Codelivery of Ponatinib and SAR302503 by Active Bone-Targeted

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Co-delivery of ponatinib and SAR302503 by active bone-targeted polymeric micelles for the treatment of therapy-resistant chronic myeloid leukemia Chao-Feng Mu, Yang Xiong, Xue Bai, Yun-Jie Sheng, and Jiajun Cui Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00872 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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Molecular Pharmaceutics

Co-delivery of ponatinib and SAR302503 by active bone-targeted polymeric micelles for the treatment of therapy-resistant chronic myeloid leukemia

Authors: Chao-Feng Mu*,†, Yang Xiong†, Xue Bai†, Yun-Jie Sheng†, Jia-Jun Cui‡ Affiliations: †

Department of Pharmaceutics, Zhejiang Chinese Medical University, Hangzhou,

Zhejiang 310053,China ‡

Department of Biochemistry, College of Medicine, Yichun University, Yichun, Jiangxi

336000, China

Corresponding Author: * Department of Pharmaceutics, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China. Phone: +86 571 61768158; Fax: +86 571 61768136; Email: [email protected]

Notes The authors declare no competing financial interest.

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For Table of Contents Use only Co-delivery of ponatinib and SAR302503 by active bone-targeted polymeric micelles for the treatment of therapy-resistant chronic myeloid leukemia Authors: Chao-Feng Mu, Yang Xiong, Xue Bai, Yun-Jie Sheng, Jia-Jun Cui

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ABSTRACT Point mutations in the BCR-ABL1 domain and primitive chronic myelogenous leukemia (CML) cells existing in the bone marrow environment insensitive to tyrosine kinase inhibitors (TKIs) have become two major challenges in the CML therapy. In this study, combined TKI ponatinib and JAK2 inhibitor SAR302503 short-term treatment effectively suppressed growth and promoted apoptosis of BaF3/T315I cells in cytokine-containing medium in vitro. SAR302503 prevented cytokine-dependent resistance to ponatinib via inhibition of JAK2/STAT5 phosphorylation. Co-delivery of ponatinib and SAR302503 by active bone-targeted polymeric micellar formulation greatly increased the drug accumulation in medullary cavity. The therapeutic efficacy of bone-targeted formulation was demonstrated in BaF3/T315I cells inoculated murine model with no dose-limited toxicity detectable in health mice. Thus, the intravenous injectable bone-homing ponatinib and SAR302503 micellar formulation represents a promising strategy for the treatment of therapy-resistant CML. KEYWORDS:

therapy

resistance,

combination

microenvironment, targeted delivery

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treatment,

bone

marrow


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INTRODUCTION Chronic myeloid leukemia (CML) is caused by a reciprocal translocation between chromosomes 9 and 22, which results in the formation of BCR-ABL1 fusion gene.1 Inhibition of BCR-ABL1 oncogene by the first and second generation of tyrosine kinase inhibitors (TKIs) triggers durable responses in most patients and considerably improves their survival suffering from CML. The high-level drug resistance to imatinib which is predominantly due to emergence of kinase domain mutations such as T315I that impair inhibitor binding can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib.2, 3 Ponatinib (Figure 1) as the third generation of TKI aims to overcome the resistance including T315I mutation.4 However, its dose-limiting toxic effect on life-threatening arterial thrombosis and hepatotoxicity limit its broad applications in clinic.5 Beyond mutation resistance, recent evidence suggests that once BCR-ABL1 was inhibited by TKIs in primitive CML stem/progenitor cells, cytokine and growth factor signaling, by stimulating pro-survival pathways in CML progenitors, still render them BCR−ABL-independent

TKIs

resistance

in

bone

marrow

microenvironment.6

Microenvironment interactions could contribute to protection and preservation of CML stem/progenitor cells during TKI treatment. JAK2 as a member of the JAK family plays an important role in the signaling pathways induced by hematopoietic growth factors such as erythropoietin, thrombopoietin (TPO) and interleukin-3 (IL-3) in bone marrow microenvironment.7 In BCR-ABL1-positive CML cells, JAK2 is activated by BCR-ABL1 and involves phosphorylation of critical Tyr1007.8, 9 STAT5 as one STAT protein, plays a critical role in BCR-ABL1 leukemia initiation and maintenance.10, 11 BCR-ABL1 directly phosphorylated and activated STAT5 by BCR-ABL1 in CML cells. Current researches 4


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demonstrated that JAK2 inhibitor had great benefits for hematological disease therapy.12 Several studies have shown that inhibition or knockdown of JAK2 diminished the viability of BCR-ABL1 CML cells including ABL TKI-resistant cells, and combination treatment of JAK2 and ABL inhibitors prolonged the survival time of BCR-ABL1 CML mice.13,

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SAR302503 (Figure 1) as selective JAK2 inhibitor is potent for inhibition of MPLW515L/K mutations commonly associated with primary myelofibrosis (PMF) ,15 and was used for combination with TKIs for CML therapy.16, 17 However, its phase 2 study was terminated for dose-limiting safety concerns.18 Combination therapy for cancer treatment is becoming more popular because it generates synergistic anticancer effects, reduces individual drug-related toxicity. In our study, with a view to designing effective and safe therapeutic strategies for therapy-resistant CML, we examined the antiproliferative capability of short-term combined treatment with ponatinib and SAR302503 and identified the key survival pathways in vitro. Both drugs were co-encapsulated in alendronate-modified polyethylene glycol–polylactic acid (PEG-PLA) polymeric micelles. Alendronate as the targeting moiety on the surface of micelles was expected to improve the entrapped drug accumulation inside bone marrow in vivo. The bone-targeting efficiency was evaluated via in vivo imaging and biodistribution study. The therapeutic efficacy of bone-targeted combination formulation was investigated in CML murine model and its safety evaluation was also conducted in health mice. MATERIALS AND METHODS Cell Lines and Culture

