CA1238043A - Water insoluble preparations of hyaluronic acid and processes therefor - Google Patents
Water insoluble preparations of hyaluronic acid and processes thereforInfo
- Publication number
- CA1238043A CA1238043A CA000460460A CA460460A CA1238043A CA 1238043 A CA1238043 A CA 1238043A CA 000460460 A CA000460460 A CA 000460460A CA 460460 A CA460460 A CA 460460A CA 1238043 A CA1238043 A CA 1238043A
- Authority
- CA
- Canada
- Prior art keywords
- hyaluronic acid
- cross
- treatment
- effected
- linking agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
Abstract
ABSTRACT OF THE DISCLOSURE
Water insoluble, biocompatible hyaluronic acid pre-parations are prepared by subjecting hyaluronic acid to treatment with a cross linking agent, particularly divinyl sulfone at room temperature in an aqueous alkaline medium.
Water insoluble, biocompatible hyaluronic acid pre-parations are prepared by subjecting hyaluronic acid to treatment with a cross linking agent, particularly divinyl sulfone at room temperature in an aqueous alkaline medium.
Description
The present invention relates to biocompatible water insoluble preparations of hyaluronic acid ("HA") which, because of their biocompatibility enables th~m to be used in numerous in vivo applications, such as various prosthetic devices in-cluding artificial heart valves, vascular grafts, etc. The water insoluble ~or cross linked) ~A can also be used to modify various polymeric articles which can likewise be used in n~merous in vivo applications. The invention also relates to processes for making these preparations at room temperature.
Hyalur~nic acid is a known, naturally occurring mate-rial which has many applications in medicine and biology. See, for example, E.A. Balazs U.S. Patent No. 4,272,522 and publi-cations cited therein.
Cross-linked gels of hyaluronic acid are known, having been described by ~aurent et al in Acta Chem. Scand. 18 (1964 No. l.; p. 274~5).
~L~3~3 , The present invention is directed to water insoluble preparativns of hyaluronic acid (HA) which are biocompatible.
As used hereinl the term HA includes not only hyaluronic acid, but the acid addition salts thereof as well, such as the sodium potassium, calciuml etc. salts. Because of their biocompati-bility, they can be used in nurnerous in vivo applications both per se, and in comhination with various polymeric materials which have been modi~ied by the inclusion therein of such water insoluble preparations.
More specifically, the invention is directed to water insoluble preparations of hyaluronic acid including the ollow- ¦
ing types of materials:
1~ cross-li.nked hyaluronic acid powder
Hyalur~nic acid is a known, naturally occurring mate-rial which has many applications in medicine and biology. See, for example, E.A. Balazs U.S. Patent No. 4,272,522 and publi-cations cited therein.
Cross-linked gels of hyaluronic acid are known, having been described by ~aurent et al in Acta Chem. Scand. 18 (1964 No. l.; p. 274~5).
~L~3~3 , The present invention is directed to water insoluble preparativns of hyaluronic acid (HA) which are biocompatible.
As used hereinl the term HA includes not only hyaluronic acid, but the acid addition salts thereof as well, such as the sodium potassium, calciuml etc. salts. Because of their biocompati-bility, they can be used in nurnerous in vivo applications both per se, and in comhination with various polymeric materials which have been modi~ied by the inclusion therein of such water insoluble preparations.
More specifically, the invention is directed to water insoluble preparations of hyaluronic acid including the ollow- ¦
ing types of materials:
1~ cross-li.nked hyaluronic acid powder
2. cross-linked hyaluxonic acid film;
3. cross linke~ gel of hyaluronic acid;
4. cross-linked hyaluronic acid film reinforced with a polyethylene terephtalate knitted fabric, r as well as other knitted fabrics; and
5. particulate materials coated with cross-linked hyaluronic acid.
The cross-linking agents that can be used to make the instant preparations include:
1. formaldehyde;
:25 2. dimethylol urea;
3. dimethylolethylene urea;
4. polyaziridinyl compound;
5. ethylene oxide;
The cross-linking agents that can be used to make the instant preparations include:
1. formaldehyde;
:25 2. dimethylol urea;
3. dimethylolethylene urea;
4. polyaziridinyl compound;
5. ethylene oxide;
6. polyisocyana~e; and 3~) 7. divinyl sulfone.
~23~3 The cross-linking agent partlcularly used to make the instant prepara-tions is di~Tinyl sulfone. It has now been found that an insoluble hyaluronic acid gel can be obtained by cross-linking hyaluronic acid with divinyl sulfone (DVS) in water solution at pH higher than 9 at room temperature, i.e., about 20C. Depending upon the concentration and molecular size of the hyaluronic acid in the solution, the hyaluronic acid/divinyl sulfone ratio and reaction time, the swelling ratio of the gel can vary over broad limits, i.e., from 20 to 2000. The swelling ratio depends 10 substantially on the ionic strength and the hydrogen ion con- I
centration of the medium and decreases with the ionic strength.
