Stability tests were further conducted on rapamycin mixed with HYADD®-4, also at different concentrations, storing the mixture at room temperature for 12 hours. The technique used involves HPLC analysis, as described above.

Rapamycin in organic solvent, such as DMSO, has a stability of 12 months, but only if stored at temperatures ranging between 5 and 20°C, whereas at ambient T it deteriorates very rapidly.

The results are set out in the table below:

Table 2

The HYADD-rapamycin mixture clearly makes the active ingredient bioavailable in an aqueous medium, and at the same time ensures its stability at room temperature for a relatively long time. This certainly represents an unexpected result, and an improvement on the state of the art.

Example 2: in vitro release into simulated synovial fluid (SSF) of a mixture of HYADD4 0.8% + rapamycin

To evaluate the real efficacy of the release system described herein, the release of rapamycin from the viscous matrix of HYADD®-4 was tested by comparison with the release of rapamycin dissolved in DMSO into a simulated synovial fluid consisting of foetal bovine serum (FBS) and hyaluronidase, with the addition of gentamicin, required to prevent contamination during the incubation stage.

The samples were prepared in 2 mL glass vials suitable for the HPLC test, each of which contained SSF with the following composition: 0.6 mL of foetal bovine serum (FBS, Gibco, code A31160801) prefiltered at 0.45 microns, hyaluronidase (BTH, 5U/mL) and gentamicin (0.1 mg/mL).

The following were added to each vial:

• for Sample 1 : 0.2 g of HYADD®-4 0.8% + rapamycin (0.2 mg/mL) mixture in PBS/DMSO (99/1% v/v)

• for Sample 2: 0.002 mL of rapamycin at 20 mg/mL in DMSO.

The final theoretical content of both formulations to be tested was equal, namely 0.05 mg/mL of rapamycin.

All the samples, except those to be evaluated at TO, were incubated at 37°C under gentle stirring; at the various time points the vials were taken up, sonicated for 3 minutes to dissolve any undissolved rapamycin, and filtered through an 0.2 micron nylon filter, which simulates the barrier effect of the synovial membrane.

The filtrate was treated with 3 volumes of ACN, thus causing the precipitation of HYADD®-4 and the FBS proteins. After filtration through an 0.45 micron RC filter, the samples were injected into HPLC, as described in Example 1. The amount of rapamycin released is given by the sum of the rapamycin signal and the rapamycin degradation peaks.

As will be seen in Figure 1, when rapamycin per se is loaded into DMSO there is a massive release after a few minutes, whereas rapamycin in the formulation with HYADD®-4 0.8% is released gradually over about 60 hours.

Due to the release system designed, rapamycin, which is insoluble and unstable in water, proved:

- stable in an aqueous carrier, which aids its bioavailability;

- less toxic, because it is in a formulation practically devoid of organic solvent; the final formulation contains a maximum of 1% v/v of DMSO, which is well within the limits of tolerability;

- to be released gradually over a long period, thus remaining at local level, specifically in the cartilage, for the time required to perform its therapeutic activity, enabling it to be administered at lower doses and/or at longer intervals;

- storable in an extempore preparation at ambient T for at least 24 hours.

The final formulation is also iso-osmotic, has a pH ranging between 6 and 8, ie. physiological, and is perfectly extrudable (18G, 20G, 22G and 25G needle), and therefore compatible with use for intra-articular infiltration. Example 3: preparation of a mixture of HYADD®-40.8% and sodium alendronate (final cone. 40 mg/mL)

482 mg of sodium alendronate trihydrate was weighed in a screw-cap glass container, 1 mL of IM NaOH was added, and complete solubilisation was awaited, using the Vortex for stirring. 9 mL of pre-prepared phosphate buffer (PBS), consisting of 9 mg of Na2HP04*2H2O, was then added, and the mixture was stirred again in the Vortex. The pH was adjusted to 7.2 with IM NaOH. Final concentration of sodium alendronate: 40 mg/mL. 80 mg of powdered HYADD®-4 was introduced, stirring with a spatula, and the mixture was left to hydrate for 2 hours at room temperature, to obtain a final HYADD®-4 concentration of 8 mg/mL. After complete hydration of the hydrogel, it was homogenised and filtered at 100 pm with a steel filter; and finally transferred to 5 mL glass syringes, filled with 4 mL and sterilised in the autoclave.

