Delivery of safe and efficacious dose of chemotherapy to the CNS for treatment of cancer

Abstract

CED-based methods, products and compositions for treating glioblastoma with anti-inflammatory therapeutics and chemotherapeutics without decreasing brain corticosterone compared to systemic delivery of dexamethasone, and not causing splenal atrophy and / or adrenal atrophy.

Description

DELIVERY OF SAFE AND EFFICACIOUS DOSE OF CHEMOTHERAPY TO THE CNS FOR TREATMENT OF CANCER

[0001] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U. S. Provisional Application No. 63 / 722,660 filed November 20, 2024, and U. S. Provisional Application No. 63 / 718,953 filed November 11, 2024, the contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under CA161404 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0004] Glioblastoma is the most common and aggressive primary brain malignancy and is associated with a dismal clinical prognosis.1Despite standard of care treatment consisting of surgical resection, temozolomide, and radiotherapy, median survival for glioblastoma is around 15 months after diagnosis, a consequence of the nearly inevitable recurrence of the disease.2

[0005] Cerebral edema and tumor-associated inflammation are among the chief clinical problems contributing to neurological decline and reduced quality of life in glioblastoma patients. Since the 1960s, these problems have primarily been managed with dexamethasone.3Patients are frequently on dexamethasone therapy for prolonged periods of time, often until death4,5As a result, Cushing syndrome is common4,6with irritability, weight gain, muscle atrophy, plethora, moon facies, ecchymoses, striae, hyperglycemia, and systemic immunosuppression.7These adverse effects likely contribute to the observed negative correlation between dexamethasone dose and overall survival in glioblastoma patients.8,9

[0006] Dexamethasone exerts its effects through both genomic and nongenomic mechanisms,10 12and its pleiotropic effects are responsible for both its potent edema-reducing effects and its harmful side effects.13Improvements in cerebral edema following dexamethasone therapy have been linked to its suppression of vascular permeability factors including VEGF and IL-ip as well as upregulation of various tight-junction proteins in endothelial cells.14 18Dexamethasone has been shown to attenuate microglial activation to inflammatory stimuli both in vitro and in vivo and decrease nitric oxide and cytokine production by activated microglia.19 22In vivo glioblastoma studies have demonstrated the immunosuppressive effects of dexamethasone on T-cells,23,24but concomitant effects on the chronic inflammatory milieu remain poorly defined.

[0007] The diffuse nature of gliomas means that neoplastic cells grow and intermingle with non-neoplastic cells in a tumor microenvironment primarily consisting of tumor-associated microglia and macrophages that comprise up to 30% of tumor bulk in primary glioblastoma.25’26This chronic inflammatory environment promotes tumor progression and is therefore a putative therapeutic target.27 30Broad inhibition of tumor-associated microglia and macrophages with CSF-1R inhibitors and targeted suppression of IL- 1 [3 have both been shown to prolong survival in glioma-bearing mice.28,29’31Unfortunately, an initial clinical trial of a CSF-1R inhibitor was unsuccessful,32and IL-1J3 inhibition has limited clinical precedent in glioblastoma. Dexamethasone remains the most clinically relevant and widely used anti-inflammatory drug in glioblastoma patients.BRIEF SUMMARY OF THE INVENTION

[0008] A method of treating a glioblastoma in a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of dexamethasone, and / or (ii) does not effect splenal atrophy and / or adrenal atrophy, the method comprising administering (a) an amount of dexamethasone to the glioblastoma of the subject by convection-enhanced delivery (“CED”), and (b) an amount of a chemotherapeutic to the subject, effective to treat a glioblastoma in a subject and not decrease brain corticosterone compared to systemic delivery of dexamethasone and / or not effect splenal atrophy and / or adrenal atrophy

[0009] A method of treating a brain tumor in a subj ect, or a method of treating a chemotherapy-associated inflammation in a brain of a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of anti-inflammatory medication, and / or (ii) doesnot effect splenal atrophy and / or adrenal atrophy, the method comprising administering an amount of an anti-inflammatory medication to the brain tumor of the subject by convection-enhanced delivery (“CED”), effective to treat a brain tumor, or treat a chemotherapy-associated inflammation in a brain of a subject, and not decrease brain corticosterone and / or not effect splenal atrophy and / or adrenal atrophy.

[0010] A pharmaceutical composition for treating glioma in a patient comprising: (a) therapeutically effective amounts of at least one chemotherapy agent, pharmaceutically acceptable salts thereof, and combinations thereof; (b) therapeutically effective amounts of at least one steroid or non-steroidal anti-inflammatory agents, and (c) at least one pharmaceutically acceptable carrier, and optionally (d) an amount of gadolinium.

[0011] A kit for the treating brain inflammation associated with chemotherapy treatment of glioma in a patient comprising: (a) a pharmaceutical composition described herein; and (b) a catheter for administering a pharmaceutical composition described herein via convection enhanced delivery.

[0012] An implantable pump with a reservoir for therapeutic(s) to be delivered to a subject in which the pump is implanted, comprising in the reservoir (a) a therapeutically effective amount of at least one steroid or non-steroidal anti-inflammatory agent, and (b) at least one pharmaceutically acceptable carrier.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1C: A - Schematic of the survival experiment. Mice were implanted with tumors on DPI 0. At DPI 21 an Alzet pump (1007D) was implanted containing either 100 ng / pl of dexamethasone or PBS (vehicle). After seven days of treatment pumps were explanted and mice were followed until death. B - Representative MRI images from mice demonstrating the volume of gadolinium distribution between control and CED dexamethasone treatment conditions. C - Survival curves of vehicle- or CED-dexamethasone-treated mice. Median survival for control mice was 34 days while median survival for CED-dexamethasone-treated mice was 42 days (logrank p = 0.03).

[0014] FIGS. 2A-2M: A - Schematic of the post-treatment tissue analysis experiment. Mice were implanted with tumors on DPI 0. On DPI 21 mice received either CED of dexamethasone (100 ng / pl), daily systemic i.p. injections of dexamethasone (10 mg / kg), or PBS (vehicle) control. All mice received both injections and pump implantation. After seven days of treatment, pumps were explanted, and the mice were sacrificed for either snRNA sequencing or histology. B -UMAP of myeloid nuclei. Nuclei clustered into macrophage-like (Msrl+, Cd74+) or microglial-like (P2ry 12+, Tmeml 19+). C - CED of dexamethasone reduced the expression of a broad set of immune activation genes in myeloid nuclei. D - GSEA of Hallmark pathways in myeloid nuclei revealed significant positive enrichment of inflammatory pathways in vehicle-treated mice. At the same time, there was significant negative enrichment of inflammatory pathways in mice treated with CED of dexamethasone. E - A GSEA of GO biophysical pathways revealed a similar downregulation of immune and inflammatory-related pathways in mice treated with CED of dexamethasone. Cytokine signaling pathways, including IL6, IL-ip, and TNFa, were significantly down-regulated. F - A survival analysis utilizing TCGA and CGGA bulk RNA sequencing showed that greater than median expression of the top 50 significantly downregulated myeloid genes following CED of dexamethasone was correlated with worse overall survival (p = 0.0018). G - Representative immunofluorescence images showing Ibal positivity in Cre-positive fields. White bar represents 20 pm. H - Ibal positive staining area was decreased throughout the tumor in mice treated with dexamethasone (CTRL vs SYS p = 0.018; CTRL vs CED - p = 0.0031; CED vs SYS - p = 0.031). I - Ibal positive staining area was decreased with CED of dexamethasone compared to vehicle-treated mice even when normalized to total cellularity as a proxy for tumor burden (p < 0.0001). J - Representative immunofluorescence images showingIbal and Msrl in the tumor core. White bar represents 20 pm. K - CED of dexamethasone led to a significant reduction in Msrl positive cells over control (p = 0.0032). Msrl positive cells trended lower in mice treated with systemic dexamethasone compared to control (p = 0.24). L -Representative microglia demonstrating ramified and ameboid morphologies. M - Automated morphological analysis of tumor-associated, Ibal-positive microglia in the various treatment conditions and in the contralateral brain (resting). Tumor-associated microglia were significantly less spherical and more branching when treated with CED of dexamethasone - statistics computed on a per microglia basis. (* = p < 0.05; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001)

[0015] FIGS. 3A-3H: A - Schematic of the iPSC-derived microglial experiment. iPSC cells were differentiated into microglia before being treated for 24 hours. RNA was then extracted from the cells differential gene expression analysis was performed. B - Venn diagram representing the number of unique and overlapping differentially expressed (p-adj < 0.05) genes in each comparison. C - The addition of dexamethasone to LPS reversed the expression of a large number of genes that were initially differentially expressed with LPS. The number of overlapping genes that were differentially expressed in opposite directions in the LPS vs CTRL and LPS+Dex vs LPS comparisons was significantly greater than would be expected by chance (p < le-10, hypergeometric test). D - Dexamethasone broadly reversed a large number of cytokines that were upregulated with the addition of LPS. The log2(FC) column represents the log-fold change in expression between the LPS and LPS+Dex conditions. An asterisk (*) next to a gene name denotes that LPS induced a significant change in expression that was significantly reversed in the LPS+Dex condition. E - Significant Inflammatory HALLMARK Pathway Enrichment; F, G, H -Normalized RNA expression for IL- 10, IL6, and TNFa.

[0016] FIGS. 4A-4E: A -Dexamethasone concentrations in the tumor-bearing quadrant (196.2 ± 56.4 ng / g) and liver (6.7 ± 3.1 ng / g) in mice treated with CED of dexamethasone. Values were obtained after 5 days of treatment with an Alzet osmotic pump (1007D). B - Dexamethasone concentration in the tumor-bearing quadrant (124.8 ± 93.4 ng / g), liver (10,999 ± 5,486 ng / g), and serum (2,411 ± 1,248 ng / g) of mice one hour after i.p. injection of 10 mg / kg of dexamethasone. C - The partitioning of dexamethasone between the brain and liver depends on the route of administration. CED of dexamethasone allowed for preferential partitioning of the drug in the brain. Intraperitoneal injection led to a partitioning coefficient of Kbrain, liver = 0.011 ± 0.010. CED led to a partitioning coefficient of Kbrain, liver = 29.2 ± 15.8. D -Dexamethasone concentrations inthe brain, liver, and serum were determined 1, 4, 8, and 24 hours post-i.p. injection of 10 mg / kg dexamethasone. E - Areas under the curve were calculated for once daily 10 mg / kg dexamethasone i.p. and for CED of dexamethasone (100 ng / yl). Assuming CED leads to a steady state concentration over 24 hours, the present disclosure found that CED delivery gave an AUC of 4,709 ± 1,327 (ng / g)-h, whereas systemic delivery had an AUC of 375 ± 75 (ng / g)·h.

[0017] FIGS. 5A-5J: A - Blood glucose levels at one day post-treatment compared to pretreatment baseline. Mice receiving systemic dexamethasone had a significant decrease in blood glucose levels compared to control and CED-treated mice (p = 0.0019; p = 0.00018, respectively). Blood glucose levels of CED-treated mice were not different from control (p = 0.95). B - Blood glucose levels at seven days post-treatment compared to pre-treatment baseline. By the end of treatment, mice receiving systemic dexamethasone still had a significant decrease in blood glucose levels compared to control and CED-treated mice (p = 0.0075; 0.0066, respectively). CED-treated mice did not have a significant difference in blood glucose levels compared to control mice (p = 0.34). C - Lymphocyte percentages after seven days of treatment for each treatment group (CTRL: 59 ± 21%, CED: 59 ± 30%, SYS: 38 ± 27%). Systemically treated mice had a significant decrease in lymphocyte percentage compared to control and CED-treated mice (p = 0.0062; p = 0.01, respectively). CED-treated mice had a similar lymphocyte percentage to control mice (p = 1). D - Monocyte percentages after seven days of treatment for each treatment group (CTRL: 5.1 ± 2.9%, CED: 5.8 ± 4.9%, SYS: 2.4 ± 2.4%). Systemically treated mice had a significant decrease in monocyte percentage compared to control and CED-treated mice (p = 0.0024; p = 0.0035, respectively). CED-treated mice had a similar monocyte percentage to control mice (p = 0.51). E -Neutrophil percentages after seven days of treatment for each treatment group (CTRL: 29 ± 25%, CED: 31 ± 31%, SYS: 50 ± 33%). Systemically treated mice had a significant increase in neutrophil percentage compared to control and CED-treated mice (p = 0.017; p = 0.038, respectively). CED-treated mice had a similar neutrophil percentage to control mice (p = 0.72). F - Mean platelet volume after seven days of treatment for each treatment group (CTRL: 5.3 ± 1.1 fL, CED: 5.3 ± 0.9 fL, SYS: 5.9 ± 0.6 fL). Systemically treated mice had a significant decrease in mean platelet volume compared to control and CED-treated mice (p=0.05; p 0.016, respectively). CED-treated mice had a similar mean platelet volume to control mice (p = 0.91). G - Average adrenal gland weight for mice in each treatment group after seven days of treatment (CTRL: 0.010 ± 0.003 g, CED: 0.011 ± 0.003 g, SYS: 0.004 ± 0.004 g). Systemically treated mice had asignificant decrease in adrenal gland weight compared to control and CED-treated mice (p < 0.0001; p < 0.0001, respectively). CED-treated mice had a similar adrenal gland weight to control mice (p = 0.25). H - Spleen weight for mice in each treatment group after seven days of treatment (CTRL: 0.084 ± 0.037 g, CED: 0.082 ± 0.022 g, SYS: 0.034 ± 0.010 g). Systemically treated mice had a significant decrease in spleen weight compared to control and CED-treated mice (p < 0.0001; p < 0.0001, respectively). CED-treated mice had a similar adrenal gland weight to control mice (p = 0.70). I, J - Endogenous corticosterone concentrations were determined in the liver (CTRL: 75.9 ± 35.4 ng / g, CED: 46.6 ± 46.6 ng / g, SYS: 0 ± 0 ng / g) and brain (CTRL: 56.8 ± 36.0 ng / g, CED: 45.1 ± 15.2 ng / g, SYS: 0 ± 0 ng / g). Systemic delivery of dexamethasone ablated endogenous corticosterone by day seven of treatment. Similar ablation of endogenous corticosterone was not seen with CED of dexamethasone. (* = p < 0.05; ** = p < 0.01, *** = p < 0001, **** = p < 0.0001)

[0018] FIG. 6: The IC50 of dexamethasone was determined to be 549 ± 289 ng / pl (1,399 ± 736 pM) for murine APCL (p53' / _, PDGFA+) cells, 202 ± 19 ng / pl (515 ± 48 pM) in human TS543 glioma cells, and 194 ± 23 ng / pl (494 ± 59 pM) in human U87 cells.

[0019] FIGS. 7A-7C: A - Major cell types assigned by SingleR. B - Dot plot of canonical marker genes present in major cell lineages. C - Microglial and macrophage gene expression across myeloid nuclei using canonical genes.

[0020] FIGS. 8A-8E: A - GSEA enrichment scores for select Hallmark inflammatory pathways. Interferon response pathways moved in the same direction, regardless of delivery method, while other inflammatory gene sets moved in opposite directions. Local delivery appears to be more uniformly anti-inflammatory than once daily systemic delivery. B, C, D, E -Programs in Mouse Glioma Myeloid cells treated with CTL vs CED and CTL vs SYS Dexamethasone. The “complement immunosuppressive” and “systemic inflammatory” pathways from Miller et al. were differentially regulated in the local vs systemic delivery groups. Local delivery led to a NES of -1.39 whereas systemic delivery had a NES of +1.21 in the “complement immunosuppressive” gene set. Similarly, local delivery led to a NES of -1.59 whereas systemic delivery had a NES of +1.91 in the “systemic inflammatory” gene set.

[0021] FIG. 9: The partitioning coefficient between brain and plasma in human contrastenhancing tumor samples, non-enhancing samples, and mouse tumor-bearing quadrants.DETAILED DESCRIPTION OF THE INVENTION

[0022] A method of treating a glioblastoma in a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of dexamethasone, and / or (ii) does not effect splenal atrophy and / or adrenal atrophy, the method comprising administering (a) an amount of dexamethasone to the glioblastoma of the subject by convection-enhanced delivery (“CED”), and (b) an amount of a chemotherapeutic to the subject, effective to treat a glioblastoma in a subject and not decrease brain corticosterone compared to systemic delivery of dexamethasone and / or not effect splenal atrophy and / or adrenal atrophy.

[0023] A method of treating a brain tumor in a subj ect, or a method of treating a chemotherapy-associated inflammation in a brain of a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of anti-inflammatory medication, and / or (ii) does not effect splenal atrophy and / or adrenal atrophy, the method comprising administering an amount of an anti-inflammatory medication to the brain tumor of the subject by convection-enhanced delivery (“CED”), effective to treat a brain tumor, or treat a chemotherapy-associated inflammation in a brain of a subject, and not decrease brain corticosterone and / or not effect splenal atrophy and / or adrenal atrophy.

[0024] In embodiments, the methods do not decrease brain corticosterone compared to systemic delivery of the same amount of dexamethasone as is being delivered via CED. CED is a method of drug delivery in which a pressure gradient is created at the tip of a catheter to use bulk flow rather than diffusion to delivery drugs into the brain. Using bulk flow to deliver a drug can mean a drug can be delivered further into a target tissue with higher pressure, resulting in lower concentrations and less risk of drug toxicity. In some embodiments, CED is effected using one or more stereotactically placed catheters, connected to mechanical pumps to provide a positivepressure micro-infusion of the desired agents through target tissues. In some embodiments, the catheter is inserted into brain parenchyma or tumor, e.g, using image-guided neuronavigation. In some embodiments, the catheter is attached to an implantable infusion pump. In some embodiments, the catheter is attached to an external infusion pump.

[0025] In some embodiments, the method further comprises (b) administering an amount of a chemotherapeutic to the subject.

[0026] In some embodiments, the chemotherapeutic comprises a temozolomide or a topotecan.

[0027] In some embodiments, the method achieves greater reduced intratumoral inflammation as compared to an otherwise identical treatment wherein the same amount of dexamethasone or anti-inflammatory medication is delivered to the subject systemically and not by CED.

[0028] In some embodiments, the method delivers 5 mg to 20 mg of DEX daily. In some embodiments, the method delivers 2 mg to 4 mg of DEX daily. In some embodiments, the method delivers Img to 2 mg of DEX daily.

[0029] In some embodiments, the brain tumor comprises glioblastoma multiforme or anaplastic astrocytoma.

[0030] In some embodiments, the method does not effect splenal atrophy.

[0031] In some embodiments, the method does not effect adrenal atrophy.

[0032] In some embodiments, the method does not decrease brain corticosterone compared to systemic delivery of dexamethasone.

[0033] In some embodiments, the method does not decrease brain corticosterone compared to systemic delivery of an anti-inflammatory medication.

[0034] In some embodiments, the method increases the survival time of the subject as compared to an otherwise identical method but wherein the dexamethasone is administered systemically to a subject.

[0035] In some embodiments, the method does not increase subject neutrophil levels and / or mean platelet volume more than compared to an otherwise identical method but wherein the dexamethasone is administered systemically to a subject.

[0036] In some embodiments, the method does not increase subject neutrophil levels.

[0037] In some embodiments, the method does not increase subject mean platelet volume.

[0038] In some embodiments, the method does not decrease subject lymphocyte percentage.

[0039] In some embodiments, the method does not decrease monocyte percentage.

[0040] In some embodiments, the method does not decrease subject lymphocyte percentage or decrease monocyte percentage more than compared to an otherwise identical method but wherein the dexamethasone is administered systemically to a subject.

[0041] In some embodiments, the method does not decrease subject blood glucose levels.

[0042] In some embodiments, CED is effected via an implantable pump. In some embodiments, CED is effected via an implantable osmotic pump. In some embodiments, the pump is implanted peripherally. In some embodiments, is implanted centrally, or in the brain.

[0043] In some embodiments, the dexamethasone or anti-inflammatory medication is coinfused with gadolinium so as to permit observer assessment of delivery of dexamethasone or antiinflammatory medication with respect to the subject’s glioblastoma or brain tumor. In some embodiments, the method further comprises assessment of delivery of dexamethasone or antiinflammatory medication via gadolinium tracking.

[0044] In some embodiments, the anti-inflammation medication is prednisone or cortisone.

[0045] In some embodiments, the chemotherapeutic is Topotecan or Etoposide.

[0046] In some embodiments, any parameter recited in a method herein which is better than a recited parameter not in the presence of the claimed method regards as compared to a premeasured or predetermined control amount / value decided or obtained, usually beforehand, as a control. The concept of a control is well-established in the field, and can be determined, in a non-limiting example, empirically from subjects treated with recited standard methods, for example, and may be normalized as desired (in non-limiting examples, for volume, mass, age, location, gender) to negate the effect of one or more variables.

[0047] A pharmaceutical composition for treating glioma in a patient comprising: (a) therapeutically effective amounts of at least one chemotherapy agent, pharmaceutically acceptable salts thereof, and combinations thereof; (b) therapeutically effective amounts of at least one steroid or non-steroidal anti-inflammatory agents, and (c) at least one pharmaceutically acceptable carrier, and, optionally, (d) an amount of gadolinium.

[0048] A kit, for the treating brain inflammation associated with chemotherapy treatment of glioma in a patient, comprising: (a) the pharmaceutical composition described herein; and (b) a catheter for administering the pharmaceutical composition described herein via convection enhanced delivery. Optionally, the kit further comprises a pump for CED delivery of (a) through (b).

[0049] An implantable pump, with a reservoir for therapeutic(s) to be delivered to a subject in which the pump is implanted, comprising in the reservoir (a) a therapeutically effective amount ofat least one steroid or non-steroidal anti-inflammatory agent, and (b) at least one pharmaceutically acceptable carrier. Optionally the pump is (c) a subcutaneously implantable.

[0050] In some embodiments, the pump further comprises in the reservoir (c) a therapeutically effective amount of at least one chemotherapy agent, pharmaceutically acceptable salt thereof, or combination thereof.

[0051] In some embodiments, the pump further comprises a catheter attached or attachable to the pump for delivery of the therapeutic to the subject.

[0052] In some embodiments of the pump or kit, wherein the catheter is a reflux -preventing catheter, multiple port catheter, and / or ballon-tipped catheter.

[0053] In some embodiments of the pump or kit the pump is subcutaneously implantable.

[0054] In some embodiments, the pump is a microinfusion pump. In some embodiments, the pump is an osmotic pump.

[0055] In some embodiments of the pump or kit the anti-inflammatory is or comprises dexamethasone. In some embodiments of the pump or kit the anti-inflammatory is prednisone or cortisone. In some embodiments of the pump or kit the chemotherapy agent is or comprises Topotecan or Etoposide.

[0056] Chemotherapeutic agents are known in the art. Chemotherapeutic agents commonly used in treating CNS tumors include temozolomide, procarbazine, lomustine, vincristine and carmustine. Steroids used in therapy are known in the art. Steroids commonly used in treating CNS tumors (for, e g., treating swelling) include dexamethasone. Non-steroidal anti-inflammatory agents used in therapy are known in the art. Non-steroidal anti-inflammatory agents commonly used in treating CNS tumors (for, e.g., treating swelling) include RAGE inhibitors and S100A9 inhibitors. In some embodiments, the anti-inflammation medication is prednisone or cortisone. In some embodiments, the chemotherapeutic is Topotecan or Etoposide.

[0057] As used herein, “pharmaceutically acceptable salt” refers to a salt of a compound that does not abrogate the biological activity and properties of the compound. Pharmaceutical salts can be obtained by reaction of a compound disclosed herein with an acid or base. Base-formed salts include, without limitation, ammonium salt (NH4+); alkali metal, such as, without limitation, sodium or potassium, salts; alkaline earth, such as, without limitation, calcium or magnesium, salts; salts of organic bases such as, without limitation, dicyclohexylamine, piperidine, piperazine, methylpiperazine, A-methyl-D-glucamine, diethylamine, ethylenediamine, tris(hydroxymethyl)-methylamine; and salts with the amino group of amino acids such as, without limitation, arginine and lysine. Useful acid-based salts include, without limitation, acetates, adipates, aspartates, ascorbates, benzoates, butyrates, caprate, caproate, caprylate, camsylates, citrates, decanoates, formates, fumarates, gluconates, glutarate, glycolates, hexanoates, laurates, lactates, maleates, nitrates, oleates, oxalates, octanoates, propanoates, palmitates, phosphates, sebacates, succinates, stearates, sulfates, sulfonates, such as methanesulfonates, ethanesulfonates, -toluenesulfonates, salicylates, tartrates, and tosylates.

[0058] Acid addition salts can be formed by mixing with a solution of a pharmaceutically acceptable non-toxic acid such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, di chloroacetic acid, or the like. Basic salts can be formed by mixing with a solution of a pharmaceutically acceptable nontoxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like. Suitable pharmaceutically acceptable salts can be composed of a compound with one or more counterions, e.g., a dichloride, or with a fraction of a counterion, e.g., a hemitartrate. Definitions

[0059] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0060] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word“or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0061] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0062] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0063] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0064] CED is a high-precision local drug infusion methodology where drugs are delivered at high concentrations directly into the tumor and peritumoral brain through a surgically implanted catheter attached to a microinfusion pump.3 35Compared to systemic drug delivery, CED achieves higher drug concentrations in the brain while minimizing systemic toxicides.36,37Initial studies of CED used an external pump and catheter which prevented continuous or repeated drug infusion without the danger of infection or risks of repeat surgery.38,39While this limited the efficacy of early CED trials, the recent successful clinical trial use of subcutaneously implantable, refillable pumps for repeated and prolonged drug infusions has expanded its clinical advantage.40,41General

[0065] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0066] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0067] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0068] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0069] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.EXAMPLES - 1

[0070] Materials and Methods

[0071] Cell Viability Assays'.

[0072] The present disclosure used Promega’s CellTiter 96 Aqeous One Solution Cell Proliferation Assay (G3582) for determining APCL cell viability. Briefly, cells were plated on the inner wells of a 96-well plate and allowed to settle overnight. Fresh media was then added with the relevant concentration of dexamethasone and cells were allowed to grow for 72 hours. All conditions were done in triplicate. CellTiter 96 Aqueous One Solution was added per themanufacturer’s directions and allowed to incubate for 90 minutes. Absorbance at 490 nm was recorded using a 96 well plate reader. IC50 values were calculated using the drc package in R.

[0073] Animal Ethics Statement.

[0074] All experiments were performed in accordance with institutional and national guidelines for the care and use of laboratory animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University. Female B6(Cg)-Tyre’2J / J mice from Jackson Laboratories (strain #000058, age 6-8 weeks) were used for all experiments. Mice were housed under standard, pathogen-free conditions with a 12-hour light / dark cycle and had unrestricted access to food and water.

[0075] Viral and Orthotopic Cell Injections.

[0076] Murine glioma cells lines were generated as previously described.72As described in Upadhyayula et al.72, “C57B1 / 6 neonatal mice with floxed p53 and stop-flox mCherry-luciferase were anesthetized using hypothermia and orthotopically injected with a PDGFA-intemal ribosomal entry site (IRES)-cyclization recombination (Cre) retrovirus (stereotaxic coordinates relative to bregma: 1 mm anterior, 1 mm lateral, 1 mm deep), resulting in tumor cells that overexpress PDGFA and mCherry -Luciferase, and have deleted p53.73End-stage tumors were harvested and tumor cells isolated and cultured in basal media (BFP), containing DMEM (Gibco™ 11965092) with 0.5% FBS (Gibco™ 16000044), antibiotic-antimycotic (Thermo Scientific 15240096), N2 supplement (Thermo Fisher Scientific, 17502-048), and lOng / ml each of recombinant human PDGF-AA (Peprotech, 100-13 A) and FGFb (Peprotech, 10018B50UG).” Resulting cell lines were grown at 37C with 5% CO2.

[0077] Orthotopic tumor implantation was performed as previously described72Briefly, mice were anesthetized with Ketamine / Xylazine (100 mg / kg and 10 mg / kg, respectively) and assessed for lack of reflexes by toe pinch. Hair was shaved and scalp skin incised. The skull was cleaned with a cotton swab and the bregma was identified. A burr hole was made with a 17-gauge needle 2 mm lateral and 2 mm anterior to the bregma. A cell suspension was made from lifted cell lines. An intracranial injection was performed under stereotactic guidance, 2 mm deep into the brain parenchyma using a Hamilton syringe at a flow rate of 0.3 pl / min to deliver 50,000 cells with a volume less than 2 uL. Tumor growth was assessed through bioluminescence imaging aspreviously described.74Mice were approximately 8 weeks old when they were injected with tumors.

[0078] CED of Dexamethasone and Systemic Administration'.

[0079] Pump implantation was performed as previously described.74Alzet osmotic pumps (1007D) were fdled with either PBS (control) or PBS with 100 ng / pl of dexamethasone-phosphate. Pumps were implanted at 21 DPI for all studies, and the catheter was implanted along the same burr hole that was used for tumor implantation. For mice receiving systemic administration of dexamethasone, a PBS containing pump was implanted and mice received an i.p. injection of 10 mg / kg of dexamethasone daily. One percent Omniscan was included in all Alzet pumps to confirm successful drug distribution by MRI.

[0080] Murine MRI Scans:

[0081] MRI scans were conducted using a Bruker BioSpec 9.4T scanner (Bruker Corp., Billerica, MA). Mice were anesthetized with 1-2% isoflurane mixed with medical air and administered via a nose cone. The isoflurane concentration was adjusted throughout the scan to maintain a stable respiratory rate between 40 and 70 breaths per minute. Respiration was monitored using a sensor pillow connected to a physiological monitoring system (SA Instruments, Stony Brook, NY). To ensure consistent body temperature, a circulating water heating pad was used to maintain the temperature at approximately 37°C.

[0082] For initial localization, low-resolution T1 -weighted scout images were acquired. High-resolution anatomical imaging was performed using a T2-weighted rapid acquisition with relaxation enhancement (RARE) sequence with the following acquisition parameters: repetition time (TR) = 3000 ms, echo time (TE) = 45 ms, field ofview(FOV) = 17 x 15 mm, matrix resolution = 225 x 198, and slice thickness = 0.7 mm, with 16 slices spanning the entire brain. Contrast-enhanced imaging was performed using a T1 -weighted sequence with the same geometric parameters but different acquisition settings: TR = 150 ms, TE = 2.2 ms, and flip angle = 70°.

[0083] Murine Survival, Post-treatment Tissue, and Dexamethasone Quantification Studies:

[0084] The survival results shown represent the combination of two survival studies, each of 10 mice per group. Tumors were implanted on DPI 0 and treatment began on DPI 21. Tumor growth was assessed through luciferase imaging. Three mice were excluded from further analysis.Two mice died during pump implantation from an anesthetic overdose, and one was excluded due to a lack of luciferase signal prior to pump implantation. Treatment with vehicle (PBS) or dexamethasone (100 ng / pl) lasted 7-days and pumps were removed on DPI 28. An MRI was conducted at this point to visualize intraparenchymal gadolinium from all pumps. Mice were followed until death or tumors reached end stage as evidenced by peri-orbital hemorrhage, epistaxis, seizures, or severe lethargy.

[0085] For post-treatment analysis of tissue, mice were implanted with Alzet pumps on DPI 21, as described above. Following 7-days of treatment, pumps were removed, and mice were anesthetized and perfused with 15 mL PBS followed by 15 mb 4% PFA at a rate of 5 mL / min. Brains were excised and further fixed in 4% PFA for 24 hours. Brains were then embedded in paraffin and five pm thick sections obtained. Sections were stained following basic immunohistochemical methods. Following deparaffinization with xylene and ethanol, antigen retrieval was performed under pressure in a 10 mM, pH 6 citrate buffer with 0.05% tween-20. Sections were incubated in primary antibody solutions overnight at room temperature. Immunoperoxidase staining was accomplished with the appropriate Vector kit (PK-6100). Fluorescent secondary antibodies were used at 1:1000.

[0086] The following antibodies were used: Cre (1:50, Cell Signaling 15036), Ibal (1:1000, Cell Signaling 17198), Ibal (1:500, Aves 1BA1-100), MSR1 (1:500, Invitrogen PA5-102519). Fluorescent secondaries were Goat Alexa Fluor anti-rabbit 488 / 568 (Al 1008 / A11036), antichicken 647 (A21449).

[0087] Images of immunoperoxidase staining were scanned at 40x using a Leica SCN 400 digital slide scanner. Immunofluorescence images were obtained on a Nikon AX confocal microscope. Images were quantified using QuPath.75

[0088] For quantification of dexamethasone, dissected tissue or plasma was flash frozen in liquid nitrogen for later analysis (see Quantification of Dexamethasone). In mice receiving systemic dexamethasone, the mice were perfused with 20 mL of PBS before the tissue was dissected and frozen.

[0089] Measurement of Physiologic Side Effects:

[0090] Blood glucose measurements were taken by briefly sedating mice with isoflurane before pricking their tail with a 20-gauge needle. Glucose levels were determined with a Accu-Chek home diabetes glucose meter from Roche.76Glucose levels were measured after 1-day, 2-days, and 7-days of treatment.

[0091] For murine CBCs, blood from the three treatment groups was collected via cardiac puncture immediately after CED pump removal prior to sacrifice and was placed into 0.5mL EDTA-anticoagulated tubes for CBC analysis. Samples (15uL) were analyzed using Heska Element HT5 Veterinary Hematology Analyzer (laser flow cytometry, colorimetric detection, and impedance technology). Maintenance and quality control procedures are performed daily. Samples were tested within one hour of collection. Samples submitted overnight and for repeated analysis (if initial counts were unanalyzable) were stored at 4°C and tested within 12 hours after acquiring. Before testing, blood samples were placed on a sample rocker and then gently inverted 4-5 times to ensure proper mixing. At this time, the samples were inspected for blood clots. Any samples with visible blood clots were rejected for analysis. Serum biochemistry was performed using the Heska Element DC5X Veterinary Chemistry Analyzer to measure glucose, triglyceride, total protein, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. Fresh blood was collected from the same three groups and placed into a 0.8mL serum separator tube. Tubes were centrifuged at 2000g spin for 10 minutes after 15 minutes incubation at room temperature in the dark. The supernatant in the form of serum was transferred to a 0.5mL polypropylene tube specific to this analyzer. The samples were collected over 1 day, stored at -20°C, and tested over two consecutive days. The serum was thawed at room temperature for 20-25 minutes before testing.

[0092] Adrenal and splenic atrophy was assessed by carefully dissecting the mouse spleen and adrenal glands from surrounding organs and adipose tissue. The mean weight of the adrenal glands was recorded along with the weight of the spleen. Corticosterone concentrations in homogenized liver and brain were measured by high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0093] iP SC -derived Microglia Differentiation and Treatment:

[0094] The differentiation of immunopluripotent stem cells (iPSCs) to primitive hematopoietic progenitor cells (HPCs) is optimized using the STEMdiff™ Hematopoietic Kit (Catalog # 05310,STEMCELL Technologies) as adapted from McQuade et al?8iPSC microglial differentiation from HPCs was done through the following process. Cells were incubated at 37°C. Briefly, HPCs were plated on 24-well Cultrex coated plates at a density of 100,000 cells per well. Cells were cultured in DMEM / F12 (brand) medium, Insulin (ITS) lOOx, B2750x, N2 lOOx, Glutamax lOOx, MEAA lOOx, Monothioglycerol, Insulin lOmg / mL, PenStrep lOOx. Cells were fed every 2 days with a tri-cytokine cocktail of these differentiation factors: lOOng / mL IL-34, 50ng / mL TGB 1, and 25ng / ml M-CSF. On day 13, cells were gently lifted with mechanical disruption to split -50% of cells into new wells as confluency of cells allowed for distribution of cells into new plate for higher yield. Differentiation with the tri-cytokine mix continued every 2 days until day 25. On day 25, the addition of lOOng / mL CD200 and lOOng / mL CX3CL1 for further maturation and ensure homeostatic state. Feeding occurred daily until day 28 when the cells were ready for functional and transcriptomic assays. On day 28, the fully matured human iPSC microglia were treated with 25 ng / pl of dexamethasone (63 pM) or 1 ng / pl of LPS, either in separately or in combination. The cells underwent RNA extraction as per the Qiagen reagent protocol as well as the creation of cDNA. Bulk RNA sequencing was then performed.

[0095] Quantification of Dexamethasone:

[0096] Dexamethasone was measured by high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) on a platform comprising of Waters Xevo TQS triple quadrupole mass spectrometer integrated with a Waters Acquity UHPLC (Waters, Milford, MA). Tissue samples were homogenized in LCMS grade water using Precyllis lysing kits with a bead homogenizer (Benchmark, NH). Dexamethasone was extracted from the tissue homogenates and serum / plasma samples spiked with deuterated Dexamethasone (d4 Dexamethasone) by liquidliquid extraction using tert butyl methy ether (MTBE). Chromatographic separation was done on a Waters Aquity UPLC BEH C18 column (1.7pm, 2.1X50mm) maintained at 50°C employing gradient elution using water and methanol with 0.1% formic acid as mobile phases. Positive ESI-MS / MS mass spectrometry under MRM mode was performed using following transitions: 393.30>355.21. Dexamethasone and 397.30>377.20 (d4 Dexamethasone). The dexamethasone concentration was measured by comparing integrated peak areas against known amounts of dexamethasone using Targetlynx V4.2. The limit of quantitation of the assay is 0.5ng / ml. The mean intra-and inter-assay imprecision was 1.68% and 3.42% respectively.

[0097] snRNA Sequencing of Murine Tissue'.

[0098] As above, mice were sacrificed following seven days of CED of dexamethasone or vehicle treatment. Mouse brains were quickly dissected and the tumor bearing quadrant of the brain was isolated. The quadrant was then flash frozen in liquid nitrogen for later analysis. Later, samples were homogenized and nuclei isolated according to the Chromium Single Cell Nuclei Isolation Kit protocol (1000494). Barcoding, library preparation, and sequencing were performed by the JP Zulzberger Columbia Genome Center. The fastq files were loaded into lOxGenomics cloud and mapped with Cell Ranger Count v9.0.0 onto Mouse library GRm392024-A.

[0099] Standard Seurat pipelines were used for the analysis of snRNA sequencing data. The fastq files were loaded into lOxGenomics cloud and mapped with Cell Ranger Count v9.0.0 onto Mouse library GRm392024-A. A feature / cell matrix (filtered) was used to create Seurat objects. The merged object was subset to include only those nuclei containing between 500 and 5000 features and mitochondria counts and less than 5% across all samples. Doublets were called and excluded by using https: / / github.com / chris-mcginnis-ucsf / DoubletFinder. Normalization, scaling and integration of the data was performed with the Seurat SCTransform() function. Nuclei were projected into UMAP space, clustered, and assigned cell lineages. Clustering of nuclei was done using the shared nearest neighbor smart local moving algorithm, dimensionality PCA reduction using Seurat’s FindNeighbors() function. Major cell types were identified using canonical cell type markers and SingleR.25,77Gene set enrichment analysis (GSEA) was performed using standard downstream analysis methods for geneset enrichment workflows, including the R packages hypeR (“hypergeometric testing”) and the SingleSeqgSet package for a Wilcoxon Mann Whitney Correlation Corrected GSEA analysis. Gene sets included those from MSigDB C2 (KEGG, REACTOME, etc) and C5 (Gene Ontology) libraries. Only significant gene sets with an adjusted p-value < 0.05 were considered.

[0100] Bulk RNAseq Survival Analysis and RNAseq Data Analysis'.

[0101] The count matrix for the TCGA GBM dataset was downloaded using the GDCquery tool in R. The Chinese Glioma Genome Atlas (“CGGA”) RNAseq datasets was downloaded from the Chinese Glioma Genome Atlas website.78,79Counts were normalized using deseq2 in R.80Only primary IDFI wildtype GBM samples were kept for downstream analyses (TCGA: 139 samples, CGGA: 179 samples). Survival analysis was performed using the survival package in R,using the enrichment of the CED-dexamethasone gene set. Kaplan-Meier survival analysis was completed using median expression values (high vs. low).

[0102] Bulk RNA sequencing data was normalized and differentially expressed genes between conditions were determined using DEseq2. Gene ontology of differentially expressed genes between iPSC conditions were determined using GSEA and Hallmark pathways.

[0103] Statistics'.

[0104] No statistical methods were used to predetermine sample sizes. At least three samples per condition were analyzed in all cases. Statistics were computed in R. Unless otherwise mentioned, p-values were computed using two-sample t-tests with unequal variance. Statistics were computed on a per animal basis, except for microglial morphology which were computed on a per microglia basis. Error bars and reported uncertainties represent the 95% confidence interval. The present disclosure exclusively examined female mice.Results

[0105] Convection-enhanced delivery of dexamethasone is well tolerated and provides a modest survival advantage in a PDGFa-driven mouse glioma model.

[0106] Local delivery of a high dose of dexamethasone (0.24 mg / day) has been shown to prolong survival in a leporine metastatic carcinoma model.43Therefore, the present disclosure performed a survival study to determine the tolerability and potential benefit of locally delivered dexamethasone (Figure 1A). Mice were randomized to receive a phosphate buffer solution (PBS) control or PBS solution containing 100 ng / pl of dexamethasone, respectively. At 21 days posttumor injection (DPI 21) dexamethasone or vehicle was delivered directly to the tumor using a seven-day, 100 pl Alzet osmotic pump, which was removed on DPI 28.

[0107] Dexamethasone treatment was well-tolerated without weight loss (Table 1) or outward signs of distress. All mice demonstrated adequate intraparenchymal distribution of the drug or vehicle as assessed by co-infused gadolinium (1% Omniscan), and a consistent volume of distribution was observed between groups (Figure IB).

[0108] Table 1. A table listing the body of weights of mice the day prior to pump implant and the day of pump explant, organized by treatment group. CTL and SYS mice received PBS andCED mice received 100 ng / pl via an Alzet osmotic pump (1007D). SYS mice also received a daily i.p. injection of 10 mg / kg of dexamethasone.

[0109] Kaplan-Meier survival analysis demonstrated a modest but statistically significant survival advantage for mice treated with CED of dexamethasone compared to vehicle-treated control mice (median survival 42 DPI vs 34 DPI, respectively; log-rank p = 0.03) (Figure 1C).

[0110] Analysis of murine tissue immediately after CED of dexamethasone shows a marked anti-inflammatory effect.

[0111] In vitro studies showed that the concentration of dexamethasone delivered by CED is below the level affecting murine tumor cell viability (FIG. 6), therefore the present disclosure performed post-treatment tissue analyses to determine the effects of CED of dexamethasone on the tumor microenvironment. Histological analysis and snRNA sequencing of tissue was performed immediately following seven days of CED of dexamethasone or vehicle with Alzet osmotic pump, starting at 21 days post injection. A systemic delivery group was included for histological analysis utilizing dosages used in previous studies of glioma bearing mice (10 mg / kg / day i.p. dexamethasone)8,17,23’24(Figure 2A).

[0112] The present disclosure performed snRNA-sequencing of tumor containing brain tissue from the CED of dexamethasone-treated and vehicle-treated samples. These nuclei were projected in UMAP space and assigned cell lineages using canonical marker genes, which were validated with SingleR (FIG. 7 A, B). Shifts in transcriptional patterns between treatment conditions in myeloid cells, the predominant inflammatory cell type captured in the present disclosure’s analysis, were assessed. UMAP visualization demonstrated that myeloid cells clustered into resting microglial (P2ryl2, Hexb, Sail!') and activated macrophage / monocyte-like subpopulations Msrl, CD206 / Mrcl, Lyz2) (Figure IB; FIG. 7C).44Microglial-like nuclei were enriched inP2ryl2 and Hexb expression, while the macrophage / monocyte-like nuclei were enriched in Msrl (Supplementary Figure 2C). Analysis of a broad set of inflammatory genes showed that CED of dexamethasone reduced reactive microglial and macrophage genes, including markers of interferon signaling (Irf7, Ifit2, Ifit3 ^, phagocytosis and antigen presentation (H2-Aa, H2-Abl'6, and chemokine / cytokine signaling (TLR4, Illb, 116, Tnf1(Figure 2C).

[0113] The anti-inflammatory transcriptional effects of CED of dexamethasone were further examined by performing gene set enrichment analysis (GSEA)48on treatment groups of present disclosure. Analysis of significant Hallmark pathways revealed that the most significantly downregulated pathways were all related to inflammation, including interferon response, IL2-STAT5 and IL6 signaling, and immune rejection pathways (Figure ID).

[0114] GSEA analysis of Gene Ontology (GO) pathways provided further resolution, highlighting the suppression of several inflammatory and immune activation pathways. Pathways involving positive regulation of innate immunity, MHCII antigen processing, inflammatory cytokine production and signaling, interferon production and signaling were all downregulated with CED of dexamethasone (Figure IE).

[0115] To assess the prognostic importance in the reduction of inflammatory transcriptional signatures, the present disclosure performed log-rank testing on the human homologues of the top 50 significantly downregulated myeloid cell genes after CED of dexamethasone (Table 2). Using VST normalized TCGA and CGGA bulk sequencing data from IDH-WT primary GBM patients25, a significantly improved survival in patients with low expression of these inflammatory genes was observed (log-rank, p=0.0018; Figure IF).

[0116] Table 2. Top downregulated genes in CED-treated, myeloid nuclei in the snRNA data compared to control. This gene set was used to assess the prognostic importance of suppressing an inflammatory transcriptional signature by correlating survival to the expression of these genes in the TCGA and CGGA datasets.

[0117] Immunohistochemical staining of post-treatment tumors demonstrated a significant decrease in Ibal staining within the tumor of mice treated with CED dexamethasone compared to tumor in control (untreated) mice and mice treated with systemic Dexamethasone (Figure 2G, H). This decrease in Ibal positivity was also observed when normalized to tumor cellularity (Figure 21). The present disclosure also examined Msrl, a marker of tumor-associated macrophages that has been associated with a poor prognosis in glioblastoma49,50, (Figure 2J). The present disclosure found a significant decrease in Msrl -positive cells with CED dexamethasone over control, whereas the effects of systemic dexamethasone were not significant (Figure 2K).

[0118] In addition to a decrease in the overall abundance of Ibal -positive myeloid cells and Msrl-positive macrophages after CED of dexamethasone, the morphology of Ibal -positive cells was significantly more ramified in mice treated with CED of dexamethasone. It has been suggested that microglial morphology (i.e. a hypertrophic, ameboid appearance) is a sign of activated microglia, while resting microglia take on a ramified appearance.51-53Using a previously developed and validated automated morphological analysis toolkit34, the present disclosure quantified sphericity and branching in a subset microglial from mice treated with systemic dexamethasone, CED dexamethasone, and vehicle. Tumor-associated, Ibal -positive cells were less spherical and had more branching nodes in mice treated with CED of dexamethasone than in the systemic- or vehicle-treated conditions (Figure 2L).

[0119] The present disclosure investigated the differential effects of local versus systemic dexamethasone therapy by comparing snRNA-seq data from myeloid cells in each group. Differential gene expression revealed 464 differentially expressed genes (Table 3), many of which are inflammatory in ontology. Genes such as Cd68 and Ccr2 are significantly downregulated in the CED group compared to systemic administration, consistent with the broad anti-inflammatory effect observed with local delivery. Not all inflammatory genes are downregulated, however, as evidenced by an upregulation in Ly86 and Statl.

[0120] Table 3. Dexamethasone remains stable at physiologic conditions for up to 10 days. Nominal dexamethasone concentration was 100 ng / pl.

[0121] Comparison of enrichment scores in Hallmark pathways between the systemic and local groups showed a consistent negative enrichment in inflammatory pathways in the CED group. Systemic delivery, on the other hand, showed a similar negative in some Hallmark pathways (e.g. interferon response and allograft rejection), whereas a positive enrichment was actually observed in some key inflammatory ontologies (e.g. IL6 and TNFa signaling) (FIG. 8A). Finally, the present disclosure assessed the clinical significance of RNA expression patterns in the present disclosure’s CED and systemic groups by comparing them to recently published myeloid cell expression programs in glioblastoma.55The present disclosure found that systemic and local dexamethasone caused opposite enrichments (positive and negative, respectively) in two of these expression program - “complement immunosuppressive,” and “systemic inflammatory.” (FIG.8B)

[0122] In vitro treatment of iP SC-derived microglia with dexamethasone inhibits LPS-induced activation.

[0123] To assess the direct effects of dexamethasone on microglia cells in-vitro, the present disclosure utilized human induced pluripotent stem cell (iPSC) derived microglia (Figure 3A)56which are thought to more closely represent resting, in vivo microglia than established myeloid cell lines.57’58Bulk-RNA sequencing was analyzed from iPSC-derived microglia treated for 24 hours with dexamethasone or lipopolysaccharide (LPS), either alone or in combination.

[0124] Differentially expressed genes between treatment conditions were calculated (Figure 3B). The present disclosure found that LPS was a robust inflammatory stimulus with over 6,000 differentially expressed genes compared to control cells (padj < 0.05). A significant overlap in the differentially expressed genes was seen when dexamethasone was added to LPS compared to LPS alone and those genes that were differentially expressed when LPS was compared to control cells. Namely, a significant number of genes that were significantly upregulated with LPS were significantly downregulated with the addition of dexamethasone and vice-versa (Figure 3C). The pool of overlapping genes was significantly greater than would be expected by random chance (p < 1z1010by hypergeometric testing).

[0125] Similarly, LPS significantly altered the transcription of a broad range of cytokines. When dexamethasone was added to LPS, cytokine transcription typically shifted back toward control levels (Figure 3D; F-H). Additionally, analysis of Hallmark Pathways shows significant enrichment in inflammatory pathways (e.g. complement, TNFa, interferon, IL6, and IL2 signaling pathways) that are correspondingly negatively enriched with the addition of dexamethasone in combination with LPS (Figure 3E).

[0126] CED of dexamethasone improves drug delivery to the brain while minimizing systemic exposure

[0127] Using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), the present disclosure quantified dexamethasone concentrations in tissue and plasma under various delivery conditions in both murine and human specimens.

[0128] The present disclosure first showed that dexamethasone is stable at 37°C for up to 10 days in the Alzet pump. (Table 3). After five days of local dexamethasone delivery in mice, the average concentration of dexamethasone in the tumor-bearing quadrant was 196.2 ng / g (Figure 4A). The concentration of dexamethasone in the liver of CED-treated mice was 6.1 ng / g, which likely results from dexamethasone efflux from the brain (Figure 4A). The dexamethasone concentrations in the liver, brain, and serum of mice were measured one hour after i.p. injection of 10 mg / kg of dexamethasone (Figure 4B). When delivered systemically, dexamethasone preferentially partitioned in the liver (10,998.9 ng / g versus 2,410.9 ng / mL of serum), with a relatively lower concentration in the brain (124.8 ng / g). The partitioning coefficient between brainand liver parenchyma of Kbrain.iiver.ip = 0.011 ± 0.010, whereas local delivery allows for preferential partitioning in the brain with a Kbrain, liver, ced = 29.2 (Figure 4C).

[0129] Additionally, the present disclosure collected tissue at 1-, 4-, 8-, and 24-hours post i.p. dexamethasone injection and observed a first-order rate of elimination in both the liver, brain, and serum consistent with a half-life of approximately 1.1 hours in all three compartments (Figure 4D). The present disclosure estimated the area under the curve (AUC) for the dexamethasone concentration over time when given via CED and via daily i.p. dosing was roughly 10-fold higher AUC with CED of dexamethasone compared to systemic delivery (4,709 ± 1,327 vs 375 ± 75 (ng / g)-h, respectively; Figure 4E).

[0130] The present disclosure also quantified dexamethasone partitioning in human brains and plasma from intraoperative GBM samples (FIG. 9). Contrast-enhancing tumor samples had a concentration of dexamethasone of 73 ± 25% of the corresponding plasma samples. Nonenhancing samples, however, had a dexamethasone concentration of 20 ± 10% of the plasma concentration. In the present disclosure’s murine studies, mice that received systemic dexamethasone had a dexamethasone concentration in the tumor bearing quadrant of 6 ± 2% of the plasma concentration.

[0131] CED of Dexamethasone avoids side effects that accompany systemic administration of dexamethasone.

[0132] Finally, the present disclosure sought to demonstrate that CED of dexamethasone was a viable strategy to avoid systemic side effects. As per prior experiments, systemically treated mice were given 10 mg / kg / day of dexamethasone via i.p. injection while locally treated mice were given dexamethasone via an Alzet pump (1007D) at an intra-pump concentration of 100 ng / pl.

[0133] Systemically delivered dexamethasone led to several measurable physiological side effects in the present disclosure’s mice. Blood glucose levels, taken 24 hours post i.p. dexamethasone dosing (a trough level), were decreased from their pretreatment baseline with systemic dexamethasone (Figure 5A), and a similar decrease was still seen on day seven of treatment (Figure 5B). When dexamethasone was given via CED, no change in blood glucose level was seen (Figure 5A, B).

[0134] Systemically delivered dexamethasone also led to a significant decrease in lymphocyte percentages (CTRL: 59 ± 21%, CED: 59 ± 30%, SYS: 38 ± 27%) and monocyte percentages (CTRL: 5.1 ± 2.9%, CED: 5.8 ± 4.9%, SYS: 2.4 ± 2.4%) after seven days of treatment (Figure 5C, D). Similarly, neutrophil percentages (CTRL: 29 ± 25%, CED: 31 ± 31%, SYS: 50 ± 33%) and mean platelet volume (CTRL: 5.3 ± 1.1 fL, CED: 5.3 ± 0.9 fL, SYS: 5.9 ± 0.6 fL) were increased following systemic treatment (Figure 5C, D). No significant alterations in blood counts were observed when dexamethasone was given by CED (5C, D, E, F). Additionally, splenic and adrenal atrophy were observed after 7 days of systemic delivery of dexamethasone and not with CED (Figure 5G, H). In line with the above findings, we observed a complete ablation of endogenous corticosterone in the brain and liver in mice with systemic dexamethasone that was not observed with CED of dexamethasone (Figure 51, J).EXAMPLES - 2

[0135] A population of subject mammals with a glioblastoma, or animal model thereof, are divided and one group is administered standard systemic DEX therapy and the other group is administered CED DEX therapy as described herein. In measurement of survival rates, the CED DEX therapy will show longer survival times than standard systemic DEX therapy.

[0136] A population of subject mammals with a glioblastoma, or animal model thereof, are divided and one group is administered a CED DEX therapy with an external infusion pump and the other group is administered a CED DEX therapy with an internal implanted infusion pump as described herein. In measurement of survival rates, the CED DEX therapy with implanted infusion pump will show longer survival times than external infusion pump CED DEX therapy.DISCUSSION

[0137] Dexamethasone is widely used to manage inflammation and cerebral edema in glioblastoma patients, but its utility is limited by significant systemic toxicities and unfavorable prognostic outcomes. To maximize intratumoral anti-inflammatory effects and minimize systemic exposure, the present disclosure evaluated convection-enhanced delivery (CED) as a strategy to infuse dexamethasone directly into the tumor microenvironment. In a syngeneic glioma mouse model, seven-day CED of dexamethasone was well tolerated and conferred a modest but significant survival benefit. Single nucleus RNA sequencing and immunohistochemical stainingdemonstrated significantly reduced intratumoral inflammation in tumors treated with CED of dexamethasone compared to systemic administration, characterized by marked downregulation of reactive microglial and macrophage-associated genes, including inflammatory cytokines and interferon signaling pathways. Complementary in vitro experiments using human pluripotent stem cell-derived microglia confirmed that dexamethasone directly suppresses inflammatory gene expression. Together, these findings establish that CED of dexamethasone exerts potent antiinflammatory effects via direct modulation of myeloid cells within the glioma microenvironment. By achieving therapeutically effective intratumoral concentrations without systemic side effects, CED of dexamethasone offers a promising approach to improve the management of glioma-associated inflammation.

[0138] Dexamethasone is the main therapeutic for controlling glioma-associated edema, but its clinical efficacy is diminished by systemic toxicities. In this study, the present disclosure used CED of dexamethasone to achieve high doses of dexamethasone in the brain with negligible systemic exposure. CED of dexamethasone led to enhanced intratumoral drug penetration and eliminated the metabolic and hematologic side effects seen with systemic drug delivery. Importantly, while suppressing tumor-associated inflammation to a greater extent than systemic delivery, CED of dexamethasone provided a significant, although modest, survival benefit, which roughly approximates the infusion duration.

[0139] CED of dexamethasone profoundly reduces inflammation in the tumor microenvironment. This anti-inflammatory effect involves broad transcriptional suppression of inflammatory pathways as well as a decrease in Ibal -positive myeloid cells in and around the tumor. Ibal -positive cells inside CED-treated tumors were also significantly more ramified in their appearance. Ramified microglial have often been interpreted as more resting, while a morphological shift towards an ameboid appearance is a sign of their response to an inflammatory stimulus.51,39-60This suggests that dexamethasone has direct effects on microglial activation. The present disclosure’s iPSC-derived microglial data validated the direct effects of dexamethasone on microglia, demonstrating the LPS-induced activation was partially suppressed by concurrent treatment with dexamethasone. The present disclosure also found a significant depletion of Msrl-positive macrophages in the tumor core. Additionally, comparative snRNA-seq analysis between the CED and systemic murine groups revealed unique differences in inflammatory gene expression depending on the route of drug administration. Recently, Miller et al. noted that a uniqueinflammatory gene expression pattern, termed “complement immunosuppressive” is upregulated in human patients receiving systemic dexamethasone therapy. In line with this, we found a positive enrichment of this pathway in mice treated with systemic dexamethasone and negative enrichment of the pathway in mice treated with CED dexamethasone. These results suggest that suggests that local dexamethasone both inhibits local microglial cell activation and reduces MSR1+ macrophages in the tumor core in a way that depends on the route of drug administration.

[0140] CED of dexamethasone effectively avoided systemic toxicities while inhibiting tumor-associated inflammation. Mice treated with CED of dexamethasone exhibited stable blood glucose levels, no changes in hematologic measurements, and no evidence of splenic or adrenal atrophy, all of which were observed in mice receiving systemic dexamethasone therapy. The decrease in monocyte and lymphocyte percentages in the blood with systemic dexamethasone mirrors reported human side effects, as does the increase in neutrophil percentage and mean platelet volume61,62The present disclosure also observed an acute decrease in blood glucose following i.p. dexamethasone administration, which has been observed in mice by other groups although its mechanism remains unclear.63,64Additionally, systemic dexamethasone eliminated endogenous corticosterone production in the present disclosure’s mice, while corticosterone levels were unaffected with local delivery of dexamethasone.

