WO1992013526A1

WO1992013526A1 - Stabilisation of fibroblast growth factor using a polysaccharide

Info

Publication number: WO1992013526A1
Application number: PCT/EP1992/000119
Authority: WO
Inventors: Pierre Frédéric MONSAN; François Marie Bernard PAUL; Didier Joseph Betbeder; Marco Adami; Roberto De Ponti; Giorgio Finetti
Original assignee: Farmitalia Carlo Erba S.R.L.
Priority date: 1991-01-31
Filing date: 1992-01-21
Publication date: 1992-08-20
Also published as: GB9102107D0

Abstract

The present invention relates to a fibroblast growth factor (FGF) stabilised by a carrageenan. The invention further provides a process for the preparation of the stabilised form of FGF according to the invention and pharmaceutical compositions containing it. An FGF stabilised with a carrageenan has fibroblast growth promoting activity.

Description

TITLE: STABILISATION OF FIBROBLAST GROWTH FACTOR USING A POLYSACCHARIDE

The present invention relates to a stabilised form o a fibroblast growth factor (FGF), to its preparation and to pharmaceutical compositions containing the stabilised FGF.

Genetic engineering techniques have allowed the availability of recombinant growth factors for therapeutic use. However, these growth factors are very unstable and sensitive to denaturation such as thermal or proteolytic degradation before reaching their target. It is known that basic fibroblast growth factor (bFGF), a strongly cationic molecule, is stabilised by heparin, an anionic polysaccharide (Sommer and Rifkin, J. Cell Physiol. 138, 215-220, 1989; Gospodarowicz and Cheng, J. Cell. Physiol. 128, 475-484, 1986; Edelman E.R. et al., WO 89/12464).

We have now found that a FGF can be stabilised by a carrageenan. Accordingly, the present invention provides a FGF stabilised by a carrageenan. The invention further provides a process for the preparation of a stabilised FGF according to the invention, which process comprises contacting the FGF with the carrageenan in an aqueous medium. The carrageenan, in particular lambda-carrageenan, stabilises a FGF as well as heparin and shows a potentialisation effect on FGF mitogenesis at least as good as that for heparin.

The FGF may be acidic FGF (aFGF) or, more preferably, bFGF. The aFGF or bFGF may be any polypeptide which has biological activity similar to that exhibited by natural human aFGF or bFGF respectively. The amino acid sequence of natural human bFGF is shown in the Sequence Listing under SEQ ID NO: 1. This is the sequence determined by Abraham et al, EMBO J. 5, 2523-2528, 1986 and is designated 155-bFGF or (1-155)bFGF.

The FGF may therefore be any mammalian FGF, for example human, bovine, murine or rodent. The FGF may be produced by recombinant DNA techniques or derived from natural sources. The amino acid sequence of the FGF may be altered from that of a natural FGF, for example by substitution, deletion, insertion or extension. Any bFGF molecule as described in, for instance, WO 86/07595; WO 87/01728; EP-A-0226181; Abraham et al, 1986; or Lobb, Eur. J. Clin. Invest. 18, 321-336, 1988 may be usefully employed.

A suitable human bFGF is (l-155)bFGF, (2-155)bFGF, (3-155)bFGF or (10-155)bFGF. A mixture of FGF's, for example of bFGF's, may be stabilised in accordance with the present invention. The invention can therefore be applied to a mixture of (2-155)bFGF and (3-155)bFGF, for example.

The carrageenan may be iota-, kappa- or lambda-carrageenan. The carrageenan is typically essentially pure. Preferably the carrageenan is lambda-carrageenan. The carrageenan may be a carrageenan obtained from G.stellata, G. acicularis, G.skottsbergii , G.pistillata, G.chamissol, C. crispus , Iridaea, E.cottonii, E.spinosum or H.muciformis. Preferably a carrageenan having a high degree of sulphation is employed.

A FGF is stabilised according to the invention by contacting it with a carrageenan in an aqueous medium. Any appropriate aqueous medium may be employed. The aqueous medium may be a physiologically acceptable aqueous medium for example water such as Water for Injection, physiological saline or a glucose solution. The pH of a solution may be adjusted by means of an acid or buffer which will be physiologically acceptable if the medium is intended for injection. Preferably the pH is from 5 to 9. Suitable buffers include phosphate buffer and Tris buffer.

