The present invention relates to the field of bio¬ compatible polysaccharide gel compositions, and more spe¬ cifically to a novel process for cross-linking such com- positions, a new gel structure thereby being obtained.

The new structure imparts improved properties to the pre¬ viously known gei compositions as well as enables new uses of said compositions, both as such and containing active ingredients. BACKGROUND OF THE INVENTION

Water-binding gels are widely used in the biomedical field. They are generally prepared by chemical cross¬ linking of polymers to infinite networks. When using bio¬ compatible polymers generally a low degree of cross- linking has to be utilized to maintain said biocompati¬ bility. However, often a more dense gel is required to have a proper effect of the active ingredients utilized, and in such a case the biocompatibility will often go lost. Another valuable property of water-binding gels, or hydrogels, is that peptides and larger biologically ac¬ tive substances can be enclosed therein to the formation of a sustained release composition. However, practical problems have been involved in accomplishing a sufficient maintenance time of the active ingredient, since gener¬ ally the active ingredient is released at the same rate with which it was dissolved or enclosed in the composi¬ tion referred to. Furthermore, if such a gel were densi¬ fied in ah attempt to maintain the active ingredient for a longer time, it would rapidly swell in an animal tissue where there is a free access of water.

One of the most widely used biocompatible polymers for medical use is hyaluronic acid. As it is present in identical composition in each living organism, it gives a minimum of reactions and allows for advanced medical

uses. As a consequence thereof it has been the subject of many modification attempts. Thus, it has been cross¬ linked with agents such as aldehydes, epoxides, polyaz- iridyl compounds and divinylsulfone (Laurent et al, Acta Chem. Scand 18 (1964) No 1, p. 274; EP 0 161 887B1; EP 0 265 116A2; and US 4,716,154).

In WO 87/07898 there is disclosed a reaction of a polysaccharide with a polyfunctional epoxide, removal of excess of said epoxide and finally drying operation to cross-link said polysaccharide into a film, powdered ma¬ terial or similar dry product. However, there is no sug¬ gestion therein to dilute the activated polysaccharide and then reconcentrate the same to the desired density or consistency which is then substantially permanent. US 5,128,326 discloses a number of modified hyaluronic acids for use as depot pharmaceuticals. The disclosed methods of "charging" the gel preparations are all based on a diffusion of the active ingredient into the gel and then a release thereof with the same diffu- sion constant. Contrary thereto the present invention in¬ volves a dissolution of the active ingredient followed by a densification or concentration of the gel composition until no or a very minor diffusion of said active ingre¬ dient takes place. US 5,399,351 discloses mixtures of gel and polymeric solutions, said solutions being utilized to improve the rheological properties of the gel. However, also in this case reversibly compressed gels are disclosed, as can be gathered from e.g. col. 6, lines 53-58. SUMMARY OF THE INVENTION

According to the present invention it has unexpect¬ edly been found that polysaccharide gel compositions hav¬ ing a novel structure and thereby new, outstanding ^' prop¬ erties can be obtained by using a new technique for the cross-linking thereof. Said new cross-linking technique enables a versatile control of the structure and proper¬ ties of the manufactured polysaccharide gel composition.

which in turn makes it possible to tailor the final com¬ position for the inteded purposes.

More specifically, one object of the present inven¬ tion is to provide a process for preparing a cross-linked polysaccharide gel composition, the biocompatibility of which can be retained in spite of a high degree of cross¬ linking or polymerisation.

Another object of the invention is to provide a polysaccharide gel composition with viscoelastic proper- ties in spite of being cross-linked to a substantial de¬ gree.

Yet another object of the invention is to provide a polysaccharide gel composition which is more or less ir- reversably densified or concentrated, i.e. which does not swell substantially or only to a limited degree when con¬ tacted with water.

Still another object of the invention is to provide a polysaccharide gel composition enclosing a biologically active substance for use as a sustained release co posi- tion or a depot composition.

Another object of the invention is to provide poly¬ saccharide gel compositions containing a variety of bio¬ logically active substances for uses as medical or pro¬ phylactic compositions for different purposes. Yet another object of the invention is to provide uses of the compositions referred to for the manufacture of medical or prophylactic compositions as well as for administration to mammals, especially humans.

