CA1341603C - Acidic polysaccharide esters and process for their preparation - Google Patents

Abstract

Processes are provided herein for the preparation of acidic polysaccharide esters. One process comprises treating a quaternary ammonium salt of the polysaccharide with an esterifying agent, preferably in an aprotic solvent, and, if desired, salifying free carboxy groups in the partial esters obtained, or releasing salified groups in them.

Description

(a) TITLE OF THE INVENTION

ACIDIC POLYSACCHARIDE ESTERS AND PROCESS FOR THEIR
PREPARATION

(b) TECHNICAL FIELD TO WHICH THE INVENTION RELATES
This invention relates to novel acidic polysaccharide esters, particularly acidic esters of hyaluronic acid, and to processes for their preparation.
This application is a division of application Serial No. 513,350, filed July 8, 1986.

(c) BACKGROUND ART

The term "hyaluronic acid" (also referred to as "HY"
hereinafter) is used in the literature to designate an acidic polysaccharide with various molecular weights constituted by residues of D-glucuronic acid and N-acetyl-D-glucosamine, which naturally occur in cellular surfaces, in the basis extracellular substances of the connective tissues of vertebrates, in the synovial fluid of joints, in the vitreous humor of the eye, in the tissue of the human umbilical cord and in cocks' combs.
Hyaluronic acid plays an important role in the biological organism, firstly as a mechanical support of the cells of many tissues, e.g., the skin, the tendons, the muscles and cartilage, and it is therefore the main component of the intracellular matrix. Hyaluronic acid also performs other functions in the biological processes, e.g., the hydration of tissues, lubrication, cellular migration, cell function and differentiation. (See, for example, A. Balazs, et al, Cosmetics & Toiletries, No.
5/84, pages 8-17). Hyaluronic acid may be extracted from the above-mentioned natural tissues, e.g., from cocks' combs, or also may be obtained from certain bacteria.
Today, hyaluronic acid may also be prepared by microbiological processes. The molecular weight of whole hyaluronic acid which is obtained by extraction is in the region of 8-13 million. However, the molecular chain of 2 1341601"

the polysaccharide can be degraded quite easily under the influence of various physical and chemical factors, e.g., mechanical influences, or under the influence of radiation, hydrolyzing, oxidizing or enzymatic agents. For this reason, or often in the ordinary purification procedures of original extracts, degraded fractions with a lower molecular weight are obtained. (See Balazs, et al cited above). Hyaluronic acid, its molecular fractions and the respective salts have been used as medicaments and their use is also proposed in cosmetics (see, for example, the above-mentioned article by Balazs, et al and French Patent No. 2478468).
As a therapeutic agent, hyaluronic acid and its salts have been used especially in therapy for arthropathies, e.g., in veterinary medicine for the cure of arthritis in horses [Acta Vet. Scand. 167, 379 (1976)].
As an auxiliary and substitutional therapeutic agent for natural tissues and organs, hyaluronic acid and its molecular fractions and their salts have been used in ophthalmic surgery (see, for example, Balazs, et al, "Modern Problems in Ophthalmology", Vol. 10, 1970, p. 3 -E.B. Strieff, S. Karger eds. Basel; "Viscosurgery and the Use of Sodium Hyaluronate During Intraocular Lens Implantation", Paper presented at the International Congress and First Film Festival on Intraocular Implantation, Cannes, 9179; U.S. Patent No. 4,328,803 with a summary of the literature on the uses of HY in ophthalmology; and U.S. Patent No. 4,141,973 issued February, 1974 to Balazs).
,In application W086/6728 published November 1986, a molecular fraction of hyaluronic acid is described which can be used, for example, as its sodium salt, for intraocular and intraarticular injections suitable for the substitution and intraarticular injections suitable for the substitution of internal fluids of the eye and in arthropathy therapies, respectively.

Hyaluronic acid may also be used as an additive for a wide variety of polymeric materials used for medical and surgical articles, e.g., polyurethanes, polyesters, polyolef ins, polyamides, polysiloxanes, vinylic and acrylic polymers and carbon fibres with the effect of rendering these materials biocompatible. In this case, the addition of HY or one of its salts is effected, for example, by covering the surface of such materials, by dispersion in the same, or by both of these procedures. Such materials may be used for the manufacture of various sanitary and medical articles, e.g., cardiac valves, intraocular lenses, vascular clips, and pacemakers (see, for, example, U.S.
Patent No. 4,500,676).
Although the term "hyaluronic acid" is commonly used in an improper sense, meaning, as can be seen from above, a whole series of polysaccharides with alternations of residues of D-glucuronic acid and N-acetyl-D-glucosamine with varying molecular weights or even degraded fractions of the same, and although the plural form "hyaluronic acids" may seem more appropriate, the discussion herein shall continue to use the singular form to refer to hyaluronic acid in its various forms, including its molecular fractions. The abbreviation "HY" will also often be used to describe this collective term.
Relative to the esters of hyaluroTiic acid, there is a description in the literature of the total methyl ester of a hyaluronic acid with a high molecular weight obtained by extraction from human umbilical cords [Jeanloz, et al., J.
Biol. Chem. 186 (1950), 495-511, and Jager, et al., J.

Bacteriology 1065-1067 (1979)] This ester was obtained by treatment of free hyaluronic acid with diazomethane in ether solution and in it substantially all the carboxylic groups proved to be esterified. Furthermore,.methyl esters of oligomers of HY with between 5 and 15 disaccharide units have also been described [see Biochem. J. (1977) 167, 711-716]. Also described is a methyl ester of hyaluronic acid etherified with methyl alcohol in a part of the hydroxyl alcohol groups [Jeanloz, et al., J. Biol. Chem.
194 (1952), 141-150; and Jeanloz, et al., Helvetica Chimica Acta 35 (1952) , 262-271]. No biological activity, and therefore no pharmaceutical use, has been reported for these esters.

(d) DESCRIPTION OF THE INVENTION

An object of one aspect of the present invention is the provision of processes for the preparation of acidic polysaccharide esters, e.g., acidic hyaluronic acid esters.
An object of a second aspect of the present invention is the provision of novel tetraethylammonium salts of such esters.
An object of a third aspect of the present invention is to provide such acidic esters of hyaluronic acid with specified organic alcohols, with metals or with organic bases, which are biocompatible or acceptable from a pharmacological point of view, such acidic esters and salts possessing bio-plastic and pharmaceutical properties, so that they may be used in innumerable fields, including cosmetics, surgery and medicine.
An object of a fourth aspect of this invention is to provide such acidic esters which are considerably more stable, especially regarding the action of the natural enzymes which are responsible for the degradation of the polysaccharide molecule in the organism, e.g., especially hyaluronidase, and they, therefore, conserve the above-mentioned physical-chemical, pharmacological and therapeutic properties, which are qualitatively the same as foz' hyaluronic acid, for very long periods.
One aspect of the invention of the present divisional application provides a process for the preparation of acidic polysaccharide esters containing car'boxyl groups.
That process comprises treating a quaternary ammonium salt of the polysaccharide with an esterifying agent, preferably in an aprotic solvent. If desired, free carboxy groups in ~3 40~

the partial esters so-obtained, are salified. The salified groups in them may thereafter be released.
A second aspect of the invention of the present divisional application provides a process for the 5 preparation of acidic polysaccharide esters containing carboxyl groups. That process comprises, in a first step, treating a quaternary ammonium salt of the polysaccharide with-an esterifying agent in a suitable solvent. In a second step, free carboxy groups in the partial esters so-obtained are salified.
By a first variant of these first and second aspects of this invention of the present divisional application, the process includes the step of releasing salified groups in the acidic salified carboxy groups in the esters.
By a second variant of these first and second aspects of this invention of the present divisional application, the acidic polysaccharides are of vegetable origin.
By a third variant of these first and second aspects of this invention of the present divisional application, the acidic polysaccharides are of animal origin.
By a fourth variant of these first and second aspects of this invention of the present divisional application, the acidic polysaccharide is hyaluronic acid or one of its molecular fractions.
By a f if th variant of these first and second aspects of this invention of the present divisional application, the quaternary ammonium salt is a lower tetraalkylammonium salt.
By a sixth variant of these first and second aspects of this invention of the present divisional application, the quarternary ammonium salt is the tetraalkylammonium salt of an acidic polysaccharide. By a variation thereof, the acidic polysaccharide also contains sulphonic acid groups.
By a seventh variant of these first and second aspects of this invention of the present divisional application, the quaternary ammonium salt is the tetralkylammonium salt of hyaluronic acid. By a variation thereof, the tetraalkylammonium salt is the tetrabutylammonium salt.
By an eighth variant of the second aspect of this invention of the present divisional application, the suitable solvent is selected from the group consisting of a polar solvent, a non-polar solvent and an aprotic solvent. By a first variation thereof, the solvent is selected from the group consisting of a dialkylsulphoxide, a dialkylcarboxamide, a lower alkyl dialkylamide of a lower aliphatic acid, an alcohol, an ether, a ketone, and an ester. Preferablyl, the solvent is dimethylsulphoxide.
By a ninth variant of these first and second aspects of this invention of the present divisional application, the salifying is carried out to form a salt with an alkali metal, with an alkaline earth metal, with magnesium, with aluminum, or with ammonia. By a first variation thereof, the salt is formed with sodium or with ammonia. By a second variation thereof, the salt is formed with a therapeutically-acceptable ammonium base, aliphatic base, araliphatic base, cycloaliphatic base or heterocyclic base.
Specific processes within the ambit of the invention provided by the present divisional application include the following:
Reacting the tetrabutylammonium salt of hyaluronic acid with n-pentyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with isopentyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with benzyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with 2-bromoethylbenzene.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-17a-ol-3,11,20-trione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with cortisone.

Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-11(3,17a-diol-3,20-dione, and then salifying with sodium ions, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with hydrocortisone.
Reacting the tetrabutylammonium salt of hyaluronic acid, with ethyl iodide and with 9R-fluoro-2l-bromo-4-pregnene-11(3,17a-diol-3,20-dione, and salifying with sodium ions, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the caroxy groups are esterified with fluorocortisone.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl chloroacetate and then salifying with sodium ions, whereby 75% of the carboxyl groups are esterified and whereby 25% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with 21-bromo-4-pregnene-17a-ol-3,11,20-trione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with 21-bromo-4-pregnene-11R,17a-diol-3,20-dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with 9-fluoro-2l-bromo-4-pregnene-11R,17a-diol-3,20 dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with 21-bromo-4-pregnene-3,20-dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.

Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-3,20-dione, and then salifying with sodium ions, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with deoxycorticosterone and whereby the remaining 40% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with deoxycorticosterone, and whereby the remaining 40% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-17a-ol-3,11,20-trione, and then salifying with sodium ions, whereby 40% of the carboxyl groups esterified with ethanol, whereby 20% of the carboxyl groups are esterified with cortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-11(3,17a-diol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl gr,oups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with hydrocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 9(3-fluoro-2l-bromo-4-pregnene-11(3,17a-diol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with fluorocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.

Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 9(3-fluoro-21-bromo-4-pregnene-11(3,17oc-tiol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with fluorocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.
By a tenth variant of these first and second aspects of this invention of the present divisional application, and of any of the specific processes above, the hyaluronic acid ester derives from integral hyaluronic acid,or from one of its salts obtained by extraction from cocks' combs and having a molecular weight of between 8 and 13 million.
By an eleventh variant of these first and second aspects of this invention of the present divisional application and of any of the specific processes above, the hyaluronic acid ester derives from hyaluronic acid obtained by first dehydrating cocks' combs with acetone and then by exposing them to enzymatic digestion with papain.
By a variation thereof, that process includes the additional steps of molecular ultrafiltration and further purification of the hyaluronic acid fraction so-obtained.
By a twelfth variant of these first and second aspects of this invention of the present divisi,onal application and of any of the specific processes above, the hyaluronic acid ester derives from a hyaluronic acid fraction with a molecular weight of between 90 - 0.23% of the molecular weight of an integral hyaluronic acid having a molecular weight of 13 million.
By a thirteenth variant of these first and second aspects of this invention of the present divisional application and of any of the specific processes above, the hyaluronic acid ester derives from a molecular fraction having a molecular weight of between 50,000 and 100,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.

By a fourteenth variant of these first and second aspects of this invention of the present divisional application and of any of the specific processes above, the hyaluronic acid ester derives from a molecular fraction 5 having a molecular weight of between 500,000 and 730,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
A third aspect of the invention of the present divisional application provides a tetraalkylammonium salt 10 of hyaluronic acid or one of its molecular fractions.
A fourth aspect of the invention of the present divisional application provides a tetraalkylammonium salt of an acidic polysaccharide containing carboxy groups deriving from alkyls with between 1 and 6 carbon atoms. By a variation thereof, the tetraalkylammonium salt is one in which the acidic polysaccharide is hyaluronic acid or one of its molecular fractions.
By a first variant of the third and fourth aspects of the invention of the present divisional application, the salt of the acidic polysaccharide is one with an alkali metal, or with an alkaline earth metal, or with magnesium, or with aluminum, or with ammonia.
By a second variant of the third and fourth aspects of the invention of the present divisional application, and of the above first variant thereof, the hyaluronic acid ester derives from a hyaluronic acid molecular fraction having a molecular weight of between 500,000 and 730,000 and which is substantially free of hyaluronic acid having a molecular weight of less than 30,000.
By a third variant of the third and fourth aspects of the invention of the present divisional application, and of the first variant thereof, the hyaluronic acid ester derives from an integral hyaluronic acid or from one of its salts obtained by extraction from cocks' combs, and having a molecular weight of between 8 and 13 million.
By a fourth variant of the third and fourth aspects of the invention of the present divisional application, and of 11 ~ 3 4 1 6 0 J
the above first variant, the hyaluronic acid derives from hyaluronic acid obtained by first dehydrating cocks' combs with acetone and the exposing them to enzymatic digestion with papain.
By a fifth variant of the third and fourth aspects of the invention of the present divisional application, and of the above first variant thereof, the hyaluronic acid ester derives from a hyaluronic acid fraction with a molecular weight of between 90 - 0.23% of the molecular weight of an integral hyaluronic acid with a molecular weight of 13 million.
By a sixth variant of the third and fourth aspects of the invention of the present divisional application, and of the above first variant thereof, the hyaluronic acid ester derives from a molecular fraction having a molecular weight of between 50,000 and 100,000 and which is substantially free of hyaluronic acid having a molecular weight of less than 30,000.
Thus, as specified hereinabove, the acidic hyaluronic esters of aspects of the invention of the present divisional application may be prepared according to a process which may be generally applied to the preparation of carboxylic esters of acidic polysaccharides with carboxyl groups. This process consists of treating a quaternary ammonium salt of an acidic polysaccharide containing carboxyl groups with an esterifying agent, preferably in an aprotic organic solvent. As starting acidic polysaccharides, it is possible to use, for example, apart from hyaluronic acid, other acidic polysaccharides of animal or vegetable origin and synthetically modified derivatives thereof, e.g., acid hemicellulose, obtainable from the alkaline extracts of certain plants and after precipitation of xylans, whose disaccharide components are made up of D-glucuronic acid and D-xlyllopyranose, (see "The Carbohydrates" by W. Pigman, pages 668-669 - R.L.
Whistler, W.M. Corbett), the pectins and acidic polysaccharides obtainable therefrom, that is, ~

12 0~

galacturonan, acidic polysaccharides which are obtainable from plant gum (exudates), e.g., gum arabic, gum tragacanth, and finally acidic polysaccharides which are derived from seaweed, e.g., agar and carrageenans. As starting material, it is, of course, possible also to use the molecular fractions obtained by degradation of any of the above-mentioned polysaccharides.
As organic solvents, it is preferable to use aprotic solvents, e.g., dialkylsuphoxides, dialkylcarboxamides, e.g, in particular lower alkyl dialkylsulphoxides, especially dimethylsulphoxide, and lower alkyl dialkylamides of lower aliphatic acids, e.g., dimethylformamide, diethylformamide, dimethylacetamide, or diethylacetamide.
Other solvents, however, which may be used are not always aprotic, e.g., alcohols, ethers ketones, esters, especially aliphatic alcohols or heterocyclic alcohols, and ketones with , a lower boiling . point, e.g., hexafluoroisopropanol, trifluoroethanol, and N-methylpyrrolidone.
The reaction is preferably effected at a temperature range of between 0 C and 100 C, especially between 25 C and 75 C, for example, at 30 C.
The esterification preferably is carried out by adding the esterifying agent by degrees to the above-mentioned quaternary ammonium salt in one of the above-mentioned solvents, for example, in dimethylsulphoxide.
As starting quaternary ammonium salts, it is preferable to use the lower ammonium tetraalkylates, with alkyl groups having preferably between 1 and 6 carbon atoms. Most preferably, the hyaluronate of tetrabutylammonium is used.
It is possible to prepare these quaternary ammonium salts by reacting a metallic salt of an acidic polysaccharide, preferably one of those mentioned above, especially the sodium or potassium salts, in aqueous solution with a salified sulphonic resin with a quaternary ammonium base.

13 ~.~ 4 0~
The tetraalkylammonium salts of the acidic polysaccharide of other aspects of the invention of the present divisional application can be obtained by freeze-drying the eluate. The tetraalkylammonium salts of acidic polysaccharides which may be preferably used as starting compounds of the process of aspects of the invention of the present divisional application and deriving from lower alkyls, especially alkyls with between 1 and 6 carbon atoms, are new and thus provide this other aspect of the invention of the present divisional application.
Surprisingly, such salts have proven to be soluble in the above-mentioned organic solvents, and for this reason the esterification of acidic polysaccharides according to the above-mentioned novel process of aspects of the invention of the present divisional application is particularly easy and gives generous yields. It is, therefore, only by using this kind of procedure that one can exactly dose the number of carboxylic groups of acidic polysaccharide which are to be esterified.
The above-described process of aspects of the invention of the present divisional application is very suitable especially for the preparation of hyaluronic acidic esters and salts thereof, according to other aspects of aspects of the invention of the present divisional application. In particular, therefore, as starting compounds for this process of aspects of the invention of the present divisional application, the quaternary ammonium salts of hyaluronic acid, especially those deriving from lower alkyls, and especially from alkyls with between 1 and 6 carbon atoms, are new and provide a particular aspect of the invention of the present divisional application. In a variant of a salt form of a partial ester of hyaluronic acid, the salt is formed with an alkali metal, or with an alkaline earth metal, or with magnesium, or with aluminum, or with ammonia. In a variation thereof, the salt is a sodium salt . ,.

In a variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, the hyaluronic acid derives from hyaluronic acid which is obtained by first dehydrating cocks' combs with acetone and then exposing them to enzymatic digestion with papain.
In a further variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, the hyaluronic acid has been obtained by first dehydrating cocks' combs with acetone and then exposing them to enzymatic digestion with papain, followed by molecular ultrafiltration, and further purification of the hyaluronic acid fraction so-obtained.
In yet another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from an integral hyaluronic acid, or from one of its salts which is obtained by extraction from cocks' combs, and which has a molecular weight of between 8 and 13 million.
In a further variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives froma hyaluronic acid fraction with a molecular weight of between 90 and 0.23%
of the molecular weight of an integral hyaluronic acid with a molecular weight of 13 million.
In still another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from a hyaluronic acid molecular fraction having a molecular weight of between 50,000 and 100,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.

3 4 1 ~0 3 In yet another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from a hyaluronic acid 5 molecular fraction having a molecular weight of between 500,000 and 730,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30, 0&0 .
For the preparation of the new acidic hyaluronic acid 10 esters according to other aspects of aspects of the invention of the present divisional application, it is possible to use hyaluronic acids of any origin, for example, the acids extracted from the natural starting materials, for example, from cocks' combs. The preparation 15 of such acids is described in the literature: preferably, purified hyaluronic acids are used. Especially useful hyaluronic acids comprise molecular fractions of the integral acids obtained directly by extraction of the organic materials, with molecular weights varying within a wide range, for example from 90% to 80% (MW = 11.7 to 10.4 million) to 0.23% (MW = 30,000) of the molecular weight of the integral acid having a molecular weight of 13 million, preferably between 5% and 0.2%. Such fractions may be obtained by means of various procedures described in the literature, e.g., by hydrolysing, oxidizing, enzymatic or physical procedures, e.g., mechanical or rotational procedures. Primordial extracts are therefore often formed during these same purification procedures (for example, see the article by Balazs, et al., quoted above in "Cosmetics & Toiletries"). The separation and purification of the molecular fractions obtained are brought about by known techniques, for example, by molecular filtration.
One fraction of purified hyaluronic acid which is suitable for use according to aspects of the invention of the present divisional application is, for example, that known as "non-inflammatory-NIF-NaHA sodium hyaluronic"
described by Balazs in the booklet "Healon - A Guide To Its Use In Ophthalmic Surgery", D. Miller & R. Stegmann, eds, John Wiley & Sons N.Y. 81983: p.5.
Particularly important as starting materials for the preparation of these hyaluronic acid acidic esters of aspects of the invention of the present divisional application are two purified fractions which are obtainable from hyaluronic acid, for example the ones extracted from cocks-' combs, known by the trade-marks HYALASTINETM and HYALECTINTM of Fidia S.p.A. The fraction HYALASTINE.,,M has an average molecular weight of 50,000 to 100,000, while the fraction HYALECTINTM has an average molecular weight of between 500,000 and 730,000. A combined fraction of these two fractions has also been isolated and is characterized as having an average molecular weight of 250,000 to 350,000. This combined fraction may be obtained with a yield of 80% of total hyaluronic acid available in the particular starting material, whi-le the fraction HYALECTINTM
may be obtained with a yield of 30% and the fraction HYALASTINETM with a yield of 50% of the starting HY. The preparation of these fractions is described in Examples A-C
hereinunder.
The salification of hyaluronic acid with the above metals, for the preparation of starting salts for the particular esterification procedure of aspects of the invention of the present divisional application described above, is performed in a manner which is known per se, for example, by reacting hyaluronic acid with the calculated base quantity, for example, with alkali metal hydrates or with alkaline earth metal hydrates or with basic salts of such metals, e.g., carbonates or bicarbonates.
In the process of aspects of the invention of the present divisional application for preparing the partial acidic esters of aspects of the invention of the present divisional application, it is possible to salify all the remaining carboxylic groups or only part of them, dosing the base quantities so as to obtain the desired stoichiometric degree of salification. With the correct 17 :3 4 1 C 0 3 degree of salification it is possible to obtain esters with a wide range of different dissociation constants and which therefore give the desired pH, in solution in situ at the time of therapeutic application.
The invention in the present divisional application also includes amongst its other aspects, modifications of the above-described processes for the production of the new acidic esters and their salts, in which a process is interrupted at any given stage, or started with an intermediate compound on which the remaining stages are carried out, or in which the starting products are formed in situ.