Example 4: preparation of a mixture of HYADD®-40.8% and sodium alendronate (final cone. 4 mg/mL)

48.2 mg of sodium alendronate trihydrate was weighed in a screw-cap glass container, 0.1 mL of IM NaOH was added, and complete solubilisation was awaited, using the Vortex for stirring. 9.9 mL of pre-prepared phosphate buffer (PBS), consisting of 9 mg of Na2HP04*2H2O and 75 mg of sodium chloride, was then added, and the mixture was stirred again in the Vortex. The pH was adjusted to 7.2 with IM NaOH. Final concentration of sodium alendronate: 4 mg/mL. 80 mg of powdered HYADD®-4 was introduced, stirring with a spatula, and the mixture was left to hydrate for 2 hours at room temperature, to obtain a final HYADD®-4 concentration of 8 mg/mL. After complete hydration of the hydrogel, it was homogenised and filtered at 100 pm with a steel filter; and finally transferred to 5 mL glass syringes, filled with 4 mL and sterilised in the autoclave.

Example 5: preparation of a mixture of HYADD®-40.8% and sodium alendronate (final cone. 0.4 mg/mL)

4.8 mg of sodium alendronate trihydrate was weighed in a screw-cap glass container, 0.01 mL of IM NaOH was added, and complete solubilisation was awaited, using the Vortex for stirring. 9.99 mL of pre-prepared phosphate buffer (PBS), consisting of 9 mg of Na2HP04*2H2O and 85 mg of sodium chloride, was then added, and the mixture was stirred again in the Vortex. The pH was adjusted to 7.2 with IM NaOH. Final concentration of sodium alendronate: 0.4 mg/mL. 80 mg of powdered HYADD®-4 was introduced, stirring with a spatula, and the mixture was left to hydrate for 2 hours at room temperature, to obtain a final HYADD®-4 concentration of 8 mg/mL. After complete hydration of the hydrogel, it was homogenised and filtered at 100 gm with a steel filter; and finally transferred to 5 mL glass syringes, filled with 4 mL and sterilised in the autoclave.

Example 6: rheological characterisation of a mixture of HYADD®-4 0.8% and sodium alendronate (final cone. 4 mg/mL)

The sample prepared as described in Example 4 underwent dynamic viscosity measurement (Anton Paar MCR92 rheometer equipped with a 1° and 50 mm cone-plate measurement system, 0.102 mm gap, temperature 25°C, measurement conducted with increase in shear rate from 0.01 to 1000 s-1, acquiring 33 points on the logarithmic scale) interpolated at 1 s’¹, at Time 0, after 6 months’ storage at 40°C and after 24 months’ storage at 25°C, and compared with HYADD®-4 0.8% subjected to the same treatments. The results are set out in Figure 2. The addition of alendronate clearly keeps the rheological characteristics of the polymer HYADD®-4 substantially unchanged, but improves them slightly without interfering with the usability of the composition, which continues to be extrudable with an 18G, 20G, 22G or 25G needle, and is therefore compatible with use for intra-articular infiltration.

Example 7: in vitro tests of release into PBS (phosphate buffer) of a mixture of HYADD®-4 0.8% and sodium alendronate (cone. 6 mM)

The test was performed using:

• Sample: mixture of sodium alendronate (1.9 mg/mL, 6 mM) in HYADD®-4 8 mg/mL in PBS pH 7.2

• CTRL: sodium alendronate in PBS pH 7.2 (1.9 mg/mL, 6 mM)

1 mL of CTRL or about 1 g of the sample to be tested was deposited in dialysis tubes with membranes having a 100 kDa cut-off. 5 mL of PBS pH 7.2 was added in the external reservoir; the reservoirs were placed in 50 mL Falcon tubes to prevent evaporation and placed at 37°C under stirring at 60 rpm. At preset times (12 h, 3, 7, 10, 14, 21 and 23 days) the contents of the reservoir were emptied into screw-cap test tubes and replaced with fresh medium.

The alendronate released at the various timepoints was analysed by HPLC-UV after derivatisation with FMOC-C1 using the method described in the literature by Kang et al., 2006 (https://doi.org/10.1080/108260706006783Q8), with adjustment of some parameters.

The results are described in Figure 3, which clearly indicates that the release of alendronate from the test sample is slow and gradual, unlike the observations made for the CTRL, thus demonstrating the efficacy of the release system described herein.

Example 8: cytotoxicity test on a mixture of HYADD®-4 0.8 mg/mL and sodium alendronate at increasing concentrations on primary bovine chondrocytes

For the optimum conduct of the test, HYADD®-4 had to be diluted with PBS 1 : 10, because at the concentration of 8 mg/mL HYADD®-4 is a highly viscous hydrogel, which prevents the correct migration and proliferation of the cells used, and thus prejudices the success of the experiment.

