WO2018191594A1

WO2018191594A1 - Methods for regulating adipocytes and treating conditions associated with excessive adipose tissue

Info

Publication number: WO2018191594A1
Application number: PCT/US2018/027454
Authority: WO
Inventors: Ching-Hsuan Tung; Myung Shin HAN
Original assignee: Cornell University
Current assignee: Cornell University
Priority date: 2017-04-13
Filing date: 2018-04-13
Publication date: 2018-10-18
Anticipated expiration: 2019-10-13

Abstract

A method for treating a condition characterized by excessive adipose tissue in a subject, the method comprising administering to said subject a compound that inhibits adipogenesis and/or induces adipocyte apoptosis in said excessive adipose tissue, said compound having the structure : (1) wherein: X¹, X², X³, and X⁴ are each independently selected from iodine and bromine atoms; X⁵, X⁶, X⁷, and X⁸ are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms; Y¹ is an -O-, -NR'-, or -CR'₂- linker, wherein R' is independently selected from hydrogen atom and methyl; Z is a hydrocarbon linking group containing 1-12 carbon atoms; R¹ is selected from hydrogen atom, methyl, -OH, and -OR groups, wherein R is an alkyl group containing one to three carbon atoms; and wherein said Formula (1) includes pharmaceutically acceptable salts and solvates of the compounds embraced by Formula (1).

Description

METHODS FOR REGULATING ADIPOCYTES AND TREATING CONDITIONS ASSOCIATED WITH EXCESSIVE ADIPOSE TISSUE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/484,954, filed April 13, 2017, which is incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. 1R01GM094880 awarded by the National Institutes of Health (NIH) and National Institute of General Medical Sciences (NIGMS). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] This invention generally relates to methods for treating conditions characterized by or associated with excessive adipose tissue, such as obesity, double chin, or cellulite. The invention more particularly relates to methods of inhibiting adipogenesis and/or inducing adipocyte apoptosis.

BACKGROUND OF THE INVENTION

[0004] Control over the unhealthy expansion of adipose tissue would provide an important advancement in the management of obesity and its associated diseases. Current anti-obesity treatments are able to change the size of the adipocytes, which results in some weight loss, although total cell number remains the same (Kusminski, C. M. et al., Nat Rev Drug Discov, 15, 639-60. (2016); Bays, H. et al., Nat Rev Drug Discov 5, 919-931, (2006); Spalding, K. L. et al., Nature 453, 783-787, (2008)). A major drawback of current methods is that, once the treatment has ended, the contracted adipocytes tend to expand and regain their original size.

[0005] The term "adipocytes" refers to terminally differentiated fat cells, which originate from committed pre- adipocytes. The term "adipogenesis" refers to the process of maturation of pre-adipocytes into adipocytes. Blocking adipogenesis has been proposed to stop the differentiation of pre-adipocytes into adipocytes, presenting one possible way to cull the number of adult fat cells in the body. Another method to achieve that result is with reagents that can push mature adipocytes into apoptosis or adipolysis, both terms referring to programmed cell death.

[0006] As is well known, obesity is characterized by an abnormally excessive amount of fatty issue in a subject. Obesity has become a major health concern in the United States and globally. According to the National Institute of Diabetes and Digestive and Kidney Diseases, more than one-third of U.S. adults suffer from obesity, which increases their chances of developing many chronic diseases and metabolic disorders, including diabetes (Kusminski, C. M. et al. Nat Rev Drug Discov, 15, 639-60. (2016)), heart disease, atherosclerosis (Bays, H. E. Am J Med 122, S26-37, (2009); Poledne, R. et al. Physiol Res 64, S395-402, (2015)), and cancer (Lazar, I. et al. Cancer Res 76, 4051-4057, (2016); Hefetz-Sela, S. & Scherer, P. E. Pharmacol Ther 138, 197-210, (2013)). Obesity as an epidemic also poses a significant financial burden on the healthcare system. The Center for Disease Control and Prevention estimates the annual medical cost of obesity as $147 billion in 2008 in U.S. dollars

(Finkelstein et al., Health Affairs, 28(5), September/October 2009). The same article also reports that the medical costs for people who are obese was reported to be significantly higher than those of normal weight.

[0007] Under normal metabolic conditions, adipocytes have an average lifespan of 10 years (Prins, J. B. & O'Rahilly, S. Clin Sci 92, 3-11, (1997)). Only 10% of all adipocytes undergo a yearly renewal process, which is tightly balanced between the adipogenesis of pre- adipocytes and the apoptosis or cell death of adipocytes. However, recent studies have suggested that a prolonged obesity period may cause the body to recruit pre-adipocytes and stimulate their differentiation into mature adipocytes; thus, increasing the number of total adipocytes (Wang, Q. A., et al. Nat Med 19, 1338-1344, (2013); Tchoukalova, Y. D. et al. Proc Natl Acad Sci U S A 107, 18226-18231, (2010)). As adipocyte numbers increase, their size simultaneously becomes enlarged with the continuous deposit of fat (Rutkowski, J. M. et al. P. E. / Cell Biol 208, 501-512, (2015)). Over-expansion of adipose tissue results in severely dysfunctional adipocytes that secrete adipokines and cytokines, such as leptin and adiponectin, and cause alterations to normal metabolism (Lehr, S. et al. Proteomics Clin Appl 6, 91-101, (2012)).

[0008] Currently-available methods for decreasing fat content of the body and managing weight are focused on dietary restrictions coupled with continuous exercise. However, these approaches have, in many instances, shown to be largely ineffective in the long run. Few people are able to adhere to strict dietary programs and calorie counting in addition to physically demanding routines. Thus, there is an urgent need for more effective anti-obesity therapies that target adipocyte regulation. A method that reduces the number of total adipocytes rather than their size would be an alternative strategy in regulating the expansion of unhealthy fat tissue and achieving sustained weight management. However, such an approach has achieved only limited success according to methods of the conventional art.

