Selective hypothalamic permeable HDAC6 inhibitors for treatment of leptin resistant obesity

Abstract

Formulations of HDAC6 inhibitors that traverse the blood brain barrier in the hypothalamus and inhibit HDAC6 in arcuate AgRP neurons in the hypothalamus are effective for causing weight loss in obese individuals. These inhibitors also restore leptin sensitivity in leptin resistant individuals.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 379,215, filed Oct. 12, 2022, and U.S. Utility Application No. 18 / 324,598, filed May 26, 2023, which are hereby incorporated by reference in their entirety.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under Grant No. R01 DK098496 - 02 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0005] This generally pertains to the field of treating obesity, particularly leptin - resistant obesity. Background Art

[0006] Since 1975, obesity has almost tripled worldwide and currently causes over 5 million deaths per year. The disease is defined as "abnormal or excessive fat accumulation that presents a risk to health" and affected one in every three adults, or approximately 36% of the population, in 2020, including 39 million children under the age of 5.

[0007] Obesity is a global epidemic that can have extremely serious health consequences and increased costs that can cripple healthcare systems. The most obvious direct costs of obesity are related to comorbid conditions and the medications required to treat these diseases. Currently, there are 236 diseases related to obesity, including type 2 diabetes, hypertension, and depression. Recent data shows that in the United States, obesity costs nearly $150 billion in healthcare annually. According to the World Health Organization, in 2016, obesity - related comorbidities (such as hypertension, dyslipidemia, type 2 diabetes, fatty liver disease, heart disease, and some types of cancer) caused approximately 3.4 million adult (over 18 years old) deaths. It is reported that 1.9 billion adults are overweight, and over 650 million overweight adults are obese. Hyperleptinemia and resistance to body mass reduction are two common features of obesity.

[0008] The peptide hormone leptin regulates food intake, body mass, and reproductive function, and plays a role in fetal growth, pro-inflammatory immune responses, angiogenesis, and lipolysis. Leptin is the product of the obesity (ob) gene, which is synthesized and secreted from adipocytes in white adipose tissue and binds to and activates its cognate receptor, the leptin receptor (LEP-R). The distribution of LEP-R facilitates the pleiotropic actions of leptin, which plays a key role in regulating body mass through a negative feedback mechanism between adipose tissue and the hypothalamus. The leptin receptor receives and transmits signals from leptin, a hormone released from adipocytes that is mainly involved in the regulation of metabolism but also plays a role in bone metabolism, immunity, and reproductive function. The leptin receptor is located in the cell membranes of various tissues in the body but is most highly expressed on neurons in the hypothalamus, a region of the brain involved in regulating hunger, body temperature, sleep, and other activities. It is a member of the cytokine receptor protein superfamily. When leptin binds to the leptin receptor, particularly in the hypothalamus, it generates chemical signals that promote satiety, thereby reducing hunger. The significance of this function is evident in the absence of leptin receptors, which is associated with elevated plasma levels of leptin and thus leads to persistent hunger and overeating, resulting in obesity.

[0009] Leptin resistance is characterized by reduced satiety, excessive consumption of nutrients, and an increase in total body mass. There is a strong positive correlation between serum leptin levels and percentage of body fat. Leptin regulates food intake, body mass, reproductive function, and plays a crucial role in fetal growth, pro-inflammatory immune responses, angiogenesis, and lipolysis.

[0010] Brain lesion and stimulation studies led to the discovery of a "satiety center" in the ventromedial hypothalamus (VMH) and a "hunger center" in the lateral hypothalamus (LH). This defined a dual-center model of feeding. Leptin mainly regulates appetite and metabolism by acting on a subset of neurons expressing AgRP in the arcuate nucleus (ARC). Depletion of LepR in these neurons almost completely abolishes the appetite-suppressing and weight-reducing effects of leptin. Thus, LepR on neurons expressing AgRP is crucial for mediating the actions of leptin. Leptin can inhibit neural pathways activated by orexigenic agents (appetite promoters) to reduce energy intake and activate pathways targeted by anorexigenic agents to suppress appetite.

[0011] Hyperleptinemia and resistance to body mass loss are two characteristics of typical obesity. In the adipose tissue of individuals with obesity, leptin is overexpressed at the gene level. Additionally, there is a strong positive correlation between plasma leptin levels and percentage of body fat. Other studies point to leptin resistance. For example, plasma leptin levels and ob mRNA content in individuals with obesity decrease at the initial time of weight loss but increase as they continue to lose weight.

[0012] Various compounds have been proposed to treat obesity, including leptin-resistant obesity, but with mixed results.

[0013] Cakir, et al. Nat Metab. 2022 January; 4(1): 44–59. doi:10.1038 / s42255-021-00515-3 report that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors (e.g., tubastatin A) reduce food intake, fat mass, hepatic steatosis, and improve systemic glucose homeostasis in an HDAC6-dependent manner. The data show that peripheral, but not central, HDAC6 inhibition confers central leptin sensitivity. Furthermore, the anti-obesity effects of tubastatin A are attenuated in animals with defective central leptin-melanocortin circuits, including db / db and MC4R knockout mice, indicating that HDAC6-regulated adipokines act as leptin sensitizers and revealing HDAC6 as a potential target for the treatment of obesity. The HDAC6 inhibitors tested included tubastatin and its inactive analog BRD3067, as well as the selective HDAC6 inhibitors CAY10603 and ricolinostat. It is claimed that tubastatin reverses leptin resistance in mice via a peripheral mechanism of action, possibly by inducing the release of leptin sensitizing factors. Due to the low permeability of the blood-brain barrier ("BBB"), the level of tubastatin in the brain is extremely low. They concluded that tubastatin acts on tubastatin-induced weight loss through a dual mode of action, in which peripheral inhibition of HDAC6 activity by a selective HDAC6 inhibitor leads to the release of systemic factors from the periphery that act in the CNS to increase leptin sensitivity.

[0014] Obesity is a persistent and difficult to treat problem, especially leptin-resistant obesity. There remains a significant need for compounds that safely achieve weight loss, especially in leptin-resistant obesity.

[0015] Therefore, the object of the present invention is to provide compounds and methods of use thereof for treating obesity, particularly leptin-resistant obesity, wherein the compounds cross the blood-brain barrier and act on a central mechanism of action. Summary of the invention

[0016] It has been found that HDAC6 inhibitors with high levels of permeability across the BBB, especially the hypothalamus, and in particular inhibitors that block the LepRb-HDAC6 interaction in hypothalamic AgRP neurons, are effective in treating leptin-resistant obesity and are effective in increasing leptin sensitivity. It has further been found that the key brain region to which the inhibitors are effective is the hypothalamus. Several compounds were tested and it was determined that the mechanism of action is central rather than peripheral and is associated with HDAC6 inhibitors having a high level of permeability to cross the blood-brain barrier and enter the hypothalamus. Analysis of hypothalamic gene expression in fed, starved, and obese mice determined the correlation with gene expression changes induced by small molecule inhibition of HDAC6 activity, establishing that pharmacological inhibition of HDAC6 activity in the hypothalamus is effective in the treatment of obesity and related disorders. Subsequent studies in high-fat diet-induced mice treated with the HDAC6 isoform-specific inhibitors ACY738 and ACY775 showed that these compounds resulted in significant reductions in food intake and total body fat mass. The more selective HDAC6 inhibitor ACY775 had a greater effect compared to ACY738. Bavarostat, as well as the compounds ACY257 and ACY1083, which inhibit HDAC6 in the hypothalamus but have low BBB permeability, also showed significant weight loss.

[0017] Only HDAC6 inhibitors that penetrate the hypothalamus and inhibit HDAC6 therein are effective in treating obesity, especially leptin-resistant obesity. Data indicate that the compound ACY 257, which inhibits HDAC6 in the hypothalamus but not in other brain regions (such as the cerebrum, frontal lobe, temporal lobe, and brainstem), significantly reduces body weight. The hypothalamus is a very small part of the brain. The hypothalamus has a blood-brain barrier with slightly higher permeability compared to other parts of the brain. Data indicate that HDAC6 inhibitors with the ability to preferentially cross the BBB and enter the hypothalamic region (especially the arcuate nucleus) are able to reduce body weight.

[0018] Based on these studies, small molecule (less than 1000 Da) selective inhibitors of HDAC6 with high permeability across the BBB and into the hypothalamus, having a brain / plasma ratio greater than 1 (inhibitors that result in α-tubulin acetylation without affecting histone acetylation), in particular inhibitors that block the LepRb-HDAC6 interaction, are most effective in treating obesity (including leptin-resistant obesity) by a central nervous system (CNS) mechanism of action. Preferred HDAC6 inhibitors have a brain or hypothalamus / plasma concentration > 0.25, > 0.5 or > 1. More preferably, the HDAC6 inhibitor has a hypothalamus / plasma concentration > 0.25, > 0.5 or > 1. Most preferably, the HDAC6 inhibitor has an arcuate nucleus / plasma concentration > 0.25, > 0.5 or > 1. The arcuate nucleus has the highest BBB permeability in the hypothalamus and is approximately 1 / 10,000 of the total brain area. Results show that inhibiting HDAC6 in AgRP neurons in the arcuate nucleus is sufficient to produce weight loss. If the HDAC6 inhibitor only reaches the arcuate nucleus at a concentration capable of inhibiting HDAC6 activity and does not reach other regions of the brain, the total brain / plasma or hypothalamus / plasma concentration may be low. However, the arcuate nucleus concentration will be higher than the total brain / plasma and hypothalamus / plasma concentrations.

[0019] Preferred compounds are ACY775 (brain / plasma ratio of 1.26) and ACY738 (brain / plasma ratio of 1.22), ACY257, ACY1083 which have high permeability to the arcuate nucleus of the hypothalamus, and bavarostat (4-(((((3r,5r,7r)-adamantan-1-yl)methyl)(methyl)amino)methyl)-3-fluoro-N-hydroxybenzamide) which has high permeability to the hypothalamus. Peripherally acting HDAC6 inhibitors are ineffective, such as ricolinostat (ACY1215) with a brain / plasma ratio of 0.01 and tubastatin A with a brain / plasma ratio of 0.18, which do not have high permeability to the hypothalamus, and citarinostat (ACY241) is also not effective as it does not show HDAC6 inhibition in AGP neurons in the hypothalamus. HDAC6 inhibition was tested by treating cells with a high concentration of HDAC6 inhibitor and analyzing tubulin acetylation (an established biomarker of HDAC6 inhibition).

[0020] The selective HDAC6 inhibitor is preferably administered to the mucosal surface, most preferably orally, buccally or nasally. These can be formulated using known excipients. The formulations can also be formulated for sustained, delayed and / or pulsatile release to deliver an effective amount of the HDAC6 inhibitor to cause weight loss. Preferably, they are administered once or twice daily. The dose is weight-based. A typical dose will be in the range of 25 to 500 mg / day.

[0021] A pharmaceutical preparation can be administered to induce weight loss in pre-obese, obese, or morbidly obese patients, reduce body fat in pre-obese, obese, or morbidly obese patients, reduce food intake in pre-obese, obese, or morbidly obese patients, improve glucose homeostasis in pre-obese, obese, or morbidly obese patients, or a combination thereof. In some cases, a pharmaceutical preparation containing one or more HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients at a therapeutically effective amount to induce weight loss, preferably at a therapeutically effective amount and for a period of time such that body mass or body fat is reduced by at least 10%, more preferably at least 15%, and most preferably at least 20% or more. In some cases, a pharmaceutical preparation containing one or more HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients at a therapeutically effective amount to reduce food intake, appetite, or a combination thereof, preferably at a therapeutically effective amount to reduce the average daily food intake (in calories). In some cases, a pharmaceutical preparation containing one or more selective HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients at a therapeutically effective amount to improve glucose homeostasis, preferably at a therapeutically effective amount to reduce the average fasting plasma glucose. In cases where the pharmaceutical preparation is administered to normalize blood glucose, the preparation is preferably administered in an amount effective to reduce the blood glucose level to less than about 180 mg / dL. If desired, the preparation can be co-administered with other anti-diabetic therapies to improve glucose homeostasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a graph showing the change of p-stat3 / stat3 over time (in minutes) for control and HDAC6-OE.

[0023] Figures 2A - 2O shows that the specific HDAC6 inhibitor (ACY775) increases leptin sensitivity (2C), reduces appetite ( Figure 2B , 2E ) and body weight. DIO mice ( Figure 2D ) were pre-treated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days, and then co-treated with ACY775 (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 9 per group). Figure 2A , 2D , % change in body weight 15 h after leptin treatment, Figure 2B , food intake (g) during the 15 h period after leptin treatment. The results were reproduced in two independent experiments. Figure 2C , 2E, DIO mice were pre-treated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days, and then received a final injection of ACY775 (or vehicle) on the morning of the fourth day. Six hours later, the mice were injected with saline or leptin (1 mg / kg, i.p.), and the hypothalamus was extracted 45 minutes after leptin injection. Quantification of the ratio of p-STAT3 Tyr705 to the total STAT3 signal in immunoblotting. The results were reproduced in three independent experiments. DIO mice were injected with HDAC6 inhibitors (ACY775, 10 mg / kg, i.p. or ACY738, 50 mg / kg, i.p., n = 3) until 3:00 am. DIO mice were treated with ACY775 (10 mg / kg / day, i.p.) for 3 weeks. During the 3-week treatment period, the daily body weight (g) of DIO mice treated with vehicle (n = 17) or ACY775 (n = 17). Figures 2F - 2O : Figure 2F , the food intake per mouse in 24 hours during the first week of treatment with vehicle or ACY775. Figure 2G , the serum leptin (ng / ml) level after 3 weeks of treatment with vehicle (n = 24) or ACY775 (n = 24). Measured by dual-energy X-ray absorptiometry (DEXA) scan after 3 weeks of treatment with vehicle (n = 17) or ACY775 (n = 17) Figure 2H , lean mass (g), Figure 2I , fat mass (g) and Figure 2J , fat percentage (%). DEXA scans were performed in two different groups. Figure 2K , glucose tolerance test (GTT) within 120 minutes after 1 week of treatment of DIO mice with vehicle (n = 33) or ACY775 (n = 26). Figure 2L , in ( Figure 2K ) area under the curve (AUC) analysis of the GTT performed. Figure 2M , blood glucose (mg / dl) level within 120 minutes after 1 week of treatment of DIO mice with vehicle (n = 17) or ACY775 (n = 16); Figure 2O . Figure 2N, Insulin tolerance test (ITT) in DIO mice treated with vehicle (n = 31) or ACY775 (n = 21) for 2 weeks. q, Serum insulin (ng / ml) levels in DIO mice treated with vehicle (n = 22) or ACY775 (n = 21) for 3 weeks. r, Homeostatic model assessment of insulin resistance (HOMA-IR) analysis in DIO mice treated with vehicle (n = 14) or ACY775 (n = 13) for 3 weeks. s, H&E staining of liver sections from DIO mice treated with vehicle or ACY775 for 3 weeks. Results were reproduced in two independent experiments. Values represent mean ± s.e.m. P values were determined by ANOVA, where Bonferroni multiple comparison test was used for curves ( Figure 2D and 2E , 2K, 2M) or Student's T test for two-group comparisons ( Figure 2A and 2B , 2C, 2F-2J, 2L, 2O, 2N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

[0024] Figures 3A - 3Q . The specific HDAC6 inhibitor (ACY738) increases leptin sensitivity, reduces appetite and body weight. ( Figures 3A - 3B ) DIO mice were pre-treated with ACY738 (50 mg / kg / day, i.p.) or vehicle for 3 days, and then co-treated with ACY738 (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 14 per group). Figure 3A , % change in body weight 15 h after leptin treatment, Figure 3B , Food intake (g) during the 15-h period after leptin treatment. ( Figure 3A and 3B ) The results in were reproduced in two independent experiments. DIO mice were pre-treated with ACY738 (50 mg / kg / day, i.p.) or vehicle for 3 days, and then the last injection of ACY738 (or vehicle) was given on the morning of the fourth day. Six hours later, mice were injected with saline or leptin (1 mg / kg, i.p.), and the hypothalamus was extracted 45 minutes after leptin injection. Figure 3C , Quantification of the ratio of p-STAT3 Tyr705 to total STAT3 signal in immunoblotting. ( Figure 3C ) The results in were reproduced in two independent experiments. Figure 3D, DIO mice were injected with HDAC6 inhibitors (ACY775, 10 mg / kg, i.p., or ACY738, 50 mg / kg, i.p.) after 24 h of fasting. Food intake was analyzed hourly until 3:00 am. ( Figures 3E - 3Q ), DIO mice were treated with ACY738 (50 mg / kg, i.p.) for 3 weeks. Figure 3E , Daily body weight (g) of DIO mice treated with vehicle (n = 27) or ACY738 (n = 24) during the 3-week treatment period. Figure 3F , Daily food intake (g) during the 3-week vehicle or ACY738 treatment. Figure 3G , 24-hour food intake per mouse during the first week of vehicle or ACY738 treatment. Figure 3H , Serum leptin (ng / ml) levels after 3 weeks of treatment with vehicle (n = 26) or ACY738 (n = 25). Measured by DEXA scan after 3 weeks of treatment with vehicle (n = 23) or ACY738 (n = 23) Figure 3I , Lean mass (g). Figure 3J , Fat mass (g) and Figure 3K , Fat percentage (%). DEXA scans were performed in two different groups. Figure 3L , GTT within 120 minutes; Figure 3M is the GTT of DIO mice treated with vehicle (n = 23) or ACY738 (n = 24) for 1 week. Figure 3N , The AOC analysis of the GTT performed in ( Figure 3L ). Figure 3O , Blood glucose (mg / dl) levels of DIO mice treated with vehicle (n = 27) or ACY738 (n = 23) for 1 week. Figure 3P , ITT of DIO mice treated with vehicle (n = 27) or ACY738 (n = 22) for 2 weeks. Figure 3Q , Serum insulin (ng / ml) levels of DIO mice treated with vehicle (n = 25) or ACY738 (n = 25) for 3 weeks. ( Figures 3E - 3Q ) The results in were reproduced in two independent experiments. Values are expressed as mean ± s.e.m. P values were determined by two-way ANOVA, where Bonferroni multiple comparison test was used for curves ( Figure 3D 、 3E 、3F、3L、3O) or Student's T test for two-group comparisons ( Figure 3A 、 3B, 3C, 3G - 3K, 3M, 3N, 3P, 3Q). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

[0025] Figures 4A - 4K . The HDAC6 inhibitor (ACY775) reduces the appetite of DIO mice. Conditioned place preference (CPP) assay under ad libitum feeding conditions: DIO mice were treated with vehicle or ACY775 (10 mg / kg, i.p., once daily) for 3 days. On the fourth day, 1 h after the start of the light cycle, mice were administered vehicle or ACY775 (10 mg / kg, i.p.), and CPP assay was performed 6 h later under ad libitum feeding conditions (n = 10 mice in both the vehicle-treated group and the ACY775-treated group). CPP assay under 20-h fasting conditions: DIO mice were treated with vehicle or ACY775 (10 mg / kg, i.p.), once daily, and fasted for 15 h after the third injection. On the fourth day, 1 h after the start of the light, mice were administered vehicle or ACY775 (10 mg / kg body weight, i.p.), and CPP assay was performed 5 h after this injection under fasting conditions (n = 10 mice in both the vehicle-treated group and the ACY775-treated group). Figure 4A , the total distance traveled by the mice during the CPP test. Figure 4B , the average locomotion speed during the assay. Figure 4C , the total time the mice spent in the dark chamber during the test. Figure 4D , the frequency of the mice going to the dark chamber during the test. Figure 4E , the total time the mice spent in the food-paired white chamber. Figure 4F , the frequency of the mice going to the food-paired white chamber. Figure 4G , the total time the mice spent in the area containing food in the food-paired side chamber. Figure 4H , the frequency of the mice going to the area containing food in the food-paired side chamber during the test. Values are expressed as mean ± s.e.m. The P values for comparisons between the two groups in this study were determined by Student's t-test ( Figures 4A - 4H ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05). Figures 4I - 4K , energy expenditure ( Figure 4I ) and respiratory exchange ratio (RER; VCO2 / VO2) ( Figure 4J)。Similar to ACY775, DIO mice treated with ACY738 showed significantly reduced RER values during both dark and light cycles, indicating a greater utilization of fat as an energy source in vivo. Figure 4K , and there was no difference in the level of physical activity between the ACY738-treated group and the vehicle-treated group during the dark and light cycles.