Primary bovine chondrocytes (PBC) were isolated from the femoral condyle cartilage of an adult bovine according to the protocol described in the literature (Mouw JK et al., Osteoarthr Cart 2005, 13: 828-836 The chondrocytes isolated were cultured in DMEM/F-12 (1 : 1) medium (Life Technologies, cat. No. 11320074, Italy) containing 10% foetal bovine serum (Life Technologies, cat. no. 10270106, Italy) and 50 pg/mL ascorbic acid under standard conditions (37°C, 5% CO2) until the cells had reached a state of semi-confluency. The cells were then seeded at the concentration of 1 x 10⁴ cells/well in 96-well Multiwell plates (Sarstedt, cat. no. 83.3924, Germany) and divided into 4 groups:

• CTRL: wherein the cells were neither treated nor stimulated;

• ALN: wherein the cells were incubated with decreasing concentrations of sodium alendronate (5 mM, 2.5 nM; 1 mM) for 24 hours;

• HYADD®-4 0.8 mg/mL + ALN (5 mM, 2.5 nM; 1 mM): wherein the cells were incubated for 24 hours.

At the end of the preset incubation times, cell viability was quantified with the alamarBlue® assay (Life Technologies, DAL1025, Italy), according to the manufacturer’s instructions, to determine the cell viability as an indicator of the ability of the mixture to effect prolonged release of alendronate over time compared with free alendronate. The results are set out in Fig. 4.

In the cytotoxicity evaluation, it is evident that sodium alendronate is strongly cytotoxic at the concentrations of 5, 2.5 and 1 mM.

The combination of HYADD®-4 (0.8 mg/mL) and alendronate is not cytotoxic at the concentrations tested, a finding which ensures the viability of at least 65% of the chondrocytes.

The use of HYADD®-4 therefore surprisingly masks the cytotoxic effect of sodium alendronate at the concentrations wherein it is toxic for PBC cells if it is used alone.

This means that the release system according to the invention allows the intra-articular administration of sodium alendronate without giving rise to the toxic effects on the chondrocytes typical of bisphosphonate.

Example 9: calcium chelation test

A further advantage of the release system disclosed herein is the synergistic effect of the mixture in preventing, or greatly reducing, the formation of calcium phosphate crystals in the joint, as demonstrated in the present example. It is known that at least 60% of osteoarthritic joints are characterised by the presence in the synovial fluid of calcium phosphate and/or pyrophosphate crystals, induced by an imbalance in the phosphate metabolism typical of the disorder. Not only do the crystals have strong inflammatory activity; pyrophosphate crystals in particular tend to deposit, giving rise to the highly undesirable phenomenon of chondrocalcinosis (Stucker et al., 2021 https://doi.Org/10.1016/j.berh.2021.101722)

The test was conducted as described in van der Akker 2023 (https://doi.Org/10.1016/j.joca.2022. l l.007) adapted for use of a microplate reader; briefly, the addition of phosphate ions to a TRIS (tris(hydroxymethyl)aminomethane) buffer containing CaCh gives rise to the formation of calcium phosphate crystals, and the samples containing them show increased absorbance on spectrophotometric reading at 620 nm. The absorbance evaluation is therefore an indicator of the amount of crystals formed.

As regards the controls, each well of the plate contained:

• CTRL-: 200 pL of TRIS-CaCh with the addition of 40 pL of 0.1M CaCl₂ and 10 pL of MQ p water. The plate was incubated for 15 min at 37°C under stirring at 50 rpm, and the absorbance was read at 620 nm at preset times.

• CTRL+: 200 pL of TRIS-CaCL with the addition of 10 pL of PBS buffer (the medium wherein the samples are dissolved) + 40 pL of 0. IM CaCL. The plate was incubated for 15 min at 37°C under stirring at 50 rpm, 40 pL of 40mM phosphate was then added, and the absorbance was read at 620 nm at preset times.

The samples containing the species to be tested

• ALN (sodium alendronate) 50 mM in PBS pH 7.2

• HYADD®-4 (8 mg/mL) in PBS pH 7.2

• HYADD®-4 (8 mg/mL) + ALN 50 mM in PBS pH 7.2 were all prepared by the same procedure, namely in each well:

40 pL of CaCL 0.1M and 10 pL of the species to be tested were added to 200 pL of TRIS-CaCh.

The plate was incubated for 15 min at 37°C under stirring at 50 rpm; 40 pL of 40mM phosphate was then added, and the absorbance was read at 620 nm after 2.5 h.

The results are presented in Figure 5, which clearly shows that the release system according to the invention acts synergistically with the individual species in counteracting the formation of calcium crystals.

