Article Cite This: J. Med. Chem. 2018, 61, 9637−9646
pubs.acs.org/jmc
Evaluation of Novel Tumor-Targeted Near-Infrared Probe for Fluorescence-Guided Surgery of Cancer Sakkarapalayam M. Mahalingam,†,∥ Sumith A. Kularatne,*,‡,∥ Carrie H. Myers,‡ Pravin Gagare,‡ Mohammad Norshi,‡ Xin Liu,† Sunil Singhal,§ and Philip S. Low*,† †
Purdue University Institute for Drug Discovery, 720 Clinic Drive, West Lafayette, Indiana 47907, United States On Target Laboratories, 1281 Win Hentschel Blvd, West Lafayette, Indiana 47906, United States § Division of Thoracic Surgery, Department of Surgery, University of Pennsylvania, 3400 Spruce Street, 6 White Building, Philadelphia, Pennsylvania 19104, United States J. Med. Chem. 2018.61:9637-9646. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 11/10/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Because the most reliable therapy for cancer involves quantitative resection of all diseased tissue, considerable effort has been devoted to improving a surgeon’s ability to locate and remove all malignant lesions. With the aid of improved optical imaging equipment, we and others have focused on developing tumor-targeted fluorescent dyes to selectively illuminate cancer nodules during surgery. We describe here the design, synthesis, optical properties, in vitro and in vivo tumor specificity/ affinity, pharmacokinetics, preclinical toxicology, and some clinical application of a folate receptor (FR)-targeted NIR dye (OTL38) that concentrates specifically in cancer tissues and clears rapidly from healthy tissues. We demonstrate that OTL38 binds FR-expressing cells with ∼1 nM affinity and eliminates from receptor negative tissues with a half-time of 300× the expected dose to be used in humans, (ii) OTL38 could be reproducibly synthesized under GMP conditions at >98% purity and >98% chemical yield, (iii) none of the residual byproducts were toxic, and (iv) the final drug product was stable for >2 years at 4 °C (see SI Figure 6). Supported by these data, successful phase 1 and/or phase 2 studies were subsequently completed on ovarian 15 and lung cancers,17,18,20,22,23 with investigator-sponsored trials on other cancers showing similar positive results.16,19,21 Representative data from one patient with nonsmall cell lung cancer 2 h following intravenous infusion of 0.025 mg/kg of OTL38 are shown in Figure 5g. The observed TBR in humans (∼4:1) was similar to TBR in the murine tumor models. Moreover, as described in the clinical trial reports,23 OTL38 was also found to repeatedly reveal additional cancer nodules that could neither be observed in a preoperative CT scan nor detected during surgery. Based on these and similar data on other
■
DISCUSSION
The objective of this study was to develop a real time fluorescent imaging agent that could enable surgeons to identify occult tumor nodules and eliminate positive tumor margins that would otherwise remain inside a cancer patient following surgery. In OTL38, we have designed, synthesized, and tested an FR-targeted NIR imaging agent that (i) demonstrates high affinity and specificity for FR+ tumors, (ii) clears rapidly from FR-negative tissues, allowing for intraoperative imaging within 2 h of infusion, (iii) is stable for days following uptake by solid tumors, (iv) is readily synthesized in multigram quantities at high purity and excellent yield, (v) is stable for long periods during synthesis and storage, (vi) enables more accurate identification of positive tumor margins,15−23 (vii) allows more accurate staging of some cancer patients,23 and (viii) facilitates localization and resection of otherwise undetectable cancer nodules. Since quantitative removal of all malignant disease constitutes the most reliable treatment for cancer, use of OTL38 to facilitate cancer removal has the potential to save lives. 9642
DOI: 10.1021/acs.jmedchem.8b01115 J. Med. Chem. 2018, 61, 9637−9646
Journal of Medicinal Chemistry
Article
imaging agent that can enable surgeons to locate and resect more cancer tissue and thereby prolong patient survival.
