Purified exosome product (PEP) for drug packaging vehicle

Abstract

Methods and materials for using purified exosome product (PEP) as a carrier in delivery of therapeutic agents to mammals are provided herein. For example, methods and materials for using drug-carrying PEP in the administration (e.g., by convection-enhanced delivery) of one or more drugs to a mammal are provided herein.

Description

[0001] Attorney Docket No. 07039-2279WO1

[0002] 2023-254

[0003] PURIFIED EXOSOME PRODUCT (PEP) FOR DRUG PACKAGING VEHICLE

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application Serial No. 63 / 635,958, filed on April 18, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0005] TECHNICAL FIELD

[0006] This document relates to methods and materials for using purified exosome product (PEP) as a carrier in delivery of therapeutic agents to mammals. For example, this document relates to methods and materials for using drug-carrying PEP to administer (e.g., by convection-enhanced delivery ) one or more drugs to the brain of a mammal.

[0007] BACKGROUND

[0008] Pediatric diffuse midline glioma (DMG) is a uniformly lethal tumor for which no cure has been elucidated. The blood-brain barrier (BBB) represents a significant hurdle to effective drug delivery to the brain (Himes et al., Front. Oncol. 9:31, 2019). Convection-enhanced delivery (CED) can bypass the BBB and deliver therapeutic agents through direct infusion (Rechberger et al., Childs. Nerv. Syst. 36:39-46, 2020). Despite safety and feasibility having been demonstrated in DMG (Power et al., Neuro-Onco. Advances. 5:vdad033, 2023; Banks et al., Nat. Rev. Drug. Disc. 15:275-292, 2016; and Arvanitis et al., Nat. Rev. Cancer. 20:26-41, 2020), however, CED has not shown a survival benefit in any brain tumors to date. Repeated acute CED delivery does not provide survival benefits in DMG tumor models, as drugs that are efflux substrates are rapidly cleared from the brainstem through BBB efflux transporters (Warren et al.. Front. Oncol. 8:239. 2018; and Veringa et al.. pLoS. One.

[0009] 8:e61512, 2013). In contrast, prolonged (7-day) drug infusion using a mini osmotic pump (an animal version of CED) can significantly improve survival and efficacy in DMG animal models. Maintaining drug-tumor residence time post CED infusionAttorney Docket No. 07039-2279WO1

[0010] 2023-254

[0011] against drug efflux over time is critical to improve survival and efficacy, and is translatable to patients with this devastating disease. However, prolonged CED in children can result in adverse effects. Thus, alternative methods of augmenting drug retention are needed to translate this work to patients.

[0012] Exosomes are lipid-membrane extracellular vesicles released by all types of cells to facilitate intercellular communication (Van et al., Nat. Rev. Mol. Cell. Biol.

[0013] 19:213, 2018). Exosomes can transfer molecular cargo between donor and recipient cells, modulating cellular signaling crosstalk without cell-to-cell contact (Mathivanan et al.. J. Proteo. 73:1907-1920, 2010). With effective stability, biocompatibility and inherent targeting properties, exosomes also can deliver drugs as natural nanoparticles (<100nm, NPs) (Pullan et al.. Mol. Pharm. 16:1789-1798, 2019). Exosomes can bypass efflux transporters in a resistant cell line expressing drug efflux transporters, and thereby may reduce drug elimination and confer increase drug efficacy in resistant tumors (Kim et al., Nanmed. Nanotech. Bio Med. 12:655-664, 2016).

[0014] However, the translation of exosome-based drug delivery to the clinic has been limited by production and standardization of exosomes (Tenchov et al., DrugDeliv. Diag. 16:17802-17846, 2022; and Herrmann et al., Nat. Nanotech. 16:748-59, 2021).

[0015] SUMMARY

[0016] This document is based, at least in part, on the discovery' that PEP alone does not promote tumor cell growth and therefore is suitable for use as a drug vehicle for cancer therapy. In addition, this document is based, at least in part, on the discovery' that drug-loaded PEP can preferentially kill tumor cells while promoting normal cell proliferation. This document provides methods and materials for using PEP to deliver therapeutic agents to the brain and other organs in mammals having solid tumors. For example, this document provides methods that can be used to treat mammals having solid tumors by administering PEP loaded with one or more therapeutic agents (e.g., chemotherapeutic agents).

[0017] As demonstrated herein, loading drugs in PEP (which is a nanoparticle) and delivering the drug-loaded PEP acutely by CED produced results similar to those obtained with extended (7-day) drug delivery in DMG animal models. In particular, the drug-loaded nanoparticles (NPs) appeared to have a profound effect against drugAttorney Docket No. 07039-2279WO1

[0018] 2023-254

[0019] efflux post-acute CED infusion. Thus, the studies described herein indicated that PEP-based CED therapy is useful to improve retention of small molecule drugs in the brain of DMG patients following direct delivery'. The results described herein can be translated directly to DMG patients, whose diagnosis remains a death sentence despite intensive study. The methods described herein also can be extended to other high grade brain tumors, such as adult glioblastoma (GBM), and other solid tumors.

[0020] In a first aspect, this document features a composition that contains (a) purified exosome product; and (b) one or more therapeutic agents. The purified exosome product can contain spherical or spheroid exosomes having a diameter of 300 nm or less. The purified exosome product can have a moisture content of 10% or less. The purified exosome product can contain CD63" (CD63neg) exosomes and CD63+(CD63pos) exosomes. The purified exosome product can contain at least 50% CD63‘ exosomes. The composition can contain the one or more therapeutic agents in an amount of about 0.1 mg / mL to about 1 mg / mL of dissolved therapeutic agent per mL of purified exosome product. The purified exosome product can contain about 200 pg / mL of purified exosomes. The one or more therapeutic agents can include a chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of alisertib, doxorubicin, topotecan, and ponatinib. The composition can further contain an efflux pump inhibitor. The efflux pump inhibitor can include everolimus and / or elacridar.

[0021] In another aspect, this document features a method for treating a mammal having a solid tumor. The method can include, or consist essentially of, administering to the mammal a composition containing (a) purified exosome product comprising spherical or spheroid exosomes; and (b) one or more therapeutic agents. The mammal can be a human. The administering can include convection enhanced delivery'. The administering can include direct delivery' to a tumor site, intraperitoneal administration, intranasal administration, or intrathecal administration. The solid tumor can be selected from the group consisting of a brain tumor, an ovarian tumor, a breast tumor, a lung tumor, a prostate tumor, and a pancreatic tumor. The mammal can have diffuse midline glioma. The mammal can be a pediatric human. The spherical or spheroid exosomes can have a diameter of 300 nm or less. The purified exosome product can have a moisture content of 10% or less. The purified exosome productAttorney Docket No. 07039-2279WO1

[0022] 2023-254

[0023] can contain CD63’ exosomes and CD63 exosomes. The purified exosome product can contain at least 50% CD63‘ exosomes. The composition can contain the exosomes and the one or more therapeutic agents in an amount of about 0.1 mg / mL to about 1 mg / mL of dissolved therapeutic agent per mL of purified exosome product. The purified exosome product can contain about 200 pg / mL of purified exosomes. The one or more therapeutic agents can include a chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of alisertib, doxorubicin, topotecan, and ponatinib. The method can further include administering an efflux pump inhibitor to the mammal. The composition can further contain the efflux pump inhibitor. The efflux pump inhibitor can include everolimus and / or elacridar.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety7. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0025] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0026] DESCRIPTION OF DRAWINGS FIGS. 1A-1E. PEP is suitable for packaging drugs with vastly different chemical properties. FIG. 1A is a distribution plot of physicochemical properties of drugs encapsulated into PEP is shown with their respective loading capacity. Despite the differences in physiochemical properties, the drugs were successfully encapsulated into PEP. This figure w as generated from DataWarrior: An Open-Source Program for Chemistry Aware Data Visualization and Analysis (Sander et al., J. Chem. Inf. Model. 55(2):460-473, 2015). Lipophilicity is denoted by cLogP. TheAttorney Docket No. 07039-2279WO1

[0027] 2023-254

[0028] loading capacity of each drug is denoted by %. The solubility of the chemical is denoted by cLogS. FIG. IB is a plot showing the size distribution of PEP before and after loading with alisertib (AliPEP), measured by nanoparticle tracking analysis (Nano Sight). FIG. 1C includes representative transmission electron microscope (TEM) images showing the size of AliPEP. FIG. ID is a graph plotting the effects of different packaging procedures on dose response curve and cell viability of AliPEP compared to alisertib (free drug). FIG. IE is a graph plotting the proliferation rate of SF8628 cells after exposure to either vehicle or native PEP for 6 days, which showed that PEP alone do not promote tumor cell proliferation. 200 pg / mL native PEP is equivalent to 40 pM of drug packaged PEP.

[0029] FIGS. 2A-2D. Effects of PEP alone and after drug-loading on cell viability were evaluated in normal or tumor cell lines. Viability' was tested using normal cell lines: normal human astrocytes (NHA; FIG. 2A) and retinal pigment epithelial (RPE) cells (FIG.2B), and pediatric DMG tumor cell lines: diffuse intrinsic pontine glioma (DIPG) XVII cells (FIG. 2C) and SF8628 cells (FIG. 2D). The following treatment groups were tested in all four cell lines: 1) alisertib (free drug); 2) sonicated PEP without drug loading; 3) ly ophilized PEP that were rehy drated from Rion’s original product and re-lyophilized; 4) alisertib-loaded in PEP using bath sonication.

