GB2245831A - Delivery system for growth factors - Google Patents

Abstract

Formulations for treating burns or wounds comprise a powder of water-insoluble and water-swellable polysaccharide microspheres loaded with a heparin-binding growth factor e.g. basic fibroblast growth factor. Polysaccharide is e.g. a cellulose, starches, dextrans, collagen, gelatin or albumin.

Description

DRUG DELIVERY SYSTEM FOR ADMINISTERING GROWTH FACTORS The present

invention relates to compositions useful for delivering growth factors to burns and wounds. The invention particularly relates to such compositions 5 comprising basic fibroblast growth factor (b-FGF).

Growth factors have recently been proposed for wound healing treatment. Slow-release pellets made of cholesterol, methy1cellulose and lactose and able to release Epidermal Growth Factor (EGF) at a rate of 10 to 20 gg/day were made by Buckley gt al (PNAS USA 82f 7340-7344, 1985; J. Surg. Res. 43, 322-328, 1987). These pellets were embedded in polyvinyl alcohol sponges.

This system has been tested in animal studies and has been found to be useful especially for implants. However, there are problems due to the presence of inethylcellulose (MC). MC may be considered biodegradable, although more exactly it may be defined as bioabsorbable, more or less depending on the degree of substitution. MC has a low biocompatibility, though. MC provokes an immune response when administered to the body (Myamoto et al, J. Biom. Mater. Research 23, 125-133, 1989).

EP-A-0267015 discloses stabilised compositions containing a growth factor such as EGF. Aqueous medicinal compositions containing a polypeptide growth factor having human mitogenic activity are stabilised against loss of biological activity in the presence of moisture by including a stabilising amount of a water-soluble polysaccharide.

Gel formations for use in wound healing and containing a polypeptide growth factor having human mitogenic or angiogenic activity, in particular EGF, have been reported in EP-A-0312208. The aqueous gel formulations also comprise a water-soluble or water-swellable pharmaceutically acceptable polymer for providing a viscosity from 1 to 12 x 104 Pas (103 to 1.2 x 107 cps) at room temperature. These gels may be made of polyacrylic acid (Carbopol, Trade Mark), poloxamers (Pluronics, Trade Mark), hydroxypropylmethylcellulose (Methocel, Trade Mark), hyaluronic acid and polyacrylamide (Cyanomer, Trade Mark). However, the use of a gel formulation, due to its high content of water, can give a stability problem for the growth factor and lead to a short product shelf-life. The gels can be lyophilised to provide a stable dosage form which can be reconstituted at the time of use (EP-A-0308238).

Another way that has been reported to stabilize b-FGF and other growth factors (11heparin binding growth factors") is to bind them to heparin, other glycosamino- glycans or sulphated and/or carboxylated macromolecules (Thomas et al "Structure and Activities of Acidic - 3 Fibroblast Growth Factor", in Angiogenesis (current communications in molecular biology), eds.: Rifkin D.B., Klagsbrun M., Cold Spring Harbor Laboratory, 9-12 (1987); Morton et al, Current Eye Research, g (10), 975- 986 (1989); Shing gt &1, Analytical Biochemistry, 185, 108-111 (1990); Lobb, European Journal of Chemical Investigation L8, 321-336 (1988)). This stabilizing action is also accompanied by protection from inactivating proteases (Rosengart et al. Fed. Proc., 46, 2116 (1987); Lobb, Biochemistry, 27, 2572-2578 (1968)).

A proposal to vehiculate b-FGF or other growth factors has been to load the growth factor in a composite graft for burn treatment made of collagen-glycosaminoglycan membranes, with a surface of the membrane covered with cultured human keratinocytes (Hansbrough et al, UAMA, 262 (15), 2125-2130 (1989)). This therapeutic system can obviously not be applied to large scale manufacture, and remains a treatment restricted to specialized centers for burn treatment.

We have now found that loading b-FGF into water insoluble, water-swellable, microspheres offers a suitable way to deliver b-FGF for burn or wound healing.

This finding has general applicability to all heparin binding growth factors.

Accordingly, the present invention provides a powder comprising water-insoluble and water-swellable - 4 microspheres loaded with a heparin-binding growth factor. The powder has gel-forming capability. It is typically a lyophilised powder. The microspheres are typically biodegradable.

The term "heparin-binding growth factor" includes not only the natural human growth factor polypeptide but also polypeptides that have biological activity similar to the natural human growth factor polypeptide. Thus, the term includes a human heparin-binding growth factor produced by recombinant DNA techniques or derived from natural sources, as well as closely related mammalian growth factor such as murine or rodent. A human heparin-binding growth factor produced by recombinant DNA techniques is preferred for use in the present invention.

