A NOVEL DRUG DELIVERY SYSTEM: USE OF HYALURONIC ACID AS A CARRIER MOLECULE FOR DIFFERENT CLASSES OF THERAPEUTIC ACTIVE AGENTS Prior art Many drugs, which are hydrophobic in character and hence show poor solubility in water have been conjugated with hydrophilic polymers to increase their water solubility and improve the bioavailability. For this purpose a number polymeric materials showing the property of biocompatibility, biodegradability have been used, some of them are bioactive, have sufficient drug loading capacity, and have drug targeting capabilities. Examples are polyglutamate, polyethylene glycole, carboxymethyl dextran and hyaluronic acid. However, PLG, PEG and CMD lack in bioactivity and targeting capabilities while HA has the advantage over the others because in addition it is bioactive and has the capability to target the drug to the diseased site. Many tumour types overexpress CD44 receptors; and HA can be used to conjugate anticancer drugs to target the delivery of the drug to the diseased site. Endocytosis of dehvatised HA has been shown in cell lines expressing CD44 HA receptor. The fluorescent labelled HA-Taxol conjugate has been shown to be selectively toxic towards human cancer cell lines which were known to overexpress HA receptors. The presence of liver receptors for HA (HARLEC) suggests that it can be used as a carrier molecule to target a drug to the liver tissue. HA has been demonstrated for liver metastases from a colon adenocarcinoma in mice.

The preparation of HA substituted at the C-6 primary hydroxyl group with dihydrofolate reductase inhibitors (DHFR) have been described in WO0168105. This conjugate has been obtained by preparing HA-6-halogen by selective halogenation reaction of HA, and followed by displacement of the halogen by the DHFR. This conjugate is still endowed with antiproliferative activity, however it still presents the problem that it contains residual halogen groups. Selective introduction of a leaving group on polysaccharide has been described in Carb. Res. 340, 2229-2235, 2005 where a tosylation of cellulose in a mixture of acetamide and lithium chloride is reported; the conditions chosen allows the

complete sulfonylation of all the primary hydroxyl groups with the aim of blocking said positions and introducing other chemical groups on the free positions.

Description of the figures

FIGURE 1 : represents the formula of DDSs: HA-6-methotrexate, HA-6-ibuprofen, HA-6-PenG

FIGURE 2: represents the DOSY NMR spectrum of HA-6-OMs obtained in example 9 (in DOSY weighed monodimensional NMR spectra only rigid macromolecules are present, furnishing evidence for polymer chemical modification) FIGURE 3: represent the ¹³ C NMR spectrum of HA-6-OMs, peaks of salifying

DIEA are present.

FIGURE 4: represents the DOSY NMR spectrum of HA-6-MTX obtained in example 24

FIGURE 5: represents the ¹³ C NMR spectrum of HA-6-MTX obtained in example 24

FIGURE 6: represents the DOSY NMR spectrum of HA-lbuprofen obtained in example 26

FIGURE 7: represents the ¹³ C NMR spectrum of HA-lbuprofen obtained in example 26 FIGURE 8: represents the DOSY NMR spectrum of HA-Penicillin G obtained in example 29

Detailed description of the invention

In a first aspect of the invention, there is provided a drug delivery system (DDS) consisting of hyaluronic acid (HA) and a therapeutic active agent, whereby this active agent is covalently linked at the C-6 position of the λ/-acetyl-D-glucosamine residue of the hyaluronic acid with the exception of active agents of formula (I):

H ₂ ) ₂ -γCOOH

R; formula (I)

wherein:

R ₂ and R ₄ independent from one another represent: -NH ₂ , -OH, -OCH ₃ , C ₁ -C ₅ alkyl, =0; X and Y represent: -C(R ₅ )=, -CH(R ₅ )-, -NH-, -N=) , wherein R ₅ represents: -H, CrC ₅ alkyl; Z represents: -CH(R ₁₀ )-, -N(R ₁₀ )-, -O-; R ₁₀ represents: -H, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkenyl, C ₁ -C ₅ alkynyl, 5-6 membered heterocyclic ring with 1 -3 heteroatoms selected in the group consisting of nitrogen, sulphur and oxygen; Ar represents: 1 ,4-phenyl group, 1 ,4-phenyl group condensed with one or more 5- 6 membered aromatic rings, 1 ,4-phenyl group condensed with one or more 5-6 membered heterocycles, wherein said Ar is possibly substituted with R ₂ ; rings A and b, independently from one another, may be aromatic or non-aromatic. The compounds of formula (I) are the dihydrofolato reductase inhibitors described in WO0168105. Hyaluronic acid (also herein indicated as HA) is composed of a disacchahdic repeating unit, consisting of D-glucuronic acid and 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) bound by β(1 → 3) glycosidic linkage; the D-glucuronic acid residue may either be in the acid form or in the form of a salt. Each repeating unit is bound to the next one by a β(1 →4) glycosidic linkage that forms a linear polymer. The term hyaluronic acid, as used in the present invention, encompasses both the acid and the salified form.

The term hyaluronic acid is commonly used to describe a general group of molecular fractions of HA with varying molecular weights or also hydrolysed fractions of said compound. For the purposes of the present invention the

hyaluronic acid has preferably an average molecular weight comprised between 10000 to 1 million and more preferably 20000 to 500000.

The therapeutic active agent is chosen from drugs belonging to a number of different therapeutic categories: analgesic, antihypertensive, anestetic, diuretic, bronchodilator, calcium channel blocker, cholinergic, CNS agent, estrogen, immunomodulator, immunosuppressant, lipotropic, anxiolytic, antiulcerative, antiarrhytmic, antianginal, antibiotic, anti-inflammatory, antiviral, thrombolitic, vasodilator, antipyretic, antidepressant, antipsychotic, antitumour, mucolytic, narcotic antagonist, hormones, anticonvulsant, antihistaminic, antifungal, antipsoriatic.

These therapeutic active agents contain a nucleophilic group. A nucleophilic group is an electron-pair donor group such as carboxylic, amino, substituted amino, hydroxyl, thiol, amide group; the carboxylic group is preferred. In the DDS the linkage between the hyaluronic acid and the active agent is an ester, an amino, an ether, a thioether, an amide. The ester linkage is preferred. The DDSs are either in the acid form or in the salt form. When they are in salt form they may be salified with alkaline metals (preferably Na or K), earth-alkaline metals (preferably Ca or Mg), transition metals (preferably Cu, Zn, Ag, Au, Co, Ag). The salification is obtained by processes known by the skilled artisan. Optionally, also the secondary hydroxyl groups on the DDSs may be derivatised to form a group selected from: -OR, -OCOR, -SO ₂ H, -OPO ₃ H ₂ , -O-CO-(CH ₂ ) _n -COOH, -O-(CH ₂ ) _n -OCOR, wherein n is 1 -4 and R is C ₁ -C ₁₀ alkyl, -NH ₂ , -NHCOCH ₃ These substitutions can be easily obtained by processes known in the art, and they may be chosen in order to modulate the hydrophilic character of the DDSs. The total amount of the therapeutic active agent in the DDSs is defined by the degree of substitution (C6-DS); the latter can alternatively indicate the % by weight of the active agent with respect to the total weight of the DDS (C6-DS _W ) or the % by mole of the active agent with respect to the mole of repeating unit of modified HA (C6-DSmoi). In the DDS of the invention the C6-DS _W is preferably comprised between 0.1 and 60%, more preferably between 1 and 50%, even more preferably between 5 and

As demonstrated in the experimental part, the invented DDSs are characterised by the presence of active agent directly linked to the primary hydroxyl groups of the λ/-acetyl-D-glucosamine units of the hyaluronic acid. No other hydroxyl groups of the HA are involved in the chemical linkage with the drug. Moreover, the DDSs are stable and free of undesired reaction by-products and impurities that can be harmful to their practical pharmaceutical use.

