阅读说明：本技术 水溶性多糖衍生物、其制备工艺及其用途 (Water-soluble polysaccharide derivatives, process for their preparation and their use ) 是由 朱利奥·比安基尼 兰弗兰科·卡莱加罗 于 2018-05-29 设计创作，主要内容包括：公开了一种由聚氨基糖阳离子和阴离子组成的中性盐、以及用于制备该中性盐的程序和该中性盐作为生物材料和作为药物组合物中的成分的用途。事实上,所述中性盐已经令人惊讶地呈现出高的水溶性和高纯度,制备程序使最终产物中的高风险污染物的含量最小化,并且减少了反应时间和纯化时间。(Neutral salts composed of polyaminosaccharide cations and anions are disclosed, as well as procedures for preparing the neutral salts and use of the neutral salts as biomaterials and as ingredients in pharmaceutical compositions. In fact, the neutral salts have surprisingly been shown to have high water solubility and high purity, the preparation procedure minimizes the content of high risk contaminants in the final product, and reduces the reaction time and purification time.)

1. A neutral salt consisting of a polyaminosugar cation and an anion, wherein the polyaminosugar cation consists of three repeating units:

a) up to 25%

b) Less than 65% of

c) Up to 90%

Wherein R is an aldose or ketose moiety,

and is

Wherein the anion is monovalent, divalent, or trivalent.

2. The neutral salt of claim 1, wherein R is a moiety of formula (1):

wherein R is₁is-CH₂-or-CO-,

R₂is-OH or-NHCOCH₃，

R₃Is H, a monosaccharide, disaccharide or oligosaccharide,

or R is a moiety of formula (2):

R₄is a group-CH-,

R₅and R₆Independently of one another, H, a monosaccharide, a disaccharide or an oligosaccharide.

3. A neutral salt according to claim 2, wherein R₃、R₅And R₆Independently of each other, H, glucose, galactose, arabinose, xylose, mannose, lactose, trehalose, gentiobiose, cellobiose, cellotriose, maltose, maltotriose, chitobiose, chitotriose, mannobiose, melibiose, fructose, N-acetylglucosamine, N-acetylgalactosamine or a combination thereof.

4. The neutral salt of claim 1, wherein R is a lactose or galactose moiety.

5. The neutral salt of any one of claims 1-4, wherein in the polyaminosaccharide cation the repeating unit a) is present in a percentage of 5% to 20%, preferably 7% to 18%.

6. The neutral salt of any one of claims 1-5, wherein in the polyaminosaccharide cation the repeating unit b) is present in a percentage of 5% to 45%, preferably 20% to 40%.

7. The neutral salt of any one of claims 1-6, wherein in the polyaminosaccharide cation the repeating unit c) is present in a percentage of 45% to 90%, preferably 50% to 70%.

8. The neutral salt of any one of claims 1-7, wherein the anion is chloride, bromide, fluoride, iodide, acetate, trifluoroacetate, carbonate, bicarbonate, sulfate, bisulfate, C1-C10 alkylsulfate, C1-C6 alkylsulfonate, C6-C10 arylsulfonate, nitrate, hydrogenphosphate, dihydrogenphosphate, orthophosphate, oxalate, fumarate, ascorbate, citrate, gluconate, lactate, formate, tartrate, succinate, mandelate, p-toluenesulfonate, carboxylate, saccharate, benzoate, or a mixture thereof.

9. The neutral salt of any one of claims 1-8, wherein the neutral salt has a weight average molecular weight of up to 2500kDa, or the neutral salt has a number average molecular weight of up to 2000 kDa.

10. A process for preparing the neutral salt of claim 1, the process comprising the steps of:

i) providing a polyaminosugar polymer consisting of repeating units a) and repeating units b),

ii) reacting the polyaminosaccharide polymer with mono-, di-or oligosaccharides in an aqueous solution,

iii) adding an aminoborane to the reaction mixture,

iv) adding an acid until the pH is below 4,

v) adding an organic solvent, thereby precipitating the neutral salt, and

vi) isolating the precipitated neutral salt.

11. The process of claim 10, wherein the polyaminosaccharide polymer consists of 5-95% of repeating units a) and 95-5% of repeating units b).

