WO2008134766A1 - Applications of ubiquinones and ubiquinols - Google Patents
Applications of ubiquinones and ubiquinols Download PDFInfo
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- WO2008134766A1 WO2008134766A1 PCT/US2008/062123 US2008062123W WO2008134766A1 WO 2008134766 A1 WO2008134766 A1 WO 2008134766A1 US 2008062123 W US2008062123 W US 2008062123W WO 2008134766 A1 WO2008134766 A1 WO 2008134766A1
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- ubiquinol
- cell
- ubiquinone
- exemplary embodiment
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- 0 C1C2(*CC3)C3CCC12 Chemical compound C1C2(*CC3)C3CCC12 0.000 description 2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Definitions
- the present invention relates to cell culture medium compositions comprising lipophilic compounds and solubilizing agent and methods of using such compositions for the culturing of cells.
- the invention also relates to the prevention and/or treatment of diseases of the nervous system by administration to a subject in need thereof with a pharmaceutical formulation including an effective amount of a ubiquinone and/or a ubiquinol mixed with an agent that renders the ubiquinone and/or a ubiquinol soluble in water.
- Cell culture media provide nutrients for maintaining and/or growing cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements and any functions for which the cells are cultured. Important parameters generally include osmolality, pH, and nutrient formulations.
- the normal environment of a cell in culture is an aqueous medium in which nutrients and other culture components are dissolved or suspended. Especially advantageous is incorporation of useable quantities of lipid soluble or other components that are only sparsely soluble in water.
- the cells are kept alive and stimulated to produce the target proteins through precise culture conditions that include a balance of temperature (which can often vary by no more than one degree Celsius), oxygen, acidity (if pH levels change by even a small fraction, cells can easily die), media components and other variables.
- temperature which can often vary by no more than one degree Celsius
- oxygen if oxygen, acidity (if pH levels change by even a small fraction, cells can easily die)
- media components if media components change by even a small fraction, cells can easily die
- the proteins are isolated from the cultures, stringently tested at every step of purification, and formulated into pharmaceutically-active products.
- Cell culture media supplemented with compounds having therapeutic efficacy provides a test environment suitable for determining the effect of these compounds on cell viability, growth, division, integrity, etc. Accordingly, conclusions can be drawn regarding the efficacy of therapeutic compounds on the treatment of a disease of a cell from the culture of that cell in the culture medium supplemented with the therapeutic compound.
- cell culture medium that includes a therapeutic compound solubilized in the aqueous medium is of use to investigate the efficacy of the therapeutic compound in treating, ameliorating or preventing a disease afflicting the cell or cell type in the culture.
- the present invention provides a novel aqueous cell culture medium including compounds solubilized in the medium, which are generally considered to be essentially insoluble in aqueous media.
- any therapeutic agent can be incorporated into the media, though the media is considered to have particular utility with respect to agents that are essentially insoluble or only sparingly soluble in aqueous media.
- the invention is exemplified by reference to the essentially water-insoluble ubiquinones, particularly coenzyme QlO. The focus on this coenzyme is intended to exemplify, and not limit, the invention. Those of skill will appreciate that the cell culture media of the invention.
- the invention provides a water-soluble cell culture medium supplement composition
- a water-soluble cell culture medium supplement composition comprising a) a solubilizing agent; and b) a ubiquinone/ubiquinol having a structure which is described herein.
- An exemplary ubiquinone/ubiquinol of use in the invention has a structure which is a member selected from Formula (I) and Formula (II):
- n is an integer from 0 to 20. In exemplary embodiments, n ranges from 1 to 13.
- Exemplary ubiquinols/ubiquinones include CoQio, ubiquinol-50, or reduced CoQio.
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy. In various embodiments, R 1 is methyl. In exemplary embodiments, R 2 and R 3 are methoxy.
- the solubilizing agent has a structure according to the following formula: wherein a, b and c are integers independently selected from 0 and 1.
- Z is a hydrophobic moiety which is a member selected from tocopherol, sterol, ubiquinone and ubiquinol.
- Y 1 and Y 2 are hydrophilic moieties, which are members selected from polyethers, polyalcohols and derivatives thereof; and L 1 and L 2 are linkers.
- the solubilizing agent is polyoxyethanyl ⁇ -tocopheryl sebacate (PTS), polyoxyethanyl ⁇ -sitosteryl sebacate (PSS) or polyoxyethanyl-ubiquinol- sebacate (PQS).
- PTS polyoxyethanyl ⁇ -tocopheryl sebacate
- PSS polyoxyethanyl ⁇ -sitosteryl sebacate
- PQS polyoxyethanyl-ubiquinol- sebacate
- the cell culture supplement can be prepared separately, or combined with other culture media, such a basal culture medium, for example, RPMI 1640, Dulbecco's modified Eagle's Medium, and Ham's F 12.
- a basal culture medium for example, RPMI 1640, Dulbecco's modified Eagle's Medium, and Ham's F 12.
- the pharmaceutical formulation including solubilized therapeutic e.g., ubiquinone and/or ubiquinol
- can be prepared and optionally combined with other components of the pharmaceutical formulation can be prepared and optionally combined with other components of the pharmaceutical formulation (dispersants, diluents, carriers, solvents, etc.).
- the cell culture supplements and culture media provided herein are of use to culture a variety of cells, e.g., neuron, glia (e.g. Schwann cell) and Chinese Hamster Ovary (CHO) cells.
- the cell can be a primary cell culture cell, or derived from a cell line.
- the present invention also pertains to pharmaceutical compositions containing, in an aqueous medium, solubilized ubiquinols, e.g., coenzyme Q, brought into aqueous solution with one or more solubilizing agents. Also provided are methods of using such compositions for the prevention and treatment of peripheral neuropathy.
- FIG. 1 shows that coenzyme QlO (H Q OTM) promotes neurite growth.
- FIG. IA depicts the concentration effect curve for Coenzyme QlO to promote DRG neurite growth over 22 days in culture.
- FIG. IB depicts the concentration effect curve for Coenzyme QlO to promote DRG neurite growth over 11 days in culture.
- FIG. 1C depicts the concentration effect curve for Coenzyme QlO to promote DRG neurite growth on day 11 in culture.
- FIG. ID shows enhanced growth starting at day 8 out to day 22.
- FIG. 2 shows that Coenzyme Qi 0 (H Q OTM) reduces neurotoxicity induced by exposure to NRTIs.
- alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated ⁇ i.e. C 1 -C 10 means one to ten carbons).
- saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.
- An unsaturated alkyl group is one having one or more double bonds or triple bonds.
- alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
- alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.”
- Alkyl groups, which are limited to hydrocarbon groups are termed "homoalkyl".
- alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
- an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
- a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
- alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
- heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
- the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
- heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 - CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
- heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R'- represents both -C(O) 2 R'- and -R 5 C(O) 2 -.
- an "acyl substituent” is also selected from the group set forth above.
- acyl subsituent refers to groups attached to, and fulfilling the valence of a carbonyl carbon that is either directly or indirectly attached to the polycyclic nucleus of the compounds of the present invention.
- cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl” and
- heteroalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
- cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like.
- heterocycloalkyl examples include, but are not limited to, 1 -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, A- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
- halo or halogen
- haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
- terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
- halo(Ci-C4)alkyl is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
- aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently.
- heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
- Non- limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquino
- aryl when used in combination with other terms ⁇ e.g. , aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
- arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like).
- alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
- an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthy
- R', R", R'" and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
- each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
- R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
- - NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
- alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g. , -CF 3 and -CH 2 CF 3 ) and acyl (e.g. , -C(O)CH 3 , -C(O)CF 3 , - C(O)CH 2 OCH 3 , and the like).
- Two of the aryl substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
- two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of from 1 to 4.
- One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
- two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')s-X-(CR"R'")d-, where s and d are independently integers of from 0 to 3, and X is -O- , -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
- the substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (Ci-C6)alkyl.
- heteroatom includes oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).
- labeling moiety refers to a moiety, which provides a signal that is detectable by a detection method known in the art.
- the signal can be used to determine the location or concentration of the labeling moiety, for example, in an organism, a tissue sample or a reaction vial.
- Exemplary signals include color, emitted light of any wavelength, radioactivity, or any other electromagnetic or quantum mechanical effect.
- Exemplary labeling moieties include but are not limited to fluorescent molecules (e.g. fluorescein), luminescent moieties (e.g., transition-metal complexes), chemo luminescent molecules, molecules used in colorimetric applications (i.e.
- targeting moiety refers to a moiety which is capable of binding to a particular tissue- or cell-type (e.g., tumor cells, neuronal or glial cells, liver cells, Chinese Hamster Ovary (CHO) cells and the like) with at least some level of specificity.
- tissue- or cell-type e.g., tumor cells, neuronal or glial cells, liver cells, Chinese Hamster Ovary (CHO) cells and the like
- exemplary targeting moieties are selected from carbohydrates, proteins, peptides, antibodies, and small- molecule ligands.
- the targeting moiety is a ligand for a biological receptor, such as a cell surface receptor.
- the targeting moiety is an antibody that is capable of binding to an antigen, such as a tissue- or tumor-specific antigen.
- drug moiety refers to pharmaceutical drugs and other biologically active molecules.
- drug moiety includes small-molecule drugs as well as biologies, including peptides, mutant and wild-type polypeptides, mutant and wild-type proteins, antibodies (e.g., humanized, monoclonal antibodies) and the like.
- water-soluble refers to moieties that have a detectable degree of solubility in water. Methods to detect and/or quantify water solubility are well known in the art.
- Exemplary water-soluble polymers include peptides, saccharides, poly(ethers), poly(amines), poly(carboxylic acids) and the like. Peptides can have mixed sequences of be composed of a single amino acid, e.g., poly(lysine), poly(aspartic acid), and poly(glutamic acid).
- An exemplary polysaccharide is poly(sialic acid).
- An exemplary poly(ether) is poly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplary polyamine, and poly(acrylic) acid is a representative poly(carboxylic acid).
- cell culture refers to the maintenance of cells in an artificial, e.g., an in vitro environment. It is to be understood, however, that the term “cell culture” is a generic term and may be used to encompass the cultivation not only of individual prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant and fungal) cells, but also of tissues, organs, organ systems or whole organisms, for which the terms “tissue culture,” “organ culture,” “organ system culture” or “organotypic culture” may occasionally be used interchangeably with the term “cell culture.”
- prokaryotic e.g., bacterial
- eukaryotic e.g., animal, plant and fungal
- the term “cultivation” refers to the maintenance of cells in an artificial environment under conditions favoring growth, differentiation, biologic production or continued viability, in an active or quiescent state, of the cells. Thus, “cultivation” may be used interchangeably with “cell culture” or any of its synonyms described above. [0038]
- the term “cell culture medium,” “culture medium” (plural “media” in each case) and “medium formulation” refer to a nutritive solution that supports the cultivation and/or growth of cells; these phrases may be used interchangeably.
- the term "contacting” refers to the placing of cells to be cultivated into a culture vessel with the medium in which the cells are to be cultivated.
- the term “contacting” encompasses inter alia mixing cells with medium, perfusing cells or organs with medium, pipetting medium onto cells in a culture vessel, and submerging cells in culture medium.
- culture vessel refers to a glass, plastic, or metal container that can provide an aseptic environment for culturing cells.
