CA2642096A1 - Coenzyme q10 production from marine bacteria - Google Patents

Abstract

Disclosed are methods, compounds, and compositions related to the production of coenzyme Q.

Description

1. CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to Provisional Application Numbers 60/771,958, filed February 9, 2006, and 60/812,740, filed June 12, 2006, which are both incorporated by reference herein in their entirety.
II. FIELD
The disclosed matter relates to compounds comprising coenzyme Q, including methods of making and using such compounds.
III.BACKGROUND
Coenzymes Q occur in the majority of aerobic organisms, from bacteria to plants and animals. Two numbering systems exist for designating the number of isoprenoid units in the terpinoid "tail": coenzyme Q, and coenzyme Q(x), where n refers to the number of isoprenoid side chains and x refers to the number of carbons in the terpinoid "tail" and can be any multiple of five. Thus, coenzyme Q]o (also termed CoQ~o) refers to a coenzyme Q
having 10 isoprenoid units in the "tail." Since each isoprenoid unit has five carbons, CoQio can also be designated coenzyme Q(50) or CoQ(50). The name CoQõ can be used to generally refer to both the oxidized form and reduced form of the compound;
alternatively, these specific forms can be individually designated CoQned and CoQõoX.
Chemically, CoQ100x is known as 2,3-dimethyoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, and its structural formula is:
O CHg O
COQio CoQ10 is a model carrier of protons and electrons. It plays a vital role in the mitochondrial respiratory chain and oxidative phosphorylation. It was first isolated by researchers working at the Enzyme Institute of the University of Wisconsin (Crane, et al., BBA 25:220-1, 1975). Currently Japanese Kaneka Corp. supplies 60 -70 % of CoQ10 sold in the USA.
The oxidized form of CoQ10 (CoQloox) has anti-atherogenic properties.
Deficiencies in CoQlooX are associated with higher incidence of heart failure and other cardiovascular problems. Although CoQIo plays an important role in the development of ardiovascular disease, there have been data that suggest that the coenzyme also plays an important role in the nervous system. For example, CoQIo is believed to have beneficial effects in the prevention and treatment of Parkinson's disease, mitochondrial myopathies, muscular dystrophy, etc.
Several attempts have been made to deliver benzendiol derivatives such as CoQlo to a subject. Selzer disclosed a liquid dietary CoQio supplement based on vegetable oil-water emulsion. The absorption of CoQio from this formulation was enhanced (U.S. Pat.
No. 6,652,891 to Selzer et al.).
Natural Health Sciences together with General Nutrition Centers developed a blend of Pycnogenol, a French maritime pine bark extract, and CoQio called PycnoQlO.
Joint research executed at Showa Medical University, Tokyo, and State University of New York suggested that the combination protected 53% of blood lipids from oxidation compared to 30% when the ingredients were used separately_ The product protected blood vessel integrity, blood lipid values, circulation, blood pressure, and platelet function. The activity is believed to be derived from the synergy of antioxidant properties.
Horrobin describes a physical mixture of CoQ10 and eicosapentaenoic acid (EPA) (Int'l. Pub. No. WO 02/096408 Al). Sears, et al., describes a composition made of CoQlo and polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA), EPA, or linolenic acid, which is intended for the prevention and/or treatment of mitochondriopathies (U.S. Pat. No. 6,417,233). Formation of the ester between PUFA and CoQIo is not disclosed. U.S. Pat. Nos. 6,300,377 and 6,441,050 to Chopra disclose a combination of CoQIo with a polysorbate surfactant, which can also be mixed with other active materials such as omega-3 fatty acids.
In light of the numerous health benefits associated coenzyme Q compounds such as CoQlo, what is needed in the art are new methods of preparing and using such compounds and compositions. The compounds, compositions, and methods disclosed herein meet these needs.

IV. SUMMARY
In accordance with the purposes of the disclosed materials, compounds, compositions, articles, and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to compounds and compositions and methods for preparing and using such compounds and compositions.
Disclosed herein is a method of preparing an isoprenoid composition, the method comprising: culturing bacteria from the marine environment, and isolating the isoprenoid.
Disclosed herein is a method of preparing an isoprenoid composition, the method comprising: culturing bacteria from Sphyingomonodales family, and isolating the isoprenoid.
Also disclosed herein is a method of preparing an isoprenoid composition, the method comprising: culturing bacteria from Bacillaceae family, and isolating the isoprenoid.
Also disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing PTA-7565 (SEQ ID NO. 1), and isolating the isoprenoid.
Also disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing PTA-7566 (SEQ ID NO. 9), and isolating the isoprenoid.
Also disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing PTA-7567 (SEQ ID NO. 13), and isolating the isoprenoid.
Also disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing PTA-7568 (SEQ ID NO. 15), and isolating the isoprenoid.
Also disclosed are nutritional supplements, delivery devices, and foodstuffs comprising the compounds disclosed herein.
Also disclosed are methods of lowering total cholesterol levels or triglyceride levels, increasing HDL levels, or a combination thereof in a subject, comprising the step of administering an effective amount of the compounds disclosed herein.
Also disclosed are methods of reducing hyperglycemia or hypercholesterolemia in a subject, comprising the step of administering an effective amount of the compounds disclosed herein.
Disclosed herein are methods for treating or preventing a mitochondrial condition or disease in a subject, comprising the step of administering to the subject an effective amount of the compounds disclosed herein.

Also disclosed are methods for increasing circulation or the immune system in a subject, comprising the step of administering to the subject an effective amount of the compounds disclosed herein.
Disclosed are methods for reducing the side effects of chemotherapy in a subject, comprising the step of administering to the subject an effective amount of the compounds disclosed herein.
Also disclosed are methods for treating or preventing degenerative heart disease in a subject, comprising the step of administering to the subject an effective amount of the compounds disclosed herein.
Also disclosed herein are pharmaceutical formulations comprising the compounds disclosed herein and a pharmaceutical carrier.
V. FIGURE DESCRIPTIONS
Figure 1 shows a neighbour-joining tree showing relatedness between the 16S
rRNA gene from microbial strains ONC-Q00001-17 corresponding to SEQ ID NOs: l-17, respectively and nearest neighbours as discussed herein.
VI. DETAILED DESCRIPTION
The materials, compounds, compositions, articles, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.
Before the present materials, compounds, compositions, articles, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
A. Methods of Makin~
Disclosed are prokaryotic marine microbes capable of producing isoprenoids, such as CoQ10. Also disclosed are the microbes themselves as well as variants and related strains having the ability to produce the disclosed compounds as disclosed herein.

Prokaryotic microbes have been found to possess the ability to produce said isoprenoid compounds. Specific examples of isoprenoid compounds include ubiquinone, including but not limited to ubiquinone-10 (CoQlo). The disclosed bacteria can also produce other antioxidants, such as, but not limited to, carotenoid compounds. Therefore, disclosed 5 herein are methods for preparing compounds such as isoprenoids and carotenoids. Also, disclosed herein are compounds prepared by the methods disclosed herein.
The prokaryotic microbes that are capable of producing the compounds disclosed herein can be found in the Suhyingomonodales family. Examples of bacteria in the Sphyingmonodales family include those from the genus Erythrobacter, such as Erythrobacter seohaensis, Erythrobacter sp. MBIC 2351, Erythrobacter sp. SD-2 1, and Erytrhobacter sp. MBIC 3953. Examples of bacteria in the Sphyingmonodales family include those from the genus Erythrobacter, such as Erythrobacter seohaensis, Erythrobacter sp. MBIC 2351, Erythrobacter sp. SD-21, and Erytrhobacter sp.
MBIC
3953. For example, Erythrobacter seohaensis (PTA-7565) (SEQ ID NO. 1) can be used to produce the compounds disclosed herein. The bacteria can also be from the genus Sphingomonas, such as Sphingomonas baekyungensis. For example, Spingomonas baekyungensis (PTA-7566) (SEQ ID NO. 9) can be used to produce the compounds disclosed herein. The bacteria can also be from the genus Lutibacterium, such as Lutibacterium anuloederans. For example, Lutibacterium anuloederans (PTA-7568) (SEQ
ID NO. 15) can be used to produce the compounds disclosed herein. The bacteria can be marine-derived. "Marine derived" is defined as any bacteria derived from a marine environment, such as leaf material growing in or near water, sediment in or near water, seawater, seaweed-derived, sea-grass derived, or growing in or near a marine animal or plant.
The bacteria can also be from the Bacillaceae family, such as those in the Exiguobacterium genus. An example of a bacteria in the Exiguobacterium genus is Exiguobacterium oxidotolerans. For example, Exiguobacterium oxidotolerans (PTA-7567) (SEQ ID NO. 13) can be used to produce the compounds disclosed herein.
This bacteria can also be marine-derived.
Also disclosed are conditions for the isolation and growth of the bacteria.
For example, disclosed are growth conditions for production of the disclosed isoprenoids and carotenoids, for example, both individually and cumulatively. It is understood that the microorganism and any clones, modified organisms or genes isolated from said microorganism set forth herein are also disclosed.

