orbital shaker at RT. Sections were protected against light during the secondary antibody incubation. Sections were washed 1x for 30s then 2x for 10 minutes in 1xPBS while continuing to protect against light exposure. Sections were poured into a 150mm petri dish filled with 1xPBS with 0.3% Triton X-100. Glass slides were submerged into solution. Fine paint brush was used to mount free floating brain sections towards the slides. Brain sections were carefully positioned on to the slides. Following mounting, sections were dried onto the slides for about 10-15 minutes while being shielded against the light. Aqueous mounting medium containing DAPI were then added to the mounted slides. Using tweezers, coverslip was placed on top of the medium. Edges of the cover slipped slides were then sealed with clear nail polish. Slides were stored in a dark slide box at 40 until visualised using a Leica Thunder fluorescent microscope at magnifications 10x, 20x, and 40x.

[0217] For individual fat pads (epididymal, inguinal and BAT), tissues were excised, weighed, placed in histology cassettes, and immediately fixed in 4% (w / v) paraformaldehyde in phosphate buffer overnight at room temperature and embedded into paraffin. Sections were cut at 5 urn thickness.Statistical analyses

[0218] Data are presented as means ± SEM. Differences between SIK3fl / fl and SIK3fl / fl;NPYcre / + group under different experimental conditions were assessed using Student-t test. Differences among groups of mice were assessed by two-way ANOVA or repeated-measures ANOVA. Correlation coefficient was calculated using Spearman’srank correlation coefficient. Statistical analyses were assessed using Prism software 9.0. Differences were regarded as statistically significant if *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001.Example (2): Pharmacological inhibition of SIK-3 in overweight mice using YKL- 05-099Animal model

[0219] All in vivo experiments were performed according to protocols approved by the Garvan / St. Vincent Institute Animal Ethics Committee (AEC No. GBNML 760 and 016 / 019). The mice used in this study were housed up to 6 per cage and raised under standard laboratory condition with 12-hour light / dark cycle, steady temperature 22°C and free access to water and food.

[0220] High-fat diet (HFD) & Streptozotocin (STZ) induced type II diabetic mice model. A cohort of wild type male C57 / BL6 mice were purchased from The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. These mice were treated with high-fat diet (46% of calories from fat, 20% calories from protein and 34% calories from carbohydrate, Specialty Feeds, Australia) for 4 weeks prior to streptozotocin (STZ, Sigma) injection. Mice were fasted for 4 hours and intraperitoneally injected 5 doses of 40mg / kg of STZ within 2 weeks. STZ was dissolved in sodium citrate buffer (pH=4.5) right before the injection. Body weight and blood glucose was measured weekly since HFD, blood was collected from mouse tail tips and blood glucose was determined via Accu-check Performa glucometers (Roche, Switzerland). Two weeks after STZ injection, the average blood glucose level spike up to 15.4mmol / L. These mice were randomly allocated into two groups, one group treated with placebo and the other group will be treated with YKL-05-099 (YKL).

[0221] YKL 05-099 Treatment. Diabetic mice were randomly allocated for either YKL or placebo. YKL (18mg / kg of body weight) or placebo (6.25mM HCL + PBS) was administrated through oral gavage once per day for 4 weeks. Blood glucose was measured weekly to monitor mice’s glycaemic control ability. Body composition such as whole-body fat mass and lean mass were measured after 4 weeks of YKL / placebo treatment using nuclear magnetic resonance imaging (Whole Body Composition Analyzer, Echo MRI, Houston, USA).

[0222] Glucose tolerance test. After 4 weeks of placebo / YKL treatment, an intraperitoneal glucose tolerance test (IPGTT) was performed to investigate glucose clearance ability. Mice were fasted for 6 hours, prior to glucose injection, mice were weighted, and basal blood glucose level was measured and recorded. Then the mice were intraperitoneally injected with 1 g / kg body weight of glucose (dissolved in saline). Blood glucose levels were measured at 15, 30, 60, 90, 120 mins time point post glucose injection. Blood glucose levels were measured by Accu-check Performa glucometers (Roche, Switzerland) by assessing blood from tail tip.

