Collagenase-iron oxide linked through a cathepsine b cleavable linker

Abstract

Compositions and methods are provided of a therapeutic nanoparticle composed of collagenase IV linked via a linker (e.g. cathepsin B cleavable linker) to ferumoxytol (iron oxide). The collagenase IV is key in the composition intended for the breakdown of the tumor wall as the collagenase. Such compositions and methods are aimed at solving at the same time two of the main challenges of current approaches in the treatment of glioblastoma multiforme (GBM) which are low specificity and poor uptake.

Description

[0001] COLLAGENASE-IRON OXIDE LINKED THROUGH A CATHEPSINE B

[0002] CLEAVABLE LINKER

[0003] FIELD OF THE INVENTION

[0004] The invention relates to delivery of therapeutic drugs. More specifically, the invention relates to compositions and methods for improving delivery of macromolecules to glioblastoma.

[0005] BACKGROUND OF THE INVENTION

[0006] Glioblastoma (GBM) is the most invasive brain tumor in humans with a median survival time of less than one year. The delivery of therapeutic drugs from the blood to the extracellular matrix of GBM is limited by a dense collagen network within the tumor, which stabilizes microvessels and serves as scaffold for tumor cell proliferation and invasion. The present invention addresses ways to improve the delivery of macromolecules to GBM.

[0007] SUMMARY OF THE INVENTION

[0008] Glioblastoma multiforme (GBM) is a devastating primary brain tumor with a grim prognosis. Standard treatments, including surgery, radiotherapy, and chemotherapy, have not significantly improved patient outcomes in years. One major challenge is the lack of specificity in current treatments, leading to side effects and limited efficacy. The present invention aims at solving at the same time two of the main challenges of current approaches: low specificity and poor uptake.

[0009] S23-523 / PCT 1 / 11 In one aspect, the invention provides a composition of a therapeutic nanoparticle composed of collagenase IV linked via a linker (e.g. cathepsin B cleavable linker) to ferumoxytol (iron oxide). The collagenase IV is key in the composition intended for the breakdown of the tumor wall as the collagenase cleaves the collagen of the tumor - hence improving iron oxide uptake inside the tumor.

[0010] Embodiments of the invention include therapeutic nanoparticle (TNP) composed of collagenase IV linked to Ferumoxytol (Iron Oxide) via a cathepsin B cleavable linker. The linker allows the nanoparticle to be cleaved only in the proximity of the tumor leveraging the cathepsin B overexpression in tumors. The collagenase cleaves the collagen strand lining the tumor vessels and present in the tumor tissue, improving the contrast agent (Ferumoxytol) uptake.

[0011] This drug has also the potential to be used in the context of combination therapies in other tumor types. Collagen is in fact one of the main components of tumor tissues, and in solid tumors it constitutes not only a barrier to treatment but also plays a role in creating a tumor microenvironment fertile for cancer development. In particular, it is the main component of interstitial fibrosis, promoting increased solid pressure and compressing blood vessels, leading ultimately to low perfusion and high resistance to therapy.

[0012] In some embodiments, the invention can be characterized as a composition of a therapeutic nanoparticle composed of collagenase IV linked via a linker to ferumoxytol iron oxide, where the composition is used for delivery of macromolecules to glioblastoma. In one example, the linker is a cathepsin B cleavable linker.

[0013] S23-523 / PCT 2 / 11 In other embodiments, the invention can be characterized as a method for improving delivery of macromolecules to glioblastoma, where the method comprises using a therapeutic nanoparticle composed of collagenase IV linked via a linker to ferumoxytol iron oxide. Similarly, in one example, the linker is a cathepsin B cleavable linker.

