FIELD OF THE INVENTION

The present invention relates to esters of hyaluronic acid functionalized with nitric oxide-releasing molecules. The hyaluronic acid derivatives of the invention are able to release nitric oxide (NO) and they can be advantageously used in ophthalmology.

TECHNICAL BACKGROUND

Hyaluronic acid (HA) is a naturally occurring, bio compatible polysaccharide with unique viscoelastic and hygroscopic properties; in particular its role as a natural lubricant and its excellent water-retaining properties make it well-suited for use in ophthalmic products.

Hyaluronic acid sodium salt solution is commonly used as a spacer in eye surgeries such as cataract removal, intraocular lens surgery, corneal transplant surgery and repair of a detached retina and other eye injuries. One common side effect of these surgical procedures is a postoperative rise in intraocular pressure which can cause permanent loss of function of optic nerve fibers and, therefore, loss of visual field function. Several pharmacological treatments are provided for reducing the postoperative intraocular pressure to normal preoperative levels while maintaining the therapeutic effects of the hyaluronic acid.

However such methods for relieving the intraocular pressure are often undesirable because of the side effects of many of these drugs. For example, carbonic anhydrase inhibitors can cause lethargy and, in some instances, disorientation. Beta-blocker medications are contraindicated in patients with breathing problems or slow heart rates. Sympathomimetic drugs can cause an increase in blood pressure.

NO is known to possess many functions that include vasodilating action, neurotransmission, blood clotting, immune responses and blood pressure control including the control of ocular blood flow. Evidence indicates that NO is involved in maintaining resting tone in the uveal and retinal circulations. Di Meo (Biomacromolecules, Vol. 7, No. 4, 2006) discloses NO releasing hyaluronic acid derivatives based on diazeniumdiolate structures (NONO-ate structures) in which spermidine was used as the nitric oxide releasing linker. The diazeniumdiolate derivatives are able to release NO in aqueous solution and the data showed that the diazeniumdiolate derivatives show a complete gas release in about 4 min. However, a prolonged NO release would be more desirable for a therapeutically application point of view in order to reduce the number of the administrations.

WO 2009/003624 discloses S-nitroso-cysteine and S-nitroso-N- acetylpenicillamine hyaluronic derivatives for dermatological and cosmetic use. In particular, WO 2009/003624 discloses the use of hyaluronic derivatives, administered in the form of an injectable gel, a hydrogel, cream, dressing or film for topical application, for the treatment of skin lesions or defects, biorevitalisation of tissues or tissue healing. The NO release is prolonged up to 500 min but any indication of completeness degree is not given.

To date there are no NO-releasing hyaluronic derivatives approved for ophthalmic therapeutic applications.

The present invention provides NO-releasing hyaluronic esters that provide a long-lasting release of nitric oxide having potential applications for topical nitric oxide release.

DISCLOSURE OF THE INVENTION

The present invention provides nitric oxide releasing hyaluronic acid derivatives of formula (I) in which the carboxylic groups of the hyaluronic acid are partially esterified with a group capable to release nitric oxide:

wherein

a) the free carboxylic acid groups are salified or at least partially salified with sodium;

b) (n-l) is the number of repeating units and it is defined by the average molecular weight of the compound of formula (I) that ranges from 100,000 to 1,500,000 Da;

c) at least one repeating unit comprises a NO-releasing group R selected from the following groups:

Al): -(CH ₂ ) _m -(CH-0N02)p-CH ₂ -0N02

A2): -(CH ₂ ) _m -[0-(CH ₂ ) _t ] _q -(CH-0N0 ₂ ) _p -CH ₂ -0N0 ₂

wherein

p is 1 or 0;

q is 1 or 0;

m is an integer ranging from 1 to 10; preferably m is from 1 to 6;

t is an integer ranging from 1 to 6; preferably n is 1 or 2;

d) the substitution degree is from 0.10 to 0.90;

preferably the average molecular weight of the hyaluronic acid is from 600,000 to 1,100,000, and the substitution degree is 0.12 to 0.4.

Preferably R is Al): -(CH ₂ ) _m -(CH-0N0 ₂ ) _P -CH ₂ -0N0 ₂ and p is 0.

Another embodiment of the invention refers to the nitric oxide releasing hyaluronic acid derivatives of formula (I):

wherein:

a) the free carboxylic acid groups are salified or at least partially salified with sodium;

b) (n-l) is the number of repeating units and it is defined by the average molecular of hyaluronic acid that ranges from 100,000 to 1,500,000 Da;

c) at least one repeating unit comprises a NO-releasing group R selected from the following group:

d) and the substitution degree is 0.10 to 0.90;

preferably the average molecular weight of the hyaluronic acid is from 600,000 to 1,100,000 Da, and the substitution degree is 0.12 to 0.4.