- the term "basal medium” refers to a solution of amino acids, vitamins, salts, and nutrients that is effective to support the growth of cells in culture, although normally these compounds will not support cell growth unless supplemented with additional compounds.
- the nutrients include a carbon source (e.g., a sugar such as glucose) that can be metabolized by the cells, as well as other compounds necessary for the cells' survival.
- DMEM Dulbecco's Modified Eagle Media
- KO-DMEM Knockout- DMEM
- DMEM/F12 Dulbecco's Modified Eagle Media
- conditioned medium refers to a growth medium that is further supplemented with soluble factors derived from cells cultured in the medium. Techniques for isolating conditioned medium from a cell culture are well known in the art. As will be appreciated, conditioned medium is preferably essentially cell-free. In this context,
- essentially cell-free refers to a conditioned medium that contains fewer than about 10%, preferably fewer than about 5%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001% than the number of cells per unit volume, as compared to the culture from which it was separated.
- defined medium refers to a biochemically defined formulation comprised solely of the biochemically-defined constituents.
- a defined medium may include solely constituents having known chemical compositions.
- a defined medium may also include constituents that are derived from known sources.
- a defined medium may also include factors and other compositions secreted from known tissues or cells; however, the defined medium will not include the conditioned medium from a culture of such cells.
- a "defined medium” may, if indicated, include particular compounds added to form the culture medium, up to and including a portion of a conditioned medium that has been fractionated to remove at least one component detectable in a sample of the conditioned medium that has not been fractionated.
- substantially remove of one or more detectable components of a conditioned medium refers to the removal of at least an amount of the detectable, known component(s) from the conditioned medium so as to result in a fractionated conditioned medium that differs from an unfractionated conditioned medium in its ability to support the long-term substantially undifferentiated culture of primate stem cells.
- Fractionation of a conditioned medium can be performed by any method (or combination of methods) suitable to remove the detectable component(s), for example, gel filtration chromatography, affinity chromatography, immune precipitation, etc.
- a "defined medium” may include serum components derived from an animal, including human serum components.
- known refers to the knowledge of one of ordinary skill in the art with reference to the chemical composition or constituent.
- complete medium refers to a medium that provides all the necessary nutrients for a cell to grow in a cell culture.
- a serum- free medium may also be a defined medium, though some serum- free medium contain discrete proteins or bulk proteins.
- binder refers to a composition that is present in granular form, which may or may not be complexed or agglomerated with a solvent such as water or serum.
- dry powder may be used interchangeably with the term “powder;” however, “dry powder” as used herein simply refers to the gross appearance of the granulated material and is not intended to mean that the material is completely free of complexed or agglomerated solvent unless otherwise indicated.
- the term "ingredient” refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the growth of proliferation of cells.
- component e.g., fetal calf serum
- ingredient e.g., calf serum, calf serum, calf serum, calf serum, calf serum, calf serum, calf serum, calf serum, calf serum, calfate, calfate, calfate, calfate, glycerol, glycerin, fitol, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate, calfate,
- cytokine refers to a compound that induces a physiological response in a cell, such as growth, differentiation, senescence, apoptosis, cytotoxicity, synthesis or transport, immune response or antibody secretion. Included in this definition of "cytokine” are growth factors, interleukins, colony-stimulating factors, interferons, thromboxanes, prostaglandins, hormones and lymphokines.
- extract refers to a composition comprising a purified, partially purified or concentrated preparation of the subgroups of a substance, typically formed by treatment of the substance either mechanically (e.g., by pressure treatment) or chemically (e.g., by distillation, solublization, precipitation, enzymatic action or high salt treatment).
- enzyme digest refers to a composition comprising a specialized type of extract, namely one prepared by treating the substance to be extracted (e.g., plant components or yeast cells) with at least one enzyme capable of breaking down the components of the substance into simpler forms (e.g., into a preparation comprising mono- or disaccharides and/or mono-, di- or tripeptides).
- substance to be extracted e.g., plant components or yeast cells
- enzyme capable of breaking down the components of the substance into simpler forms e.g., into a preparation comprising mono- or disaccharides and/or mono-, di- or tripeptides.
- hydrolysate may sometimes be used interchangeably with the term “enzymatic digest.”
- lipid has its meaning as generally understood in biochemistry.
- “Lipid” also refers to a portion of the cell or an ingredient of a medium that is soluble in non-polar or non-aqueous solvent.
- the lipid may be sparsely soluble or insoluble in water in the presence or absence of other medium ingredients.
- Lipid may be soluble in a solvent mixture that includes water and one or more organic solvents.
- Lipids may comprise fatty acids, hormones, metabolites, cytokines, vitamins, indicators, stimulators or inhibitors.
- Lipid in some contexts may refer to ingredients that are normally insoluble or sparsely soluble in water, but that have been converted, e.g., by saponification hydroxylation, etc., to a forma compound or ion that is water soluble.
- a fatty acid is a lipid, but also a salt of a fatty acid is to be included in the definition.
- lipid is used generically to mean generally any component that is advantageously introduced using organic or non- polar solvents or that is not normally soluble in water or aqueous media. Lipids may be present as dissolved molecules, or in other forms such as micelles or other loose associations of molecules.
- a lipid may be used as a free molecule or may be bound to one or more other molecules.
- proteins or peptides may be associated with one or more other lipids for stability and/or to aid in delivery to the agglomerated powder.
- Lipid may also refer to an ingredient that might act as a drug to inhibit or activate one or more functions of a cell or cell component.
- Combining refers to the mixing or admixing of ingredients in a cell culture medium formulation. Combining can occur in liquid or powder form or with one or more powders and one or more liquids.
- treatment generally refers to obtaining a desired pharmacologic, physiologic or cosmetic effect.
- the effect may be prophylactic in terms of completely or partially preventing a condition, appearance, disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition and/or adverse effect attributable to a condition or disease.
- Treatment covers any treatment of a condition, disease or undesirable appearance in a mammal, particularly a human, and includes one or more of the following: (a) preventing the disease (e.g. cancer), condition (pain) or appearance (e.g.
- wrinkles from occurring in a subject which may be predisposed to such but has not yet been observed or diagnosed as having it; (b) inhibiting the disease, condition or appearance, i.e., causing regression of condition or appearance; and (c) relieving the disease, condition or undesired appearance, i.e., causing regression of condition or appearance.
- Peripheral neuropathy refers to damage to nerves of the peripheral nervous system (PNS). Peripheral neuropathies may either be symmetrical and generalized or focal and multi-focal, which is usually a good indicator of the cause of the peripheral nerve disease.
- Distal axonopathies are the result of some metabolic or toxic derangement of neurons. They may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs.
- Myelinopathies are due to a primary attack on myelin causing an acute failure of impulse conduction.
- the most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; also known as Guillain-Barre syndrome).
- Other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophy), or toxins.
- Neuronopathies are the result of destruction of PNS neurons. They may be caused by motor neuron diseases, sensory neuronopathies (e.g., Herpes zoster), toxins or autonomic dysfunction. Neurotoxins may cause neuronopathies, such as the chemotherapy agent vincristine.
- Peripheral neuropathy can also be categorized by cause, such as being associated with systematic disease, being genetically acquired (such as inherited), or by chemical toxic agent, with the latter including antiretro viral toxic neuropathy (ATN).
- Peripheral neuropathy may be caused either by diseases of the nerve or from the side-effects of systemic illness, most often manifested as one or a combination of motor, sensory, sensorimotor, or autonomic neural dysfunction.
- Chemotherapeutic agents known to cause sensory and/or motor neuropathies include vincristine, an antineoplastic drug used to treat haematological malignancies and sarcomas.
- the neurotoxicity is dose-related, and exhibits as reduced intestinal motility and peripheral neuropathy, especially in the distal muscles of the hands and feet, postural hypotension, and atony of the urinary bladder. Similar problems have been documented with taxol and cisplatin. Mollman, New Eng Jour Med. 322:126-127 (1990), although cisplatin- related neurotoxicity can be alleviated with nerve growth factor (NGF), Apfel et al., Annals of Neurology 31 :76-80 (1992)). Although the neurotoxicity is sometimes reversible after removal of the neurotoxic agent, recovery can be a very slow process. Legha, Medical Toxicology 1 :421-427 (1986); Olesen et al., Drug Safety 3:114 (1991).
- CMT Charcot-Marie-Tooth
- HMSN Hereditary Motor Sensory Neuropathy
- Type II (80% of cases) was believed to have demyelination as its initial pathophysiology, but distal clinical involvement suggests a primary axonal degeneration, as in Type II.
- Type II (30% of cases) is primarily an axonal degeneration without demyelination, and may not be as severe as Type I.
- Nerve conduction impairment is often present at birth, though this is not a predictor of the possible age of onset or severity of progression.
- Type III and Type IV forms which are recessively-linked.
- the present invention provide a cell culture medium and medium supplements comprising a lipophilic compound (such as ubiquinone or ubiquinol) and a solubilizing agent. These supplements enhance the growth and survival of cultured cells when cells are cultured in their presence.
- a lipophilic compound such as ubiquinone or ubiquinol
- ROS include oxygen ions, free radicals and peroxides (both inorganic and organic). They are generally very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. ROSs form as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling. However, during times of environmental stress ROS levels can increase dramatically, which can result in significant damage to cell structures. This culminates in a situation known as oxidative stress.
- the effects of ROS on cell metabolism have been documented in a variety of species, such as the roles in programmed cell death (apoptosis). Cells are normally able to defend themselves against ROS damage through the use of enzymes such as superoxide dismutases and catalases. Small molecule antioxidants such as ascorbic acid (vitamin-C), uric acid, and glutathione also play important roles as cellular antioxidants. Similarly, polyphenol antioxidants assist in preventing ROS damage by scavenging free radicals.
- oxidative stress raises particular concerns in cell culture because cells are generally under stress already in culture and without the normal defense systems available in a living organism.
- One ROS hydrogen peroxide, which is lethal to cells, has been found to be produced in Dulbecco's Modified Eagle's Medium (DMEM) as photoproducts.
- DMEM Dulbecco's Modified Eagle's Medium
- Riboflavin and tryptophan or tyrosine are components necessary for formation of hydrogen peroxide during light exposure. Since most mammalian culture media contain riboflavin, tyrosine and tryptophan, toxic photoproducts are likely produced in most cell culture media.
- WO/1996/017626 discloses adding coenzyme Qi 0 to a cultured human T-cell line (CCRF-CEM), undifferentiated neuronal cell line PC 12, and human neuroblastoma cell line IMR-32. Without being bound by theory, it is believed including ubiquinone or ubiquinol, such as coenzyme Q 10 , provides protective effect against oxidative stress, thus promotes cell growth and survival in cell culture.
- ubiquinone or ubiquinol such as coenzyme Q 10
- the cell culture medium supplement can be in solid form or in solution.
- the supplement is in a solid form, and can be dissolved prior to be added to culture medium such as a basal medium or a complete medium as described herein.
- the supplement is in solution form, and can be added to a medium such as basal medium or complete medium directly.
- the amount of supplement needs to be added to the medium can vary, such as depending on the culture conditions and the type of cells being cultured. Optimal amount can be determined by running a series of pilot cultures in relative small scales.
- the medium supplement according to the present invention is advantageously used by adding it to an ordinary medium, it is desirable to dissolve the medium supplement in a small volume of the medium and then add it to the whole medium.
