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1. (WO2019064245) A COMPOSITION FOR USE IN THE PREVENTION AND/OR TREATMENT OF GLAUCOMA
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A composition for use in the prevention and/or treatment of glaucoma

The present invention relates to a composition of substances preferably obtained from natural sources, which is effective in the prevention and/or treatment of glaucoma.

Glaucoma is a form of optic neuropathy characterised by progressive degeneration of the retinal ganglion cells, which are cells of the central nervous system whose cell body is located within the retina, and the axons in the optic nerve. Degeneration of these neurons is associated with a progressive loss of vision and a characteristic morphology of the optical disc referred to as "cupping".

The biological bases of glaucoma are poorly understood and the factors contributing to its progression have not yet been characterised.

Glaucoma affects more than 70 million people worldwide, 10% of whom lose their vision because of this disease.

Glaucoma has no symptoms in the early and intermediate stages, so it is very likely that the number of people suffering from glaucoma is much higher than that of people diagnosed with this disease. In fact, several surveys have shown that less than 50% of people diagnosed with glaucoma were aware of being affected by this disease.

Glaucoma can be classified into 2 broad categories: open-angle glaucoma and closed-angle glaucoma. In the United States, more than 80% of glaucoma cases are open-angle cases, but closed-angle glaucoma is the main cause of loss of vision in patients.

These diseases may be primary, i.e. without a well-defined cause, or secondary, resulting from trauma, glucocorticoids, pigment dispersion or pseudoexfoliation syndrome.

Although, as indicated, the mechanism of pathogenesis of glaucoma is not fully understood, an increase in the intraocular pressure is known to be associated with the death of retinal ganglion cells. Intraocular pressure is determined by the balance between

aqueous humour secretion by ciliary bodies and its trabecular and uveoscleral outflow. Patients with open-angle glaucoma show a reduction in the outflow of aqueous humour due to partial obstruction of the trabecular and uveoscleral ducts, generally caused by the iris. Intraocular pressure can cause stress and mechanical strain on the rear structures of the eye. particularly the lamina cribrosa and adjacent tissues. Stress and mechanical strain -induced by increased intraocular pressure - can lead to compression, deformation and remodelling of the lamina cribrosa, with consequent impairment of axonal transport of trophic factors essential for retinal ganglion cells.

The increase in intraocular pressure is not the only risk factor, since individuals with high intraocular pressure may not develop the disease, while in some cases the hypotensive therapy alone proves ineffective in slowing down or stopping the progression of the disease. The death of retinal ganglion cells has been shown to be able to induce the death of surrounding neurons, leading to secondary and trans-synaptic neuronal degeneration, which could be of great importance in the progression of the disease. Alterations of the microcirculation and the immune system, excitotoxicity and oxidative stress may contribute to the development of optic neuropathy, both in the presence and in the absence of high intraocular pressure values.

The main objectives of glaucoma therapy are to slow down the progression of the disease and maintain the quality of life of the patients.

One of the few methods currently known and effective in the treatment of glaucoma is the reduction of intraocular pressure. Numerous multicentre studies have shown the benefit resulting from the reduction of intraocular pressure in the prevention of the onset and in the slowing down of the progression of this disease.

Currently, different classes of therapeutic agents are used for the reduction of intraocular pressure:

· Prostaglandin analogs (Latanoprost, travoprost, tafluprost, unoprostone, bimatoprost). These are therapeutic agents that increase the uveoscleral outflow of aqueous humour. These drugs are administered once a day, at night, and this restricts the action of pressure reduction to the night period only. They have many side effects, both local and systemic, including conjunctival hyperemia, thickening of the eyelashes, iris colouring, uveitis, macular edema and headache.

β-adrenergic antagonists (Timolol, levobunolol, carteolol, metipranolol, betaxolol). The mechanism by which these drugs act consists in the reduction of the production of aqueous humour. They are administered once a day. in the morning, and have serious systemic side effects due to the antagonistic action on β-adrenergic receptors. This restricts the possibilities for use in patients with asthma, chronic obstructive pulmonary disease and bradycardia.

