WO2018019996A1 - Compositions containing hyaluronic acid oligosaccharides (ha4), chondroitin sulfate (cs2-4), and heparan sulfate (hs2-4), pentacyclic triterpenes and derivatives for curative medical use, and method for preparing the compositions - Google Patents

Abstract

The present invention includes: - The association of hyaluronic acid tetrasaccharides HA4 with pentacyclic triterpenes that can be obtained from plants such as Burseraceae, Oleaceae, Apiaceae, and their derivatives. - The optional further association with sulfated oligo-glycosaminoglycans [heparan sulfate disaccharides or tetrasaccharides, preferably sulfated in C4 and C6 (H_4,6S_2-4); chondroitin sulfate disaccharides and tetrasaccharides, preferably sulfated in C4 and C6, (C_4,6S_2-4)]. - A method for preparing the products. - The use of these preparations.

Description

COMPOSITIONS CONTAINING HYALURONIC ACID OLIGOSACCHARIDES (HA₄), CHONDROITIN SULFATE (CS₂_₄), AND HEPARAN SULFATE (HS₂_₄), PENTACYCLIC TRITERPENES AND DERIVATIVES FOR CURATIVE MEDICAL USE, AND METHOD FOR PREPARING THE COMPOSITIONS

DESCRIPTION PRODUCTS USED AND THEIR CHARACTERISTICS

I. Hyaluronic acid (HA).

Hyaluronic acid (HA) is a polysaccharide of repeated units of the disaccharide D-glucuronic acid and N-acetyl-glucosamine. It is the main nonprotein component of the extracellular matrix (ECM), structurally distinct from the other glycosaminoglycans (GAGs) because it is not modified (not sulfated), linear (not branched) and without a core protein (to which the other GAGs are anchored).

HA is synthesized specifically by three hyaluronan synthase enzymes

(HASs) in cytoplasm, at the level of the plasma membrane, and is completely extruded in the extracellular space.

HA requires two distinct processes for its deposition in the ECM: synthesis of HA catalyzed by HASs in cellular cytosol, and export of HA in the ECM.

In the most common homeostatic native form, HA is a long polymeric chain (up to more than 20,000 disaccharides), which can reach a length of 25 μιη when fully extended, with a high molecular weight (HMW) comprised between 1000 and 10,000 kDa.

Native hyaluronic acid has the ability to retain a large amount of water with lubricating, hydrating and filling properties of voids such as connective tissue and joints, in which it also has biomechanical functions; it also has the ability to influence the absorption and concentration of tissue liquids and to act as a molecular sieve capable of sequestering growth factors and cytokines. HA is a negatively charged molecule and therefore it attracts positive ions; it is considerably hydrophilic (it absorbs water up to 10,000 times its own weight), and forms in an aqueous solution specific tertiary structures which also involve other glycosaminoglycans, such as chondroitin sulfates [Scott JE and Heatley F 1999]. It is present mainly in the connective tissue, in the lungs, in the kidney and in the brain, less in the liver and in serum.

Native HA can be broken down by hyaluronidases (Hyals), a family of six enzymes which are rather specialized in hydrolyzing chains of native HA into fragments of various size: intermediate (medium molecular weight, MMW); small (low MW-HA, LMW-HA); minimal (oligosaccharides, o- HA) up to two saccharides. The properties of HA reside in that its biological functions depend on its molecular weight. Hyaluronidases degrade both hyaluronic acid and chondroitin sulfates.

Any increase in the expression and activity of Hyal leads to an accumulation of bioactive HA fragments in tissues in pathological conditions. For example, increased Hyal activity, together with high levels of LMW-HA, are present in inflammatory atheromatous plaques in cardiovascular disease and in diabetes; HA fragmentation is typical of rheumatoid arthritis.

A correlation between Hyal levels and increase in HA fragments has been observed in pulmonary pathology [pulmonary hypertension, pulmonary ischemia, asthma and chronic obstructive pulmonary disease (COPD)]; in tumor pathology of the colon-rectum, of the breast, of the pancreas, of the lung, of the neck, of the prostate, of the brain, of the urinary tract, of the bladder, of the ovaries.

The present invention indeed provides for the use of triterpenes for their anti-hyaluronidase and antitumor action, as will be described hereinafter.

In the literature there is no consensus regarding the limits of HMW- MMW- LMW- and o-HA. In this text, in agreement with Monslow J et al. 2015, the various forms of HA are classified as follows: HMW-HA: >1000 kDa; MMW-HA: 250-100 kDa; LMW-HA: 10-250 kDa; o-HA: <10 kDa. These groups are in no way distinctly distributed; often they are polydispersed in multiple categories; conversely, in certain morbid conditions a specific spectrum of HA stands out [Petrey AC and de la Motte CA 2014].

High and low molecular weights of HA exhibit opposite effects on cell behavior.

- HMW-HA inhibit cell proliferation and angiogenesis, are anti- inflammatory and immunosuppressive.

- LMW-HA are involved as pro-inflammatory and pro-angiogenic. MMW-HA and LMW-HA have roles which overlap with HMW-HA and with o-HA and can be harmful and pro-inflammatory as well as beneficial and protective.

- HA₆-5o oligosaccharides have different functions from HA polysaccharides but not univocally: as regards inflammation, they can induce pro-inflammatory signals but also acceleration of the healing of inflammatory processes; as regards the progression of cancer, it appears that oligomers longer than 10 saccharides stimulate tumor growth via a link with CD 44 (Cluster Determinant 44), while oligosaccharides shorter than 10 mers are protective.

The present invention provides for the use of hyaluronic acid tetrasaccharides, HA₄, because they have a behavior that is completely different from the other fragments of hyaluronic acid and without the pro- inflammatory, pro-angiogenic and also tumor growth stimulating effects.

The HA₄ oligosaccharides according to the present invention, according to in vitro and in vivo experiments in animals, have demonstrated in the central nervous system that they are not cytotoxic; they have a neuroprotective effect and accelerate the healing process by promoting in vitro survival, growth and regeneration of neurons; axon growth and remyelination of peripheral nerves; they mitigate the symptoms of experimental immune encephalitis; they are capable of reducing the excitability of nociceptors and therefore both peripheral and central pain, proposing themselves as potential drugs against epilepsy; they have complementary functions in neurogenic inflammation and in body temperature regulation; they facilitate fear memory extinction; they improve cognitive and learning flexibility; they induce, together with other oligo- glycosaminoglycans, a resetting of the extracellular matrix in various morbid conditions, as in tumors and in neurodegenerative diseases.

HMW-HA via CD44 can induce the expression of cyclooxygenase-2

(COX-2) in endothelial cells of vessels, which induces in turn the production of PGI₂ prostacyclins and PGE₂ prostaglandins, via PGH₂, also having a vasodilator action, and are involved in the inflammation.

Nonselective COX-2 inhibitors are non-steroidal anti-inflammatory drugs (NSAID), which however have side effects at the gastrointestinal level (gastritis, bleeding) and selective COX-2 inhibitors or coxibs, which have side effects on the cardiovascular system.

Hyaluronic acid oligosaccharides with a minimum of six saccharides have been used both as carriers and as ligands on liposomes or nanoparticles which are selective against tumors which hyperexpress CD44. An additional property of HA-CD44 conjugates is in fact the internalization of the drug, with boosting of antitumor activity.

Since HA₄ do not bond to known membrane receptors such as CD44 and RHAMM, they can remain in the extracellular matrix, resetting it and contributing to keeping intact the glycocalix (i.e., the outermost layer of the plasma membrane of cells) and, in the nervous system, the perineuronal net and therefore the integrity of the tissues; they prevent the destruction of tight junctions induced by oxidative and inflammatory stimuli and therefore maintain the integrity of the barriers of the body (cutaneous, intestinal, pulmonary, hematoencephalic); moreover, they can cross the cell membrane without hindrance, also acting as carriers.

II. Boswellic pentacyclic triterpenes and their analogues

Frankincense or olibanum is a resin produced by Boswellia plants of the Burseraceae family and is known since antiquity for its curative properties.

The Boswellia genus is divided into about fifteen species. They originate from the Persian Gulf in the Indian Ocean and are cultivated in several countries, such as the South of Saudi Arabia, Somalia, Ethiopia, Eritrea, the Sudan, and Kenya. Boswellia serrata is cultivated in India.

The best resin is gathered in autumn, following incisions made in the summer, and produces what is known as white incense in contrast with red incense, which is collected in spring after incisions during winter. It is from this oleoresinous, fragrant, transparent and yellow-brownish gum that one extracts with various methods the essential oil of incense, which is composed mainly of pentacyclic triterpenes.

The phytochemical content of the resins of the various species of Boswellia depends on the botanical origin and consists of triterpenes (30- 60%), essential oils (5-10%), polysaccharides. Triterpenes are considered species-specific markers of the Boswellia genus.

The main triterpenes contained in the Boswellia serrata extracts are listed below [Catanzaro D et al. 2015]. The structures of some boswellic acids are also shown schematically.

Triterpene name Molecular weight (MW) % in the boswellic extract

1 l-keto-P-boswellic acid (KB A) 471 5.02

3-O-acet l-l l-keto- -boswellic 513 2.71

acid

9,11-dehydro-a-boswellic acid 455 4.40

9,l l-dehydro- -boswellic acid 455 4.68

a-boswellic acid 457 1.40

β-boswellic acid 457 0.94

Lupeolic acid 455 2.84

3 -O-acetyl-9, 11 -dehydro-a- 497 2.42

boswellic acid 3 -O-acet l-9, 11 -dehydro-β- 497 0.67

boswellic acid

Ursanic acid 499 0.87

3-O-acetyl-a-boswellic acid 499 1.77

3-0-acetyl-P-boswellic acid 499 1.61

Structures of boswellic acids.

The present invention also considers other pentacyclic triterpenes extracted both from Boswellia plants and from other plants, which can be compared with each other and with boswellic acids due to their chemical structure, to their pharmacological and clinical effects, and to the mechanisms of action [Safe SH et al. 2012]: in particular, oleanolic acid C30H48O3, MW 457), ursolic acid (C30H48O3, MW 457), betulinic acid (C30H48O3, MW 457), glycyrrhetinic acid (C30H46O4, MW 471), asiatic acid (C₃₀H₄₈O₅, MW 489), and their derivatives.

Conclusion

The triterpenes according to the present invention have pharmacological activities that can be compared with those of antiinflammatory, antitumor and neurological drugs of the official pharmacopeia.

They have mechanisms of action which are synergistic and complementary to disaccharides and tetrasaccharides of hyaluronic acid (HA₂ and HA₄), in particular HA₄: HA₂ and HA₄ act predominantly in the extracellular matrix, triterpenes act predominantly within the cell.

III. Sulfated oligo-glycosaminoglycans

The third group of elements is represented by the other glycosaminoglycans (GAGs) that are present in the extracellular environment and which, differently from hyaluronic acid, are attached as branches to a protein core, constituting proteoglycans.

Proteoglycans (PGs) are indeed a family of macromolecules that consist of a core protein (central core protein) bonded at one end to the hyaluronic acid chain and attached at the other end in a covalent manner to a long chain of carbohydrates known as glycosaminoglycans (GAGs) which include chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate

(KS), heparin (H) and heparan sulfate (HS). Glycosaminoglycan chains are linear, highly negatively charged, arranged on the cellular surface or in the extracellular matrix (ECM) or free-floating.

