U.S. patent application number 11/312056 was filed with the patent office on 2006-06-29 for cytochrome p450 enzyme complexes and methods of treatment using the same.
Invention is credited to Arthur M. Nonomura.
Application Number | 20060140927 11/312056 |
Document ID | / |
Family ID | 36611823 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060140927 |
Kind Code |
A1 |
Nonomura; Arthur M. |
June 29, 2006 |
Cytochrome P450 enzyme complexes and methods of treatment using the
same
Abstract
The present invention provides methods and compositions for
balancing electron reduction potentials of formulations in a manner
that reduces susceptibility to changes from xenobiotics. The
present invention also provides novel compositions of matter based
on structuring from a mobile nucleotide integral to its
architecture.
Inventors: |
Nonomura; Arthur M.;
(Litchfield Park, AZ) |
Correspondence
Address: |
NIELDS & LEMACK
176 EAST MAIN STREET, SUITE 7
WESTBORO
MA
01581
US
|
Family ID: |
36611823 |
Appl. No.: |
11/312056 |
Filed: |
December 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60638918 |
Dec 23, 2004 |
|
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|
Current U.S.
Class: |
424/94.4 ;
424/442; 435/189 |
Current CPC
Class: |
A23L 33/12 20160801;
A61K 31/7072 20130101; C12N 9/0077 20130101; A23V 2002/00 20130101;
A61K 35/60 20130101; A23L 33/13 20160801; A23V 2002/00 20130101;
A23V 2200/328 20130101; A23V 2250/638 20130101; A23V 2002/00
20130101; A23V 2250/638 20130101; A23V 2250/264 20130101; A61K
35/60 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/094.4 ;
435/189; 424/442 |
International
Class: |
A61K 38/44 20060101
A61K038/44; C12N 9/02 20060101 C12N009/02; A23K 1/165 20060101
A23K001/165 |
Claims
1. An isolated enzyme complex comprising P450:FMN, wherein there is
a plurality of FMN molecules in said complex.
2. A dietary supplement composition comprising FMN together with a
food product comprising a reductant or an oxidant.
3. The dietary supplement composition of claim 2, wherein the
oxidant is a flavinoid.
4. A formulation comprising an oxidant that induces
NADPH:cytochrome P450 reductase in animals together with a
carrier.
5. The formulation of claim 4, wherein said oxidant is selected
from the group consisting of flavin mononucleotide, riboflavin,
glycolate, combinations thereof, and salts and derivatives
thereof.
6. The formulation of claim 4, wherein said oxidant is FMN or a
salt thereof.
7. The formulation of claim 4, wherein said carrier is selected
from the group consisting of an aqueous solution, a carbonated
solution, fish oil, cod liver oil, chocolate, and a reducing
sugar.
8. The formulation of claim 4, further comprising a reductant that
induces cytochrome P450 monooxygenase in said animal.
9. The formulation of claim 8, wherein said reductant is selected
from the group consisting of reducing sugars, fish oil and cod
liver oil.
10. The formulation of claim 8, wherein said reductant is xylose or
glucose.
11. The formulation according to claim 8, wherein said oxidant is
FMN or a salt thereof present in an amount of from about 1 mcg to
about 100 mcg and said reductant is xylose present in an amount of
from about 1 gram to about 1000 grams.
12. The formulation according to claim 8, wherein said oxidant is 1
mcg to 200 mg slow release niacin and said reductant is 1 gram to
1000 grams xylose.
13. The formulation according to claim 8, wherein said oxidant is
nicotinic acid or a salt thereof in an amount of from about 1 mcg
to about 1000 mcg and said reductant is xylose in an amount of
about 1 gram to about 1000 grams.
14. A method of reducing the amount of xenobiotics in an animal,
comprising administering to an animal an effective amount of an
oxidant for inducing NADPH:cytochrome P450 reductase.
15. The method according to claim 14, wherein said oxidant is a
member selected from the group consisting of ammonium salts, flavin
mononucleotide (FMN), flavins, glycolate, liver extract, menadione,
nicotinic acids, nicotinamide adenine dinucleotide phosphate
(NADP), riboflavin, and salts, hydrates, esters, amines, and
aldehydes thereof.
16. The method according to claim 14, wherein said oxidant is a
vitamin.
17. The method according to claim 14, wherein said oxidant is a
flavin.