This process contrasts with that disclosed and claimed in parent application serial no. 561,818, filed December 15, 1983 in that here r DVS is used at ambient temperatures under alkaline conditions whereas in the parent application, DVS is used as a cross-~inking agent only under reflux (ca. 60~65C).
~8~
DETAIL~D DESCRIPTION OF THE INVENTION
_ ' The following examples (wherein all parts gi~en are by weight unless otherwise specified) illustrate the several embodiments of the invention, without however being a limita-tion thereof, the in~ention being solely defined by the claims.
Example l To a water-acetone mixture a 37% by weight water solu-tion of formaldehyde and concentrated hydrochloric acid were bdded. The mixture obtained was of the following compositions (~ by wt~: CH20, 0.27; HCl, 0.19; water/acetone ratio 1:28.
Sodium hyaluronate powder ~0.5 g) was refluxed in 50 ml of the ~ixture for 10 minutes. Then the powder was filtered off, ,~ashed thoroughly wlth a water/acetone 1:3 mixture, then with ~cetone, and dried in a vacuum oven. The hyaluronic acid powder :~btai~ed was insoluble in water and contained 1.41~ of combined ~ j Example 2 ~, The above example.was repeated with the cross-linkinq ~ixture of the following composition (~ by wt): CH20, 2.5; HCl, ~.38; wate~/acetone ratio, 1: 2 . The CH20 content of the pro-huct was 5.3%.
In Examples 1 and 2, the cross-linking of a hyaluronic cid powder was performed in water-acetone mixtures. By ~hanging the water/acetone ratio and the CH20 concentration, It is possible to control the swelling ratio of the product.
~hus, the swelling ratio was 178% for the product of Example ~ and 230~ for that of Example 2. The swelling ratio can be L educed by increasing the amount o~ acetone in the mixture and L he CH;20,concentration.
l -4- 1 i ~L~3~3 The following examples illustrate the use of a poly- ;
~aziridine compound as the cro~s~linking agent. This poly- ;
~aziridi~e type compound cross-links hyaluronic acid under dry c~nditions and at ambient temperature which is very important l¦in the case of hyaluronic acid as the latter is a heat sensitive .Ipolymer, ' Example 3 To 113.0 gm of a sodium hyaluronate solution in water ll(concentration 14.2 mg/ml), 0.42 g of polyaziridine compound -,~cross-linker CX-100 (Polyvinyl chemical~ ~as added. The molar ¦¦ratio of cross-linking agent to hyaluronic a~id was 0.5. The ¦! mixture was ca~t ln a glass plate as a 5mm thick layer and allowed to dry off at room temperature for 2 days. A clear l~film of cross-linked hyaluronic acid was obtained which was ¦¦not soluble in wat~r and had swelling ratio in water of 160%.
I! Exam le 4 :1 P ---,1 O.S g of a dry sodium hyaluronate powder was mix~d with l50 ml of a 1~ solution of cross-linXer CX-100 in acetone, kept ,for 5 minutes and filtered off. The powder was dried off in air for 2 hours, then washed several times with water and dried ~lin a ~a~uum oven at 40C for 4 hours~ The swelling ratio o~
!I the cross~linked powder in water was 135~.
¦ The following example illustrates the use of the poly-azlriaine compound for obtaining cross-linked hyaluronic acid wi~h a high degree of swelling.
Exa~le S
0.6 9 of solid sodium hyaluronate was mixed with 9.2 g of 0.5% by weight solution of cross-linker CX-100 in water.
The solution obtained had a molar ratio of CX-100 to sodium hyaluronate = ~ he sodium hyaluronate con~ent in the solution waæ 6.04~ by weight. The pH of the very viscous mix- ¦
ture obt.ained was ad~usted to 2.5 with 2~ HCL. The resultirl~ I
. 1, l _5_ "`` ~L2~ 3 fllm was readlly soluble In water. The fllm was heated at 60C
for 30 mlnutes. The heat treatment provlded a strong and water Insoluble fllm.
Example 6 Fl~er~ e sodlum hyalurona-te (0.~1093 g) was mlxed wl~h 25 ml of a 1% solutlon of polylsocyanate (supplled under the trademark Desmodur N-75, Mobay Chemical Corp.) In acetone and the ~ mlxture was refluxed for 10 mlnutes. The preclpltate was separated and washed three tlmes wlth acetone, drled In vacuum at 45 mm Hg and 60C for 30 mlnutes and, fInally, in a vacuum desslcator over phosphorus pentoxlde. The product obtalned (0.1127 g) was insoluble In water and had a degree of swelllng of 120%.
The followlng Example Illustrates the use of dlmethylolethylene urea for cross-lInklng hyaluronlc acld.
ExamPle 7 6.0 9 of sodlum hyaluronate solutlon In wa-ter (concentratlon 9.8 mg/ml) were mlxed wlth 0.017 g of N,N'-dlmethylolethylene urea and 0.005 g of ~Inc nltrate. The mlxture was cast onto a glass plate and left to dry off overnlght. The obtalned fllm was heat treated at 110C for 15 mlnutes. Itbecame Insoluble In water and had a degree of swelllng of 145%.