In the esters of aspects of this invention which are obtained according to this procedure or according to the other procedures described hereinafter, free carboxylic groups of the partial esters may be salified, by means of a procedures known per se.

Hyaluronic acid or one of its molecular particles, as described above is preferably used in aspects of the present invention. Dimethylsulphoxide is preferably used as the aprotic solvent. A lower tetraalkylammonium salt is used as the starting salt; preferably a tetraalkylammonium salt of acidic polysaccharide is used.
The invention in the present divisional application, also provide tetraalkylammonium salts of an acidic polysaccharide containing carboxyl groups deriving from alkyls with between 1 and 6 carbon atoms, especially where the acidic polysaccharide is hyaluronic acid or one of its molecular fractions.
=It is possible to form inorganic salts deriving from alkali metals, e.g., potassium and especially sodium, or from ammonium, or deriving from alkaline earth metals, e.g., calcium, or magnesium, or from aluminum salts.
Particularly interesting are the salts with organic bases, especially nitrogenized bases and therefore aliphatic, arylaliphatic, cycloaliphatic or heterocyclic amines.

18 ~- 3 4 1 6 0 7 These ammoniac salts may derive from therapeutically-acceptable but inactive amines or from amines with therapeutic action. Of the former, the aliphatic amines should be considered, e.g., mono-, di-, and tri-alkylamines with alkyl groups having a maximum of 18-carbon atoms or arylalkylamines with the same number of carbon atoms in the aliphatic part and where aryl means a benzene group, possibly substituted by 1 to 3 methyl groups or halogen atoms or hydroxyl groups. The biologically-inactive bases for the formation of salts may also be cyclic, e.g., monocyclic alkylenamines with rings of between 4 and 6 carbon atoms, possibly interrupted in , the ring by heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, e.g., piperidine or morpholine, and may be substituted, for example, by aminic or hydroxylic functions, e.g., aminoethanol, ethylendiamine, ephedrine or choline.
It is also possible to form the quaternary ammonium salts of the partial esters of HY, for example, tetralkyl-ammonium salts with the above-mentioned number of carbon atoms and preferably salts of such a type in which the fourth alkyl group has between 1 and 4 carbon atoms, for example, a methyl group.
By one broad aspect of the invention, which is disclosed and claimed in the above-identified parent application, an ester is provided of hyaluronic acid of the Formula Hy ( COOR ) ,õ
wherein HyCOO represents a hyaluronic acid radical, and R
is derived from an alcohol which is selected from the group consisting of an aliphatic alcohol having a chain of 2 to 34 carbon atoms, an araliphatic alcohol, a cycloaliphatic alcohol and a heterocyclic alcohol and n is a large member.
By important variants of such invention which is disclosed and claimed in the above-identified parent application, these esters may be in the form of a total ester, or in the form of a partial ester or in the form of 19 ~3 0 3 a salt of such partial ester with an inorganic base or with an organic base.
In either the total ester form, or the partial ester form or the salt form, by variants of such ester, the alcohol is an aliphatic alcohol which is substituted by one or two functional groups which are selected from the group consisting of amino, hydroxy, mercapto, aldehyde, keto, carboxyl, hydrocarbyl, dihydrocarbylamino, ether, ester, thioether, thioester, acetal, ketal, carbalkoxy, unsubstituted carbamide groups and carbamide groups which are substituted by one or two alkyl groups, the hydrocarbyl radicals in these functionally-modified groups having a maximum of 6 carbon atoms, and in which the carbon atom chain may be interrupted by heteroatoms which are selected from the group consisting of oxygen, sulphur and nitrogen.
In a variation thereof, the alcohol is ethyl alcohol, propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol, tert-butyl alcohol, amyl alcohol, pentyl alcohol, hexyl alcohol, octyl alcohol, ethylene glycol, propylene glycol, butylene glycol or glycerin.
In either the total ester form, or the partial ester form or the salt form, by other variants of such ester, the alcohol is an araliphatic alcohol with only one benzene residue, in which the aliphatic chain has a maximum of 4 carbon atoms, and in which the benzene residue is unsubstituted or is substituted by 1 to 3 methyl groups or by 1 to 3 hydroxy groups, or by 1 to 3 halogen atoms, and in which the aliphatic chain may be substituted by one or two functions which are selected from the group consisting of free amino groups, monoethylamino groups, diethylamino groups, pyrrolidinyl groups and piperidinyl groups. In a variation thereof, the alcohol is benzyl alcohol, phenethyl alcohol, ephedrine or adrenalin.
In either the total ester form, or the partial ester form or the salt form, by other variants of such ester, the alcohol is a cycloaliphatic alcohol or an aliphatic-cycloaliphatic alcohol and derives from a monocyclic hydrocarbide or from a polycyclic hydrocarbide with a maximum of 34 carbon atoms.
In either the total ester form, or the partial ester form or the salt form, by still other variants of such 5 ester, the alcohol is a polycyclic alcohol which is selected from the group consisting of sterols, chloric acids, steroid alcohols, groups of the estrane and pregnane series and their unsaturated derivatives. In a variation thereof, the alcohol is cortisone, hydrocortisone, 10 prednisone, prednisolone, fluorocortisone, dexamethasone, betamethasone, corticosterone, deoxycorticosterone, paramethasone, flumethasone, fluocinolone., fluocinolone acetonide, fluprednylidene, clobetasol, or beclomethasone.
In a variant of a salt form of a partial ester of 15 hyaluronic acid, the salt is formed with an alkali metal, or with an alkaline earth metal, or with magnesium, or with aluminum, or with ammonia. In a variation thereof, the salt is a sodium salt In a variant thereof, in either a total ester of 20 hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, the hyaluronic acid derives from hyaluronic acid which is obtained by first dehydrating cocks' combs with acetone and then exposing them to enzymatic digestion with papain.
In a further variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, the hyaluronic acid has been obtained by first dehydrating cocks' combs with acetone and then exposing them to enzymatic digestion with papain, followed by molecular ultrafiltration, and further purification of the hyaluronic acid fraction so-obtained.
In yet another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from an integral hyaluronic acid, or from one of its salts which is obtained by extraction from cocks' combs, and which has a molecular weight of between 8 and 13 million.
In a further variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from a hyaluronic acid fraction with a molecular weight of between 90 and 0.23%
of the molecular weight of an integral hyaluronic acid with a molecular weight of 13 million.
In still another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from a hyaluronic acid molecular fraction having a molecular weight of between 50,000 and 100,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
In yet another variant thereof, in either a total ester of hyaluronic acid, a partial ester of hyaluronic acid or a salt of a partial ester of hyaluronic acid, such hyaluronic acid ester derives from a hyaluronic acid molecular fraction having a molecular weight of between 500,000 and 730,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
Specific embodiments of such esters of hyaluronic acid of aspects of the invention, which is disclosed and claimed in the above-identified parent application, include the following:
The total ethyl ester of hyaluronic acid.
The total propyl ester of hyaluronic acid.
The total pentyl ester of hyaluronic acid.
The total isopentyl ester of hyaluronic acid.
The total benzyl ester of hyaluronic acid.
The total phenethyl ester of hyaluronic acid.

22 s 3 The mixed ethanol-cortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with cortisone.
The mixed ethanol-hydrocortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with hydrocortisone.
The mixed ethanol-fluorocortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with fluorocortisone.
The mixed ethanol-deoxycorticosterone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with deoxycorticosterone.
A salt of a partial propyl ester of hyaluronic acid with 50% of the carboxyl groups esterified and with 50% of the carboxyl groups salified with sodium..
A salt of a partial isopropyl ester of hyaluronic acid with 50% of the carboxyl groups esterified and with 50% of the carboxyl groups salified with sodium.
A salt of a partial propyl ester of hyaluronic acid with 85% of the carboxylic groups esterified and with 15%
of the carboxylic groups salified with sodium.
A salt of a partial ethyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium.
A salt of a partial methyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium.
A salt of a partial butyl ester of hyaluronic acid with 50% of the carboxylic groups salified with sodium.
A salt of the partial ethoxycarbonylmethyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium.

A salt of a partial cortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium.
A salt of a partial hydrocortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium.
A salt of the partial fluorocortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium.
A salt of a deoxycorticosterone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium.
A salt of a partial and mixed ethanol-deoxycorticosterone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with deoxycorticosterone and with the remaining 40% of the carboxyl groups salified with sodium.
A salt of a partial and mixed ethanol-cortisone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with cortisone, and with the remaining 40% of the carboxyl groups salified with sodium.
A salt of a partial and mixed ethanol-hydrocortisone ester of hyaluronic acid with 40% of -the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with hydrocortisone, and with the remaining 40%
of the carboxyl groups salified with sodium.
A salt of a partial and mixed ethanol-fluorocortisone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with fluorocortisone, and with the remaining 40%
of the carboxyl groups salified with sodium.
A salt of a hyaluronic acid ester of streptomycin with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with streptomycin.

~3 4 6 03 A salt of a hyaluronic acid ester of erythromycin with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with erythromycin.
A salt of a hyaluronic acid ester of neomycin with 75%
of the carboxyl groups esterified with ethanol and with 25%
of the carboxyls salified with neomycin.
A salt of a hyaluronic acid ester of gentamycin with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with gentamycin.
A salt of a hyaluronic acid ester of kanamycin with 75%
of the carboxyl groups esterified with ethanol and with 25%
of the carboxyls salified with kanamycin.
A salt of a hyaluronic acid ester of pilocarpine with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with pilocarpine.
A salt of a hyaluronic acid ester of pilocarpine with 85% of the carboxyl groups esterified with ethanol and with 15% of the carboxyls salified with pilocarpine.
The hyaluronic acid ester of amikacin with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with amikacin.
By another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of an ester of hyaluronic acid of the Formula Hy (COOR) rõ
wherein HyCOO represents a hyaluronic acid radical, and R
is derived from an alcohol which is selected from the group consisting of an aliphatic alcohol having a chain of 2 to 34 carbons, an araliphatic alcohol, a cycloaliphatic alcohol and a heterocyclic alcohol and n is a large number, which process comprises: (I) providing a total ester of hyaluronic acid with such alcohol by carrying out one of the following esterification reactions: (a) treating free hyaluronic acid with a sufficient amount of a selected alcohol as defined above in the presence of a catalyzing substance; or (b) treating free hyaluronic acid with a 13~16 03 sufficient amount of an etherifying agent which is capable of introducing the desired alcoholic residue of an alcohol, as defined above, in the presence of an inorganic base or an organic base; or (c) treating a metal salt of hyaluronic 5 acid or an organic azotized base of hyaluronic acid with a sufficient amount of a selected alcohol, as defined above, in the presence of a catalyzing substance; or (d) treating a quaternary ammonium salt of hyaluronic acid with a sufficient amount of an etherifying agent capable of 10 introducing the desired alcoholic residue of an alcohol, as defined above, in the presence of an inorganic base or an organic base; or (II) providing a partial ester of hyaluronic acid with such alcohol, by carrying out one of the following reactions: (a) treating free hyaluronic acid 15 with an insufficient amount of a selected alcohol as defined above in the presence of a catalyzing substance; or (b) treating free hyaluronic acid with an insufficient amount of an etherifying agent which is capable of introducing the desired alcoholic residue of an alcohol, as 20 defined above, in the presence of an inorganic base or an organic base; or (c) treating a metal salt of hyaluronic acid or an organic azotized base of hyaluronic acid with an insufficient amount of a selected alcohol, as defined above, in the presence of a catalyzing substance; or (d) 25 treating a quaternary ammonium salt of,hyaluronic acid with an insufficient amount of an etherifying agent which is capable of introducing the desired alcoholic residue of an alcohol, as defined above, in the presence of an inorganic base or an organic base; or (III) providing a salt of a partial ester of hyaluronic acid with such alcohol, by carrying out one of the following reactions: (a) treating free hyaluronic acid with an insufficient amount of a selected alcohol, as defined above, in the presence of a catalyzing substance; or (b) treating free hyaluronic acid with an insufficient amount of an etherifying agent which is capable of introducing the desired alcoholic residue of an alcohol, as defined above, in the presence of an #3 3 inorganic base or an organic base; or (c) treating a metal salt of hyaluronic acid or an organic azotized base of hyaluronic acid with an insufficient amount of a selected alcohol, as defined above, in the presence of a catalyzing substance; or (d) treating a quaternary ammonium salt of hyaluronic acid with an insufficient amount of an etherifying agent capable of introducing the desired alcoholic residue of an alcohol, as defined above, in the presence of an inorganic base or an organic base; and then salifying the remaining carboxylic groups, or part of such carboxylic groups, in the partial ester so formed, with an inorganic base or with an organic base.
By a variant of such process, which is disclosed and claimed in the above-identified parent application, the catalyzing substance is a strong inorganic acid or an ionic exchanger of the acid type.
By another variant of such process, which is disclosed and claimed in the above-identified parent application, the etherifying agent is selected from the group consisting of an ester of an inorganic acid, an ester of an organic sulphonic acid, a hydracid, a hydrocarbyl halogenide, a neutral sulphate, a hydrocarboxyl acid, an alfite, a carbonate, a silicate, a phosphite, and a hydrocarbyl sulphonate.
By still another variant of such process, which is disclosed and claimed in the above-identified parent application, the process takes place in a suitable solvent which is selected from the group consisting of a polar solvent, a non-polar solvent and an aprotic solvent.
By yet another variant of such process, which is disclosed and claimed in the above-identified parent application, the process takes place in a solvent which is selected from the group consisting of a dialkylsulphoxide, a dialkylcarboxamide, a lower alkyl dialkylamide of a lower aliphatic acid, an alcohol, an ether, a ketone, and an ester.

27 a By a further variant of such process, which is disclosed and claimed in the above-identified parent application, the base is a hydrate of an alkali metal, a hydrate of an alkaline earth metal, magnesium oxide, silver oxide, a basic salt of an alkali metal, of an alkaline earth metal, of magnesium or of silver, of a carbonate of an alkali metal, of an alkaline earth metal, of magnesium or of~silver, an organic base, a tertiary azotized base, or an ionic exchanger of the basic type. By a variation of such process, the metal salt is a salt of an alkali metal or a salt of an alkaline earth metal. By another variation of such process, the organic azotized base is an ammonium salt or an ammonium-substituted salt.
By yet another variant of such process, which is disclosed and claimed in the above-identified parent application, the process is carried out at temperature of 0 C-100 C.
By variations of any of the aspects or variants described above, which is disclosed and claimed in the above-identified parent application, the alcohol is an aliphatic alcohol which is substituted by one or two functional groups which are selected from the group consisting of amino, hydroxy, mercapto, aldehyde, keto, carboxyl, hydrocarbyl, dihydrocarbylamino, ether, ester, thioether, thioester, acetal, ketal, carbalkoxy, unsubstituted carbamide groups or carbamidic groups which are substituted by one or two alkyl groups, the hydrocarbyl radicals in these functiorially-modified groups having a maximum of 6 carbon atoms, and in which the carbon atom chain may be interrupted by heteroatoms which are selected from the group consisting of oxygen, sulphur and nitrogen.
By a specific variation thereof, the alcohol is ethyl alcohol, propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol, tert-butyl alcohol, amyl alcohol, pentyl alcohol, hexyl alcohol, octyl alcohol, ethylene glycol, propylene glycol, bu-tylene glycol or glycerin.

28 6~i By some variations of any of the aspects or variants described above, which is disclosed and claimed in the above-identified parent application, the alcohol is an araliphatic alcohol with only one benzene residue, in which the aliphatic chain has a maximum of 4 carbon atoms, and in which the benzene residue may be substituted by 1 to 3 methyl groups, or by 1 to 3 hydroxy groups or by 1 to 3 halogen atoms, and in which the aliphatic chain may be substituted by one or two functional groups which are selected from the group consisting of free amino groups, monoethylamino groups, diethylamino groups, pyrrolidinyl groups and piperidinyl groups. By a specific variation thereof, the alcohol is benzyl alcohol, phenethyl alcohol, ephedrine or adrenalin.
By other variations of any of the aspects or variants described above, which is disclosed and claimed in the above-identified parent application, the alcohol is a cycloaliphatic alcohol or an aliphatic-cycloaliphatic alcohol and derives from a monocyclic hydrocarbide or from a polycyclic hydrocarbide with a maximum of 34 carbon atoms. By a specific variation thereof, the alcohol is a polycyclic alcohol which is selected from the group consisting of sterols, colic acids, steroid alcohols, groups of the estrane and pregnane series and their unsaturated derivatives. By another specific variation thereof, the alcohol is cortisone, hydrocortisone, prednisone, predinisolone, fluorocartisone, dexamethasone, betamethasone, corticosterone, deoxycorticosterone, paramethasone, flumethasone, fluocinolone, fluocinolone acetonide, fluprednylidene, clobetasol, or beclomethasone.
By another variant thereof, which is disclosed and claimed in the above-identified parent application, the salt is formed with an alkali metal, with an alkaline earth metal, with magnesium, with aluminum, or with ammonia.
Preferably the salt is a sodium salt or an ammonia salt.
By a variation thereof, the salt is formed with a therapeutically-acceptable ammonium base, aliphatic base, araliphatic base, cycloaliphatic base or heterocyclic base.
Specific embodiments of such processes of aspects of the invention which is disclosed and claimed in the above-identified parent application, comprise the following reactions:
Reacting hyaluronic acid with ethyl alcohol.
Reacting hyaluronic acid with n-propyl alcohol.
Reacting the tetrabutylammonium salt of hyaluronic acid with n-pentyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with isopentyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with benzyl bromide.
Reacting the tetrabutylammonium salt of hyaluronic acid with 2-bromoethylbenzene.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-17a-ol-3,11,20-trione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with cortisone.
Reacting the tetrabutylammonium salt of hyaluronic acid with ethyl iodide and with 21-bromo-4-pregnene-11(3,17a-diol-3,20-dione, and then salifying with sodium ions, whereby 80% of the carboxyl groups -are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with hydrocortisone. Reacting the tet rabutyl ammonium salt of hyaluronic acid with ethyl iodide and with 9(3-fluoro-21-bromo-4-pregnene-11~,17a-diol-3,20-dione, and salifying with sodium ions, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxy groups are esterified with fluorocortisone.
Reacting hyaluronic acid with propyl alcohol and then salifying with sodium ions, whereby 50% of the carboxyl groups are esterified and whereby 50% of the carboxyl groups are salified with sodium.

;3~fi1~3 Reacting hyaluronic acid with isopropyl alcohol and then salifying with sodium ions, whereby 50% of the carboxyl groups are esterified and whereby 50% of the carboxyl groups are salified with sodium.
Reacting hyaluronic acid with n-propyl alcohol and then salifying with sodium ions, whereby 85% of the carboxylic groups are esterified and whereby 15% of the carboxylic groups are salified with sodium.
Reacting hyaluronic acid with ethyl alcohol and then salifying with sodium ions, whereby 75% of the carboxyl groups are esterified and whereby 25% of the carboxyl groups are salified with sodium.
Reacting hyaluronic acid with n-butyl alcohol and then salifying with sodium ions, whereby 50% of the carboxylic groups are salified with sodium.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with streptomycin, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with streptomycin.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with erythromycin base, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with erythromycin.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with neomycin, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with neomycin.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with gentamycin, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with gentamycin.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with amikacin, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with amikacin.