The composition and structure of glycosaminoglycan disaccharides are represented in tables 1 and 2.

Name Acid or neutral Aminosaccharide Bond geometry among monosaccharide the main monomer units

Chondroitin sulfate GlcUA or GalNAc or →4GlcUA i→3

GlcUA(2S) GalNAc(4S) or GalNAc i→

GalNAc(6S) or

GalNAc (4S,6S)

Dermatan sulfate GlcUA or GalNAc or →4IdoUAal→3

IdoUA or GalNAc(4S) or GalNAc i→

IdoUA(2S) GalNAc(6S) or

GalNAc (4S,6S)

Keratan sulfate Gal or Gal(6S) GlcNAc or →3Gal(6S) i→4GlcNA

GlcNAc(6S) C(6S) 1→

Heparin GlcUA or IdoUA(2S) GlcNAc or →4IdoUA(2S)al→4

GlcNS or GlcNS(6S)al→

GlcNAc(6s) or

GlcNS(6S)

Heparan sulfate GlcUA or GlcNAc or →4GlcUA i→4

IdoUA or GlcNS or GlcNAcal→

IdoUA(2S) GlcNAc(6S) or

GlcNS(6S)

Hyaluronic acid GlcUA GlcNAc →4GlcUA i→3

GlcNAc i→ Table 1. Composition of glycosaminoglycan disaccharides

[GlcUA = D-glucuronic acid; IdoUA = L-iduronic acid; GlcNac = N- acetylglucosamine; GalNac = N-acetylgalactosamine; GlcNac(6S): N- acetylglucosamine sulfated in C6]

Table 2. Structure of glycosaminoglycan disaccharides (o-GAGs₂)

The functions of proteoglycans are adjusted in a flexible manner by virtue of the structural variability of glycosaminoglycans, generated by multiple syntheses of glycosaminoglycans and by enzyme modifications.

Chondroitin sulfate proteoglycans (CS-PGs) and heparan sulfate proteoglycans (HS-PGs) are the main constituents of the ECM and of the cellular surface in the brain.

Chondroitin sulfate (CS) is a sulfated linear polysaccharide

(approximately 75 kDa) involved in various biological processes. The structure of CS is truly heterogeneous and contains various sulfation patterns caused by the multiple and random modifications (approximately 16 types of CS disaccharides) that occur during its biosynthesis in the Golgi apparatus.

The resulting structural microdomain in the CS chain interacts with specific biomolecules to regulate biological functions [Wakao M et al. 2015].

It is by now clear that the distribution of CS is tissue-specific, with distinct functions according to the variations of the CS chains, the degree and profile of sulfation, with expression regulated during growth, in particular of the brain [Bao X et al. 2004]. Specific CS-PG sulfation patterns can affect axonal guidance in various manners.

The present invention relates to the use of a particular type of sulfation of chondroitin sulfates, i.e., in C4 and C6 of the saccharide GalNAc (N-acetyl-galactosamine).

Numbered position of the carbon atoms in the three glycosaminoglycan disaccharides.

Heparan sulfate proteoglycans (HS-PGs) are present in all animal tissues. They can be classified into two types: HS-PGs of the cell surface and HS-PGs of the extracellular matrix ECM. Many studies suggest an important role thereof in axonal guidance and growth. Recent studies demonstrate that modifications of ECM and MMP (matrix metalloproteinase) are important regulators of plasticity, learning and memory and can be involved in various diseases such as epilepsy, schizophrenia, dementia and drug dependency addiction [Tslibary E et al. 2014]. In glioma, characterized by rapid cell proliferation, growth and aggressiveness of the tumor, there is an deregulation of the components of ECM, in particular of HS-PGs [Xiong A et al. 2014] .

The HS sulfation pattern determines the potential interaction of proteoglycans with a large number of cytokines and growth factors. The binding with growth factors requires a minimum of 4-6 saccharides [Higginson JR et al. 2012].

The content of CS/DS-GAGs in the brain is approximately 9 times higher than HS-GAGs (89% and 11%, respectively). The GAGs associated with perineuronal nets (PNN) are only a small proportion of the total of CS/DS-GAGs and HS-GAGs: 1.3 and 5.0% respectively, but in PNN the CS/DS-GAGs are 71% and the HS-GAGs are 29% of total GAGs [Deepa SS et al. 2006], i.e., approximately 10:4.

The literature regarding the actions of sulfated glycosaminoglycans is vast, but differently from hyaluronic acid it has not yet reached clear and consistent results both in terms of the mechanisms of action and in terms of the therapeutic indications. The difficulties are due to the number of subtypes of GAGs, at least twenty, to their degree of sulfation, to the ability to obtain pure saccharides, to the need to study their metabolism in the various sectors of the nervous system.

The composition HA₄ ± CS-E₂_₄ ± H_4;6S₂_₄ + triterpenes varies according to the physiopathology of the neurological disease to be treated.

With these products, which we can consider raw materials, the compositions according to claim 1 and following are prepared.

Material and method

The method according to the present invention is to utilize the hydrogen bonds that form between HA₄, terpenes and the other oligo- glycosaminoglycans in an aqueous solution when subjected to a rotating magnetic field.

Use of the following material is provided for the present invention:

1) Tetrasaccharides of hyaluronic acid HA₄ (C28H₄₄N₂0₂3, MW -776 Da, hydrogen donor bonds 14, hydrogen acceptor bonds 23) sodium salt. These products are commercially available.

2) Pentacyclic triterpenes from plants of the Boswellia genus and from various plants, extracted selectively with the supercritical C0₂ method, listed here: 1 l-keto- -boswellic acid (KBA), 3-0-acetyl-l l-keto- - boswellic acid (AKBA), 9,1 1-dehydro-a-boswellic acid, 9, l l-dehydro- - boswellic acid, a-boswellic acid, β-boswellic acid, lupeolic acid, 3-0- acetyl-9, 1 1-dehydro-a-boswellic acid, 3-0-acetyl-9, l l-dehydro- -boswellic acid, 3-O-acetyl-a-boswellic acid, 3-0-acetyl- -boswellic acid, oleanolic acid, ursolic acid, betulinic acid, glycyrrhetinic acid, asiatic acid, and the acid derivatives of all of the above.

[For all of these triterpenes, the molecular weight is comprised between 427 and 513 Da; average molecular weight 474 Da; for the beta- boswellic and oleanolic acids, taken as example: hydrogen donor bonds 2, hydrogen acceptor bonds 3].

The extract of triterpenes, at least with the desired terpene or terpenes predominant, is generally available in a water-alcohol solution and can be obtained at desired concentrations.

3) Heparan sulfate

a) disaccharides sulfated in R₂ and R₃ according to the same diagram shown in Table 2 (II-S, Ci₂Hi₆N a₃0i₆S₂ disaccharide, MW 563 Da, hydrogen acceptor bonds 17, hydrogen donor bonds 5);

or:

b) disaccharides trisulfated in Ri, R₂ and R (I-S, Ci₂Hi₅NNa₄0i₉S disaccharide, MW 665 Da, hydrogen acceptor bonds 20, hydrogen donor bonds 4),

or: c) mixtures of the two: I-S + II-S

or, preferably to disaccharides:

d) tetrasaccharides heparan disulfated in R₂ and R₃

or:

e) trisulfated tetrasaccharides, C24H₄oN₂0₃9S6, MW 1173 Da, hydrogen acceptor bonds 41, hydrogen donor bonds 16.

Commercially available.

4) Chondroitin sulfate

Disaccharides and tetrasaccharides of chondroitin sulfate, preferably CS-E (sulfated both in C4 and in C6, marked as R₂ and R in the diagram shown in Table 2 [Kiinze G et al. 2014], molecular weights, hydrogen acceptor and donor bonds similar to those of heparan disulfates.

Commercially available.

M I]

Γ Ί

Table 2. Composition and sulfation pattern of main heparin and chondroitin disaccharides [Kiinze G et al. 2014]. The provided operation occurs in four steps:

1. Preparation of the aqueous solution of HA₄ and of the hydroalcoholic solution of terpenes.

2. Preheating in a low-strength rotating magnetic field also by evaporation of the ethanol.

3. Continuation of the treatment in a high- strength rotating magnetic field to stabilize the solution with the electrostatic bonds.

4. Optional addition to the preparation of the solutions containing heparan sulfate and/or chondroitin sulfate and treatment in a low-strength rotating magnetic field.

The preparation of the aqueous solution of HA₄, of triterpenes and of sulfated glycosaminoglycans (GAGs) takes into account:

- the molecular weight of HA₄ (776 Da) and the average molecular weight of the triterpenes (474 Da) with a ratio of 10:6, in practice 2: 1. In this manner, an HA tetrasaccharide corresponds to each triterpene used.

- the final concentration of triterpenes, which in the desired product must be 20-40 mg/ml.

- the therapeutic dose in a human being of inhaled triterpenes tested by the Author in various morbid conditions, which is 10-20 mg per day, considerably lower than that practiced by other Authors [Gupta I et al. 2001 : 900 mg/day administered orally for 6 weeks in chronic colitis; Prabhavathi K et al. 2014: 250 mg administered orally in a single administration to test the analgesic effect in healthy volunteers].

- the concentration of HA₄ with respect to the sulfated GAGs (CS and HS) provided in the final 5: 1 solution, considering that in the body hyaluronic acid is in a considerably higher quantity.

- the concentration ratio between the two GAGs (CS:HS), which is

2: 1. These proportions are indicative and can be changed according to the physiopathological mechanisms involved in the various diseases, with predominant CS or HS damage.

The methods described here is merely by way of example.

Step I. - obtaining a solution containing 40 g of HA₄ in 700 ml of distilled water;

- obtaining a hydroalcoholic solution (70% ethanol, 30% water) containing 20 mg of triterpenes in 500 ml.

The two solutions are joined.

Step II. The mixture is subjected to a rotating magnetic field for a time that can vary between 60 and 120 minutes with a field strength between 30 and 100 mT (millitesla), preferably between 50 and 75 mT at a temperature of 85±5°C. Since ethanol has a boiling point of 78.4°C, it is eliminated completely by evaporation. One obtains an aqueous solution of 1000 ml containing 40 g of HA₄ and 20 g of triterpenes, i.e., respectively 40 and 20 mg/ml, a quantity which corresponds to the daily dose that is considered fully therapeutic according to the present invention.

Step III. Use of a rotating magnetic field with a strength that can vary from 100 to 300 mT, preferably between 150 and 200 mT is continued. Temperature between 60 and 90°C, preferably around 75 °C. Operation performed with an average time of 90 minutes, which oscillates between 60 and 120 minutes.

Step IV. After a variable time interval that is non-binding, it is possible to add the sulfated GAGs, approximately 8-9 g in total, of which approximately 6 g are CS and 3 g are HS, and the entire mixture is subjected to a rotating magnetic field for a variable time of 30-60 minutes with a field strength that is halved with respect to the preceding one, between 50 and 150 mT, preferably between 75 and 100 mT. Temperature between 60 and 90°C, preferably around 75 °C.

The mixture is brought to ambient temperature and the concentrations of the dissolved substances are measured.