The followlng Examples Illustrate the use of dlvlnyl sulfone for cross-llnking hyaluronlc acld.
.
Example 8 35 ml of sodlum hyaluronate solutlon contalnlng 0.401 g (1 mmole) of dlvlnyl sulfone. The p~ of the mlxture was adJusted to approxImately 8.5-9 wlth a 1% solutlon of sodlum hydroxlde. A
~; ~
fllm was obtalned from the mlxture by castlng It onto a glass plate and drylng It overnlght at room temperature. Thls - 6a -.~
\
~3 Il film was re~dily soluble in water. The ~ilm was heated at 60C
jl for 30 minutes. The heat treatment provided a strong and water insoluble film.
Example 9 1 l A dry film of non cross-linked hyaluronic acid was put ! into a soIution of 0.6 g of divinyl sulfone in a mixture of ¦ 26 g of acetone and 13 g of water and kept there for 10 minutes.
¦ The film was removed from the solution, dried in air for 10 minutes and then heated in an oven for 20 mlnutes at 65C. A
l! strong cross-linked film of hyaluronic acid was obtained.
,j Example 10 ! 2.95 gm of air-dry sodium hyaluronate were mixed with 57.35 gm of an 0. 2N solution of NaOH in water and stirred with a glass rod un~il completely dissolved. Then 1.O gm of divinyl sulfone was stirred into the mixture and the latter was left ~j for one hour at room temperature. The mixture turned into a ! hard gel. The gel was put into a Vir-Tis "45" homogenizer ~¦ along with 100 ml of H20 and treated for 5 minutes at 30,000 rpm. Highly swollen small particles were obtained. The par-I ticles were washed several times with water and filtexed of~
il with suc~ion on a glass filter. To determine the swelling ,I ratio, about 1 gm of tha gel was put in a 15 ml glass filter '¦ which, in turn, was put into a plastic centrifuge tube. The ! gel was centrifuged for 30 minutes at 3 000 rpm. The pressed out water collected at the bottom of the tube. The hyaluronic ~¦ acid concentration in the gel was found to be G.21%, i.e., ~' the swelling ratio in water was 475.
The procedure described in Example 10 was repeated but the ~el obtained was dispersed in an 0.15 M solution of Nacl ~3~3 ¦ in water, and the particles were washed in the same solution.
The hyalurQnic acid concentration in th~ gel after centrifuga-tion was 1.29% and the swelling ratio was 77.5 Il Example 12 5 ,1 1.0 gm of air-dry sodium hyaluronate was dissolved in 900 gm of 0.2 M NaOH w~ile stirring with a glass rod. 0.33 gm of divinyl sulfone was stirred into the viscou5 solut.ion ob-ll tained and the mixture was left to stand for 20 hours at room il temperature. The hard gel obtained was treated as described 1l in ~xample 10. The hyaluronic acid concentration in the qel after centrifugation was 4.30~, i.e., the swelling ratio was j 230~.
!' i .I The biocompatibility of ~he preparations according to ¦ the invention was demonstrated by the test procedure hereafter 15 ¦I de~ribed.
I¦ Example 13 - Blood Compatibility Test I,I Release of 3H-serotonin by human platelets was used ,¦ in preliminary studies to assess the blood reactivity of a l; sa~ple prepared according to Example 10. Normal human venous ¦ blood was drawn into plas~lc syringes and immediately trans-ferred to plastlc tubes containing 3 . 8~ sodium citrate (one part citrate to nine parts whole blood~. Platelet rich plasma wa~ prepared by centrifugation at 4C for 15 minutes at 125 x 9 !l and removed by serological p~pet to a plastic or siliconized ; 25 il test tube. 3H-serotonin ~3H-5-hydroxytryptamine, 3H-5HT New ~I England Nuclear, 26.3 Ci/mmol, lmCi/ml ethanol-water) was ¦1 add~d to platelet rich plasma ~PRP), 0.2-O.S ul~ml PRP, and incubated for 15 minutes at 30C. In t~e assay, silîcon1zed or polypropylene test tube5 were~used; thrombin was used as a positive control, coated and uncoated samples were tested.
l _~_ .
~3~3 ~!
1.0-2.0 ml of 3H~5MT - PRP was adaed to eacll of duplicate test tube~ containing samples to be assayed; a 50 ul aliquot was removed from the control mlxture for determination of total ll radioactivlty. Following the appropriate incubation period S l~ ~10-120 minute3~ a.2 0.5 ml aliquots of the suspension we~e I --removed and centrifuged over silicon oil in an Eppendorf micro-fuge for 2 minutes at 12~000 x g. 50 ul of the supernatant waa re~oved from each tu~e, mixed with 5 ml of liquid scintilla I tlon fluid, and r~dioactivity measured by beta-spectrometry.
