Technical Field

The invention is related to the oral use of Coenzyme Q10 in high doses for treatment purposes in the primary Coenzyme Q10 deficiencies.

Prior Art

Coenzyme Q10 (KoQlO; ubiquinone, ubidecaquinone, ubidecarenone) is not a vitamin but is a fat-soluble, antioxidant, vitamin like compound which is commonly found in nature and in every cell of the animal and human body, can be de novo synthesized by the cells [1].

The structure of Coenzyme Q10 in humans is similar to vitamin K, is formed with the addition of 10 isoprene sub-unit to the 1,4-benzoquinone ring, therefore this structure is called Coenzyme Q10 [2].

The chemical formula of Coenzyme Q10 is 2,3-dimethoxy-5-methyl-6- decaprenly- 1,4-benzoquinone and can be found in biological tissues in two forms biochemically: The reduced form (ubiquinol-10) and oxidized form (ubiquinone-10) [3].

Coenzyme Q10 is located in the inner membrane of the mitochondria and acts as an essential coenzyme and a component of lipid membranes which are required for the activities of the enzyme systems named as complex I, II and II that acts in the oxidation-reduction reactions in the electron transfer chain. Coenzyme Q10 affects the function of all cells in the body especially the ones requiring high energy as it participates in ATP synthesis. Thus, Coenzyme Q10 is essential for the health of all tissues and organs. Moreover, Coenzyme Q10 acts as an important antioxidant by interacting with free oxygen radicals [4].

The cells generally make biosynthesis in order to provide Coenzyme Q10. The endogenous levels of Coenzyme Q10 are arranged by the physiological factors regarding the oxidative activity of the organism [5]. The para-hydroxybenzoic acid in tyrosine amino acid is the first aromatic precursor in the biosynthesis pathway of Coenzyme Q10 in humans, and it forms the quinoid ring structure of Coenzyme Q10 molecule. The caudal consisting of ten isoprenoid units is derived from the mevalonate pathway [6]. Endogenous Coenzyme Q10 levels are determined by means of both the production rate and the consumption rate in the body. These levels can change during various diseases which are seen in humans such as cardiovascular diseases and degenerative muscle defects [7].

Many diseases related to Coenzyme Q10 deficiency such as primary and secondary Coenzyme Q10 deficiencies, mitochondrial diseases, fibromyalgia, cardiovascular disease, neurodegenerative diseases, cancer, diabetes mellitus, male infertility and periodontal disease etc. can benefit from Coenzyme Q10. In this application early treatment with high dose of Coenzyme Q10 for the treatment of Coenzyme Q10 deficiencies which occur due to genetic disorder is required to be started and a patent for using thereof is required to be obtained for this indication. Since current Coenzyme Q10 preparation in our country are generally available in low dose (lOOmg) for supplement purposes and are mostly in tablet form. It is required to take 2-3 grams of Coenzyme Q10 daily in the indication for Coenzyme Q10 treatment with the patent subject to application. The high dose Coenzyme Q10 usage included in the patent subject to application is used for treatment purposes in genetic-dependent Coenzyme Q10 deficiency. Different from said current Coenzyme Q10 tablets, it can be used in high doses. There are no descriptions in the current applications regarding the uses of high doses of Coenzyme Q10 for treatment purposes since the treatment protocols are not standardized yet. In the invention subject to application, for example with 20( g/ml Coenzyme Q10 syrup, treatment doses in both pediatric and adult patient populations can be provided easily. Aims of the Invention

The aim of this invention is to use high dose Coenzyme Q10 such as 2-3 grams in the treatment of primary, genetic-derived Coenzyme Q10 deficiencies.

Another aim of this invention is to use high-dose Coenzyme Q10 which provides treatment doses in both pediatric and adult patient populations easily for example with 20( g/ml Coenzyme Q10 syrup. Another aim of this invention is to use high-dose Coenzyme Q10 in powder form or liquid form orally for treating some diseases.

Another aim of this invention is to use high-dose 1-3 gr Coenzyme Q10 pill or sachet in the treatment genetic-derived Coenzyme Q10 deficiencies. Another aim or this invention is to use high-dose or liquid Coenzyme Q10 in both pediatric and adult patient population. Another aim of the invention is to use high-dose Coenzyme Q10 in syrup or tablet or capsule or sachet form such that treatment doses can be adjusted easily.