18. The method according to claim 14, wherein said oxidant has an
E.sub.0 value of between about -400 mV and about -165 mV.
19. The method according to claim 14, wherein said oxidant is
selected from the group consisting of flavins, salts thereof,
hydrates thereof derivatives thereof and combinations thereof.
20. The method according to claim 14, wherein said oxidant
comprises flavin mononucleotide.
21. The method according to claim 14, wherein said oxidant
comprises flavin mononucleotide and liver extracts.
22. The method of claim 14, further comprising administering an
effective amount of a reductant for inducing cytochrome P450
monooxygenase in said animal.
23. The method according to claim 14, wherein said animal is a
human.
24. The method according to claim 14, wherein said oxidant is
flavin mononucleotide in the dosage range of 0.1 mg to 10 mg.
25. A topical composition comprising an effective amount of an
oxidant for inducing NADPH:cytochrome P450 reductase, together with
a suitable carrier.
26. The topical composition of claim 25, further comprising an
effective amount of a reductant for inducing cytochrome P450
monooxygenase.
27. A carbonated beverage comprising FMN or a salt thereof.
28. The beverage of claim 27, further comprising a reducing sugar.
Description
[0001] This application claims priority of provisional application
Ser. No. 60/638,918, filed Dec. 23, 2004, the disclosure of which
is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to methods and
compositions for treating living organisms.
[0003] Upon utilization of a nanoscalar particle to achieve a
practical purpose, its design and replication defines the field of
nanotechnology. Indeed, over the past decade, inventions of
biomaterials have focused on synthesis, while purposeful in vivo
applications have gone wanting. Developments have resulted from the
fabrication of novel architectures that range from Bucky Balls to
octahedral DNA. Cytochromes offer some special nanometric
advantages that are realized as a result of architecture elucidated
within the current invention A diversity of cytochromes present
possibilities for multitudes of combinations. For example, linkage
of chemical reduction of dioxygen to electrogenic translocation of
protons across a membrane occurs through Cytochrome C (Cc) oxidase
(A. Namslauer, A. S. Pawate, R. B. Gennis and P. Brzezinski (2003)
Redox-coupled proton translocation in biological systems: Proton
shuttling in cytochrome c oxidase PNAS 100(26): 15543-15547) and if
Cc, for instance, is singled out from all other cytochromes for
consideration, over three score evolutionary lines are documented,
a diversity that offers many avenues of process.
[0004] Cytochromes P450 (P450) are hemoproteins that catalyze
monooxygenation of endogenous and xenobiotic hydrophobic
substrates. Families of P450 have a broad range of substrates and
are responsible for processing metabolic quantities of exogenous
compounds by inserting singlet oxygen that renders them soluble for
removal. The utilization of oxygen by P450 mediates hydroxylations,
epoxidations, dehalogenations, deaminations and dealkylations. The
predominance of literature is devoted to P450 monoxygenases (CYP);
however, electron reduction must, necessarily, be balanced by
coupling reactions with NADPH:Cytochrome P450 reductases (CPR).
P450 is understood as an enzyme complex of CYP and CPR.
[0005] The present invention generally relates to cytochromes,
particularly cytochromes P450. Specifically, the invention is of
primary application for electron reduction potential mechanisms
that activate the induction, building, and accurate architectural
reproduction by over fifty functionally different structures of
P450 Each monooxygenase is substrate specific and, coupled with
CPR, may be read spectrophotometrically at 450 nm in the presence
of carbon monoxide and, for example, nitrobenzoate.
[0006] In particular, the present inventor has determined that
small quantities of xenobiotics, such as carcinogens and drugs, can
be recognized, deactivated and prepared for removal by P450
systems. Although, some substrates are beneficial, others are
toxins, mutagens, or carcinogens.
[0007] It is in the best interest of humanity to maintain P450
systems at optimal levels to metabolize and remove physiological
concentrations of objectionable substances. The majority of
xenobiotics, being unavoidable through environmental, gustatory,
and pharmaceutical exposures, may be processed at some point of
metabolism by P450. On the other hand, inhibitors of P450 are also
unavoidable. Upon exposure to the strongest inhibitors, the
population is left without the ability to defend adequately against
xenobiotics. Until the discoveries of the present invention, a
means of re-establishing and/or fortifying P450 to process
xenobiotics in living animals had not been made available.