- the cell culture media supplements provided by the present methods may be used to support the growth of a cell, which may be a bacterial cell, a fungal cell (particularly a yeast cell), a plant cell or an animal cell (particularly an insect cell, a nematode cell or a mammalian cell, most preferably a human cell), any of which may be a somatic cell, a germ cell, a normal cell, a diseased cell, a transformed cell, a mutant cell, a stem cell, a precursor cell or an embryonic cell.
- a cell which may be a bacterial cell, a fungal cell (particularly a yeast cell), a plant cell or an animal cell (particularly an insect cell, a nematode cell or a mammalian cell, most preferably a human cell), any of which may be a somatic cell, a germ cell, a normal cell, a diseased cell, a transformed cell, a mutant cell, a stem cell, a precursor cell or an embryonic cell.
- the present invention provide as cell culture medium supplement provided further comprises an additive.
- the additives could be any compounds that could be used in cell culture, include but is not limited to growth factors.
- One more additives can be included in the supplement composition.
- the invention provides cell culture medium formulation comprising an ubiquinone or ubiquinol and a solubilizing agent described herein, and an un-supplemented cell culture medium.
- compositions of the invention contain from about 5% to about 50% by weight of ubiquinone/ubiquinol.
- the composition contains from about 10% to about 30% (w/w) ubiquinone/ubiquinol, preferably from about 15% to about 25%
- un-supplemented cell culture medium or “un-supplemented medium” here in is meant a medium does not contain a lipophilic compound and a solubilizing agent provided herein.
- Un-supplemented media include, but not limited to, basal media, defmed-media, serum-free media, conditioned media, and complete media.
- DMEM medium H. J. Morton, In Vitro, 6, 89/1970
- F12 medium R. G. Ham, Proc. Natl. Acad. Sci. USA, 53, 288/1965
- RPMI 1640 medium J. W. Goding, J. Immunol. Methods, 39, 285/1980; JAMA 199, 519/1957
- basal media usually seriously deficient in the nutritional content required by most animal cells.
- serum must be added to the basal media to overcome these deficiencies.
- FBS fetal bovine serum
- horse serum or human serum is used in significant concentrations.
- FBS FBS
- It is relatively expensive, and its use greatly increases the cost of cell culture.
- it is difficult to obtain serum with consistent growth characteristics.
- biochemical complexity of FBS can complicate the downstream processing of the proteins of interest, therefore raising the production costs.
- Serum-free medium is an excellent alternative to standard serum-containing media for the cultivation of cells. It has several advantages, which include better definition of the composition, reduced contamination and lower cost. A serum-free medium having cultivation ability comparable to that of the conventional serum-containing medium has long been sought.
- bovine serum albumin BSA
- HSA human serum albumin
- certain growth factors derived from natural (animal) or recombinant sources including epidermal growth factor (EGF) or fibroblast growth factor (FGF); lipids such as fatty acids, sterols and phospholipids; lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins; protein and steroid hormones such as insulin, hydrocortisone and progesterone; nucleotide precursors; and certain trace elements.
- BSA bovine serum albumin
- HSA human serum albumin
- certain growth factors derived from natural (animal) or recombinant sources including epidermal growth factor (EGF) or fibroblast growth factor (FGF)
- lipids such as fatty acids, sterols and phospholipids
- lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins
- protein and steroid hormones such as insulin, hydrocortisone and
- Basal media that may be used include those commercially available from Invitrogen, Lonza, Sigma Chemical Co., Life Technologies, Inc., or Bio Whittaker Co. Any basal medium may be used.
- the basal medium composition comprises carbon sources assimilatable by cells, digestible nitrogen sources and inorganic salts. More specifically, for example, inorganic salts, amino acids, glucose, and vitamins are included. If necessary, a trace substance for nutritional stimulation and an effective trace substance such as a precursor can be included in the basal medium composition. More specifically, for example, MEM medium (H. Eagle, Science, 130, 432 (1959)), DMEM medium (R.
- the cell culture media provided herein are produced by adding a lipophilic compound and a solubilizing agent described herein to a serum-free medium known in the art.
- Serum-free media are described in GIBCO Cell Culture Systems Catalog, 7-55 (Invitrogen, 2005), and U.S. Patent Application Publication No. 20060073591 (particularly, 19-39), herein all incorporated by reference.
- Other media which can be appropriately used in the present invention include serum- free medium ASF 104 (Ajinomoto Co., Inc.), serum-free medium SF-02 (Sanko Junyaku Co., Ltd.), serum-free medium Hybridoma-SFM (Lifetech Oriental), serum- free medium BIO-MPM-I (Biological Industries), serum-free medium EX-CELL. TM. 302-HDP (JRH Biosciences), serum-free medium Cosmedium 001 (Cosmo Bio), and serum- free medium SFM-101 (Nissui Pharmaceutical Co., Ltd.).
- the media that can be used by the present invention include cell culture media that support the growth of animal cells, plant cells, bacterial cells or yeast cells.
- animal cell culture media that may be used in the present invention include, but are not limited to Chu (N6) basal salt mixture, DKW/Juglans basal salt mixture, Gamborg's B-5 basal salt mixture, Gamborg's B-5 basal salt mixture with minimal organics, Hoagland's No. 2 basal salt mixture, McCown's woody plant basal salt mixture, Murashige and Skoog basal salt mixture (MS), Quoirin and Lepoivre basal salt mixture, Schenk and Hildebrandt basal salt mixture, White's basal salt mixture.
- Also can be included in the media are vitamins, antibiotics and growth factor mixes.
- animal cell culture media examples include, but are not limited to, DMEM, RPMI-1640, MCDB 131, MCDB 153, MDEM, IMDM, MEM, M 199, McCoy's 5 A, Williams' Media E, Leibovitz's L- 15 Medium, Grace's Insect Medium, IPL41 Insect Medium, TC-100 Insect Medium, Schneider's Drosophila Medium, Wolf & Quimby's Amphibian Culture Medium, cell-specific serum- free media (SFM) such as those designed to support the culture of keratinocytes, endothelial cells, hepatocytes, melanocytes, etc., FlO Nutrient Mixture and F 12 Nutrient Mixture.
- SFM serum-free media
- the cell culture media provided herein may also include media supplements including but not limited to, animal sera (such as bovine sera (e.g., fetal bovine, newborn calf and calf sera), human sera, equine sera, porcine sera, monkey sera, ape sera, rat sera, murine sera, rabbit sera, ovine sera and the like), defined replacements such as LipoMAX®,
- animal sera such as bovine sera (e.g., fetal bovine, newborn calf and calf sera)
- human sera equine sera, porcine sera, monkey sera, ape sera, rat sera, murine sera, rabbit sera, ovine sera and the like
- defined replacements such as LipoMAX®
- OptiMAb®, Knock-OutTM each available from Invitrogen Corporation, Carlsbad, Calif
- hormones including steroid hormones such as corticosteroids, estrogens, androgens (e.g., testosterone) and peptide hormones such as insulin, cytokines (including growth factors (e.g., EGF, aFGF, bFGF, HGF, IGF-I, IGF-2, NGF and the like), interleukins, colony-stimulating factors, interferons and the like), neurotransmitters, lipids (including phospholipids, sphingolipids, fatty acids, cholesterol and the like), attachment factors (including extracellular matrix components such as fibronectin, vitronectin, laminins, collagens, proteoglycans, glycosaminoglycans and the like), and extracts of animal tissues, organs or glands (such as bovine pituitary extract, bovine brain extract, chick embryo extract, bovine embryo extract, chicken tissue
- Other media supplements that may be used by the present invention include a variety of proteins (such as serum albumins, particularly bovine or human serum albumins; immunoglobulins and fragments or complexes thereof; aprotinin; hemoglobin; haemin or haematin; enzymes (such as trypsin, collagenases, pancreatinin or dispase); lipoproteins; fetuin; ferritin; etc.), which may be natural or recombinant; vitamins; amino acids and variants thereof (including, but not limited to, L-glutamine and cystine), enzyme co-factors; polysaccharides; salts or ions (including trace elements such as salts or ions of molybdenum, vanadium, cobalt, manganese, selenium, and the like); and other supplements and compositions that are useful in cultivating cells in vitro that will be familiar to one of ordinary skill.
- proteins such as serum albumins, particularly bovine or human serum albumins; immunoglobulins
- sera and other media supplements are available commercially (for example, from Invitrogen Corporation, Carlsbad, Calif, and Sigma Cell Culture, St. Louis, Mo.); alternatively, sera and other media supplements described above may be isolated from their natural sources or produced recombinantly by art-known methods that will be routine to one of ordinary skill (see Freshney, R. L, Culture of Animal Cells, New York: Alan R. Liss, Inc., pp. 74-78 (1983), and references cited therein; see also Harlow, E., and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, pp. 116-120(1988)), all incorporated herein be reference in their entireties.
- the media of the present invention may also contain buffers, include but are not limited to, phosphate -buffered saline (PBS) formulations, Tris-buffered saline (TBS) formulations, HEPES-buffered saline (HBS) formulations, Hanks' Balanced Salt Solutions (HBSS), Dulbecco's PBS (DPBS), Earle's Balanced Salt Solutions, Puck's Saline Solutions, Murashige and Skoog Plant Basal Salt Solutions, Keller's Marine Plant Basal Salt Solutions, Provasoli's Marine Plant Basal Salt Solutions, and Kao and Michayluk's Basal Salt Solutions.
- PBS phosphate -buffered saline
- TBS Tris-buffered saline
- HBS HEPES-buffered saline
- HBSS Hanks' Balanced Salt Solutions
- DPBS Dulbecco's PBS
- Earle's Balanced Salt Solutions Puck's Sa
- Formulations for these buffers which are commercially available, as well as for many other commonly used buffers, are well-known in the art and may be found for example in the GIBCO Catalogue and Reference Guide (Invitrogen Corporation, Carlsbad, Calif), in the DIFCO Manual (DIFCO; Norwood, Mass.), and in the Sigma Cell Culture Catalogues for animal and plant cell culture (Sigma; St. Louis, Mo.), all incorporated herein be reference in their entireties.
- the cell culture media provided herein can be produced by different approaches.
- the cell culture medium supplements provided herein, and optionally other desirable additives can be added to an un-supplemented medium to produce a cell culture medium suitable for the maintenance and/or growth of the cell.
- the lipophilic compound and solubilizing agent can be mixed first, and mixed with other ingredients of an un-supplemented medium known in the art.
- a lipophilic compound such as an ubiquinone or ubiquinol
- a solubilizing agent are mixed and to be in solid form. Such solid form is then added to a solid form of un-supplemented medium before being dissolved in water to produce a cell culture medium solution.
- a lipophilic compound such as an ubiquinone or ubiquinol
- a solubilizing agent are mixed and to be in solid form. Such solid form is then added to a solution form of un- supplemented medium to produce a cell culture medium solution.
- a lipophilic compound such as an ubiquinone or ubiquinol
- a solubilizing agent are mixed and dissolved in a solvent, such as water, to produce a solution.
- a solvent such as water
- This solution is then mixed with a basal medium, either in solid form or in solution form, to produce cell to produce a cell culture medium solution.
- the invention provides pharmaceutical formulations for treatment or prevention of nervous system disorders, e.g. peripheral neuropathy, comprising an ubiquinone or ubiquinol and a solubilizing agent described herein.
