Patent Description:
The most common malignant primary brain tumour in adults is glioblastoma multiforme (GBM) [<NPL>. GBM accounts for <NUM>% of primary malignant brain and CNS tumour gliomas [<NPL>], with an annual incidence rate of <NUM> per <NUM>,<NUM> worldwide [<NPL>]. It is invasive and one of the most aggressive tumours of the central nervous system, having the histological features of high cellularity, nuclear atypia, microvascular proliferation, brisk mitotic activity and necrosis. Upon initial diagnosis of GBM, the standard treatment is known as the Stupp protocol [<NPL>] and involves surgical resection ('debulking') with maximal tumour-free margins, radiotherapy with concomitant temozolomide chemotherapy and additional higher dose temozolomide chemotherapy after each radiotherapy cycle. Even with complete surgical resection of the tumour, combined with this very aggressive treatment, the overall survival of patients remains at just <NUM>-<NUM> months with a <NUM>-year survival of <NUM>% [<NPL>].

Due to GBM being an extremely infiltrative brain cancer, and thus impossible to fully remove surgically, tumour recurrence is almost inevitable and <NUM> to <NUM>% recur within <NUM> of the resection site [<NPL>]. There is no established chemotherapy regimen available to patients who recur and increasing numbers of patients with recurrent GBM are undergoing re-operation to control their disease where conventional second line therapy has failed.

The reason most GBM treatments fail is because they are administered either via the intravenous or oral route. Systemic delivery of chemotherapeutic drugs to the brain is limited by the blood-brain barrier (BBB) and administration of high systemic drug levels is required to achieve the therapeutic levels needed in the brain. Local delivery directly into the resection margin at the time of surgery using an implantable device would allow for the delivery of chemotherapeutic drugs directly to the brain offering a number of advantages such as a lower dose, increased patient tolerance, and reduced side effects due to the avoidance of systemic circulation.

The Gliadel® wafer is a local delivery device, which was approved by the Food and Drug Administration in <NUM> for the treatment of recurring GBM. It is a disc-shaped, <NUM> biodegradable wafer containing <NUM>% w/w of the chemotherapeutic agent Carmustine. Gliadel® has demonstrated a small but significant benefit in combination with surgery for patients with recurrent gliomas [<NPL>]. However, Gliadel® and other similar approaches are limited by the reliance on drug diffusion from the device into the brain parenchyma restricting penetration distances to a few millimetres.

Biodegradable implants are used for local drug delivery as they can be administered relatively easily and designed to control the release of a wide range of therapeutic agents. For instance, Ramachandran et al describe the delivery of Temozolomide from a theranostic nano brain-implant for the prolonged and localized treatment of recurrent glioma [<NPL>]. McConville et al describe the development of disulfiram-loaded poly lactic glycolic acid (PLGA) millirods for the treatment of GBM administered to the tumour via stereotactic injection [<NPL>]. Lesniak et al demonstrated the efficacy of doxorubicin-loaded biodegradable wafers using a GBM rat model [<NPL>], while Zembeko et al describe the manufacture of disulfiram-loaded PLGA wafers for the localised treatment of GBM [<NPL>].

Irinotecan (IRN) is a semi-synthetic pro-drug, whose active metabolite <NUM>-ethyl-<NUM>-hydroxycamptothecin, also referred to as SN-<NUM>, acts as an inhibitor of the Topoisomerase I group of enzymes. Topoisomerase I enzymes act within the cell to induce temporary cuts within one or both strands of DNA, allowing the DNA to uncoil for transcription and replication. During this process, Topoisomerase I forms a covalent linkage with DNA, allowing it to form a cleavable complex. SN-<NUM> binds to Topoisomerase I in this confirmation, inhibiting the enzymes from re-joining the strands of DNA, causing S-phase specific cell killing [<NPL>; <NPL>; <NPL>].

Currently, IRN is part of the standard treatment regimen for advanced colorectal cancer, when used in combination with <NUM>-fluorouracil (<NUM>-FU) and folinic acid. In treating GBM, IRN administered as an intravenous monotherapy had a response rate between <NUM> and <NUM>%, with progression free survival between <NUM> to <NUM> months; in combination with other drugs IRN had a response rate between <NUM> and <NUM>%, with progression free survival of <NUM> to <NUM> months [<NPL>].

Even though IRN crosses the BBB, high intravenous doses between <NUM> and <NUM>/m<NUM> are required to achieve therapeutic levels in the brain resulting in serious systemic side effects including gastrointestinal toxicity, leading to early and late onset diarrhoea, and severe neutropenia. The issue of severe diarrhoea and neutropenia and the need to increase the levels of IRN in the brain has spurred on recent developments in improving IRN's ability to cross the BBB.

Local delivery of IRN directly to the brain tumour resection site could improve therapeutic outcomes by allowing for the delivery of larger doses directly to the tumour site, while reducing systemic concentrations and thus alleviating the aforementioned side-effects.

Baltes et al demonstrated a significant difference in the survival of GBM rats treated with <NUM>-<NUM> Drug Eluting Beads containing IRN, when compared to the placebo group [<NPL>]. Furthermore, there was no local toxicity associated or pronounced haemorrhaging around the area of implantation of the IRN beads. In-vitro IRN elution from the Drug Eluting Beads was fast.

A Phase I clinical trial demonstrated that IRN Drug Eluting Beads were safe for the local treatment of the tumour margin in GBM patients, with preliminary survival data as good as Gliadel® when compared to historical controls [<NPL>[Clinical Trial Identifier: NCT02433392]]. However, the pharmacokinetic data demonstrated that the majority of IRN was removed from the brain within <NUM> hours, while all of it was removed by <NUM> hours.

<CIT> discloses water-insoluble, water-swellable, negatively-charged polymer microspheres for the delivery of positively-charged chemotherapeutic agents, including the preparation of IRN-loaded microspheres made from a <NUM>:<NUM> copolymer of PVA and <NUM>-acrylamido-<NUM>-methylpropane sulphonate.

<CIT> discloses polymer nanoparticles containing IRN and conjugated cisplatin, with the nanoparticles having an average diameter of about <NUM>.

There remains an unmet need to provide a drug delivery system for the treatment of brain tumours which is efficacious against even the most aggressive of tumours, whilst avoiding the unwanted side effects associated with current treatments.

The present invention was devised with the foregoing in mind.

In a first aspect, the present invention provides a chemotherapeutic seed comprising a biodegradable polymer and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>)and a pharmaceutically acceptable salt of SN-<NUM>; wherein the seed has a primary length of at least <NUM>, and wherein primary length refers to the shortest dimension of the seed.

In another aspect, the present invention provides a chemotherapeutic seed consisting of a biodegradable polymer and a drug selected from IRN, a pharmaceutically acceptable salt of IRN, SN-<NUM> and a pharmaceutically acceptable salt of SN-<NUM>; wherein the seed has a primary length of at least <NUM>.

In a further aspect, the present invention provides a seed as described herein for use in therapy.

In a further aspect, the present invention provides a seed as described herein for use in the treatment of a brain tumour, such as high grade gliomas (e.g. GBM).

In another aspect, the present invention provides a pharmaceutical composition comprising one or more seeds as described herein.

In a further aspect, the present invention provides a process for the preparation of a chemotherapeutic seed as described herein, the process comprising the steps of:.

The above and further aspects of the invention are described in further detail herein.

Particular embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:.

As described above, the present invention provides a chemotherapeutic seed comprising a biodegradable polymer and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>) and a pharmaceutically acceptable salt of a derivative of SN-<NUM>; wherein the seed has a primary length of at least <NUM>, and wherein primary length refers to the shortest dimension of the seed.

By incorporating the drug into a biodegradable seed of the above dimensions it is possible to achieve sustained drug release from the seed over a period of at least <NUM> days under physiological conditions. This makes the seeds of the present invention suitable for efficacious local drug delivery to the resection site of brain tumours, especially GBM.

Irinotecan (IRN) has the following structure:
<CHM>.

Derivatives of irinotecan include active metabolites of irinotecan, such as SN-<NUM>. SN-<NUM> is the compound <NUM>-ethyl-<NUM>-hydroxy-camptothecin, which has the following structure:
<CHM>.

Pharmaceutically acceptable salts of a drug of the present invention include an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citric or maleic acid. In addition a suitable pharmaceutically acceptable salt of a drug of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(<NUM>-hydroxyethyl)amine.

In an embodiment, the drug is irinotecan. In an embodiment, the drug is a pharmaceutically acceptable salt of irinotecan, such as the hydrochloride salt. In an embodiment, the drug is irinotecan hydrochloride. It will be understood that the invention also comprises hydrates or solvates of the drugs of the invention. In a particular embodiment, the drug is a hydrate of irinotecan hydrochloride. In a further embodiment, the drug is irinotecan hydrochloride trihydrate.

Suitable biodegradable polymers are polymers capable of breaking down under physiological conditions such that the polymer and its degradation products do not invoke any unacceptable toxicity or immune response. Such polymers may be natural or synthetic in origin. Examples of natural biodegradable polymers include polysaccharides such as chitosan, alginate and dextran, or polyesters such as polyhydroxyalkanoates. Examples of synthetic biodegradable polymers include polymers of lactic acid and glycolic acid and copolymers thereof.

