Carfilzomib Delivery by Quinic Acid-Conjugated Nanoparticles: Discrepancy Between Tumoral Drug Accumulation and Anticancer Efficacy in a Murine 4T1 Orthotopic Breast Cancer Model

Journal Pre-proof Carfilzomib delivery by quinic acid-conjugated nanoparticles: Discrepancy between tumoral drug accumulation and anticancer efficacy in a murine 4T1 orthotopic breast cancer model Yearin Jun, Jun Xu, Hyungjun Kim, Ji Eun Park, Yoo-Seong Jeong, Jee Sun Min, Naeun Yoon, Ji Yoon Choi, Jisu Yoo, Soo Kyung Bae, Suk-Jae Chung, Yoon Yeo, Wooin Lee PII:

S0022-3549(20)30013-7

DOI:

https://doi.org/10.1016/j.xphs.2020.01.008

Reference:

XPHS 1845

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 17 August 2019 Revised Date:

7 November 2019

Accepted Date: 7 January 2020

Please cite this article as: Jun Y, Xu J, Kim H, Park JE, Jeong YS, Min JS, Yoon N, Choi JY, Yoo J, Bae SK, Chung SJ, Yeo Y, Lee W, Carfilzomib delivery by quinic acid-conjugated nanoparticles: Discrepancy between tumoral drug accumulation and anticancer efficacy in a murine 4T1 orthotopic breast cancer model, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2020.01.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Carfilzomib delivery by quinic acid-conjugated nanoparticles: Discrepancy between tumoral drug accumulation and anticancer efficacy in a murine 4T1 orthotopic breast cancer model

Yearin Jun1, Jun Xu2, Hyungjun Kim2, Ji Eun Park1, Yoo-Seong Jeong1, Jee Sun Min3, Naeun Yoon1, Ji Yoon Choi1, Jisu Yoo1, Soo Kyung Bae3, Suk-Jae Chung1, Yoon Yeo 2 and Wooin Lee1,*

1

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, S.

Korea; 2Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN, USA; 3College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, Catholic University of Korea, Bucheon, S. Korea

* Corresponding author: Wooin Lee, Ph.D. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, S. Korea 08826 Phone: 82-2-880-7873 E-mail: [email protected]

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Keywords: Proteasome inhibitor, carfilzomib, nanoparticle, quinic acid

Abbreviations: carfilzomib, CFZ; nanoparticle, NP; quinic acid, QA; quinic acid-conjugated nanoparticle, QANP; CFZ-loaded nanoparticle, CFZ@NP; polydopamine-coated CFZ@NP, CFZ@NP-pD; quinic acid- conjugated nanoparticle containing CFZ, CFZ@QANP; sulfobutylether β-cyclodextrin, CD; CD-based formulation of CFZ, CFZCD; multiple myeloma, MM; sialyl Lewis X, sLeX; bortezomib, BTZ; poly(lactic-co-glycolic acid), PLGA; paclitaxelloaded nanoparticle, PTX@NP; paclitaxel-loaded nanoparticle, PTX@NP; QANP loaded with paclitaxel, PTX@QANP; N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, Suc-LLVY-AMC; dichloromethane, DCM; below the lower limit of quantitation, LLOQ; dynamic light scattering, DLS; transmission electron microscopy, TEM; polydispersity index, PDI; pharmacokinetic, PK; pharmacodynamic, PD; mononuclear phagocytic system, MPS; phosphate-buffered saline, PBS; dimethyl sulfoxide, DMSO

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ABSTRACT

Despite being a major breakthrough in multiple myeloma therapy, carfilzomib (CFZ, a second-generation proteasome inhibitor drug) has been largely ineffective against solid cancer, possibly due to its pharmacokinetic drawbacks including metabolic instability. Recently, quinic acid (QA, a low-affinity ligand of selectins upregulated in peritumoral vasculature) was successfully utilized as a surface modifier for nanoparticles containing paclitaxel. Here, we designed QA-conjugated nanoparticles containing CFZ (CFZ@QANP; the surface of poly(lactic-coglycolic acid) (PLGA) nanoparticles modified by conjugation with a QA derivative). Compared to the clinically used cyclodextrin-based formulation (CFZ-CD), CFZ@QANP enhanced the metabolic stability and in vivo exposure of CFZ in mice. CFZ@QANP however showed little improvement in suppressing tumor growth over CFZCD against the murine 4T1 orthotopic breast cancer model. CFZ@QANP yielded no enhancement in proteasomal inhibition in excised tumors despite having a higher level of remaining CFZ than CFZ-CD. These results likely arise from delayed, incomplete CFZ release from CFZ@QANP as observed using biorelevant media in vitro. These results suggest that the applicability of QANP may not be predicted by physicochemical parameters commonly used for formulation design. Our current results highlight the importance of considering drug release kinetics in designing effective CFZ formulations for solid cancer therapy.

