Field of the invention:
The invention relates to compositions comprising CoQ9 and method of enriching the CoenzymeQlO levels in mammals through supplementing CoQ9. The invention further relates to the compositions comprising CoQ9 optionally in combination with Co QIO that can be admistered to the patients to achieve potent antioxidant, cardioprotective, immunomodulating anticancer effects similar to those obtained with CoQlO supplementation..
Back ground of the invention:
Coenzyme QIO (CoQlO), an endogenously synthesized pro-vitamin present in the Mitochondrial electron transport chain, is a natural substance, belong to a family of 2, 3-diinethoxy-5-methyl-6-polyprenyl-l,4-benzoquinone compounds, widely know as ubiquinone for its ubiquitous occurrence in animal and plant tissues. It has been found to be cardio-protective and used as adjunct therapy for ischemic heart disease (Kitamura, N., et. al., In: Biochemical and Clinical Aspects of Coenzyme Q. Folkers, K., Yamamura, Y., Eds.; Elsevier, Amsterdam, Netherlands vol 4, pp 243-256). Mitochondrial respiratory chain contains several coenzymes including Coenzymes Ql, Q2, Q4, Q6, Q7, Q8, Q9 and QIO. Coenzymes Q6, Q7 and Q8 exist in yeast and bacteria, whereas, CoQlO is prevalent in humans. CoQ9, on the other hand, is found in rats and mice. The CoQ9 differs from CoQlO with respect to the number of isoprene units in the tail. CoQ9 has nine Isoprene units in the side chain in contrast to the presence of 10 units in CoQlO.
The CoQlO is an essential cofactor for the proper functioning of uncoupling proteins. In addition to its important role as a redox component for both mitochondria and lipid membrane, CoQlO in the reduced form (ubiquinol) functions as an antioxidant, which protects biological membranes and serum LDL from lipid peroxidation. CoQlO also serves as a powerful antioxidant in other organelle membranes that contain CoQ (Overvad, K., et. al., Eur. J. Clin. Nutr.1999,53,764-770).
Most of the CoQ 10 are found in mammalian hearts including human myocardium (Folkers, K., et al., Proc. Natl. Acad. Sci. USA 1985, 82(3), 901-904). CoQ 10 is not an essential nutrient because it is synthesized in the body. The CoQ 10 level in the body

decreases with age and under several pathophysiological conditions. Several food products including meat, fish, peanuts and broccoli are the rich source of CoQlO. Although the dietary intake of CoQIO is about 2-5 mg per day, it is inadequate for the body under pathophysiologic conditions and in old age.
The levels of endogenous CoQlO in the heart decreases during ischemic heart disease including heart failure and this has prompted clinical trials on CoQlO in heart patients (Otani, H., et. Al., Circ. Res. 1984, 55, 168- 175). Randomized, double-blind placebo-controlled trials on oral administration of CoQlO have confirmed the effectiveness of CoQlO in improving anginal episodes, arrhythmias, and left ventricular function in patients with acute myocardial infarction (Singh, R. B., et. al., Cardiovasc. Drug. Ther. 1998,12,347-353).
Studies involving breast cancer patients showed that CoQlO concentrations in tumor tissues significantly depleted as compared to the surrounding normal tissues. Administration of coenzyme QIO by dietary supplementation found to induce the protection against humor growth. The high risk breast cancer patients supplemented with 90 to 390 mg daily doses of CoQ 10 obtained partial to complete regression (Lockwood K, et. al. Mol Aspects Med. 15 (Suppl): s231-40, 1994). It also prevents cardio toxicity of some of the anticancer drugs. CoQ 10 is an immune modulating agent and is essential for the optimal function of the immune system (Folkers K; Drugs Exp. Clin. Res. 11(8):539-45, 1985).
A recent study showed that supplementation of CoQ 10 could reduce myocardial ischemia reperfusion injury in pigs on cardiopulmonary bypass. Additionally, recent studies also indicate a novel role of exogenous CoQ 10 in the induction and transcription of genes involved in cell signaling, metabolism and transport. Another recent study has indicated CoQ 10 as a modulator of transition pore suggesting its role in apoptosis. In most countries CoQ 10 is widely used as a nutritional supplement. In countries like Japan, it is a drug prescribed for those having suffered from heart disease. However, in USA it is a dietary supplement available from health food store or mail order business. CoQ9 is a lower homolog having nine isoprene units compared to ten present in CoQlO. A recent study has indicated that reduced CoQ9 could act as a potential antioxidant

regardless of its cellular concentration (Ernster, L., et. al., Clin. Investig. 1993, 77, S60-S65). Reduced CoQ9 together with a-tocopherol, were found to act as potential antioxidant in guinea pig hepatocytes when incubated with AAPH, while reduced CoQlO mainly exhibited its antioxidant activity in cells containing CoQlO as the predominant CoQ homolog. Another related study has demonstrated significant decrease of CoQ9 in heart mitochondria of diabetic rats suggesting reduced CoQ~ could be responsible for the increased susceptibility of diabetic heart to oxidative damage. Yet another study indicated that myocardial reperfusion decreased the mitochondrial content of ubiquinone and stimulated CoQ9 biosynthesis in young rats but not in aged rats. The synthesis of CoQ9 was found to be increased in the liver in hyperthyroidism. A recent study indicated that Coenzyme Q9 could regulate the aging process in Caenorhabditis elegans mitochondria. Similar to CoQlO, CoQ9 also participates in the mitochondrial electron transport inside of the cell, and in rodents where CoQ9 is the predominant Coenzyme Q, it serves as an essential component for the ATP synthesis.
WOO7017168A1 describes a process for the preparation of ubihydroquinones and ubiquinones by condensation of a prenol or isoprenol with a hydroquinone or derivative thereof in the presence of 0.005 -1.0 mol% of a catalyst which is a Bronsted-acid, a Lewis- acid from the group consisting of a derivative of Bi or In or an element of group 3 of the periodic table of the elements, a heteropolyacid, an NH-or a CH-acidic compound, and optionally oxidizing the ubihydroquinone obtained.
W007014392A2 comprises benzoquinone compositions of enhanced solubility and bioavailability that contain at least one benzoquinone with at least one solubility-enhancing polymer. In one embodiment, the benzoquinone is coenzyme Q 10. W007004091A2 relates to novel intermediates for the preparation of coenzymes, processes for the preparation of the intermediates and an improved process for the preparation of Coenzymes, more particularly relating to regio and stereo controlled process for Coenzyme Q9 and Coenzyme QIO.
US200600 105 19A1 describe a plant which expresses a large amount of ubiquinone- 10 and a method for producing ubiquinone-10 using the plant are provided. A dietary supplement, a food and a food additive which contains ubiquinone-10 produced by the plant or the method are provided.
JP2005041870A2 describes an external preparation having skin keratin-softening action, skin-whitening action, moisturizing action or wrinkle-removing action, comprising a