[0223] Fasting / Refed blood glucose & insulin level measurement. Glucose sensitivity was assessed through fast / refed blood glucose test and insulin secretion level was measured. Mice were fasted overnight (16 hours) and 30 mins of refed time, blood glucose was measured before and after refed by collecting blood from tail tips. To determine insulin secretion level, mouse eye bleed was collected before and after refed and centrifuged at 4°C 14,800 RPM for 10 mins to separate plasma for insulin level measurement. 10pl of neat fasting plasma and 10 pl of 1 in 3 diluted refed plasma were added to mouse ultrasensitive insulin ELISA kit (ALPCO Diagnostics, Salem, NH USA) to investigate insulin secretion level.

[0224] Insulin ELISA. Mouse ultrasensitive insulin ELISA kit (ALPCO Diagnostics, Salem, NH, USA) was used to measure insulin secretion both in vitro and in vivo. 10pl of sample was added to the ELISA plate with 75pl of conjugate buffer and shake it at room temperature with 900 RPM for 2 hours. Then, washed the ELISA plate using the ELISA wash for 6 times and 10OpI of TMB was added to each well. Shaked for another 15 mins and added stop buffer. Then ELISA result was read by Enspire Plate Reader and analysed by Prism 10.0.

[0225] Statistical analysis. Data will be presented as means ± SEM. Differences between YKL or Vehicle treated group under different experimental condition will be assessed using Student-t test. Differences between groups of mice were assessed by two-way ANOVA or repeated-measures ANOVA. Correlation coefficient will be calculated using Spearman’s rank correlation coefficient. Statistical analyses were assessed using Prism software 10.0. Differences were regarded as statistically significant if *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****p < 0.0001.

[0226] The results of the YKL inhibitor studies are shown in figure 10. These studies showed that administration of the pharmacological SIK3 inhibitor YKL-05-099 reduced body weight in diet-induced obese and diabetic mice. Mice administered with the pharmacological SIK3 inhibitor had increased post-prandial induced insulin secretion and improved glucose tolerance.

[0227] These experiments demonstrate that YKL-05-099 reduces body weight, adiposity and improves glycaemic control in high fat diet (HFD) + streptozotocin (STZ) mice a widely used model for obese and type 2 diabetes (reduced beta-cell mass).

[0228] Additionally, mice were fed on a high fat diet for up to 12 weeks to achieve a body weight of above 40g, which reflects clinical obesity with a BMI >30. Obese mice treated with YKL-05-099 exhibited reduced body weight over 4 weeks of treatment (figure 12). The body weight reduction in obese mice treated with YKL-05-099 was found to be due to reduced adiposity, with minimal reduction in lean mass (figure 13). YKL-05-099 treatment enhanced thermogenesis and UCP1 levels in brown adipose tissue in obese mice (figure 14).Example (3) Oral administration of YKL-05-099 in mice

[0229] A dose of placebo or YKL-05-099 (18mg / kg) was orally administered to mice 2 hours before sacrifice. The results are shown in figure 11. It was observed that YKL- 05-099 crosses the blood-brain barrier upon oral administration.Example (4) Pharmacological inhibition of SIK-3 in obese mice using GLPG3970

[0230] Using the same animal model as the YKL-05-099 study, WT C57BL / 6J mice were fed a high fat diet for 12 weeks starting from 8 weeks of age, and placebo or GLPG3970 treatment (50mg / kg or 25mg / kg) began at 20 weeks of age. GLPG3970 treatment led to significant reduction in body weight for both the 25 mg / kg dose and the 50 mg / kg dose compared to the placebo group (figure 15). Obese mice treated with GLPG3970 exhibit decreased adiposity (figure 16) and exhibit reduced food intake and improved glycaemic control (figure 17).

