The present invention is directed to thymoquinone or thymoquinone-comprising compositions for use in the treatment of neurodegenerative diseases such as Parkinson, Huntington and Alzheimer ^' s disease.

About 5 percent of all people over the age of 65 suffer from dementia, and Alzheimer ^' s disease (AD) is the most frequent form. In Europe 8.5 million people are affected by AD and the costs are estimated at 141 billion Euros per year. AD is characterized by an aberrant amyloidogenic pathway, i.e. aberrant fragmentation of amyloid precursor proteins (APP) and subsequent APP aggregation and deposition, hyperphosphorylation, aggregation of tau proteins as well as neurofibrillary degeneration (for a review on AD see Ewers et al., Exp. Gerontol., 45(1 ):75 2010). These processes are accompanied by inflammation in the surrounding microglia. In addition, there is a disturbance of the central nervous blood circulation. It is not known whether inflammation and the changes in blood circulation are the cause or rather the consequence of the neurodegenerative process.

At present, there is no causative therapy for AD. Therapeutic intervention primarily focuses on the treatment of etiology, on the pathogenetic mechanism and on the modification of AD symptoms. However, the medicaments clinically evaluated so far have not provided any sustainable effects.

Plants have been investigated for controlling the severity and progression of AD. Siderits scardica, melissa, rosemary, sage and ginkgo are presently being investigated experimentally in vitro or in vivo and in some cases even clinically.

Thymoquinone (TQ) is the main active constituent of the oil of Nigella sativa (NS) (also called black seed, cumin, black onion seed, nutmeg flower, black caraway, Roman coriander and fennel flower). Nigella sativa oil contains an abundance of conjugated linoleic acid, thymoquinone, nigellone (dithymoquinone), melanthin, nigilline, damasce- nine and tannins. Melanthin is toxic in large doses and nigelline is paralytic. TQ is an antioxidant that supposedly protects from heart, liver and kidney damage, it has analgesic, anti-convulsant, anti-angiogenic and anti-epileptic effects.

More specifically, NS oil and isolated TQ have demonstrated effects on blood circulation. For example, TQ induces relaxation in isolated rat pulmonary arteries that had been pre-contracted by phenylephrine, probably in part by activation of ATP- sensitive potassium channels and possibly by non-competitive blocking of serotonin, alphal and endothelin receptors (G.M. Suddek, J. Ethnopharmacol. 127(2):210-4, 2010); TQ reduces lipid peroxidation upon intraperitoneal injection immediately after global ischemia-reperfusion injury in the rat hippocampus in a rat reperfusion model (Hossei- nzadeh et al., Phytomedicine 14:621 -627, 2007); and TQ improves aging-related endothelial dysfunction in the rat mesentery artery, at least in part, through inhibition of oxidative stress and normalization of the angiotensin system (Idris-Khodja and Schini- Kerth, Naunyn Schmiedebergs Arch. Pharmacol, 385(7):749-58, 2012).

Because of its anti-oxidant and anti-inflammatory activity TQ has also shown a promising neuroprotective effect on the spatial cognitive functions of rats suffering from global cerebrovascular hypofusion (Azzubaidi et al., Acta Neurobiol. Exp., 72:154-1665, 2012).

TQ has also been associated with AD. TQ protected cultured rat primary neurons against amyloid β-induced neurotoxicity, probably because of its antioxidant and antiinflammatory effects and was suggested as a possible promising candidate for AD treatment (Alhebshi and Gotoh, Biochem. Biophys. Res. Comm., 433(4):362-7, 2013). In forty elderly but healthy human volunteers TQ improved memory, attention and cognition. However, the question whether Nigella sativa seed could be considered as a potential food supplement for preventing or slowing progression of AD was considered to need further investigations, and a study with AD patients with a large population size for a longer period of time was recommended before using Nigella sativa daily, and extensive phytochemical investigations were recommended for novel drug discovery use of Nigella sativa for treating cognitive disorders (Shahdaat et al., Journal of Ethnopharmacology, 148:780-786, 2013). In summary, whether or not the anti-oxidant and anti-inflammatory effects of NS or TQ would have positive effects on the onset or progression of neurodegenerative diseases such as AD is an interesting area to explore, the outcome of which is unpredictable and an open question, the answer of which requires clinical investigation.