- the solubilizing agent is according to Formula (II).
- the pharmaceutical formulations are of use in preventing or reducing neurotoxicity attributable to an administered medicament (e.g., anti- cancer, anti-HIV chemotherapeutic).
- an administered medicament e.g., anti- cancer, anti-HIV chemotherapeutic.
- compositions of the invention contain from about 5% to about 50% by weight of ubiquinone/ubiquinol.
- the composition contains from about 10% to about 30% (w/w) ubiquinone/ubiquinol, preferably from about 15% to about 25%
- the formulation of the invention is encompassed within a soft gelatin (soft gel) capsule.
- soft gel soft gelatin
- the method of making soft gel is disclosed in U.S. Patent Application No. 60/886,212, which is herein incorporated by reference.
- the soft gel capsules of the invention include ubiquinone/ubiquinol from about 1% to about 30% by weight.
- the soft gel capsule includes from about 3% to about 20% (w/w), and preferably from about 5% to about 20% of active component, such as CoQio.
- the formulations include from about 10% to about 30% (w/w) ubiquinone/ubiquinol, from about 15% to about 40% (w/w) solubilizing agent (e.g., PQS), from about 30% to about 60% (w/w) lipophilic carrier (e.g., fish oil) and from about 1% to about 10% (w/w) viscosity enhancer (e.g., beeswax).
- the soft gel capsule of the invention includes CoQio, PQS, beeswax and a lipophilic carrier (e.g., fish oil) enriched with omega fatty acid.
- the ubiquinone or ubiquinol is combined with solubilizing agents to obtain improved bioavailability.
- Such formulations may further contain additional active ingredients and/or pharmaceutically or cosmetically acceptable additives or vehicles, including solvents, adjuvants, excipients, sweeteners, fillers, colorants, flavoring agents, lubricants, binders, moisturizing agents, preservatives and mixtures thereof.
- the formulations may be suitable for topical (e.g., a cream, lotion, gel, ointment, dermal adhesive patch), oral (e.g., a capsule, tablet, caplet, granulate), or parenteral (e.g., suppository, sterile solution) administration.
- acceptable vehicles and solvents that may be employed for administration by injection are water, mildly acidified water, Ringer's solution and isotonic sodium chloride solution.
- Ubiquinone or ubiquinol when combined with a solubilizing agent of the invention, may be administered to a warm-blooded animal, particularly a human, in need of the prophylaxis or therapy.
- the method comprises administering to such human or warmblooded animal, an effective amount of a water-soluble composition including the solubilizing agent and ubiquinone or ubiquinol.
- the solubilizing agent is hydrolyzed by enzymes and is systemically converted back to the respective ubiquinol, which is further converted to the respective ubiquinone.
- the dose of a ubiquinone or ubiquinol for treating peripheral neuropathy varies upon the manner of administration, the age, sex, the body weight of the subject, and the condition being treated, and will ultimately be decided by the attending physician or veterinarian.
- Such an amount of the ubiquinone or ubiquinol as determined by the attending physician or veterinarian is referred to herein as a "therapeutically effective amount”.
- compositions described herein are preferably pharmaceutical compositions formulated according to known methods. Formulations are described in detail in a number of sources, which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulation, which can be used in connection with the subject invention. In general, the compositions of the subject invention are formulated such that an effective amount of coenzyme Q is provided in the composition. [0098] In accordance with the present invention, pharmaceutical compositions are provided which comprise, an active ingredient as described, supra, and an effective amount of one or more pharmaceutically acceptable excipients, vehicles, carriers or diluents.
- Such carriers include ethanol, dimethyl sulfoxide, glycerol, silica, alumina, starch, and equivalent carriers and diluents.
- acceptable carriers can be either solid or liquid.
- Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules.
- a solid carrier can be one or more substances, which may act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.
- Injectable preparations include sterile suspensions, solutions or emulsions of the active ingredient in aqueous or oily vehicles.
- the compositions can also contain formulating agents, such as suspending, stabilizing and/or dispersing agent.
- the formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.
- the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use.
- a suitable vehicle including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc.
- the compositions can be lyophilized.
- the stored preparations can be supplied in unit dosage forms and reconstituted prior to use in vivo.
- the active ingredient can be formulated as a depot preparation, for administration by implantation; e.g., subcutaneous, intradermal, or intramuscular injection.
- transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the active ingredient for percutaneous absorption can be used.
- permeation enhancers can be used to facilitate transdermal penetration of the active ingredient.
- a particular benefit can be achieved by incorporating the active agents of the invention into a nitroglycerin patch for use in patients with ischemic heart disease and hypercholesterolemia.
- the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents ⁇ e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
- binding agents ⁇ e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
- fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
- lubricants e.g., magnesium stearate, talc or silica
- disintegrants e.g
- Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
- the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
- Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
- compositions can take the form of tablets or lozenges formulated in conventional manner.
- active ingredient may be formulated as solutions (for retention enemas) suppositories or ointments.
- the active ingredient can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the disclosed pharmaceutical compositions can be subdivided into unit doses containing appropriate quantities of the active component.
- the unit dosage form can be a packaged preparation, such as packeted tablets, capsules, and powders in paper or plastic containers or in vials or ampoules.
- the unit dosage can be a liquid based preparation or formulated to be incorporated into solid food products, chewing gum, or lozenges.
- the invention provides for a cell culture composition
- n is a member selected from 0 to 13.
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy. In various embodiments, n is 9. In exemplary embodiments, R 1 is methyl. Exemplary moieties for R 2 and R 3 are methoxy.
- the ubiquinone can be CoQio or ubiquinol-50, or reduced CoQio.
- the ubiquinone or ubiquinol is coenzyme Q (or its reduced form).
- Coenzyme Q refers to a series of quinones, which are widely distributed in animals, plants and microorganisms, and are found in various biological species differ only slightly in chemical structure and form a group of related, 2-3-dimethoxy-5-methyl- benzoquinones with a polyisoprenoid side chain in the 6-position, which varies in length from 30 to 50 carbon atoms. Since each isoprenoid unit in the chain contains five carbon atoms, the number of isoprenoid units in the side chain varies from 6 to 10.
- the different numbers of the groups have been designated by a subscript following the Q to denote the number of isoprenoid units in the side chain, as in Qi 0 ⁇ e.g. coenzyme Qio). Difference in properties is due to the difference in length of the side chain.
- the members of the group known to occur naturally are Q 6 through Qio.
- Coenzyme Q functions as an agent for carrying out oxidation and reduction within cells. Its primary site of function is in the terminal electron transport system where it acts as an electron or hydrogen carrier between the flavoproteins (which catalyze the oxidation of succinate and reduced pyridine nucleotides) and the cytochromes. This process is carried out in the mitochondria of cells of higher organisms. As an electron carrier/acceptor, coenzyme Qio is continually going through an oxidation reduction cycle. As each coenzyme Qio molecule accepts electrons, it is reduced; when it gives up electrons, it becomes oxidized again.
- coenzyme Qio In coenzyme Qio's reduced form (ubiquinol), the coenzyme Qio molecule holds electrons loosely and will quite easily give up one or two electrons to neutralize free radicals. In its electron rich reduced form, coenzyme Qio is as potent an antioxidant as vitamin E. Coenzyme Q 10 1 S main role as an antioxidant is in the mitochondria where it first participates in the process by which free radicals are generated and then helps to quench the extra free radicals that threaten cellular components such as DNA, RNA, proteins, and cell membranes.
- compositions of the invention can comprised a mixture or combination of oxidized and reduced coenzyme Q.
- exemplary ubiquinones and ubiquinols include a range of isoprenoid units, e.g., n is 6-10, for example, coenzyme Qio.
- ubiquinones or ubiquinols The method of making ubiquinones or ubiquinols is known in the art. There are two general processes by which coenzyme Qio (CoQio) can be produced, 'fermentation' or 'synthesis'. Ubiquinones/ubiquinols can be purchased commercially from sources such as Kaneka (Japan) and Nisshin (Japan). Most of the world's supply of coenzyme Qio is generated in Japan using patented processes based on the fermentation of glucose by yeast and is then purified. See U.S. Patent Nos. 4,447,362 and 4,194,065, all are incorporated herein by reference in their entirety.
- the remaining supply of coenzyme Qio is made by what is referred to as the "synthetic" method.
- This method involves combining two naturally derived compounds.
- One is 'solanesol', which is isolated and purified from tobacco plants.
- the other component is TMT, derived from gallic acid, found in several different plant sources.
- TMT derived from gallic acid
- the synthetic process of coenzyme Qio involves combining two naturally occurring compounds derived from plants.
- organic chemical synthesis of coenzyme Q is described in U.S. Patent No. 6,686,485.
- the Lipshutz method for the synthesis of ubiquinones and ubiquinone analogues that is described in U.S. Patent Nos.
- the formulations of lipophilic compounds of use in the invention include a solubilizing agent.
- the solubilizing agent has a structure according to the following formula:
- a, b and c are integers independently selected from 0 and 1.
- b is 0.
- Z is a hydrophobic (lipophilic) moiety.
- Z is a sterol (e.g., beta-sitosterol, cholesterol).
- Z is a tocopherol (e.g., alpha-tocopherol, alpha-tocotrienol) or a derivative or homologue thereof.
- Z is an ubiquinol.
- the residue of the hydrophobic moiety is the entire hydrophobic molecule, except for at least one hydrogen atom, which is replaced with the hydrophilic moiety or the linker-hydrophilic moiety cassette (e.g., hydrogen atom of esterified hydroxyl group, such as 3- ⁇ -hydroxyl group of cholesterol or sitosterol or 6-hydroxyl group of ⁇ -tocopherol).
- the hydrophilic moiety or the linker-hydrophilic moiety cassette e.g., hydrogen atom of esterified hydroxyl group, such as 3- ⁇ -hydroxyl group of cholesterol or sitosterol or 6-hydroxyl group of ⁇ -tocopherol.
- Y 1 and Y 2 are linear or branched hydrophilic moieties comprising at least one polymeric moiety, wherein each polymeric moiety is independently selected.
- Y 1 and Y 2 are independently selected from hydrophilic (i.e., water-soluble) polymers.
- Y 1 and Y 2 are members independently selected from poly(alkylene oxides) (i.e., polyethers), polyalcohols, polysaccharides (e.g., polysialic acid), polyamino acids (e.g., polyglutamic acid, polylysine), polyphosphoric acids, polyamines and derivatives thereof.
- Exemplary poly(alkylene oxides) include polyethylene glycol (PEG) and polypropylene glycol (PPG).
- PEG derivatives include those, in which the terminal hydroxyl group is replaced with another moiety, such as an alkyl group (e.g., methyl, ethyl or propyl).
- the hydrophilic moiety is methyl-PEG (mPEG).
- PEG is usually a mixture of oligomers characterized by an average molecular weight.
- the PEG has an average molecular weight from about 200 to about 5000.
- PEG has an average molecular weight from about 500 to about 1500.
- PEG has an average molecular weight from about 500 to about 700 or about 900 to about 1200.
- the lipophilic moiety of the solubilizing agent is PEG-400.
- the lipophilic moiety of the solubilizing agent is PEG-600. Both linear and branched PEG moieties can be used as the hydrophilic moiety of the solubilizing agent in the practice of the invention.