Conclusion

A mixture of substances in a solution which is electrostatically stable at ambient temperature is obtained. Another advantage is that the preparation according to the invention, due to the size of the individual components of the mixture (triterpenes, hyaluronic acid, sulfated glycosaminoglycans) smaller than 1 nm, passes through the cutaneous, mucosal, pulmonary barriers and can reach the central nervous system (brain and spinal cord) transnasally.

Another advantage is that the preparation does not require particular nanoengineering methods, which require high technology and high production costs.

Another advantage is that the rotating magnetic field method allows the forming in a percentage of around 20-30% of covalent bonds between carboxylic groups of triterpene acids and amine groups of HA₄, forming a triterpene-tetrasaccharide complex.

Obtaining also a triterpene-tetrasaccharide complex would have the property of crossing also the blood-brain and intestinal barrier and not only the other barriers of the body, i.e., the cutaneous, mucosal, and pulmonary barriers.

Another advantage is the fact that the drug is carried into the cell and this further enhances the effect of the carried drug, regardless of the fact that triterpenes and glycosaminoglycans are synergistic.

Another advantage is constituted by the fact that therotating magnetic field method also allows heparan sulfate and chondroitin sulfate to act as carriers of transcription and growth factors, such as VEGF (vascular endothelial growth factor) and antineoplastic drugs, much more effectively than what has been attempted so far, eliminating the need to manufacture nanocapsules, nanogels, nanoparticles of bioactive compounds and the problems of barrier crossing.

This method differs substantially from methods that use electrostatic fields and have other purposes and provide for nanoparticles larger than 20 nm [Sun Q et al. 2014, Teong B et al. 2015]. Attempts to solve the problem of the penetration of the drug in tumor cells with other types of nanoassemblies by utilizing electrostatic and hydrophobic interactions are in progress [Sun Q et al. 2016] but do not have the ease, simplicity, and universality of use of the method according to the present invention.

The most significant literature data related to the dispensing of antitumor drugs nanoconjugated with glycosaminoglycans [Misra S et al. 2014] demonstrate that one is still in the presence of applications that are local or provided on cell cultures, with carriers of hyaluronic acid of considerable size, the smallest with oligomers of 6 saccharides (HA₆-g). Even these oligomers, as already mentioned, are captured by membrane receptors, such as CD44 and RHAMM, and are then endocytosed, without performing the beneficial activity in the extracellular matrix, synergistically with the carried drug, that is possible only with the preparation according to the present invention.

The present invention therefore provides for the use of four groups of substances (HA, CS, HS, triterpenes) which are rather known in their actions, which are complementary, but the invention is based both on the choice of the individual components (disaccharides and/or tetrasaccharides) and on their association with pentacyclic triterpenes.

The resulting preparation, modulated in its composition according to the therapeutic requirements, has the characteristic of a synergistic enhancement event and of unequaled effectiveness with respect to conventional drugs.

THE HA₄ + TRITERPENES + HS₂_₄ + CS-E₂_₄ PRODUCT

As already mentioned, a particular characteristic of the present invention, which is different from common commercially available products, is that the preparation, derived from the association of glycosaminoglycan oligosaccharides with triterpenes, has the capacity for substantially universal use and to cross the barriers of the body and therefore perform curative actions that other commercially available products do not have. Cutaneous transport of the product

The transdermal route for the administration of substances into the body has several advantages with respect to other routes, such as oral route or injection. The advantages include noninvasive treatment, self- administration, increased patient compliance, the avoidability of the metabolism of hepatic first pass metabolism or digestion. Despite these benefits, the low cutaneous penetration of nanoconstructs (such as polymers, proteins, hydrophilic drugs and nanoparticles) limit their vast transdermal use [Jung HS et al. 2014].

In order to facilitate the transdermal delivery of nanoconstructs, additional treatments have been adopted to increase penetration, such as iontophoresis, ultrasound and microinjections. However, these methods induce tissue perturbations and in some cases skin damage.

As regards the elements of this invention:

- HA₄ tetrasaccharides are absorbed well and have a trophic and protective action on the skin [Kage M et al. 2013]

- triterpenes, being liposoluble, are absorbed well through the corneal layer and cross the cutaneous barrier. Boswellic triterpenes have been used by the Author for topical applications to treat cellulite, lipomas in Dercum disease, forms of inflammatory articular pathology with and without articular effusion, hematomas, seborrhea and seborrheic dermatitis with excellent results.

- sulfated oligo-GAGs, preferably CS-E₄, have all these properties and augment the action of the triterpenes for transdermal treatment of skin and osteoarticular diseases of various nature (inflammatory, infective, allergic) and also for curing systemic diseases.

Therefore, the HA₄ + triterpenes + CS-E₂_₄ association is ideal for skin regenerative medicine, such as chronic ulcers and burns, radiodermatitis, trophic lesions to the lower limbs caused by diabetes. It can be much more useful than commonly commercially available preparations for treating inflammatory-immune diseases such as psoriasis and psoriatic arthritis, due to the joint action against TNF-a (a pathogenic element that is important in this disease [Girolomoni G et al. 2012]) of boswellic acids and stabilizing the extracellular matrix of the oligo-GAGs; in seborrheic dermatitis and keratitis; on erythematous eczema already treated effectively with boswellic acids [Togni S et al. 2014]; in cellulite, due to the combined actions on microcirculation and on lipid metabolism (personal observations); on actinic keratoses.

The invention of the HA₄ + triterpenes + CS-E₄ association in skin diseases and in transcutaneous transport is claimed.

Composition: preferably 40 mg/ml + 20 mg/ml + 8 mg/ml for the respective components.

Intestinal transport of the product

The gastrointestinal mucosal barrier has an essential role in separating the inside of the body from the outside environment. Tight junctions (TJs) are the most important components for the construction of a constitutive barrier of epithelial cells and they regulate the permeability of the barrier by sealing the cell-to-cell junctions. Destruction of their function leads to chronic inflammatory conditions and to chronic or progressive diseases.

Altered intestinal permeability can predict the risk of Crohn's disease highlighted with the sucralose/mannitol test: mannitol, MW 182 Da, passes freely through intestinal junctions; sucralose, MW 403 Da, is instead scarcely absorbed.

This explains why the intestinal permeability of boswellic acids is very low even though they are lipophilic and are not a substrate of P- glycoproteins; because (similar to what has been observed regarding the permeability of boswellic acids at the level of the blood-brain barrier) 11- keto- -boswellic acid (KBA, MW 471) has a higher permeability than AKBA, MW 512 [Weber CC et al. 2006, Kriiger et al. 2009, Bagul P et al. 2014] with blood levels of KBA three times higher than AKBA [Husch J et al. 2012].

Boswellic acids are safe and protect the intestinal epithelial barrier from oxidation and inflammation damage; they preserve the intestinal barrier from oxidative and inflammatory damage; their pretreatment prevents the destruction of tight junctions. Therefore, boswellic acids can be listed among the next natural compounds in the treatment of chronic inflammations of the intestine, and first of all in protection from damage caused by NSAIDs [Hirano K et al. 2013] and as an alternative to them.

As regards hyaluronic acid, it has been demonstrated that it can be absorbed orally and that this absorption is inversely proportional to its size, is highest below 10 kDa and is likely receptor-mediated.

We also know that disaccharides derived from chondroitin sulfate cross the intestinal barrier through a paracellular route and that sulfation does not affect the "transport rate" of o-CS₂ [Jin M et al. 2010].

The obvious conclusion is that the mixture HA₄ + triterpenes, obtained with the method of the rotating magnetic field, without the addition of the other GAGs components CS and HS, can be used in the topical treatment of gastrointestinal disorders with greater benefit and without the side effects of the drugs usually used.

Moreover, since the same rotating magnetic field method likely allows the formation of an HA₄-triterpene compound, the HA₄ + triterpenes + HS₂_₄ + CS-E₂_₄ preparation can be employed usefully also to cure systemic disorders.

Pulmonary transport of the product

The weight of a hyaluronic acid tetrasaccharide (HA₄) is approximately 770 Da with a diameter of approximately 0.5 nm. The same applies to disaccharides of the other two GAGs, CS and HS (MW -500 Da). The tetrasaccharide of the two sulfated GAGs, despite their size around 1 nm, likely pass through the pulmonary barrier, and it has been demonstrated that they pass through the blood-brain barrier [Ma Q et al. 2002]. Triterpenes, which are liposoluble, with MW -500 Da and with dimensions slightly larger than 0.5 nm on their own also can cross the alveolar-capillary barrier.

One deduces from this that the HA₄ + terpenes + HS₂-₄ + CS-E₂_₄ mixture can be used effectively both to act locally for disorders of the respiratory system [in fact, the delivery of drugs by inhaling is a noninvasive means for administration with advantages for local treatment of respiratory diseases, since one reaches the organ directly, avoiding first pass metabolism (liver and kidneys) and avoiding systemic toxicity] and for systemic delivery through a pulmonary route because it crosses the alveolar- capillary barrier.

In the lungs, GAGs are distributed in the interstice, in the subepithelial tissue of the bronchial wall, and in the secretions of the air passages. GAGs have important functions in pulmonary ECM: they regulate hydration and water homeostasis; they maintain structure and function; they modulate the inflammatory response; they influence tissue repair and remodeling [Souza-Fernandes AB et al. 2006].

Plasma increases in HA in relation to the inflammatory state and to the oxidative stress have been observed in secondary and idiopathic pulmonary hypertension. In secondary and idiopathic pulmonary fibroses and in lung transplant rejection there is an excess of deposition of extracellular matrix in the pulmonary interstice, in particular collagen, proteoglycans and HA fragments also due to an increase in hyaluronidase activity. Systemic inflammatory diseases (such as sepsis) are characterized by degradation of the endothelial glycocalyx, i.e., of the layer of GAGs (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) that lines the vascular lumen.

In respiratory therapy, hyaluronic acid is used in various dimensions, generally from HMW-HA to LMW-HA. The effects of HA on inflammation appear to be correlated to its molecular weight, the largest polysaccharide chains with anti- inflammatory action [Garantziotis S et al. 2016], the smallest with pro-inflammatory properties. Beneficial effects have been observed in infections of the upper air passages, in nasal polyposis, in bronchial asthma, in pulmonary emphysema.

The Author has administered by inhalation a solution of boswellic triterpenes in hundreds of subjects with phlogosis of the upper air passages, even severe allergic bronchial asthma, intra-thoracic chronic phlogoses, chronic obstructive pulmonary disease, after traditional medical therapy and in stable clinical conditions, achieving a further and significant clinical and functional improvement. In particular, in subjects with severe chronic obstructive pulmonary disease and pulmonary emphysema under continuous oxygen therapy and mechanical ventilation at home, in stable conditions for months, a spectacular clinical improvement has been observed, with a reduction of dyspnea and a considerable increase in functional and motor autonomy [Bevilacqua M. Terpeni, Masson Ed. 2005].

Conclusion

The association of triterpenes with HA₄ in nonsevere respiratory morbid conditions is not only effective but can lead to a further advantage with respect to traditional treatments, in particular cortisonic drugs, and without side effects.

Recommended therapy: HA₄ + triterpenes by inhalation, 40 mg +20 mg/day, of which the invention is claimed.

An even greater advantage is predictable with HA₄ + triterpenes + CS- E₂-₄ + HS₂_₄ in severe morbid conditions, such as idiopathic pulmonary fibrosis, distress syndrome and lung transplant.