[0030] FIGS.3A-3I. The potency of AliPEP (alisertib-loaded PEP) was tested in normal and tumor cell lines, using cell viability to show differential effects of drug-loaded PEP on normal cells vs. tumor cells. FIG. 3A: Drug potency of alisertib (free drug) was compared with AliPEP in normal cell lines (NHA-brain derived, ADHF-skin derived and RPE-eye derived) and in patient-derived tumor cell lines including DMG (DIPG IV, DIPG XVII, SF8628), adult glioblastoma (BT114, GBM 108), and breast cancer (MCF7). The table shows numeric ICso values calculated based on the dose-response curves. FIGS. 3B-3H: Cell viabilities were measured by CellTiter-Glo 2.0 after 72 hours of treatment. Figures show dose-response curves for each individual cell line compared between alisertib (free drug) and AliPEP (alisertib-loaded in PEP).

[0031] FIG. 31: ICso ratios were calculated for alisertib and AliPEP for each cell line. An IC50 ratio equal to 1 means there were no differences between the drug treatment groups. IC50 ratios smaller than 1 suggest a diminished drug potency compared to free drug, while IC50 ratios greater than 1 suggest improved drug potency of modified drugAttorney Docket No. 07039-2279WO1

[0032] 2023-254

[0033] (drug-loaded PEP) when compared to free drug. The table below the graph shows numeric values of the ICso ratios between alisertib and AliPEP.

[0034] FIGS. 4A-4J. Effects of PonaPEP (ponatinib-loaded PEP) on cell viability were tested in normal and tumor cell lines. FIG. 4A: Drug potency of ponatinib (free drug) was compared with PonaPEP in normal cell lines (NHA-brain derived, ADHF-skin derived and RPE-eye derived) and in patient-derived tumor cell lines for DMG (DIPG IV, DIPG XVII, SF8628, PE17), adult glioblastoma (BT 114, GBM 108) and breast cancer (MCF7). The table shows numeric values of ICso calculated based on the dose-response curves. FIGS.4B-4I: Cell viabilities were measured by CellTiter-Glo 2.0 after 72 hours of treatment. A dose-response curve was generated for each individual cell line to compare between ponatinib (free drug) and PonaPEP. An ICso ratio equal to 1 means there were no difference between the drug treatment groups. FIG. 4J: ICso ratios smaller than 1 suggest a diminished drug potency. ICso ratios greater than 1 suggest improved drug potency. The table below the graph shows numeric values of the ICso ratio between ponatinib and PonaPEP.

[0035] FIGS. 5A-5J. Effects of DoxoPEP on cell viability in normal and tumor cell lines. FIG.5A: Drug potency of doxorubicin (free drug) was compared with DoxoPEP (doxorubicin-loaded PEP) or DOXIL® (an FDA-approved nanoparticle slow-release formulation of doxorubicin) in normal cell lines (NHA-brain derived, ADHF-skin derived and RPE-eye derived cells), in patient-derived tumor cell lines for DMG (DIPG IV, DIPG XVII, and SF8628 cells), and in adult glioblastoma (GBM 108) cells. The table below the graph shows the numeric ICso values calculated based on the dose-response curves. FIGS.5B-5I: Cell viabilities were measured by CellTiter-Glo 2.0 after 72 hours of treatment. The graphs show dose-response curves for each individual cell line compared between doxorubicin (free drug) and DoxoPEP.

[0036] FIG. 5J: ICso ratios for each type of cell. ICso equal to 1 means there were no differences between the drug treatment groups. ICso ratios smaller than 1 suggest a diminished drug potency. ICso ratios greater than 1 suggest improved drug potency. The table below the graph shows numeric values of the ICso ratios between doxorubicin and DoxoPEP.

[0037] FIGS. 6A-6D. Effects of EverPEP (everolimus-loaded PEP) on cell viability was tested in normal and tumor cell lines. FIGS. 6A-6C: Drug potency of everolimusAttorney Docket No. 07039-2279WO1

[0038] 2023-254

[0039] (free drug) was compared with EverPEP in normal adult human primary dermal fibroblasts (ADHF) and a DMG cell line (DIPG XVII). FIG.6D: ICso ratios of everolimus and EverPEP were determined in normal and tumor cell lines. Cell viabilities were measured by CellTiter-Glo 2.0 after 72 hours of treatment.

[0040] FIGS.7A-7C. Loading drugs in PEP did not affect drug potency against intended protein targets compared to free drug treatment. FIG. 7A shows a representative Western blot analysis of pAURKA, AURKA, and H3S10 expression in DIPG XVII after the cells were treated with alisertib (free drug) or AliPEP for 48 hours. FIG. 7B shows a representative Western blot analysis of PDGFR-a expression in DIPG XVII after the cells were treated with 100 nM ponatinib or PonaPEP for 48 hours. FIG. 7C shows a representative Western blot analysis of y-H2A.X expression in DIPG XVII after the cells were treated with 100 nM Doxo, DoxoPEP, or DOXIL* for 48 hours.

[0041] FIG. 8A-8D. Drug potency test of drug-loaded PEP in comparison to PLGA (poly (lactic-co-gly colic acid)) nanoparticle formulation. PLGA is one of the most effective biodegradable polymeric NPs, and has been approved by the U.S. FDA for use in drug delivery' systems due to its controlled and sustained-release properties, low toxicity, and biocompatibility with tissue and cells (see, Mirakabad et al., Asian Pac J Cane Prev., 15(2):517-535, 2014; and Hua et al.. Drug Deliv., 28(1): 1342-1355, 2021). Alisertib was loaded in two different PLGA formulations (Formula 1 and Formula 2). The PLGA formulations were tested against AliPEP in both NHA and DMG cell line. Neither the PLGA formulation nor AliPEP showed toxicity against normal human cells (NHA). AliPEP showed improved potency (lower ICso) in comparison to both PLGA formulations loaded with alisertib, suggesting that PEP is a better drug delivery nanoparticle than liposomal or PLGA formulations. Fig 8A shows an ICso potency graph of Alisertib vs AliPLGA in NHA (normal cell line) and DIPG XVII (DMG cell line). The table provides numeric values of for bar graph. Dose response curves are plotted in FIG. 8B (NHA) and FIG. 8C (DIPG XVII). Cell viabilities were measured by CellTiter-Glo 2.0 following 72 hours of treatment. FIG.

[0042] 8D includes a graph and a table showing ICso ratios of alisertib (free drug) to various nanoparticle formulations loaded with alisertib.Attorney Docket No. 07039-2279WO1

[0043] 2023-254

[0044] FIG. 9. Bulking agents do not cause additional damage to brain tissues when used with drug-loaded PEP. 5% D-mannitol was used as a bulking agent for drug-loaded PEP during lyophilization, to improve drug-loaded PEP rehydration / solubility after the shelf-stable lyophilized product was created. FIG.9 shows an enlarged representative image of a mouse brain 7 days after CED infusion, demonstrating that the parenchyma surrounding the cannula tract (arrows) and all nonadj acent brainstem tissue were normal on H&E staining, with no signs of tissue necrosis, cavitary lesions, or cellular (inflammatory) infiltrate.

[0045] FIGS. 10A-10B. In solution release of drug-loaded PEP showed similar release kinetics as liposomal or PLGA NP formulations. FIG. 10A is a graph plotting the percent of alisertib released into solution using a dialysis bag for alisertib (free drug), AliPEP, and AliPLGA-NP formulation. FIG. 10B is a graph plotting the percent of doxorubicin released into solution for doxorubicin (free drug), DoxoPEP, and DOXIL®.

[0046] FIGS. 11A-11D. Distribution of free doxorubicin (FIG. 11A) and DoxoPEP (FIG. 11B) immediately after acute CED infusion in normal mouse brain as detected by fluorescence imaging. Drug-loaded PEP had a much more limited distribution compared to free drug. Post-administration MRI imaging of brains from mice receiving gadolinium (GAD) alone by CED (FIG. 11C) vs GAD-loaded PEP delivered by CED (FIG. 11D) suggested that imaging agents can be co-loaded with drug to accurately depict the volume of distribution of drug in real-time.

[0047] FIGS. 12A-12C. Doxorubicin showed higher accumulation in DMG cells when delivered via drug-loaded PEP (FIG. 12A). Patient-derived DMG cell lines were treated with 10 pM Doxo, DoxoPEP, or DOXIL® for 1 hour before the cells were harvested. Concentrations of Doxo were determined using a fluorescent plate reader with Ex / Em: 470 / 550 nm. A standard curve was used to determine the concentration of Doxo uptake by the cells. Intracellular uptake of doxorubicin was measured (FIG. 12B). DoxoPEP delivered ~20 times more doxorubicin into DMG cells compared to free doxorubicin. The presence of everolimus (an efflux transporter competitive substrate) in the media significantly increased intracellular doxorubicin compared to cells maintained in just media. n=5 replicates per data point. *p<0.05, ***p<0001 FIG. 12C is a representative image showing P-gp expression in DIPGAttorney Docket No. 07039-2279WO1

[0048] 2023-254

[0049] XIII PDX brain tissue after receiving acute CED (Ix / week) with either Doxo, DOXIL". or DoxoPEP until the animal became moribund.

[0050] FIGS. 13A-13C. Visualization of PEP in tumor-bearing animals post tail-vein i.v. delivery'. FIG. 13A. PEP loaded with DiR dye was delivered through the tail vein of a mouse inoculated with DIPG XIIIp* that were labelled with a bioluminescent tag. The tumor in the brain was visualized by bioluminescence using IVIS (In Vivo Imaging System) imaging (left panel). The PEP was visualized using fluorescence of the IVIS imaging (right panel). FIG. 13B shows fluorescent imaging of organs harvested from the mouse shown in FIG. 13A, demonstrating that most PEP were accumulated in peripheral organs when PEP was systemically (i.v.) delivered. FIG.

[0051] 13C shows fluorescent imaging of the brain harvested from the mouse shown in FIG.

[0052] 13A, demonstrating that a fair amount of PEP accumulated in the brain within 1 hour of the i.v. infusion.

