FR2556728A1 - PROCESS FOR PREPARING HYALURONIC ACID INSOLUBLE IN WATER - Google Patents

Abstract

PROCESS FOR THE PREPARATION OF A WATER INSOLUBLE HYALURONIC ACID. A PREPARATION OF BIOCOMPATIBLE AND WATER-INSOLUBLE HYALURONIC ACID IS OBTAINED BY SUBJECTING THE HYALURONIC ACID OR ONE OF ITS SALTS TO TREATMENT WITH A CROSS-LINKING AGENT. DIVINYL SULFONE CAN BE USED AS A CROSS-LINKING AGENT AT ROOM TEMPERATURE IN AQUEOUS ALKALINE MEDIUM.

Description

1. The invention relates to acid preparations

  hyaluronic acid ("AH") insoluble in water, these preparations

  Because of their biocompatibility, they can be used in many in vivo applications, for example, various prosthetic devices, such as artificial heart valves, vascular grafts, etc. Hyaluronic acid insoluble in water or crosslinked

  can also be used to modify many

  These polymers can also be used in many in vivo applications. According to another aspect, the invention also relates to a method for

  manufacture of these preparations at room temperature.

  Hyaluronic acid is a known material that meets

  naturally and with many applications

  in medicine and biology. For example, we can refer to

  US Patent 4,272,522 and the cited documents

In this one.

  Cross-linked gels of hyalic acid are also known.

  luronic, in particular by the article by Laurent and others

  in Acta Chem. Scand. 18 (1964 N 1, pp. 274-5).

  The present invention therefore relates to

  hyaluronic acid parabens insoluble in water and bio-

  compatible. The term hyaluronic acid refers,

  in the present description and in the preceding claims.

  not only with hyaluronic acid itself but also with the addition salts of the acid, for example the sodium, potassium, calcium, etc. Due to their biocompatibility, these preparations may be used in many in vivo applications and in combination with various polymeric materials that have been

  modified by the inclusion of such insolu-

in the water.

  More particularly, the invention relates to

  Water-insoluble hyaluronic acid para- meters having the following types of materials: 1) crosslinked hyaluronic acid powder; 2) cross-linked hyaluronic acid film; 3) cross-linked gel of hyaluronic acid; 2. 4) a cross-linked hyaluronic acid film reinforced with a polyethylene terephthalate knitted fabric, as well as other knitted fabrics; and) particulates coated with cross-linked hyaluronic acid. Crosslinking agents which can be used to obtain these preparations include: 1) formaldehyde; 2) dimethylol urea; 3) dimethylolethylene urea; 4) a polyaziridinyl compound; ) ethylene oxide; 6) polyisocyanate; and

7) Divinyl sulfone.

  The preferred crosslinking agent used to obtain the preparations according to the invention consists of the

  divinyl sulfone. It has been found that a hyalic acid gel

  insoluble material can be obtained by cross-linking the

  Hyaluronic acid with divinyl sulfone (SDV) in

  an aqueous solution at a pH greater than 9 and the temperature

  ambient temperature, that is to say about 20 C. According to

  concentration and molecular size of the hyaluronic acid in the solution, the hyaluronic acid / divinyl sulfone ratio and the reaction time, the melting rate of the gel

  can vary to a large extent, for example from 20 to 2000.

  The melting rate depends substantially on the ionic strength and the hydrogen ion concentration of the medium and decreases

  with the ionic strength. In this process, the sulfone of

  nyle is used at room temperature under conditions

  alkaline solutions instead of being used as

  culation only by reflux (60-65 C).

  The examples below, in which all the

  percentages are by weight, unless otherwise indicated, illustrate various embodiments of the invention,

  but do not constitute a limitation, the invention

  its scope in the claims.

Example 1

  A 37% aqueous solution of formaldehyde and concentrated hydrochloric acid was added to a water-acetone mixture. The mixture obtained had the composition

  following: CH20 0.27%; HC1 0.19%; water-acetone rap-

  port 1/28. 0.5 g of sodium hyaluronate powder was placed in 50 ml of the mixture for 10 minutes. Then

  the powder was filtered, washed completely in a

  water-acetone mixture (ratio 1/3) then with acetone,

  and it was dried in a vacuum oven. Acid powder

  hyaluronic acid obtained was insoluble in water and

held 1.41% combined CH20.

Example 2

  The previous example was repeated with the crosslinking mixture of the following composition: CH 2 O, 2.5%; HC1,

  0.38%, water / acetone 1/2 ratio. The CH20 content of the

duit was 5.3%.

  In Examples 1 and 2, the crosslinking of the

  of hyaluronic acid was carried out in water-acetone mixtures. By changing the water / acetone ratio and the CH 2 O concentration, it is possible to control the melting rate of the product. Thus, the melting rate was 178% for the product of Example 1 and 230% for that of

  Example 2. The melting rate can be reduced by increasing

  the amount of acetone in the mixture and the concentration

of CH20.