Reacting the 75% ethyl ester/25% sodium salt of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with kanamycin.
Reacting the 75% ethyl ester/25% sodium salt of hyaluronic acid with pilocarpine, whereby 75% of the carboxyls are esterified with ethanol and whereby 25% of the carboxyls are salified with pilocarpine.
Reacting the 85% propyl ester/15% tetrabutylammonium salt of hyaluronic acid with pilocarpine, whereby 85% of the carboxyls are esterified with propanol and whereby 15%
of the carboxyls are salified with pilocarpine.
By variants of any of the variations,or embodiments described above, the hyaluronic acid ester derives from hyaluronic acid which is obtained by first dehydrating cocks' combs with acetone and then by exposing them to enzymatic digestion with papain.
By other variants of any of the variations or embodiments described above, the hyaluronic acid ester derives from hyaluronic acid which is obtained by first dehydrating cocks' combs with acetone and then by exposing them to enzymatic digestion with papain, followed by molecular ultrafiltration, and further purification of the hyaluronic acid fraction obtained By still other variants of any of the variations or embodiments described above, the hyaluronic acid ester derives from integral hyaluronic acid or from one of its salts, which is obtained by extraction from cocks' combs and having a molecular weight of between 8 and 13 million.
By further variants of any of the variations or embodiments described above, the hyaluronic acid ester derives from a hyaluronic acid fraction with a molecular weight of between 90 and 0.23% of the molecular weight of an integral hyaluronic acid having a molecular weight of 13 million.
By yet further variants of any of the variations or embodiments described above, the hyaluronic acid ester derives from a molecular fraction having a molecular weight of between 50,000 and 100,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
By still further variants of any of the variations or embodiments described above, the hyaluronic acid ester derives from a molecular fraction having a molecular weight of between 500,000 and 730,000 and which is substantially-free 'of hyaluronic acid having a molecular weight of less than 30,000.
By yet still variants of any of the variations or embodiments described above, the process includes the steps of: molecular ultrafiltration; and further purification of the hyaluronic acid fraction so-obtained.
By a preferred variant, the hyaluronic acid may derive from a molecular fraction, which is identified by the Trade-mark HYALASTINETM of Fidia SpA, having a molecular weight of between 50,000 and 100,000 and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000. By another preferred variant thereof, the hyaluronic acid may derive from a molecular fraction, which is identified by the Trade-mark HYALECTINTM
of Fidia SpA, having a molecular weight of between 500,000 and 730,000, and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
To elaborate on the processes of broad aspects of the invention, which is disclosed and claimed in the above-identified parent application, described above, the esters of hyaluronic acid according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, may be prepared by novel processes including the use of procedural methods known per se for the esterification of carboxylic acids. Examples of such known procedures include treatment of free hyaluronic acid with the desired alcohol or alcohols in the presence of catalyzing substances, e.g., strong inorganic acids or ionic exchangers of the acid type, or with an etherifying agent which is capable of introducing the desired alcoholic 33 4 11~ 0 3 residue of the alcohol, in the presence of an inorganic base or an organic base. As etherifying agents, it is possible to use those known in literature, e.g., especially the esters of various inorganic acids or of various organic acids, e.g., sulphonic acids, or hydracids, e.g., hydrocarbyl halogenides, e.g., methyl iodide or ethyl iodide, or neutral sulphates or hydrocarbyl acids, alfites, carbonates, silicates, phosphites or hydrocarbyl sulphonates, e.g., methyl benzene sulphonate, p-toluene sulphonate, methyl chlorosulphonate or ethyl chlorosulphonate. The reaction may take place in a suitable solvent, for example, an alcohol, preferably that corresponding to the alkyl group to be introduced in the carboxyl group. The reaction may also take place in non-polar solvents, e.g., ketones, ethers, e.g., dioxane, or aprotic solvents, e.g., dimethylsulphoxide. As a base catalyzing substance, it is possible to use, for example, a hydrate of an alkali metal or of an alkaline earth metal, or magnesium oxide, or silver oxide, or of a basic salt of one of these metals, e.g., a carbonate, and of the organic bases, e.g., tertiary azotized base, e.g., pyridine or collidine. In the place of the base, it is also possible to use an ionic exchanger of the basic type.
Another esterification procedure involves the use of the metal salts or salts with organic azotized bases, for example, ammonium salts or ammonium-substitute salts.
Preferably, salts of the alkali metals or of the alkaline earth metals are used, but any other metallic salt may also be used. The esterifying agents which may be used are also the,same as those mentioned above, and the same applies to the solvents. It is preferable to use aprotic solvents, for example, dimethylsulphoxide or dimethylformamide.
In the esters of aspects of this invention which are obtained according to this procedure or according to the other procedures described hereinafter, free carboxylic groups of the partial esters may be salified, by means of a procedures known per se.

34 - % 4. 1 6 0 ~

By yet another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a pharmaceutical preparation is provided comprising: an effective amount of at least one ester of hyaluronic acid of the Formula Hy ( COOR ) n, wherein HyCOO represents a hyaluronic acid radical, and R
is derived from an alcohol which is selected from the group consisting of an aliphatic alcohol having a chain of 2 to 34 carbons, an araliphatic alcohol, a cycloaliphatic alcohol and a heterocyclic alcohol and n is a large number;
and a pharmaceutically-acceptable carrier.
By important variants of such pharmaceutical preparation of this aspect of the invention, which is disclosed and claimed in the above-identified parent application, the hyaluronic acid ester may be a total ester, or a partial ester or a salt of a partial ester with an organic base or with an inorganic base.
By other variants of such pharmaceutical preparation, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol in such ester is an aliphatic alcohol which is substituted by one or two functional groups which are selected from the group consisting of amino, hydroxy, mercapto, aldehyde, keto, carboxyl, hydrocarbyl, dihydrocarbylamino, ether, ester, thioether, thioester, acetal, ketal, carbalkoxy, unsubstituted carbamide groups and carbamide groups which are substituted by one or two alkyl groups, the hydrocarbyl radicals in these functionally-modified groups having a maximum of 6 carbon atoms, and in which the carbon atom chain may be interrupted heteroatoms which are selected from the group consisting of oxygen, sulphur and nitrogen.
By a specific variation thereof, the alcohol is ethyl alcohol, propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol, tert-butyl alcohol, amyl alcohol, pentyl alcohol, hexyl alcohol, octyl alcohol, 35 + 3 c o ~
ethylene glycol, propylene glycol, butylene glycol or glycerin.
By still other variants of such pharmaceutical preparation, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol in such ester is an araliphatic alcohol with only one benzene residue, in which the aliphatic chain has a maximum of 4 carbon atoms, and in which the benzene residue may be substituted by 1 to 3 methyl groups or by 1 to 3 hydroxy groups, or by 1 to 3 halogen atoms, and in which the aliphatic chain may be substituted by one or two functions which are selected from the group consisting of free amino groups, monoethylamino groups, diethylamino groups, pyrrolidinyl groups and piperidinyl groups. By a specific variation thereof, the alcohol is benzyl alcohol, phenethyl alcohol, ephedrine or adrenalin.
By yet other variants of such pharmaceutical preparation, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol in such ester is a cycloaliphatic alcohol or an aliphatic-cycloaliphatic alcohol and derives from a monocyclic hydrocarbide or a polycyclic hydrocarbide with a maximum of 34 carbon atoms. By a variation thereof, the alcohol is a polycyclic alcohol which is selected from the group consisting of sterols, colic acids, steroid alcohols, and groups of the estrane and pregnane series and their unsaturated derivative. By specific variations of such variations, the alcohol is cortisone, hydrocortisone, prednisone, prednisolone, fluorocortisone, dexamethasone, betamethasone, corticosterone, deoxycorticosterone, paramethasone, flumethasone, fluocinolone, fluocinolone acetonide, fluprednylidene, clobetasol, or beclomethasone.
By yet further variants of such pharmaceutical preparation, which is disclosed and claimed in the above-identified parent application, as described above, such ester is in the form of a salt of such partial ester with an alkali metal, or with an alkaline earth metal, or with ,4 1 fi03 magnesium, or with aluminum, or with ammonia. By a variation thereof, the salt is a sodium salt.
Preferred examples of such pharmaceutical preparations, which is disclosed and claimed in the above-identified parent application, as described above comprise the following:
An effective amount of the salt of the partial propyl ester of hyaluronic acid with 50% of the carboxyl groups esterified and with 50% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial isopropyl ester of hyaluronic acid with 50% of the carboxyl groups esterified and with 50% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial propyl ester of hyaluronic acid with 85% of the carboxylic groups esterified and with 15% of the carboxylic groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial ethyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial methyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the total ethyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.
An effective amount of the total propyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial butyl ester of hyaluronic acid with 50% of the carboxylic groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial ethoxycarbonylmethyl ester of hyaluronic acid with 75% of the carboxyl groups esterified and with 25% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial cortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial hydrocortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial fluorocortisone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the deoxycorticosterone ester of hyaluronic acid with 20% of the carboxyl groups esterified and with 80% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the mixed ethanol-cortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with cortisone, and a pharmaceutically-acceptable carrier.
An effective amount of the mixed ethanol-hydrocortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with hydrocortisone, and a pharmaceutically-acceptable carrier.
An effective amount of the mixed ethanol-fluorocortisone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the 38 6 0 ~
carboxyl groups esterified with fluorocortisone, and a pharmaceutically-acceptable carrier.
An effective amount of the mixed ethanol-deoxycorticosterone ester of hyaluronic acid with 80% of the carboxyl groups esterified with ethanol and with 20% of the carboxyl groups esterified with fluorocortisone, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial and mixed ethanol-deoxycorticosterone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with deoxycorticosterone and with the remaining 40% of the carboxyl groups salified with sodium, and a pharma-ceutically-acceptable carrier.
An effective amount of the salt of the partial and mixed ethanol-cortisone ester of hyaluronic acid with 40%
of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with cortisone, and with the remaining 40% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial and mixed ethanol-hydrocortisone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with hydrocortisone, and with the remaining 40% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the salt of the partial and mixed ethanol-fluorocortisone ester of hyaluronic acid with 40% of the carboxyl groups esterified with ethanol, with 20% of the carboxyl groups esterified with fluorocortisone, and with the remaining 40% of the carboxyl groups salified with sodium, and a pharmaceutically-acceptable carrier.
An effective amount of the total pentyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.
An effective amount of the total isopentyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.

An effective amount of the total benzyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.
An effective amount of the total phenethyl ester of hyaluronic acid, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with streptomycin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with erythromycin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with neomycin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with gentamycin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with amikacin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with kanamycin, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 75% of the carboxyl groups esterified with ethanol and with 25% of the carboxyls salified with pilocarpine, and a pharmaceutically-acceptable carrier.
An effective amount of a salt of a hyaluronic acid ester with 85% of the carboxyl groups esterified with ethanol and with 15% of the carboxyls salified with pilocarpine, and a pharmaceutically-acceptable carrier.

iJ

By variations of any of the variants and embodiments, which is disclosed and claimed in the above-identified parent application, described above, the hyaluronic ester derives from hyaluronic acid which is obtained by first 5 dehydrating cocks' combs with acetone and the exposing them to enzymatic digestion with papain.
By other variations of any of the variants and embodiments, which is disclosed and claimed in the above-identified parent application, described above, the 10 hyaluronic ester derives from hyaluronic acid which is obtained by first dehydrating cocks' combs with acetone and the exposing them to enzymatic digestion with papain, followed by molecular ultrafiltration, and further purification of the hyaluronic acid fraction obtained.
15 By still other variations of any of the variants and embodiments, which is disclosed and claimed in the above-identified parent application, described above, the hyaluronic ester derives from an integral hyaluronic acid or from one of its salts, which is obtained by extraction 20 from cocks' combs, and having a molecular weight of between 8 and 13 million.
By still further variations of any of the variants and embodiments, which is disclosed and claimed in the above-identified parent application, described above, the 25 hyaluronic ester derives from a hyaluronic acid fraction with a molecular weight of between 90 and 0.23% of the molecular weight of an integral hyaluronic acid with a molecular weight of 13 million.
By yet further variants of any of the variants and 30 embodiments, which is disclosed and claimed in the above-identified parent application, described above, the hyaluronic ester derives from a hyaluronic acid fraction having a molecular weight of between 50,000 and 100,000, and which is substantially-free of hyaluronic acid having 35 a molecular weight of less than 30,000.
By still other variant of any of the variants and embodiments, which is disclosed and claimed in the above-identified parent application, described above, the hyaluronic ester derives from a hyaluronic acid fraction having a molecular weight of between 500,000 and 730,000, and which is substantially-free of hyaluronic acid having a molecular weight of less than 30,000.
By yet another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a medicament is provided comprising: (a) at least one pharmacologically-active substance, and (b) a carrying vehicle comprising an ester of hyaluronic acid of the Formula Hy (COOR) ,,, wherein HyCOO represents a hyaluronic acid radical, and R
is derived from an alcohol which is selected from the group consisting of an aliphatic alcohol having a chain of 2 to 34 carbons, an araliphatic alcohol, a cycloaliphatic alcohol and a heterocyclic alcohol and n is a large number.
By still another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a medicament is provided comprising (a) at least one pharmacologically-active substance, (b) a carrying vehicle comprising an ester of hyaluronic acid of the Formula Hy ( COOR ) ,, , wherein HyCOO represents a hyaluronic,acid radical, and R
is derived from an alcohol which is selected from the group consi-sting of an aliphatic alcohol having a chain of 2 to 34 carbons, an araliphatic alcohol, a cycloaliphatic alcohol and a heterocyclic alcohol, and n is a large number and (c) common excipients for pharmaceutical preparations.
By variants of such aspects of medicaments, which is disclosed and claimed in the above-identified parent application, as described above, such hyaluronic acid ester may be a total ester, or a partial ester or a salt of a partial ester with an organic base or with an inorganic base.

By other variants of such aspects of medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol is an aliphatic alcohol which is substituted by one or two functional groups which are selected from the group consisting of amino, hydroxy, mercapto, aldehyde, keto, carboxyl, hydrocarbyl, dihydrocarbylamino, ether, ester, thioether, thioester, acetal, ketal, carbalkoxy, unsubstituted carbamide groups and carbamide groups which are substituted by one or two alkyl groups, the hydrocarbyl radicals in these functionally-modified groups having a maximum of 6 carbon atoms, and in which the carbon atom chain may be interrupted by heteroatoms which are selected from the group consisting of oxygen, sulphur and nitrogen.
By a specific variation thereof, the alcohol is ethyl alcohol, propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol, tert-butyl alcohol, amyl alcohol, pentyl alcohol, hexyl alcohol, octyl alcohol, ethylene glycol, propylene glycol, butylene glycol or glycerin.
By further variants of such aspects of medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol is an araliphatic alcohol with only one benzene residue, in which the aliphatic chain has a maximum of 4 carbon atoms, and in which the benzene residue may be substituted by 1 to 3 methyl groups or by 1 to 3 hydroxy groups, or by 1 to 3 halogen atoms, and in which the aliphatic chain may be substituted by one or two functions which are selected from the group consisting of free amino groups, monoethylamino groups, diethylamino groups, pyrrolidinyl groups and piperidinyl groups. By a specific variation thereof, the alcohol is benzyl alcohol, phenethyl alcohol, ephedrine or adrenalin.
By another variant of the medicament, which is disclosed and claimed in the above-identified parent application, as described above, the alcohol is a 1 3'603 cycloaliphatic alcohol or an aliphatic-cycloaliphatic alcohol and derives from a monocyclic hydrocarbide or a polycyclic hydrocarbide with a maximum of 34 carbon atoms.
By a variation thereof, the alcohol is a polycyclic alcohol which is selected from the group consisting of sterols, colic acids, steroid alcohols, and groups of the estrane and pregnane series and their unsaturated derivative. By a specific variation thereof, the alcohol is cortisone, hydrocortisone, prednisone, prednisolone, fluorocortisone, dexamethasone, betamethasone, corticosterone, deoxycorticosterone, paramethasone, flumethasone, fluocinolone, fluocinolone acetonide, fluprednylidene, clobetasol, or beclomethasone.
By yet other variants of such aspects of medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the ester is in the form of a salt of a partial ester with an alkali metal, or with an alkaline earth metal, or with magnesium, or with aluminum, or with ammonia. By a variation-thereof, the salt is a sodium salt.
By still other variants of such aspects of medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the pharmacologically-active substance (a) is selected from the group consisting of an anaesthetic, an,analgesic, an anti-inflammatory, a vasoconstrictor, an antibiotic-antibacterial, and an antiviral. By a specific variation thereof, the pharmacologically-active substance (a) is topically active.
By yet other variants of aspects of such medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the carrying vehicle (b) contains an ester of hyaluronic acid with a pharmacologically-inactive alcohol, or the carrying vehicle (b) contains an ester of hyaluronic acid with a pharmacologically-active alcohol.

44 x34 1 603 By still other variants of aspects of such medicaments, which is disclosed and claimed in the above-identified parent application, as described above, the carrying vehicle (b) is of a basic nature and contains a partial ester of hyaluronic acid, the unesterified groups of which are salified with the pharmacologically-active substance.
By still other aspects of this invention, which is discl-osed and claimed in the above-identified parent application, various uses are provided for the above described esters. Thus, one use is in cosmetic articles, as a carrying vehicle.
Another use, which is disclosed and claimed in the above-identified parent application, is in sanitary and surgical articles, as a carrying vehicle. In one variation of such use, the sanitary and surgical articles, which is disclosed and claimed in the above-identified parent application, are in the form of microcapsules for subcutaneous, intramuscular or intravenous injection. In another variation of such use, the sanitary and surgical articles, which is disclosed and claimed in the above-identified parent application, are in the form of solid inserts to be removed after a certain length of time. In still another variation of such use, the sanitary and surgical articles, which is disclosed and claimed in the above-identified parent application, are in the form of sponges for the medication of wounds and various lesions.
Yet another use, which is disclosed and claimed in the above-identified parent application, is in films or threads, as a carrying vehicle. In one variation of such use, the films or threads, which is disclosed and claimed in the above-identified parent application, are for the medication of wounds and in surgery or as suture threads in surgical operations. In another variation of such use, the films, which is disclosed and claimed in the above-identified parent application, are in the form of artificial skin for use in surgical dermatology.

4 6 0.3 Still another use, which is disclosed and claimed in the above-identified parent application, is as films or threads in the production of gauze, as a carrying vehicle.
In one variation of such use, such threads, which is 5 disclosed and claimed in the above-identified parent application, provide a gauze for the medication of wounds and in surgery. In another variation of such use, such threads, which is disclosed and claimed in the above-identified parent application, provide a gauze in the form 10 of sponges for the medication of wounds or in surgery.
A further use of such ester of hyaluronic acid, which is disclosed and claimed in the above-identified parent application, is for the preparation of films or threads which includes the steps of dissolving such hyaluronic 15 ester in an organic solvent, making the solution into sheet form or thread form respectively, and then eliminating the organic solvent by treatment with another suitable organic or aqueous solvent which is soluble in the first solvent.
Yet a still further use of such ester of hyaluronic 20 acid, which is disclosed and claimed in the above-identified parent application, is for the preparation of films or threads which includes the steps of dissolving such hyaluronic ester in an organic solvent making the solution into sheet form or thread form respectively, and 25 then eliminating the organic solvent by treatment with a current of a suitably heated inert gas.
Thus, as noted above, a first group of esters of HY
according to a first aspect of the invention, which is disclosed and claimed in the above-identified parent 30 application, useful in therapy as well as in the other above-mentioned fields, is represented by those in which the qualities of hyaluronic acid itself dominate and may be exploited. Such esters derive from alcohols of the above-mentioned series which do not themselves possess a notable 35 pharmacological action, for example, the saturated alcohols of the aliphatic series or simple alcohols of the cycloaliphatic series.

~3 41603 A second group of esters of HY according to a second aspect of the invention, which is disclosed and claimed in the above-identified parent application, also useful in therapy, is represented, on the other hand, by the esters in which the pharmacological qualities of the alcohol component dominate. That is, it relates to esters of HY
with pharmacologically-active alcohols, e.g., steroid alcohols, i.e., those of the cortisone type with an anti-inflammatory action. These compounds possess properties qualitatively similar to those of the alcohol, but with a more differentiated range of action, compared also to the already-known esters, ensuring a better balanced, constant and regular pharmacological action, and usually obtaining a marked retard effect.
A third group of esters of HY according to a third aspect of the invention, which is disclosed and claimed in the above-identified parent application, and which represent a particularly original and useful aspect, is represented by the esters with a more mixed character compared to the two previous groups. That is, it relates to esters of HY in which a part of the carboxylic groups of HY is esterified with a pharmacologically-active alcohol and in which another part or the carboxylic group of HY is esterified with a pharmacologically-indifferent alcohol, or with one whose activity is negligible. By suitably dosing percentages of the two types of alcohols as esterifying components, it is possible to obtain esters with the same pharmacological activity as the pharmacologically-active alcohol, without the specific activity of hyaluronic acid, but having those above-mentioned qualities of better stability and bioavailability, with respect to the activity desired and characteristic of the pharmacologically-active alcohol and due to the ester groups of the pharmacologically-inert alcohol.
A fourth group of esters of HY according to a fourth aspect of the invention, which is disclosed and claimed in the above-identified parent application, is represented by 47 4i %0 4 -0 ~
those of a mixed character in which the ester groups derive from two different therapeutically-active substances. In this case also the esters may be partial or total, that is, only some carboxylic groups may be esterified by two different therapeutically-active alcohols, for example, by one cortisone steroid and by an antibiotic, or by a phenothiazine, while the carboxylic groups may be free or salified, for example, with alkali metals, particularly with sodium, or all the carboxylic groups may be esterified with the above-mentioned alcohols. It is, however, also possible to prepare esters with three or more alcohol components, e.g., esters in which a part of the carboxylic groups are esterified with one therapeutically-active alcohol, another part of the carboxylic groups are esterified with another therapeutically-active alcohol, a third part of the carboxylic groups are esterified with a therapeutically-inactive alcohol and a fourth part of the carboxylic groups are possibly salified with a metal or with a therapeutically-active or inactive base or which may comprise carboxylic groups in a free form.
In the above-mentioned partial esters of HY in which some of the carboxylic acid groups remain free, these may be salified with metals or organic bases, e.g., with alkali metals or with alkaline earth metals or with ammonia or with nitrogenous organic bases.
Most of the esters of Hy, unlike Hy itself, present a certain degree of solubility in organic solvents. This solubility depends on the percentage of esterified carboxylic groups and on the type of alkyl group linked with the carboxyl. Therefore an ester of HY with all its carboxylic groups esterified presents, at room temperature, good solubility, for example, in dimethylsulphoxide (the benzyl ester of HY dissolves in DMSO in a measure of 200 mg/ml). Most of the total esters of HY also present, unlike HY and especially its salts, poor solubility in water.