[0026] Figures 5A - 5H . HDAC6 inhibitors do not reduce appetite or body weight in lean mice. WT lean mice were treated with vehicle (n = 20) or ACY775 (10 mg / kg, i.p., once daily, n = 22) or ACY738 (50 mg / kg, i.p., once daily, n = 20) for 3 weeks. Figure 5A , Daily body weight (g) of WT lean mice treated with vehicle or ACY775 or ACY738 during the 3-week treatment period. Figure 5B , Daily food intake (g) during the 3-week treatment period. Figure 5C , Serum leptin (ng / ml) level after 3 weeks of treatment. Measured by DEXA scan after 3 weeks of treatment Figure 5D , Lean mass (g), Figure 5E , Fat mass (g) and Figure 5F , Fat percentage (%). Figure 5G , GTT after 1 week of treatment. Figure 5H .

[0027] Figures 5I - 5Q and 5U-5W. HDAC6 inhibitors are ineffective in db / db mice. db / db mice were treated with vehicle (n = 20) or ACY775 (10 mg / kg, i.p., once daily, n = 22) or ACY738 (50 mg / kg, i.p., once daily, n = 20) for 3 weeks. Figure 5I , Daily body weight (g) of db / db mice treated with vehicle or ACY775 or ACY738 during the 3-week treatment period. Figure 5J , Daily food intake (g) during the 3-week treatment period. Figure 5K , Serum leptin (ng / ml) level after 3 weeks of treatment. Measured by DEXA scan after 3 weeks of treatment Figure 5L , Lean mass (g), Figure 5M , Fat mass (g) and Figure 5N , Fat percentage (%). Figure 5O , GTT after 1 week of treatment. Figure 5P , in ( Figure 5O ) AOC analysis of GTT performed. Figure 5Q , Blood glucose (mg / dl) level after 1 week of treatment. In ( Figure 5R ) AOC analysis of GTT performed.Figure 5S Blood glucose (mg / dl) levels after 1 week of treatment. Figure 5T Serum insulin (ng / ml) levels after 3 weeks of treatment. Figure 5U Blood glucose (mg / dl) levels after 3 weeks of treatment. Figure 5V Serum insulin (ng / ml) levels after 3 weeks of treatment. Figure 5W ITT after 2 weeks of treatment. Figures 5I - 5Q The results in 5U - 5W were reproduced in two independent experiments. Figures 5A - 5H The results in 5R - 5T were reproduced in two independent experiments.

[0028] Values represent mean ± s.e.m. P values were determined by two-way ANOVA, where Bonferroni multiple comparison test was used for curves ( Figure 5A , 5B , 5G, 5I, 5J, 5O, 5S, 5W) or Student's T test for two-group comparisons ( Figures 5C - 5F , 5H, 5K - 5N, 5P - 5R, 5T, 5U and 5V). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

[0029] Figures 6A - 6X . In contrast to the specific HDAC6 inhibitors ACY775 and ACY738, Tubastatin A reduced body weight and food intake in DIO HDAC6 knockout mice as well as in DIO wild-type (WT) mice. DIO WT or HDAC KO mice were treated with specific HDAC6 inhibitors (ACY775 and ACY738) and Tubastatin A for 3 weeks. Figure 6A Daily body weight of WT (n = 20) or HDAC6-KO (n = 20) DIO mice during treatment with ACY775 (10 mg / kg / day, i.p.). Figure 6B Average food intake (g) of WT or HDAC6-KO mice during the first week of ACY775 treatment. Figure 6C Blood glucose (mg / dl) levels of WT or HDAC6-KO mice treated with ACY775 (n = 20 per group) after 1 week of treatment. Figure 6F Serum insulin (ng / ml) levels of WT or HDAC6-KO mice treated with ACY775 after 3 weeks of treatment (n = 8 per group). Figure 6G Serum leptin (ng / ml) levels of WT or HDAC6-KO mice after 3 weeks of ACY775 (n = 8) treatment. Measured by DEXA scan after 3 weeks of ACY775 treatment Figure 6D Lean mass,Figure 6E , fat mass, Figure 6H , fat percentage (%).

[0030] Figure 6I , daily body weight of WT (n = 16) or HDAC6-KO (n = 20) DIO mice during ACY738 (50 mg / kg / day, i.p.) treatment. Figure 6J , average daily food intake (g) of WT or HDAC6-KO mice during the first week of ACY738 treatment. Figure 6K , blood glucose (mg / dl) levels of ACY738-treated WT (n = 18) or HDAC6-KO (n = 20) mice after 1 week of treatment. Figure 6L , serum insulin (ng / ml) levels of ACY738-treated WT or HDAC6-KO mice after 3 weeks of treatment (n = 8 per group). Figure 6M , serum leptin (ng / ml) levels of WT or HDAC6-KO mice after 3 weeks of ACY738 (n = 8) treatment. Measured by DEXA scan after 3 weeks of ACY738 treatment Figure 6N , lean mass, Figure 6O , fat mass, Figure 6P , fat percentage (%).

[0031] Figure 6Q , daily body weight of WT or HDAC6-KO DIO mice during vehicle or Tubastatin A treatment (25 mg / kg / day, i.p, WT-vehicle n = 15, WT-Tubastatin A n = 17, and HDAC6-KO-Tubastatin A n = 18). Figure 6R , average daily food intake (g) of WT or HDAC6-KO mice during vehicle or Tubastatin A treatment. Figure 6S , blood glucose (mg / dl) levels of vehicle or Tubastatin A-treated WT or HDAC6-KO mice after 1 week of treatment (WT-vehicle n = 8, WT-Tubastatin A n = 9, and HDAC6-KO-Tubastatin A n = 10). Figure 6T , serum insulin (ng / ml) levels of vehicle or Tubastatin A-treated WT or HDAC6-KO mice after 2 weeks of treatment (WT-vehicle n = 12, WT-Tubastatin A n = 12, and HDAC6-KO-Tubastatin A n = 13). Figure 6U, serum leptin (ng / ml) levels in WT or HDAC6-KO mice after 3 weeks of vehicle or Tubastatin A treatment (WT-vehicle, n = 12; WT-Tubastatin A, n = 13; HDAC6-KO-Tubastatin A, n = 14). After 3 weeks of Tubastatin A or vehicle treatment Figure 6V , lean mass, Figure 6W , fat mass, Figure 6X , fat percentage (%).

[0032] ( Figures 6A - 6X ) The results in Figures 6A - 6C were reproduced in two independent experiments. Values are expressed as mean ± s.e.m. P values were determined by two-way ANOVA, where Bonferroni multiple comparison test was used for curves ( Figures 6D - 6X ) or Student's t-test for two-group comparisons (

[0033] Figures 7A - 7I ). In contrast to the specific HDAC6 inhibitors ACY775 and ACY738, Tubastatin A improved glucose homeostasis in DIO HDAC6 knockout mice. Figure 7A , glucose tolerance test (GTT) after 1 week of ACY775 (10 mg / kg / day, i.p.) treatment in WT (n = 17) or HDAC6-KO (n = 20) DIO mice. Figure 7B , Figure 7A Area under the curve (AUC) analysis of the GTT performed in Figure 7C , insulin tolerance test (ITT) after 2 weeks of ACY775 (10 mg / kg / day, i.p.) treatment in WT (n = 14) or HDAC6-KO (n = 18) DIO mice. Figure 7D , GTT after 1 week of ACY738 (50 mg / kg / day, i.p.) treatment in WT (n = 18) or HDAC6-KO (n = 22) DIO mice. Figure 7E , in Figure 7D AUC analysis of the GTT performed. Figure 7F , ITT after 2 weeks of ACY738 (50 mg / kg / day, i.p.) treatment in WT (n = 18) or HDAC6-KO (n = 22) DIO mice. Figure 7G, GTT after 1 week of vehicle or Tubastatin A treatment in WT or HDAC6-KO DIO mice (WT-vehicle, n = 7; WT-Tubastatin A, n = 7; HDAC6-KO-Tubastatin A, n = 9). Figure 7H , AOC analysis of the GTT performed. Figure 7I , ITT after 2 weeks of vehicle or Tubastatin A treatment in WT or HDAC6-KO DIO mice (WT-vehicle, n = 7; WT-Tubastatin A, n = 7; HDAC6-KO-Tubastatin A, n = 9). ( Figures 7A - 7F ) The results in () were reproduced in two independent groups. Values represent mean ± s.e.m. P values were determined by two-way ANOVA, where Bonferroni multiple comparison test was used for curves ( Figure 7A , Figure 7C , Figure 7D , 7F , 7G, 7I) or Student's t test for two-group comparison ( Figure 7B , 7E , 7H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

[0034] Figures 8A - 8O The figure of shows that the poorly blood-brain barrier-permeable HDAC6 inhibitor Ricolinostat is not a leptin sensitizer and anti-obesity agent. Figure 8A is a figure of body weight compared with the control group ( Figure 8A ), and treatment with Ricolinostat alone (25 mg / kg, i.p.) also did not reduce the body weight of DIO mice. Figure 8B is a figure of the effect on food intake of DIO mice after administration of leptin following pre-treatment with Ricolinostat alone or in combination with leptin. Figure 8C is a figure of the effect of injecting leptin on STAT3 Tyr705 in vehicle- or Ricolinostat-treated DIO mice. Figures 8D - 8O is a figure of the results of DIO mice treated with Ricolinostat (50 mg / kg, i.p.) for 3 weeks: body weight, food intake ( Figure 8E , 8F ), serum leptin level ( Figure 8G ), lean mass ( Figure 8H ), fat mass ( Figure 8I ), fat percentage ( Figure 8J ), blood glucose level ( Figure 8K , 8L) Glucose tolerance ( Figure 8M ), insulin sensitivity ( Figure 8N ), and insulin levels ( Figure 8O ).

[0035] Figure 9A And 9B The graphs of Figure 9A and Figure 9B show that ACY257 is as effective as ACY775 in reducing body weight ( Figure 9A ) and percentage change in body weight ( Figure 9B ).

[0036] Figures 10A - 10F shows the weight-reducing effect of bavarostat. Quantification of the ratio of Ac-tubulin to total tubulin signal in immunoblots is shown in Figure 10A (liver) and Figure 10B (brain). The results show that in HDAC-6 KO mice, bavarostat is equivalent to the vehicle.

[0037] Figure 10C is a graph of the daily body weight (g) of DIO mice treated with bavarostat (10 mg / kg, i.p.) or vehicle (n = 9) or bavarostat (n = 9) during a 3-week treatment period. Figure 10D is a graph of the daily percentage change in body weight of DIO mice during a 3-week treatment period; Figure 10E is a graph of the daily food intake (g) of DIO mice during a 3-week vehicle or bavarostat treatment. Figure 10F is a graph of the average 24-hour food intake per week of DIO mice during vehicle or bavarostat treatment. The results show that bavarostat is statistically significantly effective in reducing food intake and body weight in DIO mice.

[0038] Figures 11A - 11F shows the ineffective weight-reducing effect of citarinostat compared to the vehicle. Quantification of the ratio of Ac-tubulin to total tubulin signal in immunoblots is shown in Figure 11A (liver) and Figure 11B (brain). Figure 11C is a graph of the daily body weight (g) of DIO mice treated with citarinostat (25 mg / kg, i.p.) or vehicle (n = 12) or citarinostat (n = 11) during a 3-week treatment period. Figure 11D is a graph of the daily percentage change in body weight of DIO mice during a 3-week treatment period; Figure 11E is a graph of the daily food intake (g) of DIO mice during a 3-week vehicle or citarinostat treatment. Figure 11FGraph of the average 24-hour food intake per week during treatment with the vehicle or citarinostat.

[0039] Figure 12 Graph showing weight loss over a 14-day period following oral administration of ACY1083.

[0040] Figure 13 Graph of the signaling pathway of leptin and its downstream effectors. Obr oligomerization (only dimerization is shown here for clarity) leads to phosphorylation and activation of the cytoplasmic associated jak2 kinase. These activated jaks phosphorylate tyrosine residues in the cytoplasmic tail of the receptor. Recruitment and activation of secondary signaling molecules allow obr to signal through the jak / stat, mapk, pi3k, ampk, and mtor pathways. The figure is from Wauman, et al Front. Endocrinol., Sec.Molecular and Structural Endocrinology Vol. 8 (2017) https: / / doi.org / 10.3389 / fendo.2017.00030. Inserted in the figure are the positions of HDAC6 interaction and the sites where the interaction between HDAC6 and the leptin receptor can be inhibited or disrupted.

[0041] Figures 14A - 14C Schematic diagram of the sites where the compounds of the present disclosure inhibit or disrupt HDAC6 or leptin receptor binding. Figure 14A Schematic diagram of leptin binding to LepR leading to phosphorylation of its tyrosine by Jak2 and subsequent phosphorylation and activation of Stat3. Stat3 plays a crucial role in mediating the anorectic and weight-lowering effects of leptin. Figure 14B Schematic diagram showing that the interaction between Hdac6 and LepRb during fasting or obesity reduces LepR and thus reduces Stat3 activation. This leads to inhibition of LepR signaling, increased appetite, and weight gain. Figure 14C Schematic diagram of an Hdac6 inhibitor that, by blocking the binding of Hdac6 to LepR, enhances LepR activity, suppresses appetite, increases energy expenditure, and leads to weight loss. DETAILED DESCRIPTION OF THE INVENTION

[0043] It has been determined that small molecule (less than 1000 Da) selective inhibitors of HDAC6 with high permeability across the BBB having a brain or hypothalamus / plasma ratio greater than 0.5, more preferably greater than 1 (HDAC6 inhibitors that result in α-tubulin acetylation with no effect on histone acetylation) are effective for treating obesity, including leptin-resistant obesity, by a central nervous system (CNS) mechanism of action. In a preferred embodiment, these compounds penetrate into the hypothalamus. Preferred compounds are ACY775 (brain / plasma ratio 1.26), ACY738 (brain / plasma ratio of 1.22), ACY257, ACY1083, and Bavarostat. This is in contrast to peripherally acting HDAC6 inhibitors such as ricolinostat (ACY1215) with a brain / plasma ratio of 0.01, tubastatin A with a brain / plasma ratio of 0.18, and citarinostat which does not inhibit AgRP neurons in the hypothalamus. HDAC6 inhibition is tested by treating cells with a high concentration of the HDAC6 inhibitor and analyzing tubulin acetylation (an established biomarker of HDAC6 inhibition).

[0044] I. Definitions

[0045] Obesity is defined by the Centers for Disease Control ("CDC") in the United States based on body mass index (BMI), which is a person's weight in kilograms divided by the square of their height in meters. A BMI between 25.0 and less than 30 is considered overweight. A BMI of 30.0 or higher is in the obese range. Obesity is generally subdivided into the following categories:

[0046] Class 1: BMI 30 to <35

[0047] Class 2: BMI 35 to <40

[0048] Class 3: BMI 40 or higher. Class 3 obesity is classified as "severe" obesity.

[0049] Histones are basic proteins that order and package DNA into nucleosomes (the basic subunits of chromatin). A nucleosome is an octamer composed of two of each of the four core histones (an H3(2), H4(2) tetramer and two H2A and H2B dimers, surrounded by 146 base pairs of DNA).

[0050] Histones play a crucial role in the regulation of gene expression by organizing DNA. Modifications of the four histone tails through reactions (such as methylation, acetylation, SUMOylation, and ubiquitination) at the N-terminus of relevant amino acids in H3 and H4 affect transcription, repair, and replication. Histones undergo acetylation and deacetylation through the opposing actions of two enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC superfamily consists of 18 members and is organized into four classes of two different protein families:

[0051] Zinc - dependent metalloprotein :

[0052] Class I (HDAC 1 - 3 and 8)

[0053] Class II (HDAC 4 - 7, 9, and 10, further subdivided into class IIa (HDAC 4, 5, 7, and 9) and class IIb (HDAC 6 and 10). Class IIa HDACs constantly shuttle between the nucleus and the cytoplasm, while class IIb HDACs are located in the cytoplasm and contain two catalytic sites.

[0054] Class IV (HDAC 11) shows similarities to class I and class II.

[0055] NAD+-dependent protein

[0056] Class III (SIRT1–7) shows similarity to yeast Sir2. Class III HDACs are insensitive to the inhibition of HDAC inhibitors, and their role in the cell cycle is not well understood currently.

[0057] HDACs are classified into four classes based on localization and amino acid sequence similarity: class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), class IIa HDACs (HDAC4, HDAC5, HDAC7, and HDAC9), class IIb HDACs (HDAC6 and HDAC10), class II HDACs (HDAC4 - 7, HDAC9, and HDAC10), class III HDACs (SIRT1 - 7), and histone deacetylase 11 (HDAC11). Although class I HDACs are found in the nucleus, class IIb can travel to the cytoplasm where they can interact with non-histone proteins.