The concentration of FGF in the aqueous medium preferably is from 0.005 to 5 w/v %, more preferably from 0.01 to 1 w/v %. The concentration of carrageenan in the aqueous medium preferably is from 0.0005 to 5 w/v %, more preferably from 0.01 to 1 w/v %. Suitably the FGF and carrageenan are present in the aqueous medium at a weight ratio of FGF:carrageenan of from 0.25:1 to 10:1, preferably from 0.5:1 to 5:1. The FGF may be added to an aqueous medium containing a carrageenan or vice versa. The added component may itself be in the form of an aqueous medium. Alternatively, the components in solid form may be dissolved in water. The materials are typically mixed in the aqueous medium at a temperature of from 0 to 40°C, for example from 10 to 25°C, for from 1 to 30 minutes. A suitable temperature is room temperature. The FGF and carrageenan bind together in the aqueous medium to form a complex. Complex can be isolated as desired. The complex may be isolated for example using gel filtration techniques. The isolated stabilised FGF may be used or the stabilised FGF may be used without isolation. The aqueous medium containing the FGF and the carrageenan may be lyophilised.

A stable composition may therefore be provided which contains a FGF and an effective amount of a carrageenan. The composition may be in the form of an aqueous solution or a powder. The stabilised FGF has an ability to resist trypsin hydrolysis which is at least as good as that of heparin, in particular of heparin having a molecular weight of 15000. Particularly when the carrageenan is lambda-carrageenan, it is possible to provide a stabilised bFGF according to the invention which resists thermal denaturation in aqueous solution at 37°C and 45°C and, when freeze-dried, thermal denaturation at 75°C at least as well as bFGF stabilised with heparin. A FGF stabilised according to the invention may also potentialise the mitogenic effect of bFGF at least as well as bFGF stabilised with heparin. These properties are demonstrated further in the Examples.

A FGF stabilised with a carrageenan has fibroblast growth promoting activity. It may therefore be used as a healing promoter for burns, wounds or post-operative tissue. It may be used as a therapeutic drug for thrombosis, arteriosclerosis and brain diseases. The amount of the stabilised FGF administered to a patient, in particular a human, depends upon a variety of factors including the route of administration and condition being treated. The FGF is typically formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.

The stabilised FGF may be provided on water-swellable and water-insoluble microspheres. Such microspheres may be composed of dextran, starch or chitosan. The microspheres may be loaded with e.g. from about 0.2 to about 50 mg of a FGF per g of microspheres. A suitable amount of FGF is, e.g., from about 0.8 to about 1.5 mg/g microspheres. Microspheres loaded with bFGF can typically be provided as a powder which may be applied to the site of a burn or wound or to post-operative tissue.

A useful powder may be prepared by:

(i) soaking water-insoluble and water-swellable polysaccharide microspheres in an aqueous medium containing a FGF and a carrageenan; and

(ii) lyophilising the dispersion of the microspheres in the said aqueous medium.

During the soaking the microspheres reach a certain degree of swelling depending from their chemical composition, unswollen diameter, the nature of their cross-linking agent and its relative content with reference to the microspheres, temperature, pH, ionic strength, nature of the solution and presence of surface modifiers on the microspheres. The microspheres are soaked for sufficient time so that the growth factor is adsorbed onto and/or into the microspheres. The time needed for soaking (incubation) can vary greatly. The time may be from 2 minutes to 48 hrs, preferably from 2 minutes to 2 hrs, at room temperature.

Following the soaking step, freeze-drying is carried out to eliminate water. The type of freeze-drying process can vary greatly. Suitable results are obtained with anything from very simple equipment (e.g. a microsphere suspension in a growth factor solution contained in a flask is taken from the freezer and the flask is then attached directly to a vacuum pump) to sophisticated freeze-driers where it is possible to control various temperatures ( shelfs, product, condenser), the vacuum and times.

The microspheres loaded with growth factor and obtained in the form of powder from the freeze-drier can be placed in a suitable container for use in wound or burn healing. The loaded microspheres can be diluted by mixing with microspheres which are unloaded or with microspheres loaded with other factors or another therapeutic agent, or with powders (loaded or unloaded) in general. However, it is preferred to use growth factor loaded microspheres diluted with a suitable amount of unloaded microspheres. This suitable amount depends upon the necessity to have enough powder to cover a burn or wound site completely.