[0231] GLPG3970 is well tolerated with acceptable safety profile in humans.

[0232] Compared to Placebo, obese mice treated with YKL-05-099 or GLPG3970 exhibited significant body weight loss due to reduced adiposity.

[0233] Without wishing to be bound by theory, it is proposed that a key underlying explanation for the reduction in body weight and adiposity could be a reduction in caloric intake (food intake). Mice treated with YKL-05-099 also exhibited significantly increased in thermogenesis, as indicated by upregulation of the thermogenic marker LICP1.

[0234] Collectively, these results indicate that pharmacological inhibition of SIK-3 promotes weight loss in obese subjects.Example (5) Lipid production and lipid utilization in liver specific SIK3- KO hepatocytes and SIK-3 pharmacologically inhibited hepatocytes

[0235] An assay was performed to investigate the effect of SIK-3 inhibition Primary hepatocytes were harvested from WT (Sik3lox / lox) and liver-specific SIK3-KO (AlbCre / +Sik3lox / lox) mice. Lipogenesis (lipid production) in WT and liver-specific SIK3- KO primary hepatocytes in response to basal and SIK3 inhibitor Pterosin B was determined by measuring the incorporation of radiolabelled acetate in the primary hepatocytes. The results are shown in figure 19(A) with data presented as mean ± SEM, n = 3 independent experiments (each experiment contains at least three replicates), p-values by two-way ANOVA with Sidak post-hoc test: *p < 0.05 for the difference between genotypes.

[0236] These results indicate that inhibition of SIK-3 in hepatocytes is able to reduce hepatocyte lipogenesis through either genetic manipulation (SIK-3 KO hepatocytes) or through administration of a pharmacological inhibitor (Pterosin B). Furthermore, when the SIK-3 KO hepatocytes were treated with a pharmacological SIK-3 inhibitor (Pterosin B) a further reduction lipid production was observed, likely due to inhibition of other SIK isoforms such as SIK1 and SIK2. The experiments showed decreased lipid synthesis in hepatocytes with SIK-3 inhibition, indicating that SIK-3 inhibition may be useful for reducing excessive fat synthesis, a major driver of fatty liver disease in obese subjects, and therefore reducing fat accumulation in the liver.

[0237] A second assay was performed to investigate the effect of lipid utilization in SIK-3 inhibited hepatocytes. Primary hepatocytes were harvested from WT (Sik3lox / lox) and liver-specific SIK3-KO (AlbCre / +Sik3lox / lox) mice. Rates of oxygen consumption (JO2) were determined in permeabilised primary hepatocytes isolated from WT and liver-specific SIK3-KO mice. Hepatocytes were incubated in the presence of bufferalone (basal) and 25 or 50 pM palmitoyl-CoA (P-CoA). The results of the lipid utilization assay are shown in figure 19(B). All data are shown as the mean ± SEM, n = 3-4 independent experiment per genotype, p-values by Student’s t-test: *p< 0.05.

[0238] The results of the lipid utilization assay show that hepatocytes harvested from SIK-3 KO mice had increased lipid oxidation when treated with 25pM and 50pM palmityl-CoA compared to the wild type mice. This indicates that SIK3 inhibition is able to enhance lipid utilization in the liver, and useful for treating or preventing fatty liver diseases associated with fat storage in the liver, or reversing or ameliorating fat storage in the liver.Example (6) Reversing and treating fatty liver disease by pharmacological inhibition of SIK-3 in high fat fed mice

[0239] WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 (18 mg / kg), or GLPG-3970 (50mg / kg) treatment began at 20 weeks of age. The mice were euthanized at 26 weeks of age and the livers collected for analysis. Photographs of the livers of the placebo treated and SIK-3 inhibitor treated mice are shown in figures 20 and 21 (left most insert). The placebo livers show increased discoloration (the whiteness indicates more fat accumulation and storage in the liver) compared to the livers of the mice treated with the SIK-3 inhibitor (the darker and reddish appearance indicates less fat stored in the liver).