10 ¦ The amount of 3H-5HT released by thrombin or the test samples was the increment in radioacti~ity of the supernatant (radio- l , aotivity of experimental samples minus radioactivity of control,) The te~t material did not induce signf icant platelet release of 3H-5HT fox up to 120 minute~. ¦
15 1 . I
,1 1 , ,, ,1 l _9_ ,! '
~23~3 The cross-linking agent partlcularly used to make the instant prepara-tions is di~Tinyl sulfone. It has now been found that an insoluble hyaluronic acid gel can be obtained by cross-linking hyaluronic acid with divinyl sulfone (DVS) in water solution at pH higher than 9 at room temperature, i.e., about 20C. Depending upon the concentration and molecular size of the hyaluronic acid in the solution, the hyaluronic acid/divinyl sulfone ratio and reaction time, the swelling ratio of the gel can vary over broad limits, i.e., from 20 to 2000. The swelling ratio depends 10 substantially on the ionic strength and the hydrogen ion con- I
centration of the medium and decreases with the ionic strength.
This process contrasts with that disclosed and claimed in parent application serial no. 561,818, filed December 15, 1983 in that here r DVS is used at ambient temperatures under alkaline conditions whereas in the parent application, DVS is used as a cross-~inking agent only under reflux (ca. 60~65C).
~8~
DETAIL~D DESCRIPTION OF THE INVENTION
_ ' The following examples (wherein all parts gi~en are by weight unless otherwise specified) illustrate the several embodiments of the invention, without however being a limita-tion thereof, the in~ention being solely defined by the claims.
Example l To a water-acetone mixture a 37% by weight water solu-tion of formaldehyde and concentrated hydrochloric acid were bdded. The mixture obtained was of the following compositions (~ by wt~: CH20, 0.27; HCl, 0.19; water/acetone ratio 1:28.
Sodium hyaluronate powder ~0.5 g) was refluxed in 50 ml of the ~ixture for 10 minutes. Then the powder was filtered off, ,~ashed thoroughly wlth a water/acetone 1:3 mixture, then with ~cetone, and dried in a vacuum oven. The hyaluronic acid powder :~btai~ed was insoluble in water and contained 1.41~ of combined ~ j Example 2 ~, The above example.was repeated with the cross-linkinq ~ixture of the following composition (~ by wt): CH20, 2.5; HCl, ~.38; wate~/acetone ratio, 1: 2 . The CH20 content of the pro-huct was 5.3%.
In Examples 1 and 2, the cross-linking of a hyaluronic cid powder was performed in water-acetone mixtures. By ~hanging the water/acetone ratio and the CH20 concentration, It is possible to control the swelling ratio of the product.
~hus, the swelling ratio was 178% for the product of Example ~ and 230~ for that of Example 2. The swelling ratio can be L educed by increasing the amount o~ acetone in the mixture and L he CH;20,concentration.
l -4- 1 i ~L~3~3 The following examples illustrate the use of a poly- ;
~aziridine compound as the cro~s~linking agent. This poly- ;
~aziridi~e type compound cross-links hyaluronic acid under dry c~nditions and at ambient temperature which is very important l¦in the case of hyaluronic acid as the latter is a heat sensitive .Ipolymer, ' Example 3 To 113.0 gm of a sodium hyaluronate solution in water ll(concentration 14.2 mg/ml), 0.42 g of polyaziridine compound -,~cross-linker CX-100 (Polyvinyl chemical~ ~as added. The molar ¦¦ratio of cross-linking agent to hyaluronic a~id was 0.5. The ¦! mixture was ca~t ln a glass plate as a 5mm thick layer and allowed to dry off at room temperature for 2 days. A clear l~film of cross-linked hyaluronic acid was obtained which was ¦¦not soluble in wat~r and had swelling ratio in water of 160%.
I! Exam le 4 :1 P ---,1 O.S g of a dry sodium hyaluronate powder was mix~d with l50 ml of a 1~ solution of cross-linXer CX-100 in acetone, kept ,for 5 minutes and filtered off. The powder was dried off in air for 2 hours, then washed several times with water and dried ~lin a ~a~uum oven at 40C for 4 hours~ The swelling ratio o~
!I the cross~linked powder in water was 135~.
¦ The following example illustrates the use of the poly-azlriaine compound for obtaining cross-linked hyaluronic acid wi~h a high degree of swelling.
Exa~le S
0.6 9 of solid sodium hyaluronate was mixed with 9.2 g of 0.5% by weight solution of cross-linker CX-100 in water.
The solution obtained had a molar ratio of CX-100 to sodium hyaluronate = ~ he sodium hyaluronate con~ent in the solution waæ 6.04~ by weight. The pH of the very viscous mix- ¦
ture obt.ained was ad~usted to 2.5 with 2~ HCL. The resultirl~ I
. 1, l _5_ "`` ~L2~ 3 fllm was readlly soluble In water. The fllm was heated at 60C
for 30 mlnutes. The heat treatment provlded a strong and water Insoluble fllm.
Example 6 Fl~er~ e sodlum hyalurona-te (0.~1093 g) was mlxed wl~h 25 ml of a 1% solutlon of polylsocyanate (supplled under the trademark Desmodur N-75, Mobay Chemical Corp.) In acetone and the ~ mlxture was refluxed for 10 mlnutes. The preclpltate was separated and washed three tlmes wlth acetone, drled In vacuum at 45 mm Hg and 60C for 30 mlnutes and, fInally, in a vacuum desslcator over phosphorus pentoxlde. The product obtalned (0.1127 g) was insoluble In water and had a degree of swelllng of 120%.