· a-adrenergic agonists (brimonidine, apraclonidine). These drugs lead to initial reduction in the production of aqueous humour and increase in the outflow of the latter. In this case too, numerous side effects, both local and systemic, occur, such as irritation and eye dryness, allergic reactions, effects on the central nervous system, respiratory arrest, postural hypotension, brain or coronary failure, liver and kidney damage. They must also be administered 3 times a day and this reduces patient compliance.

• Carbonic anhydrase inhibitors (Dorzolamide, brinzolamide, acetazolamide). These drugs reduce the production of aqueous humour. Side effects include eye irritation, eye burning, paraesthesia, nausea, diarrhea, loss of appetite.

• Cholinergic agonists (pilocarpine, carbachol). These drugs increase the outflow of aqueous humour. They are to be administered more than 4 times a day, with considerable reduction in patient compliance and consequent reduced effectiveness due to poor adherence to the therapeutic scheme. Side effects also occur in this case, including eye irritation, myopia, ciliary spasm, with consequent headache and loss of vision.

Therefore, there is a need to provide alternative treatments to the existing ones, which are effective in the prevention and/or treatment of glaucoma, but do not have the side effects and/or disadvantages of the state-of-the art treatments.

These and other needs are met by the present invention, which provides a composition characterised in that it comprises a synergistic combination of active ingredients obtained from natural sources, the aforesaid combination having proved particularly effective against glaucoma.

The composition of the invention is as defined in appended claim 1. Further features and advantages of the invention are defined in the dependent claims. The claims form an integral part of the present description.

A detailed description of some preferred embodiments of the invention is provided hereinafter.

The synergistic composition of the present invention is a supplement, useful for the treatment and prevention of glaucoma. In the composition of the present invention, the synergistic action occurs between a Crocus sativus extract, forskolin, and the at least one further active ingredient selected from citicoline, rutin, and combinations thereof.

Saffron is produced from dried pistils of the Crocus sativus plant, a plant belonging to the Iridaceae family. This plant is widely cultivated in Iran, Spain, France, Italy, Greece, Turkey, India, and China. Saffron contains high concentrations of the carotenoids crocine and crocetine. These chemical compounds, thanks to the abundance of double bonds, exhibit strong activity as radical scavengers. Safranal is one of the major constituents of the saffron extract and is one of the main substances responsible for its pharmacological action.

Saffron has been widely used for many years in traditional Asian medicine, for the prevention and treatment of many diseases. Clinical and preclinical studies have demonstrated the pharmacological activity of Crocus sativus: this plant is known for its anti-tumour, radical scavenger, hypolipidemic and anticonvulsant activities, and has also been widely used in traditional medicine for the treatment of depressive disorders. Other studies have shown that safranal, one of the main chemical compounds of the extract, possesses anti-platelet aggregation, antioxidant, anti-tumour and anti-inflammatory activities.

A small clinical study assessed the effect of the administration of a Crocus sativus extract on intraocular pressure (IntraOcular Pressure, IOP) of patients with open-angle glaucoma. Patients in the study were randomly assigned to 2 groups. The first was given 30 mg/day of

Crocus sativus extract, the second was given a placebo for a period of one month. After 3 weeks of treatment, the group treated with saffron showed a statistically significant reduction of the intraocular pressure, compared to the pressure at the beginning of the study, equal to -2.6 ± 2.00 mmHg (p = 0.013 ). After 4 weeks of treatment, an even greater change in intraocular pressure was found (95% CI for the mean difference: -3.25 ± 1.85 mmHg, p = 0.001 ).

Another important mechanism of action of crocine and other carotenoids contained in the Crocus sativus extract is their activity as antioxidants and radical scavengers. Since one of the mechanisms of pathogenesis of glaucoma-induced optic neuropathy involves oxidative stress, the use of these compounds may further contribute to the prevention of damage induced by high intraocular pressure. Safranal has also the important ability of stabilizing biological membranes, reacting with free radicals and decreasing membrane lipid peroxidation. These pharmacological actions should further contribute to the prevention of optic neuropathy.