48 1341 6 0~

The previously-mentioned solubility characteristics, together with particular and notable viscoelastic properties, allow the use of HY esters to obtain sanitary and medical preparations which are insoluble in saline and which have the particular desired form. These materials may be obtained by preparing a solution of an HY ester in an organic solvent, forming the very viscous solution so-provided into the form of the desired article, and then extracting the organic solvent with another solvent which mixes with the first solvent, but in which the HY ester is insoluble.
Thus, by another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of films or threads of hyaluronic esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with the exception of the total methyl ester of hyaluronic acid, which comprises dissolving such hyaluronic ester in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with another suitable organic solvent or with an aqueous solvent which is soluble in said first solvent.
By a further aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of films or threads of hyaluronic esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with the exception of the total methyl ester of hyaluronic acid which comprises dissolving such hyaluronic ester in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with a current of a suitably heated inert gas.
By yet another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of 1~4 1~0 3f films or threads of total hyaluronic esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with the exception of the total methyl ester of hyaluronic acid which comprises dissolving such hyaluronic ester in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with another suitable organic solvent or with an aqueous solvent which is soluble in the first solvent.
By a still further aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the,preparation of film or threads of salts of partial esters of hyaluronic acid with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with the exception of the total methyl ester of hyaluronic acid which comprises dissolving such hyaluronic ester in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with a current of a suitably heated inert gas.
By another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of films or threads of partial esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series which comprises dissolving the hyaluronic ester in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with another suitable organic solvent or with an aqueous solvent which is soluble in the first solvent.
By yet another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of films or threads of partial esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series which comprises dissolving such hyaluronic ester in an organic solvent, making the solution into sheet or 50 4 1 6 Q~
thread form respectively, and then eliminating the organic solvent by treatment with a current of a suitably heated inert gas.
By a still further aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the preparation of films or threads of salts of partial esters of hyaluronic acid with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with an inorganic base or with an organic base which comprises dissolving such salt in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with another suitable organic solvent or with an aqueous solvent which is soluble in the first solvent.
By yet another aspect of the invention, which is disclosed and claimed in the above-identified parent application, a process is provided for the'preparation of films or threads of salts of partial esters with an alcohol of the aliphatic, araliphatic, cycloaliphatic or heterocyclic series, with an inorganic base or with an organic base which comprises dissolving such salt in an organic solvent, making the solution into sheet or thread form respectively, and then eliminating the organic solvent by treatment with a current of a suitably heated inert gas.
The esters of hyaluronic acid, in the many various aspects as described above according to aspects of the present invention, are all new, except for the afore-mentioned total methyl ester of hyaluronic acid extracted from human umbilical cords, and the methyl esters of the above-mentioned oligomers of HY. Also, new, however, are partial esters of hyaluronic acid with methyl alcohol and their salts with metal bases or with organic bases. The biological and pharmacological activities of the above-mentioned partial methyl esters described in literature, were unknown, as were their excellent bioplastic qualities and high stability. Also unknown, therefore, was the use 51 41 60~

of such esters for the preparation of medicaments, cosmetics, sanitary and surgical articles and other new products discussed above which are provided as part of other aspects of the present invention.
A particularly interesting aspect of'the process of another aspect of the invention, which is disclosed and claimed in the above-identified parent application, is the possibility of preparing more stable drugs than those available at present. It is possible therefore, on the one hand, to prepare esters of hyaluronic acid with therapeutically-inactive alcohols for use in typical indications of hyaluronic acid itself, e.g., for intra-articular injections, where the ester acts as a lubricant.
Due to the improved stability of the esters relative to hyaluronidase as compared to the free acid, it is possible to obtain quite a considerable prolonged action. On the other hand, it is possible to obtain drugs with a"retard"
action for the above-mentioned esters of HY with therapeutically-active alcohols, possibly also salified with therapeutically-active bases. The liberation of the active alcohols due to esterase and that of the salified groups due to the hydrolytic action is very slow.
For cosmetic use, it is preferable to use total or partial esters of hyaluronic acid with pharmacologically-inert alcohols, e.g., saturated or unsaturated aliphatic alcohols, for example, non-substituted alcohols of this type with a straight or ramified chain, for example, with between 1 and 8 carbon atoms, e.g., those specifically mentioned previously. Particularly interesting are those unsaturated alcohols, e.g., with one or more double bonds, e.g., vinyl or allyl alcohols and their condensed derivatives, e.g., especially polyvinyl alcohols or polyvalent alcohols, e.g., glycerine. In this,case also it is possible to use, according to the intended purpose, mixed 'esters .
Also useful are cycloaliphatic alcohols, e.g., derivatives of cyclopentane or cyclohexane and their derivatives which are substituted by lower alkyl groups, e.g., alkyls with between 1 and 4 carbon atoms, especially by methyl groups. Of particular interest also are esters with cycloaliphatic. and aliphatic alcohols - cycloali-phatics derived from terpene, e.g., those mentioned above and from therapeutically-active alcohols, and which may also be used in cosmetics.
The alcohols which may be used preferably to make articles for sanitary and surgical use are essentially the same as those listed above for cosmetic use. In the partial esters of HY according to aspects of the present invention, the percentage of esterified groups may vary greatly in relation to the use for which the product is intended, and that is above all with regard to the use in the various fields of application mentioned above.
Of particular interest, however, are those partial esters of HY which at least 5% and at most 90% of all the carboxylic groups of HY are esterified, and especially those with an esterified percent of between 50 and 80%.
The ratio between the number of different types of ester groups may obviously also vary in the mixed partial esters. For example, in the case of two types of such groups, the ratio varies preferably between 0.1:1 and 1:01, and the same is true of total esters. For the esters intended for therapeutic use, the ratio varies preferably between 0.5:1 and 1:0.5. Such ratios are preferably also valid for total esters and, in the partial esters, they are to be taken preferably with reference to the percentages mentioned above regarding the inclusive number of esterified groups.
In the partial esters of HY of aspects of the invention, which is disclosed and claimed in the above-identified parent application, the non-esterified carboxylic groups may be kept free or may be salified. For the formation of such salts, the bases are selected according to the criterion of these for which the product is intended. It is possible to form inorganic salts 53 4b~13 deriving from alkali metals, e.g., potassium and especially sodium, or from ammonium, or deriving from alkaline earth metals, e.g., calcium, or magnesium, or from aluminum salts.
Particularly interesting are the salts with organic bases, especially nitrogenized bases and therefore aliphatic, arylaliphatic, cycloaliphatic or heterocyclic amines.
These ammoniac salts may derive from therapeutically-acceptable but inactive amines or from amines with therapeutic action. Of the former, the aliphatic amines should be considered, e.g., mono-, di-, and tri-alkylamines with alkyl groups having a maximum of 18 carbon atoms or arylalkylamines with the same number of carbon atoms in the aliphatic part and where aryl means a benzene group, possibly substituted by 1 to 3 methyl groups or halogen atoms or hydroxyl groups. The biologically-inactive bases for the formation of salts may also be cyclic, e.g., monocyclic alkylenamines with rings of between 4 and 6 carbon atoms, possibly interrupted in the ring by heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, e.g., piperidine or morpholine, and may be substituted, for example, by aminic or hydroxylic functions, e.g., aminoethanol, ethylendiamine, ephedrine or choline.
It is also possible to form the quaternary ammonium salts of the partial esters of HY, for example, tetralkyl-ammonium salts with the above-mentioned number of carbon atoms and preferably salts of such a type in which the fourth alkyl group has between 1 and 4 carbon atoms, for example, a methyl group.
Among the biologically-active amines whose therapeutic actions may be put to use, are included all the nitrogenized and basic drugs, e.g., those included in the following groups: alkaloids, peptides, phenothiazines, benzodiazepines, thioxanthenes, hormones, vitamins, anticonvulsants, antipsychotics, antiemetics, anaesthetics, hypnotics, anorexics, tranquillizers, muscle relaxants, coronary vasocilators, antineoplastics, antibiotics, antibacterials, antivirals, antimalarials, carbonic anhydrase inhibitors, non-steroid anti-inflammatory agents, 5 vasoconstrictors, cholinergic agonists, cholinergic antagonists, adrenergic agonists, adrenergic antagonists, and narcotic antagonists.
All those drugs with basic nitrogenized groups listed hereinabove and regarding the use of the esters may be 10 quoted as examples.
According to a particular aspect of the invention, which is disclosed and claimed in the above-identified parent application, the new hyaluronic esters and their salts may be used as an excellent vehicle for 15 therapeutically-active substances. To this end, it is possible to use the total esters, or the partial esters, or the salified partial esters in the remaining carboxylic groups, for example, with one of the above-mentioned substances therapeutically-acceptable but no biologically-20 active, above all with alkali metals, for example sodium.
The above-mentioned medicaments may therefore be made by two associated components, namely:
Component (1) - a pharmacologically-active substance or an association of two or more active substances; and 25 Component (2) - a carrying vehicle comprising a partial or total ester of hyaluronic acid of aspects of the invention, which is disclosed and claimed in the above-identified parent application, with an alcohol, or the salts of such partial esters with an organic base or with 30 an organic base, optionally with the addition of hyaluronic acid or a salt thereof with an inorganic base or with an organic base.
The hyaluronic esters which may be used in these medicaments, which is disclosed and claimed in the above-35 identified parent application, are those in which the esterifying alcohol is not itself pharmacologically-active, for example, a simple aliphatic alcohol, as described 13 4 1 6 Q~

above. Medicaments of this type in which the ester also is pharmacologically-active are not excluded from this aspect of the invention, which is disclosed and claimed in the above-identified parent application, as, for example, in 5 the case of one of the esters described above deriving from alcohols with pharmacological action.
In the same way, the invention, which is disclosed and claimed in the above-identified parent, application, in another of its aspects also includes medicaments of this 10 type in which the esters of Component (2) are also salified with therapeutically-active substances vehicled in the hyaluronic ester, and the mixture in this case, as described below, therefore contains salts of a partial ester of hyaluronic acid with therapeutically-active bases, 15 possibly in the presence of an excess of active base Component (1) . The vehicled substance may not be of a basic nature, and free carboxylic groups in the hyaluronic ester may still be salified with therapeutically-active bases.
20 The use of hyaluronic esters as a vehicle, which is disclosed and claimed in the above-identified parent application, allows the preparation of the new medicaments of further aspects of the invention, which is disclosed and claimed in the above-identified parent application, 25 described above, including (1) a pharmacologically-active substance or an association of two or more of such substances, and (2) a hyaluronic ester as described above or one of its salts. In such medicaments, which is disclosed and claimed in the above-identified parent 30 application, if partial esters of HY are used, the possible salification of the remaining carboxylic groups is preferably carried out with therapeutically-neutral inorganic bases or organic bases, especially with alkali metals e.g., sodium, or with ammonium. Should the active 35 substance component (1) or a corresponding association of substances have basic groups, e.g., for example, antibiotics containing amine groups, and if partial esters 56 41 fi0 3 of hyaluronic acid should be used with remaining free carboxylic groups, the corresponding salts are formed between the carboxylic groups and these basic substances.
The new medicaments, which is disclosed and claimed in the above-identified parent application, therefore include, in particular, partial esters of hyaluronic acid partially or totally salified with pharmacologically-active substances and of a basic character. As described above, particularly important are the associated medicaments of the type described here, in which Component (1) is a pharma-cologically-active substance for topical use.
The use of hyaluronic esters as a vehicle for drugs, which is disclosed and claimed in the above-identified parent application, to be applied topically is particularly useful in ophthalmology where a particular compatibility is to be observed for the new products with the corneal epithelium, and therefore excellent tolerability, without any sensitization effects. Furthermore, when the medicaments, which is disclosed and claimed in the above-identified parent application, are administered in the form of concentrated solutions with elastic-viscous characteristics or in solid form, it is possible to achieve homogeneous and stable films which are preferably transparent and adhesive on the corneal epithelium, guaranteeing prolonged bioavailability of the drug and therefore representing excellent preparations with a retard effect.
Such ophthalmic medicaments, which is disclosed and claimed in the above-identified parent application, are particularly valuable in the veterinary field, considering, for example, that at present there are no veterinary specialities for oculistic use containing chemotherapeutic agents. Indeed, preparations intended for human use are usually used, and these do not always guarantee a specific range of action if they do not make allowances for the particular conditions in which the treatment must take place. This, for example, is the case in therapy for 57 ~341 6 0 3 infective keratoconjunctivitis, pink eye or IBK, an infection which usually affects cattle, sheep and goats.
Presumably for these three species, there are specific etiological factors and more particularly: in cattle, the main microorganism involved seems to be Moraxella bovis (even though other agents of a viral origin should not be excluded, e.g., for example Rinotracheitis virus); in sheep, Micoplasma, Rickettsiae and Clamidiae; and in goats, Rickettsiae. The disease manifests itself in acute form and tends to spread rapidly: in the initial stages the symptomatology is characterized by blepharospasm and excessive lachrymation, followed by purulent exudate, conjunctivitis and keratitis, often accompanied by fever, loss of appetite and milk production. Particularly serious are the corneal lesions which in the final stages may even cause perforation of the cornea itself. The clinical progress of the disease varies from a few days to several weeks.
A vast selection of chemotherapeutic agents are used for treatment, administered both topically (often in association with steroid anti-inflammatory agents), and systemically, including: tetracyclines, e.g., oxytetra-cycline, penicillins, e.g., cloxacillin and benzyl-penicillin, sulphonamides, polymyxin B (associated with miconazole and prednisolone), chloramphenicol, tylosin and chloromycetin. Topical treatment of the disease, despite its apparent simplicity, is still an unsolved problem, since with the oculistic preparations used so far, it has not been possible for one reason or another, to obtain therapeutically-efficient concentrations of antibiotics of sulphamides in the lachrymal secretion. This is quite understandable in the case of solutions, considering the mainly inclined position of the head in these.animals, but the same is also true of semisolid medicaments, as the commonly used excipients do not possess the necessary qualities of adhesiveness to the corenal surface, since they do not usually have a high enough concentration of active substance and cannot achieve optimum distribution of the same (i.e., there is a presence of a distribution gradient). These defects of conventional colliriums in ophthalmic use have, for example, been described by Slatter, et al., Austr. Vet. J., 1982, 59 (3), pp. 69 - 72.
With the esters of HY aspects of the invention, which is disclosed and claimed in the above-identified parent application, these difficulties can be overcome. The presence of the hyaluronic acid ester as a vehicle for ophthalmic drugs, which is disclosed and claimed in the above-identified parent application, in fact allows the formulation of excellent preparations with nb concentration gradients of the active substance and they are therefore homogeneous, with transparency and excellent adhesiveness to the corneal epithelium, with no sensitization effects, with excellent vehicling of the active substance and possibly a retard effect.
The above-mentioned properties of the new medicaments of aspects of the invention, which is disclosed and claimed in the above-identified parent application, may, of course, also be exploited in fields other than ophthalmology. They may be used in dermatology and in diseases of the mucous membranes, for example, in the mouth. Furthermore, they may be used to obtain a systemic effect due to the effect of transcutaneous absorption, e.g.,in suppositories. All these applications are possible both in human and veterinary medicine. In human medicine, the new medicaments, which is disclosed and claimed in the above-identified parent application, are particularly suitable for,use in pediatrics. The invention, which is disclosed and claimed in the above-identified parent application, therefore teaches any of these therapeutic applications.
For the sake of brevity, from now on when the active substance of Component (1) according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, is mentioned, it is to be ? v i ~ 6 understood to also include the association of one or more active substances.
Component (1) described above may be defined in regard to its use in the various fields of therapy, starting with the distinction between human and veterinary medicine, and then specifying the various sectors of application with regard to the organs or tissues to be treated, e.g., with reference to topical use, ophthalmology, dermatology, otorhinolaryngology, gynaecology, angiology, neurology or any type of pathology of internal organs which may be treated with topical applications, for example, with rectal applications.
The vehicling action of the hyaluronic esters also applies to associated medicaments of the type mentioned above in which the active substance acts not only topically or by nasal or rectal absorption, for example, by nasal sprays or preparations for inhalation for the oral cavity or the pharynx, but also by oral or parenteral route, for example, by intramuscular, subcutaneous or intravenous route, as it favours absorption of the drug into the application site. The new medicaments, which is disclosed and claimed in the above-identified parent application, can therefore be applied, apart from in the fields already mentioned, in practically all sectors of medicine, e.g., internal medicine, for example in pathologies of the cardiovascular system, in infections of the respiratory system, the digestive system, the renal system, in diseases of an endocrinological nature, in oncology, and in psychiatry, etc. They may also be classified therefore from the point of view of their specific action, being perhaps anaesthetics, analgesics, anti-inflammatories, wound healers, antimicrobics, adrenergic agonists and antagonists, cytostatics, antirheumatics, antihyper-tensives, diuretics, sexual hormones, immunostimulants or immunosuppressants, for example, one of the drugs having the activity already described for the therapeutically-active alcohols which may be used as an esterifying 60 a 3 4 1 6 G'3 component according to an aspect of the invention, which is disclosed and claimed in the above-identified parent application, or for the therapeutically-active bases which may be used for the salification of the free carboxyclic groups.
Component (1) of the above-mentioned medicaments may also be, according to other aspects of the invention, which is disclosed and claimed in the above-identified parent application, an association of two or more active substances, as contained in many known medicaments.
Regarding the field of ophthalmology, the indications may be, for example the miotic, anti-inflammatory, wound healing and antimicrobial effects.
Examples of pharmacologically-active substances which may be used in ophthalmic medicaments according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, are basic and non-basic antibiotics, e.g., aminoglycosides, macrolides, tetracyclines and peptides, e.g., gentamycin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacin, tobramycin, spectinomycin, erythromycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitracin, polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, nystatin, and possibly their salts, e.g., sulphate or nitrate, or associations of the same between themselves or with other active ingredients, e.g., those mentioned below.
Other ophthalmic drugs which may be used to advantage accordingly to aspects of the invention, which is disclosed and claimed in the above-identified parent application, are other antiinfectives, e.g., diethylcarbamazine, or mebendazole; sulphamidics, e.g., sulphacetamide, sulphadiazone, or sulphisoxazole, antivirals and antitumorals, e.g., iododeoxyuridine, adenine arabinoside, trifluorothymidine, acyclovir, ethyldeoxyuridine, bromovinlyldeoxyuridine, or 5-iodo-5'-amino-2',5-dideoxy-uridine, steroid anti-inflammatories, e.g., dexamethosone, hydrocortisone, prednisolone, fluorometholone, or medrysone and possibly their esters, for example, phosphoric acid, non-steroid anti-inflammatories, e.g., indomethacin, oxyphenbutazone, or flurbiprofen; wound healers, e.g., epidermal growth factor, EGF, local anaesthetics, e.g., benoxinate, or proparacaine and possibly their salts;
cholinergic agonists, e.g., pilocarpine, methylcholine, carbomylcholine, aceclidine, physostigmine, neostigmine, or demecarium and possibly their salts, cholinergic antagonist drugs, e.g., atropine and their salts, adrenergic agonist drugs, e.g., noradrenaline, adrenaline, naphazoline, or methoxamine and possibly their salts, adrenergic antagonist drugs, e.g., propanolol, timolol, pindolol, bupranolol, atenolol, metoprolol, oxprenolol, practolol, butaoxamine, sotalol, butathrin, or labetolol and possibly their salts.
Examples of the active substances which may be used alone or in association among themselves or with other active principles in dermatology are therapeutic agents, e.g., antiinfective agents, antibiotics, antimicrobials, anti-inflammatory, cytostatic, cytotoxid, antiviral, anaesthetic agents, and propylactic agents, e.g., sun screens, deodorants, antiseptics and disinfectants. Of the antibiotics, particularly important are erythromycin, bacitracin, gentamicin, neomycin, aureomycin, gramicidin and their associations, of the antibacterials and disinfectants nitroflurozone, mafenide, chlorhexidine, and derivatives of 81-hydroxyquinonline and possibly their salts; of the anti-inflammatory agents, above all, the corticosteroids, e.g., prednisolone, dexamethazone, flumethasone, clobetasol, triamcinolone acetonide, or betamethasone and their esters, e.g., valerates, benzoated, or dipropionates of the cytotoxic group fluorouracil, or methotrexate, or the anaesthetics dibucaine, lidocain, or benzocaine.
This list, of course, only gives some examples and any other agents described in the literature may be used.