It is important to recognize that OTL38 is not the only fluorescent dye currently used for intraoperative imaging during cancer debulking surgeries. 5-Aminolevulonic acid has been employed for years to illuminate brain and other cancers, owing to the fact it is converted by some cancers (e.g., glioblastomas) into a porphyrin that fluoresces in the visible range.2 While the resulting fluorescence allows facile differentiation of exposed malignant from healthy brain tissues, the dye is unfortunately compromised by the fact that its fluorescence does not penetrate solid tissues, thereby allowing buried cancer lesions to escape detection. Similarly, EC17, a folate-targeted derivative of fluorescein has been shown in clinical trials to be highly efficient in revealing exposed FR+ cancer lesions (X, Y, Z), even down to the single cell level.11−14 However, due to its similar emission in the visible wavelength range, EC17 is likewise unable to illuminate buried tumor nodules.14 While ICG, because of its NIR excitation and emission, constitutes the first fluorescent dye to image buried tumor nodules, it unfortunately suffers from a lack of tumor specificity and, as a consequence, is more commonly employed for assessing tissue perfusion and locating bile ducts during surgery.2,6,7 In an attempt to address these limitations, multiple innovative strategies to induce tumor fluorescence have been explored. A variety of quenchable NIR dyes, for example, have been developed that become fluorescent only upon release of a quenching agent by a tumor-specific enzyme,33,34 tumorassociated decrease in pH,35,36 or cancer-dependent change in redox potential.37 While several of these activatable dyes show considerable promise, others are compromised by low tumor specificity, a slow rate of fluorescence activation, or highly variable activating conditions, even at various locations within the same tumor. A second class of tumor imaging agents exploits the established tumor specificity of certain antibodies to carry attached NIR dyes selectively to tumor nodules.2 While the specificities of these dyes are largely very good, they commonly suffer from slow rates of clearance from receptornegative tissues, requiring the antibody-dye conjugates to be injected several days prior to surgery and thereby mandating a second patient visit to the hospital. To incorporate the tumor specificities of antibodies into a low molecular weight tumortargeted NIR dye, we have designed OTL38 that exploits the overexpression of FR in many tumor types by using folic acid (MR ≈ 441) to deliver a brightly fluorescent NIR dye selectively to cancer tissues. Because of its high specificity for FR-expressing tumors and rapid clearance from FR-negative healthy tissues, OTL38 has proven capable of yielding sharp contrast images of cancer nodules within 1−3 h of infusion. With its receipt of Fast Track Status from the FDA and its recent entry into phase 3 clinical trials, OTL38 may become the first ligand-targeted NIR dye to document the value of fluorescence-guided resection of cancer tissues in humans.
■
EXPERIMENTAL SECTION
Materials. N10-Trifluoroacetylpteroic acid [Pte-N10-(TFA)-OH] was purchased from Irvine Chemistry Lab (Anaheim, CA). O-t-ButylL-tyrosine t-butyl ester hydrochloride and O-(7-azabenzo-triazol-1yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) were obtained from Chem-Impex Int (Chicago, IL). All other chemicals, cell culture materials, and animal supplies were obtained from major suppliers. General Methods. Compounds were purified by preparative reverse phase (RP)-HPLC (Waters, xTerra C18 10 μm; 19 × 250 mm) and analyzed UPLC (Acquity, BEH C18, 1.7 μm, 2.1 × 50 mm). Purity of the isolated compounds were analyzed by analytical HPLC using either achiral column (XSelect CHC PFP C18, 2.5 μm, 4.6 × 150 mm) and/or chiral column [Chiralpak ZWIX(+) 3 μm, 3.0 × 150 mm] with solvent gradient of 3% B to 30% B in 30 min with run time of 45 min run (where A = 30 mM ammonium acetate and B = acetonitrile) unless otherwise noted. All the isolated compounds demonstrated ≥95% purity at λ = 270 and/or 770 nm in the aforementioned analytical HPLC method. 