SUMMARY OF THE INVENTION
[0008] This invention elucidates a design philosophy that when
applied to cytochromes, deals with physiologically important
xenobiotics effectively. In certain embodiments, building and
increasing quantities of most of the known P450 structures is
accomplished in vivo as a consequence of cascading responses to
architectural modification, primarily, with the nucleotide, flavin
mononucleotide (FMN), within CPR. As these nucleotides are
administered into living systems, they move across the surface of
reductases as they become integral to the structure and function of
entire monooxygenase-reductase complexes, primarily as P450 plus
the added FMN (P450:FMN) (or other suitable oxidant).
[0009] Thus, a P450:FMN enzyme complex, with more than one FMN
molecule in the complex, results in a novel composition of matter
(such as in the form of a food supplement, a dietary supplement, a
beverage, a topical formulation, or a pill) that assists the body
in detoxifying xenobiotics. The direct connection of FMN, for
example, to fortify these enzyme complexes, has not been made
previously. This invention thus provides methods of detoxification
by administering compositions that are selected for accelerating
specific activity. These formulations enhance P450 in subcellular
nanoarrays. By means of compositions comprised of inducers that are
selected for regeneration, the present invention introduces methods
of activating repressed nanoscopic structures.
[0010] For these reasons, it would be desirable to provide novel
methods and compositions for the purpose of fortifying the
complexes in humans and other living organisms. It would be
particularly desirable if such methods and compositions were able
to induce these complexes in specific tissues. The present
invention further provides convenient methods resulting in
fortification of these complexes for animal and human therapy. It
is desirable that the methods and compositions of the present
invention promote eradication of undesirable compounds in a manner
that enhances the quality of life. It would be further desirable to
provide palatable treatments that are nutritious and broadly
effective. To that end, preferably, the compositions of an
embodiment of the present invention are sweet-tasting, as a
rewarding means that encourage daily upkeep of the enzymes
complexes.
[0011] As a first aspect, the present invention provides isolated
P450:FMN compositions of matter wherein there is a plurality of FMN
molecules in the enzymatic complex. The methods of isolation, such
as from urine or the liver of an animal, are well known to those
skilled in the art.
[0012] As a second aspect, the present invention provides a method
for enhancing P450 family members, numbering over 50, by treatment
with their corresponding oxidants. The method comprises
administering to an animal in need thereof oxidants that increase
the quantity of the complex in that animal.
[0013] As a third aspect, the present invention provides
compositions for inducing P450:FMN enzyme complexes.
[0014] As a fourth aspect, the present invention provides
compositions for enhancing anticarcinogenic enzyme complexes in
humans. The compositions comprise an effective amount of a
nucleotide that increases the amount of the complexes in vivo.
[0015] As a fifth aspect, the present invention provides
compositions comprising a first compound selected from the group
consisting of CPR and oxidants that induce P450:FMN enzyme
complexes.
[0016] As a sixth aspect, the present invention provides methods
for the automatic fortification of P450:FMN enzyme complexes in
living systems. The methods include treatment with a compound
selected from the group consisting of one or more structural
components of the enzyme complexes.
[0017] As a seventh aspect, the present invention provides a method
for improving the chemical defense of humans and animals using
P450:FMN enzyme complexes. The method includes the step of
administering to a human or animal in need thereof an effective
amount of a composition comprising FMN.
[0018] As another specific aspect, the present invention provides
methods for dietary supplements to come to the defense of humans
against harmful xenobiotics whilst improving nutrition,
particularly, in sweeteners.
[0019] Compositions are provided that comprise naturally sweet
reductants with natural oxidants for increasing the amount of
P450:FMN enzyme complexes in an animal.
[0020] These and other aspects of the present invention are
described further in the detailed description and examples of the
invention which follow.
BRIEF DESCRIPTION OF THE DRAWING
[0021] FIG. 1 is a simplified and integrated schematic depiction of
the enzyme complex of the invention. At the top center of the
figure is supplemental FMN, that is processed and integrated into
native P450 yielding P450:FMN of the present invention. Products
are given at the bottom center of the figure. Fortification of the
complex is achieved by administration of effective therapeutic
dosages of one or more components of the complex.
DETAILED DESCRIPTION OF THE INVENTION
[0022] According to the present invention, nanoscalar methods,
compositions, and systems are provided that induce P450. Methods
are provided for treatment, particularly for those who are
compromised by exposure that requires therapy by administration of
the compositions of the present invention.