- PEG has between 1000 and 5000 subunits. In an exemplary embodiment, PEG has between 100 and 500 subunits. In an exemplary embodiment, PEG has between 10 and 50 subunits. In an exemplary embodiment, PEG has between 1 and 25 subunits. In an exemplary embodiment, PEG has between 15 and 25 subunits. PEG has between 5 and 100 subunits. In an exemplary embodiment, PEG has between 1 and 500 subunits.
- the poly(ethylene glycol) is a branched PEG having more than one PEG moiety attached. Examples of branched PEGs are described in U.S. Patent No. 5,932,462; U.S. Patent No. 5,342,940; U.S. Patent No.
- Exemplary branched PEG moieties involve a branched core molecule having at least two PEG arms attached, each at a different attachment point.
- At least one of Y 1 and Y 2 includes a moiety having the following structure:
- Y 7 is a member selected from CH 3 and H
- n is a member selected from 1 to 5000 (e.g., 1 to 2500).
- n is a member selected from 1000-5000.
- n is a member selected from 1-500.
- n is a member selected from 5-100.
- n is a member selected from 100-500.
- n is a member selected from 10-50.
- n is a member selected from 1-25.
- Y 1 and/or Y 2 is:
- n is a member selected from 1 to 5000.
- m is a member selected from 5-20.
- m is a member selected from 8-15.
- n is a member selected from 1000-5000.
- n is a member selected from 100-500.
- n is a member selected from 10-50.
- n is a member selected from 1-25.
- n is a member selected from 5-100.
- n is a member selected from 1-500.
- Y 1 and/or Y 2 is:
- Y is a member selected from CH 3 and H
- n is a member selected from 1 to 2500.
- n is a member selected from 1000-5000.
- n is a member selected from 100-500.
- n is a member selected from 10-50.
- n is a member selected from 1-25.
- n is a member selected from 5-100.
- n is a member selected from 1-500.
- Y 1 and/or Y 2 is:
- n is a member selected from 1 to 2500.
- m is a member selected from 5-20.
- m is a member selected from 8-15.
- n is a member selected from 1000-5000.
- n is a member selected from 100-500.
- n is a member selected from 10-50.
- n is a member selected from 1-25.
- n is a member selected from 5-100.
- n is a member selected from 1-500.
- the hydrophilic molecule has a reactive functional group, which can be used to chemically attach the hydrophilic molecule to the hydrophobic moiety (e.g., sterol, tocopherol or ubiquinol), either directly or through a linker moiety.
- functional groups include esterif ⁇ able hydroxyl groups, carboxy groups and amino groups.
- the hydrophilic moiety is a polyether (e.g., polyalkylene glycol).
- polyalkylene glycol includes polymers of lower alkylene oxides, in particular polymers of ethylene oxide (polyethylene glycols) and propylene oxide (polypropylene glycols), having an esterifiable hydroxyl group at least at one end of the polymer molecule, as well as derivatives of such polymers having esterifiable carboxylic acid groups.
- the residue of the hydrophilic moiety is the entire hydrophilic molecule, except for the atom involved in forming the bond to the hydrophobic moiety or the linker moiety (i.e. hydrogen atom of an esterified hydroxyl group).
- L 1 and L 2 are linker moieties.
- L 1 and L 2 are independently selected from a single bond, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
- At least one of L 1 and L 2 includes a linear or branched C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C9, Cio, C 11 , C 12 , C 13 , C 14 , C 15 , Ci6, C 17 , C 18 , Ci9, C20, C 21 , C22, C23, C 24 or C 25 -C 30 alkyl chain, optionally incorporating at least one functional group.
- Exemplary functional groups according to this embodiment include ether, thioether, ester, carbonamide, sulfonamide, carbonate and urea groups.
- At least one of L 1 and L 2 includes a moiety having the following formula:
- Each R 50 and each R 51 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
- At least one of L 1 and L 2 includes a moiety having the following formula:
- the linker can be derived from an alkanedioic acid of the general formula HOOC-(CH 2 ) m -COOH.
- Preferred linkers include diesters derived from an alkanedioic acid.
- alkanedioic acids with m from 0 to 18 are preferred, those with m from 6 to 10 being particularly preferred.
- the solubilizing agent includes the moiety:
- m is a member selected from 4, 6, 8, 10, 12 and 14.
- linker is derived from sebacic acid.
- Other preferred linkers include diethers derived from a substituted alkane.
- the substituted alkane has the general structure X-(CH 2 )D-X' wherein X and X' independently represent a leaving group such as a halogen atom or a tosylate group.
- substituted alkanes with n from 0 to 18 are preferred, those with n from 6 to 10 being particularly preferred.
- the solubilizing agent includes a moiety, which is a member selected from:
- Y3 3 I ⁇ P D — — r 0» ——— v Y 33
- n is a member selected from O to 18.
- Y 3 is a member selected from Y 1 and Y 2 .
- Y 4 and Y 5 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl.
- the solubilizing agent includes a branched linker.
- at least one of L 1 and L 2 includes a moiety having the following formula:
- a 1 , A 2 , A 3 , A 4 , A 5 A 6 , A 7 , A 8 , A 9 , A 10 and A 11 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -NA 12 A 13 , -OA 12 and -SiA 12 A 13 .
- a 12 and A 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
- solubilizing agent is not PCS (polyoxyethanyl-cholesteryl sebacate). In another embodiment, the solubilizing agent is not TPGS (polyoxyethanyl-a- tocopheryl succinate).
- the solubilizing agent has a structure according to one of the following formulae: ⁇ 1_ Z _ ⁇ 2. ⁇ 1_
- the solubilizing agent has a structure according to Formula (IV), wherein the integer a, Y 1 , Z and L 1 are defined as herein above:
- Z is a sterol.
- Exemplary sterols include 7- dehydrocholesterol, campesterol, sitosterol, ergosterol, stigmasterol, zoosterol and phytosterol. Cholesterol and sitosterol are preferred sterols, sitosterol being particularly preferred.
- Z is member selected from a zoosterol and a phytosterol.
- Z is a sterol with an oxygen atom at the 3-position of the A-ring.
- Z is:
- R 12 and R 13 are substituted or unsubstituted alkyl.
- R 14 , R 15 , R 16 , R 17 , and R 18 are independently H, or substituted or unsubstituted alkyl.
- Z is a member selected from
- Z is a member selected from:
- sterols include episterol, cycloartol, avenasterol, 24-methylenecycloartol.
- Solubilizing Agents Wherein Z is a Tocopherol or a Tocotrienol
- Z is a member selected from a substituted or unsubstituted tocopherol and a substituted or unsubstituted tocotrienol.
- Z is an ⁇ -, ⁇ -, ⁇ -, or ⁇ -tocopherol.
- Exemplary tocopherols of use include ⁇ -(+)-Tocopherol and ⁇ -(+)- tocopherol and DL tocopherol.
- Z has a structure according to the following formula:
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
- R 2 and R 3 together with the carbon atoms to which they are attached, are optionally joined to form a 5- to 7-membered ring.
- R 20 , R 21 , R 22 , R 23 , R 24 and R 25 are members selected from H, halogen, nitro, cyano, OR 17 , SR 17 , NR 17 R 18 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
- at least one of R 24 and R 25 comprises an isoprene moiety.
- R 1 , R 2 and R 3 are members independently selected from H and methyl.
- R 3 is methyl, R 2 is methyl and R 1 is methyl.
- R 3 is methyl, R 2 is H and R 1 is methyl.
- R 3 is methyl, R 2 is methyl and R 1 is H.
- R 3 is methyl, R 2 is H and R 1 is H.
- Z has a structure according to the following formulae:
- R , 25 is a member selected from substituted or unsubstituted alkyl and substituted or uunnssuubbssttiittuutteedd hheetteerrooaallkkyyll.. IInn oonnee eexxaammppllee,, RR 2244 iiss mmeetthhyyll.. IInn aannoother example, R 25 includes a moiety having a structure selected from the following formulae:
- k is an integer selected from 1 to 20. In an exemplary embodiment, k is from 2 to 12. In an exemplary embodiment, k is from 2 to 6. In another exemplary embodiment, k is 3.
- the solubilizing agent has a structure according to the following formula:
- the moiety L ⁇ Y 1 has a structure according to the following formula:
- n is member selected from 1 to 20
- m is a member selected from 1 to 5000.
- n is 4.
- m is a member selected from 1 to 2,500.
- Z is an ubiquinol.
- one or both of the phenolic hydroxy groups of the ubiquinol are derivatized with a hydrophilic moiety of the invention.
- the solubilizing agent has a structure according to the Formula (V):
- L 1 , L 2 , Y 1 and Y 2 are defined as herein above.
- R 11 , R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
- R 16 is a member selected from OR 17 , SR 17 , NR 17 R 18 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
- R 17 and R 18 are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
- R 12 and R 13 along with the atoms to which they are attached, are optionally joined to form a 4- to 8-membered ring.
- L 1 and L 2 are linker moieties, which are members independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.
- Y 1 and Y 2 are polymeric hydrophilic moieties, which are members independently selected from polyethers, polyalcohols and derivatives thereof.
- Y 1 , Y 2 , L 1 and L 2 do not comprise a labeling moiety, a targeting moiety or a drug moiety.
- the indices a, b, c and d are members independently selected from 0 and 1 with the proviso that at least one of b and d is 1.
- ((L 2 ) c -Y 2 )b is preferably a member selected from H, a negative charge, and a salt counterion.
- ((L 1 X-Y 1 ),! is preferably a member selected from H, a negative charge, and a salt counterion.
- R 16 includes a moiety having a structure selected from the following formulae:
- k is an integer selected from 1 to 20. In an exemplary embodiment, k is an integer selected from 6, 7, 8, 9, 10, 11 and 12. In another exemplary embodiment, k is 10.
- R 11 , R 12 and R 13 are members independently selected from H, unsubstituted alkyl (e.g., methyl, ethyl), alkoxy (e.g., methoxy, t-butoxy), halogen substituted alkoxy and halogen-substituted alkyl (e.g., CF3).
- R , 11 is H.
- R 11 is a methyl group.
- one or more of the substituents R 11 , R 12 and R 13 include halogen atoms.
- the halogen is fluoro.
- Exemplary fluoroalkyl and fluoroalkoxy groups according to this aspect of the invention include but are not limited to CF 3 , OCF 3 , CHF 2 , OCHF 2 , CH 2 F, and OCH 2 F.
- the solubilizing agent has a structure according to the Formula (VI):
- An exemplary solubilizing agent according to Formula (VI) has a structure according to Formula (VII):
- one of the phenolic hydroxy groups of the ubiquinol analog is derivatized with a hydrophilic moiety of the invention.
- exemplary solubilizing agents have the structure: wherein Q is a member selected from H, a negative charge and a salt counter ion.
- Exemplary solubilizing agents have a structure according to Formula (VIII), Formula (IX) or Formula (X):
- phenolic hydroxy groups of ubiquinol are part of an ether bond with the linker moiety.
- solubilizing agents have a formula, which is a member selected from:
- the solubilizing agent is a mixture of two or more solubilizing agents described herein.
- the solubilizing agents have a structure according to Formula (V).
- the integer k is constant, but at least one of the solubilizing agents includs one hydrophilic moiety, while another includes two hydrophilic moieties.