[0014] In still other embodiments, the invention can be characterized as a method for improving delivery of macromolecules to glioblastoma, where the improvement comprises using a therapeutic nanoparticle composed of collagenase IV linked via a cathepsin B cleavable linker to ferumoxytol iron oxide.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGs. 1A-D show according to exemplary embodiments of the invention collagenase- functionalized theranostic nanoparticles (TNP-collagenase) to enhance nanoparticle and therapeutics delivery to GBM. (FIG. 1A) Chemical structure of TNP-collagenase comprising of ferumoxytol, a cathepsin-B cleavable linker, and collagenase. (FIG. IB) Schematic diagrams of a tumor with dense collagen (left), which hinders nanoparticle penetration versus a tumor with broken collagen (right) following TNP-collagenase treatment, facilitating nanoparticles and therapeutics delivery. (FIG. 1C) Representative color-coded T2 MR images of GBM-bearing mouse brains treated with PBS, ferumoxytol, or TNP-collagenase at 24 hours post-injection. (FIG. ID) Quantitative analysis of T2 relaxation times for the different treatment groups at different time point.

[0017] S23-523 / PCT 3 / 11 FIGs. 2A-D show according to exemplary embodiments of the invention an evaluation of collagenase enzymatic activity of TNP-collagenase using a collagenase / gelatinase assay kit. (FIG. 2A) and (FIG. 2C) exhibit minimal collagenase enzymatic activity in the absence of cathepsin-B, indicating the collagenase remains inactive. In contrast, (FIG. 2B) and (FIG. 2D) show significantly enhanced enzymatic activity with increasing concentration of cathepsin-B, confirming that cathepsin-B cleaves the chemical linker, releasing active collagenase.

[0018] FIGs. 3A-D show according to exemplary embodiments of the invention the digestion of collagen fibers promoting nanoparticles penetration in vitro. Confocal microscopy images show the penetration of FITC-labeled ferumoxytol and TNP-collagenase (FITC-conjugated) in 3D-cultured U87MG / RFP multicellular spheroids (MCSs). The surface of MCSs was define as 0 um, and FITC fluorescence was measured from the top to bottom with 5um intervals (Ex=490nm and Em=525nm for FITC). (FIG. 3A) and (FIG. 3C) demonstrate that TNP-collagenase penetrates deep into the spheroids, with FITC signals detectable at depths of up to 60 pm, whereas ferumoxytol-FITC remains largely confined to the periphery. (FIG. 3B) and (FIG. 3D) present quantitative radial distribution analysis of FITC fluorescence intensity, confirming enhanced penetration of TNP-collagenase compared to ferumoxytol, likely due to collagen degradation facilitating deeper diffusion of nanoparticles.

[0019] FIGs. 4A-C show according to exemplary embodiments of the invention (FIG. 4A) bioluminescence images of tumor-bearing mice during the experimental period

[0020] S23-523 / PCT 4 / 11 following different treatment interventions: PBS (control), PBS+TMZ, FMX+TMZ (ferumoxytol + temozolomide), and TNP-collagenase+TMZ. Bioluminescence intensity (FIG. 4B) and body weight (FIG. 4C) of mice after different treatments at different time points. The bioluminescence intensity of PBS group was significantly increased in 2 weeks. TNP-collagenase+TMZ treated group exhibited a lower signal compared to the groups of PBS+TMZ and FMX+TMZ. The body weight of the TMZ-treated groups was not significantly different, indicating minimal toxicity associated with TMZ and TNP-collagenase.

[0021] FIGs. 5A-C show according to exemplary embodiments of the invention (FIG. 5A) Representative color-coded T2-weighted MR images of tumor-bearing mice at different time points following different treatment interventions: PBS (control), PBS+TMZ, FMX+TMZ, and TNP-collagenase+TMZ. (FIG. 5B) Quantitative analysis of normalized tumor volume over time, showing significant tumor growth in the PBS-treated group, which led to early mortality. (FIG. 5C) Tumor T2 relaxation time measurements at different time points, indicating a reduction in T2 values in the TNP-collagenase+TMZ group compared to PBS+TMZ and FMX+TMZ, suggesting enhanced therapeutic efficacy by improving drug penetration and tumor response.

[0022] FIGs. 6A-E show according to exemplary embodiments of the invention characterization of physicochemical properties of the synthesized TNPs.

[0023] FIG. 7 shows according to an exemplary embodiment of the invention in-vitro cytotoxicity of the synthesized TNPs.

