CA1341087C - Medicaments including hyaluronic acid as a drug delivery system - Google Patents

Abstract

Novel medicaments for topical administration are provided herein. Such medicaments comprise a pharmaceutically-active substance or a mixture of pharmaceutically-active: substances which is, or are, suitable for topical administration, with the proviso that the active substance is not an ophthalmic drug, together with hyaluronic acid or a pharmaceutically-acceptable salt of the hyaluronic acid as the drug delivery system, and from 0% up to a topically-suitable amount of an additional excipient. Still further, optionally, these medicaments may also contain an additional pharmaceutically-acceptable excipient. The hyaluronic acid, which typically excludes fractions of molecular weight less than 30,000 Daltons, may be in the form of the free acid or it may be in the form of a salt with an alkali metal, with an alkaline earth metal, with magnesium, with aluminum, with ammonia or with an amine, or may be in the form of a salt with one or more of those pharmaceutically-active substances. Still further, those medicaments may be pharmaceutical compositions for topical administration and comprise, as an active ingredient, a pharmacologically-effective amount of partial salt, or a stoichiometrically-neutral salt, of hyaluronic acid, or a molecular weight fraction thereof, with at least one pharmacologically-active substance of a basic nature which is suitable for topical administration and which is capable of being absorbed intradermally or through the nasal or rectal mucosa, together with an excipient which is suitable for topical administration.

Description

The present inve;ntion relates to new medicaments which are non-ophthalmic drugs, which are suitable for topical use, the medicament also essentially including hyaluronic acid, or pharmaceutically-acceptable salts thereof, as a drug delivery system.
It also relates to processes for the use of such hyaluronic acid or pharmaceutically-acceptable salts thereof for the preparation of such new medicaments.
This application is a division of copending application Serial No. 505,909 filed April 4, 1986.
The application of a topically-active medicament may be a benefit or remedy, especially in dermatology, diseases of the mucous membranes in general and particularly membranes of the oral and nasal cavities, diseases of the outer ear, and especially diseases of the outer surface of the eye. Application of these topical medicaments is particularly advisable in. paediatrics and in the veterinary field.
Such ophthalmic medicaments are of exceptional value especially in the veterinary field, considering, for example" that there are at present no veterinary specialities for oculistic use containing chemoth.erapeutics. Indeed, preparations which are intended for human use are usually used, and these do not always guarantee a specific range of activity nor comply with the particular conditions in which the treatment should be effected.
This is the case, for example, in therapy for infectious keratoconjunctivitis, pink eye or IBK, an infection which .mainly affects cattle, sheep and goats.
Presumably, these three species have specific etiological factors in common. In particular, in cattle, the main microorganism involved seems to be Moraxella bovis (even though other agents of a viral origin should nol: be excluded, e.g., Rhir~otracheitis virus, Micoplasma, Rickettsia and Chlamydia in the case of sheep, and Rickc~ttsia in the case of goats). The disease manifests itself in an acute form and tends to spread quickly. In the initial stages, the symptomatology is char.°acterized by blepharospasm and excessive lacrimation, followed by purulent exudate, conjunctivitis and keratitis, often accompanied by fever, reduced appetite and milk production. Lesions of the cornea are particularly serious and in the final stages can even c;~use perforations of the cornea itself. The clinical course varies from a few days to several weeks.
B

__ 134108

2 A vast range of chemotherapeutic agents ~~re used for treatment, administered both topically (often in association with anti-inflammatory steroids), and systemically. Among these are the following: tetracyclines, e. g. , oxyt:etracycline, penicillins, e. g. , cloxacillin and benzylpenicillin, sulphamides, polymyxin B (associated with miconazole and prednisolone), chloramphenicol, tylosin and ch.loromycetin. Topical treatment of the disease, despite its apparent simplicity, still represents an unsolved problem, since for one reason or another it has proved impossible up until now to obtain oculistic preparations having concentrations of antibiotics or sulphamides which are therapeutically-effective in the secretion of tears.. This is quite understandable in the case of solutions, bearing in mind the: mainly reclining position of the head in these animals.
It is also true of semisolid medicaments, since the excipients normally used in them do not have the qualities necessary for adhering to the surface of the cornea, as they do not usually have a sufficiently-high concentration of pharmaceutically-active substance and cannot achieve perfect dlistribution (i.e., the preaence of a distribution gradient). These defects of conventional collyriums in ophthalmic use have been described by Slatter et al. in "Austr. vet. J.," 1982, 59 (3), pp. 69-72.
The present invention, therefore, in its broad essential aspects, is related to the combination of hyaluronic acid or a pharmaceutically-acceptable salt thereof as a vehicle in association with a pharmaceutically-active substance to provide an improved drug delivery system. New medicaments according to four aspects of this invention basically are defined as follows: 1. mixtures of hyaluronic acid with a non-ophthalmic topical drug; 2. mixtures of hyaluronic; acid with a non-ophthalmic topical drug, wherein the drug is systemically-active and is capable of being absorbed intradermally; 3.
pharmaceutically-acceptable salts of hyaluronic acid with a non-ophthalmic topical drug of a basic nature; and 4.. pharmaceutically-acceptable salts of hyaluronic acid with a non-ophthalmic topical dru~; of a basic nature, wherein the drug is systemically-active and is capable of being absorbed intradermally.
Thus, one broad aspect of the present invention provides a medicament for topical administration, which comprise, a pharmaceutically-active substance or a mixture of pharmaceutically-active: substances which is or are suitable for topical administration, B

3 ~~41~8~
with the proviso that the; active substance is not an ophthalmic drug, hyaluronic acid or a pharmaceutically-acceptable salt of the hyaluronic acid, and from 0 % up to a topically-suitable amount of an additional excipient.
By one variant of this first aspect of the present invention, the pharmaceutically-active substance is capable of being absorbed intradermally or through the nasal or rectal mucosa.
By another variant of this first aspect of the present invention, the pharmaceutically-active substance is of a basic nature and is present in the form of a salt with the hyaluronic acid.
By a second aspect of this invention, a medicament pharmaceutical composition is provided which comprises, as an active ingredient, a pharmaceutically-effective amount of a partial salt of hyaluronic: acid or a moleuclar weight fraction thereof, or a stoichiometrically-neutr;~l salt of hyaluronic acid or a molecular weight fraction thereof, with at least one pharmacologically-active substance of a basic nature, which is suitable for topical administration and which is capat>le of being absorbed intradermally or through the nasal or rectal mucosa, together with an excipient which is suitable for topical administration.
By a variant of these first and second aspects of the present invention and/or the above variants thereof, the pharmaceutically-active substance is an antibiotic, an anti-infective, an antiviral, an anti:microbial, an <~nti-inflammatory, a wound healing, a cytostatic, a cytotoxic, an anaesthetic, a cholinergic promoter, a cholinergic antagonist, an adrenergic promoter or adren.ergic an antagonist agent. By one variation thereof, the pharmaceutically-active substance is erythromycin, gentamicin, neomycin, streptomycin, dihydrostreptomycin, kanamyciti, amikacyn, tobramycin, spectinomycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitracin, polymxin B, gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, nystatin, diethylcarbamazine, mebendazole, sulphacetamide, sulph;adiazine, sulphisoxazole, idoxuridine, adenine arabinoside, trifluorothimidine, a.ciclovir, ethyldeoxyuridine, pilocarpine, metacholine, carbamylcholine, acec;lidine, fisostigmine, neostigmine, demecarium, atropine, B

p..__ ,

4 noradrenalin, adrenalin, norfaz;oline, methoxamine, propranolol, timolol, pindolol, bupranolol, atenolol, mfaoprolol, oxyprenolol practolol, butoxamine, sotalol, butadrine, labetalol, dexamethasone, triamcinolone, prednisolone, fluoromethalone, medrison, fluorocil, methotrexate, or podophyllin.
By another variant of these first and second aspects of the present invention and/or the above variants and/or the above variation thereof, the hyaluronic acid is a molecular weight fraction which is substantially-free of hyaluronic acid having a molecular weight less than 30,000 Daltons. By variations of the above variant of these first and second aspects of the present invention, the hyaluronic acid fraction has an average molecular weiight between 50,000 lJaltons and 100,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between 500,000 Daltons and 730,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between 250,000 and 350,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between 30,000 and 730,000 Daltons.
By a third aspect of this invention, a medicament is provided for topical use which comprises a partial or stoichiometrically-neutral salt of hyaluronic acid with at least one pharmaceutically-active substance of a basic nature which is suitable for topical administration.
By one variant of this third aspect of the present invention, the pharmaceutically-active substance is capable of being absorbed intradermally or through the nasal or recta mucosa.
By another variant of this third aspect of the present invention and/or the above variant thereof, the medicament contains an additional excipient which is suitable for topical administration.
By yet another variant of this third aspect of the present invention and/or the above variants thereof, the salt is a partial salt, and at least a portion of the acid groups of the hyaluronic acid are salified with an alkali metal, with an alkaline earth metal, with magnesium, with alumiinum, with ammonia or with an amine.
By still another variant of this third aspect of the present invention and/or the above variants thereof, the pharmaceutically-active substance is suitable for B

dermatological, for otorhinolaryngolical, for odontological, for angiological, for obstetrical or for neurological use.
By still a further variant of this third aspect of the present invention and/or the above variants thereof, the pharmaceutically-active substance is an antibiotic, an anti-

5 infective, an antiviral, an ant:imicrobial, an anti-inflammatory, a wound healing, a cytostatic a cytotoxic, an anaesthetic, a cholinergic promoter, a cholinergic antagonist, an adrenergic promoter or an adrenergic antagonist agent.
By yet still a further variant of this third aspect of the present invention and/or the above variants thereof, the pharmaceutically-active substance is erythromycin, gentamycin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacyn, tobramycin, spectinom.ycin, oleandomycin, c~~rbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitavacin, polymxin B, ,gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, nystatin, d.iethylcwbamazine, membendazol, sulphacetamide, sulphadiazine, sulphisoxazole, idoxuridine, adenine arabinoside, trifluorothimidine, aciclovir, ethyldeoxyuridine, pilo~carpine, metacholine, carbamylcholine, aceclidine, fisostigmine, neostigmine, demecariiun, atropine, noradrenal.in, adxenalin, norfazoline, methoxamine, propranolol, timolol, p:indolol, bupranolol, atenolol, metoprolol, oxyprenolol, practolol, butoxamine, sotalol, butadrine, labetalol, dexamethasone, triamcinolone, prednisolone, fluoromethalone, medrison, fluorocil, methotrexate, podophyllin or epidermal growth factor.
By a fourth aspect of this invention, a pharmaceutical composition is provided comprising a sterile composition comprising an aqueous composition containing hyaluronic acid or a pharmaceutically-acceptable salt or pharmaceutically-acceptable ester thereof, and a non-steroidal, anti-inflammatory drug, or a pharmaceutically-acceptable salt thereof, or a pharmaceutically-acceptable ester thereof.
By a fifth aspect of the present invention, a pharmaceutical composition is provided which comprises a mixture of hyaluronic acid or a pharmaceutically-acceptable salt thereof, and a non-steroidal, anti-inflammatory agent or a pharmaceutically-acceptable salt thereof.
B

~ 341 08 7 ~..... .

6 By a sixth aspect of the present invention, a pharmaceutical composition is provided which comprises a salt ~of hyaluronic acid with a non-steroidal anti-inflammatory agent.
By one variant of the above further fourth, fifth and sixth aspects of the present invention, the non-steroidal anti-inflammatory .agent is indomethacin, oxyphenbutazone or flurbiprofen.
By other variants of the above fourth., fifth and sixth aspects of the present invention, the hyaluroniic acid fraction has an average molecular weight between 50,000 Daltons and 100,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between 500,000 Daltons and 730,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between :?50,000 and 350,000 Daltons; or the hyaluronic acid fraction has an average molecular weight between 30,000 and 730,000 Daltons.
By a seventh aspect of the present invention, the use is provided of a mixture of hyaluronic acid, or of a pharmaceutically-acceptable salt of hyaluronic acid, as described above, with a pharmaceutically-active substance which is not an ophthalmic drug for the preparation of a medic~unent for topical administration.
By a seventh aspect of t:he present invention, the use is provided of hyaluronic acid, as described above, as a delivery system for a drug for topical administration, with the proviso that the drug for topical administration is not an ophthalmic drug.
By an eighth aspect of the present invention, the use is provided of a pharmaceutically-acceptable salt of hyaluronic acid, as described above, as a delivery system for a drug for topical administration, with the proviso that the drug for topical administration is not an ophthalmic drug.
The advantages of therapy using the medicaments according to broad aspects of the present invention are due to a more efficient vehicle for the drugs which is promoted by the acidic polysaccharide of the hyaluronic acid component, and to a better bioavailability of the pharmaceutically-active substance as compared to that obtainable with known pharmaceutical formulations. Furthermore, the new medicaments of broad aspects of the present invention. assume particular importance in the case of ophthalmic B

