Nanoparticle drug delivery system for radiotherapy in the brain

Abstract

The invention relates to the field of drug delivery of radioactive nucleosides for radiotherapy in the brain. The invention provides a composition comprising a prodrug of a radioactive Auger electron-emitting radionucleoside and a nanoparticle carrier, suitable for use in the treatment of brain tumors.

Description

[0001] P6655PC00 1Nanoparticle Drug delivery system for radiotherapy in the brainTechnical field The invention relates to the field of drug delivery of radioactive nucleosides for radiotherapy in the brain. The invention provides a composition comprising a prodrug of a radioactive Auger electron-emitting radionucleoside and a nanoparticle carrier, for usein the treatment of brain tumors.Background Glioblastoma is the most common and aggressive malignant primary brain tumor accounting for 77-81% of all primary malignancies of the central nervous system (CNS)(Ostrom et al.). Aggressive infiltrative growth of glioma cells and development of tumorangiogenesis are major impediments for effective treatment, and the high diversity of intramolecular and phenotypic heterogeneity of GBM and glioma stem cells makes complete surgical resection impossible. As a result, the median survival of patients withGMB is approximately 14.6 months with a five-year survival rate of only 6.8% (Sevastreet al.).In locally advanced tumors, surgery may be used for gross excision of as much tumoras possible without disabling the patient. However, in high-grade gliomas, the surgeon cannot eliminate individual tumor cells, microscopic tumor processes, or tumor- associated vasculature from the normal tissue surrounding the tumor excision site, dueto risk of damaging functioning areas. It is often critical to minimize the volume ofsurrounding tissue that is excised in such operations, as normal brain functions may be severely compromised as a result of tissue loss.Therefore, surgery of high-grade gliomas is accompanied by radiation therapy and / orchemotherapy in an attempt to kill cancerous cells in the surrounding brain tissue. Thechemotherapy may be delivered to the residual tumor cells by a localized or systemic route of administration. By limiting the extent of surgical excision, and relying upon theadjunctive treatments to eliminate the residual cancer cells, the function of the brain maybe preserved.A unique cell-killing mechanism is the release of Auger electrons by radionuclides thatdecay by electron capture and internal conversion, such as 125I, 123I, 77Br, 99mTc, 111In, P6655PC00 2and 67Ga among others. Auger electrons have low energy, resulting in extremely shortparticle path lengths within tissues, which is highly desirable, because it minimizes collateral damage.This makes Auger electrons a potent modality for cancer treatment, particularly when theAuger electrons are released near sensitive targets like DNA. To position Augerelectron-emitting radionuclides in close proximity to DNA, Auger-emitting radionuclidescan be incorporated into nucleoside analogs, which can be incorporated into the DNA ofcells. For example, iodine-substituted thymidine (dThd) analog, 5-iodo-2'-deoxyuridine(IUdR), can be phosphorylated to IUdR monophosphate (IdUMP) by thymidine kinaseand subsequently integrated into the of cancer cell DNA. Thus, making it a promisingand viable option for targeted destruction of DNA cancer cells. It has been shown thatwhen [125I]IUdR is integrated into DNA during cancer cell proliferation, accumulation ofAuger emitting radionuclides into the DNA in the cells and their progeny is exceedinglycytotoxic. Nonetheless, the recurrence of tumor after therapeutic intervention remains a cardinal challenge in the treatment of GBM. The main reason is that surgical treatment andcurrent chemotherapies and radiotherapies are not able to deal effectively with allcancerous cells present. Even a single glioblastoma cell can significantly invade andinfiltrate into the brain parenchyma (Breznik et al.)Brain cancer stem cells are particularly challenging to eliminate as cancer stem cells areinherently resistant to chemotherapeutic agents. These types of cells are believed to proliferate more slowly than other cell populations within the tumor thereby making them less susceptible to the toxic effects of cell cycle active agents and ionizing radiation. Inaddition, these cells have elevated expression of drug efflux transport proteins and havepreferential induction of DNA damage-response genes that repair DNA damage caused by radiation.A notable challenge when using chemotherapeutic or radiotherapeutic agents to dealwith infiltrating GBM cells and brain cancer stem cells is the fast excretion from the intracranial compartment of these agents. This leads to a short window of time where the therapeutic agents can exert their function and an inefficient use of the administereddose. Thus, despite the recognition that Auger emitting agents, such as 125I-UdR, have P6655PC00 3a unique cell killing capability, and despite extensive research aimed at exploiting thismechanism of action, the effective use of these agents to treat brain tumors has provento be extremely challenging. Thus, there is a strong need for new therapeutic strategies that are able to effectively treat brain tumors that can overcome current limitations and improve therapeutic outcomes. Summary The present invention addresses the above-mentioned limitations in the treatment ofbrain cancer, and particularly brain tumors, by providing a composition that comprises aprodrug of an Auger emitting nucleoside anchored to a nanoparticle via hydrophobic interactions. In one main aspect, the present disclosure provides a radioactive compositioncomprising a nanoparticle for use in a method of treatment, prevention, or alleviation ofbrain cancer, wherein the nanoparticle comprises: a. a hydrophobic part, andb. a radioactive agent of formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. The examples demonstrate that compositions according to the present invention are located in the intracerebral compartment for longer periods of time compared to free Auger electron-emitting nucleosides. In addition, it is demonstrated that the compositionreleases the active Auger-emitting nucleoside in the presence of endogenous brain P6655PC00 4esterases and that the Auger electron-emitting nucleoside is released over anappropriate period of time, thereby allowing for efficent incorporation of the Augerelectron-emitting nucleoside into the DNA of cancer cells. Brain delivery of radioactive nucleoside analogues, such as [125I]IUdR or [123I]IUdRpresents the unique challenge of requiring a balance between the rate of drug releaseand the radioactive decay of the radioisotope. If the drug is released too quickly, it willbe washed out from the brain before it can be incorporated into the tumor cell’s DNA. Ifthe drug releases too slowly, excessive radioactive decay occurs before the drug can be incorporated into the tumor cell’s DNA. In both cases, the outcome of therapy is reducedor sub-optimal. The need for this balance comes in stark contrast to other modalities oftherapy not involving radioactive nucleoside analogues.For therapy of brain cancer with radioactive IUdR analogs, the optimal rate of release isassessed to be achieving substantial or full release within 1 to 5 days.The present inventors, have demonstrated that liposomal formulations according to thepresent disclosure with Auger emitting IUdR prodrugs with a lipophilic chain of a certainlength produce a release rate that is optimal for treatment of brain cancer. The resultsconfirm that the formulations of the present disclosure achieve 100% survival in an animal model of glioblastoma and complete reduction of tumor size. Thus, the present invention provides for a new modality of treatment of brain cancer wherein the efficiency of Auger emitting therapeutics is increased, thus allowing for better outcomes and quality of life for patients. Advantages of the invention are for example: ^Extended biological half-life and retention of Auger emitting nucleoside agents inthe brain due to reduced wash-out from the intracerebral compartment. ^Modulate the release of active Auger emitting nucleoside agents controlled bythe release of the prodrug from the nanoparticle and posterior conversion to the active Auger-emitting nucleoside by esterase. ^Tunable release via controlling the hydrophobic properties of the prodrug and thenanoparticle, making the release rate adaptable to different Auger emittingisotopes, therapeutic modalities, or administration strategies. ^Extended window of release of active Auger emitting radionucleosides providesan advantage in dealing with resistant cancer cells or slow-dividing cancer cells P6655PC00 5 with stem-cell like properties and extends the duration of a therapeuticconcentration in the brain, making it more likely that dividing cancer cells are exposed to the Auger emitting nucleoside.^ Possibility to increase the distribution of the active Auger emitting nucleosidewithin the brain tissue. In one aspect, a radioactive agent according to formula (I) is provided: formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety.In another aspect, a radioactive composition comprising a nanoparticle is provided, thenanoparticle comprising:a. a liposome or a polymeric micelle, andb. a radioactive agent of formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. P6655PC00 6 The radioactive agents or radioactive composition according to the present disclosure may be useful in nuclear imaging with different radioisotopes. Or may be useful in radiotherapy of different conditions. In yet another aspect, the present disclosure provides a kit comprising: a. a radioactive agent as described herein;b. a composition comprising a liposome or a polymeric micelle in a secondcontainer; and c. optionally an outer package.In another aspect, the present disclosure provides a method of preparing a composition comprising a radioactive nanoparticle, the method comprising the steps of: a. providing a radioactive agent as described herein;b. providing a composition comprising a liposome or a polymeric micelle in anaqueous medium; c. mixing the radioactive agent and the composition comprising a liposome or apolymeric micelle, thereby obtaining an anchoring of the radioactive agent on the liposome or micelle; and d. purifying the composition in c. from non-anchored radioactive agent, therebyobtaining a composition comprising radioactive nanoparticles. Definitions“Hydrophobic” or “lipophilic” as used herein refers to a property of molecules, groups,moieties, parts of a molecule or parts of a nanoparticle that perform intramolecular non-covalent interactions with other “hydrophobic” or “lipophilic” molecules, groups,moieties, parts of a molecule or parts of a nanoparticle in aqueous environments.Examples of a hydrophobic part of a molecule are moieties or groups derived from fattyacids, non-polar hydrocarbon chains, or groups like cholesterol. Examples ofhydrophobic parts of a nanoparticle are the inner part of the lipid bilayer of a liposome or the core of a polymeric micelle.Hydrophobic or lipophilic groups or moieties associate with each other in aqueousenvironments through the non-polar groups / moieties due to the tendency of water to exclude non-polar groups or moieties. For example, the association of lipids and P6655PC00 7phospholipids to form lipid bilayers, such as in liposomes, or micelles is caused byhydrophobic interaction. Another example is the anchoring of fatty acids, phospholipids, or other lipids, like cholesterol, to liposomes or micelles. “Hydrophilic” refers to the capacity of a molecule, a group, a moiety, a part of a molecule or a part of a nanoparticle to interact with polar solvents, in particular with water or with other polar groups. “Amphiphilic” molecules are molecules having both hydrophobic and hydrophilic groups covalently linked. “Liposome” refers to an artificially formed lipid vesicle having at least one lipid bilayer, said vesicle being in suspension in aqueous media. The lipid bilayer is a membrane made of two layers of lipid amphiphilic molecules, like phospholipids, that self-arrange in aqueous media into a two-layered membrane with the hydrophobic groups pointing towards the center of the membrane and the hydrophilic or polar groups pointing towards the aqueous phase. Liposomes may act as carriers of hydrophobic molecules, or molecules having hydrophobic groups via anchoring of said hydrophobic molecules or groups into the lipid bilayer. “Micelle” as used herein, refers to aggregates of amphiphilic molecules of colloidaldimensions (i.e.1 nm to 1 µm), which exist in equilibrium with the molecules from whichthey are formed. Micelles may act as carriers of hydrophobic molecules, or molecules having hydrophobic groups via anchoring of said hydrophobic molecules or groups to the micelle via hydrophobic interactions. “Radioactive” refers to the property of a nuclide of undergoing spontaneous nuclear transformations with the emission of radiation. Radiation refers to electromagnetic wavesand particles emitted during a nuclear process. Radioactive is used here to describe saidproperty of compositions, compounds, or agents comprising radioactive nuclei.“Radioisotope” is a radioactive isotope of a specified element.“Auger electron-emitting radioisotopes” are radioisotopes that emit Auger electronsduring their radioactive decay. Auger electrons differ from other forms of radiationtherapy because the electrons emitted in the radioactive decay, the Auger electrons, are released in large numbers with low kinetic energy, leading to high linear-energy-transfer P6655PC00 8(LET) effects. Because of their low energy, these electrons exert their damaging effecton cellular structures over a very short nanometer scale range being less than the sizeof a single cell. This very short-range delivery of energy provides a highly targeted therapy because the radiation-emitting nuclide is located inside the cell to cause damageto the genomic DNA. Examples of Auger-emitting radioisotopes include 125I, 123I, 77Br,and 99mTc, 111In, and 67Ga among others.“Nucleoside” as used herein refers to nitrogen-containing biological compounds thatcontain a nucleobase (e.g. a purine or pyrimidine base, such as adenine, cytosine,guanine, thymine or uracil) covalently bonded to a pentose sugar ribose or deoxyribose.Examples of nucleosides are thymidine, uridine, adenosine, cytidine, or guanosine, or any of their deoxy derivatives, such as 2’-deoxy derivative: deoxythymidine, deoxyuridine, deoxyadenosine, deoxycytidine or deoxyguanosine. As used herein, the term nucleoside may refer to any nucleoside or an analog or derivative thereof formed by chemical modification of a nucleotide. For example, the nucleoside analog or nucleoside derivative may be 5-iodo-2’-deoxyuridine, also referred to Idoxuridine, or IUdR, according to the formula below: . The term “hydrocarbon” as used herein refers to molecules or parts of molecules consisting of carbon and hydrogen atoms only. A hydrocarbon group or chain may be specified by the number of carbon atoms. For example a C10 hydrocarbon chain meansa molecules or part of a molecule consisting of only of carbon and hydrogen atomshaving 10 total carbon atoms. The hydrocarbon chain may comprise any possible number of double or triple bonds; or any number of branches unless otherwise specified. The term “alkyl” as used herein refers to a linear or branched hydrocarbon moiety. The term "treatment", as used anywhere herein comprises any type of therapy, which aims at terminating, preventing, ameliorating and / or reducing the susceptibility to a P6655PC00 9 clinical condition as described herein. Thus, "treatment," "treating," and the like, as used herein, refer to obtaining a desired pharmacologic and / or physiologic effect, covering any treatment of a pathological and / or clinical condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. That is, "treatment" includes (1) preventing the disorder or clinical condition from occurring or recurring in a subject, (2) inhibiting the disorder or clinical condition, such as arresting its development,(3) stopping or terminating the disorder or clinical condition or at least symptomsassociated therewith, so that the host no longer suffers from the disorder or clinical condition or its symptoms, such as causing regression of the disorder or clinical condition or its symptoms, for example, by restoring or repairing a lost, missing or defectivefunction, or stimulating an inefficient process, or (4) relieving, alleviating, or amelioratingthe disorder or clinical condition, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and / or immune deficiency. The terms "ameliorate", "ameliorating" and ”amelioration", are also used separately herein to refer to a reduction of the severity of the occurrence of symptoms or characteristics of a disorder or clinical condition. Glioblastoma exists as a “classical” subtype, a “proneural” subtype and a “mesenchymal” subtype. Most tumors of the classical subtype have extra copies of the epidermal growth factor receptor (EGFR) gene, and have elevated expression of epidermal growth factor receptor (EGFR), while the TP53 gene, which is often mutated in glioblastoma, is rarely mutated in this subtype. The proneural subtype often has high rates of alterations in the TP53 gene, and the PDGFRA gene, which encodes a-type platelet-derived growth factor receptor, and the IDHl gene, which encodes isocitrate dehydrogenase-1. The mesenchymal subtype has mutations in the NF1 gene, which encodes neurofibromatosis type 1. Many other genetic alterations have been identified in glioblastoma, and many are clustered in three pathways involving P53, RB, and PI3K / AKT. Another important alteration is methylation of the DNA repair enzyme MGMT. P6655PC00 10 Glioblastomas usually form in the cerebral white matter, grow quickly, and can become very large before producing symptoms. Some glioblastomas form more slowly followingde-differentiation of low graded gliomas. These are called secondary Glioblastomas. Thetumor may extend into the meninges or ventricular wall, leading to high protein content in the cerebrospinal fluid (CSF) (> 100 mg / dL), as well as an occasional pleocytosis of 10 to 100 cells, mostly lymphocytes. Rarely, malignant cells carried in the CSF may spread to the spinal cord or cause meningeal gliomatosis. However, metastasis of glioblastomas beyond the central nervous system is very unusual. About half of the glioblastomas occupy more than one lobe of a hemisphere or are bilateral. Tumors of this type usually arise from the cerebrum and may rarely exhibit the classic infiltration across the corpus callosum, producing a butterfly (bilateral) glioma. The tumor may take on a variety of appearances, depending on the amount of haemorrhage, necrosis, or its age. Contrast-enhanced T1-weighted MRI (magnetic resonance imaging) will usually show an inhomogeneous mass with central necrosis and a variable ring of enhancement surrounded by edema. Mass effect from the tumor and edema may compress the ventricles and cause hydrocephalus. The molecular weight average of a polymer as used herein may refer to the numberaverage molecular weight (Mn) or the weight average molecular weight (Mw).The term “natural abundance" as used herein refers to e.g. compounds or substances having their atoms in an isotopic abundance as naturally found in nature. Hence, compounds or substances that are not of natural abundance have an isotopic abundance of an isotope of an element different from the natural abundance and are usually not found in nature and are not naturally occurring substances. The “isotopic abundance”refers to the relative number of atoms of a particular isotope of an element relative to allisotopes of the element in a compound or substance. P6655PC00 11 Description of Drawings Figure 1: Drug loading efficiency (dl%) of [125I]IUdR prodrugs into liposomes. (a) Drugloading efficiency of [125I]IUdR-C4, -C8, -C12 and -C18, pre-and post-insertion intoliposomes, and post-insertion was conducted at 35 °C with incubation for 12 hours. (b-c) Drug loading efficiency of [125I]IUdR-C12 and -C18, by post-insertion into liposomes atdifferent temperatures and incubation time. Data is given as mean ± SD.Figure 2: Release of [125I]IUdR from [125I]IUdR prodrug-loaded liposomes using esterase.A) [125I]IUdR-C12 B) [125I]IUdR-C18, C) [125I]IUdR-C8, D) [125I]IUdR-C16.Figure 3. A, B, C) Release of [125I]IUdR from [125I]IUdR prodrug-loaded liposomes usingrat brain homogenates. D) Decomposition of free [125I]IUdR.Figure 4: Release of [125I]IUdR from liposomes loaded with [125I]IUdR-C18 prodrug in atwo-compartment experiment.Figure 5. Release of [125I]IUdR from liposomes loaded with [125I]IUdR-C22 and [125I]IUdR-chol in the presence of esterase. Figure 6. Cell viability analysis based on CellTiter-Blue assay in LN229 cell line. Viabilityof cells treated with [125I]IUdR-C18 loaded liposomes ([125I]IUdR-C18-LIPs), free[125I]IUdR-C18 prodrug (with or without esterase, E), and [125I]IUdR. Radioactivity rangefrom 0.23 - 30 kBq / mL. "E" represents esterase, "LIPS" stands for liposomes.Figure 7. Colony formation of LN229 cells after exposure to [125I]IUdR, [125I]IUdR-C18 prodrug-loaded liposomes in the presence of esterase and free iodine-125. Quantification of the results shown in (a), SF represents survival factors. Data are presented as mean±SD (n=3).Figure 8. In vitro and in vivo DNA incorporation of [125I]IUdR released from [125I]IUdR-C18-LIPs. (a) DNA incorporation of [125I]IUdR released from [125I]IUdR-C18-LIPs in LN229cells after 4, 7, and 24 hours of incubation. Figure 9. A) DNA incorporation in tumor tissue of mice bearing orthotopic glioblastomas(U87 cells) that were injected with either [125I]IUdR-C18-LIPs or a non-releasing “mock”and euthanized 2 or 5 days post-injection. Data was represented in terms of specificactivities (Bq) or the percentage of incorporation (IP%), or both. B) SPECT / CT scan of P6655PC00 12 [125I]IUdR-C18-LIPs in tumor-bearing mice after 1, 48 and 120 h post-injection. Left: 1 h, middle: 48 h and right: 120 h.Figure 10. Biodistribution determined ex vivo in glioblastoma-bearing athymic nude ratsafter 6 h (n = 4) and 24 h (n = 4) post-injection of [125I]IUdR-C18-LIPs in the righthemisphere. Data are represented as activity (Bq) per gram of organ (left, A / g) andpercentage of injected activity per gram of organ (right, %IA / g). Brain: left hemisphere,right hemisphere (tumor-bearing hemisphere) and cerebellum. Two-tailed pairedStudent’s t-test P-values indicate statistical significance (*P<0.05, **P<0.01,***P<0.001). Error bars shown are SD.Figure 11. A) Loading efficiency (dl%) of prodrugs [125I]IUdR-C8, [125I]IUdR-C12 and[125I]IUdR-C18 on polymeric micelles. B) Influence of different DMF:H2O ratios (1:1, 1:2,1:5 and 1:10) on the size of PMs during PM preparation at two distinct polymer concentrations: 5 mg / mL and 10 mg / mL.Figure 12. Esterase-mediated release of [125I]IUdR from polymeric micelles loaded with[125I]IUdR prodrugs: A) [125I]IUdR-C8-PMs, B) [125I]IUdR-C12-PMs and C) [125I]IUdR-C18-PMs. Esterase concentration in all release studies was 1 U / mL. The termination of the release study was either when [125I]IUdR is completely released or after 336 hours (14 days). Error bars shown are SD, n=3.Figure 13. Viability of cells treated with polymeric micelles loaded with [125I]IUdR-C18([125I]IUdR-C18-PMs), [125I]IUdR-C18prodrug (with or with out esterase), and free [125I]IUdR based on CellTiter-Blue assay in LN229 cell line. Activity ranging from 0.115 – 3.8 kBq / mL. ”e” represents esterase, with the concentration of 0.1U / mL. Two-tailedpaired Student’s t-test P-values indicate statistical significance (*P<0.05, **P<0.01,***P<0.001, ****P<0.0001). Error bars shown are SD. The groups have been markedwith numbers (1, 2, 3, 4 and 5) to ease interpretation. Figure 14. In vitro DNA incorporation in cells treated with: polymeric micelles loaded with[125I]IUdR-C18 ([125I]IUdR-C18-PMs) and esterase (0.1U / mL) or free [125I]IUdR after 1 or4 hours of incubation. Data was represented in terms of the percentage of incorporation(IP%). Two-tailed paired Student’s t-test P-values indicate statistical significance(**P<0.01, ***P<0.001, ****P<0.001). Error bars shown are SD. P6655PC00 13 Figure 15. A) Survival plot upon treatment of a pre-clinical rat model of brain tumor with[125I]IUdR-C18-LIPs. Control group had a median survival of 87 days (range 68-107 days)while treated group demonstrated 100% survival over a period of 180 days. B) Levels ofwhite blood cells (WBC), haemoglobin (HGB), platelets (PLT), plasma creatinine (Cr)and plasma blood urea nitrogen (BUN) determined in before treatment, early responseand late response groups. Detailed description A composition for use In one aspect, the present disclosure provides a radioactive composition for use in a in a method of treatment, prevention or alleviation of brain cancer, wherein the nanoparticle comprises: a. a hydrophobic part, andb. a radioactive agent of formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety.The radioactive agent according to formula (I) acts as a prodrug of a nucleoside analogof 2’-deoxyuridine, having an ester group formed from the 5’-OH group of 2’-deoxyuridine. When the 5’-OH group is in the form of an ester, incorporation into the DNA of cells is not possible. Thus, by hydrolysis of said ester a free Auger electron-emitting nucleoside analog of 2’-deoxyuridine is released, as shown below. The P6655PC00 14 hydrolysis of the 5’ ester may be catalyzed by an enzyme, in particular an esterase. The free 2’-deoxyuridine may then be incorporated into the DNA of cells and this increases its therapeutic efficacy. The radioactive agent is anchored onto the nanoparticle via hydrophobic interaction, thus the nanoparticle acts as a carrier of the radioactive agent. RadionuclidesThe radioactive agent according to formula (I) comprises RA which comprises aradioisotope of a halogen. For example, in one embodiment, RA comprises or consistsof 123I, 124I or 125I, 77Br, 76Br, 80mBr, 80Br, 126I, 131I, 18F, or 211At. In one embodiment, RA is aradioisotope of iodine. In one embodiment, RAcomprises or consist of an Auger electron-emitting radioisotope.In one embodiment, RA is an Auger electron-emitting radioisotope of a halogen, such asan Auger electron-emitting radioisotope of I, Br, Cl, F or At. In one embodiment, RA isan Auger electron-emitting radioisotope of I or Br. In one embodiment, RAis an Auger electron-emitting radioisotope of I. In one embodiment, RAis an Auger electron-emittingradioisotope of Br. In one embodiment, RA is 123I, 125I, or 77Br.In one embodiment, RA is 123I, 124I or 125I. In one embodiment, RA is 123I, or 125I. In oneembodiment, RA is 123I. In one embodiment, RA is 125I. In one embodiment, RA is 77Br.In one embodiment, RAis not of natural abundance. In one embodiment, all elements of formula (I) are of natural abundance except for RAwhich is not of natural abundance. P6655PC00 15In one embodiment, the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 2% or more, 5% or more, 10% or more, 20% or more, 50% or more, 75% or more, 90% or more, or 95% or more.In one embodiment, the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95% or more. In one embodiment, the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95%, 96%, 97%, 98%, 99%, or 100%.Lipophilic moietyThe radioactive agent according to formula (I) comprises a lipophilic moiety R whichallows the radioactive agent to anchor and associate with other hydrophobic groups, such as hydrophobic parts of a nanoparticle, via non-covalent hydrophobic interactions. The examples demonstrate that the rate of release of free Auger electron-emitting nucleoside analog is determined by the affinity of the prodrug with the nanoparticle, such as a liposome or a polymeric micelle. The affinity of the prodrug to the nanoparticle is defined by the nature of the lipophilic moiety (R) of the radioactive agent and the nature of the hydrophobic part of the nanoparticle. Hence, the compositions according to the present disclosure have the advantage to be adapted to provide specific rate of release by changing the hydrophobic group of the radioactive agent and the composition of the nanoparticle. Thus, in one embodiment, R comprises or consists of a lipophilic moiety able to anchor to a hydrophobic part of a nanoparticle, such as the lipid bilayer of a liposome or to the hydrophobic part of a polymeric micelle. In one embodiment, R comprises or consists of a lipid. In one embodiment, R comprises or consists of: a. a C3-C21 hydrocarbon chain,b. two hydrocarbon chains, wherein each of said hydrocarbon chains isindependently selected from a C3-C21 hydrocarbon chain, or c. a cholesteryl group, or a derivative thereof. P6655PC00 16 In one embodiment, R comprises a C3-C21 hydrocarbon chain. In one embodiment, R comprises or consists of two hydrocarbon chains, wherein each of said hydrocarbonchains is independently selected from a C3-C21 hydrocarbon chain. In one embodiment,R comprises or consists of a cholesteryl group, or a derivative thereof. In one embodiment, R comprises is according to formula (Ia) or formula (Ib): formula (Ia) formula (Ib) wherein, R1a, R1band R1care each independently selected of a C3-C21hydrocarbon chain, and X is a linker. In one embodiment, in formula (Ia), X is selected from: a bond, or a C1-C6hydrocarbon chain, such as a C1, C2, C3, C4, C5or C6hydrocarbon chain, wherein optionally one or more methylene groups is independently replaced by -O-, -NH-, carbonyl, an amide or a carbamate. In one embodiment, in formula (Ib) X is selected from: a C1-C6hydrocarbon chain, such as a C1, C2, C3, C4, C5or C6hydrocarbon chain, wherein optionally one or more methylene groups is independently replaced by -O-, -NH-, carbonyl, an amide or a carbamate. In one embodiment, each of said hydrocarbon chain is a linear hydrocarbon chain. In one embodiment, each of said hydrocarbon chain comprises 0, 1, 2 or 3 double bonds, such as 0, 1, or 2 double bonds, such as 0 or 1 double bonds, such as 0 double bonds or 1 double bond. In one embodiment, R1ais: P6655PC00 17 Suitable lipophilic groups for anchoring to a nanoparticle with a hydrophobic part will be known to someone of skill in the art. For example, groups comprising or consisting ofthe aliphatic chains of common fatty acids.In one embodiment, each instance of R1a, R1band R1cis independently comprising or consisting of the carbonyl radical of any one of the carboxylic acids defined in Table A.For example, in one embodiment, each instance of R1a, R1b and R1c is comprising orconsisting of the carbonyl radical of any one of the following carboxylic acids : decenoicacid, undecenoic acid, dodecenoic acid, tridecenoic acid, tetradecenoic acid, pentadecenoic acid, hexadecenoic acid , heptadecenoic acid, octadecenoic acid, nonadecenoic acid, eicosenoic acid, heneicosenoic acid, docosenoic acid alpha- linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma- linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid.In one embodiment, each instance of R1a, R1b, and R1c is independently comprising orconsisting of the aliphatic chain radical of any one of the following carboxylic acidsdefined in Table A. For example, in one embodiment, each instance of R1a, R1b and R1cis comprising or consisting of the aliphatic chain radical of any one of the carboxylic acids: decenoic acid, undecenoic acid, dodecenoic acid, tridecenoic acid, tetradecenoic acid, pentadecenoic acid, hexadecenoic acid , heptadecenoic acid, octadecenoic acid, nonadecenoic acid, eicosenoic acid, heneicosenoic acid, docosenoic acid alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic P6655PC00 18 acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid. In one embodiment, each instance of R1a, R1bor R1cis each independently selected from: -(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3, -(CH2)8-CH3, nonen-1-yl, decen-1-yl, undecen-1-yl, dodecen-1-yl, tridecen-1-yl, tetradecen-1-yl , pentadecen-1-yl, hexadecen-1-yl, heptadecen-1-yl, octadecen-1-yl, nonadecen-1-yl, eicosen-1-yl, heneicosen-1-yl, (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl, (4Z,7Z,10Z,13Z,16Z)-nonadeca-3,7,10,13,16-pentaen-1-yl, (3Z,6Z,9Z,12Z,15Z,18Z)-heneicosa-3,6,9,12,15,18-hexaen-1-yl (8Z,11Z)-heptadeca-8,11-dien-1-yl (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl (7Z,10Z,13Z)-nonadeca-7,10,13-trien-1-yl (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraen-1-yl nonadecadien-1-yl, nonadecatrien-1-yl, nonadecatetraen-1-yl, heneicosapentaen-1-yl, and heneiocsahexaen-1-yl. Table A. Unsaturated fatty acids Unsaturated fatty acidsC10:1 General formula Decenoic acid k is any integerO from 0 to 7 HO (CH2)k CH=CH (CH2)7-k HCarbonyl radical Decenecarbon-1-ylO (CH2)k CH=CH (CH2)7-k HAliphatic chain Nonen-1-yl radical(CH2)k CH=CH (CH2)7-k H P6655PC00 19C11:1 General formula Undecenoic acid k is any integerO from 0 to 8 HO (CH2)k CH=CH (CH2)8-k HCarbonyl radical undecenecarbon-1-ylO (CH2)k CH=CH (CH2)8-k HAliphatic chain decen-1-yl radical(CH2)k CH=CH (CH2)8-k HC12:1 General formula dodecenoic acid k is any integerO from 0 to 9 HO (CH2)k CH=CH (CH2)9-k HCarbonyl radical dodecenecarbon-1-ylO (CH2)k CH=CH (CH2)9-k HAliphatic chain undecen-1-yl radical(CH2)k CH=CH (CH2)9-k HC13:1 General formula tridecenoic acid k is any integerO from 0 to 10 HO (CH2)k CH=CH (CH2)10-k HCarbonyl radical tridecenecarbon-1-ylO (CH2)k CH=CH (CH2)10-k HAliphatic chain dodecen-1-yl radical(CH2)k CH=CH (CH2)10-k HC14:1 General formula tetradecenoic acid k is any integerO from 0 to 11 HO (CH2)k CH=CH (CH2)11-k H P6655PC00 20 Carbonyl radical tetradecenecarbon-1-ylO (CH2)k CH=CH (CH2)11-k HAliphatic chain tridecen-1-yl radical(CH2)k CH=CH (CH2)11-k HC15:1 General formula pentadecenoic acid k is any integerO from 0 to 12 HO (CH2)k CH=CH (CH2)12-k HCarbonyl radical pentadecenecarbon-1-ylO (CH2)k CH=CH (CH2)12-k HAliphatic chain tetradecen-1-yl radical(CH2)k CH=CH (CH2)12-k HC16:1 General formula hexadecenoic acid k is any integerO from 0 to 13 HO (CH2)k CH=CH (CH2)13-k HCarbonyl radical hexadecenecarbon-1-ylO (CH2)k CH=CH (CH2)13-k HAliphatic chain pentadecen-1-yl radical(CH2)k CH=CH (CH2)13-k HC17:1 General formula heptadecenoic acid k is any integerO from 0 to 14 HO (CH2)k CH=CH (CH2)14-k HCarbonyl radical heptadecenecarbon-1-ylO (CH2)k CH=CH (CH2)14-k HAliphatic chain hexadecen-1-yl radical(CH2)k CH=CH (CH2)14-k H P6655PC00 21C18:1 General formula octadecenoic acid k is any integerO from 0 to 15 HO (CH2)k CH=CH (CH2)15-k HCarbonyl radical octadecenecarbon-1-ylO (CH2)k CH=CH (CH2)15-k HAliphatic chain heptadecen-1-yl radical(CH2)k CH=CH (CH2)15-k HC19:1 General formula nonadecenoic acid k is any integerO from 0 to 16 HO (CH2)k CH=CH (CH2)16-k HCarbonyl radical nonadecenecarbon-1-ylO (CH2)k CH=CH (CH2)16-k HAliphatic chain octadecen-1-yl radical(CH2)k CH=CH (CH2)16-k HC20:1 General formula eicosenoic acid k is any integerO from 0 to 17 HO (CH2)k CH=CH (CH2)17-k HCarbonyl radical eicosenecarbon-1-ylO (CH2)k CH=CH (CH2)17-k HAliphatic chain nonadecen-1-yl radical(CH2)k CH=CH (CH2)17-k H P6655PC00 22C21:1 General formula Heneicosenoic acid k is any integerO from 0 to 18 HO (CH2)k CH=CH (CH2)18-k HCarbonyl radical heneicosenecarbon-1-ylO (CH2)k CH=CH (CH2)18-k HAliphatic chain eicosen-1-yl radical(CH2)k CH=CH (CH2)18-k HC22:1 General formula Docosenoic acid k is any integerO from 0 to 19 HO (CH2)k CH=CH (CH2)19-k HCarbonyl radical docosenecarbon-1-ylO (CH2)k CH=CH (CH2)19-k HAliphatic chain heneicosen-1-yl radical(CH2)k CH=CH (CH2)19-k HALA General formula alpha-linolenic acid,(9Z,12Z,15Z)-Octadeca-9,12,15-trienoic acid Carbonyl radical (9Z,12Z,15Z)-Octadeca-9,12,15-triencarbon-1-ylAliphatic chain (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl radicalEPA General formula Eicosapentaenoic acid,(5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17- pentaenoic acid Carbonyl radical (5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaencarbon-1-yl Aliphatic chain (4Z,7Z,10Z,13Z,16Z)-nonadeca-3,7,10,13,16- radical pentaen-1-yl P6655PC00 23DHA General formula docosahexaenoic acid,(4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19- hexaenoic acid Carbonyl radical (4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19-hexaencarbon-1-yl Aliphatic chain (3Z,6Z,9Z,12Z,15Z,18Z)-heneicosa-3,6,9,12,15,18- radical hexaen-1-ylLA General formula Linoleic acid,(9Z,12Z)-Octadeca-9,12-dienoic acid Carbonyl radical (9Z,12Z)-Octadeca-9,12-diencarbon-1-ylAliphatic chain (8Z,11Z)-heptadeca-8,11-dien-1-yl radicalGLA General formula Gamma-linolenic acid,(6Z,9Z,12Z)-Octadeca-6,9,12-trienoic acid Carbonyl radical (6Z,9Z,12Z)-Octadeca-6,9,12-triencarbon-1-ylAliphatic chain (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl radicalDLGA General formula Dihomo-gamma-linolenic acid,(8Z,11Z,14Z)-Icosa-8,11,14-trienoic acid Carbonyl radical (8Z,11Z,14Z)-Icosa-8,11,14-triencarbon-1-ylAliphatic chain (7Z,10Z,13Z)-nonadeca-7,10,13-trien-1-yl radicalAA General formula Arachidonic acid,(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenoic acid Carbonyl radical (5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraencarbon-1-yl P6655PC00 24 Aliphatic chain (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraen-1-yl radicalC20:2 General formula Eicosadienoic acid, e.g. (11Z ,14Z)-icosa- 11,14-dienoic acid Carbonyl radical Eicosadiencarbon-1-yl, e.g. (11Z,14Z)-icosa-11,14-diencarbon-1-yl Aliphatic chainNonadecadien-1-yl, e.g. (10Z,13Z)-nonadeca-radical 10,13-dien-1-ylC20:3 General formula Eicosatrienoic acid,Carbonyl radical Eicosatriencarbon-1-ylAliphatic chain Nonadecatrien-1-yl radicalC20:4 General formula Eicosatetraenoic acidCarbonyl radical Eiosatetraencarbon1-ylAliphatic chain Nonadecatetraen-1-yl radicalC22:5 Name Docosapentaenoic acidCarbonyl radical Docosapentaencarbon-1-ylAliphatic chain Heneicosapentaen-1-yl radicalC22:6 Name Docosahexaenoic acidCarbonyl radical Docosahexaencarbon-1-ylAliphatic chain Heneicosapentaen-1-yl radicalIn one embodiment, R consists of a C8-C21, a C8-C20, a C9-C20, a C10-C20, a C11-C20, a C9-C19, a C10-C19, a C11-C19, a C12-C19, a C13-C19, a C14-C19 or a C15-C19 hydrocarbon chain. P6655PC00 25In one embodiment, R consists of a C11, C12, C13, C14, C15, C16, C17, C18, C19, C20 or C21hydrocarbon chain. In one embodiment, R consists of a C13-C17 hydrocarbon chain. In one embodiment, R consists of a C14-C17 hydrocarbon chain. In one embodiment, R consists of a C15-C17 hydrocarbon chain. In one embodiment, R consists of a C13-C15 hydrocarbon chain. In one embodiment, R consists of a C13 hydrocarbon chain. In one embodiment, R consists of a C14 hydrocarbon chain. In one embodiment, R consists of a C15 hydrocarbon chain. In one embodiment, R consists of a C16hydrocarbon chain. In one embodiment, R consists of a C17hydrocarbon chain. In one embodiment, RAconsists of123I and R consists of a C13-C15hydrocarbon chain. In one embodiment, R consists of a saturated hydrocarbon chain. In one embodiment, R does not comprise a double bond. In one embodiment, R comprises 1, 2 or 3 double bonds. In one embodiment, R comprises 1, or 2 double bonds. In one embodiment, Rcomprises 1 double bond.The configuration of each instance of a double bond may be cis or trans. Thus, in one embodiment, each instance of a double bond has a configuration independently selectedfrom cis or trans. In one embodiment, each instance of a double bond has a cisconfiguration.In one embodiment, R consists of a linear hydrocarbon chain. In one embodiment, Rdoes not comprise a branch. In one embodiment, R consists of a C8-C20, a C9-C20, a C10-C20, a C11-C20, a C9-C19, a C10-C19, a C11-C19, a C12-C19, a C13-C19, a C14-C19, a C15-C19, a C11-C18, a C12-C18, a C13- C18, a C14-C18, a C15-C18, a C11-C17, a C12-C17, a C13-C17, a C14-C17, or a C15-C17linear hydrocarbon chain.In one embodiment, R consists of a C13-C17, C14-C17, C15-C17, C13-C16, or a C13-C15, linearhydrocarbon chain.In one embodiment, R is selected from any one of:-(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3, -(CH2)8-CH3, nonen-1-yl, decen-1-yl, undecen-1-yl, dodecen-1-yl, tridecen-1-yl, P6655PC00 26 tetradecen-1-yl , pentadecen-1-yl, hexadecen-1-yl, heptadecen-1-yl, octadecen-1-yl, nonadecen-1-yl, eicosen-1-yl, heneicosen-1-yl, (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl, (4Z,7Z,10Z,13Z,16Z)-nonadeca-3,7,10,13,16-pentaen-1-yl, (3Z,6Z,9Z,12Z,15Z,18Z)-heneicosa-3,6,9,12,15,18-hexaen-1-yl (8Z,11Z)-heptadeca-8,11-dien-1-yl (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl (7Z,10Z,13Z)-nonadeca-7,10,13-trien-1-yl (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraen-1-yl nonadecadien-1-yl, nonadecatrien-1-yl, nonadecatetraen-1-yl, heneicosapentaen-1-yl, and heneiocsahexaen-1-yl. In one embodiment, R is selected form the group consisting of: -(CH2)8-19-CH3, -(CH2)10-19-CH3, -(CH2)10-18-CH3, -(CH2)10-17-CH3, and -(CH2)10-16-CH3. In one embodiment, R is selected from any one of the group consisting of: -(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3, and -(CH2)8-CH3. In one embodiment, R is selected from any one of the group consisting of: -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3.In one embodiment, R is -(CH2)12-16-CH3In one embodiment, R is selected from any one of the group consisting of: -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3. In one embodiment, R is –(CH2)16-CH3. In one embodiment, R is –(CH2)15-CH3. In one embodiment, R is –(CH2)14-CH3. In one embodiment, R is –(CH2)13-CH3. In one embodiment, R is –(CH2)12-CH3.In one embodiment, RA is 123I, 124I or 125I; and R consists of C11-C20 linear hydrocarbon. P6655PC00 27In one embodiment, RA is 123I, 124I or 125I; and R consists of -(CH2)8-19-CH3, suchas -(CH2)10-19-CH3, such as -(CH2)10-18-CH3, such as -(CH2)10-17-CH3, such as -(CH2)10-16-CH3.In one embodiment, RA is 123I and R consists of -(CH2)8-19-CH3, such as -(CH2)10-19-CH3,such as -(CH2)10-18-CH3, such as -(CH2)10-17-CH3, such as -(CH2)10-16-CH3. In oneembodiment, RA is 123I and R consists of –(CH2)16-CH3.In one embodiment, RA is 123I and R consists of -(CH2)12-16-CH3, such as -(CH2)14-16-CH3.In one embodiment, RA is 123I and R consists of -(CH2)14-CH3. In one embodiment, RA is123I and R consists of -(CH2)12-CH3. In one embodiment, RA is 123I and R consistsof -(CH2)16-CH3.In one embodiment, RA is 123I and R consists of -(CH2)12-14-CH3, such as -(CH2)12-CH3or -(CH2)14-CH3.In one embodiment, RA is 125I and R consists of -(CH2)8-19-CH3, such as -(CH2)10-19-CH3,such as -(CH2)10-18-CH3, such as -(CH2)10-17-CH3, such as -(CH2)10-16-CH3.In oneembodiment, RA is 125I and R consists of –(CH2)16-CH3.In one embodiment, RA is 125I and R consists of -(CH2)12-16-CH3, such as -(CH2)14-16-CH3.In one embodiment, RA is 125I and R consists of -(CH2)14-CH3. In one embodiment, RA is125I and R consists of -(CH2)12-CH3. In one embodiment, RA is 125I and R consistsof -(CH2)14-CH3. n one embodiment, RA is 125I and R consists of -(CH2)16-CH3.In one embodiment, the radioactive agent is selected from any one of the compounds shown in Table B, or a pharmaceutically acceptable salt thereof. Table B. O O 12 123I 15 O3I NH O N ON ONH HO O OHHO OHHOO P6655PC00 28 P6655PC00 29 The examples demonstrate that prodrugs with hydrocarbon chains of certain length produce a favorable drug release rates for treatment of brain cancer. P6655PC00 30 P6655PC00 31 As shown in the Examples, the rate of release of free Auger electron-emitting nucleosideanalog is determined by the affinity of the radioactive agent with the nanoparticle, suchas a liposome or a polymeric micelle. The affinity of the radioactive agent with a liposomeor a polymeric micelle can be controlled by the hydrophobicity of the radioactive agent. As it will be known to someone of skill in the art, hydrophobicity of a compound may be determined by suitable methods, such as determination of a partition coefficient or distribution coefficient between an aqueous phase and a water-immiscible organic phase. For example, hydrophobicity can be measured by the octanol-water partition coefficient (logP) or the octanol-water distribution coefficient (logD). The logD measures the distribution of a compound between octanol and water phases in a pH sensitive manner. Thus, in one embodiment, the radioactive agent has a distribution coefficient (logD) from 2.0 to 13.0, such as from 3.0 to 10.0, such as 4.0 to 7.0 at room temperature, asmeasured e.g. by partitioning method in octanol-buffer at pH 7.4.Another parameter used for characterization of hydrophobicity is the lipid-permeability coefficient (log M) which may be determined using different thermodynamic calculation and computer assisted methods, also using quantum mechanical computational methods. For example, logM may be determined by a quantum chemical software package, such as Turbomole 7.4.1 at BP-TZVP-FINE level. Liposome In one embodiment, the radioactive composition for use in a method of treatment, prevention, or alleviation of brain cancer as described herein comprises a nanoparticle, wherein said nanoparticle comprises a liposome, said liposome comprising one or more lipid bilayer(s). A lipid bilayer is an example of a hydrophobic part of a nanoparticle able to perform non-covalent interactions with other hydrophobic moieties or groups. In one embodiment the liposome comprises a single lipid bilayer, also referred to as an unilamellar lipid bilayer. In such instance, the liposome can be classified as an unilamllear vesicle (UV), which according to its size may be defined as a small unilamellar vesicle (SUV) or a large multilamellar vesicle (LUV). In one embodiment, the liposome is a SUV or a LUV. P6655PC00 32 In one embodiment, the liposome comprises more than one lipid bilayer. In such instance, the liposome can be classified as a multilamellar vesicle (MLV) or a multi vesicular vesicle (MVV). In one embodiment, the liposome is selected from any form of unilamellar, multilamellar or multivesicular vesicle. In one embodiment, the composition for use as described herein comprises a combination of more than one type of liposome, such any combination of SUV, LUV, MLV or MVV. Liposomes are well known entities in the field of drug delivery and formulation, and many suitable forms of preparation will be known to the skilled artisan. Within the present invention, any suitable form for preparation and purification of liposomes is contemplated, such as thin-film hydration, ethanol / ether injection, reverse phase evaporation, detergent depletion and can also be assisted by microfluidic methods. The size and type of liposomes (SUV, LUV, MLV or MVV) can be controlled by the formation method and / or by extrusion through membranes of different mesh size and under different conditions as will be known to the skilled artisan. The lipid bilayer of liposomes commonly comprises phospholipids. Thus, in one embodiment, the lipid bilayer of the liposome comprises a phospholipid. Other lipids may be used to impart desirable properties to the lipid bilayer such as sterols, which are commonly present in the lipid bilayer of cells. Thus, in one embodiment, the lipid bilayer of the liposome comprises: i. a phospholipid; and ii. cholesterol, ergosterol, or sitosterol. Many suitable phospholipids for the preparation of liposomes are known in the art, and may be used for the preparation of nanoparticles according to the present disclosure. In one embodiment, the phospholipid is selected from the group consisting of: a fatty acid ester derivative of sn-glycero-3-phosphocholine, a fatty acid ester derivative of sn-glycero-3-phosphoserine, a fatty acid ester derivative of sn-glycero-3- phoshpoglycerol, and a fatty acid derivative of sn-glycero-3-phosphoinositol. P6655PC00 33 In one embodiment, the phospholipid is according to any one of formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIf), and (IIg): formula formula (IIg), or a salt thereof,wherein each of RPa, RPa’, RPb, RPb’, RPc, RPc’, RPd, RPd’, RPe, RPe’, RPf, RPf’, RPg, and RPg’are independently the corresponding group resulting from forming an ester of a C12-C22fatty acid with the corresponding O atom in formulas (IIa)-(IIg). In one embodiment, the phospholipid is selected from any one of the phospholipidsdescribed in Table C or any mixture thereof.Table C. Phospholipids hydrogenated soy phosphatidylcholine (HSPC) bis(monoacyglycero)phosphate (BSMP) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC)1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) P6655PC00 34 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) 1,2-stearoyl-sn-glycero-3-phosphoglycerol (DSPG) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) 1,2-Dilauroyl-sn-glycero-3-phosphoglycerol (DLPG) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) 1,2-stearoyl-sn-glycero-3-phosphate (DSPA) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) 1,2-stearoyl-sn-glycero-3-phospho-L-serine (DSPS) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DMPI) 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DPPI), 1,2-stearoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DSPI) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (POPI) 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI) N-palmitoylsphingosylphosphorylcholine (C16SM) N-stearoylsphingosylphosphorylcholine (C18SM) N-oleoylsphingosylphosphorylcholine (C18:1SM) P6655PC00 35 In one embodiment, the lipid comprises a sphingolipid, such as N-palmitoyl-D-erythro- sphingosine (Ceramide 16:0), N-stearoyl-D-erythro-sphingosine (Ceramide 18:0), N- oleoyl-D-erythro-sphingosine (Ceramide 18:1). In addition, other excipients can be used to impart desirable properties to the lipid bilayer or the liposome. For example, addition of amphiphilic derivatives of hydrophilic polymers,or amphiphilic derivatives of oligo- or poly-saccharides, poly-carbohydrates, or poly-(ethylene glycol) can be used to prepare “stealth liposomes” to increase circulation time and reduce interaction with proteins and cells. The skilled artisan is aware of methods to prepare such stealth liposomes, for example, by inclusion of amphiphilic derivatives of poly(ethylene glycol) in the lipid bilayer. In one embodiment, the liposome is a stealth liposome. In one embodiment, the lipid bilayer comprises an amphiphilic derivative of poly(ethylene glycol). In one embodiment, the lipid bilayer comprises: i. a phospholipid, or mixture thereof, as described herein; ii. cholesterol, ergosterol, or sitosterol; and iii. an amphiphilic derivative of poly(ethylene glycol). In one embodiment, the lipid bilayer comprises a phospholipid, cholesterol and anamphiphilic derivative of poly(ethylene glycol).In one embodiment, the amphiphilic derivative of poly(ethylene glycol) comprises apoly(ethylene glycol) moiety of average molecular weight (Mn or Mw) between200-20,000 g / mol covalently linked to a hydrophobic component which allows it to be anchored to a lipid bilayer.In one embodiment, the amphiphilic derivative of poly(ethylene glycol) is according toformula (III): wherein, RPrepresents -H, -CH3 , a C1-C5 alkyl, a C1-C5 hydroxyalkyl, a C1-C5 alkylamine, RXrepresent an optional linker; RHrepresents a hydrophobic group which allows anchoring to a lipid bilayer. P6655PC00 36 In one embodiment, i. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative or RHcomprises a C10-C22 fatty acid ester derivative or a C10-C22 fatty acid amide derivative, or, ii. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative or RHcomprises a phospholipid selected from the group consisting of any one of the phospholipids described in Table C. In one embodiment, the amphiphilic derivative of poly(ethyleneglycol) is any derivative of poly(ethylene glycol) wherein the poly(ethyleneglycol) is covalently linked to: i. a C10-C22fatty acid, optionally via a linker, or ii. any phospholipid as described herein, optionally via a linker; wherein the amphiphilic derivative of poly(ethyleneglycol) has a molecular weight average from 300 to 20,000 g / mol. In one embodiment, the amphiphilic derivative of poly(ethyleneglycol) is DSPE-PEG (CAS nr. 247925-28-6), DMG-PEG (CAS nr. 160743-62-4), DPPE-PEG (CAS nr. 205494-72-0), or DOPE-PEG (CAS nr. 474922-90-2), or a salt thereof, where the amphiphilic derivative of poly(ethyleneglycol) has a molecular weight average from 1000 to 20,000 g / mol. In one embodiment, the lipid bilayer comprises:a. a phospholipid or mixture thereof as described herein;b. cholesterol; andc. the amphiphilic derivative of poly(ethyleneglycol) as described herein;wherein the mass ratio between components a:b:c is: 2-4:0.5-1.5:0.5-1.5, such as about 3:1:1. In one embodiment, the nanoparticle has an average hydrodynamic diameter of: 20 nm to 150 nm, such as an average hydrodynamic diameter of 80 to 120 nm, such as an average hydrodynamic diameter of 80 nm to 90 nm, 90 nm to 100 nm, 100 nm to 110 nm, or 110 nm to 120 nm. In one embodiment the nanoparticle has an averagehydrodynamic diameter of 80 nm, or 90 nm, or 100 nm or 120 nm. In one embodiment,the nanoparticle is a liposome having an average hydrodynamic diameter of 80 to 150 P6655PC00 37 nm, such as 80 nm to 90 nm, 90 nm to 100 nm, 100 nm to 110 nm, 110 nm to 120 nm, 120 nm to 130 nm, 130 nm to 140 nm, or 140 nm to 150 nm average hydrodynamic diameter. In one embodiment, the nanoparticle is a liposome having an average hydrodynamic diameter of 80 to 130 nm. In one embodiment, the nanoparticle is a liposome having an average hydrodynamic diameter of 100 to 130 nm, preferably the nanoparticle is a liposome having an average hydrodynamic diameter of 110 to 130 nm or 120 to 130 nm.The average hydrodynamic diameter may be measured by e.g. light scatteringtechniques such as dynamic light scattering (DLS). In one embodiment, the liposome has a neutral, positive or negative net charge. The netcharge of the liposome can be determined e.g. by Z-potential measurements. In oneembodiment, the liposome has a neutral or negative net charge. Polymeric micelle In one embodiment, the radioactive composition for use in a method of treatment, prevention, or alleviation of brain cancer as described herein comprises a nanoparticle, wherein said nanoparticle comprises a polymeric micelle. A polymeric micelle is an example of a nanoparticle with a hydrophobic part of a nanoparticle able to perform non-covalent interactions with other hydrophobic moieties or groups. In oneembodiment, the radioactive agent as described herein is anchored to the nanoparticlethrough non-covalent hydrophobic interaction with the polymeric micelle. Different methods for the production of polymeric micelles are well-described in the art. For example, direct dissolution in different solvents and posterior solvent evaporation / exchange. Purification of polymeric micelles may be achieved by differenttechniques, e.g. by dialysis. P6655PC00 38 In one embodiment, the polymeric micelle comprises one or more amphiphilic block copolymers. In one embodiment the amphiphilic block copolymer(s) comprises polymer chains with hydrophilic and hydrophobic blocks comprises a polymer chain with a hydrophilic and a hydrophobic block according to: i. (hydrophilic block)-(hydrophobic block), orii. (hydrophilic block)-(hydrophobic block)-(hydrophilic block), oriii. (hydrophobic block)-(hydrophilic block)-(hydrophobic block).In one embodiment, the hydrophobic block is a polymer selected from the group consisting of: poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), poly(glutamic acid) (PGA), poly(aspartic acid) (PAsp), and poly(lactic acid) (PLA). In one embodiment, the hydrophilic block is selected from the group consisting of: poly(ethylene glycol) and poly(propylene glycol). In one embodiment, the polymeric micelle comprises a single amphiphilic block copolymer selected from the group consisting of: Poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-PLGA), Poly(ethylene glycol)-block-poly(caprolactone) (PEG-PCL), Poly(caprolactone)-block-poly(ethylene glycol)-block-poly(caprolactone) (PCL-PEG-PCL), Poly(ethylene glycol)-block- poly(caprolactone)-block-poly(ethylene glycol) (PEG-PCL-PEG), poly(ethylene glycol)- block-poly(glutamic acid) (PEG-PGA), Poly(ethylene glycol)-block-poly(aspartic acid) (PEG-PAsp), Poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA), Poly(ethylene glycol)-block-poly(caprolactone)-block-poly(ethylene glycol) (PEG-PEL-PEG), Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG- PPG-PEG), and, Poly(ethylene glycol)-block-poly(methylmethacrylate) (PEG-PMMA). In one embodiment, the polymeric micelle comprises any one of the amphiphilic block copolymer described herein, wherein the polymer has an average molecular weight between 2000-50,000 g / mol. In one embodiment, the polymer micelle comprise PEG-PLGA, wherein the PEG blockhas an average molecular weight (Mn or Mw) between 2000 and 10,000 g / mol and thePLGA block has an average molecular weight (Mn or Mw) between 5000 and 20,000g / mol. P6655PC00 39 In one embodiment, the nanoparticle has an average hydrodynamic diameter of: 40 nm to 150 nm, such as an average hydrodynamic diameter of 40 to 100 nm, 40 nm to 80 nm, such as 40 nm to 50 nm, 50 nm to 60 nm, 60 nm to 70 nm, or 70 nm to 80 nm average hydrodynamic diameter. In one embodiment, the nanoparticle has an average such as an average hydrodynamic diameter of 50, 60, 70, or 80 nm, e.g. as measuredby DLS or any other suitable technique known in the art. The average hydrodynamicdiameter may be measured by e.g. light scattering techniques such as dynamic lightscattering (DLS). Treatment of brain cancer The methods, uses, agents, compositions and kits-of-parts provided herein are generally intended for treating and / or ameliorating any type of brain cancer, and in particular any type of brain tumor, or intracerebral neoplasm. This includes all tumors inside the human skull (cranium) or in the central spinal canal. The tumor may originate from the brain itself, but also from lymphatic tissue, blood vessels, the cranial nerves, the brain envelopes (meninges), skull, pituitary gland, or pineal gland. Within the brain itself, the involved cells may be neurons or glial cells (which include astrocytes, oligodendrocytes, and ependymal cells). Brain tumors may also spread from cancers primarily located in other organs (metastatic tumors). Numerous systems exist for grading tumors of the central nervous system (CNS), such as the “2021 World Health Organization (WHO) Classification of Tumor of the CentralNervous System” (WHO CN5). Thus, in one embodiment, the brain cancer is any typeof glioma, glioneuronal tumor or neuronal tumor according to the WHO CN5 classification. In one embodiment, the brain tumor is astrocytoma, glioblastoma, diffuse midline glioma, diffuse hemispheric glioma or diffuse pediatric-type high-grade glioma according to the WHO CN5 classification.In one preferred embodiment, the brain cancer is a brain tumor involving glial cells, andin a preferred embodiment, the methods, uses, agents, compositions and kits-of-partsprovided herein are intended for treating and / or ameliorating a high-grade glioma. In onepreferred embodiment, the high-grade glioma is glioblastoma.Another grading systems in use, is the previous World Health Organization (WHO)grading system for astrocytoma, under which tumors are graded with roman numerals P6655PC00 40from I (least advanced disease - best prognosis) to IV (most advanced disease - worstprognosis). In this grading system, high-grade gliomas are categorized by the World Health Organization (WHO) as grade III and IV gliomas. These tumors are malignant and carry a worse prognosis. Thus, in one embodiment, the brain cancer is a WHO grade III or WHO grade IV according to the classification prior to 2021. Thus, in one embodiment, the methods, uses, agents, compositions and kits-of-parts provided herein are intended for treating and / or ameliorating a high-grade glioma or glioblastoma. Cancer cells with stem cell-like properties have been found in glioblastomas. The presence of cancer stem cells is a likely cause of the resistance of glioblastomas toconventional treatments, and their high recurrence rate. These cells are harder to treatwith chemotherapeutic agent as they divide more slowly. Route of administrationThe composition for use as described herein may be administered locally to the brain bydirect intracerebral administration or to the spinal cord by intrathecal injection. In one embodiment, the composition for use as described herein, may be formulated in any physiologically acceptable liquid suitable for intracerebral administration. In one embodiment, the composition for use as described herein is formulated or provided in aisotonic saline buffer or PBS buffer. In one embodiment, the composition for use asdescribed herein is administered by intracerebral bolus injection. Convection-enhanced delivery (CED) is a local delivery approach that delivers therapeutics to the tumor site under positive pressure via an implanted catheter, therebycircumventing brain-blood-barrier (BBB) constraints. Convection-enhanced deliveryinvolves the continuous infusion of a therapeutic compound under positive pressure. One or more catheters can be placed using intraoperative neuronavigation into areas ofresidual tumor, preferably after surgical resection of the tumor. The one or morecatheters are then connected to a pump, either an internal or external pump dependingon the duration of the infusion. This convection-enhanced delivery bypasses the blood-brain barrier and allows the creation of higher concentrations of the radioactive agent in the brain with no or very little systemic toxicity. P6655PC00 41 Thus, in one embodiment, the composition for use as described herein is administeredby CED. In one embodiment, the composition for use as described herein is provided orprepared in a buffer suitable for convection-enhanced delivery. In one embodiment, the infusion rate is adjusted to a level that ensures sufficient delivery of the nanoparticles, while avoiding adverse effects resulting from increased intracranialpressure, for example, the infusion rate is from 0.1 to 5.0 mL / hour. The infusion rate isshould generally be about 0.1-5 ml / hour, and preferably between 0.1-4 ml / hour, such as 0.1-3 ml / hour, such as 0.1-2 ml / hour, such as preferably 0.1-1 ml / hour. Alternatively, the infusion rate is between 0.2-4 ml / hour, such as 0.2-3 ml / hour, such as 0.2-2 ml / hour, such as 0.3-2 ml / hour, such as 0.3-1.5 ml / hour, such as preferably 0.3-1.0 ml / hour, such as 0.3-0.9 ml / hour, such as 0.3-0.7 ml / hour, such as about 0.5 ml / hour. In one embodiment, the infusion rate is from 0.1 µL / min per catheter to 1000 µL / min per catheter. In one embodiment, the infusion rate is from 1.0 µL / min per catheter to 10 µL / min per catheter, such as from 1.0 to 2.0, from 2.0 to 3.0, from 3.0 to 4.0, such as from 4.0 to 5.0, such as from 5.0 to 6.0, such as from 6.0 to 7.0, such as from 7.0 to 8.0, such as from 8.0 to 9.0, such as from 9.0 to 10.0 µL / min per catheter. In one embodiment, the composition is administered in one or more fractions, such as in 1 to 20 fractions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fractions.The time between each fraction may vary depending on the patient's response andpractical considerations. The time between each fraction will usually vary between a few hours and several days. In a preferred embodiment, one fraction per day is provided, but the fractions may also be provided once a week. Each provided fraction may thus comprise between 1 kBq to 50 GBq activity of radioactive composition. “Bq” refers to the SI unit of measure of radioactivity theBecquerel. The radioactive composition content of each fraction can be between 1 kBqto 50 GBq, such as 100 kBq to 40 GBq, such as 1 MBq to 40 GBq, such as 500 MBq to 30 GBq, such as 0.1 GBq to 20 GBq, such as 0.1-10 GBq, such as 0.1-9 GBq, such as 0.1-8 GBq, such as 0.1-7 GBq, such as 0.1-6 GBq, such as 0.1-5 GBq, such as 0.1-4 GBq, such as 0.1-3.7 GBq, such as 0.1-3 GBq, such as 0.1-2 GBq. In a preferredembodiment, the radioactive composition content of each fraction is 0.1-3.7 GBq, more P6655PC00 42 specifically 0.1-3.0, such as 0.1-2.0 GBq, such as 0.2-2.0 GBq, such as 0.3-2.0 GBq, such as 0.4-2.0 GBq, such as 0.5-2.0 GBq, such as 0.6-2.0 GBq, such as 0.7-2.0 GBq, such as 0.8-2.0 GBq, such as 0.9-2 GBq, such as 1-2 GBq. In one embodiment, each fraction comprise 0.1-3.7 GBq, such as 0.2-3.7 GBq, such as 0.3-3.7 GBq, such as 0.4- 3.7 GBq, such as 0.5-3.7 GBq, such as 0.6-3.7 GBq, such as 0.7-3.7 GBq, such as 0.8- 3.7 GBq, such as 0.9-3.7 GBq, such as 1.0-3.7 GBq, such as 1.1-3.7 GBq, such as 1.23.7 GBq, such as 1.3-3.7 GBq, such as 1.4-3.7 GBq, such as 1.5-3.7 GBq, such as 1.6-3.7 GBq, such as 1.7-3.7 GBq, such as 1.8-3.7 GBq, such as 1.9-3.7 GBq, such as 2.0-3.7 GBq, such as 2.1-3.7 GBq, such as 2.2-3.7 GBq, such as 2.3-3.7 GBq, such as 2.4-3.7 GBq, such as 2.5-3.7 GBq, such as 2.6-3.7 GBq, such as 2.7-3.7 GBq, such as 2.8-3.7 GBq, such as 2.9-3.7 GBq, such as 3.0-3.7 GBq, 3.1-3.7 GBq, such as 3.2-3.7 GBq, such as 3.3-3.7 GBq, such as 3.4-3.7 GBq, such as 3.5-3.7 GBq, such as 3.6-3.7 GBq.In one embodiment, a further therapeutic agent is administered. In one embodiment, thefurther therapeutic agent is a chemotherapeutic agent. In one embodiment, the furtherchemotherapeutic agent is administered enterally or parenterally. As shown in the examples, the compositions according to the present disclosure combine: a) a longer biological half-life and retention of the radioactive agents in theintra-cerebral compartment; and b) release of Auger-emitting nucleosides for extendedperiods of time. This gives the opportunity to extend the window of time for incorporation into the DNA of cancer cells, and treat cancer more efficiently. Therefore, the compositions of the present disclosure may be able to deal more effectively with resistant cancer cells or slowly dividing cancer cells, such as cancer cells with stem-cell like properties. In one embodiment, at least 10% of the radioactive agent is present in the nanoparticle in aqueous solutions in the presence of esterase for at least 3 hours, such as at least 20%, 30%, 40% or 50% of the radioactive agent is present in the nanoparticles in thepresence of esterase for at last 3 hours, such as 6 hours, such as 12 hours, such as 24hours, such as 48 h, such as 72, hours, such as 96 hours, such as 120 hours. P6655PC00 43 In one embodiment, at least 10% of the radioactive agent is present in the nanoparticles 3 hours after administration, such as at least 20%, 30%, 40% or 50% of the radioactive agent is present in the nanoparticles 3 hours after administration. In one embodiment, at least at least 50% of the radioactive agent is present in thenanoparticles 24 hours after administration. In one embodiment, at least 20% or at least10% of the radioactive agent is present in the nanoparticles 96 hours after administration. In one embodiment, the activity of the radioisotope in the intracranial compartment can be detected for at least 6h, such as at least 24h, such as at least 48h, such as at least 72h, such as at least 96h, such as at least 120h upon administration.In one embodiment, the retention of activity of the radioisotope in the intracranialcompartment upon intracerebral administration of the composition for use as describedherein is increased compared to the retention of an equivalent amount of activity of thea free Auger electron-emitting nucleoside analog, particularly an equivalent amount of activity of the free Auger electron-emitting 2’-doxyuridine analog, such as free [125I]IUdR or free [123I]IUdR. In one embodiment, the biological half-life of activity of the radioisotope in the intracranialcompartment upon intracerebral administration of the composition for use as describedherein is increased compared to the biological half-life of an equivalent amount of activityof the a free Auger electron-emitting nucleoside analog, particularly an equivalent amount of activity of the free Auger electron-emitting 2’-doxyuridine analog, such as free [125I]IUdR or free [123I]IUdR. In one embodiment, the biological half-life of activity of the radioisotope in the intracranial compartment upon administration of the composition for use as described herein is higher than 100 min, such as higher than 200 min, such as higher than 5 hours, such as particularly when the radioactive agent comprises 125-iodine. In one embodiment, the biological half-life of activity of the radioisotope in the intracranial compartment upon administration of the composition for use as described herein is higher than 100 min, such as higher than 200 min, such as higher than 5 hours, particularly when the radioactive agent comprises 123-iodine. P6655PC00 44In one embodiment, the volume of distribution of activity of the radioisotope uponintracerebral administration of the composition for use as described herein is increasedcompared to the volume of distribution of an equivalent amount of activity of the a freeAuger electron-emitting nucleoside analog, particularly an equivalent amount of activity of the free Auger electron-emitting 2’-doxyuridine analog, such as free [125I]IUdR or free [123I]IUdR. In one embodiment, the present disclosure provides for a radioactive composition for usein a method of treatment, prevention, or alleviation of brain cancer as described hereinin the section “Treatment of brain cancer”, wherein the nanoparticle comprises: a. a liposome as described herein in the section “A liposome”; andb. a radioactive agent according to formula (I), formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAis123I or125I; and Ris –(CH2)12-16-CH3, most preferably R is -–(CH2)16-CH3, -–(CH2)14-CH3, or-–(CH2)12-CH3; wherein the radioactive agent is anchored to the liposome through non- covalent hydrophobic interaction. P6655PC00 45 In one embodiment, the present disclosure provides for a radioactive composition for usein a method of treatment, prevention, or alleviation of brain cancer as described hereinin the section “Treatment of brain cancer”, wherein the nanoparticle comprises: a. a liposome as described herein in the section “A liposome”; andb. a radioactive agent according to formula (I), formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAis123I or125I; and R is –(CH2)12-16-CH3, preferably R is -–(CH2)14-16-CH3, most preferably R is -–(CH2)16-CH3 or -–(CH2)14-CH3; wherein the radioactive agent is anchored to the liposome through non- covalent hydrophobic interaction.

[0002] P6655PC00 46 A radioactive agent In one aspect the present disclosure provides a radioactive agent according to formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. The radioactive agent as described herein may be useful in therapy or imaging, particularly when combined with hydrophobic carriers. Different radioisotopes may impart or provide specific properties attractive for different applications. Thus, in one embodiment, RAis as described herein in the section “Radionuclides”. In one embodiment, RAis123I,124I or125I,77Br,76Br,80mBr,80Br,126I,131I,18F or211At. In one embodiment, RAis an Auger electron-emitting radioisotope.In one embodiment, RA is 123I, 124I, 125I or 131I. In one embodiment, RA is 123I, 124I or 125I.The hydrophobic properties may be modified by suitable lipophilic groups R. Thus, in one embodiment, R is as described herein in the section “Lipophilic moiety”. In one embodiment, R is -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3. In one embodiment, R is -(CH2)12-16-CH3In one embodiment, R is selected from any one of the group consisting of: -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3. P6655PC00 47 In one embodiment, R is –(CH2)16-CH3. In one embodiment, R is –(CH2)15-CH3. In one embodiment, R is –(CH2)14-CH3. In one embodiment, R is –(CH2)13-CH3. In one embodiment, R is –(CH2)12-CH3. In one embodiment, the radioactive agent is selected from any one of the groups shown in Table B. In one aspect, the present disclosure provides for a radioactive agent according to formula (I) as described herein in the section “A radioactive agent” for use in a method of treatment, prevention or alleviation of a disease. In one aspect, the present disclosure provides a method of treatment, prevention or alleviation of a disease, said method comprising administering a radioactive agent according to formula (I) as described in the section “A radioactive agent”, to a subject in need thereof. In one aspect, the present disclosure provides for the use of a radioactive agent according to formula (I) as described in the section “A radioactive agent”, for the manufacture of a medicament for the treatment, prevention or alleviation of a disease.In one embodiment, the disease is cancer. In one embodiment, the disease is cancer. Inone embodiment, the disease is brain cancer. In one embodiment, the treatment of brain cancer as described in the section “Treatment of brain cancer”

[0003] P6655PC00 48 A composition comprising nanoparticles In one aspect, the present disclosure provides for a radioactive composition comprising a nanoparticle, the nanoparticle comprising: formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. The radioactive composition as described herein may be useful in therapy or imaging. Different radioisotopes may impart or provide specific properties attractive for different applications. Thus, in one embodiment, RAis as described herein in the section “Radionuclides”. In one embodiment, RAis123I,124I or125I,77Br,76Br,80mBr,80Br,126I,131I,18F or211At. In one embodiment, RAis an Auger electron-emitting radioisotope. In one embodiment, RAis123I,124I or125I. The hydrophobic properties may be modified by suitable lipophilic groups R. Thus, in one embodiment, R is as described herein in the section “Lipophilic moiety”. In one embodiment, R is -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3. In one embodiment, R is -(CH2)12-16-CH3In one embodiment, R is selected from any one of the group consisting of: -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3. P6655PC00 49 In one embodiment, R is –(CH2)16-CH3. In one embodiment, R is –(CH2)15-CH3. In one embodiment, R is –(CH2)14-CH3. In one embodiment, R is –(CH2)13-CH3. In one embodiment, R is –(CH2)12-CH3. In one embodiment, the radioactive agent is selected from any one of the groups shown in Table B. In one embodiment, the nanoparticle comprises a liposome as described herein in the section “Liposome”. In one embodiment, the nanoparticle comprises a polymeric micelle as described in the section “Polymeric micelle”. In one embodiment, the composition as described herein, may be formulated or provided in any physiologically acceptable liquid. In one embodiment, the composition for use as described herein is formulated or provided in a isotonic saline buffer or PBS buffer. In one embodiment, the composition as described herein is provided in an aqueous solution. In one aspect, the present disclosure provides for a radioactive composition as described herein in the section “A composition comprising nanoparticles” for use in a method of treatment, prevention or alleviation of a disease. In one aspect, the present disclosure provides for a method of treatment, prevention or alleviation of a disease, said method comprising administering a radioactive composition as described in the section “A composition comprising nanoparticles” to a subject in need thereof. In one aspect, the present disclosure provides for the use of a radioactive composition as described in the section “A composition comprising nanoparticles”, for the manufacture of a medicament for the treatment, prevention or alleviation of a disease.In one embodiment, the disease is cancer. In one embodiment, the disease is braincancer.In one embodiment, the treatment of brain cancer as described in the section “Treatmentof brain cancer” P6655PC00 50 A method of preparing a radioactive composition In another aspect, the present disclosure provides for a method of preparing a composition comprising a radioactive nanoparticle, the method comprising the steps of: a. providing a radioactive agent as described herein;b. providing a composition comprising a liposome or a polymeric micelle in anaqueous medium; c. mixing the radioactive agent and the composition comprising a liposome or apolymeric micelle, thereby obtaining an anchoring of the radioactive agent on the liposome or the micelle; andd. purifying the composition in c. from non-anchored radioactive agent, therebyobtaining a composition comprising radioactive nanoparticles. In one embodiment, the liposome in step b. is as described herein in the section “Liposome”. In one embodiment, the polymeric micelle in step b. is as described herein in the section “Polymeric micelle”. In one embodiment, the radioactive agent is as described herein in the section “A composition for use” or in the section “A radioactive agent”.In one embodiment, the radioactive agent or the liposome is provided in a suitableaqueous buffer. In one embodiment, the purifying in d. is performed in a suitable aqueousbuffer. Any suitable, physiologically acceptable liquid solution or buffer is contemplated. In one embodiment, the buffer is a isotonic saline buffer or a PBS buffer. In one embodiment, the buffer is a buffer suitable for convection-enhanced delivery. As it is known to the skilled artisan, separation of nanoparticles, such as liposomes or polymeric micelles, from small molecules can be achieved by size exclusionchromatography due to the nanoparticles’ size. Columns and matrices for purification ofnanoparticles in different scales and conditions are commercially available, and can beused according to the manufacturer’s instructions. P6655PC00 51 A kit In yet another aspect, the present disclosure provides a kit comprising: a. a radioactive agent as described herein; andb. a composition comprising a liposome or a polymeric micelle in a secondcontainer; and c. optionally an outer package.In one embodiment, the liposome is as described herein in the section “Liposome”. In one embodiment, the polymeric micelle is as described herein in the section “Polymeric micelle”. In one embodiment, the radioactive agent is as described herein in the section “A composition for use” or in the section “A radioactive agent”. In one embodiment, the kit further comprises a container with a buffer. In oneembodiment, the kit comprises a size exclusion chromatography column. In oneembodiment, the buffer is as described herein in the section “A method of preparing a radioactive composition”. The kit as described herein may be particularly useful so as to easily prepare a composition wherein the radioactive agent is incorporated into the liposomes or polymeric micelles and purified in an efficient manner so as to enable ease of manipulation by the user.

[0004] P6655PC00 52 Items1. A radioactive composition comprising a nanoparticle for use in a method oftreatment, prevention, or alleviation of brain cancer, wherein the nanoparticlecomprises: a. a hydrophobic part, andb. a radioactive agent of formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. nuclei2. The radioactive composition for use according to any one of the preceding items,wherein RA comprises or consists of 123I, 124I or 125I, 77Br, 76Br, 80mBr, 80Br, 126I, 131I,18F, or211At.3. The radioactive composition for use according to any one of the preceding items,wherein RAis an Auger electron-emitting radioisotope.4. The radioactive composition for use according to any one of the preceding items,wherein RA is 123I, 125I, or 77Br.5. The radioactive composition for use according to any one of the preceding items,wherein RAis a radioisotope of iodine.6. The radioactive composition for use according to any one of the preceding items,wherein RAis123I, or125I. P6655PC00 537. The radioactive composition for use according to any one of the preceding items,wherein RAis125I.8. The radioactive composition for use according to any one of the preceding items,wherein RAis123I.9. The radioactive composition for use according to any one of the preceding items,wherein RAis not of natural abundance.10. The radioactive composition for use according to any one of the preceding items,wherein all elements of formula (I) are of natural abundance except for RAwhich is not of natural abundance.11. The radioactive composition for use according to any one of the preceding items,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 2% or more, 5% or more, 10% ormore, 20% or more, 50% or more, 75% or more, 90% or more, or 95% or more.12. The radioactive composition for use according to any one of the preceding items,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95% or more.13. The radioactive composition for use according to any one of the preceding items,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95%, 96%, 97%, 98%, 99% or 100%.14. The radioactive composition for use according to any one of the preceding items,wherein R comprises a lipid.15. The radioactive composition for use according to any one of the preceding items,wherein R comprises or consists of:a. a C3-C21 hydrocarbon chain,b. two hydrocarbon chains, wherein each of said hydrocarbon chains isindependently selected from a C3-C21hydrocarbon chain, or c. a cholesteryl group, or a derivative thereof. P6655PC00 5416. The radioactive composition for use according to any one of the preceding items,wherein R comprises a C3-C21 hydrocarbon chain.17. The radioactive composition for use according to any one of the preceding items,wherein R comprises or consists of two hydrocarbon chains, wherein each of said hydrocarbon chains is independently selected from a C3-C21 hydrocarbon chain.18. The radioactive composition for use according to any one of the preceding items,wherein the R is according to formula (Ia) or formula (Ib): R1aX formula (Ia) formula (Ib) wherein, R1a, R1band R1care each independently selected of a C3-C21hydrocarbon chain, and X is a linker.19. The radioactive composition for use according to any one of the preceding items,wherein each of said hydrocarbon chain(s) is a linear or a branched hydrocarbonchain.20. The radioactive composition for use according to any one of the preceding items,wherein each of said hydrocarbon chain(s) is a linear hydrocarbon chain.21. The radioactive composition for use according to any one of the preceding items,wherein each of said hydrocarbon chain(s) comprises 0, 1, 2, or 3 double bonds,such as 0, 1, or 2 double bonds, such as 0 or 1 double bonds, such as 0 double bonds or 1 double bond.22. The radioactive composition for use according to any one of the preceding items,wherein in formula (Ia), X is selected from: a bond, or a C1-C6 hydrocarbon chain, such as a C1, C2, C3, C4, C5 or C6 hydrocarbon chain, wherein optionally one or more methylene groups is independently replaced by -O-, -NH-, carbonyl, an amide or a carbamate. P6655PC00 5523. The radioactive composition for use according to any one of the preceding items,wherein in formula (Ib), X is selected from: a C1-C6 hydrocarbon chain, such as a C1, C2, C3, C4, C5 or C6 hydrocarbon chain, wherein optionally one or more methylene groups is independently replaced by -O-, -NH-, carbonyl, an amide or a carbamate.24. The radioactive composition for use according to any one of the preceding items,wherein in R1ais: 25. The radioactive composition for use according to any one of the preceding items,wherein R1a, R1bor R1cis each independently selected from: -(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3,-(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3, -(CH2)8-CH3, nonen-1-yl, decen-1-yl, undecen-1-yl, dodecen-1-yl, tridecen-1-yl, tetradecen-1-yl , pentadecen-1-yl, hexadecen-1-yl, heptadecen-1-yl, octadecen-1- yl, nonadecen-1-yl, eicosen-1-yl, heneicosen-1-yl, (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl, (4Z,7Z,10Z,13Z,16Z)-nonadeca-3,7,10,13,16-pentaen-1-yl, (3Z,6Z,9Z,12Z,15Z,18Z)-heneicosa-3,6,9,12,15,18-hexaen-1-yl (8Z,11Z)-heptadeca-8,11-dien-1-yl (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl (7Z,10Z,13Z)-nonadeca-7,10,13-trien-1-yl (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraen-1-yl nonadecadien-1-yl, nonadecatrien-1-yl, nonadecatetraen-1-yl, heneicosapentaen- 1-yl, and heneiocsahexaen-1-yl. P6655PC00 5626. The radioactive composition for use according to any one of the preceding items,wherein each instance of R1a, R1b and R1c is independently comprising or consistingof the carbonyl radical of any one of the following carboxylic acids: decenoic acid, undecenoic acid, dodecenoic acid, tridecenoic acid, tetradecenoic acid, pentadecenoic acid, hexadecenoic acid , heptadecenoic acid, octadecenoic acid, nonadecenoic acid, eicosenoic acid, heneicosenoic acid, docosenoic acid alpha- linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma- linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid.27. The radioactive composition for use according to any one of the preceding items,wherein R1a comprises or consists of the aliphatic chain radical of any one of thefollowing carboxylic acids: decenoic acid, undecenoic acid, dodecenoic acid, tridecenoic acid, tetradecenoic acid, pentadecenoic acid, hexadecenoic acid , heptadecenoic acid, octadecenoic acid, nonadecenoic acid, eicosenoic acid, heneicosenoic acid, docosenoic acid alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid.28. The radioactive composition for use according to any one of the preceding items,wherein R consists of a C3-C21hydrocarbon chain.29. The radioactive composition for use according to any one of the preceding items,wherein R consists of a C8-C21, a C8-C20, a C9-C20, a C10-C20, a C11-C20, a C9-C19, aC10-C19, a C11-C19, a C12-C19, a C13-C19, a C14-C19or a C15-C19hydrocarbon chain.30. The radioactive composition for use according to any one of the preceding items,wherein R consists of a C11, C12, C13, C14, C15, C16, C17, C18, C19, C20 or C21hydrocarbon chain.31. The radioactive composition for use according to any one of the preceding items,wherein R consists of a saturated hydrocarbon chain.32. The radioactive composition for use according to any one of the preceding items,wherein R does not comprise a double bond. P6655PC00 5733. The radioactive composition for use according to any one of the preceding items,wherein R comprises 1, 2, or 3 double bonds.34. The radioactive composition for use according to any one of the preceding items,wherein R comprises 1 or 2 double bonds.35. The radioactive composition for use according to any one of the preceding items,wherein R comprises 1 double bond.36. The radioactive composition for use according to any one of the preceding items,wherein each instance of a double bond has a configuration independently selected from cis or trans or wherein each instance of a double bond has a cis configuration.37. The radioactive composition for use according to any one of the preceding items,wherein R consists of a linear hydrocarbon chain.38. The radioactive composition for use according to any one of the preceding items,wherein R does not comprise a branch.39. The radioactive composition for use according to any one of the preceding items,wherein R consists of a C8-C20, a C9-C20, a C10-C20, a C11-C20, a C9-C19, a C10-C19, aC11-C19, a C12-C19, a C13-C19, a C14-C19, a C15-C19, a C11-C18, a C12-C18, a C13-C18, a C14-C18, a C15-C18, a C11-C17, a C12-C17, a C13-C17, a C14-C17, or a C15-C17 linearhydrocarbon chain.40. The radioactive composition for uses according to any one of the preceding items,wherein R is -(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3,-(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3, -(CH2)8-CH3, nonen-1-yl, decen-1-yl, undecen-1-yl, dodecen-1-yl, tridecen-1-yl, tetradecen-1-yl , pentadecen-1-yl, hexadecen-1-yl, heptadecen-1-yl, octadecen-1- yl, nonadecen-1-yl, eicosen-1-yl, heneicosen-1-yl, (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl, (4Z,7Z,10Z,13Z,16Z)-nonadeca-3,7,10,13,16-pentaen-1-yl, (3Z,6Z,9Z,12Z,15Z,18Z)-heneicosa-3,6,9,12,15,18-hexaen-1-yl (8Z,11Z)-heptadeca-8,11-dien-1-yl (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl (7Z,10Z,13Z)-nonadeca-7,10,13-trien-1-yl P6655PC00 58 (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraen-1-yl nonadecadien-1-yl nonadecatrien-1-yl nonadecatetraen-1-yl heneicosapentaen-1-yl, and heneiocsahexaen-1-yl.41. The radioactive composition for use according to any one of the preceding items,wherein R is -(CH2)8-19-CH3, such as -(CH2)10-19-CH3, such as -(CH2)10-18-CH3, such as -(CH2)10-17-CH3, such as -(CH2)10-16-CH3.42. The radioactive composition for use according to any one of the preceding items,wherein R is selected from any one from the group consisting of: -(CH2)19-CH3, -(CH2)18-CH3, -(CH2)17-CH3, -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3, -(CH2)9-CH3,and -(CH2)8-CH3.43. The radioactive composition for use according to any one of the preceding items,wherein R is -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3,-(CH2)11-CH3, -(CH2)10-CH3.44. The radioactive composition for use according to any one of the preceding items,wherein R is –(CH2)16-CH3.45. The radioactive composition for use according to any one of the preceding items,wherein RA is 123I, 124I or 125I; andR consists of C11-C20 linear hydrocarbon.46. The radioactive composition for use according to any one of the preceding items,wherein the radioactive agent is selected from the compounds shown in Table B. P6655PC00 5947. The radioactive composition for use according to any one of the preceding items,wherein the radioactive compound has: a. a distribution coefficient (logD) from 2.0 to 13.0, such as 3.0 to 10.0, such as4.0 to 7.0 at room temperature, e.g. as measured by partitioning method inoctanol-buffer at pH 7.4 ; and / or b. a lipid-permeability coefficient (logM) from -0.5 to 1.0, e.g. as calculated bysolution thermodynamic calculations in a quantum chemical software package.48. The radioactive composition for use according to any one of the preceding items,wherein the nanoparticle comprises a liposome, the liposome comprising one or more lipid bilayer(s).49. The radioactive composition for use according to any one of the preceding items,wherein the liposome comprises a unilamellar lipid bilayer or more than one multilamellar lipid bilayers.50. The radioactive composition for use according to any one of the preceding items,wherein the liposome is a single unilamellar vesicle (SUV).51. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises a phospholipid.52. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises: i. a phospholipid, and ii. cholesterol, ergosterol, or sitosterol.53. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises: i. a phospholipid; ii. cholesterol, ergosterol, or sitosterol; andiii. an amphiphilic derivative of poly(ethylene glycol).54. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises a phospholipid, cholesterol, and an amphiphilicderivative of poly(ethyleneglycol). P6655PC00 6055. The radioactive composition for use according to any one of the preceding items,wherein the phospholipid is selected from the group consisting of: a fatty acid ester derivative of sn-glycero-3-phosphocholine, a fatty acid ester derivative of sn-glycero- 3-phosphoserine, a fatty acid ester derivative of sn-glycero-3-phoshpoglycerol, and a fatty acid derivative of sn-glycero-3-phosphoinositol.56. The radioactive composition for use according to any one of the preceding items,wherein the phospholipid is according to any one of formula (IIa)-(IIg): formula (IId), formula (IIe), formula (IIg), or a salt thereof, wherein each of RPa, RPa’, RPb, RPb’, RPc, RPc’, RPd, RPd’, RPe, RPe’, RPf, RPf’, RPg, andRPg’are independently the corresponding group resulting from forming an ester of a C12-C22 fatty acid with the corresponding O atom in formulas (IIa)-(IIg).57. The radioactive composition for use according to any one of the preceding items,wherein the phospholipid is selected from any one of the phospholipids described in Table C, or any combination thereof.58. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises a sphingolipid, such as N-palmitoyl-D-erythro- P6655PC00 61 sphingosine (Ceramide 16:0), N-stearoyl-D-erythro-sphingosine (Ceramide 18:0), N-oleoyl-D-erythro-sphingosine (Ceramide 18:1).59. The radioactive composition for use according to any one of the preceding items,wherein the amphiphilic derivative of poly(ethylene glycol) comprises apoly(ethylene glycol) moiety of average molecular weight between 200-20,000 g / molcovalently linked to a hydrophobic component which allows it to be anchored to a lipid bilayer.60. The radioactive composition for use according to any one of the preceding items,wherein the amphiphilic derivative of poly(ethylene glycol) is according to formula wherein, RPrepresents -H, -CH3 , a C1-C5 alkyl, a C1-C5 hydroxyalkyl, a C1-C5 alkylamine, RXrepresent an optional linker; RHrepresents a hydrophobic group which allows anchoring to a lipid bilayer.61. The radioactive composition for use according to any one of the preceding items,wherein: i. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative orRHcomprises a C10-C22fatty acid ester derivative or a C10-C22fatty acid amide derivative, or ii. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative or RHcomprises a phospholipid selected from the group consisting of any one of the phospholipids described in items 55 to 57.62. The radioactive composition for use according to any one of the preceding items,wherein the amphiphilic derivative of poly(ethylene glycol) is any derivative ofpoly(ethylene glycol) wherein the poly(ethylene glycol) is covalently linked to:i. a C10-C22 fatty acid, optionally via a linker, or ii. any phospholipid described in any one of items 55 to 57, optionally via a linker; P6655PC00 62 wherein the amphiphilic derivative of poly(ethylene glycol) has a molecular weightaverage from 300 to 20,000 g / mol.63. The radioactive composition for use according to any one of the preceding items,wherein the amphiphilic derivative of poly(ethylene glycol) is DSPE-PEG (CAS nr.247925-28-6), DMG-PEG (CAS nr.160743-62-4), DPPE-PEG (CAS nr.205494-72- 0), or DOPE-PEG (CAS nr. 474922-90-2), or a salt thereof, where the amphiphilic derivative of poly(ethylene glycol) has a molecular weightaverage from 1000 to 20,000 g / mol.64. The radioactive composition for use according to any one of the preceding items,wherein the lipid bilayer comprises: a. the phospholipid as described in any one of items 55 to 57;b. cholesterol; andc. the amphiphilic derivative of poly(ethylene glycol) as described in any one ofitems 59 to 62;wherein the mass ratio between components a:b:c is: 2-4:0.5-1.5:0.5-1.5, such as about 3:1:1.65. The radioactive composition for use according to any one of the preceding items,wherein the radioactive agent is anchored to the liposome through non-covalent hydrophobic interaction with the lipid bilayer.66. The radioactive composition for use according to any one of the preceding items,wherein the nanoparticle has an average hydrodynamic diameter of: 20 nm to 150 nm, such as an average hydrodynamic diameter of 80 to 120 nm, such as an average hydrodynamic diameter of 80, 90, 100, 110, or 120 nm, e.g. as measuredby dynamic light scattering (DLS).67. The radioactive composition for use according to any one of the preceding items,wherein the liposome has a neutral, positive, or negative net charge, e.g. asdetermined by Z-potential measurements.68. The radioactive composition for use according to any one of the preceding items,wherein the liposome has a neutral or negative net charge. elle P6655PC00 6369. The radioactive composition for use according to any one of items 1 to 47, whereinthe nanoparticle comprises or consists of a polymeric micelle.70. The radioactive composition for use according to any one of items 1 to 47 or 69,wherein the polymeric micelle comprises one or more amphiphilic block copolymers.71. The radioactive composition for use according to any one of items 1 to 47 or 69 to70, wherein the amphiphilic block copolymer comprises a polymer chain with a hydrophilic and a hydrophobic block according to: i. (hydrophilic block)-(hydrophobic block), orii. (hydrophilic block)-(hydrophobic block)-(hydrophilic block), oriii. (hydrophobic block)-(hydrophilic block)-(hydrophobic block).72. The radioactive composition for use according to any one of items 1 to 47 or 69 to71, wherein the hydrophobic block is a polymer selected from the group consisting of: poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), poly(glutamic acid) (PGA), poly(aspartic acid) (PAsp), and poly(lactic acid) (PLA).73. The radioactive composition for use according to any one of items 1 to 47 or 69 to72, wherein the hydrophilic block is selected from the group consisting of: poly(ethylene glycol) and poly(propylene glycol).74. The radioactive composition for use according to any one of items 1 to 47 or 69 to73, wherein the radioactive agent is anchored to the nanoparticle through non- covalent hydrophobic interaction with the polymeric micelle.75. The radioactive composition for use according to any one of items 1 to 47 or 69 to74, wherein the polymeric micelle comprises a single amphiphilic block copolymerselected from the group consisting of: Poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-PLGA) (CAS nr.952111-10-3), Poly(ethylene glycol)-block-poly(caprolactone) (PEG-PCL), Poly(caprolactone)-block-poly(ethylene glycol)-block-poly(caprolactone) (PCL- PEG-PCL), Poly(ethylene glycol)-block-poly(caprolactone)-block-poly(ethylene glycol) (PEG-PCL-PEG), poly(ethylene glycol)-block-poly(glutamic acid) (PEG- PGA), Poly(ethylene glycol)-block-poly(aspartic acid) (PEG-PAsp), P6655PC00 64 Poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA), Poly(ethylene glycol)- block-poly(caprolactone)-block-poly(ethylene glycol) (PEG-PEL-PEG), Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG), and, Poly(ethylene glycol)-block-poly(methylmetacrylate) (PEG-PMMA).76. The radioactive composition for use according to any one of items 1 to 47 or 69 to75, wherein the polymeric micelle comprises any one of the polymers described initem 75, wherein the polymer has an average molecular weight between 2000- 50,000 g / mol.77. The radioactive composition for use according to any one of items 1 to 47 or 69 to76, wherein the polymer micelle comprises PEG-PLGA, wherein the PEG block hasan average molecular weight between 2000 and 10,000 g / mol and the PLGA block has an average molecular weight between 5000 and 20,000 g / mol.78. The radioactive composition for use according to any one of items 1 to 47 or 69 to77, wherein the nanoparticle has an average hydrodynamic diameter of: 40 nm to150 nm, such as an average hydrodynamic diameter of 40 to 100 nm, 40 nm to 80 nm, such as an average hydrodynamic diameter of 50, 60, 70, or 80 nm, e.g. asmeasured by DLS. cancer79. The radioactive composition for use according to any one of the preceding items,wherein the brain cancer is a brain tumor or an intracerebral neoplasm.80. The radioactive composition for use according to any one of the preceding items,wherein the brain tumor or intracerebral neoplasm involves glial cells.81. The radioactive composition for use according to any one of the preceding items,wherein the brain tumor or intracerebral neoplasm is a glioma.82. The radioactive composition for use according to any one of the preceding items,wherein the intracerebral neoplasm is a high-grade glioma, i.e. grade III or grade IVglioma.83. The radioactive composition for use according to any one of the preceding items,wherein the glioma is astrocytoma, glioblastoma, diffuse midline glioma, diffusehemispheric glioma, or diffuse pediatric-type high-grade glioma. e of administration P6655PC00 6584. The radioactive composition for use according to any one of the preceding items,wherein the composition is administered by direct intracerebral administration or by intrathecal administration.85. The radioactive composition for use according to any one of the preceding items,wherein the composition is formulated or provided in an isotonic saline buffer or PBSbuffer.86. The radioactive composition for use according to any one of the preceding items,wherein the composition is administered by convection-enhanced delivery (CED).87. The radioactive composition for use according to any one of the preceding items,wherein the infusion rate is adjusted to a level that ensures sufficient delivery of the nanoparticles, while avoiding adverse effects resulting from increased intracranial pressure, for example, the infusion rate is from 0.1 to 5.0 mL / hour.88. The radioactive composition for use according to any one of the preceding items,wherein the infusion rate is from 0.1 µL / min per catheter to 1000 µL / min per catheter.89. The radioactive composition for use according to any one of the preceding items,wherein the composition is administered in one or more fractions, such as in 1 to 20 fractions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fractions.90. The radioactive composition for use according to any one of the preceding items,wherein the composition comprises 1kBq to 50 GBq of the radioactive agent.91. The radioactive composition for use according to any one of the preceding items,wherein a further chemotherapeutic agent is administered.92. The radioactive composition for use according to any one of the preceding items,wherein the further chemotherapeutic agent is administered enterally or parenterally.93. The radioactive composition for use according to any one of the preceding items,wherein the nanoparticle comprises:a. a liposome as described in any one of items 48 to 68; andb. a radioactive agent according to formula (I), P6655PC00 66 or a pharmaceutically acceptable salt thereof, wherein, RAis123I or125I; and R is –(CH2)10-18-CH3, preferably R is -–(CH2)10-16-CH3, most preferably R is -– (CH2)16-CH3; wherein the radioactive agent is anchored to the liposome through non-covalent hydrophobic interaction. Functional features94. The radioactive composition for use according to any one of the preceding items,wherein at least 10% of the radioactive agent is present in the nanoparticle in aqueous solutions in the presence of esterase for at least 3 hours, such as at least 20%, 30%, 40% or 50% of the radioactive agent is present in the nanoparticles in the presence of esterase for at last 3 hours, such as 6 hours, such as 12h, such as 24h, such as 48 h, such as 72, hours, such as 96 hours, such as 120 hours.95. The radioactive composition for use according to any one of the preceding items,wherein at least 10% of the radioactive agent is present in the nanoparticles 3 hours after administration, such as at least 20%, 30%, 40%, or 50% of the radioactiveagent is present in the nanoparticles 3 hours after administration.96. The radioactive composition for use according to any one of the preceding items,wherein at least 50% of the radioactive agent is present in the nanoparticles 24 hours after administration.97. The radioactive composition for use according to any one of the preceding items,wherein at least 20% or at least 10% of the radioactive agent is present in the nanoparticles 96 hours after administration. P6655PC00 6798. The radioactive composition for use according to any one of the preceding items,wherein the activity of the radioisotope in the intracranial compartment can be detected for at least 6h, such as at least 24h, such as at least 48h, such as at least 72h, such as at least 96h, such as at least 120h.99. The radioactive composition for use according to any one of the preceding items,wherein the biological half-life of activity of the radioisotope in the intracranialcompartment is increased compared to the biological half-life of an equivalent amount of activity of the free radiohalogen derivative of 2’-deoxyuridine.100. The radioactive composition for use according to any one of the preceding items,wherein the biological half-life of activity of the radioisotope in the intracranial compartment is higher than 100 min, such as higher than 200 min, such as higher than 5 hours.101. The radioactive composition for use according to any one of the preceding items,wherein the volume of distribution of the activity of the radioisotope upon intracerebral administration is increased compared to the volume of distribution ofan equivalent amount of activity of the free radiohalogen derivative of 2’-deoxyuridine.ioactive agent102. A radioactive agent according to formula (I): formula (I), or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety. P6655PC00 68103. The radioactive agent according to item 102, wherein the radioactive agent, RA orR are as described in any one of items 1 to 47.104. The radioactive agent according to any one of items 102 to 103, wherein the RA is123I, 124I or 125I, 77Br, 76Br, 80mBr, 80Br, 126I, 131I, 18F or 211At.105. The radioactive agent according to any one of items 102 to 104, wherein RA isselected from: 123I, 124I, 125I, and 131I.106. The radioactive agent according to any one of items 102 to 105, wherein RA is anAuger electron-emitting radioisotope.107. The radioactive agent according to any one of items 102 to 106, wherein RA is 123I,125I, or 77Br.108. The radioactive agent according to any one of items 102 to 107, wherein Ris -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11-CH3, -(CH2)10-CH3.109. The radioactive agent according to any one of items 102 to 108, wherein Ris -(CH2)16-CH3110. A radioactive agent according to any one of items 102 to 109, for use in a methodof treatment, prevention, or alleviation of disease. nanoparticles111. A radioactive composition comprising a nanoparticle, the nanoparticle comprising:a. a liposome or a polymeric micelle, andb. a radioactive agent of formula (I): formula (I), P6655PC00 69 or a pharmaceutically acceptable salt thereof, wherein, RAcomprises a radioisotope of a halogen; and R is a lipophilic moiety.112. The radioactive composition according to item 111 wherein the liposome is asdefined in any one of items 48 to 68.113. The radioactive composition according to item 111, wherein the polymeric micelleis as described in any one of items 69 to 78.114. The radioactive composition according to any one of items 111 to 113, wherein theradioactive agent, RA or R are as described in any one of items 1 to 47.115. The radioactive composition according to any one of items 111 to 114, wherein RAis 123I, 124I or 125I, 77Br, 76Br, 80mBr, 80Br, 126I, 131I, 18F or 211At.116. The radioactive composition according to any one of items 111 to 115, wherein RAis selected from is 123I, 124I, 125I, and 131I.117. The radioactive agent according to any one of items 111 to 116, wherein RA is anAuger electron-emitting radioisotope.118. The radioactive composition according to any one of items 111 to 117, wherein RAis123I,125I, or77Br.119. The radioactive composition according to any one of items 111 to 118, wherein Ris -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, -(CH2)11- CH3, -(CH2)10-CH3.120. The radioactive composition according to any one of items 111 to 119, wherein Ris –(CH2)16-CH3.121. A kit comprising:a. a radioactive agent as defined in any one of items 102 to 109 in a firstcontainer; and b. a composition comprising a liposomes or polymeric micelles in a secondcontainer; and P6655PC00 70 c. optionally an outer package.122. The kit according to item 121, wherein the liposomes are as defined in any oneof items 48 to 68.123. The kit according to any one of items 121 to 122, wherein the polymeric micellesare as defined in any one of items 69 to 78.124. The kit according to any one of items 121 to 123, further comprising a containerwith a buffer.125. The kit according to any one of items 121 to 124, further comprising a sizeexclusion chromatography column. a radioactive composition126. A method of preparing a composition comprising a radioactive nanoparticle, themethod comprising the steps of: a. providing a radioactive agent as defined in any one of items 102 to 109;b. providing a composition comprising a liposome or a polymeric micelle in anaqueous medium; c. mixing the radioactive agent and the composition comprising a liposomeor a polymeric micelle, thereby obtaining an anchoring of the radioactive agent on the liposomes or micelles; and d. purifying the composition in c. from non-anchored radioactive agent,thereby obtaining a composition comprising radioactive nanoparticles.127. The method according to item 126, wherein the radioactive agent in step a. issubstantially free of water, such as having less than 10% water, such as less than 5% water, such as less than 1% water.128. The method according to any one of items 126 to 127, wherein the liposome instep b. is as defined in any one of items 48 to 68.129. The method according to any one of items 126 to 128, wherein the polymericmicelle in step b. is as defined in any one of items 69 to 78.130. The method according to any one of items 126 to 129, wherein the liposome is inan aqueous buffer. P6655PC00 71131. The method according to any one of items 126 to 130, wherein the purifying instep d. is performed by size exclusion chromatography. ExamplesExample 1: Preparation of prodrugsMaterials and MethodsGeneral synthesis of iododeoxyuridine derivatives. In a flask equipped with a stirringbar was placed 5-iodo-2’-deoxyuridine (IUdR) (1, 354 mg, 1.0 mmol) and alkyl carboxylicacid (2 mmol, 2 equiv.), followed by dissolution in DMF (15 mL). After this, the mixturewas cooled down to 0 °C and N,N’-dicyclohexylcarbodiimide (DCC) (216 mg, 1.05 mmol,1.05 equiv.) was added in a single portion, together with a grain of N,N-dimethyl-4-aminopyridine (10 mg, 0.01 mmol). This flask was sealed with a rubber septum, the atmosphere was replaced with argon, and the mixture was stirred at 0 °C. After 3 hours, the reaction mixture was allowed to warm up to room temperature and stirred for another 18 hours. After complete consumption of iododeoxyuridine, the reaction was diluted with aq. LiCl (15 mL, 1.0 M), transferred to an extraction-funnel, and extracted with EtOAc (3 x 15 mL). The combined organic layers were collected and dried over MgSO4 and then concentrated under reduced pressure. The crude was then furtherpurified by silica column chromatography (0% - 10% MeOH in DCM) to yield the acylatedIUdR compounds.General synthesis of stannyl iododeoxyuridine (stannyl-IUdR, or Bu3Sn-IUdR)precursors. A flask with acylated IUdR compounds ( 0.2 mmol, 1 equiv.) in 1,4-dioxane(2 mL) and bis(triphenylphosphine) palladium dichloride (6 mg, 2.5 mol%) was prepared.The vial was sealed with a rubber septum, and argon was bubbled through the solvent for 10 minutes. To this solution was added, 1,1,1,2,2,2-hexabutyldistannane (256 mg,0.44 mmol, 2.2 equiv.). The reaction mixture was heated to 120 °C and stirred for 12hours. After this, the reaction mixture was cooled down to room temperature, diluted withEtOAc and filtered over a pad of Celite (0.5 cm). The combined organic layers wereconcentrated in vacuo, and further purified by silica column chromatography (0% - 5%MeOH in dichloromethane (DCM)) to obtain the title compound the stannyl-IUdR P6655PC00 72precursors as an off-white solid. The compounds were characterized by NMR andchromatographic methods like TLC.General radiosynthesis procedure of [125I]IUdR prodrugs. In a 2 mL HPLC-vialequipped with a small stirring bar was added a mixture of stannyl IUdR precursor (0.13- 0.17 μmol, 0.2 mg) in DMF (100 µL). To this was added in a sequential manner, aceticacid (5 μL), aq. chloramine-T solution (10 μL, 100 mg / mL) and then [125I]NaI in 0.1 MNaOH (2-50 MBq). The vial was sealed with a screw cap and stirred for 30 min at 25 °C.After this, a solution of KI in H2O (10 uL, 0.1 M) was added and the reaction was stirredfor another 10 min at 25 °C. Hereafter, the reaction was terminated using a sodium meta-bisulfite solution (2 mg in 20 μL of H2O). Hereafter, an aliquot was removed for radio-HPLC or radio-TLC analysis to give the radiochemical conversion (RCC). Then, thereaction mixture was diluted with MeCN (2 mL) and transferred through a SiO2 cartridge (Sep-Pak Silica Plus Light Cartridge, 120 mg sorbent per cartridge) and collected in different fractions of MeCN (1-3 mL) depending on the substrate or activity. The mixture was then concentrated if needed, using a flow of nitrogen, a few vent-needles at atemperature of 65 °C. Then, the reaction mixture was diluted with H2O (2-50 mL, withminimal amounts of MeCN depending on the compound, taken up and trapped on a pre- activated C18cartridge (Sep-Pak C18Plus Short Cartridge, 360 mg sorbent). The C18cartridge was slowly eluted with a series of MeCN / H2O-mixtures to release the radio-iodinated compound from the cartridge; in consecutive order H2O (2 mL), 20% MeCN inH2O (2 mL), 70% MeCN in H2O (2 mL), 100% MeCN (2 mL) or 100% EtOH (2 mL) wereslowly eluted over the C18 cartridge, and collected in different fractions. All fractions wereanalysed by radio-TLC. The desired fractions were collected and concentrated using a flow of N2(1 L / min) at 65 °C, with several ventilation needles. The obtained driedcompound was analysed by dose-CAL, radio-TLC or radio-HPLC to give the finalradiochemical yield and purity (typical yields, RCY = 65 ± 4% or higher, RCP > 97 ± 2%,n > 25) and used directly for liposome or polymeric micelle loading.Preparation of 123I and 124I IUdR prodrugs. The following prodrugs were prepared with123I or 124I; [123I]IUdR-C18, [123I]IUdR-Cholesterol, and [124I]IUdR-C18. The 123I and 124I IUdRprodrugs were prepared using the same radiolabeling procedure as stated above,starting off with the sodium iodide residue (NaI / NaOH). Iodine-124 stock, [124I]NaI / NaOH in H2O, was produced in a similar manner as iodine-125, and therefore no additional P6655PC00 73 steps were needed this radionuclide. The iodine-123 stock, however, was not provided in aq. NaOH solution, but in a NaCl / NaHCO3 buffer. Which it was subsequently dried down, using azeotropic drying with MeCN at 65 °C under a flow of nitrogen (1 L / hour) (Aloss < 5%). The resulting123I-containing residue could then be used directly for the radiosynthesis of the prodrug compounds. High activity radioiodinations (with123I,124I and125I) follow the same procedure as described above. In some instances, it is possible to protect the alcohol in the 3’ position of IUdR in order to ensure acylation at the 5’ position. The 3’ protected IUdR derivative may then beacylated at the 5’ position as described above and radiolabeled prior to deprotection toobtain 5’ IUdR prodrugs according to the present disclosure. Suitable protecting groupswill be known to the person of skill in the art and can be found the literature. For examplein Philip J. Kocienski, in “Protecting Groups”, Georg Thieme Verlag Stuttgart, New York, 1994 and, Theodora W. Greene and Peter G. M. Wuts in “Protective Groups in Organic Synthesis”, Wiley Interscience, 3rd Edition 1999.Synthesis of 3’-TBDMS-iododeoxyuridine. In a flask equipped with a stirring bar,unprotected IUdR (354 mg) was added and dissolved in anhydrous DMF (10 mL) and cooled down to 0 °C. After this, TBDMS-Cl (302 mg, 2.0 mmol, 2.0 equiv.) and imidazole(136 mg, 2.0 mmol, 2.0 equiv.) was added to the stirring reaction mixture. The reactionwas then allowed to warm up to room temperature for 2 hours, and monitored by TLC. After complete consumption of the starting material, the crude was diluted with EtOAc (20 mL) and extracted with LiCl in H2O (20 mL, 1M) three times to remove excess DMF. The organic layers were collected, dried with MgSO4, filtered and concentrated in vacuo to give intermediate bis-3’,5’-TBDMS-IUdR () as crude product. This crude can be optionally run over a short plug of silica (2% MeOH in DCM) to remove some of the impurities. The collected intermediate product was then concentrated and used directly. Hereafter the intermediate crude was placed inside a round bottom flask, equipped with a stirring bar and dissolved into anhydrous MeOH (10 mL). The solution was cooled down to -10 °C (with a NaCl ice bath), and stirred for 10 minutes. Hereafter, acetyl chloride (15 µL, 0.2 mmol, 0.1 equiv.) was added dropwise and the mixture was stirred, sealed and allowed to warm up to 0 °C and stirred for 3 hours. The reaction was quenched with sat. NaHCO3 in H2O and diluted with EtOAc, extracted with 1 M HCl, NaHCO3 in H2O and brine. The combined organic layers were dried over MgSO4, filtered P6655PC00 74 and concentrated in vacuo. The crude product was further purified by column chromatography (2% MeOH in DCM), which afforded compound 3’-TBDMS-iododeoxyuridine as an off-white solid.General synthesis of 3’-TBDMS-5’-acyl-iododeoxyuridine. In a flask equipped with a stirring bar was placed 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dione (3’-TBDMS- iododeoxyuridine) (4, 468 mg, 1.0 mmol) and alkyl carboxylic acid (2 mmol, 2 equiv.), followed by dissolution in DMF (15 mL). After this, the mixture was cooled down to 0 °C and N,N’-dicyclohexylcarbodiimide (DCC) (216 mg, 1.05 mmol, 1.05 equiv.) was added in a single portion, together with a grain of N,N-dimethyl-4-aminopyridine (10 mg, 0.01 mmol). This flask was sealed with a rubber septum, the atmosphere was replaced with argon, and the mixture was stirred at 0 °C. After 3 hours, the reaction mixture was allowed to warm up to room temperature and stirred for another 18 hours. After complete consumption of iododeoxyuridine, the reaction was diluted with aq. LiCl (15 mL, 1.0 M), transferred to an extraction-funnel, and extracted with EtOAc (3 x 15 mL). The combined organic layers were collected and dried over MgSO4 and then concentrated under reduced pressure. The crude was then further purified by silica column chromatography(0% - 10% MeOH in DCM) to yield the acylated mono-protected IUdR compounds.Deprotection. Deprotection of 3’-TBDMS group was achieved by TBAF using either THFor H2O / THF solvents as mixtures. The product was transferred into a flask equipped witha stirring bar and dissolved into THF (15 mL). To this mixture was added, tetrabutylammonium fluoride (TBAF) (288 mg, 1.1 mmol, 1.1 equiv.), sealed and stirred for 18 hours at room temperature. After consumption of the starting material, the reactionwas diluted with EtOAc, transferred to an extraction funnel, and then extracted with 1MHCl, sat. NaHCO3 in H2O and brine. The combined organic layers were collected and dried over MgSO4 and then concentrated under reduced pressure. The crude was further purified by silica chromatography to afford the title compound as a white solid.LogD measurements. Hydrophobicity analysis (LogD) was done by distributioncoefficient via a standard ionized-partitioning method (Octanol-PBS buffer, pH = 7.4, at room temperature). Briefly, PBS buffer (pH = 7.4) saturated with octanol (0.1% octanol in PBS buffer, solution 1) and octanol saturated with PBS buffer (pH = 7.4) (0.1% PBS buffer in octanol, solution 2) were prepared and degassed. Hereafter, an aliquot of P6655PC00 75 125I-prodrug (100-250 kBq) was added to a 1:1 mixture of solution 1 / solution 2 (10 mL total volume, v / v) and mixed rigorously for 30 minutes. After this, the solutions are separated and measured for activity by liquid scintillation (LSC) to determine the distribution coefficient.LogM calculation. All quantum chemical calculations were performed with Turbomole7.4.1 at BP-TZVP-FINE level. The polarization charge densities of all surface segments, the corresponding atoms, and structural information of the molecules are saved in thecorresponding cosmo files and used later for solution thermodynamics calculations. Theconformers (the number of generated conformers in parenthesis) were obtained using COSMOconf: IUdR (1), IUdR-C4 (3), IUdR-C8 (4), and IUdR-C18(8).Equipment. 1H-NMR or 13C-NMR spectra were recorded on a 400 MHz Brukerinstrument using relevant deuterated solvents (e.g. CDCl3 or DMSO-d6). Coupling constants are given in Hz.1H-NMR signals are reported in chemical shift (δ ppm), multiplicities are reported as; s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, h = hextet, hept = heptet, m = multiplet, b = broad.13C-NMR signals are reported in chemical shift (δ ppm). Mass spectrometry was measured on a Shimadzu MS-2020 with an electro spray ionization spectrometer (LC-ESI) or matrix-assisted laser desorption / ionization, coupled to a time-of-flight mass spectrometer (MALDI-TOF). Radio-HPLC was performed on a Hitachi Chromaster instrument equipped with a Hitachi 5160 manual purge quaternary gradient pump, coupled to a Hitachi 5260 thermostat loop autosampler, a Hitachi 5310 column oven, a Hitachi 5430 UV-Vis multichannel detector and a radio-detector (gamma) with analogue output and ca.0.2 min signal delay. Unless stated otherwise, routine HPLC analysis was performed using a Luna C18(2) (Ø = 2.5µm, 100 Å) column using a 20 min program, with a 0 - 100 H2O / MeCN + 0.1% TFAgradient. Routine analysis of activity was performed on a Capintec CRC-55tR dose calibrator, and reported in Becquerel (kBq or MBq). Liquid scintillation counting (LSC) measurements were performed on a HIDEX 425-034 LSC for routine analysis, or on a HIDEX 300-SL LSC for large batch analysis, and reported in Becquerel (kBq, MBq) or counts per minute (cpm). Radio TLC analysis was performed with a PerkinElmer Cyclone Plus phosphor imager on commercially TLC pre-coated aluminium sheets (4 x 10 cm, Merck Silica gel 60), and unless stated otherwise run in 10% MeOH in DCM. P6655PC00 76 Results The following compounds were prepared as described above. On Namen O I1 IUdR-butyrate (IUdR-C4)O N O5 IUdR-octanoate (IUdR-C8)NH9 IUdR-dodecanoate (IUdR-C12)HO O11 IUdR-myristate (IUdR-C14)13 IUdR-palmitate (IUdR-C16)15 IUdR-stearate (IUdR-C18)17 IUdR-icosanoate (IUdR-C20)19 IUdR-behenate (IUdR-C22)On A NameA1 125I [125I]IUdR-butyrate 125n O([ I]IUdR-C4)O5 125 125 125N OI [ I]IUdR-octanoate ([ I]IUdR-C8)NH9 125I [125I]IU 125HOdR-dodecanoate ([ I]IUdR-C12)O 13 125I [125I]IUdR-palmitate ([125I]IUdR-C16)15 125I [125I]IUdR-stearate ([125I]IUdR-C18)17 125I [125I]IUdR-icosanoate ([125I]IUdR-C20)19 125I [125I]IUdR-behenate ([125I]IUdR-C22)15 123I [123I]IUdR-stearate ([123I]IUdR-C18)15 124I [124I]IUdR-stearate ([124I]IUdR-C18)O AA NameNHI IUdR-C2-chol,N OIUdR-chol 125HHOI [12512I]IUdR-C2-chol,[5I]IUdR-chol OOH 123OI [123123I]IUdR-C2-chol,HO [ I]IUdR-chol P6655PC00 77 OA NameA OH O OI IUdR-cholic acidN125I [125I]IUdR-cholic acidH HO O N O H H H HO OH H 125I Name O [125I]IAn-C18, [125I]N-(4-iodopenyl)stearamide H35C17N H In addition, the following compounds will be prepared as described above: On A NameA11 125I [125I]IUdR-myristate ([125n OI]IUdR-C14)O11 123I [123I]I 123N OUdR-myristate ([ I]IUdR-C14)NH13 123I [123I]IUdR-palmitate ( 123HO[ I]IUdR-C16)O 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- iodopyrimidine-2,4(1H,3H)-dione (3’-OTBDMS-IUdR)1H-NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 8.22 (s, 1H), 6.16 (t, J = 6.4 Hz, 1H), 4.48 (td,J = 5.1, 3.5 Hz, 1H), 3.96 (dt, J = 6.0, 2.9 Hz, 2H), 3.79 (d, J = 11.6 Hz, 1H), 2.55 (s, 1H),2.32 – 2.22 (m, 2H), 0.88 (s, 9H), 0.08 (s, 6H).13C-NMR (101 MHz, CDCl3) δ 160.28, 150.07, 146.00, 87.95, 86.99, 71.47, 68.19, 61.84,41.44, 25.83, 18.07, -4.56, -4.73.MS (LC-ESI) calculated for C15H26IN2O5Si [M+H] 469.06, found 469.45 [M+H], 491.45[M+Na], 937.30 [2M+H]. ((2R,3S,5R)-3-hydroxy-5-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl) methyl butyrate (IUdR-C4) P6655PC00 781H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 7.96 (s, 1H), 6.09 (t, J = 6.7 Hz, 1H), 5.39(d, J = 4.2 Hz, 1H), 4.22 (dd, J = 4.2 Hz, 2H), 4.20 (p, J = 4.2 Hz, 1H), 3.96 (q, J = 4.2Hz, 1H), 2.38 (t, J = 7.4 Hz, 2H), 2.28 – 2.09 (m, 1H), 1.58 (h, J = 7.4 Hz, 2H), 0.90 (t, J= 7.4 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 172.56, 160.46, 150.02, 144.42, 84.88, 84.10, 70.10,69.78, 63.47, 40.19, 35.35, 17.95, 13.43.MS (LC-ESI) m / z calculated for C13H18IN2O6 [M+H] 425.02, found 448.75 [M+Na].MS (MALDI-TOF) m / z calculated for C13H18IN2O6[M+H] 425.0210 found 448.7050 [M+Na]. 1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 0H), 7.96 (s, 0H), 6.09 (t, J = 6.7 Hz, 0H), 5.39(d, J = 4.4 Hz, 1H), 4.20 (p, J = 4.2 Hz, 0H), 3.96 (q, J = 4.1 Hz, 1H), 2.38 (t, J = 7.3 Hz,1H), 2.28 – 2.09 (m, 1H), 1.58 (h, J = 7.4 Hz, 1H), 0.90 (t, J = 7.4 Hz, 1H).13C NMR (101 MHz, DMSO-d6) δ 172.56, 160.46, 150.02, 144.42, 84.88, 84.10, 70.10,69.78, 63.47, 40.19, 35.35, 17.95, 13.43. iodo-2,4-dioxo-3,4- octanoate 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H, NH), 7.98 (s, 1H), 6.11 (t, J = 6.3 Hz, 1H),5.41 (s, 1H, 3’-OH), 4.23 (s + s, 2H + 1H), 3.98 (d, J = 4.1 Hz, 1H), 2.40 (t, J = 7.5 Hz,2H), 2.21 (dd, J = 15.0, 8.3 Hz, 2H), 1.56 (t, J = 7.0 Hz, 2H), 1.27 (t, J = 6.6 Hz, 8H), 0.86(t, J = 6.6 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 172.71, 160.51, 150.07, 144.36, 84.84, 84.05, 70.08,69.80, 63.53, 40.25, 33.50, 31.10, 28.34, 28.33, 24.44, 22.01, 13.91.MS (LC-ESI) m / z calculated for C17H26IN2O6 [M+H] 481.08, found 503.25 [M+Na].MS (MALDI-TOF) m / z calculated for C17H26IN2O6 [M+H] 481.0836, found 502.8090[M+Na]. P6655PC00 79 tetra 2-yl)methyl octanoate (stannyl-IUdR-C8, Bu3Sn-IUdR- 1H NMR (400 MHz, Chloroform-d1) δ 8.04 (s, 1H, NH), 7.10 (s, 1H), 6.14 (t, J = 6.7 Hz,1H), 4.31 (s, 1H, OH), 4.28 (dd, J = 12.1, 4.8 Hz, 1H), 4.19 (dd, J = 12.1, 3.9 Hz, 1H),4.06 (q, J = 3.9 Hz, 2H), 2.43 – 2.32 (m, 1H), 2.27 (t, J = 7.6 Hz, 3H), 2.14 (dt, J = 13.8,6.9 Hz, 1H), 1.59 – 1.37 (m, 18H), 1.03 – 0.93 (m, 7H), 0.84 (dt, J = 12.0, 7.1 Hz, 30H).13C NMR (101 MHz, cdcl3) δ 174.00, 166.30, 150.97, 143.92, 113.35, 86.31, 84.76,72.45, 64.27, 40.85, 34.56, 32.10, 29.59, 29.42, 25.33, 23.04, 14.15, 10.35. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 8.37 (s, 1H), 6.07 (t, J = 6.5 Hz, 1H), 4.22(q, J = 4.4 Hz, 1H), 3.77 (q, J = 3.3 Hz, 1H), 3.57 (qd, J = 11.9, 3.3 Hz, 2H), 2.25 (t, J =7.4 Hz, 2H), 2.10 (t, J = 5.9 Hz, 3H), 1.48 (p, J = 7.1 Hz, 2H), 1.21 (s, 14H), 0.83 (t, J =6.6 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ 173.33, 160.45, 160.34, 150.08, 145.01, 87.52, 84.65,69.99, 69.18, 60.80, 40.18, 33.25, 31.26, 28.94, 28.83, 28.66, 28.63, 28.42, 24.40, 22.05, 13.90.MS (LC-ESI) m / z calculated for C21H34IN2O6 [M+H] 537.15, found 537.15 [M+H].MS (MALDI-TOF) m / z calculated for C21H34IN2O6 [M+H] 537.1462, found 536.9480[M+H]. dioxo-5- -3- furan-2- dodecanoate IUdR-C12, or Bu3Sn-IUdR- TLC (5% MeOH in DCM) Rf = 0.34 P6655PC00 80 2,4-dioxo-3,4- stearate 1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 7.93 (s, 1H), 6.06 (t, J = 6.7 Hz, 1H), 5.36(d, J = 4.3 Hz, 1H), 4.18 (d, J = 4.3 Hz, 2H), 4.15 (s, 1H), 3.92 (q, J = 4.3 Hz, 1H), 2.35(t, J = 7.4 Hz, 2H), 2.23 – 2.05 (m, 2H), 1.51 (t, J = 7.4 Hz, 2H), 1.20 (m, 28H), 0.82 (t, J= 6.9 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 173.16, 160.88, 150.46, 144.85, 85.37, 84.58, 70.58,70.20, 63.97, 33.94, 31.72, 29.45, 29.43, 29.39, 29.30, 29.13, 29.11, 28.83, 24.88, 22.53, 14.39.MS (LC-ESI) m / z calculated for C27H46IN2O6 [M+H] 621.24, found 621.25 [M+H], 643.25[M+Na].MS (MALDI-TOF) m / z C27H46IN2O6 [M+H] 621.2401, found 621.6948 [M+H], 643.1906[M+Na]. stearate (stannyl-IUdR-C18, or Bu3Sn-IUdR- TLC Rf = 0.37 (5% MeOH in DCM).1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 7.93 (s, 1H), 6.06 (t, J = 6.7 Hz, 1H), 5.36(d, J = 4.3 Hz, 1H), 4.18 (d, J = 4.5 Hz, 3H), 4.15 (s, 0H), 3.92 (q, J = 4.2 Hz, 1H), 2.35(t, J = 7.4 Hz, 2H), 2.23 – 2.05 (m, 2H), 1.51 (t, J = 7.1 Hz, 2H), 1.20 (d, J = 3.9 Hz, 34H),0.82 (t, J = 6.9 Hz, 5H).13C NMR (101 MHz, DMSO-d6) δ 173.16, 160.88, 150.46, 144.85, 85.37, 84.58, 70.58,70.20, 63.97, 40.64, 33.94, 31.72, 29.45, 29.43, 29.39, 29.30, 29.13, 29.11, 28.83, 22.53, 14.39. P6655PC00 81 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 6.17 (t, J = 6.5 Hz, 1H), 5.30 (t, J = 6.3 Hz,3H), 4.29 (ddd, J = 12.9, 6.0, 3.5 Hz, 3H), 4.22 (d, J = 1.8 Hz, 2H), 4.13 (s, 3H), 4.10 –4.04 (m, 1H), 3.94 – 3.83 (m, 2H), 3.59 (s, 2H), 3.45 – 3.31 (m, 8H), 3.22 (dq, J = 11.4,6.7, 5.7 Hz, 3H), 2.36 – 2.04 (m, 6H), 2.01 – 0.98 (m, 24H), 0.94 (s, 2H), 0.86 (d, J = 6.4Hz, 2H), 0.84 (s, 6H), 0.62 (s, 6H).13C NMR (101 MHz, CDCl3) δ 170.50, 144.39, 140.32, 140.23, 122.33, 122.18, 85.92,85.02, 80.36, 80.19, 77.48, 77.16, 76.84, 71.94, 67.80, 65.70, 63.73, 56.78, 56.18, 50.16, 48.90, 42.35, 39.55, 36.22, 35.82, 33.81, 32.55, 31.90, 30.69, 28.05, 26.23, 25.65, 25.63, 24.94, 23.85, 22.82, 22.56, 18.72, 11.87.MS (LC-ESI) m / z calculated for C38H58IN2O7 [M+H] 781.3289, found 781.25 [M+H]. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, J = 6.5 Hz, 1H), 8.00 (s, 1H), 6.25 (t, J = 6.2 Hz,1H), 4.50 (dd, J = 12.5, 3.4 Hz, 1H), 4.40 (s, 1H), 4.30 (dd, J = 12.5, 3.0 Hz, 1H), 4.19(q, J = 3.5 Hz, 1H), 2.48 (ddt, J = 23.8, 16.1, 7.7 Hz, 3H), 2.19 (dt, J = 13.3, 6.4 Hz, 1H),1.69 (p, J = 7.5 Hz, 2H), 1.28 (s, 13H), 0.90 (t, J = 6.9 Hz, 4H).13C NMR (151 MHz, CDCl3) δ 173.60, 164.73, 149.29, 144.07, 85.68, 84.72, 71.07,63.03, 41.10, 34.30, 31.93, 29.68, 29.66, 29.62, 29.47, 29.36, 29.29, 29.15, 24.90, 22.70, 14.13. LCMS (ESI) calculated for C35H65N2O6Sn exact mass: 729.3865 Da, found: 729.38 Da P6655PC00 82 TLC (10% MeOH in DCM) Rf = 0.551H NMR (600 MHz, CDCl3) δ 7.83 (s, 1H), 6.05 (t, J = 6.5 Hz, 1H), 4.20 (dd, J = 12.5, 3.8Hz, 1H), 4.17 – 4.09 (m, 3H), 3.97 (q, J = 3.5 Hz, 1H), 2.28 (dt, J = 16.3, 8.2 Hz, 2H),2.11 (t, J = 7.6 Hz, 2H), 1.94 (dt, J = 13.6, 6.6 Hz, 2H), 1.48 (q, J = 7.6 Hz, 2H), 1.08 (s,26H), 0.70 (t, J = 7.0 Hz, 3H).13C NMR (151 MHz, CDCl3) δ 177.75, 164.87, 154.21, 148.32, 89.68, 88.66, 74.43,72.41, 67.45, 44.78, 38.17, 35.76, 33.52, 33.48, 33.45, 33.31, 33.19, 33.13, 32.97, 28.73, 26.50, 17.78. -5-(2,4-dioxo-5- furan-2- 1H NMR (600 MHz, CDCl3) δ 7.84 (s, 1H), 7.10 (s, 1H), 6.12 (t, J = 6.7 Hz, 1H), 4.35 –4.28 (m, 3H), 4.20 (ddd, J = 18.9, 12.2, 3.4 Hz, 2H), 4.05 (q, J = 4.5 Hz, 1H), 2.36 (dtd,J = 14.0, 7.2, 4.9 Hz, 2H), 2.29 – 2.24 (m, 3H), 2.15 (dt, J = 13.5, 6.6 Hz, 1H), 1.61 –1.51 (m, 5H), 1.49 – 1.37 (m, 4H), 1.28 – 1.19 (m, 25H), 0.87 – 0.79 (m, 12H).13C NMR (151 MHz, CDCl3) δ 173.60, 149.30, 144.07, 85.68, 84.71, 71.06, 68.17, 63.03,41.09, 34.30, 31.93, 29.71, 29.69, 29.67, 29.63, 29.48, 29.37, 29.29, 29.15, 24.90, 22.70, 14.12. 2,4-dioxo-3,4- icosanoate TLC (10% MeOH in DCM) Rf = 0.651H NMR (600 MHz, DMSO) δ 11.72 (s, 1H), 7.97 (s, 1H), 6.10 (t, J = 6.7 Hz, 1H), 5.40(d, J = 4.3 Hz, 1H), 4.21 (ddd, J = 10.2, 5.7, 3.0 Hz, 3H), 3.96 (dt, J = 4.8, 3.7 Hz, 1H),2.39 (td, J = 7.3, 1.5 Hz, 2H), 2.25 – 2.11 (m, 2H), 1.55 (p, J = 7.3 Hz, 2H), 1.31 – 1.21(m, 28H), 0.86 (t, J = 7.0 Hz, 3H).13C NMR (151 MHz, DMSO) δ 173.19, 160.91, 150.50, 144.89, 85.41, 84.62, 70.62,70.22, 64.01, 33.98, 31.76, 29.50, 29.48, 29.43, 29.34, 29.17, 29.15, 28.87, 24.91, 22.56, 14.42. P6655PC00 83 Table 1. Stannylation yields and radiochemical yields Compound Stanny-IUdR yieldc RCCa RCYb[125I]IUdR 99 ± 1% (n = 4) 96 ± 2% (n = 4)[125I]IUdR-C4 55% 96 ± 4% (n = 4) 94 ± 4% (n = 4)[125I]IUdR-C8 45% 97 ± 2% (n = 4) 83 ± 2% (n = 4)[125I]IUdR-C12 66% 99 ± 1% (n = 4) 64 ± 1% (n = 4)[125I]IUdR-C16 45% 95 ± 1% (n = 2) 51 ± 1% (n = 2)[125I]IUdR-C18 61% 99 ± 1% (n = 4) 71 ± 1% (n > 25)[125I]IUdR-C20 55% 91% (n = 1) 65% (n = 1)[125I]IUdR-C22 59% 99 ± 1% (n = 4) 54 ± 5% (n = 2)[125I]IUdR-chol 35% 95 ± 2% (n = 4) 80 ± 1% (n = 2)[125I]IUdR-cholic acid 27% 61 ± 1% (n = 2) 50 ± 2% (n = 2)[125I]IUdR-IAn-C18 62% 98 ± 1% (n = 2) 95 ± 1% (n = 2)a: Radio chemical conversion (RCC) based on radio-TLC analysis. b: Radio chemicalyield (RCY) based on activity (MBq) after purification and reformulation (end ofsynthesis), and non-decay corrected (ndc).cOverall yield after multiple steps.Table 2: Hydrophobicity parameters of compounds. Compound LogDlit LogDcomp LogDmeas LogM(pH = 7.4) (pH = 7.4) (pH = 7.4) (cm s-1)[125I]IUdR -0.96 -0.60 -1.16 ± 0.05 -4.80[125I]IUdR-C4 - +0.98 +0.62 ± 0.02 -2.30[125I]IUdR-C8 - +2.76 +1.54 ± 0.05 -1.00[125I]IUdR-C12 - +4.54 +2.11 ± 0.02 -0.20[125I]IUdR-C18 - +7.21 +6.51 ± 0.07 +0.52Bis-C12 - +9.68 (+5.50)c -Bis-C18 - +15.04 (+12.80)c -[125I]IUdR-chol - (+7.10) - -LogDlit, LogDcomp and LogDmeas; are from literature, calculated, or measured, respectively.cValues between parentheses are estimated values. Bis-C12 and Bis-C18 refer tocompounds where two C12 (from lauric acid) or two C18 chains (from stearic acid) have been connected to IUdR, one to the 5’-OH and one to the 3’-OH. LogD7.4 values are taken or measured at a pH = 7.4 (PBS buffer). LogM values are calculated compound lipid-permeability (with COSMOS). P6655PC00 84Example 2: Preparation of liposomes loaded with [125I]IUdR prodrugMaterials and MethodsPreparation by pre-insertion of [125I]IUdR prodrugs. In a glass vial with a stirringmagnet, pre-mixed powder composed of HSPC:CHOL:DSPE-PEG2000 (15 mg, 3:1:1mass ratio) and [125I]IUdR-prodrugs (10 MBq, mass: 0.1 - 1 mg, moles: 0.2-2.5 µmol )were dissolved in DCM / MeOH (2:1, v / v, 1 mL) and evaporated to dryness under a flowof argon at 70 °C for 30 mins. Using the thin film hydration method, the film was rehydrated with HEPES buffer (1 mL) for 1 hour at 65 °C and extruded by an Avanti® mini-extruder with 100 nm filter for 21 times at 65 °C to form single unilamellar vesicles(SUVs). Subsequently, the mixture underwent size-exclusion separation of the prodrug-loaded liposomes from non-inserted [125I]IUdR prodrugs using a PD-10 desalting column(GE Healthcare) with HEPES buffer. The hydrodynamic diameter and size distribution ofprodrug-loaded liposomes ([125I]IUdR-CX-LIPs) were determined by dynamic lightscattering. Drug insertion efficiency (dl%) is calculated as the same as following: ^^% =^^^^^^^× 100%^^^^^^^where: Afinalis the final activity of [125I]IUdR-Cn-LIPs. Ainitialis the initial activity of [125I]IUdR-Cnprodrugs.Preparation by post-insertion of [125I]IUdR prodrugs. [125I]IUdR prodrugs in MeCN(400 µL, 2 MBq, mass: 0.01 - 1 mg, moles: 0.02-2.5 µmol) was transferred to a glass vialand evaporated to dryness under argon flow for 30 mins at 70 °C. To this mixture, thepre-made “blank” stealth liposome dispersion prepared as described above (32 mM totallipid, 1.0 mL) was added and then incubated for 6 or 12 hours at 35 °C, 45 °C, or 55 °Cunder continuous stirring. Note that the molar ratio of the prodrug to total lipid was below5% in all cases. After loading, the mixtures were passed through a PD-10 size-exclusionchromatography column (GE Healthcare) with HEPES buffer to separate the [125I]IUdRprodrug loaded liposomes from free [125I]IUdR prodrug. The hydrodynamic diameter and P6655PC00 85 size distribution of the loaded liposomes were determined by dynamic light scattering. Drug insertion efficiency (dl%) was calculated as above. The particle size (Ø) and zeta potential (ζ) of the self-assembled nanoparticles (NPs) were measured by dynamic light scattering (DLS) on a NanoBrook ZetaPALS (Brookhaven Instruments Limited, USA). Unless stated otherwise, Ø and ζ analysis were performed at 0.1 mg / mL in iso-HEPES buffer (pH 7.4) at 25°C, and were done in quintuplets. Results The results showing the drug insertion efficiency for different [125I]IUdR prodrugs and different methods are shown in Figure 1. For compounds [125I]IUdR-C12 and [125I]IUdR-C18 the insertion efficiency was high with both methods. In contrast, for [125I]IUdR-C8 and [125I]IUdR-C4, the post insertion method had poor drug insertion yields. For compound [125I]IUdR-C22, the pre-insertion method was not suitable, but similar dI% as for [125I]IUdR-C18 was achieved using the post- insertion method. Conclusion Liposomes comprising the [125I]IUdR prodrugs of the present invention anchored in the lipid bilayer were produced using two different methods. Example 3: Release of [125I]IUdR from liposomes loaded with [125I]IUdR prodrugs. Materials and MethodsLiposomes were prepared as described above. [125I]IUdR prodrugs were loaded usingthe post-insertion method.Release upon exposure to esterase. A HPLC vial containing [125I]IUdR prodrug loadedliposomes dispersed in ISO-HEPES (30 kBq – 1 MBq, 1.0 mL) were continuously stirredat 37 °C. Esterase powder (2 mg, 40 U) was dissolved in PBS (400 µL), resulting in a stock solution of 100 U / mL. Then 10 µL of esterase stock solution was added to the vial for a final concentration of 1 U / mL. A series of aliquots (5 µL) were removed at different time points (0 h, 0.5 h, 1 h, 3 h, 6 h, 1 d, 2 d, 7 d and 14 d) and then the aliquots were immediately mixed with THF (15 µL) to quench the enzymatic hydrolysis and dissolve P6655PC00 86 the liposomes, with the resulting solution then analyzed by radio-TLC. The ratios of[125I]IUdR prodrug, [125I]IUdR, and remaining unidentified spots ("others") werequantified.Release upon exposure to rat brain homogenate. To obtain brain homogenate (BH),rats were administered subcutaneous ketamine (100 mg / kg) and dexmedetomidine (0.5mg / kg) for anesthesia. When the loss of response to painful stimuli was confirmed, therats were then euthanized and their brains were promptly extracted and divided into foursections. Each section was then homogenized in 5 mL of PBS using a homogenizer. The resulting BH samples were stored at a temperature of -80 °C until further utilization.49Then BH (200 µL) was diluted 1:1 (v / v) with HEPES buffer (200 µL) in an HPLC vial.Penicillin-streptomycin solution (6 μL, 10,000 U / mL) was added, followed by [125I]IUdRprodrug loaded liposomes in HEPES buffer (200 µL, 30 kBq). Then, aliquots (5 µL) wereobtained at 0 h, 0.5 h, 1 h, 3 h, 6 h, 1 d, 2 d, 7 d and 14 days and mixed with THF (15 µL) immediately after removal to quench the enzymatic hydrolysis and dissolve theliposomes. All samples were analyzed by radio-TLC with ratios of [125I]IUdR prodrug, free[125I]IUdR and unidentified spots (‘others’) quantified. Esterase mediated release in two compartments under simulated sink conditions. A beaker with HEPES buffer (60 mL) was mixed with aq. esterase (600 µL, 100 U / mL) and stirred at 37 °C. Then a dialysis tube (MWCO 8-12 KDa) containing [125I]IUdR-C18loaded liposomes in HEPES buffer (2.0 mL, 265 kBq) was placed inside the beaker.Samples from the solution inside the dialysis tube (referred to as the "inside") and thesurrounding solution in the beaker (referred to as the "outside") were sampled at 0 h, 0.5h, 1 h, 3 h, 6 h, 1 d, 2 d, 7 d and 14 days in aliquots of 5 µL and 400 µL, respectively. The radioactivity of the samples was subsequently analyzed and calculated by liquid scintillation counting (LSC). Results The drug release using esterase is shown in Figure 2. The results show that [125I]IUdR-C8 and [125I]IUdR-C12 loaded liposomes showed a fast release with 100 % consumption of the prodrug achieved within 3 hours. For [125I]IUdR-C18 loaded liposomes, the release was slower, with 50% consumption of the prodrug achieved after 48 hours and total consumption achieved after 150 hours. It is also shown (Fig.2D) that P6655PC00 87the release from [125I]IUdR-C16 loaded liposomes was slower than the [125I]IUdR-C8 and[125I]IUdR-C12 loaded liposomes, with 44 ± 1% remaining prodrug after 2 days, and 7 ±1% after 7 days. The released [125I]IUdR from [125I]IUdR-C16-LIPs was steadily increasingand plateaued at 86 ± 3% after 7 days.Release of [125I]IUdR-C14 and [125I]IUdR-C20 willbe studied following the same experimental protocol. The drug release using rat brain homogenates is shown in Figure 3. The results confirm that endogenous tissue in the mammal brain is able to convert the prodrugs and releasethe active [125I]IUdR from the liposomes. The release from liposomes loaded with[125I]IUdR-C12 showed a fast release within a few hours, while liposomes loaded with [125I]IUdR-C18 displayed extended release over the course more than 150 hours.A two compartment experiment where [125I]IUdR-C18 loaded liposomes were keptseparated from esterase via a dialysis membrane showed that the consumption of theprodrug had a similar rate as in the one-compartment experiment. This observationsupports that hydrolysis of the prodrug takes place once the prodrug is in the medium, and not on or very near the surface of the liposome, making the affinity for the prodrug to the liposomal bilayer, and thereby the size of the lipid anchor, the key factor in the rate of [125I]IUdR release. Liposomes loaded with [125I]IUdR-C22 prodrug had a very slow release in the presenceof esterase, and the release of free [125I]IUdR could not be observed, likely due to theconsumption of free [125I]IUdR in those conditions being faster than the drug release rate.(Figure 5A). Similarly, for liposomes loaded with [125I]IUdR-C2-chol, no consumption of the prodrug was observed in the presence of esterase. In contrast, the free prodrug[125I]IUdR-C2-chol was enzymatically cleaved by esterases in solution (Figure 5B).The results highlight that there is a specific hydrocarbon chain length range where substantial drug release is achieved within 1-5 days. ConclusionThe compositions comprising prodrug loaded liposomes according to the presentdisclosure are able to produce extended release of [125I]IUdR in the presence of esterase P6655PC00 88 enzyme. Particularly, it is shown that the compositions according to the present invention are able to produce extended release in the presence of endogenous brain estearses. Prodrugs bearing hydrocarbon chains of a certain length have been identified as givingan optimal drug release for Auger emitting IUdR nucleosides in the brain. Materials and MethodsLiposomes were prepared as described above. [125I]IUdR prodrugs were loaded usingthe post-insertion method.In vitro cell viability assay. LN-229 cells were grown in DMEM growth medium with apH = 7.4, supplemented with 10% FBS, 100 U / mL of penicillin, 2 mM glutamine, and 100 μg / mL of streptomycin according to supplier instructions. Cell cultures were maintained in flasks and grown at 37 °C in a humidified atmosphere of 5% CO2 in air.In vitro cytotoxicity of [125I]IUdR-C18-LIPs against LN-229 cells was determined by a cellviability assay using CellTiter-Blue from Promega. Briefly, cells were seeded in 96-wellplates, 300 cells / well, and incubated for 24 hours at 37 °C in an incubator. Samples weregrouped to evaluate cytotoxicity as (1) [125I]IUdR-C18-LIPs with / without esterase, (2) Free[125I]IUdR-C18prodrug with / without esterase, (3) Free [125I]IUdR, and (4) Free non- radioactive IUdR-C18prodrug with / without esterase. Appropriate amounts of [125I]IUdR-C18-LIPs, [125I]IUdR-C18 (dissolved in DMSO) and [125I]IUdR were diluted with completeDMEM culture medium to achieve the desired final radioactivity concentration of 0.23,0.49, 0.98, 1.97, 3.75, 7.5, 15, and 30 kBq / mL. The concentrations (µg / mL) of thenegative control groups containing non-radiolabelled IUdR-C18-LIPs and IUdR-C18prodrug were the same as the corresponding radiolabelled groups. All groups with addedesterase (E) were introduced post sample preparation and maintained a consistentconcentration of 0.1 U / mL. The plates were incubated for 7 days before mixing withCellTiter-Blue solution (20 μL). After a 4 h incubation, the absorbance was recorded bymicroplate reader at 570 and 600 nm. The results were expressed as % cell viability =(mean optical density (OD) of treated cells / mean OD of untreated cells) × 100%. P6655PC00 89Clonogenic assay. Flasks with 2× 105 cells per flask were cultured and applied to[125I]IUdR-C18-LIPs with esterase (E), free [125I]IUdR or free iodine-125 for two days inradioactivity concentrations of 0, 0.125, 0.25, 0.5, 1.0, 2.0 kBq / mL, respectively. Thenthe flasks were harvested individually and reseeded into new flasks at densities of 100,100, 200, 250, 600, and 1000 cells per flask. While flasks treated with free iodine-125were seeded at 100 cells per new flask. These densities corresponded respectively tothe radioactivity concentrations above, ranging from 0 to 2 kBq / mL. The flasks wereincubated for another 14 days, after which the cells were fixed and stained with crystalviolet for 2 hours, the flasks were washed with water and air-dried. Visible coloniescontaining at least 50 cells were manually counted under a microscope. PlatingEfficiency (PE) and Surviving Fraction (SF) were defined as: PE = Number of coloniescounted / Number of cells seeded; SF = Number of colonies counted after treatment / (Number of cells seeded* PE).ResultsThe results are shown in Figure 6. The results show that liposomes loaded with[125I]IUdR-C18 prodrug ([125I]UdR-C18-LIPs) in combination with esterase has a dosis-dependent effect on viability. The groups treated with [125I]UdR-C18-LIPs in combinationwith esterase had lower viability compared to groups treated with [125I]UdR-C18-LIPswithout esterase. At high concentrations, the effect of [125I]UdR-C18-LIPs with esteraseand free [125I]IUdR on viability is comparable. Controls performed with non-radioactiveIUdR-C18 and IUdR-C18-LIPs showed no signs of cytotoxicity (data not shown).In the clonogenic assay, it was observed that treatment [125I]IUdR-C18 loaded liposomesin the presence of esterase had a similar ability to inhibit colony formation as free [125I]IUdR. Controls treated with iodine-125 did not show any decreased survival factors (Figure 7). Conclusion The compositions and prodrugs according to the present disclosure mediate cytotoxicity due to the esterase dependent release of free [125I]IUdR. P6655PC00 90Example 5: DNA incorporation of [125I]IUdR in vitro from liposomes loaded with [125I]IUdRprodrugs. Material and MethodsLiposomes were prepared as described above. [125I]IUdR prodrugs were loaded usingthe post-insertion method. LN229 cells were seeded in 24-well plates (200,000 cells / well). The following day,18.5 kBq / mL[125I]IUdR-C18-LIPs and 1U / mL esterase was added to the cells andincubated for 4, 7 or 24 h. After incubation, the cells were washed twice in PBS and twicein 5% trichloroacetic acid (TCA, Sigma-Aldrich). TCA fractions were collected todetermine unincorporated [125I]IUdR and cells were solubilized with 1 M sodiumhydroxide and harvested to determine incorporated [125I]IUdR. The amount ofunincorporated and incorporated [125I]IUdR was determined in a 2470 Wizard AutomaticGamma Counter (Perkin Elmer) and compared to total amount of added activity. Results The results show that incorporated [125I]IUdR increased over time with incorporationpercentage (IP%) of 3% at 4 hours, 5% at 7 hours and 11% at 24 hours, of total added[125I]IUdR-C18-LIPs activity (Figure 8) Conclusion The composition according to the present disclosure is able to release free [125I]IUdR which is then incorporated into the DNA of cells. Material and MethodsLiposomes were prepared as described above. [125I]IUdR prodrugs were loaded usingthe post-insertion method.Tumor injection. 11 weeks old CB17 SCID mice (Janvier) were acclimatized for aminimum of one week before used in experiments. For cell implantation, the mice were anesthetized with a mixture of hypnorm / midazolam subcutaneously. Animals were P6655PC00 91 placed in a stereotactic frame (Model 900, David Kopf Instruments, Tujunga, USA) and a midline scalp incision was made and a burr hole was made one mm anteriorly and two mm laterally to bregma. A 2 µL cell suspension of 300,000 U87 cells in Hank's Balanced Salt Solution (HBSS, Gibco) was slowly injected at a depth of 3 mm.Treatment. Sixteen days after, the mice were anaesthetized and fixated as describedabove and the midline incision was re-incised and the burr hole was identified. [125I]IUdR-C18-LIPs (25 µL, approx.9 MBq / mL) or [125I]IAn-C18-LIPs (15 MBq / mL, a mock compoundthat cannot incorporate into DNA), was injected slowly. Tumors were removed andhomogenized after 2 days and 5 days of injection, and the DNA was purified usingDNeasy Blood & Tissue Kit (Qiagen). The standard protocol from the kit was followed.SPECT / SCT scans were conducted only with [125I]IUdR-C18-LIPs (approx. 9 MBq / mL)after 1, 48 and 120 hours of injection, animals were euthanized before scan due to thelow amount of activity. ResultsThe results show that free [125I]IUdR was incorporated in vivo into DNA from [125I]IUdR-C18 loaded liposomes. In contrast, mock liposomes loaded with [125I]IAn-C18 did not show any incorporation into DNA (Figure 9).The biodistribution of [125I]IUdR-C18-LIPs was studied performing SPECT / CT scans(Figure 9B). [125I]IUdR-C18-LIPs showed a fast clearance from blood and normal-tissuewith a retention in brain at 1 and 48 h. Thyroid uptake was visible at 48 and 120 h, whichwas expected given that thyroid uptake was not blocked with potassium iodide. In thebrain, activity could be detected in the brain for 48 h upon injection of [125I]IUdR-C18-LIPs.ConclusionThe compositions according to the present disclosure are able to release free [125I]IUdRwhich is then incorporated into the DNA of cells in vivo. Activity in the brain after injection is retained for several days. P6655PC00 92Example 7: Ex vivo Biodistribution of activity in tumor-bearing rats upon injection ofliposomes loaded with [125I]IUdR prodrugs.Material and MethodsLiposomes were prepared as described above. [125I]IUdR prodrugs were loaded usingthe post-insertion method.Tumor injection. 5-7 weeks old male athymic nude rats (rnu / rnu) (Charles River) wereacclimatized for a minimum of one week before used in experiments. Rats were anesthetized with a mixture of hypnorm / midazolam subcutaneously. Animals were placed in a stereotactic frame (Model 900, David Kopf Instruments, Tujunga, USA) and a midline scalp incision was made and a burr hole was made one mm anteriorly and twomm laterally to bregma. A 2 µL cell suspension of 300,000 T87 cells (patient-derivedGBM cell line) in HBSS supplemented with 0.9% glucose (SAD 500 mg / mL) was slowlyinjected at a depth of 3.5 mm.Treatment. Twenty days later, the rats were anaesthetized and fixated as describedabove and the midline incision was re-incised and the burr hole was identified and 25 µLC18-[125I]IUdR-LIPs (range: 62-92 kBq, average: 82.625 kBq) was injected intracranially.Ex vivo biodistribution was performed 6 h and 24 h post-injection (p.i.). Organs weremeasured with a 2470 Wizard Automatic Gamma Counter (Perkin Elmer). To blockthyroid gland, potassium iodide (KI, Sigma-Aldrich) was added to the drinking water (1 mg / ml) from two days before treatment until euthanization. ResultsThe results show that after injection on the brain, the activity is localized and retained inthe brain. Comparison of the activity between from 6h to 24h showed that activity wasretained in the brain (left hemisphere, right hemisphere (tumor-bearing hemisphere) andcerebellum) (Figure 10). A significant decrease in uptake was found from 6 hours to 24hours in the following organs: Heart, lungs, pancreas, spleen, adrenal glands, kidneys, liver, ventricle, small intestine, large intestine (unpaired student’s t-test). ConclusionThe compositions according to the present disclosure are localized and retained in thebrain upon intracerebral injection. P6655PC00 93Example 8: Preparation of polymeric micelles loaded with [125I]IUdR prodrugsMaterial and MethodsThe [125I]IUdR prodrugs were prepared as described above.Preparation of polymeric micelles. 50 mg PLGA10k-mPEG5k was weighed anddissolved in 5 mL of DMF to achieve a 10 mg / mL solution. After stirring for 15 minutes, MilliQ water was added dropwise to achieve DMF / H2O ratios of 1:1, 1:2, 1:5, and 1:10, followed by another 15 minutes of stirring. The dispersions were then purified by dialysis with MWCO of 10 kDa.Loading of [125I]IUdR prodrugs into polymeric micelles. Into a 10 mL vial, 100 μL of[125I]IUdR prodrugs in MeCN was added and its radioactivity measured as 3 MBq. Afterevaporating the MeCN under Argon for 15 minutes, 100 μL of either PLGA5k-mPEG2kor PLGA10k-mPEG5k (both 10 mg / mL in DMF) solution was added and stirred for 10 minutes. Following the dropwise addition of 1 mL MilliQ water and 20 minutes of stirring, purification was achieved using centrifugal filters or PD-10 columns. Results Polymeric micelles with sizes ranging from 50 to 120 nm were produced. Prodrugs[125I]IUdR-C8, -C12 and -C18 were loaded into polymeric micelles. As shown in Figure 11, allprodrugs were successfully loaded into the polymeric micelles with incorporation efficiencies above 50%. Conclusion [125I]IUdR were successfully incorporated into polymeric micelles.Example 9: Release of [125I]IUdR from polymeric micelles loaded with [125I]IUdRprodrugs and in vitro cell assays.Material and MethodsRelease upon exposure to esterase. Was performed similarly as described above inExample 2.In vitro cell viability assay. Was performed similarly as described above in Example 4. P6655PC00 94 ResultsThe results for esterase release of [125I]IUdR are shown in Figure 12. The results showthat [125I]IUdR-C8 and [125I]IUdR-C12 loaded polymeric micelles showed a fast release with 100 % consumption of the prodrug achieved within 1 hour. For [125I]IUdR-C18 loaded polymeric micelles, the release was more gradual and complete consumption occurred over the course of 48h.The results of in vitro cell assays showed decrease in cell viability in cells treatedpolymeric micelles loaded with [125I]IUdR-C18 in combination with esterase compared tonon-esterase treated groups (Figure 13). Thus showing that cell survival is related to theamount of released [125I]IUdR. Negative control groups, such as free iodine-125, blank PMs and esterase were also tested and showed no significant cell killing efficacy (not shown). Conclusion This studies confirm gradual release of [125I]IUdR from [125I]IUdR prodrug loaded polymeric micelles and efficient cell killing thereafter. Thus, highlighting the ability of this drug delivery system in the treatment of intracerebral neoplasms. Material and Methods Two 6-well plates, each containing 5x105 LN229 cells per well, were incubated for 24 hours at 37°C and 5% CO2 atmosphere. Post incubation, the media was replaced with fresh media that contained either 30 kBq [125I]IUdR-C18-PMs with 0.1 U / mL esterase or a control of 30 kBq [125I]IUdR. One plate was incubated for 1 hour and the other onefor 4 hours. Finally, genomic DNA was extracted using the E.Z.N.A. tissue DNA Kit fromOmega BIO-TEK. Results The results show that there was DNA uptake of [125I]IUdR in cells treated with [125I]IUdR- C18 prodrug loaded micelles and esterase but at a slower, more gradual pace than for cells treated with equivalent activity of free [125I]IUdR prodrug. Thus, highlighting the ability of this system to more gradually release [125I]IUdR (Figure 14). P6655PC00 95 Conclusion The composition according to the present disclosure provide for a gradual release of [125I]IUdR and DNA uptake in cells. Tumor injection. 5-7 weeks old male athymic nude rats (rnu / rnu) (Charles River) wereacclimatized for a minimum of one week before used in experiments. The rats were anesthetized with a mixture of hypnorm / midazolam subcutaneously. Animals were placed in a stereotactic frame (Model 900, David Kopf Instruments, Tujunga, USA) and a midline scalp incision was made and a burr hole was made one mm anteriorly and twomm laterally to bregma. A 2 µL cell suspension of up to 300,000 T87 GBM cells(patient-derived cell line) in HBSS supplemented with 0.9% glucose (SAD 500 mg / mL)was slowly injected at a depth of 3.5 mm.Treatment. Two weeks later, the rats were anaesthetized and fixated as describedabove. The midline incision was re-incised and the burr hole was identified and 25 µLC18-[125I]IUdR-LIPs (average: 9.692 MBq, range: 8.634-10.428 MBq) (n = 4) or buffer(iso-HEPES) (n = 4) was injected slowly. To block thyroid gland, potassium iodide (KI,Sigma-Aldrich) was added to the drinking water (1 mg / ml) from two days beforetreatment until 13 days after treatment. Observation period was set to 180 days aftertreatment. Animals were euthanized when they reached pre-determined humane endpoints (neurological deficits or >20% weight-loss).After euthanization, brain was fixated and evaluated with hematoxylin & eosin (H&E)staining to evaluate morphological changes and the brain was stained for vimentin to assess human-derived brain tumor cells. To evaluate adverse effects from treatment, the rats underwent blood sampling before treatment (1 week before) and after treatment (early response: 6 weeks and late response: 12 weeks) and the following parameters were assessed: white blood cells (WBC), haemoglobin (HGB), platelets (PLT), plasma creatinine (Cr) and plasma blood urea nitrogen (BUN). P6655PC00 96 ResultsAll rats treated with C18-[125I]IUdR-LIPs survived the whole observation period of 180days after treatment, while control animals (buffer control) had a median survival of 87days (range: 68-107 days) (Figure 15A). The survival benefit of C18-[125I]IUdR-LIPs compared to buffer control was significant (log-rank p = 0.007).Post-mortem H&E histology of the brains showed that all of the control rats (buffercontrol) had large tumours, while no tumour was visible in the brain of C18-[125I]IUdR-LIPs treated rats. This was confirmed with vimentin staining of the brains. The levels of WBC, PLT, Cr and BUN were stable between groups, while HGBdecreased minimally in the C18-[125I]IUdR-LIPs treated group in the late response.Overall, no or minimal adverse side effects from the treatment with C18-[125I]IUdR-LIPswas detected from the blood samples (Figure 15B).ConclusionThe results demonstrated a significant survival benefit from C18-[125I]IUdR-LIPs treatmentcompared to control (buffer) treatment. Histological evaluation of the brain using H&Estaining and immunohistochemical staining for vimentin of the brain demonstrated largetumours in the brain of all control animals (buffer control), while no tumour was detectablein C18-[125I]IUdR-LIPs treated animals.The treatment was associated with no or minimal adverse effects on the WBC, HGB, PLT, Cr and BUN levels. Material and MethodsTumor injection. 5-7 weeks old male athymic nude rats (rnu / rnu) (Charles River) areacclimatized for a minimum of one week before used in experiments. The rats are anesthetized with a mixture of hypnorm / midazolam subcutaneously. Animals are placed in a stereotactic frame (Model 900, David Kopf Instruments, Tujunga, USA) and a midline scalp incision is made and a burr hole is made one mm anteriorly and two mm laterally to bregma. A 2 µL cell suspension of 300,000 T87 GBM cells (patient-derived cell line) P6655PC00 97 in HBSS supplemented with 0.9% glucose (SAD 500 mg / mL) is slowly injected at a depth of 3.5 mm.Treatment. Two weeks later, the rats are anaesthetized and fixated as described above.For two groups, the midline incision is re-incised, the burr hole is identified and 25 µL [123I]IUdR-C18-LIPs (up to 100 MBq) (n = 6) or vehicle (non-radioactive [127I]IUdR-C18- LIPs) (n = 6) is injected intracranially. The last group is treated with Temozolomide (first line chemotherapy treatment for GBM, n = 6) through intraperitoneal injection. Further animal groups treated with [123I]IUdR-C16-LIPs, [125I]IUdR-C16-LIPs, [123I]IUdR-C14-LIPs, or [125I]IUdR-C14-LIPs can also be included in the tests.To block the thyroid gland, potassium iodide (KI, Sigma-Aldrich) is added to the drinking water (1 mg / ml). Observation period is set to 180 days after treatment. Animals are euthanized when they reach pre-determined humane endpoints (neurological deficits or >20% weight-loss). When euthanized, the brain, thyroid gland, liver, spleen, and kidneys are extracted and formalin fixated. All tissue is hematoxylin & eosin (H&E) stained to evaluate morphological changes and the brain is stained for vimentin to assess human- derived brain tumor cells. To evaluate adverse effects from treatment, the rats will undergo blood sampling before treatment (1 week before: baseline) and after treatment (early response: 6 weeks andlate response: 12 weeks) and the following parameters will be assessed: white bloodcells (WBC), haemoglobin (HGB), platelets (PLT), plasma creatinine (Cr), plasma blood urea nitrogen (BUN), plasma T3 and plasma T4. References Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, Hawkins C, Ng HK, Pfister SM, Reifenberger G, Soffietti R, von Deimling A, Ellison DW. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol.2021 Aug 2;23(8):1231-1251. doi: 10.1093 / neuonc / noab106. PMID: 34185076; PMCID: PMC8328013. Ostrom, Q. T.; Gittleman, H.; Farah, P.; Ondracek, A.; Chen, Y. W.; Wolinsky, Y.; Stroup, N. E.; Kruchko, C.; Barnholtz-Sloan, J. S. CBTRUS Statistical Report: Primary Brain and P6655PC00 98 Central Nervous System Tumors Diagnosed in the United States in 2006-2010. Neuro-Oncology 2013, 15, 1–56. https: / / doi.org / 10.1093 / neuonc / not151.Sevastre, A. S.; Costachi, A.; Tataranu, L. G.; Brandusa, C.; Artene, S. A.; Stovicek, O.; Alexandru, O.; Danoiu, S.; Sfredel, V.; Dricu, A. Glioblastoma Pharmacotherapy: A Multifaceted Perspective of Conventional and Emerging Treatments (Review).Experimental and Therapeutic Medicine 2021, 22 (6).https: / / doi.org / 10.3892 / etm.2021.10844. Breznik, B.; Motaln, H.; Vittori, M.; Rotter, A.; Lah Turnšek, T. Mesenchymal Stem Cells Differentially Affect the Invasion of Distinct Glioblastoma Cell Lines. Oncotarget; Vol 8,No 152017.

Claims

P6655PC00 99 Claims1. A radioactive composition comprising a nanoparticle for use in a method oftreatment, prevention, or alleviation of brain cancer, wherein the nanoparticle comprises: a. a liposome, andb. a radioactive agent of formula (I):formula (I), or a pharmaceutically acceptable salt thereof, wherein, RA is 123I, or 125I ; andR consists of a C13-C17 hydrocarbon chain.

2. The radioactive composition for use according to claim 1, wherein RA is 125I.

3. The radioactive composition for use according to any one of the preceding claims,wherein RAis123I.

4. The radioactive composition for use according to any one of the preceding claims,wherein RAis not of natural abundance.

5. The radioactive composition for use according to any one of the preceding claims,wherein all elements of formula (I) are of natural abundance except for RAwhich is not of natural abundance.

6. The radioactive composition for use according to any one of the preceding claims,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 2% or more, 5% or more, 10% ormore, 20% or more, 50% or more, 75% or more, 90% or more, or 95% or more.P6655PC00 1007. The radioactive composition for use according to any one of the preceding claims,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95% or more.

8. The radioactive composition for use according to any one of the preceding claims,wherein the level of isotope enrichment in the compound of formula (I) withisotopes that are not of natural abundance is 95%, 96%, 97%, 98%, 99% or 100%.

9. The radioactive composition for use according to any one of the preceding claims,wherein R consists of a C13, C14, C15, C16, or C17 hydrocarbon chain.

10. The radioactive composition for use according to any one of the preceding claims,wherein R consists of a saturated hydrocarbon chain.

11. The radioactive composition for use according to any one of the preceding claims,wherein R does not comprise a double bond.

12. The radioactive composition for use according to any one of the preceding claims,wherein R comprises 1, 2, or 3 double bonds.

13. The radioactive composition for use according to any one of the preceding claims,wherein R comprises 1 or 2 double bonds.

14. The radioactive composition for use according to any one of the preceding claims,wherein R comprises 1 double bond.

15. The radioactive composition for use according to any one of the preceding claims,wherein each instance of a double bond has a configuration independently selected from cis or trans or wherein each instance of a double bond has a cis configuration.

16. The radioactive composition for use according to any one of the preceding claims,wherein R consists of a linear hydrocarbon chain.

17. The radioactive composition for use according to any one of the preceding claims,wherein R does not comprise a branch.

18. The radioactive composition for use according to any one of the preceding claims,wherein R consists of a C13-C17, a C14-C17, or a C15-C17 linear hydrocarbon chain.

19. The radioactive composition for uses according to any one of the preceding claims,wherein R is -(CH2)16-CH3, -(CH2)15-CH3,P6655PC00 101 -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, tridecen-1-yl, tetradecen-1-yl ,pentadecen-1-yl, hexadecen-1-yl, heptadecen-1-yl, (8Z, 11Z, 13Z)-heptadeca-8,11,14-trien-1-yl, (8Z,11Z)-heptadeca-8,11-dien-1-yl, or (5Z,8Z,11Z)-heptadeca-5,8,11-trien-1-yl.

20. The radioactive composition for use according to any one of the preceding claims,wherein R is -(CH2)12-16-CH3, such as -(CH2)13-16-CH3, -(CH2)14-16-CH3, -(CH2)15-16- CH3, -(CH2)12-15-CH3, or -(CH2)12-14-CH3.

21. The radioactive composition for use according to any one of the preceding claims,wherein R is -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3,or -(CH2)12-CH3, .

22. The radioactive composition for use according to any one of the preceding claims,wherein R is –(CH2)16-CH3.

23. The radioactive composition for use according to any one of the preceding claims,wherein R is –(CH2)15-CH3.

24. The radioactive composition for use according to any one of the preceding claims,wherein R is –(CH2)14-CH3.

25. The radioactive composition for use according to any one of the preceding claims,wherein R is –(CH2)13-CH3.

26. The radioactive composition for use according to any one of the preceding claims,wherein R is –(CH2)12-CH3.

27. The radioactive composition for use according to any one of the preceding claims,wherein the radioactive agent isP6655PC00 102 , ,28. The radioactive composition for use according to any one of the preceding claims,wherein the radioactive agent has: a. a distribution coefficient (logD) from 2.0 to 13.0, such as 3.0 to 10.0, such as4.0 to 7.0 at room temperature, e.g. as measured by partitioning method in octanol-buffer at pH 7.4 ; and / or b. a lipid-permeability coefficient (logM) from -0.5 to 1.0, e.g. as calculated bysolution thermodynamic calculations in a quantum chemical software package.

29. The radioactive composition for use according to any one of the preceding claims,wherein the liposome comprises one or more lipid bilayer(s).

30. The radioactive composition for use according to any one of the preceding claims,wherein the liposome comprises a unilamellar lipid bilayer or more than one multilamellar lipid bilayers.

31. The radioactive composition for use according to any one of the preceding claims,wherein the liposome is a single unilamellar vesicle (SUV).P6655PC00 10332. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises a phospholipid.

33. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises: i. a phospholipid, and ii. cholesterol, ergosterol, or sitosterol.

34. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises: i. a phospholipid; ii. cholesterol, ergosterol, or sitosterol; and iii. an amphiphilic derivative of poly(ethylene glycol).

35. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises a phospholipid, cholesterol, and an amphiphilic derivative of poly(ethylene glycol).

36. The radioactive composition for use according to any one of the preceding claims,wherein the phospholipid is selected from the group consisting of: a fatty acid ester derivative of sn-glycero-3-phosphocholine, a fatty acid ester derivative of sn-glycero- 3-phosphoserine, a fatty acid ester derivative of sn-glycero-3-phoshpoglycerol, and a fatty acid derivative of sn-glycero-3-phosphoinositol.

37. The radioactive composition for use according to any one of the preceding claims,wherein the phospholipid is according to any one of formula (IIa)-(IIg):P6655PC00 104 formula (IId),formula (IIe),formula (IIg), or a salt thereof, wherein each of RPa, RPa’, RPb, RPb’, RPc, RPc’, RPd, RPd’, RPe, RPe’, RPf, RPf’, RPg, andRPg’are independently the corresponding group resulting from forming an ester of a C12-C22 fatty acid with the corresponding O atom in formulas (IIa)-(IIg).

38. The radioactive composition for use according to any one of the preceding claims,wherein the phospholipid is hydrogenated soy phosphatidylcholine (HSPC), bis(monoacyglycero)phosphate (BSMP), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero- 3-phosphoglycerol (DPPG), 1,2-stearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn- glycero-3-phosphoglycerol (DOPG), 1,2-Dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-stearoyl-sn- glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),, 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn- glycero-3-phosphate (DPPA), 1,2-stearoyl-sn-glycero-3-phosphate (DSPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1,2-dioleoyl-sn-glycero-3- phosphate (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-stearoyl-sn-glycero-3- phospho-L-serine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS),, 1,2-dimyristoyl-sn-P6655PC00 105 glycero-3-phospho-(1’-myo-inositol) (DMPI), 1,2-dipalmitoyl-sn-glycero-3-phospho- (1’-myo-inositol) (DPPI),, 1,2-stearoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DSPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (POPI), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI),N-palmitoylsphingosylphosphorylcholine (C16SM), N-stearoylsphingosylphosphorylcholine (C18SM), N-oleoylsphingosylphosphorylcholine (C18:1SM), or any combination thereof.

39. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises a sphingolipid, such as N-palmitoyl-D-erythro- sphingosine (Ceramide 16:0), N-stearoyl-D-erythro-sphingosine (Ceramide 18:0), N-oleoyl-D-erythro-sphingosine (Ceramide 18:1).

40. The radioactive composition for use according to any one of the preceding claims,wherein the amphiphilic derivative of poly(ethylene glycol) comprises a poly(ethylene glycol) moiety of average molecular weight between 200-20,000 g / mol covalently linked to a hydrophobic component which allows it to be anchored to a lipid bilayer.

41. The radioactive composition for use according to any one of the preceding claims,wherein the amphiphilic derivative of poly(ethylene glycol) is according to formulaformula (III), wherein, RPrepresents -H, -CH3, a C1-C5alkyl, a C1-C5hydroxyalkyl, a C1-C5alkylamine, RXrepresent an optional linker; RHrepresents a hydrophobic group which allows anchoring to a lipid bilayer.

42. The radioactive composition for use according to any one of the preceding claims,wherein: i. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative orP6655PC00 106 RHcomprises a C10-C22 fatty acid ester derivative or a C10-C22 fatty acid amide derivative, or ii. the hydrophobic component of the amphiphilic poly(ethylene glycol) derivative or RHcomprises a phospholipid selected from the group consisting of any one of the phospholipids described in claims 36 to 38.

43. The radioactive composition for use according to any one of the preceding claims,wherein the amphiphilic derivative of poly(ethylene glycol) is any derivative of poly(ethylene glycol) wherein the poly(ethylene glycol) is covalently linked to: i. a C10-C22fatty acid, optionally via a linker, or ii. any phospholipid described in any one of claims 36 to 38 , optionally via a linker;wherein the amphiphilic derivative of poly(ethylene glycol) has a molecular weight average from 300 to 20,000 g / mol.

44. The radioactive composition for use according to any one of the preceding claims,wherein the amphiphilic derivative of poly(ethylene glycol) is DSPE-PEG (CAS nr.247925-28-6), DMG-PEG (CAS nr.160743-62-4), DPPE-PEG (CAS nr.205494-72- 0), or DOPE-PEG (CAS nr. 474922-90-2), or a salt thereof, where the amphiphilic derivative of poly(ethylene glycol) has a molecular weight average from 1000 to 20,000 g / mol.

45. The radioactive composition for use according to any one of the preceding claims,wherein the lipid bilayer comprises: a. the phospholipid as defined in any one of claims 36 to 38;b. cholesterol; andc. the amphiphilic derivative of poly(ethylene glycol) as defined in any one ofclaims 40 to 44;wherein the mass ratio between components a:b:c is: 2-4:0.5-1.5:0.5-1.5, such as about 3:1:1.

46. The radioactive composition for use according to any one of the preceding claims,wherein the radioactive agent is anchored to the liposome through non-covalent hydrophobic interaction with the lipid bilayer.P6655PC00 10747. The radioactive composition for use according to any one of the preceding claims,wherein the nanoparticle has an average hydrodynamic diameter of: 20 nm to 150 nm, such as an average hydrodynamic diameter of 80 to 120 nm, such as an average hydrodynamic diameter of 80, 90, 100, 110, or 120 nm, e.g. as measured by dynamic light scattering (DLS).

48. The radioactive composition for use according to any one of the preceding claims,wherein the liposome has a neutral, positive, or negative net charge, e.g. as determined by Z-potential measurements.

49. The radioactive composition for use according to any one of the preceding claims,wherein the liposome has a neutral or negative net charge.

50. The radioactive composition for use according to any one of the preceding claims,wherein the brain cancer is a brain tumor or an intracerebral neoplasm.

51. The radioactive composition for use according to any one of the preceding claims,wherein the brain tumor or intracerebral neoplasm involves glial cells.

52. The radioactive composition for use according to any one of the preceding claims,wherein the brain tumor or intracerebral neoplasm is a glioma.

53. The radioactive composition for use according to any one of the preceding claims,wherein the intracerebral neoplasm is a high-grade glioma, i.e. grade III or grade IVglioma.

54. The radioactive composition for use according to any one of the preceding claims,wherein the glioma is astrocytoma, glioblastoma, diffuse midline glioma, diffusehemispheric glioma, or diffuse pediatric-type high-grade glioma.

55. The radioactive composition for use according to any one of the preceding claims,wherein the composition is administered by direct intracerebral administration or by intrathecal administration.

56. The radioactive composition for use according to any one of the preceding claims,wherein the composition is formulated or provided in an isotonic saline buffer or PBS buffer.P6655PC00 10857. The radioactive composition for use according to any one of the preceding claims,wherein the composition is administered by convection-enhanced delivery (CED).

58. The radioactive composition for use according to any one of the preceding claims,wherein the infusion rate is adjusted to a level that ensures sufficient delivery of the nanoparticles, while avoiding adverse effects resulting from increased intracranial pressure, for example, the infusion rate is from 0.1 to 5.0 mL / hour.

59. The radioactive composition for use according to any one of the preceding claims,wherein the infusion rate is from 0.1 µL / min per catheter to 1000 µL / min per catheter.

60. The radioactive composition for use according to any one of the preceding claims,wherein the composition is administered in one or more fractions, such as in 1 to 20 fractions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fractions.

61. The radioactive composition for use according to any one of the preceding claims,wherein the composition comprises 1kBq to 50 GBq of the radioactive agent.

62. The radioactive composition for use according to any one of the preceding claims,wherein a further chemotherapeutic agent is administered.

63. The radioactive composition for use according to any one of the preceding claims,wherein the further chemotherapeutic agent is administered enterally or parenterally.

64. The radioactive composition for use according to any one of the preceding claims,wherein the nanoparticle comprises:a. a liposome as defined in any one of claims 29 to 49; andb. a radioactive agent according to formula (I),P6655PC00 109or a pharmaceutically acceptable salt thereof, wherein, RAis123I or125I; and R is –(CH2)12-16-CH3, preferably R is -–(CH2)14-16-CH3, most preferably R is -(CH2)16-CH3, or -–(CH2)14-CH3;wherein the radioactive agent is anchored to the liposome through non-covalent hydrophobic interaction.

65. The radioactive composition for use according to any one of the preceding claims,wherein at least 10% of the radioactive agent is present in the nanoparticle in aqueous solutions in the presence of esterase for at least 3 hours, such as at least 20%, 30%, 40% or 50% of the radioactive agent is present in the nanoparticles in the presence of esterase for at last 3 hours, such as 6 hours, such as 12h, such as 24h, such as 48 h, such as 72, hours, such as 96 hours, such as 120 hours.

66. The radioactive composition for use according to any one of the preceding claims,wherein at least 10% of the radioactive agent is present in the nanoparticles 3 hours after administration, such as at least 20%, 30%, 40%, or 50% of the radioactive agent is present in the nanoparticles 3 hours after administration.

67. The radioactive composition for use according to any one of the preceding claims,wherein at least 50% of the radioactive agent is present in the nanoparticles 24 hours after administration.

68. The radioactive composition for use according to any one of the preceding claims,wherein at least 20% or at least 10% of the radioactive agent is present in the nanoparticles 96 hours after administration.P6655PC00 11069. The radioactive composition for use according to any one of the preceding claims,wherein the activity of the radioisotope in the intracranial compartment can be detected for at least 6h, such as at least 24h, such as at least 48h, such as at least 72h, such as at least 96h, such as at least 120h.

70. The radioactive composition for use according to any one of the preceding claims,wherein the biological half-life of activity of the radioisotope in the intracranial compartment is increased compared to the biological half-life of an equivalent amount of activity of the free radiohalogen derivative of 2’-deoxyuridine.

71. The radioactive composition for use according to any one of the preceding claims,wherein the biological half-life of activity of the radioisotope in the intracranial compartment is higher than 100 min, such as higher than 200 min, such as higher than 5 hours.

72. The radioactive composition for use according to any one of the preceding claims,wherein the volume of distribution of the activity of the radioisotope upon intracerebral administration is increased compared to the volume of distribution of an equivalent amount of activity of the free radiohalogen derivative of 2’-deoxyuridine.

73. A radioactive agent according to formula (I):formula (I), or a pharmaceutically acceptable salt thereof, wherein, RA is 123I, 124I, 125I, or 131I I; andR consists of a C13-C17hydrocarbon chain.P6655PC00 11174. The radioactive agent according to claim 73, wherein the radioactive agent is asdefined in any one of claims 1 to 28.

75. The radioactive agent according to any one of claims 73 to 74 , wherein RA is 123Ior 125I.

76. The radioactive agent according to any one of claims 73 to 75, wherein Ris -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3, .

77. The radioactive agent according to any one of claims 73 to 76, wherein Ris -(CH2)16-CH3.

78. The radioactive agent according to any one of claims 73 to 76, wherein Ris -(CH2)15-CH3.

79. The radioactive agent according to any one of claims 73 to 76, wherein Ris -(CH2)14-CH3.

80. The radioactive agent according to any one of claims 73 to 76, wherein Ris -(CH2)13-CH3.

81. The radioactive agent according to any one of claims 73 to 76, wherein Ris -(CH2)12-CH3.

82. A radioactive composition comprising a nanoparticle, the nanoparticle comprising:a. a liposome, andb. a radioactive agent of formula (I):formula (I), or a pharmaceutically acceptable salt thereof, wherein,P6655PC00 112 RA is 123I, 124I, 125I, or 131I; andR consists of a C13-C17 hydrocarbon chain.

83. The radioactive composition according to claim 82 wherein the liposome is asdefined in any one of claims 29 to 49.

84. The radioactive composition according to any one of claims 82 to 83, wherein theradioactive agent, is as defined in any one of claims 1 to 28.

85. The radioactive composition according to any one of claims 82 to 84, wherein RAis 123I or 125I.

86. The radioactive composition according to any one of claims 82 to 85, wherein Ris -(CH2)16-CH3, -(CH2)15-CH3, -(CH2)14-CH3, -(CH2)13-CH3, -(CH2)12-CH3,.< / sub>87. The radioactive composition according to any one of claims 82 to 86, wherein R is–(CH2)16-CH3.

88. The radioactive composition according to any one of claims 82 to 86, wherein R is–(CH2)15-CH3< / sub>.

89. The radioactive composition according to any one of claims 82 to 86, wherein R is–(CH2)14-CH3< / sub>.

90. The radioactive composition according to any one of claims 82 to 86, wherein R is–(CH2)13-CH3< / sub>.

91. The radioactive composition according to any one of claims 82 to 86, wherein R is–(CH2)12-CH3< / sub>.

92. A kit comprising:a. a radioactive agent as defined in any one of claims 73 to 81 in a firstcontainer; and b. a composition comprising liposomes in a second container; andc. optionally an outer package.

93. The kit according to claim 82, wherein the liposomes are as defined in any oneof claims 29 to 49.P6655PC00 11394. The kit according to any one of claims 92 to 93, further comprising a containerwith a buffer.

95. The kit according to any one of claims 92 to 94, further comprising a sizeexclusion chromatography column.

96. A method of preparing a composition comprising a radioactive nanoparticle, themethod comprising the steps of: a. providing a radioactive agent as defined in any one of claims 73 to 81;b. providing a composition comprising liposomes in an aqueous medium;c. mixing the radioactive agent and the composition comprising liposomes,thereby obtaining an anchoring of the radioactive agent on the liposomes; and d. purifying the composition in c. from non-anchored radioactive agent,thereby obtaining a composition comprising radioactive nanoparticles.

97. The method according to claim 96, wherein the radioactive agent in step a. issubstantially free of water, such as having less than 10% water, such as less than 5% water, such as less than 1% water.

98. The method according to any one of claims 96 to 97, wherein the liposomes instep b. are as defined in any one of claims 29 to 49.

99. The method according to any one of claims 96 to 98, wherein the liposomes arein an aqueous buffer.

100. The method according to any one of claims 96 to 99, wherein the purifying in stepd. is performed by size exclusion chromatography.

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