The followlng Example Illustrates the use of dlmethylolethylene urea for cross-lInklng hyaluronlc acld.
ExamPle 7 6.0 9 of sodlum hyaluronate solutlon In wa-ter (concentratlon 9.8 mg/ml) were mlxed wlth 0.017 g of N,N'-dlmethylolethylene urea and 0.005 g of ~Inc nltrate. The mlxture was cast onto a glass plate and left to dry off overnlght. The obtalned fllm was heat treated at 110C for 15 mlnutes. Itbecame Insoluble In water and had a degree of swelllng of 145%.
The followlng Examples Illustrate the use of dlvlnyl sulfone for cross-llnking hyaluronlc acld.
.
Example 8 35 ml of sodlum hyaluronate solutlon contalnlng 0.401 g (1 mmole) of dlvlnyl sulfone. The p~ of the mlxture was adJusted to approxImately 8.5-9 wlth a 1% solutlon of sodlum hydroxlde. A
~; ~
fllm was obtalned from the mlxture by castlng It onto a glass plate and drylng It overnlght at room temperature. Thls - 6a -.~
\
~3 Il film was re~dily soluble in water. The ~ilm was heated at 60C
jl for 30 minutes. The heat treatment provided a strong and water insoluble film.
Example 9 1 l A dry film of non cross-linked hyaluronic acid was put ! into a soIution of 0.6 g of divinyl sulfone in a mixture of ¦ 26 g of acetone and 13 g of water and kept there for 10 minutes.
¦ The film was removed from the solution, dried in air for 10 minutes and then heated in an oven for 20 mlnutes at 65C. A
l! strong cross-linked film of hyaluronic acid was obtained.
,j Example 10 ! 2.95 gm of air-dry sodium hyaluronate were mixed with 57.35 gm of an 0. 2N solution of NaOH in water and stirred with a glass rod un~il completely dissolved. Then 1.O gm of divinyl sulfone was stirred into the mixture and the latter was left ~j for one hour at room temperature. The mixture turned into a ! hard gel. The gel was put into a Vir-Tis "45" homogenizer ~¦ along with 100 ml of H20 and treated for 5 minutes at 30,000 rpm. Highly swollen small particles were obtained. The par-I ticles were washed several times with water and filtexed of~
il with suc~ion on a glass filter. To determine the swelling ,I ratio, about 1 gm of tha gel was put in a 15 ml glass filter '¦ which, in turn, was put into a plastic centrifuge tube. The ! gel was centrifuged for 30 minutes at 3 000 rpm. The pressed out water collected at the bottom of the tube. The hyaluronic ~¦ acid concentration in the gel was found to be G.21%, i.e., ~' the swelling ratio in water was 475.
The procedure described in Example 10 was repeated but the ~el obtained was dispersed in an 0.15 M solution of Nacl ~3~3 ¦ in water, and the particles were washed in the same solution.
The hyalurQnic acid concentration in th~ gel after centrifuga-tion was 1.29% and the swelling ratio was 77.5 Il Example 12 5 ,1 1.0 gm of air-dry sodium hyaluronate was dissolved in 900 gm of 0.2 M NaOH w~ile stirring with a glass rod. 0.33 gm of divinyl sulfone was stirred into the viscou5 solut.ion ob-ll tained and the mixture was left to stand for 20 hours at room il temperature. The hard gel obtained was treated as described 1l in ~xample 10. The hyaluronic acid concentration in the qel after centrifugation was 4.30~, i.e., the swelling ratio was j 230~.
!' i .I The biocompatibility of ~he preparations according to ¦ the invention was demonstrated by the test procedure hereafter 15 ¦I de~ribed.
I¦ Example 13 - Blood Compatibility Test I,I Release of 3H-serotonin by human platelets was used ,¦ in preliminary studies to assess the blood reactivity of a l; sa~ple prepared according to Example 10. Normal human venous ¦ blood was drawn into plas~lc syringes and immediately trans-ferred to plastlc tubes containing 3 . 8~ sodium citrate (one part citrate to nine parts whole blood~. Platelet rich plasma wa~ prepared by centrifugation at 4C for 15 minutes at 125 x 9 !l and removed by serological p~pet to a plastic or siliconized ; 25 il test tube. 3H-serotonin ~3H-5-hydroxytryptamine, 3H-5HT New ~I England Nuclear, 26.3 Ci/mmol, lmCi/ml ethanol-water) was ¦1 add~d to platelet rich plasma ~PRP), 0.2-O.S ul~ml PRP, and incubated for 15 minutes at 30C. In t~e assay, silîcon1zed or polypropylene test tube5 were~used; thrombin was used as a positive control, coated and uncoated samples were tested.
l _~_ .
~3~3 ~!
