WO2018107246A1

WO2018107246A1 - Improving selective internal radiation therapy

Info

Publication number: WO2018107246A1
Application number: PCT/AU2017/051404
Authority: WO
Inventors: Ross Stephens; Gregory David Tredwell; Lee Andrew Philip; Karen Knox
Original assignee: Australian National University
Current assignee: Australian National University
Priority date: 2016-12-16
Filing date: 2017-12-15
Publication date: 2018-06-21
Anticipated expiration: 2019-06-16

Abstract

The present invention provides a particulate material, a therapeutic, a therapeutic device and a method of improving the treatment of cancer, in particular liver cancer in a patient in need thereof.

Description

IMPROVING SELECTIVE INTERNAL RADIATION THERAPY

TECHNICAL FIELD

[0001 ] The present invention provides a particulate material, a therapeutic, a therapeutic device and a method of improving the treatment of cancer, in particular liver cancer in a patient in need thereof.

BACKGROUND ART

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application in any jurisdiction.

[0003] Cancer is one of the leading causes of death in the United States and in many other countries. The disease is characterized by an abnormal proliferation of cell growth known as a neoplasm. Malignant neoplasms, in particular, can result in a serious disease state, which can threaten life. Of the vast array of malignant neoplasms, colorectal cancer is one of the most common.

[0004] Metastatic carcinoma commonly occurs in the liver from primary carcinomas of, for example, the colonic mucosa. The liver is a dominant site of metastatic spread of colorectal cancer as a result of the portal venous drainage of the gut. Metastatic hepatic lesions are often multifocal and may occur on a background of hepatitis or liver cirrhosis. Hence, liver metastases are linked to poor prognosis - recurrence and death are common outcomes.

[0005] Single tumours may be resected with the expectation of achieving prolonged survival, but multifocal or diffuse metastases are generally not operable. Surgical resection of colorectal cancer liver metastases can result in a cure, but more often than not produces a 5-year survival of 27-39% to a 10-year survival of 12-36%, as opposed to median survival of approximately 9 months if untreated.

[0006] Only 10-15% of patients are eligible for surgery and intra-hepatic and extra- hepatic relapse after liver resection is common. Further, of those patients who do undergo partial hepatectomy, a large proportion will develop recurrent tumours that are no longer resectable. . -

[0007] Significant research efforts and resources have been directed toward the elucidation of anticancer measures, including chemotherapeutic and radiotherapeutic agents, which are effective in treating patients suffering from cancer. Effective anticancer agents include those that inhibit or control the rapid proliferation of cells associated with neoplasms, those that effect regression or remission of neoplasms, and those that generally prolong the survival of patients suffering from malignant neoplasia.

[0008] Systemic chemotherapy is often used as first-line treatment in patients with non- resectable liver metastases, and in some cases, can sufficiently down-size the tumour burden in patients with previously inoperable liver metastases so that they may be converted to candidates for potentially curative resection. Internationally accepted first- line chemotherapy regimens for patients with metastatic colorectal cancer include FOLFOX (combination of bolus and infusional 5-fluorouracil [5-FU], leucovorin [LV] and oxaliplatin) and FOLFIRI (combination of bolus and infusional 5-FU, LV and irinotecan). These regimens provide median survival times of 16-20 months. [0009] One of the challenges that clinicians face in using traditional chemotherapeutic regimes is that the drugs and chemicals that they use in these regimes are delivered intravenously or sometimes orally, and thereby produce nonspecific effects on a patient, damaging non-target organs. This process can be beneficial to patients suffering from secondary metastases as the passage of such chemotherapeutics will combat secondary metastases in the vascular system. However, such regimes are not site specific and often have many secondary effects on a patient.

[0010] In the last decade, significant efforts have been made towards the development of alternate selective technologies that deliver therapies in a more site-specific manner, without distributing the treatment through the patient. [001 1 ] One group of technologies that has been attractive to many patients is the use of microparticles in the selective delivery of therapeutic agents to a patient. Such microparticles are usually bound or filled with chemotherapeutic agents or with radioactive isotopes, all of which are capable of killing neoplastic cells. The challenge with such technologies, though, has been to ensure that the microparticles are delivered specifically to a tumour to be treated without the particles being washed out of the tumour into the collecting vessels of the venous or lymphatic systems. One example of a microparticle technology that has been preferentially favoured by many patients with secondary liver metastases is Selective Internal Radiation Therapy (SIRT).

[0012] SIRT is an effective alternative treatment or adjunctive treatment for liver tumours, using radioactively loaded polymer microparticles that are delivered via a trans-femoral hepatic artery catheter. The use of microparticles to deliver SIRT has been a significant advance in the treatment of liver cancer. This treatment produces measurable tumour regression and has opened the possibility of prolonged survival. Accordingly, there is increasing interest in the use of microparticles for regional therapy of hepatic metastases. [0013] A fundamental characteristic of nearly all microparticle technologies is that it is necessary to deliver these particles predominately to a tumour rather than to normal liver parenchyma.

[0014] At least three factors influence distribution of microparticles in the liver.

[0015] First, microparticles need to be of an appropriate size to permit the microparticles to travel to a tumor once introduced into a patient. Usually this is achieved by either site specific delivery of microparticles to a tumour, which is ideal for single tumors but, becomes a challenge for multifocal or diffuse tumours. Alternatively, as is the case in SIRT, microparticles are delivered to the liver via a trans-femoral hepatic artery catheter. The microparticles then use the hepatic arterial and capillary network to make their way to the tumour where they lodge at limiting diameters of the arterial vessels. Importantly tumour blood vessels are heterogeneous with regard to organisation, function and structure. Whereas the normal vasculature is arranged in a hierarchy of evenly spaced, well-differentiated arteries, arterioles, capillaries, venules and veins, the tumour vasculature is unevenly distributed and chaotic. Tumour vessels often exhibit a serpentine course, branch irregularly and form arterio-venous shunts.

[0016] Blood flow through tumours does not follow a constant, unidirectional path. Not all open vessels are perfused continuously, and, over a few minutes, blood flow may follow different paths and even proceed in alternating directions through the same vessel network. Tumour blood vessels are more abundant at the tumour-host interface than in central regions, due to the angiogenesis induced by cytokines (e.g. VEGF) produced by tumour cells. Also, vascular density tends to decrease centrally as tumours . . grow, leading to inner zones of ischaemia and ultimately necrosis as tumours Outgrow their blood supply'. Finally, tumour blood vessels are structurally abnormal.

[0017] The lodgement, entrapment or embolization of microparticles in a tumour is fundamental to the efficacy of SIRT technology. An equally important factor in the value of that technology is microparticle size in that the microparticles must not be too small that they are washed out of the tumour into the collecting vessels of the venous or lymphatic systems, but must be large enough to travel in the hepatic arterial and precapillary network and in the tumour vasculature network so that they can lodge at limiting diameters or embolize in the finer angiogenic vessels that feed the tumour growth zone.

[0018] A second factor that influences distribution of microparticles in the liver is that the normal vessel divisions from the descending aorta to those ultimately supplying the liver have variations arising during embryological development. Third, the growth of a tumour in the liver and its associated angiogenesis can produce changes in the arterial network of the liver, sometimes resulting in significant hepatopulmonary or hepatogastric shunting.

[0019] For nearly two decades researchers have examined the extent of embolization of different sized microparticles in tumours and the homogeneity of their distribution in normal liver. [0020] In Meade VM et al, (1987) Eur J Cancer Clin Oncol. 23(1 ): 37-41 the authors examined the effect of microparticle size through the ratio of arterially introduced microparticles (injected via the ascending aorta) lodging in tumour tissue compared to the surrounding normal hepatic parenchyma measured for 15, 32.5 and 50 μιη diameter tracer microparticles. Using histological techniques, they found that the mean tumour to liver arterial perfusion ratio (T/L) for 15 and 32.5 μιη spheres was approximately 3:1 in both cases and there was no significant difference between these values. However, 50 μιη microparticles did not preferentially lodge in malignant tissue. Importantly, they also assessed the homogeneity of distribution of microparticles embolizing in the normal liver tissue for each microparticle size. They discovered that as microparticle diameter increased from 15 to 50 μιη, microparticles lodged more evenly throughout the liver. For 15 μιη microparticles the coefficient of variation was 55.5% +/- 8.3 and 32.5 μιη microparticles distributed with a coefficient of 35% +/- 16.8 while 50 μιη spheres . . distributed most evenly with a coefficient of 19.7% +/- 6.8. The authors concluded from that histological study that 32.5 μιη microparticles were the optimal size for preferentially lodging in tumour tissue. They recommended that microparticles of greater than 15 μιη should be advanced in clinical research trials for intrahepatic radiotherapy as they are most likely to distribute homogeneously within the normal liver substance, yet still provide a concentrated dose of radiation to the tumour tissue. They reasoned that particles of that size would potentially provide preferential irradiation of malignant tissue while relatively sparing the normal hepatic parenchyma in patients with metastatic liver cancer. [0021 ] Subsequently, Anderson et al., (1991 ) Br J Cancer. 64(6):1031 -4 investigated the factors influencing the distribution of regionally injected microparticles. A discreet tumour was induced in rats by subcapsular hepatic inoculations of HSN cells. At 20 days, 12.5 μιη, 25 μιη or 40 μιη diameter, radiolabelled albumin microparticles were administered, in various concentrations, via the gastroduodenal artery. Tumour to normal liver microparticle distribution ratios were determined from tissue sampling and median values ranged from 0.1 (0.2 mg/ml 12.5 μιη microparticles) to 1 .8 (20 mg/ml 40 μιη microparticles). Concentrated suspensions (20 mg/ml) of large microparticles (40 μιη) produced the most favourable tumour to normal liver distribution ratios. These results reinforced the outcome of the Meade study confirming that larger particles are better in microparticle technology with the authors concluding that delivery of a concentrated suspension of large microparticles with a relatively low drug pay load was desirable for regional therapy.

[0022] Based on these and many other studies microparticle SIRT technologies have proceeded on the basis that microparticles of between 20 and 30 μιη are conventionally ideal for tumour treatments and that particles greater than 10 to 12 μιη (being the lower limit) are absolutely required for retention in capillary networks and to avoid venous drainage to the systemic circulation.

[0023] SIR-Spheres® microparticles, for example, have a median diameter of 30 μιη and lodge at limiting diameters in the arterial vessels supplying a tumour, where their loading of Yttrium-90 radioisotope delivers cytotoxic beta radiation. Experimental studies have shown that the increased density of the angiogenic network at the periphery of metastatic tumours growing in the liver can result in significantly higher . . dose delivery to the tumour tissue compared to the normal liver parenchyma [Campbell et al., (2001 ). Phys Med Biol, 46: 487-798].

[0024] A therapeutic product that improves the treatment of liver cancer will be of significant benefit to patients. The present invention seeks to provide an improved or at least an alternative product and methods of use thereof for the treatment of cancer, and in particular, lung and or liver cancer.

SUMMARY OF INVENTION

[0025] The present invention provides an improved anticancer therapy that has utility, in cancer treatments generally but more specifically in the treatment of primary and secondary lung and or liver cancer.

[0026] The inventors have discovered, contrary to conventional wisdom, that polymeric particulate microparticles that have a size range of 6 to 12 μιη, preferably, 7 to 1 1 μιη, 8 to 10 μιη and most preferably 8 or 9 μιη, provide significant improvements in SIRT. Particles of this size and polymeric form distribute more homogeneously in tumours compared to larger microparticles, lodge preferentially in tumour tissue sparing the normal tissue in patients and yet present surprisingly little washout of isotope from the tumour site to the systemic circulation.

[0027] According to a first aspect, the invention resides in a polymeric particulate material comprising: (a) polymeric matrix, having a diameter in the size range of 6 to 12 μιη or 7 to 1 1 μιη or 8 to 10 μιη and most preferably 8 or 9 μιη, and (b) a radionuclide stably incorporated therein.

[0028] In an embodiment of this aspect of the invention the radionuclide is incorporated in the polymeric particulate material delivers a radiation dose of between about 10 and 800 Gy. In a preferred form of this embodiment the radiation dose delivered by the radionuclide is between 10 and 200Gy. More preferably it is between 10 and 150Gy, 10 and 100Gy, 20 and 80Gy, 25 and 75Gy, 30 and 70Gy, 35 and 65Gy, 40 and 60Gy or 40 and 55Gy with approximately 50 Gy being optimal, at least in the treatment of liver metastases.

[0029] According to a second aspect, the invention resides in a process for the production of a polymeric particulate material having a diameter in the range of from of . .

6 to 12 μιη, said process comprising the step of: combining a polymeric matrix and a radionuclide for sufficient time and under conditions sufficient to stably incorporate the radionuclide in the matrix.

[0030] According to a third aspect, the invention provides a method for treating a patient in need of SIRT therapy, said method comprising the step of: administering to a cancerous tissue in a patient on need of SIRT therapy a polymeric particulate material as herein described.