ubiquinone and the deep-sea water. The examples of ubiquinones, coenzyme 46, coenzyme 47, coenzyme Q8, coenzyme Q9 or coenzyme QIO is used. US20040033553A1 describes a method to assay coenzyme QIO in blood plasma or blood serum, wherein QlOin the plasma sample is oxidized by treating the sample with an oxidizing agent having a redox potential higher than the redox potential of CoQlO, such as, for example, para-benzoqulnone. Following oxidation of the CoQlO, the CoQlOin the plasma sample is extracted with an alcohol, such as, for example, 1-propanol. The alcohol extract is analyzed using direct injection into the HPLC apparatus. US20050008630A1 describes a method of stabilizing reduced coenzyme Q 10, which is useful as functional nutritive foods, specific health foods and the like. Furthermore, the present invention provides a method for efficiently obtaining reduced coenzyme QIO of high quality and by a method suitable for a commercial production. US20050 13741 OAl describes a practical, cost-effective synthesis of CoQlO, wherein the invention provides a convergent method for the synthesis of ubiquinones and ubiquinone analogues. Also provided are precursors of ubiquinones and their analogues that are useful in the methods of the invention.
EP0882450B1 describe a cholesterol-lowering composition comprising coenzyme Q [From equivalent EP0882450A21 provide an antihypercholesterolemic or antihyperlipidemic agent, hence a therapeutic and prophylactic drug for arteriosclerosis, which is safer and more potent in cholesterol-lowering action than the hitherto-available drugs, comprising a coenzyme Q or a reduced coenzyme Q, wherein the polyprenyl side chain of the ubiquinone contains 6 to 11 isoprenyl units.
EP1068805A1 describe a method for inhibiting blood coagulation in humans and warm blooded animals comprising administration by the oral route of cereal germ oil, preferably corn oil, wherein germ oil used for administration has been enriched in ubiquinone 9/10.
JP02249492A2 describe a process for the production of ubiquinone 9, to industrially and advantageously obtain the subject compound useful as a remedy for cerebrovascular disorder, cardiac insuflflciency, hypertension or diabetes, side effect preventive agent of an anticancer agent adriamycin, etc., by culturing a microorganism belonging to the genus Mucor.

JPO 111 7793 A2 describe a process for the production of ubiquinone, which is used as a remedy for cerebrovascular disorders, cardiac failure, hypertension or the like in high effi ciency by culturing a strain in Mortierella in an enriched medium. JP6 10279 14A2 provide a cosmetic free from undesirable side effect such as hormonic effect, and having excellent hair-tonic effect and acne-remedying effect, by compounding a specific ubiquinone with oxendlone (1 6P-ethyl- 17P-hydroxy-4-estren-3-one).The ubiquinone compound is selected from ubiquinone 7, ubiquinone 8, ubiquinone 9 or ubiquinone 10.
JP59013719A2 describes an effective remedy to the prevention and remedy of male pattern baldness, etc. without causing side effects such as exhaustion of sexual energy, etc. even by the continuous long-term administration, free from hormone-like action, and effective at a low dose, by using a ubiquinone as an active component. The ubiquinone of formula (n is 7W10) is used as an active component. The ubiquinone is ubiquinone-7 (n=7), ubiquinone-8 (n=8), ubiquinone-9 (n=9) or ubiquinone-10 (n=10). JP5 7202294A2 describes a process for producing coenzyme Q9, by cultivating a bacterium such as Rhodopseudomonas capsulate (FERM-P 879) belonging to the genus Rhodopseudomonas, capable of producing coenzyme Q9, when inoculated into a nutritive medium under aerobic conditions, and coenzyme Q9 is collected froin the culture mold. JP55000028A2 and JP54I3 8 19 1A2 also describe similar processes for producing coenzyme Q9, by cultivating a bacteriums Pseudomonas genus and Streptomyces sapporonensis respectively followed by purification techniques.
JP53072895A2 describes a process for producing coenzyme Q9 (which is coenzyme widely distributing in microorganisms, plants and animals) having an important role of electron transfer in vivo, useful as pharmaceuticals, by culture of plant cells. US4031205 describes a method for treating nervous bladder comprising administering to a human suffering from nervous bladder a therapeutically effective amount of Ubiquinone, wherein the polyprenyl side chain of the ubiquinone contains 0 to 10 isoprenyl units whereby nervous bladder can be treated without side-effects.
None of the prior art provides compositions comprising Co Q9 for achieving potent antioxidant, cardioprotective, immunomodulating anticancer effects by enhancing the Co QIO levels in mammals and a method for enhancing the CoQlO levels in a mammal through the supplementations of an ingredient other than CoQio itself

