PCT/CA2018/050275
`
`CURCUMIN-BASED COMPOSITIONS & METHODS OF USE THEREOF
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[001] This application claims priority to, and incorporates by reference, U.S.
`
`provisional patent application 62/469,554, filed on March 10, 2017.
`
`FIELD OF THE INVENTION
`
`[002] The field of the present invention relates to certain curcumin-containing
`
`compositions and methods of use thereof, which can be used to increase low(cid:173)
`
`density lipoprotein cholesterol (LDL) receptor expression levels and thereby
`
`lower LDL levels in a plurality of cells or subject.
`
`In addition, the field of the
`
`present invention relates to certain curcumin-containing compositions and
`
`methods of use thereof, which can be used to modulate MSK1 production and
`
`thereby ameliorate a variety of health conditions.
`
`BACKGROUND OF THE INVENTION
`
`[003] The health benefits of curcumin, particularly whole turmeric extract, are
`
`known and have been demonstrated by researchers in recent years. However,
`
`several challenges continue to exist, with respect to the formulation of curcumin(cid:173)
`
`based pharmaceuticals and dietary supplements. More specifically, the most
`
`common source of curcumin,
`
`the
`
`Indian spice
`
`turmeric (a member of
`
`Zingiberaceae), does not contain a sufficient amount of curcumin to provide an
`
`efficacious dose to a subject. In fact, the therapeutic benefits provided by natural
`
`curcumin extracts have been relatively modest, very inconsistent, and not well
`
`understood. Accordingly, there is a continuing need for improved curcumin(cid:173)
`
`based formulations, which address these current challenges.
`
`1
`
`SAB1003
`U.S. Pat. No. 10,945,970
`
`
`
`CURCUMIN-BASED COMPOSITIONS & METHODS OF USE THEREOF
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[001] This application claims priority to, and incorporates by reference, U.S.
`
`provisional patent application 62/469,554, filed on March 10, 2017.
`
`FIELD OF THE INVENTION
`
`[002] The field of the present invention relates to certain curcumin-containing
`
`compositions and methods of use thereof, which can be used to increase low(cid:173)
`
`density lipoprotein cholesterol (LDL) receptor expression levels and thereby
`
`lower LDL levels in a plurality of cells or subject.
`
`In addition, the field of the
`
`present invention relates to certain curcumin-containing compositions and
`
`methods of use thereof, which can be used to modulate MSK1 production and
`
`thereby ameliorate a variety of health conditions.
`
`BACKGROUND OF THE INVENTION
`
`[003] The health benefits of curcumin, particularly whole turmeric extract, are
`
`known and have been demonstrated by researchers in recent years. However,
`
`several challenges continue to exist, with respect to the formulation of curcumin(cid:173)
`
`based pharmaceuticals and dietary supplements. More specifically, the most
`
`common source of curcumin,
`
`the
`
`Indian spice
`
`turmeric (a member of
`
`Zingiberaceae), does not contain a sufficient amount of curcumin to provide an
`
`efficacious dose to a subject. In fact, the therapeutic benefits provided by natural
`
`curcumin extracts have been relatively modest, very inconsistent, and not well
`
`understood. Accordingly, there is a continuing need for improved curcumin(cid:173)
`
`based formulations, which address these current challenges.
`
`1
`
`SAB1003
`U.S. Pat. No. 10,945,970
`
`
`
`PCT/CA2018/050275
`
`[004]
`
`The present invention, as described further below, addresses many of
`
`the foregoing challenges.
`
`SUMMARY OF THE INVENTION
`
`[005] According to certain aspects of the present invention, methods for
`
`increasing low-density lipoprotein cholesterol (LDL) receptor expression levels in
`
`a plurality of cells are disclosed (and, by extension, methods of reducing LDL
`
`levels in the cells).
`
`In certain embodiments, the methods comprise providing to
`
`the cells a composition that includes an effective and enriched amounts of
`
`curcumin 11, curcumin 111, or a combination of curcumin II and curcumin Ill.
`
`In
`
`certain embodiments, the composition is at least 15% (w/v) curcumin II or,
`
`preferably, at least 30% (w/v) curcumin II or, more preferably, at least 50% (w/v)
`
`curcumin II or, even more preferably, at least 70% (w/v) curcumin 11, such as at
`
`least 90% (w/v) curcumin II.