  The following examples illustrate the use of a polyaziridine compound as a crosslinking agent. This

  compound of the polyaziridine type cross-links hyaluronic acid

  only in dry conditions and at room temperature

  this is very important in the case of hyacinth

  luronic because it is a polymer sensitive to heat.

  their. EXAMPLE 3 To 113 g of a solution of sodium hyaluronate in water (concentration of 14.2 mg / ml) was added 0.42 g of

  polyaziridine compound (commercial CX-100 crosslinking agent

  marketed by POLYVINYL CHEMICAL INDUSTRIES). The

  molar ratio of the crosslinking agent to the hyalic acid

  luronic was 0.5. The mixture was poured on a

  4. that in glass following a layer of 5 mm thick and

  allowed to dry at room temperature for 2 days.

  In this way, a film of hyaluronic acid was obtained.

  soluble in water and having a melting rate in water of 160%.

Example 4

  0.5 g of a dry sodium hyaluronate powder was mixed with 50 ml of a 1% solution of the crosslinking agent CX-100 in acetone, allowed to stand for 5 minutes and filtered. The powder was air dried

  for 2 hours, then washed several times with water and

  in a vacuum oven at 40 C for 4 hours. The rate

  melting of the cross-linked powder in water was 135%.

Example 5

  This example illustrates the use of a compound of

  polyaziridine for obtaining a hyaluronic acid

  with a high melting rate.

  0.6 g of solid sodium hyaluronate was mixed with 9.2 g of a 0.6% crosslinking agent solution.

  CX-100 in the water. The solution obtained presented a report

  molar port of CX - 100 versus sodium hyaluronate of 0.1. The sodium hyaluronate content in the solution was 6.04%. The pH of the highly viscous mixture that was obtained was set at 2.5% with 2% HCl solution. The film obtained was easily soluble in water. It was heated at 60 C for 30 minutes. This heat treatment gave a resistant film and

insoluble in water.

Example 6

  0.1093 g of sodium hyaluronate was mixed with

  form of fibers with 25 ml of a solution & 1% of poly-

  isocyanate (marketed by the company MOBAY CHEMICAL CORP. under the reference Desmodur N-75) in acetone

  and the mixture was allowed to stand for 10 minutes. Meadow-

  The citrate was separated and washed three times with acetone, dried under vacuum at a pressure of 6 kPa at 60 ° C.

  mn and, finally, in a drying apparatus under

  in the presence of phosphorus pentoxide. The product ob-

  5. held (0.1277 g) was insoluble in water and showed

a melting rate of 120%.

Example 7

  This example illustrates the use of dimeric urea

  thylolethylene to crosslink hyaluronic acid.

  6 g of a solution of sodium hyaluronate was mixed with

  dium in water (concentration of 9.8 mg / ml) with 0.017 g of N, N'-dimethylolethylene urea and 0.005 g of zinc nitrate. The mixture was poured onto a glass plate and allowed to dry overnight. The film obtained

  then treated at 110 ° C. for 15 minutes. It has become

  naked insoluble in water and exhibited a melting rate

145%.

Example 8

  This example illustrates the use of sulfone di-

  vinyl as a crosslinking agent for hyaluronic acid.

  35 ml of a hyaluronate solution of

  sodium containing 0.401 g (1 mmol) of divinyl sulfone.

  The pH of the mixture was adjusted to about 8.5-9 with a 1% sodium hydroxide solution. The film was obtained from the mixture by pouring it onto a glass plate and allowing it to dry overnight at room temperature.

  ambient temperature. This film was easily

  luble in the water. It was heated at 60 ° C for 30

  min. The heat treatment made it possible to obtain a film

  resistant and insoluble in water.

Example 9

  Dry film of non-cross-linked hyaluronic acid

  It was placed in a solution of 0.6 g of divinyl sulfone in a mixture of 26 g of acetone and 13 g of water and allowed to stand for 10 minutes. The film was then removed from the solution, air dried for 1 minute and then heated in an oven for 20 minutes at 65 C.

  obtained a resistant crosslinked film of hyalic acid

ronicue.

Example -10

  2.95 g of air dried sodium hyaluronate were mixed with 57.35 g of a 0.2M solution of NaOH in water and stirred with a glass rod until dissolved. complete. Then, 1 g of divinyl sulfone was stirred into the mixture and it was allowed to stand for

  one hour at room temperature. The mixture has

  formed into a hard gel. The gel was placed in a homogenous

  Vir-Tis "45" type with 10O ml of H 2 O and treated

  5 minutes at 30,000 rpm. Small parties have been

  highly fusible cells. The particles were then washed several times with water and filtered by suction on a glass filter. To determine the melting rate, about 1 g of the gel was placed in a glass filter.

  ml, which in turn was placed in a plastic tube

  than centrifugation. The gel was centrifuged for 30 minutes at 3000 rpm. The water was evacuated and recovered at the bottom of the tube. The concentration of hyaluronic acid in the gel was 0.21%, i.e. the melting rate in

the water was 476%.