[0240] The extracted livers were biopsied and images of hematoxylin and eosin (H&E) stain of biopsied liver tissue from high fat-fed obese mice treated with placebo or YKL-05-099 or GLPG-3970 are shown in figures 20 and 21. Lipid droplets appear white and round in H&E staining. In both figures 20 and 21 it can be seen that the livers of the placebo treated mice have significant fat deposits (in the white / round specifical regions indicated in the central and right hand side panels of the image) where as the livers of the SIK-3 inhibitor treated mice have significantly less fat deposits (there are less white / round regions visible in the central and right hand side panels of the images). In particular the GLPG-3970 treated mice have very few visible fat deposits in the liver samples.

[0241] These results indicate that administration of a pharmacological SIK-3 inhibitor is able to treat and reverse the build-up of fatty deposits in the liver in mice on a high-fatdiet. This indicates the utility of SIK-3 inhibition in both supressing the progression and treating fatty liver disease.

[0242] The liver fat content in high-fat fed mice was measured by looking at the liver triglyceride levels in placebo treated vs GLPG-3970 or YKL-05-099 treated mice. The results of the liver triglyceride assays are shown in figure 22.

[0243] WT C57BL / 6J mice were fed a high fat diet for 12 weeks starting from 8 weeks of age, and placebo or GLPG3970 treatment (25mg or 50mg / kg) began at 20 weeks of age. Liver triglyceride levels from high fat-fed obese mice treated with placebo or GLPG3970. These results are shown in figure 22 (A). The GLPG2970 treatment led to a significant reduction in liver triglyceride levels.

[0244] WT C57BL / 6J mice were fed a high fat diet for 11 weeks starting from 9 weeks of age, and placebo or YKL-05-099 treatment began at 20 weeks of age. Liver triglyceride levels from high fat-fed obese mice treated with placebo or YKL-05-099. The results are shown in figure 22(B). YKL-05-099 treatment led to a significant reduction in liver triglyceride levels.

[0245] These results indicate that administration of a pharmacological SIK-3 inhibitor is able to potentially reverse triglyceride accumulation in the liver of mice on a high-fat diet and inhibit the progression of fatty liver disease. This indicates the benefit of administration of a SIK-3 inhibitor to treat fatty liver disease.

Claims

CLAIMS1. A method of treating obesity in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby treating obesity in the individual.

2. A method of treating, or minimising the risk of, an obesity-related disease or disorder, the method comprising administering to an individual suffering from or at risk of suffering from an obesity-related disease or disorder a SIK-3 inhibitor to the individual, thereby treating, or minimising the risk of, an obesity related disorder in the individual.

3. A method of reducing adiposity in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby reducing adiposity in the individual.

4. A method of reducing the body weight of an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby reducing body weight of the individual.

5. A method of increasing white adipose tissue browning in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby increasing white adipose tissue browning in the individual.

6. A method of preventing or minimising the weight gain of an individual consuming a high energy / caloric diet, the method comprising administering a SIK-3 inhibitor to the individual, thereby preventing or minimising the weight gain of the individual consuming a high energy / caloric diet.

7. A method of preventing or minimising adiposity in individual consuming a high energy / caloric diet, the method comprising administering a SIK-3 inhibitor to the individual, thereby preventing or minimising adiposity in the individual consuming a high energy / caloric diet.

8. A method of enhancing energy expenditure in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby enhancing energy expenditure in the individual.

9. A method of promoting weight loss in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting weight loss in the individual.

10. A method of improving insulin secretion in an individual in need thereof, the method comprising administering a SIK-3 inhibitor to the individual, thereby improving insulin secretion in the individual.

11. A method of promoting blood glucose clearance in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting blood glucose clearance in the individual.

12. A method of promoting or improving glycaemic control in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby promoting glycaemic control in the individual.

13. A method of treating diabetes in an overweight or obese individual, the method comprising administering a SIK-3 inhibitor to the individual, thereby treating diabetes in individual in need thereof.