[0053] FIGS. 14A-14C. Acute CED infusion of drug-loaded PEP showed a survival benefit like that observed with extended drug delivery in multiple xenografts of DMG. FIG. 14A is a Kaplan-Meier plot for animals (DIPG XVII PDX, n=7-9 / group) treated with alisertib or AliPEP 2x / week for 6 consecutive weeks via acute CED infusion. AliPEP treated animals experienced survival times twice as long as the survival times observed for control (p=0.0058) and alisertib (p=0.0429) treated animals. FIG. 14B is a Kaplan-Meier plot for animals (PED 17 PDX, n=4 / group) treated with ponatinib or PonaPEP Ix / week for 6 consecutive weeks via CED. The PonaPEP treated animals showed a significant increase in survival compared to control (p=0.0101) and ponatinib (p=0.0069) treated animals. FIG. 14C is a Kaplan-Meier plot for animals (DIPG XIIIp* PDX, n=7-8 / group) treated with Doxo, DOXIL®, or doxorubicin-loaded PEP (DoxoPEP) Ix / week for 6 consecutive weeks via acute CED infusion. Only the DoxoPEP -treated animals showed significantly improved survival (p=<0.0001) compared to control or animals that received Doxo or DOXIL" Unmodified PEP was included to demonstrate that PEP as natural NPs would not promote tumor growth. Deactivated-DoxoPEP was included to demonstrate that the intrinsic property' of unmodified PEP was needed to produce therapeutic efficacy.Attorney Docket No. 07039-2279WO1

[0054] 2023-254

[0055] FIGS. 15A-15B. DrugPEP caused context-dependent potency shifting in ovarian cancer serous adenocarcinoma (OC) cells. FIG. 15A is a graph plotting the results of a clonogenic assay of PE01, COV362, and Ovcar8 OC cell lines treated with alisertib vs alisertib encapsulated in PEP (AliPEP). Three technical replicates were completed. Data were baseline-corrected. FIG. 15B includes representative images of the clonogenic assay shown in FIG. 15A.

[0056] FIGS. 16A-16G. Viability of normal and ovarian tumor cell lines treated with DoxoPEP. FIG. 16A: Comparison of drug potency between doxorubicin (free drug) and DoxoPEP in a normal ovarian cell line (FTE) and in patient-derived tumor ovarian cell lines (OVCAR8, CaOV3. OV90 and PEO1). The table shows the numeric values of IC50 calculated based on dose-response curves. FIGS. 16B-16F: Cell viabilities were measured by CellTiter-Glo 2.0 following 72 hours of treatment. Doseresponse curves were generated for each individual cell line to compared between doxorubicin (free drug) and DoxoPEP (Doxorubicin-loaded in PEP). FIG. 16G: Calculated ratio of IC50 values between doxorubicin and DoxoPEP in each cell line. An ICso value equal to 1 means there was no difference between the drug treatment groups. IC50 values smaller than 1 suggests a diminished drug potency. IC50 values greater than 1 suggests improved drug potency. The table shows numeric values of the IC50 ratio between doxorubicin and DoxoPEP.

[0057] DETAILED DESCRIPTION

[0058] This document provides methods and materials for using PEP to deliver therapeutic agents to mammals identified as having a solid tumor (e.g., in the brain or another organ or tissue). For example, this document provides compositions containing PEP loaded with one or more therapeutic agents (e.g., one or more chemotherapeutic agents), and methods for using the drug-loaded PEP to treat mammals having one or more solid tumors.

[0059] Exosomes are microvesicles (e.g., spherical or spheroid microvesicles). It is noted that despite the designation as purified exosome product, PEP used in the compositions and methods described herein can be prepared from extracellular vesicles and / or exomeres. Thus, the term ‘"exosome'’ as used herein includes not onlyAttorney Docket No. 07039-2279WO1

[0060] 2023-254

[0061] exosomes but also exomeres and extracellular vesicles, provided that the product itself has the physical, structural, and / or functional characteristics of PEP.

[0062] PEP can be obtained commercially (e.g., from Rion, LLC; Rochester, MN), or can be generated using any appropriate method and any appropriate cells or tissue. Suitable methods for preparing PEP are described in, for example, PCT Publication No. WO 2019 / 118817. See, for example, the paragraphs extending from page 7, line 12 to page 10, line 31, Example 1 at pages 23-24, and Figure 1A.

[0063] PEP can be prepared from any appropriate biological material. In some cases, the starting material for preparing PEP can be a blood product such as, without limitation, whole blood or any suitable apheretic blood product (e.g., leukopheresis products, plasmapheresis product, cryo poor plasma, fresh frozen plasma, pheresis platelet products, platelet rich plasma, platelet poor plasma, or any erythrocyte depleted and leukocyte depleted product). The blood or blood product can have been obtained from any appropriate source, such as the general population, general population age 30 or below, general population age 40 or below, post-surgical population, a pre-menopausal woman, a peripartum woman, a placenta, or umbilical cord blood. In some cases, the starting material for preparing PEP can be a suitable non-blood source, such as umbilical cord Wharton’s jelly, stromal vascular fraction of fat, apheresis bone marrow products, synovial fluid, cerebrospinal fluid, mesenchymal stem cells, endothelial cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, or the conditioned medium of these or any other cell sources. PEP can be prepared from a starting material using a method that includes one or more of the following steps: filtering or apheresing the starting material, pooling the filtered or apheresed starting material, agitating the pooled material, and cryodesiccating the agitated pooled material to generate a powdered PEP. In general, PEP contain spherical and / or spheroid exosomes have a diameter of about 300 nm or less (e.g.. about 250 nm or less, about 200 nm or less, about 150 nm or less, about 100 nm or less, about 50 nm or less, about 40 nm or less, about 40 to about 100 nm, about 50 to about 150 nm, about 100 to about 200 nm, or about 200 to about 300 nm). After lyophilization, a PEP can have a moisture content that is about 10% or less (e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 1 to about 5%, about 3 to about 7%, or about 5 to about 10%). In someAttorney Docket No. 07039-2279WO1

[0064] 2023-254

[0065] cases, PEP can be reconstituted into solution before being loaded with one or more therapeutic agents.

[0066] CD63 is an membrane surface protein found on some exosomes. In some cases, a PEP can include a mixture of CD63+exosomes and CD63" exosomes. PEP containing CD63+exosomes can promote cell growth, whereas PEP that include CD63' exosomes can engage cellular machinery that slows growth (e.g., upon reaching confluence), thereby limiting the risk that a PEP preparation will result in unrestrained cell growth. Since CD63" exosomes can inhibit unrestrained cell growth, a PEP preparation that is enriched for CD63' exosomes (e g., by sorting and removing at least a portion of the CD63+exosomes) can, in some cases, be used in anti-neoplastic therapies. In some cases, CD63+exosomes can be sorted from a PEP preparation, using any method suitable for sorting membrane-bound vesicles (e.g., affinity separation, magnetic bead separation, or flow separation), thereby yielding PEP containing mainly (or only) CD63‘ exosomes. By sorting CD63+exosomes, the ratio of CD63+exosomes to CD63" exosomes in a PEP can be adjusted by removing CD63+exosomes from the naturally -isolated PEP preparation, and in some cases, adding back a desired amount of CD63+exosomes. In some cases, a PEP can contain at least 50% CD63' exosomes (e.g., at least 60% CD63’ exosomes, at least 70% CD63‘ exosomes, at least 80% CD63' exosomes, at least 90% CD63' exosomes, at least 95% CD63" exosomes, or at least 99% CD63" exosomes). In some cases, a PEP preparation can contain only CD63" exosomes.

[0067] To generate drug-loaded PEP, one or more therapeutic agents can be combined (e.g., mixed) with a PEP. Examples of therapeutic agents that can be loaded onto a PEP include, without limitation, small molecules (e.g., chemotherapeutic agents such as alisertib, doxorubicin, topotecan, and ponatinib). In some cases, a drug-loaded PEP can include a first therapeutic agent (e.g., a chemotherapeutic agent) and an efflux pump inhibitor (e.g., everolimus and / or elacridar). The PEP and the one or more therapeutic agents can be combined in any appropriate amounts. For example, a PEP (e.g., lyophilized PEP reconstituted in saline or PBS) can be used at a concentration of about 50 pg / mL to about 500 pg / mL (e.g., about 50 to about 100 pg / mL, about 100 to about 200 pg / mL, about 150 to about 250 pg / mL, about 200 to about 300 pg / mL, about 250 to about 350 pg / mL, about 300 to about 400 pg / mL, about 400 to aboutAttorney Docket No. 07039-2279WO1

[0068] 2023-254

[0069] 500 pg / mL, about 100 pg / mL. about 200 pg / mL, about 250 pg / mL, about 300 pg / mL. about 400 gg / mL, or about 500 gg / mL). The PEP can be combined with one or more therapeutic agents to achieve a concentration of about 0.01 mg / mL to about 10 mg / mL (e.g., about 0.01 to about 0.05 mg / mL, about 0.05 to about 0.1 mg / mL, about 0.1 to about 0.25 mg / mL. about 0.25 to about 0.5 mg / mL, about 0.5 to about 1 mg / mL, about 0.1 to about 1 mg / mL, about 1 to about 3 mg / mL, about 3 to about 5 mg / mL, about 5 to about 10 mg / mL, or about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1 mg / mL) of dissolved therapeutic agent per mL of PEP.

