Hyaluronic acid fractions having pharmaceutical activity, methods for preparation thereof, and pharmaceutical compositions containing the same.

DESCRIPTIVE MEMORY

SUBMISSION IN SUPPORT OF A REQUEST

OF INVENTION PATENT

TO THE GRAND DUCHY OF LUXEMBOURG by;

FIDIA SpA

forj Hyaluronic acid fractions having pharmaceutical activity, processes for their preparation and pharmaceutical compositions containing them.

HYALURONIC ACID FRACTIONS HAVING PHARMACEUTICAL ACTIVITY, METHODS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

The invention relates to specific molecular weight moieties of hyaluronic acid (hereinafter referred to as HA) which have therapeutic applications and which are non-inflammatory. One of the HA fractions according to the invention is useful in facilitating wound healing, while the second HA fraction has intraocular application, and can be substituted for endobulbar fluids or be used as intraocular injection. joint for the treatment of damaged bone joints. HA fractions have further been shown to be useful as a carrier for drugs for ophthalmic applications, providing formulations compatible with the corneal epithelium and enhancing the activity of ophthalmic drugs.

Hyaluronic acid is a naturally occurring heteropolysaccharide consisting of alternating residues of D-glucoronic acid and N-acetyl-D-glucosamine. HA is a high molecular weight linear polymer, typically about 8 to 13 million, and is found in the lining of cells, the extracellular basic substance of vertebrate connective tissues, in the synovial fluid of the joints. , in endobulbar fluids of the eye, in human umbilical cord tissue, and in roosters' ridges.

Previous studies on the use of HA are described in the work of Balazs, (US Pat. No. 4,141,973) describing a fraction of HA useful for the replacement of endobulbar fluids as well as for the replacement of endobulbar fluids. other therapeutic applications. This patent is, however, directed specifically to a fraction of HA having an average molecular weight greater than about 750,000 and preferably greater than about 1,200,000. Balazs teaches, specifically, that HA fractions having an average molecular weight of less than 750,000 cannot be used therapeutically because of their inflammatory activity. These lower molecular weight HA fractions are rejected by Balazs. However, this amounts to rejecting about 90% of the total amount of HA available obtainable from the tissues, and amounts to using only a small amount (approx.

10%) of the HA available.

Contrary to the teachings of Balazs, we have found that the lower molecular weight HA fractions, in fact, have useful pharmaceutical activity. Thus, according to the present invention, about 80% of the HA obtainable from various sources is used. In particular, the Applicant has discovered a fraction of HA which is useful for stimulating the healing of wounds, and a second fraction of HA which can be used for intra-ocular injection, and can replace endobulbar fluids in the eye and is useful as intra-articular injections for the treatment of damaged joints.

FIG. 1 is a curve showing the different fractions of hyaluronic acid which have been identified by the Applicant.

As discussed above, previous studies on HA such as, for example, those of the Balazs patent have been directed towards the use of high molecular weight fractions having an average molecular weight greater than 750,000. Applicants have isolated and characterized two novel fractions of HA, one having a low molecular weight and the other having a medium molecular weight, which are considered to be substantially pure and not having inflammatory activity. These new HA fractions can be obtained from various connective tissues which contain extractable amounts of hyaluronic acid. The specific fractions of the product according to the invention are differentiated and separated according to molecular filtration techniques.

The first fraction isolated was named hyalastin and it has an average molecular weight of about 50,000 to about 100,000. This fraction of hyalastin has been determined to be suitable for therapeutic, veterinary and human use because of its healing activity. sores. The second fraction isolated was named hyalectin and has an average molecular weight of about 500,000 to about 730,000. This hyalectin fraction is capable of being used in eye surgery as a substitute for endobulbar fluids and in veterinary and human therapy for trauma and degenerative diseases of the joints.

Hyalastin can be administered either as an intradermal injection or as a topical application for wound healing. Hyalec5 tin, on the other hand, is suitable for intra-ocular and intra-articular injections.

We have made extensive studies on the various fractions of HA and have, significantly more precisely than before, specifically determined the therapeutically useful fractions of HA and the inflammatory and non-useful fractions of HA. These studies have had, for results, to allow the identification and the investigation of two specific characteristics of the fractions of HA, namely the mobilizing activity of the cells and the intrinsic viscosity. The wound healing process in animals is facilitated by cell mobilization and, in particular, the mobilization of fibroblasts. On the other hand, cell mobilization or proliferative activity (i.e. mitosis) should be avoided in cases of surgery inside the eyeball. This is particularly true in operations intended to correct retinal detachment where an increased rate of healing could have deleterious effects.

Intrinsic viscosity is also an important parameter to consider in determining the utility of an HA fraction. A fraction having a high intrinsic viscosity is useful in surgery for the treatment of trauma and degenerative diseases of the joints and in the replacement of endobulbar fluids. On the other hand, high viscosity is an undesirable characteristic of fractions intended for use as a medicament for facilitating wound healing. In fact, the fractions to be used in wound healing should have a low viscosity in order to be more easily used in practical application.

It has been found that the hyalastin fraction identified by the Applicant has good cell mobilization or proliferation activity and low viscosity characteristics. Therefore, hyalastin exhibits desirable characteristics for a material subsequent to promoting wound healing.

The same characteristics make the hyalastin fraction undesirable for treatments comprising intra-ocular or intra-articular injection.

It has further been found that the hyalectin fraction identified by the Applicant has negligible cell mobilization or proliferation activity, while at the same time it has a high viscosity. These characteristics therefore make the hyalectin fraction useful for treatments requiring intra-ocular or intra-articular injections. But, on the other hand, hyalectin cannot be suitable for wound healing treatments because this fraction does not exhibit cell mobilization activity.

When isolating useful fractions of hyaluronic acid, it is also important to obtain those fractions which do not have inflammatory activity. The above-mentioned Balazs patent teaches that in order to obtain fractions of hyaluronic acid without inflammatory activity, one should use only fractions having an average molecular weight of more than 750,000. Thus, Balazs rejects fractions having an average molecular weight less than 750,000 as not useful because of their inflammatory activity. Contrary to the teachings of Balazs, we have found that the inflammatory activity attributed by Balazs to fractions having an average molecular weight less than 750,000 was in fact due to impurities having an average molecular weight less than 30,000. Thus, the present invention provides a method which comprises a series of molecular filtration techniques combined with chemical methods allowing the removal of inflammatory moieties having a molecular weight of less than 30,000.

By the methods according to the invention, it is possible to obtain useful fractions of hyaluronic acid having no inflammatory activity and which, when taken together, constitute a total yield of about 80% of total hyaluronic acid available from the particular starting materials. This 80% yield of available hyaluronic acid comprises a combined fraction which is a combination of the hyalectin and hyalastin fractions and which has an average molecular weight of from about 250,000 to about 350,000. More specifically, the hyalectin fraction is. obtained with a yield of about 30% of the HA available and the hyalastin fraction is obtained with a yield of about 50% of the HA available from the starting tissues.

