JP2585216C - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Background and Field of the Invention]   The present invention relates to novel pharmaceutical compositions, more precisely,   (a) a pharmaceutically active substance or a mixture of pharmaceutically active substances and (b) hyaluronic acid or
The present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable salt of hyaluronic acid.
The present invention further relates to the use of the pharmaceutical composition for treatment or prevention.
It is.   In the case of topically active medicaments, the application is especially in dermatology, generally in mucous membranes and especially in the mouth.
Beneficial for diseases of the mucous membrane of the cavity and nasal cavity, diseases of the outer ear and especially diseases of the external surface of the eye
Treatment. In particular, the application of these topical medicines is pediatric and veterinary.
Is desirable.   The advantages of treatment with the medicament according to the invention are in comparison with those obtained with known pharmaceutical preparations.
In comparison, the hyaluronic acid component promoted by acidic polysaccharides
Good effect and better bioavailability of the active substance
Cause. Furthermore, the novel medicaments of the invention have particular importance in the case of eye drops. this
Has a more special compatibility with the corneal epithelium due to said properties, and thus a sensitizing effect
This is because they exhibit a very high level of resistance without any accompanying. Elastic-viscous drug
When administered in the form of a concentrated solution or a solid, it is homogeneous, stable,
A completely transparent and sufficiently sticky film is needed to maintain the sustained bioavailability of the drug. An excellent formulation with evidence of being formed on the corneal epithelium, thereby having a retarding effect
Can be formed.   Such eye drops are not currently available in ophthalmic veterinary products, including, for example, chemotherapy.
This is of great value, especially in the veterinary area. In fact, usually
Although human preparations are used, these preparations do not necessarily retain a certain range of activity.
Unless it proves, it does not correspond to the particular condition to be treated.   That is, for example, infectious keratoconjunctivitis, conjunctivitis or infectious bovine keratitis (IBK) (C)
(A contagious disease that mainly infects horses, sheep and goats). This
These three animals seem to have a common etiology in common. Especially parasitic on cattle
The main microorganism that appears to be Moraxella bovis
[Other viral pathogens, such as Rhinotracheit in sheep
is) virus, Mycoplasma, Rickettsia and
Also excludes Chlamydia and Rickettsia in goats
No.] These diseases appear in acute forms and often spread rapidly.
No. The characteristics of the first stage comprehensive symptoms include blepharospasm and excessive lacrimation,
Subsequent purulent exudates, conjunctivitis and keratitis appear, as well as fever, decreased appetite and
Often accompanied by reduced milk production. Corneal lesions are particularly severe,
Can even result in perforation of the cornea itself. The clinical period extends from days to weeks.
Luck.   Broad range of chemotherapeutic agents administered locally (often with anti-inflammatory steroids) and systemically
Used for dosing treatment. These drugs include tetracyclines such as oxy
Tetracycline, penicillins such as cloxacillin and benzylpenici
Phosphorus, sulfamides, polymyxin B [miconazole and
Use with redonizolone], chloramphenicol, tylosin and chloro
There is mycetin (chloromycetin). Local treatment of the disease seems easy,
There are still unresolved issues. This is for some reason so far,
Ophthalmics containing therapeutically effective concentrations of antibiotics or sulfamides in secretions This is because it has been found impossible to obtain a formulation. This means this
If we pay attention to the head tilt position mainly in these animals, it is quite natural for the solution.
is there. However, excipients commonly used in semisolid medicines also adhere to the corneal surface.
This is also the case for semi-solid pharmaceuticals, as they lack the necessary properties for
Complete distribution (i.e., the existence of a distribution gradient), as it does not normally contain active substances in sufficiently high concentrations
) Cannot be achieved. These disadvantages of traditional ophthalmic ophthalmic solutions are discussed in
"Australia Veterinary Journal (Austr. Vet. J.)"
1982, 59 (3), pp. 69-72. [Detailed Description of the Invention]   One advantage of the present invention is that it has completed a new type of eye drops that overcomes the above disadvantages.
It is. Use of hyaluronic acid as an ophthalmic carrier eliminates the active substance concentration gradient
Therefore, it is completely homogeneous, transparent, adherent to the corneal epithelium and has a sensitizing effect.
In addition, it is possible to prepare an excellent formulation that has excellent transmissibility of the active substance and can have a delay effect.
It works.   Of course, the novel medicine having the above properties can be used in fields other than ophthalmology. Already
As mentioned, these can affect dermatology and mucous membranes in the mouth, for example in dentistry.
It can be applied to disease affecting. If they are used, for example, in the case of suppositories, transdermal
A systemic effect is obtained due to the effect of reabsorption. These applications are all about people
It is possible in both the department and the veterinary department. In the case of human medicine, these
The new medicament is particularly suitable for pediatric use. The invention is also particularly suitable for any therapeutic application.
Includes   Therefore, the present invention is used in its essential aspect in combination with a pharmaceutical substance.
Related to hyaluronic acid as a new carrier, providing an improved drug delivery system.
Offer. The novel drug according to the present invention basically comprises the following two components: Ingredient (1)-a pharmaceutically active substance (as will be discussed later, a mixture of these different substances)
) And Ingredient (2)-Hyaluronic acid (as discussed below, the molecular weight fraction of hyaluronic acid
Yo And various salts of hyaluronic acid or its molecular weight fraction)
I do.   The invention further relates to a physical mixture of component (1) and component (2), the activity of component (1).
Complex of the substance with the hyaluronic acid of component (2) or various combinations thereof or
Can be characterized as including mixtures.   (Component (1)-drug substance)   First of all, component (1) is used by humans for use in various areas of therapy
Or veterinary medicine, then ophthalmology, dermatology, ENT
Internal organs that can be locally treated, such as obstetrics, vasculology, neurology or rectal application
For any organ or tissue to be treated, such as any kind of internal medicine
It is classified according to various fields of application. According to a particular aspect of the invention, a pharmacologically active substance
Quality (1) is most important when used in ophthalmology. From another perspective, pharmacologically active substances
(1) is outstanding in terms of effectiveness, for example, anesthetics, analgesics, vasoconstrictors, antibacterial
Agents, antivirals or anti-inflammatory agents. For ophthalmology this is
Particularly suitable for the purpose of miotic action, anti-inflammatory action, wound healing action and antibacterial action, for example.
Can be used.   Also according to the invention, component (1) is a mixture of two or more active substances.
It is possible. For example, in ophthalmology, antimicrobial agents may be combined with anti-inflammatory and vasoconstrictor drugs.
Or a combination of several antimicrobial agents with one or more anti-inflammatory agents
Or one or more antimicrobial agents are mydriatic, miotic, wound healing, or
It may be combined with an antiallergic agent. For example, the following combinations of eye drops,
(A) kanamycin + phenylephrine + dexamethasone phosphate, (b) kanamycin
+ Betamethasone phosphate + phenylephrine, or also, for example, lolitetracycline
Used in ophthalmology such as carcinoma, neomycin, gentamicin and tetracycline
Combinations with other antimicrobial agents can be used.   In dermatology, various antibacterial agents such as erythromycin are used as the active ingredient (1).
A mixture of gentamicin, neomycin, gramicidin, polymyxin B, Use a mixture of such antibacterial and anti-inflammatory agents such as corticosteroids
Can be. For example, the components of the mixture include (a) hydrocortisone + neomycin, (b)
Hydrocortisone + neomycin + polymyxin B + gramicidin, (c) dexa
Methasone + neomycin, (d) fluorometholone + neomycin, (e) predniso
Ron + neomycin, (f) triamcinolone + neomycin + gramicidin + na
Istatin or other mixtures used in conventional ophthalmic preparations. Of course
Mixtures of various active substances are not limited to this field, but each of the aforementioned medical
In the field, similar to those already used in known pharmaceutical formulations in the art.
These mixtures can be used.   Examples of the pharmacologically active substance (1) used in eye drops according to the present invention include basic and
And nonbasic antibiotics such as aminoglucosides, macrolides, tetracyclides
And peptides such as gentamicin, neomycin, streptomycin
, Dihydrostreptomycin, kanamycin, amikacin, tobramycin
, Spectinomycin, erythromycin, oleandomycin, carbomisis
, Spiramycin, oxytetracycline, lolitetracycline, bacit
Rasin, polymyxin B, gramicidin, colistin, chloramphenicol,
Lincomycin, vancomycin, novobiocin, ristocetin, clindamai
Syn, amphotericin B, griseofulvin, nystatin and salts thereof,
For example, sulfates or nitrates, or mixtures thereof or other active ingredients, such as
There are mixtures with those mentioned.   Other eye drops that may be useful according to the invention include other anti-infectives, such as
Tylcarbamazine, mebendazole, sulfamides such as sulfacetamide
, Sulfadiazine, sulfisoxazole, antiviral and antitumor agents, such as
For example, iododeoxyuridine, arabinosyladenine, trifluorothymidine,
Acyclovir, ethyldeoxyuridine, bromovinyldeoxyuri
Gin, 5-iodo-5'-amino-2 ', 5'-dideoxyuridine, steroid
Anti-inflammatory agents such as dexamethasone, hydrocortisone, prednisolone, fluo
B Metron, medrizone and their esters such as phosphates,
Lloyd anti-inflammatory agents such as indomethacin, oxyphenedazone, flurbip
Lofen (flurbiprofen), a wound healing agent such as epidermal growth factor EGF, a local anesthetic,
For example, Benoxinate, Proparacaine and their salts,
Agonists (promoters) such as pilocarpine, methacholine, carbairocholine,
Lysine, fusostigmine, neostigmine, demepotassium and its salts, coli
Antagonists, such as atropine and its salts, adronaline agonists,
For example, noradrenaline, adrenaline, nafozoline, methoxamine and these
Salts, and adrenergic antagonists such as propanolol, timolol,
Pindolol, bupranolol, atenolol, metoprolol, oxpreno
Roll, practol, butoxamine, sotalol, butalin, labetalol
And their salts.   As mentioned above, the active ingredient (1) forms a mixture of two or more active substances.
Possible. Active substances used alone or in mixtures or used in dermatology
Examples of mixtures with other active ingredients include therapeutic agents such as anti-infectives, antibiotics
, Antibacterial, anti-inflammatory, cytostatic, cytotoxic, antiviral, anesthetic,
And prophylactic agents, such as sunscreens, deodorants, preservatives and disinfectants. Antibiotics
Includes erythromycin, bacitracin, gentamicin, neomycin, ole
Omycin (aureomycin), gramicidin and mixtures thereof, antibacterial agents and
Poisons include nitrofurazone, maphenide, chlorhexidine, and 8-hydro
Xyquinoline derivatives and their salts, especially anti-inflammatory drugs, especially corticosteroids, e.g.
For example, prednisolone, dexamethasone, flumethasone, clobetasol (clobetasol)
sol), triamcinolone acetonide, betamethasone or their esters, e.g.
For example, valerate ester, benzoate ester, dipropionate ester, cytotoxic agent
Include fluorouracil, methotrexate, podophylline, and dibuca for anesthetics
There are in, lidocaine and benzocaine.   Of course, the above list is for illustrative purposes only, and Other agents may be used.   What can be inferred from the examples already described for ophthalmology and dermatology is that
For example, in the present invention for use in otolaryngology or dentistry or internal medicine, for example, endocrinology
The medicament may be for example rectal or nasal absorption, for example a nasal spray or oral and
Treatment may be with an intradermal or transmucosal preparation, such as a pharyngeal inhaler.
It can be used in areas where it is possible.   Thus, these preparations may be, for example, anti-inflammatory agents, as already described for ophthalmology, or
Or vasoconstrictors or vasopressors, and vitamins such as antibiotics as described above.
