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Brief Article
Analysis of Cancer-targeting Alkylphosphocholine Analog Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model Paul A. Clark, Abraham J. Al-Ahmad, Tongcheng Qian, Ray R. Zhang, Hannah K. Wilson, Jamey P. Weichert, Sean P. Palecek, John S. Kuo, and Eric V. Shusta Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00441 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016
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Molecular Pharmaceutics
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Analysis of Cancer-targeting Alkylphosphocholine Analog Permeability Characteristics
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Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model
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Paul A. Clark†, , Abraham J. Al-Ahmad∥, , Tongcheng Qian⊥, Ray R. Zhang†,¶, Hannah K.
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Wilson⊥, Jamey P. Weichert¶, , Sean P. Palecek⊥, John S. Kuo*,#,†, Eric V. Shusta*,#,⊥
‡
‡
§
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Departments of †Neurological Surgery, ⊥Chemical and Biological Engineering, and ¶Radiology,
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University of Wisconsin-Madison, Madison, WI 53792, United States
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§: Cellectar Biosciences, Madison WI 53716, United States
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∥: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center –
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School of Pharmacy, Amarillo, TX 79106, United States
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‡: these authors contributed equally to this work
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#: co-corresponding authors
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Corresponding Authors
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*Eric V. Shusta; tel: (608) 265-5103; e-mail:
[email protected] 16
*John S. Kuo; tel: (608) 262-7303; e-mail:
[email protected] 17 18 19
Conflict of interest disclosure: Jamey P. Weichert is the founder and shareholder in Cellectar
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Biosciences that provided the APC analogs used in this study. All other authors declare no
21
competing financial interest.
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Table of Contents Graphic
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ABSTRACT
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Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for
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diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC
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analogs derived from chemically-synthesized phospholipid ethers were identified and optimized
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for cancer-targeting specificity using extensive structure-activity studies. While they strongly
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label human brain cancers associated with disrupted blood-brain barriers (BBB), APC
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permeability across intact BBB remains unknown. Three of our APC analogs, CLR1404 (PET
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radiotracer), CLR1501 (green fluorescence), and CLR1502 (near infrared fluorescence), were
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tested for permeability across a BBB model composed of human induced pluripotent stem cell-
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derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system
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has reproducibly consistent high barrier integrity marked by high transendothelial electrical
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resistance (TEER>1500 Ω-cm2) and functional expression of drug efflux transporters. Our
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radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the
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iPSC-BMEC (35±5.7 (CLR1404), 54±3.2 (CLR1501), and 26±4.9 (CLR1502) x10-5 cm/min)
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compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29).
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Only our fluorescent APC analogs (CLR1501, CLR1502) underwent BCRP and MRP polarized
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drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be
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specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared
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to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact
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BBB into normal brain likely contributes to the high tumor to background ratios observed in initial
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human trials. Moreover, addition of fluorescent moieties to APCs resulted in greater BMEC
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efflux via MRP and BCRP, and may affect fluorescence-guided applications. Overall, the
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characterization of APC analog permeability across human BBB is significant for advancing
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future brain tumor-targeted applications of these agents.
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Keywords: alkylphosphocholine analogs, blood-brain barrier, brain microvascular endothelial
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cells, induced pluripotent stem cells
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ABBREVIATIONS
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GBM, glioblastoma multiforme; BBB, blood-brain barrier; APC, alkylphosphocholine; PSC,
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pluripotent stem cell; iPSC, induced PSC; BMEC, brain microvascular endothelial cell; P-gp, P-
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glycoprotein; MRP, multidrug resistance protein; BCRP, breast cancer resistance protein;
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TEER, trans-endothelial electrical resistance; PLE, phospholipid ether
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INTRODUCTION
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Efficient treatment for central nervous system (CNS) tumors is a large unmet clinical need.
