A COMPOSITION COMPRISING A COMPLEX OF COENZYME QlO AND CYCLODEXTRINS AND USE THEREOF FOR THE TREATMENT OF PATHOLOGIC STATES OF THE CENTRAL NERVOUS SYSTEM.

DESCRIPTION The present invention relates to a composition comprising a complex of coenzyme QlO and cyclodextrins and the use thereof for the treatment of various diseases, in particular of neurodegenerative pathologic states of the central nervous system, more particu- larly of Parkinson's disease.

As is known, Parkinson's disease is a neurodegenerative pathology wherein the component of protein degradation of Sommering' s substantia nigra causes a functional alteration thereof. This proteolytic stress, therefore, results in a serious deficit in the production of dopamine, a cate- cholamin neurotransmitter involved in the nervous circuit of the nuclei of the base of telencephalon. In physiological conditions, proteins having a struc- ture that is unsuitable to their function are destroyed by the ϋbiquitin Proteasome System (USP) complex. The degradation process for these proteins starts with the bond of a chain of ubiquitin molecules (a protein with 76 amino acids and MW of 8,500 Da) as signal for identification and degradation by a protea-

some .

Ubiquitin monomers are activated during an ATP- dependant thioesterification by enzyme El of UPS complex. Activated ubiquitin is transferred to enzyme E2, a specific carrier, and then bound with covalent bonds by enzyme E3 (a ubiquitin protein ligase) to free amino groups of the lysines of the proteins to be de-

I graded. Among enzymes E3, parkin plays an important role in pathogenesis.

The malfunction of UPS complex results in an accumulation of abnormal proteins, among which α-synuclein is crucial: in physiological conditions, this protein is involved in mechanisms of synaptic plasticity, since it manages the transport and exchange of vesicles in synaptic terminals.

As is known, oxidative stress belongs to a sequence of events leading to neuronal death: it acts in synergy with other malfunctions, among which mitochondrial de- ficits and alterations of UPS system of degradation of cell proteins.

Dopamine metabolism is responsible for the high levels of oxidative stress in substantia nigra: as a matter of fact, the degradation of this neurotransmitter by the enzyme monoamine oxidase produces hydrogen perox-

ide (H ₂ O ₂ ) . This leads first to an increase in the formation of oxidized glutathione (GSSG) , which means a serious damage to the most important antioxidant system of the cell. Moreover, hydrogen peroxide pro- duces hydroxyl radicals and superoxide anions that are highly toxic in the presence of high intracellular iron levels, as can be found as a rule in substantia nigra, according to the following Fenton' s reactions: H ₂ O ₂ + Fe ²⁺ —> .0H + OfT + Fe ³⁺ O ₂ ^" + Fe ³⁺ --> O ₂ + Fe ²⁺

Therefore, the global reaction according to which:

H ₂ O ₂ + O ₂ ^" —> «0H + OH ^" + O ₂ is valid.

Both these reactions occur only in the presence of free iron that is not bound to ferritin.

As a matter of fact, in pathologic conditions the presence of nitric oxide (NO) causes iron to separate from ferritin, thus increasing the concentration of free iron in the cell. This process does not only involve dopaminergic neurons, but also occurs in glial cells, which indeed express enzymes belonging to the class of monoamine oxidases . Moreover, it was shown that the activation of glial cells, and in particular of microglial cells, during

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inflammatory processes causes the death of dopaminergic neurons due to the release of reactive nitrogen and highly toxic oxygen species. Eventually, it should be pointed out that oxidative stress can contribute to damaging the UPS mechanism of protein degradation, which can in its turn cause oxidative stress: this is further evidence of the close interdependence of two key factors in the pathogenesis of Parkinson's disease. The task of the present invention is to prepare a composition having an inhibitory effect on the progress of neurodegenerative pathologic states of the central nervous system and in particular in Parkinson' s disease (PD) . In the framework of this task, an aim of the invention is to prepare a composition based on CoEnQlO, as active substance, complexed with cyclodextrins . The preparation based on coenzyme QlO, whose formula is :

is proposed as inhibitor of the progress of neurode-

generative diseases of the central nervous system, in particular of PD.

Neuroprotective treatments can be useful during the onset of PD. This would mean beginning the treatment in the initial stage of the disease.

The administration of high doses (up to 1,200 mg pro die) of coenzyme QlO has shown a slackening in the progress during 16-month observation. The form of administration proposed by the preparation according to the invention, depending on its characteristics and peculiarities, enables among other things a simpler intake for patients having also a difficult deglutition. It also enables to simply vary the dose of the active substance that is required according to the physician's opinion. Coenzyme QlO (also known as ubiquinone or ubidecarenone) is a physiological molecule with antioxidant properties to be found in all cell membranes, among which inner mitochondrial membranes, where it is part of the complex I of the so- called respiratory chain, involved in the production of energy by mitochondria.

In the past it was shown that in Parkinson' s disease there is a reduction of complex I in one of the areas subject to neurodegeneration (substantia nigra of brain stem) . This led to the innovative development in

which the traditional forms of administration of coenzyme QlO that are on the market today (at far lower doses than those used in the assay) are replaced with the present solubilized preparation. Complexes of coenzyme QlO and cyclodextrins are known in the art .