1H and 13C spectra were acquired with a Bruker 500 and 125 MHz NMR spectrometer equipped with a TXI cryoprobe. Samples were run in 5 mm NMR tubes. Presaturation was used to reduce the intensity of the residual H2O peak. All 1H signals are recorded in ppm with reference to residual DMSO (2.50 ppm), and data are reported as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, and b = broad, with coupling constants in Hz. LC/MS analyses were obtained using a Waters micromass ZQ 4000 mass spectrometer coupled with a UV diode array detector. High resolution mass spectrometric results were obtained by ESI mass using an Applied Biosystems (Framingham, MA) Voyager DE PRO mass spectrometer. Synthesis of Pteroyl-N10(TFA)-Tyr(OtBu)-OtBu (2). To a solution of N10-trifluoroacetylpteroic acid (12.0 g, 29.40 mmol, 1 equiv), HATU (13.45 g, 35.28 mmol, 1.2 equiv), and NH2-LTyr(OtBu)-OtBu.HCl (11.63 g, 35.28 mmol, 1.2 equiv) in anhydrous DMF (147 mL) at 23 °C under argon, DIPEA (20.48 mL, 117.62 mmol, 4.0 equiv) was added slowly over a period of 10 min. While progress of the reaction was monitored using LC/MS, the reaction mixture was stirred at 23 °C under argon for 2 h. The reaction mixture was cannulated as a steady stream to a stirred solution of 0.1 N aq. HCl (2.0 L, 0.28 M) over the period of 30 min to give pale yellow precipitate of compound 2. The precipitate was filtered using sintered funnel under aspirator vacuum and washed with water (8 × 300 mL) until the pH of the filtrate was between 3 and 4. The wet solid was allowed to dry under high vacuum for 12 h to obtain compound 2 (16.24 g, 96.7%). Analytical UPLC: Rt = 3.78 min [solvent gradient: 0% B to 50% B in 5 min]. UV: 231, 275, 345 nm. 1 H-NMR (500 MHz, DMSO-d6/D2O) δ 8.65 (s, 1H, Pyr−CH), 7.81−7.77 (m, 2H, Pte−Ar−CH), 7.59 (d, J = 8.1 Hz, 2H, Pte−Ar− CH), 7.19−7.13 (m, 2H, Tyr−Ar−CH), 6.88−6.81 (m, 2H, Tyr− Ar−CH), 5.12 (s, 2H, Pte-CH2), 4.49 (dd, J = 8.9, 6.8 Hz, 1H, TyrαCH), 3.01 (t, J = 7.4 Hz, 2H, Tyr-CH2), 1.30 (s, 9H, tBu-CH3), 1.20 (s, 9H, tBu-CH3). 13C-NMR (125 MHz, DMSO-d6/D2O) δ 170.88, 165.66, 160.13, 155.92, 155.63, 153.61, 153.38, 149.19, 145.37, 141.67, 134.48, 132.26, 130.11, 129.76, 128.78, 128.47, 128.20, 123.68, 117.34, 115.03, 80.81, 77.82, 54.98, 53.87, 40.09, 40.00, 39.93, 39.83, 39.76, 39.67, 39.50, 39.33, 39.17, 39.00, 35.97, 28.57, 27.66. HRMS (ESI) calcd for C33H36F3N7O6 [M + H]+ m/z 684.2758; found: m/z 684.2775. Synthesis of Pteroyl-N10(TFA)-Tyr(OH)-CO2H (3). To solid material (15 g, 21.95 mmol, 1 equiv), a solution of TFA/TIPS/H2O (95:2.5:2.5, 200 mL) was added. The reaction content was stirred at 23 °C for 1 h and was monitored by LC/MS. Upon completion product formation, the reaction mixture was cannulated as a steady stream to stirred methyl tert-butyl ether (MTBE, 1.8 L) at 23 °C to give pale yellow precipitate of compound 3. The precipitate was filtered using sintered funnel under aspirator vacuum, washed with
■
CONCLUSIONS OTL38 is an easily synthesized folate receptor-targeted dye that fluoresces brightly in the near-infrared where human tissues are largely translucent. Because OTL38 exhibits high affinity and specificity for FR+ tumors and clears rapidly from receptor-negative tissues, its fluorescence can be used to locate malignant lesions within a couple of hours of intravenous injection. Thus, when used in combination with an NIR camera, OTL38 constitutes an excellent tumor-specific optical 9643