- the mixture includes two regioisomers.
- the compounds in the mixture of solubilizing agents have structures according to Formulae (VII), (VIII), (IX), (X), (XI), (XII) and (VIII).
- the solubilizing agent has a structure, which is a member selected from:
- m is a member selected from 2-16 and Y 1 is a hydrophilic moiety.
- m is a member selected from 2, 6, 8, 10, 12 and 14.
- m is 2.
- m is 8.
- the solubilizing agent is a member selected from
- n is a member selected from 10 to 2500
- L 1 is a linker moiety
- Y 7 is a member selected from H and methyl.
- the solubilizing agent has a structure according to one of the following formulae:
- n is an integer selected from 1 to 20.
- Y 1 , R 1' , R 2' , R 3' , R 20 , R 21 , R 22 , R 23 , R 24 and R 25 are defined as herein above.
- R , 25 includes a moiety having a structure selected from the following formulae:
- k is 3, R 3 is methyl, R 2 is methyl and R 1 is methyl. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is H and R 1 is methyl. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is methyl and R 1 is H. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is H and R 1 is H. [0161] In another exemplary embodiment, n is 8, k is 3, R 3 is methyl, R 2 is methyl and R 1 is methyl. In another exemplary embodiment, n is 8, k is 3, R 3 is methyl, R 2 is H and R 1 is methyl. In another exemplary embodiment, n is 8, k is 3, R 3 is methyl, R 2 is methyl and R 1 is H. In another exemplary embodiment, n is 8, k is 3, R 3 is methyl, R 2 is H and R 1 is H. In another exemplary embodiment, n is 8, k is 3, R 3 is methyl, R 2 is H
- n is 2, k is 3, R 3 is methyl, R 2 is methyl and R 1 is methyl. In another exemplary embodiment, n is 2, k is 3, R 3 is methyl, R 2 is H and R 1 is methyl. In another exemplary embodiment, n is 2, k is 3, R 3 is methyl, R 2 is methyl and R 1 is H. In another exemplary embodiment, n is 2, k is 3, R 3 is methyl, R 2 is H and R 1 is H.
- the solubilizing agent has a structure according to the following formula:
- n is a member selected from 10 to 2500.
- L 1 , R 1 , R 2 , R 3 , R 20 , R 21 , R 22 , R 23 , R 24 and R 25 are defined as herein above.
- Y 7 is selected from H and methyl.
- R 25 includes a moiety having a structure selected from the following formulae:
- k is 3, R 3 is methyl, R 2 is methyl and R 1 is methyl. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is H and R 1 is methyl. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is methyl and R 1 is H. In another exemplary embodiment, k is 3, R 3 is methyl, R 2 is H and R 1 is H.
- the solubilizing agent is a member selected from wherein k is a member selected from 1 to 15 and n is a member selected from 1 to 20.
- Y 1 , R 11 , R 12 and R 13 are defined as herein above.
- k is 10.
- n is 8.
- the solubilizing agent is a member selected from
- k is a member selected from 1 to 15 and n is a member selected from 1 to 20.
- Y 1 , R 11 , R 12 and R 13 are defined as herein above.
- k is 10.
- n is 8.
- the solubilizing agent is a member selected from
- k is a member selected from 1 to 15 and n is a member selected from 10 to 2500.
- Y 1 , R 11 , R 12 and R 13 are defined as herein above.
- k is 10.
- Y 7 is a member selected from H and methyl.
- the solubilizing agent is a member selected from
- the solubilizing agent is a member selected from polyoxyethanyl-tocopherol-sebacate (PTS), polyoxyethanyl-sitosterol-sebacate (PSS), polyoxyethanyl-cholesterol-sebacate (PCS), polyoxyethanyl-ubiquinol-sebacate (PQS) and combinations thereof.
- PTS polyoxyethanyl-tocopherol-sebacate
- PSS polyoxyethanyl-sitosterol-sebacate
- PCS polyoxyethanyl-cholesterol-sebacate
- PQS polyoxyethanyl-ubiquinol-sebacate
- the formulations of the invention include from about 10% to about 50% by weight of a solubilizing agent, such as PTS.
- a solubilizing agent such as PTS.
- the formulations include from about 15% to about 40% (w/w) solubilizing agent, more preferably from about 20% to about 40% (w/w), and even more preferably from about 20 to about 35%
- Solubilizing agents utilized in the compositions of the invention include those described in US Provisional Patent Application 60/773,951; and US Patents: 6,045,826; 6,191,172 and 6,632,443 to Borowy-Borowski et ah, which are incorporated herein by reference for all purposes. These solubilizing agents can be purchased commercially from sources such as Zymes (New Jersey) or produced according to the methods described in the above documents.
- the solubilizing agent contributes to the formation of micelles once the composition is added to an aqueous solution.
- the size of the formed micelles in solution may be measured using a dynamic light scattering (DLS) detector.
- DLS dynamic light scattering
- smaller particle sizes are associated with a greater tendency of the human body to absorb the active ingredient contained in those particles or micelles.
- the ubiquinone/ubiquinol compositions of the invention form particle sizes in aqueous solution, which are decreased when compared with the particle sizes formed by known formulations.
- the average particle size formed by the compositions of the invention in aqueous solution is preferably below about 100 nm.
- the average particle size is from about 10 nm to about 90 nm.
- An exemplary average particle size is from about 5 nm to about 70 nm, preferably from about 10 nm to about 50 nm, more preferably from about 10 nm to about 30 nm, and most preferably from about 15 nm to about 25 nm.
- the particle size is between about 300 nm and about 1 nm, preferably between about 200 nm and about 1 nm, more preferably between about 100 nm and about 1 nm. Smaller particle sizes are generally preferred.
- the formulations of the invention include from about 5% to about 50% by weight of a solubilizing agent, such as PQS.
- a solubilizing agent such as PQS.
- the formulations include from about 10% to about 30% (w/w) solubilizing agent, more preferably from about 15% to about 25% (w/w).
- the solubilizing agent is added to the compositions of the invention at a ratio of ubiquinone/ubiquinol to surfactant from about 1 : 1 to about 1 :5 (w/w), preferably from about 1 : 1 to about 1 :3 (w/w), and more preferably from about 1 :1 to about 1 :2 (w/w).
- the upper limit of the weight ratio is not critical, and the solubilizing agent can be used in any excess. This is not desirable however, as increasing the amount of the solubilizing agent decreases the concentration of the active ingredient in the composition and in its aqueous solutions.
- the water-soluble compositions of the present invention can be prepared by two different procedures, either in the presence or in the absence of an auxiliary organic solvent.
- a lipophilic compound and a solubilizing agent are first dissolved in a predetermined molar ratio in a water-miscible organic solvent and this solution is then diluted with a predetermined amount of water, without precipitation of the lipophilic compound.
- the organic solvent and water are then removed by evaporation under reduced pressure.
- a volatile organic solvent is usually removed first, followed by water, in which case the amount of water removed from the solution may be controlled, to achieve a desired concentration of the water-soluble composition in the remaining concentrate.
- both the organic solvent and water are removed by evaporation, and the waxy residue is reconstituted with a suitable aqueous medium (such as water, physiological saline, or a buffer solution), to provide a clear aqueous solution.
- a suitable aqueous medium such as water, physiological saline, or a buffer solution
- the organic solvent used in the above procedure should be a good solvent for both the lipophilic compound and the solubilizing agent and has to be miscible with water. If a water-soluble composition is to be used in a pharmaceutical formulation, this solvent should be also pharmaceutically acceptable, as the removal of the solvent by evaporation may not always be possible.
- solvents suitable for the practice of the invention are tetrahydrofuran, ethanol, ethylene glycol, propylene glycol, and acetic acid. Solvents with a low boiling point, such as tetrahydrofuran, are preferred.
- the amount of the organic solvent is not critical, and is equal to or greater than the minimum amount of solvent necessary to dissolve the given amounts of the lipophilic compound and solubilizing agent.
- the amount of water used for the dilution is also not critical, and is preferably between 10 to 25 times the volume of the organic solvent.
- An alternative procedure for preparing water-soluble compositions according to the invention consists of preparing first a mixture of an ubiquinone or ubiquinol compound and a solubilizing agent in a predetermined molar ratio. This mixture is then heated to a temperature higher than the respective melting points of the ubiquinone or ubiquinol and the solubilizing agent, for a time necessary to obtain a clear melt, which process can be seen as dissolution of the ubiquinone or ubiquinol compound in the solubilizing agent. The melt so obtained can be reconstituted with a predetermined amount of a suitable aqueous medium, to provide a clear aqueous solution of a desired concentration.
- This method of preparing water- soluble compositions of the invention is preferred, as it is simpler and avoids limitations of the procedure that relies on an auxiliary organic solvent, such as the pharmaceutically acceptable character of the solvent required for most applications.
- solubilizing agents of the present invention to dissolve an ubiquinone or ubiquinol compounds in the absence of an auxiliary organic solvent can be used for preparing water-soluble forms of bioactive compounds, in particular coenzyme Q 1O , without purifying it by crystallization after synthesis, thus reducing the overall cost of preparing water-soluble compositions of this compound.
- compositions of the present invention show a decreasing solubility in water with increasing temperature of the solution.
- This provides an alternative method of isolation and/or purification of such compositions.
- the composition is dissolved in water at a ratio of the composition to water generally not exceeding 1 :2 by volume and the solution is heated, for example in a boiling water bath, for a time necessary to achieve the separation of the water-soluble composition as a liquid phase, usually a few minutes.
- the oily phase is then separated from the hot solution, while keeping the temperature of the solution substantially unchanged, as cooling of the solution would increase the solubility of the composition and result in a reduced yield of the recovery.
- a speedy separation of the oily phase to avoid the cooling can be achieved, for example, by centrifugation.
- the water-soluble compositions of the present invention have a waxy consistence and may be difficult to manipulate in this highly concentrated form. To make them more easily processable as solids, they may be combined with suitable solid, water-soluble additives, such as vitamin C, gelatin, a protein supplement, or a polyethylene glycol. In some embodiments, polyethylene glycol is preferred. In the latter case, a polyethylene glycol having an average molecular weight greater than about 5000 is preferred.
- the ratio of the composition and the additive is not critical, but will be usually limited to avoid an unnecessary dilution of the active ingredient by the additive.
- compositions of the present invention generally show an excellent solubility in water and allow the preparation of aqueous solutions of almost any concentration.
- concentrated solutions can be diluted with an aqueous medium in any proportion and over a wide range of pH conditions without precipitation of the lipophilic compound, the solubility of the compound is maintained in cell culture medium solutions.
- the present invention provides methods for the culturing of primary culture, cell lines, tissues, as well as organs.
- the cell could be from a variety of organisms, include, but is not limited to animal, plant, bacteria, and fungus.
- the cell-types that can be used in the present invention are derived from various tissues, can be of human origin or that of any other mammal, and may be of any suitable source, such as fibroblast cells, stem cells, ovary cell, or cells from pancreas, parotid gland, thyroid gland, parathyroid gland, prostate gland, lachrymal gland, cartilage, kidney, inner ear, liver, parathyroid gland, oral mucosa, sweat gland, hair follicle, adrenal cortex, urethra, and bladder, or portions or multiples thereof.