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Parental murine BaF3 cells were cultured in RPMI 1640 medium (GIBCO, Invitrogen, US) containing 10% fetal bovine serum (FBS) supplemented with penicillin/streptomycin and 5 ng/mL recombinant murine IL-3 (PeproTech, US). BCR-ABL1 stably transfected BaF3 cells (expressing native BCR-ABL1 or BCR-ABL1 with T315I mutation) were maintained in RPMI 1640 medium containing 10% FBS supplemented with penicillin/streptomycin at 37oC and 5% CO2 as previously described.19 Cell Proliferation Assays BaF3/T315I cells were seeded in 96-well plates (5×103 cells/well) and incubated with ponatinib (LC Laboratories) (20 or 40 nM) with or without SAR302503 (MedChem Express) (1 µM) for 2 h and following incubation in normal medium or medium containing recombinant murine IL-3 (5 ng/mL) for different time intervals (24, 48 and 72 h) after drug washout. Proliferation was measured by using Cell Counting Kit-8 (CCK8) viability assay (Dojindo Molecular Technologies Inc., Kumamoto, Japan). All values were normalized to the control wells with no drug. Cell apoptosis assay was performed by using an Apoptosis Detection kit (BD Biosciences). Briefly, different treated cells were stained with Annexin V FITC and 7-aminoactinomysin (7-AAD) (eBioscience) according to the protocol for each test condition and analyzed with BD FACSCaliburTM flow cytometer. Colony-forming Unit (CFU) Assays Pretreated BaF3/T315I cells with ponatinib (20 nM) and SAR302503 (1 µM) alone or in combination for 2 h were plated in Methocult M3234 Medium (STEMCELL Technologies) with or without addition of recombinant murine IL-3 (5 ng/mL) after drug washout. The images of colonies were captured with microscope and their numbers were counted after 7 days incubation at 37oC and 5% CO2.

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Western Blot Analysis BaF3/T315I cells were seeded in 6-well plates, and treated with ponatinib (20 nM) and SAR302503 (1 µM) alone or in combination for 2 h, and 5 ng/mL murine IL-3

was

added and incubated for 15 min after drug washout. Cells were then washed and lysed in RIPA lysis buffer (Thermo Scientific Pierce). The total pretreated whole-cell lysates were collected and subjected to Western blot analysis after the cells treatment procedures. The following were used as primary antibodies: c-Abl, phospho-c-Abl (Y412), JAK2, phospho-JAK2 (Y1007/1008), STAT5 and phospho-STAT5 (Y694) were from Cell Signaling Technologies. β-actin was also probed as a loading control. The blot bands were developed and visualized by Amersham ECL western blotting detection reagents. Preparation of Ponatinib and SAR302503 Containing Alendronate-PEG-PLA Micelles Alendronate-conjugated PEG-PLA polymer was obtained through the reaction of alendronate and N-hydroxysuccinimide (NHS) activated COOH-PEG3000-PLA2000 (Advanced Polymer Materials Inc.) at pH 8.20 Ponatinib and SAR302503 were encapsulated in alendronate-PEG-PLA micelles (AlenPM) by using a co-solvent evaporation method.21 Briefly, aliquots of ponatinib and SAR302503 were mixed with 100 mg of methoxy PEG2000-PLA2000 and alendronate-conjugated PEG3000-PLA2000 at different molar ratios and then dissolved in 1.0 mL of acetone. This solution was added drop-wise into 20 mL distilled water under vigorous stirring. The mixture was then stirred for 2 h at room temperature, and the residual acetone was removed under vacuum. Finally, the micelle solution was filtered and lyophilized. Methoxy PEG2000-PLA2000 micelles (MPPMs) were prepared as the control formulation. Coumarin-6 or

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1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR)-loaded AlenPMs were prepared in the same way. Ponatinib and SAR302503 content in micelles were detected with high performance liquid chromatography (HPLC) using a Waters 1525 dual pump HPLC System (Waters, MA, USA) with a C18 column (250 × 4.6 mm, 5µm, Kromasil). Ponatinib was under gradient elution with water containing 1% ammonium hydroxide and acetonitrile solvent at a flow rate of 1.0 mL/min and wavelength of 254 nm. SAR302503 was eluted with an acetonitrile: water mixture containing 0.1% phosphoric acid (70:30, v/v) at a flow rate of 1.0 mL/min and wavelength of 272 nm. The micelle size distribution and zeta potential were measured by dynamic light scattering (DLS) using Zetasizer 3000HSA instrument (Malvern). In Vivo Bone-targeting Evaluation Female BALB/c mice (6-8 weeks) were intravenously injected with 0.1 mL of DiR-labeling AlenPM solution with different alendronate-PEG-PLA ratio (0, 10, 20 and 40%). Mice were euthanized by isoflurane and imaged (λex: 710~760 nm, λem: 810~875 nm) using a Xenogen IVIS® 100 small animal imaging system (Caliper Life Sciences, Hopkinton, MA) at 6h after administration. The femurs and tibias from different mice groups were removed and imaged again after whole body imaging. Finally, fluorescent and photographic images were acquired and merged. Images were acquired and analyzed using Living Image® 2.5 software. The fluorescence signal intensity of each femur and tibia was quantified by creating a specific, similarly sized circular region of interest (ROI).

In the same way, female BALB/c mice were intravenously injected

with coumarin-6-labeling AlenPM solution. Mice were euthanized and femur samples were collected at 6 h after administration. The femurs were fixed in 10% formalin,

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decalcified with 0.5 Methylenediaminetetraacetic acid (EDTA) solution for 3 days and embedded in paraffin. Finally, 5 µm sections were processed for immunofluorescence observation. Images were captured with an Olympus IX81 inverted fluorescence microscope. In Vivo Pharmacokinetics and Biodistribution of Drug-loaded AlenPMs Pharmacokinetics of combined ponatinib and SAR302503 micellar formulation was evaluated in female BALB/c mice. Blood samples were collected at the 0.25, 0.5, 1, 2, 4, and 8 h timepoints for intravenous injection. The free ponatinib was dissolved in 25 mM citrate buffer solution (pH 2.75) and administrated by oral gavage (25 mg/kg). Free SAR302503 was dissolved in aqueous solution containing 30% polyethylene glycol 200/2% Tween 80 and administrated by oral gavage (100 mg/kg). For oral ponatinib, blood samples were collected at the 1, 2, 3, 4, 5, 6 and 8 h timepoints. For oral SAR302503, blood samples were collected at the 0.5, 1, 2, 3, 4, 6 and 8 h. Ponatinib was extracted from plasma by using methyl tert-butyl ether: plasma (3:1). SAR302503 was extracted by using plasma and methanol mixture (1:9, v/v) and centrifuged at 21,000×g for 10 min for protein precipitation. Ponatinib and SAR302503 content in sample solution were detected with HPLC. Bone marrow distribution studies were carried out after BALB/c mice were treated with combination micellar formulation via i.v injection. Mice dosed with the drug were euthanized at 0.5, 2, or 8 h. Femur and tibia in each treatment group were dislocated and decalcified in 0.5 M EDTA (pH 7.2) for 48 h. The samples were then homogenized and centrifuged at 21,000×g for 10 min. Ponatinib was extracted by using methyl tert-butyl ether: femur suspension mixture (3:1). SAR302503 was extracted by using femur