The substitution degree (SD) is defined as the average number of the NO-releasing groups R attached per dimer-unit of hyaluronic acid and it is calculated as reported below:

SD = molar amount of bound NO releasing groups/ molar amount of hyaluronic acid.

Preferred NO-releasing hyaluronic ester of formula (I) are 6-(nitrooxy)hexyl hyaluronate partial esters at least partially salified with sodium of formula (IA) having an average molecular weight of 600,000 - 1,100,000 Da wherein the substitutions degree (SD) are 0.43, 0.176 or 0.135.

The nitric oxide releasing hyaluronic acid derivatives of the present invention showed a long-lasting release of nitric oxide that makes them suitable compounds for reducing intraocular pressure increase or controlling intraocular pressure.

The nitric oxide releasing hyaluronic acid derivatives of the present invention may be used in combination with other drugs for the treatment of glaucoma or elevated intraocular pressure. The combination of the nitric oxide releasing hyaluronic acid derivatives of formula (I) with other drugs used for the treatment of glaucoma or elevated intraocular pressure provides an additional long-lasting intraocular pressure reducing effect allowing to reduce the doses of the administered conventional drugs for the treatment of glaucoma or elevated intraocular pressure and therefore of the undesirable the side effects of these drugs.

The nitric oxide releasing hyaluronic acid derivatives of the present invention have potential application as surgical aids in eyes because they provide localized nitric oxide release that allows to control the rising the intraocular pressure and therefore to reduce the dose of the administered post-operative drugs and therefore of the undesirable the side effects of these drugs.

An embodiment of the present invention provides the use of the nitric oxide releasing hyaluronic acid derivatives of the present invention as a surgical aid in eyes surgery such as cataract extraction, corneal transplant, glaucoma filtration and retinal attachment surgery.

Another embodiment of the present invention provides ophthalmic compositions containing the nitric oxide releasing hyaluronic acid derivatives of the present invention in combination with drugs for the treatment of glaucoma or elevated intraocular pressure.

The above combinations comprises as active principles at least a nitric oxide releasing hyaluronic acid derivatives of the present invention and a prostaglandin F2a analog, a beta blocker or carbonic anhydrase inhibitor.

Preferably the prostaglandin F2a analog is selected from travoprost, bimatoprost or latanoprost, the beta blocker is selected from timolol, carteolol, betaxolol, levobunolol or metipranolol, the carbonic anhydrase inhibitor is selected from dorzolamide or brinzolamide.

The invention also relates to pharmaceutical compositions comprising the nitric oxide releasing hyaluronic acid derivatives of the present invention of formula (I) in the form of eye drops or injectable sterile formulation for intraocular use.

Depending on its formulation, the ophthalmic pharmaceutical composition may also include pharmacologically acceptable excipients. The pharmacologically acceptable excipients for eye drops and injectable sterile formulation can be any type ordinarily used for such purposes, preferably water.

General process of synthesis

The hyaluronic acid derivatives of formula (I) can be prepared as depicted in the

Scheme below:

I (NO-HA-Na)

The nitric oxide releasing hyaluronic acid derivatives of formula (I) are prepared using a process which comprises the following steps:

1) dissolving sodium hyaluronate (HA-Na) in deionized water and eluting the solution through a column packed with activated Dowex H ⁺ resin to obtain hyaluronic acid;

2) adding under an inert atmosphere, for example a nitrogen atmosphere, and preferably at a temperature of 20-25° C, tetrabutylammonium hydroxide to the collected a percolate containing hyaluronic acid (HA) of step 1 to yield a stoichiometric mixture of HA and TBA having pH 7; and recovering the obtained tetrabutylammonium-HA (HA- TBA) by freeze-dry;

3) reacting the tetrabutylammonium-HA (HA-TBA) with a tosylalkylnitroester of formula Bl) or B2):

B 1 ): Ts0-(CH ₂ ) _m -(CH-0N02)p-CH ₂ -0N02 B2):Ts0-(CH ₂ ) _m -[0-(CH ₂ )t] _q -(CH-0N02)p-CH2-0N02

p is 1 or 0;

q is 1 or 0;

m is an integer ranging from 1 to 10; preferably m is from 1 to 6;

t is an integer ranging from 1 to 6; preferably n is 1 or 2;

in a dimethyl sulfoxide, or a dimethyl sulfoxide/water solution, whereby the nitric oxide releasing hyaluronic acid derivative of formula (I) is formed; and

4) removing of the tetrabutylammonium ion by NaCl exchange and recovering the nitric oxide releasing hyaluronic acid derivative of formula (I) (NO-HA-Na) by freeze- dry.