[0031 ] In an embodiment of this aspect of the invention the polymeric particulate material is administered to a patient at a therapeutic dose that delivers a radiation dose of between about 10 and 800 Gy to at least a tumour in the cancerous tissue. In a preferred form of the invention the radiation dose delivered by the polymeric particulate material is between 10 and 200Gy. More preferably it is between 10 and 150Gy, 10 and 100Gy, 20 and 80Gy, 25 and 75Gy, 30 and 70Gy, 35 and 65Gy, 40 and 60Gy or 40 and 55Gy with approximately 50 Gy being optimal, at least in the treatment of liver metastases.

[0032] In an embodiment of this aspect of the invention the method of treatment may be integrated into other regimens of treatment, such as chemotherapeutic treatments that are commonly applied to cancer patients. Preferably, the cancer is a metastatic carcinoma which, for example, may arise in the liver from primary carcinomas of, for example, the colonic mucosa.

[0033] According to a fourth aspect, the invention resides in the use of a polymeric particulate material as herein described, in internal radiation therapy of a patient.

[0034] According to a fifth aspect, the invention resides in the use of a polymeric particulate material as herein described, in the manufacture of medicament for the treatment of cancer in a patient. Preferably, the cancer is a metastatic carcinoma which, for example, may arise in the liver from primary carcinomas of, for example, the colonic mucosa.

[0035] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent to those skilled in the art from the following description, from a review of the ensuing description given by way of example and with reference to the accompanying drawings. This description is included . . for the purposes of exemplifying and enhancing the description of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The description is made with reference to the accompanying drawings in which:

Figure 1A is a graphical representation of FibrinLite nanoparticles (FL; US 8,778,300) pretreated with low microgram concentrations of protamine binding readily to polystyrene micro-wells.

Figure 1 B is a graph showing the results of the mean values for six separate microsphere binding experiments using FibrinLite nanoparticles pretreated with protamine.

Figure 1 C is a scanning electron micrograph showing islands of protamine treated FibrinLite bound on the surface of a microsphere.

Figure 1 D is a scanning electron micrograph showing plain microspheres for comparison with 1 C.

Figure 2 shows the results from lung retention tests of radiolabeled microparticles after intravenous injection in rabbits. The frames A - C show gamma camera images of anaesthetised normal rabbits taken 3 h after intravenous injection of a 5% dextrose suspension (5 imL) containing 15 mg (130 MBq) FL-MS30, FL-MS12, and FL-MS8 respectively, (where FL is Fibrinlite and MS refers to the median diameter (μιη) of the tested microspheres). For comparison, frame D shows a 3 h post-injection image of a rabbit injected with the lung diagnostic agent Tc99m-MAA (2.5 mg, 130 MBq); note activity in the lungs but also in the kidneys.

Figures 3A to 3D show the distribution of radiolabeled microparticles and MAA in normal rabbit livers after intra-arterial instillation. Figures 3A to 3C show gamma camera images of excised livers removed from normal rabbits 1 h after intra-arterial instillation of a 5% dextrose suspension (8 imL) containing 40 mg (130 MBq) FL-MS30 (Figure 3A), FL-MS12 (Figure 3B) and FL-MS8 (Figure 3C) respectively. For comparison, Figure 3D shows an excised liver removed from a rabbit 1 h after intra-arterial instillation of Tc99m-MAA (2.5 mg, 130 MBq). . .

Figures 4A to 4D show the distribution of radiolabeled microparticles and MAA in rabbit livers hosting a VX2 tumour implant. Figures 4A to 4C show gamma camera images of excised livers with tumours removed from rabbits 1 h after intra-arterial instillation of a 5% dextrose suspension (8 imL) containing 40 mg (130 MBq) FL-MS30 (Figure 4A), FL-MS12 (Figure 4B) and FL-MS8 (Figure 4C), respectively. For comparison, Figure 4D shows an excised liver with tumour removed from a rabbit 1 h after intra-arterial instillation of Tc99m-MAA (2.5 mg, 130 MBq).

Figure 5 is VX2 tumour imaging in an intact rabbit with FL-MS8. The frames A to C show the coronal, sagital and transaxial SPECT/CT views respectively of an anaesthetized rabbit with a liver implant of a VX2 tumour, 1 h after intra-arterial instillation of FL-MS8 (40 mg; 130 MBq).

DETAILED DESCRIPTION OF THE INVENTION

[0037] The Inventors have discovered, contrary to conventional wisdom, that microparticles in a size range of 6 to 12 μιη, preferably 7 to 1 1 μιη, 8 to 10 μιη and most preferably 8 or 9 μιη provides significant improvements in SIRT. Particles of this size, surprisingly distribute more homogeneously in tumours compared to larger microparticles and preferentially towards tumour tissue sparing normal tissue in patients without significant washout effects from a tumour. [0038] The invention is described below by reference to certain identified embodiments, nonetheless the skilled reader will appreciate that the invention so identified herein presents a principal that has broad and general application. It provides a hitherto unknown and unexpected refocusing and refinement of SIRT technology with significant advantages to both the patient and the clinician in the treatment of patients with a tumour.

[0039] The following detailed description is to be understood having regard to the following definitions and interpretations.

Definitions

[0040] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as - - described herein. Similarly, those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features.

[0041 ] The entire disclosures of all publications (including patents, patent applications, journal articles, manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

[0042] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0043] In the context of this specification the term "about" will be understood as indicating the usual tolerances that a person skilled in the field would associate with the given value. For example, a person skilled in the field will understand that a 10% variation or less (i.e. a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9%) in a number is encompassed by the term "about" and is within the usual tolerances presented by the invention.

[0044] The invention described herein includes various values (for example, size, homogeneity etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. For example, a person skilled in the field will understand that a 10% variation in upper or lower limits of a range can be totally appropriate and is encompassed by the invention. More particularly, the variation in upper or lower limits of a range will be 5% or as is commonly recognised in the art, whichever is greater. [0045] Those skilled in the art will appreciate that the term "microparticle" as used herein includes all particulate materials that meet the parameters of the present invention - - including microspheres preferably without sharp edges or points that could damage patients' arteries or catch in unintended locations. It is not limited to spheres. Nor should the term microparticle be limited to spheres. Preferably, the microparticle is substantially spherical or oval, but need not be regular or symmetrical in shape. The microparticles also need not be limited to any form or type of microparticles. Any microparticles may be used in the present invention provided the microparticles can receive a radionuclide such as through impregnation, absorbing, coating or more generally bonding the particles together.

[0046] As used herein treat, "treatment" and "treated" includes:

(i) preventing a disease, disorder or condition from occurring in a patient who may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it;

(ii) inhibiting a disease, disorder or condition, i.e., arresting its development; or

(iii) relieving a disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.

[0047] The term "therapeutically effective amount" as used herein includes within its meaning a non-toxic but sufficient amount of a polymeric particulate material as herein described for use in the invention to provide a desired therapeutic effect. The exact amount of material required to treat a disease, disorder or condition will vary from subject to subject depending on factors such as the species being treated, the age, weight and general condition of the subject, co-morbidities, the severity of the disease, disorder or condition being treated, the specific characteristics of the polymeric particulate material being administered and the mode of administration. For any given case, an appropriate "effective amount" of a polymeric particulate material may be determined by one of ordinary skill in the art using only routine methods.

[0048] Reference herein to use of microparticles in a therapy will be understood to be equally applicable to human and non-human, such as veterinary, applications. Hence it will be understood that, except where otherwise indicated, reference to a "patient", "subject" or "individual" means a human or non-human species, such as an individual of any species of social, economic or research importance including but not limited to lagomorph, ovine, bovine, equine, porcine, feline, canine, primate and rodent species. - -

[0049] As used herein the term "kit" or "device" will be understood to include devices which may be used in therapy, including prevention and treatment of an actual condition or symptom, and those which may be used in diagnosis, including where the diagnosis is performed on or in the body of a patient and where the diagnosis is performed on or with a sample obtained from the body of a patient.

[0050] Other definitions for selected terms used herein are found within the detailed description of the invention and apply throughout. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. [0051 ] Features of the invention will now be discussed with reference to the following non-limiting description and examples.

Preferred Embodiments

[0052] The present invention provides a polymeric particulate material that has utility in the treatment of various forms of cancers and tumours, particularly in the treatment of primary liver cancer and secondary liver cancer and, more specifically, in secondary liver cancer deriving from the gastrointestinal tract, such as secondary liver cancers deriving from colorectal cancer.

[0053] When radioactive microparticles or other small particles are administered into the arterial blood supply of a target organ, it is desirable to have them of a size, shape and density that results in the optimal homogeneous distribution within a target organ. If radioactive microparticles or small particles do not distribute evenly as a function of the arterial blood flow, they can accumulate in excessive numbers in some areas and cause focal areas of excessive radiation. They also may not reach the arterial micro-vessels supplying a tumour. [0054] The inventors have discovered that, contrary to conventional wisdom, the ideal polymeric particulate material for injection into the blood stream within a target organ should have a very narrow size range of approximately 6 to 12 μιη, 7 to 1 1 μιη, 8 to 10 μιη and most preferably 8 or 9 μιη. This range of particle size it should be noted is comparable with the size of normal blood cells and therefore can be fully expected to reach the fine vessels of a tumour's angiogenic growth zone - -

[0055] In an embodiment, the polymeric particulate material has a mass median diameter or d50 in the range of 6 to 12 μιη, preferably 7 to 1 1 μιη, more preferably 8 to 10 μιη and most preferably 8 or 9 μιη.

[0056] In a further embodiment, the polymeric particulate material also has a narrow particle size distribution. Preferably, the standard deviation of the sample is about 0.5 to 2 μιη. By way of illustration, the standard deviation will be 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4 or 2.5 μηπ. More preferably, the standard deviation will be about 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4 or 1 .5 μιη

[0057] Preferably, the d10 value of the particle size distribution is greater than about 1 μιη and more preferably greater than about 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 μιη. Persons skilled in the art will appreciate that the d10 value for the material may vary depending on the mass median diameter of the polymeric particulate material. For example, if the median particle size is about 8 μιη and the standard deviation of the sample is about 1 μιη then the d10 will be about 6.5 μιη to 7 μιη, preferably 6.9 μιη. Alternatively, if the median particle size is 12.4 μιη and the standard deviation of the sample is 1 .5 μιη then the d10 will be about 10 μιη to 10.5 μιη, preferably 10.4 μιη.

[0058] Accordingly, an embodiment of the invention provides a polymeric particulate material, with a mass median diameter or d50 in the range of 6 to 12 μιη wherein at least 10% of the particles have a particle size of less than about 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μηπ wherein the d10 will be within 0.5 to 2.5 standard deviations (optionally, 0.5 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4 or 2.5 standard deviations) from the d50 of the polymeric particulate material.

[0059] Preferably, the d90 value of the particle size distribution is less than about 16 μιη and more preferably less than about 15.5, 15, 14.5, 14, 13.5, 13, 12.5, 12, 1 1 .5, 1 1 , 10.5 or 10 μιη. Again, persons skilled in the art will appreciate that the d90 value for the material may vary depending on the mass median diameter of the polymeric particulate material. For example, if the median particle size is 8.18 μιη and the standard deviation of the sample is 1 .06 μιη then the d90 will be about 9.5 μιη to 10 μιη, preferably 9.7 μιη. Alternatively, if the median particle size is 12.4 μιη and the standard deviation of the sample is 1 .52 μιη then the d90 will be about 14.5 μιη to 15 μιη, preferably 14.6 μιη. - -

[0060] Accordingly, an embodiment of the invention provides a polymeric particulate material, with a mass median diameter or d50 in the range of 6 to 12 μιη wherein at least 90% of the particles have a particle size of less than about 16 μιη and more preferably less than about 15.5, 15, 14.5, 14, 13.5, 13, 12.5, 12, 1 1 .5, 1 1 , 10.5 or 10 μπι wherein the d90 will be within 0.5 to 2.5 standard deviations (optionally, 0.5 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4 or 2.5 standard deviations) from the d50 of the polymeric particulate material.

[0061 ] In a preferred embodiment of the invention the particle size distribution of the polymeric particulate material will have: (i) a d10 value for the particle size distribution that is greater than about 4, 4.5, 5, 5.5, 6 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μιη depending on the d50 of the polymeric particulate material, and (ii) a d90 value of the particle size distribution that is less than about 16 μιη, more preferably less than about 15.5, 15, 14.5, 14, 13.5, 13, 12.5, 12, 1 1 .5, 1 1 , 10.5, 10, 9.5, 9 μηπ depending on the d50 of the polymeric particulate material. For example, if the median particle size is about 8 μιη and the standard deviation of the sample is 1 .06 μιη then (i) the d10 will be about 6.5 μιη to 7 μιη, preferably 6.9 μιη and (ii) the d90 will be about 9.5 μιη to 10 μιη, preferably 9.7 μιη. In an alternate example, if the median particle size is 12.4 μιη and the standard deviation of the sample is 1 .5 μιη then (i) the d10 will be about 10 μιη to 10.5 μιη, preferably 10.4 μιη and the d90 will be about 14.5 μιη to 15 μιη, preferably 14.6 μιη. [0062] Methods of determining the size of particles are well known in the art. For example, the general method of U.S. Patent No. 4,605,517, incorporated herein by reference, could be employed. The following is a description of one non-limiting method.