[0058] Cellular HDAC6 inhibition can be verified by treating cells with a high concentration of the corresponding small molecule inhibitor and analyzing tubulin acetylation, an established biomarker of HDAC6 inhibition. HDAC6 and Sirt2 have been identified as the major cytoplasmic tubulin deacetylases (Hubbert, et al. (2002). Nature 417, 455–458. doi: 10.1038 / 417455a); Inoue, et al. (2007). Oncogene 26, 945–957. doi: 10.1038 / sj.onc.1209857).

[0059] Selective HDAC6 inhibitors result in α-tubulin acetylation but have no effect on histone acetylation.

[0060] The selective HDAC6 inhibitors for reducing or treating leptin-resistant obesity as used herein are those compounds that can penetrate into the hypothalamus and block the interaction of HDAC6 with the leptin receptor, particularly at the arcuate neurons in the hypothalamus.

[0061] The arcuate nucleus (ARC) is located in the medial basal hypothalamus and forms a morphological and functional entity with the median eminence (ME), namely the ARC-ME. The ARC contains several different types of neurons that control prolactin release, food intake, and metabolism, as well as the onset of reproduction and puberty. The arcuate nucleus (ARC) of the hypothalamus contains two groups of neurons that express proopiomelanocortin (POMC) or co-express agouti-related protein (AGRP) and neuropeptide Y (NPY). Both of these two groups of neurons express the leptin receptor (OBR) and IR.

[0062] When referring to a given compound, an "analogue" or "derivative" means another compound that is structurally similar, functionally similar, or both, to the designated compound. Structural similarity can be determined using any standard known in the art, such as the Tanimoto coefficient, which provides a quantitative measure of the similarity between two compounds based on the molecular descriptors of the two compounds. Preferably, the molecular descriptors are 2D properties, such as fingerprints, topological indices, and maximum common substructures, or 3D properties, such as overall shape and molecular fields. For dissimilar molecule pairs and identical molecule pairs, the Tanimoto coefficient ranges between 0 and 1 (inclusive of the end values). If the Tanimoto coefficient of a compound with the designated compound is between 0.5 and 1.0 (inclusive of the end values), preferably between 0.7 and 1.0 (inclusive of the end values), and most preferably between 0.85 and 1.0 (inclusive of the end values), then the said compound can be regarded as an analogue of the designated compound. If a compound induces the same pharmacological action, physiological action, or both, as the designated compound, then the said compound is functionally similar to the designated compound. An "analogue" or "derivative" may also refer to a modification of the disclosed compound, including but not limited to hydrolysis, reduction, or oxidation products. Hydrolysis, reduction, and oxidation reactions are known in the art. Generally, a derivative can be envisioned as being formed from the parent compound at least in theory by chemical and / or physical processes. For example, derivatives of HDAC6 inhibitors include compounds having one or more substituents attached to the HDAC6 inhibitor core.

[0063] A "metabolite" means a compound that can be obtained by the biochemical processing of another compound. Degradation can occur after ingestion or in vitro.

[0064] As used herein, "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0065] As used herein, a "prodrug" means a pharmacological substance (drug) that is administered to a subject in an inactive (or significantly reduced activity) form. Once administered, the prodrug is metabolized in the body (in vivo) to a compound having the desired pharmacological activity.

[0066] A heteroatom (such as nitrogen) can have a hydrogen substituent and / or any permissible substituent of an organic compound that satisfies the valence of the heteroatom as described herein. It should be understood that "substituted" or "substitution" includes the implicit condition that such substitution is in accordance with the permissible valences of the substituted atom and the substituent, and the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc.

[0067] As used herein, "obesity" means that a patient has a body mass index greater than 30 kg / m 2 . As used herein, "overweight" and "pre-obesity" mean that a patient has a body mass index greater than 25 kg / m 2 . As used herein, "morbid obesity" means that a patient has a body mass index greater than 40 kg / m 2 , a body mass index greater than 35 kg / m 2 in combination with one or more co-morbidities, a body mass index greater than 30 kg / m 2 in combination with uncontrolled diabetes, or a combination thereof.

[0068] From the American Cancer Society:

[0069]

[0070] As used herein, "effective amount" or "therapeutically effective amount" means an amount of a selective HDAC6 inhibitor that is effective to induce weight loss in pre-obese, obese or morbidly obese patients, reduce body fat in pre-obese, obese or morbidly obese patients, reduce food intake in pre-obese, obese or morbidly obese patients, improve glucose homeostasis in pre-obese, obese or morbidly obese patients, prevent weight gain and / or prevent an increase in body mass index in normal, pre-obese, obese or morbidly obese patients, or a combination thereof.

[0071] II. Pharmaceutical Formulations

[0072] Lysine deacetylase 6 (HDAC6) is a class IIB Zn2+-dependent deacetylase. Among the metal-dependent HDAC isozymes, HDAC6 is unique in that it contains two catalytic domains: CD1 and CD2. The first domain specifically deacetylates acetylated C-terminal lysine residues, while the second domain exhibits a particularly broad substrate selectivity. CD2 is a tubulin deacetylase and a tau deacetylase. The development of HDAC6-selective inhibitors has focused solely on this domain. In contrast, there is a lack of structural and functional information about CD1, which exhibits a much narrower substrate specificity compared to CD2. The active site of HDAC6 CD1 is wider than that of CD2, which is unexpected considering the narrow substrate specificity of CD1. Amino acid substitutions between HDAC6 CD1 and CD2, as well as conformational differences in conserved residues, determine the significant differences in the active site profile. Measurements of the catalytic activity of HDAC6 CD1 confirm a preference for peptide substrates containing C-terminal acetylated lysine residues. However, these measurements also indicate that CD1 exhibits weaker activity towards peptide substrates bearing certain small amino acids on the carboxyl side of the labile acetyl-lysine residue. Collectively, these results provide a basis for understanding the structural basis of HDAC6 CD1 catalysis and inhibition and for the development of HDAC6 CD1-selective inhibitors.

[0073] There is evidence that HDAC6 catalyzes the deacetylation of a variety of proteins involved in multiple cellular processes. Among these, the deacetylation of α-tubulin mediated by HDAC6 regulates microtubule stability and cell motility. Another characterized substrate, cortactin, binds to deacetylated actin filaments and is involved in the fusion of lysosomes and autophagosomes. The enzyme also plays a role in protein folding by regulating the activity of the Hsp90 chaperone protein through deacetylation. In addition, HDAC6 is an important player in innate immunity, regulating the detection of pathogen genomic material through the deacetylation of retinoic acid-inducible gene-I protein.

[0074] Studies have now determined that HDAC6 inhibitors that cross the blood-brain barrier (BBB) and enter the hypothalamus are most effective in treating obesity and reducing leptin insensitivity.

[0075] The BBB is a highly dynamic and complex structure formed by specialized endothelial cells of brain capillaries and ependymal cells of the circumventricular organs. These cells establish an interface between the blood of the cerebral vasculature on one side and the interstitial fluid of brain parenchymal cells and cerebrospinal fluid (CSF) on the other side. The tight junctions between the cells forming this interface represent a fundamental physical barrier that prevents the free movement of compounds across the intercellular spaces between the endothelial cells in the BBB and the ependymal cells in the choroid plexus blood-CSF barrier. Neurons that secrete hormones into the bloodstream must communicate openly with capillaries. In the central nervous system (CNS), there are discrete regions located in the ventricular walls, called circumventricular organs (CVOs).

[0076] These "brain windows" may have two functions, namely, allowing peptides and proteins secreted by neural tissue to reach the bloodstream and allowing nerve cells to sense the plasma. CVOs share some characteristics, namely, they are located near or within the ventricles; their blood supply consists of capillaries with fenestrations and perivascular spaces; and their ependymal cells are highly specialized in terms of transport and / or secretion. Except for the choroid plexus of the lateral ventricles, all other CVOs show the unique feature of a single, unpaired structure located along the midline of the CNS CVOs, and they can be grouped into the following categories: (i) sensory organs (organum vasculosum of the lamina terminalis, subfornical organ, area postrema): The cell bodies and dendrites of the neurons forming these CVOs are not protected by the BBB and maintain open communication with the peripheral blood, enabling them to respond to signals carried by the blood, such as subfornical neurons responding to the plasma level of angiotensin II. The axons of the neurons of these sensory CVOs project to multiple regions of the CNS; thus, these axons leave the BBB-free region of the CVO and enter the brain regions protected by the BBB. This is different from providing a direct pathway for substances to enter the CNS. (ii) neurosecretory organs (pineal gland, median eminence, and neurohypophysis): In the medial hypothalamus, the cell bodies and dendrites of the neurons secreting neuropeptides and monoamines are protected by the BBB, while their axons enter the BBB-free regions of the median eminence and neurohypophysis to deliver their secretions to the portal capillaries and systemic capillaries, respectively. (iii) ependymal secretory organs (subcommissural organ, choroid plexus): These CVOs are formed by ependymal cells highly specialized for secreting proteins into the CSF. (iv) transport CVO organs (choroid plexus, median eminence). The choroidal cells and the tanycytes of the median eminence are cells endowed with transport mechanisms to transfer substances from the blood to the CSF and from the CSF to the blood.

[0077] It has been found that HDAC6 inhibitors with high permeability levels across the BBB, particularly in the hypothalamus, and in particular inhibitors that block the LepRb-HDAC6 interaction are effective in treating leptin-resistant obesity and increasing leptin sensitivity. It has further been found that the key brain region in which the inhibitors act is the hypothalamus. Several compounds were tested and it was determined that a central rather than a peripheral mechanism of action is associated with HDAC6 inhibitors having high permeability levels across the blood-brain barrier and / or into the hypothalamus. Analysis of hypothalamic gene expression in fed, starved and obese mice determined a correlation with gene expression changes induced by small molecule inhibition of HDAC6 activity, indicating that pharmacological inhibition of HDAC6 activity in the hypothalamus is effective in treating obesity and related disorders. Subsequent studies in high-fat diet-induced mice treated with the HDAC6 isoform-specific inhibitors ACY738 and ACY775 showed that these compounds caused a significant reduction in food intake and total body fat mass. The more selective HDAC6 inhibitor ACY775 had a greater effect compared to ACY738. ACY1083, bavarostat and the compound ACY257, which inhibits HDAC6 in the hypothalamus but has low BBB permeability, also showed significant weight loss.

[0078] Only HDAC6 inhibitors that penetrate the hypothalamus and inhibit HDAC6 therein are effective in treating obesity, particularly leptin-resistant obesity. Data show that the compound ACY257, which inhibits HDAC6 in the hypothalamus but not in other brain regions such as the cerebrum, frontal lobe, temporal lobe and brainstem, significantly reduced body weight. The hypothalamus is a very small part of the brain. The hypothalamus has a slightly more permeable blood-brain barrier compared to other parts of the brain. Data show that HDAC6 inhibitors with the ability to preferentially cross the BBB and enter the hypothalamic region are able to reduce body weight.

[0079] Based on these studies, small molecule (less than 1000 Da) selective inhibitors of HDAC6 with high permeability across the BBB and hypothalamus with a brain / plasma ratio greater than 1 (inhibitors that result in α-tubulin acetylation without affecting histone acetylation), particularly inhibitors that block the LepRb-HDAC6 interaction, are most effective in treating obesity, including leptin-resistant obesity, through a central nervous system (CNS) mechanism of action. Preferred HDAC6 inhibitors have a brain or hypothalamus / plasma concentration > 0.3, > 0.5 or > 1. More preferably, the HDAC6 inhibitor has a hypothalamus / plasma concentration > 0.3, > 0.5 or > 1. Most preferably, the HDAC6 inhibitor has an arcuate nucleus / plasma concentration > 0.3, > 0.5 or > 1. The arcuate nucleus has the highest BBB permeability in the hypothalamus and is approximately 1 / 10,000 of the total brain area. Results indicate that inhibiting HDAC6 in AgRP neurons in the arcuate nucleus is sufficient to produce weight loss. If the HDAC6 inhibitor reaches the arcuate nucleus only at a concentration capable of inhibiting HDAC6 activity and does not reach other regions of the brain, the total brain / plasma or hypothalamus / plasma concentration may be low. However, the arcuate nucleus concentration will be higher than the total brain / plasma and hypothalamus / plasma concentrations.

[0080] Preferred compounds are ACY775 (brain / plasma ratio 1.26) and ACY738 (brain / plasma ratio 1.22). This is in contrast to peripherally acting HDAC6 inhibitors such as ricolinostat (ACY1215) with a brain / plasma ratio of 0.01, and tubastatin A with a brain / plasma ratio of 0.18, neither of which have high permeability into the hypothalamus and do not exhibit HDAC6 inhibition in the hypothalamus. HDAC6 inhibition was tested by treating cells with high concentrations of HDAC6 inhibitor and analyzing tubulin acetylation (an established biomarker of HDAC6 inhibition).

[0081] A. HDAC6 Inhibitors and Formulations

[0082] HDAC6 inhibitors are commercially available from suppliers such as Sigma Aldrich, Sellect Chemistry, AdooQBioscience, etc.

[0083] Preferred HDAC6 inhibitors interfere with leptin signaling and LepRb deacetylation.

[0084] Small molecule (less than 1000 Da) selective inhibitors of HDAC6 with high permeability across the BBB having a brain / plasma ratio greater than 0.5, more preferably greater than 1, are most effective for treating obesity (including leptin-resistant obesity) through a central nervous system (CNS) mechanism of action. Preferred compounds are ACY775 (brain / plasma ratio 1.26) and ACY738 (brain / plasma ratio 1.22).

[0085] This is in contrast to compounds that are ineffective, including peripherally acting HDAC6 inhibitors such as ricolinostat (ACY1215) with a brain / plasma ratio of 0.01 and tubastatin A with a brain / plasma ratio of 0.18, as well as citarinostat (ACY241) or ricolinostat (ACY1215).

[0086] HDAC6 inhibition was tested by treating cells with a high concentration of a selective HDAC6 inhibitor and analyzing tubulin acetylation (an established biomarker of HDAC6 inhibition).

[0087] Compounds that are ineffective due to a low brain / plasma ratio or inability to inhibit HDAC6 in the hypothalamus:

[0088] Ricolinostat (ACY1215) brain / plasma ratio < 0.01 https: / / actaneurocomms.biomedcentral.com / articles / 10.1186 / s40478-018-0604-3

[0089] Tubastatin A brain / plasma ratio ~0.18 https: / / www.nature.com / articles / npp2013207

[0090] Brain / plasma ratios of useful compounds include:

[0091] ACY738 inhibits HDAC6 with low nanomolar potency (IC50 = 1.7 nM), and its selectivity is 60 to 1500 times that of class I HDACs. Brain / plasma ratio is ~1.22 https: / / www.nature.com / articles / npp2013207

[0092]

[0093] ACY738

[0094] ACY775 brain / plasma ratio ~1.26 https: / / www.nature.com / articles / npp2013207

[0095]

[0096] ACY775

[0097] ACY-1083 is a selective and brain-penetrant HDAC6 inhibitor with an IC50 of 3 nM and 260-fold selectivity for HDAC6 over all other classes of HDAC isoforms.

[0098]

[0099] ACY1083

[0100] bavarostat (4-(((((3r,5r,7r)-adamantan-1-yl)methyl)(methyl)amino)methyl)-3-fluoro-N-hydroxybenzamide) is a selective inhibitor of HDAC6 with high brain permeability, Strebl, et al ACS Cent Sci. 2017 Sep 27; 3(9): 1006–1014.

[0101]

[0102] The HDAC6 inhibitor ACY-257 exhibits limited brain expression upon systemic administration (Selleck Chemical).

[0103]

[0104] ACY257

[0105] B. Additional Therapeutic Agents

[0106] In some cases, the pharmaceutical formulation may further contain one or more additional active agents. Examples include anti-obesity drugs that act through an HDAC6-independent mechanism of action, such as GLP1R agonists, GIP receptor agonists, GLP1 / GIP receptor dual agonists, etc. Compounds for the treatment of diabetes and for the control of glucose production may also be important.

[0107] The FDA has approved five long-term use drugs: orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, and semaglutide.

[0108] In certain embodiments, the pharmaceutical formulation further contains leptin, leptin analogs, or combinations thereof.

[0109] Leptin is a peptide hormone that serves as an afferent signal in the negative feedback loop that regulates food intake and body weight in the body. Unprocessed human leptin is synthesized in the body as a 167 - amino acid, 16 kDa pro - protein hormone. Unprocessed leptin contains a 21 - amino acid signal sequence at the N - terminus, which is cleaved from the rest of the polypeptide to produce mature, circulating leptin (containing 146 amino acids).

[0110] As used herein, the terms “leptin” and “leptin analogs” encompass naturally occurring human leptin, naturally occurring leptin produced by non - human species (such as mice or rats), recombinantly produced mature leptin such as metreleptin (i.e., recombinant methionyl - human leptin or r - metHuLeptin, which is produced by genetically engineering the addition of a methionine N - terminus to the N - terminal amino acid of the 146 - amino acid mature circulating human leptin to produce a 147 - amino acid leptin analog), as well as leptin fragments, leptin variants, leptin fusion proteins, and other derivatives known in the art to have biological activity. Exemplary leptin variants include those in which the amino acid at position 43 is replaced by Asp or Glu; replaced by Ala at position 48; replaced by Glu or absent at position 49; replaced by Ala at position 75; replaced by Leu at position 89; replaced by Asp or Glu at position 93; replaced by Ala at position 98; replaced by Ser at position 117, replaced by Leu at position 139, replaced by Ser at position 167, and any combination thereof.

[0111] In certain embodiments, the pharmaceutical formulation contains r - metHuLeptin (A - 100, METRELEPTIN®) available from Amylin Pharmaceuticals (San Diego, Calif.).

[0112] The pharmaceutical preparation may also contain one or more vitamins, minerals, dietary supplements, nutraceuticals, such as proteins, carbohydrates, amino acids, fatty acids, antioxidants, and plant or animal extracts, or combinations thereof. Suitable vitamins, minerals, nutraceuticals, and dietary supplements are known in the art and are disclosed, for example, in Roberts et al., (Nutriceuticals: The Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing Foods, American Nutriceutical Association, 2001). Nutraceuticals and dietary supplements are also disclosed in Physicians' Desk Reference for Nutritional Supplements, 1st Ed. (2001) and The Physicians' Desk Reference for Herbal Medicines, 1st Ed. (2001).

[0113] C. Preparation

[0114] The selective HDAC6 inhibitor is preferably administered to mucosal surfaces, most preferably orally, buccally, or nasally. These can be formulated using known excipients. The preparation can also be formulated for sustained, delayed, and / or pulsatile release to deliver an effective amount of the HDAC6 inhibitor to cause weight loss. It is preferably administered once or twice a day. The dose is weight-based. The typical dose ranges from 25 to 500 mg / day.