[0070] The combination of PEP with one or more therapeutic agents can be subjected to sonication (e.g., probe sonication or bath sonication). The sonication can be conducted for any appropriate length of time and for any appropriate number of cycles. For example, a composition containing PEP and one or more therapeutic agents can be sonicated for 1 cycle or for more than 1 cycle (e.g., 1 to 40 cycles, 2 to 30 cycles, 3 to 25 cycles, 4 to 20 cycles, 5 to 10 cycles, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, to 40 cycles). Each cycle, independently, can last for a length of time ranging from about 5 seconds to about 60 seconds (e.g., about 5 to about 10 seconds, about 10 to about 20 seconds, about 20 to about 30 seconds, about 30 to about 40 seconds, about 40 to about 50 seconds, about 50 to about 60 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 50 seconds, or about 60 seconds). The sonication can take place at room temperature or any other appropriate temperature (e.g., on ice). In some cases, the mixture of PEP and therapeutic agent(s) can be chilled (e.g., on ice) after sonication (e.g., after each cycle of sonication). The sonicated composition can then be filtered and washed to remove precipitated therapeutic agent and purify the drug-loaded PEP. In some cases, the resulting drug-loaded PEP (also referred to as DrugPEP), can be lyophilized.

[0071] In some cases, PEP that is lyophilized with drug can be difficult to reconstitute back into solution (e.g., with saline or PBS) before administration to a mammal. In such cases, the solubility of the lyophilized DrugPEP can be increased by combining the DrugPEP with one or more bulking agents. Any appropriate bulkingAttorney Docket No. 07039-2279WO1

[0072] 2023-254

[0073] agent can be used. Non limiting examples of bulking agents that can be used with DrugPEP include mannitol, lactose, sucrose, dextran, and glycine. A DrugPEP composition can include one or more bulking agents at any appropriate concentration or amount (e.g., about 1% w / w, about 2.5% w / w, about 5% w / w, about 7.5% w / w, or about 10% w / w). Without being bound by a particular mechanism of action, a bulking agent also can, in some cases, act as a filler and / or a cryoprotector of a lyophilized DrugPEP composition.

[0074] This document also features methods for treating mammals having cancer and containing one or more solid tumors. The methods can include administering, to a mammal having a solid tumor, a composition that includes drug-loaded PEP containing one or more therapeutic agents, as described herein. Any appropriate mammal can be treated using the methods provided herein. For example, humans or other primates (e g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, rats, and rabbits can be administered a DrugPEP composition provided herein. In some cases, the mammal to which a DrugPEP composition provided herein is administered can be a human (e.g., an adult human or a pediatric human). The mammal can have any type of cancer associated with a solid tumor. In some cases, the mammal can have brain cancer (e g., diffuse midline glioma), ovarian cancer, breast cancer, lung cancer, prostate cancer, or pancreatic cancer.

[0075] Any appropriate method can be used to deliver a DrugPEP composition to a mammal. In some cases, a DrugPEP composition can be administered via convection enhanced delivery (CED). In some cases, a DrugPEP composition can be administered directly to a tumor site, or can be administered intraperitoneally, intranasally, or intrathecal ly.

[0076] A DrugPEP composition can be administered to a mammal having cancer in any effective dose. Effective doses can vary depending on the severity' of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating clinician. An effective amount of a DrugPEP composition provided herein can be any amount that reduces the number of cancer cells (e.g., by at least 5, 10, 25, 35, 45, 50, 55, 65, 75, 80, 90, or 100 percent) within a mammal (e.g., a human), without producing severe toxicity in the mammal. In some cases, an effectiveAttorney Docket No. 07039-2279WO1

[0077] 2023-254

[0078] amount of a DrugPEP composition provided herein can be any amount that reduces the size (e.g., by at least 5, 10, 25, 35, 45, 50, 55, 65, 75, 80, 90, or 100 percent) of a solid tumor within a mammal (e.g., a human), without producing severe toxicity to the mammal. In some cases, an effective amount of a DrugPEP composition described herein can be any amount that reduces the number of cancer cells (e.g., by at least 5, 10, 25, 35, 45, 50, 55, 65, 75, 80, 90, or 100 percent) within a mammal (e.g., a human), without producing severe toxicity' to the mammal.

[0079] An effective amount of a DrugPEP composition can be from about 0.1 mg per kg of body weight (mg / kg) to about 100 mg / kg (e.g., from about 0.1 mg / kg to about 1 mg / kg. from about 1 mg / kg to about 5 mg / kg. from about 5 mg / kg to about 10 mg / kg, from about 10 mg / kg to about 20 mg / kg, from about 20 mg / kg to about 80 mg / kg, from about 20 mg / kg to about 60 mg / kg, from about 20 mg / kg to about 50 mg / kg, from about 20 mg / kg to about 40 mg / kg, from about 20 mg / kg to about 30 mg / kg, from about 40 mg / kg to about 100 mg / kg, from about 60 mg / kg to about 100 mg / kg, from about 80 mg / kg to about 100 mg / kg, from about 30 mg / kg to about 90 mg / kg, from about 40 mg / kg to about 80 mg / kg, from about 50 mg / kg to about 70 mg / kg, from about 30 mg / kg to about 50 mg / kg, from about 40 mg / kg to about 60 mg / kg, from about 60 mg / kg to about 80 mg / kg, or from about 70 mg / kg to about 90 mg / kg). The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, severity of cancer, or risk level for development of cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0080] If a particular mammal fails to respond to a particular amount of a DrugPEP composition, then the amount of the composition can be increased by, for example, two-fold. After receiving the higher amount of the DrugPEP composition, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Vanous factors can influence the actual effectiveAttorney Docket No. 07039-2279WO1

[0081] 2023-254

[0082] amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple DrugPEP compositions, route of administration, and severity of the cancer may require an increase or decrease in the actual effective amount administered.

[0083] The frequency of administration of a DrugPEP composition provided herein can be any frequency that reduces a symptom of cancer in the mammal, reduces the number of cancer cells in the mammal, reduces the size of a tumor containing cancer cells in the mammal, and / or delays development of cancer in the mammal, without producing significant toxicity to the mammal. For example, the frequency of administration of a DrugPEP composition can be from about once a day to about once a week, or from about once a week to about once a month (e g., from about once a week to about once every other week). The frequency of administration of a DrugPEP composition described herein can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing a DrugPEP composition described herein can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple DrugPEP compositions, route of administration, and severity of the cancer may require an increase or decrease in administration frequency.

[0084] An effective duration for administering a composition containing a DrugPEP composition provided herein can be any duration that reduces a symptom of cancer in the mammal, reduces the number of cancer cells in the mammal, reduces the size of a tumor containing cancer cells in the mammal, and / or delays development of cancer in the mammal, without producing significant toxicity to the mammal. In some cases, the effective duration can vary7from several days to several months. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of multiple DrugPEP compositions, route of administration, and severity' of the cancer being treated.

[0085] In some cases, the progression of cancer in a mammal or the severity of one or more symptoms related to the cancer in the mammal being treated can be monitored. Any appropriate method can be used to determine whether or not a mammal havingAttorney Docket No. 07039-2279WO1

[0086] 2023-254

[0087] cancer or at risk for developing cancer is effectively being treated. For example, clinical scanning techniques (e.g., computed tomography (CT), positron emission tomography (PET) / CT, bone scan, and magnetic resonance imaging (MRI)) can be used to determine the presence or absence of cancer within a mammal (e.g., a human) being treated. A reduced number of tumor cells and / or reduced tumor size can indicate effective treatment.

[0088] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0089] EXAMPLES

[0090] Example 1: PEP loading and characterization

[0091] PEP derived from blood (e.g., platelets) were obtained from Rion, LLC (Rochester, MN). To optimize the PEP drug loading process while creating a shelfstable product, probe sonication of PEP (Fisher Scientific Model 550: 1 cycle of 10 seconds) was compared with bath sonication of PEP (Elmasonic P 30H; 1, 5, 10, 20, 30 cycles of 30 seconds). To load drug onto PEP, a bath sonicator (Elmasonic P 30 H) was used to sonicate drug / PEP combinations at 37 kHz, 120 W + pulse mode (which allowed for additional ultrasonic power up to 20%) for 30 seconds, followed by 30 seconds on ice. After sonication, the DrugPEP were passed through a 0.45 pm filter to remove precipitated drug, using a 50 kDa MWCO spin column (Amicon Ultra Centrifugal Filter Unit) to purify the samples. 2x column volumes of washing were used to ensure free drug removal. Samples were then lyophilized with 5% D-Mannitol and dPBS for 72 hours.

[0092] PEP loaded with alisertib (AliPEP) without a bulking agent (220427, 220512 and 220526) showed that there was marginal to no difference in loading elficiency and loading capacity between 1 cycle (10 seconds) of probe sonication and 1 cycle (30 seconds) of bath sonication (TABLE 1). Thus, it was demonstrated that bath sonication could be used for consistent and bulk packaging of small molecules into PEP. These studies also demonstrated that a single cycle of bath sonication achieved drug-loading that was comparable to or better than probe sonication. Increasing the number of sonication cycles did improve PEP drug-loading, but actually had the opposite effect (TABLE 1).Attorney Docket No. 07039-2279WO1

[0093] 2023-254

[0094] TABLE 1: Loading capacity’ and efficiency with probe sonication vs. bath sonication

[0095]

[0096] Table 1: Loading capacity and efficiency with probe sonication and bath sonication.

[0097] Post sonication, DrugPEP was passed through a 0.45-pm filter to remove precipitated drug. The sample was then purified using a 50 kDa molecular weight cut off (MWCO) spin column (AMICON® ultra centrifugal filter unit). To ensure free drug removal, the column was washed twice. The sample was lyophilized for 72 hours. D-Mannitol was used as a cryoprotectant / bulking agent (specifically, 5% D-Mannitol with a 50 kDa MWCO spin column and dPBS), as 5% D-Mannitol facilitated rapid product reconstitution, avoided collapse over time, did not alter cell viability in vitro, and was safe for convection-enhanced delivery' (CED) in vivo. Spin column purification and lyophilization with the cryoprotectant / bulking agent yielded shelf stable DrugPEP.