In an embodiment the biodegradable polymer is poly(lactic-co-glycolic acid) or polylactic acid, wherein each polymer is end-capped with either acid or ester groups. In a preferred embodiment, the biodegradable polymer is poly(lactic-co-glycolic acid). Poly(lactic-co-glycolic acid) is referred to herein also as PLGA. In an embodiment, the biodegradable polymer is PLGA end-capped with acid groups. In a preferred embodiment, the biodegradable polymer is PLGA end-capped with ester groups.

The acidity of the biodegradable polymer may be quantified by determining the acid number via titration. This technique is well-known in the art and typically involves dissolving a known amount of a polymer sample in an organic solvent (e.g. isopropanol) and titrating it with a solution of potassium hydroxide of known concentration using phenolphthalein as a colour indicator. The acid number is expressed as the mg of KOH required to neutralize <NUM> of the sample. In an embodiment, the biodegradable polymer has an acid number less than or equal to <NUM> KOH/g. In an embodiment, the biodegradable polymer is PLGA and the PLGA has an acid number less than or equal to <NUM> KOH/g.

In an embodiment, the biodegradable polymer is PLGA, wherein the PLGA has a lactic acid weight content of about <NUM> to <NUM>% with the balance being glycolic acid. In an embodiment, the PLGA has a lactic acid weight content of about <NUM> to <NUM>%, such as about <NUM> to <NUM>%, about <NUM> to <NUM>%, or preferably about <NUM> to <NUM>%; with the balance being glycolic acid. In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM>, about <NUM>:<NUM> or about <NUM>:<NUM>. In a preferred embodiment, the PLGA has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM>, about <NUM>:<NUM> or about <NUM>:<NUM>. In a most preferred embodiment, the PLGA has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM>. In a most preferred embodiment, the PLGA is end-capped with ester groups and has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM>. In a most preferred embodiment, the PLGA has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM> and an acid number less than or equal to <NUM> KOH/g.

In an embodiment, the biodegradable polymer is PLGA, wherein the PLGA has a number average molecular weight of about <NUM> to <NUM> kDa, such as about <NUM> to <NUM> kDa, or about <NUM> to <NUM> kDa.

In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM> and a number average molecular weight of about <NUM> to <NUM> kDa. In an embodiment, the PLGA is end-capped with ester groups, has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM> and a number average molecular weight of about <NUM> to <NUM> kDa. In a preferred embodiment, the PLGA has a lactic acid:glycolic acid weight ratio of about <NUM>:<NUM>, an acid number less than or equal to <NUM> KOH/g and a number average molecular weight of about <NUM> to <NUM> kDa.

In an embodiment, the PLGA has an inherent viscosity of <NUM> to <NUM> dL/g, when measured at <NUM> in chloroform at a concentration of <NUM>/dL. In a preferred embodiment, the PLGA has an inherent viscosity of <NUM> to <NUM> dL/g, such as <NUM> to <NUM> dL/g, <NUM> to <NUM> dL/g, <NUM> to <NUM> dL/g, or <NUM> to <NUM> dL/g when measured at <NUM> in chloroform at a concentration of <NUM>/dL. In a most preferred embodiment, the PLGA has an inherent viscosity of <NUM> to <NUM> dL/g when measured at <NUM> in chloroform at a concentration of <NUM>/dL.

In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>, a number average molecular weight of about <NUM> to <NUM> kDa and an inherent viscosity of <NUM> to <NUM> dL/g, when measured at <NUM> in chloroform at a concentration of <NUM>/dL. In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM> and an inherent viscosity of <NUM> to <NUM> dL/g, when measured at <NUM> in chloroform at a concentration of <NUM>/dL. In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>, an acid number less than or equal to <NUM> KOH/g and an inherent viscosity of <NUM> to <NUM> dL/g, when measured at <NUM> in chloroform at a concentration of <NUM>/dL. In an embodiment, the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>, an acid number less than or equal to <NUM> KOH/g, a number average molecular weight of about <NUM> to <NUM> kDa and an inherent viscosity of <NUM> to <NUM> dL/g, when measured at <NUM> in chloroform at a concentration of <NUM>/dL.

The loading of drug within the chemotherapeutic seed can be chosen dependent on the required levels of active agent at the local delivery site and the period of drug cover required post-implantation. In an embodiment, the drug is present in a loading of <NUM> to <NUM> weight % (wt%) relative to the weight of the seed. In an embodiment, the drug is present in a loading of <NUM> to <NUM> wt %, such as <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, or <NUM> to <NUM> wt % relative to the weight of the seed. In an embodiment, the drug is present in a loading of <NUM> to <NUM> wt %, such as <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, or <NUM> to <NUM> wt % relative to the weight of the seed. In an embodiment, the drug is present in a loading of about <NUM> wt % or about <NUM> wt % relative to the weight of the seed.

In an embodiment, the drug is irinotecan, or a pharmaceutically acceptable salt of irinotecan and the drug is present in a loading of <NUM> to <NUM> wt %, or <NUM> to <NUM> wt % relative to the weight of the seed. In an embodiment, the drug is irinotecan, or a pharmaceutically acceptable salt of irinotecan and the drug is present in a loading of about <NUM> wt %, or about <NUM> wt % relative to the weight of the seed.

In an embodiment, the drug is a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride) and the drug is present in a loading of <NUM> to <NUM> wt %, or <NUM> to <NUM> wt % relative to the weight of the seed. In an embodiment, the drug is a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride) and the drug is present in a loading of about <NUM> wt %, or about <NUM> wt % relative to the weight of the seed.

In an embodiment, the drug is SN-<NUM>, or a pharmaceutically acceptable salt of SN-38and the drug is present in a loading of <NUM> to <NUM> wt %, <NUM> to <NUM> wt %, or <NUM> to <NUM> wt % relative to the weight of the seed.

A chemotherapeutic seed according the present invention can take various shapes, such as sphere, plate, rod, cylinder, cuboid or cube. The method of preparation of the seed may influence the shape of the seed produced. For example, the shape and size of seeds prepared by extrusion can be controlled by the choice of extrusion die. Preferably the seeds of the present invention are rods. As the skilled person will understand, a rod has a shape which is cylindrical. In a preferred embodiment, the seed is substantially cylindrically-shaped.

A seed according to the present invention has a primary length of at least <NUM>. As used herein, the term 'primary length' refers to the shortest dimension of the seed. In a spherical seed this would be the diameter of the sphere and in a cube this would be the length of the cube. In shapes, such as a cuboid or a cylinder, which have dimensions of different lengths, the primary length is the shortest length, while the secondary length is the second shortest length (see schematic representation below). <IMG>
Schematic representation of possible seed shapes (I-r: sphere, cube, cuboid and cylinder) with the primary length (i) of each shape shown, as well as the secondary length (ii) for the cuboid and cylinder.

Accordingly, when the seed is substantially cylindrically-shaped, the primary length is the diameter of the cylinder and the secondary length is the length of the cylinder. In an embodiment, the seed is substantially cylindrically-shaped, the primary length is the seed diameter and the secondary length is the seed length.

It is understood that when the seed comprises one or more pores or cavities, then the primary length is defined in terms of the dimensions of the whole seed.

The size of the seed is important to deliver the desired drug release profile. In an embodiment, the seed has a primary length of at least <NUM>, such as at least <NUM>. In an embodiment, the seed has a primary length of <NUM> to <NUM>, such as <NUM> to <NUM>, <NUM> to <NUM>, or preferably <NUM> to <NUM>. Seeds having primary lengths within the range of about <NUM> to <NUM>, are particularly suited to administration to the tumour site via a catheter. In a preferred embodiment, the seed has a primary length of about <NUM>.

In an embodiment, the seed has a secondary length of at least <NUM>, such as at least <NUM>. In an embodiment, the seed has a secondary length of <NUM> to <NUM>, such as <NUM> to <NUM>, <NUM> to <NUM>, or preferably <NUM> to <NUM>. In a preferred embodiment, the seed has a secondary length of about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM>.

In a preferred embodiment, the seed has a primary length of about <NUM> and a secondary length of about <NUM> or about <NUM>. In a preferred embodiment, the seed is substantially cylindrically-shaped and has a primary length of about <NUM> and a secondary length of about <NUM> or about <NUM>.

Seeds prepared according to the present invention may be prepared with or without plasticisers. In an embodiment, the seed does not contain a plasticizer. In order to be suitable for local implantation (e.g. in the brain parenchyma) in terms of minimizing the risk of adverse reactions, it is desirable that the seeds are non-swelling and that they have a smooth surface (characterized by an absence of so-called 'shark skinning'). It may therefore be necessary for the seeds to contain one or more plasticisers. In an embodiment, the seed further comprises a pharmaceutically acceptable plasticizer. Suitable plasticisers include poloxamers (such as Kolliphor® P188, Kolliphor® P237 and Kolliphor® P407), polyethylene glycols (such as Kollisolv® PEG <NUM> and Kollisolv® PEG <NUM>), polyvinyl acetate, stearic acid, glyceryl behenate, Triacetin, diethyl phthalate, glyceryl monostearate, triethyl citrate and macrogolglycerol hydroxystearate (such as Kolliphor® RH <NUM>). In an embodiment, the plasticiser is selected from poloxamers, polyethylene glycols, polyvinyl acetate and macrogolglycerol hydroxystearate. In a preferred embodiment, the plasticiser is chosen from Kolliphor® P188, Kolliphor® P237 and Kolliphor® RH <NUM>. In a more preferred embodiment, the plasticiser is a poloxamer, such as Kolliphor® P <NUM> or Kolliphor® P <NUM>. Most preferably, the plasticiser is Kolliphor® P <NUM>.