Nervous transport of the product

Nervous traffic of glycosaminoglycans and terpenes, i.e., of the product according to the present invention, can occur in three manners:

1. With a transpulmonary route, passage into pulmonary circulation and brain circulation; diffusion in the brain parenchyma through the blood- brain barrier (BBB), with production of interstitial fluid (ISF), and through the blood-choroid plexus barrier with production of cerebrospinal fluid (CSF);

2. With a transnasal route directly to the brain, through the peripheral olfactory system and the peripheral trigeminal system;

3. With a lymphatic route, with drainage of the interstitial fluid from the CNS to the cervical and para-aortic deep lymphatic system.

1. Transport of GAGs and terpenes to the CNS with a transpulmonary route

The blood brain barrier, BBB, is composed of a monolayer of capillary endothelial brain cells fused together by tight junctions, which limits up to 98% the free flow even of small molecules between the blood and the cerebral interstice. Accordingly, except for liposoluble molecules, with a molecular weight (MW) below 400 Da and with less than 8 hydrogen bonds with the solvent water, virtually all the substances generated both by biotechnology and by classical small molecule pharmacology are subjected to negligible transport through the BBB [Pardridge WM 2015].

BBB permeability does not increase in proportion to solubility in lipids when the molecular weight of the drug increases, quite the contrary. For example, it is reduced 100 times when the molecular surface of the drug increases from 0.52 nm (e.g., 200 Da drug) to 1.05 nm (e.g., 450 Da drug) [Pardridge WM 2005].

This is the fundamental problem that blocks progress in the development of new drugs for nervous diseases. A review of the Comprehensive Medical Chemistry Database in fact demonstrates that of over 7,000 small-molecule drugs, only 5% treat the CNS, and these drugs involve only four diseases: depression, schizophrenia, chronic pain and epilepsy. There are few effective drugs with small or large molecules for most of the other diseases of the CNS (Alzheimer's, disease, Huntington's disease, amyotrophic lateral sclerosis, neuro-AIDS, cerebral tumors, stroke, cerebrospinal trauma, autism, fragile X syndrome, hereditary ataxia, blindness, lysosomal accumulation diseases), with the exception of Parkinson's disease (e.g., 1-DOPA), and multiple sclerosis (e.g., cytokines) [Pardridge WM 2005].

Oligo-GAGs (HA₄, HS₂-₄ and CS-E₂_₄) cross the blood-brain barrier and the perineural barrier of the nerves.

As regards the boswellic acids according to the present invention, which have a molecular weight comprised between 456 Da (a-boswellic acid) and 513 Da (AKBA), a considerable reduction in BBB permeability is observed with a modest increase in molecular weight. For example, in rats, eight hours after oral administration of 250 mg/kg of Boswellia resin extract the concentrations in the brain of these two acids yielded the following result [Gerbeth K et al. 2013]:

In this case, the AKBA, which has a concentration in the boswellic extract that is substantially twice that of the α-boswellic acid, with a weight variation of 11 % with respect to the other acid, undergoes a reduction in brain blood concentration of almost 22 times. One deduces from this that paradoxically AKBA, which is the most active boswellic acid, is the terpene that is less present in the brain, since it has the highest molecular weight among boswellic acids. The same applies for the other triterpenes according to the present invention.

This demonstrates that heterocyclic triterpenes cross only partially the BBB and require a carrier, constituted in our invention by HA₄, for the likely conjugation performed by the rotating magnetic field method.

2. Transport of GAGs and terpenes to the CNS through a transnasal route. Intranasal delivery is emerging as a noninvasive and well-tolerated option for reaching the CNS with minimal peripheral exposure and therefore without side effects. Moreover, this method facilitates the delivery of substances even of large size (such as growth factors, receptor antagonists, hormones, peptides, proteins, stem cells) which cannot cross the BBB, and can treat diseases which until now were practically orphan, such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, ischemic events including strokes. The future of the transnasal route to the CNS appears to be highly promising. The best prospects for the use of drugs through a transnasal route seem to be for insulin, for oxytocin, and for many other substances which are still in the preclinical stage, such as stem cells. These are substances with a molecular weight below 10,000 Da and measuring 30 nm.

The delivery of substances to the brain can occur by means of two main cranial nerve routes, associated with the olfactory and trigeminal nerves, after paracellular or transcellular transport through the nasal olfactory or respiratory epithelium.

After transport through the olfactory or respiratory epithelium to the lamina propria, experimental data indicate that proteins can access the perineural, perivascular or lymphatic compartments associated with the olfactory and trigeminal nerves, connecting at one end to the olfactory bulb through perforations in the lamina cribrosa and from there to the anterior olfactory nucleus and to the frontal cortex; at the other end, to the peripheral trigeminal system, connecting the nasal passages to the brain stem and the caudal cerebral regions via the anterior foramen lacerum [Thorne RG et al. 2004].

Furthermore, experimentally, the same marker, administered nasally, impregnates not only the CNS but also the deep cervical lymph nodes, indicating that the intranasal route can be utilized to access both the brain and the lymphatic system with a double therapeutic intent, like with interferon-beta for multiple sclerosis [Ross TM et al. 2004].

It is argued that transnasal delivery does not require bonding the drug with a receptor and does not undergo an axonal transport [Pardridge WM 2015] but occurs mainly by diffusion of the molecules through the extracellular space. Extracellular transport is swift: many drugs with a concentration very close to that obtained with intravenous administration cross in 5-10 minutes [Chapman C et al. 2013]. For some drugs, such as benzodiazepines, intranasal administration appears to be as effective as rectal administration [Holsti M et al. 2010], if not more [Figin T et al. 2002]. For naloxone there is no substantial difference between intravenous administration and transnasal administration (response within 8.1 minutes versus 12.9 minutes, respectively) [Ashton H 2005].

The delivery of the cells is enhanced by treatments that increase nasal permeability, such as with hyaluronidase applied in the nose 30 minutes prior to the application of the cells [Danielyan L et al. 2009] and with matrix metalloproteinase-9 (MMP-9) [Lochhead JJ and Thorne RG 2012].

This justifies the possibility to utilize oligo-GAGs (HA₄, HS₂-₄ and CS-E₂_₄) as transnasal triterpene absorption activators, directed to the CNS. The invention of this is claimed.

Transnasal transport is ideal for the product according to the present invention, especially because the method of administration is simple, straightforward (a simple spray device is sufficient), has high efficiency (the dispersed and not absorbed part is minimal), and does not require particular skill and involvement on the part of the patient, especially in the case of a neurological patient. The example of preparation of the product presented in the method is aimed indeed at transnasal administration. In fact, with 40+20+8 mg/ml respectively of HA₄, HS₂_₄ and CS-E₂_₄, 10 drops (= 0.5 ml) are sufficient to reach the daily therapeutic dose. The invention of this is claimed.

The preparation according to the present invention opens new therapeutic perspectives based on its transnasal and perineural transport to the CNS and then on its lymphatic drainage from the CNS along the subarachnoid space of the optic nerve, the perilymphatic space of the inner ear, the subarachnoid space and the transverse sinuses, perivascular lymphatic drainage towards the deep and lumbar para-aortic cervical lymph nodes.

The preparation can prove itself useful in the following afflictions:

- of the meninges, such as microbial, bacterial and viral infections;

- of the vestibulocochlear nerve, such as Meniere's disease, vestibular neuronitis, herpes zoster oticus, vertigo, drug-induced ototoxicity; phlogosis of the middle ear, such as otitis and mastoiditides;

- of the optic nerve and of the eye socket, such as retrobulbar neuritis, toxic amblyopia, optical atrophy, orbital cellulitis, thrombosis of the sinus cavernosus;

- of the olfactory nerve and of the ethmoid bone, as in disorders of olfaction and in ethmoiditides;

- of the trigeminal nerve, as in trigeminal neuralgia and in other inflammatory afflictions, probably also in herpes simplex labialis, by antiinflammatory action and against the herpes virus [Bevilacqua M 2005],

- of the facial nerve, in facial nerve paralysis,

- of deglutition nerves IX, X, XII (deglutition disorders).

Morbid conditions that benefit from the present invention

1. This chapter does not consider all the morbid conditions that benefit from the preparations that derive from the present invention and relate to virtually all fields of medicine (orthopedics, plastic surgery, dermatology, ophthalmology, rheumatology, pneumology, gastroenterology, immunology, otorhinolaryngology, cardiology, urology and gynecology) and does not consider the methods of administration (topical applications, through a pulmonary route, through an enteral route, through a parenteral route, through an endovisceral route, etc.), because they would require a separate description, despite being part of the present invention, and would not add novelty with respect to what was mentioned earlier regarding the anti-inflammatory, immune-modulating, antitumor, neurotrophic activities of the components of the product according to the present invention, activities which are estimated to be surprisingly more substantial than those of common commercially available preparations, and documented by the observations of the Author already with boswellic triterpenes. Therefore, the use of the present invention applies also to these sectors.

2. A special section is dedicated to rare connective tissue diseases (juvenile hyaline fibromatosis, osteogenesis imperfecta) or infrequent ones

(scleroderma, dermatomyositis) which lack a therapy, characterized by predominant involvement of chondroitin sulfate, which can benefit from treatment with HA₄ + terpenes + HS₂-₄ + CS-E₂_₄

3. Another special section is dedicated to neuropsychiatric morbid conditions, which lack a therapy and can benefit from treatment with HA₄ + terpenes + HS₂_₄ + CS-E₂_₄ .

Disorders of connective tissue with predominant involvement of chondroitin sulfates

(Therapy: HA₄ + triterpenes CS-E₂_₄)

Juvenile hyaline fibromatosis (JHF)

Juvenile hyaline fibromatosis is a rare autosomal recessive orphan hereditary disease, characterized by multiple tumor-like nodular skin lesions, gingival hypertrophy, flexion contractures of the large articulations with osteolytic lesions. Histologically, deposition of hyaline material in the extracellular spaces of the dermis [Malathi BG et al 2006].

Recent lesions show a high density of fibroblasts-like cells interlocked in an amorphous matrix of glycoproteins and hyaluronic acid, with a modest increase in chondroitin sulfates A and C (CS-A, CS-C) and dermatan sulfates (DS) [Mayer-da- Silva A et al. 1998]; the hyaline material consists mostly of type IV collagen and a reduced increase in type I collagen (4: 1 ratio) [Katagiri K et al. 1996]. In old lesions, the matrix was constituted mainly by CS-A and CS-C [Mayer-da- Silva A et al. 1998].

Recently, it has been observed that JHF lesions exhibit a significant reduction in hyaluronic acid with a down-regulation of hyaluronan synthase HAS-1 and HAS-2, and a high increase in DS and CS compared with JHF skin tissue free from lesions [Tzellos TG et al. 2009].

The present invention provides for the administration of HA₄ associated with CS-E₂_₄ and with triterpenes to reestablish homeostasis of the extracellular matrix, reducing the production of hyaluronic acid (with the mechanism already mentioned [Hagenfeld D et al. 2010]) and the production of chondroitin sulfates.

Osteogenesis imperfecta (OI)

OI is a heterogeneous group of collagen-correlated orphan hereditary diseases which mostly affect children but also adults and manifests itself with accelerated degenerative articular alterations, kyphoscoliosis and spondylolisthesis, short stature. Other manifestations of abnormal collagen include fragile dentition, loss of hearing, cardiac valve abnormalities.