[0023] The subject invention provides tools and methodologies for
activating production of structures from nucleotides, enzymes and
their substrates. Of particular note, a number of nucleotides, most
often as several FMNs, occur as mobile components of the enzyme
complex. The subject invention represents a significant
contribution to fields of nutrition, nutraceuticals, dietary
supplements, pharmaceuticals, cosmetics, prophylaxis, and medicine.
Acceleration by formulation of nutritive oxidants with reductants
contributes to distribution.
[0024] Accordingly, the present invention provides methods for
treating animals, for increasing the amount of cytochrome P450 in
animals, and for enhancing the ability of an animal to detoxify
xenobiotics. The present invention also provides compositions and
formulations for administration to animals in need thereof, or as a
prophylaxis, in order to increase the amount of cytochrome P450 in
the body of the animal. An exemplary nutritive formulation includes
a selection of a slow-release niacin with a palatable reductant,
preferably a self-rewarding sweetener, such as glucose.
[0025] Unless otherwise defined, all technical and scientific terms
employed herein have their conventional meaning in the art. As used
herein, the following terms have the meanings ascribed to them.
[0026] Enzymatic complexes as used herein refers to the complete
and functional P450:FMN quaternary structure, wherein, the
preferred molecular complex is modified with a plurality of FMN
molecules. In accordance with the present invention, these
complexes are fortified, or strengthened, in a manner that
promotes, increases and improves rates of reaction of P450s.
[0027] "Oxidant" refers to an electron acceptor of CPR.
[0028] "Reductant" refers to electron donors or oxidase substrates
that induce CYP. Oxidase substrates, which induce CYP accelerate
the metabolism of oxidants by CPR.
[0029] "Inducer" refers to oxidants and reductants that improve the
metabolic capacity of P450s. Induction involves a given substrate,
enhancing the specific activity of the P450s.
[0030] "P450:FMN" refers to a P450 enzyme complex with more than
one FMN. "Cc " refers to Cytochrome C.
[0031] "Animal" refers to virtually all living biota of the kingdom
of animals capable of receiving treatments, particularly humans,
their pets, and livestock.
[0032] "Live organism" refers to all living organisms.
[0033] "Percent" or "%" is percent weight.
[0034] "ppm" refers to parts per million.
[0035] "g" refers to grams.
[0036] "mg" refers to milligrams.
[0037] "mcg" refers to micrograms.
[0038] "Sweetener" includes, but is not limited to, the following:
natural sugars, for example, pentose, hexose, disaccharide, and
oligosaccharide; and artificial sweeteners, for example, sucralose,
saccharin, and aspartame. Natural sugars may further include:
xylose, arabinose, fructose, glucose, mannose, galactose, sucrose,
maltose, xylitol, and the like. The terms sweeteners may also
include mixtures of one or more natural sugars and/or one or more
artificial sweeteners.
[0039] The compositions and methods of the present invention may be
administered by any therapeutically effective route of
administration, including but not limited to oral, sublingual,
rectal, parental (subcutaneous, intramuscular, intravenous),
transdermal, topical, nasal, aerosol and like forms of
administration. Dosage forms include tablets, troches, suspensions,
solutions, capsules, powders, microencapsulated systems, various
transdermal delivery systems, and the like. Topical and oral
administrations are particularly preferred.
[0040] In particular, they may be advantageously administered to
humans, animals, and protistans, alike because of the universality
of cytochromes P450. The methods and compositions of the preferred
embodiment of the present invention may be used to enhance defense
mechanisms of xenobiotics in humans and their domesticated animals.
Where metabolism of medicines may be affected by prior exposure to
P450 inhibitors, the methods and compositions of the present
invention may be used, furthermore, to assist with determination of
baselines for dosage of pharmaceuticals that may be metabolized by
P450.
[0041] As provided herein, fortification focuses on modulating
electron transfer through the enzymatic complexes in a manner that
shifts the flow of electrons. FIG. 1 is a schematic depiction of
the electron transfer to the catalytic cycle of a single complex.
By accelerating or adding to the CPR of an organ or tissue,
reductive capacity is enhanced. An enhanced pool allows increased
capacity for electron transfer. Accordingly, the compositions and
methods of the present invention may include inducers or oxidants
as integral structural components.