DOI: 10.1021/acs.jmedchem.8b01115 J. Med. Chem. 2018, 61, 9637−9646
Journal of Medicinal Chemistry
Article
MTBE (6 × 300 mL), and dried under high vacuum for 8 h to obtain 3 (11.94 g, 95.29%) as a pale yellow solid. Analytical UPLC: Rt = 2.36 min [solvent gradient: 0% B to 50% B in 5 min]. UV: 225, 275, 350 nm. 1H-NMR (500 MHz, DMSO-d6/D2O) δ 8.58 (s, 1H, Pyr−CH), 7.76 (d, J = 8.2 Hz, 2H, Pte−Ar−CH), 7.56 (d, J = 8.2 Hz, 2H, Pte− Ar−CH), 7.02 (d, J = 8.3 Hz, 2H, Tyr−Ar−CH), 6.62−6.55 (m, 2H, Tyr−Ar−CH), 5.13−5.02 (m, 2H, Pte-CH2), 4.35 (dd, J = 9.5, 4.3 Hz, 1H, Tyr-αCH), 3.07 (dd, J = 13.8, 4.3 Hz, 1H, Tyr-CHH), 2.87 (dd, J = 13.8, 9.4 Hz, 1H, Tyr−CHH). 13C-NMR (125 MHz, DMSOd6/D2O) δ 174.48, 164.86, 161.30, 156.60, 155.84, 155.44, 154.70, 149.26, 143.87, 141.33, 135.28, 130.08, 129.15, 128.78, 128.19, 128.13, 117.33, 115.03, 114.81, 55.81, 53.90, 36.27. HRMS (ESI) calcd for C25H20F3N7O6 [M + H]+ m/z 572.1506; found: m/z 572.1514. Synthesis of Pteroyl-Tyr-S0456 (OTL38, 4). Pteroyl-N10(TFA)Tyr(OH)-CO2H (13.85 g, 22.78 mmol, 1 equiv) was dissolved in water (95 mL) at 23 °C, and pH of the solution was increased to ca. 9.5 by adding aqueous 3.75 M NaOH (26.12 mL, 97.96 mmol, 4.30 equiv) dropwise to give a clear pale yellow solution. Formation of trianion was monitored by observing TFA deprotected Pteroyl-Tyr using LC/MS (m/z 476), and pH of the solution was maintained at 9.5. To a solution of S0456 (20.63 g, 21.64 mmol, 0.950 equiv) in water (180 mL) at 23 °C, a solution of Pteroyl-Tyr trianion at pH 9.5 was added dropwise. The temperature of the reaction mixture was increased to 90 °C, stirred at 90 °C for 45 min, and monitored formation of 4 by LC/MS. Upon completion of product formation, the reaction mixture was cooled to room temperature and transferred via cannula as a steady stream to a stirred acetone (5.5 L) to give green precipitate. The precipitated was filtered under aspirator vacuum on sintered funnel washed with acetone (3 × 500 mL). The green powdery solid was dried under high vacuum for 12 h to obtain 4 (31 g) quantitatively. While purity of the precipitated 4 was 92.8%, the crude material was further purified using prep-HPLC. The purity of OTL38 at wavelength 270 nm was ≥98%, and at wavelength 770 nm, ≥99% (SI Figure 8a). The chiral purity of OTL38 at 770 nm was ≥98% (SI Figure 8b). Analytical UPLC: Rt = 2.33 min [solvent gradient: 0% B to 50% B in 5 min]. UV: 225, 275, 350 nm. 1H NMR (500 MHz, DMSO-d6/D2O) δ 8.27 (s, 1H), 7.72 (d, J = 13.9 Hz, 2H), 7.66−7.56 (m, 4H), 7.41 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.2 Hz, 2H), 6.53 (d, J = 8.3 Hz, 2H), 6.15 (d, J = 14.1 Hz, 2H), 4.42−4.29 (m, 3H), 4.08 (t, J = 7.2 Hz, 4H), 3.17−3.03 (m, 2H), 2.96 (dd, J = 13.8, 8.9 Hz, 1H), 2.75−2.58 (m, 5H), 2.55 (dd, J = 13.4, 6.5 Hz, 5H), 1.88 (s, 4H), 1.80−1.65 (m, 8H), 1.10 (d, J = 14.3 Hz, 12H). 13C NMR (125 MHz, DMSO-d6/D2O) δ 174.00, 171.64, 165.44, 163.11, 161.57, 157.90, 153.85, 150.54, 149.08, 148.41, 144.54, 142.31, 140.97, 140.40, 132.68, 131.02, 128.49, 127.73, 126.28, 122.39, 119.64, 113.98, 111.22, 110.54, 100.66, 54.43, 50.75, 48.49, 45.84, 43.75, 35.86, 27.25, 27.09, 25.95, 23.79, 22.49, 21.30, 20.83. HRMS (ESI) calcd for C61H67N9O17S4 [M + H]+ m/z 1326.3616; found: m/z 1326.3660. Cell Culture. KB cells (a derivative of HeLa/human cervical cancer cell line that expresses ∼2 × 106 folate receptors/cell), FaDu (a human head and neck cancer cell line), and A549 cells (a alveolar basal epithelial carcinoma cell line) were obtained from ATCC (Rockville, MD) and grown as a monolayer using folate free or normal 1640 rpmI-1640 medium (Gibco, NY) containing 10% heatinactivated fetal bovine serum (Atlanta Biological, GA) and 1% penicillin−streptomycin (Gibco, NY) in a 5% carbon dioxide/95% air-humidified atmosphere at 37 °C for at least six passages before they were used for the assays. Animals. Athymic female nude (nu/nu) and Balb/C mice (5 weeks old, 18−20 g) were purchased from Invigo (Indianapolis, IN) and maintained on gamma-irradiated folate-deficient special diet (Teklad, WI) for at least 2 weeks before the start of the study. Animals were housed 5/cage in a barrier, pathogen-free cloaked rack. Autoclaved tap water and food were given as needed. The animals were housed in a sterile environment on a standard 12 h light−dark cycle for the duration of the study. All animal procedures were approved by Purdue Animal Care and Use Committee. Animal care
and studies were performed according to national and international guidelines for the humane treatment of animals. Animal imaging experiments were then performed using a Caliper IVIS Lumina II Imaging Station with Living Image 4.0 software (PerkinElmer Inc., MA) using imager parameters as ex = 745 nm, em = ICG, and exposure time = 1 s. ROI calculations were conducted using Living Image 4.0 software. Human Subjects. Nonsmall lung cell cancer and ovarian cancer patient images used in this article were acquired under approved clinical trials by the University of Pennsylvania or Leiden University Medical Center Institutional Review Board. In Vitro Binding. KB or A549 cells were seeded in 24-well (100,000 cells/well) Falcon plates (BD Biosciences, CA) and allowed to form monolayers over a period of 12 h. Spent medium in each well was combined with 10 nM of [3H]-folate in the presence of increasing concentration (0.1 nM to 1 μM) of the OTL38 or FA (Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 h at 37 °C, cells were rinsed with PBS (3 × 0.5 mL, Gibco, NY) to remove any unbound radioactive materials. After adding 0.25 M sodium hydroxide (0.5 mL) and incubating for 12 h at 4 °C, cells were transferred into individual scintillation vials containing Ecolite scintillation cocktail (3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillation analyzer (Packard). The relative binding affinities were calculated using a plot of present cell bound radioactivity versus the log concentration of the test article using GraphPad Prism 6. Fluorescence Microscopy. KB or A549 cells (50,000 cells/well in 1 mL) were seeded into poly-D-lysine microwell Petri dishes and allowed cells to form monolayers over 12 h. Spent medium was replaced with fresh medium containing OTL38 (100 nM) in the presence or absence of 100-fold excess FA, and cells were incubated for 1 h at 37 °C. After rinsing with fresh medium (2 × 1.0 mL) and PBS (1× 1.0 mL), fluorescence images were acquired using an epifluorescence microscopy. Whole Body Imaging and Tissue Biodistribution. Xenograft model: Seven-week-old female nu/nu or Balb/c mice were inoculated subcutaneously with 1.0 × 106 KB, FaDu, or A549 cells/mouse in RPMI1640 medium on the shoulder or neck. Growth of the tumors was measured in perpendicular directions every 2 days using a caliper (body weights were monitored on the same schedule), and the volumes of the tumors were calculated as 0.5 × L × W2 (L = longest axis and W = axis perpendicular to L in millimeters). Once tumors reached approximately 300−400 mm3 in volume, animals (3−5 mice/ group) were intravenously injected with an appropriate dose of OTL38 in PBS. For whole body imaging and biodistribution studies, animals were euthanized after 2 h of administration of OTL38 by CO2 asphyxiation. For time-dependent studies, animals were imaged under anesthesia using isoflurane. Imaging experiments