The alkaloid indirubin is the main active agent in Indigo naturalis, a dark blue powder prepared from the leaves of dye plants including Baphicacanthus cusia (Acanthaceae), Polygonum tinctorium (Polygonaceae), Isatis indigotica (Brassicaceae) and Indigofera suffrutticosa (Fabacea) and Isatis tinctoria. The blue powder of Indigo naturalis is typically produced by crushing, fermenting and adding calcium to the plant. The powder of Indigo naturalis demonstrated efficacy in a clinical study with 42 psoriasis patients, which were treated for twelve weeks with an ointment containing indigo, resulting in symptom improvements in 81 % of the patients (Reuter et al., JDDG, 10, 2010, vol. 8, p. 788-793). Indirubin-comprising extracts from Isatis tinctoria have proven effective for treating allergen-induced airway inflammation (Brattstrom et al., Phytomedicine, 17:551 - 556, 2010). Indirubin has anti-tumoral effects that appear to correlate with its antimitotic effects and it is a potent inhibitor of cyclin-dependent cyclases (CDKs). Indirubin inhibits glycogen synthase kinase^ and CDK5/P25, two proteins involved in abnormal tau phosphorylation in AD and for that reason has been suggested for further investigation in the study and treatment of neurodegenerative disorders (Leclerc et al., The Journal of Biological Chemistry, 276(1 ), 251 -260, 2001 ). lndirubin-3 ^' -oxime was shown to inhibit inflammatory activation of rat brain microglia by reducing the production of various neurotoxic molecules in activated microglia (Jung et al., Neuroscience Letters, 487:139- 143, 201 1 ).

It is the objective of the present invention to provide new compounds and compositions for the prophylactic and therapeutic treatment of neurodegenerative diseases. In particular, it is an objective of the invention to provide new compounds and compositions for improving blood circulation, for inhibiting local inflammation and/or for inhibiting aberrant protein aggregation and deposition in neurodegenerative diseases.

The objective is solved by the provision of thymoquinone or thymoquinone- comprising compositions for use in the treatment of neurodegenerative diseases.

It was surprisingly found that an extract from the seeds of Nigella sativa (NS) or the active constituent thymoquinone (TQ) can be prepared for use in the treatment of neurodegenerative diseases.

The term neurodegenerative diseases, as used herein, is defined as the umbrella term for the progressive loss of structure and/or function of neurons. Neurodegenerative diseases are incurable and result in progressive degeneration and/or death of neurons. This causes problems with mental functioning (called dementia). The neurodegenerative diseases for treatment according to the invention are preferably selected from the group consisting of Parkinson, Huntington and Alzheimer ^' s disease.

The term thymoquinone as used herein is meant to refer to the chemical substance 2-isopropyl-5-methylbenzo-1 ,4-quinone and encompasses all chemical and metabolic derivatives thereof that essentially retain the physiological activity of basic thymoquinone, e.g. nigellone (dithymoquinone). Thymoquinone is a chemically rather simple methyl- benzo-1 ,4-quinone derivative and can be produced synthetically or it can be isolated from or enriched in the volatile oil or seed extract of Nigella sativa. It is preferred that the thymoquinone-comprising composition for use in the present invention comprises extract or oil, preferably volatile oil from Nigella sativa seed.

For example and in a preferred embodiment, a Nigella sativa seed extract can be produced as hydrodestillation, steam distillation solvent extraction, supercritical fluid extraction, microwave assisted extraction or solid phase microextraction (Liu et al., Mini- Reviews in Medicinal Chemistry, 1 1 :947-955, 201 1 ). Preferably, the extract for use in the present invention is a cold-pressurized volatile oil.

In a preferred embodiment the extract solvent for producing the extract of the present invention is supercritical carbon dioxide, preferably together with at least one organic, more preferably hydrophilic, most preferably alcoholic co-solvent.

Nigella sativa oil comprises a number of constituents that are susceptible to oxidation, e.g. unsaturated fatty acids such as linoleic acid (Edris AE, J. Dietary Suppl., 8:34- 42, 2011 ).

In a preferred embodiment the thymoquinone-comprising composition for use in the invention additionally comprises an antioxidant, preferably selected from the group consisting of balm (melissa), rosemary and sage, preferably rosmarinus officinalis and salvia officinalis (sage) extract, which are high in rosmarinic acid content.

In a further preferred embodiment the thymoquinone-comprising composition for use in the present invention additionally comprises indirubin, preferably in the form of an isatis tinctoria extract.