In a preferred embodiment, the dry extract of Crocus sativus is an extract from stigmas.

Forskolin is a diterpenic compound extracted from the Coleus forskohlii plant, also known with the botanical name of Plectranthus barbatus.

Coleus forskohlii is a perennial plant belonging to the Labiatae family. It is a plant native to India and is quoted in "Ayurvedic Materia Medica" with the Sanskrit names of "Makandi" and "Mayani".

The main species of the Coleus genus in India are C. forskohlii, C. amboinicus, C. blu ei, C. malabaricus, and C. scutellaroides. These species are widely used for the treatment of various diseases, including dysentery and digestive disorders. Coleus forskohlii is used in traditional medicine in several countries of the world. In Egypt and other African countries, the leaf of this plant is used as an expectorant, emmenagogue and diuretic. In Brazil, it is used for the treatment of gastric and intestinal disorders. In Ayurvedic medicine, it has been traditionally used for treating heart disease, abdominal colic,

respiratory and intestinal disorders, constipation, epilepsy, and angina pectoris.

The extract from the tuberous roots of Cole s forskohlii contains minor diterpenoids, such as deacetylforskolin, 9-deoxyforskolin, 1 ,9-dideoxyforskolin, l ,9-dideoxy-7-deacetylforskolin, as well as the forskolin ( 7P-acetoxy-8, 13-epoxy-la,6p,9a-triidroxylabd-14-en-l 1 -one).

Forskolin is the main chemical compound of Coleus forskohlii and its pharmacological action is mediated by different mechanisms. The main mechanism of action consists in the stimulation of adenylate cyclase and the consequent increase in intracellular concentrations of cyclic AMP. The unique feature of this activation is that the site of action of forskolin is the catalytic subunit of the enzyme. All adenylate cyclase isofonns expressed in humans are activated by forskolin, with the exception of isoform IX, which is specifically expressed by spermatozoa. The activation of adenylate cyclase is considered to be the main mechanism through which forskolin induces relaxation of the smooth musculature. This action is potentially useful for the treatment of many diseases, including heart failure, hypertension, glaucoma, thrombosis, asthma, and metastatic conditions.

The effect of forskolin on the aqueous humour dynamics, and consequently on intraocular pressure, has been demonstrated for the first time in 1983 by Capriole and Sears. Topical application of forskolin was able to lower the intraocular pressure in rabbits, monkeys, and healthy human volunteers. The effect resulted from a reduction in the inflow of aqueous humour rather than an increase in its outflow.

Another study involving topical administration of eye drops with a 1% concentration of forskolin demonstrated its efficacy in reducing intraocular pressure. This makes forskolin a valid alternative to β-adrenergic antagonists for the prevention and treatment of glaucoma.

Within the scope of the present invention, roots of a plant of the genus Coleus can be used as a natural source of forskolin.

Citicoline is the International non-proprietary name for cytidine 5'-diphosphocholine. This is a compound marketed in several countries, both as a drug and as a dietary supplement.

The results of a recent meta-analysis have shown that citicoline is moderately effective in cognitive and behavioural disorders associated with brain disorders in the elderly. Experimental evidence also demonstrates the effectiveness of this molecule in the treatment of damage caused by cerebral ischemia and head trauma, in Parkinson's disease and Alzheimer's disease.

It is interesting to note the low toxicity of citicoline, which has an LD50 of 4.6 g/kg in mice and 4.15 g/kg in rats when administered intravenously, and 8 g/kg in both species when administered orally. In a phase IV clinical trial, about 3000 elderly patients with neurological disorders were treated with citicoline at a dose of 600 mg/day over a period of 15-60 days: the incidence of side effects was 5.01 % and none of them was serious.

Results obtained from investigations carried out with citicoline and isotopic markers, 3H-CDP-methyl- l C-Cho, demonstrated that citicoline, after oral or intravenous administration, undergoes rapid hydrolysis into cytidine 5'-monophosphate and phosphocholine. The latter are further dephosphorylated by phosphatases, so that they can cross the blood-brain barrier.