For wound or burn treatment the powder of the invention is used to cover the wounded or burned site. Microspheres then swell, draining fluid from the tissue and thus having a useful cleaning effect. After swelling a gel is obtained in situ from which the growth factor is progressively released, e.g., in about from 5 minutes to about 6 hrs. The gel can be easily removed by washing for another application.

A method of treating a wound or a burn therefore comprises applying to the wound or burn a therapeutically effective amount of the present powder. The powder may be sprinkled over the site of the wound or burn. A bandage can be applied to cover the site. A gel forms naturally. When the bandag is removed, for example daily, the gel is removed and the wound or burn is cleaned. The powder can then be administered again.

A bandage can in fact be employed which incorporates the powder, rather than sprinkling the powder over the wound or burn. The bandage includes the powder in or on a surface to be brought into contact with a wound or burn. Such a bandage can be replaced as necessary. A stabilised FGF according to the invention may be used in a reabsorbable bandage or sponge.

The amount of a powder which is applied to a wound or burn in a human patient depends upon a variety of factors including the severity of the wound or burn, the site of the wound or burn on the body, whether a powder is being sprinkled on the wound or whether a bandage incorporating the powder is being applied, etc. Typically, however, enough powder should be given to apply the growth factor in an amount of from 10 ng to 1 mg per cm². The following Examples illustrate the invention. A Preparation Example and two Reference Examples are also provided.

Preparation Example: Preparation of 154/153 form of bFGF The construction of the synthetic DNA sequence for b-FGF and of the expression plasmid carrying such sequence was performed according to the procedure described in EP-A-363675. The fermentation and purification process was carried out as follows:

(a) Fermentation process

A bacterial strain, E. coli type B, from the Institute Pasteur collection, was transformed with a plasmid carrying both the human gene coding for bFGF and the gene for tetracycline resistance. This transformed strain was used for the production of recombinant non-glycosylated h-bFGF (human bFGF). A Master Cell Bank (15 freeze-dried vials) and a Working Cell Bank (W.C.B.) (70 vials stored in liquid nitrogen at -190°C) of this strain were prepared. The content of one vial of W.C.B. was used as the inoculum for the fermentation phase.

The fermentation process was carried out in 10 1 fermentors filled with 4 1 of culture medium. Tetracycline hydrochloride was added to the medium in order to maintain the conditions of strain selection. After 20 hours of growth at 37°C the final biomass was 42±2 g/1 dry weight, and the production of bFGF was 2500 ± 500 mg/1 as measured by comparative gel electrophoresis.

Enrichment in pure oxygen was required during the fermentation phase in order to allow a large bacterial growth.

(b) Initial purification

The cells (microorganisms) were separated from the total fermentation broth by centrifugation. The resulting pellet was resuspended in a sodium phosphate buffer containing sodium chloride. A minimum of 3 passages through a high pressure homogenizer were necessary for efficient cell breakage. The resulting cell lysate was clarified bycentrifugation and the supernatant was collected for further processing.

(c) Purification

The clarified supernatant was loaded on a column of Sepharose (Trade Mark) S Fast Flow (cation exchanger) and the product was eluted from this column using a gradient of increasing sodium chloride concentrations in a phosphate buffer (Trade Mark). The product was further purified on a column of Heparin Sepharose 6 B by eluting with a gradient of increasing sodium chloride concentration in a phosphate buffer. Finally a buffer exchange was made on a Sephadex (Trade Mark) G25 resin to obtain the product in the bulk product buffer (Sodium phosphate -EDTA).

(d) Column sanitization

Sepharose S Fast Flow and Sephadex G25 columns were sanitized by washing with sodium hydroxide solutions. Heparin Sepharose was washed alternatively with solutions at pH = 8.5 and pH = 5.5 containing 3M sodium chloride.

In this way, a 154/153 form of bFGF was obtained. This is an approximately 50:50 mixture of:

- a 154 amino acid human bFGF [(2-255)bFGF] having the amino acid sequence of the 155 amino acid form which is reported by Abraham et al but without the N-terminal Met residue; and

- a 153 amino acid human bFGF [(3-155)bFGF] consisting of the above 154 amino acid form without the first Ala residue.