Brief Description of the Invention

Patients with any kind of Coenzyme Q10 deficiency have exhibited clinical recovery with oral Coenzyme Q10; however brain symptoms have been partially recovered due to irreversible structural brain damage possibly before the treatment and poor penetration of Coenzyme Q10 along the blood-brain barrier [8]. As defined in the first claim and the other dependent claim, in order to achieve the aim of this invention, the use of high-dose Coenzyme Q10 is performed as follows. The subject of the invention is Coenzyme Q10; it can be used in doses of 2-3 grams in the treatment of primary, genetic-derived Coenzyme Q10 deficiencies. Usage dose ratios of said Coenzyme Q10 can be controlled or can be changed based on the requirements. Coenzyme Q10 can be taken orally in powder or liquid form. Said Coenzyme Q10 can be taken in syrup form and also can be taken in tablet, capsule or sachet form. After a stable dose in applied during 3-4 weeks under stable conditions, tracking Coenzyme Q10 plasma concentrations can be considered [9]. Recommended oral supplement doses in primary genetic-derived Coenzyme Q10 deficiencies are up to 1.200-2.400 mg daily in adult patients, up to 10-50 mg/kg in pediatric patients, and the dose is divided into three daily [10]. Coenzyme Q10 can be used for a longer or a shorter period of time based on the treatment of a specific disease. Coenzyme Q10 can be used with other pharmaceutically acceptable substances and supplement food substances. Said substance with pharmaceutical feature can be selected properly according to the treatment aim.

Detailed Description of the Invention

Many diseases related to Coenzyme Q10 deficiency such as primary and secondary Coenzyme Q10 deficiencies, mitochondrial diseases, fibromyalgia, cardiovascular disease, neurodegenerative diseases, cancer, diabetes mellitus, male infertility and periodontal disease etc. can benefit from Coenzyme Q10. Coenzyme Q10 tissue deficiencies or subnormal serum Coenzyme Q10 levels regarding many medical conditions including primary Coenzyme Q10 deficiencies and secondary Coenzyme Q10 deficiencies such as mitochondrial diseases etc. are reported. Levels of Coenzyme Q10 decrease together with the increased age and this decrease may contribute to the occurrence or some signs of aging.

Coenzyme Q10 deficiency may occur based on many different reasons. Coenzyme Q10 deficiency may occur due to impaired Coenzyme Q10 synthesis based in nutritional deficiencies (for example Vitamin B6 deficiency which is an essential cofactor in the synthesis of Coenzyme Q10), a genetic-derived or acquired disorder in the synthesis or usage of Coenzyme Q10 or an increase in tissue requirements based on a specific disease. Diseases such as encephalomyopathy, severe infantile multi- systemic disease, cerebellar ataxia, Leigh syndrome which is a growth retardation and isolated myopathy are included among the clinical profiles of severe Coenzyme Q10 deficiency. Since the tissue levels may increase due to the oral administration of Coenzyme Q10, it is possible to improve Coenzyme Q10 deficiency. The approach of oral administration of high- dose Coenzyme Q10 is particularly important for the infantile encephalopathy which threatens life. The most severe Coenzyme Q10 deficiencies are due to autosomal recessive mutations, when mutations affect the Coenzyme Q10 biosynthesis gene (COQ genes), if the primary deficiency is due to other genetic deficiencies, it can be classified as a secondary deficiency. In the year 1989, Ogasahara et al. detect and publish the first example of primary Coenzyme Q10 deficiency in the skeletal muscle [11]. Currently, more than 100 diseases related to Coenzyme Q10 deficiency are identified and published.

In most of the patients with infantile prime multi-systemic variants, there is genetic-derived Coenzyme Q10 deficiency. So far nine genes [COQ1 (PDSS1 and PDSS2), COQ2, COQ4, COQ6, COQ7, COQ8A/ADCK3, COQ8B/ADCK4, and COQ9] which cause CoQlO deficiency is determined. Among the secondary deficiencies, there are diseases caused by the mutation in genes which are not related to the ubiquinone biosynthesis, for example aprataxin (APTX) gene that causes ataxia and oculomotor apraxia, electron-transfer-flavoprotein dehydrogenase gene (ETFDH) and BRAF gene mutations that causes cardiofaciocutaneous syndrome can be included. Patients having Coenzyme Q10 deficiency exhibited variable responses to the Coenzyme Q10 treatment. Clinical recovery was reported in many patients after Coenzyme Q10 supplement. For this reason, it is possible to use Coenzyme Q10 in diseases that require high doses particularly genetic Coenzyme Q10 deficiencies. Usage of high-dose Coenzyme Q10 included in one embodiment of the invention is realized as the following. The inventive Coenzyme Q10 can be used in doses of 2-3 grams in the treatment of primary, genetic-derived Coenzyme Q10 deficiencies. Doses are taken daily. Usage dose ratios of said Coenzyme Q10 can be controlled or can be changed based on the requirements, on the treatment process of the disease.