There exists a need for a method to enhance the concentration of CoQio levels in mammals by providing economically cheaper alternative to CoQlO. . It is therefore an objective of the present invention to provide a practical method, to enhance the levels of CoQio in a mammal through the supplementation of relatively cost effective ingredients or the compositions containing such ingredients.
Summary of the invention:
The present invention discloses compositions comprising Co Q9 to achieve potent antioxidant, cardioprotective, immunomodulating anticancer effects and a method for enriching the CoenzymeQlO levels in mammalian body through supplementing CoQ9 or the compositions containing CoQ9.
The present invention discloses compositions comprising CoQ9 either alone or in combination with CoQlO that can be administered as pharmaceutical or nutraceutical compositions, dietary supplements, and cosmetic preparations comprising the same.
Description of the Drawings:
Figure 1. The incidence of reperfusion-induced ventricular fibrillation (VF). Guinea pigs were orally treated with a daily dose of 5 mg/kg of CoQlO or CoQ9 or vehicle control for 4 weeks, and then hearts were excised, and isolated for perfusion via Langendorff mode and subjected to 30 min of global ischemia followed by 120 min of reperfusion. N = 12 in each group, *p<0.G5 compared to the drug-free control (C) group. Figure 2/A Effects of CoQlO and CoQ9 on infarct size in isolated guinea pig hearts subjected to 30 min of ischemia followed by 120 min of reperfusion. The bars represent meaniSD of infarct size for control, CoQlO and CoQ9 supplemented groups. *p<0.05 compared to the untreated age-matched ischemic/reperfused drug-free control (C) value. N = 12 in each group.
Figure 2/B. Effects of CoQlO and CoQ9 on cardiomyocyte apoptosis in isolated guinea pig hearts subjected to 30 min of ischemia followed by 120 min of reperfusion. The bars represent mean±SD of cardiomyocyte apoptosis for control, CoQlO and CoQ9 supplemented groups. "p<0.05 compared to the untreated age-matched ischemic/reperfused drug-free control (C) value.

Figure 3. HPLG chromatograms of a) Blank (mobile phase) b) CoQ9 standard solution c) CoQlO standard solution and d) CoQ9 heart sample. The standards and sample solution were analyzed using an Agilent 1100 HPLC. The mobile phase was methanol-2-propanol-formic acid (45:55:0.05, v/v/v) containing methylamine at the concentration of 5 mmoI/L. At a flow rate of 0.2 ml/min, 5^1 injections of the samples were done using the autosampler. The CoQ9 peak eluted at 2,39 minutes and the CoQlO eluted at 2.86 minutes. However, the CoQ9 heart sample peak eluted at 2.89 minutes, which is the same as CoQlO, thus indicating the presence of CoQlO rather than CoQ9 in the sample. Bio-conversion of Q9 into QIO is suggested and this is further verified using mass spectrometric analysis.
Figure 4. Mass Spectrometry (MS).(a) CoQlO standard, b) CoQ9 standard, c) CoQlO heart sample) Methylamine was used in the mobile phase to obtain the methyl ammonium adduct molecules of C0Q9 and CoQlO. The sensitivity of the adduct ions [M + CH~NH~]'was much higher than that of the protonated ions [M + HI' [7]. The MS spectra of both [M + CH~NH~]' at m/z 826.5 for CoQ9 and m/z 894.6 for CoQlO were observed. However, the CoQ9 heart sample indicated a mass peak at m/z 894,6, which matches the peak for CoQlO and not CoQ9. Therefore, there was evidence of CoQlO in the CoQ9 heart sample from the mass spec. data. This confined the hypothesis of bio-conversion of CoQ9toCoQ10.
Detailed description of the invention:
CoQlO is an essential component of the mitochondrial electron transport chain involved in both photosynthetic and respiratory processes. It acts as the redox link between flavoproteins and cytochromes that are essential for ATP synthesis. It also functions as an antioxidant in cell membranes and lipoproteins (Emster, L,, et al., Biochim. Biophys. ActaJ995, 1271, 195-207) and exhibits potent clinical effect in human congestive heart failure, hypertension and cancer, in addition to wide array of other medicinal application. Under the normal condition, body may not require any exogenous CoQlO since it is produced by de nova biosynthesis. However, in certain pathophysiologic conditions such as hypertension, cardiomyopathy, angina, heart failure, muscular dystrophy and cancer [Simonsen, R., et. al.. In Biochemical and Clinical Aspects of Coenzyme Q Folkers, K.,

Littarru, G. P., Yamagami, T., Eds] Elsevier, Amsterdam, Netherlands, vol 6, pp 363-373 and Beyer RE, et. al., In Pathology and Cardiovascular Injury (Stone, H. L., Wegiicki. W. B., Eds), Martinus Njhoff, Boston, MA pp 489-51 11, ^fe novo production of CoQlO is reduced and hence, tissues require exogenous supply of CoQlO. The cellular CoQlO deficiency is greatly enhanced with the advancement of age. Most importantly, heart requires additional CoQlO for maintaining optimum ATP levels under pathophysiologic conditions such as ischemic heart diseases including heart failure. Correction of deficiency requires supplementation of CoQlO at concentrations, higher than those available in the regular diet.
Although CoQ9 Is also present in the human body, CoQlO remains the only CoQ supplement that is commercially available. The CoQ 10 commercial supplies have now been available and widely being used as a dietary ingredient in many countries around the world. The CoQ 10 currently being marketed around the world is produced solely from fermentation route. Its production is acutely limited due to the monopoly. Even though several chemical processes are available for the CoQ 10 production, all of them are economically unviable. Because of the source limitation, there is a big fluctuation In the market price, i. e. US $ 30001kg in 2005 to $ 8001kg in 2007, depending upon the supply and demand. Alternative products or methods or sources are greatly needed to augment the growing demand and to provide greater access to this beneficial anti-oxidant to wider cross sections of the population.
Though the chemical production of CoQlO is economically unviable, the production of its lower CoQ homolog, i. e., CoQ9 through chemical technology is cost effective. This is possible as the C45 side chain can be derived and adopted directly from a natural product called solanesol. Solanesol can be isolated from waste tobacco raw material. Whether COQ~ can also perform the same task for the heart and offer similar health benefits as CoQlO, especially if CoQ9 supplementation can reduce myocardial ischemia reperfusion, is not known. It is also not known whether exogenous CoQ9 could be equally cardio protective as CoQlO in the animals where CoQ9 is totally absent or less predominant. The inventors performed a series of ex vivo and in vivo studies snd compared the effects of CoQ9 vs. CoQ 10 in the ischemic myocardium, and found surprisingly that CoQ9 could protect the ischemic heart to the same extent as CoQ 10 (figures 1, 2A and 2B). The inventors also found most surprisingly that when a mammal is supplemented with CoQ9,