Osteopenia is due to a deficit in the synthesis of extracellular matrix. Patients with different types of OI have reduced growth factors IGF-1 and IGFBP-3 (insulin-like growth factor- 1 and IGF binding protein-3) [Hoyer- Kuhn H, Hobing L et al. 2016].

In OI patients with respect to normal subjects of the same age, collagen synthesis in the extracellular matrix is reduced by 40%; osteonectin and three proteoglycans (CS-PG, biglycan and decorin) are reduced significantly; thrombospondin, fibronectin and hyaluronic acid are increased [Fedarko NS et al. 1996].

In type IIA and IIB OI, GAGs content increased considerably with respect to controls of the same age and less with respect to older control subjects. DS, CS, HA were higher in the medium than in the cells [Kapoor R et al. 1983]. HA synthase increased by 1.3-2 times in OI with respect to controls [Turakainen H et al. 1980]. The removal of HA with high molecular weight weight does not affect the secretion of collagen in 01 cell lines [Turakainen H 1983].

Current therapy provides for the administration of human monoclonal antibody (IgG2) [Hoyer-Kuhn H, Franklin J et al 2016], biphosphonates [Lindahl K et al. 2016].

The Author believes that the administration of HA₄, CS-E₂_₄ can be useful in this heterogeneous group of collagen- correlated diseases.

Scleroderma

Scleroderma is a chronic disease of unknown etiology, characterized by diffuse fibrosis, degenerative alterations and vascular anomalies of the skin, of the articular structures and of the internal organs.

Serum levels of hyaluronic acid and hyaluronidase (Hyal-1) are high and are associated with the severity of the disease and the immune abnormalities [Yoshizaki A et al. 2008] especially in the early stage of the disease [Neudecker BA et al. 2004]. Disaccharides of dermatan sulfate and chondroitin sulfate C are also high [Higuchi T et al. 1994]. In skin lesions there is an increase in dermatan sulfate disaccharides and a relative decrease of hyaluronic acid disaccharides [Yokoyama Y et al. 1997]. An increase in type I and III procollagen has been observed [Sondegaard K et al. 1997].

There are no drugs capable of influencing significantly the natural course of systemic sclerosis, although various preparations can be used in the treatment of specific symptoms or organ diseases, such as corticosteroids, penicillamine, immunosuppressive drugs, all burdened by considerable undesirable side effects.

A successful attempt has been made to transplant stromal cells of adipose origin in a solution of hyaluronic acid [Scuderi N et al. 2013].

A derivative of oleanolic acid has shown to improve dermal fibrosis in murine models of scleroderma and in fibroblasts explanted from human skin [Wei J et al. 2014]. These data suggest that oligosaccharides of hyaluronic acid and of other glycosaminoglycans that reset the extracellular matrix and limit the efflux of cytoplasmic HA, associated with triterpenes, that have an antiinflammatory and anti-hyaluronidase action and an action on collagen formation, may be useful in scleroderma and are a step forward in the therapy of this highly invalidating disease.

Dermatomyositis (DM)

This is a systemic disease of the connective tissue, characterized by inflammatory and degenerative alterations of the muscles and often also of the skin.

Hyaluronic acid (HA) has a role in regulating the immune response, stimulating the expression of inflammatory genes in various immune cells at the damage site; it is active in regulating the inflammatory response by recruitment of cells, migration of cells and release of cytokines and inflammation factors from fibroblasts [Fraser JR et al. 1997, Noble PW et al. 201 1].

In 75 cases of DM a significant increase in HA with respect to healthy controls [Victorino AA et al. 2015] and an accumulation of chondroitin sulfates, C₄S and C₆S, in particular of C₆S around vascular endothelial cells, have been observed [Chang LM et al. 2011]. Since C₄S and C₆S have different immunologic effects (C₄S inhibits cell adhesion, C₆S promotes it [Zou X et al. 2009]), their deregulation in skin mucinosis can contribute to the pathogenesis of these disorders. Indeed, serglycin proteoglycans have C₆S side chains in the endothelium, have a role in the adhesion of inflammatory cells, are increased by almost twice in DM vessels secreted in the sites of inflammation by monocytes/macrophages or fibroblasts, can accumulate with the degradation products in the extracellular matrix [Uhlin- Hansen L et al. 1993, Kim JS and Werth VP 201 1].

Corticosteroids are drugs of first choice in initial treatment; immunosuppressants in cases in which steroid therapy is not effective. Both drugs are burdened by considerable undesirable effects.

The Author believes that the administration of HA₄ + CS-E₂_₄ + triterpenes can yield more than traditional therapy and be truly useful in this disease for their capacity to induce a resetting of the extracellular matrix, for the anti-inflammatory action at the muscle level, for the anti-inflammatory and eutrophic action on the joints and skin.

Neurological and psychiatric diseases

Some morbid conditions are listed and described hereafter which involve in particular the nervous system according to the degree of involvement of glycosaminoglycans.

It is interesting to note that this is a new nosologic classification in the light of the therapeutic perspectives induced by this invention.

1. Diseases with prevalent involvement of hyaluronic acid

(therapy: HA₄ + triterpenes)

In this list of morbid conditions, some have already been described, others are only mentioned as they are implicit in the mechanisms of action of the constituent elements of the invention (e.g., depression, anxiety, memory reduction), others are described below.

- Somatoform disorders (somatization disorder, conversion disorder, hypochondriasis, pain disorder, body dysmorphic disorder).

- Anxiety disorders (panic attacks, stress disorder, hyperventilation syndrome).

- Mood disorders (sense of tiredness, nervousness, irritability, emotional lability, pathological laughter and crying, rage, aggressiveness, anger and violence, apathy, disorders of sexual functions, depression, dysthymic disorder, bipolar disorder, cyclothymic disorder).

- Memory disorders (psychogenic amnesia).

- Schizophrenia and correlated disorders (schizophrenia, short psychotic disorder, schizophreniform disorder, delusional disorder).

- Eating disorders (anorexia nervosa, bulimia nervosa, uncontrolled eating disorder, Dercum disease).

- Substance use and dependency.

- Headache (migraine, cluster headache, muscle tension headache).

- Pain (acute postoperative pain, neoplastic pain, neuropathic pain, psychogenic pain syndrome).

- Delirium (acute confusional state due to intoxication or abstinence).

- Neuro-ophthalmological and cranial nerve diseases (Horner's syndrome, internuclear ophthalmoplegia, gaze paralysis, paralysis of cranial nerves III, IV, VI, trigeminal neuralgia, glossopharyngeal neuralgia, facial nerve diseases).

- Neoplasms of the CNS.

In this chapter it is deemed appropriate to pay particular attention to two rare orphan diseases, which can benefit from therapy with HA₄ + triterpenes: Niemann-Pick disease and Dercum disease.

Niemann-Pick disease (NP)

Niemann-Pick disease is a lipidosis caused by an abnormality in lysosomes, which are incapable of degrading macromolecules that accumulate in these organelles to form cellular inclusions. NP comprises four types of disease (NP-A, B, C, D) which are then grouped into two distinct categories: NPA and NPB; NPC and NPD.

Niemann-Pick disease type A and type B (NPA and NPB)

This is a sphingolipidosis (a disease caused by the accumulation of sphingolipids in lysosomes), characterized by a deficit of acid sphingomyelinase (which catalyzes in lysosomes sphingomyelin in ceramide and phosphorylcholine) and by the clinical manifestation of hepatosplenomegaly and cherry red spots in the retina.

Sphingomyelinases are abundant in the brain. There is a close relationship between sphingomyelinases and cytosolic phospholipase A₂ because ceramide 1 -phosphate is a direct activator of cytosolic phospholipase A₂ [Pettus BJ et al. 2004, Huwiler A et al. 2000] and, as already mentioned, AKBA and asiatic acid inhibit phospholipase A₂ and boswellic acids inhibit the transformation of arachidonic acid into leukotrienes via 5 -lipoxygenase.

NPA and NPD therapy so far is only symptomatic. Attempts at enzyme replacement with olipudase alfa (recombinant human acid sphingomyelinase) [Mc Govern MM et al. 2016] and to increase the sensitivity of acid sphingomyelinase with a short chain C6 sphingomyelinase [Chuang WL et al. 2015] are still being discussed.

In NPA, it has been demonstrated that the increase in expression of neutral sphingomyelinase (induced by dexamethasone) removes the molecular and morphological aberrations and improves motor and memory deficits [Arroyo Al et al. 2014]. Boswellic acids have properties that are similar to those of dexamethasone [Anthoni C et al. 2006].

In NPB, the addition of apolipoprotein A-l (apoAl) cholesterol acceptor is sufficient to restore homeostasis [Lee Cy et al. 2013]. Ursolic and oleanolic acids increase significantly apoAl and the efflux of cholesterol [Zhang Y et al. 2016].

It has been demonstrated that the neutral sphingomyelinase deficit (also involved in NP) increases the synthesis of hyaluronic acid, inducing an up-regulation of hyaluron synthase 2 (HAS2), and reduces the production of ceramide by activating protein kinase B (Akt) [Qin J et al. 2012].

The administration of HA₄ may inhibit the up-regulation of HA by means of the already described mechanism for regulation of the K⁺ channel [Hagenfeld D et al. 2012].

Therefore, the association of HA₄ with triterpenes, mainly with boswellic, oleanolic and ursolic acid, has a rationale of its own and may be a step forward in the therapy of this orphan disease. Its invention is claimed. Dercum disease (MD)

This is the first case of MD of which the Author is aware in which there is an association between painful lipomatosis, familial obesity, alterations of temperature regulation and water retention phenomena. It is an extremely interesting case because it is accompanied by various symptoms which are telltales for the alteration of hypothalamic centers.

This is the first case in the literature that has been treated effectively by the Author with boswellic triterpenes.

The Author believes that:

- an association of triterpenes + HA₄ can achieve excellent results on painful lipomatosis;

- an association of triterpenes + HA₄ can achieve satisfactory results also on hypothalamic functions, such as temperature regulation, eating behavior, central and peripheral lipid metabolism.

2. Diseases with prevalent involvement of heparan sulfates

(therapy: HA₄ + triterpenes + H_4;6S₂_₄ + CS-E₂_₄)

Alzheimer's disease (MA)

MA is an age- correlated neurodegenerative disease with an extremely complicated physiopathology. In MA, neuroinflammation has an important role, characterized by the association of activated microglia and of a multitude of molecules of classic immunity with cerebral histological lesions, such as amyloid plaques (Αβ) and neurofibrillary tangles. These two lesions in MA are commonly considered as neuroinflammatory motors and native proteoglycan heparan sulfates (HS-PGs) mediate the induced oxidative stress Αβ and the hypercontractility of smooth muscle cells (VSMC) [Reynolds MR et al. 2016]. The proteoglycans chondroitin sulfate are considered the best inhibitors of axonal regeneration and take part in the activation of the inflammatory response [Rolls A et al. 2006].

While the first epidemiological studies had focused on the use of nonsteroid anti-inflammatory drugs (NSAIDs) and on stopping the progression of the disease, recent research suggests that once Αβ deposition has begun, NSAIDs are no longer effective and can be harmful due to their inhibitory effects on the activity of the microglia that mediates Αβ clearance [Imbibo BP 2009]; however, the use of these drugs in a very early stage of the disease has been demonstrated to be effective [Imbibo BP et al. 2010].