The heparin-binding growth factor is typically acidic or basic fibroblast growth factor, i.e. a-FGF or b-FGF. The use of b-FGF is preferred. Any b-FGF molecule as described in, for instance, WO 86/07595; WO 87/01728; EP-A-0226181; Abraham et al, EMBO J. 5, 2523-2528, 1986; or Lobb, Eur. J. Clin. Invest. 18, 321-336, 1988; may be usefully employed in the invention.

A mixture of b-FGFs may be employed. This may be an approximately 50:50 mixture of: - a 154 amino acid human b-FGF 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 - 5 this 154 amino acid sequence also being shown in Figure 1 of the accompanying drawings; and - a 153 amino acid human b-FGF consisting of the above 154 amino acid form without the Ala residue at position 1.

The microspheres are typically composed of cellulose, a cellulose derivative, starch, a starch derivative, gelatin, albumin, collagen, dextran or a dextran derivative. The microspheres are water-insoluble and water-swellable. The nicrospheres composed of dextran derivatives may be composed of an anionic derivative such as a carboxylated or sulphated dextran or of a cationic derivative such as diethylanino ethyl or a quaternary amino ethyl dextran. Preferably the microspheres are cross-linked starch microspheres such as Spherex (Trade Mark) nicrospheres or cross-linked dextran microspheres.

The nicrospheres are loaded with an amount of growth factor effective for use in treating a wound or burn. The amount may therefore be tailored to requirements.

Typically, however, the microspheres are loaded with from 0.2 to 5.0 mg of growth factor per gram of microspheres. A useful amount is from 0.8 to 1.5 mg of growth factor per gram of microspheres.

The powder may further comprise microspheres which.are not loaded with a therapeutic agent at all. Microspheres loaded with another therapeutic agent, such as another growth factor, may be present in addition to or as an alternative to the unloaded microspheres. The unloaded microspheres can be used to dilute the microspheres loaded with growth factor. The microspheres loaded with another therapeutic agent can be used to deliver another agent, besides the growth factor, to the site of a wound or burn. Typically, the type of unloaded microspheres or microspheres loaded with another agent is the same, in terms of composition and size, as the type of microspheres loaded with growth factor. These other microspheres therefore are generally biodegradable too.

The powder of the present invention typically has an average particle diameter of from 5 to 1000 gm, preferably from 20 to 250 pm. A powder having an average particle diameter of from 20 to 30 pm is suitable, for example from 20 to 25 gm. Indeed, a powder with an average particle diameter of from 20 to 100 gm, for example from 50 to 90 gm, is suitable too. The powder is prepared by a process which comprises: (i) soaking water-insoluble and water-swellable polysaccharide microspheres in an aqueous solution of a heparin-binding growth factor; and (ii) lyophilising the dispersion of the microspheres in the aqueous growth factor solution. 25 The growth factor is loaded into microspheres by first soaking a weighed amount of microspheres in a solution of the growth factor in water. The ratio of microspheres growth factor in solution and microspheres: solution of growth factor can vary greatly. It is preferred to employ a ratio of microspheres: growth factor of from 8:1 (w/w) to 8000:1 (w/w). Preferably, the microspheres are present with respect to the solution of growth factor in an amount of from 0.005 % (w/v) to 5 % (w/v). The pH and ionic strength can be varied within preferred ranges of from pH4 to pH8 and from I=0 to I=1.

The microspheres which are to be loaded with growth factor typically have an average particle diameter of from 20 to 250 Am. Microspheres with an average diameter of from 20 to 30 pm, for example from 20 to 25 An, are suitable as are microspheres with an average particle dirmeter of from 20 to 100 Am, for example from 50 to 90 Am.

The growth factor may be stabilized by being complexed with an anionic polymer. The anionic polymer may be a natural polymer. The anionic polymer may be a glycosaminoglycan, a cellulose, a cyclodextrin or xanthan gum. Examples of suitable polymers are heparin, heparin sulphate, chondroitin sulphate, keratin sulphate, a hyaluronate, an alginate, cellulose sulphate, carboxymethylcellulose and derivatives of a-, fland.- cyclodextrins and their polymers. The growth factor complexed with the anionic polymer is - 8 provided in the solution in which the microspheres are to be soaked. An antioxidant such as dithiothreitol may be present in the solution.