They retain the pharmaceutical effect of the therapeutic agent. Therefore, they can be successfully used in the treatment of all pathologies that are appropriate for the specific therapeutic active agent in the DDS.

Accordingly, it is a further aspect of the invention the use of the above DDSs in the manufacture of a medicament for the treatment of pathologies appropriate for each therapeutic agent. Said pathologies are selected from the group consisting of tumours, skin disorders, psoriasis, inflammatory pathologies, rheumatoid arthritis, and infectious diseases.

It is also an aspect of the invention a pharmaceutical composition containing the DDSs of the invention in admixture with pharmaceutically acceptable excipients and/or diluents. The pharmaceutical composition may be either in the liquid or in solid form; it may be administered through the oral, parenteral, topical route. Particularly interesting are the injectable pharmaceutical compositions containing the invented DDSs.

A further aspect of the invention is a technology for the preparation of the drug delivery system of HA and a therapeutic active agent with the exception of compounds of formula (I) having the features described above. It has been surprisingly found that the reaction does not only occurs with compound having the structure of formula (I) having two carboxylic groups and heterocyclic rings, but this process is widely applicable to a high number of different active agents which belong to different therapeutic categories. This technology comprises the following reaction steps: (a) introducing a leaving group at the C-6 position of the N-acetyl-D-glucosamine units of the hyaluronic acid either in the free form or in the salt form thus obtaining a HA-6-activated

(b) forming a chemical linkage between the C6 position of the HA-6-activated and the therapeutic active agent by displacing the leaving group (at the C6 position of HA) with a nucleophilic group present on the therapeutic active agent, thereby obtaining a HA-6-active agent (c) possible displacing of any un-substituted leaving group from the HA-6-active agent obtained in step (b) (d) recovering the HA-6-active agent

With this process it is possible to obtain DDSs having a C6-DS _W preferably comprised between 0.1 and 60%, more preferably between 1 and 50%, and even more preferably between 5 and 40%.

There are two different ways of carrying out the process of the invention. In a first way the HA-6-activated obtained from step (a) is isolated from the reaction mixture and then reacted with the therapeutic active agent according to step (b) to give the final HA-6-active agent that may optionally undergo step (c). In the second way of carrying out the process, the step (b) is performed directly on the reaction mixture obtained in step (a) that contains the HA-6-activated. The advantage of this second way of performing the reaction consists in the fact that the isolation step of the HA-6-activated is avoided. The starting HA may be in free form or in the form of salt, wherein the countehon is preferably an alkaline or alkaline-earth metal or is a nitrogen-containing counterion. In the latter case the countehon may contain heterocycles selected from the group consisting of pyridine, pyrazine, pyhmidine, pyrrole, pyrazole, imidazole triazole, tetrazole, possibly substituted with one or more C1 -C6 alkyl groups. Preferred examples of nitrogen-containing counterions are ammonium, tetrabutylammonium (TBA), pyhdinium or sym-collidinium ions.

Step (a) is a selective reaction carried out by adding the suitable reagent to a thoroughly stirred suspension or solution of HA (in free form or in the salified form) in an aprotic organic solvent. The leaving group which is introduced at the C-6 position of the glucosamine unit of the HA is any electron-pair acceptor group that departs during the substitution by a nucleophile group. It may be selected from the group consisting of sulfonate group, phosphonate group (thphenylphoshonate), cyanide (CN-), nitrite (NO2-),

halogen (preferably chloro), sulphate group, halogensulfate group, nitrate, halogensulfite (chlorosulfite).

When the leaving group is halogen the halogenation is carried out as described in WO9918133 and WO0168105. Among the halogen group the chlorine group is the preferred one and the preferred reagent to perform the halogenation is methanesulfonyl chloride in λ/,λ/-dimethylformamide. This step allows the formation of the HA-6-activated.

Step (b) is performed by reacting the hyaluronic acid-6-activated or one of its salt obtained form step (a) with the therapeutic active agent. It consists in the substitution of the leaving group by the nucleophilic group contained in the active agent and entails the formation of a covalent linkage between the C-6 position of hA and the active agent. The chemical nature of said linkage depends on the chemical nature of nucleophile group. It may be an ester linkage which is formed when the nucleophile is a carboxylic group. Other linkages that are formed between the HA and the therapeutic active agent are: amino, ether, thioether, amide.

Step (c) is a possible step that may be any suitable reaction that allows the displacement of any possible un-substituted leaving group. Such a displacement may be carried out for example by photolyisis, by reduction. In some case, step (c) is not necessary since some un-substituted leaving group may be destroyed during the step (b) either because of the reaction conditions or during the work-up. In step (d) the obtained the HA-6-active agent (DDS) is recovered by means of standard techniques. In a preferred embodiment of the process the leaving group is the sulfonyl group and the obtained activated HA is therefore HA-6-sulfonated. This preferred reaction comprises the following reaction steps:

(a) introducing a sulfonate group at the C-6 position of the N-acetyl-D-glucosamine units of the hyaluronic acid in the salt form thus obtaining a HA-6-sulfonated

(b) forming a chemical linkage between the C-6 position of the HA-6-sulfonated and the therapeutic active agent by displacing the sulfonated group (at the C-6 position of HA) with the nucleophilic group present on the therapeutic active agent, thereby obtaining a HA-6-active agent.

(d) recovering the HA-6-active agent

In this embodiment, the selective sulfonylation reaction of step (a) is carried out using as sulfonylating reagent an alkyl- or aryl-sulfonyl halide, preferably chloride, in presence of an organic or inorganic base, preferably an organic base. The alkyl- or aryl-sulfonyl halide may be chosen among, preferred are methylsulfonyl (mesyl), toluene-p-sulfonyl (tosyl), trifyl, trimsyl, tripsyl, 1 ,1 -sulfonyl-imidazole. The organic base is selected preferably among the different organic amines, such as diisopropylethylamine, triethylamine. The solvent is chosen from the group consisting of: dimethylformamide, dimethylacetamide, dimethylsulfoxide, formamide.

The general sulfonylation procedure is as follows. The base, preferably organic base is added to a suspension or a solution of HA in salt form, preferably in an organic base form, by stirring under nitrogen flux. Then the alkyl- or aryl-sulfonyl chloride in a suitable solvent, preferably the same solvent, is added dropwise. After a period of time ranging from 2 to 90 minutes (preferably 45-75 min), the reaction is quenched by addition of NaHCO3 to remove the formate ester groups formed during the reaction at secondary hydroxyl groups of HA. Then the reaction is allowed to continue for about 10-20 hours, preferably 18 hours. The reaction product (HA-6-sulfonated) is either directly recovered form the solution by means of known techniques, such as precipitation, drying or before recovery the solution is treated in such a way as to allow the obtainement of the HA-6-sulfonated in a suitable salt form, such as HA-6-sulfonated :TBA. The reaction conditions are mild; in fact, reaction can be successfully carried out at room temperature or at a lower temperature, no cooling-heating cycles are required, pH conditions are mild.