1.0-2.0 ml of 3H~5MT - PRP was adaed to eacll of duplicate test tube~ containing samples to be assayed; a 50 ul aliquot was removed from the control mlxture for determination of total ll radioactivlty. Following the appropriate incubation period S l~ ~10-120 minute3~ a.2 0.5 ml aliquots of the suspension we~e I --removed and centrifuged over silicon oil in an Eppendorf micro-fuge for 2 minutes at 12~000 x g. 50 ul of the supernatant waa re~oved from each tu~e, mixed with 5 ml of liquid scintilla I tlon fluid, and r~dioactivity measured by beta-spectrometry.
10 ¦ The amount of 3H-5HT released by thrombin or the test samples was the increment in radioacti~ity of the supernatant (radio- l , aotivity of experimental samples minus radioactivity of control,) The te~t material did not induce signf icant platelet release of 3H-5HT fox up to 120 minute~. ¦
15 1 . I
,1 1 , ,, ,1 l _9_ ,! '
Claims (8)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1, A method of making a water insoluble hyaluronic acid preparation, said method comprising subjecting hyaluronic acid in the form of a powder, film or gel to treatment with a cross-linking agent selected from the group consisting of formaldehyde, dimethylol ura, dimethylolethylene urea, ethylene oxide, a polyaziridine, a polyisocyanate and divinyl sulfone.
2. A method according to claim 1 wherein the cross-linking agent is formaldehyde and treatment is effected in an aqueous medium at reflux temperature.
3. A method according to claim 1 wherein the cross-linking agent is a polyaziridine and treatment is effected under dry conditions at ambient temperature.
4. A method according to claim 1 wherein the cross-linking agent is a polyisocyanate and treatment is effected in acetone at reflux temperature.
5. A method according to claim 1 wherein the cross-linking agent is dimethylolethylene urea and treatment is effected at about 110°C.
6. A method according to claim 1 wherein the cross-linking agent is divinyl sulfone and treatment is effected at about 60-65°C.
7. The product produced by the method according to claim 1.
8. A method of making a water insoluble hyaluronic acid preparation, said method comprising subjecting hyaluronic acid or an acid addition salt thereof in the form of a powder, film or jelly to treatment with divinyl sulfone at about 20°C
in an aqueous alkaline medium.
9. A method according to claim 8 wherein treatment is effected at a pH in excess of about pH 9.
10. A method according to claim 8 wherein treatment is effected for 1-20 hours.
11. A method according to claim 8 wherein the ratio of hyaluronic acid to divinyl sulfone is about 3:1 by weight.
12. The product produced by the method according to
8. A method of making a water insoluble hyaluronic acid preparation, said method comprising subjecting hyaluronic acid or an acid addition salt thereof in the form of a powder, film or jelly to treatment with divinyl sulfone at about 20°C
in an aqueous alkaline medium.
9. A method according to claim 8 wherein treatment is effected at a pH in excess of about pH 9.
10. A method according to claim 8 wherein treatment is effected for 1-20 hours.
11. A method according to claim 8 wherein the ratio of hyaluronic acid to divinyl sulfone is about 3:1 by weight.
12. The product produced by the method according to
claim 8.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US56181883A | 1983-12-15 | 1983-12-15 | |
| US561,818 | 1983-12-15 | ||
| US59807184A | 1984-04-09 | 1984-04-09 | |
| US598,071 | 1984-04-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1238043A true CA1238043A (en) | 1988-06-14 |
Family
ID=27072762
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000460460A Expired CA1238043A (en) | 1983-12-15 | 1984-08-07 | Water insoluble preparations of hyaluronic acid and processes therefor |
Country Status (6)
| Country | Link |
|---|---|
| AU (1) | AU551628B2 (en) |
| CA (1) | CA1238043A (en) |
| DE (1) | DE3434104A1 (en) |
| FR (1) | FR2556728B1 (en) |
| GB (1) | GB2151244B (en) |
| IT (1) | IT1178588B (en) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4582865A (en) * | 1984-12-06 | 1986-04-15 | Biomatrix, Inc. | Cross-linked gels of hyaluronic acid and products containing such gels |
| US4713448A (en) * | 1985-03-12 | 1987-12-15 | Biomatrix, Inc. | Chemically modified hyaluronic acid preparation and method of recovery thereof from animal tissues |
| US4851521A (en) * | 1985-07-08 | 1989-07-25 | Fidia, S.P.A. | Esters of hyaluronic acid |
