IT202300009258A1 - New association of active ingredients, pharmaceutical and/or nutraceutical compositions containing it and their use in the prevention and repair of damage to the central and peripheral nervous system - Google Patents

Description

Description of the Industrial Invention entitled:

?New association of active ingredients, pharmaceutical and/or nutraceutical compositions containing it and their use in the prevention and repair of damage to the central and peripheral nervous system?

The present invention relates to a new combination of active ingredients, useful in the prevention and repair of neuronal damage, both at central and peripheral level. The invention also refers to pharmaceutical and/or nutraceutical compositions that comprise the combination and to their use in therapy, in particular in the protection of the functionality of the nervous system and in the prevention and/or treatment of central and peripheral neuropathies.

The nervous system is made up of the central nervous system (CNS) and the peripheral nervous system (PNS).

The central nervous system is made up of the encephalon (brain) and spinal cord while the peripheral nervous system is made up of ganglia (neurons) and nerves (axons) that connect information from the central nervous system to the rest of the body.

Pathologies affecting the central and peripheral nervous system are extremely widespread and represent the first cause of disability, in addition to millions of deaths each year. Their increase is due in part to the aging of the population and the lack of definitive therapeutic treatments and the poor efficacy of preventive measures.

Damage to the central nervous system of mammals can cause serious physical damage and lead to many complications that can be fatal. Damage to the central nervous system can result, for example, from a spinal cord injury or stroke-induced injury or can be a consequence of pathologies in other organs.

In addition to pain caused by stimulation of pain receptors, neuropathic pain can be caused by damage or dysfunction of the peripheral or central nervous system. In the peripheral nervous system, peripheral neuropathy can result from deterioration and subsequent malfunction of the nerves. This deterioration can be the result of injury, trauma, or repetitive stress. Very often, however, peripheral neuropathy is the result of a disease or metabolic/endocrine disorder, such as diabetes. Peripheral neuropathy is a very common disorder among middle-aged and older people, and it has been estimated that approximately 60% of diabetic patients develop damage to the peripheral nerves. Although peripheral neuropathy is usually a non-fatal disease, unlike central nervous system diseases, it is nevertheless very painful and often disabling.

There are several substances known and commercially available that are useful for protecting or improving the health of the central and peripheral nervous system. These substances have also been combined with each other in an attempt to provide a treatment that acts through different mechanisms of action on the nervous system.

However, it is not at all obvious to combine different active ingredients with a reasonable expectation of obtaining therapeutic success. In fact, a possible association, in addition to being effective, non-toxic and well tolerated, should also satisfy several other requirements including compatibility between the components, good bioavailability of the same and long-term tolerability. Last but not least, all the requirements necessary for patient compliance must be met, especially when it comes to chronic treatment. An effective duration of action, good palatability and ease of intake are therefore further necessary requirements for a combination of active ingredients to prove to be a valid therapeutic tool for the treatment for maintaining the health of the nervous system and for the treatment of neuropathic pain.

It is therefore understood that there is a continuous need to find new therapeutic approaches to maintain the well-being of the nervous system and to limit its damage and possibly promote its regeneration in the event of injury.

Following intense research activity, the Applicant has been able to develop a new association of active ingredients that is able to respond to the needs not yet met in the field of nervous system well-being and the curative and preventive treatment of neuropathic pain.

In particular, the Applicant observed that the association of gastrodin and vitamins of group D has an interesting activity in preventing and repairing damage to the nervous system and that, thanks to an anti-inflammatory and antioxidant mechanism of action, it is able to stimulate the endogenous production of neurotrophic factors.

According to one of its aspects, the invention has as its object a combination consisting of gastrodin or a derivative thereof and at least one vitamin of group D or a derivative thereof.