62 13 4 ? 6 3 As associations of drugs to be used in dermatology, the following various antibiotics should be mentioned, e.g., erythromycin, gentamycin, neomycin, gramicidin, polymyxin B, among themselves, or associations of these antibiotices with anti-inflammatory agents, e.g., corticosteroids, for example, hydrocortisone + neomycin, hydrocortisone +
neomycin + polymyxin B + gramicidin, dexamethasone +
neomycin, fluorometholone + neomycin, prednisolone +
neomycin, triamcinolone + neomycin + gramicidin + nystatin, or any other association used in conventional preparations or dermatology.
The association of various active substances are not of course limited to this field, but in each of the above-mentioned sectors of medicine it is possible use associations similar to those already in use for the known pharmaceutical preparations of the art.
In the above case of the use of a Component (1) of a basic character, the salts which are formed with a partial hyaluronic ester (since the latter is used to excess) may be of various types, that is, all the remaining carboxylic groups may be salified or only an aliquot part, thereby producing esters - acid salts, or esters - neutral salts.
The number of acid groups which are to be kept free may be of importance for the preparation of medicaments with a particular pH. Vice versa, it is possible to use an excess of basic Component (1), in which case all the carboxylic groups available in the hyaluronic ester are salified with the base.
According to a particular aspect of the invention, which is disclosed and claimed in the above-identified parent application, it is possible to prepare the medicaments of this type starting from previously isolated and possibly purified salts, in their solid anhydrous state, as amorphous powders, which form an aqueous solution on contact with the tissue to be treated, characterized by viscosity and elastic properties. These qualities are maintained even at stronger dilutions and it is possible 63 ~3 4 16 U 3 therefore to use, in place of the above-mentioned anhydrous salts, more or less concentrated solutions in water or saline, possibly with the addition of other excipients or additives, e.g., other mineral salts to regulate the pH and osmotic pressure. It is, of course, possible to use the salts also for the preparation of gels, inserts, creams or ointments, also containing other excipients or ingredients used in traditional formulations of these pharmacological preparations.
According to a major aspect of the invention, which is disclosed and claimed in the above-identified parent application, however, the medicaments containing the hyaluronic ester or their salts with therapeutically-active or inactive substances as a vehicle are used alone (except possibly with an aqueous solvent) . Also included according to other aspects of the invention, which is disclosed and claimed in the above-identified parent application, are the mixtures obtainable from all the types of medicaments described here, mixtures of the same medicaments, and also possibly mixtures of the new hyaluronic esters with free hyaluronic acid or mixtures of their salts, for example, sodium salts.
Component (1) in the composition according to an aspect of the invention, which is disclosed and claimed in the above-identified parent application, may also be associations or mixtures of two or more such drugs and possibly also with other agents. For example, in ophthalmology, a drug may be associated with an antibiotic or antiphlogistic substance and a vasoconstrictor or with several antibiotics, one or more antiphlogistic substances, or with one or more antibiotics, a mydiatric or a miotic or wound healing or antiallergic agent, etc. For example, the following associations of ophthalmic drugs may be used:
kanamycin + phenylephrine + dexamethasone phosphate;
kanamycin + betamethasone phosphate + phenylephrine; or similar associations with other antibiotics used in 64 i-3 Li 1 `1 0 3 ophthalmology, e.g., rolitetracycline, neomycin, gentamicin, or tetracycline.
If in the place of just one active substance, namely, Component (1), associations of active substances are used, e.g., those mentioned above, the salts of the basic active substances and the partial ester of hyaluronic acid may be mixed salts of one or more of such basic substances or possibly mixed salts of this type with a certain number of other acid groups of the polysaccharides which are salified with metals or bases mentioned above. For example, it is possible to prepare salts of a partial ester of hyaluronic acid or of one of the molecular fractions HYALASTINETM or HYALECTINTM with a pharmacologically-inactive alcohol, for example, a lower alkanol and with a certain percentage of salidified acid groups with the antibiotic kanamycin, another percentage of carboxylic groups which are salified with the vasoconstrictor phenylphrine, and a remaining percentage of acid groups may be, for example, free of salified with sodium or one of the other above-mentioned metals. It is also possible to mix this type of mixed salt with free hyaluronic acid or its fractions or their metallic salts, as indicated above for the medicaments containing salts of one single active substance with the aforementioned polysaccharide esters.
From the examples discussed for ophthalmology and dermatology, it is possible to understand by analogy which medicaments according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, are to be used in the above-mentioned fields of medicine, e.g., for example, in otorhinolaryngology, odontology or in internal medicine, for example, in endocrinology. Such preparations may, therefore be, for example, anti-inflammatories, vasoconstrictors, or vasocompressors, e.g., those already mentioned for ophthalmology, vitamins, antibiotics, e.g., those mentioned above, hormones, chemiotherapics, antibacterials, etc., also as mentioned above for use in dermatology.

The associated medicaments of a hyaluronic ester with a pharmacologically-active substance may contain other pharmaceutical vehicles, e.g., those mentioned below for the pharmaceutical preparations containing only hyaluronic esters, and may appear in the form of ointments, creams, pastilles, gelatine capsules, capsules, aqueous or oily solutions, sprays, suppositories, etc. However, according to a particular aspect of the invention, which is disclosed and claimed in the above-identified parent application, it is preferable to use medicaments containing an association of Components (1) and (2), with Component (2) as the sole vehicle (apart from a possible solvent, e.g., an aqueous solvent).
Of the medicaments of aspects of the invention, which is disclosed and claimed in the above-identified parent application, the following are of particular importance, according to each case, those with a degree of acidity suitable for the environment to which they are to be applied, that is with a physiologically tolerable pH. The adjustment of the pH, for example, in the above-mentioned salts of the partial ester of hyaluronic acid with a basic active substance, may be done by suitably regulating the quantities of polysaccharide, or of its salts or of the basic substance itself. Thus, for example, if the acidity of a salt of the partial ester of hyaluronic acid with a basic substance is too high, the excess of free acid groups can be neutralized with the above-mentioned inorganic bases, for example, with the hydrate of sodium, or of potassium, or of ammonia.
Of the new products of aspects of the invention disclosed and claimed in the present divisional application, of particular importance are the esters of HY
and their salts described above and those described in the following illustrative Examples, which illustrate aspects of the invention, which is disclosed and claimed in the above-identified parent application, and of the invention 3 -14 10~03 disclosed and claimed in the present divisional application.
(e) AT LEAST ONE MODE FOR CARRYING OUT THE INVENTION
Preparation Examples The following Examples A - C describe some procedures for preparing the preferred hyaluronic acids fractions utilized in the preparation of the ester of hyaluronic acid of various aspects of the invention disclosed and claimed in the parent application and in the present divisional application.
Example A - Process for Obtaining a Mixture of HYALASTINETM
and HYALECTINTM Fractions Having No Inflammatory Activity.
Fresh or frozen cocks' combs, (3000 g) are minced in a meat mincer and then carefully homogenized in a mechanical homogenizer. The paste thus obtained is placed in a stainless steel container AISI 316 or in glass and treated with 10 volumes of anhydrous acetone. The whole is agitated for 6 hours at a speed of 50 rpm. It is left to separate for 12 hours and the acetone is discarded by syphoning. The acetone extraction is repeated until the discarded acetone has reached the correct degree of humidity (Karl-Fischer method). The whole is then centrifuged and vacuum dried at a suitable temperature for 5 - 8 hours. In this way, 500 - 600 g of dry powdered cocks' combs are obtained.
300 gr. of dry powder are exposed to enzymatic digestion with papain (0.2 g) in aqueous conditions, buffered with phosphate buffer in the presence of a suitable quantity of hydrochloride cysteine. The resultant is agitated for 24 hours at 60 rpm keeping the temperature constant at 60 - 65 C. It is then cooled at 25 C and a diatomaceous earth filter aid known by the trade-mark CELITETM of Johns Manville Product Corporation (60 gr) is added maintaining the agitation for another hour. The resulting mixture is filtered until a clear liquid is obtained. The clear liquid then undergoes molecular ultrafiltration using membranes with a molecular exclusion 67 ;34~603 limit of 30,000 in order to retain on the membrane those molecules with a molecular weight greater than 30,000.
The product is ultrafiltered from 5 to 6 original volumes adding distilled water continually to the product in ultrafiltration. The addition of water is suspended and the ultrafiltration is continued until the volume is reduced to 1/3 of the original volume.
The residue liquid is rendered 0.1M by the addition of sodium chloride and the temperature is brought to 50 C.
Under agitation at 60 rpm, 45 g of cetylpyridinium chloride are added. It is agitated for 60 minutes and then 50 g of CELITETM are added. Under agitation, the temperature of the whole is brought to 25 C and the precipitate formed by centrifugation is gathered. The precipitate obtained is suspended in a 0.01M solution in sodium chloride (5 litres) containing 0.05% of cetylpiridinium chloride. The resulting suspension is agitated for 60 minutes at 50 C; the temperature is then brought to 25 C and the precipitate is centrifuged. The washing operation is repeated 3 times after which the precipitate is gathered in a receptacle containing 3 litres of a 0.05M solution of sodium chloride containing 0.05% of cetylpyridinium chloride. It is agitated at 60 rpm for 60 minutes and the temperature is kept constant at 25 C for two hours. The supernatant is eliminated by centrifugation. The procedure is repeated several times with solutions of 0.1M sodium chloride containing 0.05% of cetylpyridinium chloride. The mixture is centrifuged and the supernatant is discarded. The precipitate is dispersed in a solution of 0.30M sodium chloride containing 0.05% of cetylpyridinium chloride (3 litres). The mixture is agitated and both the precipitate and the clear liquid are gathered. Extraction is repeated three more times on the precipitate, each time using 0.5 litre of the same aqueous solution.
Finally the precipitate residue is eliminated and the clear liquids are all placed together in a single container. The temperature of the liquid is brought to 50 C

under constant agitation. The liquid is then brought to 0.23M with sodium chloride. 1 gr of cetylpyridinium chloride is added, and it is maintained in agitation for 12 hours.
The mixture is cooled at 25 C and then filtered first on CELITETM pack and then through a filter. It then undergoes molecular ultrafiltration again, on a membrane with a'molecular exclusion limit of 30,000 ultrafiltering three initial volumes with the addition of a solution of 0.33M sodium chloride. The addition of sodium chloride solution is interrupted and the volume is reduced to 1/4 of the initial volume. The solution thus concentrated is precipitated under agitation (60 rpm) at 25 C with 3 volumes of ethanol (95%). The precipitate is gathered by centrifugation and the supernatant is discarded. The precipitate is dissolved in 1 L of 0.O1M solution in sodium chloride and the precipitation is repeated with 3 volumes of ethanol 95%.
The precipitate is gathered and washed first with 75%
ethanol (3 times) , then with absolute ethanol (3 times) and lastly with absolute acetone (3 times).
The product thus obtained (HYALASTINETM + HYALECTINTM
fractions) has an average molecular weight of between 250,000 and 350,000.
The yield of HY is 0.6% of the original fresh tissue.
Example B - Process for Obtaining the Fraction HYALATINETM
from the Mixture Obtained by the Process Described in Example A.
The mixture obtained by the process described in Example A is dissolved in twice distilled apyrogenetic water at the rate of 10 mg of product to each 1 ml of water. The solution obtained is exposed to molecular filtration through filter membranes with a molecular exclusion limit of 200,000, following a concentration technique on the membrane without the addition of water.
During the ultrafiltration process through membranes with a molecular exclusion limit of 2000,000, the molecules with a molecular weight of more than 200,000 do not pass through, while the smaller molecules pass through the membrane together with the water. During the filtration procedure no water is added, so that the volume decreases, and there is therefore an increase in the concentration of molecules with a molecular weight of more than 200,000.
The product is ultrafiltered until the volume on top of the membra7ie is reduced to 10% of the initial volume. Two volumes of apyrogenetic twice distilled water are added and it is then ultrafiltered again until the volume is reduced to 1/3. The operation is repeated twice more. The solution passed through the membrane is brought to 0.1M
with sodium chloride and then precipitated with 4 volumes of ethanol at 95%. The precipitate is washed 3 times with ethanol at 75% and then vacuum dried.
The product thus obtained (HYALASTINETM fraction) has an average molecular weight of between 50,000 and 100,000.
The yield of Hy is equal to 0.4% of the original fresh tissue.
Example C - Process of Obtaining the Fraction HYALECTINTM.
The concentrated solution gathered in the container on top of the ultrafiltration membrane with a molecular exclusion of 200,000 as in Example B, is diluted with water until a solution containing 5 mg/ml of hyaluronic acid is obtained, as determined by quantitative analysis based on the dosage of glucuronic acid.
The solution is brought to 0.1M in sodium chloride and then precipitated with 4 volumes of ethanol at 95%. The precipitate is washed 3 times with ethanol at 75% and then vacuum dried.
The product thus obtained (HYALECTINTM fraction) has an average molecular weight of between 500,000 and 730,000.
This corresponds to a specific fraction of hyaluronic acid with a defined length of molecular chain of about 2,500 to 3,500 saccharide units with a high degree of purity. The yield of HY is equal to 0.2% of the original fresh tissue.

70 i3 41 603 Example D - Preparation of the Salt of Tetrabutylammonium of Hyaluronic Acid (HY).
4.02 g of HY sodium salt (10 m.Eq.) are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at 4 C containing 15 ml of sulphonic resin (known by the trade-mark DOWEXTM 50 x 8 of The Dow Chemical Company) in tetrabutylammonium form. The eluate, free from sodium, is instantly frozen and freeze-dried.
Yield: 6.18 g.
Example 1 - Preparation of the (Partial) Propyl Ester of Hyaluronic Acid (HY) - 50% of the esterified carboxylic groups - 50% of the salified carboxylic groups (Na) 12.4 g of HY tetrabutylammonium salt with a molecular weight 170,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at C, 1.8 g (10.6 m.Eq.) of propyl iodide are added and the resulting solution is kept at a temperature o~f 30 C for 12 hours.
A solution containing 62 ml of water and 9 g of sodium 20 chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
25 The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 500 ml of acetone/water (5:1) and three times with 500 ml of, acetone and finally vacuum dried for 24 hours at 30 C, 7.9 g of the partial propyl esters compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H.,Cundiff and P.C. Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].

71 1~4 16 03 Example 2 - Preparation of the (Partial) Isopropyl Ester of Hyaluronic Acid (HY) - 50% of Esterified Carboxylic Groups - 50% of Salified Carboxylic Groups (Na).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 160,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at 25 C, 1.8 g (10.6 m.Eq.) of isopropyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 500 ml of acetone/water 5:1 and three times with 500 ml of acetone and finally vacuum dried for 24 hours at 30 C.
7.9 g of the partial isopropyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas [Anal. Chem. 33, 1028 - 1030, (1961].
Example 3 - Preparation of the (Partial) Ethyl Ester of Hyaluronic Acid (HY) - 75% of Esterified Carboxylic Groups - 25% of Salified Carboxylic Groups (Na).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 250,000 corresponding to 20 m.Eq. of a monomeric unit,are solubilized in 620 ml of dimethylsulphoxide at 25 C, 2.5 g (15.9 m.Eq.) of ethyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
A solution containing 62 ml of water and 9,g of sodium chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 500 ml of acetone/water 5:1 and three times with 500 ml of acetone and finally vacuum dried for 24 hours at 30 C.
7.9 g of the partial ethyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 4 - Preparation of the (Partial) Methyl Ester of Hyaluronic Acid (HY) - 75% of Esterified Carboxylic Groups - 25% of Salified Carboxylic Groups (Na).
12.4 of HY tetrabutylammonium salt with a molecular weight of 80,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at C, 2.26 g (15.9 m.Eq.) of methyl iodide are added and the 20 resulting solution is kept for 12 hours at 30 C.
A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three 25 times with 500 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 500 ml of acetone/water 5:1 and three times with 500 ml of acetone and f inal ly vacuum dried for 24 hours at 300C.
7.9 g of the partial methyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].

73 7 3 ~ ~ 6 0 3 Example 5 - Preparation of the Methyl Ester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 120,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at 25 C, 3 g (21.2 m.Eq.) of methyl iodide are added and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
8 g of the ethyl ester product in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 6 - Preparation of the Ethyl Ester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 85,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at C, 3.3 g (21.2 m.Eq.) of ethyl iodide are added and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of 25 ethyl acetate under constant agitation-. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
8 g of the ethyl ester product in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 7 - Preparation of the Propyl Ester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dime thyl sulphoxide at 74 ~30 3 25 C, 3.6 g(21.2 m.Eq.) of propyl iodide are added and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
8.3 g of the propyl ester product in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 8 - Preparation of the (Partial) Butyl Ester of Hyaluronic Acid . (HY) - 50% of Esterified Carboxylic Groups - 50% of Salified Carboxylic Groups (Na).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 620,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at C, 1.95 g (10.6 m.Eq.) of n-butyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
20 A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with 25 acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with,500 ml of acetone/water 5:1 and three times with 500 ml of acetone and finally vacuum dried for 24 hours at 30 C.
8 g of the partial butyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C.
Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].

~~1 610 03 Example 9 - Preparation of the (Partial) Ethoxycarbonyl-, methyl Ester of Hyaluronic Acid (HY) - 75% of Esterified Carboxylic Groups - 25% of Salified Carboxylic Groups (Na).
12.4 g of HY tetrabutylammonium salt with a molecular 5 weight of 180,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethylsulphoxide at 25 C, 2 g of tetrabutylammonium iodide and 1.84 g (15 m.Eq.) of ethyl chloroacetate are added and the resulting solution of kept for twenty-fours hours at 30 C.
10 A solution containing 62 ml of water and 9 g of sodium chloride is added and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with 15 acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice 20 with 500 ml of acetone/water 5:1 and three times with 500 ml diacetone and finally vacuum dried for 24 hours at 30 C. 10 g of the partial ethoxycarbonylmethyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the 25 method of R.H. Cundiff and P.C. Markunas [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 10 - Preparation of the (Partial) Cortisone Ester (C21) of Hyaluronic Acid (HY) - 20% of Esterified Carboxylic Groups - 80% of Salified Carboxylic Groups (Na).
30 6.2 g of HY tetrabutylammonium salt with a molecular weight of 105,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 0.850 g (2 m.Eq.) of 21-bromo-4-pregnene-17a-ol-3,11, 20-trione are added and the resulting solution is kept for 35 24 hours at 30 C.
A solution containing 100 ml of water and 5 g of sodium chloride is added and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 300 ml of water containing 1% of sodium chloride and the solution is slowly poured into 1,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for 24 hours at 30 C.
4.5 g of the partial cortisone ester compound in the title are obtained. Quantitative determination'of cortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980, p. 127.
Example 11 - Preparation of the (Partial) Hydrocortisone Ester C21 of Hyaluronic Acid (HY) - 20% of Esterified Carboxylic Groups - 80% of Salified Carboxylic Groups (Na) 6.2 g of HY tetrabutylammonium salt with a molecular weight of 80,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at C, 0.850 g (2 m.Eq.) of 21-bromo-4-pregnene-11~, 17a-diol-3,20-dione are added and the resulting solution is kept for 24 hours at 30 C.
25 A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acet,one and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in -300 ml of water containing 1% of sodium chloride and the solution is slowly poured into 1,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for 24 hours at 30 C.
4.4 g of the partial hydrocortisone ester compound in the i 36 a~

title are obtained. Quantitative determination of hydrocortisone, after mild alkaline hydrolysis with hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980, p.224.
Example 12 - Preparation of the (Partial) Fluorocortisone Ester (CL1) of Hyaluronic Acid (HY) - 20% of Esterified Carboxylic Groups - 80% of Salified Carboxylic Groups (Na).
6.2 g of HY tetrabutylammonium saltwith a molecular weight of 80,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 0.89 g (2 m.Eq.) of 9-fluoro-2l-bromo-4-pregnene-11(3-17a-diol-3,20-dione are added and the resulting solution is kept for 12 hours at 30 C.
A solution is then added containing 62 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 300 ml of water containing 1% of sodium chloride and the solution is slowly poured into 1,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for 24 hours at 300C.
4.6 g of the partial fluorocortisone compound in the title are obtained. Quantitative determination of hydrocortisone, after mild alkaline hydrolysis with hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980, p.196.
Example 13 - Preparation of the (Partial) Deoxycorticosterone Ester (C21) of Hyaluronic Acid (HY) -20% of Esterified Carboxylic Groups - 80% of Salified Carboxylic Groups (Na).
6.21 g of HY tetrabutylammonium salt with a molecular weight of 105,000 corresponding to 10 m.Eq. of a monomeric C, 3 unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 0.661 g (2 m.Eq.) of 21-bromo-4-pregnene-3,20-dione are added and the resulting solution is kept for 24 hours at 30 C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 300 ml of water containing 1% of sodium chloride and the solution is slowly poured into 1,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for 24 hours at 30 C. 4.5 g of the partial desoxycorticosterone ester compound in the title are obtained. Quantitative determination of cortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980, p. 137.
Example 14 - Preparation of the (Mixed Ethanol and Cortisone Ester (C21) of Hyaluronic Acid (HY) - 80% of the Carboxylic Groups Esterified With Ethanol - 20% of the Carboxylic Groups Esterified With Cortisone (C21).
6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 1.25 g (8 m.Eq.) of ethyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-17a-ol-3-11,20-trione are added and the solution is kept for 24 hours at 30 C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.