The present invention is a considerable and unquestionable step forward in the therapy of MA in terms of effectiveness and harmlessness.

Indeed, triterpenes are anti-inflammatory with the same effectiveness as NSAIDs and corticosteroids and without any side effects.

Moreover, an increase in the activity in the brain of isoforms of cytosolic A₂ phospholipase which catalyzes products that comprise arachidonic acid has been observed in neurological disorders which include excitotoxicity, traumatic damage to nerves and brain, cerebral ischemia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, allergic experimental encephalitis, pain, depression, bipolar disorder, schizophrenia and autism. In these conditions, oxidative stress, inflammatory reactions, alterations in lipid metabolism, accumulation of lipidic peroxides and of leukotrienes are triggered. Now:

- AKBA and asiatic acid inhibit phospholipase A₂ [Ong WY, Farooqui T et al. 2015], and boswellic acids inhibit the transformation of arachidonic acid into leukotrienes via 5-lipooxygenase [Safayhi H et al. 1992, Poeckel D et al. 2006].

- HA₄ act on nerve growth and regeneration, motor functional recovery, on a wide range of functions and behaviors of the CNS.

- Heparan sulfate tetrasaccharides likely contrast competitively the harmful action of native HS-PGs and reduce VSMC dysfunction by acting on the Ca⁺⁺ channels, in a manner similar to what hyaluronic acid tetrasaccharides do on K⁺ channels.

- Chondroitin sulfate disaccharides promote functional recovery by modulating the behavior of neurons and glia [Rolls A et al. 2004] and compete with intact CS and HS-PGs in the bond with Αβ, making it precipitate in non- toxic plaques [Fraser et al. 2001].

The invention of the association of HA₄ + triterpenes + H_4;6S₂,₄ + CS- E₂-4 in MA therapy is claimed.

Amyotrophic lateral sclerosis (ALS)

This is a hereditary disease which affects the motor neuron, characterized by diffuse denervation of the muscles and degeneration of the neurons of the anterior horns, of the motor nuclei of the brain stem and of the motor cortex, with secondary degeneration of the corticospinal tracts. A metabolic alteration of HA has been observed in ALS with increase in the tunica media of the basilar artery, in skin, in serum and in urine [Oyanagi K et al. 1999, Ono S et al. 2000]. Hyaluronic acid increases as the disease progresses while the other glycosaminoglycans (dermatan sulfate, chondroitin sulfate-4S, or chondroitin sulfate-6S) are not affected [Ono S et al. 1998]. CD44 receptors in astrocytes and microglia are involved in the progression of the disease and this suggests that the inflammatory responses that involve CD44 can have a role in this disease [Matsumoto T et al. 2012]. Studies on patients with sensitive and motor neuropathy have highlighted high anti-chondroitin sulfate antibody counts [Briani C et al. 1998].

Therefore, resorting to tetrasaccharides of HA, HA₄ for hyaluronic acid metabolic rebalancing , associated with heparan sulfate tetrasaccharides which attenuate the formation of glial scar from prevalent astrocyte reactivity [Hayashi N et al. 2004) and stimulate the regrowth of dopaminergic axons of the substantia nigra [Moon LD et al. 2002], and to triterpenes which have an anti-inflammatory, neuronal integrity maintenance, myelin stability and myelin regeneration action [Pareek TK et al. 2011 , Ding Y et al. 2014] by virtue of their action might be beneficial not only in stabilizing the extracellular matrix but also in removing its toxic products. Indeed, ursolic acid, which in mice has increased neomyogenesis, has been proposed for therapy of skeletal atrophy in ALS [Bakhtiari et al. 2015].

It is justified to hypothesize that KS and CS disaccharides also might be beneficial in ALS. The invention of the association of HA₄ + triterpenes + H_4;6S₂_₄ + CS- E₂_₄ in ALS is claimed.

Multiple sclerosis (MS)

The anatomic-pathological picture is characterized by distinct and multiple areas of myelin destruction, known as plaques, which can be located anywhere in the CNS. The neurons and most of the axis cylinders, but not all, are spared. Therapy: in the initial phase, cortisonic drugs; if relapses are frequent, interferon β and/or a polymer of myelin (copolymer I); in the chronic phase, even immunosuppressants.

In experimental autoimmune neuritis (EAN), an animal model of the

Guilliain-Barre syndrome (and, by analogy, also in experimental autoimmune encephalitis), it has been observed that there is an increase in the expression of keratan sulfate which exacerbates EAN, while the keratanase enzyme reduces it [Matsui H et al. 2013].

Boswellic acids (B. papyrifera, cps 300 mg x 2/day x 2 months) in 80 patients with multiple sclerosis have shown a significant improvement in visual-spatial memory, but without an effect on verbal memory and on information processing speed [Sedighi B et al. 2014]. As already mentioned, triterpenes have an anti- inflammatory, neuronal integrity maintenance, myelin stability and myelin regeneration action [Pareek TK et al. 201 1, Ding Y et al. 2014].

Improvements in motor function and in depression have been observed also by the Author with triterpenes by inhaling.

To conclude, in this disease, which has an unpredictable course, although it is difficult in the phase to judge the effectiveness of traditional therapy, it can be useful to associate the administration of the preparation according to the invention, i.e., HA₄ + triterpenes + H_4;6S₂_₄ + CS-E₂_₄, with the purpose of synergistic enhancement or even replacement of drugs, depending on the clinical course. The invention of this is claimed.

3. Rare genetic diseases with involvement of HA, CS, HS (therapy: HA₄ + triterpene + CS-E₂_₄ + H_4;6S₂_₄)

These are neurodegenerative diseases characterized often by severe neurological deficits, by delayed, incomplete and altered somatic development, mostly in infancy, which lack a therapy and await the provision of gene therapy. The Author has treated some cases with the simple administration of boswellic triterpenes with evident benefits on hypermotility and on agitation and aggressiveness crises, gastroesophageal reflux, adjustment of the sleep-wake rhythm, increased attention and cooperation capacity: all this has improved the quality of life of the patients and has reduced anguish of family members and the stressful need for continuous assistance. The invention of the therapeutic association HA₄ + triterpene + CS-E₂_₄ + H_4;6S₂_₄ in these diseases is claimed.

Anderson-Fabry disease (MF)

MF is a rare X-linked lysosomal accumulation disease caused by a deficit of the a-galactosidase A enzyme, with consequent accumulation of glycolipids in the vascular endothelium of many organs, including the skin, kidneys, nervous system, heart, causing inflammation and fibrosis. Neurological manifestations involve both the peripheral and the central nervous systems, with accumulation of globotriaosylceramide in the Schwann cells and in dorsal roots of ganglia with deposits in the neurons of the CNS [Tuttolomondo A et al. 2013]. Clinically, from the neurological standpoint: chronic pain, burning, itching and vasomotor disorders at the limbs, intolerance to heat and exercise; in some cases, a restless legs syndrome which also leads to sleep deprivation [Dominguez RO et al 2007].

From the therapeutic standpoint, apart from resorting to enzyme replacement [German DP 2000], which is still in the experimental phase, the Author believes that it may be beneficial to administer HA and CS oligosaccharides, associated with triterpenes, due to their capacity to induce a resetting of the extracellular matrix, promote axon integrity, perform a central and peripheral antinociceptive activity, perform an anti- inflammatory and anti-depressive action.

Huntington's disease (HM)

This is an autosomal-dominant hereditary neurodegenerative disease characterized by chorea, dystonia, parkinsonism, cognitive decline and psychiatric disorders, primarily manic-depressive, associated with prion- related hereditary diseases [Mano K et al. 2016].

The clinical symptoms have been attributed mainly to cortico striatal dysfunctions ascribed to neurotrophic deficits, such as BDNF (brain-derived neurotrophic factor), which has the role of regulating LTD (long-term depression) [Zuccato C and Cattaneo E 2014] and oxidative stress.

In the striatum of rats with induced HM, the presence of metalloproteinase 9 has been observed with vascular deterioration, absent in other cerebral regions [Duran-Vilaregut J et al. 201 1].

In the extracellular matrix of the cortex and thalamus of mice with induced HM, a reduced expression of tenascin-C [Kusakabe M et al. 2001], which is a glycoprotein which modulates adhesions and sends excitatory signals to the caudate and putamen, two regions in degeneration in HM, has been observed.

The so-called "wild-type huntingtin" is involved in the traffic of proteins between the Golgi apparatus and extracellular space and represses the expression of decorin (chondroitin sulfate proteoglycan) and of agrin and glypican, two heparan sulfate proteoglycans, which have a key role in synaptic remodeling and in neural plasticity and growth [Strehlow ANT et al. 2007].

A link has also been demonstrated between dysfunction of receptors of type A subunits of gamma-aminobutyric acid, GABA(A), and striatal extracellular matrix, which leads to behavior alterations similar to those of HM [Reinius B et al. 2015, Waldvogel HJ and Faull RL 2015]. (The GABA(A) receptor is a channel for chlorine at the post-synaptic level in many neurons. Agonist: benzodiazepines). In this severe disease, which in practice lacks a therapy and requires the administration of antipsychotic drugs (chlorpromazine or haloperidol) or reserpine with poor results and severe unwanted effects, resorting to a preparation according to the present invention can be decisive.

Kashin Beck disease (KB)

KB is a chronic osteoarticular deforming disease with altered metabolism of the cartilage matrix. Matrix metalloproteinase, aggrecanase and their inhibitors have an important role in the forming of cartilage and matrix degradation [Chen J et al. 2014].

Serum levels of NO, of TNFa were found to be significantly increased and associated with clinical manifestations of KB [Li XY et al. 2007].

In a recent study it was demonstrated that heparan sulfate 6-0- sulfotransferase has an important role in the cartilage of patients with osteoarthritis and with KB, that it regulates aggrecan metabolism and chondrocyte vitality, inducing hyperexpression of FGF-2 (fibroblast growth factor-2) [Wang W et al. 2015].

Even more recently, the effectiveness of hyaluronic acid through an intra-articular route and glucosamine sulfate orally was tested with positive results on pain, rigidity and function [Xia CT et al. 2016].

One deduces that the association HA₄ + triterpene + CS-E₂-₄ + H₄,₆S₂_₄ is rational as eutrophic, anti-inflammatory, protective and symptomatic preparations.

Marfan 's syndrome (MFS)

MFS is a dominant autosomal pathology that affects connective tissue. Since all organs contain connective tissue, manifestations of MFS affect many parts of the body, especially the skeletal system, the eyes, the heart and blood vessels, the lungs and the fibrous membranes that cover the brain and the spinal column.

MFS is a condition caused by genetic mutation of fibrillin- 1. It is associated with aneurysm of the aorta, characterized by destruction of the elastic laminae, accumulation of glycosaminoglycans, apoptosis of smooth vascular cells with minimal inflammatory response [Nataatmadja M et al. 2006].

In MFS, in Weil-Marchesani syndrome and in so-called "stiff skin syndrome", genetic mutations of fibrillin- 1 have been observed, while mutation of fibrillin-2 has been observed in congenital arachnodactyly.