The bacteria can be grown without special conditions, and one of skill in the art would know how to culture such strains. Plates containing sources of carbon, nitrogen, inorganic salt, as well as substances necessary to stimulate growth can be used. As a source of carbon, any of the following can be used alone or in combination: L-mannose, L-fructose, galactose, glycerol, succinic acid, citric acid, acetic acid, or methanol. In one example, the bacteria are grown on "full strength" Marine Agar (MA) (MA:
55.2g/L).
Alternatively, the bacteria can also be grown on Marine Broth Agar (MBA) (MBA:
15 g/L
bacteriological agar and 90% artificial seawater). Optionally, the media can be supplemented with cyclohexamide to prevent competition from fungal species.
The cyclohexamide can be added, for example, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mg/L, or any amount in between.
Collected leaf, seaweed and sea grass samples can be cut and washed with seawater. (Example 2 provides non-limiting examples of how these methods can be carried out). Washed leaves and seaweeds can be grinded in sterile marine broth with mortar, then diluted. The samples can then be plated or added to an appropriate broth for growth. For collected sediment, for example, it can plated on an appropriate media or broth. For seawater, the sample can be diluted in marine broth, then plated appropriately.
Plates and/or broth can then be incubated at an appropriate temperature, for an appropriate length of time. In one example, the temperature can range from 4-40 C. For each strain of bacteria, a suitable temperature for growth and propagation can be selected.
A suitable temperature can be determined. The pH of the culture can be from 6-8, and can vary to determine the best outcome for production. If ammonium salt is used as a source of nitrogen, as the bacteria multiply, the pH of the growth fluid drops, and so ammonium or potassium (for example) can be added to maintain a consistent pH.
The bacteria can be incubated for 1, 2, 3, 4, 5, 6, or 7 days, or 2, 3, 4, 5, or 6 weeks, or any length of time in between. Optionally, the bacteria can be checked and re-plated as necessary during this time period.
Cultivation of the biomass containing the isoprenoid/carotenoid can be periodical or continuous. In one example, CoQlo or a carotenoid can be extracted from the biomass present on the media periodically. Methods of extraction are well known in the art, and an example of such extraction can be found in Example 2. For example, coenzyme can be extracted using methanol:hexane (3:2, v/v), then hexane can be added and the sample can be centrifuged. A variety of procedures can be employed in the recovery of the resultant cellular biomass from fermentation in various culture media, such as by filtration or centrifugation. The product can then be washed, frozen, lyophilized, or spray dried, and stored under a non-oxidizing atmosphere to eliminate the presence of oxygen, prior to incorporation into a processed food or feed product.
Separation and extraction of CoQtp and carotenoids can be achieved using standard methods known in the art. For example, refining can occur by using the protocols of Long (2004) and Armenta-Lopez (2002) for carotenoid extraction. In one example, acetone can be used in the extraction process (Example 2).
Also disclosed are processes for the production of microbial biomass containing the compounds disclosed herein, along with a process for preparing these compounds utilizing the microorganisms.
In addition, disclosed are isoprenoids, including CoQ~o, and carotenoids produced by the disclosed microorganisms, as well as various feedstuffs, nutraceuticals, pharmaceutical and food supplemented with the lipids and antioxidants, as well as a process for utilizing these compounds as an additive for various feedstuffs and foods.
Disclosed are methods of preparing an isoprenoid composition, the method comprising: culturing bacteria from Svhyingomonodales family, and isolating the isoprenoid. Also disclosed are methods, wherein the bacteria is an Erythrobacter, Erythrobacter seohaensis, Sphingomonas, Sphingomonas baekyungensis, Lutibacterium, Lutibacterium anuloederans, or wherein the bacteria is marine-derived. For example, disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing bacteria from PTA-7565 (SEQ ID NO. 1), PTA-7566 (SEQ ID
NO.
9), or PTA-7568 (SEQ ID NO. 15) and isolating the isoprenoid.
Also disclosed are methods of preparing an isoprenoid composition, the method comprising: culturing bacteria from Bacillaceae family, and isolating the isoprenoid.
Also disclosed are methods, wherein the bacteria is from Exiguobacterium genus, or wherein the bacteria is Exiguobacterium oxidotolerans. For example, disclosed herein are methods of preparing an isoprenoid composition, the method comprising culturing bacteria from PTA-7567 (SEQ ID NO. 13), and isolating the isoprenoid.
Also disclosed are methods, wherein the bacteria comprises a sequence having greater than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%
identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ
ID
NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, or SEQ ID NO:17, or any combination thereof.

Also disclosed are methods, wherein the organism comprises a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ
ID NO:17, or any combination thereof.
Also disclosed are methods, wherein the isoprenoid is a coenzyme, or wherein the coenzyme is Coenzyme-Qio (CoQlo).
Also disclosed are compounds prepared by any of the methods provided herein, as well as nutritional supplements or pharmaceuticals comprising the prepared compounds or compositions. For example, provided are nutritional supplements comprising from about 0.05% to 20% by weight of the compound or from about 1% to 7.5% by weight of the compound or which comprises up to or comprises less than or equal to 100% by weight of the compound.
It is understood that the compositions can be provided wherein they are in the form of a tablet, gel-cap, capsule, liquid, or syrup. Also disclosed are delivery devices comprising the disclosed compounds, such as wherein the device comprises a microcapsule, a liposome, noisome, nanoerythrosome, solid-liquid nanoparticle, microsphere, or pulmosphere, or wherein the device comprises a microcapsule, wherein the microcapsule comprises an agglomeration of primary microcapsules, each individual primary microcapsule having a primary shell and the agglomeration being encapsulated by an outer shell, wherein the compound is encapsulated in the primary microcapsule.
The delivery device can also comprise microcaps, wherein the primary shell and the outer shell comprise gelatin type A, gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrins, cyclodextrins, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, chitin, polylactide, poly-lactide-co-glycolide, polylysine, kosher gelatin, non-kosher gelatin, Halal gelatin, non-Halal gelatin, or a mixture thereof, or wherein the primary shell and the outer shell comprises gelatine type A having a Bloom strength of from 0 to 350, or wherein the primary shell and the outer shell comprises a no bloom fish gelatin.
Also disclosed are delivery devices further comprising an additional shell surrounding the outer shell, wherein at least one of the primary, outer, and additional shells comprise a complex coacervate.