Yet another object of the invention is to provide a partially cross-linked activated polysaccharide gel com¬ position as obtained as an intermediate in the above¬ mentioned process according to the invention, which in¬ termediate can be finally cross-linked in situ at any de¬ sired site. These and further objects of the invention will be¬ come apparent by the more detailed description thereof presented below.

DETAILED DESCRIPTION OF THE INVENTION According to one aspect of the present invention a process for preparing a cross-linked biocompatible poly¬ saccharide gel composition is thus provided, which proc- ess comprises:

forming an aqueous solution of a water soluble, cross¬ linkable polysaccharide;

initiating a cross-linking of said polysaccharide in the presence of a polyfunctional cross-linking agent there¬ for;

sterically hindering the cross-linking reaction from be- ing terminating before gelation occurs, an activated polysaccharide thereby being obtained; and

reintroducing sterically unhindered conditions for said activated polysaccharide so as to continue the cross- linking thereof up to a viscoleastic gel.

In other words the new process according to the present invention involves a cross-linking of a water¬ soluble, cross-linkable polysaccharide in at least two steps or stages, where the cross-linking reaction is dis- continued before the gelation is initiated, said discon¬ tinuance being accomplished by sterically hindering said cross-linking reaction. The cross-linking reaction is then continued in a second step by reintroducing steri¬ cally unhindered conditions. Thus, firstly is has unexpectedly been found that by said sterical hindrance an activated polysaccharide is obtained, the cross-linking or polymerization of which can be continued merely by reintroducing sterically un¬ hindered conditions therefor. Secondly, it has also unex- pectedly been found that the polysaccharide gel composi¬ tion obtained thereby does not form the compact, dense structure which would have been obtained if performing

the corresponding cross-linking reaction in one single step to a fully cross-linked gel but rather a viscoleas- tic gel. Furthermore, as was mentioned above, the new gel structure obtained by the present invention represents a substantially irreversible gel structure which does not swell to any appreciable extent in contact with water or any other aqueous medium. Generally this means that said reswelling is less than 10% by volume based on the volume as obtained from the process claimed. Although the invention is not bound by any theory it may be that the new structure obtained by the present in¬ vention is a combination of cross-linking between exist¬ ing polymer chains and an elongation of existing chains rather than a very dense network giving a very rigid structure. What may suggest such a mechanism is the fact that a viscoelastic product is obtainable by the inven¬ tion.

As used herein the term "sterically hindering the cross-linking reaction" should be interpreted in a broad sense, i.e. it need not necessarily be a complete hinder- ance but in many cases rather a partial hindrance of the reaction referred to. That is, what is important is that the rate of cross-linking is substantially reduced to en¬ able the final cross-linking reaction to take place with new reaction sites involved.

Similarly, the term "reintroducing sterically unhin¬ dered conditions" should also be interpreted broadly, which generally means that said sterically unhindered conditions need not necessarily be exactly the same sterical conditions as were used when initiating the cross-linking reaction. Thus, what is generally of impor¬ tance is that said sterically unhindered conditions en¬ able more rapid reactions to take place than said steri¬ cally hindered conditions. The sterical hindrance of the cross-linking reaction should be obtainable in different ways, but a preferred embodiment of the invention in this respect is repre-

sented by the case where the sterical hindrance comprises diluting the aqueous medium in which the cross-linking reaction is performed, to accomplish a lower concentra¬ tion of the polysaccharide in said medium. To reintroduce sterically unhindered conditions should also be possible in different ways, but a pre¬ ferred embodiment in this respect is the case which com¬ prises evaporating the aqueous medium in which the cross¬ linking reaction is performed, to accomplish a higher concentration of the polysaccharide in said medium. An¬ other preferred embodiment in this respect is represented by the case comprising dialysing the aqueous medium in which the cross-linking reaction is performed.