Substances, hormonal agents, chemotherapeutic agents, antibacterial agents, etc., including those used in dermatology
obtain.   (Component (2)-hyaluronic acid carrier)   As described above, the medicament of the present invention comprises, as the component (2), hyaluronic acid and its molecular weight.
Laction or its various salts. Hyaluronic acid (hereinafter sometimes referred to as “H
Y)) is D-glucuronic acid and N-acetyl-D-glucosamine alternately
It is a natural complex polysaccharide consisting of residues arranged in the following order. HY is gel, connective tissue around cells
Extracellular matrix, spinal tissue, synovial fluid of joints, vitreous humor, human umbilical cord tissue, rooster
Present in crusts and certain bacteria. Its molecular weight is about 8-13 million
You.   The first work on HY was performed by Varaths (U.S. Pat.
No. 73), but he replaced H with a suitable fluid for other therapeutic applications.
The Y fraction was isolated. In fact hyaluronic acid and its molecules with low molecular weight
The quantity fraction has been found to have broad utility in the medical field and
Uses are also considered [for example, Varazus, "Cosmetics and Toilet"
Tries "(Cosmetics & Toiletries), Italian edition 5/84]. Especially
For example, in the treatment of arthropathy, such as when treating arthritis in horses in the veterinary field.
It has been used as a therapeutic agent ["Acta Veterinaria Scandinabinica" (Acta.
Vet. Scand.) 167: 379 (1976)].   Hyaluronic acid and its molecular fraction are used as therapeutic agents for natural organs and tissues,
It has been used in ophthalmic surgery as an adjuvant and substitute [see, for example, Varaths et al.
Problems in Ophthalmology "(Modern Problems in Opthalmo
logy), Volume 10, 3 (1970), Streaf, edited by Carger, Basel and
Varaths et al. “Sodium hyaluronate during mucosal surgery and intraocular lens transplantation
International Conference on Intraocular Transplantation and the 1st Film Festival
Nenne, 1979).   In European Patent Application Publication No. 0138572 (filed October 10, 1984)
Are intraocular and intraarticular suitable for the replacement of intraocular fluid in the eye and for the treatment of arthrosis, respectively.
A molecular fraction of hyaluronic acid that can be used for injection has been described.   In the present invention, the therapeutic or surgical or cosmetic forming aid
Different from intended use, topical pharmacological activity of hyaluronic acid or its molecular fraction
It is used as a carrier for the administration of sexual substances.   Hyaluronic acid as a carrier used as component (2) of the present invention is, for example, male
From any source, such as acids extracted from the natural starting materials, including chicken combs
Things are fine. The preparation of such crude extracts of acids is described in the literature. Preferably
Should use purified hyaluronic acid. According to the present invention, the organic material
Instead of the complete hyaluronic acid obtained directly by extraction, a molecular weight of 13 million
Approximately 90-80% of the molecular weight of the complete acid (molecular weight = 11.7 million-100.4 million)
0.23% (Molecular weight = 30000), preferably greatly changed between 5% and 0.23%
A fraction of hyaluronic acid having a possible molecular weight can be used. this
Such fractions can be obtained in various ways, such as hydrolysis, oxidation or enzymatic chemicals.
Agent, obtained by physical methods, such as mechanical methods or irradiation, but also
Are often formed by the same purification method as the original extract [eg
Etc., see “Cosmetics and Toiletries” (supra)]. Obtained
Separation and purification of fractions is achieved, for example, by molecular filtration.   When used as a carrier (2) according to the invention, for example, extracted from rooster comb Hyalastine and Hyalectin from Hyaluronic Acid
Of particular interest are two purified fractions known as Hyalectin.
The fraction known as hyalastin is about 50,000-100,000
Has an average molecular weight. Hyalectin is an average molecule of about 500,000-730000
With quantity. A fraction obtained by combining these two fractions was also isolated.
, Having an average molecular weight of about 250,000 to about 350,000.
It is. This combined fraction is the hyaluronic acid available from the specific starting material
A 80% overall yield is obtained, but the hyalectin fraction is 30% of the starting HY.
% Yield and the hyalastin fraction are obtained in a 50% yield. (these
The production method of the fraction is described in Examples 20 to 22 below).   That is, the preferred hyaluronic acid when used is from about 30,000 to about 130
Molecular weight in the wide range of 00000, preferably in the range of about 30,000 to about 730000
Is the molecular weight fraction having The most preferred hyaluronic acid fraction is
, About 50,000 to about 100,000, or about 500,000 to about 730000
And the combined fractions have a molecular weight of 250,000 to about 350,000.
Have. Importantly, these preferred fractions are substantially about 30,000
Does not contain low molecular weight hyaluronic acid having a molecular weight of less than
No inflammatory side effects. (Hyaluronic acid or HY will be described later,
Both hyaluronic acid and its molecular weight fraction should be
Included).   According to the present invention, hyaluronic acid and its molecular weight flux are used as the pharmaceutical ingredient (2).
Alkali metals (sodium, potassium, lithium), alkaline earth
Metals (calcium, barium, strontium), magnesium or aluminum
It is also possible to use salts with inorganic bases such as um. All of these salts
Is stoichiometrically neutral in that the acid function of
If the group is only partially salted with said metal, it is a partial salt or an acid.
obtain. Such salts are obtained, for example, by reacting HY or said fraction with a stoichiometric amount of base.
Sa Can be easily obtained by mixing, and mixed salts with different bases can be used.
It is.   In addition to the above salts, ammonium or substituted ammonium (amines), for example,
The alkyl group preferably has 1 to 18 carbon atoms or
The kill has the same number of carbon atoms in the aliphatic portion and the aryl optionally has 1 to 3
Mono meaning a benzene residue substituted with a methyl, halogen or hydroxy group
With a wide range of compounds such as, di, tri and tetraalkyl ammonium
Can also be used. Of these HY and ammonium or substituted ammonium
Salts may be used as primary, secondary, or primary compounds or compounds having pharmaceutical activity with hyaluronic acid.
Contains a tertiary amine moiety or an ammonium hydroxide moiety, ie, active ingredient (1).
It is produced by chemically reacting these parts of the compound having the same. The aforementioned
As in the case of the salts, these salts are also used when all of the acid functions are salified.
May be stoichiometrically neutral, or may be a partial salt or acid, with a different base
May be included.   Therefore, hyaluronic acid or its molecular fraction as component (2) is inorganic.
Bases such as alkali metals (sodium, potassium, lithium), alkaline earth gold
Genus (calcium, barium, strontium), magnesium, aluminum, aluminum
It can be replaced by salts with ammonium or substituted ammonium. Also this principle
Is partially or wholly present in the metal or ammonia or amino group.
And ammonium salts are converted to hyaluronic acid and a pharmaceutically active ingredient,
The primary, secondary or tertiary amine moiety of the compound or drug containing
Or the above-mentioned partial acid salts formed by a chemical reaction with an ammonium hydroxide moiety
This also applies to   (Pharmaceutical formulation combining components (1) and (2))   There are various possibilities to embody the medicament according to the invention.   (a) Neutral or acidic active substance, component (1) is replaced with hyaluronic acid or its molecular weight
Or a mixture thereof with a metal salt thereof,   (b) leaving an acid residue of HY released or neutralized by the aforementioned metal or base
Using a partial salt of HY and a basic active substance component (1),   (c) using a stoichiometric neutral salt of HY with a basic substance, component (1),
Or one of its partial or complete (neutral) metal salts,   (d) using a stoichiometric neutral salt of HY with a basic substance, component (1),
Adding an amount of ingredient (1), and   (e) A mixture of salts or the mixture described above is optionally used.   One specific form of the medicament according to the present invention is the case where the pharmacologically active substance, component (1), is basic.
For example, in the case of a basic antibiotic, the active substance (1) and hyaluronic acid or
Represented by a mixture with the molecular weight fraction. In this case, the hyaluronic acid component
(2) and the active substance (1) together form a stoichiometric partial salt or an acidic salt [HY
The eliminable part of all acid groups of component (2) is converted into a salt by the basic group of active component (1).
Or a stoichiometric neutral salt [if all groups of HY component (2) are
Or a mixture of these neutral salts with a small amount of the basic active substance (1).
To achieve.   Therefore, for the purpose of the present invention, when the basic active substance (1) is used,
In place of the mixture of minutes (1) and (2), the aforementioned acid salts or stoichiometric neutral salts or
Of course, a mixture of such a salt with component (1) and component (2) can be used.
.   Mixtures of the above-mentioned drugs and other ingredients may also be used as the active ingredient (1) according to the present invention.
Can be used. Instead of only one active substance (1), a mixture of active substances as described above
If a compound is used, the basic active substance and hyaluronic acid and its molecular weight
The salt of the salt may be a mixed salt or a salt comprising one or more of such basic substances.
Of this type and another few acids of the HY polysaccharide salted with said metal or base
It may be a mixed salt with a functional group. For example, acid groups salified with the antibiotic kanamycin
Some acid groups were salified with the vasoconstrictor phenylephrine, and the remaining acid groups
Is free or salted, for example with sodium or the aforementioned metals.
Hyaluronate or molecular fraction, hyalastin or hyalek
H Can be manufactured. It also contains a salt of the acidic polysaccharide and a single active substance.
As already suggested for drugs that have
It is also possible to mix with acid or its fractions or their metal salts
.   Thus, according to a particular aspect of the present invention, said salt is isolated and purified amorphous
It is possible to use a powder in a solid anhydrous state as a powder. This powder is processed
When in contact with the tissue to be treated, the powder becomes a thick and elastic gelatinous concentrate.
Form an aqueous solution. These properties are also maintained in higher dilutions
Therefore, instead of the above-mentioned anhydrous salt, dissolve it in water or saline at various concentrations.
PH with other pharmaceutically acceptable excipients or additives, such as other inorganic salts.
And the osmotic pressure can be constant. Of course, salt, gels, inserts, creams or
Is an ointment (there are also other excipients or ingredients used in common dosage forms of these pharmaceutical preparations)
) Can be generated. According to a particular aspect of the invention, a single carrier (
Hyaluronic acid, its molecular weight fraction or
Is a pharmaceutical containing its inorganic salt or its partial salt or neutral salt with the active substance (1)
Is preferred.   The quantitative weight ratio of the two components (1) and (2) according to the invention can vary within wide limits.
Of course, this ratio depends on the nature of the two components and, first of all, on the nature of the active substance. For example
The ratio of the two components (1) and (2) ranges from 0.01: 1 to 100: 1. Only
However, the variation range of the ratio of the two components is preferably from 0.01: 1 to 10: 1.
And especially 0.1: 1 to 2: 1.   The medicament according to the invention comprises only two components in a mixture or is packaged separately
Solid dosage forms, such as lyophilized powders.   In the case of a solid dosage form, such a drug may, upon contact with the treated epithelium,
The nature of the epithelium produces a more or less concentrated solution,
Has the same characteristics as a solution produced in a test tube, representing another particularly important aspect of
Things. Such solutions are preferably made with distilled water or sterile saline, and preferably
Preferably, it does not contain a pharmaceutical carrier other than hyaluronic acid or one of its salts. Again The concentration of the solution, such as, depends on each of the two components taken separately and their mixture
It can vary within a wide range, e.g., from 0.01 to 75% for the substance or salt.