- fibroblast cells such as fibroblast cells, stem cells, ovary cell, or cells from pancreas, parotid gland, thyroid gland, parathyroid gland, prostate gland, lachrymal gland, cartilage, kidney, inner ear, liver, parathyroid gland, oral mucosa, sweat gland, hair follicle, adrenal cortex, urethra, and bladder, or portions or multiples thereof.
- the media and methods provided herein can be used
- the tissue is prepared using any suitable method, such as by gently teasing apart the excised tissue or by digestion of excised tissue with collagenase via, for example, perfusion through a duct or simple incubation of, for example, teased tissue in a collagenase-containing buffer of suitable pH and tonic strength.
- the prepared tissue then is concentrated using suitable methods and materials, such as centrifugation through ficol gradients for concentration (and partial purification).
- the concentrated tissue then is resuspended into any suitable vessel, such as tissue culture glassware or plasticware.
- the resuspended material may include whole substructures of the tissue, cells and clusters of cells. For example, such substructures may include fibroblast cells.
- the initial culture of re-suspended tissue cells is a primary culture.
- the cells attach and spread on the surface of a suitable culture vessel with concomitant cell division.
- serially propagated secondary and subsequent cultures are prepared by dissociating the cells of the primary culture and diluting the initial culture or its succeeding cultures into fresh culture vessels, a procedure known in the art as passaging.
- passaging results in an expanded culture of cells of the originating tissue.
- the cell culture is passaged at suitable intervals, such as about once a week or after about two to about three cell divisions of the cultured cells.
- a dilution of the cultured cells at a ratio of from about 1 :2 to about 1 : 100 is used.
- a ratio of from about 1 :4 to about 1 :50 is used.
- a ratio of from about 1 :4 to about 1 :6 is used.
- the concentrated prepared tissue which may be in the form of free cells and/or clumps (where the clumps may constitute ordered substructures of the tissue) is re-suspended at any suitable initial cell or presumptive cell density. Suitable cell densities range from about 100 cells to about 1000 cells per square centimeter of surface area of the culture vessel. For useful vessels, see U.S. Pat. No. 5,274,084 and patents and publications cited therein, all incorporated by reference. [0189] The methods to obtain these cells are known in the art. General methods for cell cultures are known in the art. Culture of Animal Cells: A Manual of Basic Technique, Freshney Wiley-Liss (5th Ed. 2005), herein incorporated by reference in its entirety.
- Animal cells which can be cultured in a medium of the present invention are not particularly limited and they can be either established cell lines or nonestablished normal cells obtained from biological tissues. Accordingly, animal cells of the present invention can be, for example, cells which can produce proteins by themselves, cells which are transformed by genetic engineering to express heterologous proteins, or cells which are infected with various virus vectors.
- Examples of the cells which can produce proteins by themselves include hybridoma cells producing monoclonal antibodies, leukocytes producing interferon (IFN)- ⁇ , fibroblasts producing IFN- ⁇ , lymphocytes producing IFN- ⁇ , human kidney cells producing prourokinase (pro-UK) or UK, melanoma cells producing tissue plasminogen activator (tPA), In-111 cells producing insulin, HIT cells producing glucagon, HepG2 cells producing erythropoietin, and B151K12 cells producing interleukin-5.
- IFN interferon
- pro-UK human kidney cells producing prourokinase
- tPA tissue plasminogen activator
- In-111 cells producing insulin
- HIT cells producing glucagon
- HepG2 cells producing erythropoietin
- B151K12 cells producing interleukin-5.
- Examples of the cell lines transformed by genetic engineering include Vera cells, HeLa cells, CHO (Chinese hamster ovary) cells, HKG cells, NIH3T3 cells, BHK cells, COS- 1 cells, COS-7 cells, and myeloma cells.
- Examples of the cells infected with virus vectors include those infected with retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and herpesvirus vectors. These virus vectors can be genetically recombined by an ordinary genetic engineering method. Further, examples of the animal cells which are infected with these virus vectors and cultured using the medium of the present invention include HEK (human embryonic kidney) 293 cells, A549 cells, and PER.C6 cells.
- Another preferred embodiment of the present invention provides a method of culturing animal cells, which comprises the steps of adding the medium supplement of the present invention to an animal cell culture medium and culturing animal cells using the resulting medium to grow the animal cells.
- Culture conditions for this method for example, the oxygen concentration, osmotic pressure, pH, temperature of the medium, can be appropriately changed depending on the kind of the cells to be cultured, the purpose of the culture, the volume of the culture, and the kind of the basal medium composition. Any culture system such as batch culture, continuous culture or perfusion culture can be used. High density culture can also be used.
- the methods provided herein find uses in a variety of applications.
- the cells grown using culture media and culture media supplements provided herein can find use in many applications, include but not limited to, assays, testing, cell biology research, DNA production, RNA production, protein production, virus production, vaccine production, drug screening and testing, cell production, tissue engineering, organ transplantation, ex vivo transplantation, and gene therapy.
- the present invention provides a method of producing a protein, comprising the steps of adding the medium supplement of the present invention to an animal cell culture medium, culturing animal cells capable of producing the protein using the resulting medium to grow the animal cells, and recovering the produced protein from said medium and/or said animal cells.
- examples of the protein which can preferably be produced include monoclonal antibodies, IFN- ⁇ , IFN- ⁇ , INF - ⁇ , pro-UK or UK, tPA, insulin, glucagon, erythropoietin, and interleukin-5.
- the protein produced can be recovered using chemical or physical characteristics of the protein and isolated and purified by various ordinary isolation methods.
- the protein can be recovered, isolated and purified by treatment with a protein coagulant, ultrafiltration, absorption chromatography, ion-exchange chromatography, affinity chromatography, molecular sieving chromatography, dialysis or the like, singly or in combination.
- the present invention provides a method of replicating a virus vector, which comprises the steps of adding the medium supplement of the present invention to an animal cell culture medium, culturing to grow animal cells infected with the virus vectors using the resulting medium and recovering the produced virus vectors from said medium and/or said animal cells.
- Virus vectors replicable by the method of replication of the present invention are various virus vectors described above as examples and can be created by genetic recombination, if necessary. Appropriately selected animal cells are infected with the virus vectors of interest by methods known in the art. Further, the virus vectors can be recovered from grown cells by isolation and purification using various ordinary isolation methods such as ultrafiltration and centrifugation. Here it is desirable to appropriately select the method of recovering virus vectors according to the kind of virus vectors.
- gene therapies are categorized into two kinds, i.e., ex vivo gene therapy and in vivo gene therapy.
- the former is a therapeutic method in which cells derived from a patient are first cultured outside the body and then treated for gene transfer, after which the cells are administered to the patient.
- the latter is a therapeutic method in which vectors with transferred genes are directly introduced into the patient's body.
- the method according to the present invention can replicate virus vectors, into which genes used for such gene therapies are introduced, more efficiently than conventional methods. Further, the medium of the present invention exhibits excellent growth stimulating effect on the animal cells used for such a replication method, such as 293 cells.
- the present invention provides composition and methods for treating patients with cells or components of cells grown in a cell culture media of the invention.
- the cells grown via the present invention may be any type of cells including stems cells which may be embryonic stem cells or adult stem cells, cells from specific organs including but limited to heart, lung, skin, pancreas and liver.
- the different cells may be obtained from the same patient as the platelets are used to treat the same patient.
- a range of different therapeutic results can be obtained. For example, heart tissue regrown, skin grafting enhanced, and diabetic patients treated by growing cells which produce insulin. Accordingly, the term "treatment" is intended to mean providing a therapeutically detectable and beneficial effect of any kind on a patient.
- the method of the present invention utilizing solubilized ubiquinones and ubiquinols enhances the viability of cells of the nervous system in culture and in vivo. Accordingly, the present invention provides methods of treating diseases of the nervous system, such as peripheral neuropathy.
- Peripheral neuropathy refers to damage to nerves of the peripheral nervous system. It may be caused either by diseases of the nerve or from the side-effects of systemic illness, most often manifested as one or a combination of motor, sensory, sensorimotor, or autonomic neural dysfunction.
- the wide variety of morphologies exhibited by peripheral neuropathies can each be uniquely attributed to an equally wide variety of causes.
- peripheral neuropathies can be genetically acquired, can result from a systemic disease, or can be induced by a toxic agent.
- Some toxic agents that cause neurotoxicities are therapeutic drugs, antineoplastic agents, contaminants in foods or medicinals, and environmental and industrial pollutants.
- Peripheral neuropathy is the major neurological complication observed among persons infected with human immunodeficiency virus type 1 (HIV-I) in the developed world.
- the most common form of peripheral neuropathy is sensory polyneuropathy (HIV-SN), affecting as many as 35% of individuals with acquired immunodeficiency syndrome (AIDS).
- HIV-SN sensory polyneuropathy
- DSP distal sensory polyneuropathy
- ATN anti retro viral-induced toxic neuropathy
- HlV- SN is characterized by prominent small-diameter axonaj loss, following a "'dying back'' pattern of degeneration, likely driven by local inflammation within the nerve and dorsal root ganglia (DRGs) with ensuing neuronal injury.
- DRGs dorsal root ganglia
- ATN has been associated with the use of the nucleoside analogue reverse transcriptase inhibitors (NRTI) didanosine (ddl), zalcitabinc (ddC), and stavudine (d4T).
- NRTI nucleoside analogue reverse transcriptase inhibitors
- ddl didanosine
- ddC zalcitabinc
- d4T stavudine
- the small -diameter nociceptive sensory axons and their respective soma in the DRGs are the principal cellular structures affected in HIV-induced DSP or NRTI-induced ATN.
- ATN may be a synergistic consequence of HIV infection together with the neurotoxic effects of select antiretroviral drugs, because some I HV- uninfected animals, treated with neurotoxic antiretroviral drugs, do not develop an ATlS -like phcnotypc.
- select NRTIs arc associated with ATN, multiple anliretroviral regimens including those with protease inhibitors (PIs) appear to have adverse effects on metabolism with increased risks for mitochondrial toxicity, hyperglycemia. hyperlipidcmia, and the lipodystrophy syndrome. Pettersen et al, Ann Neurol 59:816-824 (2006).
- AACAR acetyl-L-carnitine
- N-4-carboxaphenyl-3-(6-oxohydropurin-9-yl)propananide AIT-082 has been disclosed for treatment of drug-induced peripheral neuropathy, particularly drug-induced peripheral neuropathy associated with the administration of oncolytic drugs.
- U.S. Patent No. 6,630,478 It also had been proposed to use insulin-like growth factor-I (IGF-I) and insulin- like growth factor-Ill (IGF-III) to treat peripheral neuropathy, such as neuropathies associated with systematic disease, genetically acquired neuropathies, and neuropathies caused by a toxic agent.
- IGF-I insulin-like growth factor-I
- IGF-III insulin-like growth factor-Ill
- the present invention provides methods for the treatment, reduction or amelioration of peripheral neuropathy, including antiretro viral toxic neuropathy (ATN) - a common and typically irreversible complication of stavudine (d4T) and didanosine (ddl).
- ATN antiretro viral toxic neuropathy
- d4T stavudine
- ddl didanosine
- the method includes administering to a subject in need of such treatment, an effective amount of a solubilized ubiquinone or ubiquinol according to the disclosure set forth in relation to any of the embodiments above.
- Peripheral neuropathy is a condition caused by damage to the peripheral nervous system. Thus, protection of nerve tissues from damages should be able to ameliorate or cure peripheral neuropathy.