The reagent is used in limited quantities, the suitable amount is 1 -10 molar equivalents with respect to the repeating HA unit (preferably 2-6 molar eq) of sulfonyl halide (such as mesylchlohde), in the presence of 2-20 molar equivalents with respect to the repeating HA unit (preferably 4-12 molar eq) of organic amine (such as DIEA).

Under the above reaction conditions the obtained hyaluronic acid-6-sulfonated has degree of substitution (DS _m0 ι), ranging from 10% to 91 % mol/mol, preferably from 20 to 90%, even more preferably from 40 to 80%. The selectivity of the mesylation reaction for the primary position (C-6) of the λ/-acetyl-D-glucosamine residue is between 50 and 100% (C6-DS _m0 ι). Some mesylation reactions also occurs at the secondary positions, such as at C-4 of λ/-acetyl-D-glucosamine and at the C-2, C-3 positions of the D-glucuronic acid residue. Their structures and the degree of mesyl group substitution in the polymer are confirmed by NMR spectroscopy. In a preferred embodiment of the sulfonylation reaction, step (b) entails the formation of an ester linkages group between the HA and the carboxylic group present on the therapeutic agent .

In this last embodiment, step (a) is carried out as described above and step (b) is usually performed according to the following procedure. A solution of the carboxylic group containing-active agent is added to a solution of the HA-6-sulfonated either in TBA or in the sodium salt form, preferably TBA, in presence of an alkaline or alkaline-earth metal salt, such as cesium carbonate. The reaction is carried out between 40-90 °C, preferably 80 °C under constant stirring, preferably under nitrogen flux for a period of time ranging form 5 to 42 hours, preferably form 8 to 20 hours (18 hours). The reaction mixture is worked up according to known techniques.

A further aspect of the present invention is a drug delivery system consisting of hyaluronic acid and a compound of formula (I), whereby the carboxylic group of compound of formula (I) is covalently linked at the C-6 position of the λ/-acetyl-D- glucosamine units of the hyaluronic acid by means of an ester linkage and said DDS is obtained by the specific process described hereunder. These new DDSs contain the compound of formula (I) directly linked at the C-6 position of the HA and are characterised by the fact and no other hydroxyl groups of the HA repeating unit is involved in chemical linkage neither with the drug nor with other chemical groups. In particular these DDSs are devoid of any residual leaving groups (such as sulfonate group) both on the primary and on the secondary positions of the HA units. The term "devoid" means that the residual leaving group is present in an amount below 0.5% w/w as determined by NMR. These features

allows the maintenance of the regularity of the original HA chemical structure and the retention of the configuration of the carbon atoms, these properties/aspects are highly important to ensure the efficacy and the interaction with the specific receptors. Differently, the conjugate of HA and methotrexate that was described in WO0168105 contains residual chlorine atoms, that are introduced on the polysaccharide during the halogenation step.

Among the different compounds having formula (I) the preferred one is methotrexate. Methotrexate (MTX) is represented by formula (I) where R ₂ and R ₄ are -NH ₂ ; ring A is aromatic; ring B is aromatic; X and Y are: -N=; Z is: -N(CH ₃ )-; Ar is: 1 ,4-phenyl group. The C6-DS _W of the DDSs is preferably comprised between 0.1 and 60%, more preferably between 1 and 50%., even more preferably between 5 and 40%, the MW is comprised between 10,000 and 500,000. The technology for the preparation of this DDS comprises the following reaction steps:

(a) introducing at the C-6 position of the N-acetyl-D-glucosamine units of the hyaluronic acid either in the free form or in the salt form a leaving group selected from the group consisting of sulfonate group, phosphonate group (triphenylphoshonate), cyanide (CN-), nitrite (NO2-), sulphate group, halogensulfate group, nitrate, halogensulfite (chlorosulfite) thus obtaining a HA-6- activated

(b) forming an ester linkage between the C6 position of the HA-6-activated and the compound of formula (I) by displacing the leaving group (at the C6 position of HA) with a carboxylic group present on compound (I), thereby obtaining a HA-6- compound of formula (I)

(d) recovering the HA-6- compound of formula (I)

In the preferred embodiment, step (a) is a sulfonylation reaction and the reagent used for introducing the sulfonate group is an alkyl- or aryl-sulfonyl halide, preferably chloride, in presence of an organic or inorganic base. The preferred the reagent is methylsulfonyl chloride or toluene-p-sulfonyl chloride and the organic base is diisopropylethylamine or thethylamine.

The DDS can be obtained with the above process according to two different ways.

In the first way the HA-6-sulfonated obtained from step (a) is isolated from the reaction mixture and then reacted with the compound of formula (I) according to step (b) to give the final HA-6-compound of formula (I). In the second way of carrying out the process, the step (b) is performed directly on the reaction mixture obtained in step (a) that contains the HA-6-sulfonated. The advantage of this second way of performing the reaction consists in the fact that the isolation step of the HA-6-sulfonated is avoided.

EXPERIMENTAL PART EXAMPLE 1 : Determination of structure

The determination of mesylate content in the HA-6-Mesylate (HA-Ms) by NMR was achieved by integration of the peaks in the region 3.10í3.32ppm (1 H of HA chain and 3H of mesylate) versus the peak at 1.95ppm (3H of HA chain).

EXAMPLE 2: Determination of structure The determination of tosylate content in the HA-6-tosylate (HA-Ts) by NMR was achieved by integration of the peaks of tosylate at 7.8ppm (2H), 7.5ppm (2H) and

2.45ppm (3H) versus the peak at 1.95ppm (3H of HA chain).

EXAMPLE 3: Determination of structure

Determination of methotrexate content in HA-6-MTX by NMR was achieved by integration of the peaks in the region 6.0í8.6ppm (5H of MTX) versus the peaks in the region 1.85í2.58ppm (3H of HA chain and 4H of MTX).

EXAMPLE 4: Determination of structure

The determination of lbuprofen in HA-6-lbuprofen by NMR was achieved by integration of the peaks of ibuprofen in the regions 7.02í7.24ppm (4H), 2.38ppm (2H), 1.40ppm (3H), 0.78ppm (6H) versus the peak of the HA chain at 1.95ppm

(3H).

EXAMPLE 5: Determination of structure

The determination of Penicillin G in HA-6-Penicillin G by NMR was achieved by integration of the peaks of Penicillin G in the regions 7.05í7.20ppm (5H), 5.55ppm (1 H), 5.40 (1 H) versus the peak of the HA chain at 1.95ppm (3H).

EXAMPLE 6: Methotrexate content by HPLC was determined by analysing the samples before and after alkaline hydrolysis according to Methotrexate Official Monograph (USP 23-p 984). The analyses conditions were: Cromatograph: Dionex DX-600. Column: Column Phenomenex Synergi 4μ Hydro-RP80, Column size:150X460mm, Column particle size : 4μ, Temperature: 40 °C Eluent: 90% 0.2M dibasic sodium posphate/0.1 M citric acid (630:270), 10% CH ₃ CN, isocratic condition: 0.5 ml_/min. Detector: Diode Array (range 200-780nm), Selected wavelength for the quantitative determination: 302 nm Injected volume:25 μl, run time 30 minutes. Solutions for free methotrexate determination were prepared by dissolving HA-MTX directly in MiIIiQ water at the appropriate concentration. Total methotrexate content was determined after alkaline hydrolysis carried out in NaOH 0.1 M, room temperature for 2 hours. After neutralization with hydrochloric acid 1 M, solutions were filtered through 0.45 μm (Sartohus Minisart RC25 17795Q) prior to injection in the HPLC system. A calibration curve was determined by using standard solutions with known concentration of methotrexate. The method gives the MTX concentration in the sample solution, which normalized by the sample concentration yields the DS _we ιght %w/w.