12. The process of claim 10 or 11, wherein the aminoborane is 2-methylpyridine borane, 5-ethyl-2-methylpyridine borane, pyridine borane, trimethylamine borane, triethylamine borane, dimethylamine borane, tert-butylamine borane, or a mixture thereof.

13. The process according to any one of claims 10-12, wherein the organic solvent is acetone, methanol, ethanol, 2-propanol or a mixture thereof, preferably 2-propanol.

14. The process as claimed in any one of claims 10-13, wherein the molar ratio between mono-, di-or oligosaccharides and the recurring units b) of the polyaminosugar polymer is comprised between 0.5 and 30, or the molar ratio between aminoboranes and the recurring units b) of the polyaminosugar polymer is comprised between 0.75 and 20.

15. A pharmaceutical composition comprising at least one neutral salt of any one of claims 1-9 and a biologically active substance selected from the group consisting of collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid), polycaprolactone, gelatin, albumin, poly (glycolide-co-caprolactone), poly (glycolide-co-trimethylene carbonate), hydroxyapatite, tricalcium phosphate, dicalcium phosphate, decalcified bone matrix and mixtures thereof.

16. The pharmaceutical composition of claim 15, wherein the neutral salt and the biologically active substance are in a ratio of 10:1 to 1: 50.

17. The pharmaceutical composition of claim 15 or 16, wherein the neutral salt and the biologically active substance are in a ratio of 5:1 to 1:1, preferably 4:1 to 1:1, more preferably 3:1 to 1: 1.

18. The pharmaceutical composition of claim 17, wherein the biologically active substance is selected from the group consisting of heparin, chondroitin sulfate, collagen, sodium alginate, potassium alginate, magnesium alginate, and mixtures thereof.

19. The pharmaceutical composition of claim 15 or 16, wherein the neutral salt and the biologically active substance are in a ratio of 1:10 to 1:45, preferably 1:20 to 1:40, more preferably 1:25 to 1: 35.

20. The pharmaceutical composition of claim 19, wherein the biologically active substance is selected from the group consisting of hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralized bone matrix and mixtures thereof.

Technical Field

The present invention relates to neutral salts consisting of polyaminosaccharide cations and anions, and processes for preparing the neutral salts and the use of the neutral salts as biomaterials and as ingredients in pharmaceutical compositions.

In fact, the neutral salts according to the present invention have surprisingly shown high water solubility and high purity, since a preparation process has been developed which minimizes the content of high risk contaminants in the final product, reduces the reaction time and purification time, and facilitates varying the degree of substitution.

Background

Naturally occurring chitins generally have about 15% of repeating units deacetylated to the corresponding glucosamine (D-glucosamine), and are generally found in the presence of chitosan, i.e. similar polysaccharides corresponding to a high degree of deacetylation (> 60%), deacetylation of the repeating units N-acetylglucosamine to glucosamine makes available primary amino groups, which confers basic properties (basic property) on the polymer (chitosan pKa ≈ 6.3), the typical solubility profiles of chitin and chitosan (i.e. they are practically insoluble in water and common organic solvents) limit their potential industrial applications.

WO 2017/211776 a1 mentions a compound known as chitlac hydrochloride, wherein the chitlac is characterized by a degree of lactose substitution between 50% and 70% and is obtained from 200kDa-400kDa chitosan having a residual acetylation degree (degree of residual acetylation) of about 15%.

It is therefore an object of the present invention to provide a product that increases the industrial applicability of chitin and chitosan by improving their solubility profiles, while also improving their acceptability profile from a medical and pharmaceutical point of view.

Summary of The Invention

The object has been achieved by a neutral salt consisting of a polyaminosaccharide cation and anion as claimed in claim 1.

In another aspect, the invention relates to a process for preparing a neutral salt consisting of a polyaminosaccharide cation and anion.

In a further aspect, the invention relates to the use of said neutral salt as a biomaterial or scaffold for cell growth in the treatment of orthopaedic diseases.

In still a further aspect, the invention relates to the use of said neutral salt as a biomaterial or scaffold for cell growth in plastic cosmetic surgery, hemodialysis, cardiology, vasculology, ophthalmology, otorhinolaryngology, dentistry, gynecology, urology, dermatology and tissue repair.

In another aspect, the present invention relates to a pharmaceutical composition comprising a biologically active substance and at least one neutral salt consisting of a polyaminosaccharide cation and anion.