`
`In other embodiments, the composition is at least
`
`5% (w/v) curcumin Ill or, preferably, at least 30% (w/v) curcumin Ill or, more
`
`preferably, at least 50% (w/v) curcumin Ill or, even more preferably, at least 70%
`
`(w/v) curcumin Ill, such as at least 90% (w/v) curcumin Ill.
`
`In still further
`
`embodiments, the composition includes a combination of the curcumin II and
`
`curcumin Ill enriched compositions summarized above. As described and
`
`exemplified further herein, the invention provides that administration of such
`
`curcumin II and curcumin Ill enriched compositions elevates LDL receptor levels
`
`in a plurality of cells - which results in lower LDL levels in the cells and/or subject
`
`(which produces a number of therapeutic and health benefits).
`
`2
`
`
`
`PCT/CA2018/050275
`
`[006] According to further aspects of the present invention, LDL receptor(cid:173)
`
`modulating therapeutic compositions are disclosed that comprise a curcumin
`
`composition that
`
`includes at
`
`least 15% (w/v) curcumin
`
`II, along with a
`
`pharmaceutically acceptable solvent, filler, or carrier. The invention provides that
`
`while the curcumin composition employed may comprise 15% (w/v) curcumin II,
`
`in certain preferred embodiments, the curcumin composition employed may
`
`comprise at least 30% (w/v) curcumin II. Still more preferably, the invention
`
`provides that the curcumin composition may comprise at least 50% (w/v)
`
`curcumin 11, at least 70% (w/v) curcumin II or, even more preferably, at least 90%
`
`(w/v) curcumin II.
`
`[007] According to still further aspects of the present invention, LDL receptor(cid:173)
`
`modulating therapeutic compositions are disclosed that include a curcumin
`
`composition that includes at least 5% (w/v) curcumin Ill and a pharmaceutically
`
`acceptable solvent, filler, or carrier. The invention provides that while the
`
`curcumin composition employed may comprise 5% (w/v) curcumin Ill, in certain
`
`preferred embodiments, the curcumin composition employed may comprise at
`
`least 30% (w/v) curcumin Ill. Still more preferably, the invention provides that the
`
`curcumin composition may comprise at least 50% (w/v) curcumin Ill, at least 70%
`
`(w/v) curcumin Ill or, even more preferably, at least 90% (w/v) curcumin Ill.
`
`[008] According
`
`to additional aspects of the
`
`invention, LDL
`
`receptor(cid:173)
`
`modulating therapeutic compositions are disclosed that include a combination of
`
`the curcumin II and curcumin Ill enriched compositions described above.
`
`3
`
`
`
`PCT/CA2018/050275
`
`[009] According
`
`to additional aspects of the present
`
`invention, certain
`
`curcumin Ill enriched compositions described herein may be used to further
`
`modulate mitogen- and stress-activated protein kinase 1 (MSK1 ), which is a
`
`nuclear kinase that plays a significant role in transcription regulation (which
`
`produces a number of therapeutic and health benefits).
`
`[001 0] The above-mentioned and additional features of the present invention
`
`are further illustrated in the Detailed Description contained herein.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0011] FIGURE 1: MTT assay results demonstrating HEK293 cell survival of
`
`approximately 80% for all three curcuminoids (ranging from 20 to 22 µg/ml of the
`
`applicable curcuminoid).
`
`[0012] FIGURE 2: MTT assay results demonstrating BV2 cell survival of
`
`approximately 80% for all three curcuminoids (ranging from 20 to 22 µg/ml of the
`
`applicable curcuminoid).
`
`[0013] FIGURE 3: measurements of cytoplasmic NFkB-p65 protein levels
`
`relative to total protein concentration in BV2 cell lines provided with curcumin
`
`extract, curcuminoid I, curcuminoid 11, curcuminoid 111, and controls.
`
`[0014] FIGURE 4: measurements of nuclear NFkB-p65 protein levels relative
`
`to total protein concentration in BV2 cell lines provided with curcumin extract,
`
`curcuminoid I, curcuminoid II, curcuminoid Ill, and controls.
`
`[0015] FIGURE 5:
`
`measurement of cytoplasmic NFkB p65
`
`that
`
`is
`
`phosphorylated at
`
`the serine 276 phosphosite,
`
`relative
`
`to
`
`total protein
`
`4
`
`
`
`PCT/CA2018/050275
`
`concentration in BV2 cell lines provided with curcumin extract, curcuminoid I,
`
`curcuminoid 11, curcuminoid 111, and controls.