[0115] A pharmaceutical preparation can be administered to induce weight loss in pre - obese, obese, or morbidly obese patients, reduce body fat in pre - obese, obese, or morbidly obese patients, reduce food intake in pre - obese, obese, or morbidly obese patients, improve glucose homeostasis in pre - obese, obese, or morbidly obese patients, or a combination thereof. In some cases, a pharmaceutical preparation containing one or more HDAC6 inhibitors is administered to pre - obese, obese, or morbidly obese patients in a therapeutically effective amount to induce weight loss, preferably in a therapeutically effective amount and for a time period such that body mass or body fat is reduced by at least 10%, more preferably at least 15%, and most preferably at least 20% or more. In some cases, a pharmaceutical preparation containing one or more HDAC6 inhibitors is administered to pre - obese, obese, or morbidly obese patients in a therapeutically effective amount to reduce food intake, appetite, or a combination thereof, preferably in a therapeutically effective amount to reduce average daily food intake (in calories). In some cases, a pharmaceutical preparation containing one or more selective HDAC6 inhibitors is administered to pre - obese, obese, or morbidly obese patients in a therapeutically effective amount to improve glucose homeostasis, preferably in a therapeutically effective amount to lower average fasting plasma glucose. In cases where the pharmaceutical preparation is administered to normalize blood glucose, the preparation is preferably administered in an amount effective to reduce the blood glucose level to less than about 180 mg / dL. If desired, the preparation can be co - administered with other anti - diabetic therapies to improve glucose homeostasis.

[0116] The pharmaceutical preparation comprises a combination of a therapeutically effective amount of a selective HDAC6 inhibitor having high permeability across the blood - brain barrier (“BBB”), resulting in a brain or hypothalamus / plasma ratio greater than 0.5, more preferably greater than 1, and one or more pharmaceutically acceptable excipients, wherein the selective HDAC6 inhibitor is present in an amount effective to cause weight loss in an individual having a BMI of 25 or greater. It should be understood that the dose can be formulated as a single unit (capsule, tablet, microparticles encapsulated or suspended in a liquid, vaginal suppository), or in a form where more than one unit needs to be administered to provide an effective dose, or in a form such as a liquid or gel for oral administration or injection, where the effective amount is determined by measuring the liquid.

[0117] Representative excipients include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials generally recognized as safe (GRAS) and can be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0118] In a preferred embodiment, the preparation is for enteral administration - oral, or administration to another mucosal surface, such as nasal, pulmonary, sublingual or buccal, vaginal, rectal, or pulmonary.

[0119] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells using techniques well known in the art for encapsulating liquid, solid, and semi-solid filling materials.

[0120] The formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. Excipients (including plasticizers, pigments, colorants, stabilizers, and glidants) can also be used to form coating compositions for enteral administration. Sustained-release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington – The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and sustained-release dosage forms of tablets, capsules, and granules.

[0121] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic polymers and copolymers, and methacrylic acid resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0122] Diluents, also known as "fillers", are generally necessary to increase the volume of solid dosage forms in order to provide practical dimensions for tablet compression or the formation of beads and granules. Binders are used to impart cohesiveness to solid dosage formulations, thereby ensuring that tablets or beads or granules remain intact after dosage form formation. Lubricants are used to facilitate tablet manufacture. Disintegrants are used to facilitate the disintegration or "breakup" of the dosage form after administration. Stabilizers are used to inhibit or retard drug decomposition reactions including, for example, oxidation reactions.

[0123] Selective HDAC6 inhibitors can be formulated for controlled release. For example, one or more compounds and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed from the drug and a controlled release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled release coatings before incorporation into the final dosage form.

[0124] In another embodiment, one or more compounds and optionally one or more additional active agents are dispersed in a matrix material that gels or emulsifies upon contact with an aqueous medium (e.g., physiological fluid). In the case of a gel, the matrix swells to entrap the active agent, and the active agent is slowly released over time by diffusion and / or degradation of the matrix material. Such matrices can be formulated as tablets or as a filling material for hard and soft capsules.

[0125] In yet another embodiment, one or more compounds and optionally one or more additional active agents are formulated into a solid oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings (e.g., delayed release coatings or extended release coatings). One or more of the coatings can also contain the compound and / or additional active agents.

[0126] The formulation can provide extended release. Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in "Remington – The science and practice of pharmacy" (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). Diffusion systems generally consist of two types of devices (reservoirs and matrices), and are well-known and described in the art. Matrix devices are generally prepared by compressing the drug with a slowly dissolving polymeric carrier into tablet form. The three main types of materials used to prepare matrix devices are insoluble plastics, hydrophilic polymers, and aliphatic compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulose polymers (such as methylcellulose and ethylcellulose), hydroxyalkyl celluloses (such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and CARBOPOL® 934), polyethylene oxide, and mixtures thereof. Aliphatic compounds include, but are not limited to, various waxes (such as carnauba wax and glyceryl tristearate) and wax-like substances (including hydrogenated castor oil or hydrogenated vegetable oil) or mixtures thereof. The plastic material can be a pharmaceutically acceptable acrylic polymer, such as acrylic or methacrylic acid copolymers known in the art. A common example is EUDRAGIT®, which is commercially available from Rohm Pharma.

[0127] The above devices with different drug release mechanisms can be combined in a final dosage form containing a single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules.

[0128] The immediate release portion can be applied on top of the extended release core by using coating or compression methods or added to the extended release system in a multi-unit system (such as a capsule containing extended release and immediate release beads).

[0129] Delayed release formulations can be produced by coating a solid dosage form with a polymeric film that is insoluble in the acidic environment of the stomach but soluble in the neutral environment of the small intestine. Delayed release dosage units can be prepared, for example, by coating the drug or drug-containing composition with a selected coating material. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and can be conventional "enteric" polymers. As will be understood by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract or erode slowly as the dosage form passes through the gastrointestinal tract; while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract (especially the colon).

[0130] The formulation can provide pulsed delivery of one or more of the compounds disclosed herein. "Pulsed" means the release of multiple drug doses at spaced time intervals. Generally, after ingestion of the dosage form, the release of the initial dose is substantially immediate, i.e., the first drug release "pulse" occurs within about one hour of ingestion. This initial pulse is followed by a first time interval (lag time) during which very little or no drug is released from the dosage form, after which a second dose is then released. Similarly, a second interval with little or no drug release can be designed between the second and third drug release pulses. The duration of the intervals with little or no drug release will vary depending on the dosage form design (e.g., twice-daily dosing curve, three-times-daily dosing curve, etc.). For a dosage form providing a twice-daily dose curve, the duration of the interval with little or no drug release between the first and second doses is about 3 hours to 14 hours. For a dosage form providing a three-times-daily curve, the duration of the intervals with little or no drug release between each of the three doses is about 2 hours to 8 hours.

[0131] These compounds can be formulated for parenteral administration. As used herein, "parenteral administration" means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration can include intravenous, intradermal, intramuscular, or subcutaneous administration to a patient.

[0132] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Generally, such compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for preparation as solutions or suspensions upon addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and their microemulsions, liposomes, or lipid bodies.

[0133] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils (e.g., vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)), and combinations thereof. Appropriate fluidity can be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and / or by using surfactants. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride.

[0134] Solutions and dispersions of the active compound as the free acid or base or its pharmaceutically acceptable salts can be prepared by appropriately mixing with one or more pharmaceutically acceptable excipients in water or another solvent or dispersion medium, the excipients including but not limited to surfactants, dispersants, emulsifiers, pH regulators, and combinations thereof.

[0135] For parenteral administration, the compound and optionally one or more additional active ingredients can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release. In embodiments where the formulation contains two or more drugs, the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsed), or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsed, etc.).

[0136] In depot formulations containing a polymeric or oligomeric carrier, the carrier and the active agent can be formulated as a solution, emulsion, or suspension. One or more selective HDAC6 inhibitors, and optionally one or more additional active agents, can also be incorporated into polymeric or oligomeric microparticles, nanoparticles, or combinations thereof.

[0137] In some cases, the formulation is fluid and is designed to solidify or gel upon injection (i.e., form a hydrogel or an organogel). This can be caused by a change in the solubility of the composition upon injection, or for example, by injecting a prepolymer mixed with an initiator and / or a cross-linking agent. The polymeric matrix, polymeric solution, or polymeric particles entrap the active agent at the injection site. As the polymeric carrier gradually degrades, the active agent is released by diffusion of the agent out of the matrix and / or by dissipation of the matrix when the matrix is absorbed. The release rate of the active agent from the injection site can be controlled by varying, for example, the chemical composition, molecular weight, cross-link density, and / or concentration of the polymeric carrier. Examples of such systems include those described in U.S. Patent Nos. 4,938,763, 5,480,656, and 6,113,943.

[0138] Depot formulations can also be prepared by using other rate-controlling excipients, including hydrophobic materials, including acceptable oils (e.g., peanut oil, corn oil, sesame oil, cottonseed oil, etc.) and phospholipids, ion exchange resins, and sparingly soluble carriers.

[0139] The above liquid formulations can also be administered using a nebulizer. A nebulizer is a liquid aerosol generator that converts the above liquid formulation (usually an aqueous-based composition) into a mist or cloud of small droplets, preferably having a mass median aerodynamic diameter of less than 5 microns, which can be inhaled into the lower respiratory tract. This process is called nebulization. When the aerosol cloud is inhaled, the droplets carry one or more active ingredients into the nose, upper respiratory tract, or deep lungs. Any type of nebulizer can be used to administer the formulation to a patient, including but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.

[0140] III. Treatment Methods

[0141] A pharmaceutical composition comprising one or more selective HDAC6 inhibitors can be administered to induce weight loss in pre - obese, obese or morbidly obese patients, reduce body fat in pre - obese, obese or morbidly obese patients, reduce food intake in pre - obese, obese or morbidly obese patients, improve glucose homeostasis in pre - obese, obese or morbidly obese patients, prevent weight gain and / or prevent an increase in body mass index in normal, pre - obese, obese or morbidly obese patients, or a combination thereof.

[0142] In certain embodiments, the pharmaceutical composition is administered to a patient having obesity (e.g., a pre - obese, obese or morbidly obese patient), an obesity - related disease or disorder, diabetes, insulin resistance syndrome, lipodystrophy, non - alcoholic steatohepatitis, cardiovascular disease, polycystic ovary syndrome or metabolic syndrome.

[0143] In cases where the pharmaceutical composition is administered to normalize blood glucose, the composition is preferably administered at an effective dose to reduce the blood glucose level to less than about 180 mg / dL. If desired, the composition can be co - administered with other anti - diabetic therapies to improve glucose homeostasis.

[0144] The pharmaceutical composition can also be administered to a patient having a disease or disorder that causes obesity or predisposes a patient to obesity (e.g., Prader - Willi syndrome (PWS), Bardet - Biedl syndrome or a mutation in the gene encoding the melanocortin 4 receptor (MC4R) protein (i.e., MC4R mutation)).

[0145] A. Dose

[0146] The precise dose administered to a patient will depend on a number of factors, including the patient's physical characteristics (e.g., weight), the severity of the disease or disorder to be treated, and the presence or absence of other concurrent diseases or disorders, and can be readily determined by a prescribing physician.

[0147] In certain embodiments, the selective HDAC6 inhibitor is administered at a dose equivalent to an oral dose of about 0.005 mg to about 500 mg per kg body weight per day, more preferably about 0.05 mg to about 100 mg per kg body weight per day, and most preferably about 0.1 mg to about 10 mg per kg body weight per day. In a specific embodiment, the selective HDAC6 inhibitor is administered at a dose equivalent to an oral dose of about 1.0 mg to 5.0 mg per kg body weight per day. Typical doses are in the range of 25 to 500 mg / day, depending on the weight of the person taking the composition. The dose can be adjusted based on factors such as the route of administration, co - administration with other agents, leptin resistance, the presence of other disorders, and the desired rate of weight loss.

[0148] In some cases, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to induce weight loss. In certain embodiments, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce body mass by at least 10%, more preferably at least 15%, and most preferably at least 20%.

[0149] In some cases, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce body fat. In certain embodiments, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce body fat by at least 10%, more preferably at least 15%, and most preferably at least 20%.

[0150] In some cases, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce food intake, appetite, or a combination thereof. In certain embodiments, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce the average daily food intake (in calories) by at least 15%, 17%, 20%, 22%, 25%, 28%, 30%, 32%, 35% or more.

[0151] In some cases, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to improve glucose homeostasis. In certain embodiments, a pharmaceutical preparation comprising one or more selective HDAC6 inhibitors is administered to a pre - obese, obese, or morbidly obese patient in a therapeutically effective amount to reduce the average fasting plasma glucose by at least 10%, 12%, 15%, 18%, 20%, 22%, 25% or more. In cases where the pharmaceutical preparation is administered to normalize blood glucose, the preparation is preferably administered in an amount effective to reduce the fasting plasma glucose level to less than about 180 mg / dL, more preferably less than about 160 mg / dL, and even more preferably less than about 140 mg / dL.

[0152] B. Therapeutic Administration

[0153] The pharmaceutical preparation can be administered, for example, as a single dose, as a continuous dose, once or more than once daily, or at a lower frequency (e.g., once a week). The pharmaceutical preparation can be administered once or more than once daily, such as twice daily, three times daily, four times daily or more. In certain embodiments, the preparation is administered orally, once or less per day.

[0154] The pharmaceutical formulation is administered in an effective amount and for an effective period of time to elicit the desired therapeutic benefit. In certain embodiments, the pharmaceutical formulation is administered daily, biweekly, weekly, bimonthly, or monthly for at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer.

[0155] The HDAC6 inhibitor can be co-administered with one or more additional therapeutic, prophylactic, or diagnostic agents. Co-administration includes administration within the same dosage form or within different dosage forms. For those embodiments in which the compounds described herein and one or more additional therapeutic, prophylactic, or diagnostic agents are administered in different dosage forms, the dosage forms can be administered simultaneously (e.g., at the same time or substantially at the same time) or sequentially. As used herein, "substantially at the same time" generally means within ten minutes, preferably within five minutes, more preferably within two minutes, and most preferably within one minute. Dosage forms administered sequentially can be administered within a few hours of each other, such as ten hours, nine hours, eight hours, seven hours, six hours, five hours, four hours, three hours, two hours, one hour, 30 minutes, 20 minutes, or 15 minutes.

[0156] In certain embodiments, the selective HDAC6 inhibitors described herein are co-administered with leptin or a leptin analog. In such cases, the leptin or leptin analog can be co-administered with the selective HDAC6 inhibitor for a portion of the treatment period or throughout the treatment period. In a preferred embodiment, the selective HDAC6 inhibitor is co-administered with r-metHuLeptin (A-100, METRELEPTIN®) available from Amylin Pharmaceuticals (San Diego, Calif.).

[0157] In certain embodiments, the patient has diabetes. In such cases, the selective HDAC6 inhibitors described herein can be co-administered with one or more therapies for diabetes.

[0158] The invention will be further understood by reference to the following non-limiting examples.

[0159] Example 1: Selection process of HDAC6 inhibitors as potential leptin sensitizers.

[0160] A method for selecting hypothalamic genes regulated by celastrol that are involved in generating satiety was tested. There was no change between the vehicle and celastrol (CS) treatment groups, but gene expression regulated in the pair-fed (PF) group relative to the CS treatment group was predicted to regulate genes that promote appetite or anorexigenic stimuli.

[0161] Materials and Methods

[0162] Four groups of diet-induced obesity (DIO) mice were examined: 1) treated with vehicle and with free access to food, or 2) pair-fed with the CS-treated group, 3) treated with CS and with free access to food, or 4) treated with CS and given the same amount of food as the PF group at the same intervals as the PF group (CS / PF).

[0163] After 4 days of treatment, RNA sequencing of the hypothalamus of the mice was performed, and principal component analysis (PCA) was performed on the RNA sequencing data. Cluster analysis was performed on the overlapping genes that were significantly different in the comparisons of PF vs. vehicle, PF vs. CS, and PF vs. CS / PF. A cutoff of false discovery rate (FDR) < 0.3 was used for the combined analysis. The gene signature regulated by celastrol was compared with PF and vehicle. The analysis of the identified gene signature was placed on the Connectivity Map (CMAP) L1000 platform. The signature of genes regulated by celastrol was used as a query.

[0164] Results

[0165] Compared with the hypothalamic gene signature regulated by celastrol, the HDAC6 inhibitor signature in the L1000 platform had the highest enrichment score. Immunostaining of endogenous HDAC6 protein in the arcuate nucleus (Arc) of WT lean mice (12 weeks old) and DIO mice (18 weeks HFD-fed) after feeding or after an overnight (15-hour) fasting condition was quantified by the mean HDAC6 fluorescence intensity from lean mice (n = 4) and DIO mice (n = 5). Immunostaining of HDAC6 and GFP in Agrp-ires-cre::L-S-L-cas9-GFP mice under feeding and fasting conditions was performed using GFP as a reporter gene for AgRP-expressing neurons in the arcuate nucleus (Arc). The ratio of the number of HDAC6 / GFP co-positive cells to the number of GFP-positive cells was determined (n = 3 for fed mice, n = 4 for fasted mice).

[0166] The arcuate nucleus contains two potentially important neuronal subpopulations: neurons expressing AgRP and POMC. Among these neurons, AgRP-expressing neurons have been shown to mediate most of the anorexigenic effects of leptin. In mice expressing GFP under copy*oter, the HDAC6 protein levels analyzed under feeding and fasting conditions showed a strong correlation, indicating that fasting-induced HDAC6 mainly occurs in AgRP-expressing neurons in the arcuate nucleus.

[0167] Example 2: HDAC6 deacetylates LepRb and reduces leptin signaling.

[0168] It was investigated whether HDAC6 regulates leptin receptor signaling.

[0169] Materials and Methods

[0170] It was hypothesized that HDAC6 might interact with leptin receptor b (LepRb). To test this hypothesis, HEK293 cells were transfected with GFP or HDAC6-myc or LepRb-flag or LepRb-flag and HDAC6-myc expression plasmids. Subsequently, the cells were treated with vehicle or the HDAC6 inhibitor (ACY775) (200 nM for 1 h). Then, Flag immunoprecipitation was performed to pull down LepRb, and these immunoprecipitates were then immunoblotted with Myc and Flag antibodies.

[0171] HEK293 cells were transfected with GFP (as a control) or HDAC6 or LepRb or LepRb together with HDAC6. Subsequently, the cells were treated with vehicle or the HDAC6 inhibitor (ACY775) (200 nM for 1 h). Flag immunoprecipitation was used to pull down LepRb, and then Myc and Flag immunoblotting were performed.