Particularly preferred are solutions having significant elastic-viscous properties, such as pharmaceuticals
Is contained in the range of 10% to 90% of each component. Drugs of this type are in anhydrous form (frozen
Especially important is the form of (dry powder) or concentrated solutions or dilutions with water or saline
And optionally metal salts as specific ophthalmic bactericides or buffers
Add additives or auxiliaries.   Among the medicaments of this invention, the following are particularly selected, and in some cases,
It has a suitable acidity for its location, ie a physiologically tolerable pH value. An example
For example, the adjustment of pH in the above-mentioned salt of hyaluronic acid and a basic active substance is performed by an appropriate method.
It can be carried out by adjusting the amounts of polysaccharides, salts and the basic substance itself by a method.
That is, for example, if the acidity of the basic substance and the hyaluronate is too high,
The acid-labile group may be an inorganic base such as sodium or potassium hydroxide or ammonia.
Neutralized by uranium.   The following formulation combines the active pharmaceutical ingredient (1) and the carrier ingredient (2) containing hyaluronic acid.
1 is a formulation example according to the present invention. Formulation Example 1 EGF-containing gel Composition per 100g HY sodium salt (hyalastin fraction), 55 g HY sodium salt (hyalectin fraction), 30g 0.5g EGF 23.5g of double distilled water Formulation Example 2   Pilocarpine nitrate containing inserts Composition per 100g HY sodium salt (hyalastin fraction), 100mg Pilocarpine nitrate, 2mg Formulation Example 3 Topical powder form containing streptomycin Composition of 100g of powder HY sodium salt (hyalastin fraction), 70g HY sodium salt (hyalectin fraction), 28.5 g 1.5 g of streptomycin Formulation Example 4   Pilocarpine-containing insert of the following components (100mg)   A mixed salt of hyaluronic acid, pilocarpine and sodium (see the production method of Example 18).
100mg) Formulation Example 5   Composition per 100 ml of gentamicin and naphazoline-containing eye drops   Mixed salt of hyaluronic acid and gentamicin, naphazoline and sodium (
(See the manufacturing method of Example 16) 2.910 g Propyl oxybenzoate, 0.050 g Sodium phosphate, 1.500 g Add an appropriate amount of distilled water to make 100 ml Formulation Example 6 Chloramphenicol, neomycin, phenylephrine, nitrofurazone content
Eye drops Composition per 00ml   Mixed salt of hyaluronic acid with neomycin, phenylephrine and sodium (
(See production method of Example 17) 2.890 g Chloramphenicol, 0.500 g Nitrofurazone, 0.02 g   Add an appropriate amount of distilled water to make 100 ml Formulation Example 7   100 ml of eye drops containing dexamethasone phosphate, kanamycin and phenylephrine
Composition per hit   Mixed salt of hyaluronic acid and kanamycin and phenylephrine (Example 15)
3.060 g) Dexamethasone phosphate sodium salt, 0.100 g methyl p-hydroxybenzoate, 0.060 g   Add an appropriate amount of distilled water to make 100 ml   (Production method)   Method A   The preparation of the salts according to the invention comprises two components (1) and (2) and optionally stoichiometric amounts.
Said alkali metal, alkaline earth metal, magnesium, aluminum or aluminum
A solution or suspension containing a base or basic salt with ammonium in water or an organic solvent
Combining the suspensions and isolating the amorphous anhydrous form of the salt according to known techniques
It can be done by a method. For example, first, an aqueous solution of the two components (1) and (2) is prepared.
Metal salts such as sulfate in the case of component (1) and sodium salt in the case of component (2), respectively.
The lithium salt is treated with a suitable ion exchanger, and the aqueous solution of these salts is acidified.
To release the two components and combine the two solutions at a low temperature, for example 0 ° to 20 °.
If the salt thus obtained is easily soluble in water, freeze-dried and poorly soluble
Is centrifuged, filtered or decanted and optionally dried with a desiccant. [Example]   The following examples are provided solely for the purpose of illustrating the method A according to the present invention.
It should not be construed as limiting the invention.   Example 1 Preparation of hyaluronic acid (HY) salt with streptomycin   2.43 g of streptomycin sulfate (10 meq, hereinafter meq is expressed as mEq)
Is dissolved in 25 ml of distilled water. This solution is^-Quaternary ammonium resin (Dow
X × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Sulfate-free elution
Collect the liquid in a 5 ° C. thermostat. Nato of hyaluronic acid having a molecular weight of 255,000
4.0 g of the lithium salt (corresponding to a monomer unit of 10 mEq) are dissolved in 400 ml of distilled water. Next
Then, put this solution in H⁺15ml of sulfonic acid resin (Dowex 50x8)
Through a constant temperature column at 5 ° C. This sodium-free eluate is
Collect in a solution of isine base with stirring. Freeze the resulting solution and freeze immediately
Dry and dry. In the salt thus obtained, all of the acidic groups of hyaluronic acid are stressed.
Salt is formed by the basic functional group of peptomycin. Yield: 5.5 g.   Bacillus subtilis (Bacillus subtilis) to be compared with standard streptomycin
tilis) The result of the microbiological assay according to ATCC 6633 corresponds to the theoretically calculated weight.
33.8% by weight of streptomycin base.   Bitter method [Analytical Biochemical Story (Anal. Biochem.)
4, 330, 1962] for the colorimetric determination of glucuronic acid bound to polysaccharides.
As a result, it contains 66.2% by weight of HY acid (theoretical percentage 66.0%).
I understood.   Example 2 Preparation of Hyaluronic Acid (HY) Salt with Erythromycin   Sodium hyaluronate with a molecular weight of 77000 corresponding to 10 mEq of monomer unit
4.0 g of salt are dissolved in 400 ml of distilled water. The solution is then⁺Shape sulfone
Pass through a 5 ° C. constant temperature column packed with 15 ml of acid resin (Dowex 50 × 8). Natori
The eluate containing no um is kept at 5 ° C. 7.34 g (10 mEq) of erythromycin base
Is added to the HY solution at 5 ° C. with stirring to dissolve completely. The resulting solution And freeze-dried. In the salt thus obtained, the acidic groups of hyaluronic acid
Are all salted by erythromycin. Yield: 10.8 g.   Staphylococcus aureus compared to standard erythromycin
coccus aureus) As a result of microbiological test using ATCC 6538p, equivalent to the theoretical value.
I found out. Of glucuronic acid bound to polysaccharides by a method such as bitter
As a result of colorimetric determination, 34.0% by weight of HY acid corresponding to the theoretically calculated percentage
It was found to contain.   Example 3 Preparation of Hyaluronic Acid (HY) Salt with Kanamycin   1.46 g (10 mEq) of dikanamycin sulfate are dissolved in 25 ml of distilled water. This solution
Is OH^-5 ° C packed with 15 ml of quaternary ammonium resin (Dowex 1 × 8)
Through a constant temperature column. Collect this sulfate-free eluate in a 5 ° C constant temperature vessel
. Sodium HY having a molecular weight of 165000 corresponding to 10 mEq of monomer units
4.0 g of salt are dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin (
Dowex 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Sodium
The eluate, which is not contained, is swirled with a vortex and swirled.
In a solution of the base. The solution thus obtained is immediately frozen and freeze-dried.
You. Yield: 4.8 g.   In the obtained salt, all the acid groups of HY are salted by kanamycin.
B. subtils ATCC6 compared to standard kanamycin
The result of the microbiological assay according to 633 is 24 corresponding to the theoretically calculated percentage.
It was found to contain 0.2% by weight kanamycin base.   The results of colorimetric determination of glucuronic acid bound to polysaccharides by methods such as bitter,
It was found to contain 75.8% by weight of HY acid, corresponding to the theoretical beam content.   Example 4 Preparation of a salt of hyaluronic acid (HY) with neomycin   1.52 g (10 mEq) of neomycin sulfate was dissolved in 20 ml of distilled water.^-Four of shape
Through a 5 ° C constant temperature column packed with 15 ml of high-grade ammonium resin (Dowex 1 × 8).
You. Collect this sulfate-free eluate in a 5 ° C. thermostat. Monomer unit 10 4.0 g of HY sodium salt having a molecular weight of 170,000 corresponding to mEq was added to 400 ml of distilled water.
Dissolved in H⁺5 ° C packed with 15 ml of sulfonic acid resin (Dowex 50 × 8)
Through a constant temperature column. The sodium-free eluate is added to the neomycin with stirring.
In a solution of the base. Separation of the resulting viscoelastic precipitate by decantation
And freeze-dried. Yield: 4.76 g.   In the obtained salt, all the acid groups of HY are salted by neomycin.
Staphylococcus aureus AT compared to standard neomycin
As a result of the quantitative microbiological assay performed by CC6538p,
A corresponding content of 21.2% by weight of neomycin base was indicated.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of
It was found to contain 8.8% by weight of HY acid.   Example 5 Preparation of a salt of hyaluronic acid (HY) with gentamicin   1.45 g (10 mEq) of gentamicin sulfate are dissolved in 25 ml of distilled water. This solution
Is OH^-5 ° C packed with 15 ml of quaternary ammonium resin (Dowex 1 × 8)
Through a constant temperature column. Collect this sulfate-free eluate in a 5 ° C. thermostat.
4.0 g of HY sodium salt having a molecular weight of 170,000 corresponding to 10 mEq of monomer unit
Is dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin (Dow
X 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Contains sodium
This effluent is vortexed and gently swirled to dissolve the gentamicin base.
Collect in liquid. The thick and extremely viscous precipitate formed is decanted
Separate and freeze dry. Yield: 4.65 g.   In the salt thus obtained, all the acid groups of HY are converted into salts by gentamicin.
ing. Staphylococcus epidermidu compared to standard gentamicin
Quantitative microbiological assay performed by S. epidermidus ATCC 12228
As a result of containing 20.0% by weight of gentamicin base corresponding to the sulfuric acid content.
I knew it was there.   As a result of the colorimetric determination of glucuronic acid bound to the polysaccharide by the method of Bitter et al. The Y-acid content was shown to be 80.0%.   Example 6 Preparation of Hyaluronic Acid (HY) Salt with Amikacin   1.47 g (10 mEq) of amikacin base is dissolved at 500 DEG C. in 100 ml of distilled water.
4.0 g of HY sodium salt having a molecular weight of 170,000 corresponding to 10 mEq of monomer unit
Is dissolved in 4000 ml of distilled water. The solution is then⁺Sulfonic acid resin (Dow
X 50 × 80) through a 5 ° C. thermostatic column packed with 15 ml. Sodium salt
This eluate free is collected in a solution of amikacin base with vortexing. Living
The thick and very viscous precipitate formed is separated by decantation and lyophilized
I do. Yield: 5.16 g.   In the salt thus obtained, all of the HY acid groups are converted into salts by amikacin.
I have. Staphylococcus aureus (S. aureus) compared to standard amikacin
Results of quantitative microbiological assay performed according to ATCC 29737, theoretical content
A content of 27.7% by weight of amikacin was indicated.   As a result of the colorimetric determination of glucuronic acid bound to the polysaccharide by the method of Bitter et al.
The Y acid content was found to be 72.3% by weight.   Example 7 Production of hyaluronic acid (HY) salt with lolitetracycline   HY sodium salt having a molecular weight of 17000 corresponding to 10 mEq of monomer unit
4.0 g are dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin (
Dowex 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Sodium salt
This eluate, containing no, is kept at 5 ° C. 5.3 g of lolitetracycline base (10 mE
Add q) to the solution of HY acid while stirring at 5 ° C while shielding from light to completely dissolve
. The solution thus obtained is immediately frozen and freeze-dried. Yield: 8.9 g.   In the salt thus obtained, all the HY acid groups are converted to lolitetracycline.
It is more salty. Bacillus pumilus compared to standard lolitetracycline
(B. pumilus) microbiological assay by ATCC 14884.