- ~'`i ~ U V ~

A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
4.6 of the mixed ethanol and cortisone ester compound in the title are obtained. Quantitative determination of cortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chlor'oform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 15 - Preparation of the (Mixed) Ethanol and Hydrocortisone Ester (C21) of Hyaluronic Acid (HY) - 80% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Hydrocortisone (LC21)-.
6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000 corresponding to 10 ml m.Eq. of a monomeric unit are solubilized in 310 ml of dimethyl-sulphoxide at 25 C, 1.25 g(8 m.Eq.) of ethyl iodide are added and the solution is kept at 30 C for 12 hours.
0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-11~,17a-diol-3,20-dione are added and the solution is kept for 24 hours at 30 C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acet,one and finally vacuum dried for eight hours at 30 C.
4.6 of the mixed ethanol and hydrocortisone ester compound in the title are obtained. Quantitative determination of hydrocortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.

80 i *w~+ e 1 6 3 Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 16 - Preparation of the (Mixed) Ethanol and Fluorocortisone Ester (C21) of Hyaluronic Acid (HY) - 80% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Fluorocortisone (C21)-.
6'.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 of dimethylsulphoxide at 25 C, 1.25 g (8 m.Eq.) of ethyl iodide are added and the solution is kept for 24 hours at 30 C.
0.89 g (2 m.Eq.) of 9a-fluoro-21-bromo-4-pregnene-11(3,17a-diol-3,20-dione are added and the solution is kept for 24 hours at 30 C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
4.6 of the mixed ethanol and fluorocortisone ester compound featured in the title are obtained. Quantitative determination of fluorocortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 17 - Preparation of the (Mixed) Ethanol and Deoxycorticosterone Ester (C11) of Hyaluronic Acid (HY) -80% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Deoxycorticosterone (C21) .
6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 81 1 v 0 5 25 C, 1.25 g (8 m.Eq.) of ethyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
0.661 g (2 m.Eq.) of 21-bromo-4-pregnene-3-20-dione are added and the solution is kept for 24 hours at 30 C. A
solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered"and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
4.6 of the mixed ethanol and desoxycorticosterone ester compound in the title are obtained. Quantitative determination of desoxycortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 18 - Preparation of the (Partial and Mixed) Ethanol and Desoxycorticosterone Ester of Hyaluronic Acid (HY) -40% of Carboxylic Groups Esterified With Desoxycorti-costerone (C21) - 40% of Salified Carboxylic Groups (Na).
6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 0.62 g (4 m.Eq.) of ethyl iodide are added and the solution is kept for 24 hours at 30 C.
0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-3,20-dione are added and the solution is kept for 24 hours at 30 C. A
solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.

4.5 g of the partial and mixed ethanol and desoxycor-ticosterone ester compound in the title are obtained.
Quantitative determination of desoxycortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 19 - Preparation of the (Partial and Mixed) Ethanol and Cortisone Ester of Hyaluronic Acid (HY) - 40% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Cortisone (C21) - 40% of Salified Carboxylic Groups (Na).
6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at C, 0.62 g (4 m.Eq.) of ethyl iodide are added and the solution is kept for 24 hours at 30 C.
20 0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-l7a-ol-3-11,20-trione are added and the solution is kept for 24 hours at C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly 25 poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
4.5 g of the partial and mixed ethanol and cortisone 30 compound in the title are obtained. Quantitative deter-mination of cortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].

Example 20 - Preparation of the (Partial and Mixed) Ethanol and Hydrocortisone Ester (LC21) of Hyaluronic Acid (HY) -40% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Hydrocortisone (C21 - 40%
of Salified Carboxylic Groups (Na).
6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000 corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 25 C, 0.62 g (4 m.Eq.) of ethyl iodide are added and the solution is kept for 24 hours at 30 C.
0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-11B-17a-ol-3-11,20-trione are added and the solution is kept for 24 hours at 30 C.
A solution is then added containing 200 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for eight hours at 30 C.
4.5 g of the partial and mixed ethanol and hydrocortisone ester compound in the title are obtained.
Quantitative determination of hydrocortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO-3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 21 - Preparation of the (Partial and Mixed) Ethanol and Fluorocortisone Esters (C21) of Hyaluronic Acid (HY) -40% of Carboxylic Groups Esterified With Ethanol - 20% of Carboxylic Groups Esterified With Fluorocortisone (C21) 40% of Salified Carboxylic Groups (Na).
6.2 g of HY tetrabutylammonium salt with a molecular weight of 65,000 corresponding to 20 m.Eq. of a monomeric unit are solubilized in 310 ml of dimethylsulphoxide at 84 0 ~

25 C, 0.62 g (4 m.Eq.) of ethyl iodide are added and the solution is kept for 24 hours at 30 C.
0.89 g (2 m.Eq.) of 9a-fluoro-2l-bromo-4-pregnene-11(3,17a-diol-3,20-dione are added and the solution is kept for 24 hours at 30 C.
A solution is then added containing 100 ml of water and 5 g of sodium chloride and the resulting mixture is slowly poured'into 2,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 100 ml of acetone/water 5:1 and three times with 100 ml of acetone and finally vacuum dried for eight hours at 30 C.
4.6 g of the partial and mixed ethanol and fluorocortisone ester compound in the title are obtained.
Quantitati-ve determination of fluorocortisone, after mild alkaline hydrolysis with a hydroalcoholic solution of Na2CO3 and extraction with chloroform, is carried out according to British Pharmacopea, 1980.
Quantitative determination of the ethoxyls is carried out according to R.H. Cundiff and P.C. Markunas, [Anal.
Chem. 33, 1028 - 1030, (1961)].
Example 22 - Preparation of the N-Pentyl Ester of Hyaluronic Acid (HY).
12.4 g of Hy tetrabutylammonium salt with a molecular weight of 620,000, corresponding to 20 m.Eq. of a monomeric units, are solubilized in 620 ml of dimethylsulphoxide at 25 C, 3.8 g (25 m.Eq.) of n-pentyl bromide and 0.2 of iodide tetrabutylammonium are added, and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for,twenty-four hours at 30 C.
8.7 g of the n-pentyl ester product in the title are obtained. Quantitative determination of the ester groups is carried out using the method described on pages 169 -85 ~03 172 of Siggia S. and Hann J.G., "Quantitative Organic Analysis Via Functional Groups" 4th edition, John Wiley and Sons.
Example 23 - Preparation of the Isopentyl Ester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000, corresponding to 20 m.Eq. of a monomeric unit,'are solubilized in 620 ml of dimethysulfoxide at 25 C, 3.8 g (24 M.Eq.) of isopentyl bromide and 0.2 g of tetrabutylammonium iodide are added, and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
8.6 g of the isopentyl ester product featured in the title are obtained. Quantitative determination of the ester groups is carried out using the method described on pages 169 - 172 of Siggia S. and Hann J.G., "Quantitative Organic Analysis Via Functional Groups" 4th edition, John Wiley and Sons.
Example 24 - Preparation of the Benzylester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000, corresponding to 20 m.Eq. of a monomeric unit, are solubilized in 620 ml of dimethylsulphoxide at 25 C, 4.5 g (25 m.Eq.) of benzyl bromide and 0.2 g of tetrabutylammonium iodide are added, and the solution is kept, for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is formed which is filtered and washed four time with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
9 g of the benzyl ester product in the title are obtained. Quantitative determination of the ester groups 86 ~34 1 6 03 is carried out according to the method described on pages 169 - 172 of Siggia S. and Hanna J.G., "Quantitative Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons.
Example 25 - Preparation of the (3-Phenylethyl Ester of Hyaluronic Acid (HY).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 125,000, corresponding to 20 m.Eq. of a monomeric unit, are solubilized in 620 ml of dimethylsulphoxide at 25 C, 4.6 g(25 m.Eq.) of 2-bromoethylbenzene bromide and 185 mg of tetrabutylammonium iodide are added, and the solution is kept for 12 hours at 30 C.
The resulting mixture is slowly poured into 3,500 ml of ethyl acetate under constant agitation. A precipitate is thus formed which is then filtered and washed four times with 500 ml of ethyl acetate and finally vacuum dried for twenty-four hours at 30 C.
9.1 g of the (3-phenylethyl ester in the title are obtained. Quantitative determination of the ester groups is carried out according to the method described on pages 169 - 172 of Siggia S. and Hanna J.G., "Quantitative Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons.
Example 26 - Preparation of the Benzyl Ester of Hyaluronic Acid (HY).
3 g of the potassium salt of HY with a molecular weight of 162,000 are suspended in 200 ml of dimethylsulphoxide;
120 mg of tetrabutylammonium iodide and 2.4 g of benzyl bromide are added.
The suspension is kept in agitation for 48 hours at 30 C. The resulting mixture is slowly poured into 1,000 ml of ethyl acetate under constant agitation. A precipitate is formed which filtered and washed four times with 150 ml of ethyl acetate and finally vacuum dried for twenty four hours at 300C.
3.1 g of the benzyl ester product in the title are obtained. Quantitative determination of the ester groups is carried out according to the method described on pages 169 - 172 of Siggia S. and Hanna J.G., "Quantitative Organic Analysis Via Functional Groups", 4th Edition, John Wiley and Sons.
Example 27 - Preparation of Streptomycin Salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol - 75% of Carboxylic Groups Esterified With Ethanol - 25% of Carbokylic Groups Salified With Streptomycin.
243 mg of streptomycin sulphate (1 m.Eq.) are solubilized in 20 ml of water. The solution is eluted in a thermostatic column at 5 C containing 2 ml of a quaternary ammonium resin (e.g., that known by the Trade-mark DOWEXTM
1 x 8) in OH- form.
The sulphate-free eluate is gathered in a thermostatic container at a temperature of 5 C.
1.6 of a 75% ethyl ester of HY and 25% sodium salt (corresponding to 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of a sulphonic resin (DOWEXTM
50 X 8) in H+ form.
The sodium-free eluate is gathered under agitation in the solution of streptomycin base. The resulting solution is instantly frozen and freeze-dried. Yield: 1.7 g.
Microbiological determination on B.subtilis ATCC 6633 in comparison with streptomycin standard, shows a content of 10.9% in weight of streptomycin base, corresponding to the theoretically calculated content.

Example 28 - Preparation of the Erythromycin salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol -75% of Carboxylic Groups Esterified With Ethanol - 25% of Carboxylic Groups Salified With Erythromycin., 1.6 of a 75% ethyl ester of HY and sodium salt at 25%
(corresponding to 1 m.Eq. of a monomeric unite relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C containing 2 ml of sulphonic resin (DOWEXTM 50 x 8) in H+ form.
To the sodium-free eluate are added 734 mg of erythromycin base (1 m.Eq.). The resulting solution is instantly frozen and freeze-dried. Yield: 2.1 g.
Microbiological determination on St. aureaus ATCC 6538 in comparison to standard erythromycin, shows a content of 31.7%~in weight of erythromycin base, corresponding to the theoretically calculated weight.
Example 29 - Preparation of the Neomycin Sal of a Hyaluronic Acid (HY) Partially Esterified With Ethanol -75% of Carboxylic Groups Esterified With Ethanol - 25% of Carboxylic Groups salified With Neomycin.
152 mg of neomycin sulphate (1 m.Eq.) are solubilized in 20 ml of water. The solution is eluted in a thermostatic column at 50C containing 2 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in OH- form.
The sulphate-free eluate is gathered in a thermostatic container at a temperature of 5 C.
1.6 g of a 75% ethyl ester of HY and sodium salt at 25%
(corresponding to 1 m.Eq. of monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of sulphonic resin (DOWEXTM 1 x 8) in H+ form.
The sodium-free eluate is gathered under agitation in the solution of neomycin base. The resulting solution is instantly frozen and freeze-dried. Yield: 1.65 g.
Microbiological determination carried out on St. aureus ATCC 6538 in comparison to standard neomycin, shows a content of 6.1% in weight of neomycin base, corresponding to the theoretically calculated value.

Example 30 - Preparation of the Gentamicin Salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol -75% of Carboxylic Groups Esterified With Ethanol - 25% of Carboxylic Groups Salified With Gentamicin.
145 mg of gentamicin sulphate are solubilized in 10 ml of water. The solution is eluted in a thermostatic column at 5 C containing 2 ml of quaternary ammonium resin (DOWEX,,h, 1 x 8) in OH- form.
The sulphate-free eluate is gathered in a thermostatic container at a temperature of 5 C.
1.6 g of a 75% ethyl ester of HY and sodium salt at 25%
(corresponding to 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of sulphonic resin (DOWEXTM 50 x 8) in H+ form.
The sodium-free eluate is gathered under agitation in the solution of gentamicin base. The resulting solution is instantly frozen and freeze-dried. Yield: 1.7 g.
Microbiological determination carried out on S.
epidermidus ATCC 12228 in comparison to standard gentamicin, shows a content of 6.5% in weight of gentamicin base, corresponding to the theoretically calculated value.
Example 31 - Preparation of the Amikacin Salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol - 75% of Carboxylic Groups Esterified With Ethanol - 25% of Carboxylic Groups Salified With Amikacin 147 mg of Amikacin Base (1 m.Eq.) are Solubilized in 20 ml of Water.
147 mg of amikacin (1 m.Eq.) are solubilized in 20 ml of water.
1.6 g of a 75% ethyl ester of HY and Sodium salt at 25%
(corresponding to 1 m.Eq.) of a monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of sulphonic resin (DOWEXTM) 50 x 8) in H+ form.

90 i3416 03 The sodium-free eluate is gathered under agitation in the solution of amikacin base. The resulting solution is instantly frozen and freeze-dried. Yield: 1.70 g.
Microbiological determination carried out on St. aureus ATCC 29737 in comparison to standard amikacin, shows a content of 8.5% in weight of amikacin base, corresponding to the theoretically calculated value.
Example 32 - Preparation of the Kanamycin Salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol -75% of Carboxylic Groups Esterified With Ethanol - 25% of Carboxylic Groups Salified With Kanamycin.
146 mg of kanamycin sulphate (1 m.Eq.) are solubilized in 20 ml of water. The solution is eluted in a thermo-static column at 5 C containing 2 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in OH- form.
The sulphate-free eluate is gathered in a thermostatic container at a temperature of 5 C.
1.6 g of a 75% ethyl ester of HY and sodium salt at 25%
(corresponding to 1 m.Eq.) of a monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of a sulphonic resin (DOWEXTM 50 x 8) in H+ form.
The sodium-free eluate is gathered under agitation in the solution of kanamycin base. The resulting solution is instantly frozen and freeze-dried. Yield: 1.5 g.
Microbiological determination carried out on B.
subtilis ATCC 6633 in comparison to standard kanamycin, shows a content of 7% in weight of kanamycin base, corresponding to the theoretically calculated value.
Example 33 - Preparation of the Pilocarpine Salt of Hyaluronic Acid (HY) Partially Esterified With Ethanol -75% of Carboxylic Groups Esterified with Ethanol - 25% of Carboxylic Groups Salified With Pilocarpine.
245 mg of pilocarpine hydrochloride (1 m.Eq.) are solubilized in 20 ml of water. The solution is eluted in a thermostatic column at 5 C containing 2 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in OH- form.
The chloride-free eluate is gathered in a thermostatic container at 5 C.
1.6 g of a 75% ethyl ester of HY and sodium salt at 25%
(corresponding to 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl), are solubilized in 400 ml of water.. The solution is eluted in a thermostatic column at 20 C and containing 2 ml of sulphonic resin (DOWEXTM 50 x 8) in H+ form.
The sodium-free eluate is gathered under agitation in the solution of pilocarpine base. The resulting solution is instantly frozen and freeze-dried. Yield: 1/89 g.
Example 34 - Preparation of the (Partial Propyl) Ester of Hyaluronic Acid (HY) - 85% of Esterified Carboxylic Groups - 15% of Salified Carboxylic Groups (Na).
12.4 g of HY tetrabutylammonium salt with a molecular weight of 165,000, corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 ml of dimethysulphoxide at 25 C, 2.9 g (17 m.Eq.) of propyl iodide are added and the resulting solution is kept for 12 hours at 30 C.
A solution is then added containing 62 ml of water 9g of sodium chloride and the resulting mixture is slowly poured into 3,500 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed three times with 500 ml of acetone/water 5:1 and three times with acetone and finally vacuum dried for eight hours at 30 C.
The product is then dissolved in 550 ml of water containing 1% of sodium chloride and the solution is slowly poured into 3,000 ml of acetone under constant agitation.
A precipitate is formed which is filtered and washed twice with 500 ml of acetone/water 5:1 and three times with 500 ml of acetone and finally vacuum dried for 24 hours at 30 C. 8 g of the partial propyl ester compound in the title are obtained. Quantitative determination of the ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas, [Anal. Chem. 33, 1038 - 1030, (1961)].

92 4 Q ~
Example 35 - Preparation of the Pilocarpine salt of Hyaluronic Acid (HY) Partially Esterified With N-Propanol -85% of Carboxylic Groups Esterified With N-Propanol - 15%
of Carboxylic Groups Salified With Pilocarpine.
245 mg of pilocarpine hydrochloride' (1 m.Eq.) are solubilized in 10 ml of water. The solution is eluted in a thermostatic column at 5 C containing 2 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in OH- form.
The chloride-free eluate is gathered in a thermostatic container at 5 C.
4.1 g of the propyl ester of HY 85% and tetrabutyl-ammonium salt at 15% (corresponding to 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl) are solubilized in 100 ml of dimethylsulphoxide. The solution is eluted in a thermostatic column at 20 C containing 2 ml of damp sulphonic resin (DOWEXTM 50 x 8) in H+ form.
The eluate is gathered under agitation in the solution of pilocarpine base. The resulting solution is precipitated with ethyl acetate (600 ml).
The precipitate is filtered and washed four times with 200 ml of ethyl acetate and finally vacuum dried for 24 hours at 30 C. 3.5 g of the compound featured in the title are obtained.
Example 36 - Preparation of the ethyl ester of an acidic polysaccharide produced by Rhinocladiella eliator.

The acidic polysaccharide produced by Rhinocladiella eliator Mangenot NRRL YB-4613 is used (P.R. Watson, P.A.
Sandford, K.A. Burton, M.C. Cadmus and A. Jeanes -Carbohydr. Res. 46, 259-265 (1976); L. Kenne, B. Lindberg, K. Peterson and P. Unger, Carbohydr. Res. 84, 184-186 (1980) . It is made up of units of 2-acetamido-2-deoxy-D-glucuronic acid connected by bonds 1 > 4.

K OH

NHAc 93 1346()3 5.2 g of the potassium salt of this acidic polysaccharide, corresponding to 20 mEq of a moonomeric unit, are suspended in 250 ml of dimethylsulphoxide. While the mixture is kept in agitation, 200 mg of tetrabutylammonium iodide are added at 35 C and then slowly 3.5 g of methyl iodide. The mixture is kept in agitation for 48 hours at 35 C, after which it is slowly poured into 800 ml of ethyl acetate, keeping it under constant agitation. A precipitate is formed which is filtered and washed four times with 150 ml of ethyl acetate and lastly vacuum dried. 4 g of the ethyl ester product in the title are thus obtained, in which all the carboxylic groups are esterified. Quantitative determination of the ester groups is carried out by the method of R.H. Cundiff and P.C. Markanas Anal. Chem. 33, 1028 - 1030 (1961).
Example 37 - Preparation of the Ethyl Ester of Acid Polysaccharide Produced by Rhinocladiella Eliator.
10.0 g of the tetrabutylammonium salt of the acidic polysaccharide used as starting substance in Example 36, corresponding to 20 m.Eq. of a monomeric unit, are treated with 800 ml of dime thyl sulphoxide at 30 C. 3.3 g (21.2 m.Eq.) of ethyl iodide are added and the solution is kept under agitation for 48 hours at 30 C. The resulting mixture is slowly poured into 4000 ml of ethyl acetate while kept under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and lastly vacuum dried.
3 g of the ethyl ester product in the title are obtained, in which all the carboxylic groups are esterified.
Quantitative determination of the ester groups is carried out by the method of R.H. Cundiff and P.C.
Markanas, [Anal. Chem. 33, 1028 - 1030, (1961)].
Example 38 - Preparation of the Ethyl Ester of the Acidic Polysaccharide Produced by Rhinocladiella Mansoni.