Fibrillin- 1, -2, -3 constitute the family of fibrillins and are the main components of microfibrils. Proteoglycans and glycosaminoglycans have been located on microfibrils and participate in their assembly: biglycan, perlecan, decorin and dermatan sulfate interact with microfibrils, forming ternary complexes with fibrillin- 1 and glycoproteins- 1 associated with microfibrils in the pericellular matrix. Their modifications regulate interaction with the microfibrils. For example, fibrillin- 1 interacts only with highly sulfated heparan sulfate. However, their exact role in the biogenesis of microfibrils has not yet been defined. The integrity of microfibers is destroyed by chondroitinase-4,6-sulfate lyase [Sabatier L et al. 2014].

In MFS, a hyperexpression of TGF-β-Ι (transforming growth factor- beta- 1) has been observed which induces HAS (hyaluronan synthase) to increase the synthesis of hyaluronic acid. HA interacts with CD44 and RHAMM and therefore on cellular behavior and on homeostasis of the extracellular matrix [Nataatmadja M et al. 2006]. The effect on HA is specific and does not occur for chondroitin sulfate and for dextran sulfate [Lamberg SI 1978].

In MFS, a hyaluronan synthase activity 3 to 10 times greater than controls and an HA degradation activity of the same extent as controls has been observed in skin fibroblasts [Appel A et al. 1979]. This leads to hypothesize that the administration of HA₄ tetrasaccharides may temper the production of HA polymers, with a mechanism already described elsewhere [Hagenfeld D et al. 2012], and together with CS-E₄ and H_4;6S₄ contribute to reset the extracellular matrix; moreover, triterpenes can affect elastogenesis beneficially.

Mucopolysaccharidosis (MPS)

Mucopolysaccharidoses derive from attenuation or loss of enzyme activity required for lysosomal degradation of hyaluronic acid, heparan sulfate, chondroitin/dermatan sulfate and keratan sulfate. Various syndromal patterns of MPS are known which are represented by a characteristic facies, dysplasia of the skeleton, mental deficit, opacity of the cornea, hepatosplenomegaly. 11 types are known, depending on the enzyme deficit and the excess of GAG in urine.

Mental retardation seems to be correlated with urinary mucopolysaccharides with high molecular weight, whereas smaller fragments are observed in light forms; obviously, in healthy subjects there are only oligofragments caused by the catalytic activity of enzymes that are absent in MPS patients [Coppa G et al 2015].

It is probable that the accumulation of pericellular HA within the articular cartilage of MPS causes both destruction in the organization of ECM and a potential increase in inflammatory cytokines [Martin DC et al. 2008].

Current therapy is based on the transplantation of hemopoietic stem cells (which is not entirely free from complications) and on enzyme replacement therapy [Franco JF et al. 2016] with encouraging but modest results and not without side effects [Vairo F et al. 2015, Capanoglu M et al. 2016]. Gene therapy research is promising [Di Natale P et al. 2002].

It is very likely that in these morbid conditions the administration of hyaluronic acid tetrasaccharides (HA₄), of chondroitin sulfate (CS₂, CS₄) and of heparan sulfate, depending on the GAG eliminated in excess with diuresis, may: a) limit the production of glycosaminoglycan involved for an already described mechanism of homeostasis on the neurotrophic and extracellular matrix; b) be synergistic with enzyme replacement, improving the entire syndrome pattern.

Triterpenes can limit the phlogistic component, give their contribution to neuronal trophism, to psychic and intellectual conditions, antinociceptive (MPS I, II, VI) and to the extraneurologic syndrome pattern.

Type C and D Niemann-Pick disease (NPC, NPD) is biochemically, genetically and clinically distinct from types A and B.

The rare types C and D of Niemann-Pick disease (autosomal recessive hereditary) are characterized by a defect in intracellular transport of LDL- cholesterol, which causes an accumulation of non-esterified cholesterol in many organs and tissues, with clinical manifestations of neurodegeneration, such as neurovegetative dystonia, ataxia, epilepsy, saccadic eye motions, cognitive and anamnestic decline, progressive dementia; hepatosplenomegaly [Santos-Lozano A et al. 2015].

Until a few years ago, the treatment of NPC was only symptomatic, based on antiepileptic, anticholinergic, antidepressant drugs. Recently Miglustat, an imino sugar which reversibly inhibits glycosphingolipids synthesis but is not free from important side effects, has been introduced and appears to be capable of delaying neurological manifestations in patients with NP, type 1 Gaucher disease, Sandhoff type GM2 gangliosidosis [Lyseng- Williamson KA 2014].

In NPC1, diffuse neuronal loss, dysmyelination and neurodegeneration, abnormal axonal swelling, activation of astrocytes and microglia have been observed. The accumulation of sphingolipids influences the autophagic metabolism of the amyloid precursor protein and promotes the generation of Αβ like in Alzheimer's disease, worsening neurodegeneration [Tamboli IY et al. 2011].

The structure of the small lysosomal glycoprotein NPC2 shows a malleable binding with cholesterol which consists of a vast range of sterol- binding specificities. It is therefore likely that it can also bind with triterpenes, which have a structure that is similar to sterols (molecular weight almost identical to that of cholesterol sulfate, hydroxyl in position 3β) [Xu S, Benoff B et al. 2007].

The conclusion is that the HA₄-triterpenes association is rational and can be a step forward in the treatment of this disease, both as sole therapy and as a support and synergy with the poorly tolerated Miglustat.

Lowe oculocerebrorenal syndrome (OCRL).

Lowe's syndrome (OCRL) and Dent disease type 2 are constituted by a mutation of inositol-5-phosphatase OCRL-1.

OCRL is characterized by congenital cataract, hypotonia, increased susceptibility to epileptic crises, mental retardation, tubular renal dysfunction, arthropathy and pathological fractures with severe osteoporosis, skin cysts, cryptorchidism, considerable reduction in statural growth, delayed puberty, symptoms which mostly develop in the neonatal and infantile period [Bokenkamp A and Ludwig N, 2016, Kim HK et al. 2014].

A reduced synthesis of chondroitin sulfates and dermatan sulfates with hyposulfation in C4 and 6 [Donnelly PV et al. 1984] has been observed. Significant reduction of heparan sulfates and of hyaluronic acid has not been observed [Fukui S et al. 1981]. Axonal degenerations and bodies included in Schwann cells and in fibroblasts have been noticed [Wisniewski KE et al. 1984]. However, these data were not confirmed subsequently [Harper GS at al. 1987].

In the Author's opinion, despite the scarcity and less-than-perfect agreement of the data in the literature, in the absence of a specific gene or enzyme replacement therapy, considering the clinical condition of OCRL patients, a therapeutic approach with CS-E₄, associated with HA₄, is justified for resetting, homeostasis of the extracellular matrix, neuroprotection, eutrophism at the CNS level; associated also with triterpenes for trophism at the level of the skeletal system, since they reduce significantly the degradation of glycosaminoglycans [Reddy GK et al. 1989, Suneela D and Dipmala P 2012] and increase the production of glycosaminoglycans in human chondrocytes [Sengupta K et al. 2011].

Refsum Disease (MR)

MR is a rare recessive familial disorder which is orphan and is caused by a deficit of phytanic acid a-oxidase, an enzyme which metabolizes phytanic acid. MR is characterized by hyperproteinorrachia, cerebellar degeneration and ataxia, anosmia, peripheral neuropathy, retinitis pigmentosa, bone and skin alterations, alterations due to the neurotoxic action of phytanic acid [Busanello EN et al. 2013].

Leukoencephalopathy involves the periventricular white matter, the subcortical area, the semioval center, the thalamus and the corpus callosum, the brainstem, with cortical and subcortical atrophy and dilation of the ventricles [Bompaire F et al. 2015]. Furthermore, magnetic resonance images are compatible with a peroxysome disorder [Kilic M et al 2015] with release of reactive oxygen substances (ROS) for the lipotoxicity of phytanic acid [Schoenfeld P and Reiser G 2016].

A treatment that keeps the levels of phytanic acid in plasma low, such as a diet without phytanic acid (without chlorophyll), helps to stop the progression of the disease and to prevent relapses.

The Author believes that the administration of oligo- glycosaminoglycans associated with terpenes for the pathogenetic mechanisms described several times at the CNS level and on the peripheral nerves (these substances cross the perineural barrier) and for the antiinflammatory action and on ROS, can slow the functional destructive phenomena of the nervous system and act on bone metabolism.

Sandhoff disease

Sandhoff disease is a rare hereditary disease which progressively destroys the nervous cells of the brain and in the spinal cord.

This disease is very similar to Tay-Sachs disease and it, too, has an impairment of catabolism of glycosaminoglycans [Stirling J 1974, Cantz M et al. 1975].

Ashkenazi Tay-Sachs disease (MTS)

This is a rare orphan recessive autosomal hereditary disease, in which neurodegeneration is caused by the accumulation of ganglioside G_M2 in neuronal lysosomes due to a deficit of the enzyme hexosaminidase A.

In this severe disease there is also a metabolic disorder of GAGs and likely a severe disarray of the extracellular matrix. There is in fact a defective ability to degrade chondroitin sulfates and hyaluronic acid [Yutaka T et al. 1982, Yukata T et al. 1984]; the enzymes β-Ν- acetylhexosaminidase A and B involved in glycosidosis G_Mi do not catabolize keratan sulfates [Ludolph T et al. 1981]; there is an abnormal urinary excretion of glycosaminoglycan sulfates, in particular heparan sulfates.

Gilles de la Tourette syndrome (ST).

This is a neurological and behavioral disorder which consists of motor and phonic tics. In addition to tics, many patients with ST have a variety of comorbidities, including attention deficit, hyperactivity disorder, obsessive-compulsive disorder, depression [Piedad JC and Cavanna AE 2016] up to suicidal ideation [Johnco C et al. 2016]. Studies that assess the physiopathology of tics have been oriented toward a dysfunction of the cortico-striatal-pallidal-thalamic-cortical circuits, changes in the interhemispheric inherent connectivity [Chelse AB and Blackburn J 2015, Liao W et al. 2016] but the mechanism of hyperkinetic movements has not been clarified. A reduction of glycosaminoglycans in serum has been observed [Kurup RK and Kurup PA 2002].

Treatment of ST is multidisciplinary and typically involves behavioral therapy, oral medications, injections of botulinum toxin. Deep cerebral stimulation can be considered useful for "malignant" ST, which is refractory to conventional therapy [Thenganatt MA and Jankovic J 2016]. It should be stressed that postmortem histological examinations of ST patients have demonstrated a decrease in striatal interneurons and FSIs (parvalbumin-expressing fast-spiking interneurons) and a deficit of FSIs causes anxiety and repetitive movements triggered by stress [Xu M et al. 2016]. In practice there is no therapy.

Werner syndrome (SW)

This is a rare recessive autosomal hereditary disease which causes early aging, reduced stature and body weight, scleroderma-like skin alterations, endocrine-metabolic abnormalities (diabetes mellitus, hypogonadism, central obesity with visceral deposition of fat). It appears that genome instability is the major mechanism of biological aging. The main causes of death are tumor and myocardial infarction, and wear and dissolution of the telomere is apparently at their origin, including cognitive decline [Goletto T et al. 2015, Ishikawa N et al. 2016].

In cultures of skin fibroblasts of SW patients, the quantity of HA secreted in the medium was 2-3 times greater than cultures of normal fibroblasts, with short-chain HA polymers [Nakumara T et al. 1992]. In atrophic and sclerotic skin of patients with SW, the total content of the main disaccharides was reduced significantly, but with a normal ratio between HA and DS [Higuchi T et al. 1994]. A considerable increase in the synthesis of GAG with 80% HA and 10-20% sulfated GAG, against 50% of both GAGs in normal fibroblasts has been found in SW fibroblasts [Fujiwara Y and Ichihashi M 1985]. The second major components are heparan sulfates [Murata K, Kudo M et al. 1985]. The turnover of GAGs in SW fibroblasts, however, is not particularly impaired [Cowles EA et al. 1987].