[0172] HEK293 cells were transfected with plasmids and treated with vehicle or ACY775. Subsequently, Flag immunoprecipitation was used to pull down LepRb, and then Ac Lys or Flag immunoblotting was performed.

[0173] HEK293 cells transfected with plasmids were pretreated with leptin (200 ng / ml) for 10 min, then the medium containing leptin was removed, and the cells were washed twice with PBS and then replaced with medium without leptin. Subsequently, Flag immunoprecipitation was used to pull down LepRb, and then P Tyr or Flag immunoblotting was performed.

[0174] HEK293 cells were transfected with plasmids and treated with leptin (250 ng / ml). STAT3 phosphorylation and control parameters were analyzed by immunoblotting. Tyr705 The ratio of p-STAT3 Tyr705 to STAT3 signals in the immunoblot was quantified.

[0175] Next, a time-course experiment was conducted to analyze the tyrosine phosphorylation of LepRb after leptin removal. HEK293 cells were transfected with LepRb and HDAC6 plasmids in the indicated order and then stimulated with leptin (200 ng / ml) for 10 minutes. Subsequently, the medium was replaced with medium without leptin, and the tyrosine phosphorylation of LepRb was analyzed at 5, 10, 20, and 30 minutes. While control cells not expressing HDAC6 maintained LepRb phosphorylation, the LepRb phosphorylation in cells expressing HDAC6 began to decline after 20 minutes.

[0176] To analyze the effect of HDAC6 expression on the downstream of LepRb signaling, HEK293 cells were transfected with plasmids expressing HDAC6 and / or LepRb, and the cells were stimulated with leptin (200 ng / ml) up to the indicated time points, and STAT3 705 tyrosine phosphorylation (p-STAT3 Tyr705 ) was analyzed.

[0177] Results

[0178] The results showed that HDAC6 deacetylates LepRb and reduces leptin signaling.

[0179] To test whether HDAC6 can deacetylate LepRb, HEK293 cells were transfected with plasmids (LepRb-Flag, HA-p300, and HDAC6-myc). LepRb was immunoprecipitated with a Flag antibody, followed by acetyl-lysine (Ac Lys ) or immunoblotting. Immunoblotting showed that HDAC6 expression led to the deacetylation of LepRb.

[0180] Next, the cells were transfected with plasmids (LepRb-Flag, HA-p300, and HDAC6-myc) and then treated with ACY775 (200 nM / ml) for 0.5, 1, 2, and 3 h. LepRb was immunoprecipitated, followed by acetyl-lysine immunoblotting, and the results showed that the specific HDAC6 inhibitor ACY775 could completely block the deacetylation of LepRb. Next, to study whether HDAC6 has any effect on the LepRb tyrosine phosphorylation indicating LepRb activation, HEK293 cells were transfected with LepRb and HDAC6-Myc plasmids in the indicated order and the cells were stimulated with leptin (200 ng / ml) for 10 minutes and 60 minutes. The Flag immunoprecipitates were exposed to phosphorylated tyrosine (p Tyr ) immunoblotting.

[0181] It was found that at an early time point (10 min) of leptin stimulation, HDAC6 expression did not alter leptin-induced LepRb tyrosine phosphorylation. However, at a later time point (60 min), LepRb tyrosine phosphorylation in HDAC6-expressing cells almost completely disappeared when compared with control cells expressing GFP.

[0182] Subsequently, it was found that HDAC6 interacted with LepRb, and the specific HDAC6 inhibitor ACY775 (2-[[1-(3-fluorophenyl)cyclohexyl]amino]-N-hydroxy-5-pyrimidinecarboxamide) blocked this interaction. ACY775 is a well-characterized HDAC6 inhibitor and increases tubulin acetylation when cells are treated with this inhibitor. After observing the interaction between HDAC6 and LepRb, it was investigated whether LepRb could be deacetylated by HDAC6. For this purpose, various acetyltransferases were tested to see whether LepRb could be acetylated by one of them. It was found that the E1A-binding protein p300 (p300) led to an increase in LepRb acetylation.

[0183] Cells that did not express HDAC6 throughout the experiment maintained p-STAT3 Y705 . However, in cells expressing HDAC6, STAT3 Tyr705 phosphorylation began to decline at approximately 40 min. See Figure 1 .

[0184] These results suggest that HDAC6 interacts with LepRb, leading to deacetylation and faster dephosphorylation of the receptor after activation.

[0185] Example 3: The specific HDAC6 inhibitor ACY775 increases energy expenditure and reduces RER and leptin sensitivity, and reduces appetite and body weight in DIO mice.

[0186] Materials and Methods

[0187] Diet-induced obesity (DIO) mice were placed in metabolic cages and received ACY775 (10 mg / kg, i.p., n = 7) or vehicle (n = 8) once daily for 3 days. Energy expenditure, respiratory exchange ratio (RER; VCO2 / VO2), and locomotor counts (physical activity) were determined for each group of mice. The bar graphs represent the means for two dark cycles (8 - 20 and 32 - 44 hr) and two light cycles (0 - 8, 20 - 32, and 44 - 48 hr).

[0188] DIO mice were pretreated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days, then co-treated with ACY775 (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 9 per group). The percentage change in body weight at 15 h after leptin treatment and the food intake (g) during the 15 h period after leptin treatment were measured. The results were reproduced in two independent experiments.

[0189] DIO mice were pretreated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days, and then received the last injection of ACY775 (or vehicle) in the morning of the fourth day. Six hours later, the mice were injected with saline or leptin (1 mg / kg, i.p.), and the hypothalamus was extracted 45 minutes after the leptin injection. STAT3 Tyr705 Representative immunoblots of phosphorylated and total STAT3 from the hypothalamus. The ratio of p-STAT3 Tyr705 to the total STAT3 signal in the quantitative immunoblot. The results were reproduced in three independent experiments. The results were analyzed hourly.

[0190] DIO mice were treated with ACY775 (10 mg / kg / day, i.p.) for 3 weeks. The daily body weight (g) of DIO mice treated with vehicle (n = 17) or ACY775 (n = 17) during the 3-week treatment period was determined. The daily food intake (g) during the 3-week treatment with vehicle or ACY775 was evaluated. The 24-hour food intake of each mouse during the first week of treatment with vehicle or ACY775 was determined. The serum leptin (ng / ml) level was evaluated after 3 weeks of treatment with vehicle (n = 24) or ACY775 (n = 24). The lean mass (g), fat mass (g), and fat percentage (%) were measured by dual-energy X-ray absorptiometry (DEXA) scan after 3 weeks of treatment with vehicle (n = 17) or ACY775 (n = 17). DEXA scans were performed in two different groups. The glucose tolerance test (GTT) was evaluated after 1 week of treatment of DIO mice with vehicle (n = 33) or ACY775 (n = 26). The area under the curve (AUC) analysis was performed for the GTT. The blood glucose (mg / dl) level was evaluated after 1 week of treatment in DIO mice with vehicle (n = 17) or ACY775 (n = 16). The insulin tolerance test (ITT) was performed after 2 weeks of treatment of DIO mice with vehicle (n = 31) or ACY775 (n = 21). The serum insulin (ng / ml) level was measured after 3 weeks of treatment in DIO mice with vehicle (n = 22) or ACY775 (n = 21). The homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed after 3 weeks of treatment of DIO mice with vehicle (n = 14) or ACY775 (n = 13).

[0191] Metabolic parameters such as energy expenditure (EE), respiratory exchange ratio (RER), and physical activity levels were measured in vehicle- and ACY775-treated mice using a comprehensive laboratory animal monitoring system (CLAMS).

[0192] Diet-induced obesity (DIO) mice were pre-treated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days and then co-treated with ACY775 (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 9 per group). The % change in body weight at 15 h after leptin treatment and food intake (g) during the 15 h period after leptin treatment were evaluated.

[0193] DIO mice were pre-treated with ACY775 (10 mg / kg / day, i.p.) or vehicle for 3 days and then received a final injection of ACY775 (or vehicle) on the morning of the fourth day. Six hours later, saline or leptin (1 mg / kg, i.p.) was injected into the mice, and the hypothalamus was extracted 45 minutes after leptin injection.

[0194] The Ac-tubulin activities of HDAC6 inhibitors (ACY775, ACY738, and Ricolinostat) were tested in vitro in HEK293 cells transfected with GFP or HDAC6 and then treated with the indicated HDAC6 inhibitor for 30 minutes, as measured by Ac-tubulin immunoblotting in cells treated with ACY775, ACY738, and ACY1215 (ricolinostat).

[0195] DIO mice were treated with vehicle or ACY775 (10 mg / kg, i.p., once daily) for 3 days. On the fourth day, 1 h after the start of the light cycle, vehicle or ACY775 (10 mg / kg, i.p.) was administered to the mice, and conditioned place preference (CPP) assay was utilized under free-feeding conditions. CPP assay was performed 6 h later under free-feeding conditions (n = 10 mice in both the vehicle-treated group and ACY775-treated group). CPP assay was also performed under 20-h fasting conditions. DIO mice were treated with vehicle or ACY775 (10 mg / kg, i.p.) once daily and fasted for 15 h after the third injection. On the fourth day, 1 h after the start of the light cycle, vehicle or ACY775 (10 mg / kg body weight, i.p.) was administered to the mice, and CPP assay was performed 5 h after this injection under fasting conditions (n = 10 mice in both the vehicle-treated group and ACY775-treated group).

[0196] The following were measured: the total distance traveled by the mice during the CPP test, the average locomotor speed during the measurement period, the total time the mice spent in the dark chamber during the test; the frequency with which the mice visited the dark chamber during the test; the total time the mice spent in the food-paired white chamber; the frequency with which the mice visited the food-paired white chamber; the total time the mice spent in the food-containing area of the food-paired side chamber; and the frequency with which the mice visited the food-containing area of the food-paired side chamber during the test.

[0197] Results

[0198] As Figures 2A - 2O shown, the specific HDAC6 inhibitor (ACY775) increases leptin sensitivity, reduces appetite and body weight, and improves glucose homeostasis.

[0199] To investigate whether HDAC6 inhibition increases leptin sensitivity in vivo, several different experimental methods were used. First, if HDAC6 increases leptin sensitivity, it should potently enhance the anorectic and weight-lowering effects of exogenous leptin in DIO mice. The response to leptin in DIO animals was measured in the presence and absence of ACY775 (10 mg / kg, i.p.).

[0200] Compared with the control group, administering leptin to DIO mice did not significantly change their body weight. Treatment with ACY775 alone significantly reduced the body weight of DIO mice, while administering leptin to DIO mice pre-treated with ACY775 led to a further decrease in body weight relative to the ACY775 + vehicle group. In addition, treatment with leptin alone did not significantly change the food intake of DIO mice. Treatment with ACY775 alone reduced the food intake of DIO mice. Administering leptin in the presence of ACY775 indicated that ACY775 further enhanced the negative effect of leptin on food intake.

[0201] Subsequently, the in vivo activation of leptin receptor signaling after ACY775 treatment was investigated. To examine whether ACY775 increases leptin sensitivity in the hypothalamus, it was analyzed how acute ACY775 treatment affects leptin-stimulated STAT3 Tyr705 phosphorylation in DIO mice while they were still obese and hyperleptinemic.

[0202] Injecting leptin into vehicle-treated DIO mice did not significantly increase STAT3 Tyr705 phosphorylation. However, when leptin was administered to DIO mice pre-treated with ACY775, the level of p-STAT3 Tyr705 in the hypothalamus was significantly increased, indicating that treating DIO mice with acute ACY775 increased leptin sensitivity.

[0203] A single dose of ACY775 or ACY738 significantly reduced food intake in DIO mice without additional leptin stimulation.

[0204] Administration of ACY775 significantly reduced the body weight of DIO mice, from 48.25 ± 0.87 g to 33.53 ± 1.19 g, equivalent to a 29.58% ± 1.79% decrease in body weight. During the experimental period, the body weight of control mice treated with vehicle did not show significant changes. The food intake of the control group was stable throughout the trial.

[0205] However, ACY775 treatment significantly reduced food intake, from 2.54 ± 0.10 g to 0.85 ± 0.06 g, and the daily food intake remained within these ranges during the first two weeks. Compared with the average food intake of the control group, the average food intake during the first week of treatment showed a very significant decrease. The decrease in food intake observed during the first two weeks gradually increased during the third week. This increase in food intake during the last week of treatment was negatively correlated with circulating leptin levels, which were significantly downregulated at the end of the 3-week treatment period.

[0206] To determine the lean and fat mass throughout the body of vehicle- and ACY775-treated DIO mice, dual-energy X-ray absorptiometry (DEXA) scans were performed after 21 days of treatment. When compared with vehicle-treated control mice, ACY775 treatment did not alter the lean mass of DIO mice. However, the total fat mass and fat percentage of ACY775-treated mice were significantly lower than those of the vehicle-treated group.

[0207] A glucose tolerance test (GTT) was administered after one week of treatment to investigate whether ACY775 improved glucose homeostasis in DIO mice. Treatment with ACY775 significantly improved glucose tolerance relative to the vehicle-treated control group. Consistent with the improvement in glucose tolerance, ACY775 significantly reduced blood glucose levels. In addition, an insulin tolerance test (ITT) performed during the third week of treatment revealed a significant difference between ACY775-treated mice and vehicle-treated mice. ACY775 significantly reduced circulating insulin levels. Combining the improved GTT, reduced blood glucose and insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR) analysis showed that ACY775 treatment significantly reduced insulin resistance in DIO mice. ACY775 treatment almost completely eliminated hepatic steatosis seen in DIO mice.

[0208] To investigate whether HDAC6 inhibitors can reduce food-seeking behavior in DIO mice, separate groups of mice treated with vehicle or ACY775 for three days in the freely fed or 20-h fasted state were placed in a (food) conditioned place preference (CPP) assay during the light cycle. When compared with ACY775-treated mice under fed and fasted conditions, the mouse-centered representative traces of vehicle-treated mice showed more activity in the food area (lower right corner of the white chamber), indicating that vehicle-treated mice showed a higher interest in food compared with ACY775-treated mice. In addition, measurements of locomotion and speed based on mouse nose analysis have shown that vehicle-treated DIO mice and ACY775-treated DIO mice had similar locomotion levels and speed levels under fed or fasted conditions. There was also no significant difference in the cumulative duration that mice spent in the dark side chamber containing mock food. The frequency of mouse appearance in the dark side chamber was also similar for vehicle- and ACY75-treated DIO mice under fed or fasted conditions. ACY775-treated mice spent significantly less time and had a lower frequency of appearance in the food-containing white side chamber compared with vehicle-treated mice under fed and fasted conditions. Notably, ACY775-treated mice also spent significantly less time in the white side chamber and showed a lower frequency of appearance in the food area compared with the vehicle group under fed and fasted conditions.

[0209] In summary, ACY775 increased energy expenditure in DIO mice and reduced the decrease in energy expenditure (“RER”). It has been reported that leptin can block RER even though it reduces food intake. Therefore, despite a severe reduction in food intake and body weight, leptin sensitizers should enhance / maintain energy expenditure and promote the use of fat as the main energy source. ACY775-treated mice had higher EE in both the dark and light cycles compared with vehicle-treated controls. In addition, the RER values of ACY775-treated DIO mice were significantly reduced in both the dark and light cycles, indicating a greater utilization of fat as an energy source in vivo. There was no difference in the level of physical activity between the ACY775-treated group and the vehicle-treated group during the dark and light cycles.

[0210] The HDAC6 inhibitor (ACY775) showed reduced appetite and foraging behavior in DIO mice, as measured using a conditioned place preference (CPP) assay under freely fed conditions.

[0211] To investigate whether HDAC6 inhibitors can reduce food-seeking behavior in DIO mice, separate groups of mice treated with vehicle or ACY775 for three days under ad libitum feeding or 20 h fasting conditions were placed in a (food) conditioned place preference (CPP) assay during the light cycle. When compared with ACY775-treated mice under feeding and fasting conditions, the mouse-centered representative tracks of vehicle-treated mice showed more activity in the food area, indicating that vehicle-treated mice showed a higher interest in food compared with ACY775-treated mice. Measurements of locomotion and speed based on mouse nose analysis showed that vehicle- and ACY775-treated DIO mice had similar locomotion levels and speed levels under feeding or fasting conditions. There was no significant difference in the cumulative duration that mice spent in the dark side chamber containing sham food. The frequency of mouse presence in the dark side chamber was also similar for vehicle- and ACY75-treated DIO mice under feeding or fasting conditions. ACY775-treated mice spent significantly less time and had a lower frequency of presence in the white side chamber containing food compared with vehicle-treated mice under feeding and fasting conditions. Notably, ACY775-treated mice also spent significantly less time in the white side chamber and showed a lower frequency of presence in the food area compared with the vehicle group under feeding and fasting conditions.

[0212] Example 4: Specific HDAC6 inhibitor (ACY738) increases leptin sensitivity, reduces appetite and body weight in DIO mice.

[0213] Methods and Materials

[0214] DIO mice were pre-treated with ACY738 (50 mg / kg / day, i.p.) or vehicle for 3 days and then co-treated with ACY738 (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 14 per group). The % change in body weight 15 h after leptin treatment and food intake (g) during the 15 h period after leptin treatment were measured.

[0215] DIO mice were pre-treated with ACY738 (50 mg / kg / day, i.p.) or vehicle for 3 days and then received a final injection of ACY738 or vehicle on the morning of the fourth day. Six hours later, mice were injected with saline or leptin (1 mg / kg, i.p.), and the hypothalamus was extracted 45 minutes after leptin injection. Immunoblot analysis of phosphorylated and total STAT3 from the hypothalamus was performed to quantify the ratio of p-STAT3 Tyr705 to total STAT3 signal in the immunoblot. The results were reproduced in two independent experiments. Tyr705

[0216] ​After 24 h of fasting, DIO mice were injected with HDAC6 inhibitors (ACY775, 10 mg / kg, i.p or ACY738, 50 mg / kg, i.p, n = 3 cages per group, 3 mice per cage). Food intake was analyzed hourly until 3:00 am. DIO mice were treated with ACY738 (50 mg / kg, i.p) for 3 weeks. The daily body weight (g) of DIO mice treated with vehicle (n = 27) or ACY738 (n = 24) during the 3-week treatment period was measured, the daily food intake (g) during the 3-week treatment with vehicle or ACY738 was measured, and the 24-hour food intake of each mouse during the first week of treatment with vehicle or ACY738 was measured. Serum leptin (ng / ml) levels, lean mass (g) were evaluated after 3 weeks of treatment with vehicle (n = 26) or ACY738 (n = 25); fat mass (g) and fat percentage (%) were measured by DEXA scan after 3 weeks of treatment with vehicle (n = 23) or ACY738 (n = 23). DEXA scans were performed in two different groups. GTT was measured and the AUC of GTT was analyzed 1 week after treatment of DIO mice with vehicle (n = 23) or ACY738 (n = 24). Blood glucose (mg / dl) levels were measured 1 week after treatment of DIO mice with vehicle (n = 27) or ACY738 (n = 23). ITT was determined 2 weeks after treatment of DIO mice with vehicle (n = 27) or ACY738 (n = 22). Serum insulin (ng / ml) levels were measured 3 weeks after treatment of DIO mice with vehicle (n = 25) or ACY738 (n = 25). HOMA-IR analysis was evaluated after 3 weeks of treatment of DIO mice with vehicle (n = 14) or ACY738 (n = 13), and H&E staining of liver sections from DIO mice treated with vehicle or ACY738 for 3 weeks was performed.