- Coenzyme Qio is one of the compounds that have been found to have such protection effects.
- Coenzyme Qio has been found to have a neuroprotective effect against glutamate toxicity, and was suggested to be used in treatment of neurological diseases associated with glutamate excitotoxcity, Sandhu et al., Journal of Neuroscience Research, 72:691-703 (2003).
- Coenzyme Qio has also been proposed to be used in treatment of neurodegenerative diseases.
- Dorsal root ganglion (or spinal ganglion) is a nodule on a dorsal root that contains cell bodies of neurons in afferent spinal nerves. All of the axons in the dorsal root convey somatosensory information, bringing sensory information into the brain and spinal cord. These neurons are of the pseudo-unipolar type, meaning they have two axons, one that conveys sensory information from the body to the soma of the neuron and one from the soma to the junction in the dorsal horn of the spinal cord.
- the present invention provides method and composition comprising ubiquinone or ubiquinol (such as coenzyme Q, especially coenzyme Q 10 ) for the prevention and treatment of neurological disorders, particularly peripheral neuropathy, such as neuropathies associated with systematic disease, genetically acquired neuropathies, and neuropathies caused by a toxic agent.
- Toxic agents include, but not limited to agents used in chemical therapy for cancer or AIDS, such as stavudine (d4T) and didanosine (ddl).
- the present invention provides a method for protecting neurons in a mammalian subject against anti-viral toxic agent, comprising administering to a subject in need of protecting a therapeutically effective amount of a pharmaceutical composition provided herein.
- the present invention provides method and composition comprising coenzyme Q (such as coenzyme Q 10 ) for the prevention of antiretroviral toxic neuropathy (ATN) - a common and typically irreversible complication of stavudine (d4T) and didanosine (ddl).
- coenzyme Q such as coenzyme Q 10
- ATN antiretroviral toxic neuropathy
- d4T stavudine
- ddl didanosine
- the methods of the invention provide for administration of a composition, described supra, with a dose, dosing schedule and dosing duration sufficient to treat, prevent or ameliorate peripheral neuropathy or symptoms thereof.
- the dose of coenzyme Q can be about 0.1 mg to about 4,000 mg.
- the dose of Coenzyme Q can be about 20 mg, about 50 mg, about 100 mg, about 200 mg, about 500 mg, about 1,000 mg, about 1,200 mg, about 1,500 mg or about 2,000 mg.
- the methods of the invention provide for administration of a composition on a dosing schedule that provides a therapeutic or prophylactic amount of the composition.
- the composition can be administered about four times daily, about three times daily, about twice daily, about daily, about every other day, about three times weekly, about twice weekly, about weekly or about every two weeks.
- the composition can be administered for a duration sufficient to provide a therapeutic or prophylactic effect.
- the composition can be administered, for example, daily for one year.
- the composition can be administered for about six months, about a year, about two years, about five years, about 10 years or indefinitely.
- the composition can be administered daily for five years.
- composition and method provided herein can be used in combination with other compositions and methods that can be used to ameliorate or cure peripheral neuropathy.
- Gabapentin an anti-seizure medication
- Neurontin For night -time pain relief, Neurontin can be combined with topical pain relievers such as Lidocaine ointment and the Lidoderm patch.
- Topical pain relievers such as Lidocaine ointment and the Lidoderm patch.
- Capsaicin available over the counter as Capzasin-P and Capzasin-HP, is another option for topical pain relief.
- compositions a ubiquinone or ubiquinol and a solubilizing agent provided herein and a second compound that can be used to ameliorate or cure peripheral neuropathy include but not limited to: Neurontin, Capsaicin, carbamazepine, phenytoin, and lamotrigine.
- the invention provides a method of reducing neurotoxicity through administering a therapeutically effective amount of a composition/formulation described herein to a mammal.
- the invention provides a method of treating peripheral neuropathy through administering a therapeutically effective amount of a composition/formulation described herein to a mammal.
- the invention provides a water-soluble cell culture medium supplement composition, which includes a ubiquinone or ubiquinol; and a solubilizing agent having the structure according to the following formula:
- Z is ubiquinol.
- Y 1 and Y 2 are hydrophilic moieties which are members selected from polyethers, polyalcohols and derivatives thereof.
- L 1 and L 2 are linkers.
- the ubiquinone or ubiquinol has a structure which is a member selected from Formula (I) and Formula (II):
- integer n is a member selected from 0 to 13.
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy.
- the ubiquinone or ubiquinol is Coenzyme Qio.
- the ubiquinone or ubiquinol is a Coenzyme Qio analog.
- the ubiquinone or ubiquinol is Coenzyme Qio and said solubilizing agent is PQS.
- the invention provides a culture medium composition, comprising: a) a basal culture medium; and b) a water-soluble cell culture medium supplement composition described herein.
- the basal medium is selected from the group consisting of RPMI 1640, Dulbecco's modified Eagle's Medium, and Ham's F 12.
- the invention provides a culture medium solution comprising: a) a basal culture medium; and b) water-soluble cell culture medium supplement composition described herein, wherein the medium and the supplement composition are dissolved in water.
- the basal medium is selected from the group consisting of RPMI 1640, Dulbecco's modified Eagle's Medium, and Ham's F 12.
- the solution further comprises serum.
- the serum is fetal calf serum.
- the solution further comprises a growth factor.
- the growth factor is nerve growth factor.
- the invention provides a method of growing a cell.
- This method comprises the step of contacting said cells with a cell culture medium comprising: a) a basal culture medium; b) a ubiquinone or ubiquinol; and c) a solubilizing agent having the structure according to the following formula:
- the index a is an integer selected from O and 1 ; the index b is an integer selected from O and 1; and the index c is an integer selected from O and 1.
- Z is ubiquinol.
- Y 1 and Y 2 are hydrophilic moieties which are members selected from polyethers, polyalcohols and derivatives thereof; and L 1 and L 2 are linkers.
- the ubiquinone or ubiquinol having has a structure which is a member selected from Formula (I) and Formula (II):
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy.
- the ubiquinone or ubiquinol is Coenzyme Qio.
- the ubiquinone or ubiquinol is a Coenzyme Qio analog.
- the ubiquinone or ubiquinol is Coenzyme Qio and said solubilizing agent is PQS.
- the cell is a neuron.
- the cell is a glia.
- the glia is a Schwann cell.
- the cell is a primary cell culture cell.
- the cell is derived from a cell line.
- the invention provides a method of culturing tissue or an organ, said method comprising the step of contacting said tissue with a tissue culture medium comprising: a) a basal culture medium; b) a ubiquinone or ubiquinol; and c) a solubilizing agent having the structure according to the following formula:
- the index a is an integer selected from 0 and 1 ; the index b is an integer selected from 0 and 1; and the index c is an integer selected from 0 and 1.
- Z is ubiquinol.
- Y 1 and Y 2 are hydrophilic moieties which are members selected from polyethers, polyalcohols and derivatives thereof; and L 1 and L 2 are linkers.
- the ubiquinone or ubiquinol has a structure which is a member selected from Formula (I) and Formula (II):
- R 1 , R 2 and R 3 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy.
- the ubiquinone or ubiquinol is Coenzyme Qio.
- the ubiquinone or ubiquinol is a Coenzyme Qio analog.
- the ubiquinone or ubiquinol is Coenzyme Qio and said solubilizing agent is PQS.
- the first CoQlO dose finding experiment found that there was no evidence of toxicity in cultured fetal rat DRGs exposed to concentrations of CoQlO of up to 5OuM. In fact, by day 11, the length of neurites around DRGs was longer if the DRGs were cultured with CoQlO (concentrations ranged from 0.1 - 5OuM) compared with control DRGs, p ⁇ 0.0001 for all.
- the aim of the second experiment was to confirm that exposure to 5OuM CoQlO is not toxic to cultured fetal rat DRGs and also to see if even higher concentrations may be used safely.
- Eight DRGs (2 plates of 4 DRGs each) were cultured under each of the following conditions:
- a third experiment measured growth of DRG 's with varying doses of H Q OTM, 0.1, 10. 100 uM CoQlO. This experiment was done in DRG 's isolated from a new rat, which accounts for the difference in neurite length compared to experiments 1 and 2. The protocol is identical to the first experiments and the data confirm the initial findings further demonstrating that H Q OTM enhances neurite growth in DRGs. (FIG. IE)
- FIG. IA shows enhanced growth in DRGs after treatment of up to 50 ⁇ M.
- FIG. IB illustrates that the growth of the DRGs is similar after different doses of H Q OTM. After 11 days of H Q OTM treatment, neurite length of H Q OTM treated DRGs is greater than controls as shown in Fig. 1C.
- FIG. ID shows data from the third set of experiments, confirming enhanced growth of DRG's with 0.1, 10, 100 uM H Q OTM beginning the 8 th day of treatment. By day 22, the growth is double that of the controls.
- Fetal rat DRGs were cultured on media containing collagen, nerve growth factor and a range of concentrations of H Q OTM (PTS :COQ in saline) and/or d4T/ddI (both are 33 ⁇ M). Neurite outgrowth and DRG survival were monitored using video image analysis.
- DRGs exposed to l-100 ⁇ M H Q OTM together with 33 ⁇ M ddl showed greater neurite outgrowth compared to those exposed to ddl alone (p ⁇ 0.0001 for all) and were growing at least as well as control DRGs. (FIG. 2 A and 2B).
- H Q OTM enhances the growth and survival of cultured fetal rat DRGs, and reduces the toxicity of d4T and ddl in this model. These data suggest that H Q OTM may effectively prevent ATN. Controlled trials are needed to confirm this in a clinical setting, and to examine the role of H Q OTM in treating established ATN.
- LAC prevents NRTI (ddl)-induced neurotoxicity of fetal rat dorsal root ganglia (DRGs)
- NRTIs Nucleoside analogue Reverse Transcriptase Inhibitors
- HAART Highly Active Anti-Retro viral Therapy
- this class of drugs causes mitochondrial toxicity leading to serious end organ damage including neuropathy, lipodystrophy, and metabolic disturbances.
- Co-administration of micronutrients that improve mitochondrial function such as coenzyme QlO and L-acetyl carnitine (LAC) reduce the mitochondrial toxicity of NRTIs.
- LAC originally studied in a validated model of neurotoxicity due to unavailable source of water soluble coenzyme QlO.
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US9351517B2 (en) | 2013-03-15 | 2016-05-31 | Virun, Inc. | Formulations of water-soluble derivatives of vitamin E and compositions containing same |
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US9861611B2 (en) | 2014-09-18 | 2018-01-09 | Virun, Inc. | Formulations of water-soluble derivatives of vitamin E and soft gel compositions, concentrates and powders containing same |
US10016363B2 (en) | 2014-09-18 | 2018-07-10 | Virun, Inc. | Pre-spray emulsions and powders containing non-polar compounds |
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US10668029B2 (en) | 2008-03-20 | 2020-06-02 | Virun, Inc. | Compositions containing non-polar compounds |
US9788564B2 (en) | 2008-03-20 | 2017-10-17 | Virun, Inc. | Compositions containing non-polar compounds |
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US20130302862A1 (en) * | 2010-12-23 | 2013-11-14 | Xiamen Kingdomway Group Company | Fermentation method for producing co-enzyme q10 |
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