In a further aspect, the invention relates to the use of said pharmaceutical composition in the treatment of orthopaedic diseases.

For the purposes of the present invention, the term "orthopaedic diseases" refers to diseases affecting the musculoskeletal system, such as osteoarticular diseases, muscular diseases, ligament diseases and tendon diseases.

In still further aspects, the invention relates to the use of said pharmaceutical composition in plastic cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otorhinolaryngology, dentistry, gynecology, urology, dermatology and tissue repair.

As will become clear from the following detailed description and embodiments provided by way of non-limiting example, the neutral salts according to the present invention have surprisingly exhibited high water solubility and high purity, since a preparation process has been developed which minimizes the content of high-risk contaminants in the final product, reduces the reaction time and purification time, and facilitates varying the degree of substitution.

Detailed Description

Accordingly, the present invention relates to a neutral salt consisting of a polyaminosaccharide cation and an anion, wherein the polyaminosaccharide cation consists of three repeating units:

a) up to 25%

b) Less than 65% of

c) Up to 90%

Wherein R is an aldose or ketose moiety,

and is

Wherein the anion is monovalent, divalent or trivalent.

In fact, it has surprisingly been found that the neutral salts according to the invention reach a high solubility in water within a drastically reduced period of time, while also being suitable for pharmaceutical and medical applications.

The definition of "neutral salt" is meant to include all polymorphic forms, amorphous or crystalline forms, or co-crystalline forms, as well as anhydrous, hydrated and solvated forms.

The repeating units b) and c) have been shown as above to have a positive charge on the nitrogen atom, however, other forms of conjugate acids in equilibrium with the most likely ammonium form shown are not excluded.

Preferably, R is a moiety of formula (1):

wherein R is₁is-CH₂-or-CO-,

R₂is-OH or-NHCOCH₃，

R₃Is H, a monosaccharide, disaccharide or oligosaccharide,

or R is a moiety of formula (2):

R₄is a group-CH-,

R₅and R₆Independently of one another, H, a monosaccharide, a disaccharide or an oligosaccharide.

Preferably, R₃、R₅And R₆Independently of each other, H, glucose, galactose, arabinose, xylose, mannose, lactose, trehalose, gentiobiose, cellobiose, cellotriose, maltose, maltotriose, chitobiose, chitotriose, mannobiose, melibiose, fructose, N-acetylglucosamine, N-acetylgalactosamine or a combination thereof.

More preferably, R₃Is H, glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine or a combination thereof.

In a particularly preferred embodiment, R is a lactose or galactose moiety.

Preferably, in the polyaminosaccharide cation, the recurring units a) are present in a percentage of between 5% and 20%.

More preferably, in the polyaminosaccharide cation, the recurring units a) are present in a percentage of 7% to 18%.

Preferably, in the polyaminosaccharide cation, the recurring units b) are present in a percentage of between 5% and 45%.

More preferably, in the polyaminosaccharide cation, the recurring units b) are present in a percentage of between 20% and 40%.

Preferably, in the polyaminosaccharide cation, the recurring units c) are present in a percentage of 45% to 90%.

More preferably, in the polyaminosaccharide cation, the recurring units c) are present in a percentage of between 50% and 70%.

In a preferred embodiment, the polyaminosaccharide cation consists of:

from 5% to 20% of recurring units a), from 5% to 45% of recurring units b) and from 45% to 90% of recurring units c).

In a more preferred embodiment, the polyaminosaccharide cation consists of:

7% to 18% of recurring units a), 20% to 40% of recurring units b) and 50% to 70% of recurring units c).

Preferably, the anion is chloride, bromide, fluoride, iodide, acetate, trifluoroacetate, carbonate, bicarbonate, sulfate, bisulfate, C1-C10 alkylsulfate, C1-C6 alkylsulfonate, C6-C10 arylsulfonate, nitrate, hydrogenphosphate, dihydrogenphosphate, orthophosphate, oxalate, fumarate, ascorbate, citrate, gluconate, lactate, formate, tartrate, succinate, mandelate, p-toluenesulfonate, carboxylate, saccharate, benzoate, or a mixture thereof.

More preferably, the anion is chloride, bromide, acetate, sulfate, trifluoroacetate, methanesulfonate, orthophosphate or nitrate or mixtures thereof.