According to a preferred embodiment of the invention the sterical hindrance of the cross-linking reaction is accomplished before the cross-linking agent has been con¬ sumed. This in turn generally also means that the rein¬ troduction of sterically unhindered conditions is initi¬ ated in the presence of said non-consumed cross-linking agent.

The sterical hindrance of the cross-linking reaction can generally be started or performed in the range of 50- 90% of the total gelation time used in the process ac¬ cording to the invention, consideration also being taken to suitable elasticity or consistency for the intended use of the composition.

The inventive idea should be applicable to any bio¬ compatible polysaccharide that is cross-linkable and soluble in an aqueous medium. Thus, the term "water solu- ble" should be interpreted in a broad sense, pure water not necessarily being necessary. That is, aqueous solu¬ tion means any solution wherein water is the major compo¬ nent. A preferred sub-group of polysaccharides in connec¬ tion with the invention is, however, a glucose amine glu- can, of which hyaluronic acid is a specially interesting example.

The cross-linking agent to be used in connection with the invention is any previously known cross-linking agent useful in connection with polysaccharides, consid¬ eration being taken to ensure that the biocompatibility prerequisites are fulfilled. Preferably, however, the cross-linking agent is selected from the group consisting of aldehydes, epoxides, polyaziridyl compounds, glycidyl ethers and dividylsulfones. Of these glycidyl ethers rep¬ resent an especially preferred group, of which 1,4- butandiol digylcidylether can be referred to as a pre¬ ferred example. In this connection it should also be men¬ tioned that "polyfunctional" includes difunctional.

The initial cross-linking reaction in the presence of a polyfunctional cross-linking agent can _. be performed at varying pH values, primarily depending on whether ether or ester reactions should be promoted. Preferably this means that said cross-linking reaction is performed at an alkaline pH, especially above pH 9, e.g. in the range of pH 9-12, when promoting ether formations. When promoting ester formations said cross-linking reaction is preferably performed at an acidic pH, especially at pH 2-6.

One interesting aspect of the invention is repre¬ sented by the case where the prepared cross-linked poly- saccharide gel composition is utilized as such as the in¬ vention enables the manufacture of a viscoelastic compo¬ sition. Such a viscoelastic composition is for instance useful in eye surgery, as a synovial fluid substitute, as eyedrops, etc, and as has been referred to above the pre- sent invention makes it possible to tailor the viscoelas¬ tic properties for such uses. Thus, by utilizing the sterical technology according to the present invention it is possible to obtain chain extensions, chain branchings, cross-links, etc, in a more controlled way that by the previously used techniques with more or less randomized coupling sites.

Furthermore, through the fact that the gels obtained in accordance with the invention do not retain their original volume in the presence of an aqueous medium, the new products do not cause any interfering or negative volume effects in these or other medical uses.

In accordance with the present invention it is also possible to include within the polysaccharide gel compo¬ sition"any biologically active substance for which a polysaccharide gel carrier is desired or accepted. In this context the dilution-concentration technique used in the process claimed enables the enclosure of said bio¬ logically active substance before subjecting the polysac¬ charide to sterically unhindered conditions. That is, while sterically unhindered conditions generally means a concentrating operation, such an operation means that the biologically active substance will be present in a phase that is more compacted than when said substance was in¬ cluded in said carrier. In other words the biologically active substance can be retained much longer as compared to previously known gel cross-linking reactions. Thereby a better sustained release profile for the active sub¬ stance is obtainable.

In connection with the incorporation of the biologi¬ cally active substance into the composition an adjustment of the conditions to physiological pH and salt conditions is preferably performed to have a preparation ready for medical use. Such a physiological adjustment is preferred also as concerns the reaction conditions as the second step of the process has been found to proceed well under suchg conditions.

The invention should not be limited in any respect as to the biologically active substance as compared to the use of said substance in prior cases. In other words the condition to be treated should be decisive for the specific substance to be selected.

However, interesting substances in connection with the invention can be selected from the group consisting

of hormones, cytokines, vaccines, cells and tissue aug¬ menting substances. Thus, the unique combination of prop¬ erties of the new gel composition according to the pres¬ ent invention makes it extremely advantageous in connec- tion with these substances, i.e. primarily thanks to out¬ standing depot or sustained release properties and non- swelling properties.