Acidic polysaccharide produced by Rhinocladiella Mansoni NRRL Y-46272 [A. Jeanes, K.A. Burton, M.C. Cadmus, 94 *J4 a603 C.A. Knutson, G.L. Rowin and P.A. Sandford - Nature (London) 233, 259 - 260 (1971); P.A. Sandford, P.A. Watson and A.P. Jeanes - Carbohydr. Res. 29, 153 - 166 (1973) ].
It is made up of units formed by one molecule of 2-acetamido-2-deoxy-D-glucuronic acid and two molecules of N-acetyl-D-glucasamine connected by bonds 1 > 3.

HO HO HO
NHAt NHAc MHAt 18.2 g of tetrabutylammonium salt of this acidic polysaccharide, corresponding to 20 m.Eq. of a monomeric unit, are treated with 1000 ml of dimethylsulphoxide at 30 C. Under agitation, 3.3 g (21.2 m.Eq.) of ethyl iodide and the solution is kept at 30 C for 24 hours, after which it is slowly poured into 4000 ml of ethyl acetate, keep it under constant agitation. A precipitate is formed which is filtered and washed four times with 500 ml of ethyl acetate and lastly vacuum dried.
11 g of the product featured in the title are obtained, in which all the carboxylic groups are esterified.
Quantitative determination of the ester groups is carried out according to the method of R.H. Cundiff and P.C.
Markanas, [Anal. Chem. 33, 1028 - 1030, (1961)].
Biological Activity Studies 2 Anti-inflammatory Activity Studies The technical effect of the esters and of the medicaments according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, may be demonstrated, for example, by placing in evidence the anti-inflammatory activity of some partial '~436 esters of hyaluronic acid with antiphlogistic corticosteroids, measured in the model of exudative phlogosis induced by dextran in rabbit eye.
Nine hyaluronic esters of cortisone, hydrocortisone and 5 fluorocortisone (9-fluorohydrocortisone) identified by the code names HYC1-HYC9 were tested. Table 1 describes these compounds and gives the percentages of the number of carboxylic groups of HY which are esterified with the above corticosteroids, and where applicable the percentage 10 esterified with simple aliphatic alcohols and those salified with alkaline metals (Na):
The activity of the compounds of Table 1 was compared with the corresponding cortisones.

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== ~= co v v -s a *r U a 3 C_ C
a n.
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m d 1 ~ 4,116 0 3 All the derivatives, except for HYC4, HYC5 and HYC6 (dissolved in DMSO) were dissolved in saline (2 mg/ml).
Method Aseptic (exudative) phlogosis was induced in 48 rabbits by intraocular injection of dextran (1% in saline, 0.1 ml).
The various products were administered by instillation in the right eye (RE) of the rabbits, while in the left eye (LE) only vehicle was instilled.
The treatment (3 drops every 6 hours) was begun immediately after the injection of dextran and was continued for 16 days.
Ophthalmic Examination Both eyes of each rabbit were observed through a slit lamp. In particular the following were examined: the state of the conjunctiva and corneal epithelium, the anterior chamber (presence of Tyndall effect), state of the iris and of the posterior segment of the eye. With a Goldmann lens, the state of the back of the eye was examined. The presence of signs of inflammation (hyperaemia, exudate, cloudiness of the liquids, etc.) was recorded. The percentage of the eyes which did not present any signs of phlogisis was then calculated.
Results As can be observed from the results reported in Table 2, hereinafter, the HYC derivatives all proved to possess a considerable anti-inflammatory activity consistently superior to that of the corresponding cortisones tested in parallel, reduced not only the percentage of eyes with phlogosis on each day of observation, but also reducing the duration of inflammation. The most efficient of these derivatives seem to be HYC4, HYC5 and HYC6, presumably because they are more lipophilic.

Table 2 Antiinflammatory effect of the HYC derivatives (hyaluronic esters) on dextran-induced aseptic (exudative) phlogosis in rabbit eye Treat:aent Days from start oE phlogosis Cortisone (4) 0.0 0.0 0.0 0.0 25.0 50.0 100.0 100.0 Vehicle (4) 0.0 0.0 0.0 0.0 0.0 25,0 25.0 50.0 Hydrocortisone (4) 0.0 0.0 0.0 25.0 25.0 50.0 100.0 100.0 Vehi.cie (4) 0.0 0.0 0.0 0.0 0.0 25.0 50.0 100.0 Flucrocortis. (4) 0.0 0.0 0.0 0.0 25.0 50.0 100.0 100.0 Venicle (4) 0.0 0.0 0.0 0.0 0.0 50.0 50.0 100.0 HYC1 (4) 0.0 0.0 0.0 25.0 50.0 50.0 100.0 100.0 Veaicle (4) 0.0 0.0 0.0 0.0 00.0 25.0 50.0 100.0 HYi;2 (4) 0.0 0.0 0.0 25.0 50.0 100.0 100.0 100.0 Vehicla (4) 0Ø 0.0 0.0 0.0 00.0 25.0 50.0 100.0 HYC3 0.0 0.0 0.0 25.0 25.0 -100.0 100.0 100.0 Venicle (4) 0.0 0.0 0.0 0.0 U0.0 25.0 50.0 100.0 HYC4 (4) 0.0 0.0 25.0 50.0 50.0 100.0 100.0 100.0 Vehicia (4) 0.0 0.0 0.0 0.0 00.0 25.0 50.0 100.0 HYC5 (-4) 0.0 0.0 25.0 50.0 50.0 100.0 100.0 100.0 Vehicle (4) 0.0 0.0 0.0 0.0 U0.0 25.0 50.0 100.0 HYC6 (4) 0.0 0.0 25.0 50.0 50.0 100.0 100.0 100.0 Vehicle (4) 0.0 0.0 0.0 0.0 00.0 25.0 25.0 50.0 Table 2 (cont'd) Traatmetic Days from the start of phlogosis HYC7 (4) 0.0 0.0 0.0 25.0 50.0 100.0 100.0 100.0 vehicle (4) 0.0 0.0 0.0 0.0 00.0 25.0 50.0 50.0 HYCB (4) 0.0 0.0 0.0 25.0 50.0 100.0 100.0 100.0 Vehicle (4) 0.0 0.0 0.0 0.0 00.0 50.0 50.0 50.0 HYC9 (4) 0.0 0.0 0.0' 25.0 25.0 50.0 100.0 100.0 Vehicle (4) 0.0 0.0 0.0 0.0 00.0 50.0 50.0 100.0 Values are expressed as percentages (number of eyes without signs of phlogosis out of the total number of eyes treated). In brackets are the number of treated eyes.

2) Absorption and Bioavailability Studies The technical effect of the new products according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, may be demonstrated by a study of the absorption and of the bioavailability of some derivatives of hydrocortisone with hyaluronic acid. The derivatives used are those described above-and identified as HYC2, HYC5 and HYC8.
Materials and Methods Animals Male Sprague-Dawley rats, with a body weight of 250 -350 gr were used, obtained from Charles River-Calco (Como), fed ad libitum with water and compound feed in pellets, with the code name of 4RF 21, produced by "Italiana Mangimi", licensee of Charles River.
Treatment Hydrocortisone was administered in the form of sodium hemisuccinate salt at the dose of 1.34 mg/kg (corresponding to 1 mg/kg of hydrocortisone base) by general intravenous route and at the dose of 1.34 mg/kg and 2.68 mg/kg (corresponding to 2 mg/kg of hydrocortisone base) by subcutaneous route (the i.v. route was considered in order to determine the pharmacokinetic parameters which serve as a comparison for the evaluation of absorption of any other administration route).
The three HYC derivatives were administered by subcutaneous route at the dose of 6.5 and 13 mg/kg (doses corresponding to 1 and 2 mg/kg in hydrocortisone base).
All the various products were dissolved in sterile saline, except for HYC5 which, being insoluble in completely aqueous solutions, was first solubilized with the addition of the minimum quantity necessary of dimethylsulphoxide, and then brought to the right volume with saline. All the compounds were injected at a constant volume of 1 ml/kg.

101 C , 03 Gathering of the Plasma Samples After administration, 0.3 ml of blood was drawn from each animal by cardiac puncture in the presence of anticoagulant (sodium heparin).
Blood drawing times were as follows: *15 mins, 30 mins, 60 mins, 120 mins, 180 mins, 300 mins, 360 mins, 420 mins, 480 mins (*limited to the intravenous route).
Dosage of Hydrocortisone The hydrocortisone was dosed by a radioimmunoassay method (using a kit known by the Trade-mark CORTISOLOTM Kit, Biodata, cod. 10394) using iodate tracing. The precision and accuracy of the method, determined on six repeats (double) of a control serum with a known control assay, proved to be 3.03% and 0.021% respectively. The linearity of the method comes between 1 and 1000 ng/ml. The observation limit is 1 ng/ml.
The dosage of the cortisolemia in the rat is not influenced either by the base levels or by the circadian rhythms of this hormone, as the metabolic pattern of the endogenous giucocorticoid and not cortisol (see E.L. Green:
"Biology of the Laboratory Mouse").
Preliminary proof has demonstrated that the dosage method is specific only for free cortisol. The anticortisol antibody does not present any form of competition towards any of the three HYC derivatives.
Results In Table 3 are reported the results of the average plasma levels of hydrocortisone, after i.v. and s.c.
injection (1 and 2 mg/kg). It should be emphasized that, after s.c. injection, there is a quite rapid absorption of the product (Tmax evaluated at 30 mins, Cmax the same as the i.v. route levels at the same dose). In Table 3 hereinbelow are reported the average levels,of cortisol after subcutaneous administration of the three HYC
derivatives at doses of 6.5 and 13 mg/kg (corresponding to 2 mg/kg in hydrocortisone base). In Table 4 hereinbelow are reported the pharmacokinetic parameters relative to ~3 fi03 cortisol calculated graphically from the plasmatic decline curves with the method of residues from the plasma absorption curve of the three HYC products.
It should be noted that the kinetics of hydrocortisone released by the three derivatives with hyaluronic acid are not linear; that is, no direct relationship exists between the dose-dependent parameters, e.g., the area beneath the plasma decline curve and the plasma levels. Since the kinetics of cortisol are themselves linear and a first rate model results, it can be deduced that the saturation process in the case of the HYC derivatives is the hydrolysis of the ester bond between hyaluronic acid and cortisolo. This phase (tending towards zero rate kinetics) is not itself connected with the absorption of the active principle and therefore the kinetics of the three HYC's were likewise resolved according to a first rate model.

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S-I cow N f='1 M tI1 %0 Ul 41 <P q= f'7 O U O u >+
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u 03 Conclusions The bioavailability, as compared to hydrocortisone, of the three products in examination, proves to be complete and even superior to that of the quick release preparation.
Regarding this, however, the absorption is slower (maximum times 2 hours) and maximum concentrations equal to those of subcutaneously administered cortisol are not reached. The plasmatic cortisolemia proves, however, on average to be higher several hours after administration. Esterification with hyaluronic acid therefore determines slower release of hydrocortisone, and this is the desired objective.
J. Skin Hydration Studies Hydrolysis of the ester bond, as has already been stated, is a saturation process; that is, it tends towards zero grade kinetics. This, for a retard form, is a very desirable factor, since, by definition, a controlled release preparation is "a preparation which determines the release of a constant aliquot of active principle in a given time" and this is the condition reached by zero grade kinetics.
The skin, due to the complex nature of its physiological functions, cannot be considered as exclusively a passive covering organ, but rather as a dynamic, polyvalent organ. The complete functional capacity of the skin is fundamentally guaranteed by the presence of an intact hydrolipidic covering and this requires a correct humidity content in the horny layer, which varies a great deal according to its storage capacity (values vary between 10% and 60% of water content). The humidity of the skin depends on a series of endogenous and exogenous factors.
Cutaneous humidity fundamentally influences the formation of the specific hydrolipidic film,of the skin which modifies and stores the substances it eliminates, thus forming the basis for the realization of its protective functions.

The means of defense used so far to restore the maximum degree of hydration for the skin involve the use of highly hygroscopic substances, e.g., glycerine, sodium lactate and propylenic glycol. These substances, however, had the disadvantage, in dry conditions, of drawing humidity from the skin itself instead of from the external environment, thus making the skin even drier.
For this reason at present there is a preference for biological substances whose origins lie, for their particular characteristics, to the natural hydrating factors mentioned before. In this context is included the considerable interest in the use of hyaluronic acid.
The hydration of the skin and its nourishment seem closely related to the HY content of the cutaneous tissue.
It has in fact been demonstrated that the exogenous contribution of HY contributes noticeably to the state of hydration of the cutaneous tissue.
These particular characteristics of hyaluronic acid are also found, and to an even greater degree, in the esterified derivative of HY according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, and for this reason they may be used to a great extent in the field of-cosmetics.
In order to establish a comparison between hyaluronic acid and its derivatives of aspects of the invention, which is disclosed and claimed in the above-identified parent application, some experiments were carried out to evaluate instrumentally, after topical application, the hydrating properties of the compounds in examination.
Materials As hyaluronic derivatives according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, the following, esters were used.
HYAFF2 hyaluronic acid esterified by 75% with methanol HYAFF, hyaluronic acid esterified by 75% with ethanol HYAFFB hyaluronic acid esterified by 50% with isopropanol 107 ~ 3 4 0 3 HYAFF9 hyaluronic acid esterified by 50% with n-propanol HYAFFlo hyaluronic acid esterified by 50% with n-butanol Hyaluronic acid sodium salt (HYALASTINETM fraction).
All the compounds were vehicled at a concentration of 0.2% in an ointment the composition of which was as follows:
Polyethyleneglycol monostearate 400, gr. 10.000 CETIOL VTM (the trade-mark for a cetyl alcohol), gr.
5.000 LANETTE SXTM (the trade-mark for a lanolin), gr. 2.000 Paraoxybenzoate of methyl, gr. 0.075 Paraoxybenzoate of propyl, gr. 0.050 Sodium dehydroacetate, gr. 0.100 Glycerine F.U., gr. 1.000 Sorbitol 70 (hexahydric alcohol), gr. 1.500 Test cream, gr. 0.050 Water for injectable prepar. q.b.a., gr. 100.00 The placebo formulation contained only vehicle.
Method Study Sample The study was carried out on 10 healthy volunteers (6 women and 4 men not suffering from any form of skin disease), aged between 20 and 60 years.
Treatment Each volunteer was treated (single administration) with all the formulations in examination, which were applied (1 gr./ointment) to the inside surface of each forearm, distinguishing, with a demographic pencil, the application zone (25 cm2) of each product and standardizing the procedure as far as possible. To the right forearm were applied the compounds identified as HYAFF2, HYAFF7, HYAFF8, HYAFF9, while to the left were applied HYAFF10, placebo and hyaluronic acid.
Evaluation Parameters At the established times (0, 3, 6 and 24 hours after treatment) the degree of hydration of the horny layer of each application zone was measured with a corneometer.

Most particularly, the dielectric strength of the water was measured (in 0.8 seconds) , after application of the sensor (condenser) to the skin surface. The value thus obtained, the measurement unit of which corresponds to 0.07 mg of water (normal values are between 90 and 100 units), was read on the dial of the instrument.
Registrations were carried out in constant humidity conditions.
Results As can be seen from the results reported in Table 5, hereinbelow, treatment with the compounds of the HYAFF
series induced, in all cases, a notable increase in the degree of hydration of the horny layer, which was particularly evident not only during the hours immediately following application, but also from the last registrations. This effect proved to be superior both to that of the placebo formulation and to the formulation containing hyaluronic acid sodium salt. Of the compounds tested, the derivatives HYAFF2 and HYAFF9 appeared particularly interesting.
Conclusions On the basis of the results obtained it was possible to conclude that the esterified HYAFF derivatives do, in fact, determine a notable and prolonged hydrating effect at the skin level, which is superior to that observed with the formulation containing hyaluronic acid, thus guaranteeing the integrity and physiological efficiency of the hydrolipidic film. These satisfactory results form therefore a valid basis for the use of these compounds in the prevention (or treatment) of chapped skin, the treatment of burns and scalds and the maintaining of physiological nourishment and elasticity of the skin.

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~3416 03 4) Enzyme Stability and Oxygen Permeability Studies Materials The valuable properties of the new esters according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, and of the invention of the present divisional application, already partially described, which form their technical advantages over the already known products in the respective fields are further illustrated by the following results on the stability of the enzymes and the permeability to oxygen of the films obtained with the following compounds:
HYAFF2 hyaluronic acid esterified by 100% with methanol HYAFF7 hyaluronic acid esterified by 100% with isopropanol HYAFF9 hyaluronic acid esterified by 100% with n-propanol HYAFF11 hyaluronic acid esterified by 100% with benzylic alcohol HYAFF20 hyaluronic acid esterified by 100% with R-phenylethylic alcohol HYAFF22 hyaluronic acid esterified by 100% with isopentylic alcohol The films may be prepared according to the procedure described in Example 39.
Stability to Enzymes of the HYAFF Films Stability to Serum Esterage Each film (weighing 20 mg.) was placed in a polyethylene capsule together with 5 ml of rabbit serum and kept at a constant temperature (37 C).
The evaluation parameter was the time taken (in hours) for the film to dissolve. The results are reported in Table 6 hereinbelow.
Stability to Hyaluronidase Each film (weighing 20 mg.) was placed.in a poly-ethylene capsule together with pH 5 buffer (acetate 0.1M, NaCl 0.15M) or pH 7.2 (phosphate 0.1M, NaCl 0.15M) containing 100 U of enzyme (testicle hyaluronidase from Miles batch 8062, activity 342 turbidometric units/mg) in 1~4 41 6 0 ~

each ml and kept at a constant temperature (37 C). The evaluation parameter was the time taken (in hours) for the film to dissolve. The results are reported in Table 6 hereinbelow.
Table 6 Stability of the Films of the HYAFF Series derivatives in the presence of serum esterase (37 C) and in the presence of Hyaluronidase (37 C; pH 5 and pH 7.2) COMPOUNDS STABILITY (hrs) SERUM ESTERASE HYALURONIDASE
pH 5 pH 7.2 112 ~ 3 4 16 03 Permeability to Oxygen of the Films of the HYAFF Series Each film was placed in a container having 2 compartments separated by the membrane, itself. One compartment (volume = 1,2 cc) was filled with partially degassed water (P02=45mm of Hg at 23 C), into the other was introduced a flow of O2 and CO2 (95% and 5% respectively), kept constant (1 bubble/second) in time. The whole system was insulated in nitrogen.
At the established times (15, 30, 60, 90, 120, 240 minutes) a suitable aliquot of water was drawn off (1.2 cc) and determination of the partial pressure of 02 was effected by an analyzer known by the Trade-mark GAS SYSTEMTM 1302 of the Instrumentation Laboratories. The saturation pressure (550 mg of Hg) was taken as reference value and calculated, in the previously described experimental conditions, by insufflating the 02 atmosphere.
The tests were carried out in comparison to an impermeable membrane and an organo-silicon synthetic rubber known by the Trade-mark SILASTICTM of The Dow Chemical Company (in Lepetit Cat. No. 500-1). The results are reported in Table 7 hereinafter.

