ZA200100514B

ZA200100514B - Polymer complexes of glucuronoglucanes.

Info

Publication number: ZA200100514B
Application number: ZA200100514A
Authority: ZA
Inventors: Ivan Santar; Frantisek Kiss; Jiri Briestensky
Original assignee: Alpenstock Holdings Ltd
Priority date: 1998-07-21
Filing date: 2001-01-18
Publication date: 2001-08-29

Description

: © WO 00/05269 PCT/IE99/00067

Polymer Complexes of Glucuronoglucanes

Introduction

Oxidised cellulose and its derivatives have been widely used in medicine and pharmacy since the first preparation by Chait and Kenyon [Shorygin P P., Chait

E.V.: Zh. obshch. chim. 7, 188 (1937); Yackel E.C., Kenyon W.O.: J Am. Chem.

Soc. 64, 121 (1942)].

Other types of haemostatics and antifibrinolytics have been introduced, however, oxidised cellulose especially in the highly pure form of a polyanhydroglucuronic acid and their copolymers (PAGA), and notably salts thereof, is used in various medicinal applications as a completely resorbable semi-synthetic polymer with minimum adverse effects in the organism. This is true for both the basic substance prepared according to GB 709684; US 4,100,341, or salts thereof prepared according to more recent patents, such as.: CS AO 242920: EP 0659440A1 and PCT IE 98/00004.

It is known that after application of oxidised cellulose to stop surface bleeding a rigid scab is formed, especially on movable parts of the body, such as knees, fingers or ankles. This may be a disadvantage because it can crack and lead to renewed bleeding. Using a haemostat according to PCT IE 98/00004 this disadvantage can be partially overcome by altering the technological conditions of the manufacture (such as increasing the amount of crosslinks) which brings about increased accumulation of the body fluids in the substance and thereby the flexibility of the wound cover 1s optimised.

Within the last two decades, during investigations of various types of polysaccharides, it was established that during their biodegradation in the living organism, certain functions of various types of cells are influenced. [Berger J,

Nemec J., Sedlmayer P., Vortel V.: Report on Toxicological Investigation of a

* '

New Drug Preparation “Mikrocel”, Internal report, Research Institute for

Pharmacy and Biochemistry, Praha, branch Pardubice~Rosice and Labem, 1984;

Burchard W.: Polysacharide, Eigenschaften und Nutzung, Springer-Verlag,

Berlin, Heidelberg, New York, Tokyo, p. 144 (1985); US 5,166,137]. Depending on the type of bond in the main glycosydic chain, on the value of the degree of polymerisation, on the presence of various functional groups, and the degree of ionisation thereof, on the type of structural units, and the type of salt or a complex salt thereof, these polysaccharides affect the immune system of the organism. It seems for instance that glucanes bonded by an 1,3 B bond have immunomodulative properties while 1,4 bonded glucanes suppress tumorous growth. There are however exceptions to these rules. An important factor underlying these properties is the presence of the glucuronic acid in the chain.

It is known that a large proportion of bonds between individual substances occurring in living organisms is of a non-covalent nature, such as hydrogen bonds, van der Waals forces, or bonds of an ionic character especially with biopolymers.

These bonds create so-called intermolecular polymeric complexes (IMC) such as for example, heparin - peptides. In general these complexes represent a new class of macromolecular substances formed by association of individual polymer chains into macromolecules through secondary bonding interactions. According to the nature of the interactions these complexes can be subdivided into polyelectrolyte : complexes, hydrogen bonded complexes, stereo complexes and charge transfer complexes. These types of complexes have a number of common properties, : notably an organised supermolecular structure and the ability to create other higher supermolecular entities. The characteristic feature is their ability to undergo restructuring depending on the conditions prevailing in their environment. Further they are capable of undergoing interpolymer substitution reactions and it is especially due to this latter ability that the IMCs in their behaviour come close to imitating biochemical processes occurring in living : 30 organisms.

Bae oo

. © WO 00/05269 PCT/IE99/00067

The invention in particular involves the use of polyanhydroglucuronic acids and salts thereof. The term polyanhydroglucuronic acid and salts there of as used herein also includes copolymers thereof, especially with anhydroglucose. This is hereinafter referred to as PAGA.

Co-pending patent application PCT IE98/00004 describes particular polyanhydroglucuronic acids and salts thereof and a method of preparing such compounds. In particular therefore, the term polyanhydroglucuronic acids and salts thereof includes the acids and salts referred to in this co-pending application.

Statements of Invention

According to the invention there is provided a biocompatible intermolecular polymer complex of: an anionic component comprising a linear or branched polysaccharide chain wherein at least 5% of the basic structural units are glucuronic acid; and a non protein cationic component comprising a linear or branched natural, semi-synthetic or synthetic oligomer or polymer.

In a preferred embodiment of the invention the cationic component contains nitrogen that either carries a positive charge or wherein the positive charge is induced by contact with the polysaccharidic anionic component.

In one case the cationic component is selected from derivatives of acrylamide, methacrylamide and copolymers thereof. Preferably the cationic component is selected from polyacrylamide, copolymer of hydroxyethylmethacrylate and hydroxypropylmetacrylamide, copolymers of acrylamide, butylacrylate, maleinanhydride and/or methylmetacrylate.

In another case the cationic component is a cationised natural polysaccharide.

Preferably the polysaccharide is a starch, cellulose or gum. The gum may be guargumhydroxypropyltriammonium chloride. :

In another case the cationic component is a synthetic or semi-synthetic polyamino acid. Preferably the cationic component is polylysin, polyarginin, or «, B-poly-[N- (2-hydroxyethyl)-DL-aspartamide].

In a further embodiment the cationic component is a synthetic anti-fibrinolytic.

