Soluble adducts of boric acid or derivatives and precursors thereof with chitosan oligosaccharide derivatives

Abstract

Disclosed are adducts of boric acid and chitosan wherein the amino groups of the D-glucosamine units are bonded to residues of polyol or alditol oligosaccharides, the molar ratio between boric acid and the repeating chitosan unit ranging between 0.001 and 4.

Claims

1. Pharmaceutical or cosmetic compositions or medical devices comprising adducts of boric acid and chitosan wherein the amino groups of the D-glucosamine units are linked to residues of polyol or alditol oligosaccharides, the molar ratio between boric acid and the repeating unit of chitosan ranging from 0.001 to 4, said adducts being in a mixture with compatible carriers or excipients and optionally with other active ingredients.

2. Adducts according to claim 1 wherein the oligosaccharides are selected from glucose, galactose, lactose, cellobiose, cellotriose, maltose, maltotriose, maltotetraose, chitobiose, chitotriose, melibiose, agarobiose and carrabiose.

3. Adducts according to claim 2 wherein the oligosaccharide is lactose.

4. Adducts according to claim 1, wherein at least 30% of the amino groups of the D-glucosamine units are bonded to oligosaccharide residues.

5. Adducts according to claim 1 wherein the chitosan has a molecular weight ranging from 300 kDa to 1500 kDa.

6. Adducts according to claim 1 having a polymer concentration viscosity scaling law (η∝C.sup.α) wherein the value of a ranges from 4.5 to 6.2.

7. Adducts according to claim 1 in the form of salts with acids.

8. Process for the preparation of the adducts of claim 1, said method comprising: a) adding boric acid, derivatives or precursors thereof, and oligosaccharide to a chitosan aqueous solution at a polymer concentration greater than or equal to 1.5%; and b) purifying the soluble adducts obtained in step a) by precipitating with non-solvents selected from methanol, ethanol, isopropanol, n-propanol, acetone or mixtures thereof and washing with acid solutions of hydrochloric acid, nitric acid, sulphuric acid, phosphoric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, formic acid or mixtures thereof.

9. Process according to claim 8 wherein the boric acid derivatives or precursors thereof are selected from sodium borohydride, sodium cyanoborohydride, sodium acetate borohydride, sodium triacetoxyborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, magnesium borohydride, tetraethylammonium borohydride, methyltrioctylammonium borohydride, bis(triphenylphosphine) copper (I) borohydride, borax, potassium tri(1-pyrazolyl) borohydride, cetyltrimethylammonium borohydride, tetrahydrofuran-borane complex, picoline-borane complex, dimethylsulphide-borane complex, pyridine-borane complex, trimethylamine-borane complex, triethylamine-borane complex, morpholine-borane complex, t-butylamine-borane complex, ammonia-borane complex, diphenylphosphine-borane complex, 4-methylmorpholine borane complex and ethylenediamine-borane complex.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. .sup.11B-NMR spectrum of an adduct between boric acid and a chitosan oligosaccharide derivative (CTL). The spectrum shows the signal of the adduct, which is very different from that of boric acid (added to the test solution for demonstration purposes only). Polymer concentration 0.9% (w/V), measurements conducted in PBS at pH 7.4.

(2) FIG. 2. Dependence of zero-shear-rate viscosity (η.sub.0) on concentration of chitosan oligosaccharide derivatives (C.sub.CTL) for soluble intramolecular aggregates at a [B]/[CTL].sub.ru ratio of 0.018 (.square-solid.) and for the chitosan oligosaccharide derivative prepared according to patents EP 2 310 448 A1, EP 2 029 629 A1, WO 2010/010122 A1 and WO 2007/135116 A1 (□). In this case the scaling law found is that expected for polysaccharides, namely η.sub.□∝C.sub.CTL.sup.3.z.

(3) FIG. 3. Relative variation in diffused light of soluble aggregates with different amounts of boron ([B]/[C.sub.CTL]) and a constant concentration of soluble adducts. Measurements conducted in PBS at pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

(4) The adducts according to the invention are prepared by introducing boric acid or derivatives and precursors thereof during the synthesis and purification process of chitosan oligosaccharide derivatives. An essential feature of the process is that the boric acid or precursors and derivatives thereof are encapsulated in a phase having a high polysaccharide concentration, and the soluble adducts are purified with non-solvents and acid solutions. Examples of non-solvents include methanol, ethanol, isopropanol, n-propanol and acetone. Examples of acids include hydrochloric, nitric, acetic, formic, trichloroacetic, trifluoroacetic, sulphuric and phosphoric acids and combinations thereof.

