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WO2006039285A2 - Compositions et methodes permettant d'induire l'apoptose des cellules du tissu adipeux - Google Patents

Compositions et methodes permettant d'induire l'apoptose des cellules du tissu adipeux Download PDF

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Publication number
WO2006039285A2
WO2006039285A2 PCT/US2005/034632 US2005034632W WO2006039285A2 WO 2006039285 A2 WO2006039285 A2 WO 2006039285A2 US 2005034632 W US2005034632 W US 2005034632W WO 2006039285 A2 WO2006039285 A2 WO 2006039285A2
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WIPO (PCT)
Prior art keywords
derivatives
precursors
ajoene
garlic extract
pharmaceutical composition
Prior art date
Application number
PCT/US2005/034632
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English (en)
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WO2006039285A3 (fr
Inventor
Changlong Li
Maryanne Dellafera
Clifton A. Baile
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The University Of Georgia Research Foundation, Inc.
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Application filed by The University Of Georgia Research Foundation, Inc. filed Critical The University Of Georgia Research Foundation, Inc.
Publication of WO2006039285A2 publication Critical patent/WO2006039285A2/fr
Publication of WO2006039285A3 publication Critical patent/WO2006039285A3/fr

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Classifications

    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives

Definitions

  • compositions and Methods for Inducing Adipose Tissue Cell Death which is incorporated herein by reference in its entirety.
  • the present disclosure is generally related to compositions and methods for
  • compositions administration of the compositions to hosts and, more particularly, is related to
  • compositions designed for treatment for inducing adipose tissue cell death and
  • Obesity represents a major public health issue that continues to grow, accounts
  • Obesity is marked by excess adipose (i.e., fat) tissue
  • diabetes e.g., decreased insulin sensitivity
  • liposuction is an invasive and painful
  • osteoporosis a disease that results in over 1.5 million bone fractures a year.
  • An illustrative embodiment of a method of treating obesity is disclosed.
  • the compound is present in a dosage level effective to initiate the release or
  • the apoptosis inducing factor leads to the
  • tissue cells in a host includes initiating the release or activation of an
  • the extract compound The apoptosis inducing factor leads to apoptotic cell death.
  • the at least one garlic extract compound is present in a dosage
  • the apoptosis inducing factor leads to the apoptotic cell death of adipose tissue cells in a host.
  • composition including at least one garlic extract compound in combination with a pharmaceutically acceptable carrier.
  • the garlic extract compound in combination with a pharmaceutically acceptable carrier.
  • osteoporosis is present in a dosage level effective to treat osteoporosis.
  • FIG. 1 illustrates the effect of ajoene on cell viability. 3T3-L1 adipocytes were
  • FIGS. 2A and 2B illustrate the effect of ajoene on apoptosis.
  • FIG. 2A 3T3-
  • Ll adipocytes were treated with ajoene at various concentrations (0, 10, 50, 200, 400
  • FIG. 2B illustrates apoptosis by gel
  • 3T3-L1 adipocytes were incubated with ajoene at various
  • Lane 1 analyzed by gel electrophoresis; Lane 2, 100 ⁇ M ajoene; Lane 3, 200
  • FIG. 3 A and 3B illustrate the effects of ajoene on intracellular hydrogen
  • FIG. 3 A illustrates the time course of the effect of ajoene on
  • 3T3-L1 adipocytes were grown in 96-well plates and treated with
  • FIG. 3B illustrates the reduction of ajoene-induced apoptosis in cells pretreated with NAC. The percentage of apoptotic cells measured
  • FIGS. 4A-4D illustrate the effect of ajoene on MAPKs phosphorylation.
  • the protein levels of unphosphorylated and phosphorylated forms of MAP kinases were
  • ERKl/2 and JNK were performed. Integrated density values (phosphorylated/total)
  • FIGS. 5A-5C illustrate the effect of ajoene on PARP cleavage and caspase3
  • FIG. 5A 3T3-L1 adipocytes were treated with ajoene (200 ⁇ M) for the
  • ⁇ -Actin lower panel was used as an equal loading control.
  • FIG. 5B 3T3-L1 adipocytes were preincubated with 10 mM NAC for 1 hr
  • FIGS. 6A-6B illustrate ajoene induced translocation of AIF from mitochondria
  • FIG. 6A 3T3-L1 adipocytes were treated with ajoene (200 ⁇ M) for 0, 1, 4, 8 and 12 hr. Equal amounts of AIF protein from nuclear fraction (upper panel) and
  • Integrated density values (AIF/actin) were calculated and expressed as % highest
  • mitochondrial means with different letters are different across time: xyz: p ⁇ 0.01.