Example 10: preparation of a gel of HYADD®-4 0.8% and mesalazine

510 mg of mesalazine (5-aminosalicylic acid) was weighed in a glass container; 4.4 ml of phosphate buffer pH 6.9 was then added, and the mixture was heated to 45°C. The resulting solution was maintained under stirring for one hour at a controlled temperature. 6 mg of methylhydroxy benzoate and 2 mg of propylhydroxy benzoate were then added, and the mixture was left under stirring for a further 30 minutes. Finally, 52 mg of powdered HYADD®-4 was introduced, stirring with a spatula, and the mixture was left to hydrate for 2 hours at room temperature.

Claims

1. Pharmaceutical composition for intra-articular or topical administration, comprising or consisting of a drug having one or more of the following characteristics:

- insolubility or low solubility in aqueous solvents;

- heat instability;

- instability in acidic or basic media;

- low bioavailability;

- toxicity, wherein said drug is homogeneously dispersed in a hydrogel matrix containing hexadecyl ami de of hyaluronic acid or a salt thereof, wherein the hyaluronic acid hexadecyl ami de possesses an average degree of amidation ranging between 1% and 3% molar, provided that said drug is not a biologically and/or therapeutically active protein with a hydrophobicity index (GRAVY) of not less than -0.5.

2. Pharmaceutical composition according to claim 1, wherein the hyaluronic acid hexadecyl ami de is prepared starting from hyaluronic acid having a weight-average molecular weight ranging between 500 and 730 kDa.

3. Pharmaceutical composition according to claims 1-2, wherein alternatively:

(i) said composition is for intra-articular administration and the hyaluronic acid hexadecyl ami de concentration ranges between 5 and 12 mg/ml, and is preferably 8 mg/ml;

(ii) said composition is for topical administration and the hyaluronic acid hexadecyl ami de concentration ranges between 0.05 and 1 mg/ml, preferably between 0.1 and 0.8 mg/ml.

4. Composition according to claim 1, wherein said drug is selected from rapamycin, sodium alendronate, mesalazine, ascorbic acid and minoxidil.

5. Composition according to claim 4, wherein said drug is rapamycin and its concentration ranges between 0.01 and 1 mg/ml, preferably between 0.01 and 0.5 mg/ml.

6. Composition according to claims 4-5, wherein the drug is rapamycin and said composition is suitable for intra-articular or topical administration, said topical administration preferably being ocular.

7. Composition according to claim 4, wherein the drug is sodium alendronate and its concentration ranges between 0.1 and 50 mg/ml, preferably between 0.3 and 45 mg/ml.

8. Composition according to claims 4 and 7, wherein the drug is sodium alendronate and said composition is suitable for intra-articular administration.

9. Method for preparing a composition according to the previous claims, comprising mixing hyaluronic acid hexadecyl ami de with a solution of the drug in organic or aqueous solvent, under stirring.

10. Method according to claim 9, wherein the drug is rapamycin and the organic solvent is dimethyl sulphoxide.

11. Method according to claims 9-10, wherein the drug is rapamycin and said method is carried out immediately before intra-articular administration.

12. Method according to claim 9, wherein the drug is sodium alendronate and the aqueous solvent is phosphate buffer (PBS).

13. Pharmaceutical composition according to any one of claims 1 to 8, wherein the drug is selected from rapamycin and sodium alendronate, for use in the treatment of osteoarticular diseases, and in particular osteoarthritis.

14. Pharmaceutical composition according to any one of claims 1-6, wherein the drug is rapamycin, for use in the treatment of eye disorders and in particular dry eye, allergic diseases, keratoconjunctivitis sicca, corticosteroid-resistant vernal keratoconjunctivitis, and Sjogren’s syndrome.

15. Pharmaceutical composition consisting of sodium alendronate at a concentration ranging from 0.3 to 45 mg/mL, homogeneously dispersed in a hydrogel matrix containing hyaluronic acid hexadecyl ami de with an average degree of amidation ranging between 1% and 3% molar and a concentration of 8 mg/mL, for use in the intra-articular treatment of osteoarthritis.

16. Pharmaceutical composition consisting of rapamycin at a concentration ranging from 0.01 to 0.5 mg/mL, homogeneously dispersed in a hydrogel matrix containing hyaluronic acid hexadecyl ami de with an average degree of amidation ranging between 1% and 3% molar and a concentration ranging from 0.1 to 0.8 mg/mL, for use in the topical treatment of eye disorders.

17. Pharmaceutical composition consisting of rapamycin at a concentration ranging from 0.01 to 0.5 mg/mL, homogeneously dispersed in a hydrogel matrix containing hyaluronic acid hexadecyl ami de with an average degree of amidation ranging between 1% and 3% molar and a concentration of 8 mg/mL, for use in the intra-articular treatment of osteoarthritis.