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INTRODUCTION Carfilzomib (CFZ, Kyprolis®) is a second-generation proteasome inhibitor drug approved for the treatment of relapsed or refractory multiple myeloma (MM). Unlike the first-in-class proteasome inhibitor drug bortezomib (BTZ, Velcade®; dipeptidyl boronic acid), CFZ harbors an epoxyketone as its pharmacophore and features an improved selectivity toward the proteasome target. As such, CFZ provides therapeutic benefits with a reduced incidence of dose-limiting toxicities such as peripheral neuropathy.1 Results from phase III clinical trials also indicated that the addition of CFZ to the combinatory regimens greatly improved the outcomes for both BTZnaïve and BTZ-resistant patients.2-4 Despite these clinical advances offered by CFZ in MM therapy, CFZ showed limited efficacy in patients with advanced solid cancers.5,6 The lack of efficacy is attributable in part to molecular/cellular features of solid cancer cells different from MM cells,7 but also to the poor metabolic stability and short circulation time of CFZ in vivo, limiting the access of active drug to solid tumor tissues.8,9 These issues cannot be overcome simply by increasing CFZ doses due to adverse events arising from on-target inhibition in non-malignant cells (the proteasomes being abundantly present in all cell types). For instance, the intravenous or intraperitoneal dosing of BTZ led to modest anticancer efficacy in a murine breast cancer model, but severe side effects prohibited further escalation of BTZ doses.10 In the same study, intratumoral BTZ dosing effectively inhibited the proteasomal activity in tumors and suppressed tumor growth without incurring dose-limiting toxicities.10 Moreover, the current injectable formulation of CFZ contains nearly 16-fold molar excess of sulfobutylether β-cyclodextrin (CD) for solubilization of practically insoluble CFZ. Thus, there is a clear need for an alternative CD-free formulation that can reduce systemic toxicities and expand therapeutic utility of CFZ against solid cancers. Quinic acid (QA) is a synthetic mimic of sialyl Lewis X (sLeX) which can reversibly bind to E-/P-selectin abundantly present in peritumoral vasculature.11-14 When QA was used as a surface modifier for nanoparticles loaded with paclitaxel (PTX@QANP), both tumoral drug delivery and anticancer efficacy significantly improved as compared to Taxol®.15 Due to the low-affinity interactions between QA and selectins, PTX@QANP likely had an increased chance of extravasating through peritumoral endothelium. CFZ displays aqueous solubility and lipophilicityrelated parameters comparable to PTX (water solubility measured in the current study: 3.3 ± 0.3 μg/mL for PTX and 1.3 ± 0.2 μg/mL for CFZ; water solubility as reported in DrugBank: 5.5 μg/mL for PTX and 4.8 μg/mL for CFZ; calculated partition coefficients (clogP) as reported in DrugBank: 3.2 for PTX and 3.2 for CFZ) (DrugBank database, DB01229 and DB08889). Based on these similarities, we reasoned that CFZ may be encapsulated in QANP in a

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similar manner and benefit from the QANP encapsulation with respect to the metabolic stability and tumoral delivery of CFZ, thereby improving its anticancer efficacy against breast cancer. Here, we prepared QANP containing CFZ (CFZ@QANP) and examined the pharmacokinetic (PK) and pharmacodynamic (PD) profiles relative to the clinically used CD-based formulation (CFZ-CD). CFZ@QANP enhanced the metabolic stability and tumoral accumulation of CFZ; however, it showed little improvement in suppressing tumor growth in a mouse 4T1 model of orthotopic breast cancer. The lack of improvement in the anticancer efficacy in vivo is likely from delayed and incomplete CFZ release from CFZ@QANP in tumors as observed in vitro. In further efforts of designing novel and effective CFZ formulations for solid cancer therapy, it would be important to optimize the formulation according to drug release kinetics in biorelevant media.

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MATERIALS AND METHODS Cell lines, reagents and antibodies 4T1 cells and MDA-MB-231 cells were from the Korean Cell Line Bank (KCLB, Korea) and maintained in RPMI1640 supplemented with penicillin (100 U/mL, ThermoFisher Scientific, Waltham, MA, USA), streptomycin (10,000 µg/ml, ThermoFisher Scientific), and 10% fetal bovine serum (Welgene Inc., Daegu, Korea). CFZ was purchased from Shenzhen Wolcase Pharmaceutical Technology Co., Ltd (Guangdong, China). Poly(lactic-coglycolic acid) (PLGA, lactic acid:glycolic acid (LA:GA) = 85:15, acid endcap, molecular weight of 150 kD) was from Akina, Inc. (West Lafayette, IN, USA). Dopamine hydrochloride was purchased from Alfa Aesar (Ward Hill, MA, USA). (2-Hydroxypropyl)-β-cyclodextrin, chlorpropamide, theophylline, ammonium formate, formic acid and other chemicals for analytical assays were from Sigma-Aldrich (St. Louis, MO, USA). For the proteasome activity assay, the fluorogenic probe substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) was obtained from Bachem (Torrance, CA, USA). Antibodies against E-selectin (catalog No. AF575) and P-selectin (catalog No. AF737) were from R&D systems (Minneapolis, MN, USA). All other reagents were from Thermo Fisher Scientific (Waltham, MA, USA). Preparation and characterization of CFZ@QANP CFZ@QANP was prepared according to the previously reported scheme with a slight modification.15,16 Briefly, PLGA (20 mg) and CFZ (2 mg) were dissolved in 1 mL of dichloromethane (DCM), homogenized in 4 mL of 5% polyvinyl alcohol solution by probe sonication for 2 min, and stirred in 20 mL of deionized water overnight to evaporate DCM. After ultracentrifugation at 34,000 g, the CFZ-loaded NP (CFZ@NP) was incubated in 2 mL of Tris buffer (10 mM, pH 8.5) containing two-fold excess of dopamine (w/w) for 3 h, to form polydopamine-coated CFZ@NP (CFZ@NP-pD). To add the QA moiety on the surface, CFZ@NP-pD was ultracentrifuged at 34,000 g, and resuspended in Tris buffer containing two-fold excess (w/w) of QA-NH2. After 30 min incubation, the QA-coated CFZ@NP-pD (CFZ@QANP) was collected and lyophilized with trehalose (an equivalent amount to CFZ@QANP) for long-term storage or subsequent in vivo studies. The physicochemical properties (the size distribution, polydispersity index, and surface charge) of CFZ@QANP (diluted in 10 mM citrate buffer, pH 3.0) were assessed using an Electrophoretic Light-Scattering 8000 Spectrophotometer (Otsuka Electronics Co. Ltd., Tokyo, Japan) or Malvern Zetasizer Nano ZS90 (Worcestershire, UK). The morphology of nanoparticles was imaged using Tecnai G2 20 (200 keV; TEM; FEI Company, Hillsboro, OR, USA) after negative staining with 1% uranyl acetate. CFZ@QANP was initially reconstituted in 10 mM citrate buffer (pH 3.0) and further diluted with deionized water. The amount of CFZ was quantified using an HPLC 6