[0217] To further investigate whether HDAC6 inhibitors could suppress the appetite of fasted DIO mice, a single dose of ACY775 (10 mg / kg) or ACY738 (50 mg / kg) was injected into 24-h fasted DIO mice at the start of the dark cycle.

[0218] Results

[0219] The results are as Figures 3A - 3P shown.

[0220] The HDAC6 inhibitor ACY738 reduced the appetite of DIO mice, as shown by CPP assays under ad libitum feeding conditions: DIO mice were treated with vehicle or ACY738 (50 mg / kg, i.p., once daily) for 3 days. On the fourth day, 1 h after the onset of the light cycle, mice were administered vehicle or ACY738 (50 mg / kg, i.p.), and CPP assays were performed under ad libitum feeding conditions 6 h later (n = 10 per group). CPP assays were also performed under 20-h fasting conditions: DIO mice were treated once daily with vehicle or ACY738 (50 mg / kg, i.p.) and fasted for 15 h after the third injection. On the fourth day, 1 h after the onset of the light cycle, mice were administered vehicle or ACY738 (50 mg / kg body weight, i.p.), and CPP assays were performed under fasting conditions 5 h after this injection (n = 10 per group).

[0221] Determine whether ACY738 can produce an effect on leptin sensitivity similar to that of ACY775. Compared with vehicle treatment, ACY738 treatment significantly enhanced the acute leptin-induced reduction in body weight and food intake. Similarly, compared with vehicle-treated mice, the ratio of leptin-stimulated hypothalamic pSTAT3 Tyr705 / STAT3 was also significantly increased in mice treated with ACY738 (50 mg / kg, i.p.) for three days.

[0222] Compared with vehicle treatment, even a single dose of ACY775 or ACY738 significantly reduced / blocked food intake in fasted DIO mice.

[0223] The HDAC6 inhibitor (ACY738) increased energy expenditure and ambulatory counts (physical activity) in DIO mice and decreased RER. As Figures 4I - 4K shown, the HDAC6 inhibitor ACY738 blocked the appetite of 24-h fasted DIO mice injected with a single dose of ACY775 (10 mg / kg) or ACY738 (50 mg / kg) at the onset of the dark cycle.

[0224] These results indicate that another specific HDAC6 inhibitor, ACY738, increased leptin sensitivity and reduced body weight. Inhibition of HDAC6 activity by ACY738 was confirmed by analyzing tubulin acetylation after ACY738 treatment.

[0225] Next, it was investigated whether ACY738 (similar to ACY775) could act as an anti-obesity agent when administered to DIO mice in a long-term manner. DIO mice were treated with vehicle or ACY738 (50 mg / kg, i.p., once daily) for 21 days.

[0226] Administration of ACY738 significantly reduced the body weight of DIO mice, from 46.43 ± 0.74 g to 37.74 ± 1, corresponding to a 20.21% ± 1.32% decrease in body weight. During the experimental period, the body weight of vehicle-treated control mice did not change significantly. Throughout the treatment, the food intake of the control group remained stable. However, ACY738 treatment significantly reduced the food intake, from 2.35 ± 0.10 g to 1.32 ± 0.05 g, and the daily food intake remained within these ranges throughout the treatment. Compared with the control group, the average food intake of the drug-treated group was severely reduced during the first week of treatment. As seen after ACY775 treatment, at the end of the 3-week treatment period, ACY738 significantly reduced the circulating leptin level, total body fat mass, and percentage of fat mass in the body. There was no change in the lean mass of either group.

[0227] Next, the effects of ACY738 on glucose homeostasis and insulin sensitivity were evaluated. The GTT performed one week after ACY738 treatment showed a significant improvement in glucose homeostasis. ACY738 treatment significantly reduced the blood glucose level. Similar to ACY775, ACY738 treatment also improved insulin tolerance, reduced the insulin level, and improved insulin resistance. Finally, ACY738 treatment also improved hepatic steatosis.

[0228] To further confirm that the HDAC6 inhibitor can reduce the food-seeking behavior of DIO mice, a CPP assay was performed in another separate group of mice administered ACY738 for three days in the freely feeding or 20 h fasting state. Similar to the case of ACY775 treatment, when compared with ACY738-treated mice under feeding and fasting conditions, the representative traces of vehicle-treated mice showed higher activity in the food area, indicating that vehicle-treated mice showed a higher interest in food compared with ACY738-treated mice. The determination of movement and speed based on mouse nose analysis showed that vehicle- and ACY738-treated DIO mice had similar movement levels and speed levels under feeding or fasting conditions. There was also no significant difference in the cumulative duration that mice spent in the dark side chamber containing simulated food. Under feeding conditions, the frequency of mouse appearance in the dark side chamber of vehicle- and ACY738-treated DIO mice was also similar, but under fasting conditions, the frequency of mouse appearance in the dark side chamber of ACY738-treated DIO mice was lower. Different from ACY775-treated mice, compared with vehicle-treated mice, ACY738-treated mice spent similar amounts of time and had similar frequencies of appearance in the white side chamber containing food under feeding and fasting conditions. Interestingly, compared with the vehicle group, ACY738-treated mice spent significantly less time and showed a lower frequency of appearance in the food area of the white side chamber under feeding and fasting conditions.

[0229] To investigate whether ACY738 enhances energy expenditure like ACY775, metabolic parameters of vehicle and ACY738-treated DIO mice were measured. Notably, ACY738-treated mice had higher energy expenditure during the light cycle, but no significant difference when compared to vehicle-treated mice during the dark cycle. Similar to ACY775, the RER values of ACY738-treated DIO mice were significantly decreased during both the dark and light cycles, indicating a greater utilization of fat as an energy source in vivo. The physical activity levels during the dark and light cycles were not different between the ACY738-treated group and the vehicle-treated group.

[0230] Example 5: HDAC6 inhibitors do not reduce appetite or body weight in lean mice, nor do they alter glucose or insulin levels.

[0231] Materials and Methods

[0232] WT lean mice were treated with vehicle (n = 20) or ACY775 (10 mg / kg, i.p., once daily, n = 22) or ACY738 (50 mg / kg, i.p., once daily, n = 20) for 3 weeks. The daily body weight (g) of WT lean mice treated with vehicle or ACY775 or ACY738 during the 3-week treatment period was measured. The daily food intake (g) during the 3-week treatment. Serum leptin (ng / ml) levels were measured after 3 weeks of treatment. Lean mass (g); fat mass (g) and fat percentage (%) were measured by DEXA scan after 3 weeks of treatment. GTT was measured after 1 week of treatment. AOC analysis was performed on GTT. Blood glucose (mg / dl) levels were measured after 1 week of treatment. ITT was measured after 2 weeks of treatment. Serum insulin (ng / ml) levels were measured after 3 weeks of treatment.

[0233] Results

[0234] Figures 5A - 5H The results comparing ACY738 and ACY775 shown indicate that HDAC6 inhibitors do not affect appetite, blood glucose or insulin levels, or body weight in lean mice.

[0235] In contrast to DIO mice, lean mice have very low levels of circulating leptin, and leptin sensitizers do not affect the body weight and food intake of lean mice. Consistent with acting as leptin sensitizers, three-week treatments with ACY775 and ACY738 at doses that would be highly effective in DIO mice did not affect the body weight of lean mice. Additionally, daily food intake was similar between mice treated with vehicle or the HDAC6 inhibitor (ACY775 or ACY738). Consistently, plasma leptin levels in lean mice were not altered by treatment with ACY738 or ACY775. After three weeks of treatment with ACY738 or ACY775, we also did not detect any changes in lean mass, total fat mass, or fat percentage in lean mice.

[0236] To evaluate whether HDAC6 inhibition could improve glucose homeostasis in lean mice, mice were treated with vehicle and ACY738 or ACY775 for one week and then subjected to a GTT. Disposition of glucose from the circulation was not different between vehicle-, ACY738-, or ACY775-treated lean mice. Blood glucose levels were not affected by ACY738 or ACY775. An ITT was performed two weeks after treatment with ACY738 or ACY775, and no significant differences in insulin sensitivity and circulating insulin levels were observed between vehicle-, ACY738-, or ACY775-treated lean mice. Additionally, liver morphology was similar among the three groups at the end of the treatment.

[0237] In summary, these findings indicate that HDAC6 inhibition does not affect body weight, food intake, or glucose homeostasis in lean mice, suggesting that the anti-obesity function of HDAC6 inhibitors requires hyperleptinemia.

[0238] Example 6: HDAC6 inhibitors are ineffective in db / db mice.

[0239] Materials and Methods

[0240] Db / db mice were treated with vehicle (n = 20) or ACY775 (10 mg / kg, i.p., once daily, n = 22) or ACY738 (50 mg / kg, i.p., once daily, n = 20) for 3 weeks. During the 3-week treatment period, the daily body weight (g) of db / db mice treated with vehicle or ACY775 or ACY738 was measured. After 3 weeks of treatment, the daily food intake (g), serum leptin (ng / ml), lean mass (g), fat mass (g), and fat percentage (%) were measured by DEXA scan. GTT was determined 1 week after treatment. One week after treatment, AOC analysis of GTT was performed and blood glucose (mg / dl) levels were measured. After 3 weeks of treatment, blood glucose (mg / dl) levels and serum insulin (ng / ml) levels were measured. ITT was evaluated 2 weeks after treatment. After 3 weeks of treatment, H&E staining of liver sections was performed.

[0241] Results

[0242] As shown by Figures 5I - 5W HDAC6 inhibitors did not reduce body weight or blood glucose in db / db mice. HDAC6 inhibitors were ineffective in db / db mice. Db / db mice were treated with vehicle (n = 20) or ACY775 (10 mg / kg, i.p., once daily, n = 22) or ACY738 (50 mg / kg, i.p., once daily, n = 20) for 3 weeks. Figure 5I , the daily body weight (g) of db / db mice treated with vehicle or ACY775 or ACY738 during the 3-week treatment period. Figure 5J , the daily food intake (g) during 3 weeks of treatment. Figure 5K , serum leptin (ng / ml) levels after 3 weeks of treatment. Measured by DEXA scan after 3 weeks of treatment Figure 5L , lean mass (g); Figure 5M , fat mass (g) and Figure 5N , fat percentage (%). Figure 5O , GTT 1 week after treatment. Figure 5P , the AOC analysis of GTT performed in ( Figure 5O ). Figure 5Q , blood glucose (mg / dl) levels 1 week after treatment. Figure 5U , blood glucose (mg / dl) levels 3 weeks after treatment. Figure 5V , serum insulin (ng / ml) levels 3 weeks after treatment. 5W, ITT 2 weeks after treatment. In ( Figure 5I -Q and 5U-W) the results were reproduced in two independent experiments.

[0243] The db / db mouse model, which is obese, hyperleptinemic but lacks LepRb function, was used to study leptin sensitizers. db / db obese mice were treated with vehicle, ACY738 (50 mg / kg, i.p., once daily) or ACY775 (10 mg / kg, i.p., once daily) for three weeks. Vehicle-treated db / db mice did not lose weight during the course of the experiment, but instead gained a small amount of weight. Importantly, treatment of db / db mice with the HDAC6 inhibitor also did not result in weight loss, and the mice gained a similar amount of weight as the vehicle-treated mice ( Figure 5I ); in fact, at the end of the treatment period, the db / db mice in the ACY775 treatment group were 3.82% ± 0.99% heavier than their initial weight ( Figure 5I ), while the ACY738 treatment group was 2.53% ± 0.97% heavier than their initial weight. Food intake in db / db mice was not affected by ACY738, but ACY775 treatment transiently reduced food intake during the first 2 days of treatment, which quickly returned to the level of vehicle-treated db / db mice ( Figure 5J ). In addition, neither of the two HDAC6 inhibitors reduced plasma leptin levels, lean mass, fat mass or percentage of fat mass in db / db mice ( Figures 5K - 5N ).

[0244] To study the effect of the HDAC6 inhibitor on glucose homeostasis in db / db mice, a GTT was performed after one week of treatment with vehicle, ACY738 or ACY775. The results showed no difference in glucose tolerance among the vehicle, ACY738 or ACY775 treatment groups ( Figure 5O and 5P ). Fasting blood glucose was not affected by ACY738, but after the first week of treatment, fasting blood glucose in mice treated with ACY775 was lower than that in vehicle-treated mice ( Figure 5Q ). However, the early lower blood glucose from ACY775 treatment completely abated by the end of the treatment period ( Figure 5U ). When compared to the control db / db group, plasma insulin levels in db / db mice treated with ACY775 and ACY738 did not show any significant changes ( Figure 5V ). The ITT showed no significant difference in blood glucose levels among the vehicle, ACY738 or ACY775 treatment groups after insulin injection ( Figure 5W ). Treatment with ACY775 or ACY738 failed to improve hepatic steatosis in db / db mice. Thus, these findings suggest that HDAC6 inhibition has no or minimal effect on body weight, food intake or glucose homeostasis in db / db mice, indicating that the anti-obesity function of HDAC6 inhibitors requires intact leptin signaling.

[0245] Example 7: In contrast to the specific HDAC6 inhibitors (ACY775 and ACY738), Tubastatin A reduced body weight and food intake in DIO HDAC6 knockout mice and DIO WT mice.

[0246] Materials and Methods

[0247] DIO WT or HDAC KO mice were treated with the specific HDAC6 inhibitors (ACY775 and ACY738) and Tubastatin A for 3 weeks and the effects on food intake, lean mass, fat mass, body weight, blood glucose and serum insulin were evaluated.

[0248] Results

[0249] Figure 6A , daily body weight of WT (n = 20) or HDAC6-KO (n = 20) DIO mice during treatment with ACY775 (10 mg / kg / day, i.p.). Figure 6B , average food intake (g) of WT or HDAC6-KO mice in the first week during ACY775 treatment. Figure 6C , blood glucose (mg / dl) levels of WT or HDAC6-KO mice treated with ACY775 after 1 week of treatment (n = 20 per group). Figure 6F , serum insulin (ng / ml) levels of WT or HDAC6-KO mice treated with ACY775 after 3 weeks of treatment (n = 8 per group). Figure 6G , serum leptin (ng / ml) levels of WT or HDAC6-KO mice after 3 weeks of treatment with ACY775 (n = 8). Measured by DEXA scan after 3 weeks of ACY775 treatment Figure 6F , lean mass, Figure 6E , fat mass, Figure 6H , percentage of fat (%). Figure 6I , daily body weight of WT (n = 16) or HDAC6-KO (n = 20) DIO mice during treatment with ACY738 (50 mg / kg / day, i.p). Figure 6J , average food intake (g) of WT or HDAC6-KO mice in the first week during ACY738 treatment. Figure 6K , blood glucose (mg / dl) levels of WT (n = 18) or HDAC6-KO (n = 20) mice treated with ACY738 after 1 week of treatment. Figure 6L , serum insulin (ng / ml) levels of WT or HDAC6-KO mice treated with ACY738 after 3 weeks of treatment (n = 8 per group). Figure 6M, serum leptin (ng / ml) levels in WT or HDAC6-KO mice after 3 weeks of ACY738 (n = 8) treatment. Measured by DEXA scan after 3 weeks of ACY738 treatment Figure 6N , lean mass, Figure 6O , fat mass, Figure 6P , fat percentage (%). Figure 6Q , daily body weight of WT or HDAC6-KO DIO mice during vehicle or Tubastatin A treatment (25 mg / kg / day, i.p., WT-vehicle n = 15, WT-Tubastatin A n = 17, and HDAC6-KO-Tubastatin A n = 18). Figure 6R , average food intake (g) of WT or HDAC6-KO mice during the first week of vehicle or Tubastatin A treatment. Figure 6S , blood glucose (mg / dl) levels of WT or HDAC6-KO mice treated with vehicle or Tubastatin A after 1 week of treatment (WT-vehicle n = 8, WT-Tubastatin A n = 9, and HDAC6-KO-Tubastatin A n = 10). Figure 6T , serum insulin (ng / ml) levels of WT or HDAC6-KO mice treated with vehicle or Tubastatin A after 2 weeks of treatment (WT-vehicle n = 12, WT-Tubastatin A n = 12, and HDAC6-KO-Tubastatin A n = 13). 6U, serum leptin (ng / ml) levels of WT or HDAC6-KO mice after 3 weeks of vehicle or Tubastatin A treatment (WT-vehicle n = 12, WT-Tubastatin A n = 13, and HDAC6-KO-Tubastatin A n = 14). After 3 weeks of Tubastatin A or vehicle treatment Figure 6V , lean mass, Figure 6W , fat mass, Figure 6X , fat percentage (%).

[0250] HDAC6 knockout mice are resistant to the anti-obesity effects of ACY775 and ACY738 but not to Tubastatin A. In contrast to the specific HDAC6 inhibitors ACY775 and ACY738, Tubastatin A improves glucose homeostasis in DIO HDAC6 knockout mice, indicating that the mechanism of action is not mediated by the inhibition of HDAC6. In contrast, ACY775 and ACY738 inhibit HDAC6 in Agrp neurons to reduce body weight and food intake in DIO mice.

[0251] Drugs based on a specific target should not display their pharmacological effects when the target is depleted or knocked out. Therefore, it was decided to test whether the anti-obesity activities of ACY775 and ACY738 are mediated entirely through HDAC6. Obesity was induced by feeding a high-fat diet for 16 weeks, and HDAC6 KO mice were first treated with ACY775 (10 mg / kg, i.p., once a day) for a period of three weeks. When compared with HDAC6 KO DIO mice, ACY775 treatment led to a very significant decrease in body weight in WT DIO mice, indicating that the anti-obesity effect of ACY775 is indeed mediated by HDAC6. The decrease in food intake mediated by ACY775 in the wt group was also blocked in HDAC6 KO mice. When compared with the wt group treated with ACY775, the blood glucose, serum insulin, and leptin levels in the HDAC6 KO group treated with ACY775 were all significantly increased. DEXA scans showed that there was no difference in lean mass between the two groups, but the fat mass and fat % in the wt group were significantly lower when compared with the HDAC6 KO group. Analysis of glucose homeostasis also showed that ACY775 was unable to improve glucose tolerance and insulin sensitivity in HDAC6 KO mice when compared with wt mice treated with ACY775.