Example 11

  The operations of Example 10 were repeated, at

  except that the resulting gel has been dispersed in a

  0.15 M NaCl in water, and the particles were washed in the same solution. The concentration of hyaluronic acid in the gel after centrifugation was

  1.29% and the melting rate was 77.5%.

Example 12

  1 g of air dried sodium hyaluronate was dissolved in 9 g of a 0.2 M NaOH solution with stirring with a glass rod. 0.33 g of sulfone from

  divinyl in the viscous solution obtained, and the mixture

  was allowed to rest for 20 hours at room temperature.

  ambient temperature. The hard gel obtained was treated as in Example 10. The concentration of hyaluronic acid in

  the gel after centrifugation was 4.3%, i.e.

  say that the melting rate was 23.6%.

  Example 13 - Compatibility test with blood This test makes it possible to demonstrate the biocompatibility of

preparations according to the invention.

  The supply of 3H - serotonin by platelets

  human blood was used in preliminary studies.

7.

  to establish the reactivity of the product according to the

  10 with the blood. Normal human venous blood has been pre-

  lifted into plastic syringes and immediately transferred to plastic tubes containing 3.8% sodium citrate, one part citrate

  te for 9 parts of whole blood. Plasma-rich plasma

  The tablets were prepared by centrifugation at 4 ° C. for 15 minutes under a centrifugal acceleration of 125 × g, then extracted with a serological pipette and transferred to a test tube made of plastic or silicone. 3H serotonin (3H-5-hydroxytryptamine, or 3H-5HT, from the company NEW ENGLAND NUCLEAR, containing 26.3 Ci / mmol

  1 mCi / ml ethanol-water) to platelet rich plasma, due to

  It was 0.2 to 0.5 l / ml of plasma and incubated for 15 minutes at 37 ° C. In this test, silicone or polypropylene test tubes were used; We used

  clots as a means of positive control, and

  coated and uncoated samples. 1-2 ml of 3H-

  HT-plasma was added to each of two tubes containing the samples to be tested; an aliquot of 50 μl was removed from the control mixture for determination of total radioactivity. After the incubation period

  (10 to 120 min), aliquots of 0.2 -

  0.5 ml of the suspension was removed and centrifuged on a silicone oil in an Eppendorf apparatus for 2 minutes under an acceleration of 12,000 x g. 50 μl of the supernatant were removed from each of the tubes, mixed with 5 ml of scintillation fluid and the radioactivity measured by beta spectrometry. The amount of 3H-5HT

  released by clots or test samples

  the radioactivity increment of the supernatant

  (radioactivity of experimental samples unless the

  control dioactivity). The test material did not show

  not, after 120 minutes, a significant release of plaquet-

are 3H-5HT.

  Of course, the invention is not limited to

  description and examples above; we could at the contrary

  to design various variants without leaving for as many

its frame.

8.

Claims (8)

  1) Process for obtaining a hyaluronic acid preparation insoluble in water, characterized in that it consists in subjecting the hyaluronic acid or one of   its addition salts, in powder, film or gel form, to a   by a crosslinking agent selected from the group consisting of: formaldehyde, dimethylol urea, urea   dimethylol ethylene oxide, ethylene oxide, a polyazidine   dine, a polyisocyanate and divinyl sulfone.   2) Process according to claim 1, characterized in that the crosslinking agent is formaldehyde and   that the treatment is carried out in an aqueous medium at a time reflux temperature.   3) Process according to claim 1, characterized in that the crosslinking agent is a polyaziridine   and that the treatment is carried out under strict conditions at room temperature.   4) Process according to claim 1, characterized in that the crosslinking agent is a polyisocyanate and the treatment is carried out in acetone at a temperature of reflux temperature.   Method according to claim 1, characterized by   the crosslinking agent is dimethyl urea   ethylene and that the treatment is carried out at about 110 ° C 6) Process according to claim 1, characterized by   the fact that the crosslinking agent is the sulfone of   vinyl and that the treatment is carried out at about 60-65 C. 7) Process according to claim 1, characterized in that the treatment is carried out with sulfone of   divinyl at about 20 ° C. in an aqueous alkaline medium.   8) Process according to claim 7, characterized in that the treatment is carried out at a pH higher than about 9.   9) Method according to one of claims 7 and 8,   characterized by the fact that the treatment is carried out 1 to 20 hours.   Method according to one of claims 7 to 9,   characterized by the fact that the ratio of the contents of the   9. Hyaluronic and Divinyl Sulfone is approximately 3/1 by weight.   11) Method according to one of claims 1 to 10,   characterized in that the salt is a salt of sodium, potassium or calcium.   12) Product characterized by the fact that it is   by the method according to one of claims 1 to 11.