For instance, US patent 6,861,447 B2 describes a method for producing complexes of coenzyme QlO and γ- cyclodextrin by thermal or ultrasonic treatment. International patent application WO 2005/111224 A2 describes water-soluble complexes of coenzyme QlO and β- cyclodextrin as well as a process for producing them, in which the temperature of β-cyclodextrin dissolution in water and of the following addition of coenzyme QlO is suitably controlled.

US patent application US 2006/0078549 describes a method for the therapeutic treatment of neurodegenerative diseases, of Huntington' s disease for instance, by administering a complex of coenzyme QlO and cyclo- dextrins .

Finally, the article by Yang H. Y. and Song J. F., published in Yao Xue Xue Bao, 2006 JuI.; 41(7): 671-4 (see abstract published in PubMed, PMID: 17007363), discloses a polarographic study on complexes of coenzyme QlO and β-cyclodextrin for determining the photosta-

bility thereof. Said article reports values of apparent photodegradation constant of 7.77 x 10 ^~3 min ^"1 for coenzyme QlO and 3.38 x 10 ^~3 min ^"1 for the complex coenzyme QlO/β-cyclodextrin. The Applicants have observed that preparations containing CoEQlO as active substance for single-dose administrations (capsules, tablets, single-dose bottles, etc.), with amounts of active substance not above 50- 100 mg pro dose, are available at present on the mar- ket . Such preparations are therefore unsuitable for the treatment of neurodegenerative diseases as described above, which require quite high doses (300 mg/die to 1,200 mg/die) . The Applicants have faced the problem of providing preparations based on coenzyme QlO at high concentrations, having a high water solubility and a high pho- tostability, so as to enable the administration of the required dose also in a single step, preferably in liquid form or as water-soluble granules. The Applicants have found that such problem can be solved by associating to a complex of coenzyme QlO and at least one cyclodextrin at least one carotene, which allows to significantly improve the photostability of the preparation, the latter keeping its activity basi- cally unchanged also after a long exposure to light.

The presence of carotene further enables to significantly improve the bioavailability of the active substance.

In a first aspect, the present invention therefore re- lates to a composition comprising:

(a) a complex of coenzyme QlO and at least one cyclo- dextrin; and

(b) at least one carotene.

In another aspect, the present invention relates to a composition as defined above for use as a medicament. In a further aspect, the present invention relates to the use of a composition as defined above in the preparation of a drug for the treatment of neurodegenerative diseases of the central nervous system, in particular of Parkinson's disease (PD).

In a further aspect, the present invention relates to the use of a composition as defined above as food supplement . Cyclodextrins are cyclic oligosaccharides containing at least 6 units of D- (+) -glucopyranose bound to one another with a α, 1-4 glucoside bond. The three natural cyclodextrins α, β, γ differ in ring size and solubility, α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin contain 6, 7 or 8 units of D- (+) -glucopyranose, re- spectively. Cyclodextrins have the appearance of fine

white, basically odourless powders, with a slight sweet taste.

According to the present invention, α-cyclodextrin, β- cyclodextrin or γ-cyclodextrin, or mixtures thereof, can be used, β-cyclodextrin is particularly preferred. Cyclodextrins have a stiff molecular structure of frusto-conical shape, with a central cavity whose size varies depending on the number of glucopyranose units present . Due to the arrangement of hydroxyl groups, the inside of the cavity is hydrophobic, whereas the outside is hydrophilic. This configuration allows cyclodextrins to house the host molecule of coenzyme QlO inside the cavity so as to form an inclusion complex ("host- guest" complex) .

The amount of coenzyme QlO in the composition according to the present invention is as a rule above 20% by weight, preferably of from 1% to 10% by weight, referred to the total weight of the composition. The composition according to the present invention comprises from 40% to 100% by weight, more preferably from 50% to 90% by weight, of at least one cyclodex- trin, the percentage being expressed referred to the weight of coenzyme QlO. The composition according to the present invention

further comprises at least one carotene. Carotene is preferably selected from: α-carotene, β- carotene, lycopene, or mixtures thereof. More preferably, carotene is β-carotene. Preferably, the composition according to the present invention comprises from 0.01% to 0.5% by weight of at least one carotene, more preferably from 0.05% to 0.3% by weight, the percentage being expressed referred to the total weight of the composition. As is known, carotenes are commonly marketed with a content of active substance of about 1-2%, the rest being made up of suitable additives and of carotene degradation products. Obviously, the above percentages refer to carotene that is actually present in the com- position.

The composition according to the present invention solves the problems of solubility and photostability of coenzyme QlO as disclosed above, and is advantageous both for therapeutic use and for its technologi- cal properties, particularly for the flowability properties of the product.