58.2% by weight of lolitetracycline base. Bitter, etc.
As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of 41.8, 41.8 weight was obtained. % HY acid content was indicated.   Example 8 Preparation of Hyaluronic Acid (HY) Salt with Polymyxin B   2.4 g (10 mEq) of polymyxin B base are suspended at 5 DEG C. in 100 ml of distilled water. Mo
4.0 g of HY sodium salt having a molecular weight of 170000 corresponding to 10 mEq
Dissolve in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin
(50 × 8) through a 5 ° C. constant temperature column packed with 15 ml. Contains no sodium
Collect the eluate in a 5 ° C. polymyxin B base suspension with vigorous stirring.
. After the first phase, when the solution became clear, the poorly soluble product formed more and more,
Complete precipitation with 5 tonnes volume. The precipitate is filtered, washed with acetone and then
Vacuum dry. Yield: 6.05 g.   In the salt thus obtained, all the acid groups of HY are converted into salts by polymyxin B.
ing. B. compared to standard polymyxin B. B. bronchi
septica) Results of quantitative microbiological assay performed by ATCC 4617, theoretical values
A content of 38.7% by weight of polymyxin B base corresponding to   The colorimetric determination of glucuronic acid bound to polysaccharides by the method of Bitter et al.
HY acid content was indicated.   Example 9 Production of hyaluronic acid (HY) salt with gramicidin S   6.7 g (10 mEq) of gramicidin S hydrochloride in ethanol / H_TwoO (80; 20) 20
Suspend in 0 ml. This solution is then^-Quaternary ammonium resin (Dowett
1 × 8) through a 5 ° C. thermostatic column packed with 15 ml. To 10 mEq of monomer unit
The corresponding 4.0 g of HY sodium salt having a molecular weight of 165000 is dissolved in 400 ml of distilled water.
I do. The solution is then⁺Shaped sulfonic acid resin (Dowex 50 × 8) 15m
l through a 5 ° C. constant temperature column.   200 ml of dimethyl sulfoxide (DMSO) was added to this eluate without sodium.
Is added and the mixture is kept at 5 ° C. with stirring. Then a solution of gramicidin base
Add slowly. The resulting solution is precipitated by 10 volumes of acetone. Precipitation
Filter, wash with acetone and dry under vacuum. Yield: 9.55 g.   In the salt thus obtained, all the acid groups of HY are converted to salts by gramicidin S.
ing. S. compared to standard gramicidin S. S. faecium AT
As a result of a quantitative microbiological assay using CC10541, 60.0 corresponding to the theoretical value was obtained.
It was found to contain gramicidin S base by weight.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of
A HY acid content of 0.0% was indicated.   Example 10 Preparation of hyaluronic acid (HY) salt with naphazoline   Pure naphazoline base is prepared as follows; naphazoline-HCl 4.9
4 g (20 mM) are dissolved at 5 DEG C. in 100 ml of distilled water. NH_Four20 ml of OH (5M)
Add and extract twice with 100 ml of ethyl acetate. Organic layer H_TwoExtract twice with 50 ml of O
And recombined with anhydrous Na_TwoSO_FourDehydrate with. The solution is concentrated to 50 ml and then cooled
Crystallize in freezer. The crystallized product is filtered, washed with ethyl acetate,
Vacuum dry. Yield: 4.0 g of pure naphazoline base.   HY sodium salt having a molecular weight of 625,000 corresponding to 10 mEq of monomer unit 4.
0 g is dissolved in 400 ml of distilled water.⁺Sulfonic acid resin (Dowex 50 × 8)
Pass through a 5 ° C. constant temperature column packed with 15 ml. 2.1 g (10 mEq) of naphazoline base
Add to the solution of HY acid and stir at 5 ° C. until the mixture is completely dissolved. Got
The mixture is immediately frozen and lyophilized. Yield: 5.72 g.   In the salt thus obtained, all of the HY acid groups are converted into salts by naphazoline.
ing. Results of quantitative spectroscopic measurements performed in comparison with the Naphazoline Standard (USP)
As a result, it was found to contain 35.7% by weight of naphazoline base corresponding to the theoretical value.
.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of bitter, etc., 6
It was found to contain 4.3% HY acid.   Example 11 Preparation of hyaluronic acid (HY) salt with phenylephrine   2.04 g (10 mEq) of L-phenylephrine-HCl is dissolved in 25 ml of distilled water.
This solution is^-Filled with 15ml of quaternary ammonium resin (Dowex 1 × 8)
Through a 5 ° C constant temperature column. This eluate containing no chloride is placed in a 5 ° C thermostat Collect inside. HY having a molecular weight of 820000 corresponding to 10 mEq of monomer units
4.0 g of naphazoline salt are dissolved in 400 ml of distilled water. The solution is then⁺
Into a 5 ° C constant temperature column packed with 15 ml of sulfonic acid resin (Dowex 50 × 8)
Let it through. This eluate without naphazoline is stirred into a solution of phenylephrine base.
Collect with stirring. The mixture obtained is immediately frozen and freeze-dried. Yield: 5.
25g.   In the salt thus obtained, all the acid groups of HY are converted into salts by phenylephrine.
ing. Results of UV spectroscopic measurement using standard addition method (USP), theoretical content
It was found to contain 30.6% by weight of phenylephrine base, corresponding to the amount.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of bitter, etc., 6
A 9.4% HY acid content was indicated.   Example 12 Preparation of a salt of hyaluronic acid (HY) with atropine   HY sodium having a molecular weight of 1300000 corresponding to 10 mEq of monomer units
4.0 g of salt are dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid in form
Pass through a 5 ° C. constant temperature column packed with 15 ml of resin (Dowex 50 × 8). Natori
This eluate without the solution is kept at 5 ° C. 2.89 g (10 mEq) of atropine base
Is added to the solution of HY acid and the mixture is stirred at 5 ° C. Freeze the resulting mixture and freeze
Dry and dry. Yield: 6.5 g.   In the salt thus obtained, all the hyaluronic acid groups are converted to the salt by atropine.
Has become. Quantitative gas chromatographic measurements performed in comparison with standard atropine
The constant (USP) indicated a content of 43.3% of atropine base corresponding to the theoretical value.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of
It was found to contain 6.7% HY acid.   Example 13 Preparation of a salt of hyaluronic acid (HY) with pilocarpine   2.45 g (10 mEq) of pilocarpine hydrochloride are dissolved in 50 ml of distilled water. This solution
, OH^-5 ° C packed with 15 ml of quaternary ammonium resin (Dowex 1 × 8)
Pass through a constant temperature column. The chloride-free eluate is collected in a 5 ° C. thermostat.
Mo 4.0 ml of HY sodium salt having a molecular weight of 170,000 corresponding to 10 mEq of the nominal unit
Dissolve in The solution is then⁺Sulfonic acid resin (Dowex 50 × 8)
Pass through a 5 ° C. constant temperature column packed with 15 ml. This eluate without sodium
Collect with stirring in a solution of pilocarpine base. The solution thus obtained is immediately
And freeze-dried. Yield: 5.25 g.   In the salt thus obtained, all of the HY acid groups are combined with the salt by pilocarpine.
Has become. Molecular optical measurements by USP performed in comparison with pilocarpine standards
As a result, it was found that 35.1% by weight of pilocarpine base corresponding to the theoretical value was contained.
Was.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of bitter, etc., 6
A HY acid content of 4.6% was indicated.   Example 14 Salt of hyaluronic acid (HY) with neomycin and polymyxin
Manufacturing of   HY sodium salt having a molecular weight of 170,000 corresponding to a monomer unit of 10 mEq
4.0 g are dissolved in 400 ml of distilled water. This solution is⁺Sulfonic acid resin (Dow
X 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Contains sodium
Collect the remaining eluate in a 5 ° C thermostat. 0.150 g of polymyxin B base (0.6
3 mEq) are added with vigorous stirring. 1.425 g of neomycin sulfate (9.37 m
Eq) is dissolved in 25 ml of distilled water. This solution is^-Quaternary ammonium resin (
Dowex 1 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Contains no sulfates
The eluate in a solution of HY acid and polymyxin B with vigorous stirring
. The precipitate formed is separated by centrifugation and dried under vacuum. Raw to residual solution
There is no loss of material. Yield: 4.85 g.   17.25 mg of this product is equivalent to 5.0 mg of neomycin sulfate neomycin
, A polymyxin equivalent to 0.63 mg (about 5000 UI) of polymyxin sulfate.
Have.   For these measurements, two active substances were analyzed by HPLC (High Performance Liquid Chromatography).
Tsu This was performed after separation.   Example 15 Hyaluronic Acid (HY) with Kanamycin and Phenylephrine
Production of mixed salt   HY sodium salt having a molecular weight of 65,000 corresponding to 10 mEq of monomer units
4.0 g are dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin (
Dowex 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Sodium
The eluate free is collected in a 5 ° C. thermostat. 0.85 g of kanamycin sulfate (5.8
2 mEq) is dissolved in 10 ml of distilled water. This solution is^-Quaternary ammonium tree in shape
Pass through a 5 ° C. constant temperature column packed with 10 ml of fat (Dowex 1 × 8).   The sulfate-free eluate is collected in a container maintained at 5 ° C. Phenylev hydrochloride
Phosphorus was dissolved in distilled water at 5 ° C. to 100 mg / ml and complete precipitation was achieved.
Until NH_FourThe phenylephrine base is produced by adding OH (6N).
You. The precipitate is filtered off, washed with distilled water until chloride disappears from the washing water, and dried under vacuum.
I do. Mix the solution of HY acid and kanamycin base and maintain at 5 ° C. Pheni
699 mg (4.18 mEq) of refrin base are added with stirring to dissolve completely. Profit
The solution obtained is frozen and lyophilized. Yield: 5.1 g.   Bacillus subtilis ATCC compared to standard kanamycin
The microbiological assay according to 6633 shows a content of 13.55% by weight of kanamycin base.
Indicated. As a result of UV spectroscopic measurement using the comparative addition method (USP),
13.45% by weight.   As a result of colorimetric determination of glucuronic acid bound to polysaccharide by the method of
A HY acid content of 3.0% was indicated.   Example 16 Hyaluronan with Gentamicin, Naphazoline and Sodium
Preparation of mixed salt of acid (HY)   4.0 g of HY sodium salt having a molecular weight of 50,000 corresponding to 10 mEq of monomer unit
Is dissolved in ml of distilled water. This solution is then H⁺Sulfonic acid resin (Dowex 5
0 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. This sodium-free solution Collect the effluent in a 5 ° C. thermostat. 1.245 g of gentamicin sulfate (8.59 mEq
Is dissolved in 25 ml of distilled water. This solution is^-Quaternary ammonium resin (da
Pass through a 5 ° C. constant temperature column packed with 1 × 8.5912 ml of Wex.   Collect this sulfate-free eluate in a container maintained at 5 ° C. Naphazoline hydrochloride
At a concentration of 50 mg / ml at 5 ° C._TwoDissolved in O and NH until pH 12_FourOH (
5M) and the solution is extracted twice with ethyl acetate to give pure napha.
Produces zoline base. Organic layer H_TwoWashed with O, anhydrous Na_TwoSO_FourDehydrate with. Living
The product is crystallized in the freezer, the precipitate is filtered, washed with ethyl acetate and dried in vacuo.
Dry. 2.5 g of sodium salt of HY and 0.297 g of naphazoline base were added to HY acid (
Add 1.41 mEq) and stir until completely dissolved. Then gentamicin base
Solution, homogenize, then freeze and lyophilize. Yield: 7.35 g.   Staphylococcus evidermidus (Se
pidermidus) As a result of a quantitative microbiological assay using ATCC 12228,
A content of 11.1% by weight of isine base was indicated. Compared to standard naphazoline (USP)
As a result of the quantitative spectroscopic measurement performed, the content of naphazoline base was 4.0% by weight.