The inventive Coenzyme Q10 can be taken orally in powder or liquid form. Said Coenzyme Q10 can be taken in syrup form and also can be taken in tablet, capsule or sachet form.

Coenzyme Q10 in the invention subject to application can be taken orally in powder or liquid form throughout life. The treatment process in other words usage period of Coenzyme Q10 can be more than 2 or 3 days in different embodiments of the invention. Said Coenzyme Q10 can be used for at least 2 or 3 days during 4 weeks or more, or less than 4 weeks orally in powder or liquid form. Coenzyme Q10 can be used for a longer or a shorter period of time based on the treatment of a specific disease. The inventive Coenzyme Q10 can be used daily at least 50 mg and at most 4 grams.

Cholesterol lowering drugs such as lovastatin and pravastatin leads to the decrease of serum CoQlO by inhibiting HMG-CoA reductase enzyme required for the synthesis of Coenzyme Q10 in addition to reducing the cholesterol. [12] It is shown that beta blockers, propranolol and metoprolol, [13] also phenothiazines and tricyclic antidepressants inhibit CoQ 10-dependent enzymes [14]. The effect of CoQlO to the thrombocyte function may increases the risk of bleeding in patients taking antiplatelet drugs such aspirin [15]. On the other hand, since it acts like Vitamin K, it can resist to the anticoagulant effects of warfarin [16]. When it is given together with the antihypertensive drugs, CoQlO may have further antihypertensive drugs [17]. CoQlO, by means of increasing beta-cell function, may develop insulin sensitivity which can reduce the insulin requirements of the diabetic patients [18].

CoQlO treatment is safe even with highest doses mentioned in the literature. Important side effects which require termination of the treatment are not reported in most clinical studies [19]. In addition to this, gastrointestinal effects such as abdominal swelling, nausea, vomiting, diarrhea and anorexia occurred; allergic eruption and headache are also reported [20]. In addition to this, the antiplatelet effect of CoQlO can increase bleeding risk [21]. CoQlO is subject to biotransformation in the liver and is removed from the body particularly with the bile, thus it can accumulate in patients with liver failure or biliary obstruction [22].

In the invention subject to application, in using said high-dose Coenzyme Q10, Coenzyme Q10 substance can be protected in a darkened bottle such that the light contact is minimized. Moreover, said Coenzyme Q10 can be applied in liquid form preferably via a scaled syringe.

The tests performed regarding the invention and the study made with a family with two-influenced was as follows. The first patient was a 26-year- old male and consanguineous marriage is in question. The parents of the patient were cousins of first degree. In the index case, the patient had slow progressing ataxia and spasticity. The patient experienced difficulty in walking at the age of 8, then after 4 years had partial epilepsy and lost his walking ability at the age of 16. Cognitive deterioration and dysarthria were detected during the neurological examination of the patient, the patient could not be able to walk even with support however could be able to stand with support. The patient was experiencing severe spasticity, spasm in the lower limbs, moderate spasticity (spasm) in the upper limbs, also moderate dysmetria and dysdiadochokinesia in the upper leg muscles. The ocumolotor motions and deep tendon reflexes with sphincter were normal in both limbs. However, the Babinski sign was positive bilaterally. Brain MRI evaluation of the patient shows that; cortical and subcortical T2 hyperintensity was not limited with a specific vascular region as defined in the previous report which describes COQ4 mutations. The patient was treated with 600 mg carbamazepine twice a day and 2 mg clonazepam twice a day.

The symptoms of the patients sister at the age 27 have started when she was 8 however it has progressed slower than that of the male patient. Sister of the patient was partially caught to epilepsy when she was 12. During the physical examination of the female patient; extremities were seen in the lower limbs more specifically dysarthria, spastic-ataxic and mild spasticticity was observed in four limbs. The female patient could be able to walk without any support, her eye motions were normal, dysmetria and dysdiadokokinesis were present. There were deteriorations in the cognitive functions of the patient. Babinski sign was positive bilaterally without sphincter dysfunction. The routine biochemistry, hemogram and metabolic tests were normal. No pathology was detected in the MR of the whole spine. Moreover, cerebral and cerebellar atrophy was seen in brain MRI. The female patient was treated with 1000 mg levetiracetam twice a day. Said patient was directed to the medical genetics department with the ataxia diagnosis (disorders in physical functions) and first of all the most common genetic disorders which cause ataxia disease were examined and genetic consultancy was recommended to the patients and the relatives of the patients. After the patients were determined as normal regarding the most common genetic disorders that cause ataxia disease, the materials of the patient for a further examination namely whole exome sequencing test were sent to the University of Michigan with the permission of the trustee of the patient.