system equipped with Waters 515 Binary Pump, Waters 717 plus autosampler, and Waters 2487 dual wavelength absorbance detector with the detection wavelength set at 254 nm (Milford, MA, USA). Chromatographic separation was performed on a Phenomenex Gemini-NX C18 (100 × 4.6 mm id, 3 µM) using the isocratic mobile phase of acetonitrile and 2 mM ammonium formate in water (70:30, v/v) at the flow rate of 1 mL/min. Drug loading efficiency was defined as the CFZ amount divided by the total mass of CFZ@QANP. In vitro metabolic stability in rat liver homogenates and whole blood Sprague-Dawley rats (8-week-old, male, body weight of ~250 g, n=3) were obtained from Saeron Bio (Uiwang, Korea) and handled in accordance with the animal protocol approved by the Seoul National University Institutional Animal Care and Use Committee (approval No. SNU-160512-5-1). The liver tissue was harvested, washed with PBS and homogenized in ice-cold PBS (2 mL/g tissue) using Ultra Turrax homogenizer (IKA, Staufen, Germany). The liver homogenates and whole blood were pre-incubated at 37°C for 1 min and incubated with CFZ@QANP (dissolved in 10 mM citrate buffer, pH 3.0) or CFZ-CD (CFZ dissolved in 10 mM citrate buffer containing 20% (w/v) 2-hydroxypropy-β-cyclodextrin, pH 3.0) at the final CFZ concentration of 1 µM. After vortexing briefly, an aliquot (40 µL) of the reaction mixture was taken at pre-determined time points (0, 5, 10, or 30 min) and was quenched with 160 µL of acetonitrile containing chlorpropamide (an internal standard, 2.5 µg/mL). The mixture was kept on ice for 30 min and subsequently vortexed for 10 min. After centrifugation at 3,000 g for 3 min at 4°C, the CFZ level in the resulting supernatant was quantified by LC-MS/MS. The analytical conditions were similar to the previous reports with slight modifications.17,18 Briefly, the LC-MS/MS system was equipped with Waters e2695 HPLC system (Milford, MA) and API 3200 Qtrap mass spectrometer (Applied Biosystems, Foster City, CA). Chromatographic separation was performed on a Phenomenex Luna C18 column (50 × 2.0 mm id, 3 µm), and the mobile phase composed of acetonitrile:water containing 0.1% formic acid (70:30, v/v) was run at the flow rate of 0.3 mL/min. The optimized source-dependent mass spectrometry parameters were as follows: ion spray voltage of 5,500 V, curtain gas of 20.0 (arbitrary units), GS1 of 40 psi and GS2 of 30 psi. The CFZ-dependent parameters were set as follows: declustering potential of 45 V, entrance potential of 8 V, collision energy of 86 V, and collision cell exit potential of 31.5 V. Plasma pharmacokinetic profiles in mice ICR mice (6-week-old, male, body weight of 25-30 g) were obtained from Saeron Bio (Uiwang, Korea) and handled in accordance with the animal protocol approved by the Seoul National University Institutional Animal Care and Use Committee (approval No. SNU-161205-2-2). Briefly, CFZ@QANP or CFZ-CD was administered via tail vein of ICR mice (CFZ dose equivalent to 3 mg/kg; n=3-4 per group). Blood samples were collected at the pre7