It has been shown.   The result of the colorimetric determination of glucuronic acid bound to the polysaccharide by the method of Bitter et al.
It was found to contain 3.0% HY acid.   Example 17 Hyaluro with neomycin, phenylephrine and naphazoli
Preparation of mixed salt of acid (HY)   HY naphazoline having a molecular weight of 65,000 corresponding to 10 mEq of monomer units
4.0 g are dissolved in 400 ml of distilled water. The solution is then⁺Sulfonic acid resin (
Dowex 50 × 8) Pass through a 5 ° C. constant temperature column packed with 15 ml. Sodium
Collect this eluate free of water in a 5 ° C. thermostat. 1.28 g of neomycin sulfate
(8.42 mEq) is dissolved in 25 ml of distilled water. This solution is^-Quaternary ammonium of shape
Through a 5 ° C. constant temperature column packed with 12 ml of a water resin (Dowex 1 × 8).   The sulfate-free eluate is collected in a container maintained at 5 ° C. Phenylev hydrochloride Dissolve phosphorus in distilled water at 5 ° C to 100 mg / ml until complete precipitation
NH_FourThe phenylephrine base is prepared by adding OH (6N). Precipitation
Filter off, wash with distilled water until chloride disappears from the washings, then dry under vacuum.   2.5 g of HY sodium salt and 0.266 g (1.58 mEq) of phenylephrine base
Is added to the solution of HY acid and stirred until completely dissolved. Then neomycin base
After homogenization, it is frozen and freeze-dried. Yield: 7.3
5g.   Spectrophotometric determination by UV using the standard addition method (USP)
3.57% by weight. Compared to neomycin standard
Determination by Staphylococcus aureus ATCC 63538p
Microbiological assay showed 11.64% by weight of neomycin base.
.   Colorimetric determination of glucuronic acid bound to polysaccharides by the method of Bitter et al.
An HY acid content of 8% was indicated.   Example 18 Preparation of a salt of hyaluronic acid with pilocarpine and sodium   HY sodium having a molecular weight of 170,000 corresponding to 245 mEq of monomer units
98.31 g of salt are dissolved in 8.5 l of distilled water.   The solution is then⁺Sulfonic acid resin (Dowex 50 × 8) 300ml
Through a 5 ° C. constant temperature column packed with. Eluate containing no sodium at 5 ℃ constant temperature
Collect in the bowl.   2.34 g (9.6 mEq) of pilocarpine hydrochloride are dissolved in 50 ml of distilled water. This solution
At 5 ° C. packed with 15 ml of quaternary ammonium resin in OH form (Dowex 1 × 8)
Pass through a constant temperature column.   This eluate, free of chlorides, is collected with stirring in a solution of HY acid. Hydroxylation
235.4 ml (1 M) of sodium solution are slowly added with stirring. Thus obtained
The solution is immediately frozen and lyophilized. Yield: 99.8 g.   100 mg of this product contains 2 mg of pilocarpine as base.   Example 19 Salt of hyaluronic acid with streptomycin and sodium Manufacture   HY sodium having a molecular weight of 255,000 corresponding to 246 mEq of monomer units
98.68 g of salt are dissolved in 8.5 l of distilled water. The solution is then⁺Shape sulfone
Pass through a 5 ° C. constant temperature column packed with 300 ml of acid resin (Dowex 50 × 8). Nato
The eluate, free of lithium, is collected in a 5 ° C. thermostat.   1.88 g (7.74 mEq) of streptomycin sulfate are dissolved in 20 ml of distilled water.
This solution is^-12ml of quaternary ammonium resin (Dowex 1x8)
Through a 5 ° C constant temperature column.   This sulphate-free eluate is collected with stirring in a solution of HY acid. NH_FourOH
238.3 ml (1 M) of the solution was slowly added with stirring, and the resulting solution was immediately frozen.
Freeze and freeze dry. Yield: 99.8 g.   100 g of this product contains 1.5 g of streptomycin as base.   Example 20 Mixture of Hyalastin and Hyalectin Fractions Without Inflammatory Activity
How to get   Fresh or frozen chicken comb (3000g) is minced with a meat grinder and then
Carefully homogenize with a mechanical homogenizer. The paste thus obtained is
And then 10 volumes in a stainless steel container (AISI 316) or glassware
Treat with anhydrous acetone. The whole is then stirred at a speed of 50 rpm for 6 hours.
This is left to separate for 12 hours, and then acetone is sucked off by siphoning.
throw away. Until the water content of the acetone to be discarded reaches the correct ratio (Karl Fish
And acetone extraction are repeated. Then, the whole amount is centrifuged, and at a suitable temperature for 5-8 hours
Dry under vacuum for a while. In this way, about 500-600 g of dry powder of chicken crest is obtained.
.   Expose 300 g of the dry powder to enzymatic digestion with papain (0.2 g) under aqueous conditions,
Buffer with a phosphate buffer in the presence of an appropriate amount of cysteine hydrochloride.   The resulting material is stirred at 60 rpm for 24 hours while maintaining the temperature at 60-65 ° C.
. Then, the mixture was cooled to 25 ° C., Celite R (60 g) was added, and the mixture was further stirred for 1 hour.
Stirring Continue. The mixture thus obtained is filtered to obtain a clear liquid. Next,
In order to capture the clear solution on the membrane with a molecular weight of 30,000 or more, the exclusion limit molecular weight
Subject to molecular ultrafiltration using a 30,000 membrane.   Distilled water is continuously added to the product during the ultrafiltration operation to bring the product to its original volume.
Ultrafiltrate 5-6 times. Stop adding water and reduce to 1/3 of the original volume.
Continue ultrafiltration with. The remaining liquid was made 0.1 M by addition of sodium chloride.
, Bring the temperature to 50 ° C. Under stirring at 60 rpm, 45 g of cetylpyridine chloride was added.
Add. The solution is stirred for 60 minutes and then 50 g of Celite R are added. Whole under stirring
At 25 ° C. and collect the resulting precipitate by centrifugation. The resulting precipitate
Was dissolved in a 0.01 M solution of sodium chloride containing 0.05% of cetylpyridinium chloride.
Suspend in liquid (5 l). The precipitate is then centrifuged at a temperature of 25 ° C. Washing operation
Is repeated three times, after which the precipitate is washed with chloride containing 0.05% of cetylpyridine chloride.
Collect in a container containing 3 l of a 0.05 M solution of sodium. 60 minutes at 60 rpm
The mixture is shaken, and the temperature is kept constant at 25 ° C. for 2 hours. The supernatant is removed by centrifugation. C
Using 0.1 M sodium chloride solution containing 0.05% of tyl pyridinium chloride
This step is repeated several times. Centrifuge the mixture and discard the supernatant. Precipitation, Seti
To a 0.03 M sodium chloride solution (3 l) containing 0.05% of lupyridinium chloride
Spread. Stir the mixture and collect both the precipitate and the clear liquid. Same aqueous solution 0
The extraction of the precipitate is repeated three more times, each time using 0.5 l.   Finally, the precipitate residue is removed and all the clear liquids are put together in one container. Absolute
The temperature of the liquid is brought to 50 ° C. with constant stirring. The liquid is then
To 0.23M. Add 1 g of cetylpyridinium chloride and add this liquid to 1
Continue stirring for 2 hours. The mixture is cooled to 25 ° C. and then first with Celite R pack
Then, the mixture is filtered with filter paper. Next, this was re-separated with a membrane having a molecular weight of exclusion limit of 30,000.
Subjected to ultrafiltration and the initial volume was added by addition of a 0.33 M solution of sodium chloride.
Ultrafiltrate three times. Suspend the addition of the sodium chloride solution and reduce the volume to the original volume.
Reduced to 1/4. The solution concentrated in this manner was prepared using 3 volumes of ethanol (95%). At 25 ° C. with stirring (60 rpm). The precipitate is washed with 0.1 of sodium chloride.
Dissolve in 1 liter of M solution and repeat the precipitation with 3 volumes of ethanol (95%). Sinking
Collect the precipitate, first with ethanol (75%) three times, then with absolute ethanol (three times), and
Finally, wash with anhydrous acetone (3 times). The product thus obtained is
Tin + Hyarestectin fraction) has an average molecular weight between 250,000 and 350,000
It is in. This HY yield is equal to 0.6% by weight of the initial fresh tissue.   Example 21 From the mixture obtained by the method described in Example 20,
How to obtain fractions   The mixture obtained by the method described in Example 20 is combined with 10 mg of product per ml of water.
And dissolved in pyrogen-free water distilled twice. The resulting solution
, Subjected to molecular filtration through a filtration membrane with a rejection limit molecular weight of 200000, and then added water
Concentrate on membranes without. Ultrafiltration using a membrane with a rejection limit molecular weight of 200000
In the process of passing, molecules with a molecular weight of more than 200,000 do not pass, while smaller
The child passes through the membrane with the water. During the filtration operation, no water was added, so the volume was
The concentration of molecules having a molecular weight of 200,000 or more increases. The capacity on the membrane is the first
Ultrafiltrate the product to 10% of the volume. Does not contain pyrogens, 2
Add 2 volumes of double distilled water and ultrafiltrate the solution again until the volume is reduced to 1/3
. This operation is repeated twice more. The solution passed through the membrane was washed with sodium chloride in 0.1.
M and then precipitated using 4 volumes of 95% ethanol. Etano the precipitate
Wash three times with a tool (75%) and then vacuum dry.   The product (hyalastin fraction) thus obtained has a content of 50,000-100,000
Having an average molecular weight of between The HY yield was equal to 0.4% by weight of the initial fresh tissue.
No.   Example 22 Method for obtaining a hyalectin fraction   According to Example 21, collection on an ultrafiltration membrane having an exclusion limit molecular weight of 200,000 in a container
The resulting concentrated solution is diluted with water and used for quantitative analysis based on glucuronic acid dose.
A solution containing 5 mg / ml of hyaluronic acid is determined, as determined by: This solution is 0.1M with respect to the aqueous lithium solution, then precipitated with 95% ethanol volume.
You. The precipitate is washed three times with ethanol (75%) and dried under vacuum.   The product (hyalectin fraction) thus obtained is between 500000 and 73000
Having a molecular weight of This is a high purity standard consisting of about 2500-3500 sugar units.
It corresponds to a specific fraction of hyaluronic acid having a constant molecular chain length.   The HY yield is equal to 0.2% by weight of the initial fresh tissue. [Method B]   The present invention further provides novel hyaluronic acid salts starting from barium hyaluronate.
It relates to a manufacturing method. This new method uses water-soluble salts, especially active substances.
For the associated hyaluronate, where all the hyaluronic acid
The boxy group is in the form of a salt or only a part thereof is in the form of a salt. Partial salt
In, the remaining carboxy groups of hyaluronic acid are free or have other activities.
Substances or alkali metals, magnesium, aluminum, ammonium
Alternatively, the salt may be formed by substituted ammonium.   This new method produces an aqueous solution of barium salt of hyaluronic acid, and
Or partially salted by more organic or inorganic bases.
The addition of an aqueous solution containing a large number of sulfates, where the number of sulfates is
It corresponds to the number of hyaluronic acid values present in the aqueous solution of the sodium salt. Hyaluronate water
The solution is concentrated by filtration of the separated barium sulfate and concentration of the salt in dry form.