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suspension and methanol mixture (1:9, v/v) and centrifuged at 21,000×g for 10 min for protein precipitation. Ponatinib and SAR302503 content in sample solution were detected with HPLC. In Vivo Efficacy Evaluation in Murine Model of Leukemia BaF3/T315I cells (1×106 cells/mouse) were inoculated into female NOD/SCID mice (4-6 weeks) via tail vein. Mice were treated with beginning 3 days after inoculation for up to 20 consecutive days. The mice were divided into 6 groups: (1) untreated, (2) free ponatinib (25 mg/kg, p.o., daily), (3) untargeted MPPMs (ponatinib, 5 mg/kg, i.v., every other day), (4) untargeted MPPMs (ponatinib, 5 mg/kg; SAR302503, 10 mg/kg; i.v., every other day), (5) AlenPMs (ponatinib, 5 mg/kg; i.v., every other day), (6) AlenPMs (ponatinib, 5 mg/kg; SAR302503, 10 mg/kg; i.v., every other day). Mice were monitored daily and euthanized at the first appearance of morbidity. Survival data were analyzed using Kaplan Meier method. To measure phosphorylated proteins in cells isolated from bone marrow, BaF3/T315I cells (1×106/mouse) suspension was injected via tail vein into female NOD/SCID mice (4-6 weeks). Mice were treated beginning 8 days after inoculation for up to 14 consecutive days. The mice were divided into 4 groups: (1) untreated, (2) ponatinib-loaded AlenPMs (5 mg/kg, i.v., every other day), (3) SAR302503-loaded AlenPMs (10 mg/kg, i.v., every other day), (4) AlenPMs (ponatinib, 5 mg/kg; SAR302503, 10 mg/kg; i.v., every other day). The femurs in each group were dislocated and bone marrow cells were collected 24 hours after the last dosing for Western blot analysis. In Vivo Toxicity Evaluation

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4-6 weeks female BALB/c mice were treated with (1) free ponatinib (25 mg/kg, p.o. daily), (2) untargeted MPPMs (ponatinib, 5 mg/kg; SAR302503, 10 mg/kg; i.v., every other day), (3) AlenPMs (ponatinib, 5 mg/kg; SAR302503, 10 mg/kg; i.v., every other day), (4) lipopolysaccharide (LPS, 5 mg/kg, i.v., once per week) for four weeks. The untreated mice were as control. Body weight of the mice was measured twice per week and they were observed for signs of toxicity throughout the experiment. Blood samples in each group were collected for hematology analysis at the end of four weeks treatment. The blood samples were analyzed for the number of total white blood cells (WBC), lymphocytes (LYMPH), monocytes (MON), granulocytes (GRAN), Red blood cell count (RBC) and platelets count (PLT). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and cardiac troponin I (cTnI) in serum samples were measured by using commercial assay kits according to manufacturer instructions. The major organs were collected and histological analyses were performed at the same time. Statistical analysis Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., CA, US). Differences were considered statistically significant when P-values of less than 0.05 and 0.01, respectively. Data are presented as means ± SD. RESULTS Combination of Ponatinib and SAR302503 Suppress Growth and Promote Apoptosis of BaF3/T315I Cells in Cytokine-containing Medium in Vitro Ponatinib is the only approved TKI capable of BCR-ABL1/T315I inhibition. However, therapy resistance and relapse are often associated with BCR-ABL1 independence factors beyond BCR-ABL1 mutation inhibition.22 CML progenitor cells are dependent on

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high levels of cytokine-mediated activation for continued viability upon BCR-ABL1 inhibition in bone marrow as compared with differentiated CML cells in peripheral blood.23 Here, we detected the anti-proliferative ability of ponatinib against BaF3/T315I cells in cytokine-containing medium. BaF3/T315I cells were treated with ponatinib (around IC50 value) with or without SAR302503 (1 µM) for 2 h following incubation in normal medium or medium containing IL-3 (5 ng/mL) for different time intervals. As the potent JAK2 inhibitor, SAR302503 did not show BaF3/T315I cell-killing effect at 1 µM treatment for 2 h (data not shown). Ponatinib is very effective on inhibition of BaF3/T315I cell growth in normal medium. However, almost all of the cells survived from ponatinib treatment when addition of IL-3 for 72 h incubation. 1 µM SAR302503 completely recovered the proliferation inhibition capability of ponatinib against BaF3/T315I cells even in the existence of IL-3 (Figure 2A). Importantly, SAR302503 promoted apoptosis of ponatinib-treated BaF3/T315I cells which was terminated via IL-3 stimuli (Figure 2B). To further investigate the role of cytokine in the transforming activity of BaF3/T315I cell, we performed colony formation assay in the presence of ponatinib with or without SAR302503. Ponatinib were observed to reduce the size of colonies (Figure 2C). IL-3 withstood the inhibition capability of colonies formation by ponatinib treatment. However, ponatinib and SAR302503 combination treatment was noted to reduce the size of colonies in the presence of IL-3. Similar effects were observed for the numbers of BaF3/T315I cell colonies. 20 nM ponatinib and 1 µM SAR302503 diminish 87.3% of cell colonies formation in comparison of control group (Figure 2D). SAR302503 completely prevented IL-3-induced resistance to ponatinib in BaF3/T315I cells.