Also disclosed are foodstuffs comprising the disclosed compounds, such as, wherein the foodstuff is a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, a poultry product, a processed fruit juice, a granulated sugar, a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, a fish product, or pet companion food, or wherein the foodstuff is bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.
Disclosed are compositions comprising a marine bacterium, wherein the marine bacterium produces an isoprenoid. For example, disclosed are compositions, wherein the organism comprises a sequence having greater than 99% identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, SEQ ID
NO:16, or SEQ ID NO:17, or any of the sequences disclosed herein, or any combination thereof.
Also disclosed are compositions, wherein the organism comprises a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17, or any combination thereof.
The composition of claim 1-50, wherein the bacterium produces at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, or 3000 g/g of the isoprenoid.
Also disclosed are compositions comprising an isolated marine bacterium and an isolated isoprenoid.
Also disclosed are phanmaceutical formulations comprising the disclosed compounds and a pharmaceutical carrier.
Also disclosed are methods, wherein the composition comprises a salt of an omega-3 fatty acids, or methods wherein the salt is a magnesium or calcium salt.
Also disclosed are compositions, wherein the composition comprises a salt of omega 3 fatty acids, or compositions wherein the-salt is a magnesium or calcium salt.
B. Compositions Disclosed herein are materials, compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be 5 explicitly disclosed, each is specifically contemplated and described herein. For example, if a compound is disclosed and a number of modifications that can be made to a number of components or residues of the compound are discussed, each and every combination and permutation that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of components or residues A, B, and C are disclosed as well 10 as a class of components or residues D, E, and F, and an example of a combination compound A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
Certain rnaterials, compounds, compositions, and components disclosed herein can be obtained commercially or can be readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH
Publishers Inc., 1989).
1. Deposit Erythrobacter seohaensis (ONC-Q00001) capable of producing coenzyme Q10 was deposited on May 15L 2006 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA. 20110 2209, USA, under accession number ATCC No. PTA-7565.
Spingomonas baekyungensis (ONC-Q00009) capable of producing coenzyme Q10 was deposited on May 1s` 2006 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA. 20110 2209, USA, under accession number ATCC No. PTA-7566.
Lutibacterium anuloederans (ONC-Q00015) capable of producing coenzyme Q10 was deposited on May 1't 2006 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA. 20110 2209, USA, under accession number ATCC No. PTA-7568.
Exiguobacterium oxidotolerans (ONC-Q00013) capable of producing coenzyme Q10 was deposited on May l" 2006 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA. 201 1 0 2209, USA, under accession number ATCC No. PTA-7567.
These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that deposits are required under 35 U.S.C. 112.
2. Microorganisms Disclosed herein are microorganisms capable of producing CoQ10. Such microorganisms include, but are not limited to, microorganisms deposited with the ATCC
as PTA-7565, PTA-7566, PTA-7567, and PTA-7568. Other examples include, but are not limted to the microorganisms described in the Examples and Tables below.
3. Supplements Disclosed herein are nutritional supplements. A nutritional supplement is any compound or composition that can be administered to or taken by a subject to provide, supply, or increase a nutrient(s) (e.g., vitamin, mineral, essential trace element, amino acid, peptide, nucleic acid, oligonucleotide, lipid, cholesterol, steroid, carbohydrate, and the like). In one aspect, disclosed herein are nutritional supplements comprising any of the compounds disclosed herein. For example, a nutritional supplement can comprise any compound comprising CoQlo. The fatty acid residues of these formulas can be any fatty acid as disclosed herein (e.g., unsaturated or saturated fatty acid residues).
The nutritional supplement can comprise any amount of the compounds disclosed herein, but will typically contain an amount determined to supply a subject with a desired dose of a CoQ10 and/or fatty acids. The exact amount of compound required in the nutritional supplement will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the dietary deficiency being treated, the particular mode of administration, and the like. Thus, it is not possible to specify an exact amount for every nutritional supplement. However, an appropriate amount can be detennined by one of ordinary skill in the art using only routine experimentation given the teachings herein. In one specific example, a nutritional supplement can comprise from about 0.05 to about 20%, from about 1 to about 7.5%, or from about 3 to about 5% by weight of the compound. In another example, the nutritional supplement can comprise from about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0% by weight of the compound, where any of the stated values can form an upper or lower endpoint when appropriate. In another aspect, when the nutritional supplement, the supplement can be composed of up to 100% of the supplement.
The nutritional supplement can also comprise other nutrient(s) such as vitamins trace elements, minerals, and the like. Further, the nutritional supplement can comprise other components such as preservatives, antimicrobials, anti-oxidants, chelating agents, thickeners, flavorings, diluents, emulsifiers, dispersing aids, and/or binders.
The nutritional supplements are generally taken orally and can be in any form suitable for oral administration. For example, a nutritional supplement can typically be in a tablet, gel-cap, capsule, liquid, sachets, or syrup form.
4. Pharmaceutical formulation Also disclosed herein are pharmaceutical formulations. In one aspect, a pharmaceutical formulation can comprise any of the compounds disclosed herein with a pharmaceutically acceptable carrier. For example, a pharmaceutical formulation can comprise a compound comprising CoQ1o and a pharmaceutically acceptable carrier. The disclosed pharmaceutical formulations can be used therapeutically or prophylactically.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical formulation in which it is contained.
The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard catriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) Gennaro, ed., Mack Publishing Company, Easton, PA, 1995, which is incorporated by reference herein for its teachings of carriers and pharmaceutical formulations. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the disclosed compounds, which matrices are in the form of shaped articles, e.g., films, liposomes, microparticles, or microcapsules. It will be apparent to those persons skilled in the art that certain carriers can be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
Other compounds can be administered according to standard procedures used by those skilled in the art.
Pharmaceutical formulations can include additional carriers, as well as thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the compounds disclosed herein. Pharmaceutical formulations can also include one or more additional active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
The pharmaceutical formulation can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed compounds can be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
Pharmaceutical formulations for oral administration include, but are not limited to, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, anti-oxidants, or binders may be desirable.
Pharmaceutical formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, fish oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Pharmaceutical formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Some of the formulations can potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
5. Foodstu s Also disclosed herein are foodstuffs comprising any of the microcapsules and emulsions disclosed herein. By "foodstufP' is meant any article that can be consumed (e.g., eaten, drank, or ingested) by a subject. In one aspect, the microcapsules can be used as nutritional supplements to a foodstuff. For example, the microcapsules and emulsions can be loaded with vitamins, omega-3 fatty acids, and other compounds that provide health benefits. In one aspect, the foodstuff is a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, 5 a poultry product, a processed fruit juice, a granulated sugar (e.g., white or brown), a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, a fish product, or pet companion food. In another aspect, the foodstuff is bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.
10 6. Sequence similarities It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking 15 at the similarity or relatedness between their nucleic acid sequences. Many of the methods for detennining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.
In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences.
This identity of particular sequences disclosed herein is also discussed elsewhere herein.
In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence.
Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
Another way of calculating homology can be performed by published algorithms.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
U.S.A. 85:
2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc.
Natl. Acad.
Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment.
It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
7. Hybridization/selective hybridization The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G
interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions.
For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25 C
below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5 C to 20 C
below the Tm.
The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies.
Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA
hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al.
Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68 C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68 C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T
richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their lcd, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their lcd.
Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules.
It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80%
hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.
It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.
8. Nucleic acids There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, the 16s RNAs disclosed in SEQ ID NOs:l-17, as well as any other nucleic acids disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
a) Nucleotides and related molecules A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an intemucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracil-l-yl (U), and thymin-l-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.
Acad. Sci.
USA, 1989,86, 6553-6556).
A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of 5 a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
The Hoogsteen face includes the N7 position and reactive groups (NH2 or 0) at the C6 position of purine nucleotides.
b) Sequences There are a variety of sequences related to, for example, SEQ ID NOs: 1-17, as 10 well as any other nucleic acids disclosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.
A variety of sequences are provided herein and these and others can be found in Genbank, at www.pubmed.gov. Those of skill in the art understand how to resolve 15 sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art.
c) Primers and probes Disclosed are compositions including primers and probes, which are capable of 20 interacting with the genes disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA
transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or complement of a region of the nucleic acid.
d) Functional Nucleic Acids Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting.
For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules. Disclosed are functional nucleic acids that interac6t with the disclosed nucleic acids and organisms disclosed herein.
Also disclosed are antibodies, monoclonal and polyclonal which specifically recognize the disclosed microorgansisms and composition C. DeGvery Devices Any of the compounds described herein can be incorporated into a delivery device.
Examples of delivery devices include, but are not limited to, microcapsules, microspheres, nanospheres or nanoparticles, liposomes, noisome, nanoerythrosome, solid-liquid nanoparticles, gels, gel capsules, tablets, lotions, creams, sprays, emulsions, Other examples of delivery devices that are suitable for non-oral administration include pulmospheres. Examples of particular delivery devices useful herein are described below.
The disclosed compounds can be incorporated into liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances.
Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The disclosed compositions in liposome form can contain, in addition to a compound disclosed herein, stabilizers, preservatives, excipients, and the like. Examples of suitable lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
Methods of forming liposomes are known in the art. See, e.g., Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, p. 33 et seq., 1976, which is hereby incorporated by reference herein for its teachings of liposomes and their preparation.

