WO2005087193A2 - Chemoembolisation involving an anthracycline compound - Google Patents

Abstract

A composition for chemoembolotherapy of colorectal metastases in liver preferably comprises particles of a water-insoluble water-swellable polymer and, absorbed therein an anthracycline in high dose. Suitably the polymer is a synthetic anionic polymer, preferably a poly (vinyl alcohol) based polymer and the drug is doxorubicin.

Description

CHE OEMBOLISATION The present invention relates to the use of cytotoxic agents in the manufacture of a medicament for use in a method of treatment of colorectal metastases in liver in which method the cytotoxic agent is released from polymeric embolic material which embolises the metastases. Embolotherapy is a growing area of interventional medicine but normally relies upon the transarterial approach of the catheter to a desired location whereupon an agent is released in order to occlude a particular vessel. This treatment has been used in order to block the blood supply to certain hypervascularised tumours such as hepatocellular carcinoma and more recently is becoming a popular choice of treatment for uterine fibroids. There is a range of embolic materials in clinical use, that require transcatheter delivery to the site of embolisation, whereupon they are released into the blood stream to block it. This is achieved either by a physical blocking of the vessel using small particles or spheres, or in the case of liquid embolic agents, require some sort of phase change or reaction to set the flowable material and form a cast within the vessel. The most popular particulate-based embolic agent is poly(vinyl alcohol) (PVA) foam particles ( e.g. Ivalon) which has been used for several decades. Recently, this material has been available in particulate, rather than sheet form, and does not require granulation by the surgeon prior to delivery. In WO-A-0168720, PVA based compositions for embolotherapy are described. The PVA is, initially, derivatised to form a macromonomer, having pendant acrylic groups. Subsequently, these acrylic groups are polymerised, optionally in the presence of comonomer, to form a water- insoluble water-swellable polymer matrix. The polymerisation reaction may be carried out t7 situ, whereby the PVA is rendered water-insoluble after delivery into the vessel, at the embolisation site. Alternatively, the polymerisation is conducted prior to delivery, generally to form microspheres, which are delivered in suspension in an aqueous vehicle. In WO-A-0168720, it is suggested that biologically active agents may be included in the embolic compositions, whereby active agent may be delivered from the formed hydrogel. One class of active agents is chemotherapeutic agents. Examples of chemotherapeutic agents are cisplatin, doxorubicin and mitomycin. Some general guidance is given as to methods of incorporating the active agents into the embolic compositions. Where the composition is a liquid which is cured in situ, the active may be simply mixed with the liquid. Where the articles are preformed, it is suggested that the active may be incorporated by "encapsulation", or by coating onto the surface. There are no worked examples in which a therapeutic agent is incorporated into any type of composition. Microspheres of hydrogel material formed from poly(hydroxyethyl methacrylate), hydrolysed poly(methyl methacrylate) and PVA crosslinked using aldehyde crosslinking agents such as glutaraldehyde, have also been used as embolic agents. Hydroxyethyl methacrylate may be copolymerised with comonomers, for instance having acidic groups. For instance, a crosslinked copolymer of hydroxyethyl methacrylate with about 1-2 mole% acrylic acid cross-linked by 0.3-1.0mole% ethylene glycol dimethacrylate, has an equilibrium water content in the range 55-60% by weight, and has been used as a contact lens formulation for many years. One embolic product on the market is marketed by Biosphere, which comprises microspheres of trisacrylgelatin having a coating of collagen. Collagen has an overall cationic charge as physiological pH's. In Ball, D.S. et al. , J. Vase. Interv. Radiol. (2003), 14, 83-88, Biosphere show that the microspheres' mechanical characteristics are not adversely affected when admixed with a range of drugs commonly administered along with the embolic compositions. Doxorubicin, cisplatin and mitomycin are specifically tested. Doxorubicin and other anthracyclines have been incorporated into a variety of polymeric matrices based delivery systems, such as microspheres of polylactides or polyglycolides and cross-linked fibrinogen and albumin microspheres. Juni, K. et a/ in Chem. Pharm. Bull. (1985), 33(1), 313-318 describe the incorporation of doxorubicin into poly(lactic acid) microspheres and the delivery of the composition intra arterially to dog liver. The composition embolised peripheral hepatic arteries. These types of microspheres are hard and are not easy to store and deliver. Doxorubicin has been covalently linked to the surface of cross-linked poly(vinyl alcohol) and tested for its cytotoxic properties (Wingard, L B et al. Cancer Research (1985) 45(8) 3529-3536). Since the drug is covalently bonded to the polymer it must be cleaved before being released from the surface and hence may not be released under physiological conditions. Jones, C. etal in Brit. J. Cancer (1989) 59(5) describe incorporation of doxorubicin into ion-exchange microspheres and the use of the compositions in the chemoembolotherapy of tumours in a rat model. In our earlier application PCT/GB04/00548 we describe a composition suitable for embolisation, which comprises particles having a matrix of water- swellable water-insoluble polymer and, absorbed in the matrix, a water- soluble therapeutic agent, and is characterised in that the polymer has an overall anionic charge at a pH in the range 6 to 8, in that the particles, when swollen to equilibrium in water have particle sizes in the range 40-1500 μm and in that the therapeutic agent is an anthracycline compound having at least one amine group. The compositions are useful to embolise hepatocellular carcinoma. The most common tumour that metastasizes to the liver is colorectal cancer which accounts for 60000 deaths and 155000 new cases of malignancy per year in the US (Choti and Bulkley 1999). Colorectal cancer arises following a process of genetic change in the epithelial cells of the colonic mucosa (Vogelstein B, Fearon ER, Hamilton SR et al. Genetic alterations during colorectal tumour development. N Eng J Med 1998; 319: 525). The first and most successful drug used in the treatment of CRC liver metastases is 5-Fluorouracil (5-FU). The standard treatment is a combination of 5-FU and leucovorin or folinic acid (Ehrlichman et al 1998). More recently, Irinotecan hydrochloride (CPT-11 ), oxaliplatin and raltitrexed (tomudex) have also become available for use in advanced colorectal cancer. Chemotherapy for CRC metastases is usually well tolerated but recognized side effects such as nausea, vomiting, diarrhoea, neurological symptoms and leucopoenia occur in about 50% of patients (Cunningham D, Pyrhonen S, James RD, Punt CJA et al. Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer. Lancet 1998; 352: 1413 - 1418). Although complete response is rare in patients with liver metastases, partial response occurs in approx 30% of patients treated with 5-FU and 50% with Irinotecan (Ehrlichman C, Fine S, Wong A et al. Randomised trial of fluorouracil and folinic acid in patients with metastatic colorectal carcinoma. J Clin Oncol 1998; 6: 469 - 475, Cunningham et al 1998 op. cit, Tourniganc C, Louvet C, Quinaux E et al Folfiri followed by Folfox versus Folfox followed by Folfiri in metastatic colorectal cancer (MCRC): final results of a phase III study. Proceedings of the Am Soc Oncol (ASCO) 2001 ; 20 no 494). Hepatic Artery Chemotherapy (HAC) is administered via a catheter inserted into the hepatic artery and connected to either a subcutaneous pump or subcutaneous port which in turn is connected to an external portable pump. Compared with systemic treatment, HAC produces higher response rates, however, randomized controlled studies have not proven a survival advantage of HAC. Transarterial chemoembolisation (TACE) involves the periodic injection of a chemotherapeutic agent, mixed with an embolic material, into selected branches of the hepatic arteries feeding a liver tumour. Normal liver receives approx 70% of its blood supply from the portal vein and 30% from branches of the hepatic artery. In contrast, HCC tumours are hypervascular and receive most of their blood supply from the hepatic artery. The rational for TACE is that the infusion of drug such as doxorubicin, mitomycin C and cisplatin with a viscous material (eg lipiodol) followed by „_-„_,,_-, PCT/GB2005/000958

embolisation of the blood vessel with gelfoam, PVA particles or spherical embolic agents will occlude arterial blood supply to the tumour causing an infarct and subsequent necrosis of the tumour. The advantage of TACE is that higher concentrations of the drug can be delivered to the tumour with decreased systemic exposure compared with systemic chemotherapy. Phase II chemoembolisation studies for metastatic colorectal cancer have been conducted in a number of centres. A single study has reported a complete response of 17% and 1- and 2-year survival rates of 68% and 37% respectively using doxorubicin as the only chemotherapeutic, and lipiodol (Lang EK and Brown C Jr. Colorectal metastases to the liver: selective chemoembolization. Radiology 1993; 189: 417-422) but it is uncertain whether the effect was attributable to drug, oil or combination as there was no control arm. In another report, chemoembolisation with 5-fluorouracil, mitomycin C and gelatin sponge showed 63% partial or minor tumour morphologic responses and 62% had decreased carcinoembryonic antigen level greater than 50% with median survival of 10 months (Sanz-Altamira PM, Spence LD, Huberman MS et al: Selective chemoembolization in the management of hepatic metastases in refractory colorectal carcinoma: A phase II trial. Dis Colon Rectum 1997; 40: 770-775). Doxorubicin is reported to be ineffective against colorectal cancer

((http:/AAΛ w.cancemetwork.com/textbook/morev17.htm "Doxorubicin, epirubicin, etoposide, carboplatin, cyclophosphamide, methotrexate, topotecan, CI 980, paclitaxel, docetaxel, and cisplatin are inactive against colorectal adenocarcinomas.")) The effectiveness of Doxorubicin as an anti-cancer agent is somewhat determinant on the ability of the tumour to take up the drug at a greater rate than the surrounding normal tissue. In the case of HCC, the tumour cells proliferate at a greater rate than the normal hepatocytes and if the drug can be placed at the tumour site for a sufficient period, uptake into the tumour cells will occur preferentially over normal cells. In the case of metastases of the liver it is clear that the biology of the tumour is such that conventional means of delivery do not provide a sufficiently high enough or long enough exposure to the drug for it to have any significant effect, even though in-vitro, the drug is active against colorectal metastases cell lines. Doxorubicin has low hepatic extraction compared to treatment of choice - 5 FU suggesting that systemic or indeed intraarterial delivery of doxorubicin will be severely limited in tumour uptake because of this.