| US5202431A (en) * | 1985-07-08 | 1993-04-13 | Fidia, S.P.A. | Partial esters of hyaluronic acid |
| IT1198449B (en) * | 1986-10-13 | 1988-12-21 | F I D I Farmaceutici Italiani | ESTERS OF POLYVALENT ALCOHOLS OF HYALURONIC ACID |
| US4937270A (en) * | 1987-09-18 | 1990-06-26 | Genzyme Corporation | Water insoluble derivatives of hyaluronic acid |
| US5017229A (en) * | 1990-06-25 | 1991-05-21 | Genzyme Corporation | Water insoluble derivatives of hyaluronic acid |
| US5527893A (en) * | 1987-09-18 | 1996-06-18 | Genzyme Corporation | Water insoluble derivatives of polyanionic polysaccharides |
| US6610669B1 (en) | 1987-09-18 | 2003-08-26 | Genzyme Corporation | Water insoluble derivatives of polyanionic polysaccharides |
| US6174999B1 (en) | 1987-09-18 | 2001-01-16 | Genzyme Corporation | Water insoluble derivatives of polyanionic polysaccharides |
| AU619760B2 (en) * | 1987-12-10 | 1992-02-06 | Genzyme Biosurgery Corporation | Hylan preparation and method of recovery thereof from animal tissues |
| US4888016A (en) * | 1988-02-10 | 1989-12-19 | Langerman David W | "Spare parts" for use in ophthalmic surgical procedures |
| ATE126071T1 (en) * | 1988-12-29 | 1995-08-15 | Chang Ann Lois | DIAPHRAGM PUMP. |
| IT1260154B (en) * | 1992-07-03 | 1996-03-28 | Lanfranco Callegaro | HYALURONIC ACID AND ITS DERIVATIVES IN INTERPENETRATING POLYMERS (IPN) |
| US5550112A (en) * | 1992-12-30 | 1996-08-27 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5679655A (en) * | 1993-08-04 | 1997-10-21 | Patent Biopharmaceutics, Inc. | Method of treating lesions resulting from genital herpes with hyaluronic acid-urea pharmaceutical compositions |
| US5531716A (en) * | 1993-09-29 | 1996-07-02 | Hercules Incorporated | Medical devices subject to triggered disintegration |
| US6294202B1 (en) | 1994-10-06 | 2001-09-25 | Genzyme Corporation | Compositions containing polyanionic polysaccharides and hydrophobic bioabsorbable polymers |
| DE69635127T2 (en) * | 1995-06-06 | 2006-06-29 | C.R. Bard, Inc. | METHOD FOR PRODUCING NETWORKED WATER-SOLUBLE POLYMER PARTICLES, THE PARTICLES AND THEIR USE |
| US6214331B1 (en) | 1995-06-06 | 2001-04-10 | C. R. Bard, Inc. | Process for the preparation of aqueous dispersions of particles of water-soluble polymers and the particles obtained |
| US6368356B1 (en) | 1996-07-11 | 2002-04-09 | Scimed Life Systems, Inc. | Medical devices comprising hydrogel polymers having improved mechanical properties |
| US6060534A (en) | 1996-07-11 | 2000-05-09 | Scimed Life Systems, Inc. | Medical devices comprising ionically and non-ionically crosslinked polymer hydrogels having improved mechanical properties |
| DE69822285T2 (en) * | 1998-02-27 | 2005-01-27 | Stichting Hyppomedics | Process for the preparation of crosslinked hyaluronic acid |
| GB9902652D0 (en) * | 1999-02-05 | 1999-03-31 | Fermentech Med Ltd | Process |
| US6521223B1 (en) | 2000-02-14 | 2003-02-18 | Genzyme Corporation | Single phase gels for the prevention of adhesions |
| US7387836B2 (en) | 2002-04-17 | 2008-06-17 | Genzyme Corporation | Aziridine compounds and their use in medical devices |
| AU2003230948A1 (en) * | 2002-04-17 | 2003-11-03 | Genzyme Corporation | Cross-linked hyaluronate compounds |
| AU2003901834A0 (en) | 2003-04-17 | 2003-05-01 | Clearcoll Pty Ltd | Cross-linked polysaccharide compositions |
| US8313765B2 (en) | 2003-12-04 | 2012-11-20 | Industrial Technology Research Institute | Biodegradable hyaluronic acid derivative, biodegradable polymeric micelle composition and pharmaceutical or bioactive composition |
| GB2408741B (en) * | 2003-12-04 | 2008-06-18 | Ind Tech Res Inst | Hyaluronic acid derivative with urethane linkage |
| US20050220721A1 (en) | 2004-03-17 | 2005-10-06 | Kablik J J | Anti-adhesion spraying |
| US7858107B2 (en) | 2004-09-10 | 2010-12-28 | Medtronic Xomed, Inc. | Flexible bioresorbable hemostatic packing and stent having a preselectable in-vivo residence time |
| FR2882366B1 (en) | 2005-02-18 | 2008-04-18 | Coletica Sa | RETICULATED CARBOHYDRATE POLYMER, IN PARTICULAR BASED ON POLYSACCHARIDES AND / OR POLYOLS |
| GB2423252B (en) * | 2005-02-18 | 2007-10-17 | Engelhard Lyon | Cross-linked polymer of carbohydrate, notably based on polysaccharides, and/or on oligosaccharides and/or on polyols |
| EP1911769B1 (en) | 2005-08-04 | 2013-10-30 | Teijin Limited | Cellulose derivative |
| FR2918377B1 (en) | 2007-07-05 | 2010-10-08 | Estelle Piron | CO-RETICLE GEL OF POLYSACCHARIDES |
| WO2009041627A1 (en) | 2007-09-28 | 2009-04-02 | Shiseido Company Ltd. | Swellable crosslinked hyaluronic acid powder and method for producing the same |