~ 341 08 7 medicaments, because, due to the above mentioned qualities, there is an additional special compatibility with the corneal epithelium and, therefore, a very high level of tolerability, with no sensitization effects. ~~Vhen the medicaments of broad aspects of the present invention are administered in the form of concentrated solutions with elastic-viscose S characteristics or in solid form, it is possible to obtain films on the corneal epithelium which are homogeneous, stable, perfectly transparent, and which adhere well, guaranteeing prolonged lbioavailability of the drug, thereby forming excellent preparations with a retard effect.
One advantage of medicaments of broad aspects of the present invention is having perfected a new type of collyrium in which the above defects have been overcome. The use of hyaluronic acid as a vehicle for ophthalmic drugs allows for the formulation of excellent preparations free from concentration gradients of the pharmaceutically-active substance and, therefore;, perfectly homogenous, transparent and adhesive to the corneal epithelium, without sensitization effects, with e:KCellent vehicling of the active substance and possibly with a retard effect.
The above-mentioned properties of the medicaments of broad aspects of the present invention may, of course, be used also in other fields besides ophthalmology.
As already mentioned, they may be applied in dermatology and in diseases affecting the mucous membranes, e.g., in thc: mouth, for instance in odontology. They may also be used to obtain a systemic effect due to the effect of transcutaneous reabsorption, for instance in suppositories. All of these applications are possible both in human and veterinary medicine. in human medicine, the new medicaments of broad aspects of the present invention are particularly suitable for use in paediatrics.
The pharmaceutiically-active substance may first of all be categorized with respect to its use in various fields of therapy, starting with the distinction between human and veterinary medicine and then specifying the various sectors of application with respect to the organs or to the tisswes to be treated, e. g. , ophthalmology, dermatology, otorhinolaryngology, olbstetrics., angiology, neurology or any type of pathology of the internal organs which nnay be topically treated.. for example, rectal applications.
B

s According to some particular aspects of the present invention, the pharmaceutically-active substance is not an ophthalmic drug, and is exclusively suitable for topical use.
The pharmaceutical compositions of broad aspects of the present invention may thus be used in other fields besides ophthalmology. As already mentioned, they may be applied in dermatology and in diseases affecting the mucous membranes, e. g. , in the mouth, for instance, in odontology. They may also be used to obtain a systemic effect due to the effect of transcutaneous reabsorption, for instance, in suppositories. All of these applications are possible both in human and veterinary medicine. In human medicine, the medicaments of broad aspects of the present invention are particularly suitable for use in paediatrics.
According to other particular aspects of the present invention, the pharmaceutically-active substance is exclusively suitable for non-topical use, and may be, for example, for ophthalmic use. For the ophthalmic field, it may particularly be indicated, for examp',le, for its miotic, anti-inflammatory, wound healing and antimicrobial effects. The pharmaceutically-active substance in all aspects of the invention must be distvlct with respect to its effect and may therefore, for example, be used as an anaesthetic, an analgesic, a vasoconstrictor, an antibacterial, an antiviral, or an anti-inflammatory agent.
In dermatology, it is possible to use, a.s the pharmaceutically-active substance, mixtures of various antibiotics, e.g., erythromycin, gentamicin, neomycin, gramicidin, polymyxin B, or mixtures of such antibiotics with anti-inflammatory agents, for example, corticosteroids. For e~;ample, synergistic mixtures within ambits of various aspects of this invention may connprise: (a) hydrocortisone + neomycin; (b) hydrocortisone +
neomycin + polymyxin B -~- gramicidin; (c) dexamethasone + neomycin; (d) fluorometholone + neomycin; (e) prednisolone + neomycin; (f) triamcinolone +
neomycin + gramicidin + nystatin, or any other mixture used in conventional preparations for dermatology. The mixtures of various pharmaceutically-active substances are not, of course, limited to this field, but in each of the above-mentioned B

-- . ~3~~p87~

fields of medicine, it is possible to use mixtures similar to those already in use for the known pharmaceutical preparations of the art.
Examples of such pha~:~maceutically-active substances for use in ophthalmic medicaments to provide synergistic compositions according to other aspects of this invention are: basic and non-basic antibiotics, for example, aminoglucosidics, macrolides, tetracycline and peptides, e.g., gentamicin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacin, tobramycin, spectinomycin, erythromycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitebracycline, bacitracin, polymyxin B, gramicidin, c:olistin, chlor~~rnphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, or nystatin, and possibly their salts, e.g., sulphates or nitrates, or mixtures of the same or with other active principles, e. g. , those mentioned hereafter.
Other ophthalmic drugs which may be used to advantage to provide synergistic compositions according to other aspects of the present invention are: other anti-infective agents, e.g., diethylcarbamazine, or mebenda~:ole; sulphamides, e.g., sulphacetamide, sulphadiazine, or sulphisoxazole; antiviral anti anti-tumour agents, e. g. , idoxuridine, adeine arabinoside, trifluorothimidine, aciclovir, ethyldeoxyuridine, bromovinyldeoxyuridine, or 5-iodo-5'-amino-2',5'-dideoxyuridine; steroid anti-inflammatory agents, e. g. , dexamethasone, hydrocortisone prednisolone, fluorometholone, or medrison, and possibly their esters, for example, esters of phosphoric acid; non-steroid anti-inflammatory agents, e.g., indomethacin, oxyphenbutazone, or flurbiprofen; wound healers, e. g. , the epidermal growth factor EGF; local anaesthetics, e.g., benoxinate, o:r proparacaine and possibly their salts;
cholinergic agonist (promoter) drugs, e. g. , pilocarpine, metacholine, carbamylcholine, aceclidine, physostigmine, neostigmine, or demecarium and possibly their salts;
cholinergic antagonist drugs, e.g., atropine and its salts; adrenergic agonist (promoter) drugs, e.g., noradrenaline, adrf;naline, naphozoline, or methoxamine and possibly their salts; or adrenergic antagonist .drugs, e. g. , propranolol, timolol, pindolol, bupranolol, antenolol, metoprolol, oxyprenolol, practolol, butoxamine, sotalol, budarine, or labetalol and possibly their salts.
B

~3'~~ X87 to As noted above, the pharmaceutically-active substance may take the form of a synergistic mixture of two or more active substances. Examples of active substances to be used alone or in admixture between themselves or with other active principles in dermatology include: therapeutic agents, e. g. , anti-infective, antibiotic, antimicrobial, anti-inflammatory, cytostatic, cytotoxic, antivi.ral, anaesthetic agents, or prophylactic agents, e.g., sun shields, deodorants, antiseptics and disinfectants. Of the antibiotics, the following should be noted: erythromycin, bacitracin, gentamicin, neomycin, aureomycin, or gramicidin and their mixtures. ~Of the antibacterial and disinfectants, the following should be noted: nitrofurazone, mafenide, or chlorhexidine, and derivatives of 8-hydroxyquinoline and possibly their salts. Of the anti-inflammatory agents, the following should be noted: the corticosteroids, e.g., prednisolone, dexamethasone, fluoromethasone, clobetasol, triamcinolone acetonide, or betamethasone or their esters, e.g., valerianates, benz~oates, or dipropionates. Of the cytotoxics, the following should be noted: fluorocil, methotrexate, podophyllin. Of the anaesthetics, the following should be noted: dibucaine, lidocaine, or benzocaine.
The list is of course only for illustrative purposes and any other agent described in literature may be used.
Of the examples mentioned for ophthalmology and dermatology, it is possible to conclude, by analogy, v~hich medicaments, according to aspects of the present invention, are to be used in the above mentioned fields of medicine, for example, in otorhinolaryngology or odontology or in internal. medicine, for example in endocrinology, where it is possible to effect treatments with preparations for intradermic absorption or absorption through the mucous, for example, rectal or intranasal absorption, e.g., nasal sprays or inhalations in the oral cavity and in the pharynx.
These preparations may therefore, for example, be anti-inflammatory, or vasoconstricting or va~opressors, e.g., those already mentioned for ophthalmology, vitamins, antibiotics, e.g., those mentioned above, hormones, chemotherapeutics, antibacterials, etc., incl',uding those mentioned above for use in dermatology.
As noted above, the medicaments of broad aspects of this invention comprise as an essential component., hyaluronic acid, molecular weight fractions of hyaluronic acid, B

11 1 341 08 7 .
or various salts thereof as a drug delivery system. Hyaluronic acid (hereinafter sometimes referred to a "HY") :is a natural heteropolysaccharide which is composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. HY is present in pericellular gels, in the fundamental extracellular substance of connective tissues, in vertebrate organisms, in the synovial fluid of the joints, in the vitreous humor, in human umbilical tissue, in cocks' combs and in some bacteria. Its molecular weight is 8-13 million.
The first research carried out on HY was by Balazs (see U.S. Patent No.
4,141,973) who isolated. a HY fraction which was able to substitute for endobulbar fluids and was suitable for other therapeutic applications. Hyaluronic acid and its molecular weight fractions with lower molecular weights have, in fact, proved widely useful in medicine; a cosmetic use has also been suggested (see for example, Balazs et al, Cosmetics & Toiletries, Italian Edition No. ~~/84). It has especially been used as a therapeutic agent in therapies for arthropathies, e.g., in the veterinary field to cure arthritis in horses (Acts Vet. Scand. 167, 379) 1976).
Hyaluronic acid and its molecular fractions have been used in ophthalmic surgery as therapeutic, auxiliary and substitutive agents. for natural organs and tissues (see, for example, E. A. Balazs et al., Modern Problems in Ophthalmology, 10, 3 (1970), E. B.
Strieff, S. Karger, eds. Basel and Balazs et al., 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, 1979).
In published European Patent Application No. 0138572, filed on October 10, 1984, there was a description of a molecular fraction of hyaluronic acid which included those of molecular wei~;ht less than 30,000 Daltons that was to be used for intra-ocular and intra-articular injections, respectively, suitable for the substitution of the endobulbar fluids in the eye and for therapy of arthropathies.
In contrast to such disclosed therapeutic use or as a plastic auxiliary in surgery or in cosmetics, in the medicament of broad aspects of the present invention, hyaluronic acid or its molecular fractions, or salts thereof, are used in compositions as vehicles for B

,.-- 1 3 4 1 0 8 7 the administration of the pharmaceutically-activf: or pharmacologically-active substances for topical use only.
As a vehicle to be used in the composition of broad aspects of the present invention, hyaluronic acid of any origin may be used, e.g., the acids which are extracted from the above-mentioned natural starting materials, including cocks' combs.
The preparation of crude extracts of such acids is described in the literature.
Preferably, purified hyaluronic acids should be used. According to aspects of the present invention, in the place of integral hyaluronic acids which are obtained directly by extraction of the organic materials, it is possible to use fractions of hyaluronic acid with molecular weights which may vary greatly, having; a molecular weight of from 30,000 Daltons up to 13 million Daltons. Such fractions may be obtained by various procedures, e.g., by hydrolysing, oxidizing or enzymatic chemical agents, physical procedures, e.g., mechanical or by irradiation, anal, therefore, are often formed in the same purification procedures of the primary extracts (see for example, Balazs et al. , Cosmetics and Toiletries, cited above). The separation and purification of the fractions obtained is achieved, for example, by molecular filtration.
Of particular importance to be utilized as the vehicle in the compositions of broad aspects of the present invention are two purified fractions which may be obtained from hyaluronic acid, for ex~unple from cocks' combs, and known as HYALASTINETM and HYALECTINTM. The fraction known as HYALASTINETM has an average molecular weight of between 50,000 Daltor~s and 100,000 Daltons. HYALECTINTM has an average molecular weight of between :>00,000 Daltons and 730,000 Daltons. A combined fraction of these two firactions has also been isolated and characterized as having an average molecular weight of between 250,000 Daltons and 350,000 Daltons. This combined fraction may lie obtained with a yield of 80 % of total hyaluronic acid available from the particular startiing material, while the fraction HYALECTINTM may be obtained with a yield of 30 % and the fiaction HYALASTINETM with a yield of 50 % of the starting HY. (The preparation of these fractions is described in Examples 20-22, hereinafter) .
B

,. . 13 ~ 34 1 08 7 Thus, the preferred hyal.uronic acid to be utilized is a molecular weight fraction having a molecular weight broadly ranging from 30,000 Daltons to 13 million Daltons and preferably from 30,000 Daltons to 730,000 Daltons. Three preferred variants of hyaluronic fractions have a molecular weight of from 50,000 Daltons to 100,000 Daltons, or from 500,000 Daltons to 730,000 Daltons, or a combined fraction having a molecular weight of 250,000 Daltons to 350,000 Daltons,. These fractions are thus substantially-free of low molecular weight hyaluronic acid having a molecular weight of less than 30,000 Daltons, and, 'therefore, are substantially free of inflammatory side reactions when administered. (Further references hereinafter to hyaluronic acid or HY
are intended to include, where consistent with the particular context, both such hyaluronic acid and such molecular weight: fractions of hyaluronic acid).
According to broad aspects of the present invention, in place of hyaluronic acids and their molecular weight fractions as the vehicle of the medicaments, it is also possible to use their salts with inorganic bases, e.g., with alkali metals, (sodium, potassium, lithium), with alkaline earth metals (calcium, barium, strontium), with magnesium or with aluminum. These; salts may be stoichiometrically-neutral in the sense that all the acid functions are salified, or partial salts or acids, in which only a certain number of the acid functions are salified with the above mentioned metals. Such salts are easily obtained, for example, by reacting HY or the above mentioned fractions, with the basic calculated quantity. It i.s also possible to use mixed salts originating from different bases.
In addition to the above salts, it is also possible to utilize salts of HY
with compounds which can. broadly be considered ammonium or substituted ammonium (amines), for example, mono-, di-, tri- and tetra- alkylammonium, where the alkyl groups have preferably between 1 and 18 carbon atoms or arylalkyls with the same number of carbon atoms in the aliphatic portion and where aryl means a benzene residue, optionally substituted v~ith between 1 and 3 methyl, halogen or hydroxy groups. These ammonium or substituted ammonium salts of HY are formed by chemical reaction between hyaluronic acid and primary, secondary or tertiary amine moieties or ammonium hydroxide moieties of compounds or drugs having pharmaceutical activity, that is, with these moieties of the compounds which comprise the pharmaceutically-active component.