The anti-fibrinolytic may be a hexadimethrindibromide (polybren).

In a still further embodiment the cationic component is a natural or semi-synthetic peptide. Preferably the peptide is a protamine, gelatine, fibrinopeptide, or derivatives thereof.

In a further case the cationic component is an aminoglucane or derivatives thereof. Preferably the aminoglucane is fractionated chitin or its de-acetylated derivative chitosan. The aminoglucane may be of microbial origin or is isolated from the shells of arthropods such as crabs.

In a preferred embodiment of the invention the anionic component is polyanhydroglucuronic acid and/or bicompatible salts thereof. :

In this case preferably the polyanhydroglucuronic acid and salts thereof contain in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen. Preferably the polyanhydroglucuronic acid and salts thereof contain in their polymeric chain at most 0.2 per cent by weight of bound nitrogen.

. © WO 00/05269 PCT/IE99/00067

In a preferred embodiment the molecular mass of the polymeric chain of the anionic component is from 1x10’ to 3x10° Daltons, ideally, the molecular mass of the polymeric chain of the anionic component ranges from 5x10° to 1.5 x 10°

Daltons.

Most preferably the content of carboxyl groups is in the range of from 12 to 26 per cent by weight, at least 95 per cent of these groups being of the uronic type.

In a preferred embodiment of the invention the anionic component contains at most 1 per cent by weight of carbonyl groups.

The carbonyl groups are preferably intra- and intermolecular 2,6 and 3,6 hemiacetals, 2,4-hemialdals and C2-C3 aldehydes.

The cationic component may be gelatine.

Alternatively the cationic component is chitosan.

The invention also provides a pharmaceutical or cosmetic composition including at least one biocompatible complex of the invention.

Preferably the composition includes at least one biocompatible biologically active substance.

The composition may alternatively or additionally include at least one biologically acceptable adjuvant.

Detailed Description

We have now found that by preparing polymeric intermolecular complexes (IMC) of glucuronoglucanes, notably microdispersed PAGA, prepared especially. according to PCT IE 98/00004 it is possible to enhance the haemostatic effect of the final products on this basis and the properties of the temporary wound cover formed after the haemostasis is achieved such as its flexibility and resistance to cracking on movable parts of the body.

It is also possible to upgrade physicomechanical properties of the final products on this basis. Such IMCs make it possible to prepare application forms whose manufacture from a pure PAGA or their simple salts is extremely difficult. Such application forms includes non-woven textile-like structures or polymeric films.

To modify or upgrade the physical mechanical properties it is sufficient to use even a relatively small amount of polymeric counterion while it is possible to obtain suitable application properties within a broad concentration range of the components. The ratio of the glucuronoglucane to polymeric counterion can be 0.99:0.01 to 0.01:0.99.

Another advantage of glucuronoglucane based IMCs is the possibility to control their biological properties such as varying the degree of haemostatis, resorption time, or immunomodulative properties, and the like.

Polymeric cations suitable to form IMCs with glucuronoglucanes prepared for example according to PCT IE 98/00004 may roughly be subdivided into the following groups: 1. Synthetic biocompatible nitrogen-containing oligomers and polymers. a) Derivatives of acrylamide and methacrylamide and their copolymers [such as polyacrylamide, copolymer of hydroxyethylmetacrylate and

© WO 00/05269 PCT/IE99/00067 hydroxypropylmetacrylamide, copolymer of acrylamide, butylacrylate, maleinanhydride, and methylmetacrylate, and the like], or else cationised natural polysaccharides such as starches, celluloses, or gums such as guargumhydroxypropyltriammonium chloride.

B) b) Synthetic or semi-synthetic polyaminoacids such as polylysin, polyarginin, a,B-poly-[N-(2-hydroxyethyl)-DL-asparamide. Synthetic antifibrinolytics hexadimethrindibromide (polybren) can also be included in this group. 2. Natural or semi-synthetic peptides such as gelatine, protamines, or fibrinopeptides, and their derivatives. 3. Natural aminoglucanes such as fractionated chitin and its de-acetylated denivative chitosan, of microbial origin or isolated from the shells of arthropods such as crabs.

In preparing IMCs on the basis of PAGA according to the invention these three groups of substances can be combined to obtain required properties of the final product.

In general it can be said that IMCs using substances from la and 1b would preferably be used to prepare various types of highly absorbant biocompatible dressing materials in the form of nonwovens, films, plasters, and pads.

IMCs using the substances from 2 and 3 may serve as efficient haemostatic agents for internal applications in the microfibrillar form, in the microdispersed form as dusting powders, in the form of films, granules, tablets or non-woven textile-like structures. Those preparations also display antiadhesive properties.

We have also found out that in the form of film-like cell culture matrices the latter

IMCs incorporating PAGA and salts thereof as prepared according to PCT IE 98/00004 have a favourable effect on the growth of fibroblasts and keratinocytes.

While it 1s also possible to create IMCs using structural scleroproteins of the collagen type as disclosed in WO 9800180A, it is preferable to use the above mentioned groups of substances because of the possibility of contamination of the final product by telopeptides, viruses or pyrogens. Collagen can affect in an uncontrolled manner, the immune response of the organism because formation of antibodies can be provoked by any portion of the collagen structure even though the main determinants occur in the terminal regions of the collagen macromolecule. Removal of telopeptides only partially solves the antigenicity problem (Michaeli et al: Science, 1969, 166, 1522).