it is bio-converted into CoQlO and lead to enhancement of CoQlO concentration over and above the un-supplemented mammal. This unexpected result is likely that CoQ9 could fill up the gap for CoQlO after being converted into CoQlO as the bioavailability of CoQlO is very poor.
Experimental studies were designed to determine if CoQ9 could protect guinea pig hearts from ischemia reperfusion injury. Myocardial ischemia reperfusion injury model is the most widely accepted experimental method for assessment of parameters related to cardio- protection. Guinea pigs were randomly divided into three groups: Group II and Group 111 were supplemented with 5 mg/kg bodyweight of CoQ9 and CoQlO, respectively, for 4 weeks while Group I served as control (C). After 4 weeks, the guinea pigs were sacrificed and the isolated hearts were perfused via working mode. The isolated hearts were subjected to ischemia for 30 min followed by 2 hours of reperfusion. Cardio protection was assessed by evaluating left ventricular function, ventricular arrhythmias, and myocardial infarct size and cardiomyocyte apoptosis. Samples of hearts were examined for the presence of Coenzyme Q. The results demonstrated that both CoQ9 and CoQlO were equally cardio protective as evidenced by their abilities to improve left ventricular performance (Table 1 and figure I), and to reduce myocardial infarct size (figure 2A) and cardiomyocyte apoptosis (figure 2B). High performance liquid chromatographic (HPLC) analysis revealed surprisingly that a substantial portion of CoQ9 had been bio-converted into CoQlO. The results indicate that CoQ9 by itself, or after being converted into CoQlO, provides cardio protection in myocardial ischemic reperfusion injury.
Several unexpected salient features are apparent from the present investigation. First, CoQ9 and CoQlO provided similar magnitude of cardio protection as evidenced from the comparable degree of the post-ischemic ventricular recovery, reduction of myocardial infarct size (figure 2A) and cardiomyocyte apoptosis (figure 2B). Both CoQ9 and CoQlO supplementation reduced the incidence of ventricular fibrillation (figure 1). LC-GC results revealed complete bioconversion of CoQ9 into CoQlO; and no CoQ9 could be detected in the heart as most of the CoQ9 was detected as CoQlO. The results thus, raises interesting possibility that nutritionally supplemented CoQ9 could be an economic alternative to CoQlO and CpQ9 could provide enhanced levels of CoQlO in the mammals and provide cardio protection after being converted into CoQlO.

The intricate details of the outcome of the experiments corresponding to different aspects of the present invention are described below. Effects of CoQ9/CoQ10 on the recovery of Left Ventricular Function. Table 1 shows the recovery of post-ischemic cardiac function in isolated heai-ts subjected to 30 min ischemia followed by 120 min of reperfusion obtained from guinea pigs treated with 5 mg/kg/day of CoQlO and CoQ9, respectively, for 4 weeks. The results clearly show that post-ischemic recovery in heart rate (HR), coronary flow (CF), aortic flow (AF), and left ventricular developed pressure (LVDP) were significantly improved in the CoQlO and CoQ9 treated groups in comparison with the drug-free control values. Thus, for instance, after 30 min of ischemia followed by 120 min of reperfusion, aortic flow (Table 1) was significantly increased from its drug-free control value of 8.0±1.0 ml/min to 18.0±2.0 ml/min (*p<0.05) and 26.0±1.0 ml/min (*p<0.05) in hearts obtained from guinea pigs treated with 5 mg/kg/day of CoQlO and CoQ9 respectively. Similar types of post-ischemic recovery of HR, CF, and LVDP were registered (Table 1) in isolated hearts obtained from guinea pigs treated with 5 mg/kg/day of CoQlO or CoQ9 for 4 weeks. The improvement in post-ischemic cardiac function (HR, CF, AF, and LVDP) was more pronounced in the CoQ9 treated group than in the CoQlO treated group. However, before ischemia, cardiac function (HR, CF, AF, and LVDP) was not significantly changed in the CoQlO or CoQ9 treated groups in comparison with the drug-free control values (Table 1). Effects of CoQ9/CoQ10 on the Development of Arrhythmias
The incidence of reperfusion-induced VF was significantly reduced by CoQlO and CoQ9. As shown in Figure 1, and compared to untreated ischemic/reperfused drug-free group, incidence of reperfusion-induced VF was reduced from 92 % to 25 % (*p<0.05) and 92% to 8 % (*p<0.05) with 5 mg/kg/day of CoQlO and CoQ9, respectively.
Effects of CoQ9/CoQ10 on Myocardial Infarct Size.
Figure 21A shows the percentage of infarct size in isolated guinea pig hearts subjected to
30 min of global ischemia followed by 120 min of reperfusion. Drug-free
ischemic/reperfused control hearts were associated with a 38 14.1 % infarct size (Figure
21A) which was consistently reduced by the dose of 5 mg/kg/day of CoQlO and CoQ9 to
21.1 i5 % (*p<0.05) and 16.3 * 3.2 % (*p<0.05), respectively.
Effects of CoQ9/CoQ10 on Myocardial Apoptosls