  • FIG. 6B 3T3-L1 adipocytes were preincubated with 10 mM NAC for 1 hr in
  • organism or "host” refers to any living entity comprised of at least
  • a living organism can be as simple as, for example, a single eukaryotic cell
  • “host” includes humans, mammals (e.g., cats, dogs, horses, chicken, pigs, hogs, cows,
  • host includes humans, companion animals (e.g., cats, dogs, and the like),
  • apoptosis inducing factor refers to any compound
  • apoptosis inducing factor activated in a pathway leading to apoptosis.
  • apoptosis inducing factor activated in a pathway leading to apoptosis.
  • AIF Apoptosis Inducing Factor protein
  • apoptosis inducing factors in the present disclosure include,
  • ROS reactive oxygen species
  • MAPKs mitogen-activated protein kinases
  • PARPs PoIy(ADP ribose) polymerases
  • terapéuticaally effective amount refers to that
  • therapeutically effective amount refers to that amount that has the effect of (1) causing
  • apoptosis of adipose cells and/or (2) reducing the mass of the adipose cells/tissue.
  • “Pharmaceutically acceptable salt” refers to those salts that retain the
  • hydrobromic acid sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,
  • ethanesulfonic acid p-toluenesulfonic acid
  • salicylic acid malic acid, maleic acid
  • esters as used herein refers to those wherein
  • esters of one or more compounds of the composition that, within the scope of sound
  • a “pharmaceutical composition” refers to a mixture of one or more of the
  • composition One purpose of a pharmaceutical composition is to facilitate
  • a "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate
  • excipient refers to an inert substance added to a pharmaceutical
  • composition to further facilitate administration of a compound examples include:
  • excipients include, without limitation, calcium carbonate, calcium phosphate, various combinations thereof
  • Treating" or “treatment” of a condition includes preventing the condition
  • prodrug refers to an agent that is converted into a biologically
  • Prodrugs are often useful because, in some situations, they may
  • the prodrug may also have improved solubility in pharmaceutical compositions over the parent
  • a prodrug may be converted into the parent drug by various mechanisms,
  • topically active agents refers to compositions of the
  • topically refers to application of the compositions
  • salt(s) as used herein.
  • Pharmaceutically acceptable ⁇ i.e., non-toxic, physiologically
  • salts are preferred, although other salts are also useful ⁇ e.g., in isolation or
  • compounds may be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt
  • the disclosed compounds that contain a basic moiety may form salts with a
  • Exemplary acid addition salts include acetates
  • benzenesulfonates bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates,
  • methanesulfonates formed with methanesulfonic acid
  • 2-naphthalenesulfonates formed with 2-naphthalenesulfonates
  • nicotinates such as nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as
  • tartrates tartrates, thiocyanates, toluenesulfonates (such as tosylates), undecanoates, and the
  • the disclosed compounds that contain an acidic moiety may form salts with a
  • exemplary basic salts include ammonium
  • alkali metal salts such as sodium, lithium, and potassium salts
  • alkaline earth salts such as sodium, lithium, and potassium salts
  • metal salts such as calcium and magnesium salts; salts with organic bases (for
  • organic amines such as benzathines, dicyclohexylamines, hydrabamines
  • Basic nitrogen-containing groups may be quaternized with agents such as
  • lower alkyl halides e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and
  • dialkyl sulfates e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates
  • chain halides e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides
  • aralkyl halides e.g., benzyl and phenethyl bromides
  • Solvates of the compounds are also contemplated herein. Solvates of the compounds are preferably hydrates.
  • isomers or may be admixed, for example, as racemates or with all other, or other
  • compositions for inducing adipose tissue cell death, and methods for treating
  • compositions include, but are not limited to, compositions having at least one garlic extract compound and, in particular, garlic thiosulfinates and
  • the method includes inducing adipose
  • tissue cell death in a host by administering a composition having at least one garlic
  • the methods include treating conditions such as, but not
  • compositions having at least one
  • Obesity is a chronic and costly condition that is increasing rapidly throughout the world. Obesity is considered a major risk factor for noninsulin-dependent diabetes
  • Adipose tissue mass is determined by processes governing adipocyte size and number (3).