This factor represents a significant improvement over the process of the Balazs patent discussed above in that Applicants have found that significantly increased amounts of the available hyaluronic acid are pharmaceutically useful. By using only the fraction having an average molecular weight greater than 750,000, the Balazs process only leads to a yield of about 10% relative to the original hyaluronic acid obtained from animal organs and it rejects about 90% of the available hyaluronic acid. Thus, according to the present invention, the use of the total hyaluronic extract is greatly increased.

A comparison of the relative yields of the extraction of the various hyaluronic acid fractions is shown below in Table I.

TABLE I

<td>type of hyaluronic acid</td><td>g per 100 g of fresh tissue</td><td> %</td><td>reference</td>

<td>total hyaluronic acid from roosters' ridges</td><td> 0,08</td><td> 100</td><td>Swann DA 1968</td>

<td>HA (Balazs type)</td><td> 0,08</td><td> 10</td><td>Biochim, Biophys. Acta 156, 17-29 US patent</td>

<td>hyalectin + hamastin</td><td> 0,6</td><td> 80</td><td>n ° 4 141 973 present invention</td>

<td>hyalectin</td><td> 0,2</td><td> 30</td><td>present invention</td>

<td>hyalastine</td><td> 0,4</td><td> 50</td><td>present invention</td>

<td>inflammatory fraction</td><td> 0,16</td><td> 20</td><td>present invention</td>

FIG. 1 represents, graphically, the different fractions of HA that the applicant has identified. The solid, bell-shaped curve in Figure 1 represents the approximate distribution of the HA fractions available from a starting tissue. Zone B of Figure 1 represents the fraction of HA identified by Balazs in US Pat. No. 4,143,973 as being useful in pharmacy. Zones A, I and II in FIG. 1 are the fractions identified by the Applicant, zone A being the inflammatory fraction having an average molecular weight of less than 30,000; zone I being the hyalastin fraction and zone II being the hyalectin fraction. From this curve, it can be seen that the Balazs method rejects the vast majority of available extractable HA by removing HA fractions having an average molecular weight less than 750,000. The present invention, on the other hand, allows the pharmacy use of a large proportion of the available HA since the fraction of low molecular weight hyaluronic acid having an average molecular weight of less than 30,000 causes the inflammatory activity that it does. previous researchers had found it with various extracts of HA.

Applicants have found that a significant percentage of available HA could, in fact, be used for therapeutic purposes if the separation was carried out according to the teachings of the present invention in accordance with the particular therapeutic application. While the Balazs patent specifically teaches the use of only about 10% of the available HA, the present invention allows the use of about 80% of the available HA, either in the form of the hyalastin moiety for them. wound healing applications, either in the form of the hyalectin fraction for intra-ocular and intra-articular applications, or in the form of a combined fraction of hyalastin and hyalectin which can also be used for wound healing applications.

The chemical and physical characteristics of the identified fractions were also studied by the Applicant and these characteristics are summarized in Table 2.

PREPARATION METHODS

EXAMPLE 1 - Process for obtaining a mixture of hyalastin and hyalectin fractions without inflammatory activity.

Roosters, either fresh or frozen (3000 g) are minced in a meat grinder and they are then carefully homogenized in a mechanical homogenizer. The resulting paste is placed in an AISI 316 stainless steel vessel or glass vessel along with 10 volumes of anhydrous acetone. The total content is then stirred for 6 hours at a speed of 50 g / min and the separation is allowed to continue for 12 hours, then the acetone is siphoned off and discarded.

Ί

This extraction process is repeated until the rejected acetone has reached the correct humidity level (KarlFisher method).

The resulting substance is then centrifuged and dried in vacuo at a suitable temperature for 5-8 hours. With this process, approximately 500-600 g of dry powder is obtained from roosters' ridges.

300 g of the dry powder are then subjected to an enzymatic digestion process with papain (0.2 g) in an aqueous medium buffered with phosphate buffer in the presence of a suitable amount of cysteine ​​hydrochloride. This mixture is then stirred for 24 hours at 60 g / min and at a constant temperature of 60 to 65 ° C. The whole mass is cooled to 25 ° C, 60 g of Celite (R) are added and stirring is continued for a further hour.

TABLE 2

CHEMICAL AND PHYSICAL CHARACTERISTICS

<td>r.</td><td> 20</td><td>Fractions</td><td>----- 1 PM</td><td>dynamic viscosity at 20 ° C</td><td>acid title hyaluronic % dry powder</td><td>protein content calculated as bovine albumin</td><td>sulfurized mucopolysaccharide content</td>

<td></td><td></td><td>hyalastine +</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 25</td><td>hyalectin</td><td> 250.000-350.000</td><td>100 mP.s</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>(conc. 1% w / v)</td><td> &gt;96%^To</td><td> &lt;0,5%</td><td> &lt;1%</td>

<td> •</td><td></td><td>hyalastine</td><td> 50.000-100.000</td><td>600 mP.s</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>(conc. 5% w / v)</td><td> &gt;96%</td><td> &lt;0,5%</td><td> &lt;1%</td>

<td></td><td></td><td>hyalectin</td><td> 500.000-730.000</td><td>170 mP.s</td><td></td><td></td><td></td>

<td></td><td> 30</td><td></td><td></td><td>(conc. 1% w / v)</td><td> &gt;96%</td><td> &lt;0,5%</td><td> &lt;1%</td>

the reported values ​​represent the HA titre after removal in water.

For example, a titre of 96% indicates that, after removing water, the powder contains 4% impurities and 96% hyaluronic acid.

The resulting mixture is filtered until a clear liquid is obtained. This clear liquid then undergoes molecular filtration by means of membranes having a molecular exclusion limit of 30,000 to retain, on the membrane, molecules having a molecular weight greater than 30,000. Five to six volumes are ultra-filtered. initially and, at the same time, distilled water is continuously added to the product. The addition of distilled water is suspended and the product is ultra-filtered until it is reduced to one third of its original volume.

The liquid residue is made 0.1M by adding sodium chloride and the temperature is raised to 50 ° C. 45 g of cethylpyridinium chloride are added while the product is stirred at 60 g / min. This mixture was stirred for 60 minutes, and then 50 g of Celite ™ was added with stirring, the temperature of the product was brought back to 25 ° C and the precipitate formed was collected by centrifugation. The precipitate thus obtained is suspended in a 0.01M sodium chloride solution (5 liters) containing 0.05% of cethylpyridinium chloride. Stir again for 60 minutes at 50 ° C. The temperature is lowered to 25 ° C and the precipitate is centrifuged.

