WO2008149096A2 - Polymeric microparticles - Google Patents

Abstract

The invention provides a microparticle which comprises a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound encapsulated in said polymer. The invention further provides a coated microparticle comprising (i) a core, which core comprises a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and (ii) an outer layer which comprises calcium phosphate. The invention further relates to processes for producing the microparticles and coated microparticles; substrates, implants, tissue engineering scaffolds and injectable formulations comprising the coated microparticles; and uses of the microparticles, coated microparticles, substrates, implants, tissue engineering scaffolds and injectable formulations as drug delivery agents and in the treatment of conditions such as bone disorders.

Description

POLYMERIC MICROPARTICLES

FIELD OF THE INVENTION

The present invention relates to microparticles, coated microparticles, processes for producing the microparticles and coated microparticles, and substrates, implants, tissue engineering scaffolds and injectable formulations which comprise the coated microparticles. The invention further relates to uses of the microparticles, coated microparticles, substrates, implants, tissue engineering scaffolds and injectable formulations as drug delivery agents and in the treatment of bone disorders.

BACKGROUND TO THE INVENTION

There is an ongoing requirement to develop improved drug delivery systems. In particular, there is a need to develop drug delivery systems which can be administered locally in order to treat bone disorders. Hydroxyapatite (HA), a form of calcium phosphate having the formula

Caio(P0₄)₆(OH)₂, is particularly useful in bone replacement and reconstruction because of its osteoconductivity, similarity with bone mineral, and bone-bonding ability. Porous monolithic ceramics based on hydroxyapatite (HA) have been shown to aid in osteoconduction of bone when implanted into a bony defect. HA is therefore a desirable material to use in the construction of drug delivery systems and/or implants designed to treat bone disorders.

Due to its poor mechanical properties, HA is often applied as a coating to other materials. A common method of coating is by immersion in simulated body fluid (SBF). However, this method can take from several days to several weeks to provide uniform and thick coatings and can be detrimental to the material being coated. If the material to be coated is a biodegradable polymer, for instance, such long periods of immersion in aqueous solution can change the polymer properties (for instance the compressive modulus, surface and matrix composition) due to polymer degradation. Furthermore, if that polymer contains a drug or other bioactive agent, the drug or agent is likely to leach out of the polymer under such conditions.

The preparation of drug delivery devices by the entrapment of a drug into polymeric microspheres is known. However, coating such microspheres with HA is likely to result in poor drug loadings and poor drug-encapsulation efficiencies, due to limitations in the nature of coating methods and microspheres used. The present invention addresses these and other problems.

SUMMARY OF THE INVENTION

The present inventors have developed a method of preparing polymeric microparticles which comprise an anionic surfactant and contain a relatively high level of a drug. This is due to the high drug encapsulation efficiency associated with the method. Advantageously, the microparticles can be quickly and easily coated with calcium phosphate, in a coating process which minimises drug-loss. Thus, both the uncoated and coated microparticles are able to contain a high level of drug, which is capable of slow release from the microparticles. The microparticles are therefore potentially useful as drug delivery agents for use in medicine and, more particularly, for use in the treatment of bone disorders or in the manufacture of medically acceptable implants, including bone implants.

In one aspect, the invention provides a microparticle which comprises: a polymer; an anionic surfactant; and a hydrophilic pharmaceutically active compound encapsulated in said polymer.

Usually the microparticle is negatively charged. It may, for instance, have a zeta-potential of less than -25.0 mV.

Typically, the encapsulation efficiency of said pharmaceutically active compound in said polymer is at least 10 %.

Typically, the pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the microparticle. Thus, in another aspect, the invention provides a microparticle which comprises: a polymer; an anionic surfactant; and a hydrophilic pharmaceutically active compound encapsulated in said polymer, which pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the microparticle.

In another aspect, the invention provides a process for producing a microparticle, which microparticle comprises: a polymer; an anionic surfactant; and a hydrophilic pharmaceutically active compound encapsulated in said polymer; which process comprises: (a) preparing a solid-in-oil-in-water emulsion, which emulsion comprises: (i) a solid phase which comprises a hydrophilic pharmaceutically active compound, (ii) an oil phase which comprises a polymer and a solvent which is immiscible with water, and (iii) an external aqueous phase which comprises water and an anionic surfactant; and

(b) evaporating said solvent which is immiscible with water to produce said microparticle.

Typically, step (b) results in the production of said microparticle in aqueous suspension. Typically, the microparticle is negatively charged.

The invention further provides a microparticle which is produced by the process of the invention, as defined above, for producing a microparticle.

Calcium phosphate may be coated on to the surface of the drug-loaded polymer microparticles which are typically, but not necessarily, negatively charged. The resulting coated particles can act as drug-delivery devices; they are capable of a high level of drug-entrapment coupled with sustained drug release over at least three days, for instance over at least one month.

Accordingly, in another aspect, the invention provides a coated microparticle comprising: (i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound which is encapsulated in said polymer; and (ii) an outer layer which comprises calcium phosphate.

Usually, the encapsulation efficiency of said pharmaceutically active compound in said polymer is at least 10 %. Typically, the pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the core. More typically, the pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the coated microparticle.

In another aspect, the invention provides a coated microparticle comprising: (i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer and is present in an amount of at least 1.0 weight % based on the total weight of the core; and

(ii) an outer layer which comprises calcium phosphate. In another aspect, the invention provides a process for producing a coated microparticle, which coated microparticle comprises:

(i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and (ii) an outer layer which comprises calcium phosphate; which process comprises:

(A) producing a microparticle by the process of the invention, as defined above, for producing a microparticle; and

(B) coating the microparticle by contacting it with a solution comprising calcium and phosphate ions.

Thus, the invention provides a process for producing a coated microparticle, which coated microparticle comprises:

(i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and (ii) an outer layer which comprises calcium phosphate; which process comprises: (A) producing a microparticle, which microparticle comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound encapsulated in said polymer, by a process which comprises:

(a) preparing a solid-in-oil-in-water emulsion, which emulsion comprises:

(i) a solid phase which comprises a hydrophilic pharmaceutically active compound,

(ii) an oil phase which comprises a polymer and a solvent which is immiscible with water, and (iii) an external aqueous phase which comprises water and an anionic surfactant; and

(b) evaporating said solvent which is immiscible with water to produce said microparticle; and (B) coating the microparticle by contacting it with a solution comprising calcium and phosphate ions.

Typically, the microparticle produced in step (A) is negatively charged. Typically, the solution comprising calcium and phosphate ions is an aqueous solution. Typically, the microparticle is contacted with the solution comprising calcium and phosphate ions for no more than 10 hours. More typically, said negatively-charged microparticle is contacted with the solution for from 3 to 8 hours, depending on the thickness of the coating required.

The outer layer of the coated microparticle may further comprise another pharmaceutically active compound.

Thus, in another aspect, the invention provides a coated microparticle comprising:

(i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and

(ii) an outer layer which comprises calcium phosphate and a further pharmaceutically active compound.

The process of the invention for producing a coated microparticle, as defined above, may further comprise: (C) incorporating a further pharmaceutically active compound into said outer layer.

The invention further provides a coated microparticle which is produced by the process of the invention, as defined above, for producing a coated microparticle.

The coated microparticles of the present invention may be used to coat solid substrates, which may then be used in the manufacture of medically-acceptable implants, for instance bone implants. Accordingly, in another aspect, the invention provides a solid substrate, which substrate has attached thereto a layer comprising the coated microparticles of the invention, as defined above.

The invention further provides a medically acceptable implant, which implant comprises a solid substrate of the invention, as defined above.

The invention further provides a tissue engineering scaffold comprising the coated microparticles of the invention, as defined above. Typically, the hydrophilic pharmaceutically active compound in the coated microparticles is a bone growth factor.

The invention further provides an injectable formulation comprising coated microparticles of the invention, as defined above, which formulation is self-setting. Typically, the injectable formulation comprises a suspension of the coated microparticles in bone cement.

In another aspect, the invention provides a microparticle, a coated microparticle, a solid substrate, a medically acceptable implant, an injectable formulation or a tissue engineering scaffold of the invention, as defined above, for use in a method of treatment of the human or animal body by therapy.

Typically, the method of treatment is the treatment of a bone disorder.

In another aspect, the invention provides the use, in the manufacture of a medicament for the treatment of a bone disorder, of a microparticle or a coated microparticle of the invention as defined above.

In another aspect, the invention provides a microparticle, a coated microparticle, a solid substrate, a medically acceptable implant, an injectable formulation or a tissue engineering scaffold of the invention, as defined above, for use in the delivery of said hydrophilic pharmaceutically active compound in a patient in need thereof. Typically, the hydrophilic pharmaceutically active compound is delivered to bone.

In another aspect, the invention provides a method of delivery of a drug, which method comprises administering a microparticle, a coated microparticle or an injectable formulation of the invention to a patient in need of said hydrophilic pharmaceutically active compound. In another aspect, the invention provides a method of delivery of a drug, which method comprises implanting a medically acceptable implant of the invention in a patient in need of said hydrophilic pharmaceutically active compound.

Typically, the methods of delivery of a drug of the present invention comprise delivery of said hydrophilic pharmaceutically active compound to bone.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a schematic illustration of the apparatus used in the "constant composition" coating process according to an embodiment of the present invention. The coating process was performed in a glass vessel (125ml) at 37 ⁰C.

Fig. 2 shows SEM images of drug-loaded PLGA microparticles prepared by the w/o/w method using SDS as a surfactant. The PLGA microparticles are shown to be spherical in shape. At higher magnification (Fig. 2b) the w/o/w microspheres were found to be porous. Fig. 3 shows SEM images of drug-loaded PLGA microparticles prepared by the s/o/w method using SDS as a surfactant.

Fig. 4 consists of SEM cross-sections under FIB which show the porosity extending throughout the microspheres prepared using the w/o/w (Fig. 4a) and s/o/w (Fig. 4b) methods. Fig. 5 shows SEM images of PLGA microparticles prepared by the w/o/w method using PVA as surfactant.

Fig. 6 shows SEM images of the microspheres prepared using the s/o/w method using DCS as the surfactant.

Fig. 7 shows SEM images of the microspheres prepared using the s/o/w method using PVA as the surfactant.

Fig 8 shows SEM images of the morphology of microparticles prepared using the s/o/w method using CTAB as surfactant; the microspheres are very smooth.

Fig. 9 consists of SEM images showing that HA crystals form on the surfaces of the microparticles of the invention within 1 h. Fig. 10 consists of SEM images showing that a complete coating of HA forms on the microparticles of the invention after 3 hours. Fig, 11 consists of SEM images showing that an approximately 3 μm thick coating of HA forms on the microparticles of the invention after 6 hours.

Fig. 12 consists of SEM cross-sections under FIB which show the coating development at 3 hours (Fig. 12a) and 6 hours (Fig. 12b). Fig. 13 shows the XRD pattern of uncoated PLGA microparticles and the XRD patterns of PLGA microparticles coated with HA (HPLG), after 1 hour, 3 hours and 6 hours of coating. A variety of peaks corresponding to HA are shown: (002), (211), (130), (222), (213) and (004).

Fig. 14 shows the FTIR spectrum of uncoated PLGA microparticles and FTIR spectra of PLGA microparticles coated with HA (HPLG), after 1 hour, 3 hours and 6 hours of coating.

Fig. 15 shows the drug release profiles of AMX from AMX-loaded PLGA and HPLGA microparticles. Data are means ± S.D. (n = 3). P>0.05 for all time points.

Fig. 16 shows SEM images of HA-coated PLGA microparticles after immersion in PBS for 2 days.

Fig. 17 shows SEM images of HA-coated PLGA microparticles after immersion in PBS for 30 days, showing that, after 30 days, the PLGA was highly degraded.

Fig. 18 shows SEM images of HA-coated PLGA microparticles after immersion in PBS for 30 days, showing that, after 30 days, HA was still present on the HPLG microspheres.

Fig. 19 is a schematic illustration of the dual constant composition coating process according to an embodiment of the present invention.

Fig. 20 is a graph of pH (y axis) of PBS media (pH 7.4) incubated with PLGA microspheres (solid circles) or HPLG microspheres (solid squares), versus time (x axis) in units of days. Data are means ± S.D. (n = 3). *, PO.01 and **, PO.001 (PLGA vs. HPLG at the set time points).

Fig. 21 a is a graph of cumulative drug (CPH) release (y axis) in units of μg/mg drug/HPLG, versus time (x axis) in units of days, for dual-antibiotic-loaded HPLG-D microspheres. Fig. 21b is a graph of cumulative drug (AMX) release (y axis) in units of μg/mg drug/HPLG, versus time (x axis) in units of days, for the same dual-antibiotic-loaded HPLG-D microspheres. Fig. 22 consists of three SEM images of HPLG-D microspheres, showing the HA coating on the PLGA-D microspheres, at (a) x250, (b) x2000 and (c) x 10000 magnification, and (d) an FIB image of the microspheres.

Fig. 23 is a bar chart of cell number (y axis) in units of xlO⁵ cells, on Matrigel (left hand side bars), PLGA-D (middle bars) and HPLG-D (right hand side bars), afjter culture for 1 and 7 days. (HPLG-D vs PLGA-D at day 7, p<0.05; HPLG-D vs Matrigel at day 7, pO.Ol)

Fig. 24 is an SEM image showing the morphology of MG-63 cells on HPLG-D.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "microparticle" means a microscopic particle whose size is measured in micrometres (μm). Typically, the microparticle has an average diameter of from 1 μm to 1000 μm. More typically, the microparticle has an average diameter of from 1 μm to 500 μm, for instance from 1 μm to 250 μm. Most typically, the microparticle has an average diameter of from 1 μm to 100 μm.

Usually, the microparticle is substantially spherical. Thus, the microparticles and coated microparticles of the invention are typically "microspheres" and "coated microspheres" respectively. As used herein, the term "microsphere" means a substantially spherical microscopic particle whose size is measured in micrometres (μm). Typically, the microsphere has a diameter of from 1 μm to 1000 μm. More typically, the microsphere has a diameter of from 1 μm to 500 μm, for instance from 1 μm to 250 μm. Most typically, the microsphere has a diameter of from 1 μm to 100 μm.

The polymer of the microparticles, coated microparticles and processes of the invention may be any biodegradable polymer. It is typically a synthetic biodegradable polymer. However, any polymer that can encapsulate a drug could also inprinciple release the drug and is therefore suitable for use in the present invention. Thus, the polymer employed may be any polymer which is capable of encapsulating a drug. Typically, the polymer is a polyester, a ρoly(orthoester) or a polyphosphazene. More typically, the polymer is a polyester. Even more typically, the polymer is an aliphatic polyester. Typically the polymer is biodegradable. Poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA) and poly-e-caprolactone (PCL) are examples of biodegradable aliphatic polyesters and may be employed in the present invention. Thus, in one embodiment, the polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly-e-caprolactone (PCL) and a copolymer of any of those polymers. Most typically, the polymer is poly(lactic-co-glycolic acid). Anionic surfactants that can be used in the microparticles, coated microparticles and processes of the present invention are generally anionic surfactants with hydrophobic groups having from about 6 to about 30 carbon atoms. Examples of anionic surfactants which could be employed are saponified fatty acids, alkyl or aryl sulphonates, alkyl or aryl sulphates, sulphate esters, phosphate esters, alkyl or aryl phosphates, alkyl or aryl phosphonates, fatty acids, naphthalene sulphonate (NAS) formaldehyde polycondensates, polystyrene sulphonates, hydrophobe-modified NAS. Typically, the surfactant comprises an -SO₃ ^" group. More typically, the anionic surfactant comprises a hydrophobic tail and an ionic head which comprises an -SO₃ ^" group. Even more typically, the surfactant is sodium dodecyl sulphate (SDS), docusate sodium (DCS) or oleate sodium. Most typically, the surfactant is sodium dodecyl sulphate (SDS) or docusate sodium (DCS). In one embodiment, the surfactant is sodium dodecyl sulphate (SDS). In another embodiment, the surfactant is docusate sodium (DCS).

Microparticles and coated microparticles according to the present invention comprise a hydrophilic pharmaceutically active compound. The hydrophilicity of the compound facilitates the preparation of the solid-in-oil-in- water emulsion when preparing the microparticles; the internal solid phase of the solid-in-oil-in- water emulsion comprises the pharmaceutically active compound. The hydrophilic pharmaceutically active compound is typically insoluble or sparingly soluble in the oil phase, and is more typically insoluble in the oil phase.

The pharmaceutically active compound may be selected from a compound which assists the binding of the microparticle or coated microparticle to existing bone (a bone growth factor), a compound which treats a specific bone disease or any diseased region adjacent to bone, or a compound which relieves pain. In particular, the microparticle or coated microparticle of the present invention may contain a compound for the treatment of tumours, for instance a P or Sr containing compound, or a compound for the reduction of pain arising from tumours, for instance a narcotic analgesic. Such compounds may be administered in lower doses according to the invention as they may be administered at the site of the tumour. Additionally or alternatively, the pharmaceutically active compound may be a compound for the reduction of osteoclast activity caused by tumour cells, for instance a prostoglandin or an interleuken 6 inhibitors, or a compound which treats a specific bone disease such as osteoporosis, for example, parathyroid hormone, a vitamin D derivative, a bisphosphanate, a bone morphogenetic protein or an antibiotic. Mixtures of any of the compounds mentioned above may be employed the microparticle or coated microparticle of the present invention. Typically, the hydrophilic pharmaceutically active compound is a protein, an anti-inflammatory drug, an antibiotic, an anti-cancer drug, a compound which treats a specific bone disorder, a bone growth factor or a compound which relieves pain. The compound which treats a specific bone disorder may be a compound which treats osteoporosis. More typically, the compound is amoxicillin, parathyroid hormone, a vitamin D derivative, a bisphosphanate, a bone morphogenetic protein, an analgesic, a ³²P- or ⁸⁹Sr- containing compound, prostoglandin, an interleukin 6 inhibitor or an antibiotic.

In one embodiment, the compound is amoxicillin.

In addition to the pharmaceutically active compound which is encapsulated in the polymer, the coated microparticles of the present invention may comprise a further pharmaceutically active compound, within the outer layer of calcium phosphate. The further pharmaceutically active compound may be the same compound as the hydrophilic pharmaceutically active compound, or a different compound.

The further pharmaceutically active compound may be any of the types of pharmaceutically active compounds, or any of the specific pharmaceutically active compounds, mentioned above. However, the further pharmaceutically active compound need not necessarily be a hydrophilic compound. Thus, the further pharmaceutically active compound may be any of the hydrophilic compounds listed above or a non- hydrophilic drug such as indomethacin. The further pharmaceutically active compound may be a protein, an antibiotic, an anti-cancer reagent or a bisphosphanate.

The use of a first, hydrophilic pharmaceutically active compound, encapsulated within the polymer of the microparticle, and a further pharmaceutically active compound, present in the outer layer of the microparticle, allows for dual drug release modes. Typically, the first, hydrophilic compound will be released over a longer period of time than the further pharmaceutically active compound present in the outer layer. Indeed, the further pharmaceutically active compound may be released in an initial "burst" and the further pharmaceutically active compound over a longer period of time. Thus, the pattern of release of one or more drugs over a period of time may be tailored to suit particular applications.

In one embodiment, the hydrophilic pharmaceutically active compound which is encapsulated in the polymer is an antibiotic and the further pharmaceutically active compound which is present in the outer layer is an analgesic.

In one embodiment, the further pharmaceutically active compound is cepholathine (CPH).

The outer layer of the coated microparticles of the invention may further comprise other ions which can be incorporated to modify the properties of the calcium phosphate including anions such as carbonate, hydrogen carbonate, hydrogen phosphate, chloride and fluoride and cations such as magnesium.

In one embodiment, the microparticles of the invention are negatively charged. Typically, the negatively-charged microparticles have a zeta-potential which is equal to or less than -25.0 mV. More typically, the zeta-potential of the microparticles is equal to or less than -30.0 mV, for instance equal to or less than -35.0 mV. Most typically, the zeta-potential of the microparticles is equal to or less than -40.0 mV.

In another embodiment, however, the microparticles of the invention which are negatively-charged have a zeta-potential which is equal to or less than -50.0 mV. More typically, in this embodiment, the zeta-potential is equal to or less than -55.0 mV, for instance is equal to or less than -60.0 mV.

The negative charge of the microparticles facilitates the coating of the microparticles with an outer layer of calcium phosphate. Without wishing to be bound by theory, it is thought that the negative charge enhances precipitation or nucleation of the calcium phosphate on the surface of the microparticle. One possible mechanism is that the negative surface charge on the microparticle alters the near-surface supersaturation of the calcium phosphate solution used in the coating process, thus aiding precipitation of the calcium phosphate on to the microparticle surface. Control of the supersaturation of the calcium phosphate solution is thought to be important in aiding the coating process.

The efficiency of encapsulation of the hydrophilic pharmaceutically active compound in the polymer of the microparticles (or coated microparticles) of the present invention (the encapsulation efficiency) may be measured using either of two different methods, as detailed under the heading below "Determination of drug content". The first method involves calculation of an encapsulation efficiency denoted "EE", using equation 2, and the second method involves calculation of an encapsulation efficiency denoted "EE_a", using equation 3. Both EE and EE_a provide a measure of the efficiency of encapsulation of the hydrophilic pharmaceutically active compound in the polymer.

Typically, the encapsulation efficiency, EE, of the hydrophilic pharmaceutically active compound in the polymer of the microparticles (or coated microparticles) of the present invention is at least 6 %. More typically, EE is at least 8 %. Even more typically, the encapsulation efficiency is at least 10 %, for instance at least 12 %. In another embodiment, the encapsulation efficiency, EE, of the hydrophilic pharmaceutically active compound in the polymer of the microparticles (or coated microparticles) of the present invention is at least 20 %, for instance at least 25 %.

Typically, the encapsulation efficiency, EE_a, of the hydrophilic pharmaceutically active compound in the polymer of the microparticles (or coated microparticles) of the present invention is at least 30 %. More typically, EE₈ is at least 35 %. Even more typically, the encapsulation efficiency is at least 36 %, for instance at least 37 %.

In another embodiment, the encapsulation efficiency, EE₃, of the hydrophilic pharmaceutically active compound in the polymer of the microparticles (or coated microparticles) of the present invention is at least 38 %, for instance at least 40 %.

The high encapsulation efficiencies allow for high levels of drug loading in the microparticles and coated microparticles of the present invention.

As regards the microparticles of the present invention, the pharmaceutically active compound is typically present in an amount of at least 1.0 weight % based on the total weight of the microparticle. More typically, the pharmaceutically active compound is present in an amount of at least 1.2 weight %, for instance at least 1.3 weight %, based on the total weight of the microparticle. In another embodiment, the pharmaceutically active compound is present in an amount of at least 2.0 weight % based on the total weight of the microparticle, and more typically in an amount of at least 3.0 weight %.

As regards the coated microparticles of the present invention, the pharmaceutically active compound is typically present in an amount of at least 1.0 weight % based on the total weight of the core of the coated microparticle. More typically, the pharmaceutically active compound is present in an amount of at least 1.2 weight %, for instance at least 1.3 weight %, based on the total weight of the core.

In another embodiment, the pharmaceutically active compound is present in an amount of at least 2.0 weight % based on the total weight of the core of the coated microparticle, and more typically in an amount of at least 3.0 weight %.

The weight of the core of the coated microparticles of the present invention can be determined by weighing the "precursor" (uncoated) microparticles before they are coated with calcium phosphate to form the coated microparticles. Additionally or alternatively, as regards the coated microparticles of the present invention, the pharmaceutically active compound is typically present in an amount of at least 0.9 weight %, more typically at least 1.0 weight %, for instance at least 1.2 weight %, and even more typically at least 1.3 weight %, based on the total weight of the coated microparticle. In another embodiment, the pharmaceutically active compound is typically present in an amount of at least 2.0 weight %, more typically at least 2.2 weight %, and even more typically at least 2.3 weight %, based on the total weight of the coated microparticle.

The process ofthe invention for producing a microparticle comprises (a) the preparation of a solid-in-oil-in-water emulsion, and (b) subsequent solvent evaporation.

Compared with more conventional water-in-oil-in- water (w/o/w) methods, this solid-in-oil-in-water (s/o/w) solvent evaporation technique has been found to improve the stability of encapsulated agents and increase drug entrapment efficiency.

Although solid-in-oil-in-water (s/o/w) solvent evaporation methods have previously been used to entrap hydrophilic agents in polymeric microspheres, such previously-used methods have employed the neutral surfactant polyvinyl alcohol in the external water-based phase. This resulted in approximately uncharged microspheres. The use of anionic surfactants was typically avoided, because it was thought to result in a low drug-encapsulation efficiency. The hydrophilic antibiotic amoxicillin (AMX), for instance, contains one carboxyl group and two amino groups and its pKa is 2.8, thus the electrostatic interaction between AMX and anionic surfactant molecules at the o/w interface was thought to enhance diffusion of the drug out to the external aqueous phase containing the surfactant, resulting in a low drug-encapsulation efficiency. Anionic microspheres prepared to date via solvent evaporation methods using anionic surfactants all have very low encapsulation efficiencies (EE).

The use of the s/o/w method of the present invention however employs an anionic surfactant which facilitates coating with calcium phosphate (e.g. HA) and results in a high drug encapsulation efficiency, which renders the microparticles potentially useful as drug delivery devices.

Typically, step (a) of the process of the invention for producing a microparticle comprises: (al) dispersing said hydrophilic pharmaceutically active compound in a mixture of

(i) said polymer and (ii) said solvent which is immiscible with water; (a2) agitating the resulting mixture, to produce a solid-in-oil dispersion; and (a3) mixing said solid-in-oil dispersion with an aqueous solution of said anionic surfactant, to produce said solid-in-oil-in-water emulsion. The agitation in step (a2) is typically achieved by ultrasonication or stirring, but ultrasonication is preferable,

Usually the mixing in step (a3) comprises homogenising said solid-in-oil dispersion with said aqueous solution of said anionic surfactant, to produce said solid- in-oil-in-water emulsion. Alternatively, the mixing can be achieved by stirring. The particle size, or average diameter, of the microparticles can easily be changed from several microns, for example from 1 μm, 2 μm, 4 μm, 5 μm or 10 μm, to about 100 μm by reducing the degree of homogenisation or reducing the stirring rate in step (a3). Omitting the homogenisation step altogether, or using stirring rather than homogenisation in step (a3), also achieves such an increase in particle size / average diameter.

Typically, step (a) further comprises: (a4) mixing the solid-in-oil-in-water emulsion with a further aqueous solution of said anionic surfactant.

Usually, the step (b) of evaporating said solvent which is immiscible with water comprises stirring said solid-in-oil-in-water emulsion and allowing said solvent to evaporate.

Typically, the evaporation of step (b) results in the formation of an aqueous suspension of the microparticles, in which case the process typically further comprises isolating the microparticles from the water in which they are suspended.

Usually, therefore, the process further comprises: (c) isolating said microparticle. The process may comprise the additional step of (d) washing said microparticle.

Typically, in the process of the invention for producing a microparticle, the encapsulation efficiency, EE, of said hydrophilic pharmaceutically active compound in said polymer is at least 6 %. More typically, EE is at least 8 %. Even more typically, EE is at least 10 %, for instance at least 12 %.

In another embodiment, the encapsulation efficiency, EE, of the hydrophilic pharmaceutically active compound in the process of the invention for producing a microparticle is at least 20 %, for instance at least 25 %.

Typically, the encapsulation efficiency, EE_a, of the hydrophilic pharmaceutically active compound in the process of the invention for producing a microparticle is at least 30 %. More typically, EE₃ is at least 35 %. Even more typically, the encapsulation efficiency EE_a is at least 36 %, for instance at least 37 %.

In another embodiment, the encapsulation efficiency, EE₈, of the hydrophilic pharmaceutically active compound in the process of the invention for producing a microparticle is at least 38 %, for instance at least 40 %.

Usually, the microparticles produced according to the process of the invention for producing a microparticle are negatively charged. More typically, they have a zeta- potential which is equal to or less than -25.0 mV. Even more typically, the zeta- potential of the microparticles is equal to or less than -30.0 mV, for instance equal to or less than -35.0 mV. Most typically, the zeta-potential of the microparticles is equal to or less than -40.0 mV. In one embodiment, however, the microparticles produced according to the process of the invention for producing a microparticle have a zeta-potential which is equal to or less than -50.0 mV. More typically, in this embodiment, the zeta-potential is equal to or less than -55.0 mV, for instance is equal to or less than -60.0 mV. Typically, the average diameter of the microparticles of the invention or those produced according to the process of the invention is from 1 to 20 μm.

The presence of an anionic surfactant in the microparticles of the invention facilitates efficient coating of the microparticles, thus the process of the invention is capable of coating the microparticles rapidly. Typically, therefore, in step (B) of the process of the invention for producing a coated microparticle, said microparticle is contacted with said solution comprising calcium and phosphate ions for no more than 10 hours. More typically, the microparticle is contacted with said solution comprising calcium and phosphate ions for no more than 8 hours, and most typically for no more than 6 hours. Even more typically, the microparticle is contacted with said solution for from 3 to 8 hours, for instance from 3 to 6 hours.

Typically, said solution comprising calcium and phosphate ions is a supersaturated solution of calcium phosphate.

The process of the invention for producing a coated microparticle typically involves coating the negatively charged microparticles using a dual constant composition method. Thus, during step (B) of the process, the calcium and phosphate ions in said solution are typically maintained at a specific molar ratio. The solution comprising calcium and phosphate ions is typically a supersaturated solution of calcium phosphate. Usually, the supersaturation of said solution is maintained during step (B). Typically, the calcium phosphate with which the microparticles are coated is hydroxyapatite, in which case the molar ratio of the calcium and phosphate ions in said solution is maintained at about 10/6.

In one dual constant composition precipitation method, a stable supersaturated solution with respect to hydroxyapatite is prepared and as precipitation on nuclei occurs the solution supersaturation is maintained by automatic additions from burettes. Typically the ratio of calcium to phosphate ions is from 1 :1 to 2 : 1 , preferably from 1.4:1 to 2:1 and more preferably about 10:6. The source of calcium ions in the solution is any water soluble organic or inorganic calcium compound, preferably calcium chloride or calcium nitrite and more preferably calcium nitrate.

The source of phosphate ions in the solution is any water soluble phosphate compound, preferably an orthophosphate, for example, a potassium orthophosphate, especially di-potassium hydrogen orthophosphate trihydrate. More preferably, the source of phosphate ions is KH₂PO₄.

As indicated above, other ions may be incorporated into the layer comprising calcium phosphate. For example, carbonate and hydrogen phosphate ions may be added to increase the resorption rate in the body whereas chloride, fluoride and magnesium ions may be added to decrease the resorption rate.

In particular, carbonate ions may be added to the aqueous solution of calcium and phosphate ions to vary the crystallinity and stoichiometry of the calcified layer. The maximum concentration of carbonate ions will depend on pH, temperature and the presence of other ions. It will be appreciated, though, that the calcified layer is preferably a calcium phosphate layer or a substituted calcium phosphate layer. The source of carbonate ions is any soluble carbonate or hydrogen carbonate compound and is preferably potassium hydrogen carbonate or sodium hydrogen carbonate.

The time for which the microparticles are contacted with the calcifying solution affects the thickness of the outer layer formed on the microparticles. Typically after about 3 hours the thickness of the layer is about 0.7 μm. Typically after about 6 hours the thickness of the layer is about 3 μm. The thickness of the layer (coupled with its porosity) may affect the rate at which the polymeric microparticles are broken down in the body and the rate of release of any pharmaceutically active compounds from within the microparticles. Typically, the outer layer thickness is from about 0.2 to about 5.0 μm, more typically from about 0.5 to about 4.0 μm and most typically from about about 0.5 to about 3.5 μm.

Typically, the calcium phosphate of the outer layer of the coated microparticle of the invention or produced according to the process of the invention is hydroxyapatite. Typically, the average diameter of the coated microparticle of the invention or produced according to the process of the invention is from 10 to 100 μm, for instance from 10 to 50 μm. The average diameter is more typically from 15 to 40 μm, and most typically from 15 to 30 μm.

The microparticles and coated microparticles of the invention may find application in the treatment of bone disorders and/or in the delivery of pharmaceutically active compounds. Typically, the delivery of the pharmaceutically active compounds is to bone.

Typically, said treatment or said delivery comprises sustained release of the pharmaceutically active compound from the microparticle or coated microparticle.

Typically, the release of the pharmaceutically active compound is sustained for at least 3 days, for instance for at least one week. More typically the release is sustained for at least two weeks and even more typically for at least one month. The release may be sustained for at least 6 months, for instance for at least 12 months. The release speed can be changed by varying, for instance, the polymer composition, the polymer molecular weight and/or the particle size of the microparticles or coated microparticles. In this way, the period for which release of the pharmaceutically active compound is sustained may be varied from several days, for instance from 3 days, to several months, for instance to 12 months.

The present invention provides a solid substrate, which substrate has attached thereto a layer comprising coated microparticles of the present invention. Such substrates find application in the treatment of bone disorders and in the delivery of pharmaceutically active compounds.

In one embodiment, the invention provides a solid substrate wherein regions of said substrate have attached thereto a layer comprising coated microparticles of the present invention with another region or other regions having no coated microparticles attached thereto.

The substrates which can be coated may be electrically conductive over all or part of their surface. They may be, for example, metals such as gold, plastics or ceramics coated with metal over all or part of their surface, metals partially coated with plastic, or semi-conductors. Preferably the substrates have non-conducting regions on their surfaces of from 10 μm to 2 mm in diameter and more preferably the regions are about 150 μm in diameter. The substrates may be coated using an electrolytic deposition process or by applying the coated microparticles of the present invention in the form of a powder. Preferably the substrates are electrolytically coated. The substrates may be electrolytically coated by the process described in WO 00/00177. Thus, in a further aspect, the present invention provides a process for producing a substrate of the invention, which process comprises electrolytically depositing the coating comprising the microparticles of the invention onto a conducting region of the substrate.

The electrolytic coating of substrates with calcium-phosphate-coated microparticles is described in WO 00/00177 and the process of the present invention may be carried out accordingly.

The electrolytic deposition process may be carried out in an aqueous solution at a pH of from 5 to 11, preferably 6 to 8, more preferably about 7.4. The form of the calcium phosphate deposited may vary with pH. For example, at high pH hydroxyapatite may be deposited whereas at low pH brushite may be deposited.

The temperature of deposition is generally below 100⁰C; preferably below 7O⁰C and more preferably about 50⁰C.

A salt such as, for example, potassium chloride may be added to the solution to maintain supersaturation by keeping a high background ionic strength and act as an electrolyte. Alternatively, calcium and phosphate can be added during the precipitation process to maintain supersaturation.

The thickness of the coating of the substrate does, of course, increase with deposition time. By varying the deposition time or by performing multiple depositions, coatings of the required thickness may be obtained. For example, after a deposition time of 1 hour the coating thickness is about 2 μm. Multiple depositions may be performed or deposition time prolonged to access thicker coatings, for example coatings of about 20 μm.

Coatings may be formed from mixtures of the coated microparticles of the present invention. Thus different regions on the surface of the substrates may be coated with different types of coated microparticles. For example, a non-conducting pattern may be applied to the substrate prior to the first deposition. After the first deposition using one or more types of coated microparticle of the present invention the non- conducting pattern may be removed and a second deposition performed using different coated microparticles according to the invention. Alternatively, for example, a metal substrate is coated. It may then be subjected to a partial etching or lithographic process and a second deposition performed in a different solution of coated microparticles. Use of a variety of coated microparticles may allow the release of pharmaceutically active compounds in the coating to be controlled. For example, compounds incorporated into coated microparticles with a thin calcium phosphate coating will be released more rapidly than compounds incorporated into coated microparticles with a thick calcium phosphate coating. The substrates of the invention may find application in the treatment of bone disorders and/or in the delivery of pharmaceutically active compounds. The delivery of the pharmaceutically active compounds is typically to bone tissue.

Thus, the invention further provides a medically acceptable implant which comprises a solid substrate of the invention. The invention further provides the use of a substrate according to the invention in the manufacture of a medically acceptable implant.

The invention further provides a substrate or an implant according to the invention for use in a method of treatment of the human or animal body by therapy.

The invention further provides a substrate or an implant according to the invention for use in: the treatment of a bone disorder; or the delivery of a pharmaceutically active compound. Typically, said treatment or said delivery comprises sustained release of the pharmaceutically active compound. The sustained release is from the microparticles or coated microparticles which the substrate or implant comprises. Typically, said sustained release is for at least one week, more typically for at least two weeks and even more typically for at least one month.

The invention further provides a tissue engineering scaffold comprising the coated microparticles of the invention, as defined above. Typically, the hydrophilic pharmaceutically active compound in the coated microparticles is a bone growth factor.

The invention further provides an injectable formulation comprising coated microparticles of the invention, as defined above, which formulation is self-setting. Typically, the injectable formulation comprises bone cement. Typically, the coated microparticles are suspended in the bone cement.

The invention will be described further in the Examples which follow.

EXAMPLES

Materials

Poly(DL-lactide-co-glycolide) (PLGA) with a 50:50 copolymer ratio (inherent viscosity of 0.67dL/g) was supplied by Birmingham Polymers Inc. Amoxicillin, sodium dodecyl sulfate (SDS), docusate sodium (DCS), cetyltrimethylammonium bromide (CTAB), polyvinyl alcohol (PVA, FW 30000), dichloromethane and phosphate buffer solution (pH 7.4, 0.1M) were supplied by Sigma Chemical Co. Calcium nitride, potassium chloride, potassium hydroxide and potassium dihydrogen phosphate were purchased from BDH Chemical Co. All reagents were of reagent grade and used as received.

Preparation of PLGA microspheres

Drug loaded PLGA microspheres were prepared by two different solvent extraction/evaporation methods: water-in-oil-in- water (w/o/w) (Comparative Examples 1 to 4) and solid-in-oil-in-water (s/o/w) (Comparative Examples 5 and 6 and Examples 1 and 2). For the w/o/w method (Lamprecht, A. et al., J Control Release 2000;69:445- 454; Ibrahim, M.A. et al., J Control Release 2005;106:241-252.) 200 mg of PLGA was dissolved in 2 ml dichloromethane, and the internal aqueous phase was added to chloromethane to form the primary emulsion by probe ultrasoni cation of 1 min (1 Os working and 10s rest) in an ice- water bath at 100 W using an ultrasound nozzle (Kerry Ultrasonics Ltd., UK). The internal phase was made up of 0.1ml 1% (w/v) PVA solution and 0.2ml of NH₃. OH (IM) solution containing 50mg AMX. This primary emulsion was added drop-wise into 5ml of 1% SDS solution and homogenised at 6500 rpm with a Turrax IKA Tl 8 homogeniser at room temperature to form the w/o/w double emulsion. This dispersion was added to 100ml of 1% SDS and the organic solvent was allowed to evaporate for at least 3 hours. 1% PVA, 0.5% DCS and 0.5% CTAB solutions were also used to prepare w/o/w PLGA microspheres. A new s/o/w method was used to prepare anionic drug-loaded PLGA microspheres (Examples 1 and 2). lOOmg of AMX were dispersed in 2 ml of dichloromethane with 400mg PLGA and ultrasonicated (Kerry Ultrasonics Ltd., UK) for 1 min (10s working and 10s rest) in an ice-water bath. This s/o dispersion was then homogenised with 10 ml 1% SDS solutions at 6500 rpm using a Turrax IKA Tl 8 basic homogenizer at room temperature for 30s. Then this s/o/w emulsion was poured into 100 ml of 1% SDS solution under stirring for 3 hours to allow the dichloromethane to evaporate. 1% PVA, 0.5% DCS and 0.5% CTAB solutions were also used to prepare s/o/w PLGA microspheres. For both w/o/w, and s/o/w methods, the PLGA microspheres were collected by centrifuging at 5000rpm (Labfuge 200, Heraeus) and washing three times with distilled water. The PLGA microspheres which were to be coated with HA were frozen at -80 ⁰C (Sayan freezer, Japan) and all others were lyophilized overnight (Micromodulyo, EC Apparatus Inc., USA), and kept in a sealed container with silica gel at 4 ⁰C until use.

Hydroxyapatite-coating of PLGA microspheres (HPLG)

Negatively charged PLGA microspheres (using SDS as the surfactant) were coated with hydroxyapatite using the dual constant composition method (Xu, Q. et al, Biomaterials 2007;28:2687-2694; Wong, A.T.C. et al.; Colloid Surf A-Physicochem Eng Asp 1993;78:245-253). A stable supersaturated solution of calcium phosphate in respect with HA was prepared and stirred with a magnetic stirrer. In this supersaturated solution, Ca/P was maintained at 1.67 by additions of Ca(NO₃)₂ (2.5xl0^"3M) and KH₂PO₄ (1.5* 10^"3M). Two titrants were prepared as follows: titrant (1) Ca(NO₃)₂ (2.5xlO^"2M); titrant (2) KH₂PO₄ (1.5xlO^"2M) and KOH (5xlO-²M). Both titrants and supersaturated solutions contained 0. IM KNO₃. The coating process was performed in a glass vessel (125ml) at 37 ⁰C as schematically illustrated in Figure 1. A radiometer PHM85 pH meter and a radiometer ION85 ion analyzer were connected to a computer for automatic monitoring. pH was measured with Radiometer GK2401 combined with glass pH electrodes. Radiometer F2002Ca²⁺ selective electrodes coupled with Radiometer K4040 calomel reference electrodes were used to measure Ca²⁺ activity (pCa). Freshly prepared PLGA microspheres were suspended in distilled water and then added to the supersaturated working solution to induce the precipitation of HA on PLGA. HA precipitation resulted in a lowering of pH and pCa in the supersaturated solution, and any drop of pH or pCa triggered the simultaneous addition of titrant solutions containing Ca(NO₃)₂, KH₂PO₄ and KOH from the respective autoburetes (radiometer ABU91 Triburete) to maintain the supersaturation of the working solution. After periods of 1 , 3 and 6 hours, the HA-coated PLGA (HPLG) microspheres of Example 3 (after 1 hour), Example 4 (after 3 hours) and Example 5 (after 6 hours) were separated by centrifugation at 5000rpm for 5min, washed with distilled water 3 times, frozen and then lyophilized overnight.

Determination of drug; content

The drug content in PLGA and the drug encapsulation efficiency were measured by extraction from the microspheres. 20 mg of drug loaded microspheres were dissolved in 2 ml dichloromethane then 5 ml of distilled water was added. The mixture was vortexed at 2500 rpm using the IKA minishaker (MS2, IKA works Inc., USA) for 1 min and then placed on the shaker for extra 2 hours at 1000 rpm and room temperature.

After centrifugation at lOOOOrpm on MSE Hawk 15/05 refrigerated centrifuge, the supernatant was analyzed. All samples were measured in triplicate. The drug content (Dc, w/w) and encapsulation efficiency (EE) of AMX into the PLGA microspheres were expressed as follows:

£>_c = -^ x l00% equation 1

M where D₁ is the total amount of the drug loaded and M is the mass of microspheres, and

EE = - x 100% _{equation 2}

S where D_s is the theoretical drug content.

The concentration of AMX was measured spectrophotometrically by UV/Vis spectroscopy (JASCO V-570, Tokyo, Japan) at a wavelength of 229 ran, It is possible that the AMX is not completely extracted from the dichloromethane thus another method also was applied to measure the EE; by measuring the free drug remaining in the external aqueous phase after 3 hours solvent evaporation. EE_a by this method can be expressed by:

EE_a ^{= x ] 00%} equation 3

LJ s

D₃ is the amount of drug measured in the aqueous phase. D₃, the theoretical drug content, is calculated as follows:

D₃ = -jr x 100% equation 4

M

D_t is the total amount of drug used for encapsulation.

In order to determine the drug content in HPLG microspheres, a similar method was used. lOmg of HPLG microspheres was ground in an agate mortar to break the HA coating and then dissolved in 2 ml of dichloromethane. AMX was extracted by shaking the HPLG in dichloromethane with 5 ml pure water for 2 hours at 1000 rpm and the drug content in the aqueous phase was spectrophotometrically determined by measuring the absorbance at 229nm in a UV- Vis spectrophotometer.

X-ray diffraction measurement

X-ray diffraction pattern (XRD) was used to determine the nature and crystal size of the coating on HPLG microspheres on a Philips 1729 X-ray generator operated at 35 kV and 50mA with Cu Ka radiation. HPLG microspheres were affixed on a piece of clean silicon wafer with silicone grease. Data were collected between 5 ° and 60 ° 2Θ at a scan rate of 0.002 ° 2θ/s. X'Pert HighScore software was used to identify the crystal structure and get the values for the Bragg angle and associated line broadening (FWHM). Assuming that there is no broadening due to lattice strain and the crystallite size crystallite size in the [002] direction is given by the Scherrer formula.

t = equation 5

B cosθ

where B is peak full width at half maximum peak intensity (FWHM) modified by removing broadening due to instrumental effects, k is a constant and assumed to be 1.0, λ is the wavelength of the X-ray (0.154 nm for Cu radiation), θ is the Bragg angle of the (002) peaks.

Fourier Transform Infra-Red CFTIR) FTIR spectra were obtained in the transmission mode using a FTIR spectrometer (Spectrum 2000, Perkin Elmer Ltd., England) from discs containing potassium bromide and PLGA or HPLG. 10 scans over the range of 400-4000 cm^"1 were performed at a resolution of 2 cm^"1 with the background scan subtracted.

Zeta-potential measurement

The surface charges on PLGA and HPLG microspheres were examined by measuring their zeta-potential in distilled water on Zetasizer Nano (Malvern

Instruments, UK). Each result was the average of 30 measurements, and all the measurements were made at 25 ⁰C and at an angle of 90°.

Particle size measurement

The particle size and distribution of PLGA and HPLG microspheres were recorded using a Malvern mastersizer 2000 (Malvern Instruments, UK). Microspheres were dispersed in 0.02% Tween-20 aqueous solution, and the dispersion was then sonicated for 10 seconds and microsphere size between 0.5 and 180μm recorded. The mean particle size represents the volume mean diameter from three batches.

Scanning electrical microscopy (SEM)

The morphology of PLGA and HPLG microparticles were analyzed by SEM using JEOL JSM 840F and JSM 6500F scanning microscopes with field emission guns operated at accelerating voltages of 3-5 kV. Dry samples were put on aluminum stubs and then sputter coated with 3nm-thick platinum using a Cressington sputter coater HR208 with MTM-20 thickness controller in an argon atmosphere.

Focused ion beam (FIB) microscopy

Cross sections of HPLG microspheres were prepared and imaged using FIB microscopy (FEI 200, Cambridge, UK). A Ga⁺ ion beam with an energy of 30 keV with a beam current of 1000 pA milled the target microspheres at selected positions using a staircase pattern. Secondary electrons formed during milling enable concurrent imaging of the sample. Tilting the sample to 45° and reducing the beam current at 10 pA helped to reduce specimen damage during ion beam scanning. (Xia, Z.D. et al. J Biomed Mater Res Part A 2006;79A:582-590; Xia, Z.D. et al., Biomaterials 2006;27:4557- 4565.)

Drug release in- vitro

In-vitro drug release experiments of AMX-loaded PLGA and HPLG were carried out in a Haake SWB25 shaking bath (Karlsruhe, Germany) at 80 rpm and 37°C . 20mg of drug-loaded microspheres were enclosed in dialysis membrane with 1 ml of PBS (pH 7.4) and then incubated in 10 ml medium of PBS (pH 7.4). The release medium was exchanged completely at regular time points. The amount of AMX released at certain set times was determined by UV/Vis spectroscopy at 229nm (with PBS as reference). Each experiment was repeated three times.

Drug encapsulation

The results of the drug entrapment experiments are shown below in Table 1. Table 1: properties of AMX loaded PLGA microspheres by w/o/w and s/o/w methods

In the w/o/w method, a high drug encapsulation efficiency (EE) of 12% for AMX was achieved when the positive surfactant CTAB was used. However, EE% was very low if a negative surfactant (SDS, DCS) or neutral surfactant (PVA) was used. When the s/o/w method was applied to entrap hydrophilic drugs, EE was high irrespective of which surfactant was used.

Drug leakage during the HA coating is very tiny, and no more than 1% of the loaded drug can be detected in the reaction solution. The final drug content in the HPLG after 6 hours reaction is about 2.32 wt%, which is lower than that of PLGA microspheres (3.12 wt%) because of the HA added to the HPLG microspheres.

SEM

The morphology of the drug-loaded PLGA microspheres was characterized by SEM. Drug-loaded microparticles prepared by w/o/w method (Fig 2) and the s/o/w method (Fig. 3) were spherical in shape. At higher magnifications the w/o/w microspheres were found to be porous (Fig 2 b); cross-sections under FIB showed the porosity extending throughout the microspheres (Fig 4). However, for the microspheres prepared using the s/o/w method the morphology was quite different. They also appeared spherical, but no apparent pores were seen under SEM. Some aggregation of the microspheres occurred when using SDS (Fig 3) and DCS (Fig 6) as surfactants, with large numbers of small particles less than 5μm in diameter. Using PVA again gives spheres with some aggregation (Fig 7). If the positive surfactant CTAB was used in s/o/w method, the final microparticles exhibited no aggregatation. Fig 8 shows the morphology of s/o/w microparticles with CTAB as surfactant, and the microspheres are very smooth.

Preparation of hydroxyapatite coating

HA coating on the most negatively charged PLGA microspheres prepared by s/o/w method using SDS as surfactant has been prepared by constant composition method. Crystals form on the microsphere surfaces within 1 h (see Fig 9). A complete coating of HA was formed on microparticles after 3 hours (Fig 10), and approximately 3 μm thick after 6 hours (Fig 11). The FIB images (Fig 12 a and b) show this coating development.

XRD

Powder XRD was used to characterize the HPLG at different reaction times. A variety of peaks corresponding to HA are shown in XRD patterns of HPLG (Fig. 13), and the amplitudes of these peaks increases with reaction time. The average crystal dimensions along [002] of the precipitated HA in HPLG were calculated from the Scherrer equation to be 20.2, 25.4 and 28.9 nm for HPLG of 1, 3 and 6 hours, respectively. The broadening and overlapping of the diffraction peaks in the XRD pattern are typical of small nonstoichiometric HA crystals with possibly poor crystallinity. The signal of diffuse halo centered at about 2Θ = 20 ° is attributed to PLGA.

FTIR

FTIR spectroscopy was used to get additional information of the precipitated coating on HPLG microparticles. FTIR spectra of HPLG of 1-, 3- and 6- hour reaction times (Fig. 14) all exhibit bands at 962, 1040 and 1091 cm-1 (stretching vibration of PO₄ ^3") and 564, 602 cm^"1 (deformation vibration of PO₄ ^3") characteristic of HA. The peak at 3570 cm^"1 derived from the stretching and libational modes of hydroxyl is only observed after 6 h reaction time (Zhang, R.Y. et al., J Biomed Mater Res 1999;45:285- 293; Du, C. et al., J Biomed Mater Res 2002;59:535-546). Peaks at 472, 564, 602, 962 and 3570 cm^"1 increase in intensity with increased reaction time indicating that the amount of HA formed on PLGA increases with time. The peaks between 1800 and 1200 cm^"1 and peaks at 3002, 2960, 2885 cm^"1 are from the PLGA. Zeta potential

The zeta potential was measured to check the surface charge on these drug- loaded PLGA microspheres (see Table 1). As expected, the microspheres of Examples 1 and 2 prepared by the s/o/w method using negative surfactants, SDS and DCS, possessed the most negative zeta-potential, and microspheres prepared by using positive surfactant CTAB was positively charged, 42.4 ± 2.5 mV. The zeta-potential was slightly negative when the 'neutral' surfactant PVA was used. Compared to s/o/w microspheres, w/o/w microspheres have more negative and more positive surface charge for anionic surfactants and cationic surfactant used. The negative surface charge of PLGA was observed to decrease greatly after

HA coating, dropping from -47.1 to -20.5mV only after lhour precipitation, and dropping to nearly neutral after βhours (see Table 2).

Particle size s/o/w method produce smaller microspheres using negatively charged surfactants, SDS and DCS, with average size of 8.2±3.5 and 8.6±4.4 μm, and the sizes are 26.2±3.7 and 23.1±9.2μm when PVA and CTAB are used respectively. At the same condition, the neutral surfactant PVA produces the large microspheres. In w/o/w method, the microspheres prepared are bigger than that prepared from w/o/w method with the same surfactant, except for CTAB. Microspheres prepared by the w/o/w method have the size of 40.0±16.0, 18.2±9.1, 24.6±11.4 and 15.4±7.8 μm when PVA₅ CTAB, SDS and DCS are used respectively.

HA coating results in a larger particle size as the reaction time is increased, as expected (Table 2). After 1 hour, the size increases from 8.2±3.5 to 19.1±8.3 μm, which is mainly due to the aggregation of microspheres in the supersaturation calcium phosphate solution during coating, as confirmed by SEM images (Fig 9, 10 and 11). After that, the particle size keeps increasing to 20.3±7.8, and then to 24.9±9.6 μm, corresponding to an increase of HA coating thickness of about 1.2 and 4.6 μm. They are close to the measured HA thickness results from FIB: 0.66±0.06 and 2.79±0.61 at 3 hours and 6 hours respectively.

Drug release The drug loaded PLGA microparticles are immersed into the supersaturated calcium phosphate solution to perform the HA coating, and some drug leakage can be expected during this period (6 hours). The drug content in the final working solution after precipitation was measured and found to be minimal. Drug release profiles for AMX loaded PLGA and HPLGA were investigated in PBS (ρH7.4) (see Fig 15). No major differences were observed in the drug release profiles of AMX between the PLGA and HPLG microspheres. Burst release was seen at the first 12 hours, but it only accounted for less than 10% of total drug encapsulated for PLGA (9.7%) and HPLG (9.8%). An increased release occurred during the first 15 days and was followed by a gradual tailing off to an approximately constant rate at 0.88 and 0.72μg/mg

(drug/formulation) for PLGA and HPLG, respectively. After 30 days, PLGA was highly degraded (Fig 17), but HA was still present on the HPLG microspheres (Fig 18).

Discussion of results A new s/o/w solvent extraction and evaporation method has been used to prepare microparticles containing anionic surfactants and entrapped drugs. It has several advantages compared to conventional s/o/w and w/o/w methods.

In order to encapsulate hydrophilic drugs into biodegradable polymeric systems (such as PLGA, PLA, PCL), the w/o/w method can be used. This involves dissolving drugs into an aqueous solution and homogenizing with the organic solvent containing the hydrophobic polymers to form the primary w/o emulsion, then pouring the primary emulsion into an external water phase containing surfactant under stirring to form the microparticles. However, this w/o/w method is greatly limited by the low encapsulation efficiency and denaturation of drugs when the hydrophilic drugs are entrapped. The low EE may be caused by the splitting of the internal aqueous phase containing the drug, by stirring, and the diffusion of the drug from the small droplets to the external water phase during the solvent extraction and evaporation steps; this diffusion is enhanced if a charged surfactant is used in the external water phase. The present inventors have found that only trace amounts of drugs (almost zero for chDG and a very low amount for AMX) can be entrapped when a negatively charged surfactant SDS or DCS is used in the w/o/w method. In the s/o/w method, however, the drug is in the solid state and dispersed into the organic phase; leakage of the drug can therefore only take place following a dissolution step, which results in a slower drug leakage and higher encapsulation efficiency.

Secondly, the s/o/w method has been successfully used to entrap sensitive components, such as proteins, and with a high EE (Takada, S. et al., Pharm Res 1997;14:1146-1150; Cleland, JL, et al., Pharm Res 1996;13:1464-1475; Castellanos, IJ. et al., J Pharm Sci 2006;95:849-858; Morita, T. et al., J. Control Release 2000;69:435- 444) and hydrophilic small drugs (Lamprecht, A. et al., J Control Release 2000;69:445- 454; Weidenauer, U. et al. J Microencapsul 2003;20:509-524). However, the usual surfactant used with the s/o/w method is PVA - which is an approximately neutral polymer. The zeta-potential is about -17 mV for AMX-loaded PLGA microspheres with PVA as surfactant, the slightly negative surface charge is attributed to hydroxyl groups in PVA molecule.

The present inventors have found that anionic surfactants can be used to prepare microparticles which are more negatively charged and which can undergo enhanced HA coating. SDS and DCS have been used as anionic surfactants to prepare drug loaded negatively charged microspheres (Examples 1 and 2). For comparison, the cationic surfactant CTAB was also used (Comparative Example 6). The EE of AMX into PLGA was found to decrease with the addition of surfactant in the order: PVA, CTAB, SDS and then DCS. AMX contains one carboxyl group and two amino groups and its pKa is 2.8, thus the electrostatic interaction between AMX and anionic surfactant molecules at the o/w interface is thought to enhance the diffusion of drugs out to the external aqueous phase containing the surfactant. In general, however, such diffusion is found to be greatly inhibited by the fact that the drug is present in the s/o primary emulsion in the solid state. This results in a high encapsulation efficiency of the drug. The core-shell structure of HA-coated PLGA microspheres (HPLG) has been synthesized rapidly using the constant composition method. The synthesis is much more rapid than alternative methods such as biomimetic coating, i.e. within a matter of hours rather than days. The nuclei used in the constant composition precipitation are negatively charged PLGA microspheres with sulfate groups anchored on the surface, so no pretreatment is needed to produce polar groups. Such pretreatment can be time consuming; it can for instance take several days to induce a negative charge on PLA or PLGA materials, by immersion in aqueous solution to form carboxyl groups by polymer degradation. Moreover, such long periods of immersion in aqueous solution can change the properties of biodegradable polymers, such as the compressive modulus, surface and matrix composition, due to polymer degradation (Zhang, R.Y. et al., J Biomed Mater Res 1999;45:285-293; Chen, Y. et al., J Biomed Mater Res B 2005;73B:68-76). Furthermore, if the polymer contains entrapped drugs or other bioactive agents, then these are most likely to have leached out (Tanahashi, M. et al. J Mater Sci-Mater Med 1995;6:319-326; Tanahashi, M. et al., J Biomed Mater Res 1995;29:349-357; Tanahashi M. et al., J Appl Biomater 1994;5:339-347). The present inventors have found that drug leakage and polymer degradation can be minimized by HA coating anionic microspheres using the constant composition method; the use of anionic microspheres decreases the reaction time of the HA coating.

Many small HA particles can be found on PLGA microparticles (SDS as surfactant) in Ih, and a complete HA coating can be achieved after 3 h.. The coating thickness increases with further deposition times. In this way, the HA coating thickness can be controlled to tailor the rate of drug diffusion, or to allow secondary drugs to be adsorbed to the coating.

Only slight drug leakage (less than 1%) was observed to occur during the 6-hour coating which may be because the PLGA microparticles are directly used in their wet state. The burst release observed during the early stages of the release experiments is thought to arise because the drug molecules diffuse to and accumulate on the near surface of the microspheres during drying. Then when the drug carrier is rehydrated the near-surface drugs can rapidly diffuse.

Negative charges may be important in HA precipitation. The amphiphilic nature of surfactants makes them strongly adhere to the surface of the particles by anchoring the hydrophobic tail into the polymer, leaving the polar or ionic head protruded from the surface. For anionic surfactants (SDS and DCS) the protrudent polar and ionic head group is -SO₃ ^", which is a very strong chelating group for calcium ions, forming - SO₃Ca⁺ and (-SO₃)₂Ca (Kawai, T. et al, Biomaterials, 2004, 25:4529-4534). The results therefore indicate that the sulfate group acts as a functional group in HA precipitation on PLGA microspheres.

-COOH and -OH groups on the PLGA can dissociate partially to form a negatively charged polymer surface, which can accumulate calcium ions through electrostatic force and hydrogen bonding (Zhang, R.Y. et al., J Biomed Mater Res 1999;45:285-293). However, the accumulation capability of COOH and OH to induce apatite coating is comparatively weak. Indeed, when the PLGA microspheres were prepared by using PVA as a surfactant, only traces of HA formed on the surface after 6 h.

The in- vitro drug release from PLGA and HPLG was characterized by a typical triphasic drug release kinetics. An initial burst is followed by drug diffusion; then, at the third stage, polymer degradation occurs. The initial burst in the first 12 hours accounts for less than about 10% of the drug for both PLGA and HPLG, which is a relatively small value compared to the burst observed from drug-loaded microspheres which had been made by the spray-drying method or w/o/w method (Friess, W.F. et al., J Pharm Sci 2002;91:845-855). The second phase lasts about 15 days, with a slowly increasing release followed by the erosion phase, in which polymer degradation is thought to have occurred (see Fig 16). Due to the HA coating and a small amount of drug leakage during the coating, the drug content in HPLG is smaller than that in PLGA, which can be seen in the final stage of drug release. The cumulative drug release at day 31 for PLGA and HPLG is 54.7 and 52.1μg/mg (drug/formulation), respectively. There is no great difference between PLGA and HPLG in drug release, despite the presence of a layer of HA coating in HPLG. During the incubation of PLGA and HPLG microspheres in PBS, the pH value in the PBS media had decreased (to approximately pH=6 after 30 days) by the degradation of PLGA microspheres. However, the pH drop was much inhibited in the presence of HA coating in HPLG microspheres (see Fig. 20). Compared to PLGA, HPLG demonstrated a statistically significant pH buffer capability after 13 days (P>0.05). After 31 days, even though the PLGA was greatly degraded (see Fig. 18), the HA coating had only partially dissolved (see Fig. 17). The PLGA had degraded more quickly than the HA coating causing the microspheres to burst, and this has controlled the overall rate of drug release. AMX has a low adsorption on HA, and this together with the more rapid degradation of the PLGA compared to HA has resulted in the similar drug release profiles observed for the PLGA and HPLG microspheres. The drug release profiles could be altered by changing the thickness of the HA coating. In conclusion, a hydrophilic drug, amoxicillin (AMX), has been encapsulated into poly(lactide-co-glycolide) (PLGA) microspheres by a solid-in-oil-in-water (s/o/w) multi-emulsion method using a range of surfactants -positive, neutral and negative. Using the negative surfactant sodium dodecyl sulfate (SDS), an AMX content in the microspheres of about 3.1% (w/w) was achieved with an encapsulation efficiency of 40.6%, and the microspheres had a negative surface charge of -47.1±2.2mV. Complete covering of hydroxyapatite (HA) using constant composition precipitation on the negatively charged PLGA microspheres was achieved within 3 hours. In- vitro drug release experiments showed a sustained release profile for HA coated and uncoated PLGA microspheres for at least one month.

Dual antibiotic delivery

Figures 21a and 21b show the release of both CPH (Fig. 21a) and AMX (Fig. 21b) from HPLG-D, in a dual antibiotic delivery.

During this dual-antibiotic release from HPLG-D (i.e. from HPLG in which the anionic surfactant is DCS), HA-absorbed CPH was quickly released in the first 2 days in a high dose (see Fig. 21a). As a control, HPLG-D microspheres with only CPH adsorbed (single release of CPH) showed a similar CPH release profile to that of the dual-antibiotic-loaded HPLG-D microspheres. The release of the absorbed CPH from HPLG-D was very quick despite the strong interaction of between CPH and HA. Such an effective initial release of an absorbed drug from HPLG-D could provide a high level of drug concentration in a localised area; such a high initial dose of a drug can be of great assistance in achieving a successful therapeutic effect. During the first 2 days, only a small amount of AMX (approximately 6%) was released from the dual-antibiotic-loaded system, followed by a sustained release of AMX with almost no plateau phase (see Fig. 21b). During days 2-31, the release of AMX was approximately linear, at a rate of 1.5 μg/mg (drug/HP LG) per day. The linear release of AMX from HPLG-D can be explained by the small particle size of PLGA-D microspheres (4.7±2.4μm). The smaller the particle size, the quicker the polymer swells in aqueous solution, and the quicker the microsphere degrades. Osteoconductivity of HPLG-D microspheres

The potential osteoconductivity of HPLG-D was examined by a primary study of the osteoblast-like MG63 cell response. Samples of HPLG-D and PLGA-D microspheres, having diameters in the order often micrometers, were immobilised on a cell culture plate before seeding the cells. Cell-culture Matrigel, a gelatinous protein mixture secreted by mouse tumour cells, was used to cover the coverclips and to immobilise the microspheres. The cellular responses of HPLG-D, PLGA-D and, as a control, Matrigel were evaluated. Cell proliferation and cell morphologies were also investigated. A cell number standard curve was obtained by comparing the results of cell counting and Almar blue assay.

About 1x10⁵ cells/well were seeded and, after one day of incubation, the cell numbers for HPLG-D and PLGA-D increased to 3.24(±0.63)xl0⁵ and 2.64(±0.89)xl0⁵ respectively. Both numbers are higher than the value for the control Matrigel sample (1.84(±0.55)xl0⁵), and HPLG-D showed the largest cell number. However, the differences in the cell numbers between the three samples, after one day's culture, were not statistically significant (P>0.05). PLGA-D and control samples underwent a slight increase in cell number after 7 days culture (p>0.05). There was however a statistically significant increase (p<0.05) in the cell number for HPLG-D at day 7 compared to day 1 , the cell number reaching 4.88(±0.29)x 10⁵. Comparing the proliferation results at day 7, the increase in cell number after 7 days for HPLG-D was significantly greater than that observed for the PLGA-D and control Matrigel samples (p<0.05 for HPLG-D vs PLGA-D and p<0.01 for HPLG-D vs control sample). The significant increase in cell number on HPLG-D after 7 days shows that good proliferation of osteoblasts occurs on HPLG-D. This can be explained by the achievement of more effective cell growth following cell attachment to HPLG-D after the first day of incubation. The good spread of cells achieved after the initial incubation is more favourable for cell proliferation and differentiation. This was confirmed by the SEM image of Fig. 24, which shows flattened cells that are well spread out, across the whole surface of the HPLG-D, their filopodia and lamellipodia being connected and in good contact with the HA coating surface.

Claims

1. A coated microparticle comprising; (i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and

(ii) an outer layer which comprises calcium phosphate.

2. A coated microparticle according to claim 1 wherein the pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the core.

3. A process for producing a coated microparticle, which coated microparticle comprises:

(i) a core, which core comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound, which pharmaceutically active compound is encapsulated in said polymer; and (ii) an outer layer which comprises calcium phosphate; which process comprises:

(A) producing a microparticle, which microparticle comprises: a polymer, an anionic surfactant, and a hydrophilic pharmaceutically active compound encapsulated in said polymer, by a process which comprises:

(a) preparing a solid-in-oil-in-water emulsion, which emulsion comprises: (i) a solid phase which comprises a hydrophilic pharmaceutically active compound, (ii) an oil phase which comprises a polymer and a solvent which is immiscible with water, and (iii) an external aqueous phase which comprises water and an anionic surfactant; and

(b) evaporating said solvent which is immiscible with water to produce said microparticle; and (B) coating the microparticle by contacting it with a solution comprising calcium and phosphate ions.

4. A process according to claim 3 wherein said microparticle is contacted with said solution comprising calcium and phosphate ions for no more than 10 hours.

5. A process according to claim 3 or claim 4 wherein said solution comprising calcium and phosphate ions is a supersaturated solution of calcium phosphate.

6. A process according to claim 5 wherein the supersaturation of said solution is maintained during step (B).

7. A process according to any one of claims 3 to 6 wherein during step (B) the calcium and phosphate ions in said solution are maintained at a molar ratio of 10/6.

8. A coated microparticle according to claim 1 or claim 2 or a process according to any one of claims 3 to 7 wherein the calcium phosphate of said outer layer is hydroxyapatite.

9. A coated microparticle according to claim 1 or claim 2 or claim 8 or a process according to any one of claims 3 to 8, wherein the pharmaceutically active compound of the coated microparticle is present in an amount of at least 1.0 weight % based on the total weight of the coated microparticle.

10. A coated microparticle according to any one of claims 1 , 2, 8 or 9 or a process according to any one of claims 3 to 9, wherein the average diameter of the coated microparticle is from 10 to 100 μm.

11. A coated microparticle according to any one of claims 1 , 2, 8, 9 or 10, or a process according to any one of claims 3 to 10, wherein the average thickness of said outer layer is from 0.2 to 5.0 μm.

12. A microparticle which comprises: a polymer; an anionic surfactant; and a hydrophilic pharmaceutically active compound encapsulated in said polymer, which pharmaceutically active compound is present in an amount of at least 1.0 weight % based on the total weight of the microparticle.

13. A process for producing a microparticle, which microparticle comprises: a polymer; an anionic surfactant; and a hydrophilic pharmaceutically active compound encapsulated in said polymer; which process comprises:

(a) preparing a solid-in-oil-in- water emulsion, which emulsion comprises: (i) a solid phase which comprises a hydrophilic pharmaceutically active compound, (ii) an oil phase which comprises a polymer and a solvent which is immiscible with water, and

(iii) an external aqueous phase which comprises water and an anionic surfactant; and

(b) evaporating said solvent which is immiscible with water to produce said microparticle.

14. A process according to any one of claims 3 to 11 and 13 wherein step (a) comprises:

(al) dispersing said hydrophilic pharmaceutically active compound in a mixture of (i) said polymer and (ii) said solvent which is immiscible with water;

(a2) agitating the resulting mixture, to produce a solid-in-oil dispersion; and (a3) mixing said solid-in-oil dispersion with an aqueous solution of said anionic surfactant, to produce said solid-in-oil-in-water emulsion.

15. A process according to claim 14 wherein step (a) further comprises: (a4) mixing the solid-in-oil-in-water emulsion with a further aqueous solution of said anionic surfactant.

16. A process according to any one of claims 3 to 11 and 13 to 15 wherein the step of evaporating said solvent which is immiscible with water comprises stirring said solid-in-oil-in-water emulsion and allowing said solvent to evaporate.

17. A process according to any one of claims 3 to 11 and 13 to 16 which further comprises:

(c) isolating said microparticle.

18. A process according to claim 17 which further comprises:

(d) washing said microparticle.

19. A process according to any one of claims 3 to 11 and 13 to 18 wherein the encapsulation efficiency of said hydrophilic pharmaceutically active compound in said polymer is at least 10 %.

20. A microparticle according to claim 12 or a process according to any one of claims 3 to 11 and 13 to 19, wherein the microparticle is negatively charged.

21. A microparticle according to claim 12 or claim 20 or a process according to any one of claims 3 to 11 and 13 to 20, wherein the microparticle has a zeta potential of less than -25.0 mV.

22. A microparticle according any one of claims 12, 20 and 21 or a process according to any one of claims 3 to 11 and 13 to 21, wherein the average diameter of the microparticle is from 1 to 20 μm.

23. A microparticle according to any one of claims 12 and 20 to 22, a coated microparticle according to any one of claims 1 , 2 and 8 to 11 or a process according to any one of claims 3 to 11 and 13 to 22 wherein the microparticle is a microsphere.

24. A microparticle according to any one of claims 12 and 20 to 23, a coated microparticle according to any one of claims 1, 2, 8 to 11 and 23, or a process according to any one of claims 3 to 11 and 13 to 23 wherein said anionic surfactant is a saponified fatty acid, an alkyl or aryl sulphonate, an alkyl or aryl sulphate, a sulphate ester, a phosphate ester, an alkyl or aryl phosphate, an alkyl or aryl phosphonate, a fatty acid, a naphthalene sulphonate (NAS), oleate sodium, a formaldehyde polycondensate, a polystyrene sulphonate, a hydrophobe-modified NAS or a surfactant which comprises a hydrophobic tail and an ionic head which comprises a -SO₃ ^" group.

25. A microparticle according to any one of claims 12 and 20 to 24, a coated microparticle according to any one of claims 1 , 2, 8 to 11 , 23 and 24 or a process according to any one of claims 3 to 11 and 13 to 24 wherein said anionic surfactant is sodium dodecyl sulphate (SDS) or docusate sodium (DCS).

26. A microparticle according to any one of claims 12 and 20 to 25, a coated microparticle according to any one of claims 1, 2, 8 to 11 and 23 to 25 or a process according to any one of claims 3 to 11 and 13 to 25 wherein said polymer is poly(DL- lactic-co-glycolic acid), poly-e-caprolactone, poly(lactic acid) or a copolymer of any of said polymers.

27. A microparticle according to any one of claims 12 and 20 to 26, a coated microparticle according to any one of claims 1 , 2, 8 to 11 and 23 to 26 or a process according to any one of claims 3 to 11 and 13 to 26 wherein said hydrophilic pharmaceutically active compound is a protein, an anti-inflammatory drug, an antibiotic, an anti-cancer drug, a compound which treats a specific bone disorder, a bone growth factor or a compound which relieves pain.

28. A microparticle according to any one of claims 12 and 20 to 27, a coated microparticle according to any one of claims 1 , 2, 8 to 11 and 23 to 27 or a process according to any one of claims 3 to 11 and 13 to 27 wherein said hydrophilic pharmaceutically active compound is amoxicillin, parathyroid hormone, a vitamin D derivative, a bisphosphanate, a bone morphogenetic protein, an analgesic, a ³²P or ⁸⁹Sr containing compound, prostaglandin, an interleukin 6 inhibitor or an antibiotic.

29. A coated microparticle according to claim 1, claim 2 or any one of claims 8 to 11 and 23 to 28 wherein the outer layer comprises a further pharmaceutically active compound.

30. A process for producing a coated microparticle according to any one of claims 3 to 11 and 14 to 28 further comprising:

(C) incorporating a further pharmaceutically active compound into said outer layer.

31. A microparticle produced by a process as defined in any one of claims 13 to 28.

32. A coated microparticle produced by a process as defined in any one of claims 3 to 11 and 14 to 28.

33. A solid substrate, which substrate has attached thereto a layer comprising coated microparticles, which coated microparticles are as defined in any one of claims 1, 2, 8 to 11, 23 to 29 and 32.

34. A medically acceptable implant, which implant comprises a solid substrate as defined in claim 33.

35. A tissue engineering scaffold comprising coated microparticles, wherein the coated microparticles are as defined in any one of claims 1, 2, 8 to 11, 23 to 29 and 32 and wherein the hydrophilic pharmaceutically active compound is a bone growth factor.

36. An injectable formulation comprising coated microparticles, wherein the coated microparticles are as defined in any one of claims 1, 2, 8 to 11, 23 to 29 and 32 and wherein the formulation is self-setting.

37. A microparticle according to any one of claims 12, 20 to 28 and 31 , a coated microparticle according to any one of claims 1, 2, 8 to 11, 23 to 29 and 32, a solid substrate according to claim 33, a medically acceptable implant according to claim 34, a tissue engineering scaffold according to claim 35, or an injectable formulation according to claim 36, for use in a method of treatment of the human or animal body by therapy.

38. A microparticle according to any one of claims 12, 20 to 28 and 31, a coated microparticle according to any one of claims 1, 2, 8 to 11, 23 to 29 and 32, a solid substrate according to claim 33, a medically acceptable implant according to claim 34, a tissue engineering scaffold according to claim 35, or an injectable formulation according to claim 36, for use in the treatment of a bone disorder.

39. A microparticle according to any one of claims 12, 20 to 28 and 31, a coated microparticle according to any one of claims 1, 2, 8 to 11, 23 to 29 and 32, a solid substrate according to claim 33, a medically acceptable implant according to claim 34, a tissue engineering scaffold according to claim 35, or an injectable formulation according to claim 36, for use in the delivery of said hydrophilic pharmaceutically active compound in a patient in need thereof.

40. A microparticle, a coated microparticle, a solid substrate, a medically acceptable implant, a tissue engineering scaffold or an injectable formulation according to any one of claims 37 to 39 wherein said treatment or said delivery comprises sustained release of the pharmaceutically active compound.

41. A microparticle, a coated microparticle, a solid substrate, a medically acceptable implant, a tissue engineering scaffold or an injectable formulation according to claim 40 wherein the release of the pharmaceutically active compound is sustained for at least 3 days.

42. A microparticle, a coated microparticle, a solid substrate, a medically acceptable implant, a tissue engineering scaffold or an injectable formulation according to any one of claims 39 to 41 wherein the delivery of said hydrophilic pharmaceutically active compound is to bone.