were then performed using IVIS Image. Following whole body imaging, animals were dissected and selected tissues were analyzed for fluorescence activity using IVIS imager, and ROI of the tissues was calculated using Living Image 4.0 software. For ImageJ analysis, the whole body imaging was acquired in gray scale and processed in ImageJ software. Either a line across the tumor or box around the tumor was drawn to define the fluorescence to be quantitated. The tumor-to-muscle ratio was analyzed using a plot of the fluorescence gray value versus distance. Pharmacokinetic Study: Ten nanomoles of OTL38 was administered to female nude mice as a single bolus intravenous injection. Blood was collected at regular intervals (0−180 min), and serumbound OTL38 was quantified by measuring the fluorescence using IVIS imager. The half-life of OTL38 was calculated as %serum bound vs time.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01115. 9644
DOI: 10.1021/acs.jmedchem.8b01115 J. Med. Chem. 2018, 61, 9637−9646
Journal of Medicinal Chemistry
■
Article
(6) Nguyen, Q. T.; Tsien, R. Y. Fluorescence-guided surgery with live molecular navigation − a new cutting edge. Nat. Rev. Cancer 2013, 13, 653−662. (7) Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van de Velde, C. J.; Frangioni, J. V. Image-guided cancer surgery using nearinfrared fluorescence. Nat. Rev. Clin. Oncol. 2013, 10, 507−518. (8) DeLong, J. C.; Hoffman, R. M.; Bouvet, M. Current status and future perspectives of fluorescence-guided surgery for cancer. Expert Rev. Anticancer Ther. 2016, 16, 71−81. (9) Low, P. S.; Kularatne, S. A. Folate-targeted therapeutic and imaging agents for cancer. Curr. Opin. Chem. Biol. 2009, 13, 256−262. (10) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nat. Rev. Drug Discovery 2015, 14, 203−219. (11) van Dam, G. M.; Themelis, G.; Crane, L. M.; Harlaar, N. J.; Pleijhuis, R. G.; Kelder, W.; Sarantopoulos, A.; de Jong, J. S.; Arts, H. J.; van der Zee, A. G.; Bart, J.; Low, P. S.; Ntziachristos, V. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results. Nat. Med. 2011, 17, 1315−1319. (12) Newton, A. D.; Kennedy, G. T.; Predina, J. D.; Low, P. S.; Singhal, S. Intraoperative molecular imaging to identify lung adenocarcinomas. J. Thorac. Dis. 2016, 8 (Suppl 9), S697−704. (13) Tummers, Q. R.; Hoogstins, C. E.; Gaarenstroom, K. N.; de Kroon, C. D.; van Poelgeest, M. I.; Vuyk, J.; Bosse, T.; Smit, V. T.; van de Velde, C. J.; Cohen, A. F.; Low, P. S.; Burggraaf, J.; Vahrmeijer, A. L. Intraoperative imaging of folate receptor alpha positive ovarian and breast cancer using the tumor specific agent EC17. Oncotarget. 2016, 7, 32144−32155. (14) Guzzo, T. J.; Jiang, J.; Keating, J.; DeJesus, E.; Judy, R.; Nie, S.; Low, P.; Lal, P.; Singhal, S. Intraoperative molecular diagnostic imaging can identify renal cell carcinoma. J. Urol. 2016, 195, 748− 755. (15) Hoogstins, C. E.; Tummers, Q. R.; Gaarenstroom, K. N.; de Kroon, C. D.; Trimbos, J. B.; Bosse, T.; Smit, V. T.; Vuyk, J.; van de Velde, C. J.; Cohen, A. F.; Low, P. S.; Burggraaf, J.; Vahrmeijer, A. L. A novel tumor-specific agent for intraoperative near-infrared fluorescence imaging: a translational study in healthy volunteers and patients with ovarian cancer. Clin. Cancer Res. 2016, 22, 2929−2938. (16) Boogerd, L. S. F.; Hoogstins, C. E. S.; Gaarenstroom, K. N.; de Kroon, C. D.; Beltman, J. J.; Bosse, T.; Stelloo, E.; Vuyk, J.; Low, P. S.; Burggraaf, J.; Vahrmeijer, A. L. Folate receptor-α targeted nearinfrared fluorescence imaging in high-risk endometrial cancer patients: a tissue microarray and clinical feasibility study. Oncotarget. 