Reference Example: Development of a standard experimental assay

1. Kinetics of bFGF trypsic hydrolysis

Assay conditions:

- 180 μg of bFGF

- Tris 50 mM, 20 mM CaCl₂ (final concentration)

- pH 8.5

- trypsin (EC 3.4.21.4) from Sigma T-0134: 18μg

- final volume: 200 μl

- temperature: 15°C

- incubation time: 15 min, 30 min, 45 min, 60 min, 75 min.

180 μg of bFGF were incubated in the Tris buffer, pH 8.5 at 15°C in the presence of trypsin. The reaction was analysed by SDS-PAGE at various incubation times. We determined that after 45 min incubation time, non-protected bFGF was completely digested. 2. Kinetics of heparin-protected bFGF trypsic hydrolysis

This experiment was performed in order to determine the protecting effect of heparin 15,000 on bFGF trypsic hydrolysis. Assay conditions: - 180 μg bFGF

- 36 μg heparin: bFGF/heparin 15,000 = 5 (w/w)

- Tris 50 mM, 20 mM CaCl₂ (final concentration)

- 18 μg of trypsin

- temperature: 15°C

- pH 8.5

- final volume: 200 μl

- incubation time: 30 min, 60 min, 90 min, 120 min, 180 min, 240 min

180 μg of bFGF were incubated in the Tris buffer pH 8.5 at 15°C in the presence of heparin (bFGF/heparin 15,000 = 5 (w/w). Then the enzymatic reaction was initiated by the addition of 18 μg of trypsin. The reaction was analysed by SDS-PAGE at various incubationtimes. We determined that bFGF was fully protected from the action of trypsin even after 240 min incubation time. Example 1: Screening of bFGF protecting agents

In order to screen any compound able to strongly protect bFGF against trypsic digestion we developed drastic conditions where compounds having a weak protecting effect would be rejected. The screening of anionic compounds was performed using the experimental assay described below:

EXPERIMENTAL ASSAY

- 180 μg of bFGF

- Tris 50 mM, 20 mM CaCl₂ (final concentration)

- bFGF/protecting agent = 5 (w/w)

- 18 μg of trypsin - final volume = 200 μl

- temperature = 15°C

- pH 8.5

- incubation time = 60 min.

In the SDS-PAGE analyses every anionic compound was compared with the protecting effect obtained with heparin 15,000 under the same conditions. In the experimental assay developed, we chose an incubation time of 60 min. The Reference Example showed that an incubation time of 45 minutes was sufficient to digest fully free bFGF. 36μg of protecting agent (bFGF/protecting agent ratio = 5(w/w)) was used which is equivalent to the lowest amount of heparin necessary to protect fully bFGF against trypsin hydrolysis.

Using this experimental assay, only compounds having a bFGF affinity as strong as heparin would have a protecting effect on bFGF trypsin hydrolysis. We screened over 40 compounds. The results are summarized in Table 1. Every compound having a protecting effect analogous to that of heparin 15,000 was described as a "good" protecting agent (+++). Those having a medium protecting, effect not as good as heparin were described as a "weak" protecting agent (+). Those having no protecting effect were marked with (0). TABLE 1: SCREENING OF PROTECTING AGENTS

COMPOUND bFGF TRYPSIN

HYDROLYSIS

A: GLUCIDS 1. Sulfated Glycans

- Heparan sulfate 0

- Heparin 3,000 + + +

- Heparin 5,000 + + +

- Heparin 15,000 + + +- Dermatan sulfate 0

- Chondroitin sulfate +

- Pentosan sulfate + + +

- dextran sulfate

Mw = 5,000 + + + - dextran sulfate

Mw = 500,000 + + +

- Tetradecasulfate

cyclodextrin + + +

- Carrageenan kappa + + +- Carrageenan lambda + + +

- Carrageenan iota + + +

- fucoidan + + + -carrageenan kappa

+ caroube + + + TABLE 1 ( CONTINUED)

COMPOUND bFGF TRYPSIN

HYDROLYSIS

2. Carboxylated

oligosaccharides

- Pectin 0

- Polygalacturonic

acid 0

- Salts of alginic acid:

- Satialgine 150 0

- Satialgine 300 +

- Satialgine 800 +

- Protanal 0

- Xanthan 0

- Carboxymethylcellulose 0

- Arabic gum 0

- Karaya gum 0

3. non-ionic oligo- saccharides

- Xylan 0

- Hydroxypropylcyclodextrin 0

- Inulin +

B: ACIDIC POLYPEPTIDES

AND PROTEINS

- Polyaspartic acid + TABLE 1 (CONTINUED)

COMPOUND bFGF TRYPSIN

HYDROLYSIS

- Polyglutamic acid 0

- Denaturated pepsin 0

C: OTHERS

- Deoxycholic acid 0

- Monooleate

(Tween 80) 0

- Deoxycholic acid+

Tween 80 0

- DNA + +

- DNA - cellulose 0

- L-α-phosphatidic

acid 0

- Gellan gum 0

- Guar gum 0

- Ghatti gum +

- Heptane sulfonic

acid +

Iota-, kappa- and lambda-carrageenan exhibited a protecting effect as good as that of heparin 15,000. For each experiment performed in this analysis we used 3 controls:

bFGF just before trypsin action

- bFGF/heparin 15,000 = 5 (w/w), (100% protection) - bFGF without protecting agent (to check the enzyme activity).

Example 2: Determination of the lowest amount of protecting agent necessary to protect fully bFGF

In order to precisely determine the lowest amount of protecting agent necessary to protect fully bFGF against trypsin hydrolysis, we performed bFGF trypsin hydrolysis assays using the experimental assay described in Example 1 but employing different bFGF/protecting agent ratios. Results are summarized in Table 2. TABLE 2: DETERMINATION OF THE HIGHEST bFGF/PROTECTING AGENT RATIO NECESSARY TO PROTECT FULLY bFGF AGAINST TRYPSIC HYDROLYSIS

COMPOUND HIGHEST bFGF/PROTECTING

AGENT RATIO

Heparin 15,000 7

Dextran sulfate 5,000 7

Dextran sulfate 8,000 7

Dextran sulfate 500,000 7

Fucoidan 7

Pentosan sulfate 7

Carrageenan iota 5

Carrageenan kappa 5

Carrageenan lambda 6

Xylan sulfate 7

Cyclodextrin sulfate 7

Reference Example 2: Kinetics of heparin protected bFGF thermal denaturation bFGF samples were submitted to thermal denaturation at 45°C in presence of heparin (bFGF/heparin 15,000 ratio = 5 (w/w)) for various incubation time . These samples were cooled at 15°C and submitted to tryps n hydrolysis for one hour. We determined that after 120 minutes of thermal denaturation, bFGF is fully protected by heparin.

Example 3: Screening of bFGF protecting agents for stabilisation of bFGF solutions against thermal denaturation

The compounds screened were the best compounds selected during the trypsin hydrolysis assays of Example 1. The experimental assay was as described in Example except that no trypsin was initially added and drastic incubation conditions were used: 60 minutes of thermal denaturation (unprotected bFGF is fully denaturated after 45 minutes at 45°C), then the samples were cooled at 15°C for 10 min and submitted to trypsin hydrolysis for 60 minutes. The results are shown in Table 3.

TABLE 3: THERMAL DENATURATION OF bFGF SOLUTIONS AT 45°

COMPOUND THERMAL DENATURATION PROTECTION

Dextran sulfate

Mw = 5,000 + + +

Dextran sulfate

Mw = 500,000 + + +

Fucoidan 0

Pentosan sulfate 0 TABLE 3 (CONTINUED)

COMPOUND THERMAL DENATURATION

PROTECTION

Cyclodextrin sulfate + + +

Xylan sulfate + + +

Carrageenan lambda + + +

Carrageenan kappa 0

Carrageenan iota 0

Heparin 15,000 + + +

Carboxymethyl- cellulose

Example 4: Determination of the lowest amount of protecting agent necessary to protect fully bFGF against thermal denaturation

The best compounds tested in Example 3, other than heparin 15 , 000 , were :

- dextran sulfate (Mw = 5,000 and 500,000),

- tjyclodextrin sulfate,

- xylan sulfate,

- carrageenan lambda.