As shown in Figure 2B, in case of ischemic control group guinea pig (IIR) cardiomyocyte apoptosis determined by Tune 1 method was about 2112 % at the end of reperfusion. Both CoQlO and CoQ9 treatment significantly reduced the number of apoptotic cardiomyocytes to 611 % (pC0.05) and 711.5 % (p<0.05) respectively.
LC Analysis of CoQ9JCoQ 10.
CoQ9 and CoQlO were observed at the retention times of 2.39 and 2.86 minutes
respectively. Figures 3b and 3c shows chromatograms of CoQ9 and CoQlO standard
solutions. However, the retention time of CoQ9 heart sample indicated a retention time of
2.86 minutes and not
2.39 minutes (figure 3d). The retention time of the CoQ9 heart sample matched with that
of CoQlO rather than CoQ9. The qualitative analysis was done by identifying the
compounds by their retention times. It was obvious that at this point CoQ9 was probably
bio-converted to CoQlO. Further investigation was conducted by using mass
spectrometry to verify the conversion of Q9 into QIO in the heart sample.
Mass Spectroscopy of CoQ9/CoQ10.
The analytical sensitivity for CoQlO is known to be very low due to poor ionization property of CoQlO [Teshima, K., et a\Aml. Biochem. 2005, 338, 12-19], Hence the optimization of the LC-MS method was done by introducing 5 mmol of methylamine (v/v/v) in the mobile phase, to enhance the sensitivity for the determination of CoQ9 and CoQlO. The standard and heart derived sample solutions were injected using an Agilent 1100 HPLC. The HPLC was interfaced with the mass spectrometer and electron spray ionization mass spectrometry (ESI-MS) was conducted for the identification of the compounds. The ion spectra of standard samples of C0Q9 and CoQlO exhibited [M +CH3NH3]'peaks at m/z 826,5 and m/z 894.6 respectively (figure 4b and 4c). However, the GoQ9 heart sample indicated a mass peak at m/z 894.6 (see fig.4), which matches the m/z peak for CoQlO and not CoQ9 (figure 4d). Therefore, there is evidence that CoQlO is present in the Q9 supplemented heart sample. This confirms the bio-conversion of CoQ9 intoCoQlO.
CoQlO is present ubiquitously in most of the mammals including humans except for rodents where CoQ9 is the predominant form of CoQ. For this reason, the inventors choose guinea pigs as experimental animals to study the effect of CoQ9 as the hearts of

this animal does not contain any CoQ9. Feeding the guinea pigs CoQ9 for 4 weeks provided similar degree of cardio protection as CoQlO. Since most of the CoQ9 was found as CoQlO, it could be possible that CoQ9 after being converted into CoQlO provided cardio protection. In addition the present invention provides valuable information that nutritional supplementation of CoQ9 should be adequate for the animals needing CoQ 10 supplementation.
The generation of CoQlO is a complex process requiring many cofactors (e.g., vitamin B6, BIZ, folic acid, etc.) and several chain reactions. In the present study, prior to subjecting the hearts to ischemia/reperfusion protocol, majority of CoQ9 was found to be present as CoQ 10.
It is generally accepted that most of the exogenously administered CoQ 10, either as nutritional supplement or derived from CoQ 10 rich foods, is taken up by the liver and blood components, and only a small amount goes to other organs such as heart. In the present study, the inventors were able to detect appreciable amount of CoQ 10 in the heart tissue and very small or no amount of CoQ9 after 4 weeks of CoQ9 supplementation. In summary, the results of the present study demonstrate for the first time that nutritional supplementation of Coo9 leads to enrichment of CoQlO levels in the mammal and also that nutritional supplementation of CoQ9 can reduce myocardial ischemia reperfusion injury to the same extent as CoQ 10. The cardio protection was achieved either directly from CoQ9 or indirectly through its bioconversion into CoQ 10. Nevertheless, the finding that CoQ9 and CoQ 10 can provide the same degree of cardio protection appears to be important due the fact that only very little exogenous CoQ 10 is taken up by the heart, while significant amount of CoQ 10 was detected in the heart after four weeks of CoQ9 feeding. It is tempting to speculate that heart may be able to better utilize CoQ9 than CoQlO.
Therefore, to obtain full benefit, the inventors found that CoQ9 ingredient can be conveniently formulated into a solid, semi-solid or liquid dosage form by adding a conventional biologically acceptable carrier or diluent to achieve the desired therapeutic benefit as discussed above. Such specific dosage form includes, for example, oral agents such as tablets, soft capsule, hard capsule, pills, granules, powders, emulsions, suspensions, syrups, and pellets; and parenteral agents such as injections, drops, suppositories and the like.

The CoQ9 ingredient may be optionally combined with suitable quantity of CoQlO and the composition obtained thereof is administered using a method described above.
The CoQ9 composition or formulation used in the present invention may be prepared by formulating CoQ9 along with the biologically acceptable carrier or diluents. The examples of the biologically acceptable carrier or diluents employed in the present inventions includes but are not limited to, surfactants, excipients, binders, disintegrators, lubricants, preservatives, stabilizers, buffers, suspensions and drug delivery systems.
Preferred examples thereof include solid carriers include glucose, fructose, sucrose, maltose, sorbitol, stevioside, com syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-.alpha.-tocopherol, glycerin, propylene glycol, glycerin fatty ester, polyglycerin fatty ester, sucrose fatty ester, sorbitan fatty ester, propylene glycol fatty ester, acacia, carrageenan, casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, flavors, and preservatives. Preferred examples of liquid carriers (diluents) include distilled water, saline, aqueous glucose solution, alcohol (e.g. ethanol), propylene glycol, and polyethylene glycol; and oily carriers such as various animal and vegetable oils, white soft paraffin, paraffin and wax.
In alternative aspects of the invention, the product of the present invention is delivered in the form of controlled release tablets, using controlled release polymer-based coatings by the techniques known in the art. The said formulation is designed for once daily administration.
In other aspects of the invention, the product of the present invention is delivered in the form of nanoencapsulated or liposomal formulation to enhance the solubility and bioavailabity.
In accordance to the present invention, the CoQ9 or the composition is formulated into any food and drink forms such as solid food like chocolate or nutritional bars, semisolid food like cream or jam, or gel. Contemplation was also done to formulate the product of the invention into a beverage and the like, such as refreshing beverage, coffee, tea, milk-contained beverage, lactic acid bacteria beverage, drop, candy, chewing gum, chocolate, gummy candy, yoghurt, ice cream, pudding, soft adzuki-bean jelly, jelly, cookie and the