Experiments using Kolliphor® P188, Kolliphor® P237 or Kolliphor® RH <NUM> plasticisers to prepare blank (i.e. drug-free) PLGA seeds via hot melt extrusion showed that low loadings (<NUM> % w/w) resulted in seeds with inconsistent average diameters, whilst high loadings (<NUM>-<NUM> % w/w) typically resulted in the seeds showing swelling when placed in water for up to <NUM> hours - see Comparative Example <NUM>. Therefore, optimal plasticizer loading is in the range <NUM>-<NUM> % w/w. In an embodiment, the seed comprises plasticizer at a loading of about <NUM>-<NUM> weight %, preferably about <NUM> weight %, relative to the weight of the seed. In a preferred embodiment, the seed comprises Kolliphor® P <NUM> at a loading of about <NUM> weight % relative to the weight of the seed.

In an embodiment, the seeds according to the present invention only contain biodegradable polymer and drug and optionally plasticizer. Therefore, in an aspect of the invention there is provided a chemotherapeutic seed consisting of a biodegradable polymer and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, a derivative of irinotecan and a pharmaceutically acceptable salt of a derivative of irinotecan; wherein the seed has a primary length of at least <NUM>. In an embodiment, there is provided a chemotherapeutic seed consisting of a biodegradable polymer (preferably PLGA) and a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride); wherein the seed has a primary length of at least <NUM>. In a preferred embodiment, there is provided a chemotherapeutic seed consisting of a biodegradable polymer (preferably PLGA) and a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride); wherein the seed has a primary length of <NUM> to <NUM>. In a further aspect of the invention there is provided a chemotherapeutic seed consisting of a biodegradable polymer, a plasticiser and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, a derivative of irinotecan and a pharmaceutically acceptable salt of a derivative of irinotecan; wherein the seed has a primary length of at least <NUM>. In an embodiment, there is provided a chemotherapeutic seed consisting of a biodegradable polymer (preferably PLGA), a plasticizer (preferably Kolliphor® P <NUM>) and a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride); wherein the seed has a primary length of at least <NUM>. In a preferred embodiment, there is provided a chemotherapeutic seed consisting of a biodegradable polymer (preferably PLGA), a plasticizer (preferably Kolliphor® P <NUM>) and a pharmaceutically acceptable salt of irinotecan (preferably irinotecan hydrochloride); wherein the seed has a primary length of <NUM> to <NUM>.

Irinotecan works during the S-Phase of the cell cycle, which occurs around day <NUM>, which means the efficacy of irinotecan could be improved if therapeutic levels could be maintained at the tumour site for at least <NUM> to <NUM> days.

As described above, a device for local delivery of a chemotherapeutic agent, such as irinotecan, directly to a solid tumour resection site could improve therapeutic outcomes by allowing for the delivery of larger doses of the drug directly in the first <NUM>-<NUM> hours, followed by a prolonged period of slower drug release for at least <NUM> to <NUM> days, post device implantation. Such a release profile (`initial burst' followed by prolonged release) is particularly suited to the efficacious delivery of irinotecan to solid tumour resection sites, as (i) the initial burst allows for a high local dose of drug to be delivered initially, enhancing diffusion of the drug through the surrounding tissue and killing off most of the residual tumour cells not removed surgically, while (ii) the sustained period of release provides a 'top-up' of drug to kill those cells that were in the G<NUM> or G<NUM> phase of the cell cycle and did not enter the S-Phase of the cell cycle until around day <NUM> post implantation.

Chemotherapeutic seeds of the present invention are capable of providing a drug release profile as described above. Experiments conducted measuring the release of drug from the seeds into aqueous sink and bio-relevant media over a period of <NUM> days, show an initial burst of drug release by day <NUM>, followed by a steady slow release of drug out to <NUM> days post-incubation (Example <NUM> and <FIG>).

Suitably, the 'initial burst' release phase is defined as the seed releasing between <NUM>% and <NUM>% of the drug after <NUM> day post-incubation in aqueous media, relative to the total amount of drug released. More suitably, the 'initial burst' is defined as the seed releasing between <NUM>% and <NUM>% of the drug after <NUM> day post-incubation in aqueous media, relative to the total amount of drug released.

Suitably, the 'prolonged release' phase is defined as the seed releasing between <NUM>% and <NUM>% of the drug between day <NUM> and day <NUM> post-incubation in aqueous media, relative to the total amount of drug released. More suitably, the 'prolonged release' is defined as the seed releasing between <NUM>% and <NUM>% of the drug between day <NUM> and day <NUM> post-incubation in aqueous media, relative to the total amount of drug released.

An example of a suitable in vitro test to measure the drug release profile of seeds according to the present invention are described below in Example <NUM>. In summary, individual seeds (n = <NUM>) were placed into a sealed flask containing either <NUM> of water (bio-relevant medium designed to mimic cerebral spinal fluid) or <NUM> of phosphate buffered (pH <NUM>) saline solution (sink conditioned media) and placed into an orbital shaking incubator at <NUM> and <NUM> rpm. Complete media replacement was performed at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days. The samples where filtered using a <NUM> filter and analysed for IRN content using HPLC.

An aqueous physiological-type environment is defined as being either bio-relevant or sink-conditioned water-based media, as described hereinabove.

In an embodiment of the present invention, there is provided a seed as described herein, wherein the seed continuously releases the drug over a period of at least <NUM> days, at least <NUM> days, at least <NUM> days, at least <NUM> days or at least <NUM> days when placed in an aqueous physiological-type environment.

In an embodiment, the seed, when placed in an aqueous physiological-type environment, releases:.

relative to the total amount of drug released.

relative to the total amount of drug released after <NUM> days.

In an embodiment, when the seed is placed an aqueous physiological-type environment, the cumulative drug release after <NUM> day is <NUM> to <NUM> and after <NUM> days is <NUM> to <NUM>. In an embodiment, when the seed is placed an aqueous physiological-type environment, the cumulative drug release after <NUM> day is <NUM> to <NUM>; after <NUM> days is <NUM> to <NUM>; and after <NUM> days is <NUM> to <NUM>.

In an embodiment, the seed, when placed in an aqueous physiological-type environment, releases at least <NUM> to <NUM>µg of drug per day for a period of at least <NUM> days, at least <NUM> days or at least <NUM> days. In a further embodiment, the seed, when placed in an aqueous physiological-type environment, releases at least <NUM> to <NUM>µg of irinotecan (or a pharmaceutically acceptable salt thereof) per day for a period of at least <NUM> days, at least <NUM> days or at least <NUM> days. In a yet further embodiment, the seed, when placed in an aqueous physiological-type environment, releases <NUM> to <NUM>µg of irinotecan (or a pharmaceutically acceptable salt thereof) after <NUM> day, followed by at least <NUM>µg of irinotecan (or a pharmaceutically acceptable salt thereof) per day for the subsequent <NUM> days.

In an embodiment, the seed comprises <NUM>-<NUM>% w/w of irinotecan hydrochloride and when placed in an aqueous physiological-type environment, the seed releases:.

relative to the total amount of irinotecan released after <NUM> days.

In an embodiment, the seed has a primary length of about <NUM>, comprises <NUM>-<NUM>% w/w of irinotecan hydrochloride and when placed in an aqueous physiological-type environment, the seed releases:.

In an embodiment, the seed has a primary length of about <NUM>, a secondary length of about <NUM> to <NUM>, comprises <NUM>-<NUM>% w/w of irinotecan hydrochloride and when placed in an aqueous physiological-type environment, the seed releases:.

In any of the above embodiments, placing of a seed in an aqueous physiological-type environment may refer to incubation of a seed in <NUM> of phosphate buffered (pH <NUM>) saline solution at <NUM> with agitation at <NUM> rpm and complete media replacement being carried out at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days post-incubation. In any of the above embodiments, placing of a seed in an aqueous physiological-type environment may refer to incubation of a seed in <NUM> of water at <NUM> with agitation at <NUM> rpm and complete media replacement being carried out at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days post-incubation.

In an embodiment, the seed, when incubated in <NUM> of pH <NUM> phosphate buffered saline solution at <NUM> with agitation at <NUM> rpm and complete media replacement being carried out at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days post-incubation, releases:.

In an embodiment, the seed, when incubated in <NUM> of pH <NUM> phosphate buffered saline solution at <NUM> with agitation at <NUM> rpm and complete media replacement being carried out at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days post-incubation, releases at least <NUM> to <NUM>µg of drug per day for a period of at least <NUM> days, at least <NUM> days or at least <NUM> days.

In any of the above embodiments describing the drug release profile, the term 'drug' may refer to irinotecan (or a pharmaceutically acceptable salt thereof), or <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>) (or a pharmaceutically acceptable salt thereof). Preferably, 'drug' refers to irinotecan (or a pharmaceutically acceptable salt thereof).

In an aspect of the present invention there is provided a pharmaceutical composition comprising one or more seeds as described herein. In an embodiment, the pharmaceutical composition comprises <NUM>-<NUM> seeds as described herein. In a preferred embodiment, the pharmaceutical composition comprises <NUM>-<NUM> seeds as described herein.

In an embodiment, the pharmaceutical composition comprises one or more seeds, wherein each seed has a primary length of at least <NUM> and comprises a biodegradable polymer in which is dispersed a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>) and a pharmaceutically acceptable salt of SN-<NUM>.

In an embodiment, the pharmaceutical composition comprises one or more seeds, wherein each seed has a primary length of about <NUM> and comprises PLGA in which is dispersed irinotecan hydrochloride.

In an embodiment, the pharmaceutical composition comprises one or more seeds, wherein each seed has a primary length of about <NUM>, a secondary length of about <NUM> to <NUM> and comprises PLGA in which is dispersed irinotecan hydrochloride.

In an embodiment, the pharmaceutical composition comprises one or more seeds, wherein each seed has a primary length of about <NUM>, a secondary length of about <NUM> to <NUM> and comprises PLGA (such as <NUM>:<NUM> w/w LA:GA) in which is dispersed <NUM>-<NUM>% w/w irinotecan hydrochloride.

The compositions of the present invention may comprise one or more additional therapeutic agents. The additional therapeutic agents may have an anti-cancer effect or any other therapeutic effect, such as being an antibiotic, anti-inflammatory, anticoagulant, analgesic, statin, antiplatelet, antifungal, angiotensin-converting enzyme (ACE) inhibitor, enzyme acetaldehyde dehydrogenase inhibitor or other suitable, complementary therapeutic agent. In an embodiment, the composition further comprises an additional anti-cancer agent. In an embodiment, the one or more additional therapeutic agents are selected from anti-cancer agents (such as bevacizumab), statins (such a pitavastatin), antiplatelet agents (such as ticlopidine), antifungal agents (such as itraconazole), angiotensin-converting enzyme (ACE) inhibitors (such as captopril), enzyme acetaldehyde dehydrogenase inhibitors (such as disulfiram) and anti-inflammatory agents (such as celecoxib).

In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein and a statin (such as pitavastatin). In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein and an angiotensin-converting enzyme (ACE) inhibitor (such as captopril). In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein and an enzyme acetaldehyde dehydrogenase inhibitor (such as disulfiram). In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein, a statin (such as pitavastatin) and an angiotensin-converting enzyme (ACE) inhibitor (such as captopril). In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein, a statin (such as pitavastatin) and an enzyme acetaldehyde dehydrogenase inhibitor (such as disulfiram). In an embodiment, there is provided a pharmaceutical composition comprising one or more seeds as described herein, an angiotensin-converting enzyme (ACE) inhibitor (such as captopril) and an enzyme acetaldehyde dehydrogenase inhibitor (such as disulfiram).

In an embodiment, the additional anti-cancer agent comprises one or more radioactive isotopes suitable for the treatment of cancer. Alternatively, the additional therapeutic agent is a radiosensitising agent for concurrent or subsequent radiotherapy treatment.

The additional therapeutic agent may be present in the composition in an admixture with the seeds. Alternatively, in an embodiment, the seeds may comprise two layers, wherein a first layer contains a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, a derivative of irinotecan and a pharmaceutically acceptable salt of a derivative of irinotecan; and a second layer contains an additional therapeutic agent. The seeds may further comprise additional layers and/or further therapeutic agents. In an embodiment, all the layers are made of biodegradable polymer, such as PLGA. In a further embodiment, the first layer is made of one biodegradable polymer, such as PLGA, and the second layer is made of a different biodegradable polymer. In an embodiment, there is provided a seed according to the present invention, the seed further comprising one or more additional therapeutic agents selected from anti-cancer agents (such as bevacizumab), statins (such as pitavastatin), antiplatelet agents (such as ticlopidine), antifungal agents (such as itraconazole), angiotensin-converting enzyme (ACE) inhibitors (such as captopril), enzyme acetaldehyde dehydrogenase inhibitors (such as disulfiram), anti-inflammatory agents (such as celecoxib) and copper gluconate. In an embodiment, there is provided a seed according to the present invention, the seed further comprising pitavastatin. In an embodiment, there is provided a seed according to the present invention, the seed further comprising captopril and disulfiram. In an embodiment, there is provided a seed according to the present invention, the seed further comprising captopril, celecoxib and itraconazole. In an embodiment, there is provided a seed according to the present invention, the seed further comprising captopril, pitavastatin and ticlopidine. In an embodiment, there is provided a seed according to the present invention, the seed further comprising pitavastatin, disulfiram and copper gluconate.

Pitavastatin refers to the compound ((3R,<NUM>,6E)-<NUM>-[<NUM>-cyclopropyl-<NUM>-(<NUM>-fluorophenyl)quinolin-<NUM>-yl]-<NUM>,<NUM>-dihydroxyhept-<NUM>-enoic acid) having the following structure and pharmaceutically acceptable salts thereof:
<CHM>.

Conveniently, as used herein, pitavastatin refers to the calcium salt of the above compound, i.e. calcium bis((3R,<NUM>,6E)-<NUM>-[<NUM>-cyclopropyl-<NUM>-(<NUM>-fluorophenyl)quinolin-<NUM>-yl]-<NUM>,<NUM>-dihydroxyhept-<NUM>-enoate).

In another embodiment, the composition comprises one or more seeds, wherein the seeds comprises a single layer of biodegradable polymer in which are dispersed:.

In one embodiment, the composition comprises one or more seeds, wherein each seed comprises two layers of biodegradable polymer in which are dispersed:.

In one embodiment, the composition comprises one or more seeds, wherein each seed comprises two layers of PLGA (such as <NUM>:<NUM> w/w LA:GA) in which are dispersed:.

In one embodiment, the composition comprises one or more seeds, wherein each seed has a primary length of about <NUM> and a secondary length of about <NUM> and comprises two layers of PLGA (such as <NUM>:<NUM> w/w LA:GA) in which are dispersed:.

wherein each of the two layers has a primary length of about <NUM> and a secondary length of about <NUM>.

In an aspect of the present invention there is provided a seed as described herein for use in therapy. In an embodiment, there is provided a seed as described herein for use in the treatment of a disease or condition treatable by a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>)and a pharmaceutically acceptable salt of SN-<NUM>.

There is also provided a method for administering a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>) and a pharmaceutically acceptable salt of <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>)to a warm-blooded animal (preferably a human), said method comprising implanting one or more seeds as described herein in the warm blooded animal.

In a further aspect of the present invention there is provided a seed as described herein for use in treating cancer. In a further aspect of the present invention there is provided a seed as described herein for use in treating solid tumours. In an embodiment, the cancer is a solid tumour selected from colorectal cancer, prostate cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, bladder cancer, lung cancer (including small cell lung cancer) or brain cancer. In a preferred embodiment, there is provided a seed as described herein for use in the treatment of a brain tumour; preferably wherein the brain tumour is a high grade (grades III and IV) glioma. In an embodiment, the seed is for use in the treatment of a grade IV glioma (such as glioblastoma multiforme).

The chemotherapeutic seeds of the present invention, or compositions comprising them, may enhance the effects of conventional chemotherapy, radiotherapy, immunotherapy, hormone therapy or gene therapy.

The chemotherapeutic seeds of the present invention, or compositions comprising them, may be placed directly in, or injected around, a solid tumour. Preferably, they may be placed into the resection cavity created after surgical debulking of a solid tumour. In an embodiment, there is provided a seed as described herein for use in the treatment of a solid tumour, wherein the treatment comprises implanting the seed (or seeds) into the resection margin of the debulked tumour. Alternatively, they may be inserted intra-tumourally in unresectable tumours by stereotaxy. In a preferred embodiment, there is provided a seed as described herein for use in the treatment of a brain tumour (preferably high grade gliomas (grades III and IV)), wherein the treatment comprises implanting the seed into the resection margin of the debulked brain tumour.

There is also provided a method for treating a brain tumour (preferably high grade gliomas (grades III and IV)), wherein the treatment comprises implanting a seed as described herein in or around the brain tumour (preferably wherein the treatment comprises implanting the seed into the resection margin of the debulked brain tumour).

Due to the size of the seeds as described herein, it is envisaged that the seeds may be administered to the tumour site via a catheter or needle. In an embodiment, a seed according to the present invention, or a composition comprising one or more of said seeds, is to be administered via a catheter or needle.

The chemotherapeutic seeds as described herein may be prepared by any suitable method, including compression, solvent casting, hot moulding, injection moulding, extrusion, or 3D printing. The nature of the biodegradable polymer and/or the drug may influence the choice of manufacturing method. Preferably, the chemotherapeutic seeds as described herein are prepared by hot melt extrusion.

According to a further aspect of the invention there is provided a process for the preparation of a chemotherapeutic seed as described herein, the process comprising the steps of:.

Preferably, the drug and biodegradable polymer are pre-mixed before being charged to the hot melt extruder. Therefore, in an embodiment, step a) comprises the steps of:.

The mixing in step a1) may be carried out by any conventional means, such as by using a pharmaceutical mixer or a speed mixer.