After the CoQ4 gene mutation was defined in the Coenzyme Q10 bio ^¬ synthesis, it was tested in the patient with Coenzyme Q10 supplement for treatment. After the Coenzyme Q10 treatment is successful, local health insurances gave approval for the treatment of Coenzyme Q10. Both patients were treated under a long term treatment and they were evaluated after 1 year.

The plasma Coenzyme Q10 (Q10, 87853) levels were analyzed by the Mayo Clinic (Rochester, MN). Since the samples were subjected to transfer more than 72 hours, only total Coenzyme Q10 values were reliable and these values were reported. Although sister of the patient were not treated with Coenzyme Q10, the levels of Coenzyme Q10 were measured at the same time with the male patient. DNA was isolated from the whole blood via Qiagen Gentra Puregen. The new generation exome sequence was performed by the DNA core facility of Michigan University. Exomes were caught by the SeqCap EZ exome v3.0 kit (Roche, Ca, USA) and matched ends were sequenced to an average 52 X deepness in HiSeq2000. The concerned variables were confirmed and mother-father was tested for determining whether they are carriers of relevant mutations and it was confirmed. DNA samples of the sister with childhood onset ataxia were sent for the next generation exome sequencing. Common SNPs were used for the link analysis using Merlin which is a family tree considering the first cousin mating. The frequency of the model parameters was selected 0.0001 for a rare recessive disease. Only one region on the distal chromosome 9 showed a LOD score under this model but above 1.0. Said region had 51 genes. We defined only one gene, COQ4, with homozygous mutation which is considered as harmful, ekson2:c.G164T:p.G55V. Both parents were heterozygous. This specific mutation was not previously reported and glycine at this point is protected in all vertebrates. Because COQ4 mutations lead to Coenzyme Q10 deficiency, plasma Coenzyme Q10 was measured. Coenzyme Q10 levels was under normal levels.

Coenzyme Q10 treatment was proposed to the patient. Ataxia disease was seen more commonly in humans and animals that have Coenzyme Q10 deficiency. The neurologist who gave the treatment proposed treatment with high-dose (2000 mg/day) Coenzyme Q10 to the patient. Following one-month treatment, the patient was evaluated again and blood sample is taken from the patient and ataxia was measured and evaluated. Following a 1-month treatment with Coenzyme Q10, although serum Coenzyme Q10 values are not recovered, the clinical evaluation and the subjective report exhibited an explicit recovery. Total SARA point increased from 30 to 10; an improvement was reported in terms of the patient, since he/she can be able to walk with a walker and remain standing without any support instead of being bound to a wheelchair, not walking even with support or not standing without support. Following approximately 1 year treatment with Coenzyme Q10, the patient achieves a better level than the basal line with a SARA score of 17. Then, a long term treatment was initiated for the sister and an improvement was detected in SARA score.

References

[1] Ernster, L, Dallner, G. "Biochemical, physiological and medical aspects of ubiquinone function." Biochim. Biophys. Acta., 1271(1), 195-204 (1995).

[2] Crane, F.L. "Biochemical functions of coenzyme Q10." J. Am. Coll. Nutr., 20(6), 591-598 (2001). Greenberg, S., Frishman, W.FI. "Co-enzyme Q10: a new drug for cardiovascular disease." J. Clin. Pharmacol., 30(7), 596-608 (1990).

[3] Parkhideh, Daryoush, "Methods and compositions that enhance bioavailability of coenzyme-QlO", United States Patent 7,438,903, Parkhideh, October 21, (2008).

[4] M. Turunen, J. Olsson, G. Dallner, "Metabolism and function of coenzyme Q", Biochi mica et Biophysica Acta (BBA), Biomembranes; 1660(1-2): 171-199, (2004).

[5] L. Ernster and G. Dallner: Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta 1271(1), 195-204 (1995). U. C. Tran and C. F. Clarke: Endogenous synthesis of coenzyme Q in eukaryotes. Mitochondrion 7 Suppl, S62-71 (2007). [6] K. Folkers: Relevance of the biosynthesis of coenzyme Q 10 and of the four bases of DNA as a rationale for the molecular causes of cancer and a therapy. Biochem Biophys Res Commun 224(2), 358-61 (1996). [7] M. Turunen, J. Olsson and G. Dallner: Metabolism and function of coenzyme Q. Biochim Biophys Acta 1660(1-2), 171-99 (2004).

[8] A. Rotig, J. Mollet, M. Rio and A. Munnich: Infantile and pediatric quinone deficiency diseases. Mitochondrion 7 Suppl, S112-21 (2007).