[0063] In preparing the particulate compound of the present invention, microparticle size is characterized using an instrument adapted to measure equivalent spherical volume diameter, a Horiba LA910 Laser Scattering Particle Size Distribution Analyzer or a Malvern Mastersizer 3000 laser diffraction particle size analyzer or equivalent instrument.

[0064] Polymeric particulate material of such a size range and particle size distribution as presented above has been found to preferentially concentrate in neoplasia in a target organ with surprisingly little washout or shunting. Retention of radiolabeled material, as - - demonstrated by imaging, was shown to be highly favourable for achieving tumour irradiation with a therapeutic microsphere.

[0065] According to first aspect of the present invention there is provided a polymeric particulate material having a diameter in the size range of 6 to 12 μιη, 7 to 1 1 μιη, 8 to 10 μιη and most preferably 8 or 9 μιη comprising a polymeric matrix and a stably incorporated radionuclide.

[0066] In a preferred embodiment of the invention the particulate polymeric material is suitable for SIRT. Ideally, the preferred particulate polymeric material is in the form of microparticles with a level of radioactivity that is between about 0.01 to 0.4 GBq (activity per particle). Preferably, the activity per microparticles is 0.10, 0.1 1 , 0.12, 0.14, 0.15, 0.16, 0.17, 0.18, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.26, 0.27, 0.28, 0.29, 0.30, 0.32, 0.33, 0.34, 0.35, 0.36, 0.38, 0.39, 0.40, GBq.

[0067] In SIRT use, polymeric microparticles loaded with Yttrium 90 will deliver a level of radioactivity to a tumor of up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3 or 3.4 GBq of tumor volume at the site of treatment.

[0068] In an embodiment of this aspect of the invention the polymeric particulate material has a mass median diameter in the size range of 6 to 12 μιη, more preferably 7 to 1 1 μιη or 8 to 10 μιη and most preferably 8 or 9 μιη and comprises a polymeric matrix in which a radionuclide is stably incorporated, wherein the polymeric particulate material incorporating the radionuclide delivers a radiation dose of between about 10 and 800 Gy. In a preferred form of the invention the radiation dose delivered by the polymeric particulate material is between 10 and 200Gy. More preferably it is between 10 and 150 Gy, 10 and 100 Gy, 20 and 80 Gy, 25 and 75 Gy, 30 and 70 Gy, 35 and 65 Gy, 40 and 60 Gy or 40 and 55 Gy with approximately 50 Gy being optimal, at least in the treatment of liver metastases.

[0069] Preferably, SIRT treatment is most effective when the activity of microparticles loaded with Yttrium 90, delivers a radiation dose of between about 10 and 800 Gy. Generally, 1 GBq of Yttrium-90/kg of tissue provides 50 Gy of radiation dose. In a preferred form of the invention the radiation dose delivered by the microparticles is between 10 and 200Gy. More preferably it is between 10 and 150 Gy, 10 and 100 Gy, - -

20 and 80 Gy, 25 and 75 Gy, 30 and 70 Gy, 35 and 65 Gy, 40 and 60 Gy or 40 and 55 Gy with approximately 50 Gy being optimal, at least in the treatment of liver metastases.

[0070] The invention is not limited to delivering the above doses of radiation. It can be used to deliver higher radiation doses. Such higher doses of radiation can be used to treat liver metastases or used to treat other forms of metastases, such as those commonly seen in the lung and kidneys. In such instances, the activity of the polymeric microparticles produced according to the invention delivers a radiation dose to a neoplasia of between about 10 and 800 Gy. Preferably, the radiation dose is 10, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 or 800 Gy.

[0071 ] More preferably, the radiation dose delivered to a neoplasia is between about 10 and 200 Gy. Illustrative radiation doses include: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 120, 130, 140, 150, 160, 170, 180, 190 or 200 Gy.

[0072] The radioactivity of the microparticles used in the SIRT can be calculated by determining the tumour volume and then adjusting the amount of the radioactive microparticles, having regard to tumour volume, to deliver to the neoplasia the desired radiation dose.

[0073] As used herein, references to the radionuclide being stably incorporated into particulate material or polymeric matrix are to be understood as referring to incorporation of the radionuclide so that it does not substantially leach out of the particulate material under physiological conditions such as in a patient or in storage. In a preferred embodiment, the radionuclide is incorporated by precipitation into a polymeric matrix forming the microparticle.

[0074] The radionuclide doped microparticles need not be limited to any particular form or type of microparticle. So, for example, the radionuclide doped microparticles suitable for use in the invention may comprise any additional material capable of receiving a radionuclide such as through impregnation, absorbing, coating or more generally - - bonding the radionuclide with the microparticle or material used to carry the radionuclide.

[0075] There are many radionuclides that can be incorporated into microparticles that can be used for cancer therapies such as SIRT. Of particular suitability for use in SIRT treatment is the unstable isotope of yttrium (Y-90). Yttrium-90 is a high-energy pure beta-emitting isotope with no primary gamma emission. The maximum energy of the beta particles is 2.27 MeV, with a mean of 0.93 MeV. The maximum range of emissions in tissue is 1 1 mm, with a mean of 2.5 mm. The half-life of yttrium-90 is 64.1 hours. In use requiring the isotope to decay to infinity, 94% of the radiation is delivered in 1 1 days leaving only background radiation with no therapeutic value. The microparticles themselves are a permanent implant and each device is for single patient use. Alternate radionuclides that can be used in the production of these microparticles, include for example, lutetium, holmium, samarium, iodine, phosphorous, iridium rhenium and terbium. [0076] The radionuclide that is incorporated into the microparticle in accordance with the present invention is preferably yttrium-90, but may also be any other suitable radionuclide which can be precipitated in solution, of which the isotopes.

[0077] Variation to the activity of the microparticle used in the SIRT and the intended radiation dose to the neoplasia are two of the variable that must be accounted for in delivering a therapy. Relevantly, any variation of the radiation dose delivered to the neoplasia will cause a consequential variation to the activity of the microparticles used in the method and vice versa.

Leaching of radionuclides

[0078] Preferably the radionuclide is stably incorporated into the particulate material or polymeric matrix such that the incorporated radionuclide does not substantially leach out of the particulate material under physiological conditions such as in the patient or in storage. The leaching of radionuclides from the polymeric matrix can cause non-specific radiation damage to the patient and damage surrounding tissue.

[0079] In particular, a radionuclide will be stably incorporated into a particulate material if less than 5% of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material. More - - preferably, a radionuclide will be stably incorporated into a particulate material if less than 4%, 3%, 2%, 1 % or 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1 % of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material. Such physiological conditions will be understood by a person skilled in the art as being those that are typically found in a patient when the particulate material is introduced into said patient. As for the life of the particulate material, generally Y-90 has a half-life is 64.1 hours. Preferably, the radionuclide will be stably incorporated into a particulate material for at least 2 days, with, 3, 4,5, 6, 7, 8, 9, 10 or 1 1 days being more preferable. In therapeutic use, requiring the isotope to decay to infinity, 94% of the radiation is delivered in 1 1 days

[0080] In an embodiment of this aspect of the invention, the polymeric particulate material has a mass median diameter in the size range of 6 to 12 μιη more preferably 7 to 1 1 μιη or 8 to 10 μιη and most preferably 8 or 9 μιη and comprises a polymeric matrix in which a radionuclide is stably incorporated, wherein (i) the polymeric particulate material incorporating the radionuclide delivers a radiation dose of between about 10 and 800 Gy; and (ii) less than 5% (optionally less than 4%, 3%, 2%, 1 % or 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1 %) of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material. [0081 ] One method of assessing leaching is by adjusting a sample to pH 7.0 and agitating in a water bath at 37°C for 20 minutes. A 100 μί sample is counted for beta emission in a Geiger-Muller counter. Another representative 100 μΙ_ sample is filtered through a 0.22 μπΊ filter and the filtrate counted for beta emission in the Geiger-Muller counter. The per cent unbound radionuclide is calculated by:

FiltrateCo unt

A WO = % UnboundRadionuclide

SampleCount

[0082] The radionuclide can be stably incorporated into the polymeric matrix by precipitating it as an insoluble salt. Where the radionuclide used is yttrium-90 the yttrium is preferably precipitated as a phosphate salt. However, the present invention also extends to precipitation of the radionuclide as other insoluble salts including, for example, carbonate and bicarbonate salts. - -

Microparticle Composition

[0083] The particulate polymeric material used in the invention are polymer based and separated by filtration or other means known in the art to obtain a cohort of microparticles of the defined specific size range that is preferred for a particular use in the herein described methods.

[0084] In a specific form, the invention provides a particulate polymeric material as described above in which the polymeric matrix is an ion exchange resin, particularly a cation exchange resin. Preferably, the ion exchange resin comprises a partially cross linked aromatic polymer, including polystyrene. One particularly preferred cation exchange resin is the sulfonated styrene/divinylbenzene copolymer resin commercially available under the trade name Aminex 50W-X4 (Biorad, Hercules, CA). However, there are many other commercially available cation exchange resins which are suitable.

[0085] In alternate forms the polymer of the present invention can be any polymer having a surface that is biocompatible with blood (i.e. does not promote blood coagulation by the so-called intrinsic pathway, or thrombosis by promotion of platelet adhesion).

[0086] In one embodiment, the polymer of the present invention is a cationic exchange resin comprising anionic substituent groups, such as sulfate, sulfonate, carboxylate and phosphate groups. For example, the polymer may be any blood biocompatible polymer known in the art, including but not limited to polystyrene, polystyrene sulfonate, polypropylene, polytetrafluorethylene (PTFE), expanded polytetraflouroethylene (EPTFE), polyurethane, polyvinyl chloride, polyamides, teflon, polyester, polyethylene terephthalate, poly(butylene terephthalate) (PBT), poly(ethylene oxide) (PEO), polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(e- caprolactone), polydioxanone, trimethylene carbonate, polyanhydride, and poly[bis(p- carboxyphenoxyl) propane:sebacic acid. Preferably, the polymer is polystyrene sulfonate.

[0087] In particular, polytetrafluorethylene (PTFE), expanded polytetraflouroethylene (EPTFE), polyurethane, polyvinyl chloride, polyamides, polystyrene and teflon may be employed as polymers in the present invention. . -

[0088] The polymer microparticles used in the present invention includes those used in the manufacture of SIR-spheres® (SIR-spheres^® is a registered trademark of Sirtex SIR-Spheres Pty Ltd) microparticles, which are resin based microparticles comprised of polystyrene sulfonate. Density

[0089] When small particles are administered into the arterial blood supply of a target organ, it is desirable to have them of a density that results in the optimal homogeneous distribution within the target organ. If the particles do not distribute evenly they may accumulate in excessive numbers in some areas and cause focal areas of excessive radiation. The particulate polymeric material is preferably low density, more particularly a density below 3.0 g/cm³, even more preferably below 2.8 g/cm³, 2.5 g/cm³, 2.3 g/cm³, 2.2 g/cm³ or 2.0 g/cm³.

[0090] It is also desirable to have the particulate material manufactured so that the suspending solution has a pH less than 9. If the pH is greater than 9 then this may result in irritation of the blood vessels when the suspension is injected into the artery or target organ. Preferably the pH is less than 8.5 or 8.0 and more preferably less than 7.5.

Method for the Production of a Radioactive Particulate Materia

[0091 ] The present invention particularly provides a method for the production of a radioactive particulate material comprising a polymeric matrix as described above, characterised by the steps of:

(i) absorbing a radionuclide onto an ion-exchange resin particulate material having a diameter in the size range of 6 to 12 μιη, 7 to 1 1 μιη, 8 to 10 μιη and most preferably 8 or 9 μιη and a specific gravity of less than 2.5; and

(ii) binding or precipitating the radionuclide as an insoluble salt to stably incorporate the radionuclide into the particulate material.

[0092] Alternate sources of yttrium-90 may be used in the production of these microparticles. For example, a highly pure source of yttrium-90 may be obtained by extracting yttrium-90 from a parent nuclide and using this extracted yttrium-90 as the source of the soluble yttrium salt that is then incorporated into the polymeric matrix of the microparticles. - -

[0093] To decrease the pH of the suspension containing the microparticles for injection into patients, the microparticles may be washed to remove any un-precipitated or loosely adherent radionuclide. The present invention provides a suspension of the required pH by precipitating the yttrium with a tri-sodium phosphate solution at a concentration containing at least a three-fold excess of phosphate ion, but not exceeding a 30-fold excess of phosphate ion, and then washing the microparticles with de-ionised water. Another approach which ensures that the pH of the microparticle suspension is in the desired range is to wash the resin with a phosphate buffer solution of the desired pH. Method of Radiation Therapy

[0094] The present invention also provides a method of radiation therapy of a human or other mammalian patient that comprises administration to the patient of particulate material as described above.

[0095] Accordingly, the present invention provides a method of treating liver or lung neoplasia in a subject in need of treatment, by subjecting the patient to SIRT.