Preferably, the neutral salts of the present invention have a weight average molecular weight (Mw) of up to 2500kDa, more preferably up to 250kDa-1500kDa, and even more preferably up to 400kDa-900 kDa.

Preferably, the neutral salts of the present invention have a number average molecular weight (Mn) of up to 2000kDa, more preferably of up to 100kDa-1000kDa, and even more preferably of up to 200kDa-500 kDa.

In certain embodiments, the neutral salt consisting of the polyaminosaccharide cation and anion is as described above, with the proviso that:

when the repeating unit c) is between 50% and 70%, R is lactose and the anion is chloride, the polyaminosaccharide cation is not obtained from 200kDa-400kDa chitosan having a residual acetylation degree of about 15%.

In another aspect, the present invention relates to a process for the preparation of a neutral salt consisting of a polyaminosaccharide cation and anion, said process comprising the steps of:

i) providing a polyaminosugar polymer consisting of repeating units a) and repeating units b),

ii) reacting the polyaminosaccharide polymer with mono-, di-or oligosaccharides in an aqueous solution,

iii) adding an aminoborane to the reaction mixture,

iv) adding an acid until the pH is below 4,

v) adding an organic solvent, thereby precipitating a neutral salt, and

vi) isolating the precipitated neutral salt.

It has been surprisingly observed that aminoboranes exhibit a significant selectivity in the reduction of the imino group compared to the carbonyl group and are compatible with aqueous environments; at the same time, the formation of salts due to reaction with the acid reduces the time required to purify the final product and neutralize excess hydride ions, thereby advantageously avoiding the use of bacteriostatic agents. Thus, from a medical and pharmaceutical point of view, the process as a whole offers the advantage of improved acceptability, since the purity of the end product and the overall speed of preparation have been significantly increased.

Preferably, the polyaminosaccharide polymer consists of 5% to 95% of recurring units a) and 95% to 5% of recurring units b).

The mono-, di-or oligosaccharides correspond to the definitions given above for the R moiety.

Preferably, the aqueous solution of step ii) is an aqueous solution of 0.5 wt% to 5 wt% acetic acid, more preferably 0.5 wt% to 2.5 wt% acetic acid.

The aminoborane is preferably 2-methylpyridine borane, 5-ethyl-2-methylpyridine borane, pyridine borane, trimethylamine borane, triethylamine borane, dimethylamine borane, tert-butylamine borane or mixtures thereof. More preferably, the aminoborane is 2-methylpyridine borane, 5-ethyl-2-methylpyridine borane, or a mixture thereof.

The aminoborane may be used as such, or may have been previously dissolved or dispersed in a water-miscible organic solvent such as an alcohol. The most preferred alcohols are methanol, ethanol, 2-propanol or mixtures thereof.

The term "acid" means the corresponding acid of the anion described above.

The term "organic solvent" means an organic water-miscible solvent capable of lowering the dielectric constant of an aqueous reaction solution. Suitable organic solvents are acetone, methanol, ethanol, 2-propanol or mixtures thereof, and preferably the organic solvent is 2-propanol.

Optionally, the precipitate separated in step vi) is washed with a mixture of water and organic solvent, the percentage of water being up to 60%, more preferably up to 40%.

Preferably, the molar ratio of the recurring units b) of mono-, di-or oligosaccharide and polyaminosaccharide polymers is from 0.5 to 30, more preferably from 1 to 20, and even more preferably from 1 to 5.

Preferably, the molar ratio of aminoborane to recurring units b) of the polyaminosaccharide polymer is from 0.75 to 20, more preferably from 1 to 10, and even more preferably from 1 to 3.

In a further aspect, the invention relates to the use of said neutral salt as a medicament.

In a further aspect, the invention relates to the use of said neutral salt as a biomaterial or scaffold for cell growth.

In still a further aspect, the present invention relates to a pharmaceutical composition comprising a biologically active substance and at least one neutral salt consisting of a polyaminosaccharide cation and anion as described above, the bioactive substance is selected from the group consisting of collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid), polycaprolactone, gelatin, albumin, poly (glycolide-co-caprolactone), poly (glycolide-co-trimethylene carbonate), hydroxyapatite, tricalcium phosphate, dicalcium phosphate, decalcified bone matrix, and mixtures thereof.