Drugs commonly used in hepatic artery infusion therapy for treatment of liver metastases* Drug Hepatio's stemtc ratio % Hepatic extraction Floxuridirse 400 95-99 5-FluorouracH 10-100 19-81 itomyαn-C 3 15-20 Cisplatin 5 10-20 Doxorubicin 2 20-30

Drugs for I patic Λrtetis.1 Infusi n (II Al) Estimated Inciωised Drug Malf-Liie fmia. fcxpesure byHAI Fkι rυunκ.1 (FU. JO 5-IO-fβW 5-Fluoro-2- eox xindmc « KUDR'l <10 lUCMXMold Bisc fe tbvlrøirosourea (BCNUϊ 5 6-7-fold

€tsjviatjn 20- 30 4- 7-fold Adπamycifl t «xo_ubιcιn 60 2-tb ! liydioeh.oride)

Ref: Bast et al (2000) Cancer Medicine , Hamilton, Ontario: BC Decker INC, pp1436-1464 Despite the widespread usage of chemoembolisation, no randomized study to date has demonstrated that the theoretical advantage of adding chemotherapy translates into a clinical advantage for colorectal metastases. Doxorubicin is thought to be ineffective as the concentration in the tumour is not high enough. In vitro evaluation of the cytotoxic effect of doxorubicin on the growth of human colorectal cancer cell lines demonstrates that the drug is effective at levels at least 1000 fold lower than those for 5-FU. This indicates the concentration of doxorubicin within a tumour in vivo does not reach sufficiently high enough levels to effect cell 5 death when administered by ordinary means. (Ref: Abaza et al (2003) Tumor Biology, Vol 24 p 241-257). The concentration of doxorubicin given cannot be raised without compromising cardiac toxicity (150mg maximum single dose; 550mg maximum lifetime dose (with very rare cases >1000mg have been reported).0 In Proc. Int. Symp. Control. Rel. Bioact. Mater. 18, 128-129 (1991) Cremers, H.F.M. et al. describe formation of albumin-heparin microspheres (cross-linked with glutaraldehyde) and loading of these with doxorubicin by a swelling-deswelling process. The microspheres had a dry size of 5 to 35 μm and the fraction with sizes 10 to 25 μm was used in the drug loading tests.5 The microspheres were administered via the portal vein to rats and tissue distribution investigated after 1 hour. This revealed good liver/heart ratio (about 3). The microsphere sizes are smaller than the minimum considered useful for embolisation. In Cardiovasc. Intervent. Radiol. 1992, 15, 535 Fischbach, W. etal. o describe TACE via the hepatic artery for primary and secondary tumours. The embolic agent was Ivalon (foamed poly(vinyl-alcohol) or Ethibloc. The chemotherapeutic agent was mitomycin C or epirubicin. The administration protocol is not described in detail but TACE generally involves administration of drug followed by_^ separate administration of embolic agent.5 The side-effects were extreme. It is not clear that epirubicin was effective. In Cancer 1998, 82, 1250-9, Tellez, C. et al describe clinical trials in which hepatic artery chemoembolisation of metastatic CRC is conducted 8.75 ml of a mixture of a cross-linked fibrous collagen material (10 mg/ml) (Angiostat) and cisplatin, doxorubicin (3mg/ml) or mitomycin C was injected 0 by microcatheter into the hepatic artery. Collagen tends to be positively charged. The authors concluded their study did not show conclusively whether there were benefits over intraarterial chemotherapy. According to the present invention there is provided a new use of an anthracycline compound in the manufacture of a medicament for use in a method of treatment of a colorectal metastasis of the liver in which method the anthracycline compound is released from a polymeric matrix forming an embolus of the metastasis in the liver, in which the release rate of the anthracycline compound from the polymeric matrix under test conditions has initial t¹ greater than 20 mins. In the method it is preferred that the polymeric matrix, when present in the embolus comprises a water-insoluble water-swellable polymer. The composition administered to the patient may comprise water-soluble polymer which is converted into water-insoluble polymer after administration into the blood vessel. Such a reaction is, for instance, a cross-linking reaction or a polymerisation reaction of monomers and/or prepolymers with which the anthracycline compound is admixed before administration. Preferably, however the composition which is administered is substantially in the form of the matrix from which the anthracycline is to be released in the method of treatment. Thus, the composition which is administered comprises the polymer in the form of a water-insoluble, water-swellable matrix in or on which the anthracycline is absorbed or adsorbed. The polymer is preferably, in the composition which is administered, in the form of a hydrogel, preferably with extra aqueous liquid, e.g. in which particles of hydrogel are suspended. Although this composition may be made up immediately before administration, it is preferred that the polymer and anthracycline composition is preformed and is substantially at equilibrium, that is the anthracycline is absorbed to equilibrium in the matrix. The initial VΛ value is the time at which half the drug load is released from the polymeric matrix. This is measured by the test used in Example 6. Generally the polymer is covalently crosslinked, although it may be appropriate for the polymer to be ionically crosslinked, at least in part. In a preferred embodiment, the polymer is preferably substantially wholly synthetic, that is it is substantially free of biological polymers such as proteins and polysaccharides. In another preferred embodiment, the polymer comprises polysaccharide and preferably is substantially free of protein. Preferably the polymer is substantially non-biodegradable, that is it remains as an embolus for a useful and therapeutically effective period after delivery. The polymer may be formed by polymerising ethylenically unsaturated monomers in the presence of di- or higher-functional crosslinking monomers. Many of the potentially active anthracyclines have at least one amine group. We have found that high doses of such compounds can be incorporated into polymeric matrices which have an overall anionic charge at a pH in the range 6 to 8. Therefore such polymers are preferred. To form such polymers the ethylenically unsaturated monomers preferably include an anionic monomer. Copolymers of hydroxyethyl methacrylate, acrylic acid and cross- linking monomer, such as ethylene glycol dimethacrylate or methylene bisacrylamide, as used for etafilcon A based contact lenses, may be used. An alternative method is to functional ise a preformed polymer by a reaction which provides anionic pendant groups. Another type of polymer which may be used to form the water- swellable water-insoluble matrix is polyvinyl alcohol crosslinked using aldehyde type crosslinking agents such as glutaraldehyde. For such products, the polyvinyl alcohol is preferably rendered anionic, for instance by providing pendant anionic groups by reacting a functional acidic group containing monomer with the hydroxyl groups. Examples of suitable reagents are di-acids, for instance dicarboxylic acids. Suitable polymeric matrices, some of which are used commercially as embolic agents are: Ivalon - polyvinyl alcohol foam SAP microspheres (copolymer of sodium acrylate and vinyl alcohol) Trufill (PVA Particles) Ionic styrene/DVB microspheres (Ion exchange resins) Alginate microspheres (such as, for example, those described in EP- A-1025869 and WO-A-00/09566) PLA/PLGA microspheres The invention is of particular value where the polymer matrix is formed of a polyvinyl alcohol macromer, having more than one ethylenically unsaturated pendant group per molecule, by copolymerisation with ethylenically unsaturated monomers preferably including an acidic monomer. The PVA macromer may be formed, for instance, by providing PVA polymer, of a suitable molecular weight such as in the range 1000 to 500,000 D, preferably 10,000 to 100,000 D, with pendant vinylic or acrylic groups. Pendant acrylic groups may be provided, for instance, by reacting acrylic or methacrylic acid with PVA to form ester linkages through some of the hydroxyl groups. Methods for attaching vinylic groups capable of polymerisation onto polyvinyl alcohol are described in, for instance, US 4,978,713 and, preferably, US 5,508,317 and 5,583,163. Thus the preferred macromer comprises a backbone of polyvinyl alcohol to which is linked, via a cyclic acetal linkage, to an (alk)acrylaminoalkyl moiety. Example 1 describes the synthesis of such a macromer. Preferably the PVA macromers have about 2 to 20 pendant ethylenic groups per molecule, for instance 5 to 10. Where PVA macromers are copolymerised with ethylenically unsaturated monomers including an acidic monomer, the acidic monomer preferably has the general formula I

Y¹BQ

in which Y¹ is selected from

CH₂=C(R)-CH₂-O-, CH₂=C(R)-CH₂ OC(O)-, CH₂=C(R)OC(O)-, CH₂=C(R)-O-, CH₂=C(R)CH₂OC(O)N(R¹)-, R²OOCCR=CRC(O)-O-, RCH=CHC(O)O-, RCH=C(COOR²)CH₂-C(O)-O-,

wherein: R is hydrogen or a 0^₄ alkyl group; R¹ is hydrogen or a C C₄ alkyl group; R² is hydrogen or a C_1J} alkyl group or BQ where B and Q are as defined below; A is -O- or -NR¹ -; K¹ is a group -(CH₂)_rOC(O)-, -(CH₂)_rC(O)O-, - (CH₂)_rOC(O)O-, -(CH₂)_rNR³-, -(CH₂)_rNR³C(O)-, -(CH₂)_rC(O)NR³-, -(CH₂)_rNR³C(O)O-, -(CH₂)_rOC(O)NR³-, -(CH₂)_rNR³C(O)NR³- (in which the groups R³ are the same or different), -(CH₂)_rO-, -(CH₂)_rSO₃ -, or, optionally in combination with B\ a valence bond and r is from 1 to 12 and R³ is hydrogen or a ( C₄ alkyl group; B is a straight or branched alkanediyl, oxaalkylene, alkanediyloxaalkanediyl, or alkanediyloligo(oxaalkanediyl) chain optionally containing one or more fluorine atoms up to and including perfluorinated chains or, if Q or Y¹ contains a terminal carbon atom bonded to B a valence bond; and Q is an anionic group. The anionic group may be, for instance, a carboxylate, carbonate, sulphonate, sulphate, nitrate, phosphonate or phosphate group, preferably a sulphonate group. The monomer may be polymerised as the free acid or in salt form. Preferably the pr . of the conjugate acid is less than 5. In the monomer of general formula I preferably Y¹ is a group

