WO2004096178A1 - Nano and microparticle drug delivery systems comprising polyesters containing aliphatic dicarboxylate residues and residues of aliphatic polyols - Google Patents

Abstract

A nano or microparticle drug delivery system comprising a pharmaceutically-active agent and nano or microparticles of a linear aliphatic polyester, the polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with the pharmaceutically-active agent. The nano or microparticle drug delivery system can be used to deliver a pharmaceutically-active agent to an animal, including human.

Description

NANO AND MICROPARTICLE DRUG DELIVERY SYSTEMS COMPRISING

POLYESTERS CONTAINING ALIPHATIC DICARBOXYLATE RESIDUES AND

RESIDUES OF ALIPHATIC POLYOLS

The present invention relates to nano and microparticle drug delivery systems. More particularly, it relates to such drug delivery systems using, as the carrier for a pharmaceutically-active agent, a derivatised functional polymer. It also relates to a method of making such delivery systems and the use of such systems in therapy.

Microparticles and nanoparticles are increasingly being researched as drug delivery systems, which can deliver drugs to specific sites in the body. Nanoparticles can potentially be used parenterally to treat diseases where vasculature is leaky, e.g. sites of tumour and inflammation, sites in the reticuloendothelial system (RES), e.g. parasitic diseases such as malaria, and sinusoidal sites in the liver and spleen. Nanoparticles also have some potential for lung delivery and for immune stimulation of lymphoid tissue accessed either orally or through mucous membranes.

For parenteral delivery, nanoparticles need to evade the reticuloendothelial system, which recognises most particulate materials and effectively removes them from the circulation. By incorporating a surface layer of poly(ethylene glycol) (PEG) onto the nanoparticles, a hydrophilic surface is provided which prevents the adsorption of opsonins and greatly reduces uptake by the RES (1).

Nanoparticles foϊ parenteral applications can be made with a core-shell structure, where the core encapsulates the drug and the shell is a hydrophilic layer producing a steric stabilisation to prevent particle aggregation and adsorption of opsonins (2,3). This core-shell arrangement can either be achieved by the adsorption of a surfactant with a hydrophilic segment which is adsorbed onto a core encapsulating the drug or by the use of block copolymers with both hydrophilic and core-forming segments. Systems with adsorbed surfactants have included the use of poloxamers and poloxamines on poly(Iactide-co-glycolide) (PLGA) particles (1), or polysorbates on polycyanoacrylate particles (4). For block copolymer systems, these usually have hydrophobic and hydrophilic segments arranged as either A-B or A-B-A configurations, typified by poly (L-lactic acid)-poly(ethylene glycol) copolymer systems (PLA-PEG copolymer systems). Depending on the length of the core-forming polymer segment, delivery systems may be micelle-like, where the micelles are thermodynamically unstable and so in an equilibrium condition, or alternatively may form particles which are essentially stable (5).

Alternative systems have also investigated the use of charge to maintain the nanoparticle structure, e.g. polyelectrolyte complexes, e.g. cationic polymeπDNA complexes (6,7). Other systems have relied on a copolymer with a block containing a chemical functionality which can be conjugated to a drug to produce a hydrophobic segment for nanoparticle formation (8,9).

Many copolymers have been investigated which have varying hydrophilic- hydrophobic block combinations. In most cases, the hydrophilic segment is PEG with a variety of hydrophobic core blocks including, poly(beta benzyl aspartate), polycaprolactone, poly(gamma-benzyl-L glutamate), polylactide, polylysine, polypropylene oxide and polybutylene oxide. These micelle-like systems each have distinct physicochemical characteristics, and the uniqueness of the system therefore largely originates from the varied hydrophobic blocks employed (10).

For effective use in drug delivery systems, microparticles and nanoparticles need to incorporate a high drug loading, need to be stable to aggregation when loaded, and need to be able to release the drug under appropriate conditions. Drug release may be designed to occur either in response to a stimulus, or in a time dependent manner as a depot formulation.

Much work on nanoparticles as drug delivery systems has been carried out on PLA-PEG and PLGA-PEG nanoparticles. However, in general, it has proven difficult to achieve high drug loading with these materials (11 ,12), and drug release tends to occur rapidly, with a large initial burst effect, with most drug being released in a relatively short time period. Materials such as PLA- PEG have been favoured for this work because they are approved for pharmaceutical use. However, the physicochemical parameters which can be adjusted to enhance drug incorporation and the control of drug release are limited.

Ideally, to produce drug delivery systems which can be used effectively in a wide variety of applications, the core should either be capable of retaining a variety of drugs, or be capable of being made with several different properties to accommodate drugs with a variety of different physicochemical properties. Consideration also needs to be given to fine tuning core-drug interactions so that release of the drug can be adequately controlled.

One way to achieve these aims is to use a biodegradable polymer with chemical functional groups which are readily modified with a variety of chemical structures. A variety of natural biomolecules exist which fit this description, such as polysaccharides (e.g. pullulan, dextran, chitin) and polyamino acids (e.g. poly-L-lysine, poly-L ornithine). Several of these biomaterials have been modified with substituents to produce nanoparticles in the form of either micelles or vesicles (13-16). However, although these are biological materials, they are not necessarily readily degradable in animal tissues, (e.g. dextran) or the functional groups may result in undesirable toxicity (e.g. poly-L-lysine, poly-L ornithine) (17). It has been recognised for many years that chemically synthesised biodegradable polymers with functional groups would be useful in drug delivery applications. Chemically synthesised biodegradable funtionalised polymers have also been prepared and used for preparation of micelles. This has been achieved by acylation of the backbone to provide a hydrophobised interior into which low levels of drug incorporation have been achieved (18,19). However, chemical synthesis of biodegradable polymers with functional groups can be difficult to achieve because useful functional groups are liable to participate in the polymerisation reaction. Such functional groups may be difficult to protect chemically in a way where deprotection is possible without hydrolysing the biodegradable polymer (20,21).

An exceptional synthesis of a functional polymer backbone has, however, been described in the literature. This is a polymer produced from biological components, a diacid and a polyol condensed in the presence of an enzyme, as a catalyst, to give a linear polyester (22). This enzyme-catalysed condensation is a single step reaction in comparison with the multiple step synthesis required for a similar chemical polymerisation. The diacid is usually used as the divinyl derivative to act as a leaving group and facilitate polymer formation (23). When the polyol is glycerol, this leaves a single pendant hydroxyl functional group per repeating unit which can be further modified to impart more and varied desirable properties to the polymer. Polyesters are naturally biodegradable by a hydrolysis mechanism.

The present invention is based on the discovery that microparticles and nanoparticles of a linear polyester having a polymer backbone containing aliphatic dicarboxylate residues and the residues of an aliphatic polyol have use as carriers for pharmaceutically-active agents in drug delivery systems.

Accordingly, the present invention provides a nano or microparticle drug delivery system comprising a pharmaceutically-active agent and nano or microparticles of a linear aliphatic polyester, the polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with the pharmaceutically-active agent.

The present invention also provides a method for preparing a nano or microparticle drug delivery system which method comprises providing nano or microparticles of a linear aliphatic polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with a pharmaceutically-active agent, and entrapping a pharmaceutically-active agent within the nano or microparticles of the linear polyester.

The present invention further provides a method of delivering a pharmaceutically-active agent to an animal, including a human, which comprises administering a nano or microparticle drug delivery system comprising a pharmaceutically-active agent and nano or microparticles of a linear polyester, the polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with the pharmaceutically-active agent.

The polyester used as the carrier in the drug delivery system for a pharmaceutically-active agent is a linear aliphatic polyester which has a polymer backbone comprising aliphatic dicarboxylate residues and the residues of an aliphatic polyol and wherein the backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with a pharmaceutically-active agent. Typically, the aliphatic dicarboxylate residues are those which may be derived from an aliphatic dicarboxylic acid having the formula I

HOOC R¹ COOH (|) wherein R¹ is a straight chain or a branched chain 1 to 12C alkylene group. Preferably, the group R¹ above is a straight chain alkylene group generally defined as -(CH₂)-_n, where n is 1 to 8. Examples of such straight chain alkylene groups include methylene, ethylene, propylene, butylene, hexylene and octylene groups. The length of the alkylene chain between the two carboxylate groups in the dicarboxylate residue has an effect on the hydrophobicity of the polyester and, of course, on the separation between one functional group and another functional group on the polymer backbone. Preferably, the two carboxylate groups in the dicarboxylate residue are separated by an alkylene group selected from methylene, ethylene, propylene_. and butylene. A particularly preferred dicarboxylic acid for use in the manufacture of the linear polyester is adipic acid.

The polymer backbone of the linear aliphatic polyester molecule comprises residues derived from at least one aliphatic polyol, i.e. polyhydric alcohol, containing at least three hydroxyl groups. The use of such polyols results in a polymer backbone provided with pendant hydroxyl groups which may be derivatised or substituted to provide pendant moieties capable of interacting with a pharmaceutically-active agent. Typically, the aliphatic polyols from which the residues in the polyester backbone are derived will have the general formula II R —(OH)_m (ii)

wherein R² is a straight or branched chain hydrocarbyl group having from 3 to 8 carbon atoms and m is 3 to 8. Preferably, the aliphatic polyol from which the residues in the polyester backbone are derived will have the general formula III

where p is 1 to 4. Such polyols according to the general formula III include glycerol, mesoerythritol and linear pentitols and hexitols. Preferably, the aliphatic polyol according to general formula III above will be one selected from glycerol, mesoerythritol, xylitol, mannitol and sorbitol. Glycerol is especially preferred as the aliphatic polyol.

As is stated above, it is essential that the polymer backbone of the linear aliphatic polyester contains at least one residue of an aliphatic polyol which residue contains at least one moiety capable of interacting with a pharmaceutically-active agent. Thus, the polymer backbone of the linear aliphatic polyester will contain at least one aliphatic polyol residue wherein at least one pendant hydroxyl group is preferably derivatised or substituted to produce a moiety which is capable of. interacting with a pharmaceutically- active agent. In certain circumstances, however, a pendant hydroxyl group which is not derivatised or substituted may, itself, be capable of interacting with a pharmaceutically-active agent.

By the expression "moiety capable of interacting with a pharmaceutically- active agent" as used herein, what is meant is a chemical entity attached to the polymer backbone which is able to engage a^■ pharmaceutically-active agent by means of one or more molecular interactions (i.e. non-covalent bonds) such that a pharmaceutically-active agent may be entrapped in nano or microparticles of the linear polyester. Examples of such interactions include electrostatic bonds, hydrogen bonds, van der Waals interactions and hydrophobic interactions. Thus, it is an essential feature of the invention that the polymer backbone includes at least one residue of an aliphatic polyol which contains a moiety that is capable of promoting at least one molecular interaction with the pharmaceutically-active agent.

A moiety capable of interacting with a pharmaceutically-active agent may, as described above, be an unmodified or unsubstituted hydroxyl group but preferably is provided on the polymer backbone of the polyester by derivatising or substituting a pendant hydroxyl group of one of the aliphatic polyol residues using an appropriate chemical reagent to react with the hydroxyl group. An example of a chemical reagent that will react with a pendant hydroxyl group is an activated acyl-containing compound. The reaction between such an activated acyl-containing compound and a pendant hydroxyl group of an aliphatic polyol residue in the polyester backbone results in a pendant group attached to an aliphatic polyol residue via an oxyacyl linkage. One example, which relates to a preferred embodiment of the present invention, is the provision of at least one pendant group attached to the polyester polymer backbone by an oxycarbonyl linkage. Such a pendant group may be produced by acylating a pendant hydroxyl group attached to the polymer backbone using an appropriate activated carboxylic acid or derivative thereof. Preferably, the moiety capable of interacting with a pharmaceutically- active agent is an optionally-substituted alkyl, alkenyl or alkynyl group formed by acylating a pendant hydroxyl group with an optionally-substituted alkyl, alkenyl or alkynyl carboxylic acid chloride. Preferably, the moiety capable of interacting with a pharmaceutically-active agent is a pendant group, attached to an aliphatic polyol residue in the polymer backbone, having the general formula IV

O

-O-CR³ (|V)

wherein R³ is an optionally-substituted 1 to 20C straight or branched chain alkyl group or is an optionally-substituted 2 to 20C straight or branched chain alkenyl or alkynyl group. More preferably, R³ is an optionally-substituted 1 to 18C straight or branched chain alkyl group or an optionally-substituted 2 to 18C alkenyl or alkynyl group. Particularly preferred is when R³ is an optionally-substituted straight chain 2 to 18C alkyl group.

Such optionally-substituted alkyl, alkenyl or alkynyl groups, for R³, may be substituted by one or more groups which may additionally promote a molecular interaction with a pharmaceutically-active agent. Typical substituent groups, for this purpose, include the groups -OR⁴, -NR⁵R⁶, -COOR⁷ and SO₂OR⁷ where each of R⁴, R⁵ and R⁶ is, independently, selected from H and 1 to 6C alkyl which alkyl may optionally be substituted by -OR⁷, -NR⁸R⁹, -COOH and -SO₃H; and wherein each of R⁷, R⁸ and R⁹ is independently selected from H and 1 to 6C alkyl groups.

The moiety capable of interacting with a pharmaceutically-active agent may comprise a heterocyclic or aromatic ring structure.

According to one preferred embodiment, the moiety capable of interacting with a pharmaceutically-active agent is selected to be hydrophobic. If it is desired to use, in the present invention, a linear aliphatic polyester having pendant hydrophobic moieties, it is preferred that the moieties have the general formula IV above wherein R³ is a long chain, typically, 8 to 18C alkyl group which is unsubstituted. If the alkyl is substituted, the substituents should not, in this particular case, substantially increase the hydrophilicity of the group. The choice of moiety used will, of course, depend on the type of, and chemical character of, the pharmaceutically-active agent to be used in the nano or microparticle drug delivery system of the invention. Thus, in some cases, it will be necessary to provide pendant moieties on the polyester backbone which themselves contain charged groups or ionisable groups. For instance, moieties containing groups which are, or can be made, positively charged, for instance tertiary amine groups, are advantageous in the case where the pharmaceutically-active agent is, or comprises, an oligonucleotide or DNA polyelectrolyte complex. The moieties may have other characteristics which will facilitate interactions between the core of the nano or microparticle and the pharmaceutically-active agent, e.g. amino acids which are acetylated on the alpha amino group, or desamino analogs of amino acids.

According to a different embodiment, the polymer backbone of the linear aliphatic polyester includes at least one aliphatic polyol residue containing a molecule of a pharmaceutically-active agent which can further interact with other molecules of pharmaceutically-active agents. An example of such an embodiment is when one or more molecules of a pharmaceutically-active agent are conjugated to a polymer, such as a poly(amino acid), which is then attached to the polymer backbone of the linear aliphatic polyester via one or more pendant hydroxyl groups. An attachment in this way creates a hydrophobic segment in the polymer which enables the formation of particles which tend to be micelle-type particles. The pharmaceutically-active agent thus conjugated to the polymer backbone is capable of interacting with non- covalently attached pharmaceutically-active agent.

The proportion of pendant hydroxyl groups on the polyester polymer backbone that are derivatised or substituted, as described above, to produce moieties capable of interaction with a pharmaceutically-active agent is in the range of from 0.1 to 1.0. In some cases, however, the presence of pendant hydroxyl groups (non-derivatised or non-substituted hydroxyl groups) on the polymer backbone may directly interact with a pharmaceutically-active agent or may enhance the amount of water that is included into the core of the nanoparticles into which the pharmaceutically-active agent can dissolve.

In addition to the residues of aliphatic polyols providing, in the polymer backbone, pendant hydroxyl groups and/or derivatised or substituted hydroxyl groups, the polymer backbone of the linear aliphatic polyester may also contain the residues of one or more alkylene diols, for example polymethylene glycols such as ethylene glycol, propan-1,3-diol and butan-1,4-diol.

According to a preferred embodiment, the polymer backbone of the linear aliphatic polyester is provided with at least one pendant group containing the residue of hydrophilic polymer to produce a copolymer having a hydrophilic segment which can form a sterically stabilising layer on the nano or microparticles. As mentioned previously, it has been found that nanoparticles provided with a surface layer of PEG have a prolonged circulation in the bloodstream, i.e. a reduced uptake by the RES, because the hydrophilic surface of PEG prevents the adsorption of opsonins and, thus, prevents the recognition of the nanoparticles, by the RES, as foreign particles. Therefore, particularly when the drug delivery system of the present invention is intended for parenteral administration, it is preferred that the polymer backbone of the polyester contains at least one residue of an aliphatic polyol to which is attached a PEG group. Attachment of a PEG group may conveniently be achieved by linking it to a pendant hydroxyl group of the polymer backbone, for instance by means of a dicarboxylate link. In general, from 1 to 90% of pendant hydroxyl groups on the polymer backbone may be attached to PEG groups to provide nanoparticles having a PEG surface layer. PEG which may be attached to the polyester backbone in carrying out the present invention will typically have a molecular weight in the range of from 500 to 20,000.

Linear polyesters with pendant hydroxyl groups for use in the present invention may be prepared, as described above (22,23), by reacting an activated diester of an aliphatic dicarboxylic acid, such as the divinyl ester, with an aliphatic polyol containing at least three hydroxyl groups in the presence of a catalytic amount of a lipase, immobilised on a support, at a temperature typically higher than 30°C. An amount of an aliphatic glycol may be used in addition to the aliphatic polyol containing at least three hydroxyl groups to make up the polyol component of the reaction mixture. If a glycol is used, the ratio of glycol to polyol containing at least three hydroxyl groups will, of course, have an effect not only on the hydrophilicity of the polymer backbone of the resulting polyester but also on the total number of pendant hydroxyl groups on the resulting polymer backbone and, therefore, on the total possible number of pendant groups that are capable of interacting with a pharmaceutically-active agent, as described above. The total amount of polyol or polyol-glycol component in the reaction mixture will typically be such as to provide an equimolar amount with respect to the activated diester used.

The reaction is, as mentioned above, carried out in the presence of a catalytic amount of a lipase. Examples of lipases that can be used in the enzyme-catalysed condensation reaction include lipase isolated from Candida antarctica (available under the trade name Novozym 435), Candida rugosa, Pseudomonas fluorescens and Mucor miehei (available under the trade name Lipozyme IM).

The reaction is preferably carried out in the presence of an organic solvent, such as tetrahydrofuran or acetonitrile, to facilitate mixing of the monomers and to keep the forming oligomers in solution, thus allowing the polymerisation reaction to continue. Upon completion of the condensation reaction, the resulting polymer may be separated from the immobilised enzyme by filtration and then washed with solvent prior to removal of the solvent, typically by evaporation.

The pendant hydroxyl groups on the polymer backbone of the linear polyester may then, if desired, be treated with appropriate reactants to produce pendant moieties that are capable of interacting with a pharmaceutically-active agent. For instance, if pendant hydroxyl groups are to be acylated, i.e. esterified, the linear polymer containing the pendant hydroxyl groups may be treated with the appropriate aliphatic carboxylic acid chloride, typically in the presence of pyridine as a catalyst and acid scavenger. The appropriate aliphatic carboxylic acid chloride may conveniently be prepared by the reaction of the parent carboxylic acid with thionyl chloride.

In the preferred embodiment of the invention described above where the polymer backbone of the linear polyester is attached to a hydrophilic polymer group, such as PEG, the hydrophilic polymer group may be attached to the linear polyester backbone by a reaction carried out prior to or subsequent to the acylation reaction using the carboxylic acid chloride described above. PEG groups may be attached to the polymer backbone by first reacting the PEG, for instance as monomethyl ether, with succinic anhydride to form the half ester (24), converting the terminal acid group of the half ester to the acid chloride by reaction with thionyl chloride, after azeotropic drying (25), and then acylating one or more pendant hydroxyl groups on the linear polyester backbone using this acid chloride. Nano or microparticles of the linear polyester may be formed according to the prior art procedures (26,27). Typically, a solution of the polymer in an organic solvent, such as acetone, is added into an excess of water with stirring. After the organic solvent has been allowed to evaporate, the produced particles can be filtered. The particles may then be cleaned by gel filtration through a sepharose column using water as eluant. As used herein, the term "nanoparticles" means particles having an average particle size up to, but not including, 1000 nm. The term "microparticles", as used herein, means particles having an average particle size equal to or greater than 1000 nm, typically up to about 50 μm.

The nano or microparticle drug delivery system is prepared by entrapping a pharmaceutically-active agent within the nano or microparticles of the linear polyester. This may be achieved, for instance, by dispersing the particles of the polymer carrier in an aqueous solution of the pharmaceutically-active agent.

A pharmaceutically-active agent which may be entrapped in the nano or microparticles of the linear polyester described above is any substance, natural or synthetic, which has a physiological action on a living body, for example a natural or synthetic drug, a protein or a nucleic acid which has a therapeutic effect on the living body.

If it is desired to include a surfactant into the nano or microparticles, the surfactant may be incorporated into the aqueous phase containing the pharmaceutically-active agent into which the particles of the linear polyester are dispersed.

The present invention also provides a method of delivering a pharmaceutically-active agent to an animal, including a human, which comprises administering the nano or microparticle drug delivery system described above to the animal body. The drug delivery system may be formulated according to methods known in the art. Typically, the system will be formulated for administration to a patient parenterally in water or saline solution, although formulations for administration to a patient by other means is possible. EXPERIMENTAL

Typical procedure for synthesis of polymers for nanoparticle formulation

Synthesis of 2% PEGylated (2000) - 40% acylated (C18) - polyester (M_w ~10kDa)

Introduction

The synthesis was carried out in three steps - backbone formation, acylation, and PEGylation - each of these shall be discussed separately below. The figures in brackets relate to the molecular weight of the original mPEG (550, 2000, or 5000), before activation, the chain length of the linear aliphatic acid chloride used (C8, C14, C16, or C18), or the approximate molecular weight of the parent backbone polyester as determined by reference to polystyrene standards. The order of the steps is important as the percentage values were calculated depending on the average unit molecular weight.

Materials and Methods

The enzyme Novozyme 435 (a lipase derived from Candida antarctica and immobilised on an acrylic macroporous resin), was stored over P₂O₅ at 5°C prior to use. Divinyl adipate was purchased from Flurochem UK, and used as received. Glycerol, polyethylene glycol methyl ether (mPEG) (M_n = 550, 750, 2000 and 5000), alditols (meso-erythritol, xylitol, D-mannitol and D-sorbitol), succinic anhydride, and anhydrous benzene were obtained from Aldrich and used as received. Acid chlorides (capryloyl, myristoyl, palmitoyl, and stearoyl) were purchased from Aldrich, or synthesised, by standard procedures, from the parent acid using thionyl chloride, and distilled prior to use. Chloroform, dichloromethane (DCM), diethyl ether, pyridine and tetrahydrofuran (THF) were obtained from Merck. THF was freshly distilled from sodium/benzophenone ketyl prior to use. Reactions requiring anhydrous conditions were conducted in oven-dried (130°C) glassware. Mechanical stirring was accomplished using a standard mechanical mixer at 200rpm, using a Teflon paddle. The activated mPEG was prepared in two steps. Firstly reaction of the monomethyl polyethylene glycol with succinic anhydride formed the half ester. Azeotropic drying, using anhydrous benzene, followed by conversion of the acid end group to an acid chloride, using thionyl chloride, yielded the activated mPEG. The NMR Spectra were recorded on a Bruker DPX 250MHz spectrometer and are expressed in parts per million (3) from internal tetramethylsilane. Gel Permeation Chromatograph (GPC) were recorded on a Polymer Labs system, using 2 mixed bed columns at 40°C, flow rate 1ml/min in THF, using an evaporative light scattering detector calibrated with ten narrow polystyrene standards.

Synthesis

Backbone - Synthesis of polyester (M_w ~10kDa)

Synthesis was carried out using a large water bath to maintain constant temperature (50°C), and a mechanical stirrer (200 rpm), using a paddle type stirrer blade. Thus an oven dried 500ml three-necked RB flask, equipped with centre stirrer guide, and an open top condenser - to act as an outlet for the acetaldehyde produced, was charged with divinyl adipate (108.82g, 0.549moles), and glycerol (50.53g, 0.549moles), 4A molecular sieves (2g), and 200ml anhydrous THF. This mixture was stirred for 30 mins to allow reactants to warm to the water bath temperature (50°C). The enzyme complex (1.75g) was then added and the mixture stirred for 24 hrs (the reaction started after 10 mins as noticed by bubble formation). The enzyme and sieves were removed by filtration, washing with 50ml THF. The solvent was removed by rotary evaporation (bath set at 50°C) to yield the polyester as a colourless viscous liquid, weight 104.19g (M_w = 12554Da). Analysis of the polyester by NMR and MALDI-TOF confirmed the linearity of the polyester (due to the inherent selectivity of the enzyme). The presence of free hydroxyl groups was confirmed by FT-IR and indirect titration (22). Acylation step - Synthesis of 40% acylated (C18) - polyester (M_w~10kDa)

The percentage acylation was calculated using the repeat unit size of the backbone polyester (Mn = 202.21 g), thus a 250ml three-necked RB, fitted with a condenser and dropping funnel, was charged with polyester (20.48g, 101.28mmoles) and 100ml THF. The mixture was warmed to reflux to dissolve the polyester and stearoyl chloride (12.27g, 40.50mmoles) was added. Pyridine (20ml) was added dropwise, producing acid fumes and a white precipitate as the reaction proceeds. The mixture was refluxed 2 hrs, and then poured onto 100ml 2M HCI, followed by extraction with 3x100ml DCM, washing 2x100ml water, dried and the solvent removed. The title product was collected, 30.21 g, as a white waxy solid. The level of acylation was confirmed by GPC, NMR, or by calculating the change in hydroxyl content using the indirect titration method.

PEGylation step - Synthesis of 2% PEGylated (2000) - 40% acylated (C18) - polyester (M_w~10kDa)

Reaction was carried out in an identical manner to the acylation step. Thus the reaction of 40% acylated (C18) - polyester (Mn = 308.81 g) (3.04g, 9.84mmoles) and activated mPEG (2000) (0.42g, 0.198mmoles) yielded the title product, 2.98g, as a white waxy solid.

Particle formation using Acylated poly(glycerol-adipate) and PEGylated, acylated poly(glyeerol-adipate)

Nanoparticles were produced by the interfacial polymer deposition method (27,28). Polymer (4mg/ml, 20mg) was dissolved in acetone, and the dissolved polymer was added dropwise into an excess of water (15ml) under stirring. After addition of the polymer, the particle solution continued to be stirred overnight to allow evaporation of the solvent. Particles were filtered to remove aggregated material and then cleaned by gel filtration through a sepharose 4B column (2.5 x 30cm) using water as the eluent. Incorporation of drug or surfactant

Hydrophilic drug, dexamethasone phosphate, was loaded into the nanoparticles by dissolution into the water (0-12.0mg) to which the polymer was added. Similarly to incorporate tween into the nanoparticles, this was also included in the aqueous phase (0.1-0.4% v/v) prior to polymer addition.

Particle Characteristics

Particle preparation was carried out with a range of polymers with different characteristics. Polymers were acylated with either C8 or C18 groups following the procedure described above at 20, 40, 60 and 80% acylation of hydroxyl groups. These polymer specifications were available either with or without PEG 2000 attached at 2% hydroxyl group substitution. Stable particles were formed by this method by polymers both with and without PEG in the absence of either drug or surfactant. This showed that the acyl groups provided sufficient interaction to cause particle formation, and also that the remaining pendant hydroxyl groups and terminal groups (either hydroxyl or carboxyl) were sufficient to stabilise the particles. Particle sizes were in the range 150-240nm diameter as measured by photon correlation spectroscopy. These particle properties are very different from those reported by other groups where polymers with pendant chemical functionality were modified with acyl groups (13,18,19). In this previously reported work, small (~20nm) micelle-like aggregates were obtained. In another formulation of chitosan it was reported that particles were not formed in the absence of cholesterol (14). These chitosan particles were made by a technique similar to that used to form liposomes. Using this method of particle formation with acylated polyglycerol-adipate polymers does not result in the production of vesicles. In yet another formulation where Poly-l-lysine was substituted with both PEG and acyl groups, particle formation was again achieved by liposome formation methods, not by use of an interfacial deposition technique (15,16). Particle size increased with increasing substitution of hydroxyl groups by acyl moieties for both non-PEGylated and PEGylated particles. Particle sizes were decreased in the PEGylated particles compared to equivalent non- PEGylated particles. The results are shown in the following Tables 1 and 2. In Tables 1 and 2, the figures given for the particle size (diameter) in rounded brackets represent standard deviation (S.D.) and the figures in square brackets represent the polydispersity, a measure of the size distribution. The figures given in rounded brackets for Z-potential represent standard deviation (S.D.).

TABLE 1

TABLE 2

Drug loading was assessed using a hydrophiiic drug dexamethasone phosphate. Drug loading varied in a non-linear way with changes in the degree of substitution by acyl groups. The presence of drug resulted in a decrease in particle size suggesting that the presence of drug increased the interactions in the core of the nanoparticle. A graph showing the change in drug loading with increased amount of drug is shown in Fig. 1 and the change in drug concentration with acyl substitution is shown in Table 3. TABLE 3

Amounts of drug incorporated reached a maximum of 31% of input drug loaded and drug entrapment of 11.45% w/w drug in particles entrapped. The entrapment of such a high proportion of hydrophilic drug compares very favourably with that reported for PLA-PEG particles, and suggests that both the hydrophobic moieties and the hydrophilic drug groups may play a part in enhancing drug incorporation. The variation in properties with polymer specification also confirms the expectation that flexibility in polymer specification will lead to more effective drug delivery systems.

In a further investigation, the drug loading in a delivery system according to the present invention was studied using, as drug models, a range of bases and nucleotides. These can be viewed as models of the closely-related cytotoxic drugs 5-fluorouracil, 5-fluorouridine and cytosine arabinoside. The study also provides an insight into the effect of drug structure on incorporation into the nanoparticles. The investigation used acylated polyesters, prepared as described above, with 80% acylation by Cι₈ groups (80% C₁₈ (4kDa) ) or with 40% acylation by Cι₈ groups (40% C₁₈ (10kDa) ). Using 20mg polymer and 4mg drug/base, the particle size, zeta potential and actual drug loadings were determined and these are set out in the following Table 4. TABLE 4

The results in Table 4 show several interesting features. Firstly, it is apparent that the relatively-hydrophilic drug moiecules incorporate well into nanoparticles of the linear aliphatic polyester. However, the drug incorporation varies both with differences in drug structure and with differences in polymer formulation. In general, the addition of a phosphate group to_. the drug vastly enhances drug incorporation (comparing uridine with uridine monophosphate and adenosine with adenosine monophosphate) but this is not entirely related to solubility since the solubilities of uridine and uridine monophosphate are very similar. For the dexamethasone and dexamethasone phosphate the steroid nucleus would be expected to interact well with the alkyl chains, and it is thought to be the water solubility of the phosphate group which allows a good incorporation using the interfacial deposition technique. However, the heterocycles of the nucleoside drugs are not particularly hydrophobic and, thus, are not expected to be predisposed to interact with the alkyl chains. The interactions are more likely to be hydrogen bonding with the free hydroxyl groups.

Overall, these result suggest that a wide range of drugs are likely to incorporate well with these polymers and that the extent of drug incorporation can be greatly influenced by the polymer structure. Furthermore, this is further evidence that unmodified hydroxyl groups in the polymer may also be important in interacting with the drugs to give a good incorporation.

REFERENCES

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Claims

1. A nano or microparticle drug delivery system comprising a pharmaceutically-active agent and nano or microparticles of a linear aliphatic polyester, the polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with the pharmaceutically-active agent.

2. A system according to claim 1 , wherein the polymer backbone of the linear aliphatic polyester includes at least one aliphatic polyol residue containing a moiety capable of interacting with a molecule of the pharmaceutically-active agent by at least one molecular interaction selected from hydrophobic interactions, van der Waals forces, hydrogen bonding and ionic bonding.

3. A system according to either claim 1 or claim 2, wherein the polymer backbone of the linear aliphatic polyester comprises aliphatic dicarboxylate residues derived from a dicarboxylic acid having the formula I

HOOC - R - COOH (I)

wherein R¹ is a straight chain or branched chain 1 to 12C alkylene group.

4. A system according to claim 3, wherein R¹ is -(CH₂)-_n, where n is 1 to 8.

5. A system according to claim 4 where n = 1 to 4.

A system according to any one of claims 1 to 5, wherein the polymer backbone of the polyester comprises residues derived from an aliphatic polyol having the formula II

wherein R² is a straight or branched chain hydrocarbyl group having from 3 to 8 carbon atoms and m is 3 to 8.

7. A system according to claim 6, wherein the aliphatic polyol is one having the general formula III

where p is 1 to 4.

8. A system according to claim 7, wherein the aliphatic polyol is selected from glycerol, mesoerythritol, xylitol, mannitol and sorbitol.

9. A system according to any one of claims 1 to 8, wherein the polymer backbone of the linear aliphatic polyester includes at least one residue of an aliphatic polyol which contains at least one pendant group having the general formula IV

O

— O-CR^J (|V)

wherein R³ is an optionally-substituted 1 to 20C straight or branched chain alkyl group or is an optionally-substituted 2 to 20C straight or branched chain alkenyl or alkynyl group.

10. A system according to claim 9, wherein the R³ group is optionally substituted by one or more substituents selected from -OR⁴, -NR⁵R⁶, -COOR⁷ and -SO₂OR⁷, wherein each of R⁴, R⁵ and R⁶ is, independently, selected from H and 1 to 6C alkyl which may be substituted by -OR⁷, -NR⁸R⁹, -COOH and -S0₃H; each of R⁷, R⁸ and R⁹ is independently selected from H and 1 to 6C alkyl.

11. A system according to any one of claims 1 to 10, wherein the polymer backbone of the linear aliphatic polyester includes at least one residue of an aliphatic polyol which contains one or more moieties comprising a heterocyclic or aromatic ring structure.

12. A system according to any one of claims 1 to 10, wherein the polymer backbone of the linear aliphatic polyester includes at least one residue of an aliphatic polyol which contains one or more moieties capable of interacting with a pharmaceutically-active agent, which moieties are hydrophobic.

13. A system according to any one of claims 1 to 10, wherein the polymer backbone of the linear aliphatic polyester includes at least one residue of an aliphatic polyol which contains one or more moieties capable of interacting with a pharmaceutically-active agent, which moieties are positively charged.

14. A system according to any one of claims 1 to 13, wherein the polymer backbone of the linear aliphatic polyester includes at least one aliphatic polyol residue to which is attached directly or through a linker group a hydrophilic polymer.

15. A system according to claim 14, wherein the hydrophilic polymer is a poly(ethylene glycol) residue.

16. A system according to claim 15, wherein the poly(ethylene glycol) residue has a molecular weight in the range of from 500 to 20,000.

17. A system according to any one of claims 1 to 16, wherein the pharmaceutically-active agent is a natural or synthetic drug, a protein or a nucleic acid.

18. A system according to any one of claims 1 to 17, wherein the average particle size of the drug delivery system is up to 1000 nm.

19. A system according to any one of claims 1 to 13, which additionally contains a surfactant.

20. A system according to claim 1 , wherein the polymer backbone of the linear aliphatic polyester includes at least one aliphatic polyol residue containing a molecule of a pharmaceutically-active agent which interacts with other molecules of pharmaceutically-active agents.

21. A method for preparing a nano or microparticle drug delivery system which method comprises providing nano or microparticles of a linear aliphatic polyester having a polymer backbone containing aliphatic dicarboxylate residues and residues of an aliphatic polyol wherein the polymer backbone includes at least one aliphatic polyol residue containing a moiety capable of interacting with a pharmaceutically- active agent, and entrapping a pharmaceutically-active agent within the nano or microparticles of the linear polyester.

22. A method of delivering a pharmaceutically-active agent to an animal, including a human, which comprises administering to the animal nano or microparticle drug delivery system of any one of claims 1 to 20.