(5) To obtain soluble adducts, the chitosan derivatives must have a degree of substitution of the amino groups with the oligosaccharide side chain ranging between 30% and 80%, preferably 60%. The average molecular weight (hereinafter called MW) of chitosan usable for the production of soluble adducts can be up to 1500 kDa, and preferably ranges between 300 kDa and 1000 kDa.

(6) The soluble adducts can be obtained by using boric acid or derivatives and precursors thereof such as sodium borohydride, sodium cyanoborohydride, sodium acetate borohydride, sodium triacetoxyborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, magnesium borohydride, tetraethylammonium borohydride, methyltrioctylammonium borohydride, bis(triphenylphosphine) copper (I) borohydride, borax, potassium tri(1-pyrazolyl) borohydride, cetyltrimethylammonium borohydride, tetrahydrofuran-borane complex, picoline-borane complex, dimethylsulphide-borane complex, pyridine-borane complex, trimethylamine-borane complex, triethylamine-borane complex, morpholine-borane complex, t-butylamine-borane complex, ammonia-borane complex, diphenylphosphine-borane complex, 4-methylmorpholine-borane complex and ethylenediamine-borane complex. The boric acid derivatives used are preferably sodium cyanoborohydride and picoline-borane complex, generally a borane complexed with a heterocyclic aromatic nitrogen compound, and more preferably picoline-borane complex.

(7) The increase in polymer concentration at the synthesis stage and the inclusion therein of boric acid and derivatives or precursors thereof gives rise to adducts which remain soluble at polymer concentrations ranging between 3% and 5%.

(8) In this way, the ratio between the moles of boric acid and the moles of repeating polymer units can be fine-tuned so that it is not less than 0.001 and not more than 4.

(9) Unlike U.S. Pat. No. 4,424,346, the presence of boric acid or derivatives and precursors thereof encapsulated in the adducts in a concentrated solution situation prevents the formation of hydrogels at polymer concentrations ranging between 3% and 5% (w/V). In fact, the adducts according to the invention remain soluble up to concentrations of 10% (w/V) without forming hydrogels.

(10) The soluble adducts were analysed with the use of rheological measurements. Surprisingly, the flow curves recorded for solutions of the soluble adducts demonstrate that the dependence of the zero-shear-rate viscosity (η.sub.0) presents a polymer concentration (C) scaling law of η ∝ C.sup.5.□ with a [B]/[C.sub.CTL] ratio of 0.018 (FIG. 2). The latter proved to be considerably greater than that normally reported for polysaccharide solutions (Morris E. R. et al., Carbohydr. Polym., 1981, 1, 5), i.e. ranging between approximately η ∝ C.sup.3.□ and η ∝ C.sup.3.a.

(11) FIG. 3 shows the relative variation in diffused light which increases, in the interval of interest for the present patent, proportionally to the boron in the soluble intramolecular aggregates, thus demonstrating that the molecular structure is more compacted.

(12) The presence of soluble aggregates between boric acid or derivatives and precursors thereof and chitosan oligosaccharide derivatives gives rise to very pronounced, lasting formation of filaments, which is particularly interesting for all applications wherein the viscoelastic response is crucial, such as viscosupplementation, wound treatment, cosmetic and ophthalmic applications, and treatment of joint disorders. The adducts according to the invention can also be used in the field of biomaterials, sports medicine, as elasticisers, viscosity-controlling agents, thickeners and adhesives, to obtain an antimicrobial barrier effect and as a medical device in wound treatment.

(13) The following examples provide a detailed description of the invention.