`
`[0016] FIGURE 6: measurement of nuclear NFkB p65 that is phosphorylated at
`
`the serine 276 phosphosite, relative to total protein concentration in BV2 cell
`
`lines provided with curcumin extract, curcuminoid I, curcuminoid 11, curcuminoid
`
`Ill, and controls.
`
`[0017] FIGURE 7: measurement of cytoplasmic MSK1 protein levels relative to
`
`total protein concentration in BV2 cell lines provided with curcumin extract,
`
`curcuminoid I, curcuminoid II, curcuminoid Ill, and controls.
`
`[0018] FIGURE 8: measurement of nuclear MSK1 protein levels relative to total
`
`protein concentration
`
`in BV2 cell
`
`lines provided with curcumin extract,
`
`curcuminoid I, curcuminoid 11, curcuminoid Ill, and controls.
`
`[0019] FIGURE 9: measurement of cytoplasmic MSK1 that is phosphorylated
`
`at the serine 376 phosphosite, relative to total protein concentration in BV2 cell
`
`lines provided with curcumin extract, curcuminoid I, curcuminoid 11, curcuminoid
`
`111, and controls.
`
`[0020] FIGURE 10: measurement of nuclear MSK1 that is phosphorylated at
`
`the serine 376 phosphosite, relative to total protein concentration in BV2 cell
`
`lines provided with curcumin extract, curcumin0id I, curcuminoid II, curcuminoid
`
`Ill, and controls.
`
`[0021] FIGURE 11: measurement of LDL receptor expression levels in HepG2
`
`cells after treatment with 22 µg/ml curcuminoid I, curcuminoid II, curcuminoid 111,
`
`and controls.
`
`5
`
`
`
`PCT/CA2018/050275
`
`[0022] FIGURE 12: measurement of LDL receptor expression levels in HepG2
`
`cells after treatment with 30 µg/ml curcuminoid I, curcuminoid 11, curcuminoid 111,
`
`and controls.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0023] The following will describe, in detail, several preferred embodiments of
`
`the present invention. These embodiments are provided by way of explanation
`
`only, and thus, should not unduly restrict the scope of the invention.
`
`In fact,
`
`those of ordinary skill in the art will appreciate upon reading the present
`
`specification and viewing the present drawings that the invention teaches many
`
`variations and modifications, and that numerous variations of the invention may
`
`be employed, used and made without departing from the scope and spirit of the
`
`invention.
`
`[0024] According to certain preferred embodiments, the present invention
`
`includes certain curcumin-enriched compositions (and methods of using such
`
`compositions). More particularly,
`
`the present
`
`invention
`
`includes certain
`
`compositions that contain elevated and concentrated levels of (1) curcumin II
`
`(relative to the amount of curcumin II found in natural curcumin extract); (2)
`
`curcumin Ill (relative to the amount of curcumin Ill found in natural curcumin
`
`extract); or (3) a combination of curcumin II and curcumin Ill (relative to the
`
`amounts of curcumin II and curcumin Ill found in natural curcumin extract).
`
`Notably,
`
`the LDL
`
`receptor-modulating compositions described herein will
`
`preferably exclude curcumin I. The invention provides that such compositions
`
`can be used to increase low-density lipoprotein cholesterol (LDL) receptor
`
`6
`
`
`
`PCT/CA2018/050275
`
`expression levels in a plurality of cells (which, in turn, results in lower LDL levels
`
`in a subject and ameliorates a variety of associated health conditions and/or
`
`impart one or more associated health benefits).
`
`[0025]
`
`In addition, the invention provides that certain curcumin Ill enriched
`
`compositions described herein may be used to modulate mitogen- and stress(cid:173)
`
`activated protein kinase 1 (MSK1 ), which is a nuclear kinase that plays a
`
`significant role in transcription regulation. As described below, the invention
`
`provides that curcumin Ill (and not curcuminoids I and II) can be used to
`
`selectively and efficaciously inhibit cytoplasmic and nuclear MSK1 production,
`
`the inhibition of MSK1 serine376 phosphorylation, and inhibition of the recruitment
`
`of MSK1 at inflammatory gene promoters. The curcumin Ill compositions
`
`described herein - and related methods of using such compositions - provide a
`
`major step in the transactivation regulation of downstream transcription factors
`
`that are key to cell survival and recruitment of inflammatory and immune system
`
`events. For example, as demonstrated in the Examples below, the ability of the
`
`curcumin Ill compositions described herein to
`
`inhibit MSK1 production (or
`
`otherwise significantly reduce MSK1 levels) indicates that such compositions may
`
`also (indirectly) be used to modulate NFkB (nuclear factor kappa-light-chain(cid:173)
`
`enhancer of activated B cells) - the aberrant expression and transactivation of
`
`which has been linked to cancer, inflammation, and autoimmune diseases. The
`
`curcumin Ill compositions (and related methods) of the present invention provide
`
`improved efficacy, reliability, and drug target selectivity, relative to natural
`
`curcumin extracts.