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Identification

of

Signaling

Pathway

Involved

in

SAR302503

Prevents

Cytokine-dependent Resistance to Ponatinib in BaF3/T315I Cells Analysis of the phosphoraylation levels of BCR-ABL1 and its downstream STAT5 demonstrated that full inhibition of BCR-ABL1 kinase activity in BaF3/T315I cells had been achieved upon treatment with ponatinib. No significant changes in either BCR-ABL1 or STAT5 levels were observed when cells were treated with SAR302503 as a single agent, which eliminate any concerns that the off-target effect of SAR302503 alone on BCR-ABL1 kinase activity, as had been suggested for other JAK2 inhibitors at higher concentrations.24 In contrast, BCR-ABL1 phosphorylation was still inhibited by ponatinib while JAK2 and STAT5 were reactivated in the presence of IL-3. This reactivation was inhibited by low concentration of SAR302503 (Figure 3A). Ponatinib and SAR302503 combination deeply inhibits JAK2 and STAT5 activities in the presence of IL-3. Figure 3B shows the proposed mechanism of JAK2/STAT5 signaling in CML progenitor cells inside bone marrow microenvironment. BCR-ABL1 activates JAK2 and STAT5 in leukemia cells. However, the cytokines such as IL-3 can activate the JAK2/STAT5 pathway even though BCR-ABL1 activities are inhibited by TKIs inside bone marrow. This may contribute to the intrinsic resistance of leukemia cells to BCR-ABL1-targeting TKIs. Furthermore, JAK2 inhibitors can disrupt the activated JAK2/STAT5 signaling via cytokines and overcome the therapy resistance to TKIs inside bone marrow. Therefore, effective delivery of combined potent BCR-ABL1 TKIs and JAK2 inhibitors into cytokine-rich bone marrow can contribute to achieve more complete eradication of CML disease.

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Preparation and Characterization of Ponatinib and SAR302503 Co-loaded AlenPMs Leukemia is a bone marrow disease and accumulation of therapeutic agents inside the malignant tissue can greatly contribute to treatment success. On the other hand, the aforementioned findings suggested the synergistic molecular mechanisms of ponatinib and

SAR302503

combination

therapy

inside

cytokine-rich

bone

marrow

microenvironment. Therefore, we developed bone-targeted formulation for co-delivery of ponatinib and SAR302503 inside bone marrow to treat CML in vivo. PEG-PLA micelles have been used as a safe vehicle to delivery single drug or combinations, especially for poorly water-soluble anticancer agents.25,

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Paclitaxel-loaded PEG-b-PLA polymeric

micelles (Genexol-PM) showed favorable efficacy in patients with metastatic breast cancer in phase II clinical trial.27 Both of ponatinib and SAR302503 are hydrophobic drugs and they were co-encapsulated inside the hydrophobic core of PEG-PLA micelles by co-solvent evaporation method. Alendronate were modified on the surface of micelles as the targeting moieties due to the calcium ion-chelating mechanism between bisphosphonates and the mineral hydroxyapatite existed in bone tissue.28 The average size of AlenPMs was 20 to 30 nm, which is identical to that of untargeted micelles (Figure 4A). However, their zeta potential values depended on the ratio of alendronate on the micelle surface (Figure 4B), which is attributed to the strong negative charge of phosphate group of alendronate. The zeta potential decreased to -42.2 mV when the alendronate-PEG-PLA polymer ratio in micelles increased to 60% (Figure 4B). Because the high surface charge of micelles increases their opsonization probability inside blood circulation, we set the alendronate-containing polymer ratio to less than 40% in the next

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step of experiments. For drug encapsulation, we found that the maximum ponatinib loading content was only 2.73% (w/w), less than that of SAR302503 (7.43% (w/w)) inside polymeric micelles (Table 1). The co-encapsulation and alendronate ratio on the surface did not influence the loading capability of each drug in the micellar formulation. In Vivo Bone-targeting Efficiency Evaluation of AlenPMs The bone-targeting efficiency of AlenPMs was evaluated in BALB/c mice via tuning the alendronate density on the surface of DiR (near-infrared fluorescent cyanine dye)-labeling micelles (from 10% to 40%) with an in vivo imaging system (IVIS). As shown in the whole mice image (Figure 5A), the fluorescence intensity in the mouse skull enhanced obviously when the alendronate ratio on the surface of micelles increased to 40%. The ex vivo femur and tibia images clearly demonstrated the alendronate modified micelles can enrich DiR inside the bone marrow (Figure 5B). The DiR accumulation content of 40%-AlenPMs was raised 3.2-fold compared with that of untargeted micelles (Figure 5C). The results indicated that small amount of targeting moieties modification (less than 10%) on the surface of micelles cannot contribute to the accumulation of loaded drugs in targeting tissue or organs. Furthermore, strong green fluorescence from coumarin-6 was founded in bone marrow cavities and along with the endosteum in the femur cross section, while there was no distribution in the cortical bone in 40%-AlenPMs treated mice. For the untargeted micelles, only weak green fluorescence was observed along with the endosteum and there was no fluorescence distribution in the cortical bone (Figure 5D). Therefore, 40%-AlenPMs were chosen to encapsulate ponatinib and SAR302503 as the bone-targeted micellar formulation in the next step of experiments. Pharmacokinetics and Accumulation of Combination Drugs Inside Bone Marrow

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We prepared micellar formulations with two different ponatinib: SAR302503 loading contentratios (1.25%:5% and 2.5%:5%, w/w) for in vivo use. 25 mg/kg of free ponatinib and 100 mg/kg of free SAR302503 via oral administration were employed as the control groups, because these dosages have been explored in preclinical studies, respectively.4, 15

Figure 6A shows the plasma concentration v.s. time curves of bone-targeted (2.5 and