The washing process is then repeated three times and the precipitate is finally collected in containers containing three liters of a 0.05M solution of sodium chloride containing 0.05% of cethylpyridinium chloride. Stir at 60 g / min for 60 minutes and maintain a constant temperature of 25 ° C for a period of two hours. The supernatant is removed by centrifugation.

This procedure is then repeated several times with a 0.1M sodium chloride solution containing 0.05% of cethylpyridinium chloride. The mixture is centrifuged and any supernatant is discarded. The precipitate is dispersed in a 0.30M sodium chloride solution containing 0.05% of methylpyridinium chloride (3 liters). The mixture is stirred and both the precipitate and the clear liquid are collected. The extraction is repeated on the precipitate three more times, each time using 0.5 liter of the same aqueous solution.

Finally, the residual precipitate is removed and the clear liquids are collected in a single container. The temperature of the liquid is increased to 50 ° C. while stirring. The liquid is then brought to 0.23M with sodium chloride. 1 g of methylpyridinium chloride chloride is added and stirring is continued for 12 hours. The mixture is cooled to 25 ° C and then filtered first through (R) through packings of this Celite.^{K 1} and then through a filter (iy).

The resulting mixture then undergoes further molecular ultrafiltration through membranes having a molecular exclusion limit of 30,000, ultrafiltering 3 original volumes with the addition of 0.33M sodium chloride solution. The addition of the sodium chloride solution is discontinued and the volume of liquid is reduced to a quarter of its original volume.

The solution thus concentrated is precipitated with stirring (60 g / min) at a temperature of 25 ° C. with three volumes of ethanol (95%). The precipitate is collected by centrifugation and any supernatant is discarded. The precipitate is dissolved in one liter of 0.1M sodium chloride solution and the precipitation process is repeated with three volumes of 95% ethanol.

The precipitate is collected and washed, first with 75% ethanol (three times), then absolute ethanol (three times) and third with absolute acetone (three times).

The product thus obtained (fraction of hyalastin + hyalectin) has an average molecular weight of between 250,000 and 350,000.

The yield of hyaluronic acid is equal to 0.6% of the original fresh tissue.

EXAMPLE 2 - Method for obtaining the hyalastin fraction from the mixture obtained by the method described in Example 1.

The mixture obtained by the method described in Example 1 is dissolved in distilled water free from pyrogen in a proportion of 10 mg of product per ml of water. The solution thus obtained undergoes molecular ultrafiltration through a membrane having a molecular exclusion limit of 200,000 with a concentration technique and without the addition of water to the membrane. During the filtration process through membranes having an exclusion limit of 200,000, molecules with a molecular weight greater than 200,000 do not pass while smaller molecules pass through the membrane at the same time. water. During the filtration process, no water is added to the compartment above the membrane; therefore, the volume in this compartment decreases and at the same time there is an increase in the concentration of molecules having a molecular weight greater than 200,000. Ultrafiltration is then carried out until the volume on the membrane. or reduced to 10% of the initial volume. Two volumes of pyrogen-free bi-distilled water are added and the solution is again ultrafiltered until the volume is reduced to one third. The operation is repeated two more times.

The solution which passes through the membrane is brought to 1.0M with sodium chloride and is then precipitated with four volumes of 95% ethanol. The precipitate is washed three times with 75% ethanol and then dried in vacuo.

The product thus obtained (fraction of hyalastin) has an average molecular weight of between 50,000 and 100,000.

The yield of hyaluronic acid is equal to 0.4% of the original fresh tissue.

EXAMPLE 3 - Method for obtaining the hyalectin fraction.

The concentrated solution, collected in a container from the ultrafiltration membrane having a molecular exclusion limit of 200,000 described in Example 2, is diluted with water until a concentration is obtained. solution containing 5 mg / ml of hyaluronic acid as determined by quantitative analysis based on a glycuronic acid assay.

The solution is brought to 0.1M in sodium chloride and it is then precipitated with 4 volumes of 95% ethanol. The precipitate is washed three times with 75% ethanol and then dried in vacuo.

The product thus obtained (hyalectin fraction) has an average molecular weight of between 500,000 and 730,000. This corresponds to a specific hyaluronic acid fraction having a defined molecular chain length of approximately 250,000 to 350,000 saccha units. with a high degree of purity.

The yield of hyaluronic acid is equal to 0.2% of the original fresh tissue.

EVALUATION OF BIOLOGICAL AND PHARMACOLOGICAL ACTIVITY 1.- Biological activity of cellular mobilization of hyaluronic acid fractions

The method consisting in determining the detachment activity of fibroblasts in a culture is used as a method for evaluating the cell mobilization activity of HA fractions.

BALB 3T3 mouse cells are grown on Dulbecco's Modified Eagle's medium with 10% calf serum, penicillin (250 units / ml) and streptomycin (0.25 mg / ml), and incubated. in 5% humidified CO ^, 95% air at 37 ° C. For experimental purposes, cells are conventionally inoculated into plastic tissue culture dishes with a diameter of 60 mm (6 x $ 10 cells / dish).

Confluent monolayers of 3T3 cells are decanted and fresh medium containing 2.0 mg / ml of different fractions of HA is added. At fixed intervals, cell detachment is observed both under the microscope and by counting the mobilized cells in a Coulter counter. Detachment tests

To measure the kinetics of detachment, cells are inoculated into plastic dishes and allowed to grow for 24 hours. After this time, the culture medium is decanted and fresh medium containing 2.0 mg / ml of HA is added. Every 24 hours, two dishes, one of test culture and one of control culture, are decanted and both the cells in the supernatant and the bound cells are counted in a Coulter counter. Table 3 gives the results obtained with tests using the fractions obtained in Preparation Examples 1 to 3 above.

TABLE 3

Results of mobilization studies

<td rowspan="2"> 25</td><td rowspan="2">Fraction</td><td rowspan="2">Concentration (mg / ml)</td><td colspan="2">Number of detached cells compared with</td><td rowspan="2">% efficiency (compared to controls)</td>

<td>the </td><td>testimonies</td>

<td></td><td>witness hyalectin + hyalas</td><td>tine 2</td><td> 2 3,5</td><td>x 10⁶x 10⁶</td><td> 75</td>

<td> 30</td><td>hyalectin</td><td> 2</td><td> 2,1</td><td>x 10⁶</td><td> 5</td>

<td></td><td>hyalastin L —————. i</td><td> 2</td><td> 5</td><td>x 10⁶</td><td> 150</td>

The data shown in Table 3 show that the hyalastin fraction exhibits high cell mobilization activity which makes this fraction useful for wound healing applications. This cell-mobilizing activity of hyalastin stimulates the migration and proliferation of new cells when a pharmaceutical preparation of the fraction is applied to an area of ​​damaged tissue.

The hyalectin fraction exhibits very limited cell mobilization activity and is therefore not useful for wound healing. However, hyalectin, because of its high average molecular weight and inherent viscosity, is useful for ocular and intra-articular injections, and the lack of appreciable cell mobilization activity is an important feature of the d fraction. 'hyalectin which makes it especially useful for intra-ocular and intra-articular injections.