Gastrodin, 4-(hydroxymethyl)phenyl ?-D-glucopyranoside, of formula (I)

(CAS RN. 62499-27-8) is the active ingredient contained in Gastrodia Elata, which is part of the Orchidaceae family and is traditionally used for the treatment of cerebrovascular damage. Its activity is based on increasing the activity of antioxidant enzymes and reducing oxidative stress, which, together with the inhibition of caspase, leads to the attenuation of the apoptosis mechanism. Gastrodin can also decrease the production of IL-6 and TNF-?, significantly reducing the expression of iNOS, resulting in a significant anti-inflammatory effect. Finally, it has a very powerful antioxidant effect, reducing the production of ROS and oxidative stress in various anatomical districts.

Derivatives of gastrodin include for example ester derivatives.

The vitamins of group D useful in the combination of the invention are the following:

- vitamin D1 compound consisting of 1:1 parts of ergocalciferol and lumisterol,

- vitamin D2 ergocalciferol,

- vitamin D3 cholecalciferol,

- vitamin D4 dihydroergocalciferol e

- vitamin D5 sitocalciferol

They are useful for the well-being of the skeletal system and teeth, and also perform functions at the brain and immune system level.

According to a preferred embodiment, the combination of the invention consists of gastrodin and at least vitamin D2 and/or vitamin D3.

According to a preferred embodiment, the combination of the invention consists of gastrodin and vitamin D3 and vitamin D, in particular a vitamin D2 and/or D3.

According to one of its aspects, the invention has as its object a pharmaceutical and/or nutraceutical composition which comprises, or alternatively consists of the association of the invention together with at least one pharmaceutically acceptable excipient and/or vehicle.

According to one embodiment, the composition of the invention comprises gastrodin and at least vitamin D2 and/or vitamin D3, together with at least one pharmaceutically acceptable excipient and/or carrier.

More preferably the composition of the invention comprises gastrodin and vitamin D3, together with at least one pharmaceutically acceptable excipient and/or carrier.

The composition of the invention is a composition for oral use, preferably a solid state composition, preferably formulated in dosage units. By ?solid state? it is meant that the composition may exist in the form of granules or powders. The granular or powder compositions are mixed with pharmacologically acceptable additives and excipients to provide a final product such as a food supplement, a medical device or a pharmaceutical composition. The final product may be in pharmaceutical dosage units such as granules in sachets, tablets, gels or capsules.

Tablets may have different shapes among those known in the field of pharmaceutical forms, such as a cylindrical or spheroidal shape. Tablets may be coated or filmed with one or more layers of coating or film capable of passing through the gastric barrier, according to known methods.

Gel capsules may be made of hard gelatin or soft gelatin or soft gel. Preferably the oral composition of the invention is a solid or semi-solid composition (gel), as described above. However, if desired or necessary, the composition may be formulated in liquid form, for example by dissolution or suspension in water.

The solid compositions of the invention may contain, as noted, conventional physiologically acceptable excipients and carriers, such as diluents, bulking agents, binders, disintegrants, flow promoters, lubricants, etc. Non-limiting examples of suitable carriers and carriers are described in Remington: The Science and Practice of Pharmacy, 21st Ed., . The compositions of the invention may, for example, include cellulose derivatives, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition may also contain pH buffering reagents and wetting or emulsifying agents. In addition to the components described above, if desired or necessary, the composition of the invention may include: include additional active components, for example components capable of assisting the active ingredients in carrying out their action in the organism of the treated subject, provided that said additional components are physically and chemically compatible with the active ingredients of the composition. Examples of additional components are vitamins of the B or E group, or hyaluronic acid.

In one embodiment, the composition is a capsule.

In one embodiment, the composition is a tablet.

In one embodiment, the composition is a gel for oral use.

In one embodiment, the composition is a granulate.

The composition of the invention, preferably formulated in dosage units, can be administered once or more times a day.

The composition of the invention preferably comprises

- 10 to 1000 mg, preferably 50 to 800 mg of gastrodin and 5 ?g to 200 ?g, preferably 10 to 100 ?g of vit. D, preferably D2 and/or D3.

The composition of the invention preferably comprises

- 100 to 400 mg of gastrodin, preferably 200 to 300 mg; and

- 10 to 50 g of at least one vitamin of group D, preferably 25 to 50 g; together with one or more pharmaceutically acceptable excipients and/or vehicles.