5 mg/kg) and untargeted (5 mg/kg) ponatinib in micellar formulation via intravenous injection, and free ponatinib (25 mg/kg) via oral administration. Ponatinib plasma concentration dropped quickly in 2 h after intravenous injection. In contrast, the maximum ponatinib concentration was 313.5 ng/mL at 4 h and dropped slowly after oral administration of 25 mg/kg ponatinib. Similarly, the maximum SAR302503 concentration was 1835.4 ng/mL at 3 h after oral administration of 100 mg/kg SAR302503 in mice (Figure 6C). Recent studies demonstrated that the high-dose TKIs short-term treatment causes the irreversible apoptosis induction in BCR–ABL-positive and primary CML progenitor cells.16 Based on this targeted CML therapy rationale, intravenous injection could achieve much higher drug concentration in the systemic circulation within 2 h after dosing in comparison to oral administration at the same dose level, which would benefit the short-term high dose exposure of TKIs for CML treatment. Figure 6B and 6D present ponatinib and SAR302503 concentrations achieved in femur at three time points after intravenous injection. For bone-targeted formulation, ponatinib concentrations are 202.3 and 157.1 ng/mL at 0.5 h and 2 h, respectively, which were around 2-fold compared with that of untargeted MPPM formulation (5 mg/kg). It was noted that ponatinib concentration of low dose targeted AlenPMs (2.5 mg/kg) almost have the same drug availability compared with that of 5 mg/kg untargeted formulation

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inside bone marrow. SAR302503 concentration of untargeted formulation inside femur decreased rapidly as the same trend in plasma. However, bone-targeted AlenPMs greatly enhanced its accumulation content around 2 to 3 folds at 2 h and 8 h compared to that of untargeted formulation. The effective enrichment of ponatinib and SAR302503 inside bone marrow may contribute to block BCR-ABL1-dependent and independent pathways for CML progenitor cells proliferation and differentiation in bone marrow environment. In Vivo Therapeutic Efficacy BaF3/T315I cells inoculated murine model of leukemia was used to investigate in vivo therapeutic efficacy of ponatinib and SAR302503 combination treatment. As shown in Figure 7A, untreated mice developed lethal leukemia and died in 20 days. Daily oral dosing of 25 mg/kg ponatinib prolonged median survival to 26 days, compared with 17 days for untreated mice. Ponatinib untargeted MPPMs (5 mg/kg) dosed via intravenous injection every other day had the same efficacy as that of oral administration (25 mg/kg). Notably, the bone-targeted AlenPMs of ponatinib (5 mg/kg) via intravenous injection significantly increased the mouse survival compared with ponatinib (25 mg/kg) orally treated mice. Interestingly, treatment of mice with ponatinib and SAR302503 untargeted MPPMs was similarly effective compared with ponatinib untargeted MPPMs in prolonging survival of the mice, while treatment of mice with bone-targeted AlenPMs of combined drugs significantly prolong survival of the mice compared with the mice treated with ponatinib AlenPMs. The bone-targeted AlenPMs shows successfully prolonged the survival than the untargeted MPPMs, which attributes to the increased accumulation of ponatinib and SAR302503 inside bone marrow.

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To test key proteins phosphorylation in bone marrow tissues, the bone marrow lysate was collected from the late stage of leukemia mice treated with drug-containing AlenPMs. BCR-ABL1 phosphorylation was inhibited both in ponatinib and combination treatment groups. However, 43% of STAT5 was still phosphorylated in ponatinib treatment group, which was resulted from the JAK2/STAT5 pathway activation in bone marrow (Figure 7B). In contrast, combination treatment with ponatinib and SAR302503 completely

inhibited

BCR-ABL1

kinase

activity

and

BCR-ABL1-independent

cytokine-activated JAK2 pathway inside bone marrow. These results also could interpret the rationale of combination treatment molecular mechanisms as shown in Figure 3B. In Vivo Toxicity Evaluation Ponatinib as a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of CML with T315I mutation. Serious dose-limiting adverse events included arterial thrombosis, hepatotoxicity, cardiovascular risks were observed in clinic.29 Therefore, we carried out in vivo studies to evaluate the potential toxicity from the bone-targeted AlenPM formulations. LPS was employed as positive control, which can cause toxicities and injuries in the liver and systemic circulation.30 Healthy BALB/c mice were treated every other day by intravenous administration with micellar formulation for four weeks. These mice were sacrificed at the end of last treatment. Whole blood and serum samples were collected and used for cell counts and biomarkers analysis to detect hematological toxicity and organ function. No hematological values were changed in the combination treatment groups compared with that in the untreated group. In contrast, treatment with LPS caused dramatic decrease in the number of white blood cells, lymphocytes and platelets (Figure 8A and 8B). For the organ function,

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aminotransferase (ALT), aspartate aminotransferase (AST) and cardiac troponin I (cTnI) were used for liver and cardiac function analysis. The AST and cTnl values were elevated in oral ponatinib (25 mg/kg) treatment group, which indicated the potential hepatic and cardiac toxicity of high-dose oral ponatinib. However, there was no change of AST and cTnl values in the micellar formulation of ponatinib and SAR302503 treatment groups (Figure 8C). These results demonstrated that the micellar formulation reduced the hepatic and cardiac toxicity of ponatinib in healthy mice. Histological analysis of the liver and heart were performed in all treatment groups. There were no obvious morphological abnormalities based on H&E staining (Figure 8D). The significantly decreased exposure of drug concentration and duration in systemic circulation resulted in minimal toxicity in vivo via co-delivery of ponatinib and SAR302503 by bone-targeted micellar formulation. DISCUSSION Chronic myeloid leukemia is a cancer which starts in bone marrow. The challenge for development of an effective and safe advanced or TKI-resistance BCR-ABL1 leukemia therapy is to develop a treatment strategy that does not solely inhibition of BCR-ABL1 protein regardless of its mutational status, but rather eliminate the most primitive CML cells are largely insensitive to TKIs existing in the bone marrow microenvironment.31 The primitive CML cells insensitive to TKIs are caused by the cytokine-activated

JAK2/STAT5

pathway

existing

in

the

bone

marrow

microenvironment.13 High levels of STAT5 were protective for BCR-ABL1 cells treated with TKI and specific targeting of STAT5 activity increased eradication of BCR-ABL1 cells, including primary CML cells and CML cells resistant to TKI. However, STAT5 is a

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difficult drug target as it lacks an enzymatic domain and a simpler approach to interfere with STAT5 function is to inhibit its predominant activating kinase, JAK2.24 Therefore, how to effective delivery of combined potent BCR-ABL1 TKIs and JAK2 inhibitors into cytokine-rich bone marrow can contribute to achieve more complete eradication of CML disease. Ponatinib as the only TKI approved for the treatment of CML patients with T315I mutation, it has the ability to inhibit native BCR-ABL1 and all known BCR-ABL1 mutants. However, its dose-limiting toxic effect on arterial thrombosis and hepatotoxicity limit its broad applications in clinic.29 Even though new generation of selective JAK2 inhibitor SAR302503 can contribute to inhibit BCR-ABL1-independent cytokine-mediated resistance to TKIs in bone marrow, it also shows dose-limiting toxicities such as reversible hyperamylasemia, cytopenias and diarrhea etc. in phase II trial.18 These adverse effects of potent BCR-ABL1 TKIs and JAK2 inhibitors were dose-dependent and can be alleviated via reducing dose and duration time in normal tissues. In this study, we have developed bone-targeted AlenPM formulation for combined ponatinib and SAR302503 intravenous injection. Bone marrow is a spongy, fatty tissue in the central medullary cavity of bone. Bone marrow microenvironment plays an important role in the development and progression of leukemia and other types of cancer metastasis.32 However, limited accessibility of TKIs into bone marrow requires a higher dose,33 leading to intolerable cytotoxicity and nonspecific multi-kinase targeting.29 The dual-drug AlenPMs carry payloads preferentially to bone marrow, release physically loaded drugs by disassembling and acting concurrently in bone marrow with minimum off-target effects. Ponatinib as a multikinase inhibitor, ilts broad spectrum of kinases

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inhibition including VEGF receptors, PDGF receptors, FLT3, enhances the potential for a high rate of cardiac and vascular adverse effects during treatment.34 The dose-limiting toxicities of ponatinib and SAR302503 were diminished greatly by lowering down the overall dosage of both drugs in circulation via bone-targeted micellar formulation encapsulation. One of the most important principles is to achieve the specific and sustained target inhibition effect in the early development of targeted cancer therapy inhibitors such as ABL, EGFR, VEGFR, and HER2 inhibitors etc. However, this rationale was inapplicable in the clinical development of BCR-ABL1 TKIs.35 Short-term BCR–ABL1 kinase inhibition (around 2 h) can cause the BCR–ABL1-positive and primary CML progenitor cells commit to apoptosis.35 The same phenomena were reported for high doses of imatinib (32.5 µM), dasatinib (100 nM) and nilotinib (1 µM).36,

37

In our study, in vivo

pharmacokinetics and bone marrow biodistribution study demonstrated that oral administration were unfavorable to attain the desirable short-term high concentration drug exposure compared to intravenous administration in the same bioavailability level. In the meantime, the rapid clearance of TKIs via i.v. injection could shorten the relative drug exposure duration in normal tissues, which contribute to reduce their systemic toxicity. CONCLUSTIONS Combined ponatinib and SAR302503 alendronate-modified micellar formulation greatly enhanced drug availability inside bone marrow. The dose-limiting side effects associated with high doses of single drug were overcome by countering different biological signaling pathways via the bone-targeted intravenous injectable formulation.

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This resulted in significant therapeutic efficacy in murine model of leukemia with no hematological and tissue toxicities to health mice. ACKNOWLEDEGMENTS The authors acknowledge financial support from the National Natural Science Foundation of China (81473434), and internal support from Zhejiang Chinese Medical University. The authors would also like to thank Dr. Bing Shen for his assistance. REFERENCES 1. Daley, G. Q.; Van Etten, R. A.; Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 1990, 247, (4944), 824-30. 2. Cortes, J.; Hochhaus, A.; Hughes, T.; Kantarjian, H. Front-line and salvage therapies with tyrosine kinase inhibitors and other treatments in chronic myeloid leukemia. J Clin Oncol 2011, 29, (5), 524-31. 3. Hochhaus, A.; Ernst, T.; Eigendorff, E.; La Rosee, P. Causes of resistance and treatment choices of second- and third-line treatment in chronic myelogenous leukemia patients. Annals of hematology 2015, 94 Suppl 2, S133-40. 4. O'Hare, T.; Shakespeare, W. C.; Zhu, X.; Eide, C. A.; Rivera, V. M.; Wang, F.; Adrian, L. T.; Zhou, T.; Huang, W. S.; Xu, Q.; Metcalf, C. A., 3rd; Tyner, J. W.; Loriaux, M. M.; Corbin, A. S.; Wardwell, S.; Ning, Y.; Keats, J. A.; Wang, Y.; Sundaramoorthi, R.; Thomas, M.; Zhou, D.; Snodgrass, J.; Commodore, L.; Sawyer, T. K.; Dalgarno, D. C.; Deininger, M. W.; Druker, B. J.; Clackson, T. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell 2009, 16, (5), 401-12. 5. Frankfurt, O.; Licht, J. D. Ponatinib--a step forward in overcoming resistance in chronic myeloid leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research 2013, 19, (21), 5828-34. 6. Dorsey, J. F.; Cunnick, J. M.; Lanehart, R.; Huang, M.; Kraker, A. J.; Bhalla, K. N.; Jove, R.; Wu, J. Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. Leukemia 2002, 16, (9), 1589-1595. 7. Zhang, X. F.; Wang, J. F.; Matczak, E.; Proper, J. A.; Groopman, J. E. Janus kinase 2 is involved in stromal cell-derived factor-1alpha-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells. Blood 2001, 97, (11), 3342-8. 8. Xie, S.; Wang, Y.; Liu, J.; Sun, T.; Wilson, M. B.; Smithgall, T. E.; Arlinghaus, R. B. Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl transformation. Oncogene 2001, 20, (43), 6188-95. 9. Samanta, A. K.; Lin, H.; Sun, T.; Kantarjian, H.; Arlinghaus, R. B. Janus kinase 2: a critical target in chronic myelogenous leukemia. Cancer Res 2006, 66, (13), 6468-72. 10. Carlesso, N.; Frank, D. A.; Griffin, J. D. Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. The Journal of experimental medicine 1996, 183, (3), 811-20. 11. Sillaber, C.; Gesbert, F.; Frank, D. A.; Sattler, M.; Griffin, J. D. STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells. Blood 2000, 95, (6), 2118-25.