One interesting group of biologically active sub¬ stances thus is tissue augmenting substances as a poly- saccharide gel is an advantageous carrier therefor. Fur¬ ther details concerning such products can be found in W094/21299. More specifically, a preferred tissue aug¬ menting substance comprises a polymer selected from col¬ lagen, starch, dextranomer, polylactide and compolymers thereof, and poly-β-hydroxibutyrate and copolymers thereof.

In connection with hormones erytropoeitin and calci¬ tonin are especially preferred.

The process according to the present invention also enables the incorporation of the biologically active sub¬ stance by chemical reaction with the polysaccharide gel structure, or the cross-linking agent therefor, ^' provided that said active substance contains functional groups re¬ active therewith. Unique properties or combinations of properties can thereby be obtained as in such a case for instance the release rate of the active ingredient will be decided by the degredation or decomposition of the polymer network rather than by the dissolution or migra¬ tion rate for the substance referred to from the gel net- work.

A modification of last-mentioned technique in accor¬ dance with the invention means that the functional groups of the active substance may have been prereacted with a cross-linking agent for the polysaccharide. Preferably the same cross-linking agent is used as is used in the cross-linking of the polysaccharide.

Since the process of the present invention provides a new polysaccharide gel composition or structure, an¬ other aspect of the invention is represented by the novel polysaccharide gel composition prepared. In this respect the scope of protection encompasses not only the polysac¬ charide gel composition whenever prepared by said process but also any polysaccharide gel composition which is ob¬ tainable by a similar technique.

Expressed in another way the present invention also provides a cross-linked biocompatible polysaccharide gel composition, which is obtainable by cross-linking of a cross-linkable polysaccharide with a polyfunctional cross-linking agent therefor in two steps, the first cross-linking step being terminating before gelation oc- curs, by a sterical hindrance of the cross-linking reac¬ tion, and the second cross-linking step being initiated by reintroducing sterically unhindered conditions for said cross-linking reaction to continue the same up to a viscoelastic gel. All those features which have been presented as pre¬ ferred or interesting features in connection with the claimed process are applicable also to said polysaccha¬ ride gel composition per se and need not be repeated once more. Still another aspect of the invention is represented by the case where an intermediate product is obtained by postponing the final step of the cross-linking reaction with sterically unhindered conditions to a later stage or site, for instance at the ultimate use of the composi- tion. Thus, it has been found that the intermediate prod¬ uct obtained after the sterical hindrance of the cross¬ linking reaction possesses such a stability that the ter¬ mination of the cross-linking reaction can be performed at a later stage. The invention also relates to the composition de¬ fined above for use a medical or prophylactic composi¬ tion.

Another aspect of the invention is the use of said composition for the manufacture of a medical or prophy¬ lactic composition for any of the above-mentioned spe¬ cific medical or therapeutical purposes, tissue augmenta- tion and hormone treatment of a mammal, especially a hu¬ man being, being preferred applications.

Finally, the invention relates to a method of medi¬ cal or prophylactic treatment of a mammal, especially a human being, which comprises administering a composition as defined above to a mammal in need of such a treatment.

EXAMPLES

The invention will now be exemplified by the follow¬ ing non-limiting examples.

Example 1 Activation of the polymer. a. Under alkaline conditions Polysaccharide in the form of 10 g of hyaluronic acid prepared by fermentation of Streptococcus were dis¬ solved in 100 ml of 1% NaOH pH >9. Cross-linking agent in the form of 1, 4-butandiol diglycidylether was added to a concentration of 0,2%. The solution was incubated at 40°C for 4 hours. b. Under acidic conditions

The experiment was performed as in la but at an acidic pH of about 2-6 by the addition of 1% of acetic acid to the solution instead of NaOH according to la. Example 2

Preparation of a viscoelastic gel.