EXAMPLE 7: Determination of weight average molecular weight (Mw). The molecular weight of the hyaluronic acid DDS was measured by HP-SEC (High Performance Size Exclusion Chromatography). The analysis conditions were: Chromatograph: HPLC pump 980-PU (Jasco Ser. No. B3901325) with Rheodyne 9125 injector. Column: TSK PWxI (TosoBioscience) G6000+G5000+G3000 6, 10, 13 μm particle size; Temperature: 40 °C Mobile phase: NaCI 0.15 M + 0.01 % NaN ₃ . Flux: 0.8 mL/min. Detector: MALS (WYATT DAWN EOS - WYATT, USA), λ= 690 nm, (dn/dc = 0.167 mL/g), UV spectrophotometry detector 875-UV (Jasco, Ser. No. D3693916), λ = 305 nm, lnterferometric Refractive Index OPTILAB REX (WYATT, USA); λ=690 nm, Sensitivity: 128x; Temperature: 35 °C Injected volume:100 μl, run time 60 minutes. The samples of HA-CI, HA-OMs, HA-MTX, HA-conjugated to different drugs to be analysed were solubilised in 0.9 % NaCI at the concentration of about 1.0 mg/ml and kept under stirring for 12 hours. Then, the solutions were filtered on a 0.45 μm porosity filter (Sartohus Minisart RC25 17795Q) and finally injected in the

chromatograph. The analysis allows the measurement of Mw (weight average molecular weight), Mn (number average molecular weight), Pl (polydispersity). Mw and Mn values are expressed as g/mole.The concentration of the polymeric samples solutions were controlled by means of the integral of the refractive index. EXAMPLE 8: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-6-Ms or HA- Ms)

To a solution of 500 mg (0.806 mmol) of TBA salt of HA (MW 20,000) in 20 ml of dimethylsulfoxide (DMSO) were added 1.1 1 ml (6.48mmol) of diisopropylethylamine (DIEA) by stirring under nitrogen. Methanesulfonyl chloride (MsCI) (314μl_; 4.03mmol) was then added dropwise at room temperature, whereupon an orange solution formed. After 1 h stirring at room temperature, one third of the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (50ml), stirring overnight at pH 9. The resulting solution was ultrafiltered, concentrated in a rotary evaporator and freeze-dried to afford 40mg of an off-white solid (total DS 83% mol/mol by NMR).

The rest of the reaction mixture was stirred overnight and then worked up as described above, to obtain 90mg of an off-white solid (total DS 86% mol/mol by

NMR).

Overall yield: 130mg of HA-Ms sodium salt (40%). ¹ H NMR (D ₂ O) ppm: 1.95 (s, 3H, NHCOCH ₃ ), 3.23 (s, 2.58H, MsO), 3.2í4.2 (m, 7.42H, HA chain), 4.3í4.7 (m, 2H, anomeric + 1.72H, CH-OMs); ¹³ C NMR (D ₂ O) ppm: 23 (NHCOCH ₃ ), 37 (MsO), 55, 61 (CH ₂ OH), 68, 69.5 (CH ₂ OMs), 72, 74, 75, 76, 80, 83, 101 , 103, 174, 175. EXAMPLE 9: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 5.00 g (8.06 mmol) of TBA salt of HA (MW 20,000) in 200 ml of DMSO were added 13.9ml (81 mmol) of DIEA by stirring under nitrogen. MsCI (3.2 ml; 41 mmol) was then added dropwise at room temperature, whereupon an orange solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (400 ml), bringing the total volume to 1 L with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was evaporated to dryness in

a rotary evaporator (100 mg) for NMR analysis: total mesylate DS 91 % mol/mol by proton NMR, primary mesylates 58% mol/mol by carbon NMR, selectivity 64% for the C6 position.

The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 4.62g of an off-white solid (HA-Ms:TBA salt).

EXAMPLE 10: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 2.50 g (4.03 mmol) of TBA salt of HA (MW 20,000) in 100 ml of DMSO were added 5.6 ml (32.7mmol) of DIEA by stirring under nitrogen. MsCI (1.3 ml; 16.7mmol) was then added dropwise at room temperature, whereupon an orange solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (200 ml), bringing the total volume to 600ml with water (resulting pH: 9.2), and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was freeze-dried (136mg) for NMR analysis: total mesylate DS 79% mol/mol by proton NMR, primary mesylates 64% mol/mol by carbon NMR, selectivity 81 % for C6 position. The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 2.1 Og of an off-white solid (HA-Ms:TBA salt). EXAMPLE 11 : Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 3.00 g (4.84 mmol) of TBA salt of HA (MW 20,000) in 100 ml of DMSO were added 8.4 ml (48.4 mmol) of DIEA by stirring under nitrogen. MsCI (1.92 ml; 24.2 mmol) was then added dropwise at room temperature, whereupon an orange solution formed. After 15min stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (200ml), bringing the total volume to 600ml with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was freeze-dryed (187mg) for NMR analysis: total mesylate DS 76% mol/mol by proton NMR, primary mesylates 58% mol/mol by carbon NMR, selectivity 76% for C6. The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 2.561 g of an off-white solid (HA-Ms:TBA salt).

EXAMPLE 12: Preparation of 6-O-Methanesulfonylhyaluronic acid (HA-Ms) To a solution of 3.0Og (4.84mmol) of HA TBA salt (MW 20.000) in DMSO (100 ml) were added 4.96ml (29.0mmol) of DIEA by stirring under nitrogen. MsCI (1.13ml; 14.5mmol) was then added dropwise at room temperature, whereupon an orange solution was formed. After 15min stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (200ml), bringing the total volume to 600ml with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was freeze-dried (248mg) for NMR analysis: total mesylate DS 55% mol/mol by proton NMR, primary mesylates 41 % mol/mol by carbon NMR, selectivity 75% for C6.