If a plasticiser is a component of the seed being produced, then the plasticiser is also charged during step a) of the above process.

Preferably, the hot melt extruder is a co-rotating twin screw hot melt extruder. In an embodiment, the screw speed is <NUM> to <NUM> RPM, such as <NUM> to <NUM> RPM or preferably <NUM> to <NUM> RPM.

Typically, the hot melt extruder comprises three zones; firstly a feeding zone, then a mixing zone and finally a metering or discharge zone. To enable efficient melting of the biodegradable polymer and homogenous mixing with the drug particles, but without causing thermal degradation, typically the three zones will be run at different temperatures. In an embodiment, the feeding zone is set at <NUM> to <NUM>. In an embodiment, the mixing zone is set at <NUM> to <NUM>. In an embodiment, the discharge zone is set at <NUM> to <NUM>. In an embodiment, the feeding zone is set at <NUM> to <NUM>, the mixing zone is set at <NUM> to <NUM> and the discharge zone is set at <NUM> to <NUM>.

In step b) the mixture is typically extruded from the discharge zone of the hot melt extruder via a die on to a haul off conveyor. After cooling on the haul off conveyor, the extrudate solidifies. Once solidified the extrudate may be formed into one or more seeds of the required dimensions (step c)). Typically, the extrudate is cut into seeds of the desired length (such as <NUM> to <NUM>, preferably <NUM> to <NUM>). The diameter of the seeds is determined by the diameter of the die aperture and the haul off conveyor speed. Preferably the diameter of the seeds is <NUM> to <NUM>, such as <NUM> to <NUM>, or most preferably about <NUM>. In a preferred embodiment, the diameter of the die aperture is <NUM> to <NUM>, such as <NUM> to <NUM>. In a preferred embodiment, the haul off conveyor speed is <NUM> to <NUM> RPM, such as <NUM> to <NUM> RPM. Preferably, diameter of the die aperture is <NUM> to <NUM> (such as <NUM> to <NUM>) and the haul off conveyor speed is <NUM> to <NUM> RPM (such as <NUM> to <NUM> RPM).

In an embodiment, the biodegradable polymer in step a) is PLGA, preferably the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>. In an embodiment, the drug in step a) is irinotecan hydrochloride. In a preferred embodiment, the biodegradable polymer in step a) is PLGA (preferably the PLGA has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>) and the drug in step a) is irinotecan hydrochloride. In a preferred embodiment, the biodegradable polymer in step a) is PLGA wherein the PLGA is ester end-capped and has a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>; and the drug in step a) is irinotecan hydrochloride. In a preferred embodiment, the biodegradable polymer in step a) is PLGA with an acid number less than or equal to <NUM> KOH/g and a lactic acid:glycolic acid weight ratio about <NUM>:<NUM>; and the drug in step a) is irinotecan hydrochloride.

In a preferred embodiment, there is provided a process for the preparation of a chemotherapeutic seed as described herein comprising PLGA and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>) and a pharmaceutically acceptable salt of SN-<NUM>, the process comprising the steps of:.

wherein the hot melt extruder has a feeding zone set at <NUM> to <NUM> (such as <NUM>), a mixing zone set at <NUM> to <NUM> (such as <NUM>) and a discharge zone set at <NUM> to <NUM> (such as <NUM>). In a further embodiment, wherein the seed further comprises a plasticiser, the screw speed is about <NUM> RPM and the mixing zone is set at about <NUM>. In a further alternative embodiment, wherein the seed comprises no plasticiser, the screw speed is about <NUM> RPM and the mixing zone is set at about <NUM>.

In a further embodiment there is provided a process for the preparation of a chemotherapeutic seed as described herein comprising PLGA, irinotecan hydrochloride and an additional therapeutic agent, the process comprising the steps of:.

In a further embodiment there is provided a process for the preparation of a chemotherapeutic seed as described herein comprising PLGA, irinotecan hydrochloride and an additional therapeutic agent, wherein the seed comprises two layers of which a first layer contains irinotecan hydrochloride; and a second layer contains an additional therapeutic agent; the process comprising the steps of:.

In the above embodiment, preferably in step c) and/or step f) the extrudate is cut into seeds of the desired length (such as <NUM> to <NUM>, preferably about <NUM>). In the above embodiment, preferably in step c) and/or step f) the extrudate is formed into seeds of diameter about <NUM>. In the above embodiment, preferably in step c) and/or step f) the extrudate is formed into seeds of diameter about <NUM> and length about <NUM>. In the above embodiment, preferably in step d) the additional therapeutic agent is pivatastatin. In the above embodiment, preferably in step g) the physical joining of the seeds is achieved by butt welding.

In an aspect of the present invention, there is provided a chemotherapeutic seed obtained, or obtainable, by a process as described herein. In an embodiment, there is provided a chemotherapeutic seed obtained, or obtainable, by a hot melt extrusion process as described herein.

The invention will now be described in more detail in relation to the following illustrative examples.

Poly(DL-lactide-co-glycolide) (PLGA) with a <NUM>:<NUM> lactide:glycolide ratio (PURASORB PDLG)) was purchased from Corbion (Amsterdam, The Netherlands). Samples '<NUM>', '<NUM>' and '<NUM>' were described as having inherent viscosities of <NUM>-<NUM> dL/g, <NUM>-<NUM> dL/g and <NUM>-<NUM> dL/g respectively when measured at <NUM> in chloroform at a concentration of <NUM>/dL. The '<NUM>' PLGA was acid end-capped (acid number ≥ <NUM> KOH/g) , while the '<NUM>' and `<NUM>' PLGA samples were ester end-capped (acid number ≤ <NUM> KOH/g). Irinotecan hydrochloride (IRN HCl) was purchased from LGM Pharma (Nashville, TN). Pitavastatin (calcium salt) was purchased from LGM Pharma (Nashville, TN). Temozolomide, Kolliphor® P188, acetonitrile, dichloromethane and sodium phosphate were purchased from Sigma-Aldrich (Dorset, England). Kolliphor® plasticisers RH40 and P237 were purchased from BASF (Ludwigshafen, Germany). GBM cell lines were retrieved from patients who received resection surgery at the Queen Elizabeth Hospital, Birmingham, UK.

Hot melt extrusion was carried out using a <NUM> <NUM>:<NUM> microlab co-rotating twin screw extruder (Rondol Technology, Stoke-on-Trent, UK).

Confocal Raman spectroscopy was used to detect and spatially resolve IRN distribution within the extruded PLGA polymer blocks. Maps and spectra were acquired using a confocal Raman microscope (Alpha300R, WITec, Ulm, Germany), equipped with an Acton SP2300 Imaging Monochromator/Spectrograph (Princeton Instruments, MA, USA), fitted with a <NUM>/mm <NUM> blaze grating, and a <NUM> 250mW diode laser (XTRA II, Toptica photonics, Munich, Germany). Raman maps were acquired a 20X (NA = <NUM>) objective lens and in continuous scan mode with an integration time of <NUM> and scan areas of <NUM> wide by <NUM> high. Data were accumulated and exported using the WITec Control software version <NUM> (WITec, Ulm, Germany) into. spc files, which were then processed using in-house MATALB Raman data processing tools. Imported hyperspectral datasets were baseline corrected before applying vector normalisation and finally generation of IRN and PLGA maps.

The IRN HCl-loaded biodegradable polymer seeds are manufactured by mixing IRN HCL with a biodegradable polymer using a pharmaceutical mixer or speed mixer. Depending on the weight % of IRN HCL within the implant, addition of a plasticiser may be required. The mix is then fed into the barrel of the hot melt extruder. The biodegradable polymer (and plasticiser if required) soften and/or melt and the IRN HCL or derivatives thereof are incorporated into the biodegradable polymer matrix. The mix is discharged from the hot melt extruder via a die, whereby the haul off conveyor speed is used to control the diameter of the extrudate strand. Upon cooling, the extrudate is cut into one or more seeds of the required dimensions.

The appropriate amount of PLGA polymer and plasticizer (<NUM>, <NUM>, <NUM> and <NUM>% w/w) - of either Kolliphor® P188, Kolliphor® P237 or Kolliphor® RH40 - were weighed into a sealed plastic container and roll mixed for <NUM> minutes. The active mix was subsequently fed into a <NUM>:<NUM> microlab extruder at a feed rate of <NUM> grams per hour. The feeding, mixing and metering zones of the extruder were set at <NUM>, <NUM> and <NUM> respectively. The melt was extruded through a <NUM> die and subsequently cut into blank PLGA seeds of <NUM> in length.

To determine the diameters and weights of the blank seeds, samples (n = <NUM>) of each blank seed were assessed for their average weight and diameter. Each seed was measured in the middle and at both ends, using a digital Vernier calliper. The three measurements were averaged to give the diameter of each individual blank seed. The results can be seen in <FIG>. The higher the plasticiser loading the more consistent the seed diameter produced, with Kolliphor® P188 and RH <NUM> requiring a minimum loading of <NUM>% w/w to produce seeds with a consistent diameter, while Kolliphor® P237 required a minimum loading of <NUM>% w/w.