determined time points (2, 5, 20, 60, 120, 360, or 600 min) from the retro-orbital plexus of the mice. To prevent excessive blood loss, the blood sampling volume and the number of sampling per mouse were limited to 20 µL and 4 times, respectively. Collected whole blood samples were centrifuged (at 3,000 g for 5 min at 4°C) and the resulting plasma samples (10 µL) was mixed with 50 µL of acetonitrile containing chlorpropamide (an internal standard, 0.5 µg/mL) and mixed by vortexing for 10 min. After centrifugation, the CFZ level in the resulting supernatant was quantified by LC-MS/MS. The PK parameters were obtained using non-compartmental methods with the sparse data option of WinNonlin (version 8.1, Certara, Princeton, NJ, USA). CFZ biodistribution profiles in various tissues in the 4T1 orthotopic breast cancer model CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg) was injected via tail vein into BALB/c mice harboring orthotopic 4T1 implants of an average volume of 100 mm3. At 0.5 or 4 h after drug dosing, tumor tissues and major organs were harvested, weighed, and snap-frozen. To quantify the CFZ levels in tissues, tissue homogenates were prepared using ice-cold PBS (4 mL/g tissue) and subject to liquid-liquid extraction by mixing with 9-fold excess volume of cold tert-butyl methyl ether containing theophylline (an internal standard, 0.5 µg/mL). After centrifugation at 3,000 g for 5 min at 4°C, the resulting organic phase was transferred to a new tube and evaporated. The samples were reconstituted with acetonitrile and analyzed by LC-MS/MS. The working concentration ranges of CFZ were typically 0.5 - 500 nM, although it varied slightly in some tissues. When samples contained CFZ levels below the lower limit of quantitation (LLOQ), the values were substituted with the half value of the LLOQ as recommended in the literature.19-21 In vivo anticancer efficacy in the 4T1 orthotopic breast cancer model To establish the orthotopic breast cancer mouse model, 4T1 cells (0.5 × 105 cells/50 µL of complete media) were inoculated into the inguinal mammary fat pad on the right side of BALB/c mice. When the tumor volume reached at ~100 mm3, the mice were divided into 3 different groups (n=3-4/group): vehicle control (10 mM citrate buffer, pH 3.0), CFZ-CD and CFZ@QANP at the CFZ dose equivalent to 3 mg/kg. Similar to clinically used dosing regimens,5 the drug was administered intravenously on two consecutive days per week for three weeks (days 0, 1, 7, 8, 14, and 15). The length (L) and width (W) of tumor were measured every 2 – 3 days using a digital caliper and used to calculate the tumor volume by the modified ellipsoid formula V=(L × W2)/2. The body weight of mice was monitored every 2 – 3 days. Three days after the last drug dosing (day 18), whole blood and major organs were collected and stored at -80°C until analysis.

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Measurement of the proteasome activity in tumors from mice treated with CFZ@QANP or CFZ-CD To measure the inhibitory extent of proteasomal activity in tumors, 4T1 implants were excised either from the mice in in vivo anticancer efficacy testing (at 72 h after the last dosing) or the mice in biodistribution studies (at 0.5 or 4 h after single dosing). Tumor tissue homogenates (prepared in PBS as described in the previous sections) were further mixed with the passive lysis buffer (Promega, WI, USA). After centrifugation at 3,000 g for 3 min at 4°C, the resulting supernatant was collected. To assess the proteasome activity, diluted samples of tissue lysates (2 µL, at 50 µg total protein/µL) were incubated with 100 μM Suc-LLVY-AMC (Bachem, Bubendorf, Switzerland) in the final volume of 50 µL assay buffer (20 mM Tris-Cl buffer containing 500 μM EDTA, pH 8.0). The fluorescence signal generated from the hydrolysis of 7-amino-4-methylcoumarine (AMC) from Suc-LLVY-AMC was monitored using a SpectraMAX M5 microplate reader (Molecular Devices, CA, USA) at the excitation/emission wavelength of 360/460 nm with cut-off filter set at 420 nm. To verify whether the signals were within the linear range, the samples were serially diluted and tested for linearity in the signals obtained. CFZ solubility and release kinetics from CFZ@NP in the buffer containing 50% FBS To gain insights into differential in vivo performance of PTX@QANP and CFZ@QANP, drug release kinetics from each core NP (i.e., PTX@NP and CFZ@NP) were compared in PBS containing 50 v/v% FBS (FBS/PBS). To establish a sink condition, the solubility of PTX and CFZ in 50% FBS/PBS was first measured. Excess PTX (0.5 mg) and CFZ (0.5 mg) were dispersed in 1 mL of 50% FBS/PBS and incubated at 37°C for 24 h with agitation. For comparison, PTX and CFZ were dispersed in 1 mL of water at room temperature and incubated for 24 h with agitation. Samples were centrifuged at 16,000 g for 20 min to separate the supernatant. PTX and CFZ dissolved in 50% FBS/PBS or water were extracted with ethyl acetate prior to HPLC analysis. Briefly, 0.5 mL of the supernatant was spiked with 10 μg of carbamazepine as an internal standard, mixed with 1.5 mL of ethyl acetate and shaken on a rotating shaker for 10 min. The mixture was then centrifuged at 16,000 g for 10 min to separate the organic phase, which was transferred to a new glass vial and dried under vacuum. The dried sample was resuspended in the mobile phase, filtered through a 0.45 µm syringe filter, and analyzed by HPLC. A calibration curve was drawn with serially diluted, known concentrations of PTX or CFZ solutions in each medium. The samples were analyzed using Agilent 1100 HPLC system (Palo Alto, CA), equipped with Ascentis C18 column (25 cm × 4.6 mm, 5 µm) and a UV detector. PTX@NP and CFZ@NP were prepared as described in the previous section.15 In brief, 20 mg of PLGA (150 kDa, LA:GA = 85:15) and 2 mg of PTX or CFZ were dissolved in 1 mL of dichloromethane, homogenized in 4 mL of 4% polyvinyl alcohol solution by probe sonication for 2 min (4 s on and 2 s off cycle at 40% amplitude), and stirred in 9