An obtainable aqueous solution of hyaluronate can be obtained.   The barium salt of hyaluronic acid has not been described in the literature and, surprisingly,
It turned out to be soluble. This is the less tolerated hyaluronic acid cetylpi
Treat the lydinium salt with an aqueous barium chloride solution and add ethanol or other suitable
The volume is reduced by precipitating the barium hyaluronate from this solution using a solvent.
It can be easily manufactured. Cetylpyridinium salts of hyaluronic acid are available in various forms.
Separation and purification of hyaluronic acid extracted from organic materials
Is a commonly used intermediate.   A number of salts, wholly or partially salted by one or more organic bases;
For an aqueous solution containing a sulfuric acid value, dissolve the neutral sulfate of the base in water or add sulfuric acid
Manufactured by The number of hyaluronic acid values present in barium salt aqueous solution
Neutral sulfuric acid of one or more organic or inorganic bases containing a corresponding number of sulfuric acid values
If there is a solution consisting of salts, this result is present in the corresponding aqueous solution (sulphate)
It will be a stoichiometric neutral salt of the base and hyaluronic acid. If hyaluronic acid
If a stoichiometric partial salt or acid salt is desired, sulfuric acid is added to the aqueous sulfate solution.
Should be used or a basic sulfate should be used.   The method B according to the present invention can be exemplified by the following examples.   Example 23 Hyalastin and barium salt in the form of no inflammatory activity
For obtaining a mixture of fractions   Fresh or frozen chicken comb (3000g) is minced with a meat grinder and then
Carefully homogenize with a mechanical homogenizer. The resulting paste is
10 volumes of anhydrous acetone in a stainless steel container (AISI 316) or glassware
To process. The whole is stirred at a speed of 50 rpm for 6 hours. Leave this for 12 hours
And discard acetone by siphoning. Aceto to be discarded
Acetone extraction until the water content of the solution reaches the correct ratio (Karl Fischer method).
to continue. Next, the whole amount is centrifuged and vacuum dried at an appropriate temperature for 5 to 8 hours. Thus chicken
Approximately 500-600 g of dry powder of the crust are obtained.   Expose 300 g of the dry powder to enzymatic digestion with papain (0.2 g) under aqueous conditions,
Buffer in a phosphate buffer in the presence of an appropriate amount of cysteine hydrochloride. Got
The material is stirred at 60 rpm for 24 hours while maintaining a constant temperature of 60-65 ° C. Then
The whole was cooled to 25 ° C., celite (60 g) was added, and stirring was continued for another hour.
I can. The mixture is filtered to obtain a clear liquid. The clear liquid is treated as the exclusion limit molecular weight.
Subject to molecular ultrafiltration using 30,000 membranes. Distilled water is continuously added to the ultrafiltration product
Ultrafiltration of the initial volume of 5-6 volumes of product, while adding. Stop adding water
Stop and continue ultrafiltration until reduced to 1/3 of original volume.   The remaining liquid is brought to 0.1 M by adding barium chloride and the temperature is brought to 50 ° C.
. Cetylpyridinium chloride is added while stirring at 60 rpm. Solution for 60 minutes
And then add 50 g of celite. Keep the temperature at 25 ° C with stirring
The precipitate formed by centrifugation is collected. The precipitate was washed with cetylpyridinium chloride 0
Suspend in 5 l of a 0.01 M solution of barium chloride containing 0.05%. At 50 ° C
Stir for 60 minutes, then centrifuge the precipitate at 25 ° C. 3 washing steps
Times and finally the precipitate is washed with chloride containing 0.05% of cetylpyridinium chloride.
Collect in a container containing 3 l of a 0.05 M solution of barium. At 60 rpm
Shake for 60 minutes and maintain at a constant temperature of 25 ° C. for 2 hours. Centrifuge the clear supernatant
Remove.   Use 0.1M barium chloride solution containing 0.05% cetylpyridinium chloride
And repeat this step several times. Centrifuge the mixture and discard the supernatant. The precipitate,
A 0.30 M solution of barium chloride containing 0.05% cetylpyridinium chloride (3 l
). The mixture is stirred and both the precipitate and the clear liquid are collected. same
The precipitate is extracted three more times, each time using 0.5 l of aqueous solution.   Finally, the precipitate residue is removed and the clear liquid is collected in one container. Stirring constantly
While the temperature of the liquid is being brought to 50 ° C. The liquid is then 0.2 bar with barium chloride.
3M. Add 1 g of cetylpyridinium chloride and keep stirring for 12 hours
You. The mixture is cooled to 25 ° C. and filtered first on celite and then on filter paper. Next
This was again subjected to molecular ultrafiltration with a membrane having a molecular weight of exclusion limit of 30,000 to obtain a 0.33M
Ultrafiltrate to 3 times its original volume by adding barium chloride solution. Barium chloride
Stop adding the solution and reduce the volume to 1/4 of the original volume. The concentrated solution is
Precipitation with stirring (60 rpm) at 25 ° C. using 3 volumes of ethanol (95%)
Let it. Collect the precipitate by centrifugation and discard the supernatant. The precipitate is treated with barium chloride
Dissolve in 1 liter of a 1M solution and repeat the precipitation using 3 volumes of ethanol (95%).   The precipitate was collected, first with 75% ethanol three times, then with absolute ethanol (three times),
And finally, it is washed with anhydrous acetone (3 times). The product thus obtained (hyalara (Stin + hyalectin fraction) has an average molecular weight between 250,000 and 350,000
Have. The yield of HY corresponds to 0.6% of the initial fresh tissue.   Example 24 Barium salt in the mixture obtained by the method described in Example 30
To Obtain a Hyalastin Fraction in the Form of   The mixture obtained by the method described in Example 23 is combined with the product 10
Dissolve in pyrogen-free distilled water in mg amounts. In the solution thus obtained,
Perform molecular filtration using a membrane with a molecular weight of exclusion limit of 200000, and then add water on the membrane.
Concentrate without adding. Ultrafiltration process using a membrane with the exclusion limit molecular weight of 200000
And molecules with a molecular weight of 200,000 or more are trapped, while smaller ones
Both pass through the membrane. During the filtration process, no water is added onto the membrane, resulting in a reduced volume.
There is a slight increase in the concentration of molecules with a molecular weight above 200,000. Ultrafiltration
, Until the volume on the membrane is reduced to 10% of the original volume. Contains pyrogens
Add 2 volumes of no distilled water and ultrafilter the whole again until the volume is reduced to 1/3
You. This operation is repeated twice more. The solution that passes through the membrane is 0.1% with barium chloride.
M and then precipitated with 4 volumes of 95% ethanol. 75% ethanol in the precipitate
And then vacuum dry. The product thus obtained (hyalastin
Fraction) has an average molecular weight between 50,000 and 100,000. HY yield
, Equivalent to 0.4% of fresh starting tissue.   Example 25 Method for obtaining a hyalectin fraction in the form of a barium salt   According to Example 24, collection on an ultrafiltration membrane having an exclusion limit molecular weight of 200,000 in a container
The resulting concentrated solution is diluted with water and used for quantitative analysis based on glucuronic acid dose.
A solution containing 5 mg / ml of hyaluronic acid is determined, as determined by: This solution is
0.1M for water and then precipitated using 4 volumes of 95% ethanol.
. The precipitate is washed three times with 75% ethanol and then dried in vacuo. Thus obtained
The product (hyalectin fraction) has a molecular weight between 500000 and 730000
. The yield of HY is equal to 0.2% of fresh starting tissue.   Example 26 Preparation of hyaluronic acid (HY) salt with streptomycin   4.47 g (10 mEq) of the HY barium salt are dissolved in 300 ml of distilled water.   2.43 g (10 mEq) of streptomycin sulfate was dissolved in 100 ml of distilled water. The solution is added dropwise to the HY salt solution while stirring. Centrifuge the mixture at 6000 rpm for 30 minutes
To separate. The solution is separated and the precipitate is washed twice with 25 ml of distilled water. Solutions and washings
And freeze-dried. In the salt thus obtained, all the acid groups of hyaluronic acid are
The salt is a basic functional group of streptomycin. Yield: 5.5 g.   B. subtilis (B. subtilis) compared to standard streptomycin
The microbiological assay according to ATCC 6633) corresponds to the theoretical calculated weight.
8% by weight of basic streptomycin. Bitter method [A
Analytical Biochemistry, Vol. 4, p. 330, 19
62], the colorimetric determination of glucuronic acid bound to the polysaccharide was 66.2 weight.
% HY acid was shown to be present (theoretical percentage was 66.0%)
.   Example 27 Preparation of a salt of hyaluronic acid (HY) with naphazoline   HY barium salt having a molecular weight of 625,000 corresponding to 10 mEq of monomer units 4.4
7 g are dissolved in 400 ml of distilled water.   2.6 g of neutral naphazoline sulfate (10 mEq of sulfate) was dissolved in 50 ml of distilled water.
Add to barium salt solution. Mix this mixture until barium sulfate is completely precipitated.
Stir at 5 ° C. After centrifugation, the resulting solution is frozen and lyophilized immediately. Income
Amount: 5.72 g.   In the salt thus obtained, all the acid groups of the HY acid are converted into salts with naphazoline.
ing. Results of quantitative spectroscopic measurements compared to standard naphazoline (USP), theory
It was found to contain 35.7% by weight of basic naphazoline corresponding to the above calculated value.
Was. Colorimetry of glucuronic acid bound to polysaccharides, performed by the method of Bitter et al.
As a result of the quantification, it was shown that the HY acid content was 64.3%.   Example 28 Preparation of partial salt of hyaluronic acid (HY) with naphazoline   4. A barium salt of HY having a molecular weight of 625,000 corresponding to 10 mEq of monomer units.
47 g are dissolved in 400 ml of distilled water.   Dissolve 1.54 g of acid, naphazoline sulfate (10 mEq of sulfate) in 50 ml of double distilled water.
Add to HY barium salt solution. Mix until barium sulfate is completely precipitated Stir at 5 ° C. After centrifugation, freeze the resulting solution immediately and freeze-dry
. Yield: 4.5 g.   In the salt thus obtained, 50% of the acid groups of the HY acid are
It is salt and 50% is free. Compared to standard naphazoline (USP)
Weight of basic naphazoline corresponding to the theoretically calculated value as a result of quantitative spectroscopic measurement
Was shown.   Example 29 Preparation of a salt of hyaluronic acid (HY) with phenylephrine   2.16 g (10 mEq) of neutral L-phenylephrine sulfate are dissolved in 25 ml of distilled water.   HY barium salt having a molecular weight of 820000 corresponding to 10 mEq of monomer unit 4.
47 g are dissolved in 400 ml of distilled water and added to the solution of phenylephrine sulfate. Sulfuric acid bath
The mixture is stirred until the ium has completely precipitated. After centrifugation, freeze the resulting solution
Freeze and freeze dry. In the obtained salt, all of the acid groups of HY are phenylephryl.
It is salted by   Result of UV spectroscopic measurement performed by the standard addition method (USP), theoretical calculation
It was found to contain 30.6% basic phenylephrine corresponding to the value.   Colorimetric determination of glucuronic acid bound to polysaccharides by the method of Bitter et al.
It showed a HY acid content of 4%.   Example 30 Hyaluronan with neomycin, phenylephrine and sodium
Preparation of a mixture of acid (HY)   7.15 g of HY barium salt having a molecular weight of 65000 corresponding to 16 mEq of monomer units
Is dissolved in 400 ml of distilled water.   1.28 g (8.42 mEq) of neomycin sulfate are dissolved in 150 ml of distilled water. this
0.34 g (1.58 mEq) of neutral phenylephrine sulfate and Na were added to the solution._TwoSO_Four0.4
3 g (6 mEq) are added. The obtained solution is added to the HY barium salt solution,
The mixture is centrifuged after the complete precipitation of the calcium.   The barium sulfate is separated and the solution is frozen and freeze-dried. Yield: 7.35 g.   (Pharmacological considerations)   The technical effects of the novel pharmaceuticals according to the present invention are based on in vivo experiments with rabbit eyes.
