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WO2018191594A1 - Procédés de régulation d'adipocytes et traitement des états associés à un tissu adipeux excessif - Google Patents

Procédés de régulation d'adipocytes et traitement des états associés à un tissu adipeux excessif Download PDF

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Publication number
WO2018191594A1
WO2018191594A1 PCT/US2018/027454 US2018027454W WO2018191594A1 WO 2018191594 A1 WO2018191594 A1 WO 2018191594A1 US 2018027454 W US2018027454 W US 2018027454W WO 2018191594 A1 WO2018191594 A1 WO 2018191594A1
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compound
hydrogen atom
adipose tissue
atoms
formula
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Ching-Hsuan Tung
Myung Shin HAN
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Cornell University
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Cornell University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0004—Applications of ultrasound therapy
    • A61N2007/0008—Destruction of fat cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0073—Ultrasound therapy using multiple frequencies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes

Definitions

  • 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.
  • adipocytes refers to terminally differentiated fat cells, which originate from committed pre- adipocytes.
  • 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.
  • adipocytes 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.
  • SD sodium deoxycholate
  • 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/cm 2 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compounds that inhibit or arrest adipogenesis and/or induce adipocyte apoptosis are within the scope of the following generic formula:
  • X 1 , X 2 , X 3 , and X 4 are each independently selected from iodine and bromine atoms;
  • X 5 , X 6 , X 7 , and X 8 are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms;
  • Y 1 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;
  • R 1 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).
  • the compound being administered is within the scope of the following sub-generic structure:
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 are defined as above under Formula (1);
  • Y 1 is an -0-, -NR.'-, or -CRV linker, wherein R' is independently selected from hydrogen atom and methyl;
  • R 2 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).
  • the compound being administered has the following specific structure, also referred to herein as MI-401:
  • 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.
  • 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. 1 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 ⁇ ) or SD (10
  • FIG. 2C is a cell viability plot showing decreased viability of 3T3-L1 cells on day 1 and day 2 following MI- 401 (10 ⁇ ) treatment compared to SD (50 ⁇ ) 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 EC5 0 for MI-401 after 1-day or 2-day of treatment was 7.9 or 5.3 ⁇ , respectively; and for SD after 1-day or 2-day of treatment was 253.8 or 52.8 ⁇ , respectively. Data is presented as mean + standard deviation (n > 3).
  • 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 ⁇ ), but only partial solubilization of membranes with diminished lipid droplets in SD (50 ⁇ ) treated cells.
  • 3D is a graph depicting time course curves of caspase 3 and 7 activity after SD treatments (50 ⁇ ) or MI-401 (10 ⁇ ).
  • 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 ⁇ 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 LipdoxTM lipid stain prior to imaging.
  • 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 IC 50 of MI-401 with 1-day or 2-day treatment was 3.2 and 2.5 ⁇ , respectively.
  • 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 ⁇ ) 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.
  • 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 10 ⁇ MI-401.
  • FIG. 6C shows a quantitative analysis of viability of NIH-3T3 fibroblast with MI-401.
  • the EC 50 at day 2 was 169.2 ⁇ .
  • FIG. 6D shows a quantitative analysis of viability of 3T3- Ll pre-adipocytes with MI-401.
  • the EC5 0 at day 2 was 48.7 ⁇ .
  • 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.
  • certain xanthene-based compounds 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.
  • the xanthene-based compound is injected (typically, dissolved in a
  • 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.
  • 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.
  • xanthene-based compounds considered herein that inhibit adipogenesis and/or induce adipocyte apoptosis are within the scope of the following generic formula:
  • the variables X 1 , X 2 , X 3 , and X 4 are each independently selected from iodine and bromine atoms. In some embodiments, at least one, two, three, or all of X ⁇ X 2 , X 3 , and X 4 are iodine atoms. In other embodiments, at least one, two, three, or all of X ⁇ X 2 , X 3 , and X 4 are bromine atoms.
  • the foregoing embodiments include the possibility that one, two, or three of X 1 , X 2 , X 3 , and X 4 are iodine atoms while one, two, or three of X ⁇ X 2 , X 3 , and X 4 are bromine atoms, i.e., where X 1 , X 2 , X 3 , and X 4 represent a mix of iodine and bromine atoms.
  • the variables X 5 , X 6 , X 7 , and X 8 are each independently selected from hydrogen atom, chlorine, bromine, and iodine atoms. In some embodiments, X 5 , X 6 , X 7 , and X 8 are selected from chlorine, bromine, and iodine atoms. In a first set of embodiments, at least one, two, three, or all of X 5 , X 6 , X 7 , and X 8 are hydrogen atoms. In a second set of embodiments, at least one, two, three, or all of X 5 , X 6 , X 7 , and X 8 are chlorine atoms.
  • At least one, two, three, or all of X 5 , X 6 , X 7 , and X 8 are bromine atoms. In a fourth set of embodiments, at least one, two, three, or all of X 5 , X 6 , X 7 , and X 8 are iodine atoms.
  • the foregoing embodiments include the possibility that one, two, or three of X 5 , X 6 , X 7 , and X 8 are hydrogen atoms while one, two, or three of X 5 , X 6 , X 7 , and X 8 are chlorine, bromine, and/or iodine atoms, or the possibility that X 5 , X 6 , X 7 , and X 8 represent a mix of halide atoms (e.g., chlorine and bromine, or chlorine and iodine, or bromine and iodine, or chlorine, bromine, and iodine).
  • halide atoms e.g., chlorine and bromine, or chlorine and iodine, or bromine and iodine, or chlorine, bromine, and iodine.
  • the variable Y 1 is an -0-, -NR.'-, or -CR' 2 - linker, wherein R' is independently selected from hydrogen atom and methyl.
  • R' is independently selected from hydrogen atom and methyl.
  • the compounds of Formula (1) contain an ester group (i.e., where -C(0)-Y 1 -Z-R 1 is -C(0)0-Z-R 1 .
  • the compounds of Formula (1) contain an amide group (i.e., where -C(0)-Y 1 -Z-R 1 is -C(0)NR'- Z-R 1 ).
  • X 1 , X 2 , X 3 , and X 4 are iodine atoms and Y 1 is an -NR'- linker.
  • the compounds of Formula (1) contain a ketone group (i.e., where -C(O)- Y ⁇ Z-R 1 is -QC CRVZ-R 1 ).
  • X 1 , X 2 , X 3 , and X 4 are iodine atoms and Y 1 is an -NR'- linker.
  • Y 1 may be -CH 2 -, -CH(CH 3 )-, or -C(CH 3 ) 2 -.
  • 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.
  • 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 -(03 ⁇ 4) ⁇ -, 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 -(03 ⁇ 4) ⁇ - can also represent an alkenyl linker by replacing two hydrogen atoms on adjacent carbon atoms with a carbon-carbon double bound.
  • 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.
  • 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).
  • 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 (N0 2 ) groups.
  • the group Z may be or include a polyalkyleneoxide (polyalkyleneglycol) moiety, such as a polyethyleneoxide group.
  • a polyalkyleneoxide polyalkyleneglycol
  • any one or more of the above heteroatoms or heteroatom-containing groups are excluded.
  • the variable R 1 is selected from hydrogen atom (H), methyl, -OH, and -OR groups, wherein R is an alkyl group containing one to three carbon atoms.
  • alkyl groups (R) containing one to three carbon atoms include methyl, ethyl, n-propyl, and isopropyl groups.
  • alkoxy groups (OR) include methoxy, ethoxy, n- propoxy, and isopropoxy groups.
  • R 1 is -OH or -OR when Z is a -(CH 2 ) n - linker, wherein n is 1-12 or a sub-range therein, as discussed above.
  • X 1 , X 2 , X 3 , and X 4 are iodine atoms
  • Y 1 is an -NR' - linker
  • R 1 is -OH.
  • R 1 and Z-R 1 do not correspond to carboxylic acid (COOH) groups or salts thereof.
  • R 1 and Z-R 1 do not correspond to carboxylic acid esters (C(O)OR).
  • the xanthene -based compounds of Formula (1) are within the scope of the following sub-generic formula:
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 are defined as above under Formula (1).
  • the variable Y 1 is an -O- or -NR'- linker, wherein R' is selected from hydrogen atom and methyl, as described above.
  • the variable R 2 is selected from hydrogen atom (H) and alkyl groups containing one to three carbon atoms.
  • the variable n is an integer of 1-12.
  • X 1 , X 2 , X 3 , and X 4 are iodine atoms.
  • Y 1 is an -NR' - linker, wherein R' is selected from hydrogen atom and methyl.
  • R 2 is H.
  • the Formulas (1) and (la) also include pharmaceutically acceptable salts and solvates of the compounds embraced by these formulas.
  • pharmaceutically acceptable salt 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 Y 1 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
  • 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,
  • 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).
  • 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):
  • the above formula for MI-401 also includes all pharmaceutically acceptable salts, solvates, enantiomers, and physical forms, as described above.
  • 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.
  • 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
  • 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).
  • a pharmaceutically acceptable carrier 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
  • 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.
  • 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.
  • suitable salt or buffering agent such as, for example, sodium chloride, or sodium or potassium phosphate, respectively.
  • additives include, for example, glycerol, benzyl alcohol, and l,l,l-trichloro-2-methyl-2- propanol (e.g., chloretone or chlorobutanol).
  • 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.
  • 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.
  • 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 ⁇ , or a concentration within a range bounded by any two of the foregoing values.
  • 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.
  • 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.
  • 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.
  • the microneedle patch can be prepared, for example, on a uniform silicone mold.
  • the microneedle array contains 121 needles in a 7 X 7 mm 2 patch with a center-to-center interval of 600 ⁇ .
  • Each microneedle may be of a conical shape, such as 300 ⁇ in diameter at the base and 800 ⁇ 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.
  • 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/cm 2 , or a power within a range bounded by any two of the foregoing values.
  • any ultrasound process suitable for medical administration is applicable herein, particularly the low-intensity ultrasound systems well known in the art.
  • 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.
  • 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.
  • the compound halts adipogenesis.
  • the compound ablates (induces apoptosis of) mature adipocytes.
  • the compound may advantageously perform both functions simultaneously.
  • 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.
  • 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 ⁇ ) or SD (10 or 50 ⁇ ). As shown by the images in FIG. 2B, microscopic examination at day 2 revealed significant morphological changes in the MI-401 (10 ⁇ ) treated cells, which were small, dense, and rough. In contrast, no difference was observed between the untreated mature adipocytes and the SD (10 ⁇ ) treated cells.
  • FIG. 2C is a cell viability plot showing decreased viability of 3T3-L1 cells on day 1 and day 2 following MI-401 (10 ⁇ ) treatment compared to SD (50 ⁇ ) 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 ⁇ ) treatment was down to 30 % on day 1 and 10 % on day 2, while SD (50 ⁇ ) 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.
  • 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 ⁇ ) but only partial solubilization of membranes with diminished lipid droplets in SD (50 ⁇ ) treated cells.
  • 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
  • FIG. 4A is a schematic representation of 3T3-L1 cells treated with MI-401 for the indicated time periods during differentiation.
  • FIG. 4B 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.
  • 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.
  • FIG. 5A 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.
  • 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 ⁇ MI-401.
  • FIG. 6C shows a quantitative analysis of viability of NIH- 3T3 fibroblast with MI-401.
  • the EC5 0 at day 2 was 169.2 ⁇ .
  • FIG. 6D shows a quantitative analysis of viability of 3T3-L1 pre- adipocytes with MI-401.
  • the EC5 0 at day 2 was 48.7 ⁇ .