[0252] Next, the same experiment was conducted in DIO HDAC6 KO and wt mice by administering ACY738 (50 mg / kg i.p., once a day). The results of ACY738-induced body weight reduction were similar to our previous observations, with a ~20% decrease in body weight in wt mice, but also a slight decrease in body weight in HDAC6 KO mice. However, the difference between ACY738-treated HDAC6 KO mice and wt mice was substantial and reached a highly significant level (p < 0.005). When compared with the wt group, the food intake in the HDAC6 KO group was significantly higher. When compared with the wt group, the blood glucose, serum insulin, and leptin levels in the HDAC6 KO group treated with ACY738 were all significantly higher. There was no difference in lean mass between the two groups, but the fat mass and fat % in the wt group were significantly lower when compared with the HDAC6 KO group. GTT and ITT studies also showed that ACY738 did not improve glucose tolerance and increase insulin sensitivity in HDAC6 KO mice when compared with wt mice treated with ACY738. These data indicate that the anti-obesity effects of ACY775 and ACY738 are mainly mediated by HDAC6, and the HDAC6 KO mouse model is a good tool for dissecting the effects of specific HDAC6 inhibitors on obesity.

[0253] Tubastatin A decreased the body weight of HDAC6 KO mice, which was the same as that of wt mice. The body weight curves of wt and HDAC6 KO mice treated with Tubastatin A were almost identical. When compared with wild-type, the food intake of Tubastatin A-treated HDAC6 KO mice was also similarly decreased. The blood glucose, serum insulin, and serum leptin levels in both the wt and HDAC6 KO groups were equally decreased. Similarly, there was no difference in lean mass among the three groups, but the fat mass and fat % in the wt and HDAC6 KO groups treated with Tubastatin A were significantly lower.

[0254] Effects of ACY 775, ACY738, and tubastatin in GTT and ITT studies ( Figures 7A - 7C , ACY 775; Figures 7D - 7F , ACY738 and Figures 7G - 7I , tubastatin) indicated that Tubastatin A improved glucose tolerance and increased insulin sensitivity in both wt and HDAC6 KO mice when compared with wt mice treated with vehicle. These data suggest that the anti-obesity effect of Tubastatin A is not mediated by HDAC6, but rather by off-target effects or through induction of toxicity.

[0255] Example 8: The blood-brain-barrier poorly permeable specific HDAC6 inhibitor Ricolinostat neither increased leptin sensitivity nor decreased body weight.

[0256] Materials and Methods

[0257] DIO mice were pretreated with ricolinostat (25 mg / kg / day, i.p.) or vehicle for 3 days, and then co-treated with ricolinostat (or vehicle) and leptin (1 mg / kg, i.p.) (or saline) (n = 32 per group). The change in body weight 15 h after leptin treatment and the food intake (g) during the 15-h period after leptin treatment were determined. The results were reproduced in two independent experiments.

[0258] DIO mice were pretreated with ricolinostat (50 mg / kg / day, i.p.) or vehicle for 3 days, and then the last injection of ricolinostat (or vehicle) was given on the morning of the fourth day. Six hours later, mice were injected with saline or leptin (1 mg / kg, i.p.), and the hypothalamus was extracted 45 minutes after leptin injection. Immunoblotting was performed for phosphorylated and total STAT3 from the hypothalamus. Quantification of p-STAT3 in the immunoblot Tyr705 and total STAT3 Tyr705Ratio to total STAT3 signal.

[0259] Subsequently, DIO mice were treated with ricolinostat (50 mg / kg, i.p.) for 3 weeks. Daily body weight (g), daily food intake (g), serum leptin (ng / ml) levels after 3 weeks of vehicle (n = 8) or ricolinostat (n = 8) treatment; Lean mass (g), fat mass, and fat percentage (%) measured by DEXA scan after 3 weeks of vehicle (n = 16) or ricolinostat (n = 17) treatment were determined.

[0260] Blood glucose (mg / dl) levels were measured 1 week after treatment of DIO mice with vehicle (n = 16) or ricolinostat (n = 16). GTT was evaluated 1 week after treatment of DIO mice with vehicle (n = 17) or ricolinostat (n = 16). AOC analysis was performed on the GTT. ITT was measured 2 weeks after treatment of DIO mice with vehicle (n = 17) or ricolinostat (n = 16). Serum insulin (ng / ml) levels were measured in DIO mice 3 weeks after treatment with vehicle (n = 8) or ricolinostat (n = 8). Liver sections from DIO mice treated with vehicle or ricolinostat for 3 weeks were stained with H&E.

[0261] Results

[0262] HDAC6 directly interacts with LepRb and reduces its activity. The HDAC6 inhibitor ACY775 blocks this interaction. LepRb is expressed in the central nervous system, and the anorectic and weight-lowering effects of leptin are mainly mediated by this isoform of LepR.

[0263] These results in Example 7 indicate that the anti-obesity effect of Tubastatin is not mediated by HDAC6. In vitro and in vivo data suggest a possible central role for HDAC6. If this hypothesis is correct, then highly specific HDAC6 inhibitors with low blood-brain barrier (BBB) permeability would be ineffective in increasing leptin sensitivity and reducing body weight and food intake in DIO obese mice. One such agent is Ricolinostat. The brain / plasma ratio of ACY775 is approximately 1.26, the brain / plasma ratio of ACY738 is approximately 1.22, but the brain / plasma ratio of Ricolinostat is 0.01, which has poor BBB permeability.

[0264] The ability of Ricolinostat to increase tubulin acetylation in cells was tested to confirm its HDAC6 inhibitor activity. After confirming the HDAC6 inhibitory activity of Ricolinostat, whether Ricolinostat could increase leptin sensitivity in DIO mice was investigated. The same experimental protocol was used for ACY738 and ACY775.

[0265] Figures 8A - 8O The results shown indicate that the poorly blood-brain barrier-permeable HDAC6 inhibitor Ricolinostat is not a leptin sensitizer and anti-obesity agent.

[0266] Compared with the control group, administration of leptin to DIO mice did not change their body weight (Figure 8A). Treatment with Ricolinostat alone (25 mg / kg, i.p.) also did not reduce the body weight of DIO mice, and administration of leptin to DIO mice pre-treated with Ricolinostat also did not reduce body weight. Treatment with Ricolinostat alone or together with leptin had no effect on food intake in DIO mice ( Figure 8B ). A study of the activation of leptin receptor signaling in vivo after Ricolinostat treatment showed that injection of leptin into vehicle- or Ricolinostat-treated DIO mice did not significantly increase STAT3 Tyr705 phosphorylation ( Figure 8C ). These results indicate that, unlike ACY738 and ACY775, acute treatment of DIO mice with Ricolinostat does not increase leptin sensitivity. These results are consistent with those of DIO mice treated with ricolinostat (50 mg / kg, i.p.) for 3 weeks, as Figures 8D - 8O shown.

[0267] Ricolinostat was administered chronically to three different groups. The first group received (25 mg / kg, i.p., once daily), and the second and third groups received 50 mg / kg of Ricolinostat once daily for three weeks. Administration of Ricolinostat (50 mg / kg, i.p., once daily) resulted in a slight decrease in body weight in the first few days, but did not produce any meaningful or significant decrease during the three-week treatment period ( Figure 8D ). Food intake was temporarily reduced in the first 2 - 3 days of treatment, but then rebounded and did not change during the course of treatment ( Figure 8E , 8F ). Consistent with the body weight or food intake that did not change during chronic Ricolinostat treatment, the serum leptin levels between vehicle- and Ricolinostat-treated mice ( Figure 8G)、 Lean meat quantity ( Figure 8H )、 Fat quantity ( Figure 8I )、 Fat percentage ( Figure 8J ) showed no difference.

[0268] Ricolinostat treatment did not reduce blood glucose levels ( Figure 8L ), did not improve glucose tolerance ( Figure 8M ) or insulin sensitivity ( Figure 8N ), and did not reduce insulin levels ( Figure 8O ). In addition, there was no difference in H&E staining of the liver, and hepatic steatosis was not reduced.

[0269] Therefore, the results indicate that Ricolinostat does not reduce body weight, thus supporting the premise that HDAC6 inhibitors must penetrate into the hypothalamus to be effective for body weight and glucose control.

[0270] Example 9: ACY257 is as effective as ACY775

[0271] Materials and Methods

[0272] A study was conducted to compare the efficacy of another HDAC6 inhibitor, ACY257, with ACY775 in reducing body weight.

[0273] The study was conducted as described above with reference to ACY775.

[0274] Results

[0275] Figure 9A And 9B are the comparisons of body weight change over time (2P) and percentage of body weight change over time of ACY775 and ACY257 with the vehicle.

[0276] The results indicate that ACY257 is as effective as ACY775 in reducing body weight and provide further evidence that only HDAC6 inhibitors that penetrate into the hypothalamus and inhibit HDAC6 therein are effective for treating obesity, especially leptin-resistant obesity. The data show that ACY257, which inhibits HDAC6 in the hypothalamus but not in other brain regions (such as the cerebrum, frontal lobe, temporal lobe, and brainstem), significantly reduces body weight. The hypothalamus is a very small part of the brain. Compared with other parts of the brain, the hypothalamus has a slightly more permeable blood-brain barrier. The data show that inhibitors of HDAC6 with the ability to preferentially cross the BBB and enter the hypothalamic region (especially the arcuate nucleus) can reduce body weight.

[0277] Example 10: HDAC6 in AgRP-expressing neurons is a direct target and mediator of HDAC6 inhibitors, and DMH neurons are required for the appetite and weight loss effects of HDAC6 inhibitors in DIO mice.

[0278] Materials and Methods

[0279] HEK293 cells were transfected with a plasmid expressing mouse HDAC6 cDNA with a myc tag at the C-terminus and Cas9, infected with AAV-sgHDAC6, and then harvested. Protein blotting was performed using an antibody against myc or tubulin.

[0280] An AAV viral vector carrying a guide RNA targeting the mouse HDAC6 genomic locus was designed, validated, and stereotaxically injected. Three designed sgRNAs were concatenated and constructed into an AAV vector carrying a cre-activated mCherry fluorescent marker.

[0281] After bilateral injection of AAV-sgHDAC6 into the arcuate nucleus of Agrp-ires-cre::LSL-Cas9-GFP (Agrp-Cre + / - ::Cas9 + / - ) or control LSL-Cas9-GFP (Cas9 + / - ) mice, immunostaining was performed to detect the expression of mCherry and GFP. Agrp-Cre + / - ::Cas9 + / - (HDAC6 Agrp-△ ) and Cas9 + / - (HDAC6 WT ) DIO mice (n = 7 per group) were fasted for 24 hours and injected with ACY775 (10 mg / kg, i.p.). After ACY775 injection, the total dark cycle food intake (g) per hour of these mice was measured (n = 7 per group). During 3 weeks of treatment with ACY775 (10 mg / kg / day, i.p.), the body weights of AAV-sg-HDAC6-injected HDAC6 Agrp-△ (n = 11) and HDAC6 WT (n = 6) mice were measured. During 3 weeks of treatment with ACY775 (10 mg / kg / day, i.p.), the daily average food intake of HDAC6 Agrp-△ (n = 11) and HDAC6 WT (n = 6) mice was measured. The daily average food intake of HDAC6 Agrp-△ and HDAC6 WT was evaluated during the 3-week ACY775 treatment period.24-hour average food intake of mice. Serum leptin levels (ng / ml) of HDAC6 Agrp-△ and HDAC6 WT mice (HDAC6 WT with n = 13 and HDAC6 Agrp-△ with n = 18) were measured. Lean mass (g), fat mass, and fat percentage (%) of HDAC6 Agrp-△ and HDAC6 WT DIO mice were measured by DEXA scan 3 weeks after ACY775 treatment. HDAC6-responsive neurons in the dorsomedial hypothalamus (DMH) were evaluated. Immunostaining and quantification of c-Fos expression were performed in the DMH of DIO mice after a single ACY775 treatment (10 mg / kg, i.p.).

[0282] Coordinates for injecting aav-sg-HDAC6 into the DMH of c-Fos-iCreER::LSL-Cas9-GFP (TRAP2-Cas9) mice were evaluated, and mCherry immunostaining in TRAP2-cas9 obese mice injected with aav-sg-HDAC6, which were treated with a single dose of ACY775 (10 mg / kg, i.p.) three weeks ago (TRAP2-HDAC6 DMH-△ ) or vehicle (TRAP2-HDAC6 WT ), followed by 4-hydroxytamoxifen (4-OHT) injection. TRAP2-HDAC6 WT and TRAP2-HDAC6 DMH-△ mice were fasted for 24 h and treated with ACY775 (10 mg / kg, i.p.). Hourly and total dark cycle food intake (g) of these mice were determined after ACY775 injection (n = 4 per group). Daily body weight of TRAP2-HDAC6 DMH-△ (n = 10) or TRAP2-HDAC6 WT (n = 11) DIO mice was measured during 2 weeks of ACY775 treatment (10 mg / kg / day, i.p.). Daily average food intake of TRAP2-HDAC6 DMH-△ (n = 10) or TRAP2-HDAC6 WT (n = 11) DIO mice was measured during 2 weeks of ACY775 treatment.

[0283] Results

[0284] Results showed that HDAC6 in AgRP-expressing neurons is a direct target and mediator of HDAC6 inhibitors (including ACY775, ACY738, and ACY257) that are effective in body weight and blood glucose control. DMH neurons are necessary for the appetite- and body weight-lowering effects of HDAC6 inhibitors in DIO mice.

[0285] Neurons expressing AgRP in the ARC of the hypothalamus (AgRP ARC neurons) have been shown to be one of the major neuronal populations in the brain that mediate the effects of leptin on feeding, body weight, and energy balance. Considering that fasting increases the expression of HDAC6 in AgRP ARC neurons, and that HDAC6 binds to LepRb and reduces its activity, and considering that HDAC6 inhibitors with low BBB permeability cannot increase leptin sensitivity and reduce body weight, it was hypothesized that HDAC6 in AgRP ARC neurons mediates the effects of HDAC6 inhibitors on appetite and body weight. To investigate this, three single-guide RNAs (sgRNAs) were tandemly designed to target three different exons of the mouse HDAC6 locus, and their efficacy was verified in cultured HEK293 cells in vitro. These three sgRNAs were constructed into an adeno-associated viral vector with a cre-dependent mCherry reporter gene (AAV-sgHDAC6) to indicate virus-transduced neurons. To evaluate the functional relevance of HDAC6 in AgRP ARC neurons, AAV-sgHDAC6 was bilaterally injected into the ARC of LSL-Cas9-GFP and Agrp-IRES-cre::LSL-Cas9-GFP DIO mice, and widespread co-localization of cre-activated mCherry with GFP immunoreactivity was observed in the ARC of Agrp-IRES-cre::LSL-Cas9-GFP (HDAC6 Agrp-△ ) mice, but not in LSL-Cas9-GFP (HDAC6 WT ) control mice.

[0286] HDAC6 WT and HDAC6 Agrp-△ DIO mice were treated with a single dose of (ACY775, 10 mg / kg, i.p.). It was found that HDAC6 Agrp-△ DIO mice showed almost complete resistance to the inhibition of food intake induced by ACY755. These results prompted long-term ACY775 treatment to observe whether all the anti-obesity effects of ACY775 are mediated by HDAC6 in AgRP neurons. HDAC6 Agrp-△ and HDAC6 WTDIO mice were treated with ACY775 (10 mg / kg / day, i.p.) for three weeks.

[0287] The effects of ACY775 on all parameters were significantly attenuated, including weight loss, long-term appetite, leptin levels, obesity, and glucose homeostasis. These data strongly suggest that HDAC6 in AgRP ARC neurons rather than in peripheral tissues is the main target of HDAC6 inhibitors for their anti-obesity effects. Similarly, deletion of HDAC6 in AgRP ARC neurons did not alter the effects of Tubastatin A on suppressing food intake or body weight.

[0288] To investigate whether the activity of other neuronal populations is altered after HDAC6 inhibitor treatment, c-fos staining was performed in the whole brains of DIO mice acutely treated with ACY775. Under fasting conditions, the number of c-fos positive cells increased significantly in the dorsomedial hypothalamic nucleus (DMH) of ACY775-treated DIO mice. Some DMH neurons express high levels of LepRb and are involved in appetite regulation (reference). Then it was tested whether HDAC6 in these ACY775-responsive DMH neurons is required or in any way involved in mediating the appetite-suppressing and body weight-lowering effects of ACY775. To deplete HDAC6 in these ACY775-responsive DMH neurons, c-fos-icreER (TRAP2) was crossed with the LSL-cas9-GFP line to generate c-fos-icreER::LSL-cas9-GFP (TRAP2-cas9) mice, and obesity was induced by 20 weeks of HFD feeding. Subsequently, aav-sgHDAC6 was bilaterally injected into the DMH of TRAP2-cas9 DIO mice, followed by a single dose of ACY775 (or vehicle, as a control) and 4-hydroxytamoxifen (4-OHT) injection to specifically allow sgHDAC6 to deplete HDAC6 only in ACY775-induced and c-fos cre-activated DMH neurons (TRAP2-HDAC6 DMH-△ ).

[0289] Twenty-four-hour fasted TRAP2-HDAC6 DMH-△ and TRAP2-HDAC6 WT DIO mice were treated with a second dose of ACY775, and it was found that mice from both groups consumed similar but small amounts of food. In addition, during the two-week period of ACY775 treatment, TRAP2-HDAC6 DMH-△ and TRAP2-HDAC6 WTThe mice in the group lost weight, and the food intake of both groups decreased equally. These results suggest that HDAC6 in DMH-responsive neurons is not essential for the appetite and weight loss effects of HDAC6 inhibitors.

[0290] The activity of ACY775-responsive DMH neurons, which were investigated as possible second-order neurons of AgRP ARC neurons, was examined to determine whether it might be involved in the food intake and weight loss activities of HDAC6 inhibitors. These neurons were ablated to study their necessity in mediating the anti-obesity effects of ACY775. AAV-flex-taCasp3-TEVp was injected into the DMH of TRAP2DIO mice, and then the mice were injected with a single dose of ACY775 and 4-OHT to ablate these neurons through caspase 3 protein activation. To ensure the effectiveness of this experimental system, the presence of ACY775-responsive DMH neurons was verified by c-fos staining after the injection of the second dose of ACY775. This experimental approach predicted that when caspase 3 was activated by administering the first dose of ACY775 in combination with 4-OHT injection, it would result in the ablation of ACY775-responsive DMH neurons and attenuate the c-fos signal that would otherwise be generated by the second dose of ACY775.