By preparing IMCs according to the invention it is possible to essentially enhance properties of the originally prepared glucoronoglucanes such as 1,4 § PAGA. For instance an intermolecular complex salt of PAGA and gelatine in one single production step can be used to prepare final products in the form of a non woven, film, microdispersed granules, or dispersions. In contrast to collagen, suitably hydrolysed gelatine is well tolerated, has no toxicity or side effects and it is a much less costly raw material. We have found out that this complex has very i good haemostatic properties being about 40% higher than the original PAGA calcium sodium salt. This is despite the fact that the gelatine itself only displays a . haemostatic effect after an addition of thrombin [Schwartz S.1. et al.: Principles of

Surgery, St.Louis: McGraw Hill Co, 1979, p. 122-123]. In this case the absorption in the organism can be controlled by changing the composition of the complex within the range from tens of hours to several months. This complex has a higher haemostatic efficiency and can be used as an embolisation or microembolisation product. It can also be used to prepare haemostatic layers of

An important advantage of these IMCs is the fact that the compounds can be prepared within a single manufacturing operation using the hydrolytic process described in PCT IE 98/00004 which makes these products cost effective. ‘These IMCs can further be modified by biologically active and/or biologically acceptable substances. Because the IMCs prepared by the present procedure are either of a microdispersed or microfibrillar nature, the active substances tend to be bound uniformly and also are uniformly released in the organism without the need for other adjuvants such as microcrystalline waxes or stearates. However, the addition of such adjuvants is not excluded.

Biologically active substances which can be incorporated into the IMC may mvolve, for instance, antibiotics carrying at least a weak positive charge in the molecule such as cephalosporins (cephotaxin), aminoglycosides (neomycin, gentamycin, amikacin), penicillins (tikarcilin) or macrolides (erythromycin, clarithromycin) and the like.

In cases where the calcium/sodium salt of PAGA or its IMC complexes according to the invention are used as microembolisation or embolisation agents in regional chemotherapy of malign tumours, suitable types of cytostatics such as adriamycin or derivatives of 1,4-diaminoanthrachinone can be incorporated. It is also possible to use the IMCs as detaching ligands for platinum(Il) based cytostatics.

Biologically acceptable substances used for modification of the IMCs include, for instance, glycerol and its polymers (polyglycerols); mono, di, and certain triglycerides; polyethyleneglycols; monopropyleneglycol; block copolymers of polyethyleneoxides and polypropyleneoxides (Pluronic); starches; cyclodextrines:

polyvinylalcohols; cellulose and its derivatives; in general, substances that, in the concentrations used, are not irritating or toxic for the living organism while being capable of further optimising the physicomechanical properties of the final product based on the IMCs according to the invention. :

The invention will be more clearly understood from the following examples of polymer complexes of glucuronoglucanes.

Example 1:

Material: long-fibre cotton - medicinal cotton wool oxidised by N,O, (proprietary)

COOH 18.8 %b/w ash content <0.1 % b/w 2.C=0 0.6 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

Ca(Cl,.6H,0O anal.grade (LLachema, a.s. Neratovice) demineralised water 2uS ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, as.) acid acetic anal.grade (IL.achema, a.s. Neratovice)

H,O, anal.grade 30% (Lachema, a.s. Neratovice) :

N-HANCE 3000 guargumhydroxypropyltriammoniumchloride (Aqualon — Hercules) :

Equipment. mixer: bottom stirring, 150 litre (duplicator), stainless steel EXTRA

S vibrating screen: stainless steel, 150 mesh rotary air pump: rotor diameter 150 mm turbostirrer: ULTRA TURAX (Janke-Kunkel) beaker: 5 litre

: © WO 00705269 PCT/IE99/00067 pH meter PICCOLO thermocouple thermometer

Procedure: : 30g of N-HANCE 3000 were placed into a 5 | beaker and 3 1 of demineralised water 2uS were added. The contents of the beaker were intensely stirred for 30 minutes. The pH value was adjusted to less than 4.5 by the addition of an acetic acid solution leading to a viscosity rise. 60 I of demineralised water 2uS were introduced into a mixer. 3 kg of

CaCl,.6H,O anal grade were added and the contents heated up to a temperature of 50°C with stirring. On dissolution of the calcium chloride the stirring was interrupted and 2.7 kg of the raw oxidised cotton wool were introduced. The mixer was closed and the contents were agitated for 120 seconds. The pH value of the contents was adjusted by addition of a 20% solution of Na,CO; to 6 — 6.5 and 13 kg of HO, 30% were introduced. The fibre suspension was slowly agitated for 10 minutes. Then the pH value was readjusted to 4.5 — 5.0 and the prepared viscous solution of N-HANCE 3000 was introduced. The contents of the mixer were stirred intensely for 30 seconds. Subsequently 60 1 of synthetic rectified ethanol conc. 98% were introduced into the mixer. 15 seconds after adding the ethanol the contents of the mixer were transferred onto a vibrating screen, and the } supernatant liquid was filtered off. The filtration cake was redispersed in the mixer mn 60 I of a mixture of 18 1 of synthetic rectified ethanol conc. 98% and 42 1 of demineralised water 2S. The fibre suspension was filtered again on the vibrating screen.

The isolated material thus prepared may further serve to prepare final products of the nonwoven type via a wet or dry process.

Analysis:

Ca content 4.0 %b/w

Na content 1.8 % b/w 2. C=0 content 0.0 % b/w

COOH content 20.7 % b/w

Example 2:

Material: oxidised short-fibre cotton (Linters ~ Temming) (proprietary)

COOH 16.8 % b/w ash content <0.15 % b/w 2C=0 2.6 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

CaCl,.6H,O anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

H,O, anal.grade 30% (Lachema, a.s. Neratovice) gelatine (PhBs 1997)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 1 litre heater 1.5 kW : laboratory centrifuge: 4000 rpm thermostated water bath pH meter PICCOLO glass thermometer rotary vacuum dryer or hot-air dryer

: © WO 00/05269 PCT/IE99/00067

Procedure:

Into a 1 litre sulphonation flask equipped with a turbostirrer and a heater, 400 ml of redistilled H,O were placed, 15.73 g of CaCl,.6H,0 were added and on dissolution, 40.0 g of 20% Na,CO; solution were introduced under stirring.