Flowability ensures a correct dose when portioning in containers, and further prevents post-granulation segregation phenomena. The composition in granular form has a controlled fri-

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ability, such as to reach an excellent compromise between mechanical resistance and solubility. The composition according to the present invention can comprise further pharmacologically acceptable excipi-

5 ents and/or adjuvants, such as carriers, dispersants, flavoring agents, sweeteners, stabilizers, preservatives, antioxidants, amino acids, vitamins, enzymes, and the like. In particular, the composition according to the present invention can include at least one com- io pound selected from: lactose, maltodextrins and lipoic acid.

The product is obtained by dividing the manufacturing process into two separate steps:

1) mixing coenzyme QlO with at least one cyclodex- I ₅ trin, so as to obtain the complex coenzyme

Q10/cyclodextrin ( ^λλ host-guest" complex) ;

2) mixing the complex coenzyme Q10/cyclodextrin with at least one carotene and, if necessary, excipients and/or adjuvants.

20 Then wetting, drying and granulation processes are carried out according to known techniques. The composition according to the present invention is used as inhibitor of neurodegenerative progress, in particular in Parkinson's disease.

25 It is believed that the active substance acts by

blocking the propagation of radical reaction, preventing the propagation thereof and therefore the lipoper- oxidation of cell membranes and thus cell death. Such action is enhanced by the presence of carotene which, beyond having such chemical and physical characteristics as to improve the bioavailability and pho- tostability of coenzyme QlO, performs an antioxidant action and promotes the inhibition of mechanisms producing free radicals. The end product can be portioned in suitable sealed containers so as to avoid any contamination from outside .

The product thus obtained has a duration not below 24 months in suitable conditions of temperature, humidity and package integrity.

Beyond the aforesaid use as inhibitor of neurodegenerative process, in particular in Parkinson's disease (PD) , the composition according to the present invention can be used in several other diseases, such as: (a) mitochondrial diseases in general, with doses of coenzyme QlO of 1 mg/die to 10,000 mg/die;

(b) mitochondrial encephalomyopathies, with doses of coenzyme QlO of 1 mg/die to 4,000 mg/die;

(c) progressive external ophthalmoplegia (PEO) , with doses of coenzyme QlO of 1 mg/die to 4,000 mg/die;

(d) neurological alterations related to an altered energy production and oxidative stresses in general, such as Friedrich' s ataxia, Alzheimer's disease, Hunt- ington' s disease, amyotrophic lateral sclerosis (ALS), with doses of coenzyme QlO of 1 mg/die to 4,000 mg/die;

(e) cardiovascular diseases in which the oxidation of low density lipoproteins (LDL) is deemed to be the essential element for the development of atherosclero- sis, with doses of coenzyme QlO of 1 mg/die to 4,000 mg/die (it is believed that coenzyme QlO, thanks to its key role in energy production, contributes to the safeguard of vascular endothelium), such as: heart diseases in general, congestive heart failure, fatigue syndrome, dyspnea, essential hypertension, angina pectoris, surgical ischemiae;

(f) immune deficits;

(g) diabetes;

(h) retinopathy of prematurity, stimulation of visual function in general;

(i) male infertility, oligospermia;

(j) periodontal disease;

(k) photoageing, derma rugosity.

The present invention shall now be further illustrated with some examples, which are provided by mere way of

example without limiting the scope of the invention. EXAMPLE 1

350 mg of β-cyclodextrin (0.31 mmol, commercial product distributed by A. C. E. F.) were dissolved in 8 ml of

5 distilled water at a temperature of 60 ⁰ C. The solution thus obtained was added with 500 mg of coenzyme QlO

(0.58 mmol, commercial product by Kyowa, fine granu- lometry: 95% of the powder gets through a 80 mesh sieve). After stirring for about 30 min. at 60 ⁰ C, the io complex began to precipitate. After about 4 hours the precipitated complex was filtered and dried in an oven at 75°C for 6-10 hours.

The complex thus obtained was added, together with the other excipients (lactose or maltodextrins) , with β-

I ₅ carotene (commercial product distributed by A. C. E. F. with a titer of 1% and fine granulometry: 95% gets through a 80 mesh sieve) in such an amount as to obtain 0.1% by weight of β-carotene referred to the total weight of the composition.

20 The actual complexation was checked by IR-FT analysis, which allowed to observe the characteristic bands of the complex coenzyme Q10/cyclodextrin.

Moreover, the complexation constant was determined according to formula:

2S ST = Sw + Kf * L _T

wherein:

S _τ = concentration of coenzyme QlO (free and bound) ; S _w = intrinsic solubility; K _f = complexation constant; L _τ = concentration of binder.

An excellent linear correlation was found between S _τ and L _τ with a K _f value of 1.26 * 10 ⁴ .

The apparent photodegradation constant (K _fa ) of coenzyme QlO was measured with the polarographic method mentioned by Yang H. Y. et al. in the above article. Measuring took place on coenzyme QlO as such, on the complex coenzyme QlO/β-cyclodextrin prepared as described above, and on the complex coenzyme QlO/β- cyclodextrin added with β-carotene (0.05% by weight and 0.1% by weight). The results are listed in the following Table 1:

TABLE 1

The remarkable decrease of the apparent photodegrada- tion constant caused by the addition of β-carotene demonstrates an improved photostability of the active substance .