`
`7
`
`
`
`PCT/CA2018/050275
`
`[0026] A natural curcumin extract comprises a mixture of curcumin
`
`I,
`
`desmethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin Ill).
`
`The term curcumin refers to the principal curcuminoid in the Indian spice turmeric
`
`plant (a member of Zingiberaceae). The IUPAC name for the curcumin I
`
`molecule
`
`is
`
`(1 E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-
`
`dione. Although curcumin I may exist in several different tautomeric forms, the
`
`enol form is illustrated below:
`
`HO
`
`~
`OH
`
`0
`
`The IUPAC name for the desmethoxycurcumin (curcumin II) molecule is (1 E,6E)-
`
`1-( 4-Hydroxy-3-methoxyphenyl)-7-( 4-hydroxyphenyl)hepta-1,6-diene-3,5-dione,
`
`and has the chemical structure shown below:
`
`0
`
`0
`
`OH
`
`The IUPAC name for bis-desmethoxycurcumin (curcumin Ill) that is used in the
`
`compositions and methods of the present
`
`invention
`
`is
`
`(1 E,6E)-1,7-bis(4-
`
`hydroxyphenyl)hepta-1,6-diene-3,5-dione, and has the chemical structure shown
`
`below:
`
`0
`
`0
`
`HO
`
`OH
`
`8
`
`
`
`PCT/CA2018/050275
`
`[0027] According to certain preferred embodiments, the invention provides that
`
`curcumin II and curcumin Ill may be extracted from turmeric plant rhizome
`
`(Curcuma
`
`longa) and subsequently concentrated
`
`to
`
`the desired
`
`levels.
`
`Alternatively, the invention provides that the curcumin II and curcumin Ill
`
`molecules may be chemically synthesized and used to formulate a therapeutic
`
`composition described herein. As explained below, the desired concentration of
`
`curcumin II is at least 15%, 30%, 50%, 70%, or 90% (w/v) curcumin II, while the
`
`desired concentration of curcumin Ill is at least 5%, 30%, 50%, 70%, or 90%
`
`(w/v) curcumin Ill.
`
`[0028] According to certain preferred embodiments of the present invention,
`
`methods for increasing LDL receptor expression levels (and thereby lowering
`
`LDL) in a plurality of cells (and subject) are provided. In such embodiments, the
`
`methods include providing to the cells (or administering to a biological system
`
`that comprises a plurality of cells) an effective amount of a LDL receptor(cid:173)
`
`modulating curcumin composition that is (1) at least 15% curcumin II; (2) at least
`
`5% curcumin Ill; or (3) a combination of (1) and (2). According to additional
`
`preferred embodiments of the present invention, methods for inhibiting MSK1
`
`serine376 phosphorylation in a plurality of cells are provided. Such methods
`
`include providing to the cells (or administering to a biological system that
`
`comprises a plurality of cells) an effective amount of a curcumin composition that
`
`is at least 5% curcumin Ill.
`
`[0029] The
`
`"effective amount" of a LDL
`
`receptor-modulating curcumin
`
`composition will preferably be sufficient to significantly increase LDL receptor
`
`9
`
`
`
`PCT/CA2018/050275
`
`expression levels ( such as by at least 10% relative to a control cell line or, even
`
`more preferably, by at least 20% relative to a control cell line), to thereby reduce
`
`the amount of LDL in the target cells (and subject). Similarly, the "effective
`
`amount" of a MSK1-modulating curcumin composition will preferably be sufficient
`
`to significantly reduce the amount of MSK1 protein being expressed in the target
`
`cells ( such as by at least 10% relative to a control cell line or, even more
`
`preferably, by at least 20% relative to a control cell line).
