Note: Descriptions are shown in the official language in which they were submitted.
1 341 48 7
1
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.
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__ 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,
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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,
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1 341 48 7
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
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1 341 08 7
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.
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~..... .
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
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~ 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.
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1 341 08 7
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
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9
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.
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~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,
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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
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12
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) .
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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.
134187
14
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
1 341 08
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
16 7
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
17
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
19 1 3 4 1 0 8 7
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
21
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 ~
22
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
24
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
12228
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
26
~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
20
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
ATCC 4617
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
.._ .
1341 08 7
27
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
1 341 08 7
~~ 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
1 341 08 7
. ,._
29
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
1
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
32 1 341 08 7
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 ~ _
34
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
60-
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
36
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
37
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
38
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
.._
1 341 08 7
39
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
40
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
41
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
PROCESS DESCRIBED IN EXAMPLE 23
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
43 1 341 08 7
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
44
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
46
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~
47
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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.
1 341 08 7
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 ) .
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50
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1 341 ~8 ~
X
51
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
52
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).
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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.
5
TABLE 4
Duration of Duration of Appearance of
Solution integral film fluorescence fluorescence
10 (min) (min) in nose (min)
1 30 100 2 - 3
2 80 150 10 - 15
3 100 150 5
15 4 120 180 15 - 20
5 140 210 50
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
S6
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
1341487
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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.
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58
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59
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~
61
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
341 08 7
62
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
1 341 08 7
63
TABLE 7 - EFFECT OF GENTAMICIN VEHICLED IN HYALURONIC
ACID H~'ALECTIN FRACTION ON SEPTIC INTRA-OCULAR
INFLAMMATION
Treatment D~a s from the start of inflammation
1 2 3 4 5 6 7
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