CH₂=CRCOA- in which R is H or methyl, preferably methyl, and in which A is preferably NH. B is preferably an alkanediyl group of 1 to 12, preferably 2 to 6 carbon atoms. One particularly preferred type of monomer is an (alk)acrylamido alkane-sulphonic acid, such as 2-acrylamido-2-methyl-1 -propane-sulphonic acid (AMPS). There may be included in the ethylenically unsaturated monomer diluent monomer, for instance non-ionic monomer. Such monomer may be useful to control the pK, of the acid groups, to control the hydrophilicity or hydrophobicity of the product, to provide hydrophobic regions in the polymer, or merely to act as inert diluent. Examples of non-ionic diluent monomer are, for instance, alkyl (alk) acrylates and (alk) acrylamides, especially such compounds having alkyl groups with 1 to 12 carbon atoms, hydroxy, and di- hydroxy-substituted alkyl(alk) acrylates and -(alk) acrylamides, vinyl lactams, styrene and other aromatic monomers. The ethylenically unsaturated monomer may also include zwitterionic monomer, for instance to increase the hydrophilicity, lubricity, biocompatibility and/or haemocompatibility of the particles. Suitable zwitterionic monomers are described in our earlier publications WO-A- 9207885, WO-A-9416748, WO-A-9416749 and WO-A-9520407. Preferably a zwitterionic monomer is 2-methacryloyloxy-2'-trimethylammonium ethyl phosphate inner salt (MPC). In the polymer matrix, the level of anion is preferably in the range 0.1 to 10 meq g^'1, preferably at least 1.0 meq g^"1. Where PVA macromer is copolymerised with other ethylenically unsaturated monomers, the weight ratio of PVA macromer to other monomer is preferably in the range of 50:1 to 1:5, more preferably in the range 20:1 to 1 :2. In the ethylenically unsaturated monomer the anionic monomer is preferably present in an amount in the range 10 to 100 mole%, preferably at least 25 mole%. Preferably the water-insoluble water-swellable polymer has an equilibrium water content measured by gravimetric analysis of 40 to 99 weight %, preferably 75 to 95%. The polymer is preferably provided in the composition which is administered to the patent in the form of particles. These may be formed in several ways. For instance, the crosslinked polymer may be made as a bulk material, for instance in the form of a sheet or a block, and subsequently be comminuted to the desired size. Alternatively, the crosslinked polymer may be formed as such in particulate form, for instance by polymerising droplets of monomer in a dispersed phase in a continuous immiscible carrier. Examples of suitable water-in-oil polymerisations to produce particles having the desired size, when swollen, are known. For instance US 4,224,427 describes processes for forming uniform spherical beads of up to 5 mm in diameter, by dispersing water-soluble monomers into a continuous solvent phase, in the presence of suspending agents. Stabilisers and surfactants may be present to provide control over the size of the dispersed phase particles. After polymerisation, the crosslinked microspheres are recovered by known means, and washed and optionally sterilised. Preferably the particles eg microspheres, are swollen in an aqueous liquid, and classified according to their size. Preferably when the particles are swollen to equilibrium in water, they have a size in the range from 40 to 1500μm. It is known that use of polymer particles less than 10Oμm in diameter may lead to excessive necrosis which can lead to abscesses which are difficult to treat. Preferably the particles when swollen in water to equilibrium have a particle size of at least 100μm. The therapeutic active used in the present invention is an anthracycline compound, which comprises an anthraquinone group to which is attached an amine sugar. The amino group on the sugar is believed to associate with anionic groups in the preferred polymer matrix, to allow high levels of loading and controlled delivery after administration. The dose range is 10 to 100mg/ml of administered composition, preferably 50mg/ml. The amount of composition used in each procedure is 1 to 8ml, preferably 4ml. The number of repeat treatments is 1 to 5 more (2-6 overall treatments), preferably 3 times (4 treatments). Preferably the composition comprises hydrogel particles in or on which the anthracycline compound is absorbed or adsorbed and suspending liquid, which may additionally comprise an imaging agent such as a radiopaque agent. Examples of suitable anthracyclines have the general formula II

(axial) Doxorubicin (axial) Daunorubicin

(axial) Idarubicin X=COCH₂OH Y=OCH₃ Z=HO (equatorial) Epriubicin We have found that doxorubicin, has particularly interesting loading and release characteristics for preferred polymers. The advantage of the present invention is that high doses of the anthracycline compound can be delivered locally to the metastasis without concomitant side effects exhibited when a compound is administered systemically. The drug appears to have a particular affinity for poly(vinyl alcohol-graft-acrylamido propane sulphonic acid), so that high levels of doxorubicin are capable of incorporation into the polymer, and release over many days. In order for microspheres of the present invention to be useful for the treatment of colorectal metastases, the device must maintain a sustained release of the doxorubicin into the tumour tissue over a number of days. This is demonstrated in-vivo using microspheres of the invention in the rabbit VX-2 tumour model in which drug was present in tumour tissue for at a least two week period. This type of release modality requires a method for slowing drug release from the microspheres other than by normal diffusion. Such method may also provide the necessary interaction to ensure high loading of the drug into the device. Methods of slowing the release may include any of the classical approaches such as the use of impermeable barriers, use of co-excipient release modifiers, cross-linking chemistries, bioerosion or degradation, covalent attachment of the drug with hydrolysable linkages or as exemplified here, the use of ionic interactions. In order that the devices are useful for embolisation of colorectal metastases and that a local delivery of doxorubicin can be sustained for a sufficient length of time in order to be efficacious, a slower release modality is necessary. It is therefore desirable that the under sink conditions (i.e. in which release is conducted in a large enough volume to overcome contribution of solubility factors), that less than 50% of the total drug is released in 20 minutes. This may alternatively be expressed as an initial VΛ (time for half the drug to be released) of more than 20 mins. The therapeutic active may be incorporated into the polymer matrix by a variety of techniques. In one method, the therapeutic active may be mixed with a precursor of the polymer, for instance a monomer or macromer mixture or a cross-linkable polymer and cross-linker mixture, prior to polymerising or crosslinking. Alternatively, the active may be loaded into the polymer after it has been crosslinked. For instance, particulate dried polymer may be swollen in a solution of therapeutic active, preferably in water, optionally with subsequent removal of non-absorbed agent and/or evaporation of solvent. A solution of the active, in an organic solvent such as an alcohol, or, more preferably, in water, may be sprayed onto a moving bed of particles, whereby drug is absorbed into the body of the particles with simultaneous solvent removal. Most conveniently, we have found that it is possible merely to contact swollen particles suspended in a continuous liquid vehicle, such as water, with a solution of drug, over an extended period, whereby drug becomes absorbed into the body of the particles. This is believed to be analogous to a cation exchange type process. The swelling vehicle may subsequently be removed or, conveniently, may be retained with the particles as part of the product for subsequent use as an embolic agent. In one particularly preferred embodiment the swollen particles are separated from swelling vehicle not absorbed into the matrix by a simple gel/liquid separation technique such as by filtration through a filter having suitable apertures, conveniently a glass filter. The slurry of swollen particles with little or no extra-particulate liquid may be pumped into suitable storage containers for sterilisation and storage as it is. It is found that the slurry is sufficiently stable, in that little exudation of liquid nor loss of drug occurs during storage in such a form. Alternatively, the suspension of particles can be filtered to remove any remaining drug loading solution and the particles dried by any of the classical techniques employed to dry pharmaceutical-based products. This could include, but is not limited to, air drying at room or elevated temperatures or under reduced pressure or vacuum; classical freeze-drying; atmospheric pressure-freeze drying; solution enhanced dispersion of supercritical fluids (SEDS). Alternatively the drug-loaded microspheres may be dehydrated using an organic solvent to replace water in a series of steps, followed by evaporation of the more volatile organic solvent. A solvent should be selected which is a non solvent for the drug. In brief, a typical classical freeze drying process might proceed as follows: the sample is aliquoted into partially stoppered glass vials, which are placed on a cooled, temperature controlled shelf within the freeze dryer. The shelf temperature is reduced and the sample is frozen to a uniform, defined temperature. After complete freezing, the pressure in the dryer is lowered to a defined pressure to initiate primary drying. During the primary drying, water vapour is progressively removed from the frozen mass by sublimation whilst the shelf temperature is controlled at a constant, low temperature. Secondary drying is initiated by increasing the shelf temperature and reducing the chamber pressure further so that water absorbed to the semi-dried mass can be removed until the residual water content decreases to the desired level. The vials can be sealed, in situ, under a protective atmosphere if required. Atmospheric pressure freeze drying is accomplished by rapidly circulating very dry air over a frozen product. In comparison with the classical freeze-drying process, freeze-drying without a vacuum has a number of advantages. The circulating dry gas provides improved heat and mass transfer from the frozen sample, in the same way as washing dries quicker on a windy day. Most work in this area is concerned with food production, and it has been observed that there is an increased retention of volatile aromatic compounds, the potential benefits of this to the drying of biologicals is yet to be determined. Of particular interest is the fact that by using atmospheric spray drying processes instead of a cake, a fine, free- flowing powder is obtained. Particles can be obtained which have submicron diameters, this is tenfold smaller than can be generally obtained by milling. The particulate nature, with its high surface area results in an easily rehydratable product. The composition which is administered to a colorectal metastasis (CRM) patient in need of embolotherapy of a colorectal metastasis in the liver is preferably an aqueous suspension of swollen particles containing absorbed drug. It is often desirable for the suspension to be mixed prior to delivery with an imaging agent such as a conventional radiopaque agent, as is used for gel type embolic compositions. For example an aqueous suspension of swollen particles containing absorbed anthracycline compound may be mixed immediately prior to administration with a liquid radiopaque agent conventionally used with embolic agents, e.g. lipiodol, in amounts in the range 2:1 to 1 :2, preferably about 1 :1 by volume. For the embodiment of the invention comprising a slurry of swollen particles with absorbed anthracycline compound but with little or no extra-particulate liquid, the slurry and contrast (radiopaque) agent may, similarly, be mixed together immediately before delivery, for instance in amounts in the range 1 :5 to 2:1 , preferably in the range 1 :2 to 1 :1 by volume. Where the compositions containing anthracycline compound are supplied for use in dried form particles may be added dry to contrast agent, or preferably, are initially swollen in an aqueous vehicle such as physiological saline, to form a slurry or suspension, before being blended with contrast agent prior to delivery. Alternatively or additionally particles may be pre-loaded with radiopaque material, in addition to the anthracycline. The composition which is administered may also be admixed with other therapeutic agents, or may be administered in separately but in combination with other therapeutic agents. Usually the composition is administered from a reservoir in a syringe using the conventional delivery devices, such as an intra-arterial catheter. The embolic composition as administered to the patient in need of CRM embolisation therapy, may be delivered as a single one-off dosage.

Embolisation is monitored by following the contrast agent using conventional techniques. It is usually found to be desirable for a second dose of an embolic composition, preferably for a second dose of the embolic composition to be delivered at a time interval after the first dose, for instance, to embolise newly formed blood vessels supplying the tumour e.g. after 4 to 10 weeks from the first treatment for a doxorubicin-containing composition. The composition will be administered in a drug dosage in the range 10-800 mg per treatment, although it may be possible to use higher dosages following adequate safety assessments. Preferred dosages per treatment for doxorubicin may be above 150mg for instance up to 1000 mg or more. The dosages are preferably as specified above. The invention further comprises a new composition comprising particles of a water-insoluble water-swellable polymer and absorbed in the matrix an anthracycline compound for use in a method of treatment in which the composition is introduced into the liver to embolise a colorectal metastasis wherein the release rate of the anthracycline from the polymeric matrix under test conditions has a VΔ of greater than 20 mins. In this aspect the features relating to the method are intended to be effective limitations on the protection sought and distinguish the claims from the prior art. The present invention is illustrated in the following examples for which the results are shown in the figures as follows: Figure 1 shows the results of example 2; Figure 2 shows the results of example 3; Figure 3 shows the results of example 4; Figure 4 shows the results of example 5; Figure 5 shows the results of example 6; Figures 6a and b show the results of example 7; Figure 7 shows the results of example 6; Figure 8 shows the results of example 12; Figure 9 shows the result of example 13; Figures 10 and 11 show the results of example 14; Figure 12 shows the results of example 15; Figure 13 shows the results of example 16; Figure 14 shows the results of example 17; Figure 15 shows the results of example 18; Figure 16 shows the results of example 19; Figure 17 shows the results of example 23; Figure 18 shows the results of example 25; Figure 19 shows the results of Example 26; Figures 20 to 22 show the results of Example 27; Figures 23 to 26 show the results of Example 28; Figures 27 and 28 show the results of Example 30; Figure 29 shows the results of Example 32; Figure 30 shows the results of Example 33; Figure 32 and 33 show the results of Example 36; Figure 34 and 35 show the results of Example 37; Figure 36 shows the results of Example 38; and Figure 37 shows the results of Example 39. Example 1 : Outline Method for the Preparation of Microspheres Nelfilcon B macromer synthesis: The first stage of microsphere synthesis involves the preparation of Nelfilcon B - a polymerisable macromer from the widely used water soluble polymer PVA. Mowiol 8-88 poly(vinyl alcohol) (PVA) powder (88% hydrolised, 12% acetate content, average molecular weight about 67,000D) (150g) (Clariant, Charlotte, NC USA) is added to a 2litre glass reaction vessel. With gentle stirring, 1000ml water is added and the stirring increased to 400rpm. To ensure complete dissolution of the PVA, the temperature is raised to 99 ±9°C for 2-3 hours. On cooling to room temperature N-acryloylaminoacetaldehyde (NAAADA) (Ciba Vision, Germany) (2.49g or 0.104mmol/g of PVA) is mixed in to the PVA solution followed by the addition of concentrated hydrochloric acid (100ml) which catalyses the addition of the NAAADA to the PVA by transesterification. The reaction proceeds at room temperature for 6-7 hours then stopped by neutralisation to pH 7.4 using 2.5M sodium hydroxide solution. The resulting sodium chloride plus any unreacted NAAADA is removed by diafiltration (step 2). Diafiltration of macromer: Diafiltration (tangential flow filtration) works by continuously circulating a feed solution to be purified (in this case nelfilcon B solution) across the surface of a membrane allowing the permeation of unwanted material (NaCl, NAAADA) which goes to waste whilst having a pore size small enough to prevent the passage of the retentate which remains in circulation. Nelfilcon B diafiltration is performed using a stainless steel Pellicon 2 Mini holder stacked with 0.1 m² cellulose membranes having a pore size with a molecular weight cut off of 3000 (Millipore Corporation, Bedford, MA USA). Mowiol 8-88 has a weight average molecular weight of 67000 and therefore has limited ability to permeate through the membranes. The flask containing the macromer is furnished with a magnetic stirrer bar and placed on a stirrer plate. The solution is fed in to the diafiltration assembly via a Masterflex LS peristaltic pump fitted with an Easy Load II pump head and using LS24 class VI tubing. The Nelfilcon is circulated over the membranes at approximately 50psi to accelerate permeation. When the solution has been concentrated to about 1000ml the volume is kept constant by the addition of water at the same rate that the filtrate is being collected to waste until 6000ml extra has been added. Once achieved, the solution is concentrated to 20-23% solids with a viscosity of 1700-3400 cP at 25°C. Nelfilcon is characterised by GFC, NMR and viscosity. Microsphere Synthesis: The spheres are synthesised by a method of suspension polymerisation in which an aqueous phase (nelfilcon B) is added to an organic phase (butyl acetate) where the phases are immiscible. By employing rapid mixing the aqueous phase can be dispersed to form droplets, the size and stability of which can be controlled by factors such as stirring rates, viscosity, ratio of aqueous/organic phase and the use of stabilisers and surfactants which influence the interfacial energy between the phases. Two series of microspheres are manufactured, a low AMPS and a higher AMPS series, the formulation of which are shown below. A High AMPS:

Aqueous: ca 21 % w/w Nelfilcon B solution (400 ±50g approx) ca 50% w/w 2-acrylamido-2-methylpropanesulphonate Na salt (140 ±10g) Purified water ( 137±30g) Potassium persulphate (5.22±0.1g) Tetramethyl ethylene diamine TMEDA (6.4±0.1 ml) Organic: n-Butyl acetate (2.7 ±0.3L) 10% w/w cellulose acetate butyrate in ethyl acetate (46±0.5g) (stabiliser) Purified water (19.0 ±0.5ml)

B Low AMPS:

Aqueous: ca 21 % w/w Nelfilcon B solution (900 ±1 OOg approx) ca 50% w/w 2-acryamido-2-methylpropanesulphonate Na salt (30.6 ±6g) Purified water (426±80g) Potassium persulphate (20.88±0.2g) TMEDA (25.6±0.5ml) Organic: n-Butyl acetate (2.2 ±0.3L) 10% w/w cellulose acetate butyrate (CAB) in ethyl acetate (92±1.0g) Purified water (16.7 ±0.5ml) A jacketed 4000ml reaction vessel is heated using a computer controlled bath (Julabo PN 9-300-650) with feedback sensors continually monitoring the reaction temperature. The butyl acetate is added to the reactor at 25°C followed by the CAB solution and water. The system is purged with nitrogen for 15 minutes before the PVA macromer is added. Cross linking of the dispersed PVA solution is initiated by the addition of TMEDA and raising the temperature to 55°C for three hours under nitrogen. Crosslinking occurs via a redox initiated polymerisation whereby the amino groups of the TMEDA react with the peroxide group of the potassium persulphate to generate radical species. These radicals then initiate polymerisation and crosslinking of the double bonds on the PVA and AMPS transforming the dispersed PVA-AMPS droplets into insoluble polymer microspheres. After cooling to 25°C the product is transferred to a filter reactor for purification where the butyl acetate is removed by filtration followed by: • Wash with 2 x 300ml ethyl acetate to remove butyl acetate and CAB • Equilibrate in ethyl acetate for 30m ins then filtered • Wash with 2 x 300 ml ethyl acetate under vacuum filtration • Equilibrate in acetone for 30mins and filter to remove ethyl acetate, CAB and water • Wash with 2 x 300ml acetone under vacuum filtration • Equilibrate in acetone overnight • Wash with 2 x 300ml acetone under vacuum • Vacuum dry, 2hrs, 55°C to remove residual solvents. Sieving: The manufactured microsphere product ranges in size from 100 to 1200 microns and must undergo fractionation through a sieving process using a range of mesh sizes to obtain the nominal distributions listed below. 1. 100 - 300μm 2. 300 - 500μm 3. 500 - 700μm 4. 700 - 900μm 5. 900 - 1200μm Prior to sieving the spheres are vacuum dried to remove any solvent then equilibrated at 60°C in water to fully re-hydrate. The spheres are sieved using a 316L stainless steel vortisieve unit (MM Industries, Salem Ohio) with 15" stainless steel sieving trays with mesh sizes ranging from 32 to 1000μm. Filtered saline is recirculated through the unit to aid fractionation. Spheres collected in the 32micron sieve are discarded. Example 2: Loading of Doxorubicin For this experiment the low AMPS microspheres prepared as in example 1 were used. For each size of bead used, 0.5 ml was transferred in to 2, 1 ml syringes, one for drug take up and the second to act as a control. The sizes chosen for the experiment were, 106 - 300 μm, 300 - 500 μm, 500 - 710 μm and 850 - 1000 μm. Additionally a further 3 syringes of the 500 - 710 μm were prepared in order to validate the procedure. 11 , 10 ml glass vials were covered in foil, to prevent degradation of the doxorubicin by light for the duration of the experiment. A standard curve was created. Using the 80 ml, 20 mg/ml drug solution, the following concentrations were prepared and their absorbances (at 483nm) measured: 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml and 3.125 μg/ml. The resulting absorbances were plotted on a graph and the equation of the line used to calculate the concentration of drug that was up-taken by the beads in the experiment. Four of the vials were filled with 5 ml of distilled water (ROMIL) to be used as controls when the beads were added. To the remaining 7 vials were added 5 ml of the drug solution at the desired concentration. The starting absorbance and therefore concentration of the solution was already known from the preparation of the standard curve. (In order to measure the absorbance of the 20 mg/ml solution it was necessary to dilute it 200 times, using the concentration 100 μg/ml. This 1 :200 dilution was carried through for the duration of measuring the uptake of the solution by the beads.) The stopwatch was started as soon as the first set of microspheres were added to the first drug containing vial, microspheres were added to each of the remaining 6 vials working from smallest to largest. Once sealed using the caps they were placed on the rotary mixer. The process was repeated for the control samples. The absorbances were measured in the same order as the vials were set up at time intervals of 0.167 hr (10 min), 0.5 hr, 1 hr, 2 hr, 24 hr and 96 hr. From the data the amount of drug (in mg) per 1 ml of microspheres and the % uptake of drug by 1 ml of microspheres could be calculated. The results are shown in Figure 1. Example 3: Effect of Drug Concentration on Loading Following the procedure outline in Example 2, it was possible to load a range of different concentrations of Doxorubicin into the high AMPS microsphere formulation. The majority of the drug was seen to load into the microspheres (500-710 μm size range) within a few hours (see Figure 2). The loading can be seen to be far higher than for the low AMPS formulation on a weight basis.

Example 4: Effect of Microsphere Size on Loading Loading of doxorubicin was conducted on several different size ranges of microspheres to enable comparison of the uptake. Whilst the smaller microspheres were seen to load drug more rapidly, continued loading over a 24 hour period suggests that an equivalent weight of microspheres will equilibrate to about the same drug loading. The more rapid uptake is attributed to increased surface area of the smaller microspheres (see fig 3). Example 5: Reproducibility of Loading The loading experiments outlined in example 2 were repeated a number of times in order to measure the reproducibility in loading of the doxorubicin. High AMPS microspheres of 500-710 μm size range were loaded from a 20mg/ml drug solution in water and the drug uptake monitored over time (Figure 4). Example 6: Elution of Doxorubicin from Microspheres High AMPS microspheres were loaded with various concentrations of doxorubicin and the microspheres eluted into 250 ml of distilled water (Figure 5). The drug eluting from the 133.2 μg/ml and 2mg/ml loaded microspheres was still below the detection limit at 3 hours. For the higher drug loadings, a burst effect is evident in the first few minutes, followed by a prolonged period of slower release. It is surmised that the burst represents the free drug eluting from the water held within the microspheres, whereas the prolonged elution results from the drug that is "bound" into the spheres essentially by ionic interaction between the charged groups. For the highest loading of drug (from the 20mg/ml loading solution), the burst effect represents some 45% of the total drug loading of the spheres, the remainder taking several days to completely elute from the carrier. Studies have shown that 100% of the drug is eventually eluted from the microspheres. Example 7: Visualisation of Doxorubicin Sequestration by High AMPS microspheres To a vial containing ca 0.5g of High AMPS microspheres in the size range 850-1000 μm (hand sieved), 1 ml of doxorubicin in phosphate buffered saline PBS (66.6ug/ml) and 3 ml of PBS was added. The microspheres were placed under a CCD camera, and images taken every 2 mins for a period of 2.5hrs. No agitation of the sample occurred in this time period, but small movements were observed due to localised thermal heating from the light source. The initial and final microspheres are thus identical, and can be compared over the time period. The uptake of drug was observed by the increase in red colour in the microspheres, and the depletion of the surrounding solution (Figure 6): Example 8: Preparation of Dried Drug-Loaded Microspheres Microspheres can be loaded with doxorubicin by the method outlined in example 2. The microspheres are dehydrated using the following procedure: The microspheres to be dehydrated were placed in a plastic container and covered with a 10% acetone (ROMIL) solution made in PBS (Inverclyde Biologicals). The microspheres were left in the solution for 10 minutes during which time they were agitated for 30 seconds several times. The solution was then decanted off and the process repeated twice more. This procedure was repeated with increasing acetone concentrations of 25%, 50%, 75% and finally 100%. After the final 100% dehydration step the acetone was decanted off and the beads placed in an oven set to 50°C and dried to constant mass. The resulting dried product can be resuspended /rehydrated in saline/contrast media prior to the embolisation procedure. Hydration is rapid taking only a few minutes to swell to >80% of the fully hydrated size. Example 9 - Preparation of microsphere slurry High AMPS microspheres produced according to Example 1 above, are swollen in a solution of 20 mg/ml doxorubicin in water for a period of 30 minutes. The extra-particulate liquid was seen to be substantially decoloured after this period, the colour (red) being substantially localised within the microspheres. The suspension was filtered through a sinterglass funnel to remove supernatant. The microspheres were washed with double their volume of distilled water while on the filter, under slight negative pressure. The microspheres were then transferred into a jar and pumped from the jar into a glass syringe using a peristaltic pump. Following removal of the pump the syringe was closed with a syringe lock and sterilised by gamma irradiation. Example 10: Loading - Target vs Actual Loaded Dose A series of doxorubicin solutions were prepared from 22-80mg/ml in water. 1 ml of these solutions were added to 1 ml of high AMPS microspheres, and uptake monitored by UV. The samples were agitated on a roller mixer. Time points were taken at 10, 20, 30, 60, mins and then at 2hr, out to 24hr. Uptake was calculated from the doxorubicin remaining in solution. The microspheres could be loaded with different doses up to 80mg per ml of hydrated microspheres, and in less than 30 minutes, 99 % of the drug solution is located in the microspheres. Example 11 : High Dose Doxorubicin In Example 10 a 80mg/ml solution of doxorubicin was prepared. This was a thick gelatinous mixture, that would not be suitable for everyday use. This high dose was repeated, but using 4 ml of a 20mg/ml Doxorubicin solution. Uptake was monitored by UV, and a final loading of 80mg of drug into 1 ml of hydrated high AMPS microspheres was again achieved. Example 12: Loading - Drug Sources Three sources of doxorubicin were used to prepare microspheres of the present invention with a loading of 25mg/ml. • Adriamycin™ PFS is a commercially (Pharmacia and Upjohn) available solution at a concentration of 2mg/ml. • Adriamycin™ RDF is a commercially (Pharmacia and Upjohn) powder formulation with lactose added for dissolution ease. • Doxorubicin EP. For the Adriamycin RDF and Doxorubicin EP solution, 2ml was added to 2 ml of microspheres, and the uptake monitored by UV. For the Adriamycin PFS solution 50ml of the 2mg/ml solution was added to 2ml of microspheres, and uptake monitored. After 30min both 25mg/ml solutions were fully loaded, the 2mg/ml solution was followed for 24hr to show full uptake (fig 8). Example 13: Loading of Other Anthracyclines Four samples of 1 ml of hydrated high AMPS microspheres (900- 1200μm) into an 8ml- glass container were prepared. 1 ml of microspheres in Phosphate buffered saline (PBS), measured with a 10ml- glass cylinder, was transferred to a glass container. Following this, all the PBS was removed with a Pasteur glass pipette in each sample. Loading solutions were prepared by the following: 1 vial of 20 mg of Daunorubicin (Beacon Pharmaceuticals) was reconstituted with 1 ml of water (ROMIL) to give a final concentration of 20mg/ml. 1 vial of 50 mg of epirubicin rapid dissolution (Pharmacia) was reconstituted with 2.5 ml of water to give a final concentration of 20 mg/ml. A solution of 20-mg/ml doxorubicin (Dabur Oncology) was prepared for comparison as in the previous examples. Once prepared, the absorbances of the solutions were read by UV at 483 nm and dilutions were made to produce a standard curve for each drug solution. 1 ml of each loading solution and 1 ml of water (as control) was added to each vial containing 1 ml of microspheres as prepared above and timing was started. The vials were placed on the roller mixer for the entire experiment. At predetermined time points (0, 10, 20, 30, 45 and 60 min) 50 μl was removed, diluted as necessary and read at 483 nm. From these readings, the concentration of the solution at each time point was calculated from the corresponding standard curve in each case. The amount of drug loaded into the microspheres was measured by the depletion of the drug in solution when extracted with the device. From the data the mg drug loaded per 1 ml of hydrated microspheres was calculated and the graph plotted (fig 9). This shows the anthracyclines tested loaded in the same manner. Example 14: Elution of Other Anthracyclines Microspheres loaded as described in example 13 were used to determine drug release profiles. 1 ml of each drug loaded microsphere type was transferred into a brown glass container filled with 100 ml of PBS and timing was started. The containers were placed in a water bath at 37 °C for the entire experiment. At predetermined time points (0, 0.16, 0.5, 1 , 2 and 72 hours) 1 ml of the solution was removed, read and then placed back into the container, so the volume remained constant. Samples were read at 483 nm and concentrations were calculated from the equation of the respective anthracycline standard curve determined in example 12. From the data the mg of drug eluted per 1 ml of microspheres were calculated and the graph plotted (fig 10+11 ). This showed the anthracyclines under study eluted from the microspheres with the same elution profile. Example 15: Effect of Anthracyclines on Microsphere Size Microspheres as described in example 13 were used and size distributions were determined using images of microspheres photographed using a CCD camera and microscope then the diameters resolved with Image Pro Plus 4.05. Microspheres loaded with different anthracyclines were transferred to small cell culture flasks; between 50 and 1500 microspheres were photographed per image. Image Pro Plus 4.05 resolved the diameters of between 100 and 1500 microspheres dependent on size range. Diameters were tabulated and converted to histograms of size range versus frequency, normalised and represented as graphs using Excel (fig 12). This showed the anthracyclines had the same effect on microsphere size. Example 16: Drug Loading of Other Commercial Microspheres A comparison was made of doxorubicin loading into high Amps PVA microspheres compared to another commercially-available embolic microsphere (Embosphere, trisacryl microsphere coated with collagen), following the procedures outlined in the previous examples. From the figure 13, the capabilities of the preferred anionic PVA microspheres and a commercial microsphere product to sequester the drug. Example 17: Loading - Physical Effects The effect of loading and elution of doxorubicin on High AMPS PVA microspheres was evaluated by measurement of size and compression after loading and elution of the drug, and deliverability of the doxorubicin loaded microspheres. The loading of drug produced a small decrease in the overall size range, as drug is effectively displacing water from the hydrated spheres (fig 14); this is accompanied by a small decrease in the compressibility.

Upon elution it was seen that the size was not permanently affected and nor was compressibility (as determined by Young's Modulus measurement using an Instron tensile tester. The deliverability of the spheres through standard catheters remained unchanged by the process of drug loading. Example 18: Elution - Effect of Bead Size Microspheres (High AMPS - described above) of each size were loaded with 70mg/ml of doxorubicin solution. Microspheres were then placed in 500ml of phosphate buffered saline, and release measured by UV. The in vitro elution profiles show that up to 40% of the loaded doxorubicin is released in the first 2 hours of elution as a burst, then the remaining drug elutes over at least a 12-day period. All size ranges release with similar characteristics and within ± 5% (fig 15). Example 19: Elution - Effect of Media Microspheres of the present invention loaded with 25mg/ml of doxorubicin were placed in various media and the elution monitored over 60 minutes. Plasma and PBS show slow release over the first 60 minutes. Release into water was below the detection limits of the UV. This suggests that the release of the drug is determinant on the presence of ions to displace the ionically-bound drug from the anionically-charged polymer matrix (fig 16). Example 20: Doxorubicin Stability The effect of the loading and release process on the stability of the drug was determined. The stability of doxorubicin solution when stored at different conditions was determined. The loading and release of doxorubicin from the microspheres (High AMPS - described above) were followed by HPLC using the USP method, to determine if the doxorubicin was affected by the process. The resulting chromatograms all show a single peak with similar retention times, showing that there is no detrimental effect on doxorubicin during loading and release from the microspheres. Example 21 : Doxorubicin-loaded Microspheres - Materials Compatibility Microspheres (High AMPS - described above) were loaded with doxorubicin at 25mg/ml. They were then suspended in contrast media and saline, and then left in a deliver catheter (Progreat™, Terumo) and syringe (Merit) for 24 hrs. AT various time points, the stability of doxorubicin, and components were measured (UV/HPLC for Doxorubicin, and Visual inspection/SEM for components). No degradation was observed in components or drug over 24 hours at room temperature. Example 22: Preloaded Product - Loaded Dose Samples were prepared at set doses 5, 10, 20, 45 mg/ml across the size range of High AMPS microspheres. Loading for each sample was determined by UV measurements. The data for 25 separate runs are presented in the following table:

Table 1: Actual Dose from UV measurement of the Loading Solution.

The measured dose ranges are:

45mg/ml 44.37- 45.77mg/ml (3.11 % Range) 20mg/ml 21.11- 21.32 mg/ml (1.05 % Range) 10mg/ml 9.93 - 9.98 mg/ml (0.5% Range) 5mg/ml 4.98 - 5.14 mg/ml (3.2% Range)

These data demonstrate that there is little variation between runs and that precise and accurate drug loadings can be achieved. Example 23: Preloaded Product - Lyophilisation Weight Loss Doxorubicin loaded microspheres (High AMPS PVA) were subjected to lyophilisation using a proprietary cycle. Percent weight loss was determined for doses of 5, 10, 20 and 45mg/ml for all of the microsphere size ranges (expressed as a % of the loaded microspheres, table 2) for 25 separate runs. A consistent weight loss was obtained, indicating that any variation in the weight of loaded microspheres prior to lyophilisation had no effect on the product post lyophilisation. The data in figure17 show that there is consistently greater than 82% weight reduction on lyophilisation due to water loss.