To investigate the influence of the plasticiser type and loading on the swelling of the blank seeds, each blank seed (n = <NUM>) was placed in a glass vial with <NUM> of distilled water and the vials placed into an orbital shaking incubator (Infors HT) at <NUM> and <NUM> RPM. Their length and width were measured using a digital Vernier calliper at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours post-incubation. The RH <NUM> plasticizer resulted in significant (P = <NUM>) swelling at all loadings (<FIG>). The P188 and P237 plasticizers had significant (P = <NUM>) swelling at loadings of <NUM> and <NUM>% w/w (<FIG>). Based on the data shown in <FIG>, the preferred plasticiser in terms of consistent seed diameter and resistance to swelling was P188 at a loading of <NUM>% w/w.

<NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA. <NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA. <NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA.

Each of the mixes was individually fed into the barrel of the hot melt extruder. For all mixes the feed rate was set at <NUM> per minute and the screw speed of the extruder was set at <NUM> RPM. The barrel of the hot melt extruder had three heating zones with heating zone <NUM> (feeding zone) at <NUM> and heating zone <NUM> (mixing zone) set at <NUM>, while heating zone <NUM> (discharge zone) was set at <NUM>. The torque of the extruder was between <NUM> and <NUM> N m, dependent on the composition of the mix being fed into the extruder. The die on the extruder was <NUM> and the haul off conveyor had a speed of <NUM> RPM to maintain the diameter of the extrudate at <NUM>. Once solidified the extrudate was cut into seeds of <NUM> in length. The extrudate containing <NUM> wt% of IRN HCl was also cut into seeds of either <NUM> or <NUM> in length.

<FIG> shows the appearance of <NUM> wt% IRN HCl seeds with <NUM> diameter and <NUM> length.

<NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA and <NUM> of Kolliphor® P188 plasticiser. <NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA and <NUM> of Kolliphor® P188 plasticiser. <NUM> wt% seeds were prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA and <NUM> of Kolliphor® P188 plasticiser.

Each of the mixes was individually fed into the barrel of the hot melt extruder. For all mixes the feed rate was set at <NUM> per minute and the screw speed of the extruder was set at <NUM> RPM. The barrel of the hot melt extruder had three heating zones with heating zone <NUM> (feeding zone) was at <NUM> and heating zone <NUM> (mixing zone) set at <NUM>, while heating zone <NUM> (discharge zone) was set at <NUM>. The torque of the extruder was between <NUM> and <NUM> N m, and depended on the composition of the mix being fed into the extruder. The die on the extruder was <NUM> and the haul off conveyor had a speed of <NUM> RPM to maintain the diameter of the extrudate at <NUM>. Once solidified the extrudate was cut into seeds of <NUM> or <NUM> in length.

<NUM> wt% IRN HCl-loaded PLGA seeds having <NUM> diameter and <NUM> length were prepared according to Example <NUM>, under the following hot melt extrusion parameters:.

<NUM> wt% IRN HCl-loaded PLGA seeds having <NUM> diameter and <NUM> length were prepared according to Example <NUM>, substituting either '<NUM>' or `<NUM>' PLGA for '<NUM>' PLGA.

A random sample (n = <NUM>) of each seed type was selected, weighed and placed into a glass vial. DCM (<NUM>) was added to each vial and left for <NUM> hour to dissolve the seeds. Once dissolved, the vials were placed in a water bath set at <NUM> to completely evaporate the dichloromethane. The remaining residue was re-suspended in HPLC mobile phase, causing the PLGA polymer to crash out and the IRN HCl to go into solution. To ensure complete dissolution of IRN HCl the solution was sonicated for <NUM> minutes. The solution was then filtered and analysed for its IRN HCl concentration using the HPLC methodology described below.

<FIG> show that the seeds tested had drug contents between <NUM>% and <NUM>% of their theoretical value, confirming that the hot melt extrusion process produced seeds with the desired drug content.

<FIG> shows the drug content variation for the <NUM>% w/w seeds prepared according to Example <NUM> (A) to (E). At <NUM> RPM and <NUM> mixing, <NUM>% w/w of a plasticiser is required to ensure the homogenous mixing of the IRN into the PLGA. As it may be desirable to produce seeds without the need for a plasticizer, alternative hot melt extrusion parameters were explored as detailed in Example <NUM>. Upon removing the plasticiser the average content of the IRN in the seeds significantly reduced, while the homogeneity was poor with a standard deviation of <NUM> (<FIG>). The screw speed was reduced to <NUM> RPM to increase the residence time and improve mixing, however the average drug content was further reduced (<FIG>) due to degradation of the IRN. By reducing the mixing temperature to <NUM> and maintaining the screw speed at <NUM> RPM it was possible to achieve an acceptable IRN content and homogeneity (<FIG> - std dev of <NUM>) similar to that of the seeds with <NUM>% plasticizer.

Raman spectroscopic mapping carried out on cross-sections of the Example <NUM> seeds (<FIG>) confirmed that in Example <NUM> (A) and Example <NUM> (E) the drug is evenly distributed across the seeds. In Examples <NUM> (B), (C) and (D) the drug is not evenly distributed across the seed and the overall intensity of the dark spots is less, indicating lower drug content. IRN HPLC methodology - HPLC analysis was performed on a Dionex Ultimate <NUM> HPLC with a Phenomenex Luna C18 <NUM> × <NUM> column having a <NUM> particle size. The mobile phase was comprised of <NUM>% pH <NUM> phosphate buffer and <NUM>% acetonitrile. The flow rate was <NUM>/min, while UV detection was performed at a wavelength of <NUM> with an injection volume of 20µL. Linearity was found to be in the range of <NUM> to <NUM>/ml with an R<NUM> of <NUM>.

<NUM> of IRN HCl was mixed with <NUM> of '<NUM>' PLGA to form mix A. <NUM> of PVT was separately mixed with <NUM> of '<NUM>' PLGA to form mix B.

Mix A was fed into the barrel of a hot melt extruder. The feed rate was set at <NUM> per minute and the screw speed of the extruder was set at <NUM> RPM. The barrel of the hot melt extruder had three heating zones with heating zone <NUM> (feeding zone) was at <NUM> and heating zone <NUM> (mixing zone) set at <NUM>, while heating zone <NUM> (discharge zone) was set at <NUM>. The torque of the extruder was between <NUM> and <NUM> N m, and depended on the composition of the mix being fed into the extruder. The die on the extruder was <NUM> and the haul off conveyor had a speed of <NUM> RPM to maintain the diameter of the extrudate at <NUM>. Once solidified the extrudate was cut into (A) seeds of <NUM> in length. The above hot melt extrusion process was repeated with mix B to give (B) seeds of <NUM> in length.

Dual layer implants were prepared by butt welding (A) seeds to (B) seeds as follows: a pallet knife was heated to <NUM> and an (A) seed and a (B) seed pushed against opposite sides of the knife. The knife was removed and the melted ends of each layer were pushed together and held for <NUM> seconds to allow the polymer to cool and the layers to weld together. The dimensions of the dual-layer seeds were <NUM> diameter × <NUM> length.

<NUM> of IRN HCl was mixed with <NUM> of '<NUM>' PLGA to form mix A. <NUM> of PVT was separately mixed with <NUM> of '<NUM>' PLGA to form mix B. Dual layer implants were prepared from these mixes in an analogous manner to the method described for Example 6A.

<NUM> wt% mix was prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA. <NUM> wt% mix was prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA. <NUM> wt% mix was prepared by mixing <NUM> of IRN HCl with <NUM> of '<NUM>' PLGA.

Each of the <NUM> mixes was individually fed into the barrel of the hot melt extruder. For all mixes the feed rate was set at <NUM> per minute and the screw speed of the extruder was set at <NUM> RPM. The barrel of the hot melt extruder had three heating zones with heating zone <NUM> (feeding zone) was at <NUM> and heating zone <NUM> (mixing zone) set at <NUM> while heating zone <NUM> (discharge zone) was set at <NUM>. The torque of the extruder was between <NUM> and <NUM> N m, and depended on the composition of the mix being fed into the extruder. The die on the extruder was <NUM> and the haul off conveyor had a speed of <NUM> RPM to maintain the diameter of the extrudate at <NUM>. Once solidified the extrudate was cut into seeds of <NUM> in length.

Each of the <NUM> mixes was individually fed into a <NUM> diameter pellet die and subsequently placed into a <NUM> Ton hydraulic KBr press. The mixes were compressed under <NUM> tonnes (<NUM> PSI) of pressure for <NUM> minutes at room temperature. The compressed implants were cut into seeds of <NUM> in length.

400µL of the IRN content solutions from Example <NUM> were diluted with 3600µL of sterile cell culture media and filtered. Control solutions were produced by dissolving the required amount (based on the actual content of IRN in each seed) of IRN in <NUM> of DCM and subsequently evaporating it off. The residual IRN was dissolved in <NUM> of PBS and 400µL of this solution was diluted with 3600µL of cell culture media and filtered. The cytotoxicity of the solutions was determined against primary GBM cell lines and using a <NUM> day exposure time.

<FIG> show the GBM cell viability data generated according to Example <NUM> and demonstrate that the IRN extracted from the various seeds retained its cytotoxicity. When compared to the unprocessed control solutions of the same concentrations, there was no significant (P = <NUM>) difference in cell viability across the samples.