Preferably, the neutral salt and the biologically active substance are in a weight ratio of 10:1 to 1: 50.

Preferably, the pharmaceutical composition of the invention comprises up to 10 wt% of said neutral salt, and more preferably up to 5 wt% of said neutral salt, based on the weight of the pharmaceutical composition. Particularly preferred are pharmaceutical compositions in which the amount of the neutral salt is 0.5 wt% to 5 wt% based on the weight of the composition.

In a first preferred embodiment of the pharmaceutical composition, the biologically active substance is selected from the group consisting of collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid), polycaprolactone, gelatin, albumin, poly (glycolide-co-caprolactone), poly (glycolide-co-trimethylene carbonate) and mixtures thereof. In these embodiments, the amount of the neutral salt is preferably higher than or equal to the amount of the biologically active substance. In these embodiments, preferably, the neutral salt and the biologically active substance are in a weight ratio of 5:1 to 1:1, preferably 4:1 to 1:1, and more preferably 3:1 to 1: 1.

The first embodiment is also preferred when the active substance is selected from the group consisting of heparin, chondroitin sulfate, collagen, sodium alginate, potassium alginate, magnesium alginate and mixtures thereof.

In a second preferred embodiment of the pharmaceutical composition, the biologically active substance is selected from the group consisting of hydroxyapatite, tricalcium phosphate, dicalcium phosphate, decalcified bone matrix and mixtures thereof. In these embodiments, the amount of the neutral salt is preferably less than or equal to the amount of the biologically active substance. In these embodiments, preferably, the neutral salt and the biologically active substance are in a weight ratio of 1:10 to 1:45, preferably 1:20 to 1:40, and more preferably 1:25 to 1: 35.

The second embodiment is also preferred when the active substance is selected from hydroxyapatite and tricalcium phosphate and mixtures thereof.

In a particularly preferred embodiment, the present invention relates to a pharmaceutical composition comprising hydroxyapatite and at least one neutral salt consisting of a polyaminosaccharide cation and anion as described above. The composition finds advantageous use in orthopaedic applications involving the skeletal system.

The pharmaceutical compositions may be administered via oral, intramuscular, intravenous, intraarticular, transdermal, subcutaneous, or topical external or internal routes, e.g., surgically.

Preferably, the pharmaceutical composition is administered via intra-articular, transdermal or local internal routes.

The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

Suitable pharmaceutically acceptable excipients are, for example, pH adjusters, isotonicity modifiers, solvents, stabilizers, chelating agents, diluents, binders, disintegrants, lubricants, glidants, colorants, suspending agents, surfactants, cryoprotectants, preservatives and antioxidants.

The invention also relates to a biomaterial comprising a neutral salt as described above, alone or in combination with at least one of the active substances described above. The biomaterial may be in the form of microspheres, nanospheres, membranes, sponges, filaments, films, gauze, catheters, hydrogels, fabrics, non-woven fabrics, or combinations thereof.

It should be understood that all aspects identified as preferred and advantageous for neutral salts should be considered as also preferred and advantageous for the above stated manufacturing processes, compositions, biological materials and uses.

It is also to be understood that all possible combinations of the preferred aspects of neutral salts, preparation processes, compositions, biomaterials and uses set out above are described herein and are therefore also preferred.

The following are working examples of the present invention provided for illustrative purposes.

Examples

General procedure for the preparation process:

mono-, di-or oligosaccharides (0.30M-0.20M), water, acetic acid (0.10M-0.20M) and chitosan (0.10M) with 5% to 20% of the repeating unit a) were loaded into the reactor. The mixture thus obtained was heated to 60 ℃ for 2 hours. Then, under the same conditions, after dispersion in alcohol (10% -20%), aminoborane (0.10M-0.25M) was gradually added and the system was left under stirring at 60 ℃ for 2 hours. Subsequently, an aqueous solution of acid (2N-4N) was added dropwise until a pH of about 2 was reached. Then, the system was cooled to room temperature and the product was precipitated by adding an organic solvent; the precipitate was decanted, the supernatant removed, and the solid portion was washed a first time with (30:70) water organic solvent mixture and then several times with (15:85) water organic solvent mixture and a last time with organic solvent. Finally, the solid thus obtained is dried under reduced pressure and under controlled temperature conditions.