[0024] FIGs. 8A-D show according to exemplary embodiments of the invention in-vitro enzymatic

[0025] S23-523 / PCT 5 / 11 activity.

[0026] FIGs. 9A-C show according to exemplary embodiments of the invention in-vivo animal imaging.

[0027] FIG. 10 shows according to an exemplary embodiment of the invention another representation of the design and synthesis of TNP-collagenase.

[0028] DETAILED DESCRIPTION

[0029] In one embodiment, the invention constitutes ferumoxytol nanoparticles conjugated with collagenase-IV via a cathepsin-B cleavable linker using DBCO-azide click chemistry and carbodiimide crosslinking (EDC / NHS coupling), followed by a size-exclusion chromatography (SEC) process for purification. In-vitro cytotoxicity and enzymatic activity were evaluated using CCK-8 and a gelatinase / collagenase assay kit. The cellular penetration of TNP-collagenase was investigated using 3D spheroids of U87MG / Luc / RFP cells. Next, U87MG / Luc / RFP tumor cells were stereotaxically implanted 4- to 6-week-old mice. 7T MRI was performed before and at 24 and 48 hours after intravenous injection of 50 mg / kg TNP-collagenase, 50 mg / kg ferumoxytol or 200 pL PBS. T2 relaxation times were calculated to assess nanoparticle accumulation in the tumor. To evaluate therapeutic efficacy, a longitudinal study was conducted in which tumorbearing mice were randomized to 5 groups (n=6): PBS, TNP-collagenase, PBS + temozolomide (TMZ, 33mg / kg), FMX+TMZ, and TNP-collagenase + TMZ. Treatments were administered once per week for 21 days. Tumor progression and nanoparticle uptake were monitored using bioluminescence imaging (BLI) and MRI. Tumor volume and T2 value were compared in different groups. Histological analysis, including H&E, Prussian-blue staining and immunofluorescence staining, was used to verify the nanoparticle accumulation and the

[0030] S23-523 / PCT 6 / 11 degradation of collagen-IV in the brain tissue.

[0031] As shown in FIGs. 1A-D, 2A-D, 3A-D, 4A-C and 5A-C, results of this method were that the

[0032] TNP-collagenase showed a hydrodynamic diameter of 28 ± 2.3 nm, zeta-potential of -8.1 ± 1.6 mV and r2 relaxivity 142.4 + 3.8 mM'1s'1. Cytotoxicity was comparable between TNP- collagenase (91.6% ± 5.4%) and FMX (97.1% ± 7.2%). TNP-collagenase showed the enhanced penetration ability compared to FITC-labeled FMX. In-vivo MRI studies demonstrated stronger T2 enhancement of GBM after intravenous injection of TNP-collagenase compared to ferumoxytol alone. At 24 hours post-injection, T2 relaxation times of tumors treated with TNP- collagenase were significantly shorter (29.3 ± 2.6 ms) compared to those treated with ferumoxytol (36.8 ± 1.2 ms, P = 0.004) and PBS (47.3 ± 1.1 ms, P < 0.0001). The in vivo longitudinal studies demonstrated that mice treated with TNP+TMZ showed the greatest tumor regression, with a 60.9% reduction in tumor size by day 21. This effect was significantly greater compared to the PBS+TMZ group (33.2% reduction) and the FMX+TMZ group (43.7% reduction). Prussian-blue staining further confirmed more iron accumulation in tumors treated with TNP-collagenase. Collagen-IV staining revealed partial degradation of the perivascular collagen network in the TNP-collagenase group, supporting the hypothesis that enzymatic cleavage of collagen enhances intratumoral penetration of both nanoparticles and coadministered TMZ.

[0033] From these results, the inventors concluded that collagenase-functionalized theranostic nanoparticles (TNP-collagenase) enhance nanoparticle delivery to GBM compared to nonfunctionalized ferumoxytol nanoparticles. This improvement is attributed to the enzymatic degradation of the collagen network by collagenase, which facilitates deeper intratumoral

[0034] S23-523 / PCT 7 / 11 penetration. As a result, co-delivery of TMZ is more effective, leading to improved therapeutic efficacy in the GBM tumor microenvironment.