like. These various preparations or foods and drinks are useful as a healthy food for the treatment and prevention of cardiac problems.
The amount of the CoQ9 or its composition to enrich CoQlO concentration in mammals to be administered or ingested to mammals in the form of above-mentioned formulations or preparations or foods and drinks is not uniform and varies depending on the nature of the formulation and suggested human or animal dosage of CoQ9, but preferably within a range of fromO.Ol to 50, more preferably from about 5 to about 50, most preferably about 5mg/kg, weight/day.
In a further variation of the invention, the CoQ9 or the composition containing CoQ9 used for the supplementation optionally may be combined with a suitable quantity of CoQlO. In this embodiment, the method of the invention preferably comprises administering from about 0.0 1 to about 50 mg/kg/day of the CoQ9 and from about 0.0 1 to about 50mg/kg/day of the CoQlO. More preferably, in this embodiment, the method comprises administering from about 5 to about 50 mg/kg/day of the CoQ9 and from about 5 to about 50 mg/kg/day of the CoQ 10. Most preferably, in this embodiment, the method comprises administering about 5 mg/kg/day of the CoQ9 and about 5 mg/kg/day of the CoQ 10. The present invention is illustrated by the following non-limiting examples;
Example 1
Protective effect of CoQ9 and CoQlO against from Ventricular Fibrillation (VF): Healthy Male Hartley guinea pigs of about 350-400 gm body weight were randomly divided into three groups, Control, CoQ9 and CoQ 10. The guinea pigs were given orally 5 mg/kg body weight [in 0.5 ml water] of vehicle only, CoQ9 or CoQlO respectively by gavage once a day. CoQ9 or CoQ 10 by gavaging once a day 5 mg/kg [in 0.5 ml water] body weight. Treatment was continued for 30 days, the animals had free access to food and water. After 30 days, all animals were anesthetized, heparinized and sacrificed. The hearts excised, and isolated for perfusion via Langendorff mode for 5 min of washout period of the Langendorff heart perfusion, the pulmonary vein was cannulated, and the heart was switched to the "working" mode via perfusion of the left atria (at a filling pressure of 17 cm of the buffer, 1.7 kPa) as it was described in detail elsewhere. Global ischemia was imposed by clamping the atrial and aortic cannulas. Eplcardial ECG was recorded through out the experiment, by attaching two silver electrodes directly to the myocardium

and data collected using a data acquisition system (ADInsruments, Powerlab, Castle Hill, Australia). ECGs were analyzed to determine ventricular fibrillation (VF) and ventricular tachycardia (VT). The first 10 min of reperfusion was done in Langendorff ('nonworking') mode in order to avoid the development of reperfusion-induced VT and VF during the 'working' heart reperfusion. After the initial 2 min of VT or/and VF (sustained VF) in Langendorff reperfusion, hearts were defibrillated (if it was necessary), reperfused for an additional 8 min In Langendorff mode, and switched to 'working heart' reperfusion, and myocardial function was recorded. The heart was considered to be in VF if an irregular undulating baseline was apparent on the ECO. The data of VT, VF and sinus rhythm show their durations (in seconds) within the first 120 s of nonworking Langendorff reperfusion. The incidences of reperfusion-induced ventricular fibrillation (VF) forCoQ9 and CoQlO are depicted in figure I. Pretreatment of CoQ9 and CoQlO significantly reduced the incidence of ischemia-reperfusion induced ventricular fibrillation (VF), compared to untreated drug free group. Incidence of VF was reduced from 92 % (control group) to 25 % ('^p<0.05) and 8 % (♦p<0.05) with 5 mg/kg/day of CoQlO and C0Q9 respectively.
Example 2
CoQ9 and CoQlO treatment protects from cardiac infarction: Animal preparation, drug pretreatment and isolated working heart preparation were done as described in example 1. Animal pretreatment and isolated heart experiments were done as mentioned in example 1. Hearts for determination of infarct size were perfused, at the end of each experiment, with 25 inl of 1 % triphenyl tetrazolium solution (TTC) in phosphate buffer (Na2HP04 88 mM, NaH2P04 1.8 mnM) via the side arm of the aortic cannula, and then stored at -70 OC for later analysis. Frozen hearts were sliced transversely in a plane perpendicular to the apico-basal axis into 3-4 min thick sections, weighted, blotted dry, placed in between microscope slides and scanned on a Hewlett-Packard Scanjet 5p single pass flat bed scanner (Hewlett-Packard, Palo Alto, CA, USA). Using the NIH Image 1.61 image processing software, Infarct zones of each slice were traced and the respective areas were calculated in terms of pixels. The areas were measured by computerized planimetry software and these areas were multiplied by the weight of each slice, then the results summed up to obtain the weight of the risk zone. Infarct size was calculated as the ratio.