2. Biological inflammatory activity of the hyaluronic acid fractions For this evaluation, the method of counting the invading cells after intraocular administration in the rabbit is used.

Method

Five (5) New Zealand or California rabbits weighing about 2 kg, having considered perfect vision, were used for this test. The exterior of the rabbit eye is checked for inflammatory processes at the macroscopic level and the interior of the eye is checked with an ophthalmoscope. If the fundus is clearly visible and normal, the test can take place.

The selected animals undergo local anesthesia by instillation of a few drops of a sterile anesthetic suitable for ophthalmology, a few drops of atropine in ophthalmological solution are also instilled.

The test is carried out under sterile conditions. The ocular gland is pushed out by exerting pressure until it is possible to inject, through the sclera about 5-6 mm from the edge of the cornea, into the center of the vitreous humor, 100 µl of the solution using a 26G needle. The other eye is taken as a witness. Two drops of the antibiotic solution are instilled into the treated eye and the animals are then placed alone in cages.

After 50 to 60 hours, the test can continue. Eyes are checked in the same way as above for animal selection. The animals are sacrificed with an intravenous injection of pentothal. Then the aqueous humor is first collected (approximately 0.2 ml) by means of an insulin syringe with a 26G needle. The eyeballs are then enuclosed, stripped of all foreign matter, washed in a saline solution, dried on paper, incised and opened in Petri dishes; the main part of the vitreous humor is separated and gathered with a sterile syringe (approximately 0.7 ml). The vitreous humor is placed in small polyethylene test tubes and 50 µl of hyaluronidase (100 U NF / ml) is added. The mixture is then maintained at a temperature of 37 ° C for about 3 hours to make the solution less viscous.

A leukocyte count is performed under a phase contrast microscope (120X) in a Burker chamber. A series of counting is carried out on each sample, the average values ​​are calculated and the result is expressed as the number of leukocytes per mm³.

The test is considered positive when:

.- the eyes examined do not show any sign of damage, and .- the average number of leukocytes from at least four of the five treated eyes does not exceed 200 per mm³ and the average number of leukocytes from each control eye does not exceed 50 per mm³.

Table 4 shows the results obtained from this evaluation using the HA fractions obtained in Preparation Examples 1 to 3 discussed above.

TABLE 4

Results of inflammatory activity studies

<td rowspan="2">Fraction</td><td>Number of invading cells</td>

<td></td>

<td>. witness</td><td> 25</td>

<td>. hyalastin + hyalectin</td><td> 32</td>

<td>. hyalastine</td><td> 20</td>

<td>. hyalectin . inflammatory fraction (mean MW</td><td> 22</td>

<td>from 30,000) . total hyaluronic acid from</td><td> 150</td>

<td>cockscombs (RF. Swann DA 1968, BBA 156, 17-29)</td><td> 120</td>

The results shown in Table 4, indicate that the hyalastin and hyalectin fractions do not show any higher inflammatory activity than the control and the combined hyalastin and hyalectin fraction shows only a negligible increase in inflammatory activity compared to the control. to the witness. Therefore, the hyalastin and hyalectin moieties are pharmaceutically useful without exhibiting undesirable inflammatory side effects. These results also confirm the Applicant's discovery that it is the fraction of HA having a low average molecular weight of less than 30,000 which is responsible for the inflammatory activity of HA preparations. Hyaluronic acid obtained from the crests of roosters prepared according to the method described in the literature by Swann (Swann DA, 1968, BBA 156, 17-29) exhibits remarkable inflammatory activity.

It has thus been shown that the hyalastin fraction having an average molecular weight of about 50,000 to 100,000 exhibits high cell mobilization activity and is therefore useful in wound healing applications without exhibiting adverse inflammatory reactions. The hyalectin fraction having an average molecular weight of about 500,000 to 730,000 has been shown to be useful for making ocular and intra-ocular injections because of its high molecular weight and inherent viscosity and because, at the same time time, it does not stimulate cell mobilization activity or inflammatory reactions which are undesirable side effects to be avoided in these pharmaceutical applications.

More specifically, the hyalastin fraction has been found to be useful as a wound healing preparation because of the following characteristics:

1. the preparation promotes a remarkably short healing time compared to conventional therapies, sanitation of the affected area being rapid, regularization of the edges of ulcers, vigorous development of granulation tissues, activation of cell migration of macrophages and fibroplasts and early epithelization.

2. the preparation promotes accelerated preparation for reconstructive surgery in more severe cases.

3. absence of keloid formation or retraction scar with final reshaping of scar tissue providing good aesthetic and functional results.

The hyalastin preparation has been found to be useful for the treatment of a variety of wounds, including pressure ulcers (bed sores), trophic ulcers, burns, dull wounds, post-traumatic ulcers, varicose ulcers and post-phlebitic ulcers. as a result of various venous spots, radiological necrosis, skin lesions, skin grafts and skin lesions due to herpes simplex.

For these wound healing treatments, the preparation of hyalastin or its sodium salt can be administered by various methods such as a gas pad, cream, spray or ampoules for intradermal injection. For topical applications in the form of a cream or a gas pad, the hyalastin is preferably combined with an emulsifying agent which absorbs the exudate from the exposed area while providing excellent acid diffusion. hyaluronic acid and a water dispersible excipient so that the wound dressing is easily removed.

The hyalectin fraction has been found particularly useful for the treatment of horses, particularly racehorses suffering from joint disorders and diseases caused by acute or chronic trauma, infections or repeated intra-articular corticosteroid injections. Specific disorders which can be treated with hyalectin are, for example, osteoarthrosis with or without inflammatory signs, acute or chronic synorith, degenerative processes in joint cartilages and dry joint diseases. The most common symptoms of these disorders are generally pain, damaged joint function, and decreased joint flexion. The hyalectin fraction according to the invention has been found to promote a marked reduction in healing time for these affected horses compared to conventional therapies, promoting early and lasting improvement in joint function and reducing pain and disability. These clinically beneficial effects are believed to be promoted by normalization of the viscoelasticity of synovial fluid and by the activation of tissue repair processes in articular cartilages.

In addition, all of the above advantageous effects are promoted by the hyalectin fraction in the absence of toxic, local and / or systemic effects. Repeated administration of hyalectin does not give any evidence of an allergic reaction or of any harmful or lasting effects.

Pharmaceutical preparations The above description has shown that the hyalastin and hyalectin fractions have good activity in pharmaceutical applications. The following examples are given only to describe pharmaceutical preparations which can be used for efficient in vivo administration of the HA fractions.