The incubates according to la and lb were diluted to a volume which was twice the volume finally desired or about 0,5-1% and were neutralised. The gel was then ro¬ tary evaporated to a viscoelastic gel. Example 3

Preparation of a gel containing dextranomer particles, The incubates according to la and lb were diluted to a strength of 1% and 20 g dry dextranomer particles (Sephadex®25, Pharmacia) were mixed with the solution,

the particles being enclosed by the cross-linking of hyaluronic acid polymer in a few minutes as a consequence of the concentration of hyaluronic acid which is accom¬ plished by an absorption of water by the dextranomer beads.

The viscoelastic gels obtained were stable, auto¬ clavable and injectable by means of thin hypodermic need¬ les.

Example 4 Preparation of a gel for use as a depot medicine containing erytropoeitin (EPO) .

The incubate obtained in Example la was diluted to a strength of 1% and the pH was adjusted by the addition of a citrate buffert according to the instructions from the manufacturer (Ortho Biotech Inc., Raritan USA) for a good stability in aqueous solution. 5 x IO ⁶ IU of EPO were added under stirring. After evaporating the solution to 1/4 of the volume the polymer had been cross-linked to a depot composition and an amount of 20 000 IU of EPO/ml was recovered.

Example 5

Preparation of a gel for use as a depot preparation containing calcitonin.

Calcitonin from salmon 100 IU/ml (Miacalcic® San- doz) were admixed with 2% of polymer solution manufac¬ tured in accordance with Example lb and the solution was concentrated to 5% (250 IU/ml) by rotary evaporation. A horse with chronic claudication in the right front leg was treated with an injection of 2 ml s.e. per week dur- ing two weeks. In the six weeks following thereafter said horse was free from pains. The serum calcium was lowered with 12% only.

Example 6

Preparation of a gel containing heparin to be released in a sustained way.

In a diluted activated polymer according to Example 4 heparin was dissolved in an amount of 5% of the poly-

mer. The mixture obtained was equilibrated for 1 hour, whereupon it was evaporated to 1/4 of the volume. A co¬ agulation inhibiting release thereof was noted during 16 days of incubation in physiological saline. Example 7

Preparation of a gel with covalently bonded heparin in a sterically controlled position. Activated polymer according to Example 1 was pre¬ cipitated in methanol under vigorous stirring. The fine- threaded precipitation obtained was dried during the night. Heparin was activated in accordance with example 1. After said incubation (4 hours at 40°C) the polymer precipitation was mixed with the activated heparin solu¬ tion. The mixture was incubated during the night and the following day the gel solution was neutralised, particu- lated and washed from reactant residues.

The gel formed was able to bind growth factor, inter alia basic Fibroblast Growth Factor (bFGF) , but did not show any inhibition of the coagulation of whole blood. Examo -le 8

Preparation of a gel containing positively charged groups of chitosan.

Incubation of a mixture of 7,5 g of hyaluronic acid polymer and 2,5 g of chitosan (See Cure® Protan) was per- formed in accordance with example 1. After a dissolution and a neutralisation a copolymerized viscoelastic solu¬ tion was obtained. Said solution possessed healing pro¬ moting properties after having been applied to a sore slow in healing. Example 9

Preparation of a gel which has been sterically coupled.

7,5 g of hyaluronic acid were activated in accor¬ dance with Example la. In the same way 2,5 g of dextran were activated. The hyaluronic acid was precipitated in methanol, the precipitation then being mixed with 500 ml ^' of a diluted activated 0,5% dextran solution. After stir-

ring and adjustment of pH and salt concentration a vis¬ coelastic solution was obtained. 5 ml of said solution was infused in an Achilles tendon sheath which repeatedly showed inflammation in the form of soreness and "creaking". After four weeks said Achilles tendon prob¬ lems had disappeared.

Example 10

Preparation of a gel for use as a medicinal depot containing GMCSF. The product was prepared in accordance with Example 5 but instead of calcitonin there was added Granulocyte macrophage - colony stimulating factor, GMCSF (Leucomax®) 1 mg/g polymer.

Example 11 Preparation of gel containing killed virus type Influenza A2.