[0009] In 2015, sodium deoxycholate (SD), a compound that triggers cell death, was approved by the FDA to reduce the unwanted submental fat (Humphrey, S. et al. J Am Acad Dermatol, (2016); Wollina, U. & Goldman, A. Expert Opin Pharmacother 16, 755-762, (2015); Dayan, S. H. et al. Dermatol Surg 42, S263-S270, (2016)). In the process, SD functions similarly to a detergent, causing adipolysis (or adipocytolysis) when injected directly into the area containing an excess of fatty tissue (Wollina, U. & Goldman, supra; Rotunda, A. M. Lasers Surg Med 41, 714-720, (2009)). The adipocyte's membrane, which is deficient in cell-associated proteins, is lysed by SD and results in necrosis (Dayan, supra; Rotunda, supra). The required active dose of SD is high, 2 mg/mL (~ 5 mM) and 0.2 ml/cm². Although SD is effective in reducing fatty tissues, the FDA only approved its usage for localized treatment under the chin. This restriction is due to the required high SD dose, so that it is near prohibitive for other applications. To treat submental fat, 0.2 mL of a 10 mg/mL (~ 25 mM) SD solution is typically injected per square centimeter. Although doable, this procedure will be difficult and possibly not even practical for treating a larger area.

SUMMARY OF THE INVENTION

[0010] The present disclosure is directed to methods for treating conditions characterized by excessive adipose tissue in a subject. The method advantageously targets adipocyte regulation. More specifically, the method operates by inhibiting or arresting adipogenesis and/or inducing adipocyte apoptosis. In the method, a compound that inhibits adipogenesis and/or induces adipocyte apoptosis is administered to a subject having excessive adipose tissue to result in a reduction of the excessive adipose tissue. In particular embodiments, the method involves injecting the compound into the excessive adipose tissue. The condition being treated may be associated with, for example, obesity, double chin, or cellulite.

[0011] For purposes of the present invention, the compounds that inhibit or arrest adipogenesis and/or induce adipocyte apoptosis are within the scope of the following generic formula:

[0012] In Formula (1) above, X¹, X², X³, and X⁴ are each independently selected from iodine and bromine atoms; X⁵, X⁶, X⁷, and X⁸ are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms; Y¹ is an -0-, -NR.'-, or -CRV linker, wherein R' is independently selected from hydrogen atom and methyl; Z is a hydrocarbon linking group containing 1-12 carbon atoms; R¹ is selected from hydrogen atom, methyl group, -OH, and - OR groups, wherein R is an alkyl group containing one to three carbon atoms; and wherein Formula (1) includes pharmaceutically acceptable salts, solvates, enantiomers, and physical forms of the compounds embraced by Formula (1).

[0013] In some embodiments, the compound being administered is within the scope of the following sub-generic structure:

[0014] In Formula (la) above, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, and X⁸ are defined as above under Formula (1); Y¹ is an -0-, -NR.'-, or -CRV linker, wherein R' is independently selected from hydrogen atom and methyl; R² is selected from hydrogen atom and alkyl groups containing one to three carbon atoms; and n is an integer of 1-12; and wherein Formula (la) includes pharmaceutically acceptable salts, solvates, enantiomers, and physical forms of the compounds embraced by Formula (la).

[0015] In further particular embodiments, the compound being administered has the following specific structure, also referred to herein as MI-401:

(la-1) BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1A-1B show the structure and function of the synthetic xanthene analog compound MI-401. FIG. 1A is the chemical structure of MI-401. FIG. IB is a drawing representing the dual functionality of MI-401 in fat cell regulation; MI-401 inhibits pre- adipocyte's adipogenesis, and stimulates apoptosis in adipocytes.

[0017] FIGS. 2A-2D show the effects of SD and MI-401 compounds on mature adipocytes. FIG. 2A is a schematic representation of the stages of differentiation protocol for 3T3-L1 cells. Drugs were added at day 3 after maturation, as indicated by the arrowhead, and treatments lasted for one or two days, as indicated by the thicker line. FIG. 2B shows representative images of mature 3T3-L1 cells treated with MI-401 (10 μΜ) or SD (10 or 50 μΜ). Morphological changes in 3T3-L1 cells were induced by MI-401 and SD at high concentration but not SD at low concentration. Scale bar = 100 μιη. FIG. 2C is a cell viability plot showing decreased viability of 3T3-L1 cells on day 1 and day 2 following MI- 401 (10 μΜ) treatment compared to SD (50 μΜ) treated adipocytes. Data is presented as mean + standard deviation (n > 3). FIG. 2D is a cell viability plot showing viability of cultured adipocytes treated with SD or MI-401. The determined EC5₀ for MI-401 after 1-day or 2-day of treatment was 7.9 or 5.3 μΜ, respectively; and for SD after 1-day or 2-day of treatment was 253.8 or 52.8 μΜ, respectively. Data is presented as mean + standard deviation (n > 3).