Which is comparable to the use of component (1) when administered in a conventional manner.
Shows its superiority. As an example, the following antibiotics
Experiments performed with salts are reported below: Streptomycin, Erythroma
Isin, neomycin, gentamicin. These are all of hyaluronic acid
Example 2 is an entire salt wherein the acid group of the salt is salted by the basic group of the antibiotic,
1, 2, 4 and 5. Of these, depending on their antibiotic content
A solution of distilled water having the following suitable concentrations was used:   Hyaluronic acid + streptomycin (HYA1) 33.8%   Hyaluronic acid + erythromycin (HYA2) 66.0%   Hyaluronic acid + neomycin (HYA4) 21.2%   Hyaluronic acid + gentamicin (HYA5) 20.0%   The activity of these antibiotics is dissolved in phosphate buffer to give the same antibiotic concentration.
Compared to the activity shown for the same antibiotic. The activity of the two groups of products is
Based on the time required for suppression of dry eye inflammation in rabbit eyes caused by fungi
It was measured. More specifically, this dry inflammation was observed in the eyes of 24 rabbits with the following:
Induced by intraocular injection of a calibrated suspension of one of the bacterial groups:
Domonas aeruginosa, Staphylococcus aus
Reus (staphylococcus aureus), Salmonella typhi (salmonella typhi) (
0.1 ml).   Various salt derivatives of antibiotics were administered to the right eye (RE) of rabbits (3 drops every 6 hours)
On the other hand, the left eye (LE) was dripped with a corresponding amount of antibiotic dissolved in phosphate buffer.
. The treatment started immediately after injection of the bacterial suspension and continued until the inflammation had disappeared.
. Both eyes of each rabbit were observed using a slit lamp. In particular, we examined the following:
The condition of the corneal and corneal epithelium, the anterior chamber (the presence of the Tyndall effect), and the posterior part of the eye
Iris state. The condition behind the eyes was examined with a Goldman lens. Signs of inflammation (congestion
, Exudation, liquid cloudiness, etc.) were recorded. Then there are no signs of inflammation
I The eye percentage was calculated.   The results of this experiment are shown in Table 1, which shows the administration of the salt derivative according to the present invention.
Followed by the administration of a corresponding antibiotic that is not salted with hyaluronic acid.
It is shown that more rapid inflammation recovery is obtained.   The practical effects of the medicament according to the present invention include miosis, anti-inflammatory, wound healing and antibacterial activity
The following other experiments with are further substantiated.   I. Miotic activity of pilocarpine nitrate supported on hyaluronic acid 〔material〕   For various formulations of pilocarpine nitrate, the following
Used substance:   Hyaluronic acid sodium salt, hyalastin fraction (molecular weight about 100,000), 10
concentrations of mg / ml and 20 mg / ml;   Hyaluronic acid sodium salt, hyalectin fraction (molecular weight 500000 to 730)
000) at a concentration of 10 mg / ml and 20 mg / ml;   5% polyvinyl alcohol as comparative ophthalmic vehicle.   Various formulations (corylium or gel) of 2% pilocarpine nitrate were prepared and two
Adding different HY sodium salt fractions at concentrations of 10 and 20 mg / ml
Therefore, it was carried. The following solutions were prepared:   Formulation 1. ……… Pilocarpine nitrate used as a control (PiNo_Three) (2%)
Saline   Formulation 2. ............ Supported on polyvinyl alcohol% used as a control (PiN
o_Three) Solution (2%).   Formulation 3. ... supported on hyalastin fraction sodium salt (10 mg / ml) (PiN
o_Three) Solution (2%).   Formulation 4. ... supported on the sodium salt of hyalastin fraction (20 mg / ml) (PiN
o_Three) Solution (2%).   Formulation 5. ... supported on sodium salt of hyalectin fraction (10 mg / ml) (PiN
o_Three) Solution (2%).   Formulation 6. ... supported on hyalectin fraction sodium salt (20 mg / ml) (PiN
o_Three) Solution (2%). 〔Method〕   Albino New Zealand rabbits were used (2-2.5 kg). The test preparation is
Each rabbit was instilled into one eye of each rabbit using an icrosy syringe (10 mcl). The other eye
Used as a control. In all cases, the pupil diameter should be
It was measured. Each solution was tested on at least eight rabbits. Each
Eyes undergo up to three treatments. At least one week rest between each treatment
Was. [Parameter]   Measurement of pupil diameter at various intervals to determine the miotic activity curve over time
Was performed. Next, the following activity parameters were calculated from the miosis / time graph.
Was:   Imax = maximum difference in pupil diameter between treated and control eyes   Maximum peak time = time required to reach Imax   Persistence = time required to return to the original state   Stabilization time = period of pure miotic activity   AUC = area under the curve of miosis / time 〔result〕   Table 2 shows the results of this experiment. For all solutions tested, miotic activity
From data obtained from various parameters determined by the time curve,
Addition of a 2% solution of pilocarpine nitrate to acid results in increased miotic activity of the drug
You can see that In fact, the bioavailability of the drug includes 2% pilocarpine nitrate.
It can increase 2.7 times of the aqueous solution (formulation 1).   Furthermore, 10 and 20 mg / ml of the hyalectin fraction of hyaluronic acid was used as a medium.
When used (formulations 5-6), pilocarpi nitrate supported on polyvinyl alcohol
Note that there is a statistically significant increase in activity compared to the control solution (Formulation 2)
This is very important. The miotic activity of pilocarpine nitrate is supported on hyaluronic acid
Use of hyaluronic acid as a medium
Is particularly interesting. That is, for preparations containing hyaluronic acid, the pupil diameter The time required for the drug to return to its original state was pilocarpine (formulation 1) in saline only.
Is 110 minutes or more, which is 190 minutes or more (formulation 6).   II. Miotic activity of pilocarpine salted with hyaluronic acid 〔material〕   The following products were used for various formulations of salted pilocarpine:   Hyaluronic acid of low molecular weight (hyalastin, m.w. 100,000 [HY₁];   High molecular weight hyaluronic acid sodium salt (Hairale) at concentrations of 10 mg / ml and 20 mg / ml
Kuching, m. w.between 500000 and 730000) [HY_Two-Na];   5% polyvinyl alcohol as an ophthalmic vehicle for a control formulation.   The various formulations produced are as follows:   1) Pilocarpine nitrate (PiNo)_Three) Saline containing 2% (used as control);   2) PiNo supported on 5% polyvinyl alcohol_Three  2% solution (as control
use);   3) Pilocarpine base / HY₁A solution of an aqueous acid solution. 2% pilocarpine base content
Equivalent to;   4) HY_Two-Pilocarpine salt supported on 10 mg / ml Na / HY₁Contains acid
solution. Pilocarpine base content equals 2%;   5) HY_Two-Pilocarpine salt supported on 20 mg / ml Na / HY₁Acid containing solution.
Pilocarpine base content equal to 2%;   6) Hyaluronic acid [HY₁HY containing pilocarpine basic salt containing_Two-Na
Insertion agent. Pilocarpine base corresponds to 6.25%. 〔Method〕   Albino New Zealand rabbits were used (2-2.5 kg). The test solution was
Each rabbit was instilled into one eye (10 μl) with an icrosy syringe while the other eye was
Used as a control. The insert was inserted into the conjunctival sac using appropriate forceps. All cases
In, the pupil diameter was measured at appropriate intervals. Each formulation contains at least 8 rabbits
Was tested. Each eye performs up to three treatments, with few during each treatment.
I had a rest period of at least one week. [Parameter]   Determine the miotic activity curve against time, and then follow the miosis / time graph
The pupil diameter at various time intervals to calculate the following activity parameters:
did:   Imax = maximum difference in pupil diameter between treated and control eyes;   Peak time = time required to reach Imax; duration = time required to return to original state
Time to do;   Stabilization time = period of pure miotic activity;   AUC = area under the curve of miosis / time. 〔result〕   As can be seen from Table 3, for each test solution, the curve of miotic activity versus time
The values of the various parameters recorded from pilocarpine 2% hyaluronic acid
Can show how salt formation by the drug results in increased miotic activity of the drug.
Its activity reaches about twice that of an aqueous solution containing 2% of pilocarpine nitrate (Formulation 1)
You.   In addition, high molecular weight hyaluronic acid was used as 10 and 20 mg / ml medium (manufactured by
When the agents 4 to 5) are used, a statistically significant increase in activity is observed.   Salinization by hyaluronic acid causes miosis of pilocarpine after loading with such a formulation
It is particularly interesting in view of its longer lasting activity. In the original state
The time required to return to normal pupil diameter was 110 for pilocarpine (Formulation 1).
The value reaches 160 minutes (formulation 3) compared to the minutes.   III. Corneal coating stability of hyaluronic acid and pilocarpine derivatives   The purpose of this experiment was to apply pilocarpine and hyaluronan after application to the cornea of the animal.
The purpose is to evaluate the adhesion and filmogenicity of the chloride derivative of the acid. 〔Method〕   This test measures the formation, stability and survival of the capsule formed by the formulation on the cornea.
It consists of a visual assessment. Sodium fluorescein for this purpose
It was added to the ophthalmic formulation (0.1%) and the eyes were examined after dripping with UV light at 366 nm.   Twelve albino rabbits were all used in both sexes (New Zealand, 2-2.5).
kg). One drop (50 μl) of each medium was dropped on one eye of each rabbit, and the other eye was used as a control.
Used. (Solution used)   1. Pilocarpine nitrate (PiNo)_Three) 2% saline;   2. PiNo increased with 5% polyvinyl alcohol_Three 2% solution (wacker
Chemie, PVA W48 / 20);   3. Pilocarpine hydrochloride / HY₁Solution containing acid. Contains pilocarpine base
The amount corresponds to 2%;   4. HY_Two-Pilocarpine hydrochloride / HY using Na as a medium 10 mg / ml₁Contains acid
Solution. Pilocarpine base content equals 2%;   5. HY_Two-Na 20 mg / ml pilocarpine hydrochloride / HY₁Contains acid
Solution. The content of pilocarpine base corresponds to 2%.   All solutions contained 0.1% sodium fluorescein. In all cases
The pH of the solution was around 5.8. 〔result〕   Parameters related to fluorescence: a) persistence of complete corneal capsule, b) persistence of fluorescence (eye
The time required for the fluorescence to completely disappear) and c) the presence of fluorescence in the nose (
Table 4 shows the time required to appear at the nasal level after administration.   Derivatives of hyaluronic acid, including pilocarpine, are stable corneas for more than 2 hours. Generate a coating. Therefore, the corneal permeability of pilocarpine depends on the medium of this drug.
Depends on the ability of hyaluronic acid to form a uniform and stable coating on the cornea
Seem.   IV. Anti-inflammatory activity of triamcinolone supported on hyaluronic acid 〔material〕   I used the following:   Hyaluronic acid sodium salt hyalectin fraction solution (mw 500000-7)
Between 3000), 10 mg / ml in saline;   Triamcinolone phosphate solution (10% in saline). 〔Method〕   The experiment was performed on male New Zealand rabbits (average body weight 1.6 kg). 5 days
After an adaptation period, dextran (10%, 0.1 ml) was injected into the anterior chamber.