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c. U _ ~ Vl O Z O r.. ~. _ Pharmaceutical Preparations The invention, which is disclosed and claimed in the above-identified parent application, also provides pharmaceutical preparations of another aspect of this invention, which is disclosed and claimed in the above-identified parent application, containing one or more of the above-mentioned esters of hyaluronic acid and salts thereof, of other aspects of this invention, or one or more medicaments resulting from the association of one of such esters with a pharmacologically-active substance for topical application, of other aspects of the invention, which is disclosed and claimed in the above-identified parent application, as described above. That is, the invention, which is disclosed and claimed in the above-identified parent application, provides medicaments in which the hyaluronic ester acts as a vehicle for the active substance.
The pharmaceutical preparations of other aspects of the invention, which is disclosed and claimed in the above-identified parent application, containing the hyaluronic esters of other aspects of the invention, which is disclosed and claimed in the above-identified parent application, as an active principle, both in the case of esters with therapeutically-inactive alcohols destined for the same uses as hyaluronic acid itself, and esters of HY
with therapeutically-active alcohols intended for the usual uses of such alcohols, contain the usual excipients and may be employed for oral, rectal, parenteral, subcutaneous, local or intradermal use. They are therefore in solid or semisolid form, for example, as pastilles, tablets, gelatin capsules, capsules, suppositories, or soft gelatin capsules. For parenteral and subcutaneous use it is possible to use forms intended for intramuscular or intradermal administration, or suitable for infusions or intravenous injections. It is possible therefore to provide solutions of the active compounds or freeze-dried powders of the active compounds to be added to one or more 115 ~ 1 6, Q 3 pharmaceutically-acceptable excipients or diluents, convenient for the above-mentioned uses and with osmolarity compatible with the physiological liquids. For local use, preparations in spray form may be used; for example, nasal sprays, creams or ointments for topical use of specially prepared sticking plasters for intradermal administration.
The solubility of the hyaluronic esters in organic solvents with low boiling points makes them particularly suitable for the manufacture of "sprays".
The preparations of aspects of the invention, which is disclosed and claimed in the above-identified parent application, may be administered to man or animals. They contain preferably between 0.01% and 10% of active component for the solutions, sprays, ointments and creams and between 1% and 100%, preferably between 5% and 50%, of active compound for the preparations in solid form. The dosage to be administered will depend on individual needs, on the desired effect and on the chosen administration route. The daily dosage of such preparations may be decided according to that use for the corresponding known preparations both of hyaluronic acid for the corresponding cures; for example, for the cure of arthritis, for example, in man or horse; and of the therapeutically-active alcohol, the action of which is to be put to use. Thus, for example, the dosage of a hyaluronic ester with cortisone may be derived from its content of this steroid and from its usual dosage in the known pharmaceutical preparations.
One particular form of pharmaceutical preparations, which is disclosed and claimed in the above-identified parent application, is represented by the above-mentioned medicaments containing the association of an hyaluronic ester and of one or more active substances. These may also be in solid form, for example, as freeze-dried powders containing only the two Components (1) and (2), together or separate. This galenic form is especially suitable for topical use. Indeed these solid medicaments form, on contact with the surfaces to be treated, more or less concentrated solutions according to the nature of the particular epithelium, with the same characteristics of the solutions previously prepared in vitro and which represent another particularly important aspect of the invention, which is disclosed and claimed in the above-identified parent application. Such solutions are preferably in distilled water or sterile saline and preferably contain no other' pharmaceutical vehicle apart from the hyaluronic ester or one of its salts. The concentrations of such solutions may also vary within ample limits, for example, between 0.01 and 75% both for each of the two components taken separately, and for their mixtures or salts.
Particular preference is given to solutions with a pronounced elastic-viscous character, for example, with a content of 10% to 90% of the medicament or of each of its components.
Particularly important are the medicaments of this type, which is disclosed and claimed in the above-identified parent application, both in anhydrous form (freeze-dried powder) or as solutions, concentrated or diluted in water or saline, possibly with the addition of additive or auxiliary substances, e.g., in particular disinfectant substances or mineral salts acting as a buffer or others, for ophthalmic use.
Of the medicaments of aspects of the invention, which is disclosed and claimed in the above-identified parent application, particularly important, as the case may be, are those with a degree of acidity suitable for the environment to which they are to be applied, that is with a physiologically-tolerable pH. Adjustment of the pH, for example in the above-mentioned salts of the esters of hyaluronic acid with a basic active substance, may be effected by suitably regulating the quantities of polysaccharide, of the salts or of the basic substance itself. Thus, for example, if the acidity of a salt of a hyaluronic ester with a basic substance is too high, the excess of free acid groups is neutralized with the above-fi03 mentioned inorganic bases, for example, with sodium, potassium or ammonium hydrate.
The preparation of the salts according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, may be effected by a procedure known per se by placing in contact solutions, aqueous solutions or organic solutions, of the two Components (1) and (2), and possibly bases or basic salts of the above-mentioned alkaline or alkaline earth metals or magnesium or aluminum in the right quantities and isolating the salts in an amorphous anhydrous form according to known techniques. It is possible, for example, to'prepare first aqueous solutions of the two Components (1) and (2), freeing such components from aqueous solutions of their salts with suitable ionic exchangers, and mixing the two solutions at a low temperature, for example, between 0 C and C. If the salt thus obtained is easily soluble in water, it can be freeze-dried, while the salts which are difficult to solubilize may be separated by centrifugation, 20 filtration or decantation and possibly subsequently dried.
For these associated medicaments, too, the dose is based on that of the active principles used singly and may therefore easily be determined by a skilled person, taking into consideration the dosage recommended for the corresponding known drugs.
In the cosmetic articles according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, the hyaluronic esters and their salts are mixed with the excipients commonly used in the field and are, for example, those already listed above for the pharmaceutical preparations, which is disclosed and claimed in the above-identified parent application. Mostly used are creams, ointments, and lotions for topical use in which the hyaluronic ester or one of its salts may represent the cosmetic active agent, possibly with the addition of other cosmetically-active agents, e.g., steroids, for example, pregnenolone, or one of the agents mentioned above. In such preparations, the hyaluronic ester is preferably an ester with an alcohol with no cosmetic action, e.g., a lower aliphatic alcohol, e.g., one of those already mentioned. The effect is due to the intrinsic cosmetic properties of the polysaccharide component, e.g., in the case of free hyaluronic acid or of its salts.
The cosmetic articles, which are disclosed and claimed in the above-identified parent application, may, however, be based on substances with specific actions other than those of hyaluronic acid, for example, disinfectant substances, sunshields, waterproofing or regenerating or antiwrinkle substances, or odourants, especially perfumes.
In this case, the hyaluronic ester may itself be the active ingredient and derives from alcohols with the same properties, for example, from higher aliphatic alcohols or terpenic alcohols in the case of perfumes or acts as a vehicle for substances with those properties associated with it.
Particularly important therefore are cosmetic compositions, according to other aspects of the invention, which is disclosed and claimed in the above-identified parent application, similar to the medicaments described above in which the pharmaceutically-active component (1) or its relative salts is substituted by a cosmetic factor.
The use of the above-mentioned esters of HY deriving from alcohols used in the perfume industry, which is disclosed and claimed in the above-identified parent application, represent an important step forward in technology, since they allow a slow, constant and protracted release of the scented ingredients.
An important application of aspects of the invention, which is disclosed and claimed in the above-identified parent application, regards sanitary and surgical articles which have already been described above, and to the procedures for their manufacture and use. The invention, which is disclosed and claimed in the above-identified parent application, therefore includes all the articles similar to those already on the market, based on the hyaluronic acid but containing a hyaluronic ester of aspects of this invention or one of its salts of aspects of this invention in place of the free acid or one of its salts, for example, inserts or ophthalmic lenses.
Completely new surgical and sanitary articles according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, are represented by the esters of hyaluronic acid which has been regenerated as such by appropriate organic solutions from which it is possible to obtain, by means of suitable procedures, films, thin sheets or threads to be used in surgery, as aids or substitutes of the skin in case of serious damage to this organ, e.g., following burns, as a suture in surgical operations. The invention, which is disclosed and claimed in the above-identified parent application, in other aspects includes particularly these uses and a procedure for the preparation of such articles consisting in the formation of a solution of hyaluronic ester of aspects of the invention, which is disclosed and claimed in the above-identified parent application, or of one of its salts of aspects of the invention, which is disclosed and claimed in the above-identified parent application, in a suitable organic solvent, e.g., an amide of a carboxylic acid, especially a dialkylamide of an aliphatic acid with between 1 and 5 carbon atoms and deriving from alkyl groups with between 1 and 6 carbon atoms, and above all from an organic sulphoxide, that is, a dialkylsulphoxide with alkyl groups with a maximum of 6 carbon atoms, e.g., dimethylsulphoxide or diethylsulphoxide and again most importantly by a fluorinated solvent with a lower boiling point such as especially hexafluoroisopropanol. The invention, which is disclosed and claimed in the above-identified parent application, in other aspects, includes turning such solutions into sheet or thread form and in removing the organic solvent by contact with another organic or aqueous solvent which can be mixed with the first solvent and in which the hyaluronic ester is not soluble, especially a lower aliphatic alcohol, for example ethyl alcohol (wet spinning), or, should a solvent with a not-too-high boiling point be used to prepare the solution of' the hyaluronic derivative, in removing such a solvent in dry conditions with a current of gas, especially with suitably heated nitrogen (dry spinning) It is also possible to obtain excellent results with dry-wet spinning.
The threads of other aspects of the invention, which is disclosed and claimed in the above-identified parent application, obtained with hyaluronic acid esters of aspects of the invention may be used for the preparation of gauze for the medication of wounds and in surgery. The use of such gauze has the exceptional advantage of the biodegradability thereof in the organism, made possible by the enzymes which they contain. These enzymes divide the ester into hyaluronic acid and the corresponding alcohol, and therefore into a compound already present in the organism, and into a harmless compound, e.g., an alcohol, should a hyaluronic ester be used which derives from a therapeutically-acceptable alcohol, e.g., ethyl alcohol, made possible by the enzymes which they contain.
These gauzes and also the aforementioned threads may therefore be left inside the organism after surgery, since they are slowly absorbed thanks to the above-described degradation.
During the preparation of the sanitary and surgical articles of aspects of the invention, which is disclosed and claimed in the above-identified parent application, described above, it is possible to add plastifying materials which improve their mechanical characteristics, e.g., in the case of the threads, to improve their resistance to knots. These plastifying materials may be, for example, alkaline salts of fatty acids, for examples, sodium stearate or sodium palmitate, the esters of organic acids with many carbon atoms, etc.

Another application of the esters of HY of aspects of the invention, which is disclosed and claimed in the above-identified parent application, using to advantage their biodegradability due to the esterases present in the organism, is represented by the preparatiori of capsules of aspects of the invention, which is disclosed and claimed in the above-identified parent application, for subcutaneous implaritation of medicaments or of microcapsules for injection, for example, by subcutaneous or intramuscular route. For the applications of subcutaneous medicaments for obtaining a slow release and therefore a "retard"
action, capsules made of silicone materials have mostly been used up till now, with the disadvantage that the capsule is liable to move about inside the organism and it is not possible to recover it. Evidently with the new hyaluronic esters of aspects of the invention, which is disclosed and claimed in the above-identified parent application, this danger is no longer exists.
Of great importance is also the preparation of microcapsules of aspects of the invention, which is disclosed and claimed in the above-identified parent application, which are made with hyaluronic esters of aspects of the invention, which is disclosed and claimed in the above-identified parent application, eliminating the problems regarding their use which up till now has been limited, for the same reasons as those mentioned above and which opens up a vast field of application where a "retard"
effect is sought be an injected route.
A further application in the sector of medicine and surgery of the esters of HY, which is disclosed and claimed in the above-identified parent application, and the acidic ester of aspects of the present divisional application, concerns the preparation of a large variety of solid inserts, e.g., plates, discs, sheets, etc. substituting those in metallic form or those made of synthetic plastic materials already in use, in the case of inserts which are intended for removal after a certain period of time.

122 13 4 1s 0 3 Preparations made of animal pollagen, being of a proteic nature, often provoke undesirable side effects e.g., inflammation or rejection. In the case of animal, and not human, hyaluronic acid, this danger does not exist, as there is no incompatibility between the polysaccharides of different animal species.
Another application relates to the use to augment and correct soft tissue defects. The need for a safe and effective biomaterial by which to replace missing or damaged soft tissue has long been recognized. Several alloplastic materials, including paraffin, a paste of the polytetrafluoroethylene known by the Trade-mark TEFLONTM of du Pont, silicone and bovine collagen have been used to replace lost soft tissue. However, these materials have been associated with permanent undesirable textural changes in the skin, with migration from the site of implantation and with adverse treatment reactions. Thus, the need for a versatile biomaterial in medicine continues. The hyaluronic acid esters of aspects of the invention, which is disclosed and claimed in the above-identified parent application, and the acidic ester of aspects of the present divisional application, can be used safely and effectively to augment and correct such soft tissue defects as acne scars, atrophy post surgical irregularities, mohs chemosurgery, cleft lip sears and age-related wrinkles.
Part of the application in the field of medicine and surgery of the esters according to aspects of the invention, which is disclosed and claimed in the above-identified parent application, and the acidic ester of aspects of the present divisional application, are represented by expansive materials, especially in the form of sponges, for the medication of wounds and various lesions.
The following are particular exemplary pharmaceutical preparations according to aspects of the invention, which is disclosed and claimed in the above-identified parent application.

Formulation 1 - Collirium containing cortisone of which 100 ml contain:
partial ester of hyaluronic acid with cortisone (Ex.10), gr. 0.200 ethyl p. hydroxybenzoate, gr. 0.010 methyl p. hydroxybenzoate, gr. 0.050 sodium chloride, gr. 0.900 water for injectable preparations/q.b.a., ml. 100 Formulation 2 - Injectable solution containing hydrocortisone of which 100 ml contain:
partial ester of hyaluronic acid with hydrocortisone (Ex. 11), gr. 0.1 sodium chloride, gr. 0.9 water for injectable preparations/q.b.a., ml. 100 Formulation 3 - Cream containing a partial ester of hyaluronic acid with ethyl alcohol (Ex. 3), of which 100 gr. contain:
partial ester of hyaluronic acid with ethyl alcohol, gr. 0.2 polyethylenglycol monostearate 400, gr. 10.000 CETIOL VTM, gr. 5.000 LANETTE SXTM, gr. 2. 0 0 0 paraoxybenzoate of methyl, gr. 0.075 paraoxybenzoate of propyl, gr. 0.050 sodium dihydroacetate, gr. 0.100 glycerine F.U., gr. 1.500 sorbitol 75, gr. 1.500 test cream, gr. 0.050 water for injectable preparations/q.b.a., gr.
100.00 The following are exemplary material products utilizing the hyaluronic esters of aspects of the invention, which is disclosed and claimed in the above-identified parent application.

Example 39 - Preparation of Films Using Esters of Hyaluronic Acid.
A solution is prepared in dimethylsulphoxide of the n-propyl ester of HY (MW 130,000) with a concentration of 180 mg/ml.
By means of a stratifier, a thin layer of solution is spread on a glass sheet; the thickness must be 10 times greater than the final thickness of the film. The glass sheet is immersed in ethanol which absorbs the dimethylsulphoxide but does not solubilize the HY ester which becomes solid. The film is detached from the glass sheet, is repeatedly washed with ethanol, then with water and then again with ethanol.
The resulting sheet is dried in a press for 48 hours at 30 C.
Example 40 - Preparation of Threads Using Esters of Hyaluronic Acid A solution is prepared in dimethylsulphoxide of the benzyl ester of HY (MW 165,000) with a concentration of 200 mg/ml. The solution thus obtained is pressed by means of a pump through a threader with 0.5 mm holes.
The threader is immersed in ethanol/dimethylsuiphoxide 80:20 (this concentration is kept constant by continuous addition of ethanol); when the solution in dimethylsuiphoxide is soaked in this way it tends to lose most of the dimethylsulphoxide and the thread solidifies.
The thread is stretched while it still has a content of dimethylsulphoxide, is then repeatedly stretched and washed with ethanol. The thread is dried in nitrogen current.
ExamDle 41 - Preparation of a Spongy Material Made With Hyaluronic Acid Esters.
1 g of benzyl ester of hyaluronic acid with a molecular weight of 170,000 in which all the carboxylic groups are esterified (obtained for example as described in Example 14) are dissolved in 5 ml of dimethylsulphoxide. To each 10 ml of solution prepared, a mixture of 31.5 g of sodium chloride with a degree of granularity corresponding to 300 p, 1.28 g of sodium bicarbonate and 1 g of citric acid is added and the whole is homogenized in a mixer.
The pasty mixture is stratified in various ways, for instance, by means of a mange consisting of two rollers which turn opposite each other at an adjustable distance between the two. Regulating this distance, the paste is passed between the rollers together with a strip of silicone paper which acts as a support to the layer of paste thus formed. The layer is cut to the desired dimensions of length and breadth, removed from the silicone, wrapped in filter paper and emerged into a suitable solvent, e.g., water. The sponges thus obtained are washed with a suitable solvent, e.g., water and possibly sterilized with gamma rays.
Example 42 - Preparation of a Sponcty Material Made With Hyaluronic Acid Esters In the manner described in Example 41, it is possible to prepare spongy materials with other hyaluronic acid esters of aspects of the invention, which is disclosed and claimed in the above-identified parent application, and with the acidic ester of aspects of the present divisional application. In the place of dimethylsulphoxide it is possible to use, if desired, any other solvent capable of dissolving the chosen ester of HY of aspects of the invention, which is disclosed and claimed in the above-identified parent application, and the chosen acidic ester of aspects of the present divisional application. In the place of sodium chloride it is possible to use any other solid compound which is insoluble in the solvent used to dissolve the hyaluronic acid ester of aspects of the invention, which is disclosed and claimed in the above-identified parent application, and the acidic ester of aspects of the present divisional application, but which is however soluble in the solvent used to precipitate the hyaluronic ester of aspects of the invention, which is disclosed and claimed in the above-identified parent application, and the acidic esters of aspects of the present divisional application, after the above-mentioned mechanical treatment, and finally which has the correct degree of granularity to obtain the type of pores desired in the sponge material.
In the place of sodium bicarbonate and citric acid it is possible to use other couples of similar compounds, that is, compounds, which react to each other in suspension or solution of the solvent used to dissolve hyaluronic acid in such a way as to form a gas, e.g., carbon dioxide, which has the effect of producing a less compact spongy material.
In this way it is possible to use, in the place of sodium bicarbonate, other bicarbonates or alkaline or alkaline earth carbonates and in the place of citric acid other acids in solid form., e.g., tartaric acid.

Claims (37)

1. A process for the preparation of an acidic polysaccharide ester containing carboxyl groups, which process comprises:
treating a quaternary ammonium salt of said polysaccharide with an esterifying agent in an aprotic solvent; and, if desired, salifying some or all of the free carboxy groups in the partial esters obtained.

2. A process for the preparation of an acidic polysaccharide ester containing carboxyl groups, which process comprises: in a first step, treating a quaternary ammonium salt of said polysaccharide with an esterifying agent in an aprotic solvent;
and in a second step, salifying free carboxy groups in the partial esters obtained.

3. The process according to claim 1 or claim 2, wherein all of the free carboxy groups in said esters are salified.

4. The process of claim 1 or claim 2, which is carried out at a temperature of 0°C to 100°C.

5. The process of claim 4, which is carried out at a temperature of 25°C to 75°C.

6. The process of claim 5, which is carried out at a temperature of 30°C.

7. The process according to any one of claims 1 to 6, wherein said acidic polysaccharide is of vegetable origin.

8. The process according to any one of claims 1 to 6, wherein said acidic polysaccharide is of animal origin.

9. A process according to any one of claims 1 to 8, wherein said quaternary ammonium salt is a C1-6 tetraalkylammonium salt.

10. The process according to any one of claims 1 to 8, wherein said quarternary ammonium salt is the tetraalkylammonium salt of an acidic polysaccharide.

11. The process according to claim 10, wherein said acidic polysaccharide also contains sulphonic acid groups.

12. The process according to any one of claims 1 to 11, wherein said solvent is a dialkylsulphoxide, a dialkylcarboxamide, a lower alkyl dialkylamide of a lower aliphatic acid, an alcohol, an ether, a ketone or an ester.

13. The process according to claim 12, wherein said solvent is dimethylsulphoxide.

14. The process according to any one of claims 1 to 13, wherein at least a portion of said carboxyl groups are salified with an alkali metal, with an alkaline earth metal, with aluminum or with ammonia.

15. The process according to claim 14, wherein said salt is formed with sodium or with ammonia.

16. The process according to any one of claims 1 to 14, wherein at least a portion of said carboxyl groups are salified with a therapeutically-acceptable ammonium base, aliphatic base, araliphatic base, cycloaliphatic base or heterocyclic base.

17. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with n-pentyl bromide.

18. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with isopentyl bromide.

19. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with benzyl bromide.

20. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with 2-bromoethylbenzene.

21. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-17.alpha.-ol-3,11,20-trione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with cortisone.

22. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-11.beta.,17.alpha.-diol-3,20-dione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with hydrocortisone.

23. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 9.beta.-fluoro-21-bromo-4-pregnene-11.beta.,17.alpha.-diol-3,20-dione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with fluorocortisone.

24. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl chloroacetate and then salifying with sodium ions, whereby 75% of the carboxyl groups are esterified and whereby 25% of the carboxyl groups are salified with sodium.

25. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with 21-bromo-4-pregnene-17.alpha.-ol-3,11,20-trione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.

26. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with 21-bromo-4-pregnene-11.beta.,17.alpha.-diol-3,20-dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.

27. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with 9-fluoro-2l-bromo-4-pregnene-11.beta.,17.alpha.-diol-3,20-dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.

28. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with 21-bromo-4-pregnene-3,20-dione and then salifying with sodium ions, whereby 20% of the carboxyl groups are esterified and whereby 80% of the carboxyl groups are salified with sodium.

29. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-3,20-dione, whereby 80% of the carboxyl groups are esterified with ethanol and whereby 20% of the carboxyl groups are esterified with deoxycorticosterone.

30. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with deoxycorticosterone, and whereby the remaining 40% of the carboxyl groups are salified with sodium.

31. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-17.alpha.-ol-3,11,20-trione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with cortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.

32. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 21-bromo-4-pregnene-11R,17.alpha.-diol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with hydrocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.

33. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 9.beta.-fluoro-21-bromo-4-pregnene-11.beta.,17.alpha.-diol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with fluorocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.

34. The process according to claim 2, or according to any one of claims 4 to 16 when appended to claim 2, comprising reacting the tetrabutylammonium salt of said acidic polysaccharide with ethyl iodide and with 9.beta.-fluoro-21-bromo-4-pregnene-11R,17.alpha.-diol-3,20-dione, and then salifying with sodium ions, whereby 40% of the carboxyl groups are esterified with ethanol, whereby 20% of the carboxyl groups are esterified with fluorocortisone and whereby the remaining 40% of the carboxyl groups are salified with sodium.

35. A C1-6 tetraalkylammonium salt of hyaluronic acid or a molecular fraction of hyaluronic acid.

36. A tetraalkylammonium salt of an acidic polysaccharide containing carboxyl groups, wherein the tetraalkylammonium ions of said salts are derived from alkyls with between 1 and 4 carbon atoms.

37. The salt of the acidic polysaccharide according to claim 35 or 36 which is a tetrabutylammonium salt.