A.- Wound healing preparations

Example 1: ampoules for intradermal injection: each ampoule contains:

. sodium salt of hyalastin. 4 mg. sodium chloride ........... 16mg. water for injection, qs ... 2 ml

Example 2: ampoules for intradermal injection: each ampoule contains:

. potassium salt of hyalastine 5 mg. sodium chloride ........... 8mg. water for injection, qsp .... 1 ml

Example 3: Spray bottle for topical application each bottle contains:

. sodium salt of hyalastine .. 20 mg. sodium chloride ........... 80mg. water for injection, qs ... 10 ml

Example 4: Spray bottle for topical application each bottle contains:

. potassium salt of hyalastine 30 mg. mannitol ..................... 100mg. water for injection, qs ... 10 ml

Example 5: cream for topical application each tube of cream contains:. potassium salt of hyalastin 25 mg. polyethylene 30 glycol monostearate 400 ................... 1000mg. cetiole (decyl ester of oleic acid) ............. 500mg. strip SX (cetylstearic alcohol + laurylsulfate 9/1) 150 mg. glycerol ..................... 200mg. sorbitol ..................... 150mg. Na hydroacetate ......... 10mg. p-oxymethylbenzoate .......... 7.5mg. p-oxypropylbenzoate .......... 5mg. redistilled water, qsp ...... 10 g

Example 6: cream for topical application each tube of cream contains: 5. sodium salt of hyalastin. 30 mg. paraffin jelly ........... 3mg. polyethylene glycol monostearate 400 ................... 1000mg. cetiole (decyl ester of oleic acid) ............. 500mg. strip SX (cetylstearic alcohol + laurylsulfate 9/1) 150mg. glycerol ..................... 200mg. sorbitol ..................... 150mg. Na dehydroacetate ......... 10mg. p-oxymethylbenzoate .......... 7.5mg. p-oxypropylbenzoate ........ 5mg. redistilled water, qsp ...... 10 g

Example 7: gas pad containing medicaments for topical application, each gas pad measuring x 10 cm, contains:. sodium salt of hyalastine .. 3mg. glycerol ..................... 1g. polyethylene glycol 2g. redistilled water, qsp ...... 3g

Example 8: drug-loaded gas pad for topical application, each gas pad measuring 10 x 10 cm, contains:. potassium salt of hyalastin 6 mg. paraffin jelly ........... 0.5mg. glycerol 1 g. polyethylene glycol 2g. redistilled water, qsp ...... 3 g

Example 9: dry powder for application in wound healing each gram of dry powder contains:. sodium hyalastin salt. 10 mg. mannitol ..................... 0.75g. glycine ...................... 0.24 g

B.- Preparations for intra-ocular use

Example 10: 1 ml vials each vial contains:

. sodium salt of hyalectin. 10 mg. sodium chloride ........... 8mg. sodium phosphate monobasic 2H₂0 ..................... 0.25 mg. 12H dibasic sodium phosphate₂0 .................... 3mg. water for injection, qs ... 1 ml

Example 11: 5 ml vials each vial contains:. potassium salt of hyalectin 60 mg. mannitol ..................... 50mg. sodium phosphate monobasic 2H₂0 ..................... 1.25 mg. dibasic sodium phosphate 121-1 ^ 0 .................... 15mg. water for injection, qs ... 5 ml

Example 12: pre-filled syringes each syringe contains:. sodium salt of hyalectin. 40 mg. sodium chloride ........... 16mg. sodium phosphate monobasic 2H₂0 ..................... 0.8 mg. sodium phosphate dibasic 12H₂0 .................... 8.16 mg. water for injection, qs ... 2 ml

C.- Preparations for intra-articular use Example 13: 2 ml vials - each vial contains:. sodium salt of hyalectin. 40 mg. sodium chloride ........... 16mg. water for injection, qs ... 2 ml

Example 14: 4 ml vials each vial contains:. potassium salt of hyalectin 60 mg. mannitol ..................... 35mg. glycine ...................... 10mg. water for injection, qs ... 4 ml

Example 15: Pre-filled syringes 5 Each syringe contains:

. sodium salt of hyalectin. 25 mg. sodium chloride ........... 12mg. mannitol ..................... 10mg. sodium phosphate monobasic 21 ^ 0 ..................... 0.5 mg. sodium phosphate dibasic 12H₂0 .................... 6mg. water for injection, qs ... 2 ml

Although the above preparations have been described by way of example, it will be understood that other pharmaceutical formulations can be prepared by combining the hyalastin and hyalectin fractions discovered by the Applicant or their potassium or sodium salts with other pharmaceutically acceptable vehicles, diluents or excipients and, at various doses, depending on the particular use of the formulation.

For wound healing uses, hyalastin fraction preparations are applied to affected skin areas in one of the dosage forms discussed above, i.e. either as a cream, spray, or as a buffer. gas, dry powder or intradermal injection. For intra-articular uses, hyalectin preparations are generally administered at a dose of 2 ml per joint, either from a vial prepared or from a pre-filled syringe as described above.

In a further study, the ability and effectiveness of hyaluronic acid as a vehicle for various molecules, especially as a vehicle for ophthalmic drugs, was examined to ensure perfect tolerability and compatibility (i.e. absence of sensitization phenomenon) with the corneal epithelium. Hyaluronic acid is considered to be of particular interest as an ophthalmic vehicle. As discussed above, HA is an aminoglycan glycose present in various connective tissues and body fluids (such as synovial fluid and, in particular, vitreous humor) or due to its physical and chemical nature and its viscous characteristics. - striking elastic, it plays a structural and biological role of fundamental importance.

Thus, a study was conducted to examine the use of HA fraction having different molecular weights, including hyalastin and hyalectin fractions and a mixture thereof for the preparation of various pharmaceutical forms such as eye drops, gels, creams, inserts or dry powders. Therefore, ophthalmic drugs of different types are being studied with a view to obtaining a broad understanding of the potential use of this biological polymer in these different fractions as a vehicle.

The experiments described below aim to determine whether formulations containing hyaluronic acid as an excipient are capable of improving the biological availability of the drugs carried or of producing a cynergic effect in combination with the drugs conveyed, in particular with drugs having ophthalmic activity or utility. These potential capacities of HA as a vehicle are studied in the rabbit eye with four ophthalmic drugs having different types and actions, in particular pilocarpine nitrate, triamcinolone, epidermal growth factor (EGF) and an antibiotic such than streptomycin and gentamicin. All of these drugs are known to have anti-inflammatory healing and anti-microbial miotic efficacy. In addition, the evaluation of the activity of the hyaluronic acid-mediated antibiotic streptomycin is very important, because it is one of the most widely used antibiotics in eye infections.

The experimental models studied and the experiments carried out are as follows:

1) miotic activity of pilocarpine nitrate mediated by hyaluronic acid in rabbit eye;

2) anti-inflammatory activity of triamcinolone mediated by hyaluronic acid in the model of inflammation induced by textran in the rabbit eye;

3) wound healing activity of epidermal growth factor (EGF) mediated by hyaluronic acid in a rabbit corneal epithelial lesion model;

4) anti-microbial activity of streptomycin mediated by hyaluronic acid against Bacillus substilus 6633 on agar-agar plates.