As with the above-discussed salts, these salts also may be stoichiometrically neutral wherein all of the acid functions are salified, or may be partial salts or acids, and may comprise mixed salts originating from different bases.
Hyaluronic acid or its molecular fractions as the vehicle may, therefore, be substituted by their salts with inorganic bases, e.g., with alkali metals (sodium, potassium, lithium), with alkaline earth metals, (calcium, barium, strontium), with magnesium, with aluminum, with ammonium or with substituted ammonium. This principle is also valid for the above-mentioned partial acid salts, in which all the acid groups present may be partially or totally neutralized with the above mentioned metals, or with ammonia or with amines, wherein the ammonium salts are formed by chemical reaction between hyaluronic acid and primary, secondary or tertiary amine moieties or ammonium hydroxide moieties of compounds or drugs having pharmaceutical or pharmacological activihr, i.e., the pharTnaceutically-active component.
There are various possibilities of realizing the medicaments in the form of the compositions according to broad aspects of the present invention including:
(a) using a neutral or acid pharmaceutically-active component mixed together with hyaluronic acid or a molecular weight fraction of hyaluronic acid, or their pharmaceutically-acceptable salts;
(b) using partial salts of HY with a basic pharmaceutically-active component leaving the residual acid groups of HY free or neutralized with the above-mentioned metals or bases;
(c) using stoichiometrically-neutral salts of HY with a basic pharmaceutically-active substance, possibly adding HY or one of its partial or total (neutral) metal salts;
(d) using stoichiometric.ally-neutral salts of HY with a basic pharmaceutically-active substance, adding further quantities of a pharmaceutically-active substance; and (e) using ad libitum mixtures of the salts or of the mixtures described hereinabove.
The present specification also teaches a process for the preparation of a salt of hyaluronic acid with a pharmaceutically-active substance, which process comprises combining an aqueous solution of a barium salt of hyaluronic acid with a sulphate of a B

pharmaceutically-active substance thereby to precipitate barium sulphate, separating the precipitated barium sulphate therefrom, thereby to obtain the salt of hyaluronic acid with the pharmaceutically-active substance in aqueous solution.
The sulphate may be added in an amount such that the number of sulphate 5 equivalents is equal to the number of hyaluronic acid equivalents, thereby producing a stoichiometrically-neutral hyaluronic acid salt; or the sulphate may be added in an amount such that the number of sulphate equivalents is less than the number of hyaluronic acid equivalents, thereby producing a partially-salified hyaluronic acid salt.
In such process, such barium salt may be further combined with a sulphate of at 10 least one member which is selected from the group consisting of an alkali metal, an alkaline earth metal, m.agnesiurn aluminum or ammonium. In such case, the sulphate may be added in an amount such that the number of sulphate equivalents is equal to the number of hyaluronic acid equivalents; or the sulphate may be added in an amount such the number of sulphate equivalents is less than the number of hyaluronic acid equivalents.
15 In any of the above variants of the process described above, the pharmaceutically-active substance is at least one member which is selected from the group consisting of erythromycin, gentamycin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacyn, tobramycin, aureomycin, spectinomycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitracin, polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin, amphotericin B, griseofulvin, nystatin, diethylcarbamazine, rnebenda~ol, sulphacet,~nide, sulphadiazine, sulphisoxazole, iododeoxyuridine, adeine arabinoside, tricarpine, metacholine, carbamylcholine, aceclidine, fisostigmin.e, neostigmine, demac;arium, stropina, propranolol, timolol, pindolol, bupranolol, a~tenolol, metoprolol, oxprenolol, practolol, butoxamine, sotalol, butadrine, labetalol, dexamethasone, triamcino:lone, prednisolone, fluorometholone, and medrysone.
The hyaluronic acid may be a molecular weight fraction having a molecular weight of between of up to 13 million Daltons., e.g., where the hyaluronic acid fraction is substantially-free of hyaluronic acid having a molecular weight less than 30,000 Daltons, namely, where the molecular weight fraction may have an average molecular B

,.-_ , 13 4 ~ 0 8 weight of between 50,000 Daltons and 100,000 Daltons, or of between 500,000 Daltons and 730,000 Daltons, or of between 250,000 Daltons and 350,000 Daltons.
The pharmaceutically-active substance is a substance which is suitable for topical administration, e.g., it may have ophthalmological or dermatological activity.
The present specification also teaches a process for the preparation of a barium salt of hyaluronic acid or of a molecular weight fraction of hyaluronic acid, the process comprising treating a cetylpyridinium salt of h.yaluronic acid or of a molecular weight fraction of hyaluronic acid with an aqueous solution of barium chloride, and separating the aqueous solution and adding ethanol thereto, thereby to precipitate the barium salt of hyaluronic acid or of a molecular weight fraction of hyaluronic acid. In this process, the hyaluronic acid fraction is substantially-free of hyaluronic acid having a molecular weight less than 30,000 Daltons, namely, where such :molecular weight fraction has an average molecular weight of between 50,000 Daltons and 100,000 Daltons, or of between 500,000 Daltons and .'30,000 Daltons, or of between 250,000 Daltons and 350,000 Daltons.
The barium salt thus provided is of a substantially-pure, non-inflammatory hyaluronic fraction having an average molecular weight of between 250,000 Daltons and 350,000 Daltons, or of between 50,000 Daltons and 100,000 Daltons or of between 500,000 Daltons and 730,000 Daltons and which is substantially-free of hyaluronic acid having molecular weig.'ht less than 30,000 Daltons, e.g., the barium salt of hyaluronic acid or of a molecular weight fraction of hyaluronic acid.
One particular form of medicament according to an aspect of the present invention is represented by synergistic mixtures of the pharmaceutically-active substance with hyaluronic acids or molecular fractions of hyaluronic acid when the pharmaceutically-active substance is of a basic nature for example, in the case of basic antibiotics. In this case, the hyaluronic acid and the pharmacewtically-active substance together form stoichiometrically-partial salts, or acid salts, in which an aliquot part of all the acid groups of the HY are salified with the basic groups of the pharmaceutically-active substance; or stoichionnetrically-neutral salts, in which all the groups of the HY are B

.~ 1341 087 salified; or mixtures of these neutral salts with a further quantity of the basic active pharmaceutically-active substance.
Therefore, for tlhe purpose of broad aspects of the present invention, if a basic pharmaceutically-active substance is used, it is possible to replace the synergistic mixtures of pharmaceutically-active substance and HY with the above mentioned acid salts or those which are; stoichiometrically-neutral, or, of course mixtures of such salts both with pharmaceutically-active substances and with HY components.
Mixtures of drugs between themselves and possibly with other agents may also be used as the active pharmaceutically-active substance in the medicament according to other aspects of the prcaent invention. If, in the place of only one pharmaceutically-active substance, mixt:u.res of pharmaceutically-active substances are used, e.g., those mentioned above, the salts of those substances and hyaluronic acid and its molecular weight fractions may be mixed salts of one or snore of such basic substances or possibly mixed salts of this type with a certain number of other acid groups of the HY
polysaccharide salified with the: above-mentioned metals or bases. For example, it is possible to prepare salts of hyaluronic acid or one of the molecular fractions HYALASTINF~.M or HYALECTINTM with a certain percentage of salified acid groups with the antibiotic ka~aamycin,, another percentage salified with the vasoconstrictor phenylephrine, while a remaining percentage acid groups are free or salified for example with sodium or another of the .above-mentioned metals. It is also possible to mix this type of mixed salt with other quantities of hyaluronic acid or its fractions or their metallic salts, as indicated above for the medicament containing salts of only one active substance with such hyaluronic acids.
It is, therefore, possible according to a particular aspect of the present invention to use the above mentioned salts, which are isolated and possibly purified to the solid anhydrous state, as an amorphous powder. When the powder comes into contact with the tissue to be treated, the powder forms a concentrated aqueous solution of a gelatinous character, of a viscous consistency, and with elastic properties. These qualities are also maintained at stronger dilutions and may therefore be used in place of the above mentioned anhydrous salts, solutions in water at various degrees of concentration or in s 1341487 .
is saline, possibly with the addition of other pharmaceutically-acceptable excipients or additives, e. g. , other mineral salts to regulate the pH and the osmotic pressure. It is also possible, of course, to u.se the salts to make gels, inserts, creams or ointments, in which there are other excipiemts or ingredients which .are used in conventional formulations of these pharmaceutical preparations. According to a particular aspect of the present invention, there is a preference for the medicaments as a composition containing hyaluronic acid, the molecular weight fractions thereof or their pharmaceutically-acceptable salts or their partial or neutral salts with the pharmaceutically-active component as the sole vehicle (with the possible exception of an aqueous solvent).
The quantitative ratios by weight of the two components to provide synergistic compositions according to aspects of the present invention may vary within ample limits and this naturally depends also on the nature of the two components and in the first case on that of the pharmacologically-active or pharmaceutically-active substance.
Such limits are, for example, the ratios of 0.01:1 and 100:1 between the two components.
The range of variation, however, is preferably between the limits of 0.01:1 and 10.1 for the two components and especially between 0.1:1 and 2:1.
The medicament, according to broad aspects of the present invention may be in solid form, for example, freeze-dried powders containing only the two components in mixture, or separately packed.
In solid form, such medicaments form., on contact with the epithelium to be treated, more or less concentrated solutions according to the nature of the particular epithelium with the same characteristics of the previously-prepared solutions in vitro which represent another particularly important aspect of the present invention. Such solutions are preferably in distilled water or sterile saline and contain preferably no other pharmaceutical vehicle besides hyaluronic acid or one of its salts. The concentrations of such solutions may also vary within ample lvnits, for example between 0.01 and 75 both for each of the two components taken separately, and for their mixtures or salts.
There is a particular preference for solutions of a pronounced elastic-viscous character, for example with a content of between 10 % arid 90 % of the medicament or of each of its components. Medicaments of this type are particularly important, both in an B

anhydrous form (freeze:-dried powders) or concentrated solutions or diluted in water or saline, possibly with tlae addition of additive or auxiliary substances, e.g., particular disinfectant substances or mineral salts acting as buffer or others for ophthalmic use.
Among the medicaments of aspects of tree present invention, the following should be chosen in particular, as the case may be, those with a degree of acidity suiting the place 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 hyaluronic acid with a basic active substance, may be effected by regulating, in a suitable manner, the quantities of polysaccharide, of its salts and of the basic substance itself.
Thus, for example, should the acidity of a hyaluronic acid salt with a basic substance be too high, the excess of the free acid groups with th.e above mentioned inorganic bases is neutralized, for example with sodium or potassium or ammonium hydrate.
The following formulations are exemplary of preparations according to aspects of the present invention.
Formulation 1 - A GEIL CONTAINING EGF OF WHICH 100 g CONTAIN:
- HY sodium salt (HYA.LASTINF~.M fraction), 55 g - HY sodium salt (HYA.LECTINTM fraction), 30 g - EGF 0.5 g - twice distilled water 23.5 g Formulation 2 - A 100 mg INSERT WITH PILOCARPINE NITRATE CONTAINING:
- HY sodium salt (HYALASTINETM fraction), 100 mg - Pilocarpine nitrate, 2 mg Formulation 3 - A POWDER FORM FOR TOPICAL APPLICATION CONTAINING
STREPTOMYCIN:
100 g of powder contain:
- HY sodium salt (HYALASTINF.rM fraction), 70 g - HY sodium salt (HYALECTINTM fraction), 28.5 g B

X341 Os~
- Streptomycin 1.5 g t Formulation 4 - A 100 mg INSERT WITH PILOCARPINE CONTAINING:
- mixed salt of h:yaluronic: acid with piloc:arpine and with sodium (see preparation 5 in Example 18, 100 mg) Formulation S - A COL,LYRIUM CONTAINING GENTAMYCIN AND
NAPHAZOLINE, OF 'JVHICH 100 ml CONTAINS:
- mixed salt of hyaluronic acid with gentamycin, with naphazoline and with 10 sodium (see preparation in Example 16), 2.910 g - propyl oxyben;aoate, 0.050 g - sodium phosphate, 1.500 g - distilled water., q.b.a. 100 ml 15 Formulation 6 - A C:OLLYRIUM WITH CHLORAMPHENICOL, NEOMYCIN, PHENYLERPHRINE, NITROFURAZONE, C>F WHICH 100 ml CONTAINS:
- mixed salt of hyaluronic acid with neomycin, with phenylephrine and with sodium (see preparation Example 17), 2.890 1;
- chloramphenicol, 0.500 g 20 - nitrofurazone, 0.02 g - distilled water, q.b.a. 100 ml Formulation 7 - A COLLYRIUM WITH DEXAMETASONE PHOSPHATE, KANAMYCIN E PHENYLEPHRINE, OF WHICH 100 ml CONTAINS:
- mixed salt of hyaluronic acid with kanamycin and phenylephrine (see preparation Example 15), 3.060 g dexametasone phosphate sodium salt, 0.100 g - methyl p-hydroxybenzoate, 0.060 g - distilled water, q.b.a. 100 ml.
B

1 3~1 48 7 PROCESS OF PRI?PARATION
The preparation of salts to provide compositions according to aspects of the present invention may be carried out in a known manner by bringing together solutions or suspensions in water or in organic solvents of the two components and possibly of bases or basic salts of the above: mentioned alk;~li metal, alkali earth metal, magnesium, aluminum, ammonium or amines, in calculated quantities and isolating the salts in an amorphous anhydrous form according to known techniques. It is possible, for example first to prepare aqueou s solution of the two components, freeing such components from aqueous solutions of their salts with acids of the metallic salts, respectively, for example, sulphates in the case of the pharmaceutically-active component and sodium salts in the case of the vehicle component for treatment with relative ionic exchangers, uniting the two solutions at a low temperature, for example, between 0'C. and 20'C. If the salt thus obtained is easily soluble in water it should be freeze-dried, while salts which are not easily soluble may be separated by centrifugation, filtration or decantation and possibly then desiccated.
The following examples are given merely as illustrative of the process of preparing certain salts of hyalu:ronic acid.
Example 1 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
STREPTOMYCIN
2.43 g of streptomycin sulphate (10 mEq) are solubilized in 25 ml of distilled H20. The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (I)OWEXTM 1 x 8) in the OH- form. The sulphate-free eluate is collected in a thermostatic container at 5°C. 4.0 g of the sodium salt of hyaluronic acid having; a molecular weight of 255,000 Daltons, used as one of the essential components e~f the medicament of an aspect of this invention, (corresponding to 10 mEq of a monomeric unit), are solubilized in 400 ml of distilled H20.
The solution is then eluted in a thermostatic column at 5 ° C, containing 151. ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The sodium-free eluate is collected under agitation in the solution is frozen and instantly freeze-dried. In the salt thus obtained, B

1 341 de ~

all of the acidic groups of hyaluronic acid are salified with the basic functions of streptomycin. Yield: .'i.5 g.
Microbiological determination on Bacillus subtilis ATCC 6633 compared to standard streptomycin shows a content of 33. 8 % by weight of streptomycin base, corresponding to the theoretically calculated weight.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. (Anal. :Biochem. 4, 330, 1962) shows a content by weight of HY acid of 66.2 % (theoretical percentage 66.0 % ) .
Example 2 - PR.EPAR.ATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
ERYTHROMYC IN
4.0 g of the sodium salt of hyaluronic acid with a molecular weight of 77,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 rnl of sulphonic resiin (DOWEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is kept at a temperature of 5°C. 7.34 g of erythromycin base (10 mEq) are added to the solution of HY under agitation at 5 ° C until complete solubilization is obtained. The resultin;; solution is frozen and freeze-dried. In the salt thus obtained, all of the acid groups o~F hyaluronic acid are salified with erythromycin.
Yield: 10.8 g.
Microbiological determination on staphylococcus aureus ATCC 6538p in comparison with standard erythromycin shows a content of 66.0 % by weight of erythromycin base, corresponding to the theoretical value. Colorimetric determination of the glucuronic acid combined in the polysaccharide according to the method of Bitter et al. shows a content of HY acid of 34.0 % by weight, corresponding to the theoretically calculated percentage.
B

23 ~ 3 4 1 0 8 7 Example 3 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
KANAM'YCIN
1.46 g of dikanaunycin sulphate ( 10 mF?q) are solubilized in 25 ml of distilled HZO. The solution is eluted in a thermostatic: column at 5°C, containing 15 ml of quaternary ammonium resin (DC1WEXTM 1 x 8) in the OH- form. The eluate, free from sulphates, is gathered in a thermostatic container at 5 ° C 4.0 g of the sodium salt of HY
having a molecular weight of 165,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled HZO. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The: eluate, free from sodium, is collected under vortex agitation in the solution of kanamycin base. The solution thus obtained is instantly frozen and freeze-dried. Yield: 4.8 g.
In the salt obtaiined, all the acid groups of HY are salified with kanamycin.
Microbiological determination on B. subtilis ATCC 6633 in comparison with standard kanamycin shows a content of 24.2 % by weight of kanamycin base, corresponding to the theoretically calculated percentage.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a content of HY acid of 75.8 %
by weight, also corresponding to the theorevtical content.
Example 4 - PREPAP;ATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
NEOMYCIN
1.52 g of neomycin sulphate ( 10 mEq) are solubilized in 20 ml of distilled HZO
and eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form. The; eluate, free from sulphates, is collected in a thermostatic container at 5"C. 4.0 g of H'Y sodium salt with a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled H20 and eluted in a thermostatic column at 5°C
containing 15 ml of B

..... , l~~~t087 sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is gathered under agitation in the solution of neomycin base. The viscoelastic precipitate which forms is separated by decantation and frt:eze-dried. Yield: 4.76 g.
In the resulting salt, all of the HY acid groups are salified with neomycin.
Quantitative microbiological determination carried out on S. aureus ATCC 6538p compared to standard neomycin shows a content by weight of 21.2 % of neomycin base, corresponding to the thc;oretically calculated value.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows ,~ HY acid content of 78.8 % by weight.
Example 5 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
GENTAr~IYCIN
1.45 g of gentamycin sulphate ( 10 mEq) are solubilized in 25 ml of distilled H20.
The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form. The eluate, free from sulphates is collected in a thermostatic container at 5°C. 4.0 g of the sodium salt of HY with a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at .5°C, containing 15 ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The elua.te, free from sodium, is collected under agitation in a vortex in the solution of gentamycin base. The thick and very viscous precipitate which forms is separated by decantatio:n and freeze-dried. Yield: 4.65 g.
In the salt thus obtained, all the HY acid groups are salified with gentamycin.
Quantitative microbiological determination carried out on S. epidermidus ATCC

compared to standard g;entamycin shows a corntent by weight of 20.0 % of gentamycin base, corresponding to the theoretical content.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 80.0 % .
B

w ~ 1 341 08 7 Example 6 - PREPAR:ATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
AMIKACIN
1.47 g of amikacin base ( 10 mEq) are solubilized in 100 ml of distilled HZO
at 500°C. 4.0 g of the sodium salt of HY with a molecular weight of 170,000 Daltons, 5 used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 m~Eq of a :monomeric unit, are solubilized in 4000 ml of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWI:XTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected under agitation in a vortex in the solution of amikacin base. The thick and 10 extremely viscous precipitate which forms is separated by decantation and freeze-dried.
Yield: 5.16 g.
In the salt thus obtained, all the HY acid groups are salified with amikacin.
Quantitative microbiological determination carried out on S. aureus ATCC 29737 in comparison to standard amikacin shows a content of 27.7 % by weight in amikacin base, 15 corresponding to the theoretical content.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 72. 3 % by weight.
Example 7 - PREPARATION OF THE SAL7.' OF HYALURONIC ACID (HY) WITH
20 ROLITE;TRACYCLINE
4.0 g of HY sodium salt: having a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 m.Eq of a monomeric unit, are solubilized in 400 ml of distilled HZO. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of 25 sulphonic resin (DOWIEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is kept at a temperature of 5°C. 5.3 g of rolitetracycline base (10 mEq) are added to the solution of HY acid under agitation at 5°C away from the light, until complete solubilization has been achieved. The solution thus obtained is instantly frozen and freeze-dried. Yield: 8.9 g.
B

~34~ X87 In the salt thus olbtained, all the HY acid groups are salified with rolitetracycline.
Microbiological determiination on B. pumilus ATCC 14884 in comparison to standard rolitetracycline shows a content of 58.2 % by weight of rolitetracycline base, corresponding to the theoretical value. Colorimtaric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY
acid content of 41. 8 % by weight.
Example 8 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
POLYM'i'XIN B
2.4 g of polymy:Kin B base ( 10 mEq) are suspended in 100 ml of distilled H20 at 5°C. 4.0 g of HY sodium salt with a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mlEq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWE;XTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected under vigorous agitation in the suspension of polymyxin base at 5°C. After an initial phase during whiich the solution becomes clear, there is a progressive formation of a difficultly soluble product which is completely precipitated by 5 volumes of acetone.
The precipitate is filtered, washed with acetone and then vacuum dried. Yield:
6.05 g.
In the salt thus .obtained, all of HY acid groups are salified with polymyxin B.
Quantitative microbiological determination carried out on B. bronchiseptica in comparison to standard polymyxin B shows a content of 38.7 % by weight of polymyxin B. base, corresponding to the theoretical value.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 61.3 % .
Example 9 - PREPAP;ATION OF THE SALT OF HYALURONIC ACID (HY) WITH
GRAMICIDIN S
6.7 g of gramicidin S hydrochloride salt ( 10 mEq) are suspended in 200 ml of ethanol/H20 (80:20). The solution is then eluted in a thermostatic column at 5°C, B

.._ .

containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form.
4.0 g of the sodium salt of HY with a molecular weight of 165,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 m:Eq of a monomeric unit, are solubilized in 400 ml of distilled HZO. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form.
200 ml of dimethyl sulphoxide (DMSO) are added to the eluate, free from sodium, and the mixture is kept under agitation at 5°C. The solution of gramicidin base is then slowly added. 'The resulting solution is precipitated by 10 volumes of acetone.
The precipitate is filtered, washed with acetont: and vacuum dried. Yield:
9.55 g.
In the salt thus obtained, all the HY acid groups are salified with gramicidin S.
Quantitative microbiological determination carried out on S. faecium ATCC
10541 in comparison to standard gramicidin S shows a content of 60.0 % by weight of gramicidin S base, corresponding to the theoretical value.
Colorimetric determination of the gluc.uronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 40.0 % .
Example 10 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
NAPHAZOLINE
Pure naphazoline base is prepared as follows: 4.94 g of naphazoline-HC1 (20 mM) are solubilized in 100 m1 of distilled H20 at 5°C. 20 ml of NH40H
(SM) are added and extracted twice with 100 ml of ethyl acetate. The organic layers are extracted twice with 50 ml of H2~0, mixed together again and anhydrified with anhydrous NaZS04.
The solution is concentrated at 50 ml and then placed in a freezer to crystallize. The crystallized product is Filtered, washed with ethyl acetate and vacuum dried.
Yield: 4.0 g of pure naphazoline base.
4.0 g of the H'~ sodium salt with a molecular weight of 625,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mlEq of a monomeric unit, are solubilized in 400 ml of distilled H20 and eluted in a thernnostatic column at 5°C, containing 15 ml of sulphonic resin B

~~ 1 28 (DOWEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is kept at a temperature of 5°C. 2.1 g of naphazoline base (10 mEq) are added to the solution of HY acid and the mixture; is agitated at 5°C until complete solubilization is achieved. The resulting mixture is instantly frozen and freeze--dried. Yield: 5.72 g.
In the salt thus obtained, all the HY acid groups are salified with naphazoline.
Quantitative spectrophotometric determination, carried out in comparison to a naphazoline standard (CUSP) shows a content of 35.7 % weight of naphazoline base, corresponding to the theoretical value.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 64.3 % .
Example 11 - PR.EPAR:ATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
PHENYLEPHRI:iVE
2.04 g of L-phe;nylephrine-HC 1 ( 10 mEq) are solubilized in 25 ml of distilled H20. The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form. The eluate, free from chlorides, is collected i.n a thermostatic container at 5°C. 4.0 g of a HY sodium salt having a molecular weight of 820,000 Daltons, used as one of the essential components of the medicament of an aspect of this invf:ntion, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled HZO. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected under agitation in the solution of phenylephrine base. The resulting; mixture is instantly frozen and freeze-dried. Yield: 5.25 g.
In the salt thus obtained, all the HY acid groups are salified with phenylephrine.
U.V. spectxophotometric determination using the standard addition method (USP) shows a content of 30. 6 % by weight of phenylephrine base, corresponding to the theoretical content.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 69.4 % .
B

. ,._ Example 12 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
ATROPINE
4.0 g of HY sodium salt having a molecular weight of 1,300,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled HZO. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWE,XTM 50 x 8) in the H+ form. The eluate, free from sodium, is kept at a temperature of 5 °C. 2.89 g of atropine, base ( 10 mEq) are added to the solution of HY acid and the mixture is agitated at 5°C. The resulting mixture is frozen and freeze-dried. Yield: 6.5 g.
In the salt thus obtained, all the hyaluronic acid groups are salified with atropine.
Quantitative gas chromatography determination (USP) carried out in comparison to standard atropine shows a content of 43.3 % in atropine base, corresponding to the theoretical value.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 56.7 % .
Example 13 - PR.EPARATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
PILOCARPINE
2.45 g of pilocaipine hydrochloride (lO:mEq) are solubilized in 50 ml of distilled HZO. The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form. The eluate, free from chlorides, is collected in a thermostatic container at 5 ° C. 4.0 g of HY sodium salt with a molecular weight of J170,000 Daltons, used a.s one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 m.l of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected under agitation in the solution of pilocarpine base. The solution thus obtained is instantly frozen and freeze-dried.
Yield: 5.25 g.
B

~34~~g7 In the salt thus obtained, all the HY acid groups are salified with pilocarpine.
Spectrophotometric dete:rminatian according to the USP carried out in comparison to a pilocarpine standard shows a content of 35.1 % by weight of pilocarpine base, corresponding to the theoretical value.
5 Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 64.6 % .
Example 14 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
NEOMYCIN AND WITH POL'~'MYXIN
10 4.0 g of HY sodium salt having a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mlEq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is eluted i:n a thermostatic column at S°C, containing 15 ml of sulphonic resin (DOWF?XTM 50 x 8) in the H+ form. The eluate, free from sodium is 15 collected in a thermostatic contaiiner at 5°C. 0.150 g of polymyxin B base (0.63 mEq) are added under vigorous agitation. 1.425 g of neomycin sulphate (9.37 mEq) are solubilized in 25 ml of distilled H20. The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH' form. The eluate, free from sulphates, is collected under vigorous agitation in the 20 solution of HY acid and polymyxin B. The precipitate which forms is separated by centrifugation and vacuum dried; there is no loss of the product in the residual solution.
Yield: 4.85 g.
17.25 mg of this product contains:
Neomycin equal to 5.0 mg of neomycin sulphate;
25 Polymyxin equal to 0.63 mg (5000 UI) of polymyxin sulphate.
These determinations were carried out after separation by HPLC (high pressure liquid chromatography) of the two active principles.
Example 15 - PREPAF;ATION OF THE MIXED SALT OF HYALURONIC ACID
30 (HY) WIfTH KA:~VAMYCIN AND WITH PHENYLEPHRINE
B

w 31 ~ 34 1 08 7 4.0 g of HY sodium salt having a molecular weight of 65,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is tlhen eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWI:XTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected in a thermostatic container at 5°C. 0.85 g of kanamycin sulphate (5.82 mEq) are solubilized in 10 ml of distilled HZO. The solution is eluted in a thermostatic column at 5°C, containing 10 rnl of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH' form.
The eluate, free from sulphates, is collected in a container kept at a temperature of 5°C. The phenylephrine base is prepared by dissolving phenylephrine hydrochloride in distilled H20 at 5 ° C at 100 mg/ml, and NH40H (6N) is added until complete precipitation is achieved. The precipitate is separated by filtration, washed with distilled H20 until the chlorides have disappeared from the washing water, and then vacuum dried. The HY acid at~d kanarrtycin base solutions are mixed and kept at a temperature of 5°C 699 mg of phenylephrine base (4.18 mEq) are added under agitation until being completely dissolved. The resulting solution is frozen and freeze-dried.
Yield: 5.1 g.
Microbiological determination on B. subtilis ATCC 6633 in comparison to standard kanamycin shows a content of 13.55 % by weight of kanamycin base. U.
V .
spectrophotometric determination using the standard addition method (USP) shows a content of 13.45 % by 'weight of phenylephrine base.
Colorimetric dexermination of the gluc:uronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 73.0 % .
Example 16 - PREPARATION OF MIXED SALT OF HYALURONIC ACID (HY) WITH C?ENTAMYCIN, WITH NAPHAZOLINE AND WITH SODIUM
4.0 g of HY sodium salt: with a molecular weight of 50,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 rr.~Eq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of B

sulphonic resin (DOWE.XTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected in a thermostatic container at 5°C. 1.2,45 g of gentamycin sulphate (8.59 mEq) are solubilized in 25 ml of distilled H20. The solution is eluted in a thermostatic column at 5 °c, containing 12 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH-form.
The eluate, free from sulphates, is collected in a container kept at a temperature of 5°C. The pure naphazoline base is prepared with naphazoline hydrochloride dissolved in distilled H20 at 5°C .at a concentration of 50 mg/ml, NH40H (SM) is added until pH
12 is achieved and the solution is extracted twice with ethyl acetate. The organic layers are washed with H20 and anhydrified on anhydrous Na2S04. The product is placed in a freezer to crystallize, and the: precipitate is filtered, washed with ethyl acetate and vacuum dried. 2.5 g o:F HY sodium salt and 0.297 g of naphazoline base are added to the HY acid (1.41 mEq) and agitated until being completely solubilized. The solution of gentamycin base is then added, homogenized and then frozen and freeze-dried. Yield:

7.35 g.
Quantitative mic;robiolog;ical determination on B. epidermidus ATCC 12228 in comparison to a gentam;ycin standard shows a content of 11.1 % by weight of gentamycin base. Quantitative spectrophotometric determination carried out in comparison to standard naphazoline (LISP) shows a content of 4.0% by weight of naphazoline base.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bittf:r et al. shows a HY acid content of 83.0 % .
Example 17 - PREPARATION OF THE MIXED SALT OF HYALURONIC ACID
(HY) WITH NF?OMYCIN, WITH PHENYLEPHRINE AND WITH
SODIUM
4.0 g of HY sodium salt: having a molecular weight of 65,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 m:Eq of a monomeric unit, are solubilized in 400 ml of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 15 ml of sulphonic resin (DOWI:XTM 50 x 8) in the H+ form. The eluate, free from sodium, is B

_. 33 1 34i 48 7 collected in a thermostatic container at 5°C. 11.28 g of neomycin sulphate (8.42 mEq) are solubilized in 25 ml of distilled HZO. The solution is eluted in a thermostatic column at 5°C, containing 12 rnl of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH-form.
The eluate, free from sulphates, is collected in a container kept at a temperature of 5°C. The phenylepluine base is prepared by dissolving phenylephrine hydrochloride in distilled H20 at 5 "C at 100 mg/ml, anti adding NH40H (6N) until complete precipitation is achieved. The precipitate is separated by filtration, washed with distilled H20 until the chlorides have disappeared from the washing water, and then it is vacuum dried.
2.5 g of HY sodium salt and 0.266 g of phenylephrine base ( 1.58 mEq) are added to a solution of HY ac:id and agitated until being completely solubilized. The solution of neomycin base is then added. and after homogenization it is frozen and freeze-dried.
Yield: 7.35 g.
Spectrophotometric determination by LJ. V . using the standard addition method (USP) shows a content of 3..57 % by weight of phenylephrine base. Quantitative microbiological determination on B.aureus AT'CC 6538p in comparison to a neomycin standard shows a content of 11.64 % by weight of neomycin base.
Colorimetric determination of the glucuronic acid combined in polysaccharide according to the method of Bitter et al. shows a HY acid content of 82. 8 % .
Example 18 - PREPARATION OF THE SALT OF HYALURONIC ACID (HY) WITH
PILOCA,RPINE AND WITH SODIUM
98.31 g of HY sodium salt having a molecular weight of 170,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 245 mEq of a monomeric unit, are solubilized in 8.5 litres of distilled HZO.
The solution is then eluted in a thermostatic column at S~C, containing 300 ml of sulphonic resin (DOWIEXTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected in a thermostatic container at 5°C.
B

~_ ~ ~ ~~+~ p8 ~ _ 2.34 g of pilocarpine hydrochloride (9.6 mEq) are solubilized in 50 ml of distilled H20. The solution is eluted in a thermostatic column at 5°C, containing 15 ml of quaternary ammonium resin (DOWEXTM 1 x 8) in the OH- form.
The eluate, free from chlorides, is collected under agitation in the solution of HY
acid. 235.4 ml of a solution of sodium hydroxide (1M) are slowly added under agitation.
T'he solution thus obtained is instantly frozen and freeze-dried. Yield: 99.8 g.
100 mg of the product contains 2 mg oi.-' pilocarpine as a base.
Example 19 - PREPARATION OF THE SALT' OF HYALURONIC ACID (HY) WITH
STR.EPT~OMYCI:~V AND WITH SODIUM
98.68 g of HY sodium salt having a molecular weight of 255,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 246 m.Eq of a monomeric unit, are solubilized in 8.5 litres of distilled H20. The solution is then eluted in a thermostatic column at 5°C, containing 300 ml of sulphonic resin (DOWF:XTM 50 x 8) in the H+ form. The eluate, free from sodium, is collected in a thermostatic container at 5 ° C .
1.88 g of streptomicin sulphate (7.74 mEq) are solubilized in 20 ml of distilled HZO. The solution is eluted in a thermostatic column at 5°C, containing 12 ml of quaternary ammonium :resin (DOWEXTM 1 x 8) in the OH' form.
The eluate, free from sulphates, is collected under agitation in the solution of HY
acid. 238.3 ml of a solution of NaOH (1 M) are slowly added under agitation and the resulting solution is instantly frozen and freeze-dried. Yield: 99.8 g.
100 g of the product contains 1.5 g of streptomicin as a base.
Example 20 - PROCESS TO OBTAIN A MIXTURE OF THE FRACTIONS
HYALA~STINE AND HYALECTIN WITH NO INFLAMMATORY
ACTIVITY
Fresh or frozen cocks' combs (3000 g ) are minced in a meat mincer and then carefully homogenized in a mechanical homo~;enizer. The paste thus obtained is then treated in a stainless steel container (AISI 316) ~or in glass with 10 volumes of anhydrous B

1 34~ ~g ~
acetone. The whole is then agitated for 6 hours at a speed of 50 rpm. It is left to separate for 12 hours after which the acetone i.s discarded by siphoning. The acetone extraction is repeated until the discarded acetone reaches the right degree of humidity (Karl-Fischer method). The whole is then centrifuged and vacuum dried at a suitable 5 temperature for 5-8 hours. 500-600 g of dry powder of cocks' combs are thus obtained.
300 g of dry powder are c;xposed to enzymatic digestion with papain (0.2 g) under aqueous conditions and buffered with phosphate buffer in the presence of a suitable quantity of cysteine hyclrochlori~de.
The resultant is agitated for 24 hours at 60 rpm, keeping the temperature at 10 65°C. It is then cooled at 2_'i°C and CELI'TF~.M (60 g) is added, maintaining the agitation for another hour. The; mixture thus obtained is filtered until a clear liquid is obtained. The clear liquid then undergoes molecular ultrafiltration using membranes with a molecular weight exclusion limit of 30,000 Da.ltons, in order to retain on the membrane those molecules with a molecul;~r weight greatc;r than 30,000 Daltons.
15 The product is ultrafiltered from 5 to 6 original volumes, adding distilled water continually to the product during the ultrafiltration procedure. The addition of water is discontinued and the ult:rafiltration is continued until the volume is reduced to 1/3 of the original volume. The residual liquid is rendered O.1M by the addition of sodium chloride and the temperature is brought to 50''C. Under agitation at 60 rpm, 45 g of 20 cetylpyridine chloride are added. The solution is agitated for 60 minutes and then 50 g of CELITF~.M are added. Under agitation, the temperature of the whole is brought to 25°C and the precipitate which is formed by centrifugation is collected. The precipitate obtained is suspended in a O.O1M solution in sodium chloride (5 litres) containing 0.05%
of cetylpyridinium chloride. The resulting suspension is agitated for 60 minutes at 50°C;
25 the temperature is then brought to 25°C and thc: precipitate is centrifuged. The washing operation is repeated 3 Mimes after which the precipitate is collected in a container having 3 litres of a 0.05M solution of sodium chloride containing 0.05 % of cetylpyridine chloride. It is agitated at 60 rpm for 60 minutes and the temperature is kept constant at 25°C for two hours. T'he supernatant is eliminated by centrifugation.
The procedure is 30 repeated several times with solutions of O.1M sodium chloride containing 0.05% of B

~._ ~ 341 08 7 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 collected. Extraction is repeated 3 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 while under constant agitation. The liquid is then brought to 0.23M with sodium chloride. 1 g of cetylpyridinium chloride ins added, and the liquid is kept under agitation for 12 hours. The mixture is cooled to 25°C and then it is filtered first on a CELITF.~.M pack and then through a filter. It then undergoes molecular ultrafiltration again, on a membrane with a molecular weil;ht exclusion limit of 30,000 Daltons, 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 collected by centrifugation and the supernatant is discarded. The precipitate is dissolved in 1 litre of a O.1M
solution of sodium chloride and the precipitation is repeated with 3 volumes of ethanol (95%). The precipitate is collected and washed first with ethanol (75 % ) 3 times, and then with absolute ethanol (3 times), and finally with absolute acetone (3 times). The product thus obtained (HYALASTITIETM + EIYALECTINTr,, fractions), used as one of the essential components of the medicament of an aspect of this invention, has an average molecular weight of between 250,000 Daltons and 350,000 Daltons. The HY yield is equal to 0.6 % by weight of the original fresh tissue.
Example 21 - PROCESS TO OI3TAIN THE FRACTION HYALASTINF~.M FROM THE
MIXTURE OBTAINED BY THE PROCESS DESCRIBED IN
EXAMPLE 20.
The mixture obtained by the method described in Example 20 is dissolved in twice distilled, apyrogenetic water at the rate of 10 mg of product to each 1 ml of water. The B

~ 341 08 7 solution obtained is exposed to molecular filtration through filter membranes with a molecular weight exclusion limit of 200,000 Daltons, following a concentration technique on the membrane without the addition of water. During the process of ultrafiltration through membranes with a molecular weight exclusion limit of 200,000 Daltons, the molecules with a molecular weight of more than 200,000 Daltons 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 Daltons. The product is ultrafiltered until the volume on top of the membrane is reduced to 10 % of the initial volume. Two volumes of apyrogenetic, twice distilled water are added and the solution is then ultrafiltered again until the volume is reduced to 1/3. The operation is repeated two :more times. The solution passed through the membrane is broul;ht to O.1M with sodium chloride and then precipitated with 4 volumes of ethanol at 95 % . The precipitate is washed 3 times with ethanol (75 % ) and then vacuum dried.
The product thus obtained (HYALASTINETM fraction), used as one of the essential components of the medicament of an aspect of this invention, has an average molecular weight of bcaween 50,000 Daltons and 100,000 Daltons. The HY yield is equal to 0.4 % by weight of the original fresh tissue.
Example 22 - PROCESS TO OBTAIN THE FRACTION HYALECTINTM
The concentrated solution collected in the container on top of the ultrafiltration membrane with a molecular weight exclusion of 200,000 Daltons, as in Example 21, is diluted with water until. a solution containing 5 mg/ml of hyaluronic acid is obtained, as determined by quantitative analyses based on the dosage of glucuronic acid.
The solution is brought to O.1M in an aqueous sodium chloride solution and then precipitated with 4 volumes of ethanol at ~~5 % . Tihe precipitate is washed 3 times with ethanol (75 %) and then vacuum dried.
The product thus obtained (HYALECTINTM fraction), used as one of the essential components of the medicament of an aspect of this invention, has a molecular weight of B

~34~ ~8 7 between 500,000 Daltons and 730,000 Daltons. This corresponds to a specific fraction of hyaluronic acid with a defined length of molecular chain of 2,500 to 3,500 saccharide units with a high degree; of purity.
The HY yield is equal to 0.2 % by weight of the original fresh tissue.
The invention also provides a new process for the preparation of hyaluronic acid salts, used as one of the essential components of the medicament of an aspect of this invention, starting with hyaluronic acid barium salt. The new process provides the salts which are soluble in water, in which all the carboxylic groups of hyaluronic acid may be salified or only a part of the groups are salified. In the partial salts, the remaining carboxylic groups of hyaluronic acid may be free or salified with alkali metals, alkaline earth metals, magnesium, aluminum, ammonia, or substituted ammonium (amines).
The new process consists of preparing an aqueous solution of the barium salt of a hyaluronic acid, and adding an. aqueous solution containing a number of sulphuric acid equivalents, totally or partially salified by one or more organic or inorganic bases;
wherein the number of sulphuric equivalents corresponds to the number of hyaluronic acid equivalents present in the barium salt aqueous solution. The aqueous solution of hyaluronic acid salt is obtained by filtration of the separated barium sulphate. That is, by filtration of the separated barium sulphate, it is possible to obtain the aqueous solution of hyaluronic acid salt from which the salt in its dry form is obtainable by concentration.
The barium salt of hyaluronic acid, used as one of the essential components of the medicament of an aspect of this invention, is not: described in literature and, surprisingly, has proved to be soluble in wager. It can be easily prepared by treating the not-very-soluble hyaluronate of c;etylpyridinium with an aqueous solution of barium chloride and precipitating from the solution the hyaluronate of barium with ethanol or another suitable solvent. The hyaluronate of cet.ylpyridinium is an intermediate which is commonly used in production procedures of hyaluronic acid to separate and purify the hyaluronic acid which has been extracted from various organic materials.
The aqueous solution, containing a number of sulphuric acid equivalents, totally or partially salified witlh one or more organic bases, is prepared by dissolving in water the neutral sulphates of the bases and possibly adding sulphuric acid. Should there be B

.._ a solution formed of neutral sulphates of one or more organic or inorganic bases, containing a number of sulphuric equivalents corresponding to the number of hyaluronic acid equivalents present: in the aqueous solution of barium salt, the end result will be a stoichiometrically neutral salt of hyaluronic acid with the bases present in the corresponding aqueous solution (;sulphates). If a stoichiometrically partial salt or acid salt of hyaluronic acid be dc;sired, sulphuric acid should be added to the aqueous solution of sulphates, or basic acid sulphates should be used.
This process is illustrated by the following examples.
Example 23 - PROCESS OF OBTAINING A MIXTURE OF FRACTIONS
HYALASTINE,.~,, AND HYALF?CTINTM IN THE FORM OF BARIUM
SALTS AND WITHOUT ANY INFLAMMATORY ACTIVITY
Fresh or frozen cocks' combs (3000 g) are minced in a meat mincer and then carefully homogenized in a mechanical homog~enizer. The paste obtained is treated in a stainless steel container (AISI 316) or in glass with 10 volumes of anhydrous acetone.
The whole is agitated for 6 hours at a speed of _'~0 rpm. It is left to separate for 12 hours and the acetone is disca.:rded by syphoning. The acetone extraction is contained until the discarded acetone has reached the right degree of humidity (Karl-Fischer method) . The whole is then centrifugc;d 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 g of dry pov~rder are exposed to enzymatic digestion with papain (0.2 g) under aqueous conditions and buffered in a phosphai:e buffer in the presence of a suitable a quantity of cysteine hydrochloride. The resultant is agitated for 24 hours at 60 rpm, keeping the temperature constant at 60-65°C. The whole is then cooled to 25°C and CELITE,.M (60 g) is added, while agitation is continued for another hour. The mixture is filtered until a clear liquid is obtained. The clear liquid undergoes molecular ultrafiltration using membranes with a molecular weight exclusion limit of 30,000 Daltons. Between 5 and 6 original volumes of the product are ultrafiltered, continuously adding distilled water to the ultrafiltered produca. The addition of water is discontinued B

and ultrafiltration is continued until the volume has been reduced to 1/3 of the original volume.
The residual liquid is brought to O.1M with the addition of barium chloride and the temperature is brought to 50°C. While agitating at 60 rpm, 45 g of cetylpyridinium chloride are added. The solution is agitated for 60 minutes and then 50 g of CELITE,.M
are added. While agitating, thE: temperature of the whole is brought to 25°C and the precipitate which is formed by centrifugation is collected. The precipitate is suspended in a O.O1M solution in barium chloride (5 litres) containing 0.05 % of cetylpyridinium chloride. It is agitated ;For 60 minutes at 50°C; the temperature is then brought to 25°C
and the precipitate is centrifuged. The washing process is repeated 3 more times and finally the precipitate is collected in a receptacle containing 3 litres of a 0.05M solution of barium chloride containing 0.05 % of ce~tylpyridinium chloride. The resulting suspension is agitated at 60 rpm for 60 minutes and the temperature is kept constant at 25°C for two hours. T'he clear supernatant is eliminated by centrifugation.
The process is repeated several times with a solution of O.1M barium chloride containing 0.05 % of c.etylpyridinium chloride. The mixture is centrifuged and the supernatant is discarded. T'he precipitate is dispersed in a 0.30M solution of barium chloride containing 0.0-'i% of cetylpyridinium chloride (3 litres). The mixture is agitated and both the precipitate and the clear liquid are gathered. The precipitate undergoes extraction 3 more time<.~, each time using 0.5 litre of the same aqueous solution.
Finally, the residue precipitate is eliminated and the clear liquids are pooled in one container. The temperature of the liquid is brought to 50°C under constant agitation.
The liquid is then brought to ~0.23M with barium chloride. 1 g of cetylpyridinium chloride is added, and agitation is maintained for 12 hours. The mixture is cooled to 25°C, filtered first wiith CELITETM and then through a filter. It then undergoes molecular ultrafiltration once more on membranes with a molecular exclusion limit of 30,000 Daltons, ultrafiltering three initial volumes with the addition of 0.33M barium chloride solution. The addition of barium chloride solution is suspended and the volume is reduced to 1/4 of the; original. The solution concentrated in this way is precipitated under agitation (60 rpm) at 25 ° C with 3 volumes of ethanol (95 % ) .
The precipitate is B

~ X41 08 7 gathered by centrifugation and the supernatant i;s discarded. The precipitate is dissolved in 1 litre of O.1M sohition of barium chloride and precipitation is repeated with 3 volumes of ethanol (95'~ ).
The precipitate is collected and washed first three times with 75 % ethanol, then with absolute ethanol (3 times), ;end finally with absolute acetone (3 times).
The product thus obtained (fractions HYAL.ASTINETM + HYALECTINTM), used as one of the essential components of the medicament of an aspect of this invention, has an average molecular weight between 250,000 Daltons and 350,000 Daltons. The yield of HY
corresponds to 0. 6 % of the original fresh tissue.
Example 24 - PROCESS OF OBTAINING THI: FRACTION HYALASTINF~.M IN THE
FORM OF BARIUM SALT OF THE MIXTURE OBTAINED BY THE

The mixture obtained with the process described in Example 23 is dissolved in apyrogenic distilled water at a .quantity of 10 mg of product per 1 ml of water. The solution thus obtained its subjected to molecular filtration through a membrane with a molecular exclusion limit of (.00,000 Daltons, following a concentration technique without the addition of water on top of the membrane. During the ultrafiltration process through membranes with a molecular exclusion limit of 200,000 Daltons, the molecules with a molecular weight of more than 200,000 Daltons are detained, while the smaller molecules pass through the membrane together with the water. During the filtration process, no water is added on top of the membrane, so that the volume diminishes and consequently the concentration of molecules with a molecular weight of more than 200,000 Daltons is increased. i:~ltrafiltration is maintained until the volume on top of the membrane is reduced to 10 % of the initial volurne. Two volumes of apyrogenic distilled water are added and thf: whole is ultrafiltered again until the volume is reduced to 1/3.
The operation is repeated twice :more. The solution which passes through the membrane is brought to O.1M with barium chloride and is then precipitated with 4 volumes of 95 ethanol. The precipitate is washed 3 times with 75 % ethanol and then vacuum dried.
The product thus obtaiined (HYALASTINETM fraction), used as one of the essential B

~.. 42 1 341 ~8 ~
components of the medicament of an aspect of this invention, has an average molecular weight of between 50,000 Daltons and 100,00() Daltons. The yield of HY is equal to 0.4 % of fresh starting tissue.
Example 25 - PROCESS OF OBTAINING HYALECTINTM FRACTION IN THE
FORM O~F BARIUM SALT
The concentrated solution gathered in th.e receptacle on top of the ultrafiltration membrane with a molecular exclusion limit of 200,000 Daltons, as in Example 24 is diluted with water until a solution containing 5 mg/ml of hyaluronic acid is obtained, as determined by quantitative analysis based on the glucuronic acid dosage. The solution is brought to O.1M in 'barium c;hloride and then precipitated with 4 volumes of 95%
ethanol. The precipitate is washed 3 times with 75 % ethanol and then vacuum dried.
The product thus obtained (HYALECTINTM fraction), used as one of the essential components of the medicament of an aspect of this invention, has a molecular weight of between 500,000 Daltons and 730,000 Daltons. The yield of HY is equal to 0.2%
of the fresh starting tissue.
Example 26 - PREPARATION OF THE SALT OF A HYALURONIC ACID (HY) WITH STREPTOMYCIN
4.47 g of HY barium salt according to one aspect of this invention ( 10 mEq) are solubilized in 300 ml oi= distilled HZO.
2.43 g of streptomycin sulphate ( 10 mE;q) are solubilized in 100 ml of distilled HZO, then added drop-wise under agitation to t:he solution of HY salt. The mixture is centrifuged for 30 minutes at 6000 rpm. T'he solution is separated, the precipitate is washed 2 times with 25 ml of distilled H20. The solution and the washings are pooled and then freeze dried. In the salt thus obtained, all the acid groups of hyaluronic acid are salified with the basic functions of streptorr~ycin. Yield: 5.5 g.
Microbiological determination on B. subtilis (ATCC 6633) compared to standard streptomycin shows a content of 33.8 % in wei~;ht of basic streptomycin, corresponding to the theoretically calculated weight. Colorvnetric determination of glucuronic acid B

combined in the polysaccharide according to the method of Bitter et al. (Anal.
Biochem.
4, 330, 1962) shows a content in weight of HY acid of 66.2% (theoretic percentage 66.0%).
Example 27 - PREPARATION OF THE SALT OF A HYALURONIC ACID (HY) WITH NAPHAZOLINE
4.47 g of the barium salt of HY with a molecular weight of 625,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mlEq of a monomeric unit, are solubilized in 400 ml of distilled HZO.
2.6 g of neutral naphazoline sulphate ( 10 mEq sulphate) are solubilized in 50 ml of distilled water and added to the solution of HY barium salt. The mixture is agitated at 5°C until the barium sulphate is completely precipitated. After centrifugation the resulting solution is fro:aen and instantly freeze dried. Yield: 5.72 g.
In the salt thus obtained, all the acid groups of HY acid are salified with naphazoline. Quantitative spectrophotometric determination compared with standard naphazoline (USP) showed a content of 35 .7 % in weight of basic naphazoline, corresponding to the theoretically calculated value. Colorimetric determination of the glucuronic acid combined in the: polysaccharide, carried out according to the method of Bitter et al. showed a HY acid content of 64.3 % .
Example 28 - PREPAP;ATION OF THE PARTIAL SALT OF A HYALURONIC ACID
(HY) WITH NA:PHAZOLINE
4.47 g of the barium salt of HY with .a molecular weight of 625,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled H20.
1.54 g of acid naphazoline sulphate (10 mEq sulphate) are solubilized in 50 ml bi-distilled water and added to the solution of btcrium salt of HY. The mixture is agitated B

at 5°C until complete precipitation of the sulphate of barium. After centrifugation, the resulting solution is instantly frozen and freeze dried. Yield: 4.5 g.
In the salt thus obtained, 50 % of the acid groups of HY acid are salified with naphazoline and 50% are free. Quantitative spectrophotometric determination in comparison to standard naph~~zoline (USP) shows a content in weight of basic naphazoline which corresponds to the theoretically-calculated value.
Example 29 - PREPAP;ATION OF THE SALT OF A HYALURONIC ACID (HY) WITH PHENYLEPHRINE
2.16 g of neutral L-phenylephrine sulphate ( 10 mEq) are solubilized in 25 ml of distilled H20.
4.47 g of the barium salt of HY with .a molecular weight of 820,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 10 mEq of a monomeric unit, are solubilized in 400 ml of distilled water and added to the solution of sulphate phenylephrine. The mixture is agitated until the barium sulphate is completely precipitated. After centrifugation the resulting solution is frozen and freeze dried. In the salt obtained, all the acid groups of HY
are salified with phenylephrine.
U. V . spectroph.otometri.c determination carried out by the standard addition method (USP) shows a content of 30.6 % of basic phenylephrine, corresponding to the theoretically calculated value.
Colorimetric determination of the glucuronic acid combined in the polysaccharide according to the method of Bitter et al. shows a HY acid content of 69.4 % .
Example 30 - PREPARATION OF THE MIXED SALT OF A HYALURONIC ACID
(HY) WITH NEOMYCIN, WI':CH PHENYLEPHRINE AND SODIUM
7.15 g of HY barium salt with a molecular weight of 65,000 Daltons, used as one of the essential components of the medicament of an aspect of this invention, corresponding to 16 rriEq of a monomeric unit, are solubilized in 400 ml of distilled HZO.
B

4s 1 341 08 7 1.28 g of neomycin sulphate (8.42 mEq) are solubilized in 150 ml of distilled H20. 0.34 g of neutral phenyl.ephrine sulphate ( 1.58 mEq) and 0.43 g of Na2S04 (6 mEq) are added to the solution. The resulting solution is added to the solution of HY
barium salt and, after complete precipitation of the barium sulphate, the mixture is centrifuged.
The barium sulphate is separated and the solution is frozen and freeze-dried.
Yield: 7.35 g.
PHARMACOLOGICAL STUDIES
The technical effect of the new medicaments according to various aspects of the inventions disclosed in the present application and the divisional applications thereof may be demonstrated in viva by experiments on the rabbit eye which show their superiority as compared to the use of the antibiotics when administered in a conventional way. As an example, hereinafter are reported experiments carried out with hyaluronic acid salts with the following antibiotics: streptomycin., erythromycin, neomycin, gentamicin.
These are the total salts in which all of the acid groups of hyaluronic acid are salified with a basic group of tlhe antibiotic, and are dcacribed in Examples 1, 2, 4 and 5. Of these, solutions in distilled water were used., having concentrations suitable to the antibiotic content, as follows:
hyaluronic acid + streptomycin (HYA1) - 33.8%
hyaluronic acid + erythromycin (HYA2) - 66. 0 hyaluronic acid + neomycin (HYA4) - 21.2 %
hyaluronic acid + gent,amicin ( RYAS) - 20.0 %
The activity of these antibiotics was compared to that given by the same antibiotics which are dissolved in phosphate buffer and which have the same concentrations of antibiotic.
The activity of the two groups of products was measured on the basis of the time necessary to suppress a dry inflammation of the: rabbit eye induced by a bacterial agent.
More precisely, the dry inflammation was determined in both eyes of 24 rabbits by intra-ocular injection of a titered suspension of one of the following bacterial groups:
pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi (0.1 ml).
B

~ 341 08 7 The various saline derivatives of the antibiotics were administered (3 drops every hours) into the right eye (RE) of the rabbits, while into the left eye (LE) was instilled the corresponding quantities of the antibiotics dissolved in phosphate buffer. The treatment was begun immediately after injection of the bacterial suspension and was continued until inflammation disappeared. Both eyes of each rabbit were observed with a slit lamp. In particular the following; were examined: the state of the conjunctiva and the corneal epithelium, anterior chamber (presence of the T'yndall effect), and the state of the iris of the posterior segment of the eye. The state of the back of the eye was examined with a Goldman lens. The presence of signs of inflammation(hyperemia, exudates, cloudiness of the liquids etc.) was registered. The percentage of eyes which did not present any signs of inflammation was then calculated.
The results of the experiments are reported in Table 1, whereby it can be observed that administration of the saline derivatives according to an aspect of the invention was followed by a more rapid recovery from inflammation as compared to the administration of the corresponding antibiotics which were not salified with hyaluronic acid.
B

-.~_ ~ 1341 ~8~

O M M O O
O O t~

.
O M M O O
'O O c~

W J M M O O
O u1 .l1 .-1 r-i r-i .~i v .,., o w .o o o v ~o M fh s~
v t~ o w .o o o o fn rn G
u~ .-~ .-mn u~ O
o M o '.~

~
o b ~

2 v0 t0 ip Q O O M O
~D fn H ~D lD . . C
O

.-i o o M o O
o ~ rn C

d ~-I r-1 fn rn ~n x a o o o o M
~~ ~ vo w . . . . .
. . .

~
>. zw o~~ o~c ono oM ro x H m ~ .-~ fn .N

m a~ w ~
x v ~ o V
v C
v o o 0 o ~n r ~w 0 .o .
u E. . . . . . 3 ~r . . . s, o 0 o o vo .t1 ~,0 0 vo ro .., A; ,--~ ~-~ ~ v c gi H

v n c . n ~

v ~ w o~0 00 00 00 w p x M
. . . o c E, o~~ 00 00 0o y, ... ,-.i 'G ~

. c o ~
U

o x ~

c C v w .~ c~. -- .,a ~, sr o ~ 00 00 00 0o r~ v~0 ro cn . . . . N ~ ro cv . . . v C E ~ o o 0 o o v m 0 O E d .,,~ o '.u N

1J r1 C N
N

w U C
v ~

a G O O O O O ~ r-~~ O

-.~

U1 ' O O O O O

.i ?, 'L7.C

c * * a a N

'~ c b E

ra o w w * * u1 w w C w 4 ~ * i.

w * ~O

t C C ~ v Cw O O

JJ ~'1 '.-1rl ~O . x ....~
* ~ .rl n-i U U* --* c v~ ~~i~

w v H >.* >.* * w* ~~v o > z E E vv .-. -- ~ : ~' ~'~n :v ~ =

o ca o o . .., ~
~ o > ~
o 1! U ~" ~ ~ U ~ ''' H H
''' v .,r H

W U v E-~ i1. ~ >, b ..7 . d u~ ~ E mn ~-a c~ o y W ~. >. O C d ~ ~
d d .= ~

w C C~ 1J 4 v v >i rt) 1-1 '~ ~1 :r~ ~

E-~w o H v~ w z t7 > ~ *
x x :~ x o B

1 341 ~8 7 -, ""~ 48 Further data is provided by the following other experiments regarding the miotic, anti-inflammatory, wound healing and antimicrobial action.
I. Miotic activity of pilocar-,pine nitrate vehicled in hyaluronic acid.
Materials The following materials were used as excipients for pilocarpine for the various formulations of pilocapine nitrate:
- hyaluronic acid sodium salt, HYALASTINF~.M fraction, (molecular weight 100,000 Daltons), at concentrations of 10 mg/ml and 20 mg/ml.
- hyaluronic acid sodium salt, HY.ALECTINTM fraction, (molecular weight 500,000 Daltons - 730,000 Daltons) at concentrations of 10 mg/ml and 20 mg/ml.
- 5 % polyvinyl alcohol as ophthalmic excipient of comparison.
Various formulations at 2 % (collyrium or gel) of pilocarpine nitrate were prepared and vehicled by adding the two different fractions of HY sodium salt at concentrations of 10 and 20 mg/ml. ':Che following solutions were prepared:
Formulation 1. - saline with pilocarpine nitrate (PiN03) (2%) used as a reference.
Formulation 2. - solution of (PiN03) (2: % ) vehicled in 5 % of polyvinyl alcohol used as a reference.
Formulation 3. - solution of ((PiN03) (2 % ) vehicled in HYALASTINETM fraction sodium salt ( 10 mg/ml) .
Method Albino New Zealand rabbits were used (2-2.5 kg). The formulation to be tested was instilled in one eye of each rabbit with a microsyringe (10 mcl). The other eye was used as a reference. 7.'he diameter of the pupil was measured in all cases at suitable intervals of time. Each solution was tested on at least 8 rabbits. Each eye was treated not more than three times and a rest period of ar least a week was observed between each treatment.

Parameters Measurements were made of the diameters of the pupils at various intervals to determine the miotic activity curve according to time. The following activity parameters were then calculated by miosis/time graphs:
1 max = maximum difference in the diameter of the pupil between the treated eye and the reference eye Time of the may;imum peak = time taken to reach the I max duration = time taken to return to basa'.l conditions plateau = period of absolute miotic activity AUC = area under the miosis/time curve Results The results of floe studies are reported in Table 2. It is possible to see from the data from the various parameters determined by the time curve of miotic activity for all the solutions studied that the addition to hyaluronic acid at 2 % of pilocarpine nitrate solutions causes an increase in the miotic activity of the drug. Indeed the bioavailability of the drug may be 2. i' times greater than that of the aqueous solution containing 2 %
pilocarpine nitrate (Formulation. 1).
It is important also to note that there is a statistically-significant increase in the activity when the HYALECTIlsfTM fraction of llyaluronic acid is used as a vehicle both at 10 and 20 mg/ml (Formulations 5-6), compared to the pilocarpine nitrate solutions vehicled in polyvinyl alcohol (Formulation 2). The use of hyaluronic acid as a vehicle is particularly interesting because the miotic activity of pilocarpine nitrate lasts longer when it is vehicled with this substance. That is, for the formulations containing hyaluronic acid, the tome necessary for the diameter of the pupil to return the basal conditions is more than 190 minutes compared to 110 minutes for pilocarpine in saline alone (Formulation 1 ) .
B

~._ a:

~a . >

U

~ o ~ o a. ~ ~

~r Q .~ .~ N r. N N

U

C) ~7 a.

N M

~-I ~I +I -h~ ~-I -H

U .-a Q: ~ +I ~ ~ o v~' N

Q' ~ .-r .-i N N N M

~:

r C) L) p C ~

. ~a .

_ a.

aS
o C) C).x. A .~ ~ ,--~.-..

~jU x a'Q v-~~z E-,~~~ N ~N N ~ ~ N~ 00 C)~r ~", M N M M M

~

O O O O O O

0 o O o ~ ~

Q'W - ~ N N N N N

Q;
~l ~.

~
C.) rir C

~ O
r~

~ O O O y a1 ~
N

> cct ' ',..,',.~a,.,...

V O A~

i-.~ ,~ ~ Vl ~. x ~. r~.

a~

Q, .x.

o '~
o u, ~ .- N M ~ ~n ~o z 1 341 ~8 ~
X

II. Miotic Activity of Pilocarpine Salified 'kith H~aluronic Acid.
Materials For the various :Formulations of salified pilocarpine, the following products were used:
- hyaluronic acid at low molecular weight (HYALASTINF~.M, m.w.
100,000 Daltons) [HY,];
- hyaluronic acid sodium salt at high molecular weight (HYALECTINTM, m.w. between 500,000 Dalton.s and 730,000 Daltons) [HYZ-Na] at concentrations of 10 mi;/ml and 20 mg/ml;
- polyvinyl alcohol 5 % as ophthalmic vehicle to obtain comparison formulations .
The various formulations prepared were: the following:
1) saline with pilocarpine nitrate (PiN03) 2% (used as a reference);
2) solution of PiNC>3 2 % vehicled with polyvinyl alcohol 5 % (used as a reference);
3) solution of pilocarpine base/HY, .acid in aqueous solution. The pilocarpine base convent corresponds to 2 % ;
4) solution containing pilocarpine salt/HY, acid vehicled with HYZ Na 100 mg/ml. The pilocarpine base content corresponds to 2 % ;
5) solution containing pilocarpine salt/HY1 acid vehicled with HYZ-Na 20 mg/ml. The pilo~carpine base content corresponds to 2 % .
6) inserts o:f HYZ-Na containing pilocarpine base salt with hyaluronic acid [HY,] . 'the pilocarpine base corresponded to 6.25 % .
Method Albino New Zealand rabbits were used (2-2.5 kg). The solution to be tested was instilled in one eye of each of the rabbits with a microsyringe (10 ~,l); the other eye was used as a reference. The insert was placed in the conjunctival sac by means of suitable pincers. In all cases the pupil diameter was measured at suitable intervals.
Each formulation was tested on at least 8 rabbits. Each eye was treated no more than three times; a rest period of at least a week was observed between each treatment.
s ,~,,-M

Parameters The pupil diameter was measured at various intervals of time in order to determine the miotic activity curve in time and subsequent calculation, from the miosis/time graphs, of the following activity parameters:
S I,I,a,~ = maximurn difference in pupil diameter between the treated eye and the Peak time = time taken to reach the Im~;
duration = time taken to return to basal conditions;
plateau = period of absolute miotic activity;
AUC = area under the miosis-time curve.
reference eye;
Results As can be seen from Table 3, where for each solution tested, the values of the various parameters regiistered from the miotic activity in time curve are reported, it is possible to show how salification with hyaluronic acid of pilocarpine at 2 %
causes an increase in miotic activity of the drug, whose .activity can reach 2 times that shown by aqueous solution with pilocarpine nitrate 2% (Formulation 1).
A statistically-significant increase in activity should also be noted when hyaluronic acid with a high molecular weight is used as a vehicle both at 10 and 20 mg/ml (Formulations 4-5).
Salification wi>xi hyaluronic acid is particularly interesting also in relation to the longer duration of miotic activity of pilocarpine after vehicling with such formulations:
the time taken to return to normal pupil diameter under basal conditions reaches values of 160 minutes (Formu.lation 3) compared to 110 minutes for pilocarpine (Formulation 1).

1341pg~
''~~ 53 CJ

~4 C N

.. >

c . ...~ o a .- m co co .. J O ~ N ehO t~

ro ro it .~ ..i ~ ..rN ~ r1 O ~.

U

N

CJ

V

.r N ~!'1 CO N N m O O

,C E av N r-1r1 v v t~

> a ~ tl +~+I +~+~ +~

U CJ r' N N O r~ N

r-1 OW-i W G eT

E ,~ ~ rl riN N fS d' r1 N

L

a ro a a~ c O ,~ .., , , o o w , ro E N a ar ,--, .

,C id.

C

w o L
u1 ~0 C O O O O O O

1r V ~ r1 V'~O CDO ('1 N 7 E .-i rir1 riN N

sJ

E

ro V N

ro E

a -.

a C o 0 0 o w o a .x -. N N N ('1ri Q' ., ~0 E

> 4J

..,p 13.
.

y,~.., U U

i0r0 tf1 00OD r'1O O

rdU eo f'1 N N v N f"'1 ro... ,n v, o o,0 0 0 0 ca. +I +~tl +~+I +I

O a x ..7 r,r., ro r-~ w n o 0 0 O ro E + ~ ov ~ N r ao r ...>" ~-~ --r1 N N N N f!1 r1 ro O

.-r Z

W O

..7 E C

a] L. O

st O

(-i .~.a aJ n-1 N ('1W !'7v0 B

..-- 1 34~ oa ~

III. Stability of the Corneal Films of the Hyaluronic Acid and Pilocarpine Derivatives.
The aim of these experiments was to evaluate the adhesive and filmogeneous properties of the derivatives of salification between pilocarpine and hyaluronic acid following application to the cornea of animals.
Method The test consisted in visually evaluating the formation, stability and duration of the film formed by the formulations on the cornea. To this end sodium fluorescein was added to the ophthalmic preparations (0.1 % ) and the eye was examined, after instillation in UV light of 366 nm.
12 albino rabbits. were used in all (New Zealand, 2-2.5 kg) of both sexes. One drop (50,1) of each vehicle was instilled in one eye of each rabbit, keeping the other eye as control.
Solutions used 1. saline at 2% of p:ilocarpine nitrate (PiN03);
2. solution at 2 % of PiN03, thickened with polyvinyl alcohol 5 % (blacker Chemie, PVA W 48/20);
3. solution containing pilocarpine base salt/HY1 acid. The pilocarpine base content corresponds to 2 % ;
4. solution c:ontainin.g pilocarpine base salt/HY, acid vehicled with HYZ Na 10 mg/ml. The pilocarpine base content corresponds to 2 % ;
5. solution containing pilocarpine base salt/HY, acid vehicled with HYz-Na 20 mg/ml. The pilocarpine base content corresponds to 2 % .
All solutions contained 0.1 % of sodium :~uorescein. The pH of the solutions was in all cases around 5.8.
Results The parameters relative to the fluorescence: a) duration of the integral corneal film, by b) duration of fluorescence (time necessary for the total disappearance of fluorescence from the eye), c) presence of fluorescence in the nose (time taken by the solution after application to appear at nose leve:l), are reported in Table 4.
s ~. 1 3 ~41 o a ~
The derivatives of hyaluronic acid with pilocarpine produce a stable corneal film for periods of more than 2 hours. Transcorneal penetration of pilocarpine seems therefore to depend on the capacity of hyaluronic acid to vehicle the drug forming a homogeneous and stable; film on the cornea.

Duration of Duration of Appearance of Solution integral film fluorescence fluorescence 10 (min) (min) in nose (min) IV. Anti-inflammatory activity of triamcinolone vehicled in hyaluronic acid Material 20 The following was used:
solution of hyaluronic acid sodium salt HYALECTINTM fraction (m.w.
between 500,000 Daltons and 730,000 Daltons), 10 mg/ml in saline;
- solution of triamc:inolone phosphate ( 10 % in saline) Method 25 The experiments were c~crried out on male New Zealand rabbits (average weight 1.6 kg). After an adaptation period of 5 days, intra-ocular inflammation was induced in the animals by injection of dextran ( 10 % , 0.1 ml) into the anterior chamber.
Administration was carried oul: in both eyes, in conditions of local anaesthetic with novesine 4 % , inserting the needle of the syringe at the edge of the cornea in the anterior 30 chamber at a distance of 2 ml.
B

1 341 4a' The test was carried out on 10 animals.
Treatment Treatment was carried out on each animal both in the right and the left eye by instillation of 3 drops 3 times a day for a total of 6 days of respectively:
- solution of triam<:inolone phosphate ( 10 % in saline) in the left eye (LE);
- solution of hyaluronic acid sodium salt HYALECTINTM fraction ( 10 mg/ml) -+- triamc.inolone phosphate ( 10 % ) in the right eye (RE) .
Parameters The anti-inflamnnatory effect on the reaction induced by dextran was evaluated by observation of the eye through a slit lamp at the following intervals: 0, lh, 3h, 24h, 48h, 3 days, 4 days, 5 days, 6 days.
The following was observed at intervals:
- state of the conjunctiva and the cornea, for the possible presence of hyperemia, edema, and in particular the iris, normally sensitive to inflammatory process after injection of inflammatory agents into the anterior chamber;
- Tyndall effect, in which the presence of opacity of varying intensity ("nubecola") is indicative of the presence of corpuscular (inflammatory) elements in the anterior chamber.
The results of the observations are reported according to subjective scoring between 0 and 3 in relation to the observed effect.
Results As reported in Table 5, administration of triamcinolone proves to have an anti-inflammatory effect on the iris .and causes the disappearance of opacity (Tyndall effect) in the anterior chamber. The inflammatory process evident from the 1st - 3rd hour until 3 - 4 days gradually disappears, returning to almost normal values, with perfect clearness of the eye, in correspondence to the 6th day. On the other hand administration associated with hyaluronic acid sodium salt HYALECTINTM fraction together with triamcinolone phosphate reduces intra-ocular inflammation observed at the above-mentioned times compared to the administration of triamcinolone phosphate alone. That B

"r~.
is, the inflammatory process in the iris and the opacity in the anterior chamber proves to be lower as soon as 24 hours later, with progressive reduction at 48 hours and total absence of inflammatory reaction from the 4th day on.
In the conjunctiva and the cornea, fundamentally no notable inflammatory reactions were observed following injection of dextran into the anterior chamber.
Thus, administration of triamcinolone phosphate together with the hyaluronic acid fraction resulted in an increase in activity of the drug, demonstrated by faster decongestion of the rabbit eye.
B

X341 D~~

a 0 "

H

L, C>

~i M
a, ~, 0 0 0 ~n c~ o c~a ~ 0 0 0 0 Ci ~ o o ~t ~n W o C> o U ~ o ~~ o o >

O O N v7 W V

Q:

.a~' O O O O
~

~az ~ o ~~ o o ~, ZO "d ; ~ o o N ~

C~E~ O O N N

Q;~ ~ O ~~ N

M O '~ O O W

C)Lh O O M M i-.~

-z ~

H ~.
~~~, a~

U
')fy., .~ O O N N ~

~, a o .~. o ~= o o W

O O M M

H

.b ) t-~

O O N N

~;Z N O ~~ O O W :~ l~
~

Q;~ O O M M

O ~,~ O l~ .~
p., ' ,~ O O M N ' c~ ceS

o ~~ o ~ W b o O O M N ~I O
O

~;z .~v ft~ O N N t~ V
n, ~

oH . o O O O

"' N ,~ O ~n W

O ,-r .-i O a .~
~ a j .~

, .
, ~
.., o ~iO o ~~ o o ~ b o 0 0 0 ~~

0 0 0 o W ~ ~ a~
~ o "

o a o o a ,~
~,, ~i m . ~:

Q U ,-, a~
'~ 'C
d.A ~
' c~

y ~ ~ ~ a ~
~/ H
b0 U ~:~ H .~ W
a X~

1 ~ 41 48 ~

V . Healing activity of EGF vehicled in hyaluronic acid Materials The following were used:
Formulation A - EGF (epidermal growth factor), dissolved in saline (0.5 mg/5 ml) Formulation B - hyaluronic acid sodium salt HYALASTINETM fraction (m.w.
100,000) dissolved in saline (10 mg/ml).
Method The experiments were carried out on male New Zealand albino rabbits (average weight 1.8 kg). The animals, after a period of adaptation of 5 days, underwent epithelial lesion of the cornea in suitable conditions of local anaesthetic with novesine (4%).
The lesion consisted of a monocular scarification of a circular area in the optic zone, carried out using a concave glass cylinder (0.3mm) with a sharp edge.
Treatment The animals were subdivided into groups, each group containing 5 animals, and they then underwent pharmacological treatment by conjunctiva) instillation as shown below:
Group Treatment Group 1 (control) Saline Group 2 EGF solution (formulation A) Group 3 HY sodium salt solution HY'ALASTINF~M fraction + EGF
solution - combination of formulation A + formulation B in the ratio 1:1 to make formulation C.
Treatment was effected in the right eye (RE) by conjunctiva) instillation of 2 drops every 8 hours for a total of 3 administrations.

~.. 134108 Parameters Healing of the corneal epithelium was evaluated by observation of the eye and photographic documentation with a slit lamp at various intervals after scarification: 0.8 hr, 16 hr, 24 hr, 32 hr, 40 hr, 48 hr.
5 Results The ophthalmic examination 1 reported in Table 4, showed that in the controls (group 1) there was complete healing (5/5 animals) 48 hours after lesion. In the animals treated with EGF (group 2) the process is apparent as soon as 24 hours after scarification with notable efficacy (4/5 animals). In the anirr~als treated with formulation C composed 10 of hyaluronic acid sodium salt, HYALASTINE,.M fraction + EGF (group 3), the healing process is complete in all the animals (5/5) as soon as 16 hours after scarification.
These results show that the use of the fraction of hyaluronic acid HYALASTINF.LM as a vehicle for EGF enhances the healing process, encouraging a faster and more efficient healing of the corneal lesions.
B

~~~~os~

TABLE 6 - iiEA:LING OF LE:iIONS OF THE CORNEAL
EPI'TIIhL IUM
Group ~TrE:atment TimE: after scarification (hr) 0 8 . 16 24 48 to Saline + + + + -+ + + + -+ + + + -+ + + + -+ + + + -2o EGF + ~ + + - -(formulation A) + + + - -+ + + - -+ + + + -+ + + - -3o EIyaluronic + + - - -~.Acid sodium + + - - -salt + EGF + + - - -(formulation + + - - -C) + + - - -f + = unhealed eye - = healed eye B

VI. Antimicrobial activi~ of Gentamicin vehicled in hyaluronic acid Materials The following were used:
- Gentamicin dissolved in saline (50 mg/ml) - hyaluronic acid sodium salt, HY,ALECTINTM fraction (2 mg/ml) Method Septic inflammation was induced in both eyes of 11 rabbits by intra-ocular injection of a titered suspension of pseudomonas aeruginosa (0.1 ml). In those rabbits showing septic inflammation, hyaluronic acid HYALECTINTM fraction in combination with gentamicin was administered by instillation in the right eye, and gentamicin in a phosphate saline vehicle was administered in the left eye. The treatment (3 drops every 6 hours) was begun immediately after injection of the infective agent and was continued until disappearance of the infection. The eyes of the rabbits were observed every day with a slit lamp.
Results Treatment with a combination of gentarnicin and hyaluronic acid resulted in the more rapid disappearance of septic infection when compared to the administration of the antibiotic alone. This conclusion is clear from the data reported in Table 7.
s ACID H~'ALECTIN FRACTION ON SEPTIC INTRA-OCULAR
INFLAMMATION
Treatment D~a s from the start of inflammation Gentamicin +
saline as vehicle 0.0 0.0 0.0 0.0 0.0 36.3 100 Gentamicin +
HA HYALECTINTM
fraction 0.0 0.1) 9 . 0 + 72. 7 + 72. 7 + 100 100 Values are expressed as percentage of the number of eyes cured of inflammation, compared to the number of eyes treated.
+ = significative differences against phosphate vehicle (- di 0.05, T test B
Fischer) B

Claims (32)

Claims

1. A medicament for topical administration which comprises:
(a) a pharmaceutically-active substance or a mixture of pharmaceutically-active substances which is or axe suitable for topical administration, with the proviso that said active substance is not am ophthalmic drug;
(b) hyaluronic acid or a pharmaceutically-acceptable salt of said hyaluronic acid;
and (c) from 0 % up to a topically-suitable amount of an additional excipient.

2. A medicament for topical administration which comprises:
(a) a pharmaceutically-active substance or a mixture of pharmaceutically-active substances which is or acre suitable for topical administration, with the proviso that said active substance is not an ophthalmic drug;
(b) hyaluronic acid consisting of a molecular weight fraction which is substantially-free of hyaluronic acid having a molecular weight less than 30,000 Daltons.

3. A medicament according to claim 1 or 2, wherein said pharmaceutically-active substance is capable of being absorbed intradermally or through the nasal or rectal mucosa.

4. The medicament according to claim 1, claim 2 or claim 3, wherein said pharmaceutically-active substance is of a basic nature and is present in the form of a salt with said hyaluronic acid.

5. A medicament pharmaceutical composition which comprises, as an active ingredient:
a pharmaceutically-effective amount of a partial salt of hyaluronic acid or a molecular weight fraction thereof, or a stoichiometrically-neutral salt of hyaluronic acid or a molecular weight fraction thereof, with, at least one pharmacologically-active substance of a basic nature;
suitable for topical administration and which is capable of being absorbed intradermally or through the nasal or rectal mucosa;
together with an excipient which is suitable for topical administration.

6. The medicament according to claims 1 to 5, wherein said pharmaceutically-active substance is an antibiotic, an anti-infective, an antiviral, an antimicrobial, an anti-inflammatory, a wound healing, a cytostatic, a cytotoxic, an anaesthetic, a cholinergic promoter, a cholinergic antagonist, an adrenergic promoter or adrenergic an antagonist agent.

7. The medicament according to claim 6, wherein said pharmaceutically-active substance is erythromycin, gentamicin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacyn, tobramycin, spectinomycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitracin, polymxin B, gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, nystatin, diethylcarbamazine, mebendazole, sulphacetamide, sulphadiazine, sulphisoxazole, idoxuridine, adenine arabinoside, trifluorothimidine, aciclovir, ethyldeoxyuridine, pilocarpine, metacholine, carbamylcholine, aceclidine, fisostigmine, neostigmine, demecarium, atropine, noradrenalin, adrenalin, norfazoline, methoxamine, propranolol, timolol, pindolol, bupranolol, atenolol, metoprolol, oxyprenolol practolol, butoxamine, sotalol, butadrine, labetalol, dexamethasone, triamcinolone, prednisolone, fluoromethalone, medrison, fluorocil, methotrexate, or podophyllin.

8. The medicament according to claims 1 to 7, wherein said hyaluronic acid fraction has an average molecular weight between 30,000 and 730,000 Daltons.

9. The medicament according to claims 1 to 7, wherein said hyaluronic acid fraction has an average molecular weight between 50,000 Daltons and 100,000 Daltons.

10. The medicament according to claims 1 to 7, wherein said hyaluronic acid fraction has an average molecular weight between 250,000 and 350,000 Daltons.

11. The medicament according to claims 1 to 7, wherein said hyaluronic acid fraction has an average molecular weight between 500,000 Daltons and 730,000 Daltons.

12. The medicament according to claims 1 to 4 and 6 to 11 which includes a partial or stoichiometrically-neutral salt of hyaluronic acid with at least one pharmaceutically-active substance of a basic nature which is suitable for topical administration.

13. The medicament according to claim 12, wherein said pharmaceutically-active substance is capable of being absorbed intradermally or through the nasal or recta mucosa.

14. The medicament according to claim 12 or claim 13, which contains an additional excipient which is suitable for topical administration.

15. The medicament according to claims 4 and claims 12 to 14, wherein said salt is a partial salt, and a second portion of the acid groups of said hyaluronic acid is salified with an alkali metal, with an alkaline earth metal, with magnesium, with aluminum, with ammonium, or with an amine.

16. The medicament according to claims 1 to 15, wherein said pharmaceutically-active substance is suitable for dermatological, for otorhinolaryngolical, for odontological, for angiological, for obstetrical or for neurological use.

17. The medicamemt according to claims 1 to 15, wherein said pharmaceutically-active substance is an antibiotic, an anti-infective, an antiviral, an antimicrobial, an anti-inflammatory, a wound healing, a cytostatic a cytotoxic, an anaesthetic, a cholinergic promoter, a cholinergic antagonist, an adrenergic promoter or an adrenergic antagonist agent.

18. The medicament according to claims 1 to 15, wherein said pharmaceutically-active substance is erythromycin, gentamycin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacyn, tobramycin, spectinomycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, rolitetracycline, bacitracin, polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin, clindamycin, amphotericin B, griseofulvin, nystatin, diethylcarbamazine, membendazol, sulphacetamide, sulphadiazine, sulphisoxazole, idoxuridine, adenine arabinoside, trifluorothimidine, aciclovir, ethyldeoxyuridine, pilocarpine, metacholine, carbamylcholine, aceclidine, fisostigmine, neostigmine, demecaxium, atropine, noradrenalin, adrenalin, norfazoline, methoxamine, propranolol, timolol, pindolol, bupranolol, atenolol, metoprolol, oxyprenolol, practolol, butoxamine, sotalol, butadrine, labetalol, dexamethasone, triamcinolone, prednisolone, fluoromethalone, medrison, fluorocil, methotrexate, podophyllin, or epidermal growth factor.

19. The medicament according to claims 1 to 15, wherein said pharmaceutically-active substance is a non-steroid, anti-inflammatory agent, or a pharmaceutically-acceptable salt thereof.

20. The medicament according to claim 19, wherein said non-steroid, anti-inflammatory agent is a member which is selected from the group consisting of indomethacin, oxyphenbutazone and flubiprotein.

21. A pharmaceutical composition comprising a sterile composition comprising an aqueous composition containing hyaluronic acid or a pharmaceutically-acceptable salt or pharmaceutically-acceptable ester thereof, and a non-steroidal anti-inflammatory drug or a pharmaceutically-acceptable salt or a pharmaceutically-acceptable ester thereof.

22. A pharmaceutical. composition which comprises hyaluronic acid or a pharmaceutically-acceptable salt thereof and a non-steroid anti-inflammatory agent or a pharmaceutically-acceptable salt thereof.

23. A pharmaceutical composition which comprises hyaluronic acid or a pharmaceutically-acceptable salt thereof and a non-steroid, anti-inflammatory agent.

24. A pharmaceutical composition according to claim 21, claim 22 or claim 23, wherein said non-steroid anti-inflammatory agent is indomethacin, oxyphenbutazone or flurbiprofen.

25. The pharmaceutical composition according to claims 21 to 24, wherein said hyaluronic acid fraction has an average molecular weight between 30,000 and 730,000 Daltons.

26. The pharmaceutical composition according to claims 21 to 24, wherein said hyaluronic acid fraction has an average molecular weight between 50,000 Daltons and 100,000 Daltons.

27. The pharmaceutical composition according to claims 21 to 24, wherein said hyaluronic acid fraction has an average molecular weight between 250,000 and 350,000 Daltons.

28. The pharmaceutical composition according to claims 21 to 24, wherein said hyaluronic acid fraction hays an average molecular weight between 500,000 Daltons and 730,000 Daltons.

29. Use of a mixture of hyaluronic acid or of a pharmaceutically-acceptable salt thereof with a pharmaceutically-active substance which is not an ophthalmic drug, said mixture being as claimed in claims 1 to 28, for the preparation of a medicament for topical administration.

30. Use of a salt of hyaluronic acid with a pharmaceutically-active substance of a basic nature which is not an ophthalmic drug, said mixture being as claimed in claims 1 to 28, for the preparation of a medicament for topical administration.

31. Use of hyaluronic acid, said hyaluronic acid being as claimed in claims 1 to 28, as a delivery system for a drug for topical administration, with the proviso that said drug for topical administration is not an ophthalmic drug.

32. Use of a pharmaceutically-acceptable salt of hyaluronic acid, said salt of hyaluronic acid being as claimed in claims 1 to 28, as a delivery system for a drug for topical administration, with the proviso that said drug for topical administration is not an ophthalmic drug.