| FR2924615B1 (en) | 2007-12-07 | 2010-01-22 | Vivacy Lab | HYDROGEL COHESIVE BIODEGRADABLE. |
| US10071186B2 (en) | 2012-03-12 | 2018-09-11 | Colorado State University Research Foundation | Glycosaminoglycan and synthetic polymer material for blood-contacting applications |
| US10266684B2 (en) | 2012-03-12 | 2019-04-23 | Colorado State University Research Foundation | Glycosaminoglycan and synthetic polymer materials for blood-contacting applications |
| ITMI20131193A1 (en) * | 2013-07-16 | 2015-01-17 | Ira Srl | RETICULATED HYALURONIC ACID, PROCESS FOR ITS PREPARATION AND AESTHETIC APPLICATIONS |
| ITUA20161822A1 (en) * | 2016-03-18 | 2017-09-18 | Indena Spa | USEFUL COMPOSITIONS IN THE PREVENTION AND / OR IN THE TREATMENT OF ORAL CABLE PATHOLOGIES, OF THE FIRST AIRWAYS AND OF THE ESOFAGO |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE361320B (en) * | 1972-03-14 | 1973-10-29 | Exploaterings Ab Tbf | |
| US4152170A (en) * | 1975-06-18 | 1979-05-01 | Sumitomo Chemical Company, Ltd. | Cross-linked pullulan |
| GB1515963A (en) * | 1975-07-15 | 1978-06-28 | Massachusetts Inst Technology | Crosslinked collagen-mucopolysaccharide composite materials |
-
1984
- 1984-08-07 CA CA000460460A patent/CA1238043A/en not_active Expired
- 1984-08-13 GB GB08420560A patent/GB2151244B/en not_active Expired
- 1984-09-17 DE DE19843434104 patent/DE3434104A1/en active Granted
- 1984-09-21 AU AU33379/84A patent/AU551628B2/en not_active Expired
- 1984-10-18 IT IT23225/84A patent/IT1178588B/en active
- 1984-11-26 FR FR8417956A patent/FR2556728B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU551628B2 (en) | 1986-05-08 |
| IT8423225A1 (en) | 1986-04-18 |
| DE3434104C2 (en) | 1991-06-20 |
| FR2556728A1 (en) | 1985-06-21 |
| GB8420560D0 (en) | 1984-09-19 |
| AU3337984A (en) | 1985-06-20 |
| GB2151244A (en) | 1985-07-17 |
| FR2556728B1 (en) | 1987-06-26 |
| IT8423225A0 (en) | 1984-10-18 |
| DE3434104A1 (en) | 1985-08-29 |
| IT1178588B (en) | 1987-09-09 |
| GB2151244B (en) | 1987-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1238043A (en) | Water insoluble preparations of hyaluronic acid and processes therefor | |
| US4500676A (en) | Hyaluronate modified polymeric articles | |
| US4487865A (en) | Polymeric articles modified with hyaluronate | |
| US4755379A (en) | Polymers substituted by groups conferring anti-coagulant properties on them, process for their preparation, articles and compositions made therefrom and uses thereof | |
| Ratner et al. | Cell adhesion to polymeric materials: implications with respect to biocompatibility | |
| EP0537292B1 (en) | Water insoluble derivatives of hyaluronic acid | |
| US4871357A (en) | Ionic heparin coating | |
| AU629467B2 (en) | Biocompatible viscoelastic gel slurries, their preparation and use | |
| KR100378109B1 (en) | Hydrophobic multicomponant heparin conjugates, a preparing method and a use thereof | |
| US3717502A (en) | Method for providing anticoagulant surfaces onto articles | |
| GB2150833A (en) | Proteolytic wounds dressing | |
| KR20080062092A (en) | Hyaluronic acid derivatives for cell delivery carriers and their process for preparing the same | |
| US7345117B1 (en) | Sulphated hyaluronic acid and sulphated derivatives thereof covalently bound to polyurethanes, and the process for their preparation | |
| AU2004303599B2 (en) | Compositions of semi-interpenetrating polymer network | |
| Mason et al. | Thrombogenicity of some biomedical materials: Platelet‐interface reactions | |
| US3865723A (en) | Method to form stable complexes of polyanions occurring in biological liquids | |
| JP2003523459A (en) | Chitosan condensation products, their production and use | |
| US4082727A (en) | Method for manufacture of non-coagulative organosilicone polymer | |
| JPS60130601A (en) | Manufacture of hyaluronic acid water-insoluble composition | |
| Raghunath et al. | Some characteristics of collagen‐heparin complex | |
| Plate et al. | On the mechanism of enhanced thromboresistance of polymeric materials in the presence of heparin | |
| JPS61130302A (en) | Ribofuranan sulfate having anticoagulant activity and its production | |
| Kashiwagi et al. | Non-thrombogenicity of organic polymers by blending with alkylamine-heparin complexes | |
| US2729633A (en) | Sulfated pectic acid blood anticoagulant | |
| Dincer et al. | Covalent immobilization of heparin on polymethylacrylate surfaces |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |