WO1994003269A1

WO1994003269A1 - Microspheres of polyhydroxylic materials

Info

Publication number: WO1994003269A1
Application number: PCT/GB1993/001692
Authority: WO
Inventors: Paul Finch; Chui Sheung Leung; Ann Denise Wheeler; Helmut Weigel
Original assignee: Royal Holloway And Bedford New College
Priority date: 1992-08-10
Filing date: 1993-08-10
Publication date: 1994-02-17
Also published as: GB9216925D0

Abstract

Biodegradable polymeric microspheres useful for medical and other purposes are made of a polyhydroxylic polymer cross-linked by 1,1'-carbonyldiimidazole, phosgene or ethylchloroformate, the microspheres being directly reactive with nucleophiles. The microspheres are made by cross-linking an aqueous emulsion of the polymeric material using one of the above cross-linking agents. The polymer is usually a polysaccharide, and the microspheres can be directly linked to drugs, proteins and other diagnostic reagents, for example.

Description

MICROSPHERES OF POLYHYDROXYLIC MATERIALS

The present invention relates generally to microspheres of polymeric material, particularly of a polyhydroxylic material, especially but not exclusively of polysaccharide, and to methods for their synthesis and derivatisat on.

Biodegradable polymeric microspheres have been developed in recent years for use in medicine. They are particularly useful as jLri vivo carriers for drugs or diagnostic agents which may be covalently bound thereto and/or encapsulated therein or may even be an integral part of the polymer itself. One way in which these microspheres can be made is by an emulsion preparative technique. An aqueous solution of the polymer is dispersed as micelles in an organic phase and the polymer then cross-linked to form water-insoluble microspheres. In one known application of this process to the preparation of polysaccharide microspheres, the polysaccharide is reacted via its hydroxyl groups with an aliphatic chloroepoxide to yield microspheres of the polysaccharide having ether-bonded cross-linking bridges of a hydroxyl-substituted straight or branched chain aliphatic saturated hydrocarbon. Examples of these cross linking bridges include -OCH₂-CH(OH)CH2θ-,

-OCH₂CH(OH)CH(OH)CH₂0- and -OCH₂CH(OH)CH₂OCH(CH₃)CH₂CH₂0- CH₂CH(0H)CH₂0-. Another known method for microsphere synthesis involves first reaction of a polysaccharide with a glycidyl ester or acyl chloride of acrylic acid followed by a free radical polymerisation to form the cross-linking bridges. If necessary, the microspheres can then be activated, in order to form covalent links with appropriate drugs/diagnostic agents.

The use of 1 , 1' -carbonyldiimidazole as an agent for the activation of the hydroxyl groups of poly- saccharides , including polysaccharide microspheres, is known. However, such reactions are always carried out in an organic medium, as the 1 , 1* -carbonyldiimidazole is known to be moisture-sensitive and decomposes readily in water. Therefore, when using 1 , 1' -carbonyldiimidazole as an activating agent it has been necessary first to synthesise polysaccharide microspheres by one of the prior art methods and subsequently to activate them with 1,1' -carbonyl¬ diimidazole in an organic medium.

However, we have now found that, surprisingly, this reagent can be employed in an emulsion preparation to cross-link and simultaneously activate the hydroxyl groups of polyhydroxylic materials, e.g. polysaccharides , in aqueous micelles to form activated microspheres in one reaction step. Thus, during the reaction with 1,1' - carbonyldiimidazole, the polyhydroxylic material is cross- linked as well as activated for further reaction in one single step, instead of the previous two. Phosgene and ethylchloroformate can also be similarly utilised and the invention includes the use of such materials.

According to a first aspect of the present invention, therefore, there is provided a method of making reactive polyhydroxylic microspheres which comprises the steps of preparing an emulsion of an aqueous solution of the polyhydroxylic material in an organic solvent, and treating the emulsion with 1 , 1' -carbonyldiimidaxole , phosgene or ethylchoroformate to form cross-linked water-insoluble microspheres.

The invention also includes reactive polyhydroxylic microspheres cross-linked by using 1 , 1 '-carbonyl¬ diimidazole, phosgene or ethylchloroformate.

Whilst we do not wish to be bound by this theory, we believe that the cross-linking agent, e.g. 1,1'- carbonyldiimidazole activates the hydroxyl groups of the polyhydroxylic material in the aqueous micelles to form P-O-CO-N2C3H3 (where P is the polyhydroxylic backbone). Subsequent displacement of the imidazole group by the oxygen atom of another hydroxyl group of the polyhydroxylic material can lead to the formation of carbonate as the cross-linker. The provision of the diester of carbonic acid as a cross-linking group (i.e. one single carbon atom as the cross-linking bridge) has the advantage over prior art bridges of being more susceptible to chemical or enzymic hydrolysis to form carbon dioxide and glucose. Consequently, it is to be expected that upon degradation of these microspheres iji vivo no aliphatic hydrocarbon chains will remain as residue. In contrast, the breakdown of polysaccharide microspheres made by prior art methods can result in fragments containing glucose units attached to hydrocarbon chains of varying length which are only degraded slowly ij vivo.

The preferred polyhydroxylic material for the microspheres of the invention is polysaccharide material, and the preferred cross-linking/activating agent is 1,1'- ^~ carbonyldiimidazole (hereinafter "CDI"). For simplicity, the present invention will be described hereafter generally with reference to these two preferred materials only. It is to be understood, however, that the teaching is also applicable mutatis mutandis to the use of other less preferred materials.

In the method of the invention, the cross-linking agent is brought into contact with an emulsion of an aqueous solution of the polysaccharide in an organic solvent. Preferably, the emulsion is prepared by firstly mixing the surfactant and organic solvent, and then adding the aqueous solution of polysaccharide and homogenising the mixture. The polysaccharide is thus held within aqueous micelles encased by surfactant molecules, and the surfactant molecules form a stable emulsion of the aqueous micelles in the organic solvent. The subsequent addition of cross- linker provides cross-linking bridges between the polysaccharide molecules within each aqueous micelle to form a cross-linked microsphere in each micelle. The spherical shape and limited size range (e.g. 0.5 microns to 2 microns dry) of the microspheres are dictated by the forms of the aqueous micelles which are, in turn, determined by the molar ratio of surfactant to water in the reaction chamber. Both ionic and neutral surfactants can be used, and the solvent will generally be chosen in dependence on the surfactant.

In the method of the present invention, "we believe that the 1 ,1 '-carbonyldiimidazole firstly reacts with a hydroxyl group of the polysaccharide to form an -O-CO-N2C3H3 substituent, and that a proportion of such substituents will then each react further with hydroxyl group of another polysaccharide molecule in the microsphere by replacement of the second imidazole group (N2C3H3) to create a carbonate cross-linking bridge. Those -O-CO-N2C3H3 which do not form cross-linking bridges are reactive, being susceptible to further derivatisation. Thus, the polysaccharide microspheres so formed may be derivatised by direct reaction with nucleophiles such as primary or other amines e.g. alkyl amines, aryl amines, arylalkyl amines, or other nucleophiles such as alkyl alcohols, aryl alcohols or alkyl thiols. The derivatisation process may be carried out with or without first isolating the microspheres from the other emulsion constituents.

The nucleophilic substituent bound to the microsphere in the derivatisation process may comprise, for example, a drug, protein, or a diagnostic agent such as an X-ray opaque substance. Release of the substance carried by the microsphere may be effected by enzymic hydrolysis of the polysaccharide comprising- the microsphere. Instead of covalently binding the drug or diagnostic agent to the microsphere, the microsphere could be formed encapsulating the drug or diagnostic agent. In this case, release could be effected simply by diffusion. Furthermore, the drug/diagnostic agent could even form an integral part of the polysaccharide itself.

In the method of the invention, it can be advantageous to include in the reaction mixture one or more bi- or polyfunctional reactive compounds in addition to the CDI and the polysaccharide. These compounds can, for example, act to extend the length of the cross-linking bridges which can improve biodegradability provided the compound itself is appropriately chosen. Polyols are generally useful, of which glycerol is one example. In addition to the possibility of advantageously extending the bridge length, it is also possible for the compounds to react to increase the number of hydroxyl groups in the polysaccharide and thus the number of CDI units linked thereto, so making the cross-linked microspheres more active for bonding to other substances. '

The polysaccharide microspheres of the invention as freshly prepared contain reactive groups, evg. carbonyl imidazole groups. Furthermore, even after derivatisation, there may be a few such groups still remaining. These reactive groups can be selectively removed if desired by raising the pH, eg. to about pH 10.

The nature of the polyhydroxylic material eg. polysaccharide, chosen for the formation of the microspheres should suit the intended application. For example, microspheres of cellulose derivatives might be adequate if diffusion release were intended. Starch is generally the most preferred polysaccharide but other potentially suitable polysaccharides include, for example, starch derivatives, dextran, heparin and hyaluronic acid. For use in NMR imaging, an iron-containing polysaccharide would be useful. The reactive microspheres of the invention can be stored in an organic solvent and then brought into contact with a nucleophile as desired. Alternatively, and more preferably, the reactive microspheres are stored lyophilised. The derivatised microspheres can likewise be stored in suspension or lyophilised. If desired, the microspheres can be successfully sterilized by, for example, autoclaving or ^-irradiation.

In order that the invention may be more fully understood, the following Examples are given by way of illustration only. Example 1 describes starch microsphere synthesis and isolation, Example 2 describes derivatisation of the isolated starch microspheres, Example 3 describes starch microsphere synthesis and derivatisation without isolation of the activated microspheres, Example 4 describes selective removal of carbonylimidazole groups from activated microspheres and Examp.le-5 illustrates sterilisation. In the accompanying drawings, Figure 1 is an electron micrograph of the starch microspheres of Example 1, and Figure 2 is an electron micrograph of the X-ray opaque iodine-containing starch microspheres of Example 3.

EXAMPLE 1

An aqueous solution of starch (10%, 10ml), having a molecular weight of 10,000, is homogenised for 3 min. with ethylene dichloride (100 ml) and Gafac PE510 (trade mark; 2.5 g), acting as surfactant, using as agitator the Ultra- Turrax T25 at a speed of 13,500 rp . A solution of 1,1'- carbonyldiimidazole (1 g) in ethylene dichloride (25 ml) is added in portions to the emulsion at room temperature with stirring. The reaction mixture is stirred for a further 30 min. Acetone (400 ml) is added to precipitate the starch. Filtration yields a white solid (1.5 g) which is soaked in water (200 ml) for 30 min., sonicated and then screened using Whatman falter paper No. 41. The filtrate is centrifuged at 2750g for 10 min., and the sediment washed with water (3 x 40 ml). Freeze-drying yields activated microspheres (220 mg) having a diameter of-_^-1 μm, as established by scanning electron microscopy (Fig. 1). The yield is not optimised an-d may be increased several fold. The microspheres of this Example have a swelling factor of 2.9 when suspended in water.

EXAMPLE 2

Activated starch microspheres (100 mg) are suspended in dioxan (4 ml). The suspension i> sonicated for 30 min. A solution of methyl 3-amino-2,4,6-triiodpbenzoate (250 mg) in dioxan (4 ml) is added and the mixture stirred at room temperature for -16 h. The mixture is centrifuged-at 2750g and the sediment washed with dioxan (3 x 50 ml) and water (3 x 50 ml). Freeze-drying affords pale mauve coloured spheres (100 mg). Alkaline hydrolysis, followed by spectroscopic analysis shows the spheres to contain 12% iodine. The material has no obvious toxic effect in rats.

EXAMPLE 3

An aqueous solution of starch (10%, 10 ml) is homogenised for 3 min. with ethylene dichloride (100ml) and Gafac PE510 (2.5 g) as described in Example 1. A solution of 1 , 1 ' -carbonyldiimidazole (1 g) in ethylene dichloride (25 ml) is added in portions to the emulsion. Microscopy shows the formation of microparticles. A solution of methyl 3- amino-2 ,4 ,6-triiodobenzoate (1.5 g) in dioxan (5 ml) is added and the mixture stirred for a further 3 hr. Acetone (450 ml) is added, the precipitate filtered using Whatman No. 2 (Qualitative 1) filter paper, washed with acetone and then soaked in- water (200 ml) for 30 min. with sonication. The aqueous suspension is filtered through Whatman filter paper No. 41, the filtrate centrifuged at 2750g, and the sediment washed with dioxan (3 x 50 ml) followed by washing with water (3 x 50ml). Freeze-drying of an aqueous suspension affords mauve- coloured spheres (360 mg), having a diameter of~>--l um as established by scanning electron microscopy (Fig. 2). Alkaline hydrolysis, followed by spectroscopic analysis, shows the spheres to contain 20% iodine.

Example 4

A portion (1.5 g) of freeze-dried reactive starch microspheres of- the invention is dried in vacuo at 50°C. Its -CO.N2C3H3 content, is determined by complete hydrolysis, followed by colorimetric determination of the imidazole liberated.

Another portion of the same batch of reactive starch microspheres (1.5 g) is suspended in a buffer of pH 10 (100 ml), and the suspension stirred for 16 hr at room temperature. The solid is removed by centrifugation, resuspended in fresh buffer of pH 10, and the suspension stirred for a further 60 hr at room temperature. The solid is removed by centrifugation, washed with water (3 x 50 ml), freeze-dried and then dried in vacuo at 50°C. Its -CO.N2C3H3 content is determined as described above. This partial hydrolysis removes 99.85% of the -CO.N2C3H3 groups. Scanning electron microscopy shows that the spherical shape of the material is retained. E X AMP LE 5

Sterilisation b'y autoclaving

Reactive starch microspheres (from Example 1) or derivatised starch microspheres from Example 2 (100 mg) are suspended in water (50 ml) and autoclaved at 115°C for 15 min and at 121°C for 30 min. No significant change in the integrity of the spheres was observed by scanning electron microscopy.

Sterilisation by -irradiation

Dried reactive starch microspheres (from Example 1) or derivatised starch micro-spheres (from Example 2) were subjected to ^-irradiation with a dose rate of 2.5 megarads, No change in physical appearance was observed by scanning electron microscopy.

Claims

CLA I MS :

1. A method of making reactive polyhydroxylic microspheres which comprises the steps of treating an emulsion of an aqueous solution of the polyhydroxylic material in an organic solvent, with 1 , 1' -carbonyl¬ diimidazole, phosgene or ethylchloroformate to form cross- linked, water-insoluble microspheres.

2. A method according to claim 1, wherein the polyhydroxylic material is a polysaccharide.

3. A method according to claim 2, wherein the polysaccharide is starch, a starch derivative, dextran, heparin or hyaluronic acid.

4. A method according to claim 1,2 or 3, wherein the emulsion is prepared by firstly mixing a surfactant with the organic solvent, and then adding the aqueous solution of polyhydroxylic material and homogenising the mixture.

5. A method according to claim 1,2,3 or 4, wherein there is also included in the reaction mixture one or more bi- or poly-functional reactive compounds.

6. A method according to claim 5, wherein the reactive compound is a polyol.

7. A method according to any of claims 1 to 6, wherein the aqueous phase contains a substance which becomes encapsulated by the microsphere.

8. A method according to any of claims 1 to 7, which includes the further step of lyophilising the microspheres for storage .

9. A reactive polyhydroxylic microsphere cross-linked by using 1,1' -carbonyldiimidazole, phosgene or ethylchloroformate .

10. A microsphere according to claim 9, which is a polysaccharide.

11. A polyhydroxylic microsphere according to claim 9 or 10, or made by the method of any of claims 1 to 8, which has been reacted directly with a nucleophile.

12. A microsphere according to claim 11, wherein the nucleophile is an amine , an alcohol or a thiol.