Citicoline increases the formation of phosphatidylcholine and other phospholipids in the brain, as revealed by in vitro and in vivo studies, and at the same time prevents ischemia-induced accumulation of free fatty acids. These two effects should be anti-apoptotic and neuroprotective. The neuroprotective effect was observed in several experimental models of cerebral ischemia, stroke, hypoglycaemic hypoxia and neuronal apoptosis induced by deposition of β-amyloid precursor protein, and hypoperfusion. The neuroprotection that follows the administration of citicoline could be associated with stimulation of phosphatidylcholine synthesis or stimulation of S-adenosylmethionine synthesis, which stabilizes the membrane and prevents the release of arachidonic acid.

A further mechanism that contributes to the neuroprotective action of citicoline is the prevention of excitotoxicity. In fact, citicoline administration has shown to be able to

prevent kainic acid-induced excitotoxicity. The mechanism is based on the prevention of nitrosative stress and inhibition of activation of kainic acid-induced ERK kinase.

In addition to neuroprotective properties, citicoline is known to increase the synthesis of acetylcholine, dopamine noradrenaline and serotonin in several brain areas. The cholinergic effect of citicoline is due to the fact that choline is the precursor of acetylcholine. The mechanism leading to increased catecholamine synthesis is less known.

Several studies confinned the effectiveness in the use of citicoline for the treatment of glaucoma by demonstrating decreased apoptosis of retinal ganglion cells following citicoline administration, both in rats and in cell cultures.

The first study evaluating the effectiveness of citicoline intramuscular administration for the treatment of glaucoma was performed in 1989. The follow-up of this study showed an excellent outcome of this therapy over a period of ten years. Ten years after this first study, a randomized, placebo-controlled clinical study based on electrophysiological methods (PERG and VEP) for assessing retinal and supraretinal function, showed a statistically significant improvement of these parameters following intramuscular administration of citicoline.

Since administration by injection is not ideal for long-term therapy, clinical studies have been conducted that included oral administration of citicoline. In an Italian investigation performed in 2008, the effectiveness of intramuscular (1 g/day) and oral (1.6 g/day) administration of citicoline was assessed. No statistically significant differences were reported in the efficacy of the two treatments. Another study carried out in 2013, performed on patients diagnosed with glaucomatous optic neuropathy despite having normal intraocular pressure values, demonstrated the efficacy of a long-term treatment with citicoline. The patients enrolled in the study were treated with 500 mg of citicoline, administered orally, for a period of 4 months. The study showed a significant reduction in the progression rate of glaucoma, from -1.1 dB/year to -0.15 dB/year.

Cotinus coggygria Scop., also known with the botanical name of Rhus cotinus, commonly

known as "sumach" or "smoke-tree", is a shrub belonging to the Anacardiaceae family. It is widely distributed in southern Europe, Caucasus, and China. The plant has been used for centuries in traditional medicine, both for its high content of essential oils and for a number of important pharmacological properties.

The main pharmacological activities include the antioxidant, anti-tumour, antigenotoxic, antimicrobial, antiviral, anti-inflammatory and hepatoprotective activities.

Several carbonic anhydrase inhibitors approved for glaucoma exist, including metazolamide, dorzolamide, and brinzolamide. But there are also compounds of plant origin that can have an inhibitory effect on this enzyme, sometimes even in a selective manner for the hCA-II isoform, which is the one mainly involved in the increase in intraocular pressure.

Sahin et al. calculated the IC50 of carbonic anhydrase inhibition by several isolated secondary metabolites, as well as plant extracts. This study identified several particularly active compounds. However, the inhibitory activity against the enzyme is not sufficient for these compounds to be effective in the treatment of glaucoma. The logP value is essential for the action of these inhibitors on the carbonic anhydrase of the eye: the ideal logP is around 0, since the inhibitors must have good water solubility in order to be able to interact with the enzyme, but of course a sufficient lipophilicity to be able to cross the blood-brain barrier and reach the eye.