In other examples, the liposomes can be cationic liposomes (e.g., DOTMA, DOPE, DC cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
Regarding liposomes, see e.g., Brigham, et al., Am JResp Cell Mol Bio11:95-100, 1989;
Felgner, et al., Proc Natl Acad Sci USA 84:7413-7, 1987; and U.S. Pat. No.
4,897,355, which are incorporated by reference herein for their teachings of liposomes.
As one example, delivery can be via a liposome using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM
(Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. Liposomes where the diffusion of the compound or delivery of the compound from the liposome is designed for a specific rate or dosage can also be used.
As described herein, niosomes are delivery devices that can be used to deliver the compositions disclosed herein. Noisomes are multilamellar or unilamellar vesicles involving non-ionic surfactants. An aqueous solution of solute is enclosed by a bilayer resulting from the organization of surfactant macromolecules. Similar to liposomes, noisomes are used in targeted delivery of, for example, anticancer drugs, including methotrexate, doxorubicin, and immunoadjuvants. They are generally understood to be different from transferosomes, vesicles prepared from amphiphilic carbohydrate and amino group containing polymers, e.g., chitosan.
As described herein, nanoerythrosomes are delivery devices that can be used to deliver the compositions disclosed herein. Nanoerythrosomes are nano-vesicles made of red blood cells via dialysis through filters of defined pore size. These vesicles can be loaded with a diverse array of biologically active molecules, including proteins and the compositions disclosed herein. They generally serve as ideal carriers for antineoplastic agents like bleomycin, actinomycin D, but can be used for steroids, other lipids, etc.
Artificial red blood cells, as described herein, are further delivery devices that can be used to deliver the compositions disclosed herein. Artificial red blood cells can be generated by interfacial polymerization and complex emulsion methods.
Generally, the "cell" wall is made of polyphtaloyl L-lysine polymer/polystyrene and the core is made of a hemoglobin solution from sheep hemolysate. Hemoglobin loaded microspheres typically have particle sizes of from about 1 to about 10 mm. Their size, flexibility, and oxygen carrying capacity is similar to red blood cells.
Solid-lipid nanoparticles, as described herein, are other delivery devices that can be used to deliver the compositions disclosed herein. Solid-lipid nanoparticles are nanoparticles, which are dispersed in an aqueous surfactant solution. They are comprised of a solid hydrophobic core having a monolayer of a phospholipid coating and are usually prepared by high-pressure homogenization techniques. Immunomodulating complexes (ISCOMS) are examples of solid-lipid nanoparticles. They are cage-like 40 nm supramolecular assemblies comprising of phospholipid, cholesterol, and hydrophobic antigens and are used mostly as immunoadjuvants. For instance, ISCOMs are used to prolong blood-plasma levels of subcutaneously injected cyclosporine.
Microspheres and micro-capsules, as described herein, are yet other delivery devices that can be used to deliver the compositions disclosed herein. In contrast to liposomal delivery systems, microspheres and micro-capsules typically do not have an aqueous core but a solid polymer matrix or membrane. These delivery devices are obtained by controlled precipitation of polymers, chemical cross-linking of soluble polymers, and interfacial polymerization of two monomers or high-pressure homogenization techniques. The encapsulated compound is gradually released from the depot by erosion or diffusion from the particles. Successful formulations of short acting peptides, such as LHRH agonists like leuprorelin and triptoreline, have been developed.
Poly(lactide co-glycolide (PLGA) microspheres are currently used as monthly and three monthly dosage forms in the treatment of advanced prostrate cancer, endometriosis, and other hormone responsive conditions. Leuprolide, an LHRH superagonist, was incorporated into a variety of PLGA matrices using a solvent extraction/evaporation method. As noted, all of these delivery devices can be used in the methods disclosed herein.
Pulmospheres are still other examples of delivery devices that can be used herein.
Pulmospheres are hollow porous particles with a low density (less than about 0.1 gm/mL).
Pulmospheres typically have excellent re-dispersibility and are usually prepared by supercritical fluid condensation technology. Co-spray-drying with certain matrices, such as carbohydrates, human serum albumin, etc., can improve the stability of proteins and peptides (e.g., insulin) and other biomolecules for pulmonary delivery. This type of delivery could be also accomplished with micro-emulsions and lipid emulsions, which are ultra fine, thin, transparent oil-in-water (o/w) emulsions formed spontaneously with no significant input of mechanical energy. In this technique, an emulsion can be prepared at a temperature, which must be higher than the phase inversion temperature of the system. At elevated temperature the emulsion is of water-in-oil (w/o) type and as it cools at the phase inversion temperature, this emulsion is inverted to become o/w. Due to their very small inner phase, they are extremely stable and used for sustained release of steroids and vaccines. Lipid emulsions comprise a neutral lipid core (i.e., triglycerides) stabilized by a monolayer of amphiphilic lipid (i.e., phospholipid) using surfactants like egg lecithin triglycerides and miglyol. They are suitable for passive and active targeting.
There are other oral delivery systems under investigation that are based on osmotic pressure modulation, pH modulation, swelling modulation, altered density and floating systems, mucoadhesiveness etc. These formulations and time-delayed formulations to deliver drugs in accordance with circadian rhythm of disease that are currently in use or investigation can be applied for delivery of the compositions disclosed herein.
In one particular aspect disclosed herein, the disclosed compounds, including nutritional supplement and pharmaceutical formulations thereof, can be incorporated into microcapsules as described herein.
In one aspect disclosed herein, the disclosed compounds can be incorporated into microcapsules. In one aspect, the microcapsule comprises an agglomeration of primary microcapsules and the CoQ1o compounds described herein, each individual primary microcapsule having a primary shell, wherein the CoQ10 compound is encapsulated by the primary shell, wherein the agglomeration is encapsulated by an outer shell.
These microcapsules are referred to herein as "multicore microca.psules."
In another aspect, described herein are microcapsules comprising a CoQio compound, a primary shell, and a secondary shell, wherein the primary shell encapsulates the chromium compound, and the secondary shell encapsulates the loading substance and primary shell. These microcapsules are referred to herein as "single-core"
microcapsules.
Optionally, other loading substances can be encapsulated with the CoQlo compound. The loading substance can be any substance that is not entirely soluble in the aqueous mixture. In one aspect, the loading substance is a solid, a hydrophobic liquid, or a mixture of a solid and a hydrophobic liquid. In another aspect, the loading substance comprises a grease, an oil, a lipid, a drug (e.g., small molecule), a biologically active substance, a nutritional supplement (e.g., vitamins), a flavour compound, or a mixture thereof. Examples of oils include, but are not limited to, animal oils (e.g., fish oil, marine mammal oil, etc.), vegetable oils (e.g., canola or rapeseed), mineral oils, derivatives thereof or mixtures thereof. The loading substance can be a purified or partially purified oily substance such as a fatty acid, a triglyceride or ester thereof, or a mixture thereof. In another aspect, the loading substance can be a carotenoid (e.g., lycopene), a satiety agent, a flavor compound, a drug (e.g., a water insoluble drug), a particulate, an agricultural 5 chemical (e.g., herbicides, insecticides, fertilizers), or an aquaculture ingredient (e.g., feed, pigment).
In one aspect, the loading substance can be an omega-3 fatty acid. Examples of omega-3 fatty acids include, but are not limited to, a-linolenic acid (18:30), octadecatetraenoic acid (18:4co3), eicosapentaenoic acid (20:5(03) (EPA), 10 docosahexaenoic acid (22:6(o3) (DHA), docosapentaenoic acid (22:5c)3) (DPA), eicosatetraenoic acid (20:40), uncosapentaenoic acid (21:5o)3), docosapentaenoic acid (22:5co3) and derivatives thereof and mixtures thereof. Many types of derivatives of omega-3 fatty acids are well known in the art. Examples of suitable derivatives include, but are not limited to, esters, such as phytosterol esters, branched or unbranched Ct-C30 15 alkyl esters, branched or unbranched C2-C30 alkenyl esters, or branched or unbranched C3-C30 cycloalkyl esters such as phytosterol esters and CI-C6 alkyl esters.
Sources of oils can be derived from aquatic organisms (e.g., anchovies, capelin, Atlantic cod, Atlantic herring, Atlantic mackerel, Atlantic menhaden, salmonids, sardines, shark, tuna, etc) and plants (e.g., flax, vegetables, etc) and microorganisms (e.g., fungi and algae).
20 In one aspect, the loading substance can contain an antioxidant other than CoQlo.
Examples of antioxidants include, but are not limited to, vitamin E, tocopherols, lipid soluble derivatives of more polar antioxidants such as ascorbyl fatty acid esters (e_g., ascorbyl palmitate), plant extracts (e.g., rosemary, sage and oregano oils), algal extracts, and synthetic antioxidants (e.g., BHT, TBHQ, ethoxyquin, alkyl gallates, hydroquinones, 25 tocotrienols).
A number of different polymers can be used to produce the shell layers of the single and multicore microcapsules. Examples of such polymers include, but are not limited to, a protein, a polyphosphate, a polysaccharide, or a mixture thereof. In another aspect, the shell material used to prepare the single- and multicore microcapsules further comprises gelatin type A, gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrins, cyclodextrins, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, chitin, polylactides, poly-lactide-co-glycolides, derivatized chitin, chitosan, poly-lysine, various inorganic-organic composites, or any mixture thereof. It is also contemplated that derivatives of these polymers can be used as well. In another aspect, the polymer can be kosher gelatin, non-kosher gelatin, Halal gelatin, or non-Halal gelatin.
In one aspect, one or more of the shell layers in the single and multicore microcapsules comprises gelatin having a Bloom number less than 50. This gelatin is referred to herein as "low Bloom gelatin." The Bloom number describes the gel strength formed at 10 C with a 6.67% solution gelled for 18 hours. In one aspect, the low Bloom gelatin has a Bloom number less than 40, less than 30, less than 20, or less than 10. In another aspect, the gelatin has a Bloom number of 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0, where any two values can be used to produce a range. In another aspect, the low Bloom gelatin is in both the primary shell and the outer shell of the multicore microcapsule. In one aspect, the low Bloom gelatin is gelatin type A. In another aspect, the low Bloom gelatin is gelatin type A produced by Kenney & Ross Ltd., R.R.
#3 Shelburne, NS Canada. In another aspect, gelatin having a Bloom number of zero is in both the primary shell and the outer shell of the multicore microcapsule.
In one aspect, the material used to make the shells of the single- or multicore microcapsules is a two-component system made from a mixture of two different types of polymers. In one aspect, the material is a complex coacervate between the polymer components. Complex coacervation is caused by the interaction between two oppositely charged polymers. In one aspect, the shell material used to produce the single and multicore microcapsules is composed of (1) low Bloom gelatin and (2) gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, carboxymethylcellulose, whey protein, soy protein, canola protein, albumin, or a mixture thereof. The molar ratio of the different polymers can vary. For example, the molar ratio of low Bloom gelatin to the other polymer component is from 1:5 to 15:1. For example, when low Bloom gelatin and polyphosphate are used, the molar ratio of low Bloom gelatin to polyphosphate is about 8:1 to about 12:1; when low Bloom gelatin and gelatin type B
are used, the molar ratio is 2:1 to 1:2; and when low Bloom gelatin and alginate are used, the molar ratio is 3:1 to 8:1.
Processing aids can be included in the shell material (e.g., primary or outer shells).
Processing aids can be used for a variety of reasons. For example, they may be used to promote agglomeration of the primary microcapsules, stabilize the emulsion system, improve the properties of the outer shells, control microcapsule size and/or to act as an antioxidant. In one aspect, the processing aid can be an emulsifier, a fatty acid, a lipid, a wax, a microbial cell (e.g., yeast cell lines), a clay, or an inorganic compound (e.g., calcium carbonate). Not wishing to be bound by theory, these processing aids can improve the barrier properties of the microcapsules. In one aspect, one or more antioxidants can be added to the shell material. Antioxidant properties are useful both during the process (e.g. during coacervation and/or spray drying) and in the microcapsules after they are formed (i.e. to extend shelf-life, etc). Preferably a small number of processing aids that perform a large number of functions can be used. In one aspect, the antioxidant can be a phenolic compound, a plant extract, or a sulphur-containing amino acid. In one aspect, ascorbic acid (or a salt thereof such as sodium or potassium ascorbate) can be used to promote agglomeration of the primary microcapsules, to control microcapsule size and to act as an antioxidant. The antioxidant can be used in an amount of about 100 ppm to about 12,000 ppm, or from about 1,000 ppm to about 5,000 ppm.
Other processing aids such as, for example, metal chelators, can be used as well. For example, ethylene diamine tetraacetic acid can be used to bind metal ions, which can reduce the catalytic oxidation of the loading substance.
In one aspect, the primary microcapsules (primary shells) have an average diameter of about 40 nm to about 10 m, 0.1 pm to about 10 m, I m to about 10 m, 1 m to about 8 m, 1 m to about 6 m, 1 m to about 4 m, or 1 pm to about 2 m, or 1 m. In another aspect, the multicore microcapsules can have an average diameter of from about 1 m to about 2000 m, 20 m to about 1000 m, from about 20 m to about m, or from about 30 m to about 80 m. In another aspect, the single-core microcapsules have an outer diameter of from 1 m to 2,000 m.
The microcapsules described herein generally have a combination of high payload and structural strength. For example, payloads of loading substance can be from 20% to 90%, 50% to 70% by weight, or 60% by weight of the single or multicore microcapsules.
In one aspect, the methods disclosed in U.S. Patent Application Publication No.
2003/0193102, which is incorporated by reference in its entirety, can be used to encapsulate the CoQio compounds described herein. It is also contemplated that one or more additional shell layers can be placed on the outer shell of the single or multicore microcapsules. In one aspect, the techniques described in International Publication No.