The preparation was performed as in Example 4 but instead of EPO 40 960 HAU killed influenza horse virus per 100 ml of diluted active 1% polymer solution were added. After contraction 4x the preparation contained 1 600 HAU per ml. By a vaccination of more than 100 horses in connection with an epidemic influenza the preparation was found to be highly effective as to protection against infection, which protection was maintained for a long time (more than 6 months) .

Example 12

Preparation of a fresh gel containing a living cell suspension.

A 5 ml fibroblast culture was mixed with 100 ml of a neutralized solution according to example la. The mixture was oxygenated and dried to half the volume. A viscoelas¬ tic solution containing living cells was obtained.

Example 13

Preparation of a dense micronised gel containing small peptides.

To an activated neutralized gel according to Example la there was added 5 mg of a peptide having 12 amino ac-

ids. The gel was evaporated during stirring to 10% and was suspended in mineral oil. After addition of methanol the dry gel particles were filtered off and washed clean from oil residues. Example 14

Preparation of a gel containing the dense micronised gel with small peptides according to Example 13.

To a 1% solution of neutralized polymer activated according to Example la microspheres from Example 13 were added. The gel was then evaporated to half its volume. A homogenous injectable and stable gel containing finely dispersed microspheres was formed.

Example 15

Preparation of a gel containing spherical poly- methylmethacrylate (PMMA) beads having a size of

40 - 120 um.

To 5 g of a polymer diluted to 1% and neutralized and activated according to Example la 100 mg of spheres of polymethylmethacrylate (PMMA) were added. Evaporation to 3% polymeric gel gave a stable injectable viscoelastic gel.

Example 16

Preparation of a gel containing PMMA fragments of

500 nm to which hydrophobic antigen has been added. Haemagglutinin antigen prepared from A2 virus ac¬ cording to Example 11 was absorbed by hydrophobic inter¬ action on 500 nm PMMA particles. Said particles were added to 1% solution according to Example 15 and a reduc¬ tion to half the volume was accomplished. A stable ho- mogenous viscoelastic gel was formed which was useful as a vaccine having a high adjuvant effect.

Example 17

A comparison between the degree of reswellinq at free availability of water between conventionally prepared gels and gels prepared according to the present invention.

Hyaluronic acid gels prepared according to Laurent et al 1963 and according to Examples 1 and 2 above were dried to half their swelling volumes. Then they were re¬ introduced into their original solutions. The previously known gels swelled to their original volume while the gel compositions according to the present Examples 1 and 2 swelled marginally only (10%) . Example 18

Comparison between biological activity of EPO copolymerized with hyaluronic acid to a gel and the gel according to Example 1 into which EPO had been enclosed by a concentration of said gel. Four patients under treatment with Eprex® (CILAG) for their anaemia caused by chronic uraemia were treated for two months with a dose each month according to the following regimen:

Patient no 1 2 3 4 Dose IU 60 000 70 000 70 000 50 000 Month 1 Directly Directly Control Control gelled depot, gelled depot,

Month 2 Control Control Concen- Concen¬ trated trated

Directly gelled depot: Epoxide cross-linking under mild conditions according to Example 11 in the presence of

EPO.

Control: EPO dissolved in 4% hyaluronic acid MW about

6xl0 ⁶ from cock's comb prepared according to US 4 141 973

(Healon® Pharmacia) .

Concentrated: EPO enclosed within activated gel which was gelled through concentration.

The dose was selected as the total dose per month which was normally required by the patient to maintain

the haemoglobin level. The serum level of EPO was ana¬ lysed at regular intervals by means of an immunochemical method.

Results A common method of expressing the functionary effect of depot preparations is to calculate the curve area (units of EPO x days) . This study also gives the bioavailability in the form of haemoglobin level in blood as 0 = retained, + = increased and - = reduced. Table

Patient no. /month 1/1 1/2 2/1 2/2 3/1 3/2 4/1 4/2 Area under the curve 41 424 57 534 224 952 567 656 Haemoglobin control - + - + 0 + + + Conclusion

An enclosure of EPO in a contracted depot gives the highest possible release during the analysis. Attempts to perform the gelling reaction in the presence of EPO de- stroyed the hormones such that a very low release could be registered.