Methods and Compositions
[0042] The present invention provides methods for administering
substrates that are designed to modify the architecture of P450s
and fortify P45:FMN complexes in animals, including humans. These
methods typically involve administration of an oxidant component
and may, preferably, include administration of an oxidant that is a
component of the complex. Oxidants may be formulated with
appropriate reductants for balanced therapy and co-induction of
monooxygenases and reductases.
Oxidants
[0043] Suitable oxidants are compounds that induce NADPH:cytochrome
P450 reductase. Any compound capable of inducing such reductases
will be useful as the oxidant component in the methods,
compositions, and systems of the present invention. Accordingly,
reductases, particularly those capable of receiving FMN and
nicotinic acids, may be utilized as the oxidant component of the
methods, compositions, and systems of the present invention. In
addition, a number of other suitable oxidants will be readily
determinable by those skilled in the art.
[0044] Preferred oxidant compounds exhibit a one-electron reduction
potential (E.sub.0) between about -400 mV and about -165 mV
inclusive, more preferably between about -396 mV and about -240 mV.
Multiple electron reductions are biologically important with P450
and oxygen. Examples of suitable oxidants include, but are not
limited to, ferredoxin-NADP+ reductases and NADPH:cytochrome P450
reductase including the reductases listed hereinabove, as well as
flavins, nucleotides, nitrobenzoates, nicotinic acids, nitrobenzoic
acids, ammonium salts, menadione, amine oxides, formamidines,
cytochrome reductases, and slow-release formularies, salts,
hydrates, aldehydes, esters, amines, amides, alcohols, derivatives,
dietary supplements and other biologically or chemically equivalent
derivatives thereof and combinations thereof.
[0045] Specific examples of flavins which are useful as oxidants in
the methods and compositions of the present invention include, but
are not limited to, riboflavin, flavin mononucleotide (FMN),
menadione, deazaflavin, lumichrome, lumizine, flavin adenine
dinucleotide (FAD), alloxazine, salts, hydrates, derivatives and
combinations thereof. Specific examples of ammonium compounds
include, but are not limited to ammonium sulfate, ammonium
chloride, ammonium hydroxide, urea, amines, and the like. Specific
examples of cytochrome reductases include, but are not limited to,
cytochrome f, Cc, cytochrome b5, flavocytochrome P450, nitric oxide
synthase, and combinations thereof. Specific examples of nicotinic
acids include but are not limited to NicoSpan, NiaSpan, niacin,
NAD, NADH, NADP, NADPH and combinations thereof. Examples of
preferred oxidants, whose selection is based on E.sub.0 (values)
and beneficial metabolism, include nitrobenzoate (-396 mV), FMN
(-313 mV), glycolate (.alpha.) (-290 mV), riboflavin (-292 mV), and
salts, hydrates and derivatives of any of the above.
[0046] Currently preferred oxidants for use in the methods and
compositions of the present invention include but are not limited
to FMN, NiaSpan, NAD/NADH, menadione, ammonium sulfate, a, and
salts, hydrates and derivatives thereof FMN and nicotinic acids are
particularly preferred oxidants in the compositions, methods and
systems of the present invention, primarily because they are cost
effective and generally regarded as safe. Additionally, organ
products such as whole liver, dry liver pills, liver oils and liver
extracts are excellent sources of undefined cytochromes, oxidants,
and reductants. Where organ products are unpalatable or are
problematic because of, for instance, cholesterol or gout, it would
be advisable to avoid these types of products. Moreover, in the
course of processing, proteins and nucleotides may be denatured;
thus, organ-derived products may require supplementation with
oxidants. FMN is the particularly preferred oxidant.
[0047] As noted above, oxidants are employed solely or, they may
comprise any one or more of the foregoing oxidants in combination
with a reductant or other oxidants. For example, in one preferred
embodiment, the oxidant comprises a combination of FMN and fish oil
concentrate. In the embodiment of the invention wherein two or more
oxidants are combined, they are typically equimolar provisions of
the oxidant component of the compositions and methods of the
present invention.
