Note: Descriptions are shown in the official language in which they were submitted.
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).
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, 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;
6. polyisocyana~e; and
3~) 7. divinyl sulfone.
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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).
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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.
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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,
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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
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fllm was obtalned from the mlxture by castlng It onto a glass
plate and drylng It overnlght at room temperature. Thls
1 0
1 5
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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
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¦ 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.
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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
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