  • Reduction of adipocyte number can result from preadipocyte and adipocyte apoptosis
  • apoptosis may be an important
  • the garlic extract compound of the present disclosure e.g., ajoene
  • apoptosis inducing factor leads to the apoptotic cell death of adipose tissue cells.
  • This apoptotic cell death is unexpectedly caspase-independent, which is in
  • apoptosis refers to a physiological process wherein selected cells
  • Apoptosis involves a sequence of
  • ROS are recognized to play a key role in cell signaling.
  • the cellular level
  • oxidant injury elicits a broad spectrum of responses ranging from proliferation to growth arrest, to senescence, to cell death (for review see 9).
  • oxidant injury elicits a broad spectrum of responses ranging from proliferation to growth arrest, to senescence, to cell death (for review see 9).
  • MAPK activated protein kinases
  • ERKl/2 Jun-N-teraiinal kinase
  • JNK Jun-N-teraiinal kinase
  • JNK and p38 MAPK activation is associated with apoptosis induction, whereas ERK activation is cytoprotective (14).
  • PARP which contributes to the pathogenesis of various diseases (15, 16).
  • Apoptosis Inducing Factor was more recently cloned and identified as a
  • ATF mitochondrial intermembrane space protein. In response to apoptotic stimuli, ATF is
  • adipocytes is initiated by the generation of hydrogen peroxide, which leads to
  • ROS reactive oxygen species
  • MAPK protein kinases
  • ERK extracellular signal-regulating kinase 1/2
  • c-Jun-N protein kinases
  • JNK N-terminal kinase
  • PARP polymerase
  • ROS reactive oxygen species
  • N-acetyl- L -cysteine NAC
  • Annexin V (AV)
  • FITC-conjugated AV is
  • chromosomal DNA to be exposed.
  • Propidium iodide a fluorescent dye that binds to
  • DNA can be used in conjunction with FITC-conjugated AV to identify subpopulations
  • the TUNEL assay is used to detect
  • Both of these assays can be used in conjunction with laser scanning cytometry (LSC) to provide both quantitative and morphological analysis of apoptosis.
  • LSC laser scanning cytometry
  • Scanning Cytometry uses lasers to excite fluorochromes in cellular specimens and
  • LSC can additionally find and quantitate events by multiple filter settings, for example, making it possible to distinguish cytoplasmic fluorescence from nuclear
  • LSC has been used to study apoptosis of adipocytes and has been shown to
  • AV Annexin V
  • PI propidium iodide
  • both dyes can be detected at the same time in a single sample.
  • the microscope stage moves the slide (e.g., incubation dish) automatically through
  • the computer For each segment that is scanned, the computer stores information
  • Another method to detect apoptosis is based on the staining of cell suspensions
  • MAbs monoclonal antibodies to single-stranded DNA
  • TUNEL detects low-mol-wt DNA fragmentation associated with late apoptosis
  • MAbs to ssDNA detect the early stages of apoptosis and stain apoptotic cells
  • adipocytes in bone marrow is a major factor contributing to age-related bone loss.
  • mesenchymal stem cells within bone marrow can differentiate to form adipocytes or
  • osteoclastogenic cytokines such as JX-6 (J Clin Endo Metab 83(3): 847-850 (1998), and adipocytes can inhibit osteoblast activity in culture ( " Bone 26(5): 485-9 (2000)).
  • fat cell development and hypertrophy can compress intraosseous capillaries
  • the present disclosure provides compounds, such as, garlic extract
  • Garlic extract compounds of the formula (1) for inducing apoptosis of adiposites.
  • present disclosure can include, but are not limited to, garlic thiosulfinates and transformation products thereof, ajoene (E and Z isomers) (4,5,9-trithiadodeca-l,6,ll- triene-9-oxide), precursors thereof, and derivatives thereof; allicin, precursors thereof,
  • the garlic extract compound can include, but is not limited to, S-
  • SAMC allylmercaptocysteme
  • SMC S-methylcysteine
  • SACS S-allyl cysteine sulfoxide
  • DADS diallyl disulfide
  • SAC S-allylcysteine
  • SEC S-ethylcysteine
  • extract compound includes ajoene (E and Z isomers), and/or precursors thereof, and/or
  • garlic extract compounds may include garlic extract compound analogues, homologues, isomers, or derivatives thereof, that function to
  • garlic extract compounds can include pharmaceutically acceptable salts, esters, and prodrugs of the garlic extract compounds described above.
  • embodiments of this disclosure include methods to treat conditions such as, but not
  • compositions and dosage forms of the disclosure include a
  • mucosal e.g., nasal, sublingual, vaginal, buccal, or rectal
  • parenteral e.g., oral, mucosal, nasal, sublingual, vaginal, buccal, or rectal
  • dosage forms include,
  • tablets but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft
  • ointments ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters;
  • solutions for solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms
  • suspensions e.g.,
  • aqueous or non-aqueous liquid suspensions oil-in-water emulsions, or water-in-oil
  • liquid emulsions liquid emulsions
  • solutions liquid elixirs
  • liquid dosage forms suitable for parenteral
  • sterile solids e.g., crystalline or amorphous solids
  • compositions of the invention are administered to a patient.
  • shape, and type of dosage forms of the compositions of the invention are administered to a patient.
  • the acute treatment of a condition or disorder may contain larger amounts of the active
  • parenteral dosage form may contain smaller amounts of the active ingredient than an
  • oral dosage form used to treat the same condition or disorder.
  • Typical pharmaceutical compositions and dosage forms include one or more
  • excipients are well known to those skilled in the art of pharmacy
  • composition or dosage form depends on a variety of factors well
  • oral dosage forms such as tablets or
  • capsules may contain excipients not suited for use in parenteral dosage forms.
  • the decomposition of some active ingredients can be any suitable active ingredients.
  • the decomposition of some active ingredients can be any suitable active ingredients.
  • ingredients that include primary or secondary amines are particularly susceptible to
  • the disclosure further encompasses pharmaceutical compositions and dosage
  • antioxidants include, but are not limited to, antioxidants such as ascorbic acid, pH
  • the disclosure may contain one or more solubility modulators, such as sodium
  • exemplary solubility modulator is tartaric acid.
  • the amounts and specific type of active ingredient in a dosage form may differ depending on various factors. It will be understood that the amounts and specific type of active ingredient in a dosage form may differ depending on various factors. It will be understood that the amounts and specific type of active ingredient in a dosage form may differ depending on various factors. It will be understood
  • compositions of the present disclosure are preferably formulated in dosage
  • Dosage unit form for ease of administration and uniformity of dosage.
  • Each dosage should contain the quantity of composition calculated to produce the desired therapeutic affect either as such, or in association with the
  • Preferred unit dosage formulations are those containing a daily dose or unit
  • the approximation includes host factors such as surface area, weight,
  • compositions of the disclosure that are suitable for oral
  • administration can be presented as discrete dosage forms, such as, but not limited to,
  • tablets including without limitation scored or coated tablets
  • pills including without limitation scored or coated tablets
  • caplets including without limitation caplets, capsules
  • chewable tablets powder packets, cachets, troches, wafers, aerosol sprays, or liquids
  • liquid a non-aqueous liquid, an oil-in- water emulsion, or a water-in-oil emulsion).
  • compositions contain a predetermined amount of the pharmaceutically
  • compositions of the disclosure are prepared
  • Excipients can take a wide variety of forms, depending on the form of the composition desired for administration.
  • excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to,
  • tablets can be coated by standard aqueous or nonaqueous techniques. These dosage forms can be prepared by any of the methods of
  • a tablet can be prepared by compression or molding.
  • Compressed tablets can be prepared by compressing, in a suitable machine, the active
  • ingredient(s) in a free- flowing form such as a powder or granules, optionally mixed
  • Molded tablets can be made by molding, in a suitable
  • Binders include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders
  • compositions and dosage forms suitable for use in pharmaceutical compositions and dosage forms include, but are not
  • gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose
  • Suitable forms of microcrystalline cellulose include, but are not limited to, the
  • suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or
  • additives include AVICEL-PH- 103TM and Starch 1500 LM.
  • dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate
  • granules or powder e.g., granules or powder
  • microcrystalline cellulose e.g., microcrystalline cellulose, powdered cellulose, dextrates
  • binder or filler in pharmaceutical compositions of the disclosure typically includes
  • Disintegrants may be used in the compositions of the disclosure to provide
  • Tablets that disintegrate when exposed to an aqueous environment Tablets that contain too much disintegrant may swell, crack, or disintegrate in storage, while those
  • the amount of disintegrant used varies, based upon the type
  • Typical pharmaceutical compositions comprise from about 0.5 to 15 weight percent of disintegrant, or from about 1 to 5 weight percent of
  • Disintegrants that can be used to form pharmaceutical compositions and
  • dosage forms of the disclosure include, but are not limited to, agar-agar,, alginic acid,
  • pre-gelatinized starch clays, other algins, other celluloses, gums, and mixtures thereof.
  • Lubricants that can be used to form pharmaceutical compositions and dosage
  • forms of the disclosure include, but are not limited to, calcium stearate, magnesium
  • glycol other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable
  • oil e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and
  • soybean oil soybean oil
  • zinc stearate ethyl oleate
  • ethyl laureate ethyl laureate
  • agar agar
  • Additional lubricants include, for example, a syloid silica gel (AEROSIL 200,
  • lubricants are typically used in an amount of less than about 1 weight
  • compositions preferably contain little, if any, lactose
  • lactose-free means that the amount of lactose present, if any, is insufficient to substantially increase the
  • Lactose-free compositions of the disclosure can comprise excipients that are
  • lactose-free compositions comprise a
  • Preferred lactose-free dosage forms comprise a pharmaceutically acceptable salt of the
  • microcrystalline cellulose microcrystalline cellulose
  • pre-gelatinized starch pre-gelatinized starch
  • magnesium stearate magnesium stearate
  • compositions and dosage forms are provided.
  • lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or
  • An anhydrous pharmaceutical composition should be prepared and stored such
  • anhydrous compositions are provided.
  • compositions of the disclosed compounds can be administered by controlled- or delayed-release means. Controlled-release
  • controlled-release formulations include: 1)
  • Conventional dosage forms generally provide rapid or immediate drug release
  • controlled-release formulations can be used to control a drug's onset
  • ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions
  • hydroxypropylmethyl cellulose examples include hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable
  • multilayer coatings microparticles, liposomes, or microspheres, or a
  • ion exchange materials can be used to prepare immobilized, adsorbed
  • anion exchangers examples include, but are not limited to, Duolite® A568 and Duolite® AP 143 (Rohm & Haas, Spring House, Pa. USA).
  • a pharmaceutically acceptable salt of the disclosed compounds e.g., a
  • form is formulated for controlled-release.
  • Specific dosage forms utilize an osmotic valve
  • a particular and well-known osmotic drug delivery system is referred to as
  • OROS® that can be used to administer compounds and compositions of the disclosure include, but are not limited to, the OROS® Push- PullTM, Delayed Push-PullTM, Multi-Layer Push-PullTM, and Push-StickTM Systems, all
  • compositions of the disclosure include, but are not limited to, OROS®-CT and L-
  • delivery rate of the drug is not influenced by physiological or experimental conditions.
  • a specific dosage form of the compositions of the disclosure includes at least
  • a wall defining a cavity, the wall having an exit orifice formed or
  • a dry or substantially dry state drug layer located within the cavity adjacent the exit orifice and in direct or indirect contacting relationship with the expandable layer; and a flow-promoting layer
  • drug layer located within the cavity, wherein the drug layer includes the compound of
  • Another specific dosage form of the disclosure includes at least the following: a wall defining a cavity, the wall having an exit orifice formed or formable therein and
  • the drug layer comprising a liquid, active-agent formulation absorbed in porous
  • active agent formulation includes the compound of the disclosure, a salt thereof, or a
  • the dosage form also optionally has a placebo layer between the exit orifice
  • Parenteral dosage forms can be administered to patients by various routes,
  • parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
  • parenteral dosage forms include, but are not limited to, solutions
  • controlled-release parenteral dosage forms can be prepared for
  • administration to a patient including, but not limited to, administration DUROS®-
  • sterile water water for injection USP; saline solution; glucose solution;
  • aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's
  • polyethylene glycol, and propylene glycol polyethylene glycol, and propylene glycol
  • non-aqueous vehicles such as, but not
  • Topical dosage forms of the disclosure include, but are not limited to, creams,
  • lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and other forms know to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences,
  • topical dosage forms viscous to semi-solid or solid forms comprising a carrier or one
  • excipients compatible with topical application and having a dynamic viscosity
  • Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders,
  • dosage forms include sprayable aerosol preparations wherein the active ingredient,
  • a pressurized volatile e.g., a gaseous propellant, such as Frean®
  • a pressurized volatile e.g., a gaseous propellant, such as Frean®
  • Fragrance and/or other cosmetic ingredients such as
  • tint, light reflectors, firming agents, and the like can also be added to topical dosage
  • ophthalmic solutions include, but are not limited to, ophthalmic solutions, patches, sprays, aerosols, creams,
  • lotions for example, lotions, suppositories, ointments, gels, solutions, emulsions, suspensions, or other
  • Dosage forms suitable for treating mucosal tissues within the oral cavity can be
  • dosage forms include "reservoir type” or “matrix type” patches, which can be applied'
  • transdermal dosage forms and methods of administration that can be used to produce transdermal dosage forms and methods of administration.