Induced intraocular inflammation in animals. Administration is under the condition of local anesthesia with Novesin 4%
The procedure was performed on both eyes by inserting the needle of the syringe into the anterior chamber of the eye at the end of the cornea at a distance of 2 mm.   The test was performed on 10 animals. 〔treatment〕   Treatment consisted of three drops on the right and left eyes of each animal three times daily for a total of 6 days.
Performed by:   A solution of triamcinolone phosphate (10% in saline) to the left eye (LE),   Hyaluronic acid sodium salt hyalectin fraction (10 mg / ml) +
Solution of triamcinolone acid (10%). [Parameter]   The anti-inflammatory effect on the response elicited by dextran
Evaluation was made by observing the eyes through a light: 0, 1 hour, 3 hours, 24 hours,
48 hours, 3 days, 4 days, 5 days, 6 days.   The following were observed at intervals:   Conjunctiva and cornea (in case of hyperemia, edema,
About), and especially, the condition of the iris, which is usually sensitive to inflammatory processes;   The presence of the Tyndall effect (opacity of varying intensity (hemicloud) indicates the presence of body (flame) in the anterior chamber.
Symptomatic component).   Observations are scored by a subjective score of 0-3 associated with the observed effect.
. 〔result〕   As reported in Table 5, administration of triamcinolone had an anti-inflammatory effect on the iris.
And the loss of opacity (Tindal effect) in the anterior chamber of the eye
You. The inflammatory process, which is evident from 1-3 hours to 3-4 days, gradually disappears and 6
On day one, the eyes return to a completely transparent, almost normal degree. In contrast, phosphorus
Hyaluronic acid sodium salt hyalectin fraction with triamcinolone acid
The administered dose was observed at the time indicated above compared to the administration of triamcinolone phosphate alone.
It reduces the apparent intraocular inflammation. That is, the progression of inflammation in the iris and in the anterior chamber
The opacity of the decay decreased as early as 24 hours, gradually decreased at 48 hours, and after 4 days
The inflammatory response proved to be completely abolished.   In the conjunctiva and cornea, noteworthy after injection of dextran into the anterior chamber
No inflammatory response was basically observed.   Thus, administration of triamcinolone phosphate with the hyaluronic acid fraction
Increases the activity of the drug as evidenced by more rapid decongestion of the rabbit eye Is brought.   V. Healing activity of EGF supported on hyaluronic acid 〔material〕   I used the following:   Formulation A: EGF (epidermal growth factor) dissolved in saline (0.5 mg / ml),   Formulation B: Hyaluronic acid sodium salt Hyalus dissolved in saline (10 mg / ml)
Tin fraction (mw 100,000). 〔Method〕   The experiment was performed on male New Zealand albino rabbits (average weight 1.8 kg).
Was. After an adaptation period of about 5 days, the animals are given appropriate local anesthesia with Novesin (4%)
Wound the corneal epithelium under conditions.   This wound is present by monocular keratotomy of a circular part in the visual field, which has a sharp blade.
This was performed using a concave glass cylinder (0.3 mm) having 〔treatment〕   The animals were subdivided to contain 5 animals in each group, and these were then
It was subjected to pharmacological treatment by instillation into the conjunctiva:   Group treatment   1 group (control) saline   Group 2 EGF solution (Formulation A)   Group 3 HY sodium salt solution hyalastin fraction +               EGF solution: Formulation A + Formulation B in a 1: 1 ratio   Formulation C is made in combination.   The right eye (RE) is treated by administering 2 drops every 8 hours and 3 drops in total to the conjunctiva.
Was placed. [Parameter]   At various intervals after the ablation, eye observation using slit lamps and photographic considerations are performed.
The healing of the corneal epithelium was assessed as follows: 0.8 h, 16 h, 24 h, 3 h
2 hours, 40 hours, 48 hours. 〔result〕   The eye test 1 reported in Table 6 shows that the control (group 1) was completely 48 hours after injury
Healing (5/5 animals). In animals treated with EGF (Group 2)
This effect is accompanied by a noteworthy effect as early as 24 hours after the morbidity (4/5 animals)
Appear. Formulation consisting of hyaluronic acid sodium salt, hyalastin fraction + EGF
In the animals treated with C (group 3), the healing effect was as early as 16 hours after the truncation.
Is also complete for all animals (5/5).   These results demonstrate the use of the hyaluronic acid hyalastin fraction as a medium for EGF.
Can accelerate the healing process and promote faster and more effective healing of corneal wounds
Is shown.   VI. Antibacterial activity of gentamicin supported by hyaluronic acid 〔material〕   I used the following:   Gentamicin dissolved in saline (50 mg / ml),   Hyaluronic acid sodium salt, hyalectin fraction (2 mg / ml). 〔Method〕   A calibrated suspension of pseudomonas aeruginosa (0
.1 ml) induced bacterial inflammation in both eyes of 11 rabbits
did. Hyaluron combined with gentamicin in rabbits with bacterial inflammation
The acid hyalectin fraction is administered by instillation into the right eye and the gel in phosphate medium is administered.
Tantamycin was administered to the left eye. This treatment (3 drops every 6 hours) is performed after injection of the infectious agent
Started immediately and continued until the infection disappeared. Watch the rabbit's eyes every day using a slit lamp
I thought. 〔result〕   Treatment with a combination of gentamicin and hyaluronic acid is a treatment of this antibiotic alone.
This led to a more rapid elimination of the bacterial infection when compared to administration. This conclusion is reported in Table 7.
It is clear from the data.   From the foregoing, it will be apparent that the invention can be varied in many ways.
U. Such modifications should not be deemed to depart from the spirit and scope of the present invention and
All such variations as would be apparent to one skilled in the art are included within the scope of the appended claims.
Is intended to be   The present invention includes the following embodiments. (1) Ophthalmology using active substances absorbed intradermally or through the nasal or rectal mucosa
Treat symptoms, dermatological symptoms, oral and nasal or outer ear mucosal disorders and
14. A medical device according to any one of claims 1 to 13 for treating a governmental disease.
A therapeutic or prophylactic method comprising administering a drug.

Claims (1)

Claims: (1) A drug for topical administration, which comprises a salt of hyaluronic acid and a basic pharmaceutically active substance as an active ingredient and has improved bioavailability of the pharmaceutically active substance. (2) The medicament according to claim 1, wherein the salt is present in the form of a partial or stoichiometric neutral salt between the pharmaceutically active substance and hyaluronic acid. (3) The drug according to any one of claims 1 to 2, which is absorbed transdermally or nasally or via the rectal mucosa. (4) The drug according to any one of claims 1 to 3, further comprising an excipient suitable for topical administration. (5) The method according to any one of claims 1 to 4, wherein the salt is a partial salt, and at least a part of the acid groups of the hyaluronic acid is salified with an alkali metal or an alkaline earth metal, magnesium, aluminum or ammonium. The drug according to any one of the above. (6) Claims 1 to 1, wherein the pharmaceutically active substance is suitable for ophthalmic use.
Item 6. The drug according to any one of Items 4 to 5. (7) The medicament according to any one of claims 1 to 5, wherein the pharmaceutically active substance is suitable for dermatology, otolaryngology, obstetrics, vasculature or neurological use. (8) pharmaceutically active substances are antibiotics, anti-infectives, antibacterials, antivirals, cytostatics, antitumors, anti-inflammatorys, wound healing agents, anesthetics, cholinergic agents, cholinergic antagonists, The drug according to any one of claims 1 to 7, comprising an adrenergic drug or an adrenergic antagonist. (9) The pharmaceutically active substance is aureomycin, gentamicin, neomycin, streptomycin, dihydrostreptomycin, kanamycin, amikacin, tobramycin, spectinomycin, erythromycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, lolitetracycline, bacitracin, Polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin, vancomycin, ristocetin, clindamycin, diethylcarbamazine, mebendazole, sulfacetamide, sulfadiazine, sulfisoxazole, iodooxyuridine, arabinosyladenine, trifluoroimidine , Acyclovir, ethyl deoxyuridine, pyrocarbine, methacholine, cal Bamylcholine, aceclidine, physostigmine, neostigmion, deme potassium, atropima, noradrenaline, adrenaline, norfazoline, methoxamine, propanolol, timolol, pindolol, bupranolol, atenolol, metroprolol, oxyprenolol, plactorol, butoxamine, sotalol, sotalol 9. The drug according to claim 8, which is selected from the group consisting of labetalol, methotrexate and podophylline. (10) The drug according to any one of claims 1 to 7, wherein the pharmaceutically active substance is selected from the group consisting of streptomycin, erythromycin, kanamycin, neomycin, gentamicin, pilocarpine and epidermal growth factor. (11) The agent according to any one of claims 1 to 10, wherein the hyaluronic acid is a fraction substantially free of hyaluronic acid having a molecular weight of less than 30,000. (12) The drug according to claim 11, wherein the fraction has an average molecular weight of 50,000 to 100,000. (13) the average molecular weight of the fraction is from 500,000 to 730,000,
The medicament according to claim 11, wherein: (14) The salt is prepared by combining (a) an aqueous solution of barium hyaluronate and a sulfate of a pharmaceutically active substance, and (b) removing the precipitated barium sulfate to obtain a hyaluronate in the aqueous solution. The drug according to claim 1. (15) The stoichiometrically neutral hyaluronate is obtained in (a) by adding the sulfate in an amount such that the equivalent number of the sulfate is equal to the equivalent number of the hyaluronic acid. Item 14. The drug according to Item 14. (16) In (a), a sulfated salt is added in an amount such that the equivalent number of the sulfate is less than the equivalent number of the hyaluronic acid to obtain a partially salified hyaluronate;
The medicament according to claim 14, wherein the medicament is used. (17) The barium salt of hyaluronic acid further comprises at least one selected from the group consisting of alkali metals and alkaline earth metals, aluminum and ammonium.
17. A medicament according to any of claims 14 to 16 in combination with a species sulphate. (18) The agent according to claim 17, wherein the sulfate is added in such an amount that the equivalent number of total sulfates is equal to the equivalent number of hyaluronic acid. (19) The drug according to claim 17, wherein the sulfate is added in such an amount that the equivalent number of total sulfate is less than the equivalent number of hyaluronic acid. (20) The pharmaceutically active substance is erythromycin, gentamicin, neomycin,
Streptomycin, dihydrostreptomycin, kanamycin, amikacin, tobramycin, aureomycin, spectinomycin, oleandomycin, carbomycin, spiramycin, oxytetracycline, lolitetracycline, bacitracin, polymyxin B, gramicidin, colistin, chloramphenicol, lincomycin , Diethylcarbamazine, mebendazole, sulfacetamide, sulfaziazine, sulfisoxazole, iodooxyuridine, arabinosyladenine, tricarpine, methacholine, carbamylcholine, aceclidine, fusostigmine, neostigmine, demepotassium, atropine,
Propanolol, timolol, pindolol, bupranolol, atenolol,
Metoprolol, oxprenolol, practol, butoxamine, sotalol, butadrine, butatarol, selected from the group consisting of methotrexate and podophylline, according to any one of claims 14 to 19, wherein Drugs. (21) The hyaluronic acid has a total hyaluronic acid molecular weight of 13,000,000 and is about 90 to 90%.
20. An agent according to any of claims 14 to 19, which is a fraction having a molecular weight between 80% and 0.23%. (22) The agent according to claim 21, wherein the fraction is substantially free of hyaluronic acid having a molecular weight of less than 30,000. (23) The average molecular weight of the fraction is from 50,000 to 100,000, 500,
23. The agent according to claim 22, wherein the agent is from 000 to 730,000 or 250,000 to 350,000.