Table 2: Weight Loss on Lyophilisation for Doxorubicin Loaded Microspheres

Example 24: Preloaded Product - Residual Water Content Microspheres (High AMPS PVA) with doxorubicin at 5, 10, 20 and 45 mg/ml across the entire size range were prepared, lyophilised and then subjected to gamma irradiation for sterilisation. The residual water content of the samples was then determined by a gravimetric method involving heating the microspheres at 70°C until a constant weight was achieved. A residual water content of less than 5% was determined for all samples.

Table 3

Example 25: Preloaded Product- Release Following Rehydration Microspheres (High AMPS PVA) loaded with doxorubicin at 5, 20 and

45 mg/ml across the entire size range were prepared, lyophilised and then subjected to gamma irradiation. Samples were then re-hydrated in water, and the release of the drug into PBS followed by UV out to 100hr (fig 18). Example 26: Size of Rehydrated Doxorubicin-loaded Microspheres Microspheres (High AMPS PVA) (300-500μm) loaded with doxorubicin at 20mg/ml were prepared, lyophilised and some samples then subjected to gamma irradiation. Samples were re-hydrated in water, and then sizing carried out using calibrated image analysis equipment (Image Pro-Plus, fig 19). Non-drug loaded samples were likewise treated and sized. The data in figure 20 show there are some slight shifts in size upon various treatments, but non that would take the product outside of the acceptable specification range of 250-5Q0μm. Example 27: Transcatheter Arterial Embolisation using Microspheres of the present invention in a Rabbit Model of Liver Cancer (Vx-2). The aim of this study was to evaluate the performance of microspheres of the present invention in a rabbit liver model. The Vx-2 tumour is a model of malignant hypervascular tumours; colorectal metastases are malignant tumours that may be hypervascular in nature. The Vx-2 tumour model may therefore be used as a predictor of outcome when embolised with microspheres of the present invention containing an anthracycline compound. The objectives were to demonstrate: 1. Embolisation of the tumour by the device. 2. Reduced systemic availability of doxorubicin delivered from the device. 3. Presence of doxorubicin in tumour. For the second and third objectives, the comparison was made to intra-arterial delivery of doxorubicin. The study was carried out at The John Hopkins Hospital, Baltimore. 27.1 Materials and Methods: Animals were divided into 6 groups (groups 1 ,2,3,4,5,6) of 5 animals each (4 study animals, 1 control). Control animals in all groups received an intraarterial injection of doxorubicin (same concentration as the treated animals), whereas treatment animals were treated with modified chemoembolisation protocol with the drug eluting spheres containing doxorubicin. Animals in the various groups were sacrificed at the following time points: Group 1 : 1 hour after the chemoembolisation procedure Group 2: 12 hours after the chemoembolisation procedure Group 3: 24 hours after the chemoembolisation procedure Group 4: 3 days after the chemoembolisation procedure Group 5: 7 days after the chemoembolisation procedure Group 6: 14 days after the chemoembolisation procedure 27.2 Animal Preparation: The VX2 tumour cell line were injected into the hindlegs of carrier rabbits (New Zealand White) and grown for 14 days. Resultant tumours were harvested from each rabbit carrier and a tumour brei prepared from each by dissection of viable tumour tissue, aseptic mincing, and passage through a stainless steel sieve. The rabbits were pre-anesthetized with a mixture of intramuscular acepromazine (1 mg/kg) and Ketamine hydrochloride (20 mg/kg). After approximately 15 minutes, IV access was established via a marginal ear vein and the animal was given sodium Pentothal IV (40mg/kg) to maintain a surgical plan of anaesthesia. The abdomen was shaved and prepped with Benzidine and a midline incision made. The liver of each rabbit was exposed by median laporotomy, then an aliquot of brei (0.2 ml) was injected directly using a 21 G angiocath into the left lobe of the exposed liver in order to develop a solitary lesion with adequate surrounding liver parenchyma. One tumour brei was used for each two test rabbits. The tumour was allowed to grow in the rabbit livers for 14 days, to a size based on previous experiments expected to range between 2.5 and 3.5 cm in diameter. Any bleeding was controlled with electrocautery. The abdomen was then closed with running suture, and the skin closed with sutures and bandaged. Proper aseptic techniques were observed throughout the procedure. Following surgery, the animals were placed in cages, kept warm with blankets and monitored for end tidal CO2 until they recover from anesthesia. Analgesic buprenorphine 0.02-0.05 mg/kg administered SQ q12 hours for 3 days was given if it was apparent that the animals were in pain or physical distress. 27.3 Preparation of Doxorubicin Eluting Microspheres: Microspheres of the present invention of the size range 100-300 microns and loaded with 45mg/ml of doxorubicin were prepared as in example 22, lyophilised as in example 23 and sterilised using gamma irradiation. Immediately prior to use, the microspheres were hydrated in 1 ml of sterile water, to which 2 ml of omnipaque and 1ml of saline were added. The solution was prepared at least 10 minutes before it was to be injected and 1 ml of the total solution was injected intraarte ally in each rabbit (as described below). 27.4 Chemoembolisation Procedure: Two weeks after implantation of the tumour in the rabbit liver, the animals were brought back for "chemoembolisation". Administration of pre- anaesthesia, IV access and sodium pentothal anaesthesia were carried out as described above. Access was obtained into the common femoral artery, after which a catheter was manipulated into the common hepatic artery.