In SW, HA is increased in serum, especially if compared with subjects of the same age, and is increased also in urine [Tanabe M and Goto M 2001]. In urine, HA is characterized by large fragments; heparan sulfates are also increased [Kieras FJ et al 1986, Satake S et al. 1983, Murata K 1982, Murata K 1985]. In Hutchinson- Gilford syndrome (progeria), HA is increased in urine but the total quantity of GAGs is within normal ranges [Tokunaga M et al. 1978]. These results, however, are questioned by other researchers [Gordon LB et al. 2003].

A considerable increase in inflammatory cytokines from Th2 lymphocytes (IL-4, IL-6, IL-10, GM-CSF), from Thl lymphocytes (IL-2, TNFa, IL-12, IFNy) and an increase in products from monocytes/macrophages (MCP-1, basic FGF and G-CSF) has been demonstrated in SW. These changes can induce a systemic chronic inflammation, such as skin ulcer and diabetes mellitus, and more complicated immune changes with respect to normal subjects of the same age [Goto M et al. 2015]. An activation of p38MAP kinase, which accelerates senescence, was also demonstrated [Davis T et al. 2016].

It should be stressed in this regard that boswellic acids inhibit the expression of telomerase [Khan S et al. 201 1], have an anti-inflammatory action, an action on lipid metabolism, neurotrophic action, and that GAGs oligosaccharides have an action on the homeostasis of the extracellular matrix in the nervous system and a neurotrophic action on water metabolism, antidepression. There is no therapy.

Cerebrotendinous xanthomatosis (XC).

XC, or Van Bogaert disease, is a rare recessive autosomal hereditary disorder of lipid metabolism, characterized by progressive ataxia, dementia, cataract, tendon xanthomas, intractable diarrhea, premature atherosclerosis and osteoporosis. The disease becomes manifest at around 20 years of age with epilepsy and parkinsonism and acquires complications with psychiatric manifestations of depression, agitation, hallucinations and suicidal ideas [Grandas F et al. 2002, Fraidakis MJ 2013, Nie S et al. 2014].

Cholestanol (didihydrocholesterol), which generally is hardly measurable in the body, occurs in increased concentrations in the CNS, in the lungs, in blood and in xanthomas. The underlying defect of this disorder is the deficit of a hepatic enzyme (27-hydroxylase) which catalyzes the hydroxylation of an intermediate sterol of the biosynthetic route of biliary acids, thus causing the accumulation of cholesterol in most tissues, including the brain, and in bile and the forming of xanthomas. Diffuse abnormalities of white matter, cerebral and cerebellar atrophy, alterations of the dentate nucleus, deposits of lipids in Schwann cells with polyneuropathy, demyelinating peripheral neuropathy have been observed [Argov Z et al. 1986, Voiculescu V et al. 1987, Razi SM et al. 2016].

In this disaster, with destruction and disarray of neurons, glia and extracellular matrix in the brain and disaster in the structure of peripheral nerves, the administration of oligo-glycosaminoglycans associated with terpenes (HA₄ + triterpene + CS-E₂_₄ + H_4;6S₂_₄) for the pathogenetic mechanisms already described several times at the CNS level, on peripheral nerves, on lipid metabolism, and on bone metabolism can only provide benefits and slow the functional destructive phenomena of the nervous system.

The disclosures in Italian Patent Application No. 102016000079773

(UA2016A005630) from which this application claims priority are incorporated herein by reference.

Claims

1. The present invention is based on the association of at least two components:

a) tetrasaccharides of hyaluronic acid HA₄;

b) the following pentacyclic triterpenes: 1 l-keto-P-boswellic acid

(KBA), 3-O-acetyl-l l-keto- -boswellic acid, 9, 11-dehydro-a-boswellic acid, 9,1 1 -deny dro-P-boswellic acid, a-boswellic acid, β-boswellic acid, lupeolic acid, 3-0-acetyl-9, l l-dehydro-a-boswellic acid, 3-0-acetyl-9, l l- dehydro- -boswellic acid, 3-O-acetyl-a-boswellic acid, 3-0-acetyl- - boswellic acid, oleanolic acid, ursolic acid, betulinic acid, glycyrrhetinic acid, asiatic acid.

2. The compound according to claim 1 , characterized in that the associated terpenes can be always the same or can be chosen among the ones listed above, depending on the morbid condition to be treated.

3. The compound according to claim 1, characterized in that it provides preferably for the equivalent association of hyaluronic acid tetrasaccharides with triterpenes, for example:

1 HA₄ + 1 triterpene

2 HA₄ + 2 triterpenes

3 HA₄ + 3 triterpenes

and so forth.

Since the molecular weight of 1 HA₄ is 766 Da and the average weight of pentacyclic triterpenes is 474 Da, the weight ratio of the two substances in the product is preferably at least 2: 1.

4. The compound according to claim 1, characterized in that in the product the final concentration of HA₄ in the solution is 40-80 mg/ml and the final concentration of terpenes is 20-40 mg/ml.

5. The compound according to claim 1 , characterized in that the therapeutic dose of triterpenes in the adult human in the various morbid conditions is preferably around 10-20 mg/day and in that the range of the triterpenes might vary from 0.10 to 0.50 mg/kg of body weight.

6. The compound according to claim 1, characterized in that it provides, depending on the pathologies to be treated indicated in the text and summarized hereinafter, also for an additional association with the two components, HA₄ + triterpenes, of disaccharides or tetrasaccharides of chondroitin sulfate (CS₄), in particular disaccharides and tetrasaccharides sulfated both in C4 and in C6, indicated as CS-E₂-₄, or the association of heparan sulfate disaccharides or tetrasaccharides, preferably disulfated or trisulfated (I-S and II-S), indicated as H_4;6S₂_₄,₄, or the association of both, CS-E₂_₄ and H₄,₆S₂_₄, with HA₄ + triterpenes.

7. The compound according to claim 1 , characterized in that the concentration of HA₄ with respect to the sulfur GAGs (CS and HS) in the final solution is preferably 5: 1, considering that hyaluronic acid is in a considerably higher quantity in the body.

8. The compound according to claim 1 , characterized in that the concentration ratio between the 2 GAGs (CS:HS) is 2: 1.

9. The compound according to claim 1, characterized in that it provides for a pretreatment of the aqueous solution of HA₄ + the hydroalcoholic solution of triterpenes in a rotating magnetic field for a variable time of 60-120 min with a field strength between 30 and 100 mT (millitesla), preferably between 50 and 75 mT at a temperature of 85 ± 5°C for approximately one hour.

10. The compound according to claim 1, characterized in that the treatment of the solution obtained above continues with the use of a rotating magnetic field with a strength that can vary from 100 to 300 mT, preferably between 150 and 200 mT. Temperature between 60 and 90°C, preferably around 80°C. Operation performed with an average time of 90 minutes, oscillating between 60 and 120 min.

11. The compound according to claim 1, characterized in that it provides for the optional addition to the preparation cited above of CS and HS in the proportions according to claims 7 and 8 and treatment in a rotating magnetic field for a variable time of 30-60 min with a field strength that is halved with respect to the preceding one, between 50 and 150 mT, preferably between 75 and 100 mT, at a temperature between 60 and 90°C, preferably around 75°C.

12. The compound according to claim 1, characterized by an aqueous solution with a composition preferably thus composed:

HA₄ 40 mg/ml, triterpenes 20 mg/ml, CS-E₂_₄ 6 mg/ml, H_4;6S₂_₄ 3 mg/ml or

HA₄ 40 mg/ml, triterpenes 20 mg/ml, CS-E₂_₄ 8 mg/ml

or

HA₄ 40 mg/ml, triterpenes 20 mg/ml, H_4;6S₂_₄ 8 mg/ml

13. The compound according to claim 1, characterized in that the composition HA₄ ± CS-E₂_₄ ± H_4;6S₂_₄ + triterpenes varies according to the physiopathology of the disease to be treated.

14. The compound according to claim 1, characterized in that it provides for obtaining a mixture of substances in an aqueous solution which is electrostatically stable at ambient temperature.

15. Use of the HA₄ + triterpenes [+ any addition of oligo-GAGs (CS-

E₂_₄ and/or H_4;6S₂_₄)] compound administered nasally with any provided means (e.g., aerosol, spray, ointment, drops, nanoconstructs).

16. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound administered through a pulmonary route with any provided means (e.g., aerosol, spray, endotracheal instillations, nanoconstructs) for disorders of the respiratory system.

17. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound administered through a pulmonary route with any provided means (e.g., aerosol, spray, endotracheal instillations, nanoconstructs) for systemic disorders.

18. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound administered orally with any provided means (e.g., tablets, suspensions, sachets, nanoconstructs) for gastrointestinal disorders.

19. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound according to claim 17 orally also for treating systemic disorders.

20. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound through a parenteral route (intravenous, intramuscular, subcutaneous) or endovisceral route (e.g., endovesical, endorectal) or endoarticular route, provided with any means (e.g., nanoconstructs, solutions, suppositories).

21. Use of the HA₄ + triterpenes (+ any addition of oligo-GAGs) compound administered through a cutaneous, mucosal, intradermal route with any provided means for curative or aesthetic purposes (e.g., nanoconstructs, hydrogel, ointment, cream, patches, infiltrations).

22. Use of the compound in disorders with a predominant involvement of hyaluronic acid, in which a treatment with HA₄ + triterpenes is indicated: somatoform disorders, anxiety disorders, mood disorders, memory disorders, schizophrenia and correlated disorders, food behavior disorders, substance use and dependence, cephalea, pain, delirium, neuro- ophthalmological and cranial nerve diseases, Niemann-Pick disease type A and type B, Dercum disease, obstructive sleep apnea syndrome, chronic fatigue syndrome, CNS neoplasms.

23. Use of the compound in connective diseases with predominant involvement of chondroitin sulfates in which treatment with HA₄ + triterpenes + CS-E₂_₄ is indicated: juvenile hyaline fibromatosis, osteogenesis imperfecta, scleroderma, dermatomyositis.

24. Use of the compound in diseases with predominant involvement of heparan sulfates in which a treatment with HA₄ + triterpenes + H_4;6S₂_₄ is indicated: peripheral neuropathies: Guillain-Barre syndrome, sensory and motor hereditary neuropathies type I, II, III; neurofibromatosis, neurodegenerative diseases: Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, leukoencephalopathy, cerebellar degeneration, aging neurology, cardiovascular diseases.

25. Use of the compound in rare genetic diseases with CNS involvement of HA, CS and HS and in which treatment with HA₄ + triterpene + CS-E₂-₄ + H₄,₆S₂_₄ is indicated: Anderson-Fabry disease, Huntington's disease, Kashin Beck disease, Marfan syndrome, mucopolysaccharidosis, Niemann-Pick disease C and D, Lowe oculocerebrorenal syndrome, Refsum disease, Sandhoff disease, Ashkenazi Tay-Sachs disease, Gilles de la Tourette's syndrome, Werner syndrome, cerebrotendinous xanthomatosis.