Kevin N. Sill

IT-143, A Polymer Micelle Nanoparticle, Widens Therapeutic Window of Daunorubicin

Background: Daunorubicin is an anthracycline family chemotherapeutic indicated for the treatment of acute myelogenous and acute lymphoblastic leukemia. Daunorubicin has a narrow therapeutic window. Objective: To extend circulation time, decrease toxicity and improve the efficacy of daunorubicin, we encapsulated the drug in our nanoparticle drug delivery platform. Method: IT-143 is a lyophilized formulation of daunorubicin, non-covalently encapsulated in the hydrophobic core of a polymer micelle. Hydroxamic acid-containing triblock polymers (ITP-102) support ferric crosslinking between the polymer chains, increasing stability for improved drug circulation and allowing a tumor targeted pH dependent release of the encapsulated daunorubicin. Results: Formulation characterization demonstrates a 3.7% weight loading (w/w) of daunorubicin and an average particle diameter of 58 nm. IT-143 has an in vitro cytotoxicity of 60-100 nM, comparable to free drug cytotoxicity of 67-114 nM. We compared daunorubicin pharmacokinetics between free drug and IT-143 in vivo and the maximum serum concentration of daunorubicin from IT-143 was increased 50-fold. At equivalent doses IT-143 eliminated in vivo gross toxicity observed at daunorubicin’s maximum tolerated dose of 7.5 mg/kg, and increased the equitoxic dose to 17.5 mg/kg. Furthermore, IT-143 improved anti-tumor efficacy. Studies in 3 xenograft models (HCT116, HT-1080 and MNNG-HOS) compared intravenous bolus administration of IT-143 at equivalent and equitoxic doses to daunorubicin treatment. IT-143 increased the inhibition of tumor volume growth in all models. Conclusion: These studies indicate that the encapsulation of daunorubicin by IT-143 widens the therapeutic window of daunorubicin treatment with reduced toxicity and increased antitumor efficacy.

Synthesis and facile end-group quantification of functionalized PEG azides

ABSTRACT Azido‐functionalized poly(ethylene glycol) (PEG) derivatives are finding ever‐increasing applications in the areas of conjugation chemistry and targeted drug delivery by their judicious incorporation into nanoparticle‐forming polymeric systems. Quantification of azide incorporation into such PEG polymers is essential to their effective use. 1H Nuclear Magnetic Resonance (NMR) analysis offers the simplest approach; however, the relevant adjacent azide‐bearing methylene protons are often obscured by the PEG manifold signals. This study describes the synthesis of 1,2,3‐triazole adducts from their corresponding PEG azides via a convenient, mild click reaction, which facilitates straightforward NMR‐based quantitative end‐group analysis.This method was found to be compatible with many examples of bifunctional azido PEGs with molecular weights ranging from 2 to 18 kDa bearing a variety of functional groups.

Imaging the delivery of drug-loaded, iron-stabilized micelles

Nanoparticle drug carriers hold potential to improve current cancer therapy by delivering payload to the tumor environment and decreasing toxic side effects. Challenges in nanotechnology drug delivery include plasma instability, site-specific delivery, and relevant biomarkers. We have developed a triblock polymer comprising a hydroxamic acid functionalized center block that chelates iron to form a stabilized micelle that physically entraps chemotherapeutic drugs in the hydrophobic core. The iron-imparted stability significantly improves the integrity of the micelle and extends circulation pharmacokinetics in plasma over that of free drug. Furthermore, the paramagnetic properties of the iron-crosslinking exhibits contrast in the tumors for imaging by magnetic resonance. Three separate nanoparticle formulations demonstrate improved anti-tumor efficacy in xenograft models and decreased toxicity. We report a stabilized polymer micelle that improves the tolerability and efficacy of chemotherapeutic drugs, and holds potential for non-invasive MRI to image drug delivery and deposition in the tumor.

Synthesis and Characterization of Micelle-Forming PEG-Poly(Amino Acid) Copolymers with Iron-Hydroxamate Cross-Linkable Blocks for Encapsulation and Release of Hydrophobic Drugs

Described is the development of a polymeric micelle drug delivery platform that addresses the physical property limitations of many nanovectors. The system employs triblock copolymers comprised of a hydrophilic poly(ethylene glycol) (PEG) block, and two poly(amino acid) (PAA) blocks: a stabilizing cross-linking central block, and a hydrophobic drug encapsulation block. Detailed description of synthetic strategies and considerations found to be critical are discussed. Of note, it was determined that the purity of the α-amino acid-N-carboxyanhydrides (NCA) monomers and PEG macroinitiator are ultimately responsible for impurities that arise during the polymerization. Also, contrary to current beliefs in the field, the presence of water does not adversely affect the polymerization of NCAs. Furthermore, we describe the impact of poly(amino acid) conformational changes, through the incorporation of d-amino acids to form mixed stereochemistry PAA blocks, with regard to the physical and pharmacokinetic properties of the resulting micelles.

Stabilized Polymer Micelles for the Development of IT-147, an Epothilone D Drug-Loaded Formulation

Epothilones have demonstrated promising potential for oncology applications but suffer from a narrow therapeutic window. Epothilone D stabilizes microtubules leading to apoptosis, is active against multidrug-resistant cells, and is efficacious in animal tumor models despite lack of stability in rodent plasma. Clinical development was terminated in phase II due to dose limiting toxicities near the efficacious dose. Taken together, this made epothilone D attractive for encapsulation in a stabilized polymer micelle for improved safety and efficacy. We have designed a library of triblock copolymers to develop IT-147, a lead formulation of epothilone D that extends plasma circulation for accumulation in the tumor environment, and potentially decrease systemic exposure to reduce dose limiting toxicities. The drug loading efficiency for IT-147 exceeds 90%, is 75 nm in diameter, and demonstrates pH-dependent release of epothilone D without chemical conjugation or enzymatic activation. Administration of IT-147 at 20 mg/kg increases exposure of epothilone D to the plasma compartment over 6-fold compared to free drug. At the same dose, 20 mg/kg epothilone D from IT-147 is considered the no observed adverse effect level (NOAEL) but is the maximum tolerated dose for free drug. Consequently, IT-147 is positioned to be a safer, more effective means to deliver epothilone D.

Large-scale synthesis of α-amino acid-N-carboxyanhydrides

ABSTRACT Hetero- and homopolymers prepared from α-amino acid-N-carboxyanhydrides (NCAs) monomers are widely useful products. The preparation of pure NCA monomers has been extensively studied in the past. Purification methods including repeated crystallizations, extraction, and flash column chromatography have been devised. However, these methods are not easily amendable to large-scale NCA preparations. This article describes the synthesis of numerous highly purified NCAs on a >100 g scale using a simple filtration step through diatomaceous earth (celite). The resulting NCAs provided polyethylene glycol (PEG)–amino acid triblock polymers devoid of low-molecular-weight by-products that were routinely observed when unfiltered batches of NCAs were used. Also disclosed is the preparation of NCAs at ambient temperature. Traditionally, NCA reactions using a phosgene source are heated. This study shows these reactions can be driven by the slight exotherm that forms upon reagent mixing. This eliminates the need for an external heating source, simplifying large-scale reactions. GRAPHICAL ABSTRACT

Abstract B22: IT-141, a stabilized polymer micelle formulation, prolongs the pharmacodynamic effect of SN-38

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 irinotecan9s 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-1419s 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.

Abstract LB-190: IT-141 and IT-147, iron stabilized micellar nanoparticles for therapeutic and diagnostic applications

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.

Abstract 1321: IT-147: A stabilized polymer micelle formulation of epothilone D for treatment of solid tumors

IT-147 is a formulation of epothilone D encapsulated in a stabilized polymer micelle. Epothilones are a class of cytotoxic chemotherapeutics that induce apoptosis via microtubule stabilization. Epothilone D is a poor substrate for P-glycoprotein efflux pumps and has demonstrated equivalent activity against drug-sensitive and multidrug-resistant human cancer cell lines. This offers a distinct advantage over other microtubule stabilizing agents such as taxanes, however clinical utility is limited by a narrow therapeutic window due to dose limiting toxicities such as peripheral sensory neuropathy, GI toxicities, and cognitive abnormalities. We have optimized a lead formulation comprising 2% epothilone D (w/w) encapsulated in a polymer micelle that is crosslinked with iron to provide micelle stability upon dilution. IT-147 has an average diameter of 70 nm. The iron-mediated crosslinking provides pH-dependent release of epothilone D such that drug can be released in the tumor microenvironment as well as in the endosome/lysosome upon cellular entry. In addition, the geometrical arrangement of iron molecules around the core of the micelle imparts superparamagnetic properties that allow for imaging of intact nanoparticles by MRI. IT-147 demonstrates a low nanomolar IC50 against a panel of human cancer cell lines in vitro. In vivo, IT-147 demonstrates a 4-fold increase in maximum tolerated dose in a mouse model compared to epothilone D free drug. Pharmacokinetics (PK) in a cannulated rat model for a 20 mg/kg epothilone D dose demonstrates a 6.5-fold increase in area under the time versus concentration curve (AUC), and a 14.3-fold increase in the terminal half-life compared to free drug. Dose escalation of IT-147 to 30 and 40 mg/kg epothilone D in the rat PK model results in linear increases in AUC from 53.5 h*μg/mL at 20 mg/kg to 82.9 and 110.8 h*μg/mL at 30 and 40 mg/kg, respectively. Treatment with IT-147 at the MTD in an HCT116 tumor xenograft model led to tumor stasis during the course of treatment, and MRI imaging of mice treated with IT-147 demonstrates tumor accumulation over 24 and 48 hours post administration. Taken together, data for IT-147 demonstrates successful encapsulation of epothilone D leading to a safer, more effective formulation with the potential for MRI imaging as a predictor for response. Citation Format: Adam Carie, Taylor Buley, Bradford Sullivan, J.Edward Semple, Tyler Ellis, Tara Lee Costich, Jyothi Sethuraman, Rick Crouse, Kevin Sill, Suzanne Bakewell. IT-147: A stabilized polymer micelle formulation of epothilone D for treatment of solid tumors. [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 1321.

Abstract 1317: IT-143: A nanoparticle delivery platform that encapsulates daunorubicin and widens therapeutic window

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.