[0141] Systemic dexamethasone administration led to high concentrations of drug in the liver with relatively poor partitioning in the brain parenchyma. This aligns with other murine studies that have shown that dexamethasone is a substrate for the p-glycoprotein efflux pump encoded by Mdrla on the blood-brain barrier and is actively transported out of the brain parenchyma.65,66

[0142] CED of dexamethasone delivers drug directly into the brain parenchyma, which allows for better partitioning of the drug in the brain compared to peripheral organs. Through continuous infusion, CED leads to a roughly constant concentration of drug throughout the treatment period,33,67compared to peaks and troughs with systemic dosing68Dexamethasone had a short half-life in the present disclosure’s albino B6 mice, approximately 1.1 hours, which is faster than the half-life of 2.3 hours reported in Wistar rats and 4 hours in human plasma.69,70The present disclosure also showed that CED of dexamethasone leads to a significantly higher 24-hour AUC, making it far more effective than systemic delivery. Dexamethasone’s relatively rapid clearancefrom the brain parenchyma is likely responsible for the far more potent anti-inflammatory effects seen with CED, which constantly infuses new drug into the parenchyma, over systemic delivery.

[0143] A prior study by Nestler et al. assessed the degree to which systemically delivered dexamethasone accumulates in the tumor microenvironment in human glioblastoma patients.71Nestler et al. reports a mean brain tumor dexamethasone concentration of 225 ng / g, nearly tenfold higher than the present disclosure’s finding in contrast enhancing biopsies. Importantly, Nestler et al. included tumor samples from patients with gliomas, meningiomas, and metastases who received 12-24 mg / day of dexamethasone for several days prior to their operation. In contrast, the present disclosure collected biopsies from glioblastoma patients who received a 10 mg dexamethasone bolus as part standard of care for a craniotomy, to examine how dexamethasone partitions between tumor tissue and plasma. Drug partitioned more effectively into contrastenhancing tumor biopsies compared to non-enhancing biopsies, suggesting that an intact bloodbrain barrier limits dexamethasone’s brain penetrance. Thus, the non-enhancing tumor that remains post-resection achieves the lowest concentration of systemic dexamethasone, providing additional justification for CED methodology.

[0144] The present disclosure utilized a syngeneic glioma mouse model to compare systemic toxicities and the effects on the tumor-associated inflammatory microenvironment when dexamethasone is delivered locally via CED versus systemically via daily i.p. injection. The present disclosure first demonstrated that CED of dexamethasone is not deleterious to gliomabearing mice and provides a modest survival benefit. The present disclosure also observed a reduction in inflammatory transcriptional signatures and histologic microgliosis with CED of dexamethasone, and these anti-inflammatory effects are recapitulated in-vitro using human induced pluripotent stem cell-derived (iPSC) microglia. Finally, the present disclosure showed the CED of dexamethasone safely facilitates greater drug concentrations in the tumor and peritumoral brain without the systemic toxicities accompanying systemic dosing.

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Claims

CLAIMS1. A method of treating a glioblastoma in a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of dexamethasone, and / or (ii) does not effect splenal atrophy and / or adrenal atrophy, the method comprising administering (a) an amount of dexamethasone to the glioblastoma of the subject by convection-enhanced delivery (“CED”), and (b) an amount of a chemotherapeutic to the subject, effective to treat a glioblastoma in a subject and not decrease brain corticosterone compared to systemic delivery of dexamethasone and / or not effect splenal atrophy and / or adrenal atrophy.

2. A method of treating a brain tumor in a subject, or a method of treating a chemotherapy- associated inflammation in a brain of a subject, wherein the method (i) does not decrease brain corticosterone compared to systemic delivery of anti-inflammatory medication, and / or (ii) does not effect splenal atrophy and / or adrenal atrophy, the method comprising administering an amount of an anti-inflammatory medication to the brain tumor of the subject by convection-enhanced delivery (“CED”), effective to treat a brain tumor or treat a chemotherapy-associated inflammation in a brain of a subject and not decrease brain corticosterone and / or not effect splenal atrophy and / or adrenal atrophy.

3. The method of Claim 2, further comprising administering an amount of a chemotherapeutic to the subject.

4. The method of Claim 1 or 3, wherein the chemotherapeutic comprises a temozolomide or a topotecan.

5. The method of any of Claims 1 to 4, wherein dexamethasone or anti-inflammatory medication administered by CED achieves greater reduced intratumoral inflammation as compared to an otherwise identical treatment wherein the same amount of dexamethasone or anti-inflammatory medication is delivered to the subject systemically and not by CED.

6. The method of any of Claims 2 to 5, wherein the brain tumor comprises glioblastoma multiforme or anaplastic astrocytoma.

7. The method of any of Claims 1 to 6, wherein the method does not effect splenal atrophy.

8. The method of any of Claims 1 to 7, wherein the method does not effect adrenal atrophy.

9. The method of any of Claims 1 to 8, wherein the method does not decrease brain corticosterone compared to systemic delivery of dexamethasone or anti-inflammatory medication.

10. The method of any of Claims 2 to 8, wherein the method does not decrease brain corticosterone compared to systemic delivery of an anti-inflammatory medication.

11. The method of any of Claims 1-10, wherein the method increases the survival time of the subject as compared to an otherwise identical method but wherein the dexamethasone or anti-inflammatory medication is administered systemically to a subject.

12. The method of any of Claims 1-11, wherein the method does not increase subject neutrophil levels and / or mean platelet volume more than compared to an otherwise identical method but wherein the dexamethasone or anti-inflammatory medication is administered systemically to a subject.

13. The method of any of Claims 1-11, wherein the method does not increase subject neutrophil levels.

14. The method of any of Claims 1-13, wherein the method does not increase subject mean platelet volume.

15. The method of any of Claims 1-14, wherein the method does not decrease subject lymphocyte percentage.

16. The method of any of Claims 1-1, wherein the method does not decrease monocyte percentage of the subject.

17. The method of any of Claims 1-15, wherein the method does not decrease subject lymphocyte percentage or decrease monocyte percentage more than compared to an otherwise identical method but wherein the dexamethasone or anti-inflammatory medication is administered systemically to a subject.

18. The method of any of Claims 1-17, wherein the method does not decrease subject blood glucose levels.

19. The method of any of Claims 1 to 18, wherein CED is effected via an implanted pump.

20. The method of any of Claims 1 to 19, wherein CED is effected via an implanted osmotic pump.

21. The method of any of Claims 1 to 20, wherein the dexamethasone or anti-inflammatory medication is co-infused with gadolinium so as to permit observer assessment of delivery of dexamethasone or anti-inflammatory medication with respect to the subject’s glioblastoma or brain tumor.

22. The method of any of Claims 2 to 21, wherein the anti -inflammation medication is prednisone or cortisone or dexamethasone.

23. The method of any of Claims 1 to 22, wherein the chemotherapeutic is Topotecan or Etoposide.

24. A pharmaceutical composition for treating glioma in a patient comprising: (a) a therapeutically effective amount of at least one chemotherapy agent, pharmaceuticallyacceptable salt thereof, or combination thereof; (b) a therapeutically effective amount of at least one steroid or non-steroidal anti-inflammatory agent, and (c) at least one pharmaceutically acceptable carrier.

25. The pharmaceutical composition of Claim 24, further comprising an amount of gadolinium.

26. An implantable pump with a reservoir for therapeutic(s) to be delivered to a subject in which the pump is implanted, comprising in the reservoir (a) a therapeutically effective amount of at least one steroid or non-steroidal anti-inflammatory agent, and (b) at least one pharmaceutically acceptable carrier.

27. The pump of Claim 26, further comprising in the reservoir (c) a therapeutically effective amount of at least one chemotherapy agent, pharmaceutically acceptable salt thereof, or combination thereof.

28. The pump of Claim 26 or 27, further comprising a catheter attached or attachable to the pump for delivery of the therapeutic to the subject.

29. A kit for the treating brain inflammation associated with chemotherapy treatment of glioma in a patient comprising: (a) the pharmaceutical composition of Claim 24 or 25; and (b) a catheter for administering the pharmaceutical composition of Claim 24 or 25 via convection enhanced delivery, and optionally (c) a subcutaneously implantable pump for CED delivery of (a).

30. The pump of any of Claims 26 to 28 or the kit of Claim 29, wherein the catheter is a reflux-preventing catheter, multiple port catheter, and / or ballon-tipped catheter.

31. The pump of any of Claims 26 to 28 or 30, or the kit of Claim 29, wherein the pump is subcutaneously implantable.

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