20 mL of deionized water overnight. CFZ@NP or PTX@NP were collected via ultracentrifugation at 34,000 g for 15 min and washed three times prior to lyophilization. The lyophilized samples were dissolved in 50% acetonitrile to measure the drug loading via HPLC-UV as reported previously.15,18 The drug loading content was defined as the measured drug amount divided by NP mass. PTX@NP or CFZ@NP were suspended in 50% FBS/PBS at a concentration equivalent to 13.7 µg/mL of PTX and 3.1 µg/mL of CFZ (corresponding to one-third of the solubility, respectively). PTX@NP and CFZ@NP suspensions were divided into multiple 1 mL aliquots and incubated at 37°C with constant agitation. At the pre-determined time points (up to 72 h), the aliquots were collected and centrifuged at 16,000 g for 10 min. The resulting supernatant was used to quantify the levels of PTX or CFZ after extraction with ethyl acetate and via HPLC-UV analysis. The pellet was also analyzed in the same manner as the drug loading determination to assess the recovery by quantifying the levels of PTX or CFZ remaining in the NPs. Statistical analysis The results were presented as the mean ± standard deviation (SD). Statistical significance between the groups was determined using student’s t-test or analysis of variance (ANOVA) using GraphPad Prism (GraphPad Software, version 7.0.3). A p value less than 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION Characterization of CFZ@QANP The transmission electron microscopy (TEM) images of CFZ@QANP displayed a wrinkled surface similar to the previous study (Fig. 1A).15 By dynamic light scattering (DLS) analysis, the average size and polydispersity index (PDI) of CFZ@QANP were 248.3 ± 18.7 nm and 0.19 ± 0.07, respectively. The size and PDI of the empty QANP without CFZ was 217.4 ± 2.0 nm and 0.22 ± 0.01, respectively (Fig. 1B, n=5 independently prepared batches). Although the drug loading slightly increased the particle size of CFZ@QANP, it still fell within the range where the particles can extravasate at tumors.22 The particle sizes were also assessed from the TEM images: a mean diameter of 110.6 ± 22.5 nm for CFZ@QANP and 107.8 ± 21.6 nm for the empty QANP (n=45 particles from the TEM images). These diameters based on the TEM images are smaller than those measured by DLS, with the difference exceeding the typical hydration layer of no more than a few nanometers.23 These results suggest that a mild degree of aggregation might have occurred in the medium for the DLS measurement. CFZ@QANP had a mean zeta potential of -5.7 ± 0.2 mV, less negative than the empty QANP of -12.7 ± 0.4 mV (Table 1). The reduction of negative surface charge of CFZ@QANP may be attributable to the presence of CFZ molecule on the particle surface (the morpholino functional group at the N-terminus of CFZ has a lone electron pair which may recruit protons from the buffer). For the drug content, CFZ@QANP contained 8.43 ± 2.10% (w/w), close to the targeted drug loading of 9% (calculated based on the starting amounts of PLGA (20 mg) and CFZ (2 mg)). In vitro metabolic stability and in vivo plasma pharmacokinetics of CFZ@QANP In the presence of rat liver homogenates, CFZ@QANP showed an improved metabolic stability over CFZ-CD; the remaining CFZ at 5 min were 17.4% and 10.0% for CFZ@QANP and CFZ-CD, respectively (p = 0.0045, Fig. 2A). This trend was maintained at 10 and 30 min (p < 0.0001). In the presence of whole blood, CFZ@QANP and CFZ-CD displayed comparable stability of CFZ up to 10 min (Fig. 2A). At 30 min, the remaining CFZ level was slightly higher with CFZ@QANP than with CFZ-CD (p = 0.0454, Fig. 2A). In the ICR mice that received the intravenous injection of CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg), the plasma concentrations of CFZ rapidly declined in both groups, similar to the previous reports.8,9,18 At all sampling timepoints, the plasma CFZ concentrations were higher in the mice receiving CFZ@QANP than in those receiving CFZ-CD (Fig. 2B). As such, the CFZ@QANP group displayed approximately 3-fold greater systemic exposure than the CFZ-CD group: the plasma area-under-the-curve from time 0 to infinity (AUCinf) values were 76.2 ± 5.6 and 24.9 ± 1.4 nmol⋅min/mL for CFZ@QANP and CFZ-CD, respectively (Table 2). Consequently, 11

