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IT-141 is a formulation of SN-38 encapsulated in an iron-stabilized polymer micelle. SN-38 is the active metabolite of irinotecan (CPT-11) which in combination with 5-FU and leucovorin is first-line FDA approved therapy for metastatic colorectal cancer. Although SN-38 is 1,000 times more potent than irinotecan alone, there is about 100-fold lower concentration of SN-38 in plasma from irinotecan. In the clinic only 2% to 10% of the administered dose of irinotecan is converted by carboxylesterases to SN-38 and there is great interpatient variability with toxicity. In vitro, IT-141 demonstrated nanomolar IC50s against a panel of human cancer cell lines in comparison to irinotecan's micromolar IC50 concentrations. SN-38 binds to the topoisomerase I-DNA complex resulting in double stranded breaks and cell death. We compared the mechanism of action of IT-141 compared to irinotecan treatment in HT-29 xenografts tumors. We demonstrated the incidence of double stranded breaks by immunohistochemistry (IHC) of γ- H2AX expression in tumors treated with IT-141 compared to irinotecan treatment at different time points (24, 48, 72 and 144 hours). In irinotecan treated tumors, γ- H2AX expression peaked at 72 hours followed by a sharp decrease in expression at 144 hours. In IT-141 treated tumors, γ- H2AX positive staining increased steadily from 24 through 144 hours. This shift in the kinetics of the mechanism corroborates the biodistribution studies where IT-141 delivered 34-fold more SN-38 to the tumor compared to irinotecan. The AUC and Cmax in IT-141 treated tumors was 30.9 μg*h/g and 12.2 μg/g respectively compared to an AUC of 1.1 μg*h/g and a Cmax of 0.2 μg/g in the irinotecan treated tumors. In an HT-29 xenograft model IT-141 inhibited tumor growth by 157% compared to a 57% with irinotecan. IT-141 demonstrates successful encapsulation of SN-38 leading to a safer, more effective formulation. Our DNA damage assay demonstrated that IT-141 extended the pharmacodynamic effect over irinotecan in treated tumors. Further studies are required to determine the duration of IT-141's pharmacodynamics effect. This data validates the increased tumor accumulation of SN-38 and increased efficacy of IT-141 over irinotecan. Citation Format: Jyothi Sethuraman, Tara Lee Costich, Adam Carie, Taylor Buley, Tyler Ellis, J. Edward Semple, Tomas Vojkovsky, Kevin Sill, Suzanne Bakewell. IT-141, a stabilized polymer micelle formulation, prolongs the pharmacodynamic effect of SN-38. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B22.

Daunorubicin is an anthracycline family chemotherapeutic with a narrow therapeutic window. Employing our nanoparticle drug delivery platform we encapsulated daunorubicin non-covalently in the hydrophobic core of a polymer micelle (IT-143). IT-143 is a nanoparticle formulation comprised of a hydroxamic acid triblock polymer with ferric crosslinking that increases stability for improved drug circulation and results in a pH dependent release of the encapsulated daunorubicin. Formulation characterization demonstrates a 3.7% weight loading (v/v) of daunorubicin resulting in an average micellar size of 58 nm. In vitro cytotoxicity is comparable to daunorubicin free drug with an IC50 ranging between 60-100 nM, dependent on cell line, compared to 67-114 nM for daunorubicin. In vivo we compared IT-143 plasma pharmacokinetics to daunorubicin administered as free drug and the Cmax was extended from 0.18 μg/mL for daunorubicin to 27.0 μg/mL for IT-143 at the 3 mg/kg dose, and to 49.4 μg/mL at the 6 mg/kg dose. We increased the maximum tolerated dose (MTD) from 7.5 mg/kg to 17.5 mg/kg in mice and improved anti-tumor efficacy. Furthermore, IT-143 eliminated in vivo toxicity compared to equivalent daunorubicin dosed at its MTD. Efficacy studies in 3 xenograft models (HCT116, HT-1080 and HOS-MNNG) compared intravenous bolus administration of IT-143 at equivalent and equitoxic dose to daunorubicin treatment. IT-143 inhibited HCT116 colorectal tumor volume growth by 21% compared to 7% at the equivalent daunorubicin dose, and by 51% at the 17.5 mg/kg dose. In the HT-1080 fibrosarcoma model IT-143 inhibited tumor volume by 38% compared to 37% inhibition at the equivalent daunorubicin dose, and by 94% at the 17.5 mg/kg dose. In the HOS-MNNG osteosarcoma model IT-143 did not inhibit tumor volume compared to 21% inhibition at the equivalent daunorubicin dose, but inhibited tumor volume by 34% at the 17.5 mg/kg dose. The encapsulation of daunorubicin as IT-143 exhibits increased dosage of daunorubicin with decreased toxicities not possible with free daunorubicin. These studies indicate that IT-143 widens the therapeutic window of daunorubicin treatment, providing a safer way to reduce patient toxicity yet increasing antitumor efficacy. Citation Format: Tara Lee Costich, Rick Crouse, Adam Carie, Taylor Buley, Tyler Ellis, Lauren Repp, J.Edward Semple, Tomas Vojkovsky, Suzanne Bakewell, Kevin Sill. IT-143: A nanoparticle delivery platform that encapsulates daunorubicin and widens therapeutic window. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1317.