The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 2.41 g of an off-white solid (HA-Ms:TBA salt). EXAMPLE 13: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 3.0Og (4.84mmol) of HA TBA salt (MW 20.000) in DMSO (100 ml) was added DIEA (4.96ml; 29.0mmol) by stirring under nitrogen. MsCI (1.13ml; 14.5mmol) in dichloromethane (20ml) was then added dropwise during 20 min, at room temperature, whereupon an orange solution formed. The reaction mixture was then immediately quenched by pouring into saturated NaHCO ₃ solution (200ml), bringing the total volume to 600ml with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was freeze-dried (172mg) for NMR analysis: total mesylate DS 85% mol/mol by proton NMR, primary mesylates 50% mol/mol by carbon NMR, selectivity 59% for C6. The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 2.78g of an off-white solid (HA-Ms:TBA salt). EXAMPLE 14: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a suspension of 3.0Og (7.48mmol) of HA sodium salt (MW 20.000) in DMSO (100ml) were added DIEA (12.8ml; 74.8mmol) and MsCI (2.90ml; 37.4mmol), observing the formation of a dark orange colour within one minute. After 1 h and 15min stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (200ml), bringing the total volume to 800ml with

water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. A small portion was freeze-dried (0.15g) for NMR analysis: total mesylate DS 5% mol/mol by proton NMR. EXAMPLE 15: Preparation of 6-O-p-toluenesulfonylhvaluronic acid

A solution of HA:TBA salt (1.018 g; 1.64 mmol) (MW 20000) in 30ml of dry DMF was treated with Et ₃ N (3.2 ml_; 23.0 mmol) and TsCI (2.24 g; 1 1.7 mmol) at room temperature; the reaction mixture turned orange-red and the solution became viscous. After 1 hour, 6ml of the reaction mixture was concentrated to half volume in a rotary evaporator and the sample was precipitated with acetone. A little amount of solid was dissolved in DMSO-d ₆ and ¹ H NMR and DOSY NMR spectra were obtained, which showed that the DS of the tosyl group was 16% mol/mol; 95 mg of the formylated sample were recovered. ¹ H NMR (d ₆ -DMSO) ppm: 1.95 (s, 3H, NHCOCH ₃ ), 2.45 (s, 0.49H, tosylate CH ₃ ), 3.0í5.4 (m, 12.3H, HA chain and anomehc), 7.5 (d, 0.34H, tosylate aromatics), 7.85 (d, 0.30H, tosylate aromatics), 8.0í8.5 (m, 2.14H, O-CHO formyl ester groups).

The rest of the reaction was heated to 50 °C for a further hour, quenched in a saturated NaHCO ₃ solution at pH 9, stirred for 24 hours, neutralised and filtered to remove solids. Than the solution was ultrafiltered and freeze-dried. ¹ H NMR and DOSY NMR spectra in DMSO-d ₆ were obtained, which showed that the DS of the tosyl group was 12% mol/mol. 55 mg of sample were recovered. EXAMPLE 16: Preparation of 6-O-p-toluenesulfonylhvaluronic acid A solution of HA:TBA salt (1.053 g; 1.70 mmol) (MW 20000) in 30ml of dry DMF was treated with Et ₃ N (3.2 mL; 23.0 mmol) and TsCI (2.24 g; 1 1.7 mmol) at 0°C; the reaction mixture turned orange-red and the solution became viscous. After 30 minutes, It was then brought to room temperature and after a further hour, the reaction mixture was concentrated to half volume in a rotary evaporator and the sample was precipitated with acetone. A little amount of solid was dissolved in DMSO-d ₆ and ¹ H NMR and DOSY NMR spectra were obtained, which showed that the DS of the tosyl group was 45% mol/mol; 700 mg of the formylated sample were recovered.

¹ H NMR (Cl ₆ -DMSO) ppm: 1.95 (s, 3H, NHCOCH ₃ ), 2.45 (s, 1.36H, tosylate CH ₃ ), 3.0í5.4 (m, 13.0H, HA chain and anomeric), 7.5 (d, 0.97H, tosylate aromatics), 7.85 (d, 0.90H, tosylate aromatics), 8.0í8.5 (m, 2.33H, O-CHO formyl ester groups) EXAMPLE: 17 Preparation of 6-O-Methanesulfonylhyaluronic acid (HA-Ms)

To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of DMSO were added 829μl_ (4.84mmol) of DIEA by stirring under nitrogen. MsCI (188μl_; 2.42mmol) was then added dropwise at room temperature, whereupon an orange solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (40ml), bringing the total volume to 100ml with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. The solution was freeze-dried to afford 329mg of a white solid. Total mesylate DS 77% mol/mol by proton NMR, primary mesylates 59% mol/mol by carbon NMR, selectivity 77% for the C6 position.

EXAMPLE 18: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of DMSO were added 414μL (2.42mmol) of DIEA by stirring under nitrogen. MsCI (94μL; 1.21 mmol) was then added dropwise at room temperature, whereupon an orange solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by pouring into saturated NaHCO ₃ solution (40ml), bringing the total volume to 100ml with water (resulting pH: 9.5) and maintaining stirring overnight. The resulting solution was ultrafiltered under a hood and concentrated in a rotary evaporator. The solution was freeze-dried to afford 310mg of a white solid. Total mesylate DS 34% mol/mol by proton NMR, primary mesylates 22% mol/mol by carbon NMR, selectivity 65% for the C6 position. EXAMPLE 19: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of DMF were added 829μL (4.84mmol) of DIEA by stirring under nitrogen. MsCI (188μL; 2.42mmol) was then added dropwise at room temperature, whereupon a yellow solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by adding saturated NaHCO ₃ solution (40ml) and bringing

the total volume to 100ml with water (resulting pH: 9.5); stirring was maintained overnight. The pH was raised to 10 and the suspension was stirred for 3 days, whereupon most of the solids dissolved. Then it was filtered and the resulting solution was ultrafiltered and concentrated in a rotary evaporator. The solution was freeze-dried to afford 277mg of a white solid. Total mesylate DS 42% mol/mol by proton NMR, primary mesylates 40% mol/mol by carbon NMR, selectivity 95% for the C6 position.

EXAMPLE 20: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of DMF were added 829μl_ (4.84mmol) of DIEA by stirring under nitrogen at -10°C. MsCI (188μl_; 2.42mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated NaHCO ₃ solution (40ml) and bringing the total volume to 100ml with water (resulting pH: 9.5); stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. The solution was freeze-dried to afford 207mg of a white solid. Total mesylate DS 37% mol/mol by proton NMR, primary mesylates 37% mol/mol by carbon NMR, selectivity 100% for the C6 position. EXAMPLE 21 : Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms) To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of N-methyl-2-pyrrolidone were added 829μL (4.84mmol) of DIEA by stirring under nitrogen. MsCI (188μL; 2.42mmol) was then added dropwise at room temperature, whereupon a yellow solution was formed. After 1 h stirring at room temperature, the reaction mixture was quenched by adding saturated NaHCO ₃ solution (40ml) and bringing the total volume to 100ml with water (resulting pH: 9.5); stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. The solution was freeze-dried to afford 310mg of a white solid. Total mesylate DS 50% mol/mol by proton NMR, primary mesylates 31 % mol/mol by carbon NMR, selectivity 62% for the C6 position. EXAMPLE 22: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms)

To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of N-methyl-2-pyrrolidone were added 829μL (4.84mmol) of DIEA by stirring under

nitrogen at -10°C. MsCI (188μl_; 2.42mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated NaHCO ₃ solution (40ml) and bringing the total volume to 100ml with water (resulting pH: 9.5); stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. The solution was freeze-dried to afford 250mg of a white solid. Total mesylate DS 41 % mol/mol by proton NMR, primary mesylates 39% mol/mol by carbon NMR, selectivity 95% for the C6 position. An HSQC NMR spectrum confirmed the selectivity. EXAMPLE 23: Preparation of 6-O-Methanesulfonylhvaluronic acid (HA-Ms)

To a solution of 500mg (0.806mmol) of TBA salt of HA (MW 20,000) in 20 ml of N-methyl-2-pyrrolidone were added 829μl_ (4.84mmol) of DIEA by stirring under nitrogen at 0°C. MsCI (188μl_; 2.42mmol) was then added dropwise and the resulting mixture was stirred for 1 h at 0°C. The reaction mixture was quenched by adding saturated NaHCO ₃ solution (40ml) and bringing the total volume to 100ml with water (resulting pH: 9.5); stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. The solution was freeze-dried to afford 275 mg of white solid. Total mesylate DS 44% mol/mol by proton NMR, primary mesylates 40% mol/mol by carbon NMR, selectivity 90% for the C6 position.