CFZ@QANP showed a slower clearance than CFZ-CD (54.4 and 167 mL/min/kg for CFZ@QANP and CFZ-CD, respectively). The volume of distribution at steady state (Vss) in the CFZ@QANP group was smaller than that in the CFZ-CD group (1.05 and 2.60 L/kg, respectively). Together, these results support that CFZ@QANP enhanced in vivo stability of CFZ and prolonged its circulation in blood compared to CFZ-CD. Comparison of CFZ biodistribution profiles in tumors and other major organs In the syngeneic breast cancer mouse model of 4T1, E-selectin was reported to be highly expressed in peritumoral vasculature.24 Via immunohistochemical staining, we verified that E-selectin was expressed in peritumoral vasculature while P-selectin was located mainly in cancer cells with a diffuse pattern in mice carrying orthotopic 4T1 implants (3 weeks after the inoculation of 4T1 cells to the mammary fat pad of BALB/c mice) (Supplementary Fig. S1A-B). Based on these results, the orthotopic 4T1 breast cancer model was used to test the potential utility of CFZ@QANP in enhancing tumoral accumulation and anticancer efficacy. To compare the biodistribution profiles of CFZ after drug dosing, the mice carrying 4T1 orthotopic implants received a single dose of CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg) via tail vein. Whole blood, tumor implants and other major organs were collected at 0.5 and 4 h after single dosing. In tumor tissues, the CFZ@QANP group displayed approximately 3-fold greater accumulation of CFZ compared to the CFZ-CD group: at 0.5 h, 3.10 ± 1.68 vs 0.708 ± 0.749 ng CFZ/g tissue for the CFZ@QANP and CFZ-CD group, respectively (p = 0.0195). At 4 h post-dosing, the tumor tissues from the CFZ-CD group showed CFZ levels below the lower quantitation limit (less than 0.360 ng CFZ/g tissue) for all five samples. In contrast, the tumor tissues from the CFZ@QANP group had the CFZ level of 3.04 ± 0.96 ng CFZ/g tissue (Fig. 3A). Together, these results indicated an enhanced tumoral accumulation of CFZ in the CFZ@QANP group over in the CFZ-CD group. In the major organs (liver, lung and spleen) from the mice receiving CFZ@QANP or CFZ-CD, the levels of CFZ detected by LC-MS/MS were much greater than those in plasma. For the CFZ@QANP group, the substantial distribution of CFZ was found in the organs of the mononuclear phagocytic system (MPS) with the following rank order: liver > lung > spleen (Fig. 3C-E). These results were not entirely surprising as it has been commonly observed with nanoparticle formulations.25,26 On the other hand, the results of the CFZ-CD group (showing the substantial distribution of CFZ to the major organs with the extent nearly comparable to CFZ@QANP) were notable. It is currently unknown to what extent the observed biodistribution profiles of CFZ-CD are driven by CFZ itself and by the complexation with CD (currently there is no alternative (CD-free) solvent system that allows the dosing of the same CFZ dose). In vivo anticancer efficacy of CFZ@QANP 12

The mice carrying the orthotopic 4T1 implants received CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg) on two consecutive days per week, similar to the dosing regimens used in clinical settings and previous preclinical studies.5,18 Near the end of the experiment, a loss in the total body weight was noted in the mice receiving CFZ-CD, but not in those receiving CFZ@QANP (Fig. 4A). These results may suggest potential advantages of CFZ@QANP in terms of gross systemic toxicity profiles. However, when the tumor growth suppression was assessed, the groups of CFZ@QANP and CFZ-CD showed a slower tumor growth than the control group receiving the vehicle only (Fig. 4B), but with no differences between the two groups. The lack of differences was also confirmed when the tumor tissues were excised and weighed at 72 h after the last drug dosing: 1.44 ± 0.69, 1.01 ± 0.07 and 2.12 ± 0.68 g for the groups receiving CFZ@QANP, CFZ-CD and vehicle only, respectively (data not shown, p > 0.05, one-way ANOVA). In vivo proteasomal inhibition in tumors by CFZ@QANP To probe possible reasons for no observed improvement in anticancer efficacy by CFZ@QANP, the extent of proteasomal inhibition in tumors was compared between the groups of CFZ@QANP and CFZ-CD. Both groups showed only a modest degree of proteasomal inhibition in the excised tumor tissues (harvested 72 h after the last drug dosing: by 27.4% ± 11.8 and 13.8% ± 6.2 for CFZ@QANP and CFZ-CD, respectively (Fig. 5A, p > 0.05). As these results were obtained using tumor tissues collected 72 h after the last drug dosing, we wanted to examine whether that the observed partial proteasomal inhibition is due to the recovery of the proteasome activity via de novo proteasome synthesis and assembly after the last drug dosing. Using the same samples of tumor homogenates prepared for CFZ biodistribution studies (collected 0.5 or 4 h after single dosing), the extent of proteasomal inhibition was assessed. The results indicated a modest inhibition of the proteasome activity with no difference between the CFZ@QANP and CFZ-CD groups: at 0.5 h, 13.6% ± 13.5 vs 14.3% ± 17.2; at 4 h, 11.1% ± 5.8 vs 22.1% ± 12.3 for CFZ@QANP and CFZ-CD, respectively (Fig. 5B). The extent of proteasomal inhibition was similar to that of tumor tissues collected 72 h after the last drug dosing (Fig. 5A). No enhancement in tumoral proteasomal inhibition in the CFZ@QANP group is in apparent disagreement with the results showing increased tumoral CFZ levels in the CFZ@QANP group measured by LC-MS/MS (Fig. 3A). However, this is not entirely surprising based on the following reasons. First, the remaining CFZ levels (detected by LC-MS/MS) include not only the released drug, but also the drug still encapsulated in CFZ@QANP. Secondly, the extent of proteasomal inhibition (assessed by the proteasome activity assay) reflects the cumulative formation of covalent CFZ-proteasome complex up to the sampling timepoints, thus not necessarily correlating with the remaining CFZ levels at a given timepoint.

13

For comparison, we also measured the extent of proteasomal inhibition in whole blood samples of the mice that received CFZ@QANP or CFZ-CD for CFZ biodistribution studies. Although these results do not allow us to quantitatively compare drug release rates between CFZ@QANP and CFZ-CD, we can interpret that both CFZ-CD and CFZ@QANP released the sufficient amount of the active drug to inhibit the proteasome in blood during 30 min post-dosing. CFZ release kinetics from PLGA NPs in serum-containing medium Our current results using CFZ@QANP showed an improvement in PK/biodistribution profiles, but not in anticancer efficacy, different from the previous results obtained with PTX-loaded QANP. These results were surprising as the two drugs, CFZ and PTX, have similar physicochemical properties such as water solubility and partition coefficient, which are commonly used in designing formulations and estimating the hydrophobicity of compounds, thereby predicting the compatibility with hydrophobic polymeric matrix and deciding the encapsulation method (single emulsion vs. double emulsion). As both drugs have comparably poor aqueous solubility and similar logP values, we initially expected that CFZ may be encapsulated in PLGA NP (core) and released in a similar manner to PTX. To compare the drug release kinetics, we prepared the core PLGA NPs with PTX and CFZ (PTX@NP and CFZ@NP) and evaluated their drug release profiles in 50% FBS/PBS, which simulates blood protein content. The drug loading efficiency of PTX@NP and CFZ@NP was 6.8 ± 0.2% and 9.8 ± 0.1%, respectively. For poorly water-soluble drugs, the solubility limitation can be a potential confounding factor in assessing the drug release kinetics.27,28 To avoid that, the saturation solubility of PTX and CFZ in 50% FBS/PBS was measured to be 41.2 ± 3.9 µg/mL and 9.3 ± 0.2 µg/mL, respectively. PTX@NP and CFZ@NP were then suspended in 50% FBS/PBS to their concentrations equivalent to the one third of the respective solubility (13.7 μg/mL PTX equivalent and 3.1 μg/mL CFZ equivalent), satisfying sink conditions. The extent of drug release in 72 h from CFZ@NP was 19.7 ± 1.0%, much lower than 37.1 ± 2.3% from PTX@NP (Fig. 6). To check whether there is degradation or unaccounted drug loss, we measured the total recovery by measuring both the drug released in the buffer and the drug remaining in the NP fraction). The results were comparable between CFZ@NP (78.4 ± 2.0%) and PTX@NP (79.8 ± 1.2%). Overall, these results support delayed and incomplete release of CFZ from CFZ@QANP, likely due to the poor solubility of the drug in 50% FBS/PBS. These results may account for no enhancement in proteasomal inhibition by CFZ@QANP in tumors despite having an elevated CFZ level detected by LC-MS/MS over CFZ-CD. It is currently unknown what contributes to the differential solubility of CFZ and PTX in 50% FBS/PBS, but at least differing affinity for serum proteins may be ruled out as both drugs show a similar extent of plasma protein binding (88-98% for PTX29; 98% for CFZ8). 14

Although PLGA has been extensively utilized in controlled drug delivery systems, our understanding is rather limited regarding the mechanisms governing the drug release kinetics and impact descriptors for the drugs, polymers, and their interactions.30-32 At least during early time period, the drug release is likely governed by diffusion from the polymer matrix rather than polymer degradation.30 Our current results show that the drug release kinetics in vivo likely deviates from the assumption that drugs with similar solubility and lipophilicity may diffuse out of the PLGA nanoparticles with comparable kinetics and have similar benefits from the same carrier. Instead, we find that drugs with similar properties can show different release kinetics, impacting in vivo efficacy. In practice, the drug release kinetics is typically evaluated in buffered saline, for rank-ordering formulations rather than predicting in vivo release. Given that drugs with similar physicochemical properties can show different in vivo release kinetics, it is important to evaluate the release kinetics of the drug cargo in conditions simulating physiological settings, in particular for poorly water-soluble drugs.27,28 Prior to our current study, there have been other efforts to extend the therapeutic utility of CFZ by developing nanoformulations for CFZ. Polymer micelles of CFZ were produced using the block copolymers poly(ethyleneglycol) (PEG) and poly(caprolactone) (PCL); however, they showed no improvement in the PK and anticancer efficacy profiles in mice despite an enhanced metabolic stability in vitro.18,33 In this case, the lack of improvement in anticancer efficacy was attributed in part to the burst release of CFZ from the formulation in vivo. Conversely, our current study using CFZ@QANP further indicates that an incomplete, delayed drug release from nanoparticles can lead to the lack of improvement in the anticancer efficacy despite the enhancement in tumoral drug accumulation. On the other hand, our recent study using albumin-conjugated nanocrystal formulation of CFZ demonstrated an improvement in the PK and anticancer efficacy profiles in an orthotopic breast cancer mouse model without an increase in drug detectable in tumors.26 This discrepancy between the remaining drug levels in tumor tissues and target engagement activity (proteasomal inhibition) can be attributed to the unique mechanism of action of CFZ, where the drug forms covalent, irreversible complex with proteasomes (the detected CFZ levels do not include the amount that interacted with the target). Here, the remaining drug levels at a given timepoint represent both the drug released and the drug present as nanocrystal while the extent of proteasomal inhibition reflects the cumulative amount of the drug released and engaged with the proteasomal target. Together, these results illustrate that an elevated CFZ level detected at tumor tissues (biodistribution based on the total, detected drug levels) does not necessarily warrant enhancement in target engagement and improvement in anticancer efficacy, due to the release kinetics and/or covalent mode of drug-target interactions. In summary, the results in this study showed that CFZ@QANP enhanced the systemic exposure and tumoral accumulation of CFZ in vivo, yet to no improvement in anticancer efficacy compared to CFZ-CD. The lack of 15

improvement in the anticancer efficacy by CFZ@QANP is likely from delayed drug release and incomplete proteasomal inhibition in tumor tissues. Our current results differ from the marked improvement of in vivo anticancer efficacy observed with PTX@QANP, suggesting the effectiveness of QANP may well be drugdependent. However, it is not feasible to directly compare the efficacy between CFZ@QANP and PTX@QANP as the two drugs differ in terms of their mechanisms of action and dosing regimens (for PTX@QANP, every three days for 2 weeks at 20 mg/kg15; for CFZ@QANP, two consecutive days per week for three weeks at 3 mg/kg). Overall, our current results suggest the importance of considering drug release kinetics in designing novel and

effective CFZ delivery strategies.