2018, 9, 791−801. (17) Predina, J. D.; Newton, A. D.; Keating, J.; Dunbar, A.; Connolly, C.; Baldassari, M.; Mizelle, J.; Xia, L.; Deshpande, C.; Kucharczuk, J.; Low, P. S.; Singhal, S. A Phase I clinical trial of targeted intraoperative molecular imaging for pulmonary adenocarcinomas. Ann. Thorac Surg. 2018, 105, 901−908. (18) Predina, J. D.; Newton, A. D.; Connolly, C.; Dunbar, A.; Baldassari, M.; Deshpande, C.; Cantu, E., 3rd; Stadanlick, J.; Kularatne, S. A.; Low, P. S.; Singhal, S. Identification of a folate receptor-targeted near-infrared molecular contrast agent to localize pulmonary adenocarcinomas. Mol. Ther. 2018, 26, 390−403. (19) Lee, J. Y. K.; Cho, S. S.; Zeh, R.; Pierce, J. T.; Martinez-Lage, M.; Adappa, N. D.; Palmer, J. N.; Newman, J. G.; Learned, K. O.; White, C.; Kharlip, J.; Snyder, P.; Low, P. S.; Singhal, S.; Grady, M. S. Folate receptor overexpression can be visualized in real time during pituitary adenoma endoscopic transsphenoidal surgery with nearinfrared imaging. J. Neurosurg. 2017, 129, 1−14. (20) Keating, J. J.; Runge, J. J.; Singhal, S.; Nims, S.; Venegas, O.; Durham, A. C.; Swain, G.; Nie, S.; Low, P. S.; Holt, D. E. Intraoperative near-infrared fluorescence imaging targeting folate receptors identifies lung cancer in a large-animal model. Cancer 2017, 123, 1051−1060. (21) Shum, C. F.; Bahler, C. D.; Low, P. S.; Ratliff, T. L.; Kheyfets, S. V.; Natarajan, J. P.; Sandusky, G. E.; Sundaram, C. P. Novel use of folate-targeted intraoperative fluorescence, OTL38, in robot-assisted
SI Tables 1−2, SI Figures 1−8, synthesis of OTL38, procedure for in silico docking studies, and procedure for patient tumor imaging (PDF) Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Sumith A. Kularatne: 0000-0002-7130-8518 Philip S. Low: 0000-0001-9042-5528 Author Contributions ∥
These authors contributed equally to this work
Notes
The authors declare the following competing financial interest(s): S.A.K., P.S.L., C.H.M., M.N., and P.G. are (were) employed by On Target Laboratories during the project. P.S.L. and X.L. are employed by Purdue University, which owns the patents for OTL38. S.M.M. was employed by Purdue University during this project. P.S.L. is one of the co-founders of On Target Laboratories. S.S. is employed by the University of Pennsylvania School of Medicine.
■
ACKNOWLEDGMENTS The authors thank Tim Biro and Sean Bradly for useful discussion involving the manufacturing, stability studies, INDenabling studies, and clinical trials of OTL38. We also thank Dr. Mini Thomas for obtaining excitation and emission spectra for S0456. The authors thank Dr. Alex L. Vahrmeijer, his team, and UNLV Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands, for providing ovarian cancer images taken using OTL38. We acknowledge Dr. Sunil Sinhal, his team, and Division of Thoracic Surgery, Department of Surgery, University of Pennsylvania, Philadelphia, PA, for providing ovarian cancer images obtained using OTL38.
■
ABBREVIATIONS USED FR, folate receptor; FGS, fluorescence-guided surgery; NIR, near-infrared; RA, relative affinity; TAMs, tumor-associated macrophages; MDSCs, myeloid-derived suppressor cells; PK, pharmacokinetics; TBR, tumor-to-background; IND, investigational new drug; FDA, Food and Drug Administration; NOAEL, no-observed-adverse-effect level; GMP, good manufacturing practice; OTL, On Target Laboratories
■
REFERENCES
(1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics 2018. CaCancer J. Clin. 2018, 68, 7−30. (2) Nagaya, T.; Nakamura, Y. A.; Choyke, P. L.; Kobayashi, H. Fluorescence-guided surgery. Front. Oncol. 2017, 7, 314−330. (3) Aliperti, L. A.; Predina, J. D.; Vachani, A.; Singhal, S. Local and systemic recurrence is the achilles heel of cancer surgery. Ann. Surg Oncol. 2011, 18, 603−607. (4) Kelsey, C. R.; Marks, L. B.; Hollis, D.; Hubbs, J. L.; Ready, N. E.; D’Amico, T. A.; Boyd, J. A. Local recurrence after surgery for early stage lung cancer: an 11-year experience with 975 patients. Cancer 2009, 115, 5218−5227. (5) Sandgren, K.; Westerlinck, P.; Jonsson, J. H.; Blomqvist, L.; Thellenberg, K. C.; Nyholm, T.; Dirix, P. Imaging for the detection of locoregional recurrences in biochemical progression after radical prostatectomy-a systematic review. Eur. Urol Focus. 2017, 30257− 30262. 9645
