Intrathecal nanoparticle delivery for the treatment of leptomeningeal tumors using core-shell particles made of hyperbranched polyglycerol and polylactic acid

Abstract

Bioadhesive and biodegradable polymeric nanoparticles can be administered intrathecally, for example, via the cisterna magna, into the spinal column to allow widespread distribution for the treatment of tumors such as leptomeningeal metastases. The nanoparticles rapidly spread to all cerebrospinal fluid (CSF) compartments, including the brain parenchyma and spinal column. The bioadhesive nanoparticles penetrate and are retained for long periods of time, during which time they can continue to release drugs. These nanoparticles can be loaded with different therapeutic, preventive, or diagnostic agents, most preferably DNA repair inhibitors, to enhance the killing of leptomeningeal tumors, such as leptomeningeal metastases, and disseminated tumors, such as medulloblastoma. In a preferred embodiment, patients are treated with a combination of a PARP inhibitor and temozolomide.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 383,211, filed November 10, 2022, which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under CA149128 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION This technology generally relates to formulations for the treatment of brain cancer by direct intrathecal administration into the cerebrospinal fluid. [Background technology]

[0004] Background of the Invention Medulloblastoma (MB) is the most common malignant brain tumor in children. It is a malignant brain tumor that begins in the lower back part of the brain, called the cerebellum. The cerebellum is involved in muscle coordination, balance, and movement. Medulloblastoma tends to spread to other areas around the brain and spinal cord via cerebrospinal fluid (CSF), the fluid that surrounds and protects the brain and spinal cord. This tumor rarely spreads to other parts of the body. Medulloblastoma can occur at any age, but it occurs most often in young children. Although rare, medulloblastoma is the most common cancerous brain tumor in children.

[0005] Treatment for medulloblastoma usually involves surgery followed by radiation or chemotherapy, or both. Age and overall health, tumor subtype and location, tumor grade and extent, and other factors influence treatment decisions. Options include surgery to remove the medulloblastoma. However, because medulloblastoma forms deep within the brain near vital structures, it may not be possible to completely remove the tumor. All patients with medulloblastoma require additional treatment after surgery to target remaining cells.

[0006] Craniospinal radiation therapy (CSI) is another option. Pediatric or adult radiation oncologists administer radiation therapy to the brain and spinal cord using high-energy beams, such as X-rays or protons, to kill cancer cells. While standard radiation therapy can also be used, proton therapy delivers a higher targeted dose of radiation to brain tumors while minimizing radiation exposure to nearby healthy tissue. Proton therapy offers the greatest survival benefit, but IQ declines by 30–40% at an average dose of 1000 mGy and further at 10 Gy or more. The average CSI is 23–36 Gy. Furthermore, 40% of children experience tumor recurrence, with a 5-year survival rate of only 60%. CSI causes DNA damage in all cells, not just tumor cells.

[0007] Chemotherapy is another option. Typically, children and adults with medulloblastoma are given these drugs by injection into a vein (intravenous chemotherapy). Chemotherapy may be recommended after surgery or radiation therapy, or in certain cases, at the same time as radiation therapy. In some cases, stem cell rescue (a stem cell transplant using the patient's own stem cells) may be used after high-dose chemotherapy.

[0008] Leptomeningeal spread commonly occurs in multiple subsets of pediatric central nervous system tumors and as metastases from solid tumors in adults. Current treatment regimens have undesirable side effects and do not significantly improve leptomeningeal prognosis. While cells harboring cerebrospinal fluid (CSF) and leptomeningeal cancers can be treated, severe late effects on cognition result from exposure of the brain parenchyma to radiation therapy. However, because the most common sites of recurrence are the CSF and leptomeninges, direct delivery of therapeutic agents to these sites can reduce brain and tissue toxicity. Intrathecal delivery has the advantage of avoiding the blood-brain barrier and limiting systemic normal tissue toxicity, but intrathecally delivered drugs are cleared too rapidly from the CSF to be effective. Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Polymeric nanoparticles (NPs) are administered intrathecally (via the cisterna magna) to deliver radiosensitizers or other chemotherapeutic agents and / or diagnostic / imaging agents to the central nervous system for the treatment of cancers such as medulloblastoma and leptomeningeal tumors. Fluorescently labeled NPs, when injected into the cisterna magna, rapidly spread to all CSF compartments, including the brain parenchyma and spinal column. Bioadhesive nanoparticles can also penetrate and be retained for long periods, during which time they can continue to release drugs. These nanoparticles can be loaded with different DNA repair inhibitors to enhance the killing of leptomeningeal tumors (such as leptomeningeal metastases) and disseminated tumors (such as medulloblastoma).

[0010] The NPs are made of hyperbranched polyglycerol-polyhydroxy acid polymers with bioadhesive properties, which allow them to selectively bind to tumor cells compared to normal microglia and neurons. A comparison of the zeta potential (mV) of bioadhesive HPG NPs ("BNPs") with amine groups on their surfaces demonstrated that BNPs have a significantly more negative potential (-28.4 mV for nonadhesive PLA-HPG NPs ("NNPs") versus -47.2 mV for PLA-HPG BNPs). Low-molecular-weight compounds, such as the imaging agent DFO, can be conjugated to the BNP surface, altering their stability or bioadhesion. The loading capacity of small molecule drugs ranged from 3% to 5%, depending on the drug-to-polymer or solvent ratio.

[0011] The results described herein demonstrate the use of a PLA-HPG NP platform that exhibits prolonged retention in the subarachnoid space, in contrast to the fate of small molecules administered in the free state. Long-term retention of NPs in the CSF space of tumor-free mice, as well as preferential accumulation and retention in CSF-adjacent tumors, was demonstrated using PET / CT and fluorescent whole-body imaging. Increased accumulation of nanoparticles in tumors early in the circulation reduces the probability of clearance by the mononuclear cell phagocytic and renal systems.

[0012] The vast majority of polymeric NPs exhibit significant absorption in the spleen and clearance organs, such as the liver and kidney, potentially limiting their therapeutic applications. The results demonstrated that PLA-HPG NPs retain their ideal properties even in the CSF space by forming a steric barrier around the NPs, extending their circulation time and allowing for greater accumulation at tumor sites. Significant accumulation of NPs at tumor sites in the meninges and cerebellum of tumor-bearing mice was also observed. Prolonged retention of NPs through controlled drug release could lead to extended drug exposure at tumor sites and provide a means to improve the overall drug half-life after intra-CSF administration. Furthermore, activity in the CNS was at least 75% of the total activity at all measured time points, significantly reducing the risk of widespread systemic toxicity.

[0013] PARP inhibitors are limited by BBB penetration and widespread toxicity. While PARPi were initially developed to sensitize tumor cells to conventional DNA-damaging agents, increasing evidence indicates that PARPi are effective in sensitizing cells to radiation therapy, temozolomide, and topoisomerase poisons and inhibitors. Talazoparib (BMN-673) is a potent PARP1 scavenger but is limited by its inability to evade the BBB in meaningful amounts. Studies with BMN-673, the first known preclinical study of intrathecal delivery of a PARPi, demonstrated unacceptable levels of toxicity with both the free drug alone and in combination with TMZ. Nanoencapsulation significantly improves the therapeutic index of BMN-673. Significantly higher doses (10x) were possible with lower systemic toxicity compared to the free drug, as measured by blood counts, weight loss, and organ toxicity. Single-agent efficacy in an orthotopic model of MB was measured at equitoxic doses of either BMN-673 or BMN-NP. Only encapsulated BMN-NPs resulted in consistent tumor regression and an overall reduction in leptomeningeal metastases. Furthermore, BMN-NPs were administered with low-dose TMZ, and the combination produced durable responses and was well tolerated by mice. This integrated therapeutic approach offers novel opportunities for PARPi combination therapy without compromising tolerability. Furthermore, this approach may offer promising therapeutic avenues for other diseases associated with widespread leptomeningeal metastases, such as leptomeningeal metastases from primary malignancies, such as lung cancer, breast cancer, and melanoma. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1A is a schematic cross-sectional view of the brain showing the skull, blood vessels, cancer cells, cerebrospinal fluid (CSF), and the brain. Figure 1B is a schematic cross-sectional view of the periarterial space where NPs are dispersed. These images show the location of the dye-loaded nanoparticles injected and distributed within the cisterna magna of the subarachnoid space. CSF drains through both the arachnoid granulations and lymphatic vessels. [Figure 2A]Figure 2A is a cross-sectional schematic of polylactic acid (PLA)-hyperbranched polyglycerol (HPG) NPs bearing bioadhesive aldehyde groups on their surface, and Figure 2B is a schematic of the synthesis of the NPs in Figure 2A. [Figure 2B] Same as above. [Figure 3A] Figures 3A-3C are schematic diagrams of the chemical structure of PLA-HPG NPs with DFO conjugates and DFO-89Zr attachment (Figure 3A); the reaction process showing the DFO conjugates on the NPs (Figure 3B); and the stability of the NPs (consistent PDI and Z-average) measured by dynamic light scattering (n=3) (Figure 3C). The DFO conjugates did not affect the bioadhesive properties of the NPs, as measured by the poly-lysine assay. [Figure 3B-C] Same as above. [Figure 4A-B] Figures 4A-4C show PET / CT images of 89Zr-DFO-NPs and 89Zr-DFO delivered via ICM to tumor-free mice. Two healthy mice (n=2) were injected with either 89Zr-DFO-NPs or 89-Zr-DFO and imaged continuously for 2 hours. The pharmacokinetic curves of 89Zr-DFO-NPs and 89-Zr-DFO in the CNS are shown. *P=0.0243 (Figure 4A). Separate pharmacokinetic curves of 89Zr-DFO-NPs and 89Zr-DFO in the brain and spinal cord are shown. ****P<0.0001 (brain), ns (spinal cord) (Figure 4B). Based on whole-body sagittal PET images of 89Zr-DFO and 89Zr-DFO-NPs over a 2-hour continuous dynamic scan. 89Zr-DFO penetrated the interstitial parenchyma more than 89Zr-DFO-NPs, and the biodistribution of Zr89-DFO-NPs in healthy mice at various time points from 3 hr to 12 days after administration (n = 4 at days 4, 7, and 12, and n = 8 at 3 hr and 24 hr) (Figure 4C). Percentage of total (%) was calculated as activity in the ROI relative to total injected activity. Data are presented as mean ± SD; significance determined by two-tailed Mann-Whitney U test. [Figure 4C] Same as above. [Figure 5A-B]Zr-DFO-NPs preferentially accumulate in tumors. Tumor-bearing mice were injected with 89Zr-DFO-NPs and imaged continuously for 2 h. Pharmacokinetic curves for the brain, spinal cord, and tumor site (Figure 5A). Pharmacokinetic curves for the cervical lymph nodes, bladder, and liver (Figure 5B). Biodistribution of 89Zr-DFO-NPs at various time points from 6 h to 21 days after administration in tumor-bearing mice (n=4). Percent of total (%) is calculated as activity in the ROI relative to total injected activity (Figure 5C). Bioluminescence intensity graph of tumor signal in mouse brain and spinal cord (Figure 5D). Quantification graph of Cy5-NP area fraction in 10 representative sections of the whole brain at 24 h post-administration (n=5) (Figure 5E). P=0.0005, one-way ANOVA. [Figure 5C] Same as above. [Figure 5D-E] Same as above. [Figure 6]Figures 6A-6B are graphs showing the toxicity of BMN-673 free drug and BMN-NP in mice. The tolerability of various doses of free BMN-673 and BMN-NP in J:Nu mice was assessed by monitoring body weight and overall health after a single IT treatment. Doses were initially administered to one mouse and, if tolerated, escalated to n = 5. Red asterisks indicate animals died or were sacrificed due to excessive weight loss. Free BMN-673 was lethal at any dose of 0.06 mg / kg or higher. Animals experienced less than 15% weight loss at 0.05 mg / kg. 0.03 mg / kg was determined to be the maximum tolerated dose (MTD) (Figure 6A). BMN-NP was lethal at 1.25 mg / kg, but lower doses were tolerated without greater than 10% weight loss. (Figure 6B) 0.5 mg / kg was determined as the MTD. J:Nu mice were treated with BMN-673 or BMN-NP at the MTD, and complete blood counts, differential white blood cell counts, and platelet cell counts were performed to assess hematotoxicity. On day 3 of treatment, free BMN-673 induced a decrease in all white blood cells except monocytes, and also showed a reduction in platelet counts. BMN-NP showed a lower level of decrease in all cell counts. By day 7 of treatment, BMN-NP showed improvements in platelet, white blood cell, basophil, red blood cell counts, and hemoglobin concentrations. Free BMN-673 showed no improvement except for basophil counts. Each group in this study had an n=6. [Figure 7A]Figures 7A–7F show graphs of tumor-bearing mice treated via a CM catheter. These mice were also treated with IT administration of either free BMN-673 (0.05 mg / kg, 1 dose) or BMN-NP (0.25 mg / kg, 1 dose) using the same catheter (Figure 7A). Two mice were excluded from the entire study because tumor growth could not be observed. Survival curves of BMN-673-treated, BMN-NP-treated, and control mice (n = 6; one mouse was removed from the control and free BMN groups due to lack of tumor growth) (Figure 7B). Body weight changes for all groups (Figure 7C). Data are shown as mean ± SD. Region-of-interest analysis of bioluminescence intensity from the whole brain (Figure 7D). Whole-body bioluminescence images of a DAOY tumor-bearing mouse. The bioluminescence scale in the first column is different from all the remaining images (NP, Figure 7E; free drug, Figure 7F). [Figure 7B-C] Same as above. [Figure 7D-F] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention I. Definition "Nanoparticles," as used herein, generally refer to nanoparticles of any shape having a diameter of from about 1 nm to about 1 micron (but not including), more preferably from about 5 nm to about 500 nm, and most preferably from about 5 nm to about 100 nm. Nanoparticles having a spherical shape are commonly referred to as "nanospheres."

[0016] "Average nanoparticle size," as used herein, generally refers to the statistical average nanoparticle size (diameter) of nanoparticles in a population of nanoparticles. The diameter of a substantially spherical nanoparticle may also be referred to as the physical diameter or hydrodynamic diameter. The diameter of a non-spherical nanoparticle may primarily refer to the hydrodynamic diameter. The diameter of a non-spherical nanoparticle, as used herein, may refer to the maximum linear distance between two points on the surface of the nanoparticle. The average nanoparticle size can be measured using methods known in the art, for example, dynamic light scattering.

[0017] "Monodisperse" and "uniform size distribution" are used interchangeably herein to describe a plurality of nanoparticles having the same or nearly the same diameter or aerodynamic diameter. A monodisperse distribution, as used herein, refers to a distribution of nanoparticles in which 80, 81, 82, 83, 84, 85, 86, 86, 88, 89, 90, 91, 92, 93, 94, 95% or more are within 5% of the mass median diameter or aerodynamic diameter.

[0018] "Hydrophilic," as used herein, refers to the property of having an affinity for water. For example, a hydrophilic polymer (or hydrophilic polymer segment) is a polymer (or polymer segment) that is primarily soluble in aqueous solutions and / or has a tendency to absorb water. Generally, the more hydrophilic a polymer is, the greater its tendency to dissolve, mix, or become wetted by water.

[0019] "Hydrophobic," as used herein, refers to a substance that lacks an affinity for water; that repels water and does not absorb water, and that tends not to dissolve or mix with water.

[0020] An amphiphilic polymer, as used herein, is a polymer that has one end formed by a hydrophilic polymer and one end formed by a hydrophobic polymer, such that when dispersed in a mixture of water and a poorly water-soluble solvent, such as many organic solvents, the hydrophilic end faces toward the water and the hydrophobic end faces toward the poorly water-soluble solvent.

[0021] Self-assembly refers to the use of amphiphilic polymers, alone or mixed with hydrophilic and / or hydrophobic polymers, that orient in a mixture of aqueous and non-aqueous solvents to form nanoparticles, with the hydrophilic ends orienting with other hydrophilic ends and the hydrophobic ends orienting with other hydrophobic ends.

[0022] Unless otherwise specified, "molecular weight," as used herein, generally refers to the relative average chain length of a bulk polymer. In practice, molecular weight estimation or characterization can be performed using a variety of methods, including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as weight-average molecular weights (Mw) compared to number-average molecular weights (Mn). Capillary viscometry provides an estimate of molecular weight as the intrinsic viscosity determined from a dilute polymer solution using a specific set of concentration, temperature, and solvent conditions.

[0023] The term "therapeutic or prophylactic agent" refers to an agent that can be administered to prevent or treat one or more symptoms of a disease or disorder. The therapeutic agent can be a nucleic acid, a nucleic acid analog, a small molecule (less than 2000 D, less than 1500 D, or less than 1000 D), a peptidomimetic, a protein, a peptide, a carbohydrate or sugar, a lipid, or a surfactant, or a combination thereof.

[0024] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective to alleviate, delay the onset of, or prevent one or more symptoms of a disease or disorder. The terms "treating" or "preventing" as used herein can include preventing a disease, disorder, or condition from occurring in an animal that may be predisposed to, but has not yet been diagnosed with, the disease, disorder, or condition; inhibiting a disease, disorder, or condition, e.g., preventing its progression; and alleviating a disease, disorder, or condition, e.g., causing the disease, disorder, and / or condition to regress. Treatment of a disease, disorder, or condition can include ameliorating at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, for example, if a subject's pain is treated by administering an analgesic, but the cause of the pain is not treated by such an agent.

[0025] "Pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and in accordance with agency guidelines such as the Food and Drug Administration.

[0026] "Biocompatible" and "biologically compatible," as used herein, typically refer to a material that, together with any metabolic or degradation products thereof, is generally not toxic to or does not cause significant adverse effects in the recipient. Generally, a biocompatible material is one that does not cause a significant inflammatory or immune response when administered to a patient.

[0027] "Biodegradable," as used herein, means that a material will break down or degrade into its constituent subunits, or that the material will be digested, for example, by biochemical processes, into smaller (e.g., non-polymeric) subunits.

[0028] II. Nanoparticle Formulations A. HPG-PLA nanoparticles The nanoparticles contain a core and a shell or coating. The shell is formed from hyperbranched polyglycerol (HPG). The HPG is covalently attached to a hydrophobic polymer that forms the core, so that the hydrophilic HPG faces the outside of the nanoparticle and the hydrophobic polymer is oriented to form the core.

[0029] The HPG coating can be modified to tailor the nanoparticle's properties. For example, an unmodified HPG coating imparts stealth properties to the nanoparticle, preventing nonspecific protein absorption, and is referred to as a nonbiodegradable nanoparticle (NNP). As used herein, hydroxyl or other groups on the HPG coating react with functional groups on tissue or are chemically modified to form functional groups that interact with tissue, attaching the nanoparticle to tissue, cells, or extracellular materials, such as proteins. Such functional groups include aldehydes, amines, oximes, and O-substituted oximes, most preferably aldehydes. Nanoparticles with an HPG coating chemically modified to form functional groups are referred to as bioadhesive nanoparticles (BNP). The chemically modified HPG coating of BNP forms a bioadhesive corona of the nanoparticle surrounding the hydrophobic polymer core. See, e.g., WO2015 / 172149, WO2015 / 172153, WO2016 / 183209, and U.S. Published Application Nos. 2017 / 0000737 and 2017 / 0266119. Figure 2A is a cross-sectional schematic of HPG NPs, and Figure 2B is a schematic showing the synthesis of stealth and sticky nanoparticles.

[0030] The core of the NP is preferably formed from polymers fabricated from polylactide (PLA) and copolymers of lactide and glycolide (PLGA), which have established commercial use in humans and a long track record of safety (Jiang, et al., Adv. Drug Deliv. Rev., 57(3):391-410; Aguado and Lambert, Immunobiology, 184(2-3):113-25 (1992); Bramwell, et al., Adv. Drug Deliv. Rev., 57(9):1247-65 (2005)).

[0031] Hyperbranched polyglycerol (HPG) is a highly branched polyol containing a polyether backbone. Hyperbranched polyglycerol can be prepared using techniques known in the art. It can be formed by controlled etherification of glycerol via cationic or anionic ring-opening hyperbranching polymerization of glycidol. For example, an initiator having multiple reactive sites is reacted with glycidol in the presence of a base to form hyperbranched polyglycerol (HPG). Suitable initiators include, but are not limited to, polyols, such as triols, tetraols, pentaols, or higher, and polyamines, such as triamines, tetraamines, and pentaamines. In one embodiment, the initiator is 1,1,1-trihydroxymethylpropane (THP).

[0032] The formula of the hyperbranched polyglycerol described in EP2754684 is: [ka] and In the formula, o, p, and q are independently an integer of 1 to 100. A1 and A2 are independently [ka] and In the formula, l, m, and n are independently integers of 1 to 100. A3 and A4 are defined similarly to A1 and A2, except that in the terminal residues, A3 and A4 are hydrogen, and n and m are each 1.

[0033] The surface properties of HPG can be tailored based on the chemical properties of the vicinal diol. For example, the surface properties can be adjusted to provide: stealth nanoparticles, i.e., nanoparticles that are not rejected by MPS due to the presence of hydroxyl groups; adhesive (sticky) nanoparticles, i.e., nanoparticles that adhere to the surface of tissues due to the presence of one or more reactive functional groups, such as aldehydes, amines, oximes, or O-substituted oximes, that can be prepared from vicinal hydroxyl moieties; or targeting by introducing one or more targeting moieties that can be directly or indirectly conjugated to the vicinal hydroxyl moieties. Indirect refers to converting the hydroxyl groups into reactive functional groups that can react with functional groups on molecules attached to the surface, such as active agents and / or targeting moieties.

[0034] The hyperbranched nature of polyglycerol allows for much higher densities of hydroxyl groups, reactive functional groups, and / or targeting moieties than can be achieved with linear polyethylene glycol. For example, nanoparticles can have at least about 1, 2, 3, 4, 5, 6, 7, or 8 groups / nm 2 The surface may have a density of surface functional groups (e.g., hydroxyl groups, reactive functional groups, and / or targeting moieties).

[0035] The molecular weight of the HPG can vary. For example, in embodiments in which the HPG is covalently attached to the core material or polymer, the molecular weight can vary depending on the molecular weight and / or hydrophobicity of the core material. The molecular weight of the HPG is generally between about 1,000 and about 1,000,000 daltons, about 1,000 and about 500,000 daltons, about 1,000 and about 250,000 daltons, or about 1,000 and about 100,000 daltons. In embodiments in which the HPG is covalently attached to the core material, the weight percent of the HPG in the copolymer is about 1% to about 50%, e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

[0036] In some embodiments, HPG is covalently coupled to a hydrophobic or more hydrophobic material, such as a polymer. Upon self-assembly, nanoparticles are formed that contain a core containing a hydrophobic material and a shell or coating of HPG. HPG coupled to the polymer PLA is shown below: [ka]

[0037] HPG-coated nanoparticles can be modified by covalently attaching PEG to the surface. This can be achieved by converting the vicinyl diol group to an aldehyde and then reacting the aldehyde with functional groups on the PEG, such as aliphatic amines, aromatic amines, hydrazines, and thiols. The linker has terminal groups such as aliphatic amines, hydrazines, thiols, and O-substituted oxyamines. The bond inserted into the linker can be a disulfide, orthoester, or protease-sensitive peptide.

[0038] PEG with functional groups or linkers can form bonds with aldehydes on PLA-HPG-CHO, restoring the bioadhesive state of PLA-HPG-CHO to a stealth state. This bond or linker can be altered by pH changes or high concentrations of peptides, proteins, and other biomolecules. After systemic or local administration, the bond attaching PEG to PLA-HPG-CHO can be reversed or cleaved, releasing PEG depending on the environment and exposing the bioadhesive PLA-HPG-CHO nanoparticles to the environment. The nanoparticles then interact with tissues, attaching to tissues or extracellular materials, such as proteins. This environment can be the acidic environment in tumors, the reducing environment in tumors, or the protein-rich environment in tissues.

[0039] HPG can be covalently attached to the polymer that forms the core of the nanoparticle using methodologies known in the art. For example, HPG, such as HPG, can be covalently coupled to a polymer bearing carboxylic acid groups, such as PLA, PGA, or PLGA, using DIC / DMAP.

[0040] HPG can be initiated from hydroxyl, amine, and carboxylate-terminated molecules, such as alcohols with one or more long hydrophobic tails. In another example, HPs, such as HPG, can be initiated from specialized functionalized initiators to facilitate conjugation to more materials. These specialized initiators include disulfides (Yeh et al., Langmuir. 24(9):4907-16(2008)).

[0041] HPG can be functionalized to introduce one or more reactive functional groups that alter the surface properties of the nanoparticles. The surface of the nanoparticles can be further modified with one or more targeting moieties or by covalently attaching HPG to HPG via an organic coupling agent or spacer, such as dichloromethane (DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), diisopropylcarbodiimide (DIC), 4-(N,N-dimethylamino)pyridine (DMAP), dicyclohexylcarbodiimide (DCC), DIC / DMAP, DCC / DMAP, or acyl chloride / pyridine. In some embodiments, the polymer is functionalized / modified prior to nanoparticle formation.

[0042] HPG-coated NPs can be converted to aldehyde-terminated NPs by NaIO4 treatment (or to carboxylic acid-terminated NPs by NaIO4 treatment followed by sodium chlorite treatment), so that targeting moieties can be directly covalently attached to the NPs via the aldehyde (or carboxylic acid) groups on the NPs and functional groups on the targeting moiety (amine, hydrazine, amino-oxy, and their derivatives) or indirectly attached to the NPs via coupling agents or spacers (e.g., amino-oxy-modified biotin and cysteine).

[0043] Low molecular weight components can be removed from the synthesized HPG by multiple solvent precipitations and dialysis, as high molecular weight HPG is less susceptible to nonspecific adsorption to biomolecules.

[0044] In a preferred embodiment, polyhydroxy acids such as PLA are chosen as the hydrophobic core material because they are biodegradable and have a long history of clinical use. To covalently attach PLA to HPG, previous approaches have involved first functionalizing HPG with amines and then conjugating carboxyl groups on the PLA to the amines. While this approach is effective, it cannot be used to coat HPG on surfaces because the amines that do not react with PLA result in a net positive charge on the neutral HPG surface, reducing the ability of HPG to adsorb other molecules to the surface. To avoid this, a one-step esterification between PLA and HPG can be employed, which maintains the charge neutrality of HPG. Alternatively, PLGA can be used as the hydrophobic core material for covalent attachment to HPG.

[0045] B. Molecules Encapsulated or Attached to the Surface of Nanoparticles The nanoparticles can contain one or more types of molecules encapsulated within the nanoparticles and / or attached to the surface of the nanoparticles. The molecules can be attached to the nanoparticles by covalent or non-covalent bonds. The molecules can be attached to the hydroxy groups on the HPG before or after nanoparticle formation. Representative methodologies for conjugating the molecules to the hydroxy groups on the HPG are described below.

[0046] One useful protocol involves "activating" hydroxyl groups with carbonyldiimidazole (CDI) in an aprotic solvent, such as DMSO, acetone, or THF. CDI forms an imidazolyl carbamate conjugate with the hydroxyl group, which can then be displaced by binding to free amino groups on ligands, such as proteins. The reaction is an N-nucleophilic substitution, resulting in a stable N-alkyl carbamate linkage of the ligand to the polymer. "Coupling" of the ligand to the "activated" polymer matrix is ​​maximized in the pH range of 9-10 and typically requires at least 24 hours. The resulting ligand-polymer conjugate is stable and resistant to hydrolysis for extended periods.

[0047] Another coupling method involves the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or "water-soluble CDI" with N-hydroxylsulfosuccinimide (sulfo-NHS) to couple exposed carboxyl groups of a polymer to free amino groups of a ligand in a fully aqueous environment at physiological pH 7.0. Briefly, EDAC and sulfo-NHS form an activated ester with the carboxylic acid group of the polymer, which reacts with the amine terminus of the ligand to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. The use of sulfo-NHS in the reaction increases the efficiency of EDAC coupling by 10-fold and provides very mild conditions that ensure the feasibility of ligand-polymer conjugates.

[0048] Using either of these protocols, it is possible to "activate" almost any polymer containing either hydroxyl or carboxyl groups in a suitable solvent system that does not dissolve the polymer matrix.

[0049] A useful coupling procedure for attaching ligands with free hydroxyl and carboxyl groups to polymers uses the crosslinker divinyl sulfone. This method is useful for attaching sugars or other hydroxyl compounds with bioadhesive properties to hydroxyl matrices. Briefly, activation involves reacting the hydroxyl groups of the polymer with divinyl sulfone to form the vinyl sulfonyl ethyl ether of the polymer. The vinyl groups couple with alcohols, phenols, and even amines. Activation and coupling occur at pH 11. The linkage is stable over a pH range of 1 to 8.

[0050] Alternatively, the hydroxyl groups can be converted to reactive functional groups that can react with reactive functional groups on the attached molecule. For example, the hydroxyl groups on HPG can be converted to aldehydes, amines, or O-substituted oximes that can react with reactive functional groups on the attached molecule. Such conversions can be performed before or after nanoparticle formation.

[0051] Any suitable coupling method known to those skilled in the art for coupling a ligand and a polymer having a double bond can be used to attach the molecule to the polymer, including the use of UV crosslinking.

[0052] C. Therapeutic, Preventive, and Diagnostic Agents Chemotherapeutic agents for the treatment of brain tumors include Afinitor (everolimus), Afinitor Disperz (everolimus), Avastin (bevacizumab), velzutifan, bevacizumab, BiCNU (carmustine), carmustine, carmustine implant, Danyelza (naxitamab-gqgk), everolimus, Gliadelwafer (carmustine implant), lomustine, Mvasi (bevacizumab), naxitamab-gqgk, Temodar (temozolomide), temozolomide, Welireg (velzutifan), and Zirabev (bevacizumab).

[0053] In a preferred embodiment, the agent delivered is a radiosensitizer, most preferably a PARP inhibitor (PARPi). Poly(ADP-ribose) polymerase-1 or PARP-1 is a multifunctional regulator of transcription, chromatin structure and genome integrity that uses NAD as a substrate and is activated by DNA cleavage.

[0054] "PARP inhibitors" (PARPi) are inhibitors of PARP-1, PARP-2, or PARP-3, but currently are primarily PARP-1 inhibitors.

[0055] Because radiation induces DNA damage in the DNA of all cells, selective targeting of PARP inhibitors to cancer cells but not brain cells increases damage to cancer cells at lower radiation doses than would be required to target all cells, cancer cells, and the brain in the absence of a PARP inhibitor acting as a radiosensitizer. PARP binds to DNA and synthesizes PAR strands, which recruit repair enzymes and repair DNA damage. PARPi inhibits repair by interfering with base excision repair, converting single-strand breaks (SSBs) to double-strand breaks (DSBs).

[0056] Medulloblastoma is sensitive to PARPi combined with radiation.For example, when medulloblastoma cell line D283 is exposed to 1 micromolar concentration of olaparib and radiation, high level of DSB occurs, and its effect increases according to radiation intensity.See van Vuurden, et al. Oncotarget 2:984-996 (2011).

[0057] Non-limiting examples of PARP inhibitors, which can also be classified as PARP-1 inhibitors, include olaparib, veliparib, CEP-8983 (11-methoxy-4,5,6,7-tetrahydro-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dione) or a prodrug thereof (e.g., CEP-9722), rucaparib, E7016 (10-((4-hydroxypiperidin-1-yl)methyl)chromeno-[4,3,2-de]phthalazine-3 (2H)-one), INO-1001 (4-phenoxy-3-pyrrolidin-1-yl-5-sulfamoyl-benzoic acid), niraparib, talazoparib (BMN673), NU1025 (8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroisoquinoline, 4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, and their analogs, isosteres, and derivatives (Curtin NJ, et al. Therapeutic applications of PARP inhibitors: anticancer therapy and beyond. Molecular aspects of medicine 2013; 34:1217-56). Olaparib was one of the first PARPi drugs to enter clinical trials for indications including patients with breast, prostate, and ovarian cancer harboring mutations in the BRCA1 or BRCA2 genes, and demonstrated antitumor activity and grade 1 or 2 side effects (Fong, Boss et al. 2009, N Engl J Med 361(2):123-134; Tutt, Robson et al. 2010, Lancet 376(9737):235-244).

[0058] III. Treatment method A. Patients to be treated The primary class of patients treated are children with cancers such as medulloblastoma and leptomeningeal tumors. However, this technology is suitable for use in treating any cancer of the CNS and may be useful for delivery of other therapeutic, prophylactic, or diagnostic agents for delivery to the CNS where release over a period of up to several days is desired.

[0059] B. Methods of Administration of Nanoparticle Formulations By encapsulating the drug in nanoparticles optimized for sustained drug release and retention in the CSF, cytotoxic levels of drug can be released into the CSF over an extended period to treat occult tumor cells.

[0060] In intrathecal delivery, the drug is administered by injection into the subarachnoid space of the spinal column using a local anesthetic. The drug then travels through the CSF to the brain CSF, as shown in Figure 1A.

[0061] As shown in Figure 1A, the central nervous system includes the brain 10, spinal cord 12, cerebellum 14, skull 16, blood vessels 18, and cerebrospinal fluid (CSF) 20. As shown in Figure 1B, intrathecally administered drugs travel via CSF 20 in the periarterial space containing arteries 24 and veins 26 to meningeal lymphatic vessels 28 and arachnoid granulations 22.

[0062] In clinical settings, intrathecal delivery is used to treat patients with chronic pain by delivering opioids or to treat leptomeningeal metastases by delivering chemotherapy drugs. Although intrathecal chemotherapy is the standard of care, median patient survival is limited to a few months. DEPOCYT®, an FDA-approved sustained-release lipid formulation of cytarabine, has been used in patients with leptomeningeal metastases. However, these are "microparticles" and not NPs, and therefore have limited diffusion and permeation properties. Therefore, biodegradable, biocompatible drug-loaded NPs that can uniformly penetrate throughout the CNS would be an improvement over current treatment paradigms.

[0063] The invention will be further understood with reference to the following non-limiting examples. [Example]

[0064] Example 1 Preparation of nanoparticles Methods and Materials Blank PLA-HPG NP, Cy-5 NP, and aldehyde-NP synthesis Blank PLA-HPG nanoparticles (NPs) were made using a single emulsion, solvent evaporation process (Deng, Y. et al. Biomaterials 35, 6595-6602 (2014)). Briefly, 50 mg of polymer was dissolved in 2.4 mL of ethyl acetate, followed by 0.6 mL of DMSO. This polymer solution was added dropwise to 4 mL of deionized water, sonicated, and then evaporated on a rotary evaporator for 15 min. The NPs were then transferred to a centrifugal filter (Amicon Ultra-15, 100 kDa MWCO, Sigma-Aldrich) and centrifuged three times at 4000 × g to remove excess polymer and solvent. After the final spin, the nanoparticles were resuspended in deionized water (DI water), flash-frozen in liquid nitrogen, and stored at -20 °C until use. Different PLA-HPG copolymers of varying MW were used to prepare NPs of different sizes.

[0065] To prepare Cy5-NPs, 50 mg of PLA-HPG polymer was combined with poly(D,L)lactic acid-cyanine 5 endcap (PolySciTech) at 10% (w / w) and processed using the same steps as before.

[0066] To prepare aldehyde-NPs, NPs were diluted to a concentration of 25 mg / mL, combined with an equal volume of 10x PBS and 0.1 M NaIO4, and incubated on ice. After 20 min, 1 volume of 0.2 M Na2SO3 was added to stop the reaction. The aldehyde-NPs were then transferred to a centrifugal filter and washed three times at 4000 x g.

[0067] BMN-673 loaded NP synthesis For BMN-673-loaded NPs, 20 mg of BMN-673 was used per 100 mg of PLA-HPG. 50 mg of polymer was dissolved in 1.2 mL of ethyl acetate, and 20 mg of drug dissolved in DMSO was added to the polymer solution. After this step, BMN-673 NPs were prepared in the same manner as blank NPs. The NPs were washed multiple times with DI water in a centrifugal filter and then filtered using a 1.5 μm glass microfiber filter (Whatman #6827-1315) before use in vivo or in vitro.

[0068] Quantification of BMN-673 loading BMN-673 loading was calculated as follows: 25-50 μL aliquots (triplicate) of BMN-NPs were prepared and lyophilized in pre-weighed tubes. The solutions were redissolved in DMSO and then analyzed by Agilent LC-MS 6120B (Agilent Technologies, Santa Clara, CA, USA) using a standard curve to determine the drug loading in the NPs. BMN-673 release from the NP formulations was measured for up to 7 days. NPs loaded with 5% (w / w) drug were dispersed at 50 mg / mL in either artificial CSF (aCSF) or PBS and incubated at 37°C. The suspensions were centrifuged using a 3 kD filter at various time points (4 h, 8 h, 24 h, 48 h, 72 h, day 5, and day 7). The filtrate was collected for HPLC analysis as previously described for BMN-673 loading, and the pellet was resuspended in the same volume of PBS or aCSF to continue release.

[0069] DFO-NP synthesis Aldehyde-NP is 89 For further labeling with Zr, the nanoparticles were surface-conjugated to the chelating agent deferoxamine mesylate (DFO, CAS# 138-14-7, Sigma). Surface conjugation of DFO was achieved by reductive amination. After aldehyde conversion, NPs (25 mg / mL) were incubated with 1 molar equivalent of DFO mesylate at room temperature for 4 h. 40 molar equivalents of NaCNBH3 were added, and the NPs were incubated for an additional 40 h. The DFO-conjugated NPs were washed four times to remove excess DFO mesylate and NaCNBH3 and resuspended at a concentration of 25 mg / mL.

[0070] 89 Zr-DFO-NPs and 89 Zr-DFO preparation DFO-NP was neutralized in 0.25 M HEPES (pH 7.4). 89 Labeling was performed with Zr-oxalate for 30 min at room temperature, with a specific activity of 10 μCiμg -1 The radiochemical yield was assessed using radio-thin layer liquid chromatography (radio-TLC). 89Zr-DFO-NPs were washed three times using a centrifugal filter (Amicon Ultra-0.5, 100 kDa MWCO, Sigma-Aldrich) and resuspended to a final concentration of approximately 40 μCi / μL (decay correction for delivery time). To determine uptake in the absence of NPs, 89 Zr-DFO was prepared; 0.1 mg / mL DFO mesylate was neutralized in 0.25 M HEPES (pH 7.4). 89 Labeled with Zr-oxalate, specific activity 10 μCi μg -1 The radiochemical yield was determined by radio-TLC. 89 The Zr-DFO was then loaded onto an activated SEP-PAK PLUS C18 cartridge (Waters Corp), washed twice with deionized water, and eluted with 95% ethanol. Excess ethanol was evaporated at 90°C for 1 h. 89 Zr-DFO was resuspended to a final concentration of approximately 40 μCi / μL.

[0071] 89 To determine the stability of Zr-DFO-NPs, particles were incubated in aCSF for 7 days at 37°C. Each day, particles were spun down through a 100 kDa centrifugal filter, and activity measurements of both the filtrate and retentate were performed using a Hidex AMG automated gamma counter (Hidex, Turku, Finland).

[0072] NP characterization All NPs were characterized using dynamic light scattering (hydrodynamic diameter, PDI, zeta potential) at a concentration of 5 mg / mL in DI water (Malvern, Zetasizer APS). For transmission electron microscopy, samples were prepared at 1.0 μg / mL, XYZ. Particle stability measurements were performed in artificial cerebrospinal fluid using a Malvern Nano-ZS at 37°C (Harvard Apparatus, Holliston, MA, USA) and measured every minute using standard operating procedures. 100 ml of NP solution was lyophilized in pre-weighed Eppendorf tubes to determine particle yield.

[0073] Xenografts and cell lines DAOY, D341, and D283 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the supplier's instructions.

[0074] mouse All procedures were approved by the Yale University Institutional Animal Care and Use Committee and performed in accordance with the guidelines and policies of the Yale Animal Resource Center. BALB / C mice (Charles River, 8 weeks old, female) or J:Nu mice (Jackson Labs, 8 weeks old, female) were used in all studies unless otherwise indicated.

[0075] Intracisternal (ICM) injection and catheter placement Mice were anesthetized intraperitoneally with ketamine / xylazine, eye drops were applied to prevent drying, and the head of the mouse was fixed in a stereotaxic frame. After incising the skin, the muscle layer was retracted to expose the cisterna magna. For bolus injections, a Hamilton syringe (connected to a 33-gauge needle) was used to deliver the desired volume of solution into the CSF-filled cisterna magna compartment. The needle was left in place for 1–2 min to avoid backflow. The mouse was then glued and sutured with Vet bond and allowed to recover on a heating pad until active.

[0076] Catheterization was performed using a 32G IT catheter (0046EO; ReCathCo). In mice with catheterization, the trimmed end of the catheter was inserted into the cisterna magna and secured along the superficial lateral muscle layer with tissue adhesive (Histoacryl), and the outer muscle layer was sutured. Mice were allowed to recover on a heating pad until active, and local anesthetic ointment was applied to the wound. Animals also received buprenorphine (0.06 mg / kg every 12 h) for pain management. Buprenorphine injections were repeated for 3 days after surgery, and mice were monitored for signs of pain and / or difficulty. For catheter administration, a 27G needle was attached to the outer catheter tube and used to deliver up to 6 μL of NP or drug.

[0077] immunohistochemistry Cy5-NP was dissolved in DI water at 50 mg / mL and 6 μL was delivered via ICM to BALB / C female mice (n=5). At various time points, mice were imaged in vivo and ex vivo using an IVIS Lumina II In Vivo Imaging System (PerkinElmer) to confirm Cy5-NP signal. Whole-body transcardial perfusion with 10 mL of PBS and 10 mL of 4% PFA was performed before tissue dissection. Tissues were fixed overnight in 2% PFA, left in 30% sucrose solution for 5 days, and then cryopreserved in Tissue-Plus OCT compound (Thermo Fisher Scientific). Tissues were cut coronally into 20 μM sections using a cryostat (Leica), collected onto gelatin-coated SUPERFROST® Plus slides (Thermo Fisher Scientific), stained with DAPI, and stored at -20°C. Samples from mice administered with 3 kD FITC-dextran and 3000 kD FITC-dextran were also prepared in the same manner (n=5).

[0078] Images were acquired and stitched together using an EVOS microscope. For quantitative analysis using Fiji software, 10–15 representative brain sections were imaged using a wide-field microscope. For all sections, the area fraction was assessed by dividing the area covered by labeled NPs by the area of ​​the brain section.

[0079] Toxicity analysis To assess toxicity, healthy female nude mice were first administered specific doses of either free BMN-673 or BMN-NP ranging from 0.03 mg / kg to 1.25 mg / kg. Mice were monitored for weight loss or other signs of distress, and if the dose was well tolerated, four mice were further dosed and monitored (n=5).

[0080] For hematological analysis, healthy nude mice (n = 5 per time point) were administered either free BMN-673 (0.05 mg / kg) or BMN-NP (0.5 mg / kg). On days 3 and 7 after administration, 100 μL of blood was collected by cardiac puncture using a 15 μL EDTA-coated tube. Blood count levels were measured using a HEMAVET® HV950 multispecies hematology analyzer (Drew Scientific, Oxford, CT, USA). Spleens and livers were harvested, fixed in formalin overnight, and stained with H&E for histopathological evaluation.

[0081] Medulloblastoma model Eight-week-old J:Nu female mice were administered 10 5DAOY cells (ATCC) were inoculated into the tumors. Tumor growth was tracked twice weekly after implantation by bioluminescence imaging. For bioluminescence imaging, mice were injected with 200 μL of D-luciferin solution (15 mg / ml, Caliper Life Science) and imaged 5 min after injection using an IVIS200 in vivo imaging system (Xenogen, Caliper Life Science). Images showing light intensity (photons / second) were generated, and the signal for each mouse was quantified using Living Image 4.0 software (Xenogen). All mice were randomized 7 days after tumor injection and treated with BMN-673-loaded NPs or free BMN-673 drug either once a week or once every two weeks for 2 weeks. Tumor regression or progression was monitored using BLI and mice were left until neurological symptoms appeared or the humane endpoint was reached (sluggish movement and weight loss >20%).

[0082] Histological examination Mice exhibiting end-stage neurological brain tumor symptoms were sacrificed, and CNS tissues for histological analysis were collected, fixed overnight in formalin, and embedded in paraffin. Brains were then cut along the coronal plane, and spinal cords were transversely sectioned into cervical, thoracic, lumbar, and sacral regions. The location and extent of the primary tumor and associated metastases were analyzed by standard H&E staining.

[0083] PET imaging In an initial biodistribution study, BALB / C female mice (n=2) 89 Zr-DFO-NPs (50 μCi), or 89 Zr-DFO (50 μCi) was administered intraperitoneally, followed immediately by a 120-min dynamic scan using an Inveon PET / CT scanner (Siemens Healthcare Global) under an isoflurane / oxygen gas mixture (2% for induction and maintenance). CT images were acquired in addition to PET for anatomical delineation. Half-lives were determined using XYZ statistical analysis in Prism.

[0084] Long-term studies in healthy mice showed 89 Zr-DFO-NPs (150 μCi) were administered intravenously (n = 4) and 3 h later, 10-minute PET scans were performed. CT images were acquired in addition to PET for anatomical delineation. PET scans were repeated at later times (24 h, on days 4, 7, and 12), with scan times ranging from 10 to 60 minutes.

[0085] For tumor-bearing mouse studies, xenografts were generated using DAOY cells as described above. Three cohorts of mice (n = 2) bearing tumors in both the brain and spinal cord, visualized by IVIS imaging, were selected and administered 89Zr-DFO-NPs (200 mCi) intraperitoneally via catheter. PET imaging was performed in the first cohort with a 120-min continuous dynamic scan. PET and CT images were acquired in the remaining cohorts at 6 h, 24 h, and on days 4, 7, 12, and 21.

[0086] All images were reconstructed using the subsetting expectation-maximization (OSEM-3D) algorithm and analyzed using Inveon Research software (Siemens Healthcare Global). Regions of interest (ROIs) were manually defined on the CT images for the following regions: brain, spinal cord, all lymph nodes, liver, spleen, heart, lungs, stomach, kidneys, bladder, and bone. Radioactivity concentrations within the ROIs were reported in units of Bq / mL, which were converted to mean standardized uptake value (SUV) and % injected dose / gram.

[0087] After the final PET / CT scan, animals were euthanized and tissues of interest (brain, spinal column, liver, kidneys, spleen, lungs, muscle, heart, bone, stomach, cervical lymph nodes, and peripheral lymph nodes) were collected, blotted, and weighed. Radioactivity was measured by gamma counting and normalized as Bq / gram.

[0088] statistical analysis Data analysis and visualization were performed using Prism 7.0 (GraphPad Software). Graphs represent the mean ± sd of either group (as indicated in the figure legend) or individual values. P values ​​were calculated using the log-rank statistic followed by repeated measures analysis of variance (ANOVA) for survival analysis. One-way ANOVA was used for multiple comparisons, and the Mann-Whitney U test was used for comparisons between two groups. P < 0.05 was considered statistically significant. P values ​​are indicated with an asterisk: P > 0.05, NS; * P < 0.05; ** P < 0.01; *** P<0.001; and **** P<0.0001.

[0089] result Engineering NPs for optimal CNS retention Particle size and surface charge were determined for a wide range of particle compositions (Figures 3A-3C). To synthesize covalently tethered fluorescent dye nanoparticles for effective microscopic tracking, NPs were fabricated using a blend of PLA-HPG copolymer and 5% Cy5-PLA conjugate. Unlike dyes, which can leak from the NPs and complicate measurement interpretation, the Cy5-PLA conjugate ensures that the fluorescent signal originates from the NPs themselves. We hypothesized that PLA-HPG NPs would similarly demonstrate distinct advantages in the CSF environment after direct CSF injection via the cisterna magna (CM). Cy5-PLA-HPG NPs (Cy5-NP) were administered to healthy mice, and frozen sections of the brain and spinal cord were prepared 24 and 48 h after injection. At 48 h, significant accumulation of Cy5-NP was detected in the leptomeningeal and perivascular spaces, but no parenchymal uptake was observed in all coronal brain sections. A uniform deposition of Cy5-NPs surrounding the outer layer of the spinal cord was also performed.

[0090] The CNS retention of three distinct sizes: 90 nm, 150 nm, and 210 nm PLA-HPG NPs was assessed by imaging in mouse brain and spinal cord sections 6 hours after delivery into the CSF via the cisterna magna. FITC-conjugated dextran (M W NPs (either 3,000 or 3,000,000) served as size-fractionated controls (estimated diameters of <4 nm and >60 nm, respectively). All mice receiving NPs showed leptomeningeal retention in the brain and spinal cord, but the 90 nm NPs provided the greatest percent area coverage, as quantified by fluorescent signal in fresh-frozen coronal brain sections. In contrast, while both dextrans produced strong fluorescent signals 30 min after injection, no signal was detected in brain or spinal cord sections from mice dosed with both 3 kD and 3,000 kD dextrans 6 h after injection.

[0091] Next, we evaluated the effects of surface charge and surface chemistry on PLA-HPG NPs. HPG on the NP surface was converted to an aldehyde-rich corona (referred to as aldehyde-NPs) with enhanced bioadhesive properties. The conversion of vicinal diols to aldehydes on the HPG coating was achieved by short-term sodium periodate treatment and monitored by changes in zeta potential (which is lower for aldehyde-NPs). Despite the fact that surface charge and surface chemistry significantly affected plasma half-life and organ accumulation after iv or ip delivery, CSF distribution in the leptomeningeal region was minimally affected. CNS retention was not significantly different between PLA-HPG aldehyde-NPs and PLA-HPG NPs during 7-day ex vivo whole-body imaging using the XENOGEN® in vivo imaging system (IVIS). Both NP formulations were well retained in the tumor-free brain and spinal cord of healthy mice at all time points examined, with no difference in fluorescence radiance flux measured in vivo or ex vivo. To account for differences in accumulation along the lymphatic clearance pathway, the percentage of Cy5-NP-positive cells and the percentage of Cy5-NP-aldehyde-positive cells were assessed in the mandibular and deep cervical lymph nodes of tumor-free and tumor-bearing mice. No significant differences in NP accumulation were detected based on surface chemistry in macrophages (CD11b+ CD11c-) and dendritic cells in lymph nodes.

[0092] NPs with optimal properties for retention in the CNS exhibited a spherical morphology under electron microscopy and an average hydrodynamic diameter of 90-100 nm. The zeta potential of the NPs used in the remaining studies averaged approximately -10 mV.

[0093] NPs exhibit higher CNS retention compared to small molecules This study was carried out to clarify the detailed in vivo distribution kinetics of NPs in the CSF space. To image NPs with PET, a NP PET probe with similar properties was developed. To label aldehyde-NPs, positron-emitting zirconium ( 89 Zr) due to its stable and long half-life (72h) and 89 Deferoxamine (DFO), a well-characterized chelator of Zr, was used due to its availability. The aldehyde-NP surface was first functionalized with DFO-mesylate via a Schiff base reaction. No change in hydrodynamic diameter was detected, but a slight change in zeta potential was observed, from -10 mV to -5 mV. DFO-grafted aldehyde-NPs were then used to etch the surface of the NPs by incubation for 30 min. 89 The stability of the complex was examined by radio-thin layer chromatography. 89 Zr-DFO-NPs were thoroughly washed in a 300 kD filter tube to isolate unconjugated 89 Zr or 89 Zr-DFO was removed from the surface of the aldehyde-NPs. Stability studies in artificial CSF (aCSF) at 37°C showed that Zr-DFO was removed from the NP surface over a 7-day period. 89 It was shown that no Zr was lost.

[0094] The quantitative biodistribution of NPs over time to all major organs was measured. In tumor-free mice, free NPs were used as a control to determine the distribution and timing of NPs after IT administration. 89 A 2-h continuous scan was performed using Zr-DFO. Within 5 min of injection (denoted as the 0-h time point), NPs were observed to distribute from the injection site (CM) to the cervical region of the brain and spinal cord (Figures 4A and 4B). 89 The Zr-NP levels dropped slightly and then remained constant for the next 2 h, with limited distribution of the signal to the systemic circulation or other organs. 89Zr-DFO was immediately distributed throughout the subarachnoid space, similar to NPs, but then distributed from the CNS to the systemic circulation, and at 2 h, less than 30% of the PET signal was detectable in the CNS. 89 Zr-DFO has a CNS half-life of approximately 60 min, which is comparable to known half-lives of small molecules after IT delivery. 89 Zr-DFO-NP levels remained relatively stable in the brain and spinal cord during the first 2 hours. 89 Zr-DFO-NPs showed a different brain distribution pattern, as evidenced by a clearer signal shape. 89 It accumulated more in the olfactory bulb than Zr-DFO (Fig. 4C), which is related to the size difference between the two materials (100 nm vs. <1 nm).

[0095] The accumulation of PET signal in the bladder immediately after delivery of NPs was 89 This indicates that some of the Zr reached the bladder. This bladder signal is unlikely to be due to extravasated NPs. The size limit for renal glomerular filtration is 48 kDa, or 5 nm for polymers such as PEG and dextran, making it unlikely that NPs would be degraded quickly enough to accumulate in the bladder within the first 5 min after delivery. 89 After IT delivery of Zr, a comparable immediate spike and drop in bladder signal was observed. 89 Zr accumulates in bones and joints. NP preparations contain significant levels of free 89 Subsequent studies showed that no signal was observed in the heart, lungs, joints, or muscle at any time point, and no signal was observed in the kidneys or bladder after the first 24 hours, suggesting that no signal was present in the renal system. 89 It was shown that Zr-DFO was eliminated rapidly.

[0096] next, 89Zr-DFO-NPs were administered IT to tumor-free individuals, and radioactivity signals were monitored over 12 days using serial PET / CT scans at designated time points (n=4). At 3 h, at least 60% of the signal was found in the brain and at least 20% in the spinal cord (less than 80% in the CNS). The extent of activity in the brain gradually decreased over the first 4 days, eventually reaching 30%. This level of activity was observed in the brain for at least 12 days (the final day of imaging). In the spinal cord, the decrease in activity was slower during the first 4 days, with similar levels of activity, approximately 25%, observed at these time points. The signal decreased to approximately 20% and approximately 10% on days 7 and 12. Ex vivo gamma counting after 12 days confirmed the in vivo imaging findings, showing the highest Bq / g signal in the brain (44% of total mass-normalized Bq activity), followed by the liver (16%), spleen (34%), and cervical lymph nodes (3%). No discernible signal was detected in the heart, GI organs, kidneys, or bladder.

[0097] NPs exhibit higher CNS retention in xenograft tumor models 89CNS retention of Zr-DFO-NPs was evaluated in tumor-bearing mice. A leptomeningeal metastatic medulloblastoma model was performed using DAOY cells stably expressing luciferase administered intravenously to the CM. By day 14, BLI demonstrated tumor growth throughout the CNS, including widespread disseminated metastases in both the cerebellum and spinal cord. To verify that the presence of tumors was not causing BBB disruption, mice were intravenously injected with Gd-DTPA MR contrast agent and imaged using MRI. Results confirmed the absence of BBB penetration compared with controls. Two mice were intravenously injected with Gd-DTPA and imaged by MRI 20 minutes later. No detectable Gd-DTPA signal was present in the brain, confirming an intact BBB. However, acute hydrocephalus was observed in both mice, likely due to tumor obstruction of CSF flow. H&E images of the brain revealed high tumor burden in the cerebellum, ventricles, and spinal leptomeninges of the brain.

[0098] 89Zr-DFO-NPs were assessed for biodistribution and uptake in tumor and normal tissues over a 21-day period in two cohorts of mice using PET / CT imaging (Figures 5A–5E). The first cohort (n = 2) underwent serial scans for 120 min after administration. Within the first 5 min, 30% of the total injected dose was detected in the tumor, gradually increasing to 40% over the next 2 h. The next cohort (n = 4) was scanned at predefined intervals (6 h, 24 h, 4 d, 7 d, 12 d, and 21 d). Tumor uptake was significantly greater than any other normal tissue, with 50% of the total injected dose identified in the tumor site (cerebellum). Over the next 21 d, brain and spinal cord levels remained stable at approximately 15% and 10% of the total activity, respectively. Higher levels of accumulation in peripheral organs were observed in tumor-bearing mice compared with healthy mice, with accumulation observed in the cervical lymph nodes, peripheral lymph nodes, liver, and spleen. None of these levels exceeded 20% of total activity at any time point. Overall, tumor-bearing mice were shown to retain NPs longer in the CNS, likely due to uptake of NPs by tumor cells. At day 21, 40% of PET activity remained detectable at the tumor site, which could be visualized using maximum intensity projection of PET activity.

[0099] NP accumulation was assessed at the cellular level by administering Cy5-NP to tumor-bearing mice and observing them under a microscope. Seven days after injection, dense NP accumulation and uptake were detected at the tumor site. NP retention in this location in the cerebellum was not observed in healthy mice. NP accumulation density in the leptomeninges of the brain and spinal cord was also lower than in healthy individuals, indicating that NPs circulate similarly throughout the perivascular space but accumulate at significantly higher densities in tumor-bearing sites than in healthy brain or spinal cord tissue.

[0100] The presence of Cy5-NPs in the parenchymal tumor and their accumulation in deep and superficial cervical LNs detected by PET raised the possibility that there may be several routes of NP delivery to the tumor microenvironment and cervical LNs. NP accumulation at the tumor site was promoted by the presence of immune cells. Cy5-NPs were injected into tumor-bearing mice, and 48 h later, brains were harvested, sectioned, and stained for F4 / 80 (a macrophage marker) and Iba1 (a microglia marker). At 48 h, the presence of activated microglia (red) and tumor-associated macrophages (green) was detected within the tumor mass, and significant colocalization of NPs with both cell types was detected. In non-tumor-bearing WT mice, NP accumulation was not observed in the brain parenchyma but only in CSF-bathed areas such as the choroid plexus, without association with microglia or macrophages. These results indicate that NP penetration and delivery into the tumor mass are promoted by tumor-associated immune cells. Some of the NPs in the tumor microenvironment were not associated with either macrophages or microglia, and these NPs may have been taken up specifically by medulloblastoma tumor cells or by other cells in the brain parenchyma, such as astrocytes.

[0101] To further investigate the pathway by which NPs are eliminated from the CNS, the meninges of tumor-bearing mice were isolated 48 h after intravenous injection of Cy5-NPs and stained for either Lyve-1 and CD45 or Lyve-1 and CD31. NP accumulation occurred throughout the meninges, but particularly along the venous sinuses (higher density in the transverse sinus than in the superior sagittal sinus). Closer examination of the lymphatic and blood vessels in the transverse sinus revealed that NP accumulation colocalized with areas of Lyve-1 and CD31 staining, but clusters of NPs existed that were not associated with either meningeal lymphatic or blood vessels. CD45-stained transverse sinuses revealed that NP accumulation frequently, but not always, colocalized with areas of Lyve-1 and CD45 immunocytochemical staining.

[0102] Polymer encapsulation of BMN-673 alters the drug's toxicity profile in animal studies As a proof-of-concept, it was determined whether the PK characteristics and activity of a novel class of drugs, PARPi, could be improved. BMN-673 was selected for its potent PARP-scavenging properties and ability to induce toxicity at very low doses. BMN-673 loading on NPs varied from 1% to 5% (w / w) depending on the solvent ratio and drug-to-polymer ratio used during NP preparation. NPs without aldehyde-modified surfaces were chosen because they exhibited similar persistence in the brain. BMN-673 release rates from these NPs were similar in both CSF and PBS at 37°C, with an average release rate of 60% over 3 days. The relative cytotoxic activity of free BMN and BMN-NPs was determined in three MB cell lines: DAOY, D341, and D283. Both agents were cytotoxic in the 10 nM to 1 μM range, but the NPs were more potent with lower IC50 values.

[0103] To characterize the in vivo safety of BMN-NPs, toxicity studies were performed in healthy female nude mice. The maximum tolerated dose (MTD) was determined for single IT administration of BMN-NPs or free BMN in nude mice (Figures 6A-6B). Increasing doses of either BMN-NPs or free BMN-673 were administered to individuals. Weight loss and overall health of the individuals were closely monitored. The MTD value for BMN-NPs was 10-fold higher than that of free BMN. The median lethal dose of IT administration of free BMN-673 was 0.125 mg / kg. Individuals treated with doses above 0.06 mg / kg developed various toxicity-related symptoms, including lethargy, respiratory distress, and occasionally death. The MTD (single dose) was determined to be 0.05 mg / kg. A slightly lower dose of 0.03 mg / kg twice weekly was tolerated with less than 10% weight loss. In contrast, BMN-NPs were well tolerated at all doses tested, with doses at or below 0.5 mg / kg, the maximum dose permitted in a single infusion due to IT volume dosing limitations. To achieve IT doses above 0.5 mg / kg, mice were dosed multiple times within 3 h on the same day, and a lethal dose of 1.25 mg / kg was determined. At 1.25 mg / kg, the onset of acute toxicity symptoms was delayed, likely due to delayed drug release from the NPs.

[0104] To further examine the difference in systemic toxicity between BMN-NPs and free BMN, red blood cell, white blood cell, and platelet levels were monitored in mice on days 3 and 7 after two biweekly doses at the MTD (Figure 5C). Mice treated with 0.05 mg / kg / week free BMN had progressive leukopenia and thrombocytopenia on day 3, with no appreciable improvement on day 7. When NPs were dosed at a 10-fold higher dose of 0.5 mg / kg / week, WBC, RBC, and PLT levels remained significantly more normal during the course of treatment, and all cell counts except eosinophils returned to normal levels by day 7.

[0105] BMN-673 NPs exhibit superior activity compared to free BMN-673 in xenograft tumor models BMN-NPs demonstrated improved efficacy in an in vivo tumor xenograft model, with an improved therapeutic index relative to BMN. Intracisternal implantation of DAOY cells stably expressing luciferase was used for in vivo studies. A surgical catheter was placed in the cisterna magna of the mice and used for both cell implantation and IT dosing during the procedure. Mice developed tumor luminescence of 10 at 7 days post-implantation. 5 Treatment was performed when tumors became detectable by BLI (units). Mice were treated once with either BMN-NP or free BMN-673 at the same dose level of 0.1 mg / kg (Figure 7A). The growth rate of BMN-NP-treated tumors was significantly slower than that of BMN-673 (free drug)-treated tumors. During the week following dosing, tumors showed a maximal decrease in BLI, followed by growth retardation for several weeks, which was not observed in the free BMN-673 group. With free BMN-673, tumor reduction was observed in only one of five mice, even though the dose exceeded the MTD. Furthermore, the weight loss of mice treated with free BMN-673 was significantly greater than that of mice treated with BMN-NP (Figure 7C). Consistent with the BLI findings, mice treated with BMN-NP survived significantly longer than those treated with free drug alone, with a median survival time of 56 days (Figure 7B).

[0106] This study was repeated using a 0.25 mg / kg twice-weekly dose of BMN-NP or a 0.03 mg / kg dose of free BMN-673 (Figures 7D–7F). These equitoxic doses corresponded to approximately 50% of the MTD, and similar levels of mean body weight loss were observed in both treatment groups. More dramatic tumor regressions were observed in the NP-treated group. NP treatment had substantial antitumor effects in all mice, particularly one week after the doses were administered. BMN-NP induced complete regression in several treated mice, while tumors continued to progress in those treated with free drug. NP-treated mice with tumor progression achieved a median survival benefit of 5 weeks compared to the free drug group and 6 weeks compared to the control group. In this study, median survival times for all treatment groups generally reflected tumor growth rates assessed by bioluminescence, suggesting that mice succumbed to cancer rather than drug-induced side effects. Furthermore, the treatment group showed a significant improvement in the rate of leptomeningeal metastasis formation, as measured by bioluminescence imaging. BMN-NPs, and to a lesser extent, free drug BMN-673, significantly reduced the incidence of CNS metastases. In contrast, over 80% of untreated animals developed overt spinal disseminated metastases and required euthanasia due to severe hydrocephalus around week 4.

[0107] BMN-673 NPs synergize with temozolomide when given together in a xenograft model To demonstrate the sensitivity of medulloblastoma cell lines to the synergistic action of TMZ and BMN-673, DAOY, D341, and D283 cells were treated with free BMN and BMN-NPs and compared with conventional IC. 50Analysis was performed using these two drugs to calculate a combinatorial index. It was demonstrated that cells treated with incremental log-fold increases of BMN-673 in the presence of TMZ exhibited reduced viability, as quantified using the CellTiter-Glo viability assay, compared with cells treated with BMN-673 alone. Using the classical Loewe synergy model, combinatorial index values ​​between BMN-673 and TMZ were calculated, demonstrating a high level of synergy. Drug interaction assays with other commonly used brain-penetrating chemotherapeutic agents (lomustine, topotecan, irinotecan, and cyclophosphamide) did not demonstrate consistent synergy with BMN-673 in all cell lines.

[0108] Synergy was observed in vitro using a D341 xenograft model inoculated with cells in a manner identical to that of the DAOY cell line. Because most mice in the D341 model develop spinal metastases detectable by IVIS imaging within 7 days of inoculation, we tested the efficacy of BMN-NP and free BMN against existing spinal metastases by initiating treatment on day 7 in the presence of leptomeningeal metastases. Four repeated doses of BMN-NP were effective in reducing brain and spinal tumor burden in the D341 model, whereas free BMN-673 treatment (at an equitoxic dose to NP) was ineffective. This treatment strategy was improved by a temozolomide (TMZ) combination approach. Synergy between TMZ and PARPi was detailed in vitro, where PARP1 capture sensitized cells to the DNA methylation mechanism of TMZ, but in vivo, the PARPi dose needed to be reduced to prevent acute toxicity. The combination of BMN-NP and TMZ was well tolerated in mice without the need for dose reduction, leading to tumor clearance in 4 of 6 mice tested. The combination of free BMN-673 and TMZ was not tolerated even at the lowest dose of BMN-673.

[0109] Collectively, these results demonstrate that intraCSF delivery of nanoparticle-encapsulated drugs is a viable treatment regimen for MB, which offers significant therapeutic advantages over intrathecal administration of free drug alone in terms of significant and sustained in vivo activity.

[0110] Consideration Direct drug infusion into the CSF has emerged as a promising method to bypass the BBB in the treatment of medulloblastoma. Given that leptomeningeal recurrence remains a major cause of patient mortality, a compelling rationale exists for delivery routes that increase CSF drug exposure. Despite the potential advantages of CSF delivery, maintaining high drug concentrations in the subarachnoid space against the rapid convection and clearance of the CSF remains a persistent challenge. The results described herein use a PLA-HPG NP platform that exhibits prolonged retention in the subarachnoid space, in contrast to the fate of small molecules administered in the free state. PET / CT and fluorescent whole-body imaging demonstrated long-term retention of NPs in the CSF space of tumor-free mice, as well as preferential accumulation and retention in CSF-adjacent tumors. Increased accumulation of nanoparticles in tumors early in the circulation reduces the probability of clearance by mononuclear cell phagocytosis and the renal system.

[0111] The vast majority of polymeric NPs exhibit significant absorption in the spleen and clearance organs, such as the liver and kidney, potentially limiting their therapeutic applications. Even with functionalized BBB penetration modalities, NP accumulation in the brain after intravenous delivery is typically limited to less than 1% of total activity, with the majority of delivered NPs being processed in the spleen and liver. Systemically delivered HPG-coated NPs exhibit reduced recognition and clearance by the reticuloendothelial system compared to other commonly used polymeric NPs. Results demonstrated that PLA-HPG NPs retain their ideal properties even in the CSF space by forming a steric barrier around the NPs, extending their circulation time and allowing for greater accumulation at tumor sites. PLA-HPG NPs showed less than 15% accumulation in clearance organs at all time points, as measured by PET / CT. The highest accumulation outside the CNS occurred in the cervical lymph nodes rather than the liver or spleen. Surprisingly, a greater shift from CSF to systemic clearance was observed in tumor-bearing mice compared with tumor-free mice, which may be due to the abnormally leaky vasculature in the tumor microenvironment and dysfunctional lymphatic drainage. Significant accumulation of NPs at tumor sites in the meninges and cerebellum of tumor-bearing mice was also observed. It is unclear whether NPs are phagocytosed by tumor-associated immune cells in the brain before being transported to the meninges, or whether NPs are taken up by resident immune cell types once they reach the meninges. It is likely that a combination of the large flow of CSF draining into the meninges and then into cervical LNs and the active transport of NP-associated immune cells provides an important drainage route for NPs in the CSF to LNs.

[0112] The controlled release of drugs allows for prolonged retention of NPs, leading to extended drug exposure at tumor sites and providing a means to improve the overall half-life of drugs after intra-CSF administration. Furthermore, activity in the CNS is at least 75% of total activity at all measured time points, significantly reducing the risk of widespread systemic toxicity. PARP inhibitors are limited by BBB penetration and widespread toxicity. While PARPi were initially developed to sensitize tumor cells to conventional DNA-damaging agents, increasing evidence suggests that PARPi are effective in sensitizing cells to radiation therapy, temozolomide, and topoisomerase poisons and inhibitors. However, clinical efforts to combine PARPi have been hampered by their high toxicity profile, and no PARPi combinations have been approved. In pediatric CNS tumors, veliparib is the most clinically advanced PARPi due to its ability to cross the BBB. Combinations with temozolomide and temozolomide plus radiation therapy have been evaluated, but progress has been hampered by difficulties in escalating doses without causing toxicity and a lack of survival benefit. TMZ sensitization is induced by PARP1 trapping, consistent with the lack of success of veliparib, which has relatively poor PARP1 trapping capacity. In contrast, talazoparib (BMN-673) is a potent PARP1 trapping agent but is limited by its inability to evade the BBB at meaningful doses. Studies with BMN-673, the first known preclinical study of intrathecal delivery of a PARPi, demonstrated unacceptable levels of toxicity with both the free drug alone and in combination with TMZ. Nanoencapsulation significantly improves the therapeutic index of BMN-673. Significantly higher doses (10x) were possible with lower systemic toxicity compared to the free drug, as measured by blood counts, weight loss, and organ toxicity. Single-agent efficacy in an orthotopic model of MB was measured at a given equitoxic dose of either BMN-673 or BMN-NPs, and it was observed that only encapsulated BMN NPs resulted in consistent tumor regression and an overall reduction in leptomeningeal metastases.Furthermore, BMN-NPs were administered with low-dose TMZ, and the combination produced durable responses and was well tolerated by mice. This highlights the applicability of this approach and its potential to overcome the high tumor heterogeneity often observed in MB. These data suggest that direct delivery of NPs into the CSF enhances drug exposure to the NPs and, therefore, cancer cells, resulting in favorable antitumor effects while minimizing damage to healthy tissues. In clinical settings, repeated administration of NPs, such as via an Ommaya reservoir (an intraventricular catheter used to deliver drugs to the CSF), may improve the therapeutic efficacy of therapy compared with intravenous or free drug administration. This integrated therapeutic approach may offer novel opportunities for PARPi combination therapy without compromising tolerability. Furthermore, this approach may offer a promising therapeutic avenue for other diseases associated with widespread leptomeningeal metastasis, such as leptomeningeal metastases from primary malignancies, such as lung cancer, breast cancer, and melanoma.

Claims

1. A formulation for intrathecal delivery to the central nervous system, comprising nanoparticles having a core of a hydrophobic polymer and a shell of hyperbranched polyglycerol having tissue-adhesive functional groups on its surface, in a medicamentally and pharmaceutically acceptable carrier, wherein the nanoparticles comprise a therapeutic, prophylactic or diagnostic agent.

2. The formulation of claim 1 comprising a chemotherapeutic agent.

3. 3. The formulation of claim 1 or 2, having a median diameter between 10 and 500 nm, more preferably between 25 and 250 nm, most preferably between 100 and 250 nm.

4. The formulation of any one of claims 1 to 3, comprising a diagnostic agent.

5. The formulation of any one of claims 1 to 4, wherein the adhesive functional group is selected from the group consisting of aldehydes, amines, oximes, and O-substituted oximes.

6. The formulation according to any one of claims 1 to 5, wherein the functional group is an aldehyde.

7. The chemotherapeutic agent is an anticancer agent, preferably a PARP inhibitor, and is preferably olaparib, veliparib, CEP-8983 (11-methoxy-4,5,6,7-tetrahydro-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dione) or a prodrug thereof (e.g., CEP-9722), rucaparib, E7016 (10-((4-hydroxypiperidin-1-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one), INO 7. The formulation of any one of claims 2 to 6, wherein the benzodiazepine is selected from the group consisting of -1001 (4-phenoxy-3-pyrrolidin-1-yl-5-sulfamoyl-benzoic acid), niraparib, talazoparib (BMN673), NU1025 (8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroisoquinoline, 4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, and analogs, isosteres, and derivatives thereof.

8. The formulation according to any one of claims 1 to 7, wherein the hydrophobic polymer is a polyhydroxy acid, preferably poly(lactic acid) or poly(lactide-co-glycolide).

9. 3. The formulation of claim 2, comprising a combination of a PARP inhibitor and temozolomide.

10. 1. A method for delivering a therapeutic, prophylactic or diagnostic agent to the central nervous system, comprising intrathecally administering the agent in a formulation for intrathecal administration, the formulation comprising nanoparticles comprising a core of a hydrophobic polymer, preferably a polyhydroxy acid, more preferably poly(lactic acid) or poly(lactide-co-glycolide), and a shell of hyperbranched polyglycerol having tissue-adhesive functional groups on its surface, in a pharmaceutically acceptable carrier.

11. 11. The method of claim 10, wherein the agent is a chemotherapeutic agent for treating cancers of the central nervous system, preferably leptomeningeal tumors such as leptomeningeal metastases and disseminated tumors such as medulloblastoma.

12. The nanoparticles comprise a PARP inhibitor, and the inhibitor is preferably olaparib, veliparib, CEP-8983 (11-methoxy-4,5,6,7-tetrahydro-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dione) or a prodrug thereof (e.g., CEP-9722), rucaparib, E7016 (10-((4-hydroxypiperidin-1-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one), INO- 12. The method of claim 10 or 11, wherein the compound is selected from the group consisting of 1001 (4-phenoxy-3-pyrrolidin-1-yl-5-sulfamoyl-benzoic acid), niraparib, talazoparib (BMN673), NU1025 (8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroisoquinoline, 4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, and analogs, isosteres, and derivatives thereof.

13. The method of any one of claims 10 to 12, comprising administering to a patient having a tumor a combination of a PARP inhibitor and temozolomide.

14. The method according to any one of claims 9 to 11, wherein the nanoparticles have a median diameter between 10 and 500 nm, more preferably between 25 and 250 nm, most preferably between 100 and 250 nm.

15. The method of any one of claims 10 to 14, wherein the nanoparticles comprise a diagnostic agent.

16. 16. The method of any one of claims 10 to 15, wherein the nanoparticles comprise adhesive functional groups selected from the group consisting of aldehydes, amines, oximes, and O-substituted oximes, preferably aldehyde and / or amine functional groups.

17. The nanoparticles according to any one of claims 10 to 16, wherein the functional group is an aldehyde.

18. The method of any one of claims 10 to 17, further comprising administering radiation to the brain after administration of the nanoparticles.

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