Inhibitors
[0048] A partial list of inhibitors includes the following common
items: foods and beverages, including grapefruit, turmeric
(curcuminoids), bergamottin (flavoring in Earl Grey Tea), and
well-water (arsenic); drugs, such as, antibiotics (ciprofloxacin,
erythromycin, chloramphenicol, and the like), antivirals
(ritonavir, etc.); contraceptives (ethynylprogesterone, and the
like), antimycotics (ketoconazole, itraconazole, terconazole,
miconazole, and the like), chemotherapies (aminoglutethimide,
etc.); acid reflux (cimetidine), bioassays (metyrapone,
diethyldithiocarbamate), barbiturates and mood drugs (secobarbital,
Prozac, Zoloft, Luvox and the like); cosmetics (hydrogen peroxide,
dental whitener, bleach, etc.); air pollution from carbon monoxide
and other components of smog; insecticides (cyanides, parathion,
piperonyl butoxide, etc.); and acyl hydrazine, alkyl hydrazine,
aryl hydrazine, allylisopropylacetamide, carbon disulfide, carbon
tetrachloride, dichloroacetamides, dihydropyridine, disulfiram,
isothiocyanate, mercaptosteroid, phenylimidazole, phenelzine,
phenylphenanthridinone, quinolones, syndones, thiourea, tienilic
acid, and undecynoic acid.
[0049] In a physiological sense, constant exposure to inhibitors
renders the general population susceptible to xenobiotics. When
P450 is inhibited, it is imperative to resurrect dysfunctional
P45:FMN complexes by the compositions and methods of the present
invention, if for no other reason than to reduce susceptibility to
carcinogens. Inhibitors include compounds that degrade or bind to
the heme iron atom or to the prosthetic heme group. Others may be
competitive inhibitors of P450, the effects of which may last the
half life of a drug.
Application
[0050] The oxidants are, by themselves useful in methods of
treatment and in methods of fortifying the P40 enzyme complexes.
For example, flavins, by themselves, or together with suitable
excipients and/or carriers, are useful in the methods of the
present invention. Although the oxidant components may be applied
in a solid form, it is sometimes advantageous to provide oxidants
in liquid form, such as by solubilizing the component in an aqueous
or suitable organic solvent or carrier to produce aqueous or
organic solutions of the oxidants. The amount of oxidant that is
solubilized in the carrier will depend upon the particular oxidant
selected and the method of application. The oxidant may be
solubilized in the carrier by adding the oxidant to the carrier and
allowing the oxidant to dissolve. In some instances, the
application of stirring, agitation, or even heat may facilitate the
dissolution of the oxidant in the carrier.
[0051] For example, suitable formulations are not particularly
limited, and include solutions of the oxidant dissolved in a
suitable carrier. For example, FMN can be dissolved in a carbonated
beverage with or without a reducing sugar such as xylose and/or
glucose. The carbon dioxide inhibits premature oxidation of the
FMN. FMN in combination with chocolate is another example, such as
a formulation comprising 0.2 to 0.5 mg FMN per 30-60 grams of
chocolate, preferably dark chocolate. Yet other suitable
formulations include the oxidant and one or more reducing sugars,
artificial or natural, as discussed above. Still further suitable
formulations include emulsions, such as FMN and fish oil, cod liver
oil or both. These emulsions can be formulated in a capsule and
administered orally. Another suitable formulation is FMN and red
wine.
[0052] The oxidant can be administered in a carrier to create a
formulation having an oxidant concentration in the range between
about 0.0001% and about 100% by weight of the composition
inclusive, preferably between about 0.01% and about 100% inclusive.
For example, because ethanol is an inducer of CYP2E1, a
flavin:alcohol beverage ratio of 1:1000 is preferred to match
ratios generally found to induce the enzyme complex. An exemplary
skin cream comprising 0.1% to 1% .alpha.-hydroxy is suitable for
topical application when formulated with 0.001% to 0.005%
riboflavin.
[0053] Compositions of the present invention may also include any
of a variety of excipients, which improve or at least do not hinder
the beneficial effects of the compositions of the present
invention. While the compositions of the present invention may
consist essentially of oxidants, compounds may be formulated in
suitable excipients. In the embodiment wherein the oxidant is a
single composition for use in the methods of the present invention,
the composition may include excipients having solubilized,
dispersed, supported, or otherwise contained therein, an amount of
the oxidant that induces cytochrome P450. A solution containing
oxidants may be prepared using the general techniques set forth
above.