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Abstract

La présente invention concerne un procédé de traitement d'un état caractérisé par le tissu adipeux excessif chez un sujet, le procédé consistant à administrer audit sujet un composé qui fait preuve d'adipogenèse et/ou qui induit l'apoptose des adipocytes dans ledit tissu adipeux excessif, ledit composé ayant la structure : (1) où : X1, X2, X3, et X4 sont chacun indépendamment sélectionnés parmi les atomes d'iode et de brome ; X5, X6, X7, et X8 sont chacun indépendamment sélectionnés parmi l'atome d'hydrogène, les atomes de chlore, brome, et iode ; Y1 est un lieur -O-, -NR'-, ou -CR'2-, où R' est indépendamment sélectionné parmi l'atome d'hydrogène et le groupe méthyle ; Z est un groupe de liaison hydrocarbure contenant de 1 à 12 atomes de carbone ; R1 est sélectionné parmi l'atome d'hydrogène, les groupes méthyle, -OH, et -OR, où R est un groupe alkyle contenant de un à trois atomes de carbone ; et où ladite formule (1) comprend des sels et des solvates pharmaceutiquement acceptables des composés indiqués par la formule (1).
PCT/US2018/027454 2017-04-13 2018-04-13 Procédés de régulation d'adipocytes et traitement des états associés à un tissu adipeux excessif Ceased WO2018191594A1 (fr)

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US20100267576A1 (en) * 2007-05-02 2010-10-21 University Of Utah Research Foundation Compositions And Methods For Identifying And Treating Subjects At Risk Of Developing Type 2 Diabetes
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US11975071B2 (en) * 2018-04-16 2024-05-07 Cornell University Tumor ablation using low-intensity ultrasound and sound excitable drug

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