WO 2004/041251 A1, which is incorporated by reference in its entirety, can be used to add additional shell layers to the single and multicore microcapsules.
1. Targeted delivery The disclosed liposomes and microcapsules can be targeted to a particular cell type, such as islets cells, via antibodies, receptors, or receptor ligands.
The following references are examples of the use of this technology to target specific tissue (Senter, et al., Bioconjugate Chem 2:447-51, 1991; Bagshawe, Br J Cancer 60:275-81, 1989;
Bagshawe, et al., Br J Cancer 58:700-3, 1988; Senter, et al., Bioconjugate Chem 4:3-9, 1993; Battelli, et aL, Cancer Immunol Immunother 35:421-5, 1992; Pietersz and McKenzie, Immunolog Reviews 129:57-80, 1992; and Roffler, et al., Biochem Pharmacol 42:2062-5, 1991). These techniques can be used for a variety of other specific cell types.
D. Methods of use The compounds disclosed herein also have a wide variety of uses. In the disclosed compounds, the one or more fatty acids are bonded to the coenzyme Q and are therefore an integral part of the complex. Thus, while not wishing to be bound by theory, it is believed that the fatty acids (e.g., DHA, DPA, and/or EPA) play at least two roles, i.e., they make the benzenediol biologically available and they also contribute with their inherent biological activity. Thus, the disclosed compounds (including the nutritional supplements, pharmaceutical formulations, microcapsules, liposomes, and foodstuffs) can deliver fatty acids (e.g., omega-3 fatty acids), lowering triglycerides and influencing prevention or treatment of neurodegenerative diseases (Calon, et al., Neuron 43:633-45, 2004), and other benzenediol derivatives.
In one particular aspect, disclosed herein are methods of lowering total cholesterol levels, triglyceride levels, and increasing HDL levels, or a combination thereof in a subject by administering an effective amount of any of the compounds described herein to the subject. In still another aspect, disclosed herein are methods of improving insulin sensitivity in a subject by administering an effective amount of any of the compounds described herein to the subject. In a further aspect, disclosed herein are methods of reducing hyperglycemia in a subject by administering an effective amount of any of the compounds described herein to the subject. In yet another aspect, disclosed herein are methods of reducing hypercholesterolemia in a subject by administering an effective amount of any of the compounds described herein to the subject.
Also disclosed herein, in one aspect, are methods of preventing a mitochondrial condition or disease in a subject by administering an effective amount of any of the compounds described herein to the subject. An example of a mitochondrial condition includes, but is not limited to, mitochondriopathy. Mitochondriopathy can be characterized by a CoQ10 deficiency, ubiquinone-cytochrome c oxidoreductase deficiency, cytochrome c oxidase deficiency, chronic progressive external ophthalmoplegia syndrome, age-related macular degeneration, neuropathy, ataxia, or retinis Pigmentosa.
In another aspect, disclosed herein are methods of increasing circulation in a subject by administering an effective amount of any compound comprising any of the compounds described herein to the subject. In still another aspect, disclosed herein are methods of increasing the immune system in a subject by administering an effective amount of any compound comprising any of the compounds described herein to the subject. Principles and examples of use of immunostimulants and iImmunomodulators are described for instance in: "Immunostimulants Now and Tomorrow" (Azuma I, Jolles G, eds.), Japan Scientific Societies Press, Tokyo, 1987; Hadden JW (1992) "Classification of Immunotherapeutic Agents In Developments of Biological Standardization," Vol 77, (eds Brown F, Revillard JP): 5-15; Karger, Basel; Galeotti M (1998) "Some Aspects of the Application of Immunostimulants and a Critical Review of Methods for their Evaluation"
JApplIchtuol 189-199; Halperin SA, Smith BS, Nolan C, Shay J, Kralovec J
(2003) "Randomized, Double-Blind, Placebo-Controlled Trial of the Safety and Immunostimmulatory Effect of a Chlorella-Derived Food Supplement in Healthy Adults Undergoing Influenza Immunization" Can Med Assoc J 169: 111-117), which are incorporated by reference in their entireties. In yet another aspect, disclosed herein are methods of reducing the side effects of chemotherapy in a subject by administering an effective amount of any compound comprising any of the compounds described herein to the subject. In still another aspect, disclosed herein are methods of treating or preventing degenerative heart disease in a subject by administering an effective amount of any compound comprising any of the compounds described herein to the subject.
Further, disclosed herein are methods of treating other conditions or diseases in a subject by administering an effective amount of CoQ,oto the subject. Such other conditions or diseases include, but are not limited to, cystic fibrosis, asthma, periodontal (gum) disease, Alzheimer's disease, poor athletic performance, breast cancer, chronic obstructive pulmonary disease (COPD), HIV, male infertility, insulin resistance syndrome (Syndrome X), lung cancer, and prostate cancer.
The disclosed compounds herein can be used neat or in combination with some other component. For example, the compounds can be used in the disclosed methods in the form of any of the nutritional supplements disclosed herein. In another example, the compounds can be used in the disclosed methods in the form of any of the pharmaceutical formulations disclosed herein. In still another example, the compounds can be encapsulated in any of the microcapsules or liposomes disclosed herein, or incorporated 5 into any foodstuff disclosed herein and used in the disclosed methods.
It is contemplated that the methods disclosed herein can be accomplished by administering various forms of the compounds disclosed herein. For example, one can administer any of the pharmaceutical formulations with any of the foodstuffs disclosed herein. In another example, one can administer any of the microcapsules with any of the 10 nutritional supplements disclosed herein. In yet another example, one can administer any of the pharmaceutical formulations with any of the microcapsules and nutritional supplement disclosed herein, and the like.
Disclosed are methods of lowering total cholesterol levels or triglyceride levels, increasing HDL levels, or a combination thereof in a subject, comprising the step of 15 administering an effective amount of the disclosed compounds.
Also disclosed are methods of reducing hyperglycemia in a subject, comprising the step of administering an effective amount of the disclosed compounds.
Also disclosed are methods of reducing hypercholesterolemia in a subject, comprising the step of administering an effective amount of the disclosed compounds.
20 A method for treating or preventing a mitochondrial condition or disease in a subject, comprising the step of administering to the subject an effective amount of the disclosed compounds, such as wherein the condition is a mitochondriopathy, such as wherein the mitochondriopathy is Coenzyme Qlo deficiency, ubiquinone-cytochrome c oxidoreductase deficiency, cytochrome c oxidase deficiency, chronic progressive external 25 ophthalmoplegia syndrome, age-related macular degeneration, neuropathy, ataxia, or retinis Pigmentosa.
Also disclosed are methods for increasing circulation in a subject, comprising the step of administering to the subject an effective amount of the disclosed compounds.
Disclosed are methods for increasing the immune system in a subject, comprising 30 the step of administering to the subject an effective amount of the disclosed compounds.
Disclosed are methods for reducing the side effects of chemotherapy in a subject, comprising the step of administering to the subject an effective amount of the disclosed compounds.