in percent, of the infarct zone to the risk zone. Effects of CoQlO and CoQ9 on infarct size in isolated guinea pig hearts are depicted in figure 2A.
Pretreatment of CoQ9 and CoQlO significantly reduced global ischemia induced cardiac infarction compared to untreated drug free group. Drug-free ischemic/reperfused control hearts were associated with a 38 -+ 4.1 % infarct size which was consistently reduced by the dose of 5 mg/kg/day of CoQlO and CoQ9 to 21.1 ± 5 % (*p<0.05) and 16.3 ± 3.2 % (*p<0.05), respectively.
Example 3
CoQ9 and CoQlO treatment reduces apoptosis of cardiomyocytes: Animal preparation, drug pretreatment and isolated working heart preparation were done as described in example 1. Iminunohistochemical detection of apoptotic cells was carried out using TUNEL assay, using APOPTAG' kit (Oncor, Gaithersburg, MD). The heart tissues were immediately put in 10% formalin and fixed in an automatic tissue-fixing machine. The tissues were embedded in the molten paraffin in metallic blocks. Prior to analyzing tissues for apoptosis, tissue sections were deparaffinized with xylene and washed in succession with different concentrations of ethanol (absolute, 95%, 70%). Then tissues were incubated with mouse monoclonal antibody recognizing cardiac myosin heavy chain to specifically recognize apoptotic cardiomyocytes. The fluorescence staining was viewed with a confocal laser microscope. The number of apoptotic cells was counted and expressed as a percent of total myocyte population. Effects of CoQlO and CoQ9 on cardiomyocyte apoptosis in isolated guinea pig hearts are depicted in figure 2B Pretreatment with CoQ9 and CoQlO significantly reduced the incidence of ischemia-reperfusion induced apoptosis of cardiomyocytes compared to untreated drug free group. Apoptosis of cardiomyocytes determined by Tunel method in control group was about 21f2 % at the end of reperfusion. Both CoQlO and CoQ9 treatment significantly reduced the number of apoptotic cardiomyocytes to 6± 1 % and 7± 1.5 % respectively.
Example 4
CoQ9 and CoQlO treatment improves post-ischemic cardiac function (HR, CF, AF, and LVDP): Animal preparation, drug pretreatment and isolated working heart preparation were done as described in example 1. The isolated hearts obtained from group 11 and Group III guinea pigs treated with 5 mg/kg/day of CoQlO and CoQ9, respectively, for 4

weeks were subjected to 30 min ischemia followed by 120 mln of reperfusion. The recovery of post- ischemic cardiac function in isolated hearts was evaluated by measuring various parameters including Coronary Flow (CF), Aortic Flow (AF), Left Ventricular Developed Pressure (LVDP), and Heart rate (HR) before ischemia, after 60 min of reperfusion and after 120 mln of reperfusion using Langendroff apparatus. The results are summarized in table 1. The pretreatment with CoQ9 and CoQlO significantly improved the post-ischemic recovery in HR, CF, AF, and LVDP compared to the drug-free control group. Pretreatment with CoQ9 and CoQlO significantly protected the heart from decrease In all functional parameters induced by ischemia-reperfusion. The improvement in post-ischemic cardiac function (HR, CF, AF, and LVDP) was more pronounced in the CoQ9 treated group than it was registered in the CoQlO treated group.
a. Coronary flow: The reduction in coronary flow due to ischemia-reperfusion was
significantly protected in CoQlO (19±1) and CoQ9 (25±2) treated groups in comparison
to drug free control group (15±1). However CoQ9 completely improved CF to its normal
value recorded before ischemic reperfusion (Before ISA 23±2 After RE 25±2) where as
CoQlO did not improve CF to normal state (Before ISA 25±2 After RE 19±1).
b. Aortic flow: The reduction in aortic flow due to ischemia-reperfusion was significantly
protected in CoQlO (18±2) and CoQ9 (26± 1) treated groups in comparison to drug free
control group (8±1).
c. Left ventricular developed pressure: The reduction in LVDP due to ischemia-
reperfusion was significantly protected in CoQlO (64± 3) and CoQ9 (75± 2) treated
groups in comparison to drug free control group (45±3).
d. Heart rate: The reduction in heart rate due to ischemia-reperfusion was significantly
protected in CoQlO (217±3) and CoQ9 (233±4) treated groups in comparison to drug free
control group (182±4). Statistics: The values of HR, CF, AF, LVDP, and infarct size were
expressed as mean value k SEM. A two-way analysis of variance was first carried out to
test for any differences in mean values between groups. If differences were established,
the values of the drug-treated groups were compared with those of the drug-free group by
Dunnett's test. A different procedure, because of the nonparametric distribution, was used
for the distribution of discrete variables, such as the incidence of VF. Thus, the chi-square
test was used to compare the incidence of VF between untreated-control and treated
groups.