[0018] FIGS. 3A-3D elucidate the mechanism by which SD and MI-401 lead to cell death in treated adipocytes. FIG. 3A is a schematic representation of the stages of differentiation and maturation of 3T3-L1 cells. SD and MI-401 were each added at day 3 after maturation, as indicated by the arrowhead, and the drug treatments lasted two days, as indicated by the thicker line. FIG. 3B shows representative images of induction of apoptosis in mature 3T3-L1 cells treated with MI-401 (10 μΜ), but only partial solubilization of membranes with diminished lipid droplets in SD (50 μΜ) treated cells. The cell membrane was stained with CellMask™ Plasma Membrane Stain (Green), and the nucleus was stained with DAPI (Blue). The top row fluorescence images showed membrane and nucleus stains, and the bottom row bright field images showed cell morphology and lipid droplets. Scale bar = 100 μιη. FIG. 3C is a graph showing significant elevation of necrosis associated LDH release 4 hrs after SD (50 μΜ) but only a modest increase in necrosis after MI-401 (10 μΜ) treatment. Data is presented as mean + standard deviation (n = 3). (** P < 0.005, * P < 21 0.05). FIG. 3D is a graph depicting time course curves of caspase 3 and 7 activity after SD treatments (50 μΜ) or MI-401 (10 μΜ). The fluorescence signal representing caspase 3/7 activity was determined in real time using a fluorescence plate reader over 18 hours. Data is presented as mean + standard deviation (n = 3). (**** P≤ 0.001).

[0019] FIGS. 4A-4C elucidate the inhibitory effect of MI-401 during the early stage of adipogenic differentiation. FIG. 4A is a schematic representation of 3T3-L1 cells treated with 10 μΜ of MI-401 for the indicated time periods during differentiation. The cells were continuously cultured in an adipocyte maintenance medium for an additional three days post- differentiation. To highlight the accumulated lipid droplets, the cells were then stained with HCS Lipdox™ lipid stain prior to imaging. FIG. 4B clearly shows a near complete inhibition of lipogenesis in the treated cells. The top row bright field images showed cell morphology and lipid droplets, and the bottom row fluorescence images showed lipid accumulation. Scale bar = 100 μιη. Numerous lipid droplets were formed under the normal culture condition, but not with MI-401. FIG. 4C is a graph showing decreased triglyceride accumulation with MI- 401 treatment (black bars and left Y-axis) and no change in cell viability after treatments. Data is presented as mean + standard deviation (n > 3). Based on triglyceride content, the IC₅₀ of MI-401 with 1-day or 2-day treatment was 3.2 and 2.5 μΜ, respectively.

[0020] FIGS. 5A and 5B show the effect of MI-401 on adipogenic gene expression in different stages of growth. FIG. 5A is a schematic representation of the experiments. MI- 401 (10 μΜ) was added to the pre-adipocyte maintenance medium or differentiation medium, as indicated by the thicker lines. The cells were harvested immediately after the treatment. In a separated set of cells, MI-401 was added to the differentiation medium for two days, and then the cells were cultured in the adipocyte maintenance medium for an additional three days. FIG. 5B is a Western blot analysis showing the effect of MI-401 on PPARg, C/EBPa, FAS and FABP4.

[0021] FIGS. 6A-6D demonstrate cytotoxicity of MI-401 on 3T3-L1 pre- adipocytes and NIH3T3 fibroblast. FIG. 6A is a schematic representation of the experiment. After seeding, 3T3-L1 pre-adipocytes were treated with MI-401 (thick arrowhead) in a 22 pre-adipocyte maintenance media for 1 or 2 days (thicker line). NIH3T3 cells were also treated with MI- 401 but in DMEM medium with 10% FBS. FIG. 6B shows in representative images that both cell types remained unharmed and healthy when exposed to 10μΜ MI-401. However, the 3T3-L1 pre-adipocyte cells were negatively affected in a 50 μΜ concentration, while NIH3T3 cells retained their health. FIG. 6C shows a quantitative analysis of viability of NIH-3T3 fibroblast with MI-401. The EC₅₀ at day 2 was 169.2 μΜ. Data are presented as mean + standard deviation (n = 3). FIG. 6D shows a quantitative analysis of viability of 3T3- Ll pre-adipocytes with MI-401. The EC5₀ at day 2 was 48.7 μΜ. Data is presented as mean + standard deviation (n = 3).

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is directed to methods for treating conditions (e.g., diseases or disorders) characterized by excessive adipose tissue in a subject. The subject is typically human, although the method should be effective in treating mammals in general. In the method, certain xanthene-based compounds, as further described below, are administered to the subject to inhibit or arrest adipogenesis and/or to induce apoptosis of adipocytes (i.e., adipolysis) in bodily tissue containing excessive adipose tissue (fat cells). The condition being treated may be associated with, for example, obesity, double chin, or cellulite. In many of the conditions, the subject has obesity or an obesity-associated disorder. In particular embodiments, the xanthene-based compound is injected (typically, dissolved in a

pharmaceutically acceptable carrier) into or within the vicinity of the bodily tissue containing excessive adipose tissue. It may also be formulated as an ointment or gel for topical application. The xanthene-based compound is administered in a therapeutically effective amount, which is an amount effective to inhibit adipogenesis and/or to induce apoptosis of adipocytes, which corresponds to an amount effective to reduce or otherwise regulate the number of fat cells in the adipose tissue, which corresponds to an amount effective to reduce or otherwise regulate the mass of adipose tissue. In the case of regulating the number of fat cells or mass of adipose tissue, the compound may function to maintain the number of fat cells at or below an excessive threshold. To do this, the compounds may prevent or slow the production and/or growth of new fat cells.

[0023] The xanthene-based compounds considered herein that inhibit adipogenesis and/or induce adipocyte apoptosis are within the scope of the following generic formula:

[0024] In Formula (1) above, the variables X¹, X², X³, and X⁴ are each independently selected from iodine and bromine atoms. In some embodiments, at least one, two, three, or all of X^ X², X³, and X⁴ are iodine atoms. In other embodiments, at least one, two, three, or all of X^ X², X³, and X⁴ are bromine atoms. The foregoing embodiments include the possibility that one, two, or three of X¹, X², X³, and X⁴ are iodine atoms while one, two, or three of X^ X², X³, and X⁴ are bromine atoms, i.e., where X¹, X², X³, and X⁴ represent a mix of iodine and bromine atoms.