[0054] Compositions containing oxidants in a single solution may
include any combination of oxidants selected from those described
hereinabove. Preferred oxidants for one-step compositions include,
but are not limited to FMN and nicotinic acids. For example, one
composition according to the present invention includes menadione
and niacin. Another composition according to the present invention
includes FMN and cod liver oil. Another composition according to
the present invention includes daily 50 mg riboflavin and 5 mg
NADP. Another composition according to the present invention
includes 1 mg FMN cloaked in carbon dioxide as a stabilizing
excipient and to be taken up to 5 times daily.
[0055] Compositions of oxidants will typically be administered at a
concentration ranging between about 1 mg and about 1000 mg per
dose. Preferred combined oxidant compositions include: (1) 5 mg
NADH and 25 mg niacin; (2) 5 mg FMN and 50 mg slow-release niacin;
and (3) 3 mg FMN and 500 mg fish oil concentrate. Macromolecules
such as CPR and CYP pose problems of stability and may require cold
storage and inert environments. Formulation with carbonates or
bicarbonates prevents oxidation. The compositions of the present
invention may also be applied topically in appropriate
dermatological or optical formulations.
[0056] Compositions according to the present invention may find
specifically tailored utilization, including enhanced performance
of NADPH:cytochrome P450 reductase; enhanced quantity of
NADPH:cytochrome P450 reductase; fortification of P450:FMN enzyme
complexes; inducing such complexes after exposure to inhibitors;
reduced susceptibility to carcinogens; and for therapy.
Compositions may also be formulated at very low concentrations of
FMN or in sustained release dosages of niacin for daily
enhancement.
[0057] The aqueous solutions employed in the systems of the present
invention may be formulated in the same manner as described
hereinabove for compositions, using the same types of aqueous
carriers. Preferably a pharmaceutically acceptable carrier is used.
One preferred formulation according to the present invention
includes flavin mononucleotide as the oxidant. Another preferred
formulation according to the present invention includes niacin as
the oxidant. Another preferred system according to the present
invention includes flavin mononucleotide and liver extracts as
oxidants. Another formulation according to the present invention
includes oxidants formulated with reductants such as, reducing
sugars; the preferred formulation utilizing treatment with 1 mg to
100 mg NiaSpan, sustained release niacin, as an oxidant formulated
with 10 g to 1000 g of a pentose reductant such as xylose.
[0058] The following examples are provided to further illustrate
the present invention, and should not be construed as limiting
thereof. The present invention is defined by the claims, which
follow.
[0059] In these examples, reagents, biochemicals, and dietary
supplements were obtained in purest form available, and were
obtained as reagent grade and USP chemicals where possible.
Purified P450s including recombinant human NADPH:Cytochrome P450
Reductase (hCPR) were obtained from PanVera.
[0060] In these examples, "L" means liter; "ml" means milliliter;
"cm" means centimeter; "cm.sup.2 means centimeters squared; "n"
means nanometer; "M" means molar; "mM" means millimolar; ".mu.M"
means micromolar; "nM" means nanomolar; "mol" means moles;
".mu.mol" means micromoles; "mg/ml" means milligrams per
milliliter; "ml/cm.sup.2" means milliliters per centimeter squared;
"kDa" means kiloDaltons; "L/min" means liters per minute; "d" means
days; "h" means hours; "min" means minutes; "s" means seconds; "g"
means multiple of centrifugal gravitational force; ".degree." means
degrees centigrade; CYP is a cytochrome P450 monooxygenase; CPR is
NADPH:cytochrome P450 reductase; CPR is microsomal-CPR.
[0061] The following exemplary compositions are intended to provide
further guidance to those skilled in the art, and do not represent
an exhaustive listing of compositions within the scope of the
present invention.