Example 5
High Performance Liquid Chromatography [HPLC] and Mass Spectroscopy [MS] for the Determination of Coo9 and CoQ 10:
Preparation of CoQ9 and CoQ 10 heart samples: Animal preparation, drug pretreatment and isolated working heart preparation were done as described in example 1. The study animals at the end of four week period were anesthetized by heparin administration and then the animals were scarified, and the hearts excised. The ground heart samples provided for analysis were centrifuged at 3000 rpm for 10 minutes. The supernatant was then transferred to another centrifuge tube and was evaporated to dryness using nitrogen, in order to obtain a more concentrated solution. The residue after dryness was then dissolved using 2 ml of mobile phase, and was then transferred to an auto sampler injection vial. The samples were analyzed immediately after preparation, and the remainder of the standard solutions was stored at 50C for future analysis. Preparation of CoQ9 and CoQ 10 standard solutions: Standard solutions were prepared by weighing approximately 10 mg of CoQ9 and CoQlO standards respectively into a 100 ml volumetric flask and then dissolving it by using the mobile phase as a diluent. The stock solution was further diluted 1:10 to attain a final working concentration of 0.01 mg/ml. The CoQlO stock solution was sonicated for 5 minutes for complete dissolution of the powder into solution. HPLC Analysis of CoQ9 and CoQ 10: The modular HPLC system consisted of an Agilent 1100 quaternary pump, Agilent 1100 autosampler, Agilent 1100 column heater, and Agilent 1100 UV detector. The analysis of CoQ9 and CoQ 10 was performed by using a YMC Pro CI8, 3pm, 120°A, 2.0 x 50 mm column and the mobile phase consisted of methanol-(2-propanol)-formic acid (45:55:0.05, v/v/v), containing methylamine at the concentration of 5 mmol/L. The flow rate was 0.2 ml/min and the column compartment was maintained at 40°C. The injection volume was 5nl
[13].
The HPLC chromatograms for the standard sample of CoQ9 and CoQ 10 are depicted in figures 3b and 3c respectively. CoQ9 and CoQ 10 were observed at the retention times of 2.39 and 2.86 minutes respectively. The samples obtained from the ground hearts of control group (group I), CoQ9 supplemented group (group II) and CoQ 10 supplemented group (group III) were analyzed and the control chromatogram was subtracted from that of the CoQ9 fed sample and CoQlO fed sample and the HPLC chromatograms for the heart samples of CoQ9 and CoQlO are depicted in figures 3d and 3e respectively. The HPLC chromatogram for the heart sample from the animals supplemented with CoQ9 showed significantly high enrichment in the CoQlO content over and above the natural concentration as indicated by an intense peak at 2.86 (figure 3d). Its identity was further conformed by mass spectrometric analysis. It showed a mass peak at m/z 894.6 for CoQlO [M + CH~NH~]'and it matches with that observed for a standard sample of CoQlO.
Example 6
Mass Spectroscopy for the Identification of the Peaks: Finnigan LCQ ion trap bench top mass spectrometer (Thermo Fischer Scientific, MA, USA) interfaced with an Agilent 1100 HPLC system was used for analysis. Data processing was done in the Finnigan Xcalibur data system operating on Windows@ NT PC-based system. The turbo ion spray interface and mass spectrometer were operated under the following conditions: positive ionization polarity, 4.8 kV spray voltage, 425" C probe temperature, collision gas pressure, 2.8 x 10-5 Ton [13]. All parameters were adjusted for each analyte, using the tune method CoQlO EP07I002 created by the analyst at the time of analysis with the Xcalibur software. Divert valve and contact closure were not used during the run. Optimization of the LC-MS method was done by introducing 5 mmol of methylamine (v/v/v) in the mobile phase, to enhance the sensitivity for the determination of CoQ9 and CoQlO. The standard and sample solutions were injected using an Agilent 1100 HPLC. The flow rate of 0.2 ml/min was maintained. A YMC Pro C18, 3nm, 120°A, 2.0 x 50 mm column was used. The HPLC was interfaced with the mass spectrometer. Electron spray ionization mass spectrometry (ESI-MS) was conducted for the identification of the compounds. A full MS scan from 50 to 1000 units was sun to obtain the m/z ratios for the compounds of interest, namely CoQ9 and CoQlO. No MSIMS or fragmentation was done at this point. In the presence of methylamine in the mobile phase, the product ion spectra of both [M + CH3NH3]^ at m/z 826.5 for C0Q9 and m/z 894.6 for CoQlO was observed (see figures 4a and 4b). However, the CoQ9 heart sample indicated a mass peak at m/z 894.6 (see figure 4c), which matches the m/z peak for CoQlO and not CoQ9.
It will be evident to those skilled in the art that the invenfion Is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and It is

therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

We Claim,
1. A pharmaceutical/nutraceutical composition or dosage forms /food supplement for administration comprising CoQ9 in an amount of about 0.01 to about 50 mg/kg body weight in combination with one or more pharmaceutically/ biologically acceptable carrier or diluent for increasing the CoQl0 levels in humans/animals.
2. The composition or dosage form as claimed in claim 1, wherein the amount of the CoQ9 is about 0.01 to 5 mg/kg body weight.
3. The dosage form or composition as claimed in claim 1, wherein the pharmaceutically/ biologically acceptable carrier or diluent comprising one or more additives selected from the group consisting of surfactants, excipients, binders, disintegrants, lubricants, preservatives, stabilizers, and buffers.
4. The dosage form or composition as claimed in claim 1, wherein the composition is orally administered as a dosage form selected from the group consisting of tablets, soft capsules, hard capsules, pills, granules, powders, emulsions, suspensions and pellets.
5. The dosage form or composition or supplement as claimed in claim 1, wherein the composition is parenterally administered as a dosage form selected from the group consisting of injections, drops, and suppositories.
6. The dosage form composition or supplement as claimed in claim 1, wherein the composition is administered in the form of a drug-delivery system selected from the group consisting of microencapsulated drug-delivery systems, nanoparticle-based drug-delivery systems, liposome-based drug-delivery systems, biodegradable block copolymer drug-delivery systems, and polymeric surfactant-based drug-delivery systems.
7. The dosage form or composition or supplement as claimed in claim 1, wherein the composition is orally administered in the form of a dietary supplement composition, a food composition, or a nutraceutical composition.
8. The composition as claimed in claim 1, wherein the composition is orally administered in the form of a composition selected from the group consisting of nutritional bars, creams, jams, gels, candies, chewing gums, cookies, and beverages.
9. The dosage form or composition or supplement as claimed in claim 1, wherein the composition is topically administered in the form of a cosmetic composition.
10. The dosage form or composition or supplement as claimed in claim 1, wherein said composition further comprising CoQlO in an amount of about 0.01 to about 50 mg/kg body weight of the CoQ 10.
11. The dosage form or composition or supplement as claimed in claim 10, wherein said composition comprising about 0.01 to about 50 mg/body weight of CoQ9 and from about 0.01 to about 50 mg/body weight of CoQ 10.
12. The composition as claimed in claim 11, wherein said composition comprising about 5 mg/kg body weight of the CoQ9 and about 5 mg/kg body weight of the CoQl0.