14. The method of any one of the preceding claims, wherein the SIK-3 inhibitor is more potent for inhibition of SIK family kinases, preferably SIK-3 kinase, compared inhibition of other kinases.

15. The method of any one of the preceding claims, wherein the SIK-3 inhibitor does not exhibit significant inhibitory activity for kinase ABL and / or the SIK-3 inhibitor does not exhibit significant activity for Bruton’s tyrosine kinase and / or Src kinase.

16. The method of any one of the preceding claims, wherein the SIK-3 inhibitor demonstrates higher inhibitory activity for SIK-3 over SIK-1 or SIK-2 or both.

17. The method of any one of the preceding claims, wherein the SIK-3 inhibitor has at least 2, preferably at least 5, times higher inhibitory activity for SIK-3 over SIK- 1.

18. The method of any one of the preceding claims, wherein the SIK-3 inhibitor has at least 2, preferably at least 5, times high inhibitory activity for SIK-3 over SIK-2.

19. The method of any one of the preceding claims, wherein the individual is overweight or obese.

20. The method of any one of the preceding claims, wherein the individual has a BMI greater than 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 kg / m2.

21. The method of any one of the preceding claims, wherein the individual in need thereof has been overweight or obese for at least 6 months prior to starting treatment.

22. The method of any one of the preceding claims, wherein the individual is insulin resistant.

23. The method of any one of the preceding claims, wherein the individual has been diagnosed with insulin deficiency.

24. The method of any one of the preceding claims, wherein the individual has a moderate, severe or absolute insulin deficiency.

25. The method of any one of the preceding claims, wherein the individual has been diagnosed as pre-diabetic or diabetic.

26. The method of any one of the preceding claims, wherein the individual has one or more obesity related diseases or disorders.

27. The method of claim 2 or claim 26, wherein the obesity-related disease or disorder is selected from the group consisting of obesity, pre-obesity, morbid obesity, Prader-Willi Syndrome, Hypothalamic Injury Associated Obesity, hyperlipidemia, hypertension, diabetes, lipodystrophy, lipodema, Bardet-Biedl Syndrome, Cohen Syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, hypothyroid, and MOMO Syndrome.

28. The method of claims 2 or 26 or 27, wherein the method of treatment commenced after the onset of the disease or disorder.

29. Use of a SIK-3 inhibitor in the manufacture of a medicament for treating obesity or an obesity related disorder in an individual in need thereof.

30. Use of a SIK-3 inhibitor in the manufacture of a medicament for reducing adiposity in an individual in need thereof.

31. Use of a SIK-3 inhibitor in the manufacture of a medicament for reducing the body weight of an individual in need thereof.

32. Use of a SIK-3 inhibitor in the manufacture of a medicament for increasing white adipose tissue browning in an individual in need thereof.

33. Use of a SIK-3 inhibitor in the manufacture of a medicament for preventing or minimising the weight gain, or preventing or minimising adiposity, in an individual consuming a high energy / caloric diet.

34. Use of a SIK-3 inhibitor in the manufacture of a medicament for enhancing energy expenditure in an individual in need thereof.

35. Use of a SIK-3 inhibitor in the manufacture of a medicament for promoting weight loss in an individual in need thereof.

36. Use of a SIK-3 inhibitor in the manufacture of a medicament for improving insulin secretion in an individual in need thereof.

37. Use of a SIK-3 inhibitor in the manufacture of a medicament for promoting blood glucose clearance in an overweight or obese individual.

38. Use of a SIK-3 inhibitor in the manufacture of a medicament for promoting or improving glycaemic control in an overweight or obese individual.

39. Use of a SIK-3 inhibitor in the manufacture of a medicament for treating diabetes in an overweight or obese individual.