16

Table 1. Surface charge and drug-loading efficiency of CFZ@QANP and empty QANP

CFZ@QANP

Zeta potential (mV) -5.7 ± 0.2

Drug-loading efficiency (%) 8.43 ± 2.10

Empty QANP

-12.7 ± 0.4

-

Mean ± SD (n=5 independent batches)

17

Table 2. Pharmacokinetic (PK) parameters following the intravenous administration of CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg) to ICR mice PK parameter (unit)

CFZ@QANP (n=3)

CFZ-CD (n=4)

AUCinf (nmol⋅min/mL)†

76.2 ± 5.6

24.9 ± 1.4

CL (mL/min/kg)

54.4

167

Vss (L/kg)

1.05

2.60

t1/2 (min)

221

167

MRT (min)

19.8

15.2

AUCinf, area under the concentration time curve from time 0 extrapolated to infinity; Vss, volume of distribution at steady state; t1/2, terminal half-life; MRT, mean residence time; CL, clearance. Mean ± SE. †

The PK parameters were obtained using non-compartmental methods with the sparse data option of

WinNonlin (version 8.1, Certara, Princeton, NJ, USA) and the output included SE values only for AUCinf.

18

Figure legends Figure 1. Morphology and physicochemical properties of CFZ@QANP and empty QANP. (A) Representative TEM image of CFZ@QANP and empty QANP. (B) The average size and polydispersity index (PDI) of CFZ@QANP and empty QANP. Mean ± SD (n=5 independently prepared batches) Figure 2. Enhanced in vitro and in vivo stability of CFZ@QANP over CFZ-CD. (A) The remaining CFZ levels were measured following the incubation with CFZ@QANP or CFZ-CD (at a CFZ concentration of 1 µM) in the presence of rat liver homogenates or whole blood. (B) Plasma CFZ concentration-time profiles after the intravenous administration of CFZ@QANP (n=3) or CFZ-CD (n=4) at a CFZ dose equivalent to 3 mg/kg in ICR mice. P values were from the analysis using Student’s t-test. Results are shown as mean ± SD. Figure 3. CFZ biodistribution profiles in BALB/c mice bearing orthotopic 4T1 implants after the single intravenous dosing of CFZ@QANP or CFZ-CD. Tumors and other major organs were harvested 0.5 and 4 h after single dosing (n = 5-7 per group). The levels of the remaining CFZ in tumors and other major organs were measured by LCMS/MS (representing CFZ released and encapsulated in CFZ@QANP). P values were from the analysis by Student’s t-test. Results are shown as mean ± SD. Figure 4. In vivo anticancer efficacy of CFZ@QANP in BALB/c mice bearing orthotopic 4T1 implants. The total body weight (A) and tumor growth curves (B) were monitored in the mice receiving the intravenous injections of CFZ@QANP, CFZ-CD or vehicle only on two consecutive days per week for three weeks. P values were from Twoway ANOVA followed by Tukey’s post hoc test. Results are shown as mean ± SD. Figure 5. In vivo proteasomal inhibition of CFZ@QANP in BALB/c mice bearing orthotopic 4T1 implants. (A) Tumors excised from the mice used in anticancer efficacy experiments (harvested 72 h after the last drug dosing). (B) Tumors excised from the mice used in biodistribution studies (harvested 0.5 or 4 h after a single drug dosing). The extent of proteasomal inhibition was expressed as % inhibition of proteasome activity compared to the control group that receiving vehicle only. Figure 6. In vitro drug release profiles of CFZ@NP or PTX@NP. Percentages of the released CFZ or PTX from nanoparticles were assessed in PBS containing 50% FBS for 72 h (n=3 replicates with representative batches). The results are shown as mean ± SD.

19

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22

Table 1. Surface charge and drug-loading efficiency of CFZ@QANP and empty QANP Zeta potential (mV)

Drug-loading efficiency (%)

CFZ@QANP

-5.7 ± 0.2

8.43 ± 2.10

Empty QANP

-12.7 ± 0.4

-

Mean ± SD (n=5 independent batches)

Table 2. Pharmacokinetic (PK) parameters following the intravenous administration of CFZ@QANP or CFZ-CD (CFZ dose equivalent to 3 mg/kg) to ICR mice PK parameter

CFZ@QANP (n=3)

CFZ-CD (n=4)

AUCinf (nmol×min/mL)†

76.2 ± 5.6

24.9 ± 1.4

CL (mL/min/kg)

54.4

167

Vss (L/kg)

1.05

2.60

t1/2 (min)

221

167

MRT (min)

19.8

15.2

(unit)

AUCinf, area under the concentration time curve from time 0 extrapolated to infinity; Vss, volume of distribution at steady state; t1/2, terminal half-life; MRT, mean residence time; CL, clearance. Mean ± SE. †

The PK parameters were obtained using non-compartmental methods with the sparse data option of WinNonlin

(version 8.1, Certara, Princeton, NJ, USA) and the output included SE values only for AUCinf.