Injection of contrast demonstrated the location of the tumour after which a 2F JB1 catheter was advanced as close to the tumour as possible. If necessary, a Transsend guide wire was used to guide the catheter into the target artery. Once the catheter was adequately positioned, the doxorubicin eluting spheres were injected into the tumour bed as describes above. Controls were injected with equivalent concentration of doxorubicin only without embolisation. After completion of the "chemoembolisation", the catheter was removed, and the artery ligated using resorbable suture material to obtain hemostasis. Proper aseptic techniques were observed throughout the procedure and post procedure. Every effort was made to minimize discomfort and pain including limiting the surgical incision for tumour implantation, subcutaneous injections of buprenorphine for minimizing pain, placing clinical calls whenever required for evaluation of the animal's condition and level of pain and discomfort. The animals were then returned to their cages. All animals were sacrificed according to the time points described above. Animals were sacrificed according to veterinary regulatory rules. All animals in each animal group as per its time point after treatment were sacrificed under deep anesthesia by IV injections of 100mg/kg of thiopental IV. 27.5 Pathological and Histological Evaluation: The liver of the rabbits were dissected, carefully removed and placed in a container containing 5% formaldehyde. Livers were sliced at 5mm intervals for gross examination. Each section was embedded completely in paraffin, after which 4μ sliced and treated with H&E stain. Tumour viability was estimated by visual inspection and expressed as a percentage of viable tumour area for each slice. Concentration of doxorubicin within the tumour and nontumourous liver tissue was determined using HPLC. 27.6 Blood Collection for Doxorubicin Analysis: 3ml of whole blood was collected at 20', 40', 60', 120' and 180' each, from the time of injection of the drug eluting beads, via an arterial catheter inserted in ear. It was then transferred to a heparinized vacutainer and centrifuged at 2000g for 10 minutes at room temperature. Plasma was separated out and transferred to a labeled polypropylene capped tube. It was the snap frozen in methanol/ice. And stored at -20°C until time of analytical analysis. 27.7 Processing of Tissue for Determination of Doxorubicin Concentrations in Tumour and Non-tumourous Liver Tissue: Tumour and non-tumourous liver tissue (approx 10Omg) were excised and removed from the overlying skin and debris. The weight of the tissue was accurately determined and recorded using a preweighted tube and immediately placed on dry ice, and later stored at -80°C until time of analytical analysis. Results The microspheres of the present invention were hydrated into 4ml of the saline, water and contrast media solution. Stasis of flow to the liver was achieved with 1ml of this suspension for all of the animals. This equates to using 0.25ml of the microspheres of the present invention, which gives a total dose of 11 mg per animal, each approximately 2kg in weight (equivalent dosing - 6mg/kg). By angiography, the delivery of the microspheres of the present invention was seen into the tumour implant, and this was confirmed in the later histology in that the microspheres were all found to have remained within the arterioles at the expected vessel size i.e. 100 to 300 microns. No evidence of microspheres escaping the intravascular space was observed. In parallel with the dose that would be delivered by the microspheres of the present invention, the intra-arterial injections were carried out using 11 mg of doxorubicin solution. The blood samples were analyzed, and minimal concentrations of doxorubicin and the metabolite, doxorubicinol, within the plasma at all the time points were observed for the microspheres of the present invention. The intra-arterial injection animals showed a profile that is commonly seen, a peak within 60 minutes as the drug is washed out systemically, then a tail off as the drug is removed from the circulation. This differences between the two treatments was especially obvious at 20 minutes after administration, with a 90%+ difference between the two treatments. Following the 20 minute peak, the doxorubicin concentration in plasma declined almost linearly from 20 to 180 minutes in the intra-arterial animals. Doxorubicin concentration was between 10 and 17 times greater in plasma when injected intraarterially without the beads (Figure 20). The concentration of doxorubicin within the tumour itself remained high even in the 14-day group, suggesting continuous elution of doxorubicin from Microspheres. The greatest concentration of doxorubicin within the tumour was obtained 7 days post treatment (Figure 21 ). Histologic analysis demonstrated the efficacy of the microspheres of the present invention in killing cancer cells. From multiple previous experiments (Johns Hopkins) intra-arterial injections of doxorubicin or carboplatin (in the same concentration) only result in 30% necrosis of the tumour, which is roughly equal to that of untreated (i.e. control) animals. In contrast, treatment of the rabbits with the microspheres of the present invention caused between 50 and 100% necrosis, with necrosis being most complete at 14 days, which is significantly greater than the intraarterial injection alone (Figure 22). This study supports that microspheres of the present invention can: • Embolize the hepatic artery at the tumour site. • Reduce systemic level of doxorubicin. • Deliver high levels of doxorubicin in a controlled manner locally to a tumour model of malignant cancers such as colorectal metastases. Example 28 Pre-clinical Assessment in Non-diseased Yucatan Pigs As there is no suitable liver cancer model for CRM in larger animals, an experiment was performed to determine whether the doxorubicin-loaded embolisation microspheres of the present invention had an effect on healthy liver cells. In healthy liver cells, we have seen that these cells do not actively take-up doxorubicin at a high rate. If significant necrosis could be caused in the normal tissue, this would be a good indication that the device may be effective against metastases, assuming the order of sensitivity to doxorubicin is HCC > Metastases > Normal cells. A healthy porcine liver model was chosen (Yucatan minipigs, 40- 50kg) in which one control and one test animal were embolised by an intra- hepatic artery injection of a slow bolus of plain microspheres and doxorubicin-loaded microspheres respectively. The femoral artery was accessed by the percutaneous approach and an appropriately sized guidewire and 6F guidecatheter advanced into the common hepatic artery. A microcatheter was advanced through the guidecatheter into the target region (hepatic lobe or segment). The guidewire was removed and digital subtraction angiography using contrast medium used to deliver the appropriate microsphere mixture. After embolisation of the segment, the wire and catheters were removed and the animal allowed to recover. After 7 days post-procedure, the animal was euthanised and the liver removed and sectioned in order to observe the gross effects of the embolisation on tissue necrosis. Figures 23 & 24 show sections of the liver embolised with ordinary microspheres and the small white areas around the blood vessels demonstrate the small degree of necrosis caused. Figures 25 & 26 demonstrate the effectiveness of the device to deliver doxorubicin to the healthy liver tissue, with large areas of necrosis surrounding the vessels and even evident on the outside of the capsule of the liver. This indicates that this device is very efficient at delivering high doses of doxorubicin to the target site of embolisation sufficient to cause cell death even in cells of lesser sensitivity to the agent than primary liver cancer cells. Example 29 Clinical Assessment in a Patient with CRMs An embolisation procedure using doxorubicin-loaded microspheres was performed on an 85kg female (area 1.97m²) with extensive bilobar unresectable colorectal metastasis (CRM). The patient was successfully treated with 4ml of 300-500 μm Microspheres of the present invention loaded with 100 mg of doxorubicin (25mg/ml beads). The microspheres were delivered into the right lobule of the liver after mixing with non-ionic contrast medium using a microcatheter (Progreat 2.7Fr). The treatment is to be repeated. Example 30 Release Kinetics of the Doxorubicin from Various Microspheres In this example we investigated the loading and release profiles for doxorubicin from various microspheres. The loading and release methods were as follows: Sink conditions were provided by eluting 1ml of hydrated microspheres loaded from 2ml of a 10mg/ml dox solution (theoretical max uptake 20mg) into 200ml of PBS. After 24hrs all of the eluent was removed and replaced with 100ml of fresh PBS. All performed with agitation (roller) and at room temperature. PBS is used as it contains ions and displaces the dox in a timeframe that allows the release to be monitored. Release is monitored over a two hour period. The following microsphere products were tested: 1. High AMPS microsphere (made as in Example 1 ) particle size fraction 595-71 Oμm. Equilibrium water content 94%. 2. Contour SE, a commercially available embolic product comprising non-ionic polyvinylalcohol microspheres particle size fraction 500-700μm and equilibrium water content 91 %. 3. Amberlite 36 an ion exchange medium comprising particles of cross-linked sulphonic acid functional poly (styrene-co-divinyl benzene) with particle size in the range 351 to 863 (average 667)μm having equilibrium water content of 55-60% at 37°C in distilled water. 4. Embosphere - a commercially available embolic agent comprising particles of N-acryloyl-2-amino-2-hydroxy methyl- propane-1 ,3-diol-N,N-bisacrylamide) copolymer cross-linked with gelatin and glutaraldehyde having particle size range 500 to 700μm and equilibrium water content of 90%. This polymer at neutral pH has a net positive charge from the gelatin component. (FR-A-7723223). 5. Amberlite IRA-400 an ion-exchange medium comprising particles of quaternary ammonium functional poly(styrene-co- divinyl benzene) with particle size (dry) 230 to 81 Oμm (average 512) and equilibrium water content 52%. 6. Amberlite IRP 69 an ion-exchange medium comprising particles of sulphonic acid functional poly(styrene-co-divinyl benzene) with particle size (dry) 25 to 150μm, equilibrium water content 57% at 37°C in distilled water. The results of doxorubicin loading are shown in Figure 27. The results of doxorubicin release for the six microsphere types are shown in Figure 28. Figure 27 shows that when 1ml volume of hydrated beads are placed in contact with 2ml of a 10mg/ml solution of doxorubicin, those beads with ionic interaction capability are able to load significant amounts of drug. Some microspheres used in the invention are capable of loading all of the drug from solution. Commercial embolic agents such as Contour SE and Embosphere are not able to load appreciable amounts of the drug. When subjected to release by placing the 1 ml volume of beads in 200ml of PBS, the drug is released very quickly from the microspheres that have no method of slowing release (Figure 28). Microspheres that allow ionic interaction are able to hold the drug and release is dictated by ionic exchange mechanisms. This clearly demonstrates two very different release profiles between those microspheres capable of slowing the release of doxorubicin such that less than 50% of the total drug is released in 20 minutes in this elution test and those that are not. Example 31 Outline Method for the Preparation of Small Microspheres In this example, a microsphere product with a size of 75 μm with a standard deviation of 25 μm is produced. The synthesis is similar to that detailed in Example 1. The Nelfilcon B macromer synthesis and Diafiltration of monomers steps are carried out in the way described in Example 1. Rather than producing two series of microspheres as in Example 1 , only a high AMPs series is manufactured, the formulation of which is as shown in Example 1. A jacketed 4000ml reaction vessel is heated using a computer controlled bath (Julabo PN-9-300-650) with feedback sensors continually monitoring the reactor temperature. The butyl acetate is added to the reactor at 60 °C followed by the CAB solution and water. The stirrer speed is set to 550rpm. The system is purged with nitrogen for 15 minutes before the PVA macromer is added. The macromer solution contains approximately 18% macromer. After addition of the PVA macromer, the mixture is stirred at a speed of 550 rpm for 10 minutes at a temperature of 60°C. Crosslinking of the dispersed PVA solution is initiated by the addition of TMEDA and raising the temperature to 55°C for three hours under nitrogen. After cooling to 25°C, the product is retained in the reaction vessel rather than being transferred, in order to avoid blocking problems on the sinter. The product is purified according to the steps detailed in Example 1 with the exception that after washing with 2x300ml acetone under vacuum, the dried microsphere product is filtered directly in acetone. The manufactured microsphere product has a size of 75 μm + 25 μm as obtained through a sieving process. Prior to sieving the spheres are vacuum dried to remove any solvent and then equilibrated at 60°C in water to fully re-hydrate. The spheres are sieved using a 316L stainless steel vortisieve unit (M M Industries, Salem Ohio) with 15" stainless steel sieving trays with mesh sizes ranging from 32 to 1000 μm. Filtered saline is recirculated through the unit to aid fractionation. Spheres collected in the 32μm sieve are discarded. Example 32 Loading of Doxorubicin into Small Microsphere For this experiment, the high AMPs microspheres prepared as in example 31 were used. The loading was achieved according to the method detailed in example 2. The loading solution was examined by UV measurement at wavelength 483nm and the amount of drug loaded into the beads was calculated by the depletion method. The results are shown in Figure 29. Figure 29 shows that the small beads load to 18.75mg of doxorubicin rapidly and completely (a profile of the 100-300 μm product is shown for comparison) but that at 37.5mg dox only 80-90% of the drug is loaded as the maximum loading capacity in this instance is slightly reduced. Example 33 Elution of Doxorubicin from Small Microsphere In this example, 1ml of the loaded microspheres produced in example

32 were placed in 250ml of PBS. The eluent was sampled over time and the results are shown in Figure 30. The results show that the percentage of doxorubincin eluted from the small microspheres over time is similar for both the 18.75mg dose and 37.5mg dose. A plateau is observed in the elution curve where the maximum solubility of the drug in solution is attained. Example 34 Loading - Physical effects on small microspheres As detailed in example 17, the effect of loading the small microspheres of example 31 was evaluated by measurement of size. The loaded drug produced a small shift in the size distribution as shown in 5 Figure 31. Example 35 Chemoembolisation of rat liver metastases with Drug Eluting Beads The purpose of this example is to evaluate the effectiveness of drug eluting beads in the chemoembolisation in a rat liver metastasis model. In0 order to determine the effectiveness of doxorubicin loaded beads as compared to controls for the treatment of diffuse colorectal disease in terms of the residual tumour cell load, tumour cell reduction will be evaluated using chemiluminescence. 35.1 Outline Protocol - Pilot Study5 A pilot study will first be carried out wherein a small number of rats are used to determine the amount of microspheres that can be injected into the liver using this technique. This will also allow the calculation of the drug dosages required for the main study based on the maximum volume of embolic that can be administered. Samples of the product with size range o 100-300μm will be used 35.2 Outline Protocol - Main Study Due to the small size of vessels in rats and in order to be consistent with earlier studies, the microspheres product with a size of 75 μm + 25 μm will also be used. The microspheres will be made specifically for the study5 by the method detailed previously in example 31 and tinted and sterilized as per normal procedures. The doxorubicin will be mixed with the microspheres immediately prior to embolisation. 35.2.1 Loading of Microspheres0 A small bore needle (26G) or a filter needle is used to carefully remove the PBS solution from a vial of microspheres containing 2ml of microspheres. 1.6 ml of a 18.75 mg/ml solution of doxorubicin is transferred to the vial of microspheres to give a dose of 30 mg. The doxorubicin and microspheres are left for 30 to 60 minutes to load and agitated every 5 to 10 minutes to load. Alternatively they are placed on a rotary mixer to aid loading. The solution will turn clear when all the doxorubicin is loaded. 35.3 The methods for the culture of the colorectal cell line, their assay and transplantation into the rat are as described in the material and methods section of J. Cancer Res Clin Oncol (2004) 130: 203-210. 35.4 Basic Procedure Tumour cells are injected into the portal vein of the rat model on day 0. A relaparatomy is performed on day 8 which allows a visual control of the presence of tumour cells in the liver. Animals found to be tumour positive will receive the embolisation treatment through the hepatic artery on day 8. On day 21 , the experiment is to be terminated. The liver weight of the animals will be determined and the livers deep frozen until the time when tumour cell number is to be determined by luminometry. 35.5 Although the drug doses to be used in the main study are to be determined in the pilot study, it is anticipated that drug levels in the range from 10 to 60mg kg will be attainable. Two doses of the doxorubicin will be compared, separated by a factor in the range from 1.5 to 2. 35.6 In the main study, a comparison between a control arm (no treatment, n=20), an embolisation only control arm (n=20) and treatment groups consisting of medium and high doses of doxrubicin loaded beads (n=15 each) will be carried out. As detailed above, the exact dosages will be determined based on the results of the pilot study. Approximately 80 rats in total will be tested to allow for an approximate tumour take failure rate of 10% 35.7 The surgery and treatment will be randomised and completed in blocks. It is anticipated that the study will take of the order of 5 to 6 months to complete. The study is currently ongoing and the results are awaited. Example 36 Loading of Doxorubicin into Spherex Starch Microspheres This example was carried out to provide a direct comparison in the loading characteristics of doxorubicin into Spherex starch microspheres as compared to the 100 to 300μm microsphere product according to the present invention. The doxorubicin loading was carried out by adding 2 ml of doxorubin solution 27.09 mg/ml (Dabur oncology) to a vial containing 0.3 mg of Spherex particles (45μm) into 5ml of saline. As a control, 2 ml of doxorubin solution (27.09 g/ml) were added to a vial containing 2ml of the (100-300 μm microsphere product of the present invention into 5ml of saline. The loading solution was examined by UV measurement at wavelength 483 nm and the amount of drug loaded into the beads was calculated by depletion method. The loading results are given in Table 5 and illustrated in Figures 32 and 33.

Table 5. Comparison between Doxorubicin loading into Spherex and microspheres according to the present invention.

These results do not suggest any active loading mechanism for the Spherex particles, whereas they suggest an active loading process for the microspheres of the present invention. Example 37 Elution of Doxorubicin from Spherex Starch Microspheres Elution of the loaded microspheres of example 36 was carried out into 500ml of HPLC water for 24 hours. Again, as a comparison, the 100-300μm microspheres of the present invention loaded with doxorubicin were also eluted in the same manner. Figure 34 shows the amount of doxorubicin eluted from both systems. Figure 35 shows the percentage eluted from the microspheres at each time point. The results obtained confirm that the doxorubicin is ionically bound to the 100-300μm microspheres of the present invention (as none is released in the HPLC water). There is not noticeable bond between doxorubicin and the Spherex particles, with more than 90% of the drug eluted within the first 10minutes. No more drug was extracted from the beads after this time. Example 38 Loading of Doxorubicin into Alginate Microspheres A slurry of 1 ml of alginate microspheres with a size in the range from 550 to 650 μm was mixed with 1 ml of 18.75 mg/ml doxorubicin solution. of 18.75 mg/ml doxorubicin solution. The mixture was roller-mixed for 24h, and the loading solution was examined by Perkin-Elmer Lamda 25 UV spectrometer at wavelength 483 nm. The loading results were calculated by depletion method and are given in Table 6.

Table 6 - Doxorubicin loading into Alginate beads

This experiments showed that ~ 17 mg of doxorubicin were loaded amount into 1 mL of alginate beads. This amount of drug was loaded within the first 10 minutes. Example 39 Elution of Doxorubicin from Alginate Microspheres The elution of alginate beads loaded with doxorubicin produced in example 38 was carried out into 100 ml of PBS for 24h. The samples were placed onto a rollermixer at room temperature (RT). The elution solution was examined by Perkin-Elmer Lamda 25 UV spectrometer at wavelength 483 nm. The results are illustrated in Figures 36 and 37. Figure 36 shows that less than 25% of the doxorubicin was eluted in 24 hours into PBS. Most of this amount was eluted within the first 3 hours (Figure 37).

48

Table 5. Comparison between Doxorubicin loading into Spherex and microspheres according to the present invention.

These results do not suggest any active loading mechanism for the Spherex particles, whereas they suggest an active loading process for the microspheres of the present invention. Example 37 Elution of Doxorubicin from Spherex Starch Microspheres Elution of the loaded microspheres of example 36 was carried out into 500ml of HPLC water for 24 hours. Again, as a comparison, the 100-300μm microspheres of the present invention loaded with doxorubicin were also eluted in the same manner. Figure 34 shows the amount of doxorubicin eluted from both systems. Figure 35 shows the percentage eluted from the microspheres at each time point. The results obtained confirm that the doxorubicin is ionically bound to the 100-300μm microspheres of the present invention (as none is released in the HPLC water). There is not noticeable bond between doxorubicin and the Spherex particles, with more than 90% of the drug eluted within the first 10minutes. No more drug was extracted from the beads after this time. Example 38 Loading of Doxorubicin into Alginate Microspheres A slurry of 1 ml of alginate microspheres (CellBeads from CellMed, Germany) with a size in the range from 550 to 650 μm was mixed with 1 ml 49 of 18.75 mg/ml doxorubicin solution. The mixture was roller-mixed for 24h, and the loading solution was examined by Perkin-Elmer Lamda 25 UV spectrometer at wavelength 483 nm. The loading results were calculated by depletion method and are given in Table 6.

Table 6 - Doxorubicin loading into Alginate beads

This experiments showed that ~ 17 mg of doxorubicin were loaded amount into 1 mL of alginate beads. This amount of drug was loaded within the first 10 minutes. Example 39 Elution of Doxorubicin from Alginate Microspheres The elution of alginate beads loaded with doxorubicin produced in example 38 was carried out into 100 ml of PBS for 24h. The samples were placed onto a rollermixer at room temperature (RT). The elution solution was examined by Perkin-Elmer Lamda 25 UV spectrometer at wavelength 483 nm. The results are illustrated in Figures 36 and 37. Figure 36 shows that less than 25% of the doxorubicin was eluted in 24 hours into PBS. Most of this amount was eluted within the first 3 hours (Figure 37).

Claims

50

CLAIMS 1. Use of an anthracycline compound in the manufacture of a medicament for use in a method of treatment of a colorectal metastasis of the liver in which method the anthracycline compound is released from a polymeric matrix forming an embolus of the metastasis in the liver, in which the release rate of the anthracycline compound from the polymeric matrix under test conditions has initial VΔ greater than 20 mins. 2. Use according to claim 1 in which the polymeric matrix, when present in the embolus, comprises a water-insoluble water-swellable polymer. 3. Use according to claim 2 in which the composition which is administered comprises particles having a matrix of water-swellable water- insoluble polymer and, absorbed in the matrix, the anthracycline compound. 4. Use according to any preceding claim in which the polymer has an overall anionic charge at a pH in the range 6 to 8. 5. Use according to claim 3 in which the particles, when swollen to equilibrium in water have particle sizes in the range 40-1500 μm. 6. Use according to claim 2 in which the polymer is covalently cross-linked. 7. Use according to claim 6, in which the polymer comprises polysaccharide and is preferably substantially free of protein. 8. Use according to claim 6 in which the polymer is substantially free of naturally-occurring polymer, and preferably comprises cross-linked poly(vinyl alcohol). 9. Use according to claim 8 formed by copolyme sing poly (vinyl alcohol) macromer having at least two pendant ethylenically unsaturated groups per molecule with ethylenically unsaturated monomers including anionic monomer. 10. Use according to claim 9 in which the anionic monomer has the general formula I 51 Y¹ BQ

in which Y¹ is selected from

CH₂=C(R)-CH₂-O-, CH₂=C(R)-CH₂ OC(O)-, CH₂=C(R)OC(O)-, CH₂=C(R)-O-, CH₂=C(R)CH₂OC(O)N(R¹)-, R²OOCCR=CRC(O)-O-, RCH=CHC(O)O-, RCH=C(COOR²)CH₂-C(O)-O-,

wherein: R is hydrogen or a C C₄ alkyl group; R¹ is hydrogen or a C_rC₄ alkyl group; R² is hydrogen or a C_1-4 alkyl group or BQ where B and Q are as defined below; A is -O- or -NR¹ -; K¹ is a group -(CH₂)_rOC(O)-, -(CH₂)_rC(O)O-, - (CH₂)_rOC(O)O-, -(CH₂)_rNR³-, -(CH₂)_rNR³C(O)-, -(CH₂)_rC(O)NR³-, -(CH₂)_rNR³C(O)O-, -(CH₂)_rOC(O)NR³-, -(CH₂)_rNR³C(O)NR³- (in which the groups R³ are the same or different), -(CH₂)_rO-, -(CH₂)_rSO₃ -, or, optionally in combination with B\ a valence bond and r is from 1 to 12 and R³ is hydrogen or a C_rC₄ alkyl group; B is a straight or branched alkanediyl, oxaalkylene, alkanediyloxaalkanediyl, or alkanediyloligo(oxaalkanediyl) chain optionally containing one or more fluorine atoms up to and including perfluorinated chains or, if Q or Y¹ contains a terminal carbon atom bonded to B a valence 52 bond; and Q is an anionic group. 11. Use according to claim 10 in which Y¹ is CH₂=CRCOA in which R is H or methyl, A is NH, and in which B is C.,.₁₂ alkanediyl. 12. Use according to claim 10 in which Q is a carboxylate, carbonate, sulphonate, sulphate, nitrate, phosphonate or phosphate group, preferably a sulphonate group. 13. Use according to any of claims 8 to 12 in which the PVA macromer has average molecular weight in the range 1 ,000 to 500,000 D preferably in the range 10,000 to 100,000 D. 14. Use according to any of claim 9 to 12 in which the pendant ethylenic groups are linked via cyclic acetal linkages with oxygen atoms from adjacent hydroxyl groups, preferably formed by the reaction of N-(alk)acrylamino-substituted aldehyde, usually in the form of the dialkyl acetal, preferably N-acrylaminoacetaldehyde dimethyl acetal. 15. Use according to any preceding claim in which the anthracycline compound has the general formula II

(axial) Doxorubicin (axial) Daunorubicin

(axial) Idarubicin X=COCH₂OH Y=OCH₃ Z=HO (equatorial) Epriubicin and is preferably doxorubicin. 16. Use according to claim 3 in which the particles are microspheres. 17. Use according to claim 3 or 16 in which the particles are swollen with and suspended in an aqueous liquid. 53 18. Use according to claim 17 further comprising an imaging agent, preferably a radiopaque agent. 19. Use according to any preceding claim in which, in the method an initial dose of embolic composition is administered, preferably followed by 1 to 5 repeat doses. 20. Use according to claim 19 in which each said dose comprises 10 to 800 mg of the anthracycline, preferably 20 to 100 mg. 21. Use according to claim 19 or 20 in which the total amount of anthracycline administered in the method is more than 200 mg preferably more than 500 mg. 22. A composition comprising particles of a water-insoluble water- swellable polymer and absorbed in the matrix an anthracycline compound for use in a method of treatment in which the composition is introduced into the liver to embolise a colorectal metastasis wherein the release rate of the anthracycline from the polymeric matrix under test conditions has a VΔ of greater than 20 mins. 23. A composition according to claim 22 having the further features defined in any of claims 2 to 21.