I.- Myotic activity of pilocarpine nitrate conveyed by hyaluronic acid

Materials

The following materials are used as pilocarpine excipients for the various pilocarpine nitrate formulations: - sodium salt of hyaluronic acid, hyalastine fraction (mw approximately 100,000) at a concentration of 10 mg / ml and 20 mg / ml;

- sodium salt of hyaluronic acid, fraction of hyalectin (mw 500,000 to 730,000) at a concentration of 10 mg / ml and 20 mg / ml;

- 5% polyvinyl alcohol as a comparative ophthalmic excipient.

Various 2% formulations (eye drops or gel) of pilocarpine nitrate are prepared and conveyed by adding the two different fractions of sodium salts of HA at a concentration of 10 and 20 ml / ml. The following solutions are prepared:

Formulation 1 - saline solution with pilocarpine nitrate (PiNO ^) (2%), used as a reference.

Formulation 2 - solution of PiNO4 (2%) carried in 5% polyvinyl alcohol (used as a reference).

Formulation 3 - solution of PiNO 4 (2%) carried in sodium salt of the hyalastin fraction (10 mg / ml).

Formulation 4 - solution of PiNO 4 (2%) carried in sodium salt of the hyalastin fraction (20 mg / ml).

Formulation 5 - solution of PiNO, (2%) carried in sodium salt O of the hyalastin fraction (10 mg / ml).

Formulation 6 - solution of PiNO4 (2%) carried in sodium salt of the hyalastin fraction (20 mg / ml).

Method

New Zealand albino rabbits are used (weight 2 to

2.5 kg). The formulation to be tested is instilled into an eye with a micro-syringe (10 1); the other eye serves as a witness. In all cases, the pupil diameter is measured at suitable time intervals. Each solution is tested on at least 8 rabbits. Each eye is not treated more than three times and a rest period of at least one week is observed between each treatment.

Parameters measured

Pupil diameters are measured at various intervals to determine the curve of miotic activity over time. The following activity parameters are then calculated from the miosis / time curve:

I = maximum difference in pupil diameter between the treated eye and the reference.

Peak time = time required to reach I.

Duration = time required to restore basic conditions. Plateau = period of absolute miotic activity.

AUC = area under miosis / time curve. Results

The results of the tests are shown in Table 5. It can be seen from the data of the various parameters measured from the miotic activity time curve for all the solutions tested that the addition of hyaluronic acid to a 2% pilocarpine nitrate solution results in an increase in the miotic activity of the drug. In fact, the bioavailability of the drug can be up to 2.7 times greater than that of the aqueous solution containing 2% pilocarpine nitrate (formulation 1).

It should also be noted that there is a statistically significant increase in activity when the hyalectin fraction and the hyaluronic acid fraction are both used at 10 and 20 mg / ml as vehicles (formulations 5 and 6), in contrast to the solution of pilocarpine nitrate conveyed by polyvinyl alcohol (formulation 2).

The use of hyaluronic acid as a vehicle is of particular interest because the miotic activity of pilocarpine nitrate lasts longer when carried with this substance. That is, for formulations containing hyaluronic acid, the time required to restore pupil diameter to baseline conditions is up to 190 minutes (formulation 6) 35 compared to the 110 minutes required. for pilocarpine with saline alone (formulation 1).

II.- Anti-inflammatory activity of triamcinolone conveyed by hyaluronic acid

Materials

The following materials are used:

- a fraction of sodium salt of hyaluronic acid-hyalectin,

pm between 500,000 and 730,000, 10 mg / ml in saline solution;

- triamcinolone solution (10% in saline solution). Method

The experiments are carried out on male New Zealand rabbits (average weight 1.6 kg). After an adjustment period of five. days, intra-ocular inflammation is induced in rabbits by intra-ocular injection of dextran (10%, 0.1 ml). Administration is carried out in both eyes, under local anesthesia with 4% Novesina, by inserting the needle of the 4 mm syringe into the anterior chamber at a distance of 2 mm from the periphery of the cornea. The test is carried out on ten animals.

TABLE 5

Biological activity of ophthalmic formulations containing pilocarpi ne-nitrate mediated by hyaluronic acid⁹

<td>Formu- „. , vehicle</td><td>I, mm max</td><td>peak time '</td><td>duration</td><td>tray</td><td>AUC, cm²</td><td>AUC</td>

<td>lation</td><td>(at LF 95%)</td><td>(in mn)</td><td>(in mn)</td><td>(in mn)</td><td>(± LF 95%)</td><td>relative</td>

<td>1 saline solution</td><td> 1,93 ± 0,35</td><td> 20</td><td>¹¹⁰</td><td> -</td><td> 117 ± 28</td><td> 1</td>

<td>2 polyvinyl alcohol</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>lique Λ 5%</td><td> 2,33 ± 0,28</td><td> 20</td><td> 140</td><td> -</td><td> 192 ± 32</td><td> 1,64</td>

<td>3 hyalastine (lOmg / ml)</td><td> 2,50 ± 0,42</td><td> 20</td><td> 120</td><td> -</td><td> 240 ± 40</td><td> 2,05</td>

<td>4 hyalastine (20mg / ml)</td><td> 2,58 + 0,38</td><td> 30</td><td> 150</td><td> -</td><td> 208 ± 41</td><td> 1,78</td>

<td>5 hyalectin (10ifig / ml)</td><td> 2,50 ± 0,38</td><td> 15</td><td> 170</td><td> -</td><td> 242 ± 48</td><td> 2,06</td>

<td>6 hyalectin (20mg / ml)</td><td> 2,10 + 0,38</td><td> 20</td><td> 190</td><td>⁴⁵</td><td> 320 + 45</td><td> 2,73</td>

the values ​​indicated represent the average value of eight tests.

Processing

Treatment is performed on each animal, both in the right eye and in the left eye by instillation of three drops, three times a day for six days in all of the following:

- a solution of triamcinolone (10% in saline solution) in the left eye (LE);

- a solution of sodium salt of hyaluronic acid, fraction of hyalectin, (10 mg / ml) + triamcinolone (10%) in the right eye (RE);

Settings

The anti-inflammatory effect on the phlogiston reaction induced by dextran is evaluated by observing the eye with a slit lamp at the following intervals: 0, 1 hour, 3 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days and 6 days.

At these intervals, examination of the eye leads to the following observations:

the state of the cornea and the conjunctiva to detect the possible presence of hypermia, edema and in particular observation of the iris which is normally sensitive to the phlogistic process after intra-ocular injection of inflammatory agents;

- Tyndall effect in which the presence of a more or less intense opacity (nubecula) is indicative of the presence of corpusculent elements (inflammatory) in the anterior chamber.