Also disclosed are methods for treating or preventing degenerative heart disease in a subject, comprising the step of administering to the subject an effective amount of the disclosed compounds.
1. Dosa~e When used in the above described methods or other treatments, or in the nutritional supplements, pharmaceutical formulations, microcapsules, liposomes, or foodstuffs disclosed herein, an "effective amount" of one of the disclosed compounds can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient, carrier, or other additive.
The specific effective dose level for any particular subject will depend upon a variety of factors including the condition or disease being treated and the severity of the condition or disease; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject;
the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
The dosage can be adjusted by the individual physician or the subject in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical daily dosage of the compounds disclosed herein used alone might range from about 10 mg to up to 500 mg (benzenediol content only) or more per day, depending on the factors mentioned above.
2. Administration and delivery In one aspect, disclosed herein are uses of a microcapsule to deliver a loading substance to a subject, wherein the microcapsule contains any of the compounds disclosed herein. Also disclosed are methods for delivering a compound to a subject by administering to the subject any of the microcapsules disclosed herein.
Further, disclosed are methods for delivering a compound disclosed herein to a subject by administering to the subject any of the nutritional supplements, pharmaceutical formulations, liposomes, and/or foodstuffs disclosed herein.
The compounds disclosed herein (including nutritional supplements, microcapsules, liposomes, and pharmaceutical formulations) can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
E. General Definitions In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:
Throughout the description and claims of this specification the word "comprise"
and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.
As used in the description and the appended claims, the singular forms "a,"
"an,"
and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of two or more such compounds, reference to "an unsaturated fatty acid" includes mixtures of two or more such unsaturated fatty acids, reference to "the microcapsule" includes mixtures of two or more such microcapsules, and the like.
"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed, then "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
As used herein, by a "subject" is meant an individual. Thus, the "subject" can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.
"Subject" can also include a mammal, such as a primate or a human.
The term "increase," or other forms of increase, such as "increasing," refers to an increase in an event or characteristic above basal levels, e.g., as compared to a control.

The terms "reduces" or "lowers," or other forms the words, such as "reducing,"
"reduction," or "lowering," refers to a decrease in an event or characteristic below basal levels, e.g., as compared to a control.
By "control" is meant either a subject, organ, tissue, or cell lacking a disease or injury, or a subject, organ, tissue, or cell in the absence of a particular variable such as a therapeutic agent. A subject, organ, tissue, or cell in the absence of a therapeutic agent can be the same subject, organ, tissue, or cell before or after treatment with a therapeutic agent or can be a different subject, organ, tissue, or cell in the absence of the therapeutic agent. Comparison to a control can include a comparison to a known control level or value known in the art. Thus, basal levels are normal in vivo or in vitro levels prior to, or in the absence of, the addition of an agent (e.g., a therapeutic agent) or another molecule.
By "prevent" or other forms of prevent, such as "preventing" or "prevention,"
is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevention does not require comparison to a control as it is typically more absolute than, for example, reduce or lower.
As used herein, something could be reduced or lowered but not prevented, but something that is reduced or lowered could also be prevented. Likewise, something could be prevented but not reduced or lowered, but something that is prevented could also be reduced or lowered.
It is understood that where reduce, lowered, or prevent are used, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. Thus, if lowering cholesterol levels is disclosed, then reducing and preventing cholesterol levels are also disclosed, and the like.
By "treat" or other forms of treat, such as "treated" or "treatment," is meant to administer a composition disclosed herein or to perform a method disclosed herein in order to reduce or prevent a particular characteristic or event (e.g., mitochondrial disease).
Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.
VII. EXAMPLES
The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.
Unless 5 indicated otherwise, parts are parts by weight, temperature is in C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process.
10 Only reasonable and routine experimentation will be required to optimize such process conditions.
1. Example 1: Isolation of Marine Coenzyme 010 Produ Microbial Strains Samples of sediments, seawaters, seaweeds and sea grass were collected from 15 Advocate Bay Nova Scotia, Canada and processed for microbial isolations within 24 hours of collection, with serial dilutions in sterile seawater between 100 and 10"5 carried out.
Two media were employed: full strength Marine Agar (MA: 55.1 g/L) and 10%
Marine Broth Agar (MBA: 3.74 g/L, 15 g/L bacteriological agar and 90% artifical seawater) to target both fast and slow growing strains. 10 mg/L cyclohexamide was added to each in 20 order to prevent competition from fungal species during the isolation process.
Collected seaweed (SWD isolates) and sea grass samples were cut into 1 cm2 discs and washed three times with sterile seawater (75%). Washed leaves (L isolates) and seaweeds were grinded in sterile marine broth with mortar. 0.1 mL supemant of each sample was diluted in 0.9 mL marine broth. Further ten-fold dilution (10'2-10-5) of each 25 sample was made and plated on MA and MBA. Second wash of leaf and seaweeds also was diluted (10 -10-) and plated on MA and MBA. In addition to, 1cm2 of leaf and seaweed were cut into 1mm2 and placed on to MA (six/plate). From collected sediment (S
isolates), 0.1 gram sample was diluted in 0.9 mL seawater. Further ten-fold dilution (10"2-10"5) of sample from each site was made and plated on MA and MBA. From seawater, 30 lmL of water sample was diluted in 9mL marine broth. Further ten-fold dilution (10"2-10"
5) of sample from each site was made and plated on MA and MBA.
MA and MBA plates were cultured at 25 C and monitored for a period of 1 month and 3 months, respectively, with colonies that produced pigments (ranging from red, through to pink and orange) selected of purification by serial culture on MA.
Pure cultures of each isolates were then heavily streaked on new MA and incubated for 3-4 weeks to extract coenzyme and analysize via HPLC.
2. Example 2: Identification of Cenzyme 010 Production Rates Individual isolates were grown on MA for another 3-4 weeks, and transferred to pre-weighed 15 ml centrifuge tubes containing 7.5 ml of 50 mM phosphate buffer with 1 mM ferricyanide, vortexed and collected via centrifugation. Pellets were subsequently freeze-dried overnight and dry weight determined. Coenzyme extraction was then performed as follows: 1.5 ml methanol:hexane (3:2, v/v) was added to each tube and vortexed for 1 minute, followed by the addition of 4 ml hexane and another vortex for 1 minute. Samples were then centrifuged (4,000 RPM for 20 mins) to separate the hexane layer, repeated and then transferred to a scintillation vial and evaporated to dryness under N2. Dried samples were then weighed, resuspended in 0.25 ml acetone and transferred to amber vials through a 0.45 m syringe filter. Subsequently, 10 l of each sample was analyzed for coenzyme content (see table 1) using an Agilent 1100 HPLC
(Phenomenex bondclone C18, 10 m, 3.9 x 300 mm column, Waters sentry, symmetry C18, 5 m, 3.9 x mm guard column; mobile phase: methanol:isopropanol (7:3; v/v); 0.6 ml/min flow rate, at 275 nm run for 35 minutes). Calibration curve was created using a 33.33 g/ml CoQ6, 9 & 10 standard mix and an individual 10 g/ml CoQ10 solution (both in acetone).
20 91 isolates were purified and freeze-dried for coenzyme analysis. 45 isolates showed appreciable levels (> 0 g/g) of coenzyme Q10. Of these 17 strains showed significant levels of coenzyme Q 10 production (> 300 g/g) (Table 1).
3. Example 3: Identification of Marine Coenzyme 010 Producing Microbial Strains Species were identified via the sequencing of the 16S rRNA gene using universal primers (27F, 1492R, 1221R & 357F) and comparison to sequence databases (GenBank, NCBI). Results of coenzyme production analysis shows production at quantities similar to or better than those found in terrestrial strains (for example: Escherichia coli BL21/pACDdsA [Appl. Microbil. Biotech. 67:192-6] produced 247.6 g/g;
Paracoccus denitrificans [Biochem. Eng. J. 16:183-190] produced 256.0 g/g; Protomonas extorquens BP-41 [JP62083895] produced 1.29 mg/g; Sporifiobolus ruineniae [US 2004209368]
produced 1.38 mg/g).