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Approximately 25,000 new primary malignant brain tumors are annually diagnosed in the United
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States, accounting for 1.4% of all cancer-related deaths and the second leading cause of death
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in pediatric cancers1, 2. Glioblastoma (GBM) is the most frequently diagnosed primary adult brain
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tumor that confers a median survival of less than two years despite maximal surgery,
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temozolomide chemotherapy, and radiation3. Furthermore, brain metastases from other cancer
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types such as melanoma, breast, and lung are approximately ten times more prevalent than
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primary CNS tumors in adults, and often herald advanced cancer stages due to limited effective
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treatments 4. Chemotherapeutic delivery to brain neoplasms is hampered by the presence of the
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blood-brain barrier (BBB), formed by specialized brain microvascular endothelial cells (BMECs)
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and surrounded by other cells of the neurovascular unit including astrocytes, pericytes, and
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neurons 5, 6. At the BBB, BMECs incorporating specific tight junction proteins form a “tight”
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barrier defined by high trans-endothelial electrical resistance (TEER)5, 6 and low paracellular
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permeability to water and hydrophilic solutes. Additionally, the physical barrier function is
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augmented by the presence of an active functional barrier due to multiple types of drug efflux
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pumps including breast cancer resistance protein (BCRP), multidrug resistance proteins (MRPs)
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and p-glycoprotein (P-gp) at the BMEC interface that limit diffusion of small lipophilic molecules
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into the CNS 5, 7, 8.
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Historically, the blood-brain tumor vasculature is described as ‘leaky’ due to immature or
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disrupted BBB vasculature9-13. However, recent studies demonstrated intra- and inter-tumoral
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heterogeneity of BBB properties both in primary gliomas13 and metastases11, with different
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tumor regions exhibiting variable and sometimes highly restricted drug permeability13-16.
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Additionally, gliomas notoriously infiltrate normal brain parenchyma with intact BBB, making
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complete surgical resection very difficult and highlighting the need for more effective
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chemotherapeutic agents or delivery approaches that cross intact BBB13.
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Cancer-targeting alkylphosphocholine (APC) analogs were recently described as tumor-specific
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labels for a broad range of cancer types including gliomas and brain metastases in cultured cell
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lines, preclinical models, and human cancer patients17, 18. The observation that naturally
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occurring phospholipid ethers (PLEs) selectively accumulate in human cancer cells compared to
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normal tissue 19-21 prompted our group to examine structural activity relationships for
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radioiodinated aryl PLEs and a subset of APC derivatives to determine how their molecular
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structures affected tumor retention. From these studies, we found that the glycerol backbone
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was not required for tumor avidity in glycerol-derived PLE analogs, the alkyl chain must contain
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>11 methylene groups, and the position of iodine on the phenyl ring did not influence tumor
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uptake or specificity 22-25. Ultimately, the APC molecular backbone named CLR1404 [18-(p-
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iodophenyl) octadecylphosphocholine] (Figure 1A) was identified as the best cancer-targeting
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APC analog exhibiting both tumor-specific uptake and long-term retention. CLR1404 can be
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labeled with iodine radioisotopes for positron emission tomography (PET) imaging (124I isotope)
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or radiotherapy (131I or 125I isotopes)17. For this APC analog, the iodine can also be replaced with
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fluorescent moieties (green fluorescent CLR1501 or near infrared CLR1502) to yield similar
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tumor selectivity and retention, enabling potential intra-operative applications to improve tumor
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cell visualization and surgical resection18. All APC analogs exhibit particularly high tumor to
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normal brain ratios in vivo17, 18 that is promising for brain tumor applications. However, BBB
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permeability properties of APC analogs are currently unknown.
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In the current study, we used our recently described human induced pluripotent stem cell (iPSC)
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derived BMECs as an in vitro BBB model26, 27 to analyze and characterize the BBB permeability
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profiles of our APC analogs. The iPSC-derived BMECs exhibit well-developed BBB-BMEC tight
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junctions and a correspondingly high TEER with functionally polarized drug efflux transporters.
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Together, these properties have yielded in vitro permeability measurements for a cohort of small
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molecules that correlate to in vivo observations 26, 27. Permeability measurements with the APC
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analogs indicated a similar intermediate permeability across the in vitro BBB for all compounds;
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whereas efflux transporter activity was only observed for fluorescent APC analogs. These
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results suggest that limited BBB permeability of APC analogs may contribute to low APC uptake
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and retention in the normal brain parenchyma observed clinically, and that moieties added to
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the APC backbone can significantly affect BBB permeability.
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MATERIALS AND METHODS
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Chemicals
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The APC analogs CLR1404, CLR1501 and CLR1502 were provided by Cellectar Biosciences,
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Inc. (Madison, WI, USA). These APC analogs consist of a cancer-targeting alkylphosphocholine
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scaffold with an attached cancer imaging, visualization or therapeutic moiety (refs 17, 18, 25, 28 and
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Figure 1): iodine isotopes for CLR1404; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (i.e.
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BODIPY, green fluorescence) for CLR1501; and 2-[2-[2-Chloro-3-[2-(1,3-dihydro-1,3,3-trimethyl-
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2H-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,3,3-trimethyl-3H-indolium chloride
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(i.e. IR-775, near infrared fluorescence) for CLR1502. Pharmacological cellular efflux
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transporter inhibitors were purchased: PSC833 and Ko143 from Tocris Biosciences, and MK571
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from Sigma-Aldrich (St. Louis, MO, USA). [14C]-sucrose and [3H]-diazepam were purchased
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from American Radiolabeled Chemicals (St Louis, MO, USA).
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LogD determination
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For APC analogs CLR1404 (MW=637.6 g/mol), CLR1501 (MW=701.7 g/mol), and CLR1502
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(MW=994.8 g/mol), the SMILES sequences were determined using ChemDraw Pro 13.0
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(Perkin-Elmer, Waltham, MA), and calculated LogD (cLogD) was calculated using the ALOGPs
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2.1 algorithm29, 30. Experimental LogD (pH 7.4) values were obtained by adding 124I-CLR1404,
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CLR1501, and CLR1502 to octanol followed by equilibration overnight. Equal volumes of buffer
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(pH 7.4) were added and the mixture was rotated at room temperature shielded from light for 48
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hrs. The octanol and water phases were separated, and then analyzed using the gamma
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counter (Wizard2 Automatic Gamma Counter, Perkin Elmer, Waltham, MA) for 124I-CLR1404 or
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Safire II (Tecan Group Ltd, Männedorf, Switzerland) for CLR1501 and CLR1502 to determine
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relative concentrations. Finally, the octanol:water partition coefficients were calculated31. LogD
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values for diazepam and sucrose were obtained from the literature32.
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Differentiation of iPSC into BMEC
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BMECs were differentiated from iPSCs following protocols previously established by our
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group26, 27. IMR90-c4 iPSC cell line33 was purchased from WiCell (Madison, WI) and passaged
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on 6-well tissue culture plates (Corning, Corning, NY) coated with 84 µg/mL of growth-factor
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reduced Matrigel® (BD Biosciences) and grown in mTeSR1 (Stem Cell Technologies,
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Vancouver, BC). Cells were fed on a daily basis. After 3-4 days in mTeSR medium, cells were
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switched to a unconditioned medium (UM): 50:50 Dulbecco’s Modified Eagle Medium: F12
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(Gibco, Life Technologies, Carlsbad, CA), 15mM HEPES (Sigma-Aldrich, St Louis, MO), 20%
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knockout serum replacement (Life Technologies), 1% non-essential amino acids (Life
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Technologies), 0.5 mM GlutaMax (Life Technologies) and 0.1mM β-mercaptoethanol (Sigma-
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Aldrich). Cell medium was changed daily for 6 consecutive days. The BMEC population was
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further enriched by replacing the incubation medium with endothelial cell (EC) medium: human
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endothelial cell serum-free medium (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF,
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R&D Systems, Minneapolis, MN), 1% platelet-poor derived bovine serum (PDS, Biotechnologies
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Inc, Boston, MA) and 10 µM retinoic acid (RA) for 48 hours. Following endothelial cell
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enrichment, cells were dissociated using Accutase (Stempro, Life Technologies) for 30 minutes
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and seeded onto Transwell® polycarbonate inserts (1.12cm2 surface area) coated with 0.4
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mg/mL human collagen from human placenta (Sigma) and 0.1 mg/mL bovine fibronectin
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(Sigma). After allowing 24 hr attachment, medium was removed from the insert and replaced
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with EC medium without bFGF and RA. Experiments were carried out 48 hours after plating
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transwell monolayers.