DOI: 10.1021/acs.jmedchem.8b01115 J. Med. Chem. 2018, 61, 9637−9646
Journal of Medicinal Chemistry
Article
laparoscopic partial nephrectomy: report of the first three cases. J. Endourol Case Rep. 2016, 2, 189−197. (22) Predina, J. D.; Newton, A.; Deshpande, C.; Low, P.; Singhal, S. Utilization of targeted near-infrared molecular imaging to improve pulmonary metastasectomy of osteosarcomas. J. Biomed. Opt. 2018, 23, 1−4. (23) Predina, J. D.; Newton, A. D.; Keating, J.; Barbosa, E. M., Jr; Okusanya, O.; Xia, L.; Dunbar, A.; Connolly, C.; Baldassari, M. P.; Mizelle, J.; Delikatny, E. J.; Kucharczuk, J. C.; Deshpande, C.; Kularatne, S. A.; Low, P.; Drebin, J.; Singhal, S. Intraoperative molecular imaging combined with positron emission tomography improves surgical management of peripheral malignant pulmonary nodules. Ann. Surg. 2017, 266, 479−488. (24) Chen, C.; Ke, J.; Zhou, X. E.; Yi, W.; Brunzelle, J. S.; Li, J.; Yong, E. L.; Xu, H. E.; Melcher, K. Structural basis for molecular recognition of folic acid by folate receptors. Nature 2013, 500, 486− 489. (25) Puig-Kroger, A.; Sierra-Filardi, E.; Dominguez-Soto, A.; Samaniego, R.; Corcuera, M. T.; Gomez-Aguado, F.; Ratnam, M.; Sanchez-Mateos, P.; Corbi, A. L. Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 antiinflammatory/regulatory macrophages. Cancer Res. 2009, 69, 9395− 9403. (26) Yang, L.; Zhang, Y. Tumor-associated macrophages: from basic research to clinical application. J. Hematol. Oncol. 2017, 10, 58−70. (27) Yang, L.; Zhang, Y. Tumor-associated macrophages, potential targets for cancer treatment. Biomark Res. 2017, 5, 25−31. (28) Shen, J.; Putt, K. S.; Visscher, D. W.; Murphy, L.; Cohen, C.; Singhal, S.; Sandusky, G.; Feng, Y.; Dimitrov, D. S.; Low, P. S. Assessment of folate receptor-β expression in human neoplastic tissues. Oncotarget. 2015, 6, 14700−14709. (29) Yang, J.; Chen, H.; Vlahov, I. R.; Cheng, J. X.; Low, P. S. Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 13872−13877. (30) Varghese, B.; Vlashi, E.; Xia, W.; Ayala Lopez, W.; Paulos, C. M.; Reddy, J.; Xu, L. C.; Low, P. S. Folate receptor-β in activated macrophages: ligand binding and receptor recycling kinetics. Mol. Pharmaceutics 2014, 11, 3609−3616. (31) Ahn, G. O.; Tseng, D.; Liao, C. H.; Dorie, M. J.; Czechowicz, A.; Brown, J. M. Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 8363−8368. (32) Vatner, R. E.; Formenti, S. C. Myeloid-derived cells in tumors: effects of radiation. Semin Radiat Oncol. 2015, 25, 18−27. (33) Gulec, S. A. PET probe-guided surgery. J. Surg. Oncol. 2007, 96, 353−357. (34) Kamiya, M.; Urano, Y. Rapid and sensitive fluorescent imaging of tiny tumors in vivo and in clinical specimens. Curr. Opin. Chem. Biol. 2016, 33, 9−15. (35) Mohamed, M. M.; Sloane, B. F. Cysteine cathepsins: multifunctional enzymes in cancer. Nat. Rev. Cancer 2006, 6, 764− 775. (36) Mochida, A.; Ogata, F.; Nagaya, T.; Choyke, P. L.; Kobayashi, H. Activatable fluorescent probes in fluorescence-guided surgery: practical considerations. Bioorg. Med. Chem. 2018, 26, 925−930. (37) Lacivita, E.; Leopoldo, M.; Berardi, F.; Colabufo, N. A.; Perrone, R. Activatable fluorescent probes: a new concept in optical molecular imaging. Curr. Med. Chem. 2012, 19, 4731−4741.
9646
DOI: 10.1021/acs.jmedchem.8b01115 J. Med. Chem. 2018, 61, 9637−9646