Rutin is one of the flavonoids most active towards the two carbonic anhydrase isoforms (0.72 and 0.55 μg/mL, respectively) and has a LogP of - 1 that should be close to the ideal value for optimal distribution within the eye. This makes rutin a potential active ingredient for the reduction of intraocular pressure and the consequent glaucomatous optic neuropathy.

Within the scope of the present invention, the bark of a plant of the genus Cotinus can be used as a natural source of rutin.

The present composition is effective in the treatment and/or prevention of glaucoma. The effectiveness of the composition derives from the activities of the following components:

Forskolin, thanks to its ability to increase the concentration of cAMP, is capable of reducing the secretion of aqueous humour, with consequent reduction of intraocular pressure and prevention of damage to retinal ganglion cells.

The Crocus sativus extract, thanks to its antioxidant and radical scavenger activities, helps to prevent oxidative stress damage generated by the increase in intraocular pressure.

Citicoline, thanks to its neuroprotective and anti-apoptotic action, prevents glaucoma-induced degeneration of retinal ganglion cells.

- Rutin, thanks to its ability to prevent oxidative stress, slows down the degeneration process leading to glaucomatous optic neuropathy. In addition, thanks to its inhibitory action against carbonic anhydrase, it prevents the secretion of aqueous humour and the consequent increase in intraocular pressure.

As previously stated, in the present invention the synergistic action takes place between the Crocus sativus extract and forskolin, with the further contribution of citicoline and/or rutin, if present.

In a preferred embodiment, the Crocus sativus extract is administered in an amount comprised within the range of from 0.1 mg to 300 mg, for example by administering a dosage form wherein the Crocus sativus extract is present at a concentration of between 0.1% and 40% w/w, more preferably in an amount of between 0.5% and 30% by weight based on the total weight of the composition. Further preferred concentrations of the Crocus sativus extract are from 1% to 25%, from 2% to 20%, from 3%> to 10% (w/w based on the total weight of the composition). Forskolin, preferentially extracted from a plant of the genus Coleus, is administered in an amount comprised within the range of from 1 mg to 500 mg, for example by administering a dosage form wherein forskolin is present at a concentration of between 1% and 70% w/w, more preferably in an amount of between 5% and 50% by weight based on the total weight of the composition. Further preferred concentrations of forskolin are from 6% to 40%, from 7% to 30%, from 8% to 20%, from 9% to 15% or 10% (w/w based on the total weight of the composition). Citicoline, if present, is administered in an amount comprised within the range of from 10 mg to 5000

mg, for example by administering a dosage form wherein citicoline is present at a concentration of between 1 % and 90% w/w, more preferably in an amount of between 5% and 80% by weight based on the total weight of the composition. Further preferred concentrations of citicoline, if present, are from 4% to 70%, from 5% to 60%, from 6% to 50%, from 7% to 40%, from 8% to 30%, from 9% to 20%, from 10% to 15% (w/w based on the total weight of the composition). Rutin, if present, preferentially extracted from a plant of the genus Cotinns, is administered in an amount comprised within the range of from 10 mg to 2000 mg, for example by administering a dosage form wherein rutin is present at a concentration of between 5% and 80% w/w, more preferably in an amount of between 10% and 60% by weight based on the total weight of the composition. Further preferred concentrations of rutin, if present, are from 15% to 50%, from 20% to 40%, from 25% to 30% (w/w based on the total weight of the composition). Any combination of the above-mentioned concentration values of each principle falls within the scope of the present description.

The dosage form can be a composition of substances including the above-mentioned active ingredients mixed together. Such a composition of substances, for example, can be formulated as a dietary supplement, a nutraceutical composition, a dietetic composition, a nutritional composition, a food product, a beverage, a food for special medical purposes, a medicated food, a pharmaceutical composition, a medical device or a cosmetic composition.

Oral dosage forms are particularly prefeired within the scope of the present invention. Other preferred dosage forms within the scope of the present invention are topical forms, for example eye drops.

The following examples are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.

EXAMPLES

Below are some examples of oral formulations, with the quantities of the relevant active

ingredients occurring in each dosage unit.