[0025] The variables X⁵, X⁶, X⁷, and X⁸ are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms. In some embodiments, X⁵, X⁶, X⁷, and X⁸ are selected from chlorine, bromine, and iodine atoms. In a first set of embodiments, at least one, two, three, or all of X⁵, X⁶, X⁷, and X⁸ are hydrogen atoms. In a second set of embodiments, at least one, two, three, or all of X⁵, X⁶, X⁷, and X⁸ are chlorine atoms. In a third set of embodiments, at least one, two, three, or all of X⁵, X⁶, X⁷, and X⁸ are bromine atoms. In a fourth set of embodiments, at least one, two, three, or all of X⁵, X⁶, X⁷, and X⁸ are iodine atoms. The foregoing embodiments include the possibility that one, two, or three of X⁵, X⁶, X⁷, and X⁸ are hydrogen atoms while one, two, or three of X⁵, X⁶, X⁷, and X⁸ are chlorine, bromine, and/or iodine atoms, or the possibility that X⁵, X⁶, X⁷, and X⁸ represent a mix of halide atoms (e.g., chlorine and bromine, or chlorine and iodine, or bromine and iodine, or chlorine, bromine, and iodine). [0026] The variable Y¹ is an -0-, -NR.'-, or -CR'₂- linker, wherein R' is independently selected from hydrogen atom and methyl. When Y¹ is -0-, the compounds of Formula (1) contain an ester group (i.e., where -C(0)-Y¹-Z-R¹ is -C(0)0-Z-R¹. When Y¹ is -NR'-, the compounds of Formula (1) contain an amide group (i.e., where -C(0)-Y¹-Z-R¹ is -C(0)NR'- Z-R¹). In some embodiments, X¹, X², X³, and X⁴ are iodine atoms and Y¹ is an -NR'- linker. When Y¹ is -CRV, the compounds of Formula (1) contain a ketone group (i.e., where -C(O)- Y^Z-R¹ is -QC CRVZ-R¹). In some embodiments, X¹, X², X³, and X⁴ are iodine atoms and Y¹ is an -NR'- linker. In the case where Y¹ is -CR'₂-, Y¹ may be -CH₂-, -CH(CH₃)-, or -C(CH₃)₂-.

[0027] The variable Z is a hydrocarbon linking group containing one to twelve (i.e., 1-12) carbon atoms. Z may, in some embodiments, be more particularly defined as having precisely one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve carbon atoms, or a particular range of carbon atoms therein, e.g., 1-10, 1-8, 1-6, 1-4, 1-3, 2-12, 2-10, 2-8, 2-6, 2-4, 3-12, 3-10, 3-8, or 3-6 carbon atoms. Z can be saturated or unsaturated, straight-chained (linear) or branched, and either cyclic or acyclic. In some embodiments, Z is composed solely of carbon and hydrogen. The hydrocarbon linking group composed solely of carbon and hydrogen can be, for example, an alkyl, alkenyl, cycloalkyl, cycloalkenyl (aliphatic), or aromatic linking group. The alkyl linkers can be linear or branched. The linear or branched alkyl linkers can be conveniently represented by the formula -(0¾)_η-, wherein n is 1-12 or a sub-range therein, and wherein one or more of the shown hydrogen atoms (H) may (optionally) be substituted with a methyl or ethyl group while maintaining 1-12 carbon atoms in Z. The formula -(0¾)_η- can also represent an alkenyl linker by replacing two hydrogen atoms on adjacent carbon atoms with a carbon-carbon double bound. In the case of Z being a cyclic hydrocarbon group, the cyclic hydrocarbon group can be conveniently represented by the formula -A-, where A represents a saturated or unsaturated (e.g., aromatic) ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl,

cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and phenyl rings.

[0028] In some embodiments, Z is a hydrocarbon linker containing 1-12 carbon atoms and at least one heteroatom (i.e., non-carbon and non-hydrogen atom), such as one or more heteroatoms selected from oxygen, nitrogen, sulfur, and halide atoms (e.g., F, CI, Br, or I atoms). In some embodiments, Z includes one or more ether (-0-) linking groups, hydroxy (OH) groups, carbonyl-containing groups (e.g., ketone, amide, carbamate, or urea functionality), amine, or nitro (N0₂) groups. If more than one ether group is present in Z, the group Z may be or include a polyalkyleneoxide (polyalkyleneglycol) moiety, such as a polyethyleneoxide group. In some embodiments, any one or more of the above heteroatoms or heteroatom-containing groups are excluded.

[0029] The variable R¹ is selected from hydrogen atom (H), methyl, -OH, and -OR groups, wherein R is an alkyl group containing one to three carbon atoms. Some examples of alkyl groups (R) containing one to three carbon atoms include methyl, ethyl, n-propyl, and isopropyl groups. Thus, some examples of alkoxy groups (OR) include methoxy, ethoxy, n- propoxy, and isopropoxy groups. In particular embodiments, R¹ is -OH or -OR when Z is a -(CH₂)_n- linker, wherein n is 1-12 or a sub-range therein, as discussed above. In some embodiments, X¹, X², X³, and X⁴ are iodine atoms, Y¹ is an -NR' - linker, and R¹ is -OH. For purposes of the present invention, R¹ and Z-R¹ do not correspond to carboxylic acid (COOH) groups or salts thereof. In some embodiments, R¹ and Z-R¹ do not correspond to carboxylic acid esters (C(O)OR).