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 tine needed for soaking (incubation) can vary greatly. The time may be from 2 minutes to 48 hrs, preferably from 2 minutes tc 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 - 9 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 released. 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 bandage 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. 5 Such a bandage can be replaced as necessary.

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 is a'-so provided. Prenaration ExamiDle: PreDaration of b-FGF (FCE 26184) The construction of the synthetic DNA sequence for bFGF 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, F. coli type B, from the Institute Pasteur collection, was transformed with a plasmid carrying both the human gene coding for b-FGF and the - 11 gene for tetracycline resistance. This transformed strain was used for the production of recombinant nonglycosylated h-b-FGF (human b-FGF). A Master Cell Bank (15 freeze-dried vials) and a Working Cell Bank (W.C.B. ) (70 vials stored in liquid nitrogen at -1900c) 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 370C the final biomass was 42+2 g/l dry weight, an the production of b-FGF was 2500 500 mg/l as measured by comparative gel electrophoresis. I Enrichment in pure oxygen was required during the fermentation phase in order to allow a large bacterial growth. (b) Initial purification 0 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 by centrifugation and the supernatant was 2 - 12 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 was, there was obtained b-FGF designated FCE 26184. This is an approximately 50:50 mixture of: - a 154 amino acid human 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, this 154 amino acid sequence also being shown in Figure i of the accompanying drawings; and a 153 amino acid human bFGF consiting of the above 154 amino acid form without the Ala residue at position 1.

Example i mg of FCE 26184 and 10 ID9 of dithiothreitol were dissolved in 200 mi of double distilled water which had previously been brought to pH=6.0 with NaOH 0.01 N, into which 4 g of starch microspheres had previously been transferred. The starch particles were cross-linked starch particles of the type Spherex which had an average diameter of 21 5 Am as determined by optical microscopy. The suspension was then allowed to stand at room temperature for 30 minutes and then Iyophilised. A white powder was obtained.

Exanple 2 5 mg of FCE 26184 and 10 mg of dithiothreitol were dissolved in 200 ml of double distilled water which had previously been brought to pH=6.0 with NaOH 0.01 N, into which 4 g of dextran microspheres had previously been transferred. The dextran microspheres were cross-linked nicrospheres of the type Sephadex G-50 superfine which had a diameter of 28 + 7 Am as determined by optical microscopy. The suspension was then allowed to stand at room temperature for 30 minutes and then 1yophilised. A white powder was obtained.

The stability of the dextran microspheres loaded with the b-FGF was tested at 250C. The results are shown in - 14 Table 1 below. The percentage values represent the percentage stability with respect to stability at time zero, i.e. the amount of residual b-FGF with respect to that at time zero, as measured by the high pressure liquid chromatography (HPLC) method.

TABLE 1

Tine 1 week 1 month 2 months HPLC Method Materials -1 96.2 86.3 78.8 b-FGF bulk frozen solution, working standard Acetonitrile, HPLC grade Water, HPLC grade 0.9% sodium chloride injection, pharmaceutical grade Trifluoroacetic acid, analytical grade 1,4-dithiothreitol, analytical grade Equipment. Liquid chromatograph Milton Roy model CM 4000, or equivalent, equipped with:.. chromatographic column: (length 250 mm, internal diameter 4.6 mm) filled with Vydac 218TP54 300 A - 15 (average particle size 5 mcm), or equivalent.. injection valve: Rheodyne model 7125, or equivalent, fitted with a 100 mcl sample loop detector: Shimadzu model SPD 6A, or equivalent integrating recorder: SP 4270 (Spectra-Physics), or equivalent Membrane filter, 0.22 pm porosity, Millipore Durapore GVWP, or equivalent High precision laboratory glassware 10. Plastic pipet tips (Gilson) Automatic pipets (Gilson) Disposable plastic microtubes, capacity 2.5 ml (Eppendorf) Solutions 15. Mobile phase (A) consiting of water, containing 0.1% of trifluoroacetic acid (w/v), filtered through the membrane filter and deaerated.

Mobile phase (B) consisting of 95% acetonitrile-5% water containing 0.1% of trifluoroacetic acid (w/v), filtered through the membrane filter and deaerated.

1,4-dithiothreitol solution Prepare a solution containing about 1 ng/ml in HPLC grade water Standard solution Transfer a suitable volume of bulk solution of b-FGF working standard, accurately measured, into a disposable plastic microtube. Dilute with a suitable volume of 1,4-dithiothreitol solution in order to obtain a final solution containing about 50 ncg/ral of b-FGF. The standard solution must be freshly prepared and used within a working day.