More preferably, the composition of the invention comprises

- 200 to 300 mg of gastrodin, preferably 200 to 250 mg; and

- 20 to 50 g of vitamin D3, preferably 25 to 35 g;

together with one or more pharmaceutically acceptable excipients and/or vehicles

The compositions of the invention can be prepared by mixing the individual components and any conventional excipients and/or vehicles.

The daily dosage depends on the patient's health status, weight, sex and age. In general, the compositions of the invention are well tolerated and can be administered once or twice a day, even for a prolonged period of time.

As will be shown in the Experimental Section that follows and from the figures illustrating the results of the experimental tests conducted, the association of the invention has proven to be particularly effective in the treatment of nervous system disorders, in maintaining the state of health and in the regeneration of the central and peripheral nervous system.

In particular, the combination, in addition to being non-toxic and well tolerated, showed an activity much higher than that of the individual components, exerting a powerful synergistic effect. Furthermore, the combination proved to be much more effective than the combination of vitamin D with 6-shogaol, which is a compound extracted from ginger (Zingiber officinale Roscoe) well known for its powerful neuroprotective activity.

The association and composition of the invention are therefore useful for maintaining the well-being and health of the nervous system and for preventing and/or treating pathologies related to it, as well as for preventing and/or treating neuropathic pain.

According to another of its aspects, the invention has as its object the association and/or composition of the invention in therapy, in particular in the prevention and repair of damage to the central and peripheral nervous system and to prevent and/or treat pathologies connected to them, in the protection of the functionality of the nervous system and in the prevention and/or treatment of central and peripheral neuropathies, to prevent and/or treat neuropathic pain and to stimulate the endogenous production of neurotrophic factors.

According to another of its aspects, the invention has as its object a method for the prevention and repair of damage to the central and peripheral nervous system and for preventing and/or treating pathologies connected thereto, for the protection of the functionality of the nervous system and for the prevention and/or treatment of central and peripheral neuropathies, as well as for preventing and/or treating neuropathic pain and for stimulating the endogenous production of neurotrophic factors, which comprises administering, to a subject who needs it, the association and/or composition of the invention, preferably in a dose effective for the purpose.

Representative forms of the compositions of the invention are reported in the Experimental Section that follows, for purely illustrative purposes and in no way limiting.

Brief description of the Figures

Figure 1 shows an assay of intestinal epithelial cell viability over time.

Figure 2 shows the assessment of ROS production in the intestinal epithelium.

Figure 3 shows the assessment of transepithelial resistance.

Figure 4 shows the permeability analysis.

Figure 5 shows plasma bioavailability.

Figure 6 shows the activity analysis of Zo-1; claudin and occludin.

Figure 7 shows the assessment of cell viability and ROS production in terms of inhibition for the reduction of oxidative stress related to degeneration.

Figure 8 shows the assessment of TNFα and IL-1b production.

Figure 9 shows the assessment of APP, pTAU and SIRT1 activity.

Figure 10 shows the stimulation of endogenous BDNF production.

Figure 11 shows the assessment of GABA and NGF activity.

Experimental section

Example 1

Composition in tablet form containing

- 200 mg of gastrodin

- 25 ?g of vitamin D3

Example 2

Composition in tablet form containing

- 400 mg of gastrodin

- 50 ?g of vitamin D3

Example 3

The activity of the association of the invention has been evaluated on the basis of various experimental tests.

In particular, absorption and bioavailability were evaluated using a 3D intestinal system, and the following tests were conducted:

- Analysis of absorption at the small intestine level from 1h to 24h

- Toxicity analysis (product safety)

- Analysis of ROS production

- Absorption and bioavailability (TEER and Papp analysis)

- Tight junction (TJ) analysis using kit

- Statistical analysis of data (5 independent experiments conducted in triplicate)

CNS activity was also evaluated using a brain organoid and the following tests:

Treatment for 24h to study the effect on the CNS

- Toxicity analysis (product safety)

- Analysis of ROS production and inflammatory picture

- Analysis of the main markers involved in the protective processes of neuronal damage (APP, pTAU and SIRT1)

- Analysis of endogenous BDNF production with microvesicle formation - Statistical analysis of data (5 independent experiments conducted in triplicate)

Finally, the activity at the CNS level was evaluated using a 3D model composed of Schwann cells and motor neurons and the following tests:

- Treatment for 24h to study the effect on SNP

- Analysis of the main markers involved in the processes of protection of the myelin sheath and in the formation of neurites (NGF and GABA)

- Statistical analysis of data (5 independent experiments conducted in triplicate) Toxicity?