[0030] In some embodiments, the xanthene -based compounds of Formula (1) are within the scope of the following sub-generic formula:

[0031] In Formula (la) above, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, and X⁸ are defined as above under Formula (1). The variable Y¹ is an -O- or -NR'- linker, wherein R' is selected from hydrogen atom and methyl, as described above. The variable R² is selected from hydrogen atom (H) and alkyl groups containing one to three carbon atoms. The variable n is an integer of 1-12. In particular embodiments, X¹, X², X³, and X⁴ are iodine atoms. In further particular embodiments, Y¹ is an -NR' - linker, wherein R' is selected from hydrogen atom and methyl. In further particular embodiments, R² is H.

[0032] The Formulas (1) and (la) also include pharmaceutically acceptable salts and solvates of the compounds embraced by these formulas. The term "pharmaceutically acceptable salt," as used herein, refers to the relatively non-toxic, inorganic or organic addition salts of compounds of the present invention. A salt form of compounds of Formula (1) or (la) is possible when the compound contains an amino group, such as when Y¹ is an -NR'- linker or in the event Z contains an amine functionality. In that case, a pharmaceutically acceptable salt form can be produced by reaction of the amino-containing compound with a

pharmaceutically acceptable acid. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt formed during subsequent purification. Some representative salts include those generated by reaction of the free base with hydrobromic, hydrochloric, sulfuric, sulfamic, bisulfuric, phosphoric, nitric, acetic, propionic, benzoic, 2-acetoxybenzoic, malic, glycolic, valeric, oleic, palmitic, stearic, lauric, benzoic, lactic, toluenesulfonic,

methanesulfonic, ethanedisulfonic, citric, ascorbic, maleic, oxalic, fumaric, phenylacetic, isothionic, succinic, tartaric, glutamic, salicylic, sulfanilic, naphthylic, lactobionic, gluconic, laurylsulfonic acids, and the like. (Berge et al. (1977) "Pharmaceutical Salts", /. Pharm. Sci. 66: 1-19). As also known in the art, a solvate can be produced by contacting, dissolving, or otherwise treating the active compound with a solvent under conditions where one, two, or more solvent molecules remain associated with each molecule of the active ingredient. When the solvent is or includes water, the solvate may be a hydrate form of the compound. The formulas also encompass all enantiomeric, crystalline, polycrystalline, and amorphous forms of the compounds within the scope of Formulas (1) and (la).

[0033] In some embodiments, the compound being administered has the following specific structure, also referred to herein as MI-401 (2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7- tetraiodo-3-oxo-3H-xanthen-9-yl)-N-(2-hydroxyethyl)-benzamide):

[0034] The above formula for MI-401 also includes all pharmaceutically acceptable salts, solvates, enantiomers, and physical forms, as described above.

[0035] The above-described xanthene -based compounds may be synthesized using chemical preparative methods well known in the art, or, in some cases, the compound may be commercially available. The synthesis of some xanthene derivatives, such as Rose Bengal and some of its derivatives, are described in, for example, Y.-S. Kim et al., Journal of Controlled Release, 156, pp. 315-322 (2011), which is herein incorporated by reference in its entirety. The synthesis of MI-401 (RB4) is described in C.-H. Tung et al., Journal of Controlled Release, 258, pp. 67-72, 2017, which is herein incorporated by reference in its entirety. In particular embodiments, the synthesis of MI-401 involves the following steps: (i) providing 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein (508.82 mg, 0.5 mmol) as a first reactant in DMF (3 mL); (ii) providing a NN-diisopropylethylamine (DIEPA, 2 mL) as a second react; (iii) activating first and second reactants by contact with the coupling agent HBTU (189.62 mg, 0.5 mmol) with stirring at room temperature (RT) for 4 hours; (iv) providing 2-aminoethanol (91 iL, 1.5 mmol) as a third reactant, and reacting the mixture overnight at room temperature; (v) removing solvent under reduced pressure; (vi) extracting the resulting residue with dichloromethane; (vii) washing the residue with brine; (viii) drying the residue over anhydrous sodium sulfate; (ix) concentrating the residue; (x) purifying the residue by a silica gel column; and (xi) eluting the residue with DCM, DCM/MeOH = 10/0.5 and 10/1 (v/v). [0036] Generally, the xanthene-based compound according to Formula (1), (la), or (la-1) is administered in the form of a liquid pharmaceutical composition wherein the xanthene-based compound is dissolved in a pharmaceutically acceptable carrier (diluent or excipient). The phrase "pharmaceutically acceptable carrier" or equivalent term, as used herein, refers to a pharmaceutically acceptable solvent which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, particularly human beings, without excessive toxicity, irritation, allergic response, or other problem or complication

commensurate with a reasonable benefit/risk ratio. In the pharmaceutical composition, the compound is generally dispersed in the physiologically acceptable carrier, by being dissolved or emulsified in a liquid carrier. The carrier should be compatible with the other ingredients of the formulation and physiologically safe to the subject. Any of the carriers known in the art can be suitable herein depending on the mode of administration. Some examples of suitable carriers include aqueous solutions, alcohols, vegetable fats or oils, propylene glycol, glycerol, and the like.

[0037] The pharmaceutical composition can also include one or more auxiliary agents, such as stabilizers, surfactants, salts, buffering agents, additives, or a combination thereof, all of which are well known in the pharmaceutical arts. The stabilizer can be, for example, an oligosaccharide (e.g., sucrose, trehalose, lactose, or a dextran), a sugar alcohol (e.g., mannitol), or a combination thereof. The surfactant can be any suitable surfactant including, for example, those containing polyalkylene oxide units (e.g., Tween 20, Tween 80, Pluronic F-68), which are typically included in amounts of from about 0.001% (w/v) to about 10% (w/v). The salt or buffering agent can be any suitable salt or buffering agent, such as, for example, sodium chloride, or sodium or potassium phosphate, respectively. Some examples of additives include, for example, glycerol, benzyl alcohol, and l,l,l-trichloro-2-methyl-2- propanol (e.g., chloretone or chlorobutanol). If required, the pH of the solutions can be suitably adjusted by inclusion of a pH adjusting agent. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, liquids and powders. The pharmaceutical formulation may be in the form of a sterile aqueous solution that contains one or more buffers, diluents, and/or other suitable additives such as, but not limited to, penetration enhancers and carrier compounds.