EXAMPLE 24: Preparation of 6-O-Methotrexylhvaluronic acid A solution of HA-OMs:TBA salt from Example 10 (500mg; 0.73mmol) in DMSO (15 ml) was treated with a solution of methotrexate (833mg; 1.83mmol) in DMSO (10ml) in the presence of solid cesium carbonate (596mg; 1.83mmol). The mixture was stirred under nitrogen at 80°C for 18h, whereupon it darkened with formation of solids. It was then cooled to ambient temperature, poured into 100ml of water (pH 6.5), treated with 15ml of saturated NaCI solution, and stirred for 1.5h. Then solids were filtered off and the solution was ultrafiltered, concentrated and freeze- dried to give 131 mg of a yellow-brownish solid. DS of MTX by NMR: 40% mol/mol; ¹³ C NMR shows that 40% of C6 is modified. HPLC analysis gave 32% w/w, corresponding to 40% mol/mol. In addition, the NMR revealed the absence of any residual secondary mesylate group and that the basic structure of HA was

unchanged, except some of the C-6 position because of the substitution by MTX. This demonstrates that any possible leaving (mesylate) groups introduced at the secondary positions (C-4,C-2',C-3') during the mesylation reaction have been hydrolysed during the displacement reaction under the basic conditions either directly or by way of 2',3'-anhydhde formation followed by hydrolysis with the retention of configuration at those positions. The NMR spectrum was repeated on the same sample after 3-months storage at room temperature, it provides the same peaks and the same intensity as those obtained on the freshly prepared product, thus indicating that the substitution degree is maintained and no by- products are formed.

EXAMPLE 25 Preparation of HA-CI: TBA salt.

5Og of hyaluronan sodium salt were suspended in 900 ml_ of dry dimethylformamide under nitrogen, with mechanical stirring at 20°C. The suspension was then cooled to -10°C and 97 ml_ of methanesulfonyl chloride were added during 30min. After additional 30min at -10°C, the temperature was raised to 20°C. After 1 h the temperature was gradually raised (during 1 h) to 60°C and stirring was continued for 18h. The reaction mixture was then poured in portions into a mixture of ice and sodium carbonate solution (4 L, initial pH=1 1 ) with vigorous mixing, maintaining the pH around 9 by addition of 1.5 M NaOH when required. The resulting brownish suspension (final volume 6 L) was stirred at pH 9.5 at room temperature for about 48h, whereupon a clear solution formed. This was filtered to remove solids and then ultrafiltered (10 KDa cut-off membrane). The resulting solution was concentrated in a rotary evaporator to a final volume of about 1 litre and treated with amberlite IRA-120 loaded with TBA. Then it was freeze-dried to afford 46.7g of HA-6-CI: TBA salt as an off-white solid (DS 64% mol/mol, determined by ¹³ C NMR). EXAMPLE 26 : Preparation of HA-lbuprofen

HA-Ms:TBA salt (400mg; 0.64mmol) as prepared in example 12 and ibuprofen (333mg; 1.61 mmol) were dissolved in DMSO (16ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (264mg; 0.81 mmol) was added and the suspension was heated at 70 °C for 2Oh with stirring. The resulting yellow-orange solution was poured into 150ml of water (pH was 6.5) and 10ml of saturated NaCI

solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dhed to give 0.15g of a white solid. DS by proton NMR: 27% mol/mol.

EXAMPLE 27 : Preparation of HA-lbuprofen HA-CI:TBA salt (1 g; 1.6mmol) as prepared in example 25 and ibuprofen (670mg; 3.2mmol) were dissolved in DMSO (50ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (264mg; 0.81 mmol) was added and the suspension was heated at 80 °C for 4Oh with stirring. The resulting dark yellow solution was poured into 100ml of water (pH was 8) and then ultrafiltered, concentrated and freeze-dhed to give g of a light brown solid. DS by proton NMR: 20% mol/mol.

EXAMPLE 28: Preparation of HA-Penicillin G

A solution of HA-Ms:TBA salt (400mg; 0.64mmol) as prepared in example 12, 18- crown-6 (338 mg; 1.28mmol) and Penicillin G sodium salt (574mg; 1.61 mmol) in DMSO (16ml) was heated at 70 °C for 2Oh with stirring.

The resulting yellow solution was poured into 150ml of water (pH was 6.5) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dhed to give 0.29g of a white solid. DS by proton NMR: 26% mol/mol. EXAMPLE 29: Preparation of HA-Penicillin G

HA-CI:TBA salt (1 g; 1.6mmol) as prepared in example 25, 18-crown-6 (840 mg; 3.2mmol) and Penicillin G sodium salt (1.13g; 3.2mmol) were dissolved in DMSO (50ml) by stirring at room temperature. The solution was heated at 80 °C for 4Oh with stirring, then it was poured into 100ml of water (pH was 7.4) and ultrafiltered, concentrated and freeze-dhed to give 1 g (yield 64%) of a pale yellow solid. DS by proton NMR: 6% mol/mol. EXAMPLE 30: Preparation of HA-Albumin

HA-CI:TBA salt (1 g; 1.6mmol) as prepared in example 25 and Human serum Albumin (300mg) were dissolved in DMSO (50ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (264mg; 0.81 mmol) was added and the suspension was heated at 80 °C for 40h with stirring. The resulting brown solution

was poured into 100ml of water (pH was 9.5) and then ultrafiltered, concentrated and freeze-dhed to give 0.9 g of a light brown solid. DS by HPLC RP: 5% mol/mol. EXAMPLE 31 : Preparation of 6-O-Methotrexylhvaluronic acid HA:TBA salt (250mg; 0.403mmol; MW 20,000) was dissolved in DMSO (10ml) by stirring and gentle heating under nitrogen; thethylamine (452μL; 3.22mmol) was then added at room temperature followed by dropwise addition of MsCI (157μL; 2.02mmol), whereupon a yellow solution formed. After 1 h stirring at room temperature, further 0.50ml of thethylamine were added, the reaction flask was connected to the vacuum and gently heated up to 50° C (bath temperature), until gas evolution ceased. Then 1.1 Og (2.43mmol) of methotrexate and 792mg (2.43mmol) of cesium carbonate were added and the mixture was stirred at 80° C overnight. Half of the reaction mixture was quenched by pouring into water (20ml); pH 6.3. The pH was adjusted to 6.8 with saturated NaHCO ₃ solution and then 10ml of saturated NaCI solution were added. After stirring for 10min, the solution was ultrafiltered, concentrated in a rotary evaporator and freeze-dried to give 60mg of a yellow solid. The DS of MTX was found to be 1 1.6% w/w by HPLC and was confirmed by NMR analysis (12% mol/mol), which also showed a small percentage of left mesylates on the polymer. The rest of the reaction mixture was worked up after further 24h at 80° C (40h overall) as described above, to afford 103mg of a yellow solid. DS in MTX 14.4% w/w by HPLC, confirmed by NMR analysis (15% mol/mol), which did not show any mesylate left on the polymer. MW 269610, Pl 10.5. Cross-linking ester bonds were cleaved by hydrolyzing the freeze dried product in 10ml of a carbonate buffer (pH 10) for 8h. After neutralization and dialysis, freeze-drying afforded 96 mg of a yellow solid. The DS of MTX in the product was 12.6% w/w by HPLC, which was confirmed by NMR analysis (13% mol/mol); MW 27,120, Pl 1.9. EXAMPLE 32: Preparation of 6-O-methotrexylhvaluronic acid To a solution of HATBA (5Og, Mw 70,000) in 1000 ml of dry DMF under stirring, mesylchloride (10 eq) was added dropwise in 1 hr time at -10°C under N ₂ flow. The mixture was maintained for 1 hour at room temperature and then heated at 60 °C for 16 hr. The work-up allow the obtainment of 46.9 g of HA-CL having chlorine content 4.2% w/w ( ¹³ C-NMR).