EXAMPLE 1

Synthesis of Soluble Aggregates with a [B]/[C.SUB.CTL.] Molar Ratio of 0.002—(CTL Hydrochloride)

(14) 2.2 g of lactose monohydrate is introduced into a 250 ml 3-necked flask, and solubilised in 21 ml of water. 750 mg of chitosan is introduced, and 270 μl of glacial acetic acid is added. The system is then placed under heating in an oil bath at 60° C. with mechanical stirring until the polymer is completely solubilised. 370 mg of picoline borane complex (pic-BH.sub.3) is dissolved in 3.0 ml of methanol and added to the chitosan and lactose solution. The reaction proceeds under stirring at 60° C. for 4 hours. At the end of the reaction, the crude reaction product is diluted with 27 ml of water. The bath is heated to 40° C., and a solution containing 400 mg of NaOH in 100 ml of methanol is dripped into it to precipitate the polymer. The solid is left to decant, the supernatant is removed, and 6 washes are performed with a 90% V/V solution of methanol/0.1N HCl, followed by a last wash in methanol only. The solid is dried at low pressure. 1.4 g of dry solid is recovered, and used to determine the residual boron content by complexing with azomethine-H and spectrophotometric analysis (“Boron Analysis from Dietary Supplements” by Anca-Michaela Bulearca et al., REV.CHIM. (Bucharest) 65, No. 1, 2014); said content amounts to 60 ppm. The degree of substitution of lactitol residue per repeating unit of chitosan (DS) is 0.53.

EXAMPLE 2

Synthesis of Soluble Aggregates with a [B]/[C.SUB.CTL.] Molar Ratio of 0.009—(CTL Hydrochloride)

(15) 2.2 g of lactose monohydrate is introduced into a 250 ml 3-necked flask, and solubilised in 21 ml of water. 750 mg of chitosan is introduced, and 270 μl of glacial acetic acid is added. The system is then placed under heating in an oil bath at 60° C. with mechanical stirring until the polymer is completely solubilised. 370 mg of picoline borane complex (pic-BH.sub.3) is dissolved in 3.0 ml of methanol and added to the chitosan and lactose solution. The reaction proceeds under stirring at 60° C. for 4 hours. At the end of the reaction, the crude reaction product is diluted with 27 ml of water. The bath is heated to 40° C., and a solution containing 400 mg of NaOH in 100 ml of methanol is dripped into it to precipitate the polymer. The solid is left to decant, the supernatant is removed, and 6 washes are performed with a 95% V/V solution of methanol/0.4N HCl, followed by a last wash in methanol only. The solid is dried at low pressure. 1.3 g of dry solid with a DS of 0.50 is recovered and used to determine the residual boron content by complexing with azomethine-H and spectrophotometric analysis according to the method described in example 1; said content amounts to 240 ppm.

EXAMPLE 3

Synthesis of Soluble Aggregates with a [B]/[C.SUB.CTL.] Molar Ratio of 0.001—(CTL Hydrochloride)

(16) 2.2 g of lactose monohydrate is introduced into a 250 ml 3-necked flask, and solubilised in 21 ml of water. 750 mg of chitosan is then introduced, and 270 μl of glacial acetic acid is added. The system is then placed under heating in an oil bath at 60° C. with mechanical stirring until the polymer is completely solubilised. 400 mg of picoline borane complex (pic-BH.sub.3) is dissolved in 3.0 ml of methanol and added to the chitosan and lactose solution. The reaction proceeds under stirring at 60° C. for 4 hours. At the end of the reaction, the crude reaction product is diluted with 27 ml of water. The bath is heated to 40° C., and 10 ml of water containing 0.6 ml of 12N HCl is added. The system is adjusted to room temperature, and acetone is dripped into it to induce the precipitation of the polymer. The solid is left to decant, the supernatant is removed, and 6 washes are performed with an 80% V/V solution of acetone/water, followed by a last wash in acetone only. The solid is dried at low pressure. 1.5 g of dry solid with a DS of 0.58 is recovered, and used to determine the residual boron content by complexing with azomethine-H and spectrophotometric analysis according to the method described in the previous examples; said content amounts to 30 ppm.

EXAMPLE 4

Synthesis of Soluble Aggregates with a [B]/[C.SUB.CTL.] Molar Ratio of 0.05—(CTL Acetate)