The term indirubin, as used herein, is meant to refer to the chemical substance as such, i.e. 3-(1 ,3-dihydro-3-oxo-2H-indol-2-yliden)-1 ,3-dihydro-2H-indol-2-on, and encompasses all chemical and metabolic derivatives thereof that essentially retain the physiological activity of basic indirubin, e.g. indirubin-3 ^' -oxime. Indirubin is a chemically rather simple indol derivative and can be produced synthetically or it can be isolated from or enriched in an extract from various plants (see above). It is preferred that the thymoquinone-comprising composition for use in the present invention comprises indirubin from an Isatis tinctoria exctract.

For example and in a preferred embodiment, an indirubin-containing extract can be produced as a lipophylic extract (Recio et al., Planta Med, 72:539-546, 2006; Mohn et al., Planta Med, 73:151 -156, 2007).

In a preferred embodiment the extract solvent for producing the indirubin extract of the present invention is supercritical carbon dioxide, preferably together with at least one organic, more preferably hydrophilic, most preferably alcoholic co-solvent.

It is preferred that both thymoquinone and indirubin, either in the form or (a) chemically pure substance(s) or (a) volatile oil(s) or (an) extract(s), are present in the composition in physiologically effective amounts each. Preferably, the ratio of thymoquinone and indirubin (TQ:IR) in the composition for use in the present invention is at least about 1 :1 - 10:1 , preferably 4:1 - 6:1 , most preferably 5:3 - 7:3 or about 6:4. Preferred TQ:IR ratios are 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 (TQ:IR).

The thymoquinone, the thymoquinone-comprising composition and preferably the thymoquinone and indirubin-comprising compositions for use in the present invention have utility for the prophylactic and/or therapeutic treatment of neurodegenerative diseases. The term treatment and prophylaxis of neurodegenerative diseases relates to the treatment of an existing neurodegenerative disease for alleviating, reducing or getting rid of the disease or at least one or more of its symptoms as well as the treatment of future neurodegenerative diseases for preventing their occurrence, recurrence or the occurrence or recurrence of symptoms thereof.

Therefore, in a further aspect, the present invention relates to the use of thymoquinone, a thymoquinone-comprising composition and preferably a thymoquinone and indirubin-comprising composition as described above for the preparation of a medicament for the prophylactic and/or therapeutic treatment of neurodegenerative diseases, preferably selected from the group consisting of Parkinson, Huntington and Alzheimer ^' s disease.

A further aspect of the present invention relates to a pharmaceutical composition comprising thymoquinone, preferably thymoquinone together with an antioxidant, preferably an antioxidant selected from the group consisting of balm, rosemary and sage, preferably balm extract, rosmarinus officinalis and salvia officinalis (sage) extract, which are high in rosmarinic acid content. More preferably, thymoquinone and indirubin, most preferably, wherein at least one, preferably all of thymoquinone, indirubin and/or the antioxidants) are present as a plant extract.

In a further aspect, the present invention pertains to a method for the prophylactic and therapeutic treatment of neurodegenerative diseases, preferably selected from the group consisting of Parkinson, Huntington and Alzheimer ^' s disease, comprising the step of administering a composition of the invention in a physiologically effective amount to a patient in need thereof.

For therapeutic or prophylactic use the compositions for use in the invention may be administered enteral, parenteral, local, e.g. topical including a transdermal patch, in any conventional dosage form in any conventional manner. The preferred mode of administration is preferably in a solid or liquid dosage form.

The compositions may be administered alone or in combination with adjuvants that enhance stability of the active components, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, support bioavailability, provide adjunct therapy, and the like, including other active ingredients. Advantageously such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

As mentioned above, dosage forms of the compounds and extracts described herein include pharmaceutically acceptable carriers and adjuvants known to those of ordinary skill in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, buffer substances, water, salts or electrolytes and cellulose-based substances. Also, maltodextrin can be used to produce a free-flowing dry extract which is particularly suitable for further processing. Preferred dosage forms include solid, liquid, solution, suspension, emulsion, reconstitutable powder and transdermal patch. Controlled release dosage forms with or without immediate release portions are also envisaged. Methods for preparing such dosage forms are known (see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 ^th ed., Lea and Febiger (1990)). Dosage levels and requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. Although one dose per day may be sufficient, up to 5 doses per day may be administered. As the person skilled in the art will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific doses and treatment regimens will depend on factors such as the patient's general health profile, the severity and course of the patient's disorder or disposition thereto, and the judgment of the treating physician which includes interaction potentials with other needed medication.

For example, the extracts of the present invention can be administered the same way as other chemical drugs or plant extracts and pharmaceutical compositions thereof.