`
`[0030] According to certain preferred embodiments of the present invention,
`
`methods for preventing and/or ameliorating the effects of certain diseases
`
`associated with high cholesterol (LDL) levels are provided. Such methods
`
`generally include providing to a subject an effective amount of the curcumin II
`
`and/or curcumin
`
`Ill enriched compositions described herein. Non-limiting
`
`examples of such diseases include cardiovascular diseases (including heart
`
`disease, stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis,
`
`and serum LDL elevation), diabetes, and high blood pressure.
`
`[0031] According to yet further preferred embodiments of the present invention,
`
`methods for preventing and/or ameliorating the effects of an adverse medical
`
`condition in which MSK1 is implicated are provided, including glucocorticoid(cid:173)
`
`resistant inflammatory diseases and chemotherapy-resistant cancers.
`
`In such
`
`embodiments,
`
`the methods
`
`include providing
`
`to a subject a curcumin
`
`composition that is at least 5% curcumin Ill (or, alternatively, at least 30%, 50%,
`
`70%, or 90% (w/v) curcumin Ill). According to certain related embodiments of
`
`the present invention, therapeutic compositions are provided that include a
`
`10
`
`
`
`PCT/CA2018/050275
`
`curcumin composition consisting of at least 5% (w/v) curcumin Ill; optionally,
`
`glucocorticoids; and a pharmaceutically acceptable solvent, filler, or carrier. As
`
`used herein, "glucocorticoids" refers to certain steroid hormones that are known
`
`to bind
`
`to glucocorticoid
`
`receptors
`
`(RCEs).
`
`Non-limiting examples of
`
`glucocorticoids
`
`include:
`
`cortisol,
`
`cortisone, prednisone, prednisolone,
`
`methylprednisolone,
`
`dexamethasone,
`
`betamethasone,
`
`triamcinolone,
`
`beclometasone, fludrocortisone, deoxycorticosterone, and aldosterone.
`
`In the
`
`foregoing embodiments of the
`
`invention, while the curcumin composition
`
`employed may comprise 5% (w/v) curcumin Ill, in certain preferred embodiments,
`
`the curcumin composition employed may comprise at least 30% (w/v) curcumin
`
`Ill. Still more preferably, the invention provides that the curcumin composition
`
`may comprise at least 50% (w/v) curcumin Ill, at least 70% (w/v) curcumin Ill or,
`
`even more preferably, at least 90% (w/v) curcumin Ill - - depending on the
`
`desired potency.
`
`[0032] The invention provides that the concentrated forms of the curcumin Ill
`
`based compositions ( and methods of using such compositions) described herein
`
`exhibit many benefits -
`
`for humans, canines, cats and equine.
`
`First, as
`
`demonstrated below and described herein, the invention provides that elevated
`
`levels of curcumin Ill will selectively inhibit MSK1 production, which thereby
`
`produces desirable anti-inflammatory activity.
`
`In addition, the invention provides
`
`that the compositions and methods described herein may be used for therapeutic
`
`nutrition; anti-inflammatory therapy for autoimmune disease and other chronic
`
`and acute inflammatory ailments; treatment of pain, swelling and inflammation;
`
`11
`
`
`
`PCT/CA2018/050275
`
`nutritional supplementation; superbug treatments; and antimicrobial, antifungal,
`
`antibacterial, and antiviral therapies.
`
`[0033] Still further, according to certain additional embodiments, the present
`
`invention encompasses therapeutic compositions (and methods of use thereof)
`
`that include a curcumin composition consisting of at least 15% curcumin II, along
`
`with a pharmaceutically acceptable solvent,
`
`filler, or carrier.
`
`In such
`
`embodiments, as described above, the curcumin II-enriched compositions can be
`
`used to increase LDL receptor expression levels and thereby lower LDL levels in
`
`target cells and/or a subject. Indeed, as shown in the Example below, curcumin
`
`II-enriched and curcumin Ill-enriched compositions can be used to increase LDL
`
`receptor expression levels (and, therefore, lower LDL levels in target cells and/or
`
`a subject).
`
`[0034]
`
`In certain specific embodiments,
`
`the compositions and methods
`
`described herein may also be used to ameliorate the effects of autoimmune
`
`diseases (and other inflammatory conditions), such as rheumatoid arthritis,
`
`colitis, non-specific inflammatory bowel diseases, crohn's disease, lupus, multiple
`
`sclerosis, psoriasis, type-I diabetes, diabetes, myocarditis, thyroiditis, uveitis,
`
`systemic lupus erythromatosis, myasthenia and gravis.
`
`Furthermore,
`
`the
`
`compositions and methods described herein may be used to ameliorate the
`
`effects of autoimmune syndromes, such as the sources of immune-mediated
`
`inflammation (which can promote chronic inflammation, Alzheimer's, asthma,
`
`allergies, obesity, chronic fatigue, fibromyelia, premature aging, and general
`
`memory impediments). Still further, the compositions and methods may be used
`
`12
`
`
`
`PCT/CA2018/050275
`
`for the purpose of performance enhancement; recovery from physical exercise;
`
`and
`
`to help neutralize
`
`lactic acid, oxidation and associated
`
`inflammatory
`
`responses to workload to improve recovery rate, anabolism, reduce post-workout
`
`soreness and associated fatigue (and allow for repeat workout sessions earlier
`
`than could otherwise be executed in typical workout and training cycles).
`
`[0035] The invention provides that the compositions described herein may be
`
`administered in any desired and effective manner, e.g., as pharmaceutical
`
`compositions or nutritional supplements for oral ingestion. More particularly, for
`
`example, pharmaceutically acceptable compositions or nutritional supplements of
`
`the invention may comprise one or more of the compositions described herein
`
`with one or more acceptable carriers. Regardless of the route of administration
`
`selected, the compositions may be formulated into acceptable dosage forms by
`
`conventional methods known to those of skill in the art. For example, acceptable
`
`carriers include, but are not limited to, sugars (e.g., lactose, sucrose, mannitol,
`
`and sorbitol), silicon dioxide, starches, cellulose preparations
`
`(such as
`
`microcrystalline cellulose), calcium phosphates (e.g., dicalcium phosphate,
`
`tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water,
`
`aqueous solutions, alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl
`
`alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),
`
`organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers
`
`(e.g.,
`
`polylactide-polyglycolide,
`
`poly(orthoesters),
`
`and
`
`poly(anhydrides)),
`
`elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive,
`
`13
`
`
`
`PCT/CA2018/050275
`
`castor, sesame, cottonseed, and groundnut), cocoa butter, waxes, paraffins,
`
`silicones, talc, silicylate, etc.
`
`[0036] Each acceptable carrier used
`
`in a pharmaceutical composition or
`
`nutritional supplement of the invention must be "acceptable" in the sense of being
`
`compatible with the other ingredients of the formulation and not injurious to the
`
`subject. Carriers suitable for a selected dosage form and intended route of
`
`administration are well known in the art, and acceptable carriers for a chosen
`
`dosage form and method of administration can be determined using ordinary skill
`
`in the art.
`
`[0037] The pharmaceutical compositions and nutritional supplements of the
`
`invention may, optionally, contain additional
`
`ingredients and/or materials
`
`commonly used in pharmaceutical compositions and/or nutritional supplements.
`
`Such ingredients and materials include (1) fillers or extenders, such as starches,
`
`lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as
`
`carboxymethylcellulose,
`
`alginates,
`
`gelatin,
`
`polyvinyl
`
`pyrrolidone,
`
`hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as
`
`glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato
`
`or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross(cid:173)
`
`linked sodium carboxy methyl cellulose and sodium carbonate; (5) solution
`
`retarding agents, such as paraffin; (6) absorption accelerators, such as
`
`quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and
`
`glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9)
`
`lubricants, such as
`
`talc, calcium stearate, magnesium stearate, solid
`
`14
`
`
`
`PCT/CA2018/050275
`
`polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as
`
`ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
`
`microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and
`
`tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars,
`
`polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa
`
`butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones,
`
`bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium
`
`silicates, and polyamide powder; (13) inert diluents, such as water or other
`
`solvents; ( 14) preservatives; ( 15) surface-active agents; ( 16) dispersing agents;
`
`(17) control-release or absorption-delaying agents, such as hydroxypropylmethyl
`
`cellulose, other polymer matrices, biodegradable polymers,
`
`liposomes,
`
`microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying
`
`agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending
`
`agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl
`
`alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
`
`propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,
`
`corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
`
`polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as
`
`chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as
`
`butane and propane; (24) antioxidants; (25) agents which render the formulation
`
`isotonic with the blood of the intended recipient, such as sugars and sodium
`
`chloride; (26) thickening agents; (27) coating materials, such as lecithin; (28)
`
`vitamins and minerals; (29) proteins that carry therapeutic or nutritional benefits,
`
`15
`
`
`
`PCT/CA2018/050275
`
`such as whey protein and other milk-derived proteins; and (30) sweetening,
`
`flavoring, coloring, perfuming and preservative agents. Each such ingredient or
`
`material must be "acceptable" in the sense of being compatible with the other
`
`ingredients of the formulation and not injurious to the subject.
`
`Ingredients and
`
`materials suitable
`
`for a selected dosage
`
`form and
`
`intended
`
`route of
`
`administration are well known in the art, and acceptable ingredients and
`
`materials for a chosen dosage form and method of administration may be
`
`determined using ordinary skill in the art.
`
`[0038] Pharmaceutical compositions and nutritional supplements suitable for
`
`oral administration may be in the form of capsules, cachets, pills, tablets,
`
`powders, granules, a solution or a suspension in an aqueous or non-aqueous
`
`liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, or a paste.
`
`These formulations may be prepared by methods known in the art, e.g., by
`
`means of conventional pan-coating, mixing, granulation or
`
`lyophilization
`
`processes.
`
`[0039] Solid dosage forms for oral administration ( capsules, tablets, pills,
`
`powders, granules and
`
`the
`
`like) may be prepared by mixing the active
`
`ingredient(s) with one or more acceptable carriers and, optionally, one or more
`
`fillers, extenders, binders, humectants, disintegrating agents, solution retarding
`
`agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or
`
`coloring agents. Solid compositions of a similar type may be employed as fillers
`
`in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be
`
`made by compression or molding, optionally with one or more accessory
`
`16
`
`
`
`PCT/CA2018/050275
`
`ingredients. Compressed tablets may be prepared using a suitable binder,
`
`lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing
`
`agent. Molded tablets may be made by molding in a suitable machine. The
`
`tablets, and other solid dosage forms, such as capsules, pills and granules, may
`
`optionally be scored or prepared with coatings and shells, such as enteric
`
`coatings and other coatings well known in the art. The tablets, and other solid
`
`dosage forms, may also be formulated so as to provide slow or controlled release
`
`of the active ingredient therein. They may be sterilized by, for example, filtration
`
`through a bacteria-retaining filter. These compositions may also optionally
`
`contain opacifying agents that release the active ingredient only, or preferentially,
`
`in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
`
`The active ingredient can also be in a microencapsulated form.
`
`[0040] Liquid dosage
`
`forms
`
`for oral administration
`
`include acceptable
`
`emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. The
`
`liquid dosage forms may contain suitable inert diluents commonly used in the art.
`
`Besides inert diluents, the oral compositions may also include adjuvants, such as
`
`wetting agents, emulsifying and suspending agents, sweetening, flavoring,
`
`coloring, perfuming and preservative agents.
`
`Suspensions may contain
`
`suspending agents.
`
`EXAMPLES
`
`[0041] Example 1 - MTT Assay. MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-
`
`diphenyltetrazolium bromide) assays are routinely used to measure cell viability
`
`and survival.
`
`In this Example, cytotoxicity of curcuminoids on two cell types -
`
`17
`
`
`
`PCT/CA2018/050275
`
`HEK293T and BV2 microglia - was measured. The MTT assay quantifies the
`
`formazan production by live cells from the tetrazolium ring cleavage of MTT.
`
`Reduction of MTT is directly proportional to metabolic activity and therefore
`
`relatable to cell viability and survival. A first MTT assay was performed on
`
`HEK293T cells in a 96-well plate requiring 3 x 104 cells per well. The MTT assay
`
`was also performed using BV2 microglia cells, pursuant to the same protocol
`
`(utilizing a 96-well plate requiring 3 x 104 cells per well). Dimethyl sulfoxide
`
`(DMSO) was used in the test drug (curcuminoid) preparation at 0.2%. The MTT
`
`assay was used to measure the health of the cells in culture with various
`
`treatment concentrations of various curcumin preparations.
`
`[0042] As shown in Figure 1 (HEK293T cells) and Figure 2 (BV2 microglia), the
`
`MTT assay results revealed that the selected cell models are relatively resilient to
`
`the curcuminoid drugs at the tested concentrations. Cell survival was shown to
`
`begin to decline below 80% survival at a drug ( curcuminoid) concentration
`
`around 40 µg/ml. Accordingly, a final test concentration of 22 µg/ml was selected
`
`and employed in the Examples that follow.
`
`[0043] Example 2 - Western Blot Analysis of Cytoplasmic and Nuclear
`
`MSK1 Levels. Western blot analysis was performed in multiple varying formats
`
`before optimization was achieved. The BV2 microglia cell line was cultured in
`
`Dulbecco's Modified Eagle's Medium (DMEM) - complete medium.
`
`The
`
`complete medium consisted of DMEM, 1 % Ampicillin, and 10% Fetal Bovine
`
`Serum (FBS). BV2 microglia cells, at a cell count of approximately 2 x 106
`
`, were
`
`seeded in each well (6 wells per plate) with 2.0 ml complete medium and cultured
`
`18
`
`
`
`PCT/CA2018/050275
`
`overnight in a ThermaForma HepaFilter Series II Co2 Incubator at 37° Celsius.
`
`Upon establishing confluence, subconfluent cells were washed out and the wells
`
`were prepared with drug pre-treatment after overnight incubation.
`
`[0044] The test drugs (curcuminoids) were procured as follows: curcumin I
`
`research standard (03926) (ChromaDex
`
`Irvine, CA USA) (97.7% purity);
`
`curcumin II research standard (04230) (ChromaDex Irvine, CA USA) (97.3%);
`
`curcumin Ill research standard (86938) (Sigma-Aldrich St. Louis, Missouri, USA)
`
`(97.7% purity); curcumin extract (curcumin I - 77.7%, curcumin II - 16.9%,
`
`curcumin Ill - 0.9%) research standard (03928) (ChromaDex Irvine, CA USA)
`
`(95.3% purity); and Lipopolysaccharide (LPS) from E. Coli (L2630) (Sigma(cid:173)
`
`Aldrich St. Louis, Missouri, USA).
`
`[0045] Curcuminoids are not soluble
`
`in aqueous medium due
`
`to
`
`their
`
`hydrophobic characteristic. However, curcuminoid extracts are soluble in polar
`
`organic solvents, such as DMSO and acetone.
`
`In
`
`this Example, each
`
`curcuminoid preparation was first dissolved in DMSO. DMSO was used in the
`
`drug preparation at 0.2%. The drug/DMSO solution was subsequently dissolved
`
`in DMEM to achieve a final drug concentration for each curcuminoid preparation
`
`tested - 22.0 µg/ml curcuminoid. The DMEM/drug solution was used to replace
`
`the culture DMEM well medium and incubated for 30 minutes at 37-degrees
`
`Celsius in a ThermaForma incubator. At 31 minutes, lipopolysaccharide (LPS at
`
`1.0 µI/ml final well concentration) induction of the cells was executed, except for
`
`the DMSO-only well to stimulate cell response amidst drug pre-treatment and
`
`without drug treatment.
`
`19
`
`
`
`PCT/CA2018/050275
`
`[0046] The plates were then incubated for another 30 minutes after LPS
`
`stimulation. Upon removal from incubation, the cell medium was carefully
`
`removed and cells were washed, scraped, and collected with phosphate-buffered
`
`saline
`
`(PBS).
`
`Using a ThermoFisher Scientific NE-PER Nuclear and
`
`Cytoplasmic E
Accessing this document will incur an additional charge of $.
After purchase, you can access this document again without charge.
Accept $ Charge
Still Working On It
This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.
Give it another minute or two to complete, and then try the refresh button.
A few More Minutes ... Still Working
It can take up to 5 minutes for us to download a document if the court servers are running slowly.
Thank you for your continued patience.
This document could not be displayed.
We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.
Your account does not support viewing this document.
You need a Paid Account to view this document. Click here to change your account type.
Your account does not support viewing this document.
Set your membership
status to view this document.
With a Docket Alarm membership, you'll
get a whole lot more, including:
- Up-to-date information for this case.
- Email alerts whenever there is an update.
- Full text search for other cases.
- Get email alerts whenever a new case matches your search.
One Moment Please
The filing “” is large (MB) and is being downloaded.
Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.
Your document is on its way!
If you do not receive the document in five minutes, contact support at support@docketalarm.com.
Sealed Document
We are unable to display this document, it may be under a court ordered seal.
If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.
Access Government Site