EXAMPLE I
[0062] First Exemplary Composition: FMN TABLE-US-00001
Concentration per dose Composition Broad Range Narrow Range FMN
0.01-50 mg 3-5 mg Cod liver oil 1-2500 mg 100-1000 mg
[0063] Second Exemplary Composition: Drink TABLE-US-00002
Concentration per 100 ml dose Composition Broad Range Narrow Range
Xylose 0.1-1% 1-1000 g FMN 1-1000 ppm 1 mcg-5 mg
[0064] Third Exemplary Composition TABLE-US-00003 Concentration
Composition Broad Range Narrow Range FMN 0.01-20 mg 1-5 mg
Analgesics 1-2000 mg 5-100 mg
[0065] Fourth Exemplary Composition TABLE-US-00004 Concentration
Composition Broad Range Narrow Range FMN 1-5 mg 2-5 mg NADP 10-100
mg 10-50 mg Bicarbonate excipient excipient
[0066] Fifth Exemplary Composition TABLE-US-00005 Component Dose
Range FMN 5 mg 1-5 mg Liver extract 1 mg 10 mcg-2 g
[0067] Sixth Exemplary Composition: Drink TABLE-US-00006
Concentration Composition Broad Range Narrow Range FMN 1 mcg-50 mg
10 mcg-5 mg D-Xylose 1-1000 g 5-50g EtOH excipient excipient Carbon
dioxide excipient excipient Water excipient excipient
[0068] Seventh Exemplary Composition: Natural Sweetener Suitable
for People with Diabetes TABLE-US-00007 Concentration Composition
Broad Range Narrow Range Folate 1-100 mcg 1-3 mcg FMN 1-8000 mcg
1-100 mcg D-Xylose 1-1000 g 5-50 g
[0069] Eighth Exemplary Composition: Blended Diet Sweetener
TABLE-US-00008 Concentration Composition Broad Range Narrow Range
FMN 1-1000 mcg 2-200 mcg D-Xylose 1-1000 g 5-50 g Sucralose 1-1000
g 5-50 g
EXAMPLE 2
[0070] The following example illustrates an application of human
P450 compositions according to the present invention. Procedures
followed previously described methods, e.g., U.S. Pat. No.
6,020,288; M. Markwell, et al., Methods of Enzymology 72: 296-303
(1981); C. A. Mihaliak, et al., Methods in plant biochemistry 19:
261-279 (1993); R. Donaldson, et al., Arch. Biochem. Biophys. 152:
199-215 (1972); and M. Persans, et al., Plant Physiol. 109:
1483-1490 (1995)). The results demonstrate the efficacy of the
methods and compositions of the present invention for the
enhancement of P450 enzyme complexes.
[0071] Methods. CPR substrates have been assayed on various tissues
(e.g., U.S. Pat. No. 6,020,288); therefore, response to human CPR
(hCPR) was examined under controlled conditions on live index
organisms. The direct effects of substrates on hCPR were measured
by preparation of microsomes for quantification against CPR and Cc.
Controls included equal concentrations of each individual treatment
or substrate in surfactant and water. Untreated controls were
maintained under identical conditions of culture. Oxidase activity
of Cc was inhibited by potassium cyanide. The reaction was
initiated by addition of Cc wherein NADPH-dependent reduction of Cc
was monitored for increases in absorbance.
[0072] Results and Discussion. FMN showed over 4.5 times the
induction of components tested. Cytochromes P450 enzyme complexes
have defined catalytic electron transfer functions. See, C. von
Wachenfeldt, et al., Structures of Eukaryotic Cytochrome P450
Enzymes, P. R. Ortiz de Montellano, ed. (1995) Cytochrome P450:
Structure, Mechanism, and Biochemistry (Second Ed.), Plenum Press,
New York, pp 183-223 and H. Strobel, et al., NADPH Cytochrome P450
Reductase and Its Structural and Functional Domains, P. R. Ortiz de
Montellano, ed. (1995) Cytochrome P450: Structure, Mechanism, and
Biochemistry (Second Ed.) Plenum Press, New York, pp 225-244.
Combinations of cytochromes P450 are numerous and underscore the
potential of the field. Selection of oxidants based on one electron
reduction of compounds (see, e.g., Wardman, P. 1989, J Phys. Chem.
Ref Data 18(4): 1637-1755) within potentials associated with CPR
reductase (see, e.g., Butler, J. et al. 1993 Biochimica et
Biophysica Acta 1161: 73) proved successful, test results showing
that specific activity may be enhanced by FMN. Oxidants accelerate
cytochrome P450 and, FMN, in particular, may fortify its integral
structure. In some instances, enhancement of cytochromes other than
CPR may be key. For example, Cc may accelerate response where CPR
does not, and for those exceptional cases, broad spectrum dosage or
specific agents will be the subject of further investigations. Even
though the most potent treatment may be nanomolar cytochromes, of
the integral components of cytochromes, FMN showed activity at
sufficiently low concentrations to be a prime selection for
practical, safe and effective therapies. P450 provides widespread
applicability of compositions and methods for selection of
components which may be utilized to endow animals with a means of
resistance to xenobiotic stresses while gaining ever greater
health.
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