Furthermore, analysis of species which were found to produce these compounds (Table 1& 2) showed that these 17 strains represented five different genus (Erythrobacter seohaensis, Sphingomonas baekyungensis, Lutibacterium anuloederans, Exiguobacterium oxidotolerans and Bacillus sp.) and were found to produce coenzyme Q10 quantities above 300 m/g. Eight of those strains were found to belong to the genus Erythrobacter, with all strains (except Exiguobacterium oxidotolerans and Bacillus sp.
GD0402), identified as being members of the gram-negative, a-proteobacteria family, Sphingomonodales (Figure 1). This is the first instance where marine derived examples have been isolated for coenzyme Q10 production. Exiguobacterium oxidotolerans, an alkaliphilic member of the gram-positive, Bacillales family of bacteria, along with another Bacillales species, have also never been linked to coenzyme Q10 production (Figure 1).
All strains mentioned here have been cryopreserved at -80 C in a medium of 10%
glycerol supplemented marine broth.

Table 1 shows both co-enzyme results and 16S rRNA identifications for all 17 strains listed after comparison to BLAST
Identification Codes ( g/g values) Closely related species ............ ............ .... .. ........................................
........------- .. ..... - ---- ............_.... ---........... Source Strain Sequence .(Q9 CoQ10 ONC-SWD4+2 SEQ ID NO. 1 60.11 1602.53 Erythrobacter seohaensis ONC-SWD4-4 SEQ ID NO. 2 128.30 1372.66 Erythrobacter seohaensis ONC-Ll 9 SEQ ID NO. 3 68.04 879.95 Erythrobacter sp. SD-21 ONC-L1+7 SEQ ID NO. 4 30.52 806.46 Erythrobacter sp. SD-21 ONC- Erythrobacter sp.
L1-45 (Lw) SEQ ID NO. 5 123.48 720.54 ONC-SVWD1-1 SEQ ID NO. 6 189.32 702.86 Erythrobacterseohaensis ONC-SWD3-14 SEQ ID NO. 7 107.73 679.60 Erythrobacter sp. SD-21 ONC-L1-21 SEQ ID NO. 8 50.00 586.35 Erythrobacter sp. MBIC 2351 ONC-Llw+5 SEQ ID NO. 9 39.40 1183.10 Sphingomonas baekyungensis ONC- SEQ ID NO.
L1-10 0 878.66 Sphingomonas baekyungensis ONC- SEQ ID NO.
L1-14 43.57 866.94 Sphingomonas baekyungensis ONC- SEQ ID NO.
SWD2+1 30.05 778.34 Sphingomonas baekyungensis ONC- SEQ ID NO. Exiguobacterium L1-37 98.86 828.24 Q00013 13 oxidotolerans ONC- SEQ ID NO. Exiguobacterium S2-1 30.09 370.82 Q00014 14 oxidotolerans ONC- SEQ ID NO.
Ll+l 33.67 951.86 Lutibacterium anuloederans ONC- SEQ ID NO.
S 1+3 29.80 598.04 Lutibacterium anuloederans ONC- SEQ ID NO.
SWD2-6 39.47 335.24 Bacillus sp. GD0402 Table 2. Results of similarity search (BLAST) on aD ONC-Q00001-17 strains Strain Most closely related species Accession No. % similari ONC-Q00001 Erythrobacter seohaensis AY562219 99 ONC-Q00002 Erythrobacter seohaensis AY562219 99 ONC-Q00003 Erythrobacter sp. SD-21 AF325445 99 Erythrobacter sp. MBIC-2531 AB012061 ONC-Q00004 Erythrobacter sp. SD-21 AF325445 99 Erythrobacter sp. MBIC-2531 AB012061 ONC-Q00005 Erythrobacter sp. 121 X/A01 / 170 AY612770 99 ONC- 00006 Erythrobacter seohaensis AY562219 99 ONC-Q00007 Erythrobacter sp. SD-21 AF325445 99 ONC-Q00008 Erythrobacter sp. MBIC-2531 AB012061 99 ONC-Q00009 Sphingomonas baekyungensis AY608604 100 ONC- 010 S hin omonas bae un ensis AY608604 99 ONC- 0011 S hin omonas bae un ensis AY608604 93 ONC- 00012 S hin omonas bae un ensis AY608604 100 ONC-Q00013 Exiguobacterium oxidotolerans AB105164 100 Exi obacterium sp. ARTIC-P28 AY573050 ONC-Q00014 Exiguobacterium oxidotolerans AB 105164 100 Exi obacterium sp. ARTIC-P28 AY573050 ONC- 00015 Lutibacterium anuloederans AY026916 99 ONC-Q00016 Lutibacterium anuloederans AY026916 98 ONC-Q00017 Bacillus sp. GD0402 DQ084536 99

Claims (91)

1. A method of preparing an isoprenoid composition, the method comprising:
culturing bacteria from Sphyingomonodales family, and isolating the isoprenoid.

2. The method of claim 1, wherein the bacteria is an Erythrobacter.

3. The method of claim 1, wherein the bacteria is Erythrobacter seohaensis.

4. The method of claim 1, wherein the bacteria is a Sphingomonas.

5. The method of claim 1, wherein the bacteria is Sphingomonas baekyungensis.

6. The method of claim 1, wherein the bacteria is a Lutibacterium.

7. The method of claim 1, wherein the bacteria is Lutibacterium anuloederans.

8. The method of claim 1, wherein the bacteria is marine-derived.

9. The method of claim 1, wherein the isoprenoid is a coenzyme.

10. The method of claim 1, wherein the isoprenoid is Coenzyme-Q10 (CoQ10).

11. A method of preparing an isoprenoid composition, the method comprising:
culturing bacteria from Bacillaceae family, and isolating the isoprenoid.

12. The method of claim 11, wherein the bacteria is from Exiguobacterium genus.

13. The method of claim 11, wherein the bacteria is Exiguobacterium oxidotolerans.

14. The method of claim 11, wherein the bacteria is marine-derived.

15. The method of claim 11, wherein the isoprenoid is a coenzyme.

16. The method of claim 11, wherein the isoprenoid is Coenzyme-Q10 (CoQ10).

17. The method of claim 1, wherein the bacteria comprises a sequence having greater than 99% identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID
NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, or any combination thereof.

18. The composition of claim 1, wherein the organism comprises a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, or SEQ ID NO:17, or any combination thereof.

19. A compound prepared by the method of claim 1.

20. A nutritional supplement comprising the compound of claim 19..

21. The nutritional supplement of claim 20, comprising from about 0.05% to 20%
by weight of the compound.

22. The nutritional supplement of claim 20 comprising from about 1% to 7.5% by weight of the compound.

23. The nutritional supplement of claim 20, wherein the supplement comprises less than or equal to 100% by weight of the compound.