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Differentiation of iPSCs to BMECs was verified using immunocytochemistry, as previously
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described26, 27. Briefly, cells were fixed in either 100% ice-cold methanol or 4%
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paraformaldehyde, rinsed with PBS, and blocked for 30 min with either 10 or 40% goat serum
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(Sigma) in PBS. Particularly for p-glycoprotein and MRP1 immunolabeling, it is important to note
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that methanol-fixed cells are permeabilized during fixation. Cells were incubated with primary
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antibody diluted in 10% or 40% goat serum overnight at 4°C, rinsed, and then incubated with
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goat anti-mouse or anti-rabbit Alexa Fluor 488 (1:200; Life Technologies) in 10 or 40% goat
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serum. Cell nuclei were counterstained with 4′,6-Diamidino-2-pheny-lindoldihydrochloride (DAPI;
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Sigma). Primary antibodies used: PECAM-1 (Thermo Fisher, polyclonal, 1:25), Claudin-5
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(Invitrogen, clone 4C3C2, 1:50), Occludin (Invitrogen, clone OC-3F10, 1:200), ZO-1 (Invitrogen,
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polyclonal, 1:100), VE-Cadherin (Santa Cruz Biotechnology, clone F-8, 1:25), BCRP (Millipore,
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clone 5D3, 1:50), MRP1 (Millipore, clone QCRL-1, 1:25), and p-glycoprotein (Neomarkers, clone
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F4, 1:25). Mouse IgG (BD Biosciences, clone HOPC-1) was used as immunolabeling isotype
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control.
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APC analog permeability studies
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BMEC transwell monolayer tightness was assessed by measuring transendothelial electrical
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resistance (TEER) using a chopstick electrode EVOM2 system (World Precision Instruments,
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Sarasota, FL), and high tightness (>1500 Ω-cm2) verified prior to permeability experiments. EC
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medium was replaced with PM20 medium (50:50 DMEM:F12+B27 supplement+1%
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antibiotics+20 ng/ml each of EGF and bFGF) and allowed to equilibrate with the monolayers for
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60 minutes, and TEER measured again. APC analogs or control compounds ([14C]-sucrose and
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[3H]-diazepam) were then added to PM20 medium and allowed to diffuse across the monolayers
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for 2 hours. At the end of the experiment, barrier integrity was assessed by TEER to verify no
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significant decrease for experimental duration. Control compounds [14C]-sucrose and [3H]-
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diazepam were diluted in PM20 medium at a dilution of 0.4 µCi/mL. For absolute permeability
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analyses, each APC analog was diluted to 1 µM in PM20. For comparative analyses (i.e.
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temperature, polarity, and inhibitor experiments), the following APC analog concentrations were
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used that optimized equipment and compound sensitivity and reproducibility: CLR1404 at 5-10
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µCi/mL (average 0.3 µM), CLR1501 at 10 µg/mL (14 µM), and CLR1502 at 1 µg/mL (1.0 µM). At
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30, 60, 90 and 120 minutes interval, an aliquot of 100 µL from the bottom chamber was
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collected and replaced by an equal volume of fresh PM20 medium. Fluorescence of CLR1501
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and CLR1502 was assessed using a Safire II (Tecan Group Ltd, Männedorf, Switzerland).
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Radioactivity was measured using a TRICARB-PACKARD β-counter (Perkin-Elmer, Waltham,
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MA) or gamma-counter (Wizard2 Automatic Gamma Counter, Perkin Elmer, Waltham, MA).
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Permeability values were calculated as previously described34. Clearance volumes were plotted
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as a function of time and linear regression used to determine a clearance rate denoted PSt for
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iPSC-derived BMEC coated filters and PSf for cell-free collagen-fibronectin coated filters. The
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PS value ascribed to the BMEC monolayer denoted PSe was subsequently calculated using
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1/PSe=(1/Psf)-(1/PSt). Finally, the permeability, Pe (cm/min), was calculated by dividing by the
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surface area, S, of the filter Pe=PSe/S.
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Inhibition studies
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Pharmacological inhibition experiments were carried out by pre-incubating BMEC monolayers
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for 60 mins with 1 µM PSC833 (P-gp inhibitor), 1 µM Ko143 (BCRP inhibitor) or 10 µM MK571
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(pan-MRP inhibitor) in EC medium. Inhibition was maintained during the permeability
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experiments by adding these inhibitors at the same concentration in the PM20 medium.
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Otherwise permeability experiments were performed exactly as described above.
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Statistical analysis
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All data in this study were expressed as mean ± s.e.m. of at least three independent
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experiments. For comparison of two groups, a two-tailed unpaired Student’s t-test was used.
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With multiple experimental groups, a normal data distribution was verified and then analysis of
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variance (ANOVA) performed to determine statistical differences. Post-hoc comparisons were
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performed upon ANOVA significance to look for specific differences between groups: Tukey test
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if equal sample sizes and similar population variance or Games-Howell if unequal sample sizes
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and high population variance35. Statistical significance was defined as p