The result of the observations is expressed in terms of subjective scores (from 0 to 3) related to the progressivity of the observed effect. Results

It can be seen from the results reported in Table 6 that the administration of triamcinolone has an anti-inflammatory effect on the iris and causes the disappearance of opacity (Tyndall effect) in the anterior chamber. The inflammatory process which is evident from the first to the third hour until the fourth day gradually decreases until substantially normal conditions are restored with perfect clarity of the eye on the sixth day. On the other hand, the administration of the sodium salt of hyaluronic acid, hyalectin fraction, together with the triamcinolone reduces the intra-ocular inflammation observed at the time discussed above compared to the administration of the. triamcinolone alone. That is, the phlogiston process in the iris and the opacity in the anterior chamber seem to have diminished at the 24th hour, with a gradual reduction until the 48th hour and with a total absence of reaction. inflammatory from the 4th day.

In the conjunctiva and the cornea, substantially no noticeable reaction is observed after intra-ocular injection of dextran.

Thus, the administration of triamcinolone, at the same time as the hyaluronic acid fraction leads to an improved activity of the drug, which demonstrates the more rapid healing of the rabbit eye.

TABLE 6

Effect of Hyaluronic Acid and Triamcinolone Combination on Dextran Induced Intraoccular Inflammation

<td rowspan="2"> —·</td><td rowspan="2"> 15</td><td rowspan="2"> 0</td><td colspan="8">Observation interval</td><td colspan="2" rowspan="2">5 days</td><td colspan="2" rowspan="2">6 days</td>

<td> 1</td><td>h</td><td> 3</td><td>h 24 h 48 h</td><td> 3</td><td>j</td><td> 4</td><td>j</td>

<td></td><td></td><td>THE RE</td><td>THE</td><td>D</td><td>THE</td><td>has rated rating RE LE RE LE RE</td><td>THE</td><td>D</td><td>THE</td><td>D</td><td>THE</td><td>D</td><td>THE</td><td>D</td>

<td></td><td> 20</td><td>conjunctiva 0.0 0.0</td><td> 0,2</td><td> 0,0</td><td> 0,0</td><td> 0,0 0,0 0,0 0,0 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td>

<td></td><td></td><td>cornea 0.0 0.0</td><td> 1,0</td><td> 0,2</td><td> 0,0</td><td> 0,7 0,1 0,0 0,0 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td>

<td></td><td></td><td>lyndall 0.0 0.0</td><td> 1,0</td><td> 1,2</td><td> 3,0</td><td> 3,0 3,0 2,1 3,0 1,2</td><td> 3,0</td><td> 0,2</td><td> 2,2</td><td> 0,0</td><td> 1,2</td><td> 0,0</td><td> 0,4</td><td> 0,0</td>

<td>V</td><td> 25 30</td><td colspan="8">Iris 0.0 0.0 0.5 0.7 2.7 2.7 3.0 2.5 3.0 1.2 3.0 0.4 2.4 LE = left eye treated with triamcinolone. RE = right eye treated with triamcinolone and hyalectin. To each value is the average of seven observations out of a total (it is expressed in terms of a subjective rating between 0 and 3 the progressivity of the observed effect.</td><td colspan="4">0.0 1.5 0.0 0.5 of seven animals and , in relationship with</td><td> 0,0</td>

III.- Healing activity of EGF conveyed in hyaluronic acid

Materials

The following materials are used:

Formulation A - EGF (Epidermal Growth Factor) dissolved in saline solution (0.5 mg / 5 ml).

Formulation B - sodium salt of hyaluronic acid, hyalastine fraction (approximately 100,000 mw) dissolved in saline solution (10 mg / ml).

Methods

The experiments are carried out on male New Zealand albino rabbits (average weight 1.8 kg). The animals, after an adaptation period of about 5 days, undergo an epithelial lesion of the cornea under suitable conditions of local anesthesia with Novesina (4%). The lesion consists of a monocular scarification of a circular area of ​​the optic area effected by a concave glass cylinder (3 mm) having a sharp edge.

Processing

The animals are subdivided into groups, each group consisting of five animals and they are subjected to pharmacological treatment.

<td> 15</td><td>logic by instillation in the</td><td>conjunctiva of the following:</td>

<td></td><td>Groups</td><td>Processing</td>

<td></td><td></td><td></td>

<td></td><td></td><td></td>

<td> -</td><td>group 1 (control)</td><td>saline solution</td>

<td> 20</td><td>group 2</td><td>EGF solution (formulation A)</td>

<td></td><td>group 3</td><td>sodium acid salt solution</td>

<td></td><td></td><td>hyaluronic, fraction of hyalas-</td>

<td></td><td></td><td>tine + EGF solution - combination</td>

<td></td><td></td><td>formulation sound A + formulation</td>

<td> 25</td><td></td><td>B in a 1/1 ratio, to obtain</td>

<td> *</td><td></td><td>end a formulation C</td>

The treatment is carried out on the right eye (RE) by instilling into the conjunctiva two drops every eight hours for three administrations in total.

Settings

The healing of the corneal epithelium is assessed by observation of the eye and by photographic documentation with a slit lamp at various intervals after scarification: 0.8 hours, 16 hours, 24 hours, 32 hours, 40 hours and 48 hours.

Results

Ophthalmological examination 1, as indicated in Table 7, reveals that for the controls (group 1) complete healing is obtained (5 animals out of 5) 48 hours after the lesion. In the animals treated with EGF (group 2), the healing process is apparent already 24 hours after scarification with considerable efficiency (4 out of 5 animals). In animals treated with formulation C comprising the sodium salt of hyaluronic acid, hyalastin fraction + EGF (group 3), the healing process is complete in all animals (5 out of 5) already 16 hours after scarification.

These results show that the use of the hyalastin fraction of hyaluronic acid as a vehicle for EGF accelerates the healing process by promoting faster efficient healing of corneal lesions.