[0098] A variety’ of drugs were packaged in PEP and then evaluated for loading capacity; loading efficiency, and size distribution (TABLE 2 and FIG. 1A). In particular, everolimus and ponatinib were loaded at 0.25 mg / mL, and alisertib and doxorubicin were loaded at 1 mg / mL The lyophilized products were stable at room temperature for months and were easily reconstituted in seconds to yield the desired DrugPEP concentrations. (FIGS. 1B-1C) The size distribution (FIG. IB) of and size (FIG. 1C) of PEP and AliPEP were measured. The size distribution did not show any significant change in the particle size. The effects of different packaging procedures on cell viability' also were evaluated (FIG. ID). No difference was observed in cell viability’ with AliPEP, but AliPEP had higher loading efficacy with lower ICsoAttorney Docket No. 07039-2279WO1

[0099] 2023-254

[0100] compared to alisertib. By purifying drug-PEP using MWCO / size exclusion (e.g.. with a 50 kDa cutoff), most of the secreted molecules thought to promote tissue regeneration were removed. To demonstrate that PEP itself does not promote tumor cell proliferation during drug treatment, SF8628 cells (a human glioma cell line derived by surgical biopsy from a pediatric H3.3K27M DI patient) were exposed to either vehicle or native PEP for 6 days. A growth kinetic assay in the SF8628 cells demonstrated that native PEP (untreated without drug) did not promote additional tumor cell proliferation (FIG. IE).

[0101] Loading capacity was calculated as:

[0102] (Mass of drug in DrugPEP) / (Mass of DrugPEP) Loading efficiency was calculated as:

[0103] (Mass of drug in DrugPEP) / (Mass of drug added for loading)

[0104] TABLE 2: Evaluation of drugs packaged in PEP.

[0105]

[0106] Cell viability and on-target effects: Studies were conducted to evaluate cell viability and on-target effects of PEP and drug-loaded PEP (AliPEP), demonstrating that simple physical modifications to PEP (sonication or lyophilization) with increased concentration did not alter viability or proliferation of either normal cell lines or tumor cell lines (FIGS. 2A-2D). Drug dose response curves showed that the drug-loaded PEP had higher loading efficacy with lower ICso compared to free drugs in DIPG XVIII cells and SF8628 cells (FIGS. 2C-2D). In contrast, normal cells were somewhat protected against drug toxicity with the drug-loaded PEP (FIGS. 2A-2B).

[0107] In further studies of the effects of drug-loaded PEP on cell viability (FIGS.

[0108] 3A, 4A, 5A, and 6A), AhPEP (FIGS. 3E-3I) and PonaPEP (FIGS.4E-4I) showed selective, enhanced, and efficacious tumor killing. Compared to free alisertib and freeAttorney Docket No. 07039-2279WO1

[0109] 2023-254

[0110] ponatinib, AliPEP and PonaPEP showed a shift in their IC50 in different cancer cell lines, including GBM cell lines, breast cancer cell lines, and DMG cell lines (FIGS.

[0111] 3E-3I and FIGS. 4E-4J). Diminished drug potency (right IC50 shift) was observed with AliPEP and PonaPEP in all normal cell lines (FIGS.3B-3D and 4B-4D).

[0112] Similarly, compared to doxorubicin (free drug) and everolimus (free dug), doxorubicin-loaded PEP (DoxoPEP) and everolimus-loaded PEP (EverPEP) showed a shift in potency in different cancer cell lines (FIGS.5A-5J and 6A-6D). DOXIL® is an FDA-approved liposomal doxorubicin formulation that is considered to be one of the strongest chemotherapies for breast cancer, but the dose-response studies showed that DOXIL® did not improve IC50 of the drugs in the tumor cell lines that were tested (FIGS.5F and 5G), most likely due to its delayed-release properties. Further, loading drugs in PEP did not affect drug potency against the intended protein targets compared to free drug treatment. As shown in FIGS. 7A-7C, western blot analyses demonstrated that drug-loaded PEP had the same effect as the free drugs on the intended protein target expression.

[0113] Drug potency testing of drug-loaded PEP in comparison to a PLGA (poly (lactic-co-gly colic acid)) nanoparticle formulation also was conducted. PLGA is one of the most effective biodegradable polymeric nanoparticles (NPs), and has been approved by the FDA for use in drug delivery systems due to its controlled and sustained-release properties, low toxicity, and biocompatibility with tissue and cells (Sadat Tabatabaei Mirakabad et al., Asian Pac J Cancer Prev. 15(2):517-535, 2014; and; Hua et al., Drug Deliv. 28(1): 1342-1355, 2021). Similar to DOXIL®, neither AliPEP nor alisertib-loaded PLGA formulations (formula 1 and formula 2) showed toxicity against normal human astrocytes (FIG.8A). Compared to free alisertib, AliPLGA-NP formula 1 and AliPLGA-NP formula 2, however, AliPEP showed a shift in the IC50 values in a cancer cell line (DIPG XVII) (FIG. 8C) but not in normal astrocytes (FIG. 8B). IC50 ratio analysis demonstrated that AliPEP had a greater than 10-fold shift compared to the AliPLGA-NP formulations (FIG.8D), similar to the observation that DoxoPEP outperformed DOXIL® in tumor cells (FIGS. 5F and 5G).

[0114] Safety’ in animals: A single CED (20 pL / 40-minute infusion) of 5% D-mannitol AliPEP (200 LIM: bath sonication; 1 cycle) was administered to mice (n=3). which were then examined daily for neurological clinical signs. All animals wereAttorney Docket No. 07039-2279WO1

[0115] 2023-254

[0116] bright, alert, and responsive for 7 days after CED. Mice were euthanized 7 days after infusion for histopathological analysis. The 5% D-mannitol was used as a bulking agent, and it did not cause additional damage to brain tissues when used with drug-loaded PEP. Parenchyma surrounding the cannula tract (FIG.9. arrows) and all nonadj acent brainstem tissue were normal on H&E staining, with no signs of tissue necrosis, cavitary lesions, or cellular (inflammatory) infiltrate (FIG. 9). The DrugPEP exhibited extended-release properties similar to those of FDA approved liposomes (DOXIL®) and synthetic nanoparticles (AliPLA-HPG) (FIG. 10A-10B). In addition, these studies demonstrated that an imaging agent can also be packaged in PEP (FIGS.

[0117] 11A-11D)

[0118] Example 2: Improved drug-uptake of drug-loaded PEP One of the hurdles for CED delivery is drug efflux. As described elsewhere, CED resulted in no survival benefit with acute delivery of various drugs to DMG animal models (Power et al., supra', and Oh et al., J. Pharma. Exp. Ther. 383:44-55, 2022). However, a significant increase in drug accumulation was observed in DMG cell lines when the drugs w ere delivered with PEP, as shown for Doxo-loaded PEP compared to free Doxo or DOXIL® (FIG. 12A). Once the drug was released from the PEP, it appeared to be subjected to drug efflux from the tumor cell lines by efflux transporters expressed in the tumor cell lines. Concurrent inhibition of efflux transporters using everolimus increased the amount of intracellular drug retention by 20-fold (FIG. 12B). Immunohistochemical staining of brains from tumor-bearing mice that received DoxoPEP also showed decreased expression of efflux transporter (FIG. 12C)

[0119] Example 3 : Clearance of drug-loaded PEP after acute CED delivery The rate of drug clearance in mice is evaluated after direct delivery of drug-loaded PEP with and ithout efflux transporter inhibitors to determine whether direct PEP-based drug delivery7to the brainstem decreases in vivo clearance compared to free drug. Characterizing drug delivery7and in vivo brainstem clearance rates of drug-loaded PEP allows for a more accurate assessment of the translational potential of direct NP delivery. These studies evaluate the safety, feasibility, and volume ofAttorney Docket No. 07039-2279WO1

[0120] 2023-254

[0121] distribution of drug-loaded PEP. with a drug or a drug and GAD, in murine brainstem. Furthermore, pharmacological synergism between the direct drug-loaded PEP and inhibition of efflux transporters is evaluated. The drugs used in these studies are alisertib and topotecan, chosen based on their pharmacological properties (TABLE 3) and on the results of studies described elsewhere (Power et al., supra,- and Zhang et al.. Neuro. Oncol. 24:1700-1711, 2022).

[0122] TABLE 3. Efflux substrate and fluorescent properties of target drugs and efflux transporter inhibitors.

[0123]

[0124] Non-tumor bearing, normal mice (C57BL / 6J) are used for these studies.

[0125] Animals are divided into the following groups (n = 4 for each group): (a) sham CED / vehicle, (b) free drug, (c) PEP alone, and (d) drug-loaded PEP. All mice undergo intracranial implantation with a standard CED device (Rechberger et al., Neuro. Focus. 48:E2, 2020) and receive a 20 pL infusion of the relevant treatment over 40 minutes (ramping rate protocol from 0.2 pL / minute to 0.8 pL / minute). Mice are then sacrificed, follow ed by the collection of CSF, plasma, and brain (forebrain and hind / midbrain) at the following timepoints after CED: 0 hour. 30 minutes, 1 hour, and 4 hours. Drug concentrations are quantified in plasma, CSF. and brain tissue using high-performance liquid chromatography (HPLC) as described elsew ere (Powder et al., supra and Oh et al., supra). Prior to extraction, samples are homogenized in 5% bovine serum albumin solution (w / v). MLN8054, camptothecin and daunorubicin are used as internal standards (for alisertib, topotecan and Doxo respectively). Mass spectrometry is used to measure drug concentrations in plasma and tissue. Samples are dried, evaporated, and extracted using formic acid in water and formic acid inAttorney Docket No. 07039-2279WO1

[0126] 2023-254

[0127] acetonitrile. A gradient elution is used with a flow rate of 0.5 mL / minute. TSQ Quantum Classic and a TSQ Vantage triple stage quadrupole mass spectrometer equipped with an electrospray ionization source are used to measure plasma concentration and tissue concentration, respectively. Analysis is performed using a single reaction monitoring system with transitions of m / z 519.1 ->328.1 and m / z 477.1 -> 316.0 for alisertib and MLN8054; m / z 422.17 ^377.11 and m / z 349.12 -> 219.09 for topotecan and camptothecin; m / z 544.18 -> 361.07 and m / z 528.19

[0128]

[0129] 321.07 for Doxo and daunorubicin respectively, in positive electrospray ionization mode.