(17) 2.2 g of lactose monohydrate is introduced into a 250 ml 3-necked flask, and solubilised in 21 ml of water. 750 mg of chitosan is then introduced, and 270 μl of glacial acetic acid is added. The system is then placed under heating in an oil bath at 60° C. with mechanical stirring until the polymer is completely solubilised. 400 mg of picoline borane complex (pic-BH.sub.3) is dissolved in 3.0 ml of methanol and added to the chitosan and lactose solution. The reaction proceeds under stirring at 60° C. for 4 hours. At the end of the reaction, the crude reaction product is diluted with 27 ml of water. The bath is heated to 40° C., and 4.2 ml of glacial acetic acid is added. The system is adjusted to room temperature, and acetone is dripped into it to induce the precipitation of the polymer. The solid is left to decant, the supernatant is removed, and 6 washes are performed with a 90% V/V solution of methanol/water, followed by a last wash in methanol only. The solid is dried at low pressure. 1.5 g of dry solid with a DS of 0.55 is recovered, and used to determine the residual boron content by complexing with azomethine-H and spectrophotometric analysis according to the method described in the previous examples; said content amounts to 1400 ppm.

EXAMPLE 5

Synthesis of Soluble Aggregates with a [B]/[C.SUB.CTL.] Molar Ratio of 0.007—(CTL Nitrate)

(18) 2.2 g of lactose monohydrate is introduced into a 250 ml 3-necked flask, and solubilised in 21 ml of water. 750 mg of chitosan is then introduced, and 270 μl of glacial acetic acid is added. The system is then placed under heating in an oil bath at 60° C. with mechanical stirring until the polymer is completely solubilised. 350 mg of picoline borane complex (pic-BH.sub.3) is dissolved in 3.0 ml of methanol and added to the chitosan and lactose solution. The reaction proceeds under stirring at 60° C. for 4 hours. At the end of the reaction, the crude reaction product is diluted with 27 ml of water. The bath is heated to 40° C., and 10 ml of water containing 1.2 ml of 6N HNO.sub.3 is added. The system is adjusted to room temperature, and acetone is dripped into it to induce the precipitation of the polymer. The solid is left to decant, the supernatant is removed, and 1 wash is conducted with an 80% V/V solution of methanol/water followed by 2 washes with a 90% V/V solution of methanol/water and a last wash in methanol only. The solid is dried at low pressure. 1.1 g of dry solid with a DS of 0.45 is recovered, and used to determine the residual boron content by complexing with azomethine-H and spectrophotometric analysis according to the method described in the previous examples; said content amounts to 180 ppm.

COMPARATIVE EXAMPLE 6

(19) A chitosan conjugated with lactose was prepared by the procedures reported in EP 2310448, EP 2029629, WO 2010/010122 and WO 2007/135116.

(20) 1.5 g of commercial chitosan is solubilised with 110 ml of a solution obtained by mixing 55 ml of an aqueous solution of 1% w/V acetic acid at pH=4.5 with 55 ml of methanol. 60 ml of a solution pre-prepared with 2.2 g of lactose, 0.90 g of NaCNBH.sub.3, 30 ml of methanol and 30 ml of a 1% w/V aqueous solution of acetic acid at pH=4.5 is added to said solution. The crude reaction product is maintained under magnetic stirring for 24 hours at room temperature and then transferred to a dialysis bag with a 12 kDa cut-off and dialysed at 4° C. Two changes of external solution are performed, first with a solution of 0.1M NaCl and then with deionised water only, until a residual conductivity of 4 μS is reached. The polymer solution is filtered through a filter with a porosity of 0.45 μm and then freeze-dried. The boron residue is determined on the freeze-dried product, and amounts to 28 ppm, which corresponds to a boric acid/chitosan-lactose molar ratio<0.001. This sample was used for rheological characterisation by comparison with the CTL according to the invention (FIG. 2).

COMPARATIVE EXAMPLE 7

(21) For comparative purposes, the preparation of glycated chitosans was replicated according to the synthesis procedure described in U.S. Pat. No. 5,747,475 (column 9, lines 30-59 and column 10, lines 1-57), using lactose as model oligosaccharide. As U.S. Pat. No. 5,747,475 does not specify the pH conditions used, the proof of synthesis was repeated at three different pH values (2.5, 5.5 and 7), obtained by using chitosan hydrochloride, chitosan acetate and commercial chitosan alone respectively; in the latter case, the polymer granules swell, but do not solubilise during the reaction. By applying the process described in U.S. Pat. No. 5,747,475 to chitosan-lactose, a DS<5% is obtained in the products of the two reactions in homogeneous (acid) phase, and no structural modification is observed when the reaction is conducted in heterogeneous phase at a neutral pH. The samples obtained proved to be insoluble in water at a neutral pH, and therefore unsuitable for the applications according to the invention.