TABLE 7

Healing of corneal epithelial damage

<td rowspan="2"> 15</td><td rowspan="2">Group</td><td rowspan="2">Processing</td><td colspan="3">Hours after</td><td colspan="2">scarification</td>

<td> 0</td><td> 8</td><td> 16</td><td> 24</td><td> 48</td>

<td></td><td> 1</td><td>saline</td><td> +</td><td> +</td><td> +</td><td> +</td><td> —</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td> 20</td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> +</td><td></td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td> —</td>

<td></td><td> 2</td><td>EGF</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>(formulation A)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td>

<td> 25</td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> —</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td></td><td> +</td><td> -</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td>

<td></td><td> 3</td><td>hyaluronic acid +</td><td></td><td></td><td></td><td></td><td></td>

<td> 30</td><td></td><td>EGF (formulation C)</td><td> +</td><td> +</td><td> -</td><td> —</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td></td><td></td><td></td><td> +</td><td> +</td><td> —</td><td> —</td><td> —</td>

<td> 35</td><td>+ = eye</td><td>unhealed.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>- = eye</td><td>scarred.</td><td></td><td></td><td></td><td></td><td></td>

IV.- Antimicrobial activity of gentamicin conveyed by hyaluronic acid

Materials

The following materials are used:

- gentamicin dissolved without saline solution (50 mg / ml);

- sodium salt of hyaluronic acid, hyalectin fraction (2 mg / ml). Methods

Septic phlogosis was caused in each eye of 11 rabbits by intra-ocular injection of a standard suspension of Pneumonas aerugi10 nosa (0.1 ml). In rabbits with septic phlogosis, hyaluronic acid, hyalectin fraction in combination with gentamicin is administered by instillation into the right eye and gentamicin in a vehicle consisting of a buffered saline solution is administered in the left eye. Treatment (three drops every six hours) begins immediately after injection of the infecting agent and is continued until the infection subsides. The eyes are observed daily under a slit lamp. Results

Treatment with the help of the gentamicin hyaluronic acid combination leads to a more rapid resolution of the septic infection compared to the case of administration of the antibiotic alone. This conclusion emerges from the data presented in Table 8.

TABLE 8

Effects of gentamicin mediated by hyaluronic acid, hyalectin fraction on spectral intra-ocular phlogosis

<td rowspan="2">Processing</td><td colspan="7">days from the onset of phlogosis</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td>gentamicin + saline buffer vehicle</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 0,0</td><td> 36,3</td><td> 100</td>

<td>gentamicin + hyaluronic acid hyalectin fraction</td><td> 0,0</td><td> 0,0</td><td> 9,0+</td><td> 27,2+</td><td> 72,7+</td><td> 100</td><td> 100</td>

The values ​​are expressed as a percentage (number of eyes cured of phlogosis relative to the number of eyes treated).

+ = significant difference compared to the buffer vehicle (less than

0.05, Fisher's exact T-test).

Additional samples, although this list is not exhaustive, of ophthalmic drugs which can be carried with HA fractions according to the present invention are as follows:

antibiotics ...................

hormones ........................

anesthetics (local) ..........

antiviral .......................

anti-inflammatory drugs .............

vasopressors and vasoconstrictors

CONCLUSIONS chloromphenicol neomycin aureomycin myxin and polymyxin bacitrincine mycetins nandrolone and nandrolone sulfate henoxinate and its hydrochloride iodocéoxyuridine iodocéoxycytidine dexamethasone and its phosphate synephrine and neosynephrine

Based on the results obtained from the experiments discussed above, it can be concluded that solutions of hyaluronic acid salts, (both in the hyalastin and hyalectin fractions) can be used as vehicles for ophthalmic drugs and s are found to be efficient as such for various types of drugs having different biological actions. For example, drugs comprising anti-glaucoma agents such as pilocarpine nitrate, anti-allergic and anti-inflammatory agents such as triamcinolone, agents for promoting tissue healing and cell proliferation, agents for promoting wound healing. eye tissues such as EGF and antibiotics such as streptomycin and gentamicin, the anti-inflammatory miotic activities of which are respectively indicated, Wounds and antimicrobials can all be administered effectively using HA as a vehicle.

The formulations of ophthalmic drugs conveyed in the hyaluronic acid fractions having various molecular weights are perfectly tolerated by the host and compatible with the corneal epithelium without giving rise, however, to sensitization phenomena.

It is also possible, from the data, to observe that this biological product, hyaluronic acid, is an effective vehicle capable of improving the biological availability in vivo of the drugs conveyed, and of strengthening the pharmacological activity of these drugs. . The use of hyaluronic acid fractions as a drug vehicle has particularly the following results:

- an increase in the miotic activity of pilocarpine nitrate, while prolonging the activity time of the drug;

- an increase in anti-inflammatory activity of triamcinolone in intra-ocular inflammation induced by dextran, with a regression of the phlogistic process in shorter times than those obtained with triamcinolone alone;

- increased protective action of epidermal growth factor (EGF) on superficial corneal lesions with evident cynergism and reduced healing times compared to the recovery time observed with EGF alone; and

- increase in the in vivo biological activity of antibiotics such as gentamicin.

The results obtained by using this biological polymer, hyaluronic acid, as a vehicle for drugs with effects of such diverse nature, allow the extrapolation of its potential as a vehicle for many other ophthalmic drugs.

Various examples of various formulations of ophthalmic drugs which may be in the form of powders, eye drops, gels, creams or inserts, in which the hyaluronic acid in the form of these different hyalastin and hyalectin fractions is indicated below are indicated below. only excipient used:

Example 1

Eye drops that can be used as artificial tears containing:

. sodium salt of hyaluronic acid, hyalectin fraction ................. 10 mg. saline buffered with phosphate at pH, 6 .................... 10 ml

Example 2

Eye drops that can be used as artificial tears containing:

. sodium salt of hyaluronic acid, hyalectin fraction ................. 20mg. saline solution buffered with phosphate at pH, 6 .................... 10mg

Example 3

A gel containing EGF in which 100 g contain:

. sodium salt of hyaluronic acid, hyalastin fraction 55 mg. sodium salt of hyaluronic acid, hyalectin fraction 30 mg. EGF ................................. 0.5g. bi-distilled water .................... 25.5g

Example 4

A 100 mg insert with pilocarpine nitrate containing:. sodium salt of hyaluronic acid, hyalastine fraction ................. 100mg. pilocarpine nitrate .............. 2mg

Example 5

A powder for topical application containing streptomycin.

100 g of powder contain:

. sodium salt of hyaluronic acid, hyalastine fraction ................. 70mg. sodium salt of hyaluronic acid, hyalectin fraction ................. 28.5 mg. streptomycin ....................... 1.5g

Although the above preparations have been given by way of example, it will be appreciated that pharmaceutical formulations can be prepared by combining the hyaluronic moieties, especially the hyalectin or hyalastin moieties or the combined hyalectin / hyalastin moiety or their sodium salts. or potassium with other drugs active in various doses depending on the particular use of the formulation.

Hyaluronic acid, especially in substantially pure hyalectin and hyalastin fractions, has therefore been demonstrated to be an effective vehicle or excipient which can be used in combination with various drug drugs having ophthalmic utility or activity. Pharmaceutical compositions containing the HA moieties as drug carriers are particularly useful because the HA moieties exhibit a high level of tolerability to the eye and high compatibility with the corneal epithelium.

The use of the HA moieties further provides a means of effectively improving the in vivo biological activity of ophthalmic drugs. The use of the particular hyalectin and hyalasti fractions derived from HA is further useful and important because these fractions when administered to the eye do not exhibit undesirable inflammatory side reactions.