[0096] The amount of microparticles used in the method and which will be required to provide effective treatment of a neoplastic growth will depend on the radionuclide used in the preparation of the microparticles. By way of example, an amount of yttrium-90 activity that will result in an inferred radiation dose to the normal liver of approximately 10 to 200 Gy may be delivered. Because the radiation from SIRT is delivered as a series of discrete point sources, the dose of 10 Gy to 200 Gy is an average dose with many normal liver parenchymal cells receiving much less than this dose. Alternate doses of radiation may be delivered depending on the disease state and the physician's treatment needs. Such variation of radiation doses obtained by altering the amount of microparticles used will be something that a skilled artisan will know how to determine.

[0097] In an embodiment of this form of the invention the polymeric particulate material administered to a patient in a therapeutic dose that delivers a radiation dose of between about 10 and 800 Gy to the tumor. In a preferred form of the invention the radiation dose delivered by the polymeric particulate material is between 10 and 200Gy. More preferably it is between 10 and 150 Gy, 10 and 100 Gy, 20 and 80 Gy, 25 and 75 Gy, . -

30 and 70 Gy, 35 and 65 Gy, 40 and 60 Gy or 40 and 55 Gy with approximately 50 to 80 Gy being optimal, at least in the treatment of liver metastases.

SIRT

[0098] There are many potential advantages of SIRT over conventional, external beam radiotherapy. Firstly, the radiation is delivered preferentially to the cancer within the target organ. Secondly, the radiation is slowly and continually delivered as the radionuclide decays. Thirdly, by manipulating the arterial blood supply with vasoactive substances, it is possible to enhance the percentage of radioactive particles that go to the cancerous part of the organ, as opposed to the healthy normal tissues. This has the effect of preferentially increasing the radiation dose to the cancer while maintaining the radiation dose to the normal tissues at a lower level.

[0099] SIRT, which may also be known as radio-embolization or microparticle brachytherapy involves two procedural components:

a. Embolization: injection into the arterial tumour feeding vessels of permanently embolic microparticles which act as the delivery vehicle for the therapeutic moiety, and

b. Irradiation: embolization of microparticles in the distal microvasculature of the tumour delivers high dose irradiation to the tumour microvascular plexus and to tumour cells themselves. [00100] Relevantly, direct irradiation of tissue and microvascular bed destruction, rather than pure embolization is responsible for the tissue destructive effects of SIRT therapy.

[00101 ] Broadly speaking radioactive microparticles do not exhibit pharmacodynamics in the classic sense, but induce cell damage by emitting radiation. Once implanted, radioactive microparticles remain within the vasculature of tumours. They are not phagocytised nor do they dissolve or degrade after implantation. High radiation emitted from the radioactive microparticles is preferably cytocidal to cells within the range of the radiation. After the radioactive microparticle has decayed, the non-radioactive microparticles remain intact and are not removed from the body. [00102] Intrinsic to the concept of SIRT is the preferential placement of the radioactive microparticles selectively into the distal microvascular supply of tumours. . -

This may be achieved by direct injection of the microparticles or through the manipulation of blood flow into and out of the target organ. The person skilled in the art will appreciate that SIRT may be delivered by any of a range of different methods, some of which are described in US patents 4789501 , 501 1677, 5302369, 6296831 , 6379648, or WO applications 200045826, 200234298 or 200234300 (incorporated herein by reference). Accordingly, administration of radionuclide doped microparticles may be by any suitable means, but preferably by delivery via the relevant artery. For example, in treating liver cancer, administration is preferably by insertion of a catheter into the hepatic artery. Pre or co-administration of another agent may prepare the tumour for receipt of the particulate material, for example a vasoactive substance, such as angiotension-2 to redirect arterial blood flow into the tumour. Delivery of the particulate matter may be by single or multiple doses, until the desired level of radiation is reached.

[00103] The technique of SIRT has also been previously reported (see, for example, Chamberlain M, et al (1983) Brit. J. Surg., 70: 596-598; Burton MA, et al (1989) Europ. J. Cancer Clin. Oncol., 25, 1487-1491 ; Fox RA, et al (1991 ) Int. J. Rad. Oncol. Biol. Phvs. 21 , 463-467; Ho S et al (1996) Europ J Nuclear Med. 23, 947-952; Yorke E, et a/ (1999) Clinical Cancer Res, 5 (SuppI), 3024-3030; Gray BN, et al. (1990) Int. J. Rad. Oncol. Biol. Phvs, 18, 619-623).

[00104] Treatment with SIRT has been shown to result in high response rates for patients with neoplastic growth in particular with colorectal liver metastases (Gray B.N. et al (1989) Surg. Oncol, 42, 192-196; Gray B, et al. (1992) Aust NZ J Surgery, 62, 105- 1 10; Gray B N et al. (2000) Gl Cancer, 3(4), 249-257; Stubbs R, et al (1998) Hepato- gastroenterology SuppI II, LXXVII).

[00105] Other studies have shown that SIRT therapy can also be effective in causing regression and prolonged survival for patients with primary hepatocellular cancer (Lau W, et al (1994) Brit J Cancer 70, 994-999; Lau W, et al. (1998) Int J Rad Oncol Biol Phvs. 40, 583-592).

Compositions

[00106] Desirably the microparticles of the invention are allowed to have an additive effect with other cytotoxic agents and are typically administered for the treatment of neoplasm. Preferably microparticles of the invention are delivered to a patient concomitantly with either systemic or loco-regional chemotherapeutic agents . - such as oxiplatin, 5-Fluorouracil or Leucovorin. This interaction may be exploited to the benefit of the patient, in that there can be an additive toxicity on tumour cells, which can enhance the tumour cell kill rate. This interaction can also lead to additive toxicity on non-tumourous cells. [00107] In addition to the identified chemotherapeutic agents and radionuclide doped microparticles the invention may also include an effective treatment with immunomodulators and other agents as part of therapy. Illustrative immunomodulators suitable for use with the invention are alpha interferon, beta interferon, gamma interferon, interleukin-2, interleukin-3, tumour necrosis factor, granulocyte-macrophage colony stimulating factors and the like.

[00108] The present invention further provides a synergistic combination of antineoplastic agents and an amount of radionuclide-doped microparticles suitable for use in SIRT for treatment of a neoplastic growth.

Combination therapy [00109] The inventors have identified that the co-administration to a patient of SIRT using the microparticles of the invention and systemic chemotherapy at doses and in a delivery regime that minimize hepatotoxicity from the chemotherapy, potentiates the radiation effect from the SIRT on liver cancer, while delivering a beneficial effect on extra-hepatic disease. [001 10] In the method of the present invention, an amount of 5-FU and LV that is "effective to treat the neoplasia" is an amount that is effective to ameliorate or minimize the clinical impairment or symptoms of the neoplasia, in either a single or multiple dose of 5-FU and LV when combined with SIRT. For example, the clinical impairment or symptoms of the neoplasia may be ameliorated or minimized by diminishing any pain or discomfort suffered by the subject; by extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment; by inhibiting or preventing the development or spread of the neoplasm; or by limiting, suspending, terminating, or otherwise controlling the maturation and proliferation of cells in the neoplasm. Notably, the amounts of 5-FU and LV effective to treat neoplasia in a subject in need of treatment will vary depending on the type of SIRT used, as well as the particular factors of each case, including the type of neoplasm, the stage of neoplasia, . - the subject's weight, the severity of the subject's condition, and the method of administration. These amounts can be readily determined by the skilled artisan.

[001 1 1 ] As an example only, doses of 5-FU administered intraperitoneal^ may be between 100 and 600mg/m²/day, or between 200 mg/m²/day and 500 mg/m²/day. More preferably doses of 5-fluorouracil administered intraperitoneal^ will be between 300 and 480 mg/m²/day, or between 400 mg/m²/day and 450 mg/m²/day. An example being 425 mg/m²/day. Doses of LV administered intraperitoneal^ will usually be about one twentieth of the dose of 5-FU. So, for example if the dose of 5-FU is 425 mg/m²/day then the dose of LV will be about 20 mg/m²/day. A skilled artisan will recognise appropriate levels of LV.

[001 12] 5-FU and LV treatment according to the present invention may be administered to a subject by known procedures, including, but not limited to, oral administration, parenteral administration (e.g., intramuscular, intraperitoneal, intravascular, intravenous, or subcutaneous administration), and transdermal administration. Preferably, the 5-FU and LV agents are administered parenterally.

[001 13] For parenteral administration, the formulations of 5-FU and LV (whether individual or combined) may be combined with a sterile aqueous solution that is preferably isotonic with the blood of the subject. Such formulations may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile.

[001 14] The formulations may be presented in unit or multi-dose containers, such as sealed ampoules or vials. Moreover, the formulations may be delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracutaneous, intramuscular, intraorbital, intraperitoneal (particularly in the case of localized regional therapies), intraspinal, intrasternal, intravascular, intravenous, parenchymatous, or subcutaneous.

[001 15] Further, in an embodiment the method includes a step of treating the patient with one or more biological anticancer agents. Desirably that step is included at either cycle 1 or cycle 4 of the treatment regime. Preferably, the biological anticancer - - agent is an antibody or antibody fragment or antibody like molecule that is targeted against cells or the blood vessels supplying the cancer cells. For example, the agent may be an antibody or fragment thereof that targets EGF and VEGF, may also be used. Preferably, the anticancer agent is bevacizumab. [001 16] Accordingly, the present invention provides a method of treating neoplasia in a subject in need of treatment, by administering to the subject an amount of a combination of 5-fluorouracil and leucovorin effective to treat a neoplasia, in combination with SIRT using the microparticles of the invention, wherein a synergistic antineoplastic effect results. [001 17] The invention further relates to a kit for killing neoplastic cells in a subject having neoplastic cells. The kit comprises an effective antineoplastic amount of 5-FU and LV and an amount of radionuclide-doped microparticles suitable for use in SIRT for treatment of a neoplastic growth. The kit may further comprise an instructional material. Preferably, the kit is prepared for use in treating a patient with colorectal liver metastases.

[001 18] The invention still further relates to use of an effective antineoplastic amount of 5-FU and LV and an amount of radionuclide-doped microparticles suitable for use in SIRT using the microparticles of the invention, for manufacture of a medicament for killing neoplastic cells in a subject having neoplastic cells. Preferably, the medicament is prepared for use in treating a patient with colorectal liver metastases.

[001 19] The invention yet further relates to the use of an effective antineoplastic amount of 5-FU and LV and an amount of radionuclide-doped microparticles suitable for use in SIRT using the microparticles of the invention, for manufacture of a kit for killing neoplastic cells in a subject having neoplastic cells. Preferably, the 5-FU and LV and radionuclide-doped microparticles are manufactured for use in a kit for treating a patient with colorectal liver metastases.

[00120] In the method of the present invention, 5-FU and LV is administered to a subject in combination SIRT using the microparticles of the invention, such that a synergistic antineoplastic effect is produced. A "synergistic antineoplastic effect" refers to a greater-than-additive antineoplastic effect that is produced by a combination of chemotherapeutic drugs and SIRT, which exceeds that which would otherwise result - - from individual therapy associated with either therapy alone. Treatment with 5-FU and LV in combination with SIRT unexpectedly results in a synergistic antineoplastic effect by providing greater efficacy than would result from use of either of the antineoplastic agents alone. [00121 ] In the method of the present invention, administration of 5-FU and LV "in combination with" SIRT using the microparticles of the invention refers to coadministration of the two antineoplastic treatments. Co-administration may occur concurrently, sequentially, or alternately. Concurrent co-administration refers to administration of both 5-FU and LV and SIRT at essentially the same time. For concurrent co-administration, the courses of treatment with 5-FU and LV and with SIRT may also be run simultaneously. For example, a single, combined formulation of 5-FU and LV, in physical association with SIRT, may be administered to the subject.

[00122] In the method of the present invention, 5-FU and LV therapy and SIRT also may be administered in separate, individual treatments that are spaced out over a period of time, so as to obtain the maximum efficacy of the combination. When spaced out over a period of time, administration of 5-FU and LV is preferably given to a patient for a period of time such as 1 to 10 days, but more preferably about 3 to 5 days following which SIRT is applied. This cycle may be repeated as manner times as necessary and as long as the subject is capable of receiving said treatment. [00123] Accordingly, to another embodiment of the invention there is provided a method of treating neoplasia in a subject in need of treatment, by administering to the subject an amount of a combination of 5-FU, LV and OXA effective to treat a neoplasia, in combination with SIRT using the microparticles of the invention, wherein a synergistic antineoplastic effect results. [00124] In the method of the present invention, the amount of 5-FU, LV and OXA that is effective to treat the cancer is an amount that at least ameliorates cancer.

[00125] Accordingly, to this embodiment of the invention there is provided a method for treatment of a neoplasia patient in need of such treatment, which comprises the steps of: (i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 to 80 mg/m²; . -

(b) a 2-hour infusion of LV at a dose of about 1 00 to 400 mg/m²;

(c) followed by a bolus of 5-FU at a dose of about 300 to 500 mg/m² and then an infusion of 5-FU for about 40 to 50 hours at a dose of about 2.0 to 2.6 g/m²; and then

(ii) delivering SIRT using the microparticles of the invention to said patient on day 3 or 4 following the commencement of step (i);

(iii) repeating step (i) for three cycles at an interval of one to three weeks between treatment cycles; then

(iv) following about two weeks from the final treatment delivered in step (iii) delivering to said patient the following treatment:

(a) a 2-hour infusion of OXA at a dose of about 80 to 1 00 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 1 00 and 400 mg/m²;

(c) followed by a bolus of 5-FU at a dose of about 300 to 500 mg/m² and then an infusion of 5-FU for about 40 to 50 hours at a dose of about 2.0 to 2.6 g/m²; and

(v) repeating step (iv) every 2 to 3 weeks, until the cancer is treated.

[001 26] Desirably step (v) in the method of the invention is repeated until either liver hepatotoxicity becomes a problem or peripheral neuropathy becomes an issue for the patient. Hepatotoxicity of tissues peripheral to neoplasia tissue may become apparent as a result of excessive chemotherapy in a subject.

[001 27] The assessment of liver toxicity is a rather complex process particularly when using chemotherapeutic agents. The current methods usually comprise clinical investigations (e.g. ultrasonography), pathological and histo-pathological investigations as well as a biochemical analysis. A state-of the-art evaluation of the drug-induced liver toxicity is described in the CDER/CBER Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation, July 2009 as well as in the EMEA (CHMP) Reflection paper on non-clinical evaluation of drug-induced liver injury (DILI), 24 Jun. 201 0 (Doc Ref EMEA/CHMP/SWP Π 501 15/2006).

[001 28] According to this method the doses of OXA administered to a patient in the initial three cycles of the invention will be less than the dose of OXA administered in the fourth and subsequent cycles of drug administration. The primary safety concern is that the OXA in the chemotherapy regimen is a radio-sensitising agent, which when . - used in combination with SIRT using the microparticles of the invention results in toxicity at doses greater than initially delivered.

[00129] Further according to the invention OXA doses in the fourth and subsequent cycles should be more than the first three cycles but minimized as much as possible to maximise the time that patients can receive protocol chemotherapy before peripheral neuropathy becomes an issue (which necessitates the removal of OXA).

[00130] The method contemplates either a single or multiple doses of 5-FU, LV and OXA delivered according to the treatment regime to impair the symptoms of the cancer being treated. For example, impairment of symptoms of the cancer may be ameliorated by diminishing pain or discomfort suffered by the patient; by extending the survival of the patient beyond that which would otherwise be expected in the absence of such treatment; by inhibiting or preventing the development or spread of the cancer; or by limiting, suspending, terminating, or otherwise controlling the maturation and proliferation of cells in the cancer. [00131 ] Notably, the amounts of 5-FU, LV and OXA effective to treat neoplasia in a patient in need of treatment will vary depending on the type of SIRT used, as well as the particular factors of each case, including the type of cancer, the stage of the cancer, the patient's weight, the severity of the patient's condition, and the method of administration. These amounts can be readily determined by the skilled artisan. [00132] Desirably, OXA is delivered to the patient in the initial three treatment cycles at a dose of about 60 to 80 mg/m². Reference to the use of the term "about" in this statement seeks to import a level of variability into the treatment regime that is consistent with the manner in which a doctor might vary the OXA regime depending on the needs of a patient. For example, a dose of OXA at 54, 55, 56, 57, 58 or 59 mg/m² can appropriately be used in the treatment regime and such doses should be considered within the scope of the present invention. Similarly, doses of OXA at 81 , 82, 83, 84, 85, 86, 87, or 88 mg/m² can also appropriately be used in the first treatment cycle. Preferably, the dose will reside within the range of 60 to 80 mg/m². In a preferred form of the invention the dose of OXA will be closer towards the lower end of the stipulated range. Such doses of OXA include 60, 61 , 62, 62, 64, 64, 66, 67, 68, 69, 70 mg/m². . .

[00133] According to the method of the invention, in the fourth cycle of treatment, the dose of OXA is increased to at least about 80 to 100 mg/m². Reference to the use of the term "about" in this statement seeks to import a level of variability into the treatment regime that is consistent with the manner in which a doctor might vary the OXA regime depending on the needs of a patient. For example, a dose of OXA at 77, 78 or 79, 80, 81 , 82, 83 or 84 mg/m² can appropriately be used in the treatment regime and such doses should be considered within the scope of the present invention. Similarly, doses of OXA at 101 , 102, 103, 104, 105, 106, 107, or 108, 109 or 1 10 mg/m² can also appropriately be used in the first treatment cycle. Preferably, the dose will reside within the range of 80 to 100 mg/m². In a preferred form of the invention the dose of OXA will be closer towards the lower end of the stipulated range. Such doses of OXA include 80, 81 , 82, 82, 84, 84, 85, 86, 87, 88, 89, 90 mg/m².

[00134] In an embodiment of the invention, the dose of OXA administered in the initial three cycles of the invention is about 60 mg/m² while the dose administered in the fourth cycle is 85 mg/m². According to this embodiment of the invention OXA is delivered at a dose of 60 mg/m² for the first three cycles of chemotherapy, and in subsequent cycles is increased to a dose of 85 mg/m². The primary safety concern is that the OXA in the chemotherapy regimen is a radio-sensitising agent, which when used in combination with the present invention results in toxicity at doses >60 mg/m². [00135] Further according to this embodiment of the invention OXA doses in the fourth and subsequent cycles should be 85 mg/m² rather than the dose of 100 mg/m², this will maximise the time that patients can receive protocol chemotherapy before peripheral neuropathy becomes an issue (which necessitates the removal of OXA).

[00136] Desirably, the dose of LV delivered to the patient in the initial three treatment cycles and in the fourth cycle is at a dose of about 100 to 400 mg/m². Reference to the use of the term "about" in this statement seeks to import a level of variability into the treatment regime that is consistent with the manner in which a doctor might vary the LV regime depending on the needs of a patient. For example, a dose of LV at 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 mg/m² can appropriately be used in the treatment regime and such doses should be considered within the scope of the present invention. Similarly, doses of LV at 401 , 402, 403, 404, 405, 406, 407, up to 428 mg/m² inclusive, can also appropriately be used in the first treatment cycle. Preferably, the dose will reside within the range of 100 and 400 mg/m². In a preferred form of the - - invention the dose of LV will be closer towards the lower end of the stipulated range, eg 100 to 200 mg/m². By way of illustration doses of LV include 100, 1 10, 120, 130, 140, 150, 160, 170, 180 and 190 mg/m² as well as every dose in between these specified doses. [00137] Desirably, the bolus of 5-FU delivered to the patient in the initial three treatment cycles and in the fourth cycle is at a dose of about 300 to 500 mg/m². Reference to the use of the term "about" in this statement seeks to import a level of variability into the treatment regime that is consistent with the manner in which a doctor might vary the LV regime depending on the needs of a patient. For example, a dose of 5-FU in the bolus can be at 250, 260, 270, 280, 290 mg/m² can appropriately be used in the treatment regime and such doses should be considered within the scope of the present invention. Similarly, doses of 5-FU in the order of 510, 520, 530, 540 and 550 mg/m² inclusive, can also appropriately be used in the first treatment cycle. Preferably, the dose will reside within the range of 300 and 500 mg/m². In a preferred form of the invention the dose of 5-FU in the bolus will be about 400 mg/m².

[00138] Desirably, the continuous infusion of 5-FU that is delivered to the patient in the initial three treatment cycles and in the fourth cycle is at a dose of about 2.0 to 2.6 g/m². Reference to the use of the term "about" in this statement seeks to import a level of variability into the treatment regime that is consistent with the manner in which a doctor might vary the LV regime depending on the needs of a patient. For example, a dose of 5-FU in the continuous infusion can be at 1 .5, 1 .6, 1 .7, 1 .8, 1 .9 or 2.0 g/m² can appropriately be used in the treatment regime and such doses should be considered within the scope of the present invention. Similarly, doses of 5-FU in the continuous infusion in the order of 2.6, 2.7, 2.8 or 2.9 g/m² inclusive, can also appropriately be used in the first treatment cycle. Preferably, the dose will reside within the range of 2.0 to 2.6 g/m². In a preferred form of the invention the dose of 5-FU in the bolus will be about 2.4 g/m².

[00139] The time period over which the continuous infusion of 5-FU is delivered to the patient may vary from about 40 to 50 hours. A physician period will preferably determine the delivery time. In a desirable form of the invention the delivery time is selected from the group consisting of 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 hours. Most preferably the infusion is for 46 hours. . .

[00140] It will be appreciated that the dose of chemotherapeutic agent delivered to the patient according to the above treatment regime may vary within the various dose ranges specified. Moreover, the dose of chemotherapeutic agent delivered to a patient may vary between treatment cycles. Ideally, variation of the dose of drug delivered accommodates for hepatotoxicity. In this respect, the dose of drug delivered in a treatment cycle should seek to keep to a minimum the hepatotoxicity in that treatment cycle.

[00141 ] In an embodiment of this aspect of the invention there is provided a method of treatment of a neoplasia patient in need of such treatment, which comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(c) followed by a bolus of 5-FU at a dose of 400 mg/m² and then a 46-hour infusion of 5-fluorouracil at a dose of 2.4 g/m²; and then

(ii) delivering SIRT to said patient on day 3 or 4 following the commencement of step

(i);

(iii) repeating step (i) three times at an interval of one to three weeks between treatment cycles; then

(a) a 2-hour infusion of OXA at a dose of about 85 mg/m²;

(b) a 2-hour infusion of LV at a dose of between 200 mg/m²;

(c) followed by a bolus of 5-FU at a dose of 400 mg/m² and then a 46-hour infusion of 5-FU at a dose of 2.4 g/m²; and

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

[00142] Chemotherapeutic agents used in the treatment according to the present invention may be administered to a patient by known procedures, including, but not limited to, oral administration, parenteral administration (e.g., intramuscular, intraperitoneal, intravascular, intravenous, or subcutaneous administration), and transdermal administration. Preferably, the 5-FU, LV and OXA agents are administered parenterally. . .

[00143] Further, the method may also include a step of treating the patient with one or more biological anticancer agents such as antibodies, fragments thereof or antibody like molecules targeted against a variety of cancer cells or the blood vessels supplying the cancer cells. For example, antibodies, fragments thereof or antibody like molecules target EGF or VEGF. Preferably, the anticancer agent is bevacizumab.

[00144] Accordingly, in an embodiment of this form of invention there is provided a method for treatment of a neoplasia patient in need of treatment, which comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 to 80 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 to 400 mg/m²;

(i);

(iv) two weeks after the final treatment delivered in step (iii) delivering to said patient the following treatment:

(a) a 2-hour infusion of OXA at a dose of about 85 to 100 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 and 400 mg/m²;

(c) followed by a bolus of 5-FU at a dose of about 300 to 500 mg/m² and a 15 to 90 minute infusion of bevacizumab at about 5 to 15 mg/kg, followed by an infusion of 5-FU at a dose of about 2.0 to 2.6 g/m² for about 40 to 50 hours; and

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

[00145] The biological anticancer agent(s) may be administered at any dose that is recommended for treating patients with cancer. Where the biological anticancer agent is bevacizumab preferably a dose of about 5 to 10 mg/kg is delivered to said patient.

[00146] The time over which the agent is delivered to a patient will be varied depending on the patient and severity of treatment required. In a preferred form of the . . invention where bevacizumab is administered per the above dose regime the agent treatment time is 30 to 60 minutes.

[00147] Bevacizumab therapy may be delivered at any one or more of the various cycles of treatment. Desirably, bevacizumab therapy is delivered with the first cycle of therapy or in the last cycle. In a highly preferred form of the invention bevacizumab therapy is delivered in the last cycle of therapy immediately after OXA therapy.

[00148] In a highly preferred embodiment, the invention resides in a method for treatment of a neoplasia patient in need of treatment, which comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(c) followed by a bolus of 5-FU at a dose of about 400 mg/m² and then an infusion of 5-FU for about 46 hours at a dose of about 2.4 g/m²; and then

(ii) delivering SIRT to said patient on day 3 or 4 following the commencement of step (i);

(a) a 2-hour infusion of OXA at a dose of about 85 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(c) followed by a bolus of 5-FU at a dose of about 400 mg/m² and a 30 to 60 minute infusion of bevacizumab at about 5 to 10 mg/kg, followed by an infusion of 5-FU at a dose of about 2.4 g/m² for about 46 hours; and (v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

[00149] According to another aspect, the invention resides in the use of a polymeric particulate material as herein described, in internal radiation therapy of a patient.

[00150] According to still another aspect, the invention resides in the use of a polymeric particulate material as herein described, in the manufacture of medicament for the treatment of neoplasia in a patient. Preferably, the neoplasia is a metastatic . . carcinoma which, for example, may arise in the liver from primary carcinomas of, for example, the colonic mucosa.

EXAMPLES

[00151 ] Further features of the present invention are more fully described in the following non-limiting example. It is to be understood that this description is included solely for the purposes of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the invention as set out above.

EXAMPLE 1 Radiolabeled microparticles imaging in vivo, using adhesive attachment of protamine-coated nanoparticles comprising a carbon encapsulated Tc99m core

Methods and Materials

Nanoparticle Synthesis

[00152] Carbon-caged Tc-99m, (FibrinLite; FL) was synthesised as described in detail in US 8,778,300. The nanoparticle technology employed was based on Technegas™, a radioactive aerosol preparation developed for diagnostic ventilation imaging of the lungs [US 7,722,856]. Vapour-phase particle sizing using an electrostatic particle classifier (TSI Inc, MN USA) showed the aerosol comprises log- normal distributed particles with the bell curve centred on 150-350 nm, and negligible particles below 100 nm or above 400 nm. Electron microscope characterisation shows metallic platelets surrounded by multiple lamellae of carbon [Senden et al., (1997). J Nucl Med; 37: 1327-1333].

[00153] Using the above FL process, sodium Tc99m-pertechnetate solution was loaded into a graphite crucible and after removing sodium chloride by sublimation at 1650 ^QC, the isotope was plasma ablated at 2750 ^QC into an argon gas stream. Aerosol nanoparticles were collected into water (6.0 mL) from the gas stream using a Browitt sonicating precipitator [US 5,792,241 ], thus producing a stable colloidal dispersion of FL. The radioactive FL colloid was filtered through a 450 nm hydrophilic membrane (mixed cellulose ester (MCE); Millipore) before use. A typical preparation of FL contained approximately 5 μg/mL of graphitic carbon with a specific activity of 20 . .

ΜΒς/μς. FL nanoparticles are highly stable, and integrity of the isotope encapsulation is preserved under standard autoclave conditions of 20 min at 120 ^QC.

In vitro assays of FL binding to polystyrene micro-wells

[00154] The inventors have previously shown using a membrane filtration model and micro-well binding assays that polycations such as polylysine bind to the surface of FL with high affinity [Lobov et al., (2013). Biomate als: 34: 1732-1738]. Binding of these polycations to FL is also stable under in vivo conditions. The inventors have previously proposed [see Freeman CG, et al. (2013). Biomatehals; 34:5670-5676] that binding is mediated by multi-site pi-cation interactions between the positively charged amino groups of the amino acid side-chains and the pi-electrons of the planar carbon rings of the graphite surface. The protamine family of proteins are also polycations due to a high content of arginine and lysine residues. In the present study the change in adhesion properties of FL after surface treatment with protamine sulfate (PS; Sigma- Aldrich, Sydney, Australia) was tested by micro-well binding as follows. [00155] FL (5 MBq) was added to serial dilutions of PS (20 to 0 μg/mL) in 0.5 mM Tris acetate buffer (pH 7.2) and allowed to stand for 1 h at 20^QC. Each treatment mixture was then added to triplicate polystyrene micro-wells (LockWell MaxiSorp; Nunc, Denmark) and the FL was allowed to bind to the plastic surface for 30 min at 37^QC with shaking. After rinsing the micro-wells 3x with water, the bound radioactivity was measured by gamma counting of the separated individual wells in a Capintec well counter (Ramsey NJ, USA).

FL and LT binding to polystyrene sulfonate microparticles (MS)

[00156] The preparation of FL-MS microparticles using protamine were prepared in accordance with the method in co-owned US patent 9,381 ,262, which is incorporated herein by reference in its entirety. In summary, polystyrene sulfonate (MS) of 3 different sizes were used in this study; 30 μιη (MS30), 12 μιη (MS12) and 8 μιη (MS8) median diameter (Sirtex Technology Pty Ltd, Sydney). The MS (40 mg) were washed three times with water (6 mL) by centrifugation (2000 rpm for 2 min) and resuspension before radiolabelling. Freshly prepared FL (260 MBq in 6 mL) was first treated with PS (20 μg/mL) for 30 min before addition to the final centrifuged pellet of washed MS. The PS treated FL was then allowed to bind to the MS with gentle mixing for 30 min at 20^QC, . . during which the colour of MS changed from pale yellow to dark grey and the supernatant cleared.

[00157] After separation of the resulting FL-MS by centrifugation, it was washed 3x with water (6 imL) by centrifugation and resuspension. At each step the radioactivity was measured in the supernatant, and the radioactivity in the final washed FL-MS was expressed as a percentage of the original radioactivity applied.

Scanning electron microscopy (SEM) of FL adherent on the surface of microparticles

[00158] FL-MS30 prepared by the method above was prepared for SEM using sputter coating with gold and imaged using a Jeol model 840 SEM instrument at the Westmead Centre for Oral Health, Sydney. Unlabelled and labelled microparticles were scanned for direct comparison of their surface features.

Retention tests of FL-MS and Tc99m-MAA after entrapment in normal rabbit lungs

[00159] All rabbit procedures adhered to the National Health and Medical Research Council's Animal Welfare Code for the appropriate use of animals for scientific purposes (Australian Government, 8^th Ed.), and the experimental protocols were approved by the Australian National University (ANU) Animal Ethics Committee. Rabbit imaging studies were carried out using intubation to deliver ventilation anaesthesia with isoflurane, so that bio-distribution of radiolabel in live animals could be followed for up to 3 h.

[00160] Imaging of the anaesthetised rabbits and their excised organs after 3 h was performed with a GE Hawkeye Infinia SPECT-CT camera. To test the lung retention of the radiolabeled MS, suspensions of FL-MS30, FL12 and FL-MS8 (1 10-170 MBq on 15 mg MS in 5 imL 5% dextrose) were injected intravenously into an ear vein, so that the MS were mechanically arrested at limiting diameters in the arterial network of the lungs. When FL is coated with PS the coated nanoparticles are not retained in the lungs by binding to the heparan sulphate in the vascular glycocalyx [Freemann et al., (2013) Biomaterials: 34: 5670-5676].

[00161 ] The lung retention obtained with the three sizes of MS were compared with lung retention of the clinical lung diagnostic agent, Tc99m-MAA (1 10-170 MBq on 2.5 mg in 5 imL 5% dextrose) (Draxlmage, Quebec, Canada). Static 5 min acquisitions . . were made on a 1024 x 1024 matrix approx. 10 min after injection of MS and again every hour up to 3 h. A blood sample (5 imL) was taken from an ear vein before the rabbit was euthanized by lethal injection while still under anaesthesia. The lungs and liver were then excised after tying off blood vessels to prevent leakage of isotope, and the excised organs and blood sample were imaged separately using a 5 min acquisition on a 1024 x 1024 matrix, and utilising the camera's zoom function (4x). Counts registered in the acquisitions were corrected for the background activity of the corresponding field, and the corrected counts were used for calculation of the percentage activity in the lungs, liver, blood and carcass. The total blood volume and radioactivity were calculated assuming 60 imL of blood per kg of rabbit body weight. Activity levels in the images shown in the Figures were assigned false colours using the Xeleris XT21 Brainl colour map.

Arterial distribution of FL-MS in rabbit livers

[00162] Intrahepatic artery instillations of FL-MS30, FL-MS12, FL-MS8 (40 mg each) and Tc99m-MAA (2.5 mg) were performed by catheterisation of the cystic artery and using pulses of the particle suspension (total 5 imL 5% dextrose, 1 10-170 MBq) interspaced with normal hepatic artery blood flow, so as to disperse the radiolabeled material throughout the liver with close to normal arterial blood perfusion conditions as previously described [Moroz P et al., (2001 ) J Surg. Oncol; 78:22-29]. The MS were kept suspended by gentle agitation during instillation. Static 5 min acquisitions were made on a 1024 x 1024 matrix approx. ten (10) minutes after administration of the imaging agent and again after one hour, when the rabbit was euthanized by lethal injection while still under anaesthesia. One hour was considered sufficient for this test to be relevant to the time between instillation of Tc99m-MAA and imaging of human patients in clinical SIRT procedures. The lungs and liver were excised after tying off blood vessels to prevent leakage of isotope, and the excised organs were imaged separately using a 5 min acquisition on a 1024 x 1024 matrix, and utilising the zoom function. Counts registered in the acquisitions were corrected for the background activity of the corresponding field, and the corrected counts were used for calculation of the percentage activity in the organs. Activity levels in the images shown in the Figures were assigned false colours using the XT21 Brainl colours in the Hawkeye Infinia SPECT/CT Xeleris colour maps.

Rabbit VX2 liver tumour model . .

[00163] The transplantable rabbit VX2 tumour as previously described [Lee et al., (2009). J Vase Interv Radiol: 20: 1 13-1 17] was a kind gift of Dr J Geschwind (Johns Hopkins University, Baltimore, USA) and was maintained as a serial transplant on the hind limbs of New Zealand white rabbits. Liver implants of tumour tissue were made at a single site in one lobe by keyhole surgery under ventilation anaesthesia with isoflurane, and allowed to grow for 18 days before use of the rabbit in imaging experiments. At this stage of growth, the tumour was an oblate ellipsoid of maximum diameter 2 cm, still contained within the liver lobe and not involving the body wall or other organs. Macroscopically, the tumour usually had a white necrotic centre, surrounded by a prominently vascularised peripheral growth zone.

Results

FL binding to polystyrene micro-wells and microparticles

[00164] FL pretreated with low microgram concentrations of PS readily bound to polystyrene micro-wells as shown in Figure 1 A. Increasing concentrations of PS produced more binding until excess PS competed with coated FL for binding to the plastic microwell surface, so that there was an optimal protamine concentration of approximately 5 μg/mL before binding of FL decreased. Similarly, FL pretreated with PS bound strongly to polystyrene sulfonate microparticles (MS), changing their colour from pale yellow to dark grey. The resulting radiolabeled FL-MS was stable to subsequent repetitive washing with centrifugation and resuspension (Fig 1 B). Closely similar results were obtained for the binding of PS treated FL to the different sizes of MS tested, i.e. MS30, MS12 and MS8. Scanning electron microscopy of FL-MS30 showed distributed islands of FL adherent on the surface of the MS (Fig 1 C), compared to the smooth surface of untreated MS (Fig 1 D). Retention tests of FL-MS following entrapment in the vascular network of normal rabbit lungs

[00165] Intravenous injection of rabbits with suspensions of FL-MS30, FL-MS12, and FL-MS8 resulted in retention of the majority of radiolabel in the lungs over at least 3 h, as shown by gamma camera imaging of the anaesthetised animals (Figs 2A-D). FL- MS30 appeared to be more centralised in the lung images (Fig 2A), while the two smaller MS were widely dispersed in the lungs (Figs 2B and 2C). For comparison, . . rabbits were also injected intravenously with the clinical diagnostic agent used for imaging lung perfusion, Tc99m-MAA. As expected, this agent gave well-distributed imaging of the lungs (Fig 2D), but after 3 h considerable label was apparent in the kidneys, presumably from free pertechnetate. [00166] More quantitative data for the distribution of label from each MS was obtained from imaging of a blood sample and the excised lungs, liver and carcass after dissection of animals at 3 h, as shown in Table 1 .

Table 1

Retention of radiolabeled MS and Tc99m-MAA in rabbit lungs following

intravenous injection

[00167] Table 1 shows the biodistribution of radioactivity in rabbits 3 h after intravenous (ear vein) injection of suspensions of FL radiolabeled microparticles and the particulate imaging agent, Tc99m-MAA. The polymer microparticles (15 mg) had median diameters of 30 μηπ (FL-MS30), 12 μηπ (FL-MS12) and 8 μηπ (FL-MS8) and each 5 imL injection contained 130-170 MBq Tc99m. The clinical diagnostic imaging agent Tc99m-MAA (2.5 mg; 1 10-170 MBq) was also injected intravenously for comparison. A blood sample (5 imL) was taken prior to dissection for calculation of the total blood radioactivity, assuming a blood volume of 60 imL per kg body weight. The carcass values shown are corrected for the total blood activity. Images for measurement of radioactivity in the excised liver, lungs, blood sample and carcasses were acquired using a GE Hawkeye Infinia gamma camera. The results shown are the means and SEM of triplicate experiments for each type of MS/particle. All radioactivity measurements were corrected for background in the corresponding acquisition field. Note transit of some FL-MS8 to the liver and label from Tc99m-MAA in the blood and carcass. - -

[00168] For FL-MS30, the mean lung proportion at dissection for 3 animals was 92.9 +/- 1 .5% of the total body activity and only 4.3% was in the excised liver and 0.84% in the total blood volume. After intravenous injection of 3 rabbits with FL-MS12, the mean lung proportion was 87.6 +/- 2.5%, while 9.8% was in the liver and 0.66% in the total blood volume. The same test with FL-MS8 showed that 72.8 +/- 1 .9% of activity had been retained in the lungs after 3 h, while 23.1 % had been taken up the liver, and 1 .1 % was in the total blood volume (Table 1 ). After injection of Tc99m-MAA and dissection at 3 h, the lungs accounted for just 66.8 +/- 5.7% of the total radioactivity from this agent, with 4.5% in the liver, 8.2% in the whole blood volume and 20.6% in the carcass (Table 1 ). While escape of FL-MS8 from the lungs appeared to be in the form of particles captured by the reticuloendothelial system in the liver (Table 1 ), escape of Tc99m-MAA from the lungs produced visible labelling of the kidneys (Fig 2D) that was measured in the carcass (Table 1 ). This renal uptake was suggestive of free Tc99m- pertechnetate from the Tc99m-MAA preparation. Bio-distribution of FL radiolabelled microparticles instilled intra-arterially in normal rabbit liver

[00169] Imaging of intact rabbits 1 h after arterial instillation of FL-MS30 in the liver showed virtually complete retention of radiolabel in the organ. Imaging of two dissected animals showed a mean retention of 99.9% of the total radioactivity in the excised liver, while barely detectable levels in the excised lungs and a blood sample verified that escape of radiolabel to other organs was negligible (Table 2).

Table 2:

Retention of radiolabelled MS and Tc99m-MAA in rabbit liver following intraarterial instillation

Excised Excised

Blood Carcass

Liver MS/particle Liver Lungs

[%Total] [%Total]

FL-MS30 99.9 0.008 0.09 0.039

FL-MS12 99.3 0.20 0.16 0.36

Normal

FL-MS8 99.2 0.28 0.17 0.39

Tc99m-

97.2 0.10 1 .20 1 .50

MAA

VX2 FL-MS30 99.2 0.04 0.31 0.50 - -

[00170] Imaging of the excised livers also revealed a pronounced, coarsely segmented distribution of the radiolabel within the organ (Fig 3A), which was markedly different from the previously reported uniform liver uptake of radiolabelled FL nanoparticles by the reticuloendothelial system following intravenous or intra-arterial administration. Distribution of label did not extend throughout all areas of the organ and was highly variable between different livers. The appearance was of restricted distribution in which dispersal of FL-MS30 by the blood flow had been arrested at limiting diameters of the arterial network extending from the main feeder vessels, so that MS distribution was incomplete and clearly could not transit to the venous side.

[00171 ] Retention of radioactivity in normal rabbit livers and livers hosting VX2 tumour implants 1 h after intra-arterial instillation of radiolabelled microparticles and Tc99m-MAA. The polymer microparticles (40 mg) had median diameters of 30 μιη (FL- MS30), 12 μηπ (FL-MS12) and 8 μηπ (FL-MS8) and each 8 ml_ instillation contained 130- 170 MBq Tc99m. The clinical diagnostic imaging agent Tc99m-MAA (2.5 mg; 1 10-170 MBq) was also instilled intra-arterially for comparison. A blood sample (5 imL) was taken prior to dissection for calculation of the total blood radioactivity, assuming a blood volume of 60 imL per kg body weight. The carcass values shown are corrected for the total blood activity. Images for measurement of radioactivity in the excised liver, lungs, blood sample and carcass were acquired using a GE Hawkeye Infinia gamma camera. The results shown are the means of duplicate experiments for each type of MS/particle (16 experiments altogether). All radioactivity measurements were corrected for background in the corresponding acquisition field. Note high rates of retention in the liver 1 h post-instillation, for normal and tumour livers. [00172] Arterial instillation of FL-MS12 in the livers of two rabbits also showed strong retention of radiolabel after 1 h; imaging of the excised livers gave a mean of 99.3% (Table 2). Distribution of radiolabel within the liver was noticeably different from FL-MS30, with more complete dispersal into the organ (Fig 3B). Interestingly, even the smallest MS, FL-MS8, was also well retained inside the liver for 1 h (99.2%, Table 2), . . but the imaging of excised livers clearly showed a considerably more detailed and fine distribution of label extending throughout the organ, consistent with arrest of MS dispersal at a more distal limit of the liver's arterial network (Fig 3C). Escape of label to the lungs in these normal rabbits after 1 h was less than 0.2% for all three sizes of MS (Table 2).

[00173] As expected from its median diameter of 30 μιη, Tc99m-MAA was also well retained in the normal rabbit liver 1 h after arterial instillation (97.2%; Table 2), however imaging of the excised liver (Fig 3D) showed that the distribution of label was considerably more extensive and diffuse within the liver lobes than FL-MS30 (cf Fig 3A), and more comparable with the finer structure revealed by FL-MS12 (cf Fig 3C). The appearance of such extensive distribution within the organ suggested that particles of this agent had reached finer vessels of the arterial network than predicted by a particle diameter of 30 μιη. While escape of label from Tc99m-MAA to the lungs after 1 h remained very low at 0.1 %, there was noticeable activity in the blood (1 .2%) and carcass (1 .5%) (Table 2).

Biodistribution of FL radiolabelled microparticles instilled intra-artehally in rabbit livers hosting an implant of VX2 tumour

[00174] The above tests with FL-MS30, FL-MS12, FL-MS8 and Tc99m-MAA were then repeated in rabbit livers hosting implants of the VX2 tumour, after 18 days of tumour growth. At the time of MS administration tumours typically appeared as a single oblate ellipsoid of up to 2 cm diameter, thickening but still contained within the liver lobe and not involving the body wall or other organs. On sectioning, they usually had a white necrotic centre, surrounded by a prominently vascularised peripheral zone. Hepatic artery instillation of FL-MS30 in 2 rabbits hosting such liver VX2 tumours resulted in 99.2% retention of radiolabel in the liver after 1 h (Table 2); it was not noticeably less than the retention by a normal liver. Accordingly, leakage to the systemic circulation in these tumour rabbits was still very low, as shown in Table 2. The imaging of excised livers showed coarsely segmented features within the organ as in normal livers but the lobe hosting the tumour had accumulated noticeably more radiolabel than the rest of the liver (Fig 4A). The accumulation of label in the lobes hosting VX2 tumours in 6 different VX2 host rabbits represented an average 33.1 % (range 23.6 to 50.8%) of the total liver uptake, but the respective host lobes represented on average just 15.8% (range 1 1 .9 to 24.4%) of the liver weight. Thus, on a tissue weight basis, the tumour lobe received - - approximately double the radioactivity of FL-MS30 per gram of tissue compared to the rest of the liver.

[00175] Arterial instillation of FL-MS12 also showed strong retention of radiolabel in the tumour bearing liver after 1 h; imaging of the excised liver showed a mean of 98.7% of the total body activity (n=2; Table 2). As in normal livers, it was noticeable that dispersal of FL-MS12 label was more extensive in the organ than FL-MS30, reaching out to most of the lobes and occupying a larger proportion of the liver, but with a prominent accumulation of label at the tumour site (Fig 4B). Intra-arterial instillation of FL-MS8 into rabbit livers hosting VX2 tumours showed even finer structure of the arterial network after 1 h (Fig 4C) compared to FI-MS30, and yet 98.5% of the total activity was still retained in the organ (n=3; Table 2). Accumulation of label at the tumour site featured prominently, and assumed the form of a bright, complete annulus at the angiogenic growth margin of the tumour, surrounding a lower intensity (necrotic) centre (Fig 4C). Thus, clearer definition of the tumour site in the whole animal (Fig 5) and its growth zone (Fig 4C) was considerably facilitated by use of the smaller MS, and without degradation of the liver retention.

[00176] Intra-arterial instillation of Tc99m-MAA into livers with tumours showed 98.1 % retention of the total activity as in normal livers (n=2; Table 2) and accumulation of radioactivity at the tumour site (Fig 4D). While definition of the tumour growth zone was comparable with FL-MS12, it was not as clear as with FL-MS8.

Discussion

[00177] Surface coating of FL with PS has previously been shown to produce a marked change in the adhesion properties of these nanoparticles, clearly evident in a membrane filtration model. This observation was exploited to bind FL to sulfonated polystyrene microparticles of the same type as used in SIRT. PS coated FL bound avidly to the MS so that radiolabel was well retained after successive washing steps, and this simple process provided specific activity levels of radioisotope that were more than sufficient to enable in vivo gamma camera imaging of three sizes of FL-MS after administration to anaesthetised rabbits. [00178] Leach tests of FL-MS30 lodged in the vascular network of rabbit lungs demonstrated that the radiolabel had excellent stability in vivo, and persisted longer in . . the lungs than the lung diagnostic agent Tc99m-MAA. Retention of label was virtually complete under these in vivo blood flow conditions so that very little radioactivity reached other organs over a 3 h time course. The same tests with FL-MS12 and FL- MS8 showed that retention diminished somewhat with decreasing microparticle diameter, and slow loss of FL-MS8 from the lungs was observed, with significant label appearing in the liver after 3 h. Nevertheless, lung retention of even FL-MS8 was appreciable, given that 10 μιη is commonly accepted as being the lower limit of particle size required for retention in the lung capillary network.

[00179] Hepatic artery instillation of FL-MS30 into the rabbit liver showed very efficient retention of this preparation at limiting diameters of the liver arterial network, producing a coarse segmented distribution in imaging, consistent with the distributing arterioles within the organ. Arterial instillation of the smaller microparticles produced a noticeably different distribution of label, with finer features extending out to fill more of each liver lobe. The diameter of the microparticles was clearly an important property determining distribution in the arterial network of the organ. While the larger microparticles reached a relatively proximal limiting diameter in the arterial supply, the smaller microparticles were carried further on to more distal limits, producing a finer featured distribution image. However, retention of even the smallest microparticles in the liver was surprisingly efficient; transit of label from the liver to other organs was very low.

[00180] Tc99m-MAA, while nominally of similar particle size to FL-MS30, and well retained in the liver after arterial instillation, nevertheless produced an image showing more extensive dispersal of label within the liver than that obtained with FL-MS30. This could suggest that the particle integrity of Tc99m-MAA was not maintained under blood flow conditions in the liver and that it was disaggregated by shear forces to produce smaller particles.

[00181 ] When a VX2 tumour was present in the liver, instillation of all radiolabeled particles resulted in varying degrees of accumulation at the tumour site compared to the rest of the liver; the presence of a tumour effectively remodelled the arterial supply so as to somewhat favour tumour perfusion at the expense of arterial circulation to the rest of the liver. Approximately 33% of instilled radiolabel was measured in excised liver tumours following intra-arterial administration of FL-MS30. Tumour definition on imaging however was most evident using FL-MS8, when the tumour featured as a remarkably . . intense annulus around the tumour set against a fine-featured background. This provided superior visual distinction of the active growth zone of the tumour compared to the coarse segmented pattern obtained with FL-MS30, and also compared to imaging with Tc99m-MAA. [00182] In the above series of rabbit liver tumour experiments hepatopulmonary or hepatogastric shunting were not in evidence and retention of radiolabel within livers hosting a tumour was maintained at a very high level. Systemic release of radiolabel was not appreciably more than that seen in the case of normal livers, and even with the smallest diameter microparticles tested, the lungs had received only 1 % of the radioactivity 1 h after instillation of these microparticles in the tumour-bearing liver. This was comparable with systemic escape of label to the blood from Tc99m-MAA administered intra-arterially in normal and tumour-bearing livers.

[00183] Polymer microparticles of the type used for internal radiation therapy in liver cancer patients were conveniently radiolabeled at a specific activity suitable for imaging in vivo, using adhesive attachment of protamine-coated nanoparticles comprising a carbon encapsulated Tc99m core. This method of radiolabelling provided a stable composite in vivo under the shear stress conditions experienced in arterial networks of the lungs and liver in rabbit models. In the normal rabbit liver, arrest of radiolabeled microparticles at limiting diameters of arterial vessels showed increasing dispersal and definition of network structure with decreasing size of the microparticles used. In livers hosting model tumours the radiolabeled microparticles clearly demonstrated preferential uptake at tumour sites due to the increased arterial perfusion produced by angiogenesis. This result was most evident with microparticles of 8 μιη diameter, that also provided clearest definition of the tumour growth zone while still being well retained within the liver. We did not find evidence of hepatopulmonary shunting from livers hosting model tumours.

EXAMPLE 2

Comparison of chemistries of binding the isotope to the resin and different isotopes on various sizes of resin sphere, soluble and unbound isotope. [00184] Studies of the leaching of labelled microspheres from rabbits treated with the microspheres were conducted in 52 rabbit experiments. These tests investigated . . three different sizes of microspheres, using two different isotopes bound with different chemical methods.

[00185] The isotope-microsphere complex was made according to the following methods. Protamine-FibrinLite-Microsphere Tc label

[00186] As a first experiment, normal rabbit livers were used to test for leaching of the radiolabeled microspheres from the liver after intra-arterial administration.

[00187] In the first set of experiments, different sized microspheres were labelled with Protamine-FibrinLite-Microsphere Tc label (PS-FL-MS) using the method of US 9,381 ,262 and compared to Tc99m-MAA.

[00188] Polystyrene sulfonate microparticles (MS) of 3 different sizes were used in this study; 30 μηπ (MS30), 12 μηπ (MS12) and 8 μηπ (MS8) median diameter (Sirtex Technology Pty Ltd, Sydney). The MS (40 mg) were washed three times with water (6 imL) by centrifugation (2000 rpm for 2 min) and resuspension before radiolabelling. Freshly prepared FL (260 MBq in 6 ml_) was first treated with PS (20 μg/mL) for 30 min before addition to the final centrifuged pellet of washed MS. The PS treated FL was then allowed to bind to the MS with gentle mixing for 30 min at 20^QC, during which the colour of MS changed from pale yellow to dark grey and the supernatant cleared.

[00189] After separation of the resulting FL-MS by centrifugation, it was washed 3x with water (6 imL) by centrifugation and resuspension. At each step the radioactivity was measured in the supernatant, and the radioactivity in the final washed FL-MS was expressed as a percentage of the original radioactivity applied.

[00190] The leaching of isotope label from three different sized labelled microspheres was tested and the results compared to Tc99m-MAA, as shown in Table 3. Table 3 Normal Rabbit Liver Leach Tests for PS-FL-MS Preparations administered intra-arterially, compared with Tc99m-MAA. MS dose = 40 mg for all experiments.

% Label in Liver Lun s, Blood and Carcass at 1 h ost-instillation via he atic arter

D5W = 5% dextrose in water; ND^* = not detectable above background; ^**Carcass includes blood activity; ^Amostly kidneys The mean for FL-MS30 does not include 141 126 084N, where there was atypical leakage to lungs

A t-test shows no significant difference in the lungs/liver ratio for FL-MS12 and FL-MS8 (P = 0.218)

. .

[00191 ] Four rabbits received PS-FL-Tc labelled microspheres of size 30 micron; five rabbits received PS-FL-Tc labelled microspheres of size 12 micron; five rabbits received PS-FL-Tc labelled microspheres of size 8 microns, and three rabbits received Tc99m-MAA. [00192] With the exception of one atypical result for 30-micron (rabbit 084N) breakthrough of label outside the liver did not occur with either of the smaller microspheres, the mean of all percentage label in the liver being greater than 99%. No significant difference was observed between liver retention for 12 microns and 8 microns. The highest "escape" or leaching from the liver consistently occurred with MAA, which was still less than 3%.

[00193] A second study was conducted in rabbits with tumours, in accordance with the VX2 rabbit model (see above and [Lee et al., (2009). J Vase Interv Radiol: 20: 1 13- 1 17]). Two rabbits received PS-FL-Tc labelled microspheres of size 30 micron; four rabbits received PS-FL-Tc labelled microspheres of size 12 micron; four rabbits received PS-FL-Tc labelled microspheres of size 8 microns, and two rabbits received Tc99m-MAA. The results of this study are set out in Table 4.

Table 4 Rabbit VX2 Liver Leach Tests for PS-FL-MS Preparations administered intra-arterially, compared with Tc99m-MAA. MS dose = 40 mg for all experiments.

T = VX2 tumour in liver, D5W = 5% dextrose in water, ^Amostly kidneys, ^*Mean excluding 150127 091 T, ^**Carcass includes bl activity

- -

[00194] When tumours are present in the livers, the results for PS-FL-MS do not significantly change with respect to the "escape" or leaching of the label from the liver. As can be seen from Table 4, the mean percentage label in the liver is greater than 98%, with one atypical result observed at 12 microns showing leakage to the lung (rabbit 091 T). The percentage uptake by the tumour varied between the sizes of the microspheres. However, we believe this is mainly due to the size/stage of the growth of the tumour rather than the size of the microsphere. We also note that the activity in the lungs appears to be higher for several rabbits, (091 T and 143T) where the tumour uptake was very high, most likely due to tumour angiogenesis. Gallium- Tannic acid-Microspheres

[00195] A third study was conducted to investigate the use of Gallium-Tannic acid- Microspheres (Ga67-TA-MS).

[00196] The microspheres were prepared by the method of PCT/AU2013/001510

[00197] The leaching of isotope label from three different sized labelled microspheres was tested and the results are shown in Table 5. Labelled microspheres were tested in normal rabbit livers leaching after treatment with Ga67-TA-MS. Three rabbits received Ga67-TA labelled microspheres of size 30 micron; two rabbits received Ga67-TA labelled microspheres of size 12 micron; and three rabbits received Ga67-TA labelled microspheres of size 8 microns.

Table 5 Normal Rabbit Liver Leach Tests for Ga67-TA-MS Preparations administered intra-arterially in 5% dextrose MS dose 40 mg for all experiments

% Label in, Liver, Lun s, Blood and Carcass at 1 h ost-instillation via he atic arter

NB: The calculated blood volume activity accounts for all of the carcass activity

. .

[00198] Leaching from these tests was in general a little higher than with PS-FL- MS in normal rabbits as discussed above (Table 3). However, the mean percentage leach of label from the liver was low, with a liver retention of above 93%. In one rabbit (141 ), using a microsphere of 8 microns, there was more lung uptake than all other rabbits tested.

[00199] A fourth study was conducted in rabbits with tumours, in accordance with the VX2 rabbit model (see above and [Lee et al., (2009). J Vase Interv Radiol: 20: 1 13- 1 17]). Two rabbits received Ga67-TA labelled microspheres of size 30 micron; four rabbits received Ga67-TA labelled microspheres of size 12 micron; and five rabbits received Ga67-TA labelled microspheres of size 8 microns. The results of this study are set out in Table 6.

Table 6 Rabbit VX2 Liver Leach Tests for Ga67-TA-MS Preparations administered intra-arterially in 5% dextrose.

MS dose 40 mg for all experiments

% Label in Lun s, Liver, Blood and Carcass at 1 h ost-instillation via he atic arter of autoclaved and resus ended re aration

^*Mean excluding 160406 206T

. .

[00200] When tumours are present in the livers, the results for Ga67-TA-MS show generally very good liver retention, with the exception of rabbit 206, which showed more lung uptake of the label, representing escape of isotope from the liver.

[00201 ] Nine (9) liver VX2 rabbit models were investigated using Ga67-TA-MS8 (i.e. 8 micron microspheres). In the first series, rabbits 167, 169, 166 and 168 (see Table 6) were observed to show poor tumour growth, with a size range of 148-1000 mm³.

[00202] In the second series of tests (see Table 6), the tumours grew well in each of the rabbits tested, covering a range of tumour size of 4046-9912 mm³. In these rabbits, (189, 185, 206, 201 and 210), it was observed that larger tumours took up more of the 8 micron microspheres than smaller tumours, and mostly without breakthrough from the liver to the lung, the one exception being rabbit 206.

[00203] Rabbits receiving the labelled microspheres showed a percentage retention of greater than 92%, comparable with the gallium results above (Table 6). Breakthrough to the lung occurred in the case of one large tumour (rabbit 156) that had taken up 67% of the microspheres.

[00204] The biodistribution results shown above for microspheres radiolabeled using two different chemical methods (FL-PS and TA), with two different imagable isotopes (^99mTc and ⁶⁷Ga) demonstrated that superior dispersion of microspheres within livers can be obtained when the microsphere size is reduced to 8 microns, independent of the labelling chemistry used. This increase in coverage of the organ and accumulation in tumours is not accompanied by unacceptable washout of isotope to non-target organs.

Claims

1 . A polymeric particulate material having a diameter in the size range of 6 to 12 μιη, comprising a polymeric matrix and a substantially stably incorporated radionuclide.

2. A polymeric particulate material according to claim 1 wherein the diameter of the particulate material is in the size range of 7 to 1 1 μιη.

3. A polymeric particulate material according to claim 1 wherein the diameter of the particulate material is in the size range of 8 to 10 μιη.

4. A polymeric particulate material according to claim 1 wherein the diameter of the particulate material is 8 μιη.

5. A polymeric particulate material according to claim 1 wherein the polymeric

material has a level of radioactivity that is between about 0.1 to 0.4 GBq (activity per particle).

6. A polymeric particulate material according to claim 1 wherein the polymeric

material is loaded with Yttrium 90 7. A polymeric particulate material according to claim 7 wherein the polymeric

material delivers a level of radioactivity to a tumor of up to 2.6, 2.

7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3 or 3.4 GBq at the site of treatment.

8. A polymeric particulate material having a diameter in the size range of 6 to 12 μιη and a stably incorporated radionuclide wherein the polymeric particulate material incorporating the radionuclide delivers a radiation dose of between about 10 and

800 Gy.

9. A polymeric particulate material having a diameter in the size range of 6 to 12 μιη and a stably incorporated radionuclide wherein the polymeric particulate material incorporating the radionuclide when administered to a patient with a tumor delivers a radiation dose of between about 10 and 800 Gy to the tumor in said patient.

10. A polymeric particulate material according to claim 1 wherein less than 5% of the radionuclide leaches from the particulate material under physiological conditions.

1 1 . A polymeric particulate material according to claim 10 wherein less than 4% of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material.

12. A polymeric particulate material according to claim 10 wherein less than 2% of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material.

13. A polymeric particulate material according to claim 10 wherein less than 0.5% of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material.

14. A polymeric particulate material according to claim 10 wherein less than 0.2% of the radionuclide leaches from the particulate material, under physiological conditions, over the radioactive life of the particulate material.

15. A polymeric particulate material according to claim 1 wherein the particles have a density below 3.0 g/cm³.

16. A pharmaceutical composition comprising a polymer particulate material as

defined by any one of claims 1 to 14 and at least a systemic or loco-regional chemotherapeutic agent, an antineoplastic agent or an immunomodulator.

17. A method for the production of a radioactive particulate material comprising a

polymeric matrix as described above, characterised by the steps of: (i) absorbing a radionuclide onto an ion-exchange resin particulate material having a diameter in the size range of 6 to 12 μιη, and a specific gravity of less than 2.5; and

18. A method of radiation therapy of a human or other mammalian patient that

comprises the step of administration to a patient of particulate material as defined by any one of claims 1 to 14.

19. A method according to claim 17 wherein the particulate material is administered to a patient at a therapeutic dose that delivers a radiation dose of between about 10 and 800 Gy to the patient's tumor.

20. A method according to claim 17 wherein the particulate material is administered to a patient at a therapeutic dose that delivers a radiation dose of between about 10 and 200Gy.

21 . A method according to claim 17 wherein the particulate material is administered to a patient at a therapeutic dose that delivers a radiation dose of between about 10 and 150 Gy, 10 and 100 Gy, 20 and 80 Gy, 25 and 75 Gy, 30 and 70 Gy, 35 and 65 Gy, 40 and 60 Gy or 40 and 55 Gy.

22. A method according to any one of claims 17 to 21 wherein the particulate material is administered concomitantly with at least a systemic or loco-regional

chemotherapeutic agent, an antineoplastic agent or an immunomodulator.

23. A method for treatment of a neoplasia patient in need of such treatment, which comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 to 80 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 to 400 mg/m²;

(ii) delivering SIRT to said patient on day 3 or 4 following the commencement of step (i), wherein the SIRT uses a polymeric material according to claims 1 to 14 or a pharmaceutical composition according to claim 16;

(a) a 2-hour infusion of OXA at a dose of about 80 to 100 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 and 400 mg/m²;

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

24. A method according to claim 23 wherein step (v) is repeated until either liver hepatotoxicity becomes a problem or peripheral neuropathy becomes an issue for the patient.

25. A method of treatment of a neoplasia patient in need of such treatment, which

comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 mg/m²;

(d) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(e) followed by a bolus of 5-FU at a dose of 400 mg/m² and then a 46-hour infusion of 5-fluorouracil at a dose of 2.4 g/m²; and then

(ii) delivering SIRT to said patient on day 3 or 4 following the commencement of step (i) wherein the SIRT uses a polymeric material according to claims 1 to 14 or a pharmaceutical composition according to claim 16;

(a) a 2-hour infusion of OXA at a dose of about 85 mg/m²;

(b) a 2-hour infusion of LV at a dose of between 200 mg/m²;

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

26. A method for treatment of a neoplasia patient in need of treatment, which

comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(a) a 2-hour infusion of OXA at a dose of about 60 to 80 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 to 400 mg/m²;

(ii) delivering SIRT to said patient on day 3 or 4 following the commencement of step (i) wherein the SIRT uses a polymeric material according to claims 1 to 14 or a pharmaceutical composition according to claim 16; WO 2018/107246 . QQ _ PCT/AU2017/051404

(a) a 2-hour infusion of OXA at a dose of about 85 to 100 mg/m²;

(b) a 2-hour infusion of LV at a dose of about 100 and 400 mg/m²;

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.

27. A method for treatment of a neoplasia patient in need of treatment, which

comprises the steps of:

(i) delivering to said patient on day one of a treatment regime:

(d) a 2-hour infusion of OXA at a dose of about 60 mg/m²;

(e) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(f) followed by a bolus of 5-FU at a dose of about 400 mg/m² and then an infusion of 5-FU for about 46 hours at a dose of about 2.4 g/m²; and then

(b) a 2-hour infusion of OXA at a dose of about 85 mg/m²;

(c) a 2-hour infusion of LV at a dose of about 200 mg/m²;

(d) followed by a bolus of 5-FU at a dose of about 400 mg/m² and a 30 to 60- minute infusion of bevacizumab at about 5 to 10 mg/kg, followed by an infusion of 5-FU at a dose of about 2.4 g/m² for about 46 hours; and

(v) repeating step (iv) every 2 to 3 weeks, until the neoplasia is treated.