Individual seeds of <NUM> × <NUM> dimensions with varying (<NUM>, <NUM>, <NUM>, <NUM> and <NUM>% w/w) IRN HCl loadings and individual seeds of <NUM>% w/w IRN HCl loading with varying (<NUM>, <NUM> and <NUM>) lengths (n = <NUM>) were placed into a sealed flask containing either <NUM> of water (bio-relevant media) or <NUM> of phosphate buffered (pH <NUM>) solution (sink conditioned media) and placed into an orbital shaking incubator (Unitron HT infors) at <NUM> and <NUM> rpm. Complete media replacement was performed at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days. The samples where filtered using a <NUM> filter and analysed for IRN content using the IRN HPLC method described above.

<FIG> and <FIG> show the measured in vitro release profiles under both sink (A) and bio-relevant (B) conditions. All sample demonstrated an 'initial burst' release over the first <NUM> hours, followed by a prolonged period of slower release until day <NUM>. The burst release was proportionate to both the loading of IRN HCl and the seed length. Under sink conditions, an increase in drug loading from <NUM> to <NUM>% significantly (P = <NUM>) increased release on day one from <NUM> to <NUM>, while the total release over the <NUM> days increased (P = <NUM>) from <NUM> to <NUM> (<FIG>). Under bio-relevant conditions, however, there was no significant (P = <NUM>) difference in release between the <NUM> and <NUM>% w/w seeds. This is potentially due to the large amount of IRN on the surface and within the seeds being released and saturating the diffusion layer at the seed/release media interface. An increase in drug loading from <NUM> to <NUM>/<NUM>% significantly (P = <NUM>) increased release on day one from <NUM> to approximately <NUM>, while the total release over the <NUM> days increased (P = <NUM>) from <NUM> to approximately <NUM> (<FIG>).

Increasing the dimensions of the seeds from <NUM> × <NUM> to <NUM> × <NUM> had no significant influence on drug release (<FIG>). However, increasing the dimensions to <NUM> × <NUM> significantly increased release, with an increase on day one from <NUM> to <NUM> and an increase in the total release from <NUM> to <NUM> under sink conditions (<FIG>) and an increase on day one from <NUM> to <NUM> and an increase in the total release from <NUM> to <NUM> under bio-relevant conditions (<FIG>).

These results demonstrated that IRN release can be controlled by both drug loading and the seed dimensions, which facilitates accurate control of IRN levels in vivo. Under sink conditions all of the seed formulations released greater than <NUM>% of their drug-loading over <NUM> days of release. Under the bio-relevant release conditions the seed formulations released between <NUM> and <NUM>% of their drug-loading over <NUM> days of release.

The IRN levels released in the bio-relevant media were lower than those released under sink conditions. It is possible that the increased drug release under sink conditions was due to the increased volume (<NUM> compared to <NUM>) of the media rather than its composition. It is postulated that the increased volume decreases the thickness and concentration of the IRN diffusion layer at the seed/release media interface, which increases drug release.

It is believed that this type of release profile (an initial burst in the first <NUM> hours, followed by a sustained period of release over <NUM> days) is clinically advantageous in the treatment of resected brain tumours, as the burst allows for a high local dose of IRN HCl to be delivered initially, enhancing diffusion of the IRN HCl through the brain tissue and killing off most of the residual tumour cells, while the sustained period of release provides a 'top-up' of IRN HCl to kill those tumour cells that were in the G<NUM> or G<NUM> phase of the cell cycle.

The in vitro daily release profiles for the seeds released under bio-relevant conditions are presented in <FIG>, and also illustrate this drug release profile. There was a large day one burst, which increased significantly (P = <NUM>) with an increase in drug loading. The seeds then demonstrated a typical matrix release profile, with a decrease in drug release over time. The <NUM> × <NUM> <NUM>%, <NUM>% and <NUM>% w/w and the <NUM> × <NUM> <NUM>% w/w seed formulations were capable of releasing at least <NUM> to <NUM>µg of IRN per day for the full <NUM> days (as represented by the lower and upper dotted lines in <FIG>). Accordingly, in terms of the drug release profile the preferred drug loading is <NUM>% to <NUM>% w/w and the preferred seed size is <NUM> × <NUM> or <NUM> × <NUM>.

The <NUM>% w/w IRN HCl PLGA seeds prepared according to Example <NUM> were also assessed for their in vitro drug release profile under sink conditions (<FIG>). Although the same overall profiles (initial burst followed by sustained release over <NUM> days) were seen for the <NUM> different PLGA viscosity formulations, the '<NUM>' seeds surprisingly released significantly less drug than the '<NUM>' or '<NUM>' seeds. It is postulated that this is a result of the '<NUM>' PLGA being acid-terminated, whereas the '<NUM>' and `<NUM>' PLGA samples were ester-terminated. The parent drug in the salt IRN HCl is positively charged and so it is possible that interactions between the polymer acid groups and the drug result in a stabilisation effect, such that release of the drug into the aqueous dissolution media is retarded.

A <NUM> sample of the bio-relevant release media from day <NUM> and day <NUM> of the Example <NUM> drug release experiments was evaluated for cytotoxicity against primary GBM cells. 400µl of each bio-relevant release sample was added to 3600µl of cell culture media and filtered using a syringe filter to ensure sterility. 200µl was subsequently added to the wells of a <NUM>-well plate containing the cultured primary GBM cells and <NUM>µl of cell culture media. Following <NUM> days of exposure an MTT assay was performed to assess % cell viability.

An increase in IRN loading from <NUM> to <NUM>% w/w resulted in an a decrease in the cell viability from <NUM> to <NUM>% (<FIG> - day <NUM> release), while an increase in the length of the seeds from <NUM> to <NUM> resulted in a decrease in cell viability from <NUM> to <NUM>% (<FIG> - day <NUM> release). In all seeds the cell viability on day <NUM> is higher than that observed on day <NUM>, which is to be expected due to the lower amount of IRN released on day <NUM> (c. These experiments demonstrate that the drug retains its cytotoxicity during formulation, storage and release.

For the samples derived from the release experiments using Example <NUM> seeds (<FIG>), it can been seen that the PLGA '<NUM>' sample is not efficacious in killing the GBM cells at either day <NUM> or day <NUM>, which is not surprising given the low drug release profile seen for '<NUM>' in <FIG>. PLGA `<NUM>', on the other hand, provides a large burst of IRN HCl on day <NUM> (see <FIG>), however, due to the almost exhaustion of drug release come day <NUM>, `<NUM>' releases less IRN HCl on day <NUM> than '<NUM>'. The optimal drug release profile over the <NUM> day period was shown by the '<NUM>' seeds, resulting in less than <NUM>% cell viability with both the day <NUM> and day <NUM> release. For this reason, the preferred PLGA is a <NUM>:<NUM> lactide:glycolide PLGA with ester end-capping, having an inherent viscosity of <NUM>-<NUM> dL/g (`<NUM>' PLGA).

GBM cells were extracted from tissue taken from the resection margin of GBM patients, cultured and seeded onto <NUM> well plates. Steam-sterilised <NUM> long PLGA seeds were placed directly onto the GBM cells to mimic the clinical scenario. <NUM> of media was used to cover the implants. The media was changed every day to represent the turnover of cerebral spinal fluid as well as the removal of IRN via diffusion, metabolic elimination and permeation into nearby vasculature, which will reduce the concentration of IRN at the resection margin. The study was designed so that MTT assays could be performed on the cells at days <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

<FIG> shows that all of the seed implants tested reduced the tumour cell viability with the reduction in cell viability being proportional to the loading of IRN HCL in the seed. The <NUM> and <NUM> weight % IRN HCl seeds reduced the cell viability to <NUM> and <NUM>% by day <NUM> respectively; however, significant cell regrowth (representing recurrence) occurred at day <NUM> and <NUM>. The <NUM> weight % IRN HCL seeds reduced cell viability to <NUM>% by day <NUM>, while the <NUM> and <NUM> weight % IRN HCL seeds achieved this by day <NUM>. Furthermore with the <NUM>, <NUM> and <NUM> weight % seeds there was no sign of cell regrowth (recurrence), which suggests that all of the GBM cells were killed off.

These results indicate that the <NUM> wt%, <NUM> wt% and <NUM> wt% seeds deliver sufficient irinotecan over a <NUM> day period to completely kill off the entire residual tumour cells remaining after resection surgery.

To assess their toxicity, <NUM> × <NUM> seed formulations with <NUM>, <NUM>, <NUM> and <NUM>% w/w IRN loadings were implanted into sham resection cavities of non-tumour bearing immunocompetent C57/BL6 mice. Mice were sacrificed via transcardial perfusion at <NUM>, <NUM>, <NUM>, and <NUM>-days post-implant. Brains were stored in <NUM>% formalin upon collection, then moved to <NUM>% formalin after <NUM> hr. Brains were cut in coronal orientation at the rostral and caudal edges of the resection cavity, then embedded in paraffin blocks. Blocks were sectioned into <NUM> thick slices and stained with H&E. The resulting histological slides (<FIG>) were examined by a blinded clinical pathologist. Degree of acute inflammation, chronic inflammation, macrophage infiltration, and necrosis were individually scored on a scale of <NUM>-<NUM> (low, medium, high) and summed to achieve a 'toxicity score' for each seed formulation at each time point (<FIG>). The in vivo toxicity study adhered to the NIH Guide for the Care and Use of Laboratory Animals.

The toxicity results presented in <FIG> shed light on the toxicity profile of the seeds relative to a placebo control. Regardless of IRN loading, moderate levels of acute inflammation are present one week after implantation, however, this inflammation subsides over time. This behaviour is attributable to the wound healing response to resection-induced surgical brain injury. Chronic inflammation due to persistent drug release from the seeds is likewise highest at the onset of implantation as a result of the initial burst of IRN from the seeds and is generally resolved over time, with the exception of <NUM>% which still has detectable levels on day <NUM>. While only mild chronic inflammation is observed in the <NUM>% and <NUM>% groups, moderate inflammation is seen on day <NUM> in both the <NUM>% and <NUM>% groups, meaning these higher concentrations of IRN elicit a more intense inflammatory response. This observation is further supported by the presence of necrosis in both the <NUM>% and <NUM>% groups. In summary, the <NUM>% seeds do not appear any more toxic than the placebo control in any substantive way, whereas the <NUM>% and <NUM>% seeds cause elevated immune activity and temporary damage to parenchymal tissue.

The U-<NUM> human GBM cell line has been characterized as a glial tumour with the histopathological appearance of a GBM. The cell line was obtained from ATCC, Manassas, Virginia, USA. The GBM cell line was maintained in culture in DMEM media in an incubator with <NUM>% CO2 at <NUM>. Flasks were kept in the logarithmic growth phase and cells were passaged every <NUM>-<NUM> days.

Orthotopic GBM tumours were established in the brains of immunodeficient athymic nude mice (n = <NUM> in each group) via stereotaxic injection as previously described [<NPL>; <NPL>]. Briefly, <NUM> × <NUM><NUM> U87 mCherry-Fluc (U87 mChFI) cells in <NUM>µl serum-free DMEM were loaded into a <NUM>µl capacity Hamilton syringe. The needle was positioned at stereotaxic coordinates [<NUM>, -<NUM>, -<NUM>] from the bregma point. Tumour cells were then injected at <NUM>µl/min, allowed to settle for <NUM>, then the needle was retracted at <NUM>/min. The tumours were given one week to engraft and grow. Established tumours were then resected under fluorescent guidance, and then <NUM> × <NUM> seeds with either <NUM>, <NUM> and <NUM> wt% IRN HCl loadings were implanted into the resulting resection cavity (<FIG>). The mice were monitored for survival over <NUM> days and Kaplan-Meier survival curves produced (<FIG>). Changes in tumour volume were tracked by bioluminescence. Mice were injected with <NUM>/kg luciferin IP, and then imaged <NUM> later in an IVIS Kinetic imager under isoflurane anesthesia. Identically-sized regions of interest were drawn over the heads of each mouse, and average radiance was recorded (<FIG> - imaging of surviving mice at day <NUM> post implantation). The in vivo efficacy study adhered to the NIH Guide for the Care and Use of Laboratory Animals.

The efficacy data (<FIG>) demonstrates that <NUM>% of the mice in the sham (no implant) and placebo groups had died by day <NUM> and <NUM> respectively. However, <NUM>% of the mice in the <NUM> wt% implant group died by day <NUM>, which is due to the level of toxicity observed in the toxicity study for this drug loading (Example <NUM>). The mice in the <NUM> wt% and <NUM> wt % implant groups showed long-term survival with <NUM>% of the mice still alive at day <NUM>. Furthermore, the surviving mice show no expected symptoms associated with a recurring brain tumour and when imaged using Fluc bioluminescence imaging none of them showed any sign of brain fluorescence, which would have been indicative of tumour recurrence (<FIG>). The in vivo testing indicates that the preferred drug loadings in terms of a balance between tumour cytotoxicity and systemic toxicity are the <NUM> wt% and <NUM> wt % IRN drug loadings.

<NUM>%, <NUM>% and <NUM>% w/w IRN HCl-loaded seeds were prepared by both hot melt extrusion (HME) and compression as described in Example <NUM>.

Individual seeds were placed into a sealed flask containing <NUM> of phosphate buffered (pH <NUM>) solution (sink conditioned media) and placed into an orbital shaking incubator (Unitron HT infors) at <NUM> and <NUM> rpm. Complete media replacement was performed at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days. The samples where filtered using a <NUM> filter and analysed for IRN content using the irinotecan HPLC method described above.

<FIG> shows the measured in vitro release profiles. The seeds prepared by compression (<FIG>) delivered a very large 'initial burst' of IRN over the first <NUM> hours and by day <NUM> almost all the IRN had been released, with very limited release of IRN between days <NUM> and <NUM> (<FIG>). The seeds prepared by HME (<FIG>) delivered an 'initial burst' release over the first <NUM> hours, followed by a prolonged period of slower release until day <NUM>. The <NUM>% and <NUM>% w/w HME seeds were capable of releasing at least <NUM> to <NUM>µg of IRN per day for the full <NUM> days (as represented by the lower and upper dotted lines in <FIG>) indicating coverage matching the desired therapeutic window for the treatment of gliomas. In this regard, HME is superior to compression in delivering IRN-loaded seeds with the desired properties for treating solid tumours.

TMZ, IRN and IRN + PVT were added at increasing concentrations up to <NUM> log nm to cultured primary GBM cells taken from eight patients with recurrent GBM. After <NUM> days of exposure an MTT assay was performed to assess % cell viability.

<FIG> shows the cytotoxicity of the three treatments. All eight patient cell lines evaluated did not respond significantly to TMZ treatment. Six of the eight patient cell lines responded to treatment with IRN and all eight patient cell lines responded to treatment with IRN + PVT. In the combination arm, at a <NUM> log nm concentration of both IRN and PVT there were no viable GBM cells in six of the eight patient lines.

In a separate experiment, <NUM> log nm TMZ, <NUM> log nm IRN or <NUM> log nm IRN + PVT (the exposure concentrations used were based on the average IC50 values determined from <FIG> - as temozolomide did not produce an IC50, a value of <NUM> Log nM was used) were added to cultured primary GBM cells taken from four patients with recurrent GBM. Average % cell viability was assessed by MTT assay after <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days exposure.

<FIG> shows the cytotoxicity of the three treatments over the differing exposure times. In all three treatment arms for all four patient cell lines, the cell viability was lowest after <NUM> days, with viability increasing out to <NUM> days. In the TMZ arm the cell viability was between <NUM> and <NUM>%, while in the IRN alone arm, cell viability was between <NUM> and <NUM>%. However, in the IRN + PVT arm, the cell viability at day <NUM> was between <NUM> and <NUM>%. This data suggests that the IRN + PVT combination is better at slowing down recurrence compared to TMZ or IRN alone.

IRN-HCl (<NUM>% w/w) and PVT (<NUM>% w/w) loaded dual-layer seeds were prepared according to Example 6A and IRN-HCl (<NUM>% w/w) and PVT (<NUM>% w/w) loaded dual-layer seeds were prepared according to Example 6B.

Individual seeds were placed into a sealed flask containing <NUM> of phosphate buffered (pH <NUM>) solution (sink conditioned media) and placed into an orbital shaking incubator (Unitron HT infors) at <NUM> and <NUM> rpm. Complete media replacement was performed every day for <NUM> days. The samples where filtered using a <NUM> filter and analysed for IRN content and PVT content over <NUM> days using the irinotecan HPLC method described above.

<FIG> shows the measured in vitro release profiles as both daily (<FIG>) and cumulative (<FIG>) API release. The dual-layer seeds gave an initial burst of both IRN and PVT over the first <NUM>-<NUM> days, followed by a sustained release of API out to <NUM> days. The dashed lines in <FIG> represent the potential daily amounts of IRN and PVT (<NUM> and <NUM> respectively) that need to be released in order to be effective based on the cytotoxicity data from Example <NUM> (<FIG> and <FIG>). It was observed that both seeds (<NUM>:<NUM>% w/w and <NUM>:<NUM>% w/w IRN:PVT) released at least these amounts of both APIs continuously over at least <NUM> days.

GBM cells were extracted from tissue taken from the resection margin of a recurrent GBM patient, cultured and seeded onto <NUM> well plates. Steam-sterilised seeds [(A) <NUM> × <NUM> PLGA seeds with no API added - control; (B) IRN-HCl (<NUM>% w/w) and PVT (<NUM>% w/w) loaded dual-layer seeds prepared according to Example 6A; and (C) IRN-HCl (<NUM>% w/w) and PVT (<NUM>% w/w) loaded dual-layer seeds prepared according to Example 6B] were placed directly onto the GBM cells to mimic the clinical scenario. <NUM> of media was used to cover the implants. The media was changed every day to represent the turnover of cerebral spinal fluid as well as the removal of API via diffusion, metabolic elimination and permeation into nearby vasculature, which will reduce the concentration of API at the resection margin. The study was designed so that MTT assays could be performed on the cells at days <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Claim 1:
A chemotherapeutic seed comprising a biodegradable polymer and a drug selected from irinotecan, a pharmaceutically acceptable salt of irinotecan, <NUM>-ethyl-<NUM>-hydroxy-camptothecin (SN-<NUM>), and a pharmaceutically acceptable salt of SN-<NUM>; wherein the seed has a primary length of at least <NUM>, and wherein primary length refers to the shortest dimension of the seed.