[0038] The xanthene-based compound is administered in a therapeutically effective amount. The therapeutically effective amount is an amount effective to inhibit adipogenesis and/or to induce apoptosis of adipocytes, which corresponds to an amount effective to reduce the number of fat cells in the adipose tissue, which corresponds to an amount effective to reduce the mass of adipose tissue. The effective amount is generally determined by the physician on a case-by-case basis. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex, and weight of the patient, the condition being treated, the severity of the condition, as well as the route of administration, dosage form, regimen, and the desired result. For purposes of the invention, the therapeutically effective amount is generally an amount that is effective to reduce the level of fatty tissue in bodily tissue containing an excessive amount of adipose tissue. The dosage level may be, for example, about 1-1000 mg, which may be administered in one or more injection volumes of 0.1-10 mL. The dosage may alternatively be expressed in terms of a concentration of the compound in bodily tissue, typically about 1, 2, 5, 10, 20, 30, 40, or 50 μΜ, or a concentration within a range bounded by any two of the foregoing values.

[0039] Typically, the xanthene -based compound, described above, is injected directly into or within the vicinity of bodily tissue where excess adipose tissue is present. A typical regimen is an injection, in any of the dosage levels provided above, once or twice a day, or once every two or three days, or once a week, for at least two, three, four, five, or six weeks. The biological tissue containing the excess adipose tissue may be in, for example, the lower abdomen (panniculus), chest, hips, chin, thighs, ankles, back, or buttocks.

[0040] In other embodiments, the xanthene-based compound is administered via a patch placed on the skin. Administration via a patch is highly advantageous since this eliminates the need for a patient to receive repetitive multi-needle injections. The patch beneficially provides a localized convenient and painless administration method. In particular embodiments, the patch is based on a transdermal micro-size needle array device. A degradable microneedle patch containing a drug-loaded and cross-linked matrix for sustained drug delivery into subcutaneous adipose tissues can be used. Such a patch is described in G. Kogan, et al., Biotechnol Lett 2007, 29, 17, the contents of which are herein incorporated by reference.

[0041] The microneedle patch can be prepared, for example, on a uniform silicone mold. In a particular embodiment, the microneedle array contains 121 needles in a 7 X 7 mm² patch with a center-to-center interval of 600 μιη. Each microneedle may be of a conical shape, such as 300 μηι in diameter at the base and 800 μιη in height. The drugs may be embedded into a methylated hyaluronic acid (HA) polymer crosslinked with NN'-methylenebis (acrylamide) under UV light (365 nm) (e.g., Kogan et al., supra; and Y. Zhang et al., ACS Nano, 11, 9223, 2017). The cross-linked HA-based matrix can enhance the stiffness of the microneedle for efficient penetration through the skin as well as enable sustained release of drug from the tips, which helps maintain local constitutive high drug concentrations in adipose tissues.

[0042] In some embodiments, the xanthene -based compound, described above, is administered in combination with exposure of the excessive adipose tissue (being treated with the xanthene-based compound) to ultrasound. The ultrasound considered herein is typically low-intensity (gentle) ultrasound, which typically corresponds to a frequency of about 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 MHz, or a frequency with a range bounded by any two of the foregoing values, at a power (intensity) of 1, 2, 3, 4, or 5 W/cm², or a power within a range bounded by any two of the foregoing values. Any ultrasound process suitable for medical administration, as widely known in the art, is applicable herein, particularly the low-intensity ultrasound systems well known in the art. Reference is made to C.-H. Tung et al., Journal of Controlled Release, 258, pp. 67-72, 2017, which is herein incorporated by reference in its entirety and which provides ample details on the equipment and conditions typically used in administering low-intensity ultrasound. When applying the ultrasound, the ultrasound may be applied during or immediately after (e.g., 1-5 minutes after) injection of the xanthene- based compound into the tissue. The ultrasound is typically applied for 1-5 minutes.

[0043] Examples have been set forth below for the purpose of illustration and to describe the best mode of the invention at the present time. However, the scope of this invention is not to be in any way limited by the examples set forth herein.

EXAMPLES

[0044] The following experiment employed MI-401, a novel synthetic xanthene analog with the chemical structure shown in FIG. 1A. FIG. IB schematically shows the mode of operation of MI-401 in reducing the number of fat cells, thereby controlling the extent of fat tissue. By one mechanism, the compound halts adipogenesis. By another mechanism, the compound ablates (induces apoptosis of) mature adipocytes. The compound may advantageously perform both functions simultaneously. [0045] 3T3-L1 pre-adipocytes were grown in an adipocyte differentiation medium for three days. These cells were then maturated in an adipocyte maintenance medium for an additional three days. Once matured, MI-401 and sodium deoxycholate (SD) were added to the adipocyte's maintenance medium. The cells were then analyzed at one or two days after treatments. FIG. 2A is a schematic representation of the stages of differentiation protocol for 3T3-L1 cells. FIG. 2B shows representative images of mature 3T3-L1 cells treated with MI- 401 (10 μΜ) or SD (10 or 50 μΜ). As shown by the images in FIG. 2B, microscopic examination at day 2 revealed significant morphological changes in the MI-401 (10 μΜ) treated cells, which were small, dense, and rough. In contrast, no difference was observed between the untreated mature adipocytes and the SD (10 μΜ) treated cells. The morphology was clearly different once the administered concentration of SD was raised to 50 μΜ. FIG. 2C is a cell viability plot showing decreased viability of 3T3-L1 cells on day 1 and day 2 following MI-401 (10 μΜ) treatment compared to SD (50 μΜ) treated adipocytes. Data is presented as mean + standard deviation (n > 3). As shown by the data in FIG. 2C, the viability of the adipocytes in the MI-401 (10 μΜ) treatment was down to 30 % on day 1 and 10 % on day 2, while SD (50 μΜ) treated groups still had 70 % or 53 % of viable cells one or two days after treatment. FIG. 2D is a cell viability plot showing viability of cultured adipocytes treated with SD or MI-401. Data is presented as mean + standard deviation (n > 3). As shown by the data in FIG. 2D, the subsequent systematic viability studies showed the EC5₀ of MI-401 and SD at post treatment day 2 to be 5.3 and 53 μΜ, respectively. The EC5₀ of MI-401 on day 1 was 7.9 μΜ.

[0046] Cells were co-stained with CellMask™ Plasma membrane stains and DAPI nuclear stains two days after treatment. FIG. 3A is a schematic representation of the stages of differentiation and maturation of 3T3-L1 cells. FIG. 3B shows representative images of induction of apoptosis in mature 3T3-L1 cells treated with MI-401 (10 μΜ) but only partial solubilization of membranes with diminished lipid droplets in SD (50 μΜ) treated cells. The cell membrane was stained with CellMask™ Plasma Membrane Stain (Green) and the nucleus was stained with DAPI (Blue). Scale bar = 100 μιη. When compared to untreated mature adipocytes, SD-treated cells were about the same in size, but possessed a substantially lower membrane fluorescence intensity. The lowered fluorescence shown in FIG. 3B suggests a partial solubilization of the membrane. Additionally, the lipid droplets appear to have diminished from the cytoplasm. In contrast, MI-401 treated cells became smaller particles. Apoptotic characteristics, such as cytoplasm shrinkage, nucleus condensation, cell debris, and the disappearance of lipid droplets were observed. SD is known to destroy adipocytes by breaking down or solubilizing cell membranes. Therefore, a lactate dehydrogenase (LDH) release assay was selected to study the integrity of the plasma membranes post treatment. FIG. 3C is a graph showing significant elevation of necrosis associated LDH release 4 hrs after SD (50 μΜ) but only a modest increase in necrosis after MI-401 (10 μΜ) treatment. Data is presented as mean + standard deviation (n = 3). (** P < 0.005, * P < 21 0.05). As shown the data in FIG. 3C, SD-treated cells exhibited a significant release of cytosolic LDH, which was -70 % over the background value. MI-401- treated cells, however, were only about 25 % higher (FIG. 3C). The results of this assay suggest that that the MI-401 does not affect adipocytes by lysing the plasma membrane as in the case of SD. An apoptosis fluorescence assay was then conducted to determine the activity of triggered apoptotic enzymes, caspase 3 and caspase 7. FIG. 3D is a graph depicting time course curves of caspase 3 and 7 activity after SD treatments (50 μΜ) or MI-401 (10 μΜ). As shown by the data in FIG. 3D, the caspases' activity of the MI-401 treated cells was steadily increased over the measuring period, and the determined signal was significantly higher than the SD treated cells. The fluorescence signal representing caspase 3/7 activity was determined in real time using a fluorescence plate reader over 18 hours. Data is presented as mean + standard deviation (n = 3). (**** P < 0.001). These experimental results further indicate that MI-401 is an effective apoptosis initiator.

[0047] MI-401 was checked for its inhibition potential of adipogenesis. 3T3-L1 pre- adipocytes were seeded and grown to confluence in a pre- adipocyte maintenance medium for

2- 3 days. Thereafter, the cells were cultured in a differentiation medium containing different amounts of MI-401 for one or two days. FIG. 4A is a schematic representation of 3T3-L1 cells treated with MI-401 for the indicated time periods during differentiation. Following a

3- day differentiation period, the cells were further cultured in an adipocyte maintenance medium for an additional three days and analyzed for their lipid contents. As shown by the micrographs in FIG. 4B, the fully differentiated 3T3-L1 adipocytes were rich in large lipid droplets. A two-day incubation with 10 μΜ of MI-401 in a differentiation medium almost completely stopped the occurrence of lipogenesis (FIG. 4B) in the treated cells. A triglyceride (TG) quantification assay was performed to determine its IC₅₀ after 1-day and 2- day treatments. FIG. 4C is a graph showing decreased triglyceride accumulation with MI- 401 treatment (black bars and left Y-axis) and no change in cell viability after treatments. Data is presented as mean + standard deviation (n > 3). Based on triglyceride content, the IC₅₀ of MI-401 with 1-day or 2-day treatment was 3.2 and 2.5 μΜ, respectively (FIG. 4C). Significantly, under testing conditions, this differentiation- arresting drug was found to be non-toxic to differentiating pre-adipocytes (FIG. 4C).