EXAMPLE 1 : 250 mg tablet

Active ingredient Daily Dose

Coleus forskohlii, root d.e. 150 mg

Crocus Sativus, stigmas d.e. 30 mg

EXAMPLE 2: 520 me tablet

Active ingredient Daily Dose

Coleus forskohlii, root d.e. 300 mg

Crocus Sativus, stigmas d.e. 100 mg

EXAMPLE 3 : 150 me capsule

Active ingredient Daily Dose

Crocus Sativus, stigmas d.e. 30 mg

Forskolin 15 mg

EXAMPLE 4: 900 me capsule

Active ingredient Daily Dose

Citicoline 500 mg

Coleus forskohlii, root d.e. 150 mg

Crocus Sativus, stigmas d.e. 30 mg

EXAMPLE 5: 1400 me sachet

Active ingredient Daily Dose

Citicoline 500 mg

Coleus forskohlii, root d.e. 300 mg

Crocus Sativus, stigmas d.e. l OO mg

EXAMPLE 6: 2000 me sachet

Active ingredient Daily Dose

Citicoline 1000 mg

Coleus forskohlii, root d.e. 300 mg

Crocus Sativus, stigmas d.e. 100 mg

EXAMPLE 7: 3200 me sachet

Active ingredient Daily Dose

Citicoline 2000 mg

Coleus forskohlii, root d.e. 300 mg

Crocus Sativus, stigmas d.e. 100 mg

EXAMPLE 8: 3700 ma sachet

Active ingredient Daily Dose

Cotinus coggygria, bark d.e. 2000 mg

Citicoline 500 mg

Coleus forskohlii, root d.e. 150 mg

Crocus Sativus, stigmas d.e. 30 mg

EXAMPLE 9: 1200 me tablet

Active ingredient Daily Dose

Citicoline 500 mg

Rutin 200 mg

Coleus forskohlii, root d.e. 150 mg

Crocus Sativus, stigmas d.e. 30 mg

EXAMPLE 10: 2300 me sachet

Active ingredient Daily Dose

Cotinus coggygria, bark d.e. 1000 mg

Citicoline 500 mg

Coleus forskohlii, root d.e. 150 mg

Crocus Sativus, stigmas d.e. 30 mg

Experimental Section

To evaluate in vitro the efficacy of the composition object of the present invention it is necessary to establish the cell culture model to be used. Retinal ganglion cells (RGC) or astrocytes, Miiller cells or microglial cells can be used.

The action of the combination of active ingredients against glaucoma can be observed by monitoring various representative parameters of the disease: cell viability, antioxidant action, neuroprotection.

An in vitro model suggests to test the present composition on RGC cells to evaluate the action against glaucoma by increasing the hydrostatic pressure of the cells. Cell cultures are subjected to an increase in hydrostatic pressure (EHP) in a range of 30 to 100 mmHg for certain time intervals. Cell pressure increase causes various effects such as cell apoptosis, morphological changes, cell migration, synthesis of elastin and other parameters. After treating the cells with the single substances to be tested and their combination, the effectiveness is assessed, for example, by monitoring cell viability or other parameters (cell morphology, cell migration, enzyme synthesis, etc.).

Damage to reference cells can also be induced by ischemia, hypoxia, chemical hypoxia, hypoglycemia, and the like.

On the other hand, in order to demonstrate the antioxidant effectiveness of the composition of the present invention, of particular interest for the prevention and/or treatment of glaucoma, in vitro assays are suitable such as, for example, the hydrogen peroxide assay used to induce oxidative stress in cells and the DPPH assay. In this assay, the antioxidant activity of the subject composition is proportional to the reduction of the 517-nm peak of the DPPH radical (2,2-diphenyl- 1 -picrylhydrazyl).

An in vivo model, instead, suggests to test the present composition for its neuroprotective efficacy. Oral administration of the individual components and their combination is carried out in mice daily for a certain number of days, while intravitreal injection of NMDA (N-methyl-D-aspartate) or similar compounds, is performed on the seventh day of this therapy. The purpose of this model is to demonstrate that administration of the present composition prevents the cellular damage induced in mice by the intravitreal injection of NMDA, to a much extent than the single components.