Example 1

Preparation of 6-(nitrooxy)hexyl hyaluronate partial ester (SD = 0.43)

MW= 600,000 - 1,100,000 Da; SD = 0.43

Preparation of 6-hydroxyexyl nitrate

Hexanediol (2.00 g, 16.95 mmol) in DCM (100 ml) was cooled to -30 °C and a mixture of HNO3 (0.36 ml) in Ac ₂ 0 (2.4 ml) was added dropwise under vigorous stirring. The mixture was left reacting at -30 °C for 2h. The mixture was then poured on to ice and diluted with AcOEt. The two phases were separated, and the organic phase washed with NaHCCE (3x); H ₂ 0 (2x) and brine. The organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered and carefully concentrated to afford a reddish oil (760 mg). The final product (360 mg, 2.20 mmol, 26% yield) was obtained after purification by flash chromatography (Cyclohexane/ AcOEt from 5 % AcOEt to 80% AcOEt).

z,

To a stirred solution of 6-hydroxyexyl nitrate (807 mg, 4.94 mmol) and tosyl chloride (989 mg, 5.19 mol) in DCM cooled to 0°C, TEA (600 mg, 5.93 mmol) was added dropwise and a catalytic amount of DMAP was introduced. The mixture was stirred overnight. The reaction was quenched with saturated NH ₄ Cl solution and extracted 3 times with DCM. The combined organic phases were washed with brine and then dried over Na ₂ S0 ₄ , filtered and the solvent evaporated under vacuum, affording reddish oil. The final product (344 mg, 1.08 mmol, 22% yield) was obtained after purification by flash chromatography (Cyclohexane/ AcOEt from 100 % Cyclohexane to 7:3 Cyclohexane /AcOEt).

NMR (300 MHz, Chloroform-^) d 7.79 (d, J= 8.0 Hz, 2H), 7.35 (d, J= 8.0 Hz, 2H), 4.40 (t, J= 6.6 Hz, 2H), 4.02 (t, J= 6.3 Hz, 2H), 2.45 (s, 3H), 1.71 - 1.61 (m, 2H), 1.42 - 1.30 (m, 4H).

Preparation of hyaluronic acid tetrabutylammonium salt (HA-TBA)

Sodium hyaluronate (l.Og) (Hyasis® 850P, MW 600,000-1,100,000 Da) was obtained from Novozymes Biopharma DK A/S, Bagsvaerd, Denmark lg of hyaluronic acid Na+ salt was dissolved in 500 ml of H ₂ 0, filtered over activated Dowex H+ resin (50W X8 strongly acidic, 50-100 mesh, Sigma- Aldrich). The acidic solution was then neutralized (pH 7) using a tetrabutylammonium hydroxide solution (1.5M in water) and finally freeze-dried.

Preparation of 6-(nitrooxy)hexyl hyaluronate partial ester (SD = 0.43)

HA-TBA salt (155 mg, equivalent to 0.343 mmol of disaccharide unit) was dissolved in 45 ml of DMSO by sonication at 40°C for lh. Then 6-(nitrooxy)hexyl tosylate (52 mg, 0.164 mmol, 48% of total disaccharide) was added. The reaction was stirred overnight at 30°C, after this time 1 ml of NaCl solution (14.5 %) was added. The mixture was added dropwise to 50 ml of cold acetone and kept under stirring. A white precipitate was observed which was centrifuged at 6000 RPM for 5 minutes. The supernatant is removed and the solid washed 3 times with 8 ml (8x3) of a 5:1 mixture acetone/H20, vortexing vigorously each time. The process was repeated using pure acetone (x3). At the end the solid was vacuum dried 8h at 30 °C, then suspended in 8.8 ml of NaCl solution 0.1%. The suspension is added to cold acetone, the mixture is centrifuged and the solid washed with acetone/water 5 : 1 until the AgNCh test for chloride ions is negative, finally the solid is treated with acetone and dried. 139 mg of hyaluronic acid partial ester (SD= 0.43) were obtained. The apparent degree of esterification (43%) (SD= 0.43) was determined by ¹ H NMR as follow: the ratio between 1/8 of the sum of the integrals of the eight aliphatic protons of the 6-(nitrooxy)hexyl ester (1.95 and 1.65 ppm) and 1/3 of the integral of the methylacetamido from hyaluronic acid at 2.2 ppm gives the degree of esterification.