Proceeding as described in Example 3, we determined that for bFGF/protecting agent ratios of 5 and

6 bFGF in solution at 45°C was fully protected against thermal denaturation using each of these protecting agents.

Example 5: Protection of bFGF against thermal denaturation at 37°C

The compounds screened in this analysis, were the best compounds selected during the trypsin hydrolysis assays of Example 1. The results are shown in Table 4. The assay conditions were:

- 180 μg of bFGF

- Tris buffer 50 mM, 20 mM CaCl₂ (final concentration)

- bFGF/protecting agent: 5 (w/w)

- final volume: 200 μl

- pH 8.5

- temperature: 37°C

-incubation time: 20 h

TABLE 4: THERMAL DENATURATION OF bFGF SOLUTIONS AT 37°C

COMPOUND THERMAL DENATURATION PROTECTION

Dextran sulfate

Mw = 5,000 + + +

Mw = 8,000 + + +

Mw = 500,000 + + +

Fucoidan +

Pentosan sulfate + + +

cyclodextrin sulfate + + +

Carrageenan lambda + + +

Carrageenan kappa 0

Carrageenan iota 0

Heparin 3,000 + + +

Heparin 5,000 + + +

Xylan sulfate + + +

Example 6: Stabilisation of freeze-dried bFGF against thermal denaturation at 75°C

In order to determine the stability of freeze- dried bFGF preparations, we developed heat-accelerated denaturation studies. The bFGF formulations used in this study were prepared from:

- 50 μg of bFGF

- 20 mg of mannitol

- 200 μg of dithiothreitol

- protecting agent: 1 mg

- phosphate buffer 10mM, 0.1 mM EDTA, pH 6.0

- final volume: 200 μl

These samples were freeze dried and kept at -30°C before use. The formulations were heated at 75°C for 3 days. The stability of bFGF samples in terms of oxidation (dimers, aggregates) was studied by SDS-PAGE analysis. The freeze-dried samples were solubilised in 100 μl H₂O and mixed with 100 μl of buffer; 0.02 M Tris HCl, 0.002 M EDTA, 2 % SDS, pH 8.0. The samples were heated at 95°C for 5 min. The SDS-PAGE analysis was performed on Phast Gel Homogeneous 20% (Pharmacia). The results are summarised in Table 5.

TABLE 5: HEAT-ACCELERATED FREEZE-DRIED bFGF DENATURATION PROTECTING PRESENCE OF

AGENT OXIDATIVE FORMS

none + + +

Cyclodextrin sulfate + + +

Dextran sulfate

- Mw = 500,000 0

- Mw = 5,000 0

Carrageenan lambda 0 TABLE 5 (CONTINUTED)

PROTECTING PRESENCE OF

AGENT OXIDATIVE FORMS

Fucoidan 0

FICE formulation:

Carboxymethylcellulose + + +

Na-CMC; TF/23634

Heparin 15 , 000 0

0: full protection; + + +: no protection

Example 7 : Bioactivity assays of stabilised bFGF

ABAE cells were used to test the bioactivity of stabilised bFGF formulations. The ABAE cells were isolated from aortic arch as described by Gospodarowicz et al (1976), Proc. Natl. Acad. Sci., USA 73, 4120-24. The cells were cloned and routinely subcultured according to Darbon et al (1986), J. Biol. Chem., 261, 8002-8008.

First, we performed the bioactivity assay for unstabilised 154/153-bFGF in comparison with unstabilised recombinant(r-) 146 bFGF. A culture of 5 x 10⁴ cells was seeded into 5 ml wells (Nunc) in DMEM supplemented with 10% calf serum. bFGF was added, at concentrations of 0.5, 1, 5, 10, 20 ng/ml, on days 0, 2 and 4 of incubation. The cells were counted on day 5 (triplicates). 154/153-rbFGF samples were prepared by freeze-drying a sample composed ^of:

- 50 μg of 154/153-rbFGF

- 20 mg of mannitol

- 200 μg of dithiothreitol - phosphate buffer 10 mM, 0.1 mM EDTA, pH 6.0

- final volume : 200 μl

We determined that 1 ng/ml was the lowest

146-bFGF concentration necessary to obtain the highest growth cell induction. Using 154/153-bFGF, 5-10 ng/ml was the lowest bFGF concentration necessary to obtain the highest mitogenic effect on ABAE cells.