[0035] The following embodiments discuss collagenase-iron oxide linked through cathepsin B cleavable linker. FIG. 10 shows design and synthesis enzyme activable theranostics nanoparticles (TNPs), ferumoxytol-collagenase, via DBCO-Azide click chemistry and carbodiimide method (EDC / NHS coupling). Ferumoxytol was used as T2-weighted contrast agent and vehicle for tumor diagnosis, and drug delivery, respectively. Collagenase was used to degrade and break extracellular matrix (ECM) in tumor basement membrane and / or interstitial space, therefore enhancing NPs / drug penetration. Cleavable linker Val-Cit was used for enzyme cathepsin-B cleavage and prodrug activation. Fluorescein isothiocyanate (FITC) was used for in- vitro and in-vivo NPs tracking.

[0036] FIGs. 6A-E show characterization of physicochemical properties of the synthesized TNPs. FIG. 6A shows hydrodynamic diameter (Dh) and poly dispersity index (PDI) of ferumoxytol and ferumoxytol-collagenase TNPs measured by dynamic light scattering (DLS). FIG. 6B shows FITC fluorescence signal, measured by fluorescence spectrophotometer, can be detected after the chemical conjugation. FIG. 6C shows particle size and size distribution of ferumoxytol and ferumoxytol-collagenase TNPs were characterized by transmission electron microscopy (TEM). Inset shows the crystalline of the core of superparamagnetic iron oxide. FIG. 6D shows T2- weighted MR phantom study under 3T MRI scanner. FIG. 6E shows r2 relaxivity of ferumoxytol and TNPs calculated from T2 relaxation decay model.

[0037] FIG. 7 shows in-vitro cytotoxicity of the synthesized TNPs. Cell viability of GBMs (U87 and

[0038] S23-523 / PCT 8 / 11 C6) treated with different concentration of TNPs (0 ~ 1 mg Fe / mL) was evaluated using CCK-8 assay. Ferumoxytol NPs were used as control. High cell viability (> 85%) of ferumoxytol- conjugated collagenase TNPs represents low cytotoxic effects after incubation with TNPs.

[0039] FIGs 8A-D show in-vitro enzymatic activity. The enzyme activity of TNPs was determined by using a collagenase activity assay kit in the absence and presence of cathepsin-B. Different concentration of TNPs was prepared (50 ug / mL ~ 500 ug / mL), buffer solution, and free collagenase were used as negative control, and positive control, respectively. The enzyme activity of TNPs was enhanced after incubation with cathepsin-B.

[0040] FIGs. 9A-C show in-vivo animal imaging. FIG. 9A shows schematic diagram of experimental design, including U87 MG cells intracranial xenografts, tumor status and body weight monitoring, MRI baseline scan, TNPs intravenous injection, etc. FIG. 9B shows representative T2-weighted MR images of mice brain before and after administrating PBS, ferumoxytol and ferumoxytol-collagenase TNPs. FIG. 9C shows quantification of T2* relaxation time. TNPs show higher T2 darkening and shorter T2 relaxation time compared to ferumoxytol.

[0041] S23-523 / PCT 9 / 11

Claims

CLAIMSWhat is claimed is:

1. A composition of a therapeutic nanoparticle composed of collagenase IV linked via a linker to ferumoxytol iron oxide, wherein the composition is used for delivery of macromolecules to glioblastoma.

2. The composition as set forth in claim 1, wherein the linker is a cathepsin B cleavable linker.

3. A method for improving delivery of macromolecules to glioblastoma, wherein the method comprises using a therapeutic nanoparticle composed of collagenase IV linked via a linker to ferumoxytol iron oxide.

4. The method as set forth in claim 3, wherein the linker is a cathepsin B cleavable linker.

5. A method for improving delivery of macromolecules to glioblastoma, wherein the improvement comprises using a therapeutic nanoparticle composed of collagenase IV linked via a cathepsin B cleavable linker to ferumoxytol iron oxide.S23-523 / PCT 10 / 11

Images