NMR (300 MHz, Deuterium Oxide, 60°C) d 4.82-3.58 (m, HA), 2.24 (s, relative integration 3.0 H, HA-NHCO.CH3), 1.95 (m, relative integration 1.6 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0), 1.65 (m, relative integration 1.83 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0). Example 2

Preparation of 6-(nitrooxy)hexyl hyaluronate partial ester SD = 0.176

MW= 600,000 - 1,100,000 Da SD = 0.176

6-Hydroxyexyl nitrate and hyaluronic acid tetrabutylammonium salt (HA-TBA) were prepared as disclosed in Example 1

HA-TBA salt (348 mg, equivalent to 0.770 mmol of disaccharide unit) was dissolved in 45 ml of DMSO by sonication at 40°C for lh. Then 6-(nitrooxy)hexyl tosylate (58 mg, 0.18 mmol, 24% of total disaccharide) was added. The reaction was stirred overnight at 30°C, after this time 1 ml of NaCl solution (14.5 %) was added. The mixture was added dropwise to 50 ml of cold acetone and kept under stirring. A white precipitate was observed which was centrifuged at 6000 RPM for 5 minutes. The supernatant is removed and the solid washed 3 times with 8 ml (8x3) of a 5:1 mixture acetone/H ₂ 0, vortexing vigorously each time. The process was repeated using pure acetone (x3). At the end the solid was vacuum dried 8h at 30 °C, then suspended in 8.8 ml of NaCl solution 0.1%. The suspension is added to cold acetone, the mixture is centrifuged and the solid washed with acetone/water 5 : 1 until the AgNCh test for chloride ions is negative, finally the solid is treated with acetone and dried. 200 mg of hyaluronic acid partial ester (SD= 0.176) were obtained. The apparent degree of esterification (17.6%) (SD= 0.176) was determined by ¹ H NMR as described in example 1.

NMR (300 MHz, Deuterium Oxide, 60°C) d 4.82-3.58 (m, HA), 2.25 (s, relative integration 3.0 H, HA-NHCO.Cft), 1.96 (m, relative integration 0.69 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0), 1.65 (m, relative integration 0.72 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0).

Example 3

Preparation 6-(nitrooxy)hexyl hyaluronate partial ester (SD = 0,135)

MW= 600,000 - 1,100,000 Da; SD = 0.135

6-hydroxyexyl nitrate and hyaluronic acid tetrabutylammonium salt (HA TBA) were prepared as disclosed in Example 1.

HA-TBA salt (264 mg, equivalent to 0.583 mmol of disaccharide unit) was dissolved in 45 ml of DMSO by sonication at 40°C for lh. Then 6-(nitrooxy)hexyl tosylate (25.0 mg, 0.0787 mmol, 13.5% of total disaccharide) was added. The reaction was stirred overnight at 30°C, after this time 1 ml of NaCl solution (14.5 %) was added. The mixture was added dropwise to 50 ml of cold acetone and kept under stirring. A white precipitate was observed which was centrifuged at 6000 RPM for 5 minutes. The supernatant is removed and the solid washed 3 times with 8 ml (8x3) of a 5:1 mixture acetone/H ₂ 0, vortexing vigorously each time. The process was repeated using pure acetone (x3). At the end the solid was vacuum dried 8h at 30 °C, then suspended in 8.8 ml of NaCl solution 0.1%. The suspension is added to cold acetone, the mixture is centrifuged and the solid washed with acetone/water 5 : 1 until the AgNCL test for chloride ions is negative, finally the solid is treated with acetone and dried. 202 mg of hyaluronic acid partial ester SD= 0.135 were obtained. The apparent degree of esterification (13.5%, SD= 0.135) was determined by ¹ H NMR as described in example 1.

NMR (300 MHz, Deuterium Oxide, 60°C) d 4.80-3.58 (m, HA), 2.25 (s, relative integration 3.0 H 3H, HA-NHCO.CH3), 1.95 (m, relative integration 0.54 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0), 1.65 (m, relative integration 0.54 H, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ 0).

Example 4

Determination of nitric oxide release by the compounds of the invention.

The nitric oxide nitric oxide release of the invention and of isosorbide mononitrate (ISMN), used as reference compound, was evaluated by UV spectroscopy using the Griess test in the presence of cysteine.

General method

A stock solution of the nitric oxide releasing hyaluronic acid derivative is prepared by dissolving a weighted amount (5-7 mg) of the nitric oxide releasing hyaluronic acid derivative of formula (I) (NO-HA-Na) in 2.0 ml of DMSO using sonication (lh, 40°C). 560 pL of the stock solution were added to 2.24 ml of PBS buffer containing 5 mM cysteine and incubated at 37 °C. At the designated time-point, an 840 pL aliquot of the incubated solution was added to 175 pL of Griess’ reagent (Fluka). The sample was further incubated in the dark for 30’ at room temperature. The UV absorbance of the sample was measured at 540 nm. The amount of nitric oxide released was determined against a standard curve obtained with sodium nitrite.

The percentage of NO release was calculated as the ratio between the experimental mmoles measured by UV using the Griess method and the theoretical mmoles of NO, calculated considering the substitution number.

The results reported in Table show that the nitric oxide release of the hyaluronic acid derivatives of the invention require 6-7 hours to achieve a plateau release of NO, while the NO release by ISMN is almost complete after 2 hours.

nd = not determined