  • Suitable excipients e.g., carriers and diluents
  • other materials that can be
  • isopropyl myristate isopropyl palmitate, mineral oil, and mixtures thereof.
  • enhancers can be used to assist in delivering the active ingredients to or across the
  • Suitable penetration enhancers include, but are not limited to: acetone; various others.
  • alcohols such as ethanol, oleyl, an tetrahydrofuryl; alkyl sulfoxides such as dimethyl
  • pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone);
  • TWEEN 80 polysorbate 80
  • SPAN 60 sorbitan monostearate
  • pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied may also be
  • solvent carrier its ionic strength, or tonicity can be adjusted to improve delivery.
  • compositions can also be added to pharmaceutical compositions or
  • stearates can serve as a lipid
  • the active ingredient(s) of the pharmaceutical compositions of the invention preferably, the active ingredient(s) of the pharmaceutical compositions of the
  • disclosure are preferably not administered to a patient at the same time or by the same
  • kits which, when used
  • a typical kit includes a unit dosage form of a pharmaceutically acceptable salt
  • kits may further include ⁇ a device
  • syringes include, but are not limited to, syringes, drip bags, patches, and inhalers.
  • Kits of the disclosure can further include pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients.
  • pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral
  • the kit can include a sealed container of a suitable vehicle in which the
  • active ingredient can be dissolved to form a particulate-free sterile solution that is
  • vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles
  • water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol,
  • non-aqueous vehicles such as, but not limited to, corn oil,
  • PBS Phosphate-buffered saline
  • DMEM medium purchased from GIBCO (BRL Life Technologies, Grand Island, NY). Ajoene was
  • NAC cysteine
  • polyclonal anti-AIF, caspase-3, and PARP-I were from Santa Cruz
  • ERKl/2 (ThT 202 ZTyT 204 ), and total ERK1/2 were from Cell Signaling Technology
  • DMEM fetal calf serum
  • BCS bovine calf serum
  • All media contained 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, and of 292
  • MTS cell viability assay Tests were performed in 96-well plates. For mature
  • adipocytes adipocytes
  • Adipocytes were incubated with either DMSO or increasing concentrations of
  • the absorbance was measured at 490 nm in a plate reader ( ⁇ QuantTM
  • Apoptosis assays For the assessment of apoptosis, we used the ApoStrandTM
  • Kit detects single stranded DNA, which occurs in apoptotic cells but not in necrotic
  • adipocytes were incubated with either DMSO or increasing concentrations of ajoene
  • Caspase-3/7 activity was measured using the substrate DEVD- aminoluciferin from the Caspase-GloTM 3/7 assay kit according to the manufacturer's
  • DCHF Molecular Probes, Eugene, OR
  • a lysis buffer (20 mM Tris, pH 7.5, 150 niM NaCl, 1 niM
  • glycerophosphate 1 mM Na 3 VO 4 , and 100 ug/ml phenylmethylsulfonyl fluoride.
  • Tris/glycerol buffer pH 8.5
  • alkaline-phosphatase-conjugated secondary antibody was added.
  • the target proteins became visible following the addition of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), a substrate of alkaline phosphatase.
  • BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
  • PVDF membranes after Western blotting with various antibodies was performed using
  • isotonic homogenization buffer 250 mM sucrose, 10 mM
  • KCl 1.5 mM MgCl 2 , 1 mM Na-ethyleneglycotetraacetic acid [EGTA], 1 mM NaOH
  • nuclei and heavy mitochondria fractions were fractionated at 75Og for 10 minutes and
  • mitochondrial and nuclear fractions was measured by Western blot as described above.
  • Tris/EDTA buffer (10 mM Tris-HCl
  • the number of live cells was determined by MTS assay. As shown in FIG. 1, ajoene time and dose-dependently reduced viability in adipocytes. Ajoene at 200
  • Ajoene triggers apoptosis by oxidative stress: To determine the involvement of
  • ROS in ajoene-induced apoptosis ROS levels were determined in ajoene-treated
  • adipocytes As shown in FIG. 3A, ajoene increased ROS production by 2.1 fold after
  • catalase (a scavenger for hydrogen peroxide) did not prevent the ajoene-induced
  • Ajoene induces activation of MAPKs: Since the apoptotic effects of ajoene
  • ROS ROS
  • FIG. 4A shows that activation of JNK occurred as late as 60 min with maximum
  • FIG. 4B shows the time-dependent phosphorylation
  • Ajoene induces PARP-J cleavage and AIF-mediated cell death in a caspase-
  • AIF apoptosis-inducing factor
  • the present example elucidates the biological effect of ajoene, a component of
  • ERK activation is associated with apoptosis induction, whereas ERK activation is
  • ROS levels peaked 20 min after ajoene treatment, whereas ERKl/2 was activated at 30 min and JNK was activated 180 min, which suggests that within this context, ROS
  • JNK protein expression suggests that ROS directly influences MAPK signaling.
  • Catalase is a scavenger for hydrogen peroxide and did not block ajoene-induced ROS generation.
  • NAC is a thiol-reducing agent in addition to its action as a free
  • radical scavenger (31) Ajoene, which contains thiol groups, may be reduced by NAC and thereby lose its ability to induce apoptosis through generation of ROS and subsequent MAPK activation.
  • treatment is caspase independent.
  • NAC inhibited AIF translocation to the nucleus, suggesting that the increased intracellular ROS level is critical for the AIF relocalization after ajoene treatment.
  • PARP is a nuclear enzyme that facilitates DNA repair in response to DNA
  • PARP poly(ADP-ribose) polymerase
  • ROS Reactive oxygen species
  • intracellular ROS level The enhancement of intracellular ROS level promotes
  • ajoene may be a new therapeutic tool for the treatment of obesity by regulating fat cell number through the induction of adipocyte apoptosis.

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Abstract

L'invention concerne des méthodes permettant de traiter l'obésité, des méthodes permettant d'induire l'apoptose des cellules du tissu adipeux chez un hôte, des compositions pharmaceutiques et des méthodes permettant de traiter l'ostéoporose chez un hôte.
PCT/US2005/034632 2004-09-30 2005-09-29 Compositions et methodes permettant d'induire l'apoptose des cellules du tissu adipeux WO2006039285A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053487A2 (fr) 2006-11-01 2008-05-08 The Medical Research Fund At The Tel-Aviv Sourasky Medical Center Assemblage d'adipocytes spécifiques et procédés inhibiteurs d'expression de 12-lipoxygénase de type plaquette

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2815865B1 (fr) * 2000-10-31 2003-03-21 Innovaderm Utilisation d'une composition therapeutique ou cosmetique pour le traitement topique de la cellulite, et composition en comportant l'application
US7867525B2 (en) * 2002-08-14 2011-01-11 Bionutrigen Co., Ltd. Powder or extracts of plant leaves with anti-obesity effects and anti-obesity food comprising them
US20050013921A1 (en) * 2003-07-17 2005-01-20 Peticca Andrea Lynn Novel multi-component garlic food supplement

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053487A2 (fr) 2006-11-01 2008-05-08 The Medical Research Fund At The Tel-Aviv Sourasky Medical Center Assemblage d'adipocytes spécifiques et procédés inhibiteurs d'expression de 12-lipoxygénase de type plaquette
EP2078079B1 (fr) * 2006-11-01 2011-05-04 The Medical Research and Infrastructure Fund of the Tel-Aviv Sourasky Medical Center Assemblage d'adipocytes spécifiques et procédés inhibiteurs d'expression de 12-lipoxygénase de type plaquette
US9279127B2 (en) 2006-11-01 2016-03-08 The Medical Research Fund At The Tel-Aviv Sourasky Medical Center Adipocyte-specific constructs and methods for inhibiting platelet-type 12 lipoxygenase expression
US9663790B2 (en) 2006-11-01 2017-05-30 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Adipocyte-specific constructs and methods for inhibiting platelet-type 12 lipoxygenase expression

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