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12. Quintas-Cardama, A.; Kantarjian, H.; Cortes, J.; Verstovsek, S. Janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond. Nature reviews. Drug discovery 2011, 10, (2), 127-40. 13. Gallipoli, P.; Cook, A.; Rhodes, S.; Hopcroft, L.; Wheadon, H.; Whetton, A. D.; Jorgensen, H. G.; Bhatia, R.; Holyoake, T. L. JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of chronic phase CML CD34+ cells in vitro and in vivo. Blood 2014. 14. Traer, E.; MacKenzie, R.; Snead, J.; Agarwal, A.; Eiring, A. M.; O'Hare, T.; Druker, B. J.; Deininger, M. W. Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors. Leukemia 2012, 26, (5), 1140-3. 15. Wernig, G.; Kharas, M. G.; Okabe, R.; Moore, S. A.; Leeman, D. S.; Cullen, D. E.; Gozo, M.; McDowell, E. P.; Levine, R. L.; Doukas, J.; Mak, C. C.; Noronha, G.; Martin, M.; Ko, Y. D.; Lee, B. H.; Soll, R. M.; Tefferi, A.; Hood, J. D.; Gilliland, D. G. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell 2008, 13, (4), 311-20. 16. Asmussen, J.; Lasater, E. A.; Tajon, C.; Oses-Prieto, J.; Jun, Y. W.; Taylor, B. S.; Burlingame, A.; Craik, C. S.; Shah, N. P. MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction. Cancer Discov 2014, 4, (2), 200-215. 17. Okabe, S.; Tauchi, T.; Katagiri, S.; Tanaka, Y.; Ohyashiki, K. Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells. J Hematol Oncol 2014, 7, (1), 37. 18. Rosenthal, A.; Mesa, R. A. Janus kinase inhibitors for the treatment of myeloproliferative neoplasms. Expert opinion on pharmacotherapy 2014, 15, (9), 1265-76. 19. Zhang, H.; Trachootham, D.; Lu, W.; Carew, J.; Giles, F. J.; Keating, M. J.; Arlinghaus, R. B.; Huang, P. Effective killing of Gleevec-resistant CML cells with T315I mutation by a natural compound PEITC through redox-mediated mechanism. Leukemia 2008, 22, (6), 1191-9. 20. Chen, H.; Li, G.; Chi, H.; Wang, D.; Tu, C.; Pan, L.; Zhu, L.; Qiu, F.; Guo, F.; Zhu, X. Alendronate-conjugated amphiphilic hyperbranched polymer based on Boltorn H40 and poly(ethylene glycol) for bone-targeted drug delivery. Bioconjugate chemistry 2012, 23, (9), 1915-24. 21. Mu, C. F.; Balakrishnan, P.; Cui, F. D.; Yin, Y. M.; Lee, Y. B.; Choi, H. G.; Yong, C. S.; Chung, S. J.; Shim, C. K.; Kim, D. D. The effects of mixed MPEG-PLA/Pluronic copolymer micelles on the bioavailability and multidrug resistance of docetaxel. Biomaterials 2010, 31, (8), 2371-9. 22. O'Hare, T.; Zabriskie, M. S.; Eiring, A. M.; Deininger, M. W. Pushing the limits of targeted therapy in chronic myeloid leukaemia. Nat Rev Cancer 2012, 12, (8), 513-26. 23. Nievergall, E.; Ramshaw, H. S.; Yong, A. S.; Biondo, M.; Busfield, S. J.; Vairo, G.; Lopez, A. F.; Hughes, T. P.; White, D. L.; Hiwase, D. K. Monoclonal antibody targeting of IL-3 receptor alpha with CSL362 effectively depletes CML progenitor and stem cells. Blood 2014, 123, (8), 1218-28. 24. Hantschel, O.; Warsch, W.; Eckelhart, E.; Kaupe, I.; Grebien, F.; Wagner, K. U.; Superti-Furga, G.; Sexl, V. BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia. Nat Chem Biol 2012, 8, (3), 285-93. 25. Shin, H. C.; Cho, H.; Lai, T. C.; Kozak, K. R.; Kolesar, J. M.; Kwon, G. S. Pharmacokinetic study of 3-in-1 poly(ethylene glycol)-block-poly(D, L-lactic acid) micelles carrying paclitaxel, 17-allylamino-17-demethoxygeldanamycin, and rapamycin. Journal of controlled release : official journal of the Controlled Release Society 2012, 163, (1), 93-9. 26. Wang, Y.; Yang, T.; Wang, X.; Dai, W.; Wang, J.; Zhang, X.; Li, Z.; Zhang, Q. Materializing sequential killing of tumor vasculature and tumor cells via targeted polymeric micelle system. Journal of controlled release : official journal of the Controlled Release Society 2011, 149, (3), 299-306. 27. Lee, K. S.; Chung, H. C.; Im, S. A.; Park, Y. H.; Kim, C. S.; Kim, S. B.; Rha, S. Y.; Lee, M. Y.; Ro, J. Multicenter phase II trial of Genexol-PM, a Cremophor-free, polymeric micelle formulation of paclitaxel, in patients with metastatic breast cancer. Breast cancer research and treatment 2008, 108, (2), 241-50. 23



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28. Giger, E. V.; Castagner, B.; Leroux, J. C. Biomedical applications of bisphosphonates. Journal of controlled release : official journal of the Controlled Release Society 2013, 167, (2), 175-88. 29. Valent, P.; Hadzijusufovic, E.; Schernthaner, G. H.; Wolf, D.; Rea, D.; le Coutre, P. Vascular safety issues in CML patients treated with BCR/ABL1 kinase inhibitors. Blood 2015, 125, (6), 901-6. 30. Ajuwon, O. R.; Oguntibeju, O. O.; Marnewick, J. L. Amelioration of lipopolysaccharide-induced liver injury by aqueous rooibos (Aspalathus linearis) extract via inhibition of pro-inflammatory cytokines and oxidative stress. BMC Complement Altern Med 2014, 14, 392. 31. Ng, K. P.; Manjeri, A.; Lee, K. L.; Huang, W.; Tan, S. Y.; Chuah, C. T.; Poellinger, L.; Ong, S. T. Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition. Blood 2014, 123, (21), 3316-26. 32. Meads, M. B.; Hazlehurst, L. A.; Dalton, W. S. The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance. Clinical Cancer Research 2008, 14, (9), 2519-2526. 33. Swami, A.; Reagan, M. R.; Basto, P.; Mishima, Y.; Kamaly, N.; Glavey, S.; Zhang, S.; Moschetta, M.; Seevaratnam, D.; Zhang, Y.; Liu, J.; Memarzadeh, M.; Wu, J.; Manier, S.; Shi, J.; Bertrand, N.; Lu, Z. N.; Nagano, K.; Baron, R.; Sacco, A.; Roccaro, A. M.; Farokhzad, O. C.; Ghobrial, I. M. Engineered nanomedicine for myeloma and bone microenvironment targeting. Proc Natl Acad Sci U S A 2014, 111, (28), 10287-92. 34. Mian, A. A.; Rafiei, A.; Haberbosch, I.; Zeifman, A.; Titov, I.; Stroylov, V.; Metodieva, A.; Stroganov, O.; Novikov, F.; Brill, B.; Chilov, G.; Hoelzer, D.; Ottmann, O. G.; Ruthardt, M. PF-114, a potent and selective inhibitor of native and mutated BCR/ABL is active against Philadelphia chromosome-positive (Ph+) leukemias harboring the T315I mutation. Leukemia 2015, 29, (5), 1104-14. 35. Shah, N. P.; Kasap, C.; Weier, C.; Balbas, M.; Nicoll, J. M.; Bleickardt, E.; Nicaise, C.; Sawyers, C. L. Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 2008, 14, (6), 485-93. 36. Hiwase, D. K.; White, D. L.; Saunders, V. A.; Kumar, S.; Melo, J. V.; Hughes, T. P. Short-term intense Bcr-Abl kinase inhibition with nilotinib is adequate to trigger cell death in BCR-ABL(+) cells. Leukemia 2009, 23, (6), 1205-6. 37. Snead, J. L.; O'Hare, T.; Adrian, L. T.; Eide, C. A.; Lange, T.; Druker, B. J.; Deininger, M. W. Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis. Blood 2009, 114, (16), 3459-63.

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Page 25 of 33

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Table 1 Physical characteristics of drug-loaded micelles (mean ± SD, n = 3) Micelles MPPM

Loading (%) Size (nm) Zeta potential (mV) Ponatinib SAR302503 2.50 ± 0.37

6.11 ± 0.67

23.5 ± 3.8

-3.85 ± 0.7

AlenPM 2.69 ± 0.17

6.57 ± 0.23

26.4 ± 6.9

-22.8 ± 2.1

25



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Figure 1. Chemical structures of ponatinib and SAR302503

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Figure 2. SAR302503 (SAR) restores the activity of ponatinib (PN) in inhibition of BaF3/T315I cell viability, apoptosis and colony formation in the existence of IL-3. (A) BaF3/T315I cell viabilities after treatment with ponatinib (20 or 40 nM) with or without SAR302503 (1 µM) for 2 h and following incubation in normal medium or medium containing IL-3 (5 ng/mL) for different time intervals (24, 48 and 72 h) after drug washout. (B) Apoptosis of BaF3/T315I cells treated with ponatinib (20 nM) and SAR302503 (1 µM) for 2 h and following incubation in normal medium or medium containing IL-3 (5 ng/mL) for 48 h. (C) Representative pictures of the morphology of forming coloies in each treatment arm (ponatinib, 20 nM; SAR302503, 1 µM; IL-3, 5 ng/mL). (D) Total colonies number was recorded after 7 days culture. 27



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Figure 3. Effects of ponatinib and SAR302503 combination on BCR-ABL1 kinase and related proteins activity in the existence of IL-3. (A) Western blot analysis on ponatinib and SAR302503 treated BaF3/T315I cells for 2 h and stimulated with IL-3 stimulation (5 ng/mL) for 15 min. (B) Targeting BCR-ABL1-dependent and -independent pathways via BCR-ABL1 TKI ponatinib and JAK2 inhibitor SAR302503 in cytokine-rich bone marrow microenvironment.

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Figure 4. Physical characterization of alendronate-modified polymeric micelles (AlenPMs). (A) Size distribution of AlenPMs. (B) changes in zeta potential of micelles with different ratios of alendronate surface modification.

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Figure 5. In vivo bone-targeting efficiency of AlenPMs. (A) Fluorescence images of whole mouse body injected with DiR-labeling micelles with different alendronate surface modification (acquired 6 h post-injection). (B) Fluorescence images of dissected mouse femurs and tibias with different alendronate surface modification (acquired 6 h post-injection). (C) Fluorescence intensity analysis of dissected mouse femurs and tibias. (D) Coumarin-6 labeling micelles distribution in femur of mouse (►, bone marrow cavity; ■, cortical bone; Green, coumarin-6; blue, DAPI).

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Figure 6. In vivo pharmacokinetics and drug availability inside bone marrow in BALB/c mice. (A) Time-plasma concentration profiles of ponatinib (PN). (B) Ponatinib (PN) concentration in femur at 0.5, 2 and 8 h after i.v. injection. (C) Time-plasma concentration profiles of SAR302503 (SAR). (D) SAR302503 (SAR) concentration in femur at 0.5, 2 and 8 h after i.v. injection.

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Figure 7. In vivo therapeutic efficacies of different formulations in BaF3/T315I leukemia model. Ponatinib (PN), SAR302503 (SAR). (A) Kaplan-Meier plot of NOD/SCID mice with BaF3/T315I leukemia disease (n=8). Treatment was initiated on the third day after leukemia cell inoculation. Treatment schedules are as labeled (Compared to the free ponatinib treatment group, *: p

Codelivery of Ponatinib and SAR302503 by Active Bone-Targeted

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