24. The nutritional supplement of claim 20, wherein the supplement is in the form of a tablet, gel-cap, capsule, liquid, or syrup.

25. A delivery device comprising the compound of claim 19.

26. The delivery device of claim 25, wherein the device comprises a microcapsule, a liposome, noisome, nanoerythrosome, solid-liquid nanoparticle, microsphere, or pulmosphere.

27. The delivery device of claim 25, wherein the device comprises a microcapsule, wherein the microcapsule comprises an agglomeration of primary microcapsules, each individual primary microcapsule having a primary shell and the agglomeration being encapsulated by an outer shell, wherein the compound is encapsulated in the primary microcapsule.

28. The delivery device of claim 25, wherein the primary shell and the outer shell comprise gelatin type A, gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrins, cyclodextrins, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, chitin, polylactide, poly-lactide-co-glycolide, polylysine, kosher gelatin, non-kosher gelatin, Halal gelatin, non-Halal gelatin, or a mixture thereof.

29. The delivery device of claim 25, wherein the primary shell and the outer shell comprises gelatine type A having a Bloom strength of from 0 to 350.

30. The delivery device of claim 25, wherein the primary shell and the outer shell comprises a no bloom fish gelatin.

31. The delivery device of claim 27, further comprising an additional shell surrounding the outer shell, wherein at least one of the primary, outer, and additional shells comprise a complex coacervate.

32. A foodstuff comprising the compound of claim 19.

33. The foodstuff of claim 32, wherein the foodstuff is a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, a poultry product, a processed fruit juice, a granulated sugar, a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, a fish product, or pet companion food.

34. The foodstuff of claim 32, wherein the foodstuff is bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.

35. A method of lowering total cholesterol levels or triglyceride levels, increasing HDL
levels, or a combination thereof in a subject, comprising the step of administering an effective amount of the compound of claim 19.

36. A method of reducing hyperglycemia in a subject, comprising the step of administering an effective amount of the compound of claim 19.

37. A method of reducing hypercholesterolemia in a subject, comprising the step of administering an effective amount of the compound of claim 19.

38. A method for treating or preventing a mitochondrial condition or disease in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 19.

39. The method of claim 38, wherein the condition is a mitochondriopathy.

40. The method of claim 39, wherein the mitochondriopathy is Coenzyme Q10 deficiency, ubiquinone-cytochrome c oxidoreductase deficiency, cytochrome c oxidase deficiency, chronic progressive external ophthalmoplegia syndrome, age-related macular degeneration, neuropathy, ataxia, or retinis Pigmentosa.

41. A method for increasing circulation in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 19.

42. A method for increasing the immune system in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 19.

43. A method for reducing the side effects of chemotherapy in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 19.

44 44. A method for treating or preventing degenerative heart disease in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 19.

45. A pharmaceutical formulation comprising the compound of claim 19 and a pharmaceutical carrier.

46. A composition comprising a marine bacterium, wherein the marine bacterium produces an isoprenoid.

47. The composition of claim 46, wherein the organism comprises a sequence having greater than 99% identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID
NO: 17, or any combination thereof.

48. The composition of claim 46, wherein the organism comprises a sequence set forth in SEQ ID NO:1, SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:15, SEQ ID NO:16, or SEQ ID NO:17, or any combination thereof.

49. The composition of claim 46, wherein the bacterium produces at least 200 µg/g of the isoprenoid.

50. A composition comprising an isolated marine bacterium and an isolated isoprenoid.

51. A method of producing a dietary supplement comprising combining fish oil and CoQ10, wherein the CoQ10 is produced using the method of claim 1.

52. The method of claim 51, wherein the dietary supplement comprises a soft gel cap.

53. The method of claim 1, wherein the composition comprises a salt of an omega-3 fatty acids.

54. The method of claim 53, wherein the salt is a magnesium or calcium salt.

55. The composition of claim 11, wherein the composition comprises a salt of omega 3 fatty acids.

56. The composition of claims 55, wherein the salt is a magnesium or calcium salt.

57. A compound prepared by the method of claim 11.

58. A nutritional supplement comprising the compound of claim 57.

59. The nutritional supplement of claim 58, comprising from about 0.05% to 20%
by weight of the compound.

60. The nutritional supplement of claim 58 comprising from about 1% to 7.5% by weight of the compound.

61. The nutritional supplement of claim 58, wherein the supplement comprises less than or equal to 100% by weight of the compound.

62. The nutritional supplement of claim 58, wherein the supplement is in the form of a tablet, gel-cap, capsule, liquid, or syrup.

63. A delivery device comprising the compound of claim 57.

64. The delivery device of claim 63, wherein the device comprises a microcapsule, a liposome, noisome, nanoerythrosome, solid-liquid nanoparticle, microsphere, or pulmosphere.

65. The delivery device of claim 63, wherein the device comprises a microcapsule, wherein the microcapsule comprises an agglomeration of primary microcapsules, each individual primary microcapsule having a primary shell and the agglomeration being encapsulated by an outer shell, wherein the compound is encapsulated in the primary microcapsule.

66. The delivery device of claim 63, wherein the primary shell and the outer shell comprise gelatin type A, gelatin type B, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbiton, maltodextrins, cyclodextrins, cellulose, methyl cellulose, ethyl cellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, canola protein, albumin, chitin, polylactide, poly-lactide-co-glycolide, polylysine, kosher gelatin, non-kosher gelatin, Halal gelatin, non-Halal gelatin, or a mixture thereof.

67. The delivery device of claim 63, wherein the primary shell and the outer shell comprises gelatine type A having a Bloom strength of from 0 to 350.

68. The delivery device of claim 63, wherein the primary shell and the outer shell comprises a no bloom fish gelatin.

69. The delivery device of claim 65, further comprising an additional shell surrounding the outer shell, wherein at least one of the primary, outer, and additional shells comprise a complex coacervate.

70. A foodstuff comprising the compound of claim 57.

71. The foodstuff of claim 70, wherein the foodstuff is a baked good, a pasta, a meat product, a frozen dairy product, a milk product, a cheese product, an egg product, a condiment, a soup mix, a snack food, a nut product, a plant protein product, a hard candy, a soft candy, a poultry product, a processed fruit juice, a granulated sugar, a sauce, a gravy, a syrup, a nutritional bar, a beverage, a dry beverage powder, a jam or jelly, a fish product, or pet companion food.

72. The foodstuff of claim 70, wherein the foodstuff is bread, tortillas, cereal, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, a dry beverage powder, rolls, cookies, crackers, fruit pies, or cakes.

73. A method of lowering total cholesterol levels or triglyceride levels, increasing HDL
levels, or a combination thereof in a subject, comprising the step of administering an effective amount of the compound of claim 57.

74. A method of reducing hyperglycemia in a subject, comprising the step of administering an effective amount of the compound of claim 57.

75. A method of reducing hypercholesterolemia in a subject, comprising the step of administering an effective amount of the compound of claim 57.

76. A method for treating or preventing a mitochondrial condition or disease in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 57.

77. The method of claim 76, wherein the condition is a mitochondriopathy.

78. The method of claim 77, wherein the mitochondriopathy is Coenzyme Q10 deficiency, ubiquinone-cytochrome c oxidoreductase deficiency, cytochrome c oxidase deficiency, chronic progressive external ophthalmoplegia syndrome, age-related macular degeneration, neuropathy, ataxia, or retinis Pigmentosa.

79. A method for increasing circulation in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 57.

80. A method for increasing the immune system in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 57.

81. A method for reducing the side effects of chemotherapy in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 57.

82. A method for treating or preventing degenerative heart disease in a subject, comprising the step of administering to the subject an effective amount of the compound of claim 57.

83. A pharmaceutical formulation comprising the compound of claim 57 and a pharmaceutical carrier.

84. An isolated microorganism deposited with the ATCC as PTA-7565.

85. An isolated microorganism deposited with the ATCC as PTA-7566.

86. An isolated microorganism deposited with the ATCC as PTA-7567.

87. An isolated microorganism deposited with the ATCC as PTA-7568.

88. The method of claim 1, wherein the bacteria is bacteria cutured from the microorganism deposited with the ATCC as PTA-7565.

89. The method of claim 1, wherein the bacteria is bacteria cutured from the microorganism deposited with the ATCC as PTA-7566.

90. The method of claim 1, wherein the bacteria is bacteria cutured from the microorganism deposited with the ATCC as PTA-7568.

91. The method of claim 11, wherein the bacteria is bacteria cutured from the microorganism deposited with the ATCC as PTA-7567.