[0291] The results showed that in ACY775- and 4-OHT-injected TRAP2 DIO mice (caspase 3 on-DMH ), the ACY775-stimulated increase in c-fos-positive cells in the DMH was almost completely abolished. However, in vehicle- and 4-OHT-injected TRAP2DIO mice (caspase 3 off-DMH ), c-fos-positive DMH neurons were still detectable after ACY755 treatment. These results confirmed that ACY755-responsive DMH neurons were successfully ablated in caspase 3 on-DMH mice. After confirming the ablation, fasted caspase 3 on-DMH and caspase 3 off-DMH mice were treated with ACY775 (10 mg / kg, i.p.), and the results showed that the food intake measured hourly or during the entire dark cycle in caspase 3 on-DMH mice was significantly higher than that in caspase 3 off-DMH mice.

[0292] To rule out the possibility that caspase 3 on-DMH mice might have a general problem in hunger perception, caspase 3 on-DMH and caspase 3 off-DMHMice were refed after an overnight fast, and it was confirmed that mice in all groups consumed similar levels of food. Then, caspase 3 on-DMH and caspase 3 off-DMH mice were treated chronically with ACY775 (10 mg / kg / day, i.p.), and it was found that ACY775 treatment neither reduced body weight nor food consumption in caspase 3 on-DMH mice in which ACY775-responsive DMH neurons were ablated. In caspase 3 off-DMH mice, ACY775 treatment did reduce food intake and body weight. Consistently, when compared to caspase 3 off-DMH mice, the blood glucose levels were significantly higher in caspase 3 on-DMH mice. These data suggest that ACY775-responsive DMH neurons are required for mediating the appetite and weight loss effects of ACY775 in DIO mice.

[0293] Next, chemogenetic activation of ACY775-responsive DMH neurons was performed to investigate whether activation of these neurons was sufficient to reduce food intake and body weight. An adeno-associated virus (AAV) carrying Cre-dependent hM3Dq-DREADD fused to the mCherry transgene (AAV-DIO-hM3Dq-mCherry) was bilaterally injected into the DMH of TRAP2 DIO mice. Four weeks after injection, a single dose of ACY775 (10 mg / kg, i.p.) or vehicle was injected to induce c-fos expression. Subsequently, in fasted DIO mice, 4-OHT was injected 5 h later to activate c-fos-driven Cre. mCherry signal was expressed only in ACY775-treated TRAP2 DIO mice and not in vehicle-treated TRAP2 DIO mice.

[0294] Next, AAV-DIO-hM3Dq-mCherry-injected, ACY775-injected, and vehicle-treated TRAP2DIO mice were treated with saline for 3 days and with clozapine N-oxide (CNO) for 4 days. During saline treatment, body weight and food intake were similar in all groups and showed no significant differences between groups. However, mice from the ACY775-induced group showed a significant decrease in body weight and food intake during CNO treatment, while this was not the case for vehicle-induced TRAP2 DIO mice. Blood glucose levels were also measured at the end of saline and CNO treatments. The results showed that CNO treatment significantly reduced blood glucose levels only in AAV-DIO-hM3Dq-mCherry-injected and ACY775-induced TRAP2 DIO mice. The blood glucose-lowering effect of CNO was absent compared to vehicle-induced TRAP2 DIO mice and all saline-treated mice. In summary, the data suggest that ACY775-responsive DMH neurons are necessary and sufficient to mediate the appetite and body weight-lowering effects of ACY775 in DIO mice.

[0295] Example 11: Bavarostat as an HDAC6 inhibitor in the hypothalamus

[0296] Materials and Methods

[0297] Bavarostat (4-(((((3r,5r,7r)-adamantan-1-yl)methyl)(methyl)amino)methyl)-3-fluoro-N-hydroxybenzamide) is a selective inhibitor of HDAC6 with high brain permeability, Strebl, et al. ACS Cent Sci. 2017 Sep 27; 3(9): 1006–1014.

[0298] DIO mice were injected intraperitoneally with bavarostat (10 mg / kg), and 30 minutes later, protein from liver and hypothalamus tissues was immunoblotted for Ac-tubulin to study the activity of the HDAC6 inhibitor in the liver and hypothalamus.

[0299] Immunoblotting of Ac-tubulin in liver and hypothalamus tissues from bavarostat-treated DIO mice was performed. The ratio of Ac-tubulin to total tubulin signal in immunoblots of liver and brain was quantified.

[0300] Subsequently, DIO mice were treated with bavarostat (10 mg / kg, i.p.) for 3 weeks, and the daily body weight (g) of DIO mice treated with vehicle (n = 9) or bavarostat (n = 9) was measured during the 3-week treatment period. The daily food intake (g) was also measured during the 3-week vehicle or bavarostat treatment.

[0301] Results

[0302] Figures 10A - 10F It was shown that treatment of wild-type mice but not HDAC6 knockout mice with bavarostat resulted in weight loss in obese mice. Appetite was initially suppressed but then increased over time.

[0303] Example 12: Citarinostat is ineffective as an HDAC6 inhibitor in the hypothalamus

[0304] Citarinostat is a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor with known pharmacological and ADMET properties and is able to improve performance during memory in a mouse model of FXS, Fmr1− / − mice. This small molecule exhibits good brain permeability by inhibiting the α-tubulin deacetylase domain (CD2) of HDAC6, inhibits HDAC6 with low nanomolar potency (IC50 = 2.3 nM), and has at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms Kozikowski, et al. ACS Chem Neurosci. 2019 Mar 20; 10(3): 1679–1695。C Citarinostat (ACY-241, HDAC-IN-2) is an orally available selective HDAC6 inhibitor with IC50 values of 2.6 nM and 46 nM for HDAC6 and HDAC3, respectively. It has 13- to 18-fold selectivity for HDAC6 compared to HDAC1-3.

[0305] Materials and Methods

[0306] Citarinostat (25 mg / kg) was intraperitoneally injected into DIO mice for 30 minutes. Protein from liver and brain (hypothalamus) tissues was immunoblotted for Ac-tubulin to study the activity of the HDAC6 inhibitor in the liver and brain (hypothalamus). Immunoblotting of Ac-tubulin in liver and brain (hypothalamus) tissues from citarinostat-treated DIO mice. Quantification of the ratio of Ac-tubulin to total tubulin signal in immunoblots of the liver and brain was evaluated in DIO mice treated with citarinostat (25 mg / kg, i.p.) for 3 weeks. The daily body weight (g) of DIO mice treated with vehicle (n = 12) or citarinostat (n = 11) was also measured during the 3-week treatment period. The daily body weight change (%) of DIO mice during the 3-week treatment period, and the daily food intake (g) and weekly average 24-h food intake during vehicle or citarinostat treatment.

[0307] Results

[0308] As Figures 11A - 11F The results shown indicate that only an HDAC6 inhibitor with inhibitory effects in the hypothalamus can be used for obesity treatment. Citarinostat inhibits HDAC6 in other brain regions (such as the cerebrum, frontal lobe, temporal lobe, brainstem, etc.), but not HDAC6 in the hypothalamus, so it cannot effectively reduce body weight.

[0309] Example 13: Administration of ACY1083 is effective in restoring leptin sensitivity, as evaluated by weight loss.

[0310] Materials and Methods

[0311] ACY1083 was administered to DIO mice as described in Example 3.

[0312] Results

[0313] The results are as Figure 12 shown. ACY1083 caused a significant decrease in body weight within 14 days.

[0314] Summary and Conclusions

[0315] Figure 13A diagram of the signaling pathway of leptin and its downstream effectors. Obr oligomerization (only dimerization is shown here for clarity) leads to phosphorylation and activation of the cytoplasmic associated jak2 kinase. These activated jaks phosphorylate tyrosine residues in the cytoplasmic tail of the receptor. Recruitment and activation of secondary signaling molecules allow obr to signal through the jak / stat, mapk, pi3k, ampk, and mtor pathways. Figure from Wauman, et al Front. Endocrinol., Sec. Molecular and Structural Endocrinology Vol. 8 (2017) https: / / doi.org / 10.3389 / fendo.2017.00030

[0316] Data in the examples show that HDAC6 inhibitors with high BBB, especially hypothalamic permeability levels, especially inhibitors that block the LepRb-HDAC6 interaction in AgRP neurons in the hypothalamus are effective in treating leptin-resistant obesity and increase leptin sensitivity. The key brain region where the inhibitor acts is the hypothalamus, where HDAC6 in arcuate neurons must be inhibited. Several compounds were tested and a central rather than peripheral mechanism of action was determined to be associated with HDAC6 inhibitors with high permeability levels that cross the blood-brain barrier and enter the hypothalamus. Analysis of hypothalamic gene expression in fed, starved, and obese mice determined a correlation with gene expression changes induced by small molecule inhibition of HDAC6 activity, establishing that pharmacological inhibition of HDAC6 activity in the hypothalamus is effective for treating obesity and related disorders. Subsequently, studies in high-fat diet-induced mice treated with the HDAC6 isoform-specific inhibitors ACY738, ACY775, ACY257, and ACY1083, and bavrostat showed that these compounds led to significant reductions in food intake and total body fat mass. The more selective HDAC6 inhibitor ACY775 had a greater effect compared to ACY738. Significant weight loss was demonstrated by ACY1083, bavrostat, and the compound ACY257 that inhibits HDAC6 in the hypothalamus but has low BBB permeability.

[0317] Only HDAC6 inhibitors that can penetrate the hypothalamus and inhibit HDAC6 therein are effective in treating obesity, especially leptin-resistant obesity. Data show that ACY257, which inhibits HDAC6 in the hypothalamus but not in other brain regions (such as the cerebrum, frontal lobe, temporal lobe, and brainstem), significantly reduces body weight. The hypothalamus is a very small part of the brain. Compared with other parts of the brain, the hypothalamus has a slightly higher permeability blood-brain barrier. Data show that HDAC6 inhibitors with the ability to preferentially pass through the BBB and enter the hypothalamic region, especially into the arcuate nucleus, can reduce body weight.

[0318] Based on these studies, small molecule (less than 1000 Da) selective inhibitors of HDAC6 with high permeability through the BBB and hypothalamus having a brain / plasma ratio greater than 1 (inhibitors that cause acetylation of α-tubulin without affecting histone acetylation), especially inhibitors that block the LepRb-HDAC6 interaction, are most effective in treating obesity (including leptin-resistant obesity) by a central nervous system (CNS) mechanism of action. Preferred HDAC6 inhibitors have a brain or hypothalamus / plasma concentration > 0.25, > 0.5, or > 1. More preferably, the HDAC6 inhibitor has a hypothalamus / plasma concentration > 0.25, > 0.5, or > 1. Most preferably, the HDAC6 inhibitor has an arcuate nucleus / plasma concentration > 0.25, > 0.5, or > 1. The arcuate nucleus has the highest BBB permeability in the hypothalamus and is about 1 / 10,000 of the total brain area. Results show that inhibiting HDAC6 in AgRP neurons in the arcuate nucleus is sufficient to produce weight loss. If the HDAC6 inhibitor reaches the arcuate nucleus only at a concentration capable of inhibiting HDAC6 activity and cannot reach other regions of the brain, the total brain / plasma or hypothalamus / plasma concentration may be low. However, the arcuate nucleus concentration will be higher than the total brain / plasma and hypothalamus / plasma concentrations.

[0319] Preferred compounds are ACY775 (brain / plasma ratio of 1.26) and ACY738 (brain / plasma ratio of 1.22), ACY257 with high permeability to the hypothalamic arcuate nucleus, ACY1083, and bavarostat with high permeability to the hypothalamus. Peripherally acting HDAC6 inhibitors are ineffective, such as ricolinostat (ACY1215) with a brain / plasma ratio of 0.01 and tubastatin A with a brain / plasma ratio of 0.18, which do not have high permeability to the hypothalamus, and citarinostat (ACY241) is also ineffective as it does not show HDAC6 inhibition in AGP neurons in the hypothalamus. HDAC6 inhibition was tested by treating cells with a high concentration of the HDAC6 inhibitor and analyzing tubulin acetylation (an established biomarker of HDAC6 inhibition).

[0320] Figures 14A - 14C Schematic diagram of the site where the compounds disclosed therein inhibit or disrupt HDAC6 or leptin receptor binding. Figure 13 A is a schematic diagram of leptin binding to LepR, leading to phosphorylation of its tyrosine by Jak2 and subsequent phosphorylation and activation of Stat3. Stat3 plays a crucial role in mediating the effects of leptin in suppressing appetite and reducing body weight. Figure 13 B is a schematic diagram of the interaction between Hdac6 and LepRb during fasting or obesity, which reduces LepR and thus Stat3 activation. This results in inhibition of LepR signaling, increased appetite, and weight gain. Figure 13 C is a schematic diagram of an Hdac6 inhibitor that, by blocking the binding of Hdac6 to LepR, increases LepR activity, suppresses appetite, increases energy expenditure, and leads to weight loss.

[0321] Selective HDAC6 inhibitors are preferably administered to mucosal surfaces, most preferably orally, buccally, or nasally. These can be formulated using known excipients. The formulations can also be formulated for sustained, delayed, and / or pulsatile release to deliver an effective amount of the HDAC6 inhibitor to cause weight loss. Preferably, it is administered once or twice a day. The dose is weight-based. Typical doses will be in the range of 25 to 500 mg / day.

[0322] A pharmaceutical formulation can be administered to induce weight loss in pre-obese, obese, or morbidly obese patients, reduce body fat in pre-obese, obese, or morbidly obese patients, reduce food intake in pre-obese, obese, or morbidly obese patients, improve glucose homeostasis in pre-obese, obese, or morbidly obese patients, or a combination thereof. In some cases, a pharmaceutical formulation containing one or more HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients in a therapeutically effective amount to induce weight loss, preferably in a therapeutically effective amount and for a duration of time to reduce body mass or body fat by at least 10%, more preferably at least 15%, most preferably at least 20% or more. In some cases, a pharmaceutical formulation containing one or more HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients in a therapeutically effective amount to reduce food intake, appetite, or a combination thereof, preferably in a therapeutically effective amount to reduce the average daily food intake (in calories). In some cases, a pharmaceutical formulation containing one or more selective HDAC6 inhibitors is administered to pre-obese, obese, or morbidly obese patients in a therapeutically effective amount to improve glucose homeostasis, preferably in a therapeutically effective amount to reduce the average fasting plasma glucose. In cases where the pharmaceutical formulation is administered to normalize blood glucose, the formulation is preferably administered in an amount effective to reduce the blood glucose level to less than about 180 mg / dL. If needed, the formulation can be co-administered with other anti-diabetic therapies to improve glucose homeostasis.

Claims

1. A preparation for inducing weight loss in obese individuals, comprising a selective HDAC6 inhibitor in a pharmaceutically acceptable excipient for administration to a mucosal surface or for injection, the selective HDAC6 inhibitor having a molecular weight of 1000 Da or less and producing a hypothalamus / plasma ratio greater than 0.25, more preferably greater than 0.5, most preferably greater than 1, and inhibiting HDAC6 in arcuate AgRP neurons, wherein the selective HDAC6 inhibitor is in an effective amount to cause weight loss in an individual having a body mass index of 25 or higher.

2. The preparation according to claim 1, wherein the selective HDAC6 inhibitor blocks the LepRb-HDAC6 interaction.

3. The preparation according to claim 1, wherein the selective HDAC6 inhibitor having high blood-brain barrier or blood-hypothalamus barrier permeability is selected from ACY775, ACY738, ACY257, ACY1083, Bavarostat, their prodrugs, analogs, and derivatives.

4. The preparation according to claim 3, wherein the HDAC6 inhibitor is selected from ACY775, ACY738, ACY257, ACY1083, and Bavarostat.

5. The preparation according to any one of claims 1-4, wherein the HDAC6 inhibitor penetrates the hypothalamus and induces weight loss in obese individuals but not in lean individuals.

6. The preparation according to claim 1, which is formulated for oral, buccal, nasal, pulmonary, vaginal, or rectal administration.

7. The preparation according to any one of claims 1-6, which is for administration by injection into a tissue.

8. The preparation according to any one of claims 1-7, which provides a controlled release of the selective HDAC6 inhibitor.

9. The preparation according to any one of claims 1-8, which provides a dosage unit containing a therapeutically effective amount of one or more of the HDAC6 inhibitors, when administered to a pre-obese, obese, or morbidly obese patient, which induces weight loss, preferably reducing body mass or body fat by at least 10%, more preferably at least 15%, most preferably at least 20% or more, in terms of a therapeutically effective amount and administration time, which reduces food intake, appetite, or a combination thereof, preferably reducing the average daily food intake (in calories) in terms of a therapeutically effective amount, or which improves glucose homeostasis, preferably reducing the average fasting plasma glucose in terms of a therapeutically effective amount.

10. The preparation according to claim 9, which provides an effective dose to cause weight loss in an individual having a body mass index of 25 or higher when administered once daily.

11. A method for causing weight loss in an individual having a body mass index of 25 or higher, comprising administering to an obese individual a preparation for inducing weight loss, the preparation comprising a selective HDAC6 inhibitor in a pharmaceutically acceptable excipient for administration to a mucosal surface or for injection, the selective HDAC6 inhibitor having a molecular weight of 1000 Da or less and producing a hypothalamus / plasma ratio greater than 0.25, more preferably greater than 0.5, most preferably greater than 1, and inhibiting HDAC6 in arcuate AgRP neurons, wherein the selective HDAC6 inhibitor is in an effective amount to cause weight loss in an individual having a body mass index of 25 or higher.

12. The method of claim 11, wherein the selective HDAC6 inhibitor blocks the LepRb-HDAC6 interaction.

13. The method of claim 11, wherein the selective HDAC6 inhibitor having high blood-brain barrier or blood-hypothalamus barrier permeability is selected from ACY775, ACY738, ACY1083, ACY257, Bavarostat, their prodrugs, analogs, and derivatives.

14. The method of any one of claims 11-13, wherein the HDAC6 inhibitor induces weight loss in an obese individual but not in a lean individual.

15. The method of any one of claims 11-14, which is formulated for oral, buccal, nasal, pulmonary, vaginal, or rectal administration.

16. The method of any one of claims 11-14, which comprises administering the HDAC6 inhibitor by injection into a tissue.

17. The method of any one of claims 11-16, wherein the individual has leptin resistance.

18. The method of claim 11, which comprises administering a second agent to improve or maintain whole-body glucose homeostasis.

19. The method of claim 11, wherein the individual has diabetes.

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