Subsequently, 50 g of oxidised Linters were added to the white emulsion formed and the contents were heated up to 95°C with the stirring intensity set to a maximum. After 10 minutes, 30 g of 30% H,0, were added into the flask and the hydrolysis continued for another 10 minutes. The contents were then cooled down to 60°C on a water bath and the pH of the system was adjusted to a value of 4.5 - 5.0 by the addition of a 20% solution of Na,COs. A gelatine solution (10 gof gelatine in 70 g of redistilled H,O) warmed to 50°C was added and left to react for another 20 minutes. The flask contents were then cooled to 30°C in a water bath and 626 ml of synthetic rectified ethanol conc. 98% were added gradually under intense stirring. The suspension of IMC thus formed was isolated using a laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged again and after the separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and alllowed stand for 4 hours. It was then centrifuged again, redispersed into 99.9 % isopropanol, and left to stand for a } minimum of 10 hours at 20°C. The gel formed was centrifuged and the product was dried in a rotary vacuum dryer or a hot-air dryer.

The product can be used, for instance, for microembolisation, for preparation of haemostatic dusting powders, for manufacture of polymer drugs, e.g. based on cytostatics, or for preparation of spheric particles for macroembolisation.

Analysis: content Ca 44 %b/w content Na 2.7 % b/w content 3, C=0 0.0 % b/w content COOH 20.5 % b/w content N 1.8 % b/w

Example 3:

Material: oxidised short-fibre cotton (Linters —- Temming) (proprietary)

Cs«OOH 16.8 % b/w ash content <0.15 % b/w 2 C=0 2.6 %b/w

NaOH anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

HO; anal.grade 30% (Lachema, a.s. Neratovice) gelatine (PhBs 1997)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 1 litre ) heater 1.5 kW laboratory centrifuge: 4000 rpm thermostated water bath pH meter PICCOLO glass thermometer rotary vacuum dryer or hot-air dryer

Procedure:

Into a 1 litre sulphonation flask equipped with a turbostirrer and a heater, 400 ml of redistilled H,O were placed, and 8 g of NaOH were added. On dissolution, 50 g of oxidised Linters were added, the contents were heated up to 70°C and the stirring intensity set to a maximum. After 20 minutes, 40 g of 30% H,0, were added into the flask, the temperature was increased to 85°C, and maintained for another 10 minutes. The contents were then cooled to 50°C on a water bath and gelatine solution (10 g of gelatine in 70 g of redistilled H,0) warmed 50°C was added to the hydrolysate. The temperature was decreased to 25 — 30°C and the pH of the system was checked and adjusted to a value of 6.0 — 6.5. Subsequently, 626 ml of synthetic rectified ethanol conc. 98% were added gradually under intense stirring. The suspension of IMC thus formed was isolated using a laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged and after the separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and left to stand for 4 hours. It was then centrifuged, redispersed into 99.9 % isopropanol, and allowed stand for a minimum of 10 hours at 20°C. The gel formed was centrifuged and the product was dried in a rotary vacuum dryer or a hot-air dryer.

Analysis:

Na content 3.8 %b/w 2 C=0 content 0.0 %b/w

COOH content 21.5 % b/w

N content 2.7 %b/w

Example 4:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

COOH 16.8 % b/w | ash content < 0.15 %b/w 2C=0 2.6 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

CaCl,.6H,0 anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

H,0, anal.grade 30% (Lachema, a.s. Neratovice) chitosan, degree of deacetylation 92% (Henkel)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 1 litre heater 1.5 kW laboratory centrifuge: 4000 rpm thermostated water bath pH meter PICCOLO glass thermometer : rotary vacuum dryer or hot-air dryer

Procedure:

Into a sulphonation flask, 250 ml redistilled H,O were placed, and 5 g of NaOH were added. On dissolution, 25 g of oxidised Linters were introduced under stirring, the temperature increased to 50°C and the stirring intensity set to a maximum. After hydrolysing for 15 minutes, 35 g of 30% H,0, were gradually added to the system and the temperature was maintained at 50°C for another 20

: © WO 00/05269 PCT/IE99/00067 minutes. The contents were cooled to 30°C and 400 g of highly viscous 5% solution of chitosan were added. The flask contents were then intensely stirred for another 10 minutes, and the pH of the system was adjusted by the addition of

NaOH to a value of 7.0. Subsequently 300 ml of synthetic rectified ethanol conc. 98% were added with stirring. The suspension of IMC thus formed was isolated using a laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged again and after the separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and left to stand for 4 hours.

It was then centrifuged again, redispersed into 99.9 % isopropanol, and left for a minimum of 10 hours at 20°C. The gel formed was centrifuged and the product was dried in a rotary vacuum dryer or a hot-air dryer.

The product can be used, for instance, for microembolisation, for preparation of haemostatic dusting powders, for manufacture of polymer drugs, e.g. based on

Ccytostatics, or for preparation of spheric particles for macroembolisation.

Analysis:

Na content 1.8 % b/w 2 C=0 content 0.0 % b/w

COOH content 104 % b/w

N content 28 %b/w

Example 5:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

C«OOH 16.8 %b/w ash content <0.15 % b/w 2 C=0 2.6 %b/w

NaOH anal.grade (Lachema, a.s. Neratovice)

HCl 39% anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

H,O, anal.grade 30% (Lachema, a.s. Neratovice) gelatine (PhBs 1997)

Ambroxol (H. Mack, Germany)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 2 1 heater 1.5 kW laboratory centrifuge: 4000 rpm laboratory pin mill ALPINE (35 000 rpm) thermostated water bath pH meter PICCOLO glass thermometer rotary vacuum dryer or hot-air dryer

Procedure:

Into a sulphonation flask, 400 ml redistilled H,O were placed, and 8 g of NaOH were added. On dissolution, 50 g of oxidised Linters were introduced under : stirring, the temperature increased to 70°C and the stirring intensity was set to a maximum. After hydrolysing for 20 minutes, 40 g of 30% H,0O, were gradually . added to the system and the temperature was increased to, and maintained at, 85°C for another 10 minutes. The content were cooled down to 50°C in a water bath, and gelatine solution (2 g of gelatine in 70 g of redistilled H,O) warmed up to 50°C was added to the hydrolysate. The temperature was decreased to 25 — 30°C and the pH of the system was checked and adjusted to a value of 1.6 — 1.8 by addition of 39% HCI. Under intense stirring, a solution of Ambroxol (25g of ambroxolium hydrochloride in 500 ml of redistilled H,O) was added gradually.

After agitating for 5 minutes the pH value was adjusted to 4.3 —4.6 by adding 5%

: © WO 00/05269 PCT/IE99/00067

NaOH solution, and 626 ml of synthetic rectified ethanol conc. 98% were added under intense stirring. The suspension of Ambroxol containing IMC thus formed was isolated using a laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into, subsequently, 800 ml of 60% ethanol and 250 ml of 98% ethanol, wherein it was let to stay for a minimum of 10 hours. The system was centrifuged again and the product was dried at 40°C in a rotary vacuum dryer or a hot-air dryer. A white to slightly yellowish powder was obtained and further desagglomerated on an Alpine pin mill.

The product serves for the preparation of a mucoregulatory drug with a prolonged action.

Analysis:

Na content 4.6 %b/w 2. C=0 content 0.0 % b/w

COOH content 14.8 % b/w

N content 1.9 % b/w

Example 6:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

CsO0OH 16.8 % b/w . ash content <0.15 % b/w 2 C=0 26 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

H,0, anal.grade 30% (Lachema, a.s. Neratovice)

gelatine (PhBs 1997) gentamycin sulphate (MERCK)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 2 litre heater 1.5 kW laboratory centrifuge: 4000 rpm laboratory pin mill ALPINE (35 000 rpm) thermostated water bath pH meter PICCOLO glass thermometer hot-air dryer lyophiliser (Leibold Heraus, Germany)

Procedure:

Into a 2 litre sulphonation flask equipped with a turbostirrer and a heater, 400 ml of redistilled H,O were placed, 15.73 g of CaCl,.6H,0 were added and on dissolution, 40.0 g of 20% Na,CO; solution were introduced under stirring.

Subsequently, 50 g of oxidised Linters were added to the white emulsion formed and the contents were heated up to 95°C and the stirring intensity set to a - maximum. After 10 minutes, 30 g of 30% H,0, were added into the flask and the hydrolysis was continued for another 10 minutes. The contents were then . cooled down to 60°C on a water bath and the pH of the system was adjusted to a value of 4.5 - 5.0 by addition of 20% solution of Na,CO;. A gelatine solution (10 g of gelatine mn 70 g of redistilled H,0) warmed up to 50°C was added and let to react for another 20 minutes. The flask contents were then cooled down to 30°C in a water bath and 40 g of gentamycin sulphate in 600 ml of redistilled H,O were added gradually within 10 minutes. 626 ml of synthetic rectified ethanol conc. 98% were then added gradually with intense stirring to the antibiotic containing

IMC suspension formed. The suspension of IMC thus formed was isolated using a

. © WO 00/05269 PCT/IE99/00067 laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged and after the separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and left for 4 hours. It was then centrifuged, redispersed into 99.9 % isopropanol, and left for a minimum of 10 hours at 20°C.

The gel formed was centrifuged and the product was dried in a rotary vacuum dryer or a hot-air dryer.

The product can be used, for instance, for the manufacture of a dusting powder or a powder spray for the treatment of infected wounds.

Analysis:

Ca content 24 % b/w

Na content 1.6 % b/w 2. C=0 content 0.0 % b/w

COOH content 9.6 % b/w

N content 2.7 % b/w

Example 7:

Material: long fibre cotton — medicinal cotton wool oxidised by N,O, (proprietary)

CsOO0H 18.8 % b/w ash content <0.1% b/w 2.C=0 0.6 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

CaCl,.6H;0 anal.grade (Lachema, a.s. Neratovice) demineralised water 2uS ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

acid acetic anal.grade (Lachema, a.s. Neratovice)

H;O; anal.grade 30% (Lachema, a.s. Neratovice)

N-HANCE 3000 guargumhydroxypropyltriammoniumchloride (Aqualon — Hercules) polybren (hexadimethrindibromide) (FLUKA) chlorhexidindigluconate

Equipment: mixer: bottom stirring, 1501 (duplicator), stainless steel EXTRA S vibrating screen: stainless steel, 150 mesh rotary air pump: rotor diameter 150 mm turbostirrer;: ULTRA TURAX (Janke-Kunkel) beaker: S litre pH meter PICCOLO thermocouple thermometer

Procedure: 30g of N-HANCE 3000 were placed into and 5 1 beaker and 3 1 of demineralised water 2uS were added. Contents of the beaker were stirred intensely for 30 minutes. The pH value was adjusted to less than 4.5 by the addition of an acetic acid solution leading to a viscosity rise. 60 1 of demineralised water 2uS were introduced into a mixer. Then 3 kg of

CaCl,.6H,O anal.grade were added and the contents heated up to a temperature of 50°C under stirring. On dissolution of the calcium chloride the stirring was interrupted and 2.7 kg of the raw oxidised cotton wool were introduced. The mixer was closed and the contents were agitated for 120 seconds. Then the pH value of the contents was adjusted by the addition of a 20% solution of Na,CO; to 6 —-6.5 and 13 kg of H,O, 30% were introduced. The fibre suspension was slowly agitated for 10 minutes. The pH value was readjusted to 4.5 — 5.0 and the prepared viscous solution of N-HANCE 3000 was introduced. The contents of the mixer were stirred intensely for 30 seconds. A solution of 35 g of chlorhexidine digluconate in 350 ml of demineralised water 2uS was then introduced slowly within 10 minutes. Within another 10 minutes, a solution of polybren containing 120 g of polybren in 1000 ml of demineralised water 2uS was added. Subsequently 60 1 of synthetic rectified ethanol conc. 98% were introduced into the mixer. 15 seconds after adding the ethanol, the contents of the mixer were transferred onto a vibrating screen, and the supernatant liquid was filtered off. The filtration cake was redispersed in the mixer in 60 | of a mixture of 18 I of synthetic rectified ethanol conc. 98% and 42 1 of demineralised water 2uS. The fibre suspension was filtered again on the vibrating screen.

The isolated material thus prepared may further serve to prepare, via a wet or dry process, final products of the non-woven type having an enhanced haemostatic activity and a bactericidal effect.

Analysis:

Ca content 3.6 %b/w

Na content 1.9 % b/w 2. C=0 content 0.0 % b/w

COOH content 18.1 % b/w : N content 0.35 % b/w : Example 8:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

COOH 16.8 %b/w ash content < 0.15 % b/w 2 C=0 26 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

Ca(Cl,.6H,O anal.grade (Lachema, a.s. Neratovice)

redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

HO, anal.grade 30% (Lachema, a.s. Neratovice)

Chitosan, degree of deacetylation 92% (Henkel)

Clarithromycin lactobionan (Abbott Laboratories, Italy)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 1 litre heater 1.5 kW laboratory centrifuge: 4000 rpm thermostated water bath pH meter PICCOLO glass thermometer rotary vacuum dryer or hot-air dryer dialysing bag (regenerated cellulose) lyophiliser (Leybold Heraus, Germany) laboratory pin mill ALPINE (35 000 rpm)

Procedure:

Into a sulphonation flask 250 ml redistilled H,O were placed and 5 g of NaOH : were added. On dissolution, 25 g of oxidised Linters were introduced under stirring, the temperature increased to 50°C and the stirring intensity set to a . maximum. After hydrolysing for 15 minutes, 35 g of 30% H,O, were gradually added to the system and the temperature was maintained at 50°C for another 20 minutes. The content were cooled down to 30°C and 400 g of highly viscous 2% solution of chitosan, having a pH value of 3.5, were added. The flask contents were then intensely stirred for another 10 minutes, and the pH of the system was adjusted, by addition of NaOH, to a value of 7.0. During another 10 minutes, a solution of clarithromycin (44 g of clarithromycin in 456 ml of redistilled H,0) was introduced and the pH of the system was adjusted to a value of 7.0-7.5.

: © WO 00/05269 PCT/IE99/00067

Stirring was interrupted, the flask contents were transferred into a dialysing bag and dialysed against water for 48 hours. Subsequently the product was isolated by centrifugation, lyophilised, and disintegrated using the laboratory pin mill

ALPINE.

The product can be used, for instance, to prepare tablets or granules efficient against Helicobacter pylori occurring in the gastrointestinal tract.

Analysis:

Na content 4.8 %b/w 2. C=0 content 0.0 % b/w

COOH content 18.8 % b/w

N content 0.7 % b/w

Example 9:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

CsOOH 16.8 % b/w ash content <0.15 % b/w 2 C=0 26 %b/w } NaOH anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) isopropanol 99.9% (Neuberg Bretang)

H,O; anal.grade 30% (Lachema, a.s. Neratovice) gelatine (PhBs 1997)

Bi(NO;).5H,0O (MERCK)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 2 litre heater 1.5 kW laboratory centrifuge: 4000 rpm thermostated water bath pH meter PICCOLO glass thermometer rotary vacuum dryer or hot-air dryer

Procedure:

Into a sulphonation flask 400 ml redistilled H,O were placed and 8 g of NaOH were added. On dissolution, 50 g of oxidised Linters were introduced under stirring, the temperature increased to 70°C and the stirring intensity was set to a maximum. After hydrolysing for 20 minutes, 40 g of 30% H,O, were gradually added to the system and the temperature was increased to, and maintained at, 85°C for another 10 minutes. The contents were cooled down to 50°C in a water bath, and a gelatine solution (0.5 g of gelatine in 50 ml of redistilled H,O) warmed up to 50°C was added to the hydrolysate. The temperature was decreased to 25 — 30°C and the pH of the system was checked and adjusted to a value of 1.6 - 1.8 by addition of 39% HCI. A freshly prepared solution of BiNO; (54 g of

BiNO:.5H,O in 746 ml of H,0) was introduced and the temperature maintained for another 15 minutes. The temperature was decreased to 25 — 30°C and the pH of the system was checked and readjusted to a value of 5.5 — 6.0. 626 ml of . synthetic rectified ethanol conc. 98% were then added gradually with intense stirring. The BiO" containing IMC suspension thus formed was isolated using a : laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged again and after the separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and left for a minimum of 4 hours. It was then centrifuged again, redispersed into 99.9 % isopropanol, and left for a minimum of 10 hours at 20°C. The suspension formed was then centrifuged again and the product was dried in a rotary vacuum dryer or a hot-air dryer.

The product can be used, for instance, to prepare dusting powders for wound treatment or tablets for treatment of gastrointestinal tract malfunctions.

Analysis:

Na content 19 % b/w >. C=0 content 0.0 %b/w

COOH content 20.0 % b/w

N content <0.3 %b/w

Bi content 4.7 % b/w

Example 10:

Material: oxidised short-fibre cotton (Linters — Temming) (proprietary)

C«OOH 16.8 % b/w ash content <0.15 % b/w 2 C=0 26 %b/w 20% solution Na,CO; (Lachema, a.s. Neratovice)

CaCl,.6H,0 anal.grade (Lachema, a.s. Neratovice) redistilled water (PhBs 1997) ethanol, synthetic rectified conc. 98% (Chemopetrol Litvinov, a.s.) : isopropanol 99.9% (Neuberg Bretang)

H,O, anal.grade 30% (L.achema, a.s. Neratovice) : gelatine (PhBs 1997) cimetidine hydrochloride (SPOFA)

Procedure:

Into a 11 sulphonation flask equipped with a turbostirrer and a heater, 400 ml of redistilled H,O were placed. 15.73 g of CaCl,.6H,O were added and on dissolution 40.0 g of 20% Na,CO; solution were introduced with stirring. 50 g of oxidised Linters were added to the white emulsion formed and the contents were heated up to 95°C and the stirring intensity set to a maximum. After 10 minutes, 30 g of 30% H,0, were added into the flask and the hydrolysis was continued for another 10 minutes. The contents were then cooled down to 60°C on a water bath and the pH of the system was adjusted to a value of 4.5 — 5.0 by addition of 20% solution of Na,COs;. A gelatine solution (10 g of gelatine in 70 g of redistilled

H,0O) warmed up to 50°C was added and allowed react for another 20 minutes.

The flask contents were then cooled down to 30°C in a water bath and a solution of cimetidine (36 g of cimetidine hydrochloride in 400 ml of redistiiled H,0) were added with intense stirring. The contents were intensely agitated for 10 minutes and 800 ml of synthetic rectified ethanol conc. 98% were added gradually. The suspension of IMC thus formed was isolated using a laboratory centrifuge. The supernatant liquid was filtered away and the cake was redispersed into 250 ml of 50% ethanol. The system was centrifuged and after separation of the supernatant liquid, the IMC was redispersed into 250 ml of synthetic rectified ethanol conc. 98% and left for 4 hours. It was then centrifuged again, redispersed into 99.9 % . isopropanol, and left for a minimum of 10 hours at 20°C. The gel formed was centrifuged again and the product was dried in a rotary vacuum dryer or a hot-air dryer.

The product can be used, for instance, to manufacture tablets or granulates for the treatment of the gastrointestinal tract or other non-malignant ulcerations.

Analysis:

Ca content 44 %b/w

Na content 2.7 % b/w 2. C=0 content 0.0 % b/w

COOH content 20.5 % b/w

N content 2.1 %b/w

Example 11:

Material: IMC-microdispersed oxidised cellulose (MDOC) complex (as per above Example 2) [(2S;2R)-3-amino-2-hydroxy-4-phenylbutenoyl]-L-leucin (Bestatin) (Boehringer Mannheim, Germany) redistilled water (PhBs 1997) methanol, conc. anal.grade (Chemopetrol Litvinov, a.s.) diethylether (Lachema, a.s. Neratovice)

Equipment: turbostirrer: ULTRA TURAX (Janke-Kunkel) sulphonation flask 2 1 laboratory centrifuge: 4000 rpm hot-air dryer : Procedure:

The IMC-MDOC complex as prepared in Example 2 above was redispersed into redistilled water in a sulphonation flask using a turbostirrer. A solution of Bestatin in methanol was added to the flask in an amount sufficient to yield a 10% b/w concentration of Bestatin in the resulting Bestatin-gelatine-MDOC complex. After thorough homogenisation, the suspension formed was isolated by centrifugation,

The supernatant liquid was filtered away and the filtration cake was redispersed into concentrated methanol, centrifuged, redispersed in diethylether, and after being left for 1 hour, it was dried in a hot-air dryer.

The product, a microdispersed form of a Bestatin-gelatine-MDOC complex, can be used, for instance, to prepare microembolisation agents used in regional chemotherapy of malignant tumours or flat dressing structures for wound treatment.

The following are application examples illustrating some of the uses of the products produced in examples 1 to 11.

Example A: Preparation of tablets and pellets from IMC-MDOC complex

MDOC = Microdispersed Oxidised cellulose

Material: IMC-MDOC complex — see Example 2 magnesium stearate (SIGMA) ascorbic acid (MERCK) a-tocoferol acetate (Slovakofarma Hlohovec) ethanol synthetic rectified (Chemopetrol Litvinov, a.s.) ’

Equipment: tabletting machine (KORSCH EK 0, Berlin ) blender (Nautamix 300) counter-flow drier BINDER

Procedure: 10 kg of IMC-MDOC complex of composition according to Example 2 were placed into the blender , and 660 g of micronised ascorbic acid, 1660 g of a- : tocoferol acetate emulgated in 2500 ml of ethanol, and 1000 g of magnesium

Claims

1. Biocompatible intermolecular polymer complex of: an anionic component comprising a linear or branched polysaccharide chain wherein at least 5% of the basic structural units are glucuronic acid; and a non protein cationic component comprising a linear or branched natural, semi-synthetic or synthetic oligomer or polymer.

2. A complex as claimed in claim 1 wherein the cationic component contains nitrogen that either carries a positive charge or wherein the positive charge is induced by contact with the polysaccharidic anionic component.

3. A complex as claimed in claim 2 wherein the cationic component is selected from derivatives of acrylamide, methacrylamide and copolymers thereof.

4. A complex as claimed in claim 3 wherein the cationic component is selected from polyacrylamide, copolymer of hydroxyethylmethacrylate and hydroxypropylmetacrylamide, copolymers of acrylamide, butylacrylate, maleinanhydride and/or methylmetacrylate.

5. A complex as claimed in claim 2 wherein the cationic component is a cationised natural polysaccharide.

6. A complex as claimed in claim 5 wherein the polysaccharide is a starch, cellulose or gum.

7. A complex as claimed in claim 6 wherein the gum is guargumhydroxypropyltriammonium chloride.

8. A complex as claimed in claim 1 wherein the cationic component is a synthetic or semi-synthetic polyamino acid.

9. A complex as claimed in claim 8 wherein the cationic component is polylysin, polyarginin, or a, B-poly-[N-(2-hydroxyethyl)-DL-aspartamide].

10. A complex as claimed in claim 1 wherein the cationic component is a synthetic anti-fibrinolytic.

Il. A complex as claimed in claim 10 wherein the anti-fibrinolytic is a hexadimethrindibromide (polybren).

12. A complex as claimed mn claim 1 wherein the cationic component is a natural or semi-synthetic peptide.

13. A complex as claimed in claim 12 wherein the peptide is a protamine, gelatine, fibrinopeptide, or derivatives thereof.

14. A complex as claimed in claim 1 wherein the cationic component is an : aminoglucane or derivatives thereof.

15. A complex as claimed in claim 14 wherein the aminoglucane is fractionated chitin or its de-acetylated derivative chitosan.

16. A complex as claimed in claim 14 or 15 wherein the aminoglucane is of microbial origin or is isolated from the shells of arthropods such as crabs. x -

17. A complex as claimed in any preceding claim wherein the anionic component is polyanhydroglucuronic acid [PAGA].

18. A complex as claimed in claim 17 wherein the polyanhydroglucuronic acid and salts thereof contain in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen.

19. A complex as claimed in claim 18 wherein the polyanhydroglucuronic acid and salts thereof contain in their polymeric chain at most 0.2 per cent by weight of bound nitrogen.

20. A complex as claimed in claim 18 or 19 wherein the molecular mass of the polymeric chain of the anionic component is from 1x10° to 3x10° Daltons.

21. A complex as claimed in claim 20 wherein the molecular mass of the polymeric chain of the anionic component ranges from 5x10° to 1.5 x 10° Daltons. ]

22. A complex as claimed in any of the claims 18 to 21 wherein the content of carboxyl groups is in the range of from 12 to 26 per cent by weight, at least 95 per cent of these groups being of the uronic type.

23. A complex as claimed in any of claims 18 to 22 wherein the anionic component contains at most 1 per cent by weight of carbonyl groups.

24. A complex as claimed in any of claims 18 to 23 wherein the carbonyl groups are intra- and intermolecular 2,6 and 3,6 hemiacetals, 24- hemialdals and C2-C3 aldehydes.

25. A complex as claimed in any of claims 1 to 2, 11, 12 or 17 to 23 wherein the cationic component is gelatine.

26. A complex as claimed in any of claims 1, or 13 to 23 wherein the cationic component is chitosan.

27. A biocompatible intermolecular polymer complex substantially as hereinbefore described with reference to the examples.

28. A pharmaceutical or cosmetic composition including at least one biocompatible complex as claimed in any preceding claim.

29. A composition as claimed in claim 28 including at least one biocompatible biologically active substance.

30. A composition as claimed in claims 28 or 29 including at least one biologically acceptable adjuvant.

31. A composition substantially as hereinbefore described with reference to the examples.

32. A pharmaceutical composition for the prophylaxis or treatment of peptic ulcers including a complex as claimed in any of claims 1 to 27.

33. A slow release formulation including a complex as claimed in any of oo claims 1 to 27. -

34. An anulipemic composition including a complex as claimed in any of claims 1 to 27.

35. A suppository formulation including a complex as claimed in any of claims 1 to 27.

36. A tablet including a complex as claimed in any of claims 1 to 27. :

37. A pelletincluding a complex as claimed in any of claims 1 to 27.

38. A granule including a complex as claimed in any of claims 1 to 27.

39. A microsphere including a complex as claimed in any of claims 1 to 27.

40. A flat flexible material including a complex as claimed in any of claims 1 to 27.

41. A textle-like fabric including a complex as claimed in any of claims 1 to

27.

42. A foam including a complex as claimed in any of claims 1 to 27. _

43. A dental pin including a complex as claimed in any of claims 1 to 27.

44. Biocompatible intermolecular polymer complex as claimed in any one of claims 1 to 27, including any new and inventive integers or combination thereof herein described.

45. A pharmaceutical or cosmetic complex as claimed in any one of claim 28 to 32, including any new and inventive integers or combination thereof herein described.

46. A slow release formulation as claimed in claim 33, including any new and inventive integers or combination thereof herein described. AMENDED SHEET

56 AMENDED SHEET

47. An antilipemic composition as claimed in claim 34, including any new and inventive integers or combination thereof herein described.

48. A suppository formulation as claimed in claim 35, including any new and inventive integers or combination thereof herein described.

49. A tablet as claimed in claim 36, including any new and inventive integers or combination thereof herein described.

50. A pellet as claimed in claim 37, including any new and inventive integers or combination thereof herein described.

S51. A granule as claimed in claim 38, including any new and inventive integers or combination thereof herein described.

52. A microsphere as claimed in claim 39, including any new and inventive integers or combination thereof herein described.

53. A flat flexible material as claimed in claim 40, including any new and inventive integers or combination thereof herein described.

S54. A textile-like fabric as claimed in claim 41, including any new and inventive integers or combination thereof herein described.

55. A foam as claimed in claim 42, including any new and inventive integers or combination thereof herein described.

57 AMENDED SHEET

56. A dental pin as claimed in claim 43, including any new and inventive integers or combination thereof herein described.