40. A SIK-3 inhibitor for use in treating obesity or an obesity related disorder in an individual in need thereof.

41. A SIK-3 inhibitor for use in reducing adiposity in an individual in need thereof.

42. A SIK-3 inhibitor for use in reducing the body weight of an individual.

43. A SIK-3 inhibitor for use in increasing white adipose tissue browning.

44. A SIK-3 inhibitor for use in preventing or minimising the weight gain, or preventing or minimising adiposity, in an individual consuming a high energy / caloric diet.

45. A SIK-3 inhibitor for use in enhancing energy expenditure in an individual in need thereof.

46. A SIK-3 inhibitor for use in promoting weight loss in an individual in need thereof.

47. A SIK-3 inhibitor for use in improving insulin secretion in an individual in need thereof.

48. A SIK-3 inhibitor for use in promoting blood glucose clearance in an overweight or obese individual.

49. A SIK-3 inhibitor for use in promoting or improving glycaemic control in an overweight or obese individual.

50. A SIK-3 inhibitor for use in treating pre-diabetes or diabetes in an overweight or obese individual.

51. A method of decreasing food intake in an individual in need thereof comprising the step of administering to the individual a SIK-3 inhibitor.

52. A method of reducing or supressing appetite in an individual in need thereof, the method comprising administering to the individual a SIK-3 inhibitor, thereby supressing appetite in the individual.

53. The method of claim 51 or 52, wherein the individual is an overweight or obese individual.

54. The method of any one of claims 51 to 53, wherein the individual has prediabetes or diabetes.

55. Use of a SIK-3 inhibitor in the manufacture of a medicament for decreasing food intake in an individual in need thereof.

56. Use of a SIK-3 inhibitor in the manufacture of a medicament for reducing or supressing appetite in an individual in need thereof.

57. A SIK-3 inhibitor for use in decreasing food intake in an individual in need thereof.

58. A SIK-3 inhibitor for use in reducing or supressing appetite in an individual in need thereof.

59. A method of treating fatty liver disease in an individual in need thereof, the method comprising administering to the individual a SIK-3 inhibitor, thereby treating fatty liver disease in the individual in need thereof.

60. The method of claim 59 wherein the SIK-3 inhibitor is more potent for inhibition of SIK family kinases, preferably SIK-3 kinase, compared inhibition of other kinases.

61. The method of claim 59 or 60 wherein the SIK-3 inhibitor does not exhibit significant inhibitory activity for kinase ABL and / or the SIK-3 inhibitor does not show significant activity for Bruton’s tyrosine kinase and / or Src kinase.

62. The method of any one of claims 59 to 61 , wherein the SIK-3 inhibitor demonstrates higher inhibitory activity for SIK-3 compared to SIK-1 or SIK-2, or both SIK-1 and SIK-2.

63. The method of any one of claims 59 to 62, wherein the SIK-3 inhibitor has at least 2, preferably at least 5, times higher inhibitory activity for SIK-3 than SIK-1 .

64. The method of any one of claims 59 to 63, wherein the SIK-3 inhibitor has at least 2, preferably at least 5, times high inhibitory activity for SIK-3 than SIK-2.

65. The method of claim any one of claims 59 to 64, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

66. The method of claim any one of claims 59 to 64, wherein the fatty liver disease is non-alcoholic steatohepatitis (NASH).

67. The method of any one of claims 59 to 66, wherein the individual is an overweight or obese individual.

68. The method of any one of claims 59 to 67, wherein the individual with a fatty liver disease also has insulin resistance.

69. The method of any one of claims 59 to 67, wherein the individual with a fatty liver disease has pre-diabetes or diabetes.

70. The method of any one of claims 59 to 66, wherein the individual is not an overweight or obese individual.

71. The method of any one of claims 59 to 67, wherein the individual with a fatty liver disease does not have insulin resistance.

72. The method of any one of claims 59 to 67, wherein the individual with a fatty liver disease does not have diabetes.

73. Use of a SIK-3 inhibitor in the manufacture of a medicament for treating fatty liver disease in an individual in need thereof.

74. A SIK-3 for use in treating fatty liver disease in an individual in need thereof.

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