Site-specific delivery of oncology drugs using nanoparticle technology has been a decades-long goal. IT-141 and IT-147 are polymer micelles that encapsulate (i.e. physically entrap without covalent bonds) hydrophobic chemotherapeutics in the core of the micelle. IT-141 incorporates SN-38, the active moiety of irinotecan, in its core with a weight loading (v/v) of 2%. IT-141 shows increased pharmacokinetics in rat plasma, increased maximum tolerated dose (MTD) and improved anti-tumor efficacy in HCT116 and HT-29 xenograft models over irinotecan in all studies. IT-147 incorporates epothilone D, a microtubule-stabilizing anti-metabolite with a weight loading (v/v) of 2%. IT-147 shows increased pharmacokinetics in rat plasma, increased MTD and improved anti-tumor efficacy in HCT116 colorectal, A549 lung and NCI-H460 lung xenograft models over epothilone D free drug treatment. Both micelles are 70-100 nm diameter clusters of surfactant triblock copolymers stabilized by the interaction between iron and multiple polymer chains. The iron-polymer dative bonds are unstable at low pH, providing a mechanism for environment-dependent micelle stability and subsequent drug release. Furthermore, these stabilized micelles in vivo possess relaxivity constants suitable to provide contrast in magnetic resonance imaging (MRI). The spin-lattice relaxivity value (r1) was 7-16 mM-1s-1 and the spin-spin relaxivity values (r2) were 65-80 mM-1s-1. Small molecule complexes of iron do not typically provide sufficient MR contrast. Because contrast is not observed with individual iron complexes, and the MR contrast is directly related to the properties of the iron-stabilized nanoparticle, only intact nanoparticles provide contrast in MRI. When these iron-stabilized micelle formulations are administered to tumor bearing xenograft mice, increased contrast in the tumor is observed, peaking between 24 and 48 hours. MRI was performed with IT-141 in HCT116, HT-29, and A549 subcutaneous tumor models. IT-147 contrast imaging was performed in HCT116 and NCI-H460 subcutaneous, and MCF-7 orthotopic tumor models. Our technology has produced stable micelles that encapsulate chemotherapeutic drugs to include SN-38, daunorubicin, epothilone D, panobinostat, paclitaxel, and aminopterin with improved pharmacokinetics, decreased toxicity and increased efficacy. The MRI imaging results hold potential for use in the clinic where delivery of the chemotherapeutic-loaded nanoparticle can be monitored non-invasively. Citation Format: Kevin Sill, Tara Lee Costich, Adam Carie, Jyothi Sethuraman, Taylor Buley, Tyler Ellis, Tomas Vojkovsky, Bradford Sullivan, J. Edward Semple, Suzanne Bakewell. IT-141 and IT-147, iron stabilized micellar nanoparticles for therapeutic and diagnostic applications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-190.

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is a first in class small molecule that has successfully completed a phase I clinical trial against solid refractory cancers. 11 of 41 patients achieved stable disease with manageable side effects and 8 patients showed tumor size reduction including a durable partial response. Preclinical studies suggest IT-139 down-regulates the chaperone GRP78, but the mechanism of action is not fully understood. GRP78 (also known as BiP and HSPA5) is the primary sensor and regulator of the endoplasmic reticulum (ER). In cancer cells it is preferably expressed on the cell surface, but also translocates to the mitochondria and cytosol where it regulates critical oncogenic signaling pathways, thus making GRP78 an excellent target for therapy. Here we show in vitro results of IT-139 treatment in a panel of cancer cell lines. IC50 levels measured at 72 hour varied ranging from 15 μM to 180 μM. Annexin V/PI staining by flow cytometry in HCT-116, HT-29, A375 and A549 cell lines at 24 hour show early and late apoptosis, which corresponds to their relative IC50s. UPR induction and overexpression of GRP78 has been reported to cause cell cycle arrest in G1. But in IT-139 treated cell lines HCT116, HT-29, A549 and LnCAP, cell cycle arrest occurred in G2, whereas in A375 cells IT-139 induced cell cycle arrest at G1. These data suggest an inhibition of GRP78 levels and that cell cycle arrest status is also dependent upon the cell line. Electron microscopic (EM) images of HCT-116 and HT-29 cells treated with IT-139 at 24 hours showed significant vacuolization, ER expansion and disorganization of intracellular organelles, strongly suggesting ER stress. However, there is an absence of double membrane vacuoles or autophagosome formation in response to the ER stress. Also, immunofluorescent analysis displayed no LC3-II punctate distribution, although protein immunoblots showed LC3 and p62 protein levels increasing in IT-139 treated cells. These data suggest an inhibition of autophagy. Additionally, EM images showed vacuolated mitochondria and distortion of cristae. In all cell lines treated with IT-139 even below their corresponding IC50 concentration, JC-1 staining showed an increased loss of mitochondrial membrane potential. These observations hypothetically indicate an inhibition of stress-induced GRP78. Quantitative real-time PCR showed IT-139 treatment in combination with thapsigargin exhibits a significant fold change decrease in the GRP78 mRNA expression. These results suggest that IT-139 downregulates GRP78 leading to an increase in ER stress, mitochondrial damage, decreased autophagy, increased apoptosis and cell death. Citation Format: Jyothi Sethuraman, Tara Lee Costich, Tomas Vojkovsky, Rick Crouse, Valentin Cognet, Suzanne Bakewell. IT-139 targets GRP78 in stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2996.

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-β, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies. [Mol Cancer Ther 2006;5(7):1774–82]