[9] K. Hosoe, M. Kitano, H. Kishida, H. Kubo, K. Fujii and M. Kitahara: Study on safety and bioavailability of ubiquinol (Kaneka QH) after single and 4-week multiple oral administration to healthy volunteers. Regul Toxicol Pharmacol 47(1), 19-28 (2007).

[10] J. N. Flathcock and A. Shao: Risk assessment for coenzyme Q10 (Ubiquinone). Regul Toxicol Pharmacol 47(1), 282-8 (2006). V. Emmanuele, L. C. Lopez, A. Berardo, A. Naini, S. Tadesse, B. Wen, E. D'Agostino, M. Solomon, S. Dimauro, C. Quinzii and M. Hirano. Heterogeneity of Coenzyme Q10 Deficiency: Patient Study and

Literature Review. Arch Neurol 69(8), 978-83 (2012). M. V. Miles, B. J. Patterson, M. B. Schapiro, F. J. Hickey, M. Chalfonte-Evans, P. S. Horn and S. L. Hotze: Coenzyme Q10 absorption and tolerance in children with Down syndrome: a dose-ranging trial. Pediatr Neurol 35(1), 30-7 (2006). Mei Lu, Yulin Zhou, Zengge Wang, Zhongmin Xia, Jun Ren, Qiwei Guo. Clinical phenotype, in silico and biomedical analyses, and intervention for an East Asian population-specific c.370G>A (p.G124S) COQ4 mutation in a Chinese family with Koenzim Q10 deficiency-associated Leigh syndrome. Journal of Human Genetics. DOI: 10.1038/sl0038-019-0563-y. [11] S. Ogasahara, A. G. Engel, D. Frens and D. Mack: Muscle coenzyme Q deficiency in familial mitochondrial encephalomyopathy. Proc Natl Acad Sci U S A 86(7), 2379-82 (1989).

[12] S. A. Mortensen, A. Leth, E. Agner and M. Rohde: Dose-related decrease of serum coenzyme Q10 during treatment with HMG-CoA reductase inhibitors. Mol Aspects Med 18 Suppl, S137-44 (1997). [13] T. Kishi, T. Watanabe and K. Folkers: Bioenergetics in clinical medicine XV. Inhibition of coenzyme Q10- enzymes by clinically used adrenergic blockers of beta- receptors. Res Commun Chem Pathol

Pharmacol 17(1), 157-64 (1977). [14] A. M. Moreno-Fernandez, M. D. Cordero, J. Garrido- Maraver, E.

Alcocer-Gomez, N. Casas-Barquero, M. I. Carmona -Lopez, J. A. Sanchez-

Alcazar and M. de Miguel: Oral treatment with amitriptyline induces coenzyme Q deficiency and oxidative stress in psychiatric patients. J Psych iatr Res 46(3), 341-5 (2012).

[15] V. L. Serebruany, J. V. Ordonez, W. R. Herzog, M. Rohde, S. A. Mortensen, K. Folkers and P. A. Gurbel: Dietary coenzyme Q10 supplementation alters platelet size and inhibits human vitronectin (CD51/CD61) receptor expression. J Cardiovasc Pharmacol 29(1), 16-22 (1997).

[16] U. Singh, S. Devaraj and I. Jialal: Coenzyme Q10 supplementation and heart failure. Nutr Rev 65(6 Pt 1), 286-93 (2007). [17] R. A. Bonakdar and E. Guarneri: Coenzyme Q10. Am Fam Physician 72(6), 1065-70 (2005).

[18] J. M. Hodgson, G. F. Watts, D. A. Playford, V. Burke and K. D. Croft: Coenzyme Q10 improves blood pressure and glycaemic control: a controlled trial in subjects with type 2 diabetes. Eur J. Clin Nutr 56(11), 1137-42 (2002).

[19] T. Hidaka, K. Fujii, I. Funahashi, N. Fukutomi and K. Hosoe: Safety assessment of coenzyme Q10 (CoQlO). Biofactors 32(1-4), 199-208

(2008).

[20] T. Hidaka, K. Fujii, I. Funahashi, N. Fukutomi and K. Hosoe: Safety assessment of coenzyme Q10 (CoQlO). Biofactors 32(1-4), 199-208 (2008).

[21] S. Greenberg and W. H. Frishman: Co-enzyme Q10: a new drug for cardiovascular disease. J Clin Pharmacol 30(7), 596-608 (1990). [22] S. Greenberg and W. H. Frishman: Co-enzyme Q10: a new drug for cardiovascular disease. J Clin Pharmacol 30(7), 596-608 (1990).