The cellular viability of the intestinal epithelium was evaluated over time to exclude toxicity with the following tests and results (Figure 1, the data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicates. For all the forms tested the results obtained between 1h and 5h are statistically significant on the control (p<0.05)).

An analysis of single and combined substances over time (from 1h to 24h) was conducted to determine differences in mitochondrial metabolism. Among the substances examined, it was observed that all induced high cell viability compared to the untreated control (p<0.05). All forms, single and combined, showed a similar trend over time with a peak around 4 hours, which was maintained over time. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

ROS Production

Evaluation of ROS production in the intestinal epithelium over time to exclude irritability, confirming the safety of the product (Figure 2, the data reported are expressed as mean /-SD of 5 independent experiments conducted in triplicates. For all the forms tested, the results obtained between 1h and 5h are statistically significant on the control (p<0.05)).

An analysis of single and combined substances over time (from 1h to 24h) was conducted to determine the possible production of ROS. Among the substances examined, it was observed that the single substances produce ROS within the physiological range in compliance with the trophism of the tissue (p<0.05). All the combined forms showed a similar trend over time with a time-dependent increase in the production of ROS. Among the combined forms, substantial differences were observed between Gastrodin + Vit D3 compared to 6-Shagaol + Vit D3 (p<0.05). These data confirm the synergistic antioxidant effect of Gastrodin and VD

TEER

Evaluation of transepithelial resistance to determine whether the integrity of the intestinal tissue is preserved (Figure 3, the data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicates. For all the forms tested the results obtained between 1h and 5h are statistically significant on the control (p<0.05)).

An analysis of single and combined substances over time (from 1h to 24h) was conducted to determine the integrity of the absorption mechanisms. The analysis also supported absorption in terms of intestinal wall permeation kinetics. It was observed that gastrodin combined with VD was able to improve absorption leading to higher values than the other combinations tested. The peak at 4h confirms the data obtained previously although an increase was observed after 2h of stimulation. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

Permeability analysis?

Figure 4 shows the permeability analysis.

The values are derived from the application of the formula for calculating permeability.

Jmax [C]/(Kt [C])

C: initial concentration of the probe

Jmax: fluorescence step.

Kt: Michaelis-Menten constant

Plasma bioavailability

Plasma bioavailability is shown in Figure 5.

The values are derived from the application of the formula for calculating Papp

- Papp values <0.2?10<-6 >cm/s indicate very poor absorption with bioavailability <1%

- data between 0.2?10<-6 >and 2?10<-6 >cm/s a bioavailability between 1 and 90%.

Tight joints

An analysis of the activity of Zo-1 (mediates adhesion); claudin (maintains structure) and occludin (contributes to stabilization) was performed (Figure 6, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

The test is crucial for the integrity of the barrier and its functionality (avoid irritability or conditions that may lead to leaky gut syndrome). The analysis of Zo-1, claudin and occludin confirmed the active role of TJs in the absorption of the compounds. The analysis was conducted 6 hours after the end of the stimulation, to verify that there were no alterations in the intestinal structures. The results showed that the combination of Gastrodin+VD has a greater influence on TJs. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

CNS toxicity

Evaluation of cell viability and ROS production in terms of inhibition for the reduction of oxidative stress related to degeneration (safety and antioxidant effect) (Figure 7, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

An analysis of the viability and ROS production of the different formulations that passed the intestinal barrier after 24 hours of treatment was conducted. All the tested combinations increased cell viability and reduced ROS production, allowing to exclude toxicity in their use at the brain level after intestinal metabolism. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