A solution of the HA-CI TBA salt (20 g) in DMSO (1.25 L) is treated with MTX (29.3g) and cesium carbonate (21 g) at 80 °C for 40 h, giving 5.6 g of a yellow solid.

¹³ C-NMR spectrum confirmed the occurrence of the linkage in position 6 of N- acetyl-D-glucosamine: the peak at 64 ppm is assigned at CH ₂ O-MTX and its intensity corresponds to the decrease of the peak at 44 ppm (CH ₂ CI) compared to the parent chlorine derivative. The MTX content was 18.8% w/w (HPLC); free MTX was 0.1 % w/w, water content: 8.2% w/w; MW: 11.000; Pl: 1.4. In addition, the NMR reveals the presence of residual chlorine which amounts to 1.76% w/w. EXAMPLE 33: Preparation of 6-O-Methanesulfonylhvaluronic acid TBA salt (HA-Ms:TBA)

To a solution of 5.Og (8.1 mmol) of HA:TBA (MW 5,000) in 100 mL of dry DMF were added 3.1 mL of DIEA (5.62 mL, 33.9 mmol) under stirring and N ₂ flow at -10°C. MsCI (1.25 mL; 16.1 mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated Na ₂ CO ₃ solution (20OmL) and bringing the total volume to 1 L with water; pH was adjusted to 10.5 with dilute HCI solution and stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. A small portion was freeze-dhed (100mg) for NMR analysis: primary mesylates 30% mol/mol by NMR, selectivity 100% for C6. (3.2 g; Mw: 6,925; P.I. 1.87)

EXAMPLE 34: Preparation of 6-O-Methanesulfonylhvaluronic acid TBA salt (HA-MsTBA) To a solution of 10.0g (16.1 mmol) of TBA salt of HA (MW 20,000) in 250 ml of DMF were added 7.58ml (44.3mmol) of DIEA by stirring under nitrogen at -10 ⁰ C. MsCI (1.56mL; 20.1 mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated Na ₂ CO ₃ solution (40OmL) and bringing the total volume to 2L with water; pH was adjuasted to 10.5 with dilute HCI solution and stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. A small portion was freeze-dhed (100mg) for NMR analysis: primary mesylates 24% mol/mol by NMR, selectivity 100% for C6.

The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 9.92g of a white solid (HA-Ms:TBA salt). EXAMPLE 35: Preparation of HA-CI: sodium salt

5g of hyaluronan sodium salt (MW 200,000) were suspended in 90 ml_ of dry dimethylformamide under nitrogen, with mechanical stirring at 20°C. The suspension was then cooled to -10°C and 9.7 ml_ of methanesulfonyl chloride were added during 30min. After additional 30min at -10°C, the temperature was raised to 20 °C. After 1 h the temperature was gradually raised (during 1 h) to 60 °C and stirring was continued for 18h. The reaction mixture was then poured in portions into a mixture of ice and sodium carbonate solution (400 ml_, initial pH=1 1 ) with vigorous mixing, maintaining the pH around 9 by addition of 1.5 M NaOH when required. The resulting brownish suspension (final volume 500 ml_) was stirred at pH 9.5 at room temperature for about 48h, whereupon a clear solution formed. This was filtered to remove solids and then ultrafiltered (10 KDa cut-off membrane). The resulting solution was concentrated and freeze-dried to afford 4.05g of HA-6-CI: sodium salt as an off-white solid (DS 17% mol/mol, determined by ¹³ C NMR). MW 79,560, P.I. 3.5. EXAMPLE 36: Preparation of HA-CI: sodium salt 5g of hyaluronan sodium salt (MW 500,000) were suspended in 90 mL of dry dimethylformamide under nitrogen, with mechanical stirring at 20°C. The suspension was then cooled to -10 ⁰ C and 9.7 mL of methanesulfonyl chloride were added during 30min. After additional 30min at -10°C, the temperature was raised to 20°C. After 1 h the temperature was gradually raised (during 1 h) to 70°C and stirring was continued for 21 h. The reaction mixture was then poured in portions into a mixture of ice and sodium carbonate solution (400 mL, initial pH=1 1 ) with vigorous mixing, maintaining the pH around 9 by addition of 1.5 M NaOH when required. The resulting brownish suspension (final volume 500 mL) was stirred at pH 9.5 at room temperature for about 48h, whereupon a clear solution formed. This was filtered to remove solids and then ultrafiltered (10 KDa cut-off membrane). The resulting solution was concentrated and freeze-dried to afford 3.56g of HA-6-CI: sodium salt as an off-white solid (DS 10% mol/mol, determined by ¹³ C NMR). MW 53,830, P.I. 4.02.

EXAMPLE 37: Preparation of 6-O-Methanesulfonylhyaluronic acid TBA salt

(HA-Ms:TBA)

To a solution of 10.Og (16.1 mmol) of TBA salt of HA (MW 180,000) in 500 ml of

DMF were added 9.16ml (53.4mmol) of DIEA by stirring under nitrogen at -10°C. MsCI (1.87ml_; 24.2mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated

Na ₂ CO ₃ solution (40OmL) and bringing the total volume to 2L with water; pH was adjuasted to 10.5 with dilute HCI solution and stirring was maintained overnight.

The resulting solution was ultrafiltered and concentrated in a rotary evaporator. A small portion was freeze-dhed (150mg) for NMR analysis: primary mesylates 30% mol/mol by NMR, selectivity 100% for C6.

The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 9.8Og of a white solid (HA-Ms:TBA salt).

EXAMPLE 38: Preparation of 6-O-Methanesulfonylhvaluronic acid TBA salt (HA-MsTBA)

To a solution of 20.Og (32.2mmol) of TBA salt of HA (MW 180,000) in 1000 ml of

DMF were added 36.7ml (214mmol) of DIEA by stirring under nitrogen at -10°C.

MsCI (7.48mL; 97mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10°C. The reaction mixture was quenched by adding saturated Na ₂ CO ₃ solution (80OmL) and bringing the total volume to 4L with water; pH was adjuasted to 10.5 with dilute HCI solution and stirring was maintained overnight.

The resulting solution was ultrafiltered and concentrated in a rotary evaporator. A small portion was freeze-dried (120mg) for NMR analysis: primary mesylates 55% mol/mol by NMR, selectivity 100% for C6. The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 19.95g of a white solid (HA-Ms:TBA salt).

EXAMPLE 39: Preparation of 6-O-Methanesulfonylhvaluronic acid TBA salt

(HA-MsTBA)

To a solution of 2.0Og (3.22mmol) of TBA salt of HA (MW 500,000) in 150 ml of DMF were added 3.67ml (21.4mmol) of DIEA by stirring under nitrogen at -10°C.