[0130] Drug efflux prevents drug therapies from reaching their maximal clinical utility' especially after direct delivery'. Tumors are guarded by efflux pumps expressed on brain endothelial cells and tumor cells (de Gooijer et al., Cell. Reports. Med. 2: 100184, 2021); and Griffith et al., Trends. Pharm. 42:426-428, 2021). Knocking out P-gp and BCRP in mice significantly increased alisertib concentration in the brainstem of treated mice by over 6-fold compared to wild-type mice, as described elsewhere (Oh et al., supra). To evaluate the impact of efflux transporters on drug clearance w ith direct delivery' of drug-loaded PEP in transporter deficient mice and systemically decipher contributions of P-gp and BRCP in drug clearance, a genetically engineered mouse model (GEMM) created in Friend leukemia virus strain B (FVB) mice is used (Oh et al., supra). Human P-gp corresponds to murine homologue Mdrla / b44. PK studies are conducted using four genoty pes: FVB wildtype, Mdrla / b (P-gp knockout). BCRP I (BCRP knockout), and Mdrla / b / BCRP1 (triple knockout of P-gp and BCRP) mice (Oh et al., supra). Mice are divided into 2 groups (n = 4 each): (a) free drug, and (b) drug-loaded PEP. The drugs are administered to the animals via CED to determine the rate of drug clearance post direct infusion for each drug (e g., AliPEP, DoxoPEP, and PonaPEP). Since the drugs have different lipophilicity’ and hydrophobicity, and different properties against the efflux pumps, the clearance rate for each DrugPEP is individually evaluated. The mice are then sacrificed, follow ed by the collection and analysis of CSF, plasma, and brain (forebrain and hind / midbrain). Drugs of interest and efflux transporters are listed in TABLE 3.Attorney Docket No. 07039-2279WO1

[0131] 2023-254

[0132] Further studies are conducted to determine the brainstem clearance of CED delivered drug-loaded PEP in presence of targeted efflux transporter inhibition, and to assess whether combining direct delivery of drug-loaded PEP with efflux transporter inhibition / saturation (ETI) improves drug retention by reducing rapid efflux thus enhancing drug retention and efficacy. Because systemic delivery of ETI may not be sufficient to reduce the drug efflux (Yang et al.. Nature. 603:885-892, 2022), different delivery routes are evaluated for maximal efflux inhibition by elacridar and everolimus in combination with direct drug-loaded PEP. Oral administration of everolimus can enhance the accumulation of the target drug (delivered systemically) in mice’s brain (Miklja et al., J. Clin. Invest. 130:5313-5325, 2020; Minocha et al.. Int. J. Pharm. 434:306-314, 2012; and Carvalho et al.. Cancer. Disc. 12:416-431, 2022), but it has not been investigated via direct CED delivery. Pharmacological synergy is examined between preconcerted treatment of ETI, delivered orally (systemic) or through co-infusion (local) with drug-loaded PEP delivered via CED.

[0133] Both ETIs (everolimus and elacridar) are delivered orally or are co-infused with the target drug loaded in the PEP. The drug-loaded PEPs (alisertib, topotecan or Doxo) are delivered only using CED in C57BL / 6J mice. The mice are divided into 7 groups (n=4 in each group): (a) Vehicle only, (b) free drug via CED, (c) drug-loaded PEP via CED, (d) oral ETI + free drug, (e) oral ETI + drug-loaded PEP, (f) coinfusion of free drug + ETI, and (g) co-infusion of drug-loaded PEP + ETI. Drug concentration often fluctuates in oral administration due to complex systemic processes. To ensure the ETI in oral administration (Groups d and e) sustains a steadystate concentration in systemic circulation, the animals are administered ETI for 5 consecutive days prior to the CED infusion. All mice undergo intracranial implantation with the CED device prior to oral dosing. Two hours after the last oral dosing on day 5, mice receive a 20 pL infusion of the relevant treatment over 40 minutes (ramping rate protocol from 0.2 pL / minute to 0.8 pL / minute). The mice are then sacrificed, followed by the collection and analysis of CSF, plasma, and brain (forebrain and hind / midbrain) at the following timepoints after CED: 0-hour, 30-minute, 1-hour, and 4-hour intervals.

[0134] All animal studies are conducted in accordance with the ARRIVE guidelines 2.0. Multiple DMG PDXs and a genetically modified mouse model (GEMM) are usedAttorney Docket No. 07039-2279WO1

[0135] 2023-254

[0136] to establish consistent effects in drug-loaded PEP delivery. Both male and female mice are used to address sex as a biological variable.

[0137] Data analysis'. Drug quantification is analyzed by quantitative analytical HPLC utilizing the AUC and calibration curves for each drug. Each quantification includes standardization of drug curves (ranging from 1 ng / mL to 5000 ng / mL). The limit of quantification is 1 ng / mL for plasma or CSF, or 0.2 ng / mL for brain homogenate. To ensure rigor and reproducibility; three-point calibrations (4 ng / mL, 80 ng / mL, 1600 ng / mL) are included in each of the drug quantification assays. All sample analysis is performed in technical duplicates. Data are summarized via descriptive statistics and plots.

[0138] Statistical analysis'. An acceptable coefficient of variation is defined as <15% and acceptable accuracy is defined as relative error <15%. All data are presented as mean ± SD except for the AUClast, which is presented as mean ± SE calculated by Bailer’s method using the Phoenix NCA module (Bailer et al., J. Pharm. Biopharm.

[0139] 16:303-309, 1988). The standard deviation of AUCinf is calculated using Yuan’s method as reported (Y uan et al., J. Pharm. Sci. 82:761-763, 1993). Unpaired t tests are performed to compare groups. Plasma / CSF and brain drug concentration data collected are analyzed by one-way ANOVA and two-way ANOVA followed by Tukey’s post-hoc test, respectively. If modeling assumptions necessitate, nonparametric approaches are considered.

[0140] Example 4: Delivery of dye- and drug-loaded PEP in DMG murine model Although systemic drug delivery is the most convenient and least invasive approach for treatment, systemic delivery of drugs often suffers from insufficient dosing concentration at the target site due to factors such as the blood brain barrier (BBB) and drug efflux. Experiments were conducted to demonstrate that PEP could be visualized in tumor-bearing mice when the animals were given PEP -loaded with DiR dye, delivered intravenously (i.v.) through the tail vein (FIG. 13A) The tumors in the brain were visualized by bioluminescence using IVIS imaging. The i.v. delivered PEP accumulated mostly in peripheral organs (FIG. 13B), although a fair amount also accumulated in the brain within 1 hour of i.v. infusion (FIG. 13C). However, the accumulated amount diminished rather rapidly post infusion, with visible loss by 2Attorney Docket No. 07039-2279WO1

[0141] 2023-254

[0142] hours, suggesting that systemic delivery may not be a good route of drug delivery for brain tumors.

[0143] Convection enhanced delivery (CED) is a drug delivery system that bypasses the BBB by delivering drug direct through infusion at the target site. Direct delivery of drug-loaded PEP 1-2 times per week via acute CED infusion was sufficient to significantly extend survival in multiple DMG PDXs (FIGS. 14A-14C). Above all, these studies demonstrated that PEP loaded with doxorubicin (Doxo) outperformed both Doxo and DOXIL®24, an FDA-approved slow-release NP formulation for Doxo, via CED in preclinical models of DMG (FIG. 14C), suggesting that PEP-based CED can improve patient survival when translated into the clinic.

[0144] Example 5: Efficacy of direct drug-loaded PEP delivery7in DMG murine model Increasing drug tumor residence time increases the efficacy of the drug in DMG tumors. To determine whether NP -based direct drug delivery improves survival, the therapeutic efficacy of drug-loaded PEP with and without ETI is evaluated in the DMG mouse model. For these studies, everolimus is used in combination with the drug-loaded PEP in patient derived xenograft (PDX) models (Peds 17 and DIPGXVII), which are derived from surgical specimens of H3K27M DMG tumors. These tumor cells also express luciferase, allowing for a serial evaluation of tumor growth with bioluminescence (BLI) using IVIS imaging. Athymic nude (Crl:NU(NCr)-Foxnlnu) mice are used for these studies. For each PDX, mice undergo stereotactic implantation of tumor cells in the brainstem and are monitored for tumor growth weekly by the BLI. Once the tumor burden is readily detectable (i.e., >lxl05p / s), stratified randomization by BLI group is used to assign mice to respective treatment groups. The results are validated in a spontaneous DMG GEMM (Power et al., supra,' and Yuan et al.. J. Pharm. Sci. 82:761-763, 1993).

[0145] Additional studies are conducted to determine a clinically relevant dosing regimen for CED delivered drug-loaded PEP. Combination therapy of CED with efflux inhibition (systemic or locally delivered) with high frequency infusion can lead to toxicities. To show a survival benefit that can be translated to the clinic, both a single treatment and the combination therapy using everolimus are investigated. DIPG XVII is used for these studies, as they provide sufficient time (-100 days) to exploreAttorney Docket No. 07039-2279WO1

[0146] 2023-254

[0147] different treatment regimens before reaching moribund. Drug-loaded PEPs (alisertib. topotecan or Doxo) are delivered only via the CED. The animals are divided into 5 treatment groups (n = 12 for each group): (a) sham CED / placebo, (b) CED with drug-loaded PEP lx / 2 weeks, (c) CED with drug-loaded PEP 1 x / 4 weeks, (d) everolimus + CED lx / 2 weeks, and (e) everolimus + CED lx / 4 weeks. For each group, mice undergo intracranial implantation with the CED device and receive a treatment infusion of 20 pL / 40 minutes. All animals receive a maximum of 6 infusions and are evaluated for neurological deficits after each infusion. Mice are monitored twice a week for tumor growth using BLI and are euthanized once moribund. The brain and the tumor are removed and evaluated for mitotic progression (H&E staining for mitotic index) and apoptosis (TUNEL). On-target drug effects are determined by immunohistochemistry (IHC) staining of H3K27M, H3K27me3, and Ki67.

[0148] The PDXs (Peds 17) or DMG GEMM are used to determine the survival rate in animals treated with drug-loaded PEP delivered via CED and with ETI. Animals are divided into 6 treatment groups (n = 12 for each group): (a) sham CED / placebo, (b) CED with free drug, (c) CED with free PEP, (d) CED with drug-loaded PEP, (e) everolimus with free drug, and (f) everolimus with drug-loaded PEP via CED. Free drugs (alisertib, topotecan or Doxo) or drug-loaded PEP are delivered only using the CED. For each group, mice undergo the intracranial implantation with the CED device and receive a treatment infusion of 20 pL / 40 minutes. All animals receive a maximum of 6 infusions and are evaluated for neurological deficits after each infusion. Mice are monitored twice a week for tumor growth using BLI and animals are euthanized once moribund. The brain and the tumor are removed and evaluated for mitotic progression (H&E staining for mitotic index) and apoptosis (TUNEL). On-target drug effects are determined by immunohistochemistry (IHC) staining of H3K27M, H3K27me3. Ki67, P-gp, and BCRP.

[0149] Data analysis'. Data are summarized via descriptive statistics and plots.

[0150] Reproducibility' of findings are confirmed using multiple PDX and each line is analyzed separately and a two-sided a<0.05 is used for all tests.

[0151] Statistical analysis: With 12 mice per group, >80% power is used to detect a hazard ratio of 3.3. The Kaplan-Meier statistical approach is used to evaluate the overall survival effect of the treatments. All modeling assumptions are verified; two-Attorney Docket No. 07039-2279WO1

[0152] 2023-254

[0153] sided a<0.05 is used for all tests. Assuming o = 0.2. >80% power is used to detect a difference in means of tumor size of 0.6.

[0154] Example 6: Volume distribution of drug-loaded PEP following CED to the brainstem Studies are conducted to evaluate the volume of distribution and clearance of drug-loaded PEP after direct delivery to the brainstem in a large animal. One of the reasons for CED trial failure in adult GBM is the inadequate ability to determine the volume of distribution (Va) of drug following the CED injections in humans (Kunwar et A.. Neuro. Oncol. 12:871-881, 2010; and Sampson et al., J. Neuro. 113:301-309.

[0155] 2010). These studies co-infused the drug with GAD (an MR1 tracer) and inferred Va from the spread of GAD with the MRI. How ever, drugs convect within and clear the brain parenchyma according to their inherent physico-chemical properties such as molecular weight, efflux liability, charge, and lipophilicity. Lipophilic drugs (e.g., alisertib) can distribute differently in the brain than GAD. which is primarily hydrophilic. Rodent brains are too small for proper investigation of Va, but the pig brain can mimic clinical-like scenarios and thus can be used for studying drug delivery' in neurosurgical approaches (Sonabend et al., Neuro Oncol. 13:886-893. 2011: D'Amico et al., J. Neurosur. 133:614-623, 2019; and Sauleau et al., Animal. 3:1138-1151, 2009). Therefore, the safety, feasibility, and volume of drug-loaded PEP (with drug or with drug and GAD) distribution are evaluated in the brainstem of a porcine model.

[0156] The spatial distribution of drugs in tissues can be visualized using drug with intrinsic fluorescence such as Doxo or topotecan (Patel et al.. Cancer. Chemo. Pharm.

[0157] 72:127-138, 2013). However, this approach does not accurately track the real-time distribution of drugs in vivo. In these studies, drugs (alisertib, topotecan and Doxo) are loaded into PEPs that are covalently tagged to GAD (to avoid loading efficiency¬ issues) on the lipid membrane to follow their distribution with MRI in real-time after acute CED injections, using methods described elsewhere (Patel et al., supra). In a non-recovery study, domestic white pigs undergo stereotactic cannula placement into the brainstem. There are 4 groups (n = 2 for each group): (a) GAD only, (b) GAD + free drug, (c) GAD-tagged PEP only, and (d) GAD-tagged, drug-loaded PEP. The pigs receive a single 6-hour CED infusion at a rate of 0.17 mL / hour (scaled from humanAttorney Docket No. 07039-2279WO1

[0158] 2023-254

[0159] clinical trials; (Oh et al., supra, and Mueller et al.. Neuro-oncol. 22:iii306, 2020). During the infusion, the animals are imaged using a 7T MRI at 1 -hour, 2-hour, 4-hour and 6-hour intervals. To confirm the localization of the drug and the GAD, animals are sacrificed and plasma, CSF, and brain are harvested. Brains are flash frozen and sectioned via cryostat. Drug distribution is visualized using fluorescence imaging and the GAD is visualized using Imaging Mass Cytometry'. Mass spectrometry is used to determine the drug concentrations.

[0160] In further studies, the clearance rate of drug-loaded PEP is examined in porcine brainstem. Topotecan loaded in PEP (without GAD) is used. Surgical and infusion procedures are as described above. Three groups of domestic white pigs (n = 3 for each group) undergo stereotactic cannula placement into the brainstem, in a non-recovety study: (a) sham / vehicle, (b) free drug, and (c) drug-loaded PEP. Brains are collected at 1-hour, 4-hour and 24-hour intervals, post infusion. Animals are sacrificed, and plasma and brain (forebrain and hind / midbrain) are collected. Drug concentration is determined using mass spectrometry.

[0161] To determine the safety of drug-loaded PEP in the porcine model, domestic white pigs are subjected to stereotactic cannula placement into the brainstem in a nonrecovery study. Surgical and infusion procedures are as described above. There are 2 study groups (n = 3 for each group): (a) vehicle only and (b) drug-loaded PEP.

[0162] Animals receive a weekly infusion for 4 consecutive weeks and are individually assessed for neurological examination pre- and post-infusion each week. Plasma and brain are collected at the end of the study. Tissue sections are subjected to punched biopsy to determine drug concentrations, and pathology analysis is performed to test tissue integrity post multiple infusions.

[0163] Data analysis'. Volumetric analysis of MRI images is conducted using a 3D slicer (wvvw.slicer.org). Serial sectioning and slide imaging are performed to validate the MRI volumetric analysis. Images of drug and GAD distribution are integrated.

[0164] Statistical analysis'. For PK, Unpaired t tests are performed to compare groups. Plasma / CSF and brain drug concentration data collected are analyzed by oneway ANOVA and two-way ANOVA followed by Tukey's post-hoc test, respectively. If modeling assumptions necessitate, non-parametric approaches are considered.Attorney Docket No. 07039-2279WO1

[0165] 2023-254

[0166] Example 7: DrugPEP studies in brain and ovarian cancer OC is among the most lethal gynecologic cancers. Most cases are discovered in late stages, as early detection tests are ineffective. Rates of recurrence are about 70-90% for stage III and about 90-95% for stage IV (Cortez et al., Cancer Chemother Pharmacol, 81 : 17-38, 2018), and most deaths result from complications caused by disseminated disease in the peritoneal cavity.

[0167] Packaging chemotherapeutics in PEP increased the potency of the packaged drug (DrugPEP) in cancer-derived cell lines and reduced their toxicity' in non-cancerous cell lines. This distinct selectivity of DrugPEP is referred to herein as "‘bidirectional context-dependent potency shifting.’7The bi-directional shift caused a dramatic increase in the TI with increased anti-cancer potency and less toxicity.

[0168] In tumors of neuronal linage, PEP was derived from platelets, which were presumed to be nonspecific, and neuro-selectivity was not expected. Like brain tumors, which allowed for direct drug delivery’ to the site of disease via CED, OC peritoneal metastases were situated for intraperitoneal drug delivery'. Both delivery mechanisms took advantage of DrugPEP’ s benefits while avoiding systemic pharmacokinetic limitations of nanoparticle drug delivery.

[0169] Studies of AliPEP were conducted in several OC cell lines, indicating that two of the three tested OC cell lines (OVCAR8 and PEO1) were more sensitive to AliPEP than to alisertib alone (FIGS. 15A-15B). Additional viability studies were conducted for normal and ovarian tumor cell lines treated with DoxoPEP. Drug potency was compared between doxorubicin (free drug) and DoxoPEP in a normal ovarian cell line (FTE) and in patient-derived tumor ovarian cell lines (OVCAR8, CaOV3, OV90 and PEO1) (FIG. 16A). The table shows the numeric values of ICso calculated based on dose-response curves. Cell viabilities were measured in FTE, OCARV8, CaOV3, OC90, and PEO1 (FIGS. 16B-16F, respectively) by CellTiter-Glo 2.0 following 72 hours of treatment. Dose-response curves were generated for each individual cell line to compare between doxorubicin (free drug) and DoxoPEP (Doxorubicin-loaded in PEP). Calculated ratios of IC50 values between doxorubicin and DoxoPEP in each cell line are shown in FIG. 16G, showing improved drug potency for DoxoPEP in the tumor cell lines.Attorney Docket No. 07039-2279WO1

[0170] 2023-254

[0171] Example 8: DrugPEP formulations in ovarian cancer Studies are conducted to evaluate AliPEP and DoxoPEP in OC in vitro, and also to develop and evaluate OC-relevant gemicitabine (GEM) packaged in PEP (GernPEP). as gemcitabine is commonly used in recurrent OC. The effects of AliPEP, DoxoPEP and GernPEP are compared with free drugs in high grade serous OC cell lines and in immortalized fallopian tube epithelial cells (normal control). In addition, it is determined whether the efficacy of DrugPEP observed in brain cancer cell lines is parallel with OC cell lines in these in vitro experiments.

[0172] Cell viability assays (MTS and CellTiterGlo) are used to test the TI profile of DrugPEP vs Drug alone in ovarian cancer cell lines (e.g., the OVCAR8, PEO1.

[0173] CAOV3, OV90, and COV362 cell lines), in addition to an immortalized fallopian tube epithelial cell line (FTE192) and / or normal fallopian tube epithelium (FTE). In addition, dose response assays are performed in the ovarian cancer cell lines and the FTE cells cell lines to test DoxoPEP and GernPEP in parallel. Drug extraction and quantification processes use HPLC quantification with a 10-point calibration curve, followed by liquid-liquid extraction of the drug of interest utilizing an optimal organic solvent.

[0174] Example 9: Mechanism of DrugPEP bi-directional context-dependent potency shifting To elucidate the mechanism of the DrugPEP bi-directional context-dependent potency shifting and mechanisms of resistance, the efficacy of PFA-treated, biologically inactive PEP (DeadPEP) and free drug + native PEP in delivering alisertib, gemcitabine, and doxorubicin to OC cell lines (OVCAR8. PEO1, COV362. and FTE192) in vitro are evaluated and compared to the efficacy of loaded DrugPEP in the ovarian cancer cell lines in vitro. Using DoxoPEP, which can be tracked using flow cytometry, the uptake of the above treatment constitutions is evaluated alongside DOXIL® in OC and normal immortalized fallopian tube epithelial cell lines.

[0175] Furthermore, a proteomics study is performed to compare the effects of DrugPEP, drug, and diluent on OVCAR8 cells. To further assess the mechanism of action of PEP, DeadPEP and native PEP + Drug cotreatment are compared in OC using cell viability assays. Doxorubicin’s fluorescent properties readily allow for tracking by flowcytometry. An uptake assay is performed using DeadPEP(doxo). doxorubicin +Attorney Docket No. 07039-2279WO1

[0176] 2023-254

[0177] native PEP (vehicle). DoxoPEP. and DOXIL® in the OC cell lines and the immortalized fallopian tube epithelial cell line. COV362, a cell line resistant to PEP, is used for comparison with OVCAR8 and PEO1 to determine whether a difference in the uptake contributes to the resistance. In addition, to elucidate the DrugPEP mechanism and mechanisms of resistance, a proteomics study is performed using the OVCAR8 cell line in which a context-dependent potency shift is observed. In particular, total proteomics and global phosphor proteomics (phospho- Ser / Thr and phospho-Tyr) are assessed after treatment of OVCAR8 cells with diluent, drug, drug + native PEP, drug + DeadPEP, and DrugPEP. Cells that do not show context-dependent potency shifting are also included. Observed differences in response are confirmed by immunoblotting after the same treatments and are followed by appropriate siRNA or overexpression studies.

[0178] Example 10: Translate the DrugPEP treatment into OC animal models To determine if the DrugPEP bidirectional context-dependent potency shifting improves the efficacy of the packaged drug against intraperitoneal OC in vivo, AliPEP and GemPEP are tested in at least two orthotopic xenograft models of OC. The Ovarian SPORE Animal Models Core is used to complete a maximal tolerated dose (MTD) study for the DrugPEP formulations as well as the parent drugs (alisertib and gemcitabine). Following MTD determination, the DrugPEP responsive OVCAR8 cell line is engrafted. The animals are treated via intraperitoneal dosing with vehicle, Drug + native PEP, or DrugPEP. In addition, when the DrugPEP formulations result in a significant survival benefit in the OVCAR8 orthotopic xenograft model, studies in PDXs engrafted with paired platinum-sensitive and -resistant PDX models are conducted using the Ovarian SPORE animal model core.

[0179] Example 11: DrugPEP bi-directional context dependent potency shift in breast cancer derived cells

[0180] Breast cancer is among the deadliest cancers and is responsible for 15% of all brain metastases (Lowen,' and Yu, Biochimica et Biophy sica Acta (BBA) - Reviews on Cancer, 1867:49-57, 2017), with more than 40,000 deaths each year resulting from metastatic breast cancer to the brain (Giaquinto et al.. CA: A Cancer Journal forAttorney Docket No. 07039-2279WO1

[0181] 2023-254

[0182] Clinicians. 72:524-541, 2022). For patients whose breast cancer metastasizes to the central nervous system (CNS), the one-year survival rate is just 20% (Malone and Tsirka, Journal of Cancer Metastasis and Treatment, 7:40, 2021). This low survival rate is the result of several factors, including limited chemotherapeutic access to the brain due to the blood brain barrier (BBB) and side effects from systemic administration. CED can bypass the BBB, enabling the infusion of drugs directly to the brain, which increases tumor cell drug exposure while reducing systemic exposure and toxicity. The bi-directional shift results in a dramatic TI increase, allowing administration of greater doses of chemotherapeutics with increased anti-cancer potency and less toxicity.

[0183] Studies are conducted to determine whether the unique bi-directional context dependent potency shift induced by DrugPEP can improve efficacy in animal models of CNS breast cancer metastases. Breast cancer cell lines (MCF-7 and T47D, which both are ER+ / PR+) and MDA-MB-231 (which is triple negative), along with normal cell line controls (Human Mammary Epithelial Cells and Normal Human Astrocytes) are treated with different DrugPEP formulations (alisertib, doxorubicin and ponatinib) or free drug to determine their corresponding cell viability and the IC50 values. In addition, the bi-directional context dependent potency shift is evaluated. Furthermore, to test whether CED delivery of DrugPEP in a metastatic CNS breast cancer animal model results in an improved survival benefit over free drug delivered in a similar manner, the CNS breast cancer metastasis intracranial orthotopic xenograft model MDA-MB-231 (Bander et al., J. Neurosurg. Pediatr. 26:661-666, 2020) is treated with DrugPEP formulation, free drug or vehicle, and survival is assessed.

[0184] OTHER EMBODIMENTS

[0185] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 07039-2279WO12023-254WHAT IS CLAIMED IS:

1. A composition comprising:(a) purified exosome product; and(b) one or more therapeutic agents.

2. The composition of claim 1, wherein said purified exosome product comprises spherical or spheroid exosomes having a diameter of 300 nm or less.

3. The composition of claim 1 or claim 2, wherein said purified exosome product has a moisture content of 10% or less.

4. The composition of any one of claims 1 to 3, wherein said purified exosome product comprises CD63’ exosomes and CD63+exosomes.

5. The composition of any one of claims 1 to 4, wherein said purified exosome product comprises at least 50% CD63’ exosomes.

6. The composition of any one of claims 1 to 5, wherein said composition comprises said one or more therapeutic agents in an amount of about 0.1 mg / mL to about 1 mg / mL of dissolved therapeutic agent per mL of purified exosome product.

7. The composition of any one of claims 1 to 6, wherein said purified exosome product comprises about 200 pg / mL of purified exosomes.

8. The composition of any one of claims 1 to 7, wherein said one or more therapeutic agents comprise a chemotherapeutic agent.

9. The composition of claim 8, wherein said chemotherapeutic agent is selected from the group consisting of alisertib, doxorubicin, topotecan, and ponatinib.

10. The composition of claim 8 or claim 9, wherein said composition further comprises an efflux pump inhibitor.Attorney Docket No. 07039-2279WO12023-25411. The composition of claim 10, wherein said efflux pump inhibitor comprises everolimus and / or elacridar.

12. A method for treating a mammal having a solid tumor, said method comprising administering to said mammal a composition comprising (a) purified exosome product comprising spherical or spheroid exosomes; and (b) one or more therapeutic agents.

13. The method of claim 12, wherein said mammal is a human.

14. The method of claim 12 or claim 13, wherein said administering comprises convection enhanced delivery.

15. The method of claim 12 or claim 13, wherein said administering comprises direct delivery to a tumor site, intraperitoneal administration, intranasal administration, or intrathecal administration.

16. The method of any one of claims 12 to 15, wherein said solid tumor is selected from the group consisting of a brain tumor, an ovarian tumor, a breast tumor, a lung tumor, a prostate tumor, and a pancreatic tumor.

17. The method of any one of claims 12 to 16, wherein said mammal has diffuse midline glioma.

18. The method of claim 17, wherein said mammal is a pediatric human.

19. The method of any one of claims 12 to 18, wherein said spherical or spheroid exosomes have a diameter of 300 nm or less.

20. The method of any one of claims 12 to 19, wherein said purified exosome product has a moisture content of 10% or less.

21. The method of any one of claims 12 to 20, wherein said purified exosome product comprises CD63' exosomes and CD63+exosomes.Attorney Docket No. 07039-2279WO12023-25422. The method of any one of claims 12 to 21, wherein said purified exosome product comprises at least 50% CD63‘ exosomes.

23. The method of any one of claims 12 to 22, wherein said composition comprises said exosomes and said one or more therapeutic agents in an amount of about 0.1 mg / mL to about 1 mg / mL of dissolved therapeutic agent per mL of purified exosome product.

24. The method of any one of claims 12 to 23, wherein said purified exosome product comprises about 200 pg / mL of purified exosomes.

25. The method of any one of claims 12 to 24, wherein said one or more therapeutic agents comprise a chemotherapeutic agent.

26. The method of claim 25, wherein said chemotherapeutic agent is selected from the group consisting of alisertib, doxorubicin, topotecan, and ponatinib.

27. The method of any one of claims 12 to 26, further comprising administering an efflux pump inhibitor to said mammal.

28. The method of claim 27, wherein said composition further comprises said efflux pump inhibitor.

29. The composition of claim 27 or claim 28, wherein said efflux pump inhibitor comprises everolimus and / or elacridar.

Images