Sample solution Prepare the sample solution using at least five freezedried vials. The content of each vial dosed at 50 iacg of b-FGF loaded nicrospheres is dissolved in 1.0 ial of 0.9% sodium chloride injection, then a pool is made with the all prepared solutions. Allow the pooled sample to decant for at least half an hour, then inject it into the liquid chromatograph.

Chromatographic (HPLC) conditions The standard and sample solution were alternatively injected at least 3 times into the liquid chromatograph under the following experimental conditions:

Column temperature Mobile phase flow-rate Analytical wavelength Gradient conditions Detector sensitivity Injection volume Integrating recorder 15 attenuation Chart speed room temperature (22 + 20C) 1 ml/min 210 + 1 nm,tine (min) 0 20 25 A% 75 60 60 B% 75 25 the detector Hcomputer" output was connected to integrator for maximum sensitivity 100 mcl 256 0. 5 cm/min Example 3 The powder obtained in Example 1 was tumble mixed for 10 minutes with 6 g of Spherex microspheres having an average diameter of 21 5 gm. Nothing had been loaded on these Spherex microspheres. ExamiDle 4 The powder obtained in Example 2 was tumble mixed-for 10 minutes with 6 g of Sephadex G-50 superfine microspheres having an average diameter of 28 + 7 An.

- is - Nothing had been loaded onto these Sephadex G-50 superfine microspheres. ExamiDle 5 The powder obtained in Example 1 was tumble mixed for 5 10 minutes with 6 g of Sephadex G-50 superfine microspheres having an average diameter of 28 7 gm. Nothing had been loaded onto these Sephadex G-50 superfine nicrospheres. Example-6 The powder obtained in Example 2 was tumble mixed for minutes with 6 g of Spherex microspheres having an average diameter of 21 5 gm. Nothing had been loaded onto these Spherex microspheres. ExamiDle 7 Proceding in an analogous way to that described in the previous Examples, analogous preparations of comparable stability could be obtained with: - a human b-FGF comprising the 153 and 154 amino acid forms mentioned in the Preparation Example in different proportions, in particular a b-FGF wherein the 154 amino acid form is present in an amount of from about 50% to about 100%; - the human b-FGF having 146 amino acid residues, i.e. that having the amino acid sequence shown in Figure 1 starting from amino acid 9; and - the human b-FGF form of 155 amino acid residues 2 - 19 reported by Abraham &-t- al, i.e. that having the amino acid sequence shown in Figure I but with a N-terminal Met residue.

Claims (11)

1. A powder comprising water-insoluble and water swellable polysaccharide microspheres loaded with a heparin-binding growth factor.
2. 2. A powder according to claim 1, wherein the growth factor is a basic fibroblast growth factor (b-FGF).
3. 3. A powder according to claim 1 or 2, wherein the microspheres are composed of cellulose, a cellulose derivative, starch, a starch derivative, gelatin, albumin, collagen, dextran or a dextran derivative.
4. 4. A powder according to claim 3, wherein the microspheres are crosslinked starch or cross-linked dextran microspheres.
5. 5. A powder according to any one of the preceding claims, wherein the growth factor is complexed with an anionic polymer.
6. 6. A powder according to any one of the preceding claims, further comprising microspheres which are not loaded with a therapeutic agent. 20
7. 7. A process for the preparation of a powder as defined in claim 1, which process comprises: (i) soaking water-insoluble and water- swellable polysaccharide microspheres in an aqueous solution of a heparin- binding growth factor; and 25 (ii) lyophilising the dispersion of the microspheres T 1 - 21 in the aqueous growth factor solution.
8. 8. A process according to claim 7, wherein step (i) is effected for from 2 minutes to 2 hours at ambient temperature.
9. 9. A process accolrding to claim 7 or 8, wherein the ratio in step (i) of microspheres: growth factor in solution is from 8:1 to 8000:1 (w/w) and the microspheres are present in step (i) with respect to the solution of growth factor in an amount of from 0.005% to 5% (w/v).
10. 10. A powder as defined in any one of claims 1 to 6 for use in the treatment of a wound or burn.
11. 11. A bandage incorporating a powder as defined in any one of claims 1 to 6.

    Published 1991 at The Patent Office. Concept House. Cardiff Road. Newport. Gwent NP9 I RH. Further copies may be obtained from Sales Branch. Unit 6. Nine Mile Point. Cwnifelinfach. Cross Kevs. Newport. NPI 7HZ. Printed by Multiplex techniques ltd. St Marv Crav, Kent.