Inflammation at the CNS level

Evaluation of TNFα and IL-1b production to establish anti-inflammatory capacity (Figure 8, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

An analysis of the activation of transcription factors and proinflammatory cytokines was conducted, probably due to the administration of the different formulations that passed the intestinal barrier after 24 hours of treatment. All the tested combinations reduced (p<0.05 vs control) the inflammatory markers, allowing to exclude the activation of inflammatory processes in their use at the cerebral level after intestinal metabolism. Gastrodin significantly reduced inflammation, having an excellent anti-inflammatory activity. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05)

CNS biological markers

Evaluation of the activity of APP, pTAU and SIRT1 to maintain brain homeostasis (Figure 9, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

An analysis of the biological activity of the different formulations that passed the intestinal barrier after 24 hours of treatment was conducted. SIRT1 plays a key role in brain aging, neuroprotection against oxidative stress, neuroinflammation and ischemic damage, central control of energy homeostasis, circadian clock modulation and neuroendocrine functions. APP is a transmembrane protein involved in synapse destruction, neuronal plasticity and ion transport. pTAU is a protein associated with microtubule dysfunction and axonal transport. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

BDNF production

Stimulation of endogenous BDNF production (Figure 10, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

An analysis of the production of endogenous BDNF by the different formulations that passed the intestinal barrier after 24 hours of treatment was conducted. All the tested combinations increased the production of BDNF allowing to stimulate neuronal activity thanks to their use at the astrocytic level. Gastrodin showed a greater production of endogenous BDNF compared to the other concentrations tested. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

SNP Biological Markers

Evaluation of GABA and NGF activity to maintain PNS homeostasis (Figure 11, data reported are expressed as mean /- SD of 5 independent experiments conducted in triplicate. All substances p<0.05 vs control; ** p<0.05 vs single substances; bars p<0.05 vs 6-Shagaol+VD).

NGF and GABA play a key role in establishing myelination and the formation of compact myelin; they also stimulate neuronal repair after oxidative trauma.

Gastrodin significantly induced the activity of these two markers having a good biological activity at the SNP level. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 compared to 6-Shagaol+Vit D3 (p<0.05).

Claims (10)

1. Pharmaceutical and/or nutraceutical combination consisting of gastrodin or its derivative and at least one vitamin of group D, preferably at least vitamin D3 or its derivative. 2. Combination according to claim 1, consisting of gastrodin and at least vitamin D2 and/or vitamin D3. 3. Association according to claim 1, consisting of gastrodin and vitamin D3. 4. Pharmaceutical and/or nutraceutical composition comprising the association of any of claims 1 to 3, together with one or more pharmaceutically acceptable excipients and/or vehicles. 5. Composition according to claim 4, which comprises, or alternatively consists of - 100 to 400 mg of gastrodin, preferably 200 to 300 mg; and - 10 to 50 g of at least one vitamin from group D, preferably 25 to 50 g, preferably of vitamin D3; together with one or more pharmaceutically acceptable excipients and/or vehicles. 6. Composition according to claim 3 or 4, which comprises, or alternatively consists of - 200 to 300 mg of gastrodin, preferably 200 to 250 mg; and - 20 to 50 g of vitamin D3, preferably 25 to 35 mg; together with one or more pharmaceutically acceptable excipients and/or vehicles. 7. Composition according to any of claims 4 to 6, characterised in that it is for oral use, preferably formulated in the solid state, more preferably in dosage units. 8. Composition according to any of claims 4 to 7, characterised in that it is a granule, a tablet, a gel or a capsule. 9. Association according to any of claims 1 to 3 or composition according to any of claims 4 to 8, for use in therapy. 10. Association or composition for use according to claim 9, to maintain the well-being and health of the peripheral nervous system and to prevent and/or treat pathologies connected to it, in the protection of the functionality of peripheral nerves, in the prevention and/or treatment of peripheral neuropathies, and to prevent and/or treat neuropathic pain and to stimulate the endogenous production of neurotrophic factors.