MsCI (750μL; 9.7mmol) was then added dropwise and the resulting mixture was stirred for 1 h at -10 ⁰ C. The reaction mixture was quenched by adding saturated

Na ₂ CO ₃ solution (8OmL) and bringing the total volume to 1 L with water; pH was adjuasted to 10.5 with dilute HCI solution and stirring was maintained overnight. The resulting solution was ultrafiltered and concentrated in a rotary evaporator. A small portion was freeze-dhed (90mg) for NMR analysis: primary mesylates 70% mol/mol by NMR, selectivity 100% for C6.

The rest of the solution was treated with amberlite IRA-120 loaded with TBA and freeze-dried to afford 1.94g of a white solid (HA-Ms:TBA salt).

EXAMPLE 40: Preparation of 6-O-Methotrexylhvaluronic acid

A solution of HA-OMs:TBA salt from Example 34 (8.Og; 12.9mmol) in DMSO (250 ml) was treated with a solution of methotrexate (14.66g; 32.3mmol) in DMSO (150ml) in the presence of solid cesium carbonate (10.5g; 32.2mmol). The mixture was stirred under nitrogen at 80 °C for 2Oh. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 9.7 and the volume to 2L. After stirring for 18h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 4.7Og of a yellow solid. DS of MTX by NMR: 7.5% mol/mol; MW 27,960, P.I. 1.92. EXAMPLE 41 : Preparation of 6-O-Methotrexylhvaluronic acid A solution of HA-OMs:TBA salt from Example 37 (7.Og; 11.3mmol) in DMSO (670 ml) was treated with a solution of methotrexate (12.79g; 28.1 mmol) in DMSO (120ml) in the presence of solid cesium carbonate (9.15g; 28.1 mmol). The mixture was stirred under nitrogen at 75 °C for 18h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 8.8 and the volume to 2.5L. After stirring for 24h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 4.3Og of a yellow solid. DS of MTX by NMR: 13% mol/mol; MW 208,400, P.I. 2.18.

EXAMPLE 42: Preparation of 6-O-Methotrexylhvaluronic acid A solution of HA-OMs:TBA salt from Example 38 (13.2g; 21.3mmol) in DMSO (1270 ml) was treated with a solution of methotrexate (24.13g; 53.1 mmol) in DMSO (120ml) in the presence of solid cesium carbonate (17.26g; 53.1 mmol). The mixture was stirred under nitrogen at 75 °C for 18h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 10.0 and the volume to 5L. After stirring for 18h the solution was neutralized, filtered,

ultrafiltered, concentrated and freeze-dried to give 6.52g of a yellow solid. DS of MTX by NMR: 20% mol/mol; MW 217,300, P.I. 2.02. EXAMPLE 43: Preparation of 6-O-Methotrexylhyaluronic acid A solution of HA-OMs:TBA salt from Example 39 (500mg; 0.74mmol) in DMSO (80ml) was treated with a solution of methotrexate (1.01 g; 2.23mmol) in DMSO (10ml) in the presence of solid cesium carbonate (726mg; 2.23mmol). The mixture was stirred under nitrogen at 80 °C for 22h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 10.0 and the volume to 40OmL. After stirring for 18h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 260mg of a yellow solid. DS of MTX by NMR: 1 1 % mol/mol; MW 460,100, P.I. 2.21. EXAMPLE 44: Preparation of 6-O-Methotrexylhvaluronic acid A solution of HA-OMs sodium salt from Example 33 (1.Og; 2.0 mmol) in DMSO (40 ml) was treated with a solution of methotrexate (1.83 g; 4mmol) in DMSO (40ml) in the presence of solid cesium carbonate (1.30; 2mmol). The mixture was stirred under nitrogen at 80 °C for 2Oh. The solution was neutralized using Na ₂ CO ₃ saturated solution bringing the final volume to 500 mL, filtered, ultrafiltered, concentrated and freeze-dried to give 500 mg of a yellow solid. DS of MTX by HPLC: 7.8% w/w; MW 16,000, P.I. 2.4. EXAMPLE 45 : Preparation of HA-lbuprofen

HA-Ms:TBA salt (500mg; 0.80mmol) as prepared in example 20 and ibuprofen (416mg; 2.01 mmol) were dissolved in DMSO (20ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (330mg; 1.01 mmol) was added and the suspension was heated at 70 °C for 2Oh with stirring. The resulting solution was poured into 200ml of water (pH was 6.5) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dried to give 0.22g of a white solid. DS by proton NMR: 30% mol/mol. EXAMPLE 46 : Preparation of HA-Naproxen HA-Ms:TBA salt (500mg; 0.80mmol) as prepared in example 20 and naproxen (463mg; 2.01 mmol) were dissolved in DMSO (20ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (330mg; 1.01 mmol) was added and the suspension was heated at 70 °C for 2Oh with stirring. The resulting solution was

poured into 200ml of water (pH was 6.6) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dried to give 0.27g of a white solid. DS by proton NMR: 28% mol/mol. EXAMPLE 47: Preparation of HA-ϋsinopril HA-Ms:TBA salt (500mg; O.δOmmol) as prepared in example 20 and lisinopril (887mg; 2.01 mmol) were dissolved in DMSO (25ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (655mg; 2.01 mmol) was added and the suspension was heated at 70 °C for 2Oh with stirring. The resulting solution was poured into 200ml of water (pH was 6.5) and 10ml of saturated NaCI solution were added. After stirring for 30min, the mixture was filtered, ultrafiltered, concentrated and freeze-dried to give 0.2Og of a white solid. DS by proton NMR: 26% mol/mol. EXAMPLE 48 : Preparation of HA-Nalidixate

HA-Ms:TBA salt (500mg; 0.80mmol) as prepared in example 20 and nalidixic acid (467mg; 2.01 mmol) were dissolved in DMSO (20ml) by stirring under nitrogen at room temperature. Solid cesium carbonate (330mg; 1.01 mmol) was added and the suspension was heated at 70 °C for 2Oh with stirring. The resulting solution was poured into 200ml of water (pH was 6.6) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dried to give 0.26g of a white solid. DS by proton NMR: 30% mol/mol. EXAMPLE 49: Preparation of HA-Penicillin G

A solution of HA-Ms:TBA salt (400mg; 0.64mmol) as prepared in example 20, 18- crown-6 (338 mg; 1.28mmol) and Penicillin G sodium salt (574mg; 1.61 mmol) in DMSO (16ml) was heated at 70 °C for 2Oh with stirring. The resulting yellow solution was poured into 150ml of water (pH was 6.5) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dried to give 0.27g of a white solid. DS by proton NMR: 31 % mol/mol. EXAMPLE 50: Preparation of HA-Cefazolin A solution of HA-Ms:TBA salt (400mg; 0.64mmol) as prepared in example 20, 18- crown-6 (338 mg; 1.28mmol) and cefazolin sodium salt (767mg; 1.61 mmol) in DMSO (18ml) was heated at 70 °C for 2Oh with stirring.

The resulting solution was poured into 180ml of water (pH was 6.7) and 10ml of saturated NaCI solution were added. After stirring for 30min, the solution was ultrafiltered, concentrated and freeze-dried to give 0.25g of a white solid. DS by proton NMR: 29% mol/mol.