[0048] Following the observation of a strong suppression of lipid deposition during the differentiation, the regulation of four key factors, including two transcription factors:

peroxisome proliferator-activated receptor-gamma (PPARg) and CCAAT/enhancer-binding protein alpha (C/EBPa), and two lipogenesis players: fatty acid synthase (FAS) and fatty acid binding protein 4 (FABP4), that participated in the adipogenic progression, were analyzed by Western blot to understand the possible mechanism of adipogenesis arrest. MI-401 (10 μΜ) was added to the pre-adipocyte phase or to the differentiation phase. FIGS. 5 A and 5B show the effect of MI-401 on adipogenic gene expression in different stages of growth. FIG. 5 A is a schematic representation of the experiments. MI-401 (10 μΜ) was added to the pre- adipocyte maintenance medium or differentiation medium, as indicated by the thicker lines. The cells were harvested immediately after the treatment. In a separated set of cells, MI-401 was added to the differentiation medium for two days, and then the cells were cultured in the adipocyte maintenance medium for an additional three days. The cells were collected at different stages and subjected to analysis (FIG. 5A). FIG. 5B is a Western blot analysis showing the effect of MI-401 on PPARg, C/EBPa, FAS and FABP4. As shown by the data in FIG. 5B, all four factors had low values at the normal pre-adipocytes stage; however, when the cells began differentiating the transcription factors, PPARg and C/EBPa, were highly upregulated. FAS and FABP4 were also observed to be upregulated but to a lesser extent. After the cell's completion of their differentiations, FAS and FABP4, which participated in lipid synthesis, were further expressed for lipid synthesis and deposition. No difference of expression was observed when pre-adipocytes were treated with MI-401. Nonetheless, when MI-401 was included in the differentiation medium, the expressions of PPARg, C/EBPa, and FAS by the differentiating pre-adipocytes were completely blocked. The expression of FABP4 was also significantly down-regulated. The suppression of these factors was maintained even after media was switched to an adipocyte maintenance medium.

[0049] FIGS. 6A-6D demonstrate cytotoxicity of MI-401 on 3T3-L1 pre-adipocytes and NIH3T3 fibroblast. FIG. 6A is a schematic representation of the experiment. After seeding, 3T3-L1 pre-adipocytes were treated with MI-401 (thick arrowhead) in a 22 pre-adipocyte maintenance media for 1 or 2 days (thicker line). NIH3T3 cells were also treated with MI- 401 but in DMEM medium with 10% FBS. Fibroblasts (NIH3T3) were also treated with MI- 401 in its optimal culture condition. FIG. 6B shows in representative images that both cell types remained unharmed and healthy when exposed to ΙΟμΜ MI-401. However, the 3T3-L1 pre-adipocyte cells were negatively affected in a 50 μΜ concentration, while NIH3T3 cells retained their health (FIG. 6B). FIG. 6C shows a quantitative analysis of viability of NIH- 3T3 fibroblast with MI-401. The EC5₀ at day 2 was 169.2 μΜ. Data are presented as mean + standard deviation (n = 3). FIG. 6D shows a quantitative analysis of viability of 3T3-L1 pre- adipocytes with MI-401. The EC5₀ at day 2 was 48.7 μΜ. Data is presented as mean + standard deviation (n = 3).

[0050] While there have been shown and described what are at present considered the preferred embodiments of the invention, those skilled in the art may make various changes and modifications which remain within the scope of the invention defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a condition characterized by excessive adipose tissue in a subject, the method comprising administering to said subject a compound that inhibits adipogenesis and/or induces adipocyte apoptosis in said excessive adipose tissue, said compound having the following structure:

wherein: X², X³, and X⁴ are each independently selected from iodine and bromine atoms;

X⁵, X⁶, X⁷, and X⁸ are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms;

Y¹ is an -0-, -NR.'-, or -CRV linker, wherein R' is independently selected from hydrogen atom and methyl;

Z is a hydrocarbon linking group containing 1-12 carbon atoms;

R¹ is selected from hydrogen atom, methyl group, -OH, and -OR groups, wherein R is an alkyl group containing one to three carbon atoms; and wherein said Formula (1) includes pharmaceutically acceptable salts and solvates of the compounds embraced by Formula (1).

2. The method of claim 1, wherein said condition is associated with obesity.

3. The method of claim 1, wherein said condition is double chin, and said compound reduces excessive adipose tissue under the chin.

4. The method of claim 1, wherein said condition is cellulite, and said compound reduces cellulite.

5. The method of claim 1, wherein said compound is administered to the subject by injecting said compound into said excessive adipose tissue.

6. The method of claim 1, wherein said compound is administered in combination with exposure of said excessive adipose tissue to ultrasound.

7. The method of claim 1, wherein said compound is dissolved in a pharmaceutically acceptable carrier.

8. The method of claim 1, wherein X¹, X², X³, and X⁴ are iodine atoms.

9. The method of claim 1, wherein Y¹ is an -NR'- linker, wherein R' is selected from hydrogen atom and methyl.

10. The method of claim 1, wherein R¹ is -OH.

11. The method of claim 1, wherein Z is an alkyl linking group.

12. The method of claim 1, wherein X¹, X², X³, and X⁴ are iodine atoms, and Y¹ is an -NR'- linker, wherein R' is selected from hydrogen atom and methyl.

13. The method of claim 12, wherein R¹ is -OH.

14. The method of claim 1, wherein said compound has the following structure:

wherein:

X\ X², X³, and X⁴ are each independently selected from iodine and bromine atoms;

Y¹ is an -0-, -NR'-, or -CRV linker, wherein R' is independently selected from hydrogen atom and methyl;

R² is selected from hydrogen atom (H) and alkyl groups containing one to three carbon atoms; and n is an integer of 1-12; wherein said Formula (la) includes pharmaceutically acceptable salts and solvates of the compounds embraced by Formula (la).

15. The method of claim 14, wherein X¹, X², X³, and X⁴ are iodine atoms.

16. The method of claim 14, wherein Y¹ is an -NR' - linker, wherein R' is selected from hydrogen atom and methyl.

17. The method of claim 14, wherein R² is H.

18. The method of claim 14, wherein X¹, X², X³, and X⁴ are iodine atoms, and Y¹ is an -NR'- linker, wherein R' is selected from hydrogen atom and methyl.

The method of claim 18, wherein R

The method of claim 1, wherein said compound has the following structure:

and wherein said Formula (la-1) includes pharmaceutically acceptable salts and solvates of the compounds embraced by Formula (la-1).