Next, the same experiment was repeated using 10 μg of the following 154/153-bFGF protecting agents:

- heparin 15,000 (Hep),

- carrageenan lambda (CL),

- cyclodextrin tetradecasulfate (CS),

- dextran sulfate Mw = 5,000 (DS).

The results are shown in Table 6. The protecting agents potentialise the mitogenic effect of 154/153-bFGF on ABAE cells. We obtained a full mitogenic response at a bFGF concentration of 0.5 ng/ml.

TABLE 6: Bioactivity assay of formulated bFGF

0.5 ng/ml

155 75/3 91/3 84/3 5,55 × 10⁵

Hep 152/3 160/3 148/3 154/3 148/3 150/3 1,01 × 10⁵

CL 193/3 201/3 210/3 203/3 191/3 188/3 1,31 × 10⁶

CS 228/3 204/3 188/3 171/3 188/3 197/3 1,30 × 10⁶

DS 183/3 187/3 185/3 171/3 172/3 163/3 1,15 × 10⁶

10 ng/ml

155 74/1 78/1 68/1 78/1 88/1 68/1 1,51 × 10⁶

Hep 80/1 77/1 78/1 73/172/1 65/1 1,48 × 10⁶

CL 78/1 75/1 63/1 92/170/1 73/1 1,5 × 10³ TABLE 6: Bioactivity assay of formulated bFGF (CONTINUED) CS 74/1 58/1 72/1 55/1 67/1 64/1 1,3 × 10⁶ DS 47/1 48/1 46/1 69/1 63/1 56/1 1.09 × 10⁶ 20 ng/ml

155 118/3 110/3 122/3 126/3 7,9 × 10⁵

Hep 126/3 131/3 155/3 142/3 9,2 × 10⁵

CL 103/3 104/3 111/3 123/3 7,33 × 10⁵

CS 131/3 148/3 157/3 162/3 9,96 × 10⁵

DS 74/3 70/3 75/3 80/3 4,9 × 10⁵bFGF 146 (1 ng/ml) 1,4 × 10⁶

SEQUENCE LISTING SEQ ID NO: 1

SEQUENCE TYPE: amino acid

SEQUENCE LENGTH: 155 amino acids

TOPOLOGY: linear

MOLECULE TYPE: protein

ORIGINAL SOURCE ORGANISM: human

PROPERTIES: fibroblast growth factor

Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp

5 10 15

Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys

20 25 3H

Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro

35 40 43Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile

50 55 80

Lys Leu Gin Leu Gln Ala Glu Glu Arg Gly Val Val Ser He Lys 65 70 75 Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg

80 85 90

Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu

95 100 105

Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr

110 115 120

Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gin Tyr Lys Leu

125 130 135 Gly Ser Lys Thr Gly Pro Gly Gin Lys Ala Ile Leu Phe Leu Pro

140 145 150 Met Ser Ale Lys Ser

155

Claims

1. A fibroblast growth factor (FGF) stabilised by a carrageenan.

2. A stabilised FGF according to claim 1, wherein the FGF is acidic FGF.

3. A stabilised FGF according to claim 1, wherein the FGF is basic FGF (bFGF).

4. A stabilised FGF according to claim 3, wherein the bFGF is a human bFGF.

5. A stabilised FGF according to claim 4, wherein the human bFGF is (1-155)bFGF, (2-155)bFGF, (3-155)bFGF, a mixture of (2-155)bFGF and (3-155)bFGF, or (10-155)bFGF.

6. A stabilised FGF according to any one of the preceding claims, wherein the carrageenan is lambda-carrageenan.

7. A process for the preparation of a stabilised FGF as defined in any one of the preceding claims, which process comprises contacting the FGF with the carrageenan in an aqueous medium.

8. A process according to claim 7, further comprising lyophilising the resulting preparation.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a stabilised FGF as defined in any one of claims 1 to 6.

10. A composition according to claim 9, which comprises water-insoluble and water-swellable polysaccharide microspheres loaded with the stabilised FGF.

